Members of this gene family are extracellular matrix glycoproteins that play a role in neural cell adhesion and neurite outgrowth. Suggestive evidence of associations with ADHD was observed for an additional 111 SNPs (⩽9.91E−05). Although intriguing, the association between DNA variation in the TNR gene and ADHD should be viewed as preliminary given the small sample size of this discovery dataset.It is well-established that there is a strong genetic contribution to the aetiology of attention deficit hyperactivity disorder (ADHD). Here, we employed a hypothesis-free genome-wide association study (GWAS) design in a sample of 480 clinical childhood ADHD cases and 1208 controls to search for novel genetic risk loci for ADHD. DNA was genotyped using Illumina’s Human Infinium PsychArray-24v1.2., and the data were subsequently imputed to the 1000 Genomes reference panel. Rigorous quality control and pruning of genotypes at both individual subject and single nucleotide polymorphism (SNP) levels was performed. Polygenic risk score (PGRS) analysis revealed that ADHD case–control status was explained by genetic risk for ADHD, but no other major psychiatric disorders. Logistic regression analysis was performed genome-wide to test the association between SNPs and ADHD case–control status. We observed a genome-wide significant association ( The global prevalence of ADHD has been estimated at 5.2–7.2%4. Although the aetiology of ADHD is not well defined, genetic and environmental factors have been implicated in the disorder. Despite minor disparities across individual samples and study designs, the overall heritability of ADHD is estimated at 70–90%6.Attention deficit hyperactivity disorder (ADHD) is the most commonly diagnosed neurodevelopmental disorder of childhood. It is typically characterised by a persistent pattern of inattention, impulsivity and/or hyperactivity. Longitudinal studies indicate that approximately 66–77% of individuals who experience childhood ADHD continue to experience at least subthreshold symptoms of ADHD that significantly impact adulthood functioning7. Putative disruption of other monoamines such as noradrenaline and serotonin in ADHD has also been proposed. Accordingly, genetic markers mapped to these monoamine pathways have historically been pursued as candidate genes for ADHD. Although a number of replicated findings exist within this candidate gene literature9, a key limitation remains the a priori selection of genes based upon incomplete knowledge of the biology of ADHD, which may ultimately impede the identification of novel risk markers.The efficiency of indirect dopamine agonists in reducing the symptoms of ADHD led to the development of the ‘dopamine hypothesis of ADHD’, which postulated that dysregulated dopamine signalling is central to the pathophysiology of ADHD16, two were family-based analyses18, and three examined the association between quantitative ADHD symptom measures and genetic markers21. A GWAS meta-analysis was also performed in 2010 22. Overall, these previous ADHD–GWAS had limited success in identifying associations. However, preliminary evidence of associations  were identified for genes that function in biological processes relevant to ADHD aetiology. For example, pathway analysis highlights a potential role for potassium channel genes and activation Ras Homologue Family Member A (RhoA) signalling genes, lending further support to hypothesised dysregulation of neurotransmitter release in ADHD23. Additional pathway analysis utilising data arising from five ADHD–GWAS using the Ingenuity and BiNGO tools, showed significant enrichment of genes mapped to a network involved in neurite outgrowth whose targets are modulated by drugs used to treat ADHD24. Most recently, a large meta-analysis of GWAS data arising from 20,183 ADHD cases and 35,191 controls yielded the first 12 independently significant ADHD–GWAS loci25.Genome-wide association studies (GWAS) allow the identification of novel risk variants without prior knowledge of the biology of a trait or disorder. Further, this approach is aligned with the polygenic nature of complex disorders whereby the small role of individual single nucleotide polymorphisms (SNPs) can be considered in aggregate to better understand how genetic susceptibility may arise. To date, 13 ADHD–GWAS have been published. Of these, seven were case–control studiesHere, we conducted a GWAS on a rigorously diagnosed clinical ADHD cohort collected across Australia, England and Ireland in an attempt to clarify further the genetic architecture of ADHD and to potentially identify novel genetic risk factor(s). An additional important purpose of the current study was to contribute to the expansion of the international GWAS community. In this context, GWAS data derived from this study can be combined with those of the international ADHD–GWAS community  for subsequent GWAS analyses.N = 567) children with ADHD of European ancestry were recruited from Australia, the United Kingdom and Ireland. None of the ADHD probands were included in previously published GWAS. All cases met the DSM-IV diagnostic criteria for ADHD at the time of sample collection. ADHD status was determined through parental semi-structured interview and completion of the Conners’ Parent Rating Scale for ADHD, which has demonstrated internal reliability and criterion validity for use in assessing ADHD26. Children with an IQ less than 70 as determined using the Wechsler Intelligence Scale for Children version 