Hemp Growing

Over-expression of WRKY2 in wheat enhanced the tolerance of transgenic wheat to drought stress,while over-expression of WRKY44 and WRKY46 enhanced the tolerance of both drought and salt stress,respectively.In addition,it is reported that WRKYs can also participate in the response of hormone signals.For example,salicylic acid  can activate the expression of WRKY70 transcription factor,while jasmonic acid  inhibits its expression.ABA can induce the WRKY gene to regulate the abscisic acid and gibberellin signaling pathway.Previous studies have proved that WRKY genes can regulate stress,respond to hormone signals to affect plant growth and development,and also plays an important role in fiber development.Gibberellins,as a type of important plant growth regulator,play a key role in fiber development and stem elongation.However,there is no report on the WRKY  gene in seed hemp so far.It is imperative to identify and analyze WRKY transcription factors in seed hemp based on its whole genome and transcriptome data.The aims of this study were to identify CasWRKY genes at the whole genome-scale,analyze its expression patterns in different tissues,and analyze the effect of CasWRKYs on plant height according to GA3 stress in seed hemp.The diversity of gene structure can reflect the evolution of polygenic families.To gain the structural evolution,the exon–intron structures of the CasWRKY genes were determined by comparing the cDNA sequences of WRKYs with their corresponding genomic DNA sequences and then visualized by GSDS  program..Gene structure analysis showed that the number of exons ranged from 2 to 6.More than half number  of the CasWRKY genes had three exons.This indicates that exon loss and gain events occurred in the CasWRKY gene family during evolution,which may be the reason for the functional diversity among CasWRKY genes.

In addition,it was found that CasWRKY genes of the same group had similar exon/intron structures.Members of II-c and II-e had fewer exons and simple structures,while,members of I、II-a and II-b had many exons.To gain insights into the conservation and diversification,marijuana grow system motif structures on 39 CasWRKY proteins were constructed using the MEME program.A total of 10 conserved motifs were identified in the 39 CasWRKY protein sequences.The length of ten identified conserved motifs of CasWRKYs ranged from 6 to 50 amino acids.The number of motifs in CasWRKY proteins ranged from 5 to 15.As expected,most CasWRKYs that have been observed to be in the same group or subgroup usually shared highly similar motif compositions.Among them,motifs 1 and 3 contained the heptapeptide stretch WRKYGQK,with motif 3 being present only in group I.Motifs 1 and 2 were distributed all over the groups and motif 2 was immediately downstream of motif 1.In addition,five genes have similar protein lengths and same motif distributions.Motif 8 was present only in groups II-a and II-b,while Motif 6 was found mainly in subgroup II-B.Motif 4 was present in most genes in groups I and II-c.Furthermore,although CasWRKY27 belonged to subgroup II-d,it had similar conserved motifs with subgroup II-c.Totally,the analysis of CasWRKY motifs showed that every group or subgroup of CasWRKYs had similar motif compositions,which further validated the taxonomic and phylogenetic relationship ofCasWRKY.The cis-acting elements in the promoter region are essential for gene expression.The 2000 bp upstream promoter regions were searched using Plant CARE,and it was found that there are many types of cisacting elements.Among them,twenty cis-acting elements contain hormone-related elements,promoter-related elements,stressrelated elements,development-related elements.the 20P-box  were distributed on the promoter region of 13 CasWRKY genes.The genes that contain P-box were concentrated in group I,group II-b,group II-c,III.Particularly,CasWRKY05 promoters had four gibberellin-responsive elements.In addition,cis-acting regulatory elements involved in light responsiveness were analyzed.17 CasWRKY promoters contain part of a light-responsive elements,which are cis-regulatory elements essential for light induction.It was noted that the three genes  contain the common cisacting elements in promoter and enhancer regions.These results showed that most of CasWRKYs might play a crucial role in multiple stress responses in seed hemp.To investigate the plant in response to hormones at the growth boom in seed hemp,several phenotype responses induced by hormones were studied.

Changes in plant height after treatment with different exogenous hormones were shown in Fig.S.1.After the first exogenous hormone treatment,the plant height changes of different concentrations of GA3 treatments were significantly different.After 5 consecutive treatments,the plant height variation of high concentration  GA3 treatment was significantly higher than that of the other two low concentration GA3 treatments and other exogenous hormones.No significant phenotypic differences were observed after treatment with exogenous hormones IAA and BR compared with the control.These results showed that GA3 at different concentrations could promote the plant height of “Yushema”,while BR and IAA had no significant effect.The above results indicated that “Yushema” was a gibberellin-sensitive variety.Since bio-active GA3 plays the key roles in the development of the stem,the RNA-seq data sampled from different time after GA3 treatment were used to study the differential expression pattern of CasWRKY genes under the GA3 stress.At 24 h of GA3 stress,most genes showed different expression levels and expression patterns,but some genes in the same subfamilies showed the same expression patterns.The expression levels of CasWRKY01,CasWRKY02,CasWRKY06,CasWRKY24,and CasWRKY29 were significantly up-regulated at 3 h after GA3 treatment.Moreover,thirteen genes exhibited the opposite trend with exposure to GA3 stress.In addition,among these 39 CasWRKY genes,the transcription levels of 4 CasWRKY genes s were continuously up-regulated in the stem barks at different time points under the GA3 stress.It should be noted that the expression of CasWRKY09 was significantly down-regulated after GA3 treatment,and then gradually up-regulated.Interestingly,nine  out of the 39 CasWRKY genes were not expressed at all-time points,indicating that they might be pseudogenes or may not be involved in expressed in the stem barks.These results suggest that CasWRKY genes in seed hemp could respond to GA3 stress and form a complicated network to regulate hormone regulatory network.To verify the accuracy of RNA-seq data,9 CasWRKYs were randomly selected for RT-qPCR analysis.The results showed that the RT-qPCR assays of the expression patterns of the 9 CasWRKY genes were similar to the results of FPKM.As one of the largest transcriptional regulatory factor families in plants,WRKY gene family plays important role in plant growth and development and abiotic stress.To date,WRKY genes have been widely studied in a variety of plants,but similar studies have not yet been performed in seed hemp.we identified a total of 39 WRKY genes in the whole genome of seed hemp.The number of WRKY genes in seed hemp is significantly lower than that in A.thaliana ,soybean ,cabbage.hypogaea ,and cotton ,close to that in E.ulmoides ,C.capsularis ,sugar beet ,and canola.

By comparing with different species,it was found that the number of WRKY genes in different species is not directly related to their genome size.Previous studies believe that gene replication,fragment replication,and whole-genome replication play crucial roles in the large-scale expansion of gene families.With the continuous progress of high-throughput sequencing technology and the continuous optimization of data analysis methods,WRKY gene family members have been comprehensively studied.In this study,we conducted a comprehensive genome-wide analysis of WRKY gene family based on the seed hemp genome for the first time.Therefore,the results of this study will contribute to further functional analysis of WRKY genes in seed hemp and provide a reference for gene family analysis of other species.In Arabidopsis,a widely accepted WRKY gene family classification system was established by analyzing the genome data of WRKY gene family.According to the classical classification on the basis of sequence alignment and phylogenetic tree analysis,39 WRKY proteins identified in seed hemp were divided into three main groups,and group II of proteins was further clustering into five subgroups.Among the 39 CasWRKY proteins,there are five proteins in Group III,eight proteins are in Group I,and the remaining 26 CasWRKY proteins belong to group II.This classification results were similar to that in Eucommia ulmoides,Brassica napus  and Brassica rapa,and the maximum numbers of group II proteins in this gene family,which means that this group may have experienced more gene duplications during the process of evolutionary.However,group III has the largest number of WRKY genes in rice,while group I has the largest number in Arabidopsis andpoplar.In this study,three domain loss events were found and concentrated in group III,which is a common phenomenon in the plant WRKY gene family and is considered to be a divergent force for the expansion of this gene family.In addition,it was also found that majority of the CasWRKY genes contain a highly conserved heptapeptide WRKYGQK motif followed by a C2H2 or C2HC type zinc finger motif except for several variants.WRKYGQK,a highly conserved domain,was replaced with WRKYGKK in CasWRKY20 and CasWRKY21.Both genes belong to subgroup II-c.Similar phenomena have been reported in Arabidopsis and cucumber.Previous reports have shown that the WRKYGKK domain failed to bind to W-box,and it needs further verification about whether CasWRKY20 and CasWRKY21 can bind to the W-box.

In addition,previous studies indicated that variations in the WRKYGQK motif of WRKY domain might influence normal interactions of WRKY genes with downstream target genes,cannabis vertical farming and therefore the molecular mechanism of these family members in plants still needs to be further explored.During the process of evolution,tandem and segmental duplication contribute to the expansion of the WRKY gene family.These replicated WRKY genes may form new gene functions to adapt to various conditions required in the process of plant growth and development.Chromosomal distribution and synteny analyses revealed that six  tandem duplication events with 12 CasWRKY genes and five segmental duplication events with 10 CasWRKY genes were observed.The number of tandem duplications was more than segmental duplications,which increased the number of members in the WRKY gene family of seed hemp.Therefore,it could be deduced that tandem and segmental duplication were the main evolutionary driving force of the WRKY gene family in the evolution of seed hemp.In general,analyzing the distribution of introns and exons can help us better to understand the phylogenetic relationship,because they play crucial rolse in the evolution process of many gene families.Here,the comprehensive analysis of the distribution and length of exons and introns of CasWRKY genes were conducted.By analyzing the gene structures of CasWRKYs,it was found that exon loss and gain events occurred in the evolution of CasWRKY gene family,which may increase the functional diversity of genes in the gene family of CasWRKY.Cis-acting elements of promoters are essential for gene transcription and expression.Twenty cis-acting elements involved in hormone-related elements,promoter-related elements,stress-related elements,development-related elements,and cis-acting regulatory elements involved in light responsiveness were analyzed.It should be noted that 20P-box  were distributed on the promoter region of 13 CasWRKY genes,the genes that contain these response elements are concentrated in group I,group II-b,group II-c,group III,and CasWRKY05 promoters had four gibberellin-responsive elements.These results suggest that these genes may produce corresponding expression responses under gibberellin stress,which will be conducive to the functional study of CasWRKYs.It is well known that gene expression pattern is closely related to gene biological function.In the present study,all 39 tested CasWRKY genes were found to be expressed in at least one of those tissues,except that CasWRKY22 and CasWRKY39 were not expressed in the three tissues examined and might be expressed in other tissues.

In addition,some CasWRKY genes showed preferential expression in all tissues tested.For example,six genes have high expression levels in stems and are mainly concentrated in group I.The CasWRKY genes which exhibited preferentially expression among different tissues are likely to have a unique role in organ development.In addition,CasWRKY15 was tissue-specific and is expressed only in stems.Its orthologous gene Atwrky12 in Arabidopsis has been reported to participate in the formation of secondary cell walls which could significantly increase plant biomass.These results suggest that the expression of WRKY transcription factor may be conducive to the regulation of fiber development,at least there is a certain relationship between them.WRKY transcription factors not only play vital roles in plant growth and development but also play key roles in response to various hormone regulations,which provides a favorable guarantee for crop genetic improvement.Phytohormones play an irreplaceable role in plant cellulose biosynthesis.As a key hormone,gibberellin participates in a variety of regulatory processes of plant life activities and almost accompanies entire plant life.Gibberellin can greatly affect fiber initiation,differentiation,and development,and promote fiber elongation by inducing more fiber initial cells.In this study,spraying gibberellin on plants of “Yushema” significantly promoted the elongation of plant heights,which proved that “Yushema” was the typical gibberellinsensitive germplasm.Therefore,the expression patterns of 39 CasWRKYs under GA3 stress were systematically analyzed.Expression pattern showed that most genes showed different expression levels at 24 h of GA3 stress,but some genes in the same subfamilies showed the same expression patterns.

Hemp’s central woody portion contains 36% cellulose and 27% lignin,whereas bast fiber contains 72% cellulose and 4% lignin.The whole hemp stem contains 47% cellulose and 18% lignin,which is more favorable than pine and birch wood.Hemp stalk contains the highest percentage of cellulose,with the lowest lignin content over almost all non-woody stalks.However,hemp bast fiber secures second for alpha-cellulose after cotton.The lignin and cellulose content in hemp stalks considerably vary among cultivars and growing seasons.The morphology and low lignin content of hemp bast fiber allow easy penetration of processing chemicals resulting faster pulping process with less harsh chemical use.Hemicellulose enhances swelling of the pulp and bonding capacity among the fibers and provides the necessary strength to paper.The hemp core has a higher ability to supply alpha-cellulose to fibrous suspensions and a similar amount of hemicellulose than that of kenaf and hardwood.Hemp fiber exhibited high yield and high tear strength of paper obtained by organosolv pulping process due to having long fibers in pulp.It allows a safer oxygen delignification process with a higher yield than that of birch or pine pulps.The organosolv pulping uses organic solvents to solubilize lignin and hemicellulose,which avoids environmentally harmful sulfur used in conventional pulping techniques,offers high-quality lignin recovery,and easy removal of solvents with less water consumption compared to kraft pulping process.The organosolv process,trim tray for weed which utilizes ethanolamine,produces high quality hemp core pulp.