4 27 were excluded. One thousand two hundred and ninety-six control (N = 1296) participants were also recruited. All patients and controls were European by descent based on self-report ethnicity of all four grandparents28. All control participants were recruited in Australia and had no self-reported personal history of psychiatric or neurological disorders including ADHD. Written informed consent was provided by the individual in the case of adults, or the primary caregiver or guardian in the case of children/adolescents.Five hundred and sixty seven  and supplemented with an additional ~50,000 SNPs implicated in psychiatric and neurodevelopmental disorders. To avoid spurious GWAS findings, we adopted a stringent quality control (QC) protocol using PLINK 1.9 software at both the individual subject and SNP levelThe following subject-level QC was employed: (1) We initially removed individuals with low-genotyping score by excluding participants with ⩾0.03 of missing data; (2) we performed identity by descent analysis to detect and remove possible sample contaminations, duplications as well as unknown familial relationships (such as relatedness); (3) we applied principal components analysis to identify any potential sources of population stratification and removed outlier subjects; (4) we tested for unusual heterozygosity, which refers to the presence of more or less heterozygous SNPs across the genome than would be expected by the population mean, and removed individuals displaying outlying mean heterozygosity (greater than ±3 SDs from the sample mean); and (5) disparities between recorded and observed sex status were determined through X-chromosome homozygosity were removed.M = 10.27 years, AgeSD = 3.03). Of the ADHD cases 87% were male and 13% were female. The high frequency of male participants in the ADHD cohort is reflective of the sex ratio in clinical populations31. For controls, 1208 participants aged between 7 and 60 years  were carried forward to the final statistical association analysis. Of these participants, 49% were male and 51% were female.Following the above rigorous QC, 480 ADHD probands  remained in the final set for analysis. All children were aged between 5 and 18 years  missing genotype rates between cases and controls were also removed. Finally, SNPs with a minor allele frequency (MAF) < 0.01 were removed leaving 290,265 in the final set taken forward to imputation.Prior to imputation, additional filtering steps were conducted by removing 3516 SNPs with genotyping call rate <95. In addition, genotyped SNPs departing from Hardy–Weinberg (H–W) equilibrium were also excluded. This resulted in the removal of 76 SNPs. A further 1855 SNPs with significantly different 34. PGRS for each subject and disorder were estimated as a sum of risk alleles weighted by their effect size as defined by data arising from the latest publically available GWAS38. For each disorder PGRS were calculated at a 1000 p thresholds (pT) ranging from 0.0005 to 0.5. To find the most predictive pT logistic regression was applied at each threshold using ADHD status as a regressor and Nagelkerke’s R2 and the corresponding p values were estimated. Analysis of PGRS here served to demonstrate that the current ADHD cohort replicated the published and publically available genetic risk profile for ADHD.Polygenic risk scores (PGRS) for the five major psychiatric disorders including ADHD, autism spectrum disorder (ASD), schizophrenia (SCZ), bipolar disorder (BD) and major depressive disorder (MDD) were calculated using PRSice software package2, age × gender, and the top five eigenvectors accounting for population stratification.Association analysis was performed in 480 ADHD cases and 1208 controls using logistic regression analysis implemented in PLINK 1.9. The following covariates were included: age, agepT values ranging from 0.0005 to 0.5. Age, gender, age2, age × gender, along with the top five eigenvectors accounting for population stratification were used as covariates. As expected, only ADHD PGRS was significantly predictive of ADHD case–control status . PGRS for the other four psychiatric disorders did not reach the recommended statistical significance threshold of p = 0.001at any p-threshold , MDD , ASD , SCZ .Here, we utilised PGRS analysis to determine whether our ADHD cohort replicated the published ADHD genetic risk profile. Logistic regression was used to examine the relationship between PGRS for each of the 5 major psychiatric disorders and ADHD case–control status, for 1000 p values (λ = 1.08) relative to expectation under the null distribution  and the distribution is skewed at the extreme tail of low p values, as expected. As can be seen from the Manhattan plot , was observed (p = 3.15E−08). The imputation quality of rs6686722 is very high (r2 = 0.98). Interestingly, and as presented in the regional association plot , which is mapped into a gene desert region . The second SNP is rs61975260 (p = 5.972E−07), which is mapped to the spermatogenesis associated 7 gene (SPATA7). This suggestive association signal is located within a subregion of Chr14 (88788507–89355721) that comprises several genes including SPATA7, ZC3H14, PTPN21, EML5 and TTC8. Some of these genes, such as SPATA7 have been implicated in psychiatric conditions, including schizophrenia.Three other genomic loci also showed strong trends towards association (p = 9.647E−07) which is mapped 58.4 kbp upstream of the Interferon Alpha and Beta Receptor Subunit 2 gene (IFNAR2). This region of the genome is enriched for genes that function in the immune system . Further, five SNPs from the current study were nominally significant in the PGC-iPSYCH meta-analysis (Supplementary Table The third of these SNPs is rs77224013 (p = 7.6E−15). These data demonstrate that the current dataset is enriched for genetic risk for ADHD and thus makes a worthwhile contribution to the international GWAS effort.Here, we report the results of a GWAS of 480 probands with childhood ADHD and 1208 controls. PGRS analysis revealed that our sample replicated the published genetic risk profile for ADHD. In fact, PGRS of ADHD, but not other major psychiatric disorders , explained 3.25% of variance in ADHD case–control status in our cohort (p = 3.15E−08). This SNP is located 22.8 kbp upstream of the Tenascin R (TNR) gene. The TNR gene is a member of the Tenascin family of the neural extracellular matrix glycoproteins and is highly expressed in the central nervous system39. TNR is known to function in biological processes such as neural cell adhesion, neurite outgrowth and modulation of sodium channel function40 that have been implicated in the aetiology of psychiatric disorders. In addition, TNR interacts with Fibronectin 1 (FN1), the latter being involved in cell adhesion and migration processes including embryogenesis. This interaction may modulate the adhesive properties of TNR during synapse maintenance, a process that is suggested as a risk mechanism for complex psychiatric disorders41. Moreover, genetic association studies have implicated the genomic region where TNR is located in several brain disorders including schizophrenia, Alzheimer’s disease, narcolepsy and neurological sleep disorder42. Further, an additional SNP (rs875326) mapped 2.5 kbp at the 3′untranslated region of the TNR was reported to marginally associate with drug response in schizophrenia43. Although indirect, our TNR ADHD–GWAS finding, combined with findings reported for other psychiatric disorders, provides tentative support for a role of TNR in the aetiology of psychiatric conditions.Genome-wide association analysis further revealed a significant association with rs6686722 that survived the stringent GWAS correction for multiple comparisons (DLC1). Recent studies have revealed common genetic susceptibilities to ADHD and smoking behaviour44. Adults with ADHD have higher rates of substance abuse, including higher rates of tobacco smoking44. In this context, a recent GWAS analysis of nicotine dependence reported genome-wide significant association with rs289519 (mapped to DLC1)45. Analysis of rare CNVs across two independent studies of ASD47, identified rare exonic loss within DLC1 as risk variants for the disorder. Specifically, Prasad et al.46 identified one ASD individual who possessed a rare CNV deletion of 22 kbp across a non-specified exonic region of DLC1. Furthermore, Woodbury-Smith et al.47 identified a 25 kbp deletion encompassing exon nine on DLC1 in two unrelated ASD-affected individuals. The above lines of evidence suggest that DLC1 is a candidate gene worthy of further investigation as a potential susceptibility locus for ADHD and its comorbid disorders (either ASD or substance abuse).The second top SNP in our ADHD–GWAS analysis is mapped 300.2 kbp upstream of the Rho GTPase Activating Protein  of the spermatogenesis associated 7 (SPATA7) gene which is expressed in the retina and cerebellum. This gene functions in the localisation of retinitis pigmentosa GTPase regulator interacting protein 1 to the photoreceptor connecting cilium (CC), as well as protein trafficking across the CC. Processing speed is a cognitive function that is compromised in psychiatric conditions including ADHD49. In this context, GWAS analysis49 of information processing speed measured using digit symbol, simple reaction time (RT), and 2 and 4-choice RT showed suggestive evidence of association between DNA variation in SPATA7 and 2-choice RT (p = 2.71E−06).An additional subthreshold association with ADHD was observed for rs61975260 (IFNAR2) and interleukin 10 receptor subunit beta (IL10RB), respectively. Other immune modulator genes such as IL10RB-As1 and IFNAR1 also map to this region. Immune imbalance has been suggested as a predisposing factor for ADHD in genetically susceptible individuals50. Significantly increased transmission of IL-1Ra 4-repeat allele and decreased transmission of 2-repeat allele of a variable number tandem repeat polymorphism to ADHD-affected children was reported by Segman et al.51 Further, immune dysregulation is supported by the finding that ADHD individuals have four times higher concentrations of Interleukins (IL-1 and IL-6) than typically developing children52. This led Verlaet et al.50 to hypothesise that “overproduction of these cytokines could lead to chronic inflammation in brain tissues”. This is consistent with brain anomalies in children with ADHD. For example, Nopoulous et al.53 reported increased frequency of gray-matter heterotropia (ectopic nodules of neurons) and posterior fossa abnormalities in ADHD patients compared to controls. Further, reduced cortical volume associated with reduced surface area and gyrification were also reported in ADHD compared to controls54. However, the correlation between overproduction of cytokines and brain anomalies in ADHD requires further evidence to establish a causal link.Finally, suggestive evidence of association between ADHD and rs77224013 (9.65E−07) was also observed. Rs77224013 is located 58.4 and 94.8 kbp upstream of the immune cytokine receptors interferon alpha and beta receptor subunit 2 , mapped to intron 3 of semaphorin 6D (SEMA6D) gene which sits within 70 kbp genomic region of significant LD. Members of the semaphorin gene family have been implicated as inhibitors or chemo-repellents in axon pathfinding and fasciculation and branching. More recently, Klein et al.55, examined if the genetic risk markers  mediate alteration in brain structure. They observed that rs281323 (in perfect LD with rs281324) is significantly associated with increased risk for ADHD and putamen volume. Further, the SEMA6D rs281323 is strongly associated with the expression level of SEMA6D55. Together, these finding clearly implicate SEMA6D as a susceptibility locus for ADHD.Our sample has provided replication evidence for some of the results arising from the recent and largest ADHD meta-analysis by the PGC-IPSYCH consortiaTNR (rs6686722) is not significant within the much larger PGC-IPSYCH meta-analysis of ADHD (p = 0.07). Notwithstanding these limitations, our ADHD cohort is clearly enriched for genetic risk for ADHD, as evinced by our strong PGRS results for ADHD (but not the other major psychiatric disorders). As such, our study therefore makes an important contribution to the international genetics effort for ADHD.Finally, it is important to emphasise that our study has a number of major limitations. First, the discovery sample is small and has limited statistical power to detect a reliable genome-wide association signal. Further, our leading SNP association for Supplimentry Table 1


Z- and E-alkenes in high stereochemical purity  starting from stereochemically pure Z- or E-alkenes. The aim of this tutorial review is to organize the reported information concerning ruthenium dithiolate catalysts in a logic manner, thus providing an "operators handbook" for chemists who wish to apply this methodology in synthesis.Stereoretentive olefin metathesis based on ruthenium dithiolate complexes has become a very active field of research within the past years. This unique catalyst class is able to kinetically produce both  Fur. FurRu-1atalysts . The samkenes Ru- 6]. Fur. FurRu-1Ru-10) . Theand Ru-2 . A highland Ru-2 . ROCM re:12 Z/E) . Cyclobuectivity  17]. It. ItRu-1  review) . Cross m Hoveyda  4]. The. TheRu-1itutions . Anotherene (18) . The resd Grubbs , stericaZ)- and (E)-methyl 9-octadecenoate (20) was studied by Grubbs in 2017  . The. TheZ)- .g., 24) –15. As pZ-alkenes react faster than E-alkenes and therefore require a lower catalyst loading .5Ru-A . This 1,2-sulfide shift generates a new ruthenium complex Ru-B which is probably catalytically inactive.Hoveyda proposed that the catalyst degradation in the presence of terminal olefins is due to the generation of unstable methylidene-ruthenium species  4]. Ter. Ter4]. Ru-A . Once coRu-13 which was formed by nucleophilic attack of a sulfide ligand onto the electron-poor benzylidene ligand [Ru-2) by an electron-deficient dichloro catecholthiolate (Ru-3) should render the sulfide ligand less nucleophilic and therefore less prone for nucleophilic attack. This hypothesis gained credence by increased isolated yield for the cross metathesis of allylbenzene with cis-butenediol: Ru-2 (42% yield) versus Ru-3 (61%) yield (This assumption is supported by the isolation of ruthenium complex e ligand . Hoveyda%) yield  4]..Ru-13 wh6A and B into methylene capped olefins C and D by applying a large excess of (Z)-2-butene -2-butene (Z-25) and propene E are then removed in vacuo (100 Torr) and a new portion of catalyst is added for the cross metathesis of C and D to give desired product F with excellent stereoisomeric purity along with side products G and H which require chromatographic removal. A major drawback of this strategy is that (Z)-2-butene (Z-25) is not commercially available in many countries .Experimental observations clearly indicate that terminal alkenes are detrimental for stereoretentive metathesis reactions with ruthenium dithiolate catalysts. In 2017 Hoveyda proposed the in situ methylene capping strategy as a solution to this problem . The trine Z-25, . (Z)-2-bA/B was applied. Practical limitations are that A and B have to be of significantly different polarity for easy column chromatographic separation and that sterically hindered olefins are not tolerated. For some alkenes, e.g., styrenes, the homodimerization is too fast leading to stilbene formation. Replacing styrenes by (Z)-β-methylstyrenes  allowed for successful reactions with methyl ester 33 (34) are not suitable cross-metathesis partners for (Z)-β-methylstyrenes. Hoveyda reasoned that with the sluggishly reacting styrene 32 the protonation and loss of the catechothiolate ligand by Brønsted acid 34 is a faster process leading to catalyst degradation. It should be noted that stereoretentive CM and RCM with (E)-2-butene (E-25) as capping reagent were also reported, however, these reactions required a significantly higher catalyst loading (10.0–12.5 mol %) [Z)-butene (Z-25) was also studied affording 14–21-membered macrocycles  in good yield and high stereoretention  and alkyl containing metathesis partners (products Z-46 and E-47) to standard reaction conditions [More recently Hoveyda disclosed his findings concerning the synthesis of atalysts  22]. In. InZ- oratalysts , Ru-7 watathesis . This wa748 is deprotonated with Et2Zn to provide Zn-dithiolate 49  to give the product in high yields and excellent