Moreover,hemp paper processing does not require harmful chlorine bleach; instead,it can use peroxide,which is safer for the environment.In searching for a sustainable pulping process from non-wood feedstock,BioRegional MiniMill technology has been claimed as a promising zero-emission environmentally friendly process for small-scale production.Alkaline pulping of woody hemp core enhances fibrillation and forms inter-fiber bonding which imparts strength in paper and can produce smooth printing grade paper as produced from straw and hardwood pulp.A combination of hemp hurd pulp with hardwood exhibited improved tensile index,bursting strength,softness with identical water absorbency of hand tissue.Blending hemp fiber or woody core with pine and eucalyptus demonstrates higher fiber length in pulp,hence the sufficient paper strength.In most cases,only hemp bast fibers are used for paper-making purposes because of outstanding tearing strength,although lower tensile strength.The successful use of hemp as paper-making materialdepends on the technical viability of exploitation of both fiber and woody core,not simply one since their differences in morphology and chemical compositions offer suitability for paper-making raw materials.The bleached kraft pulps from hemp stalk were found useful raw material than hemp bast fiber or woody core alone .Organosolv pulping process of whole hemp stalks exhibited the strength between commercial hardwood and softwood pulps.Hemp with core and sheath showed a slightly higher kappa number  than eucalyptus,indicating that it is more difficult to delignify in kraft cooking process due to lower degree of core fiber polymerization.Strengths are affected due to low hemicellulose content and a lower degree of internal fibrillation.Hemicellulose helps the pulp sheet by strengthening its tensile,bursting,tear strength,specific surface,folding endurance and opacity.The elementary chlorine-free bleaching method with enzyme treatment is not suitable for hemp stalks since it deteriorates the physical properties.It was suggested that separate pulping of fibers and cores followed by blending could yield a pulp with desired properties for multipurpose applications.

Unlike wood paper,hemp papers survive hundreds of years and do not get into decomposition and yellowing due to aging.Hemp paper’s high tear strength and wet strength make it ideal for currency paper,art paper,cigarette paper,tea bags,specialty non-wovens,carbon tissue,grease-proof paper,and other applications.The paper made from hurds can also be used as low-grade printing paper without compromising the quality of the printing surface.Hemp paper showed better oil/air filtration performance than cotton paper in practical application.As a non-wood fiber,hemp also has considerable shortcomings for industrial-scale paper making.Existing paper-making technology is not well capable of handling non-woody crops,and it significantly decreases productivity.In most cases,fibers are separated from the woody core and go through the process separately.The processing technology of hemp woody core and even the separation method of bast fibers must be introduced with state-of-the-art knowledge to reduce costings.Technological advancement is also required to process the whole stalk without separation of fibers and woody core for achieving more yield.Since hemp is an annual crop,and its stalks are very bulky,some difficulties are associated with transportation,storage capacity,and smooth handles.Long-time storage can damage the fibers and cause color change.Pulp quality and yield is related to morphology and chemical composition; they can be affected by harvesting time,geographic location,plant breeding,and even the variation are observed within the biomass,which may lead to a possible lack of consistency to paper quality.The globally produced agricultural residue could produce five times more paper than demand,but technology fails to exploit those in the paper industry.According to the study on different pulping methods of hemp bast fiber,it is found quite acceptable in place of wood from the technical point of view,and even woody cores can also be a promising raw material for paper.Hemp pulps are generally used by mixing with wood pulp,and currently,100% hemp paper production is not seen.Hemp is speculated to take the opportunity to outvie the other non-wood plants by its yield and grow ability in moderate or boreal climates.Easy pulping,good quality bleached paper and specialty papers are the key advantages of hemp.Hemp,the second largest grown bast fiber after jute,has gained considerable attention as a reinforcement in the polymer matrices in the last decade due to its renewable,biodegradable,and recycling properties.

Several composite processing methods have been investigated,including hand lay-up,film stacking,vacuum infusion,manual winding,filament winding,resin transfer molding,pultrusion and injection molding,to name a few.Hemp fiber composites with thermoset,thermoplastic and biodegradable matrices have demonstrated strong mechanical properties due to higher tensile strength of fiber up to 1110 MPa.Table 2 summarizes some important physical and mechanical properties of vegetable-based natural fibers.Thermoplastic matrix composites perform better over thermoset matrix composites concerning high specific strength,corrosion resistance,cost efficiency,recyclability and design versatility.But the main drawback of natural fiber-reinforced thermoplastic composite is that their processing temperature needs to be kept below 230 C to protect them from thermal degradation .Hemp fiber begins to degrade at a temperature above 150  C,hemicellulose and pectin decompose at around 260 C,while cellulose decomposes at about 360  C .Hemp fiber-reinforced thermoplastic composites are typically made of polyethylene,polyurethane,or polypropylene matrices.It shows the potential to replace synthetic fiber composites in many lightweight and low-cost applications .The frequently used thermoset matrices for hemp fiber-reinforced composites are epoxy resin,phenolic,vinyl ester,or unsaturated polyester resin.Composites made from hemp fiber with thermoset matrices are creep resistant,solvent resistant,and tough.W€ otzel et al.investigated a life cycle study on materials reinforced with hemp fiber against ABS  for car parts.Their study revealed that cumulative energy demand for producing hemp composite was half compared to the ABS basic component and hemp composite for an inner lining of cars showed more ecological benefits.Green composites are referred as bio-composites,the combination of natural fibers with biodegradable polymeric materials.Researchers focused on green composite due to dwindling fossil fuel resources and their negative impacts on environment.The biodegradable polymer matrices such as epoxidized soy oil ,acrylated epoxidized soybean oil,starch-based emulsion,cashew nutshell,euphorbia oil,cellulose acetate and polylactic acid were used to develop hemp fiber-reinforced green composites in number of studies and outstanding mechanical properties for primary structural applications were observed.Though cost and service longevity due to decomposition in nature are associated as drawbacks with green composites further study may open new door to overcoming these barriers.

Mechanical properties of fiber-reinforced composites strongly depend on fiber length,diameter,orientation,degree of dispersion,aggregate formation and fiber-matrix compatibility.Hemp is susceptible to thermal and oxidative degradation during processing.Another key impediment to manufacturing hemp-reinforced composites is the inability of hydrophilic lignocellulose fibers to adhere to hydrophobic matrices.Due to their polar surface character,natural fiber reinforcement shows lower compatibility with strongly apolar thermoplastic matrices.Again,vapor and void creation during processing due to moisture content,photodegradation due to UV radiation,and poor resistance to moisture can significantly affect the performance of the composite for outdoor application.A summary of the performance of hemp composite against moisture can be seen in Table 3.Hemp composites absorb moisture,and when dry,the constituent fibers shrink rapidly.Thus interfacial bonding of composites no longer can retain due to cyclic absorption and desorption of moisture and eventually debonding cracks occur inside the structure which allows room for further water penetration.The schematic diagram of the mechanism is illustrated in Figure 2.Alongside the formation of cracks,moisture uptake also encourages colonial fungal growth that further accelerates the degradation of the composite.Several researchers studied the relationship between fiber fraction and amount of moisture absorption and concluded that for natural fiber,moisture absorption increases with increasing fiber loading.For solving these inherent limitations,many studies were carried out on physical and chemical modification of natural fibers to enhance surface characteristics and effectiveness as a reinforcement material.Coupling agents were used in some experiments in chemical modification and better compatibility was achieved between cellulose fibers and hydrophobic polymers.Treating hemp fibers with methacrylic anhydride increases interfacial adhesion between hemp and polyester matrices.Mwaikambo and Ansell found that hemp fibers with a 4% & 6% alkalized treatment had the highest modulus and tensile strength.Propionylation and acetylation treatments on hemp fibers resulted in a reduced hydrophilicity but at the same time,decreased crystallinity slightly.The Scanning electron microscope  results  showed that the esterified materials’ surfaces were smoother than the untreated hemp fibers.Dayo et al.studied the influence of different chemical treatments on hemp composite and recorded the lowest water absorption for silane treated fiber.The same author also reported a similar result from another experiment where only washed,trimming tray weed alkaline treated and silane treated hemp fibers were compared.

Among the resins,Polybenzoxazine polymers showed better resistance against moisture absorption with additional advantages like good mechanical and thermal properties.Oza and Lu investigated the effects of silane and NaOH treatment on the thermal and thermomechanical properties of hemp fiber-reinforced high-density polyethylene composites.They observed that thermal stability decreases as fiber loading increases,and treated fiber composites show higher thermal stability than untreated fiber composites.The storage modulus of treated composites was higher than that of untreated composites in dynamic mechanical analysis.Up to 40% fiber loading,the storage modulus value increased,while it dramatically decreased at 50% fiber loading.It was found that the storage modulus of Silane-treated composites was higher than NaOH-treated fiber composites.A similar achievement was also reported by Dayo et al..A summary of hemp composites’ mechanical,thermal and thermomechanical performance can be seen in Tables 4 and,respectively.Hemp fibers have significant advantages over synthetic fibers in reinforcing composites and can be used efficiently for a variety of applications because of their high specific strength,low density,low production cost,bio-renewable nature and eco-friendly behavior.The applications of hemp-reinforced composites had been traced in the automotive industry in the 1940s,where Henry Ford produced car components from hemp fiber with soybeans-based bio-matrices.It is predominantly used in the automotive sector to reinforce door panels,passenger rear decks,pillars,and boot linings .Compared to other natural fibers,its uses have remarkably increased in the German and Austrian automotive industries.Due to the higher vibration damping capacity of hemp fibers,researchers also focused on hemp composites in manufacturing sporting goods and musical instruments.Claudio and Marco developed electronic racks for the helicopter by utilizing hemp fabric/epoxy composite materials.The study revealed that this electronic rack from hemp composite was 55.6% lighter than existing steel electronic racks.Hemp fiber reinforced with polycaprolactone composites proved their potential application in fabricating orthotic devices .Hemp chair was developed from hemp yarns with epoxy resin,and Xia et al.innovated a hybrid composite from hemp fiber mats and aluminum sheet with epoxy resin which offered excellent electromagnetic interference  shielding performances.The term “plastic” refers to a material’s flflexibility or ability to deform into any shape without breaking.Plastic is a carbon chained polymer allowing it to be molded into any shape; that is why they are the most adaptable material.The majority of monomers used to make plastics like ethylene and polypropylene are derived from fossil fuel hydrocarbon.As a result,they are neither biodegradable nor easily decomposable; instead,they accumulate in the landfill and the natural environment.According to literature,roughly 9% of all plastics produced are recyclable,while the remaining 79% end up in landfills and the atmosphere.Petroleum-based plastics and its by-product have a devastating effect on the land,water,and wildlife.For this reason,increasing demand has been started in the world for the usage of high-performance bio-based plastics capable of being environmentally friendly and compensating depleting of petroleum resources.Hemp plastic which is 100% biodegradable,can be a better alternative to synthetic plastic.The cellulose of the hemp plant is rated 60–70%,which can be extracted for making a different range of plastics,including rayon,celluloid and cellophane.

All cannabinoids are structurally very similar and thus the individual solubility of a particular cannabinoid  is possibly also dependent on the concentration of other cannabinoids in the water.Zoller et al. detected 1 mg/l THC analyzing the infusions from herbal hemp samples containing 1,250 μg/g THC.Theoretically adjusting their THC content in leaves to ours would correspond to 0.063 mg/l THC in the tea.The tea prepared from a drug-type C.sativa with a THC content of 6 mg/g contained 10 mg THC per liter tea.Again adjusting their THC leaf content to our THC leaf content would correspond to 0.132 mg/l THC in the tea.In another study of a fiber-type hemp,0.040 mg/l THC were detected in an infusion from hemp leaves containing 80 μg/g THC,so the same amount of THC as in our leaves.The same level of THC in the tea  were found from a fiber-type hemp tea with 15 μg/g THC in raw material.That comparisons demonstrate the difficulty of analysing lipophilic compounds in herbal teas and the overestimation by concluding from herbal residuals to compounds indeed present in the tea.In view of these results and relatively little conversion of THCA to THC even at higher water temperatures,the assumption of complete transfer of THC from the starting material into the tea – as practiced by the German BfR to overcome analytical uncertainties  – must lead to a substantial overestimation of the true THC content of infusions from hemp leaves.Even our approach,- analyzing the residual THC content in the hemp leaves after extraction and assuming the difference to have migrated into the tea,is likely still an overestimation of the true THC concentration in the infusion but at least allows a more realistic estimation of the concentration that may at worst be present in the tea.

Certainly the approach is preferable to the proposed and entirely unsubstantiated assumption of total transfer.The overall difference of cannabinoids that can be observed between tea leaves before and after tea preparation may be the combined effect of cannabinoids dissolved in water,grow tent for sale cannabinoids adsorbed onto particles suspended in solution,cannabinoids undissolved or precipitated,degradation of cannabinoids and cannabinoids bound to the pot/cup surfaces.Garrett and Hunt  determined the binding of THC on a glass surface of a 50 ml flask to 20% and 40% at 0.1 and 0.05 μg/ml,respectively,figures indicating a binding saturation at rather low cannabinoid levels.Compared to the high levels of cannabinoids in our experiment,surface binding was not a significant factor.However,this part should not be neglected in further studies of transfer rates from plant material into a specific matrix.Thus,the concentration of a cannabinoid that can be determined in solution will be the result of a dynamic equilibrium between dissolution and phase joining.Cannabinoids may be adsorbed back out of solution onto plant material or the inner surfaces of cups or pots.Considering all of these factors that potentially influence analyses,estimation of lipophilic amounts in tea based on differences in plant residues before and after tea brewing are at best approximations.Nevertheless,the values received may serve as useful reference for further improvements in herbal tea analyses and a realistic basis for risk assessment in food control.Modern civilization has extensive utilization of multiple pharmaceutical drugs such as Non-steroidal anti-inflammatory drugs  for the reprieve of pain,as analgesics and antipyretics,sex hormones,antiepileptic,blood lipid-lowering and b-blocker agents.NSAIDs are the class of drugs that are used more abundantly because these are over the counter drugs and can be easily purchased from the market without specifific prescription.More common drugs in this class are acetylsalicylic acid,paracetamol,ibuprofen,naproxen and diclofenac.Paracetamol is a white crystalline solid and is used as a mild painkiller and for temperature reduction in case of fever.

Moreover,many common pharmaceuticals are available with extensive utilization in medical care having paracetamol as a base ingredient and are used with different formulations and considered safe,except for high dosage.The structural formula is given in Scheme 1.As the use of these drugs is unavoidable and these pharmaceutical compounds are excreted in urine and other biological wastes as active metabolites,either directly or indirectly,in high fractions.These wastes are constantly being discharged into municipal waste waters which results in contaminated aquatic surroundings,surface and ground waters,and finally into the drinking water supplies.Despite their very low concentrations,these are hazardous for human beings especially for infants,and cannot be removed employing conventional water treatment techniques such as chlorination.Advanced oxidations,reverse and forward osmosis can be used to remove these contaminants but these processes are expensive; hence,large scale application for municipal water treatment is uneconomical.Up to now,membrane filtration,UV-degradation,ultrasonic degradation and electrochemical degradation are the reported processes for the removal of NSAIDs from surface or drinking water.The combination of catalytic decomposition along with ultrasonic degradation was studied by Soltani et al.,.Results elucidated that the dispersion of stone waste  improved the pore volume and specific surface area of ZnO catalyst which significantly improved the paracetamol degradation efficiency up to 98.1%.Mirzaee et al.,investigated the electrochemical decomposition of paracetamol in an ultrasound environment.Using Iron anode improved the degradation potential of the modified hybrid process  as compared to individual processes.However,catalytic and electrochemical processes have some limitations and are preferably considered for low volume and specified treatment processes.Furthermore,the conventional 2D electrochemical reactors have the limitations of low mass transfer.Therefore,the oxidation/reduction of compounds takes place at the surface of the electrode which increased the energy consumption.

Additionally,chlorides are generally present in wastewater,and oxidation of these chlorides produces chlorine  and hypochlorites.These compounds are reactive toward organic matters and produce chlorinated derivatives which are toxic and persistent pollutants.Whereas the adsorption process is more selective since it has design flexibility,simplicity,easy operation,and doesn’t need any specialized human resources.The application of biospecies derived adsorbents for the removal of different contaminations has long been used.AC impregnated with carbon moieties has a changeable aptitude to form a complex with other molecules.The procedure of activating results in particles with minute pores,extended surface area,well developed pore structure,and active surface properties.These key features developed the ability to adsorb smaller organic compounds competently.Results reported by Mestre et al.,showed that the adsorbent prepared from waste biomass is more selective for the removal of ibuprofen whereas Terzyk et al.,reported the surface modification with H2SO4 increased the drug removal efficiency as compared to HNO3 and NH3.The surface modification of AC prepared from olive stones with H3PO4 improved the active surface area up to 1000 m2 /g due to the well-developed porous structure.The recorded adsorption capacity for paracetamol removal was 100 mg/g which slightly decreased with an increase in temperature.It was observed that the adsorption capacity and drug removal efficiency of the adsorbent is generally influenced by the system pH and increased with the hydrophilicity of the adsorbent material.Therefore,the adsorption of paracetamol on AC has an inverse relation with pH,and the removal capacity of paracetamol increased to 560 mg/g at 25℃ by decreasing the pH to 3.Kinetic investigation elucidated that decrease in pH increase the paracetamol ionization as well as the surface charge of the adsorbent.Furthermore,it has been proved that materials with well-interconnected pore networks have high drug removal efficiencies.Adsorptive removal of paracetamol with dextropropoxyphene hydrochloride,N-acetylcysteine,and sorbitol were studied using activated charcoal.The vitro adsorption of paracetamol using Langmuir Isotherm showed 96.6% adsorption at a charcoal-drug ratio of 6:1,and a slight increase of 2 % and 2.9 % was noted by varying the ratio to 8:1 and 10:1,respectively.

The incorporation of magnetism eases the recovery and reusability of the adsorbent materials.Synthetically prepared nanocomposite when applied for the removal of paracetamol gives the 399.9 mg/g adsorption capacity with 96.8% removal efficiency at 50℃.The impact of H3PO4 concentration  on chemical modification and adsorption capacity was investigated employing AC prepared from kenaf by Macı´as-Garcı´a et al.,.The maximum adsorption capacity of paracetamol  was noted when 60% H3PO4 was used because of the highest active surface area of 2270 m2 /g.Therefore,it can be concluded from the above discussion that the selection and optimization of reactive agents for chemical modification is a crucial factor for designing an adsorption process.It is mostly observed that mineral acids are highly efficient for AC modification; however,the concentration is case sensitive.Wastewater treatment is mostly considered an added expense; hence,the cheapest process is always selected.Industries,as well as communities,are continuously struggling to adopt economical and robust treatment methodologies.Therefore,investigation of cheaply available adsorbent and economical routes for the substraction of paracetamol is the objective of this research.Cannabis Sativum Hemp  was selected as a natural cellulosic material for the manufacturing of AC.CSH is an annual herbaceous crop that has been cultivated by mankind for millennia for its fibers and seeds.It is the most abundant drug of abuse cultivated worldwide for psychoactive cannabinoids,durable fiber,rope,canvas,textiles,paper,and nutritious seed for oil,and can be divided into psychoactive and non-psychoactive cultivars according to the ratio of D9-tetrahydrocannabinol  and cannabidiol  present in it.As a crop,it is categorized as drug type,Intermediate type,and fiber type.This study was aimed at using CSH for the manufacturing of the adsorbent through thermochemical treatment.The developed adsorbent was used to remove the paracetamol from the aqueous solution.Process efficiency was optimized by studying the consequences of experimental features,such as pH,size of the particle,temperature,contact time,stirring speed and adsorbent dose.Kinetic,isothermal,and thermodynamic analyses were performed for in depth understanding of the process mechanism and equilibrium.Adsorption is a surface phenomenon and adsorption efficiency is strongly affected by the particle size of absorbent,similar to other surface induced reactions.Generally,indoor grow tent the adsorption efficiency is inversely proportional to the particle size of the adsorbent material.The smaller the particle size,the greater would be the efficiency of adsorption because the particles having a small size have a larger capacity to absorb the drug due to their larger total surface area.The size of particles of the adsorbent was varied from 50 mm to 150 mm to check the effect of particle size on paracetamol removal efficiency and the results are shown in Fig.3A.

The best result was shown by the particle size of 74 mm with 40% removal efficiency.Results elucidated that adsorption efficiency increases with a decrease in particle size up to a certain limit and then remains constant after achieving the peak value.The particle size of 74 mm was selected as the optimum size and used in the rest of the experiments.Adsorption is a time based process and impact of time can be positive or negative depending upon the process dynamics.This experimental evidence provides us the information about the minimum time required to achieve the adsorption equilibrium.Contact time varied from 30 min to 480 min and obtained results are provided in Fig.3B.As evident from fig.3B,a relatively low adsorption rate was observed during the first two hours of the process which accelerated after 120 min,and equilibrium was ratified in 240 min.Thereafter,no further increase in the sorption rather desorption was noted.This phenomenon can be explained by the fact that initially,more adsorption occurred due to the availability of more active surface sites that decreased with adsorption proceeding.After 240 min saturation occurred.Hence,further adsorbent molecules could not be entered into the inner surface due to a high degree of saturation.This sluggish adsorption illuminated an increased competition of adsorbate for the adsorption sites.Hence sorption rate decreased with time.Provision of the adsorbent required proper selection because the availability of active adsorption sites and active surface area is a crucial parameter that affects the removal efficiency significantly.The effect of adsorbent dose on the sorption phenomenon was investigated by varying the amount of AC from 0.02 g to 0.12 g while keeping all the other parameters constant.The results elucidated that sorption efficiency and adsorption capacity  varied inversely.An increase in sorption efficiency with an increase in sorbent dose was recorded whereas adsorption capacity decreased from 11.0 mg/g to 8.0 mg/g.An increase in the amount of adsorbent increased the number of active sites which is reciprocated in the value of S% increases.However,all the sites are not readily available for binding the drug molecules,thus overlapping in between the sites occurs.As a result,drug uptake capacity  decreased with increasing adsorbent doses.Additionally,low drug uptake can also be attributed to the low availability of drug molecules as compared to the available adsorbent surface because of high dosage.The relation between the rate of agitation and adsorption efficiency was investigated by navigating stirring speed from 100 rpm to 300 rpm.Results elucidated that the highest sorption efficiency  was achieved with 200 rpm.

The increase of iron and magnesium content in the studies by Naumova et al.  was similar to our results despite the much higher amount of hemp flour used , which again proves that the hemp flour differs between the producers. It was assumed that hemp protein or hemp flour due to their high protein content could improve the products’ water holding capacity similar to soy proteins. However, the cooking losses were comparable with the other hemp ingredients tested. A possible reason may be the presence of edestin, which is an abundant protein in hemp. The typical pH of meat is 5.6–6.0 and it was proved that edestin aggregates in pH lower than 7.0 , therefore its water holding capacity or fat absorption capacity may be low in meat batters. The poor functional properties of hem protein isolate resulting from protein aggregation are probably caused by the presence of covalent disulphide bonds between proteins and high free sulfhydryl content . The slightly lower cooking losses in the meat loaves containing hemp ingredients may be still a promising result from a technological point of view. The hardness of the meat loaves increased in all of the samples containing the hemp ingredient, except for the DHS product which was comparable with the control. As this was the only variant in which the hemp shell is almost fully removed it may be assumed that any constituents increasing the water holding capacity are located in the hemp shell. No other TPA parameters were affected by the hemp ingredients . The texture parameters changes depend on the type and amount of ingredient used and the interactions between the additive and the meat batter. Walnut green husk addition increased the hardness of the meat samples, but at the level of 3 g per 100 g of meat the chewiness decreased . Sausages with lower chickpea protein concentrate addition were significantly harder and more chewy compared to those with higher amount of the same isolate . In another experiment, trim tray pollen the hardness and shear force decreased in sausages in which the fat was replaced by two types of tragacanth gum .

The colour parameters changed depending on the hemp ingredient used . All of the additives are dark in colour. Hemp flour and hemp protein are green, hemp seeds are brownish, even the de-hulled seeds contain small shell particles. That is why, the control product was lighter and its redness was higher compared to the other products. Hemp flour and hemp protein decreased the a* value the most significantly compared to the other products, because of their green colour. Parameter b* increased significantly only in the product with hemp flour. Green colour in meat products may be associated with the spices used during the production process, but generally it is not acceptable if the origin of such a colour is unknown for a consumer. As it is shown on Fig. 1, the addition of hemp flour and protein created lumps in the middle of the meat and fat particles giving unusual, odd cross section image.It is clear, that some modififications in grinding should be made in order to get a more even cross-section image of a product.The fatty acids profile changed significantly depending on the hemp ingredient used. PUFA amount doubled after the addition of hemp seed or de-hulled hemp seed. ALA was almost 5 times higher in these products compared to the control. The LA content also doubled in HS and DHS. The presence of both ALA and LA, which are essential fatty acids, in such amounts is important from a physiological point of view. Without a source of linoleic and α-linolenic many normal metabolic processes in the human organism may be affected. The lack of essential fatty acids may cause skin scaling or poor wound healing. It was proved that n3 fatty acids positively affect mental health parameters, insulin metabolism, total testosterone, hirsutism, few inflammatory markers and oxidative stress. CLA is a term describing various isomers of linoleic acid, which have two double bonds. These isomers are considered to be biologically active. The CLA content in non-ruminant meat is usually lower than 1 mg/g of lipid . The percentage of CLA in the analysed pork loaves was generally low and even slightly lower in the samples with the hemp additives. This might be due to heating  of the samples since thermal treatment affects CLA oxidation resulting in creation of some significant volatile compounds which depends on the temperature and time of heating . PUFA protect from the development of cardiovascular diseases and they help to keep a proper composition of sperm, retina or brain lipids .

Very low levels of n3 acids are found in Western diets together with higher n6 acids intake, and meat products contain very low amounts of essential fatty acids . Therefore, any improvement of a product in this aspect should be beneficial. The content of both n6 and n3 fatty acids increased significantly in meat loaves with hemp seed and de-hulled hemp seed. As the hemp flour and hemp protein are defatted products , the increase of the unsaturated fatty acids was not significant. The specific values of the n6 to n3 ratio in the human diet are mentioned in literature, but it has been emphasised that this ratio may be associated with very low or very high overall PUFA intake. The absolute values of these fatty acids should be considered rather in a healthy diet composition . Nutritional indexes based on the fatty acid composition: AI, PI, TI and HH were calculated for the meat loaves. Oxidation has negative nutritional and organoleptic effects caused by changing the chemical structure of various substances and creation of toxic compounds . Products with high PI number are more susceptible for oxidation. Both HS and DHS had significantly higher PI indexes compared to the other products. Ulbricht and Southgate  proposed two indexes: AI and TI which could characterise artrogenic and trombogenic fat properties. These indexes should be kept at low level to decrease the risk of cardiovascular diseases. Again, both HS and DHS had significantly lower AI and TI indexes than the other meat loaves. The HH index show the fatty acids influence on cholesterol metabolism. Higher HH values indicate better products from the nutritional point of view . HH values obtained in this study ranged from 1.64 in HF to 2.38 in DHS. All the calculated values prove that both HS and DHS are the additives which improve the nutritional value of the tested products.Fat stability and rancidity problems occur in products containing unsaturated fatty acids . One of the products of lipid degradation is malondialdehyde described as a very toxic substance. The products created in its reactions with nucleic acids bases induce mutations in bacterial and mammalian cells .