stereochemical purity. We applied the in situ generated catalyst to several reactions including cross metathesis, self-metathesis and RCM reactions. The selectivities are in general very high (Z/E = 98:2 or higher).Very recently, our group developed an in situ synthesis of dithiolate catalysts with the aim to avoid tedious isolation of Ru-dithiolate catalysts and to render this class of catalyst available to every practicing chemist . A very olate 49 . Then Ho853, Z-olefin is indicated. Given the high stereoisomeric purity of the obtained products we can expect many other examples to be reported in the near future.Several biologically active compounds, fragrance molecules and natural products were synthesized utilizing stereoretentive metathesis based on Ru-dithiolate catalysts, for example (+)-neopeltolide 53,  24]. Fo. Fo53, 2Z-stereoretentive method is much more efficient and practical compared to stereoretentive methods for E-alkenes. Certainly making the precursors of Ru-6, Ru-7, Ru-8 and Ru-9 commercially available would significantly help to further promote E-stereoretentive metathesis. Nevertheless, it can be stated that the field has come a long way compared to where it was 5 years ago and certainly further important improvements will be reported in the near future.Within only a few years the field of stereoretentive metathesis using ruthenium dithiolate catalysts has attained a remarkable level of maturity. The fast development in this field is due to the complementary contributions of the Hoveyda and Grubbs groups who developed a set of general and highly stereoretentive Ru-dithiolate catalysts. A major limitation at the moment is that the


Sorbus commixta Hedl. (Rosaceae) has been traditionally used in oriental countries for the treatment of asthma and other bronchial disorders. In this study, a chalcone glycoside was isolated from the ethyl acetate extract of the fruits of this plant. The compound was identified as neosakuranin based on the spectroscopic analysis and comparion with literature data. This is the first report of isolation of neosakuranin from Sorbus commixta. Sorbus commixta Hedl. (Rosaceae) has been used for the treatment of cough, asthma, and other bronchial disorders in East Asian countries, including Korea, China and Japan + ; 1H and 13C-NMR data for 1. 1H-NMR: δ 8.01 , 7.70 , 7.62 , 6.84 , 6.34 , 6.15 , 5.19 , 3.39 - 3.45 and 3.48-3.54 , 3.72 , 3.91 , 3.83 ; 13C-NMR: δ 124.45 (C-α), 143.31 (C-β), 193.43 (C=O), 127.11 (C-1), 130.58 , 159.88 (C-4), 115.60 , 107.00 (C-1’), 160.06 (C-2’), 93.72 (C-3’), 165.87 (C-4’), 95.23 (C-5’), 166.27 (C-6’), 100.63 (C-1"), 73.74 (C-2"), 77.17 (C-3"), 69.94 (C-4"), 77.26 (C-5"), 61.11 (C-6"), 54.85 (OMe).Sorbus commixta fruits led to the isolation of the known chalcone glycoside neosakuranin. This is the first report of the presence of this compound in this species. Phytochemical study of


We describe methods to improve the efficiency with which HIV-1 Envelope glycoprotein SOSIP trimer immunogens can be produced by transient transfection of ExpiCHO-S cells and then affinity purified using the trimer-specific human monoclonal antibody PGT145. The specificity of PGT145 for properly folded trimers allows for the facile, one-step, isolation of these immunogens in research laboratories. PGT145 columns are also valuable as a component of more complex purification processes in current Good Manufacturing Practice programs. However, we found that PGT145 purification was highly variable and markedly inefficient when used to process supernatants from transiently transfected ExpiCHO-S cells expressing the BG505 SOSIP.664 and other trimeric Env proteins. In contrast, no such problems arose when the same Env proteins derived from a stable CHO cell line were processed on the same PGT145 columns, or with transient transfection supernatants from 293F cells. An investigation of the ExpiCHO-S transfection system identified the presence of polyanions, including but perhaps not limited to dextran sulfate, in the Enhancer component of the transfection system. We hypothesized that these polyanions bound to the cationic PGT145 epitope on the trimers and impeded their ability to bind to the PGT145 affinity column. We found that replacing the Enhancer component with alternative culture medium supplements substantially increased the yield of PGT145-purifiable trimers, and we also confirmed that both dextran sulfate and the Enhancer component were indeed inhibitors of PGT145 binding to BG505 SOSIP.664 trimers in immunoassays. The presence of polyanions, including but not limited to nucleic acids, should be considered in other circumstances where PGT145 columns are less efficient than expected at purifying native-like trimers. Native-like, recombinant soluble HIV-1 envelope glycoprotein (Env) SOSIP trimers are widely used as a vaccine development platform in programs aimed at inducing neutralizing antibodies (NAbs) . The proThe ExpiCHO-S transient transfection system offers advantages over 293F transfections, particularly as it allows the use of CHO cells for pre-clinical research that might advance to a cGMP program that is based on a stable CHO cell line . We noteenv and furin genes  of 10 kDa) (Thermo Fisher Scientific) and further concentrated using a 100-kDa cutoff Vivaspin column . The 2G12 affinity-purified proteins were further purified by size-exclusion chromatography (SEC) on a Superdex 200 26/60 column  to isolate the trimer fractions and eliminate dimers and monomers. When