That is why it is important to control the level of malondialdehyde not only from the sensory or technological perspective, but most importantly because of the consumers’ health. The TBARS changes in the analysed samples during storage are presented in Fig. 2. There were no statistical differences between the samples on the first or the 8th day after production. A surprisingly high increase of fat oxidation products occurred after 15 days of storage in the control and the sample with de-hulled hemp seed. TBARS in the other samples were comparable and stable throughout the whole storage period. This effect could be caused by the substances present in the hemp shell. Both hemp protein and flour are by-products of fat extraction in which the hull is present. Comparing the studies by Vonapartis et al. , who analysed ten hemp cultivars and some additional studies on hemp oil , it is clear that the phenolics are mainly located in the hull. The average value obtained by Vonapartis et al.  was 2224 mg/100 g, while the oils contained from 44 to 188 mg/100 g. That relation was confirmed by Chen et al. , who indicated two specific antioxidants: trans-caffeoyltyramine and cannabis in B. Both of them exhibited strong radical scavenging activity and were found in high amounts in the hemp seed hull.Various food additives – especially of plant origin – may increase the microbial load because of their high contamination. One of the sources of microbiological contamination of meat products, may be the addition of dried herbs and spices . In all of the analysed products the storage time had a significant effect on the multiplication of mesophilic aerobic microorganisms . None of the preparations inhibited or increased the multiplication of the total number of microorganisms in relation to the control; there were also no differences between the variants. The results helped to confirm that the hemp additives used in this study do not introduce any additional microbial contamination. Moreover, those ingredients do not stimulate the microbial growth in any way over the 15 d refrigerated storage.The consumer tests results are presented in Fig. 4. The texture of all the products was comparable for the consumers.

Despite the fact, that there were visible differences between the products in appearance , they were statistically comparable in the consumers’ rating. It is possible that in a product containing minced meat, non-meat  ingredients may be associated with spices which are usually used and do not decrease consumers’ evaluation. Juiciness and taste of the control sample were graded significantly higher than the same attributes of most of the samples with hemp ingredients. However, the difference between the control and the meat loaf with de-hulled hemp seed was not significant. The overall acceptability results clearly show that the products with hemp ingredients are not acceptable with some tendency to grade the product with de-hulled hemp seed higher. Quite the opposite results were obtained by Naumova et al.  who used 5, 10 and 15% of hemp flour in beef cutlets. The products with 10% of this ingredient were acceptable to the panellists while in our study the 5% addition decreased the products’ quality significantly. It is possible that the products’ sensory perception strongly depends on their other constituents. Verbeke  stated that the functional product is acceptable if it is tasty, although the willingness to compromise on the taste for health decreased. In this study, the consumers’ willingness to buy the products with hemp seed additives increased after providing the information about the healthy ingredient . The question “would you buy?” was asked of each product twice. The second time it was developed into: “would you buy if you knew that the product contains omega 3 acids?”, trim bin tray giving a signal to a consumer that probably this tested product  actually contains that healthy ingredient. Omega 3  acids are among the ingredients that are recognised by consumers as health promoting . For the control sample, the amount of positive answers  did not change after asking the second question. However, for the products with hemp ingredients there was a substantial increase.

The meat loaf with de-hulled hemp seed was generally graded higher and the consumers declared their willingness to buy such a product, so the positive answers to the second question changed by only 12%. The most significant increase was noted for the product with hemp flour from 34 to 64 positive answers – which is over an 88% increase. For hemp seed and hemp protein products the change was comparable: 57 and 54% respectively. These results clearly show that functional meat products which are not fully acceptable in terms of sensory attributes have a chance to be implemented on the market only if the information about their health properties  is provided. As was confirmed by Oliveira et al. , the consumers pay attention to the label, but the graphics seem to be its key component. In terms of increasing the role of the functional foods in influencing society’s healt hand well-being, a proper marketing strategy, including the label design, would be crucial.Fats and oils are major lipid components of human diet, generally obtained from plant seeds. In the last decades the interest in using non-drug hemp  seeds for oil and flour production is growing , increasing its cultivating all over the world. In fact, in addition to the traditional uses , other innovative industrial applications  are developing together with the interest to the hemp seeds as source of bio-active compounds for production of value-added products and supplemented food, as well as cosmeceuticals and nutraceuticals .

In this study, five distinct hemp seed protein concentrates , produced at industrial scale using dry or wet protein enrichment technologies , were comparatively investigated in terms of their functionality, structuring potential during wet extrusion, and the molecular properties of their most abundant component, protein. In addition, their macro-nutrient composition , mineral profile, and phenolic fractions  were comparatively investigated to elaborate on the underlying mechanisms for protein molecular properties and structuring behaviour of hemp protein concentrates during processing. Finally, selected hemp protein concentrates were subjected to wet extrusion and the resultant High Moisture Meat Analogues  were investigted for anisotropy, viscoelasticity, and water mobility  as a proxy for mouthfeel. Field pea protein, one of the most common plant-based protein alternatives, was also included in this study for comparative purposes. Pea protein is also made up of albumins and globulins, it is abundant and cheap, and pea plants are grow in moderate climates . Dietary pea and hemp proteins are less connected to GMO questions and are not listed as allergenic. Nevertheless, compared to other seeds, the content of phytic acid in the hemp seed cotyledon is reported to be higher , with values as high as 22.5 mg/g in hemp meal . The presence of phytic acid could further reduce the bioavailability of multivalent cations and its separation from the plant tissue is challenging due to its low solubility in water. Although phytic acid can be vastly reduced during pilot scale AE-IP , this would not be the case for dry fractionated hemp protein concentrates, which could limit their nutritional interest. Therefore, in this work, phytic acid content was measured in all flour samples and HMMA prototypes. Protein powders were analysed for moisture, ash, protein, and fat,drying rack cannabis and then the total carbohydrate content was obtained by weight difference with the other components. Moisture was analysed using an automated moisture analyser  following AACC method 44–15.02 .

Ash content was analysed according to AOAC 923.03 method  using a muffle furnace at 550 ◦C for 4.5 h. Protein content was determined using AACC method 46–30.01  with an automated Dumatherm N Pro protein analysis system . The released nitrogen was converted into protein by multiplying the value of nitrogen by a factor of 6.25. Lipid content was determined by Low-Field Proton Nuclear Magnetic Resonance  following the AOAC method 2008.06 . All analyses were run in triplicate. Since hemp seeds are very low in starch , total dietary fibre  was analysed using the AOAC official method 991.43 . The extraction and analysis of extractable polyphenols  and non-extractable polyphenols , the latter consisting of non-extractable hydrolysable polyphenols and non-extractable proanthocyanidins , were performed as described by Pico et al. , with modifications.Specifically,the quantification of EPP and HPP in the solutions was performed using the highly phenolic selective Fast Blue BB  reaction, developed by,to account for the lack of specificity of the Folin–Ciocalteu assay due to numerous interferences . NEPA content in the solution obtained by depolymerisation by butanolysis was measured at 555 and 450 nm to detect anthocyanins and xanthylium compounds, respectively . EPP and HPP fractions were expressed as mg of gallic acid equivalents /100 g dry matter, while the anthocyanins-NEPA fraction was expressed as mg of delphinidin equivalents /100 g dry matter. All analyses were performed at least in triplicate. The main processing steps used to obtain the hemp seed protein concentrates are detailed in Table 1. All hemp protein materials were first subjected to cold pressing-expelling  for lipid extraction, although they differed in their protein enrichment steps . Hemp 1 and Hemp 3 were subjected to dry fractionation steps, with Hemp 3 being the only sample coming from dehulled seeds. Meanwhile, Hemp 1, Hemp 4 and Hemp 5 were fractionated through AE-IP. The proximate composition of the resulting hemp protein concentrates as well as of pea protein is shown in Table 2, which was very similar to the data provided by the manufacturers.

The oil , carbohydrate  and protein  content of the hemp samples were distinctly different from each other. Compared to the pea sample used in this study, hemp protein concentrates were significantly richer in oil, carbohydrates and ash, and they exhibited a lower protein content. In terms of macro-nutrient abundance, Hemp 1, Hemp 2, and Hemp 5 were characterized by a lower lipid content  than Hemp 3 and Hemp 4 . The lower lipid content of Hemp 1 compared to Hemp 3 , and of Hemp 2 versus Hemp 4 , confirms a very efficient oil extraction performed by the manufacturer of Hemp 1 and Hemp 2. Hemp 1 contained the highest amount of carbohydrates , which can be attributed to the choice of using whole seeds and dry fractionation. Conversely, Hemp 5 presented the lowest carbohydrate and the highest protein content, which is explained by the double AE-IP. The ash content was rather similar for the Hemp 1–4 samples, ranging from 7.3 to 8.8% d.b. Hemp 5 exhibited a significantly lower ash content than the rest , which could suggest that minerals were removed during the isoelectric precipitation step , which was performed twice for Hemp 5. The mineral profile revealed that all hemp samples were generally rich in the macro-elements potassium , phosphorous  and magnesium , which are needed in the amount of >50 mg/day in the human diet. Generally, hemp samples were also richer in the macro-elements calcium and the micro-elements  manganese, copper, and zinc than the pea counterpart. Hemp samples had a significantly lower sodium content  than pea . Interestingly, lower sodium content for dry fractionated hemps, Hemp 1 and 3, was observed  compared to wet-fractionated hemps and pea, suggesting that the sodium in is coming from the alkaline extraction step, normally done with sodium hydroxide. The high amount of potassium along with a relatively low sodium content leads to a high K/Na ratio, which is believed to be related to cardio protective effects as it promotes a high K intake that is considered to be inversely related to blood platelet aggregation and stroke incidence . Nonetheless, compared to other seeds, the content of phytic acid in hemp seeds is reported to be higher , which could further reduce the bioavailability of multivalent cations, including Zn2+, Fe2+/3+, Ca2+, Mg2+, Mn2+, and Cu2+. Hemp 3 was significantly richer in magnesium and phosphorus than the other hemp samples, which could be due to differences in genotype, environmental and soil conditions as well as higher ash content as a consequence of the protein enrichment processing .

Since phytic acid naturally stores phosphorous in plants , the high phosphorous content in dry-fractionated samples  might be indicative of high levels of phytates in these particular samples. In fact, the phytic acid content of Hemp 1  and Hemp 3  was significantly higher than that of pea  and wet-fractionated hemp samples , as shown in Table 2. These values are similar to those reported previously for hemp protein concentrates  and pea . The higher values of Hemp 3 compared to Hemp 1, both being dry-fractionated samples, could be explained by the fact that only Hemp 3 seeds were hulled and that the majority of phytate is found in the cotyledon fraction . Therefore, pearling, or mechanical fractionation to remove coarse fractions, would get rid of those fractions low in phytic acid  and, therefore, increase the weight percentage of phytic acid in protein concentrates. In contrast, a significant reduction of phytic acid during the precipitation step was already reported for wet-fractionated hemp, which was partially explained by a potential activation of plant phytases at the isoelectric pH of proteins  or by the higher solubility of inositol phosphates during acidic precipitation of proteins. To the best of our knowledge, there are no regulations in the European Union regarding acceptable levels of phytic acid in hemp proteins. The total content of phenolics  and the phenolic fractions  of the pea and hemp protein flours were also studied, and the results are summarized in Table 2. Hemp protein concentrates possessed higher content of TP  than pea protein concentrate . Our values are higher than those reported by Izzo et al.  in hemp inflorescences using high resolution mass spectroscopy. However, it is noted that our study also focused on those phenolics not extracted during the aqueous-organic treatments commonly performed to analyse polyphenol content in foods. In fact, HPP  was the main phenolic fraction found in all samples. Phenolic acids and monomeric flavanols are usually reported to remain mostly un-extracted , which could represent an important fraction of HPP since hemp inflorescences are rich in both . Several studies showed that hemp seeds are rich in various phenolic acids, lignanamides, phenolic amides and flavonoids . The comparison between samples revealed that Hemp 1 was significantly richer in EPP and HPP than the rest of samples, which suggest that many of these compounds remain attached to the abundant carbohydrate fraction in Hemp 1, which was not removed during processing.

Conversely, Hemp 3 exhibited the lowest EPP to HPP ratio, followed by Hemp 5, indicating that even more phenolics were present in bound form. Interestingly, these two hemp samples presented the highest protein content and relatively low presence of carbohydrates, which may have contributed to the loss of EPP during processing. However, further studies are needed to understand why free phenolics may have been lost during the pearling used for Hemp 3 dehulling or dual AE-IP processing. It should also be noted that differences found between phenolic fractions in the samples could also be attributed to other factors different to protein enrichment processing, commercial greenhouse supplies including but not limited to phenotype, seed maturity, growth and post harvest environmental conditions.The proanthocyanidins in hemp seed cake/meal have been identified to be essentially catechin polymers, i.e., procyanidins, with total values of 245–262 mg proanthocyanidins/100 g in non-treated hemp seed cake , which generally aligns with the reported results in this work. Nonetheless, some proanthocyanidins could be extracted as EPP fraction, as reported by P´erez-Jim´enez and Saura-Calixto  with fruit peels. Hemp 1 and Hemp 2, followed by Hemp 5, displayed significantly lower NEPA than the rest of the hemp samples, suggesting that NEPA could have been co-extracted during the extraction of oil in Hemp 1 and Hemp 2 and during the more aggressive aqueous protein extraction of Hemp 5 . Hemp 4 followed by Hemp 3 possessed the highest NEPA content. Similar to flavan-3-ols, anthocyanins are highly susceptible to degradation during hydrothermal treatment . Hence, any protein enrichment technology that did not elevate the temperature of the samples could have partially protected some NEPAs from thermal degradation. The hemp protein fraction is comprised mainly by globular proteins in the form of globulins and albumins, whose relative abundance might impart different functionality. Reversed-Phase Chromatography has confirmed the quantitative dominance of the edestin fraction, accounting for up to 70% total hemp protein content . The edestin molecule is composed of six identical subunits, and each subunit consists of acidic subunits  and basic subunits  linked by one disulphide bond . Fig. 1 depicts the SDS-PAGE  and Native-PAGE profiles of pea and hemp proteins. In reducing SDS-PAGE , edestin appeared to dissociate into its acidic and basic subunits, corresponding to bands at around 35 and 20 kDa, respectively. These results are consistent with previous studies by Shen et al.  and Wang and Xiong  which found roughly the same molecular weights for these fractions. Besides the bands of edestin acidic and basic subunits, three visible but less abundant bands appeared between 50 and 70 kDa. Tang, Ten, Wang, and Yang  reported that the band at about 48.0 kDa was similar to the β-subunit of the trimeric β-conglycinin found in soybean, suggesting the presence of a 7S-vicilin-like protein. Nevertheless, it was a minor fraction compared to edestin  and the albumins seen at <18 kDa. These findings are corroborated by the results from Mamone et al.  using proteomic analyses, indicating that hemp protein isolate consisted of essentially three major storage proteins, 11S edestin, 7S vicilin-like protein, and albumin. In the absence of reducing agents, the disulfide bonds between AS and BS of edestin are not disrupted and an intense higher molecular weight band of ~55–60 kDa was instead visible .

Responses collected during the most recent interview were used for these analyses.To examine the dimensionality of the 13 subjective experiences examined for each substance, we conducted Mokken Scale Analysis  using the statistical software STATA.Mokken scaling analysis extends traditional factor analysis by systematically hierarchically ordering items that are highly correlated. Mokken analysis provides a nonparametric, iterative scale-building technique that identifies the smallest set of internally consistent scales from a given item pool. This model assumes the presence of one or more latent traits that can be measured by subject responses to a set of items.MSA is probabilistic and hierarchical, meaning that the items can be ordered by a degree of “difficulty”;individuals who agree with a more difficult item will tend to agree with less difficult items.Scales from MSA are formed by taking pairs of items with the highest correlation and including other items until there is no further improvement.Loevinger’s H coefficients, which indicate the fit of an item to the scale, are computed for each item  within a scale and for the scale as a whole.H coefficients ranging between 0.3 and 0.4 indicate a weak scale, 0.4–0.5 a medium scale, and 0.5–0.9 a strong scale. In MSA, an item can remain “unscaled” because it could not be added to one of the alternative scales without weakening the scale’s homogeneity. Based on our previous analyses,we used the scaling derived from the CADD sample. Positive and negative scales were standardized by age, sex, and clinical status with two groups.Pairwise correlations between the resulting two scales were then determined for alcohol, tobacco,pot drying and marijuana.In the current report, we examined subjective experiences to three commonly used drugs of abuse among young adults from the general community and an area treatment program.

In these data, we obtained results that supported previous observations indicating positive and negative subjective experiences for a particular drug were predictive of problem use of that same drug. We then extended this relationship in two ways. First, we obtained results that supported the notion that positive and negative experiences to one drug are similar to those experienced for another drug and second, that subjective experiences to a drug are predictive of the risk for problem use of other drugs. We interpret these findings to suggest that subjective experiences may be a useful indicator of a common liability towards use and problem use of multiple substances. Following on our previous work on marijuana and subjective experiences,we used Mokken scaling to simultaneously examine whether subjective experiences to three drugs are associated with drug use outcomes. From these analyses we observed that the subjective experience scales for each of the three drugs were comparable to those found in previous studies despite using a different methodology.We observed differences in item means and hierarchical ordering of the items by substance suggesting that subjects are reporting drug specific subjective experiences. This interpretation is consistent with findings from laboratory based studies which have shown that subjects can differentiate between a placebo and a drug or between different drugs based on subjective experiences.As different combinations of alcohol, tobacco, and marijuana use are commonly reported in epidemiological studies, we investigated the relationship between subjective experiences to different drugs in poly-substance users. We observed that subjective experiences to one drug were significantly correlated with experiences to another drug, though the strength of the relationship varied for different drug combinations. The strongest relationships were between alcohol and marijuana, replicating two previous studies,and between alcohol and tobacco. These particular drug combinations target similar neuronal receptor systems and are reported to enhance the overall drug experience when taken together.Further, as subjective experiences are thought to reflect the underlying physiology of a drug’s actions,these cross-substance relationships may provide a closer approximation of a common risk factor suitable for molecular genetic investigation. In this sample of community and clinical subjects, subjective experiences for one drug were associated with outcomes related to a different drug. Though our results replicate findings that relate positive experiences with greater use of other drugs, we also identified that negative experiences were predictive of abuse and dependence status of a different drug. In particular, negative effects of alcohol and marijuana were associated with misuse of these same drugs as well as tobacco. Although this may appear counter-intuitive, a possible explanation could be that subjects who needed greater amounts of a drug in order to feel its effects drove the observed association.

Findings from laboratory-based drug discrimination studies suggest that some subjects are unable to differentiate between drug and placebo at a standard training dose.Differences between the two conditions could, however, be reported as non-discriminators were exposed to greater doses of a drug. Interestingly, those who were able to discriminate between non-exposure and exposure to a drug reported stronger positive and negative subjective experiences, often simultaneously,at greater doses. This underscores the importance of dose in determining individuals’ drug sensitivity as assessed by subjective experiences. The relationship between drug dose, the resulting subjective experiences, and problem drug use has also been examined using self-ratings to the effects of alcohol [SRE; 28]. The SRE primarily assesses negative experiences to alcohol such as dizziness and passing out as related to the dosing levels needed to feel the sedative effects of alcohol. Among adolescent and adult samples of both sexes and family-history positive studies of alcoholics,low levels of response, as measured by the SRE have been implicated as a risk factor for alcohol use disorders. This notion that some drinkers need to ingest greater amounts of alcohol to feel its sedative effects and that this effect is related to greater drinking quantities has been recently supported  and extended to include the observation that this relationship is also relevant to those reporting lower levels of stimulant effects during the first five drinks. Our finding that negative alcohol experiences were predictive of problem alcohol use is consistent with this research, despite using a different questionnaire, and extends it to include the potential prediction of other drug use problem behaviors.The prevelance of PVCs is increased in patients diagnosed with hypertension especially when it is associated with left ventricular hypertrophy, dilated cardiomyopathy and heart failure,post acute myocardial infarction,and congenital heart disease. PVCs often cause no symptoms. In many patients, the presence of PVCs could result in the sensation of fluttering, pounding, skipped beats, palpitations, dizziness, and/or near syncope. The etiology of PVCs is not well known. Many mechanisms may explain the origin of PVCs, including enhanced normal or abnormal automaticity inside the heart, triggered activity in Purkinje cells of the ventricular myocardium, or reentry. Anxiety, alcohol, caffeine, tobacco, exercise, illicit drugs, hypokalemia, HTN, ischemia, infarction, excessive calcium, drug toxicity,or an underlying heart disease could result in PVCs through previously mentioned mechanisms.

ECG is the mainstay of diagnosis of PVCs. This includes standard ECG, exercise stress ECG, holter monitor, and event recorder depending primarily on the frequency of PVCs which helps to decide the best way to detect them. Only in symptomatic patients do PVCs need to be diagnosed and treated. Beside eliminating previously mentioned possible triggers, beta blockers and calcium channel blockers are recommended as first-line therapy for symptomatic PVCs, especially with outflow tract morphology in a structurally normal heart. Antiarrhythmic medications, such as amiodarone can sometimes be tried but with caution because of its side effects. Frequent PVCs may be associated with worsening of systolic heart failure in patients with a dilated cardiomyopathy. Small studies have suggested that in selected patients, radio frequency ablation of ectopic ventricular foci is associated with an improvement in left ventricular function and clinical improvement in symptoms.The 2006 American College of Cardiology/American Heart Association/European Society of Cardiology guidelines for the management of ventricular arrhythmias included suggestions regarding ablation therapy for PVCs. They note that ablation therapy of PVCs may be useful if they are frequent, symptomatic, and monomorphic, if they are refractory to medical therapy, if the patient chooses to avoid long-term medical therapy, or if they consistently provoke ventricular arrhythmia storm of a similar morphology. SCFP, as a separate entity, has a widely diverse presentation including chest discomfort, unstable angina, non ST elevation MI, ST elevation MI or non-sustained ventricular tachycardia. It usually presents with recurrent rest pain requiring urgent admission. The etiology of SCFP is not completely understood. It is speculated that it is caused by acute but recurrent perturbations of microvascular function. Histopathological examination  of left and right ventricular endomyocardial biopsies taken from some patients showed fibromuscular hyperplasia, myofibrilar hypertrophy, endothelial degeneration with swollen endothelial cells encroaching on the lumen, luminal size reduction, mitochondrial abnormalities, lipofuscin deposition, and glycogen content reduction, which can cause the elevation in resting coronary artery resistances, especially toward microvasculature beds, found in SCFP. Normal and pathological zones often coexisted in the same specimen.Thus, in some patients with slow coronary flow and patent coronary arteries, functional obstruction of microvessels seems to be implicated, as it is relieved by dipyridamole infusion.

This shows also that small-vessel CAD can cause classic angina pectoris. The diagnosis can be suspected when the coronary angiogram shows large patent arteries with slow flow of the angiographic contrast medium and it can be confirmed by endomyocardial biopsy. Another study suggested that elevation in plasma homocysteine, evenifitismild,may play a role in the pathogenesis of SCFP by severely disturbing vascular endothelial function and subsequently impairing coronary blood flow, and showed that patients with SCFP have statistically significant raised level of plasma homocysteine compared to control subjects with normal coronary flow.As a treatment, dipyridamole, which has dilatator properties on coronary microvessels, cannabis drying proved to be useful in most patients with SCFP. It abolishes functional obstruction in coronary arteries with diameters less than 200 m and is considered far superior in treating SCFP as compared to nitroglycerine. Other therapies proved to be effective also include simvastatin, atorvastatin, nebivolol, and mibefradil although the use of the latter is limited because of drug interactions caused by its inhibition of the cytochrome P450 3A4 pathway. Beside mean arterial pressure, blood flow in coronary arteries depends on heart contractility. During systole, and because of the big muscular mass of the left ventricle,extravascular compression prevents any antegrade blood flow in left coronary artery  particularly at the end of isovolumetric contraction. Conversely, and during diastole and especially early diastole which represents isovolumetric relaxation, coronary blood flow in LCA becomes maximal.In right coronary artery,blood flow is maximal during peak systole, because extravascular compression within the right ventricle is less than its counterpart in the LV preserving antegrade blood flow in RCA in both systole and diastole. The fact that the highest blood flow in LCA occurs throughout different stages of diastole, which is not the case in RCA, assumes that any PVC happening through diastole will have a bigger impact on blood flow in LCA compared to RCA. This, in turn, will be reflected as a slow blood flow  in LCA more than in RCA. In patients with SCFP, resting coronary artery resistances, as mentioned above, are abnormally elevated, however, these resistances respond normally to vasodilator stimuli such as papaverine and adenosine and during exercise. Marijuana affects the heart in different ways. The acute cardiovascular effects of marijuana, which are palpitations, tachycardia, elevated blood pressure, and a greater myocardial oxygen demand, are mainly caused by increased release of catecholamines. Chronic use of marijuana can worsen any underlying disease through prolonged vasoconstriction and causes digital clubbing.After a thorough review of the literature, it was noted that there was a lack of description of any specific time frame, after which marijuana consumption will have effects on the heart, resulting in myocardial ischemia or heart failure. Patient’s age and the presence of underlying CAD are key players in determining how fastthe results of smoking marijuana will take effect on the heart, especially when it comes to chronic use. The mechanisms by which marijuana affects the heart are diverse including increased cardiac work through elevated catecholamines and carboxyhemoglobin levels, as well as possible episodes of intense postural hypotension. Moreover, smoking cannabis was rarely found to trigger MI. Among cannabis users who sustained an acute MI, the risk was nearly five times higher within the first hour after smoking compared to periods of nonuse.