PGT145 affinity columns were used without SEC, a modest amount of aggregated trimers was also sometimes observed, but their presence did not substantially affect the quantitative estimates of trimer recovery. When specified in Results, 100 ml of culture supernatant was buffer-exchanged into phosphate-buffered saline, pH 7.0 (PBS) using SnakeSkin dialysis tubing (10 kDa MWCO) prior to passage down the PGT145 column. Protein concentrations were determined using a bicinchoninic acid-based assay . Env proteins purified by the 2G12/SEC or PGT145 methods were verified to be trimeric by blue-native polyacrylamide gel electrophoresis (BN-PAGE), as described previously [Env proteins were purified by immunoaffinity chromatography using a 2G12 or PGT145 column , 8. The 2 pH .2. The eThe ELISA used to detect and quantify the production of PGT145-reactive SOSIP trimers has been described previously , 16. BriRL values) of 250 RU (response units). The 2G12 and PGT145 MAbs were injected at 500 nM with or without a prior injection of dextran sulfate  at 1000 mg/L (25 μM) for 30 s. Association and dissociation were each monitored for 200 s at a flow rate of 50 μl/min. After each cycle, the anti-His surface was regenerated by injecting a single pulse of 10 mM glycine (pH 2.0) for 60 s [SPR analysis of antibody binding to immobilized SOSIP trimers has been described previously , 17. BriThe data shown in Figs Our original method for purifying SOSIP trimers from the supernatants of transiently transfected 293T or 293F cells was based on the 2G12 anti-Env MAb followed by SEC . The 2G1vs. 31 μg) implies that the 2G12 column had removed an inhibitor that was preventing trimer association with the PGT145 column. The lack of benefit of the pre-dialysis stage indicates that the inhibitor’s mass was > 10 kDa.The problems with PGT145 affinity purification were particularly pronounced when we evaluated the ExpiCHO-S transfection method. The greater volume of cells and the more prolonged culture conditions allow SOSIP trimers to be produced and purified in multi-mg amounts from a single ExpiCHO-S transfection, whereas 293F transfections typically yield < 1 mg. The outcome is that an ExpiCHO-S transfection can provide enough SOSIP trimer for, e.g., an entire macaque immunization experiment, a useful feature. We soon found, however, that trimer yields from PGT145 affinity columns could be unacceptably low, compared to the 2G12/SEC method. An example is provided by an initial investigation of the problem . Here, 1The outcome of the ExpiCHO-S transfection experiments was inconsistent with our experience with CHO cell lines and 293F cell transfections. Thus, when the same BG505 SOSIP.664 Env protein was produced in the latter two systems, the amounts of trimers recovered by the 2G12/SEC and PGT145 column methods were comparable .Our initial hypothesis to explain the unusual problem associated with ExpiCHO-S transfection was that a nucleic acid such as DNA acted as an inhibitor of trimer binding to the PGT145 column. We were aware that CpG ODNs could bind to SOSIP trimers and occlude the PGT145 epitope . We therThe ExpiCHO-S transfection method involves the addition of Enhancer and Feed components to the CHO cells to maintain the viability of the cells. Following the manufacturer’s instructions, we next assessed whether these components affected PGT145 recovery. While the unsupplemented transfection supernatant again had no substantial effect, the addition of the Enhancer plus Feed components or only the Enhancer component reduced trimer recovery to 40% and 33% of the input, respectively .inter alia, an unspecified amount of dextran sulfate, a polycation that prevents cell aggregation into clumps during the multi-day span of the post-transfection culture [A review of the patent literature revealed that the ExpiCHO-S Enhancer component contains,  culture . Dextran culture . Dextran culture . We therTo improve the production of SOSIP trimers in transfected ExpiCHO-S cells, we expressed a BG505 SOSIP.664-His construct in these cells using four different transfection conditions: ExpiFectamine CHO, ExpiFectamine CHO with Feed and Enhancer, FectoPRO and FectoPRO with Booster. Using the ExpiFectamine CHO reagent and with its supplements for ExpiCHO-S cells is recommended by the manufacturer (Thermo Fisher Scientific); the FectoPRO reagents were purchased from a different supplier, Polyplus-transfection SA. Then the four ExpiCHO-S cultures were monitored over time. In the ExpiFectamine cultures, cell viability crashed/cells died after day-4 unless Feed and Enhancer were included, in which case the culture remained viable until day-14 . HoweverWe next expressed the BG505 SOSIP.664-His trimer in ExpiCHO-S cells for 14 days, using either the ExpiFectamine CHO with Feed and Enhancer or the FectoPRO with Booster systems, and then purified the trimers by the 2G12/SEC or PGT145 column methods . For comHere, we show that the otherwise considerable benefits of the ExpiCHO-S cell transient transfection method for producing multi-milligram amounts of PGT145-purifiable SOSIP trimers are compromised by the presence of an inhibitor in the Enhancer component of the manufacturer-recommended culture system. This inhibitor impedes the binding of SOSIP trimers to the PGT145 affinity column. The most likely culprit is the polyanionic anti-clumping agent dextran sulfate, which is present in the Enhancer component and which we showed does act as an inhibitor of PGT145 binding to