Alcohol likely has similar deleterious consequences on the brain. The present dose-dependent associations are consistent with our previous findings, as Squeglia et al., found decreases in cortical thickness estimates associated with heavy episodic alcohol use in males,and accelerated declining brain volume trajectories in a large prospective investigation examining individuals  who transitioned to heavy drinking .Alcohol likely interferes with neural development of the cerebral cortex, and thinner cortices observed with more cumulative use reported may represent non-beneficial pruning and/or inhibition of cell generation or cell death.Limitations of the present study include self-report of substance use, which can introduce measurement error. Further, while this study was prospective, participants were not assessed prior to initiation of substance use. However, previous work in our laboratory finds marijuana-related associations with white matter integrity in a sample of individuals assessed pre- and post-initiation of substance use.Nevertheless, future work should determine the influence of pre-existing differences on cortical metrics. The current investigation included users of both marijuana and alcohol, and despite controlling for alcohol use, it remains unclear what is precisely the result of marijuana as compared to the combination of co-occurring marijuana and alcohol use. Our sample was predominately male,however gender should be evaluated and future studies will focus on differential gender effects on brain morphometry in adolescent marijuana users. Group did not statistically differ on days since last use of cannabis square pot and alcohol use,likely influenced by the monitored abstinenceb period,therefore acute effects may not have been captured in our reported findings.

Astatistically significant within-subjects effect was not widely observed,which may be attributed to the smaller sample size combined with a more restricted age range. We tried to reduce the number of correlational analysis that were conducted, however given that effects were modest, future work should replicate findings. Studies should continue to follow existing adolescent cohorts to understand neural and behavioral changes that occur into young adulthood. Understanding how co-occurring marijuana and alcohol use influences both macrostructural and microstructural brain development, along with structural and functional connectivity, will help clinical interventions target neural vulnerabilities to develop novel and effective interventions to reduce marijuana misuse as prevalence rates of marijuana continue to increase .To guide recruitment, the Adolescent Brain Cognitive Development  Study required a method for identifying children at high risk for early-onset substance use that may be utilized during the recruitment process.In this context, childhood risk refers to characteristics identified at ages 9 or 10 years that predict adverse outcomes in adolescence, and “high risk” refers to a categorical classification of some children as having increased risk compared to others. The construction of a brief measure for childhood substance use risk involves the identification of characteristics that predict early-onset substance use in mid to late adolescence. The identification and evaluation of optimal items for a brief childhood measure to serve as a high-risk screener ideally involves data from several large prospective studies with assessments initiated prior to the typical age of onset of substance use. To inform ABCD Study recruitment, secondary analyses are needed with data-sets collected prior to ABCD Study initiation.

In this context, a set of analyses with available data focused on a specific substance use outcome was determined to be most likely to be informative and feasible. While other substance use outcomes are also important, early-onset marijuana use is a relevant target.Marijuana is the most commonly used illicit drug by adolescents, and regular marijuana use identifies youth likely to develop cannabis use disorder.In these secondary data analyses, the definition of early-onset marijuana use was defined by the initiation of regular use as indicated in the available data-sets.The studies contributing data-sets were the Center for Education and Drug Abuse Research,the Pittsburgh Youth Study,the Pittsburgh Girls Study,and the Michigan Longitudinal Study.In the studies contributing data to the secondary analyses described here, the definitions of regular marijuana use differed by sample due to measurement variations. The variations in the definitions of regular marijuana use were as follows:five or more use occasions in the past year  and;six or more occasions in the past year.By efficiently identifying children at high risk for early-onset marijuana use,a brief and effective measure of childhood risk measure could be utilized as a screen to identify high risk children in prevention research, primary medical care, and mental health clinic settings. The present analyses were specifically undertaken to inform the development a childhood high risk screen for use in the ABCD Study.The ABCD Study is the National Institute of Healths’ large-scale prospective population study of the biological and environmental factors that influence young people’s ability to successfully navigate adolescence. The study has a special emphasis on the risk and protective factors that influence marijuana and other substance use, and subsequent health problems including substance use disorders. Utilizing data from previously conducted studies, the present study was thus undertaken to develop and establish the efficiency of a short measure  to identify youth at high risk for early-onset marijuana use with optimal features for use in the ABCD Study.

To achieve this goal, the risk level of a potential participant needs to be determined at the time of recruitment and prior to their scheduling for the extensive ABCD Study assessment protocol. Consequently, the optimal ABCD Study high risk screen has several characteristics: extreme brevity, including less than ten items;lack of sensitive items that may raise confidentiality concerns at this early stage of considering participation; consistency with prior research. These characteristics were taken into consideration in the analyses that follow. Historically, studies focusing on mental disorders such as schizophrenia, alcohol use disorder, and major depressive disorder, have used positive family history as a risk marker.Family history has been demonstrated to identify children at high risk of later substance use disorders in many prospective studies.However, a detailed family history may involve the parent being asked to disclose their own socially undesirable, embarrassing or, in some cases, illegal behavior. There have been alternative strategies to acquire this information, such as the use of publicly available records of drunk driving or other drug offenses, or the use of hospital records to identify parental diagnosis.Obtaining such records would not be feasible in the initial recruitment phase of the ABCD Study. Regardless of the method for obtaining this information, requesting this information at the point of introducing the ABCD Study raises the real possibility that the parent  will decline study involvement. Few longitudinal studies have formulated and tested measures for identifying high risk children likely to exhibit early-onset marijuana use. There have been several approaches developed for predicting substance use disorders, but relatively few have targeted the adolescent developmental period. One of the risk measures developed to identify high risk children is the SUD Transmissible Liability Index  developed by Vanyukov, Tarter, Clark and colleagues,using longitudinal data from the CEDAR study. Although the TLI is sophisticated in its development, it is long,uses different portions of existing instruments, and is under copyright. In addition, the TLI did not focus on the age 15 outcome of marijuana use, and the publications did not use Receiver Operating Characteristic  Area Under the Curve  analyses to determine an optimal threshold score.

Another screening instrument, the DSM Guided Cannabis Screen  has unknown predictive value because it was constructed using cross-sectional data from a small clinical sample aged 14–59. Therefore, the current study fills a significant gap in the empirical literature. This report describes the process and results of secondary data analyses to prospectively identify a brief screening measure applicable to age 9–10-year-old children that would predict early-onset marijuana use in the 5–7 years following the initial screening measurement. To acquire data useful for developing this screening measure, we needed to identify population-based prospective studies which  began assessments in late childhood,  had been continued at least through ages 14–17,included marijuana use variables at both age periods, measured domains previously identified in the literature as predictive of adolescent substance use disorder outcomes, and  had a sufficient number of measures in these domains that were shared across these studies so that screening validation could be replicated across different demographic groups .The objectives of these secondary data analyses were as follows:To develop a brief screener for 9–10-year-old boys and girls to predict early-onset marijuana and other substance use in mid adolescence with demonstrated predictive utility across four longitudinal data sets; To dichotomize the outcome variable, which will reduce shrinkage,improve replicability and practical utility.;  To replicate findings across construction and validation samples.The advantage of this dual analysis approach is that we could construct a screener that considers shrinkage  that typically happens between construction of a screener and subsequent validation in another sample. In summary,trim tray the objective was to develop a brief and feasible approach to the identification of children at increased risk  for early onset  marijuana use that may inform the ABCD Study recruitment procedures.The potential items for analyses were identified by examining prior research,prior analyses with the available data-sets, particularly the extensive analyses with CEDAR data,identifying pertinent items available in the four longitudinal projects used in these secondary analyses, and deliberations on the acceptability of areas of inquiry for potential participants during the recruitment process. Based on these considerations, the constructs represented by the pool of items to be considered included child externalizing behaviors, child internalizing behaviors, and parent tobacco smoking. Child externalizing behaviors. In the case of the ABCD Study design, we are projecting from ages 9–10, when marijuana use typically is minimal and not a viable risk item for screening purposes. Therefore, for candidate items on child externalizing behaviors, we considered non-substance use characteristics that other studies have found to predict early-onset substance use in mid adolescence, particularly child externalizing behaviors.

Potential externalizing behaviors considered were vandalism, lying, and disobedience at school.Child internalizing behaviors. In addition, we examined whether selected internalizing behaviors augmented predictions. After examining potential internalizing items’ correlations with both the tentative screener  and with the outcome variable, we initially focused on the following items :  unhappy, sad or depressed;  too fearful or anxious;  secretive or keep things to oneself;  self-conscious or easily embarrassed. After considering which internalizing items correlated with the externalizing screener at that point, we finally focused on:  unhappy, sad or depressed;  too fearful or anxious.Parent smoking. For candidate items on parent behaviors, parent smoking  was also considered a viable candidate. This candidate item for the screener  was available in the 4 study data sets.We searched for equivalent predictor items of interest in each data-set. This is very important because we needed construct convergence among the four longitudinal data-sets. We used prorating in cases where there were missing items  so that we would maximize the numbers of participants. Note that sample sizes varied somewhat due to missing cases for each analysis. In the PYS data-set, we combined parent and child information on child predictor variables to obtain a best estimate of the child behavior. For example, a behavior was counted when either the parent or child reported the behavior. Item scores were recoded as “Yes” or “No” where necessary to make them uniform across studies. For example, the Child Behavior Checklist [CBCL] has response options of 0, 1 or 2  then item scores were recoded as Yes or No. We undertook separate analyses for each gender. We first determined which items were predictive of the outcome. We next summed significant items into an index, examined AUC, and computed sensitivity, specificity, and positive predictive power for the summary screening score. If the variance accounted for by these indicators proved too low, we repeated the procedure for “new items”. In the final analyses, three of the studies used CBCL items,and one study  used data based on self-reported antisocial behavior,MFQ,and the Child Symptom Inventory.The items from the CBCL, the MFQ, and the CSI were highly comparable.The intercorrelation results of the predictor items showed that some items were significantly negatively correlated with the outcome variable, and other items correlated with the outcome non-significantly across all three data-sets. This reduced the number of viable items in the Pittsburgh data-sets to 14. The Michigan group derived their own scale of 9 items.In brief, a procedure very similar to that described here for the three Pittsburgh data-sets was used. We intercorrelated available predictor variables that overlapped with those originally identified across externalizing, hyperactivity/impulsivity, internalizing, and temperament items  with the outcome variable. This method was used to reduce the item pool, based on predictive accuracy.

Additionally, we found that activation in the occipital cortex to negative emotional stimuli mediated the relationship between marijuana use and later resiliency. Specifically, activation in the cortical region surrounding the calcarine fissure, including portions ofthe right cuneus and lingual gyrus, was lower in heavy marijuana users than controls. This activation was further associated with decreased resiliency at follow-up, when controlling for resiliency at the time of scan. Although the cuneus and lingual gyrus are classically considered as visual processing and integration regions, there is a large literature associating both regions with aspects of emotion functioning, including the processing of emotional faces,high-arousal emotional words,and emotional film clips,as well as the evaluation of one’s own emotional state.Cuneus activation has also been associated with the ability to attribute mental states to others, termed “theory of mind”. A recent study reported that adult marijuana users had differences in brain activation compared with controls during a ToM task, including lower activation in the right cuneus.Therefore, an impact of heavy marijuana use during adolescence on the functioning of occipital regions involved in the evaluation of emotional stimuli with respect to oneself and to others may impair self-regulation of emotional processes.In addition to the regions found to mediate later outcome, heavy marijuana users had less activation than controls in the insula to negative words. These findings are consistent with previous work showing adolescent marijuana users had reduced cerebral blood flow in the insula compared with controls.Furthermore, studies of adult cannabis grow racks users found less activation in the insula to loss outcomes during a monetary incentive task  and to errors in an inhibitory control task  compared with controls.

The insula is critical to the integration of emotional and homeostatic information, and may be involved in translating interoceptive signals into conscious feelings.For example, the magnitude of insula activation while participants evaluated their own emotional and bodily states was found to be associated with social anxiety and neuroticism.Insula activation has also been associated with self-report measures of anxiety  and anticipation of aversive exposure in anxiety-prone individuals. Here we found less insula activity to negative words in heavy marijuana users compared with controls, which was further associated with more negative emotionality at the time of scan. Together, this evidence suggests that heavy marijuana use may lead to impairment in the integration of emotional experience.Along with the insula, the amygdala is part of a network involved in translating interoceptive responses to emotional stimuli into emotional experience.Blunted amygdala response has been observed in individuals with difficulties experiencing and processing emotions.Acutely, cannabidiol, a psychoactive component of cannabis, has been shown to decrease amygdala activation to anxiety-inducing emotional stimuli; this effect was further associated with a reduction in electrodermal activity,supporting links among marijuana, amygdala functioning, and interoceptive response to emotion. Furthermore, prior evidence indicates that the impact of marijuana use on amygdala-mediated emotional responding is not restricted to negative stimuli. Gruber et al. reported less amygdala activation in adult heavy marijuana smokers compared with controls to both happy and angry faces presented below the level of conscious processing. Here we found less amygdala activation to both positive and negative words in heavy marijuana users compared with controls, which further correlated with negative emotionality. Therefore, marijuana may have an impact on amygdala functioning that impairs general emotional arousal and integration. The finding of an association between negative emotionality and reduced activation of the insula and amygdala is opposite to effects described in the depression and anxiety literature, which reports enhanced activation to negative stimuli.

However, a longitudinal study of individuals with comorbid major depression and marijuana dependence found that greater marijuana use was associated with reduced amygdala activation to emotional stimuli.This suggests that the mechanism through which marijuana impacts negative emotionality differs from the mechanism underlying depression and anxiety. For example, the associations between insula and amygdala functioning and negative emotionality in the current study may be more pertinent to differences in the experience and processing of emotions  rather than depression and anxiety. Finally, heavy marijuana users showed reduced activity in the right inferior parietal lobule and greater activation in the right dlPFC during the viewing of positive words. The inferior parietal cortex is part of an attentional system involved in the automatic allocation of attention to task-relevant information,whereas the dlPFC is involved in more effortful attentional control.Thus, the current results suggest a decrease in automatic attention to positive words in heavy users with a corresponding increase in effortful attentional control necessary to attend to the task. This is consistent with prior work demonstrating heightened activation of right-hemisphere prefrontal attentional control circuitry in adolescent marijuana users,which may reflect the need for increased effort in attending to task-related stimuli.This study extends our prior work by providing evidence of reliability and generalizability of a surveillance tool for assessing the marketing practices and socio-contextual characteristics of recreational marijuana retailers. While this study is limited by its focus on a convenience sample of 25 retailers in Seattle chosen from Weed maps, this data builds on prior tool utilization among 20 Denver retailers. This study also helped to identify distinct variables relevant within differing policy contexts.In the current and previous studies,high compliance with age requirement/verification practices, as well as use of security measures, was documented. In terms of promotion, novel products  were frequently advertised, likely in an attempt to familiarize customers with newer products.Unlike the Colorado study,however, bud was also frequently advertised among Seattle retailers. Loyalty club memberships and daily/weekly deals were prevalent, similar to the Denver study.

However, using social media was not as common in this sample of Seattle retailers compared to the sample of Denver retailers,which may reflect more conservative policies regarding online promotion and sales in Washington  relative to Colorado.Similar to our prior work,this study also documented little product and price variability among the shops. This lack of variability in product offerings and price suggests that other shop characteristics  might be used to differentiate retailers from one another. Indeed, unlike the tobacco and alcohol industry, building strong brand affiliation with shops rather than products may be strategic in the marijuana industry, potentially given limited variability in product offerings and price across marijuana retailers.This study highlighted that assessments of the marijuana retail environment should be informed by policies and regulations given activities that may be differentially prohibited or restricted in differing jurisdictions. For example, while marijuana retailers are allowed to sell clones and seeds in Colorado, retail sale of clones and seeds is prohibited in Washington. Similarly, Washington retailers are prohibited from selling branded apparel or other merchandise  in the retail store.However, the Colorado market is not restricted in this way. This is particularly relevant given that this study noted violations of these regulations, specifically in relation to the sale of branded apparel in Seattle. Furthermore, this study documented retailers being proximal  to schools, parks, and playgrounds, despite regulations limiting them to further than 1000 ft ; however, our assessment tool lacked the specificity to capture if retailers were indeed within 1000 ft. Attempts to circumvent policies are also noteworthy. This study noted that some Seattle retailers had separate storefronts adjacent to the marijuana shop where they could sell branded apparel and justify larger exterior signage. Our previous study in Denver also noted other attempts to circumvent policies; for example, publicizing “private” parties where marijuana use would be allowed despite prohibition of marijuana in public places.Surveillance of such activities is critical to informing regulatory and enforcement efforts. The MRST demonstrated perfect inter-rater reliability in two-thirds of items and ≥0.73 congruence in the remaining items. Incongruence occurred in assessments of the external environment, marketing and promotion, and price.

Greater rigor in training regarding the use of the MRST, including standardized protocols that include examples and practice assessments,cannabis grow system is needed. Additionally, in assessing products, complexities in mode of consumption, tetrahydrocannabinol versus cannabidiol, and strain  make thoroughly assessing each product category cumbersome and complicated. Moreover, assessing lowest price across marijuana product categories is complex given the diversity of product offerings in any single product category  and the ranges in volume, potency, strain, etc. Thus, this approach will need to be further refined over time and adapted as differing policy contexts may prohibit certain types of products  or regulate how they are packaged.Finally, studies involving larger sample sizes could examine differences between recreational only retailers versus those with a medicinal endorsement.A 2015 conference on medical use of marijuana indicated that 23 US states have legalised medical marijuana with some also legalising marijuana for recreational use.Although there is moderate evidence for efficacy of cannabinoids for chronic pain and spasticity, and some evidence for Multiple Sclerosis and treatment-resistant epilepsy, there is not good evidence for its use to treat nausea and vomiting associated with chemotherapy,perhaps the best known indication for medical marijuana. Some pregnant women report using marijuana to alleviate nausea and vomiting in pregnancy with success but evidence for its efficacy is mostly anecdotal. However, reports of adverse events for non-pregnant populations using medical marijuana raise concerns for pregnant marijuana users. According to the National Drug Strategy Household Survey in Australia, 7.6% of females aged ≥14 years used marijuana during 2010,with 34.8% of the female population having used marijuana at least once in their lifetime. A similar trend has also been observed in New Zealand and Europe, with 47.2% of women aged ≥16 years in NZ, 24.6% in the United Kingdom and 17.5% in Ireland having used marijuana at least once. Apart from reported negative impacts on fetal growth and brain development, marijuana has been associated with adverse pregnancy outcomes, including preterm birth,small for gestational age,placental abruption and antepartum haemorrhage. Specifically, studies have shown that using marijuana during pregnancy is associated with low birthweight and increases the risk of PTB and SGA, with an odds ratio of at least 1.5 when adjusted for age, BMI and smoking.

The association between marijuana use and pregnancy outcomes is often confounded by other known risk factors including cigarette smoking, body mass index,and socioeconomic index. Women who use marijuana also tend to smoke cigarettes and are more likely to use other drugs and alcohol, for whom national statistics have shown that amongst Australian women aged ≥14years who used marijuana in 2010, 82.7% also consumed alcohol, and 68.5% were cigarette smokers, with similar patterns of prevalence in New Zealand. There have been inconsistent results reported from American prospective cohort studies, in which associations of marijuana use with adverse pregnancy outcomes were either found or not found. Hence, this study aimed to examine the association of maternal marijuana use  in a multi-centre cohort with major pregnancy complications, amongst both cigarette smokers and non-smokers, controlling for well-known risk factors including age, SEI and BMI, as well as its effects on length of gestation.Data from this analysis were obtained from the SCreening fOr Pregnancy Endpoints  study, which aimed to build a clinical database and pregnancy biobank to screen candidate markers of pregnancy complications. The SCOPE study recruited nulliparous women with singleton pregnancies between November 2004 and February 2011 from one centre in each of Australia, New Zealand, and Ireland, and three centres in the United Kingdom. Ethical approval was obtained from local ethics committees and all women provided written informed consent. Women were invited to participate prior to 15 weeks’ gestation when attending hospital antenatal clinics, obstetricians, general practitioners or community midwives, and were interviewed and examined by a research midwife at 15 ± 1 and 20 ± 1 weeks of gestation. The exclusion criteria included women who were considered to be at high risk of PE, SGA or PTB due to underlying medical conditions,previous cervical knife cone biopsy, three terminations or three miscarriages or if their pregnancy was complicated by a known major fetal anomaly or abnormal karyotype, or if they received interventions that may modify pregnancy outcome.Details of maternal age, BMI and socioeconomic index1,medical and family history, along with dietary and lifestyle questionnaires with self-reported marijuana and cigarette smoking were recorded at 15 weeks’ and 20 weeks’ gestation and entered into an internet-accessed, password-protected centralised database with a complete audit trail.

Given a high rate of overlap between marijuana and tobacco use among the participants, our study findings provide further support for implementing comprehensive tobacco control programs and underscore the importance of target interventions among high risk populations, including those using marijuana, in order to enhance the reach and effectiveness of prevention. Third, multiple unhealthy behaviors tend to co-occur but they are amenable to concurrent or sequential interventions. A successful change in one unhealthy behavior may lead to increased self-efficacy to modify other co-occurring unhealthy behaviors for which individuals may have low motivation to change.Finally, the difference in the prevalence of unhealthy behaviors  across a number of sociodemographic subgroups highlights the need for evidence-based research to identify interdisciplinary intervention strategies that integrate science, practice, and policy to address health disparities among the population. Our study results also have several important clinical implications. SRH is an assessment tool for Patient-Reported Outcomes Measurement Information System  that measures patient–reported health status for physical, mental, and social well-being.In light of the legalization of medical and recreational marijuana use in some US states, patients may be more likely to ask their healthcare providers about potential health effects of marijuana use. Many of the proposed health benefits and unintended consequences of marijuana use have not been fully explored. The findings of our study suggest that, in addition to receiving counseling about marijuana, patients with a history of marijuana use should obtain firm advice and support for not using tobacco. For those who plan to quit, evidence suggests that simultaneously quitting both tobacco and marijuana may yield important psychological and neurobiological benefits.Until more results from experimental research are forthcoming to provide guidance, it is important to encourage dual cessation. Our study has several limitations.

It was cross-sectional and cannot establish cause and effect. With the ongoing changes in medical cannabis grow setup law in a growing number of states, people with certain health conditions might be drawn into marijuana use. It might be possible that poor perceived health resulted in marijuana use rather than marijuana use caused poor perceived health. Research shows that the predictive power of SRH for mortality is robust across many subgroups of country of origin even after extensively controlling for numerous covariates. However, validation study has not been conducted among the population of US adult ever users of marijuana. We adopted serum cotinine as a biomarker to define current tobacco use, occasional tobacco users who had used tobacco beyond 3–5 days prior to examination may be included as non-current tobacco users. In addition, data on marijuana use were self-reported and were subject to potential recall bias or under-reporting of less socially desirable behaviors. Our study did not identify the use of specific tobacco products, including electronic cigarettes or electronic nicotine delivery systems that have been gaining popularity in recent years. Due to data constraints, we were unable to account for several potential confounders such as medication use, drug abuse, and other co-morbid conditions, as well as the difference in patterns of recreational versus medical marijuana use that might have had an effect on suboptimal SRH.In the present study, we assessed the impact of recent 30-day and 60-day regular marijuana use on suboptimal SRH among regular marijuana smokers. Because of the limited analytical sample size, we could not explore additional harmful marijuana and tobacco usage patterns related to quantity, frequency, timing, and duration of usage. It is worth noting that although several subgroups of the adult population  were not evaluated in the current study due to a limited sample size, such adults are especially vulnerable to a multitude of health consequences associated with unhealthy behaviors even at low threshold levels for exposure. Long-term change of unhealthy behaviors is challenging and may require multifaceted efforts to effectively address the interplay of behaviors with biological, health, and social factors across various subgroups and environmental settings over persons’ life course .

Previous epidemiological studies have revealed strong negative impacts of marijuana use, suggesting that marijuana has similar potential for abuse as other illicit substances,is associated with respiratory illnesses, and leads to cognitive impairment.However, several focused empirical studies have countered these results, finding instead no significant effect of marijuana use on subcortical brain morphometry and only an uncertain effect on cognition.The past two decades have seen shifts in legal and societal attitudes toward marijuana use, with 23 states and the District of Columbia legalizing medical marijuana and four states legalizing recreational marijuana ; moreover, perceptions of the risk of regular marijuana use have decreased, even amongst adolescents, particularly in Colorado, recreational marijuana is now legal.As increases in the potency of marijuana have accompanied these shifts in attitudes,it is becoming increasingly important to understand the precise neural effects of long-term marijuana use and the impact of the age of first use. Adolescence is a sensitive period for brain development with white matter myelination and gray matter pruning, and, critically, an increase in the number of cannabinoid receptors that respond to marijuana.While preliminary studies of the effects of marijuana use on white matter integrity showed no significant effects in adolescents or adults,a growing body of research suggests that an adolescent onset of heavy marijuana use can have neurotoxic effects on developing white matter, reflected in decreased white matter coherence as assessed by measures of diffusivity, e.g., fractional anisotropy  and radial diffusivity.Importantly, these effects have been observed longitudinally, suggesting a causation between marijuana use and white matter changes.However, most of these studies have relied on small sample sizes,so their ability to generalize to a broader population is limited. Moreover, the majority of these studies all examined the effects of heavy use,and much less is known about the effects of casual marijuana use on white matter integrity. As many white matter tracts continue to develop in adolescence and young adulthood,with maximal change in such development during this time frame,it is important to understand how the age of onset of marijuana use impacts neurodevelopment not only in heavy users but more casual users, especially considering that adolescence is often a time of experimentation with substances of abuse.

Studies of the effects of marijuana use on cortical and subcortical morphometrics in humans have typically focused on the amygdala and hippocampus  and, to a lesser extent, the nucleus accumbens and orbitofrontal cortex. These structures are known to have important roles in reward processing and their function/structure is known to be disrupted by drugs of abuse.At least some, but far from all, of the evidence suggests an influence of marijuana on brain structure. For example, marijuana users compared to nonusers have been found to have reduced amygdala volume,and amygdala volume reductions have been correlated with increased levels of self-reported craving and relapse in consumption after 6-months from detoxification from alcohol dependence.On the other hand, a recent meta analysis of 14 studies of marijuana users compared to nonusers found no summary changes in amygdala volume, but did observe a consistent pattern of reduced hippocampal volume.In addition, a large number of studies with animals and humans have shown that marijuana affects the structure of the nucleus accumbens.Hence, there is evidence in the existing literature to suggest the possibility that marijuana influences the structure of these regions, all of which are known to be affected in addiction.Nonetheless, a recent well-controlled study by Weiland et al.  found no evidence of an effect of marijuana on the morphometry of these structures. They compared morphometry in a sample of adult and adolescent daily users of marijuana to nonusers,while controlling for other confounding variables of tobacco use, depression, impulsivity, age, and gender. Importantly, they found no group differences in measures of brain morphometry for the nucleus accumbens, amygdala, hippocampus, cerebellum, or 35 cortical regions in each hemisphere. Interestingly, when they simply controlled for the amount of alcohol use, rather than matching users and nonusers, they replicated several findings of Gilman and colleagues. Furthermore, when examining effect size across previous studies, they found that the literature demonstrates a mean lack of effect. Given the discrepancies in the literature, we wanted to re-examine this issue using a large representative sample.