SOSIP trimers in immunoassays. The mechanism is most probably a charge-based interaction between the polyanion and the cationic apex of the trimer where the PGT145 epitope is located. A similar PGT145 epitope-occluding effect was previously seen with CpG ODNs, a DNA-based polyanionic adjuvant .We consider it possible that DNA, or perhaps RNA, released from dead or moribund cells could also be problematic for PGT145 affinity columns under some conditions, although we did not observe an inhibitory effect in a spiking experiment when purified DNA (an expression plasmid) or a mock transfection culture supernatant was mixed with SOSIP trimers. The impact of nucleic acids should be explored if PGT145 affinity columns work poorly under circumstances when a high level of cell death is seen, for example as a result of electroporation-based transfection . The preThe solution we identified was to eliminate the need for the ExpiCHO-S cell Enhancer component by using instead a different transfection medium culture system, FectoPRO with Booster, with the ExpiCHO-S cells. Other such systems could probably also be identified, but we did not conduct an exhaustive survey of available commercial products. We also note that the FectoPRO with Booster system is ~40% cheaper than its ExpiFectamine CHO with Feed and Enhancer counterpart. This methodology change would therefore also provide cost savings when producing multi-millgram amounts of SOSIP trimers by transient transfection on a research laboratory scale, under circumstances when the creation of a stable cell line is not justified.


N = 192) completed self-report questionnaires for symptoms of ADHD and criminogenic cognitions. Symptoms of inattention were consistently and strongly related to criminogenic cognitions. In particular, inattention was significantly related to cutoff, cognitive indolence, and discontinuity. There was also evidence that impulsivity was positively related to criminogenic cognitions, and specifically, to the power orientation subscale. In contrast, and contrary to expectations, symptoms of hyperactivity were not related to criminogenic cognitions. These results indicate that in community-recruited adults, inattention rather than hyperactivity is related to criminogenic cognitions. We discuss the implications of these findings contrasting with those of previous studies that used forensic and clinical samples.The relationship between ADHD—in particular hyperactivity—and criminal behavior is well documented. The current study investigated the role of criminogenic cognitions in the explanation of this relationship by examining which symptoms of ADHD are associated with criminogenic cognitions. Community-recruited adults ( A disproportionately high percentage of people with ADHD become involved with the criminal justice system. For example, a meta-analysis investigating ADHD and criminal behavior—including both minor offenses and crimes leading to incarceration—indicated a moderate-to-robust association . Up to tADHD symptom clusters that are most closely related to criminogenic cognitions in adult non-offenders.There is a wide range of factors have been implicated in the ADHD-crime relationship  ,15. The There is some controversy concerning ADHD symptom clusters. For example, Babinski, Hartsough, and Lambert  found incriminogenic cognitions, that is, the problematic thought patterns  that precede criminal behavior. Thus, a large body of research indicates that the proportion of individuals with ADHD in the criminal justice system exceeds that in the general population. There is also substantial evidence to suggest that children with ADHD are more likely to engage in criminal activity throughout the course of development, including into adulthood. Moreover, the majority of the literature points toward impulsivity, and to a lesser extent hyperactivity, as the key factors in the ADHD–crime relationship . HoweverWalters  defined lifestyle model of crime, according to which criminal behavior is accompanied by a corresponding system of criminogenic cognitions, such as poor decision-making and blaming others for one’s own behavior. Walters argued that these cognitions maintain the criminal lifestyle, and that only by modifying them can we realistically hope to reduce criminal behavior. To measure criminal thinking quantitatively, Walters [Subsequently, Walters ,32,33,34 Walters  develope Walters  demonstrWhile the literature reviewed above has established a strong link between ADHD and criminal behaviour, relatively little is known about the reasons for the link . AccordiIdentification of these risk factors is likely to have important implications for the development of interventions . If praIn the statistical analyses, we included age and gender in all models. With respect to gender, females tend to commit far fewer, and less severe and violent crimes, compared with males ,39,40,41The sample consisted of 192 participants . Demographic information about the sample is provided in Each participant was given a pack of questionnaires including a demographic questionnaire, the Psychological Inventory of Criminal Thinking Styles (PICTS) ,32, and The PICTS consists of 80 questions. However, for the purposes of this study the two validity subscales were removed, leaving 64 items related to criminal thinking. Some questions implied that the individual had already committed serious crime. We re-worded these questions for use with a community-recruited  sample. Participants rated how strongly they agreed or disagreed with each item on a 4-point Likert scale ranging from 1 (disagree) to 4 (strongly agree). There are eight subscales , and a “r = 0.80 to 0.91) [The CAARS consists of 66 items. This scale contains DSM-IV symptom indices for inattention and hyperactivity/impulsivity, as well as four factor-derived subscales: Inattention/memory problems , hyperactivity/restlessness , impulsivity/emotional lability , and problems with self-concept . Participants rated how strongly they agreed or disagreed with each item on a 4-point Likert scale ranging from 0  to 3 . The CAARS has been found to have high internal consistency (α = 0.86 to 0.92) and test-retest reliability (to 0.91) .Participants completed the questionnaires in their own time, and it was estimated to take each participant approximately 40 minutes. Once completed, questionnaires were returned to the experimenter and a debrief form was provided explaining the purpose of the study. Ethical clearance for the study was provided by the University of East Anglia Research Ethics Committee, and conformed to the protocols governing the use of human research participants outlined by the British Psychological Society. Data were first checked for outliers, which were defined (based on sample size) as values greater than four SDs from the mean. One PICTS score was more than five SDs from the mean, and so further scrutiny of this individual was undertaken. After ensuring that results were not due to any errors, we ran the main regression analyses twice; first with the participant in, and second, out of the dataset. Despite their high deviation from the mean, the case did not exert any substantive influence on the main patterns of findings. Therefore, we elected to retain the participant in the dataset.  were then applied to skewed variables, which were defined as skew values exceeding twice the standard error. The transformations applied to each of the variables are reported in r-value for a significant bivariate correlation for N = ~200) or greater were interpreted; and second, we focused on results that patterned similarly (in terms of significance) for the factor-derived symptom domains and the DSM-IV indexes. In addition, for all regression analyses, the assumptions of regression  were examined. For the total PICTS scores, two backward multiple regressions were run: the first examined the factor-derived subscales , and the second examined the DSM-IV symptom indices (inattentive symptoms and hyperactive/impulsive symptoms). As mentioned previously, we also included age and gender. For the PICTS subscales, an additional set of backwards multiple regressions used each PICTS subscale as a criterion variable. Two steps were taken to avoid problems of multiple testing: first, as recommended by Stevens , only reF = 52.13, p < 0.001. The R2 was 0.53, and age, gender, inattention/memory problems, and impulsivity/emotional lability were all retained as predictors  = 44.75, p < 0.001. The R2 of the model was 0.49. Age, gender, inattentive symptoms, and hyperactive/impulsive symptoms were all retained (see A second (backwards) multiple regression using the two DSM-IV symptom indices showed that the overall model was significant ined see . SimilarAge was a consistent predictor of entitlement  and super-optimism, and impulsivity was a consistent predictor of power orientation, which is defined by aggression and manipulation see . HoweverT-scores > 60 . The results for the sub-group differed from those of the full sample only in that neither factor-derived inattention/memory problems, nor DSM-IV index hyperactivity/impulsivity, was a significant predictor when regressed on to total criminogenic cognitions. We acknowledge that this analysis is likely underpowered given the number of predictor variables included in the model. However, when we examined T-scores > 55, the same pattern of results emerged, and the number of participants meeting this criterion was N = 47. Thus, examining even sub-impairment levels of ADHD symptoms showed little-to-no relationship between hyperactivity and criminogenic cognitions.To investigate whether hyperactivity was related to criminogenic cognitions only at problematic levels, we ran a sub-group analysis including only the 37 participants with DSM-IV index > 60 see  regulation and lack of self-control, and second, strong reactions to frustration and perceived threats [The goal of this study was to investigate how symptoms of ADHD relate to criminogenic cognitions. Previous studies have tended to focus either on people diagnosed with ADHD or on individuals within the criminal justice system ,48,49. I threats . Consist threats  has implcognitive indolence  and discontinuity (inability to follow through on thoughts and actions)—fit well with the diagnostic criteria of inattention and with theories of executive dysfunction in ADHD [cut off—ignoring common psychological deterrents, such as anxiety and guilt) does not readily associate with symptoms of ADHD. However, these three PICTS subscales have been consistently identified in factor analysis studies on the PICTS [thoughtlessness and problem avoidance, which again tends to fit well with descriptions of inattentive symptoms. However, for the DSM-IV indices, the standardized regression coefficient for inattention was three times larger than for hyperactivity/impulsivity. Similarly, the strongest ADHD subscale predictor of total criminal thinking was inattention/memory problems. This