To this end, we analyzed extremely high-quality multi-modal neuroimaging data from 466 participants in the Human Connectome Project  who reported using marijuana at least once during their lives.The participants in this sample consist of twins and their non-twin siblings who have no history of major psychiatric illness, but vary greatly in terms of race, education, income, BMI, and the degree of recreational drug use. A recent study used this HCP data-set to disentangle causal effects of marijuana use on regional brain volume from shared genetic effects and found that it was mainly shared genetic effects explained differences in bran volumes.However, this study did not investigate the effects of marijuana use on white matter integrity or the shape of subcortical regions, which was the focus of the current study. Rather than investigating extremes of marijuana use  like most previous studies, we leveraged the large sample size to take a parametric approach, examining marijuana use along a spectrum, so as to search more specifically for dose-dependent effects. Nevertheless, a comparison of users and nonusers was also performed as a replication of prior work.Analyses of voxelwise gray matter morphometry were carried out with FSL-VBM  an optimized VBM protocol carried out with FSL tools.First, structural images were brain-extracted and gray matter-segmented before being registered to the MNI 152 standard space using non-linear registration.The resulting images were averaged and flipped along the x-axis to create a left-right symmetric, study-specific gray matter template. Second, all native gray matter images were non-linearly registered to this study-specific template and “modulated” to correct for local expansion  due to the non-linear component of the spatial transformation. The modulated gray matter images were then smoothed with an isotropic Gaussian kernel with a sigma of 3 mm. Finally, voxelwise GLM was applied using permutation-based non-parametric testing, correcting for multiple comparisons across space, using Threshold-Free Cluster Enhancement.Following Weiland et al.,we also performed a multivariate analysis on the effects of outdoor cannabis grow use on subcortical and cortical volumes and cortical thickness extracted with Free Surfer.

Rather than analyzing whether marijuana showed a multivariate effect across all 35 cortical regions contained in this table as did Weiland et al.,we chose an a priori approach, focusing on prefrontal regions and subcortical regions where marijuana has been shown to have significant effects.Regions of interest included 15 prefrontal cortical regions : medial and lateral orbitofrontal cortex, caudal anterior cingulate, caudal middle frontal, inferior frontal gyrus,rostral middle frontal, superior frontal, and frontal pole. Subcortical regions included nucleus accumbens, hippocampus, cerebellum cortex and white matter, thalamus, and amygdala.White matter volumes were included for the anterior and mid anterior corpus callosum.We then examined the effects of marijuana use on white matter diffusion parameters.The group comparison showed no significant effects, possibly suggesting that the frequency of marijuana use in the HCP sample is not severe enough to replicate previous studies, which largely focused on comparisons of non-users and daily marijuana users. In line with this possibility, there were no linear effects of the number of times used on white matter coherence in users.As shown in Fig. 2, age of first use had a positive association with FA as well as a negative association with RD, such that an earlier age of first use was associated with lower FA and greater RD in a large cluster of right hemisphere white matter. These tracts primarily subsisted of the Superior Longitudinal Fasciculus,Inferior Longitudinal Fasciculus,and Forceps Major and Minor. The SLF connects the prefrontal cortex and parietal cortex and is involved in executive functions,and the ILF connects the temporal and occipital cortices, has been shown to affected by adolescent marijuana abuse.The Forceps Major and Minor are extensions of the corpus callosum connecting the left and right occipital and frontal lobes, respectively. Thus, even though most of the effects on FA and RD were found in the right hemisphere, communication between the left and right hemispheres may be impacted by marijuana age of onset. When examining the subset of unrelated marijuana users, we confirmed the negative effect of an earlier age of first use on FA and RD in the SLF, as shown in Fig. 3.

Participants were also told that some decks were worse than others and were asked to treat the money in the game as real money. Following card selection, participants were given feedback about monetary gain or loss displayed on the computer screen. Participants began the task with $2000 in their bank. After card selection, participants could win $100 in decks A and B or $50 in decks C and D. In some instances, however, participants were credited with money, but were required to pay a penalty. For each card chosen, there was either an immediate gain or an immediate gain followed by a penalty.Unknown to participants, card selections in decks A and B were classified as disadvantageous decisions because although larger winnings were possible by selecting cards from these decks, selection from these decks was also associated with larger losses, decreasing net earnings during the task. Card selections in decks C and D were classified as advantageous because although smaller winnings were possible by selecting cards from these decks, selection from these decks was also associated with smaller losses, increasing net earnings during the task. Participants completed 100 trials without interruption or caps on deck selections. At the end of administration, the net earnings were displayed on the computer screen. Total net scores were derived by subtracting the total number of cards selected from disadvantageous decks A and B from the total number of cards selected from advantageous decks C and D. The majority of studies in MJ users have used net IGT scores to examine decision making between and within groups across the task .This strategy of analysis allows researchers to compare decision-making differences between and within groups across the task by examining differences of advantageous and disadvantageous card selections.

Additional analyses for the IGT include comparing the total amount of money lost by each group,or examining net earnings at the end of the task,as well as measuring the amount of time needed to complete each task administration for multiple IGT sessions.However, these strategies do not account for the possibility of detecting between group differences across time. Therefore, we chose to focus the analyses on net IGT scores using a mixed-model analysis of covariance,cannabis grow supplies as outlined below .This study examined the relationship between frequent MJ use and risky decision-making in young adult college students using the IGT. To our knowledge, only one other study has examined risky decision making using the IGT in a similar and narrow age range of young adults.In the current study, MJ+ were older and had significantly lower IQ scores relative to HC. As both age and IQ were related to IGT performance, they were included as covariates in the analyses.Although MJ+ made advantageous card selections as indicated by the positive net IGT scores, they made less advantageous choices compared to HC. This effect is consistent with prior research examining group differences between MJ users and healthy controls in young adults.Research suggests that MJ users are more likely to make risky judgments despite subsequent monetary punishment than healthy controls  and exhibit increased impulsive decision-making by selecting more disadvantageous cards than healthy controls.Additionally, the current findings support prior research that found young adult MJ users made more selections from disadvantageous decks A and B compared to healthy controls.However, in the current study, MJ+ also made fewer card selections than HC from deck C, an advantageous deck, but one that is associated with frequent punishments relative to deck D.This could suggest MJ users may prefer decks that are associated with frequent rewards and infrequent losses, which could drive reward-driven behavior.

This observed performance difference in reward-driven behavior may be attributed to differences in utilization of the prefrontal cortex during strategy and choice selection. Future studies that utilize the IGT in young adults during fMRI are needed to explore this question. Furthermore, we found that the effect of group on net IGT scores was significant when including sex as a factor in the model. Overall, MJ + had lower net IGT scores compared with HC.Additionally, there was a trend for female participants to have lower net IGT scores than male participants.In the current study, the trend towards poorer net IGT performance in female relative to male participants appears to be driven by females tending to make more disadvantageous selections from deck B, where rewards are frequent and losses are infrequent, while at the same selecting fewer cards from advantageous deck C in which loss frequency is equal to gain frequency. Females may also be performing worse than males due to differences in the time needed to develop decision-making strategies towards advantageous choices. Male participants may be better at suppressing reward-driven behaviors due to activity in the right dorsolateral prefrontal cortex activity that has been shown in males but not females completing the IGT.A previous study that examined sex differences between young adult male MJ and female MJ users found that lifetime MJ use was associated with poorer decision-making performance in male but not female participants.However, this study did not perform an interaction between group and sex on net IGT scores due to the absence of healthy controls. Thus, it is unknown whether similar findings would have also been seen if female and male non-MJ users had been included. The observed trend for sex differences on the IGT may also be attributed to the possible influence of sex hormones on executive functioning. A study examining the interactive effects of dopamine base levels and cycle phase on executive functions found that women were significantly faster on the Stroop during the luteal phase compared to menses and pre-ovulatory phases.

This suggests women have improved verbal skills during the luteal phase when levels of progesterone and estradiol are high. Another study found that women ovulating were more likely to choose risky options than men.In the current study, females may have performed worse on the IGT because we may have unknowingly sampled a high percentage of women in a stage of their menstrual cycle where they are more likely to take risks. However, since we did not ask female participants to report menstrual cycle stage at the time of the study visit, we are unable to confirm whether hormone levels may have influenced IGT performance. No differences were observed between MJ+ and HC mean reaction times during the IGT, which is inconsistent with our initial hypothesis. To our knowledge, no studies in MJ users have examined mean reaction times on the IGT. While risky decision-making may be related to impulsivity, it may be important to utilize other neurocognitive measures that assess motor impulsivity and response inhibition. In a fMRI study investigating the relationship between MJ use and inhibitory control processing, MJ users tended to have faster reaction times than healthy controls.Additionally, brain activity differences were observed in the dorsal anterior cingulate cortex, a region of the brain thought to be involved in impulse control. In the present study, as mean reaction time was not significantly related to IGT performance, MJ+ took the same amount of time as HC to make decisions during card selection. This finding suggests that lower net IGT scores in MJ+ relative to HC may be related to maladaptive decisions that are not associated with motor impulsivity during card selection. Although age at first MJ use, 30 day MJ use and lifetime MJ use were not significantly related to IGT performance among MJ+, between group differences on the IGT suggests there may be potential differences between MJ+ and HC that could be related to pre-morbid vulnerability for risk-taking tendencies and/or the effects of substance use itself.For example, a previous study showed that early-onset frequent marijuana users had a thicker prefrontal cortex than late-onset frequent MJ users, which could indicate reductions in normative grey matter pruning in the prefrontal cortex in participants who begin using MJ at a younger age.While previous studies have found associations between early adolescent MJ use and impairments in executive functioning,we did not find a relationship between age at first MJ use and risky decision-making.

In the current study, we asked participants to report their age at first MJ use instead of age at regular MJ use, which may be more closely associated with patterns of MJ use that could predict neurotoxic consequences of use. Age at first use can be a difficult variable to assess, especially in young adults aged 18–22 years, since age at first MJ use may have occurred very recently in this population and thus, participants may have only had a year or two of substance use prior to the study visit.One limitation of the current study is the modest sample size. Although our sample was relatively well matched in the number of participants in each group, our findings may not be readily generalizable to young adult college students. Another related issue is the overrepresentation of males in the MJ group. Although the prevalence of MJ use is higher in males than females,our findings may not be generalizable to female MJ users. Although onset of cannabis grow facility withdrawal symptoms typically occur in frequent MJ users after 24 h of abstinence, and peak 2–6 days post cannabis abstinence,we cannot confirm whether or not participants were in active withdrawal during the study visit. Future studies should administer the Marijuana Withdrawal Symptoms checklist  to assess withdrawal symptoms in participants at the time of the study visit. In addition, the potency of MJ is not standard and our study design does not take into account dose-response associations in MJ+. Future studies will need to assess other indicators of MJ use, such as asking participants to report THC content of the MJ they typically use. Another limitation is that we utilized a laboratory task of decision making and provided participants with hypothetical earnings rather than tangible incentives. In future studies, it will be important to use other real-life decision-making measures to determine if our findings are specific to the IGT, are associated with non-monetary risk-taking behaviors, or are associated with decision-making in general.

As we only used one task of decision-making, our findings may not generalize across a wide range of decision-making tasks. Future studies may want to utilize additional tasks to assess risky decision-making, such as the Balloon Analogue Risk Task  or Cambridge Risk Task.Additionally, as most MJ users are also alcohol users, alcohol was not used as exclusionary criteria for MJ+. While post-hoc analyses suggested alcohol use was not related to IGT performance, we cannot rule out the possibility that the neurotoxic effects of alcohol may play a role in the observed group differences on decision-making performance. In models examining the effects of both MJ use and alcohol use on net IGT scores, neither significantly predicted decision-making performance in MJ+, which may be due to lack of refined measure to assess frequency of these substances and premorbid characteristics that distinguish MJ+ from HC. Other studies that reported group differences on the IGT between MJ users and healthy controls either did not examine relationships between marijuana use variables and IGT performance,only examined other substance use variables in relation to IGT performance,or did not find associations between substance use variables and IGT performance.One study by Verdejo-Garcia et al.  reported greater joints smoked/week was associated with lower net IGT scores in abstinent marijuana users, but did not examine other substance use characteristics in relation to IGT scores within the same model. We believe future studies should consider the relationship between MJ use and decision-making performance, while accounting for poly-substance use. Finally, while we observed a trend for MJ+ to report greater recent anxiety on the Beck Anxiety Inventory,compared with HC,the main effect of group remained significant when controlling for BAI scores in the ANCOVA models with  and without sex  included as a factor. As anxiety levels may affect decision-making, future studies should ascertain that anxiety levels in MJ users are not driving any observed decision-making differences between MJ users and healthy controls. In summary, the current study examined the effects of frequent MJ on risky decision-making in college-aged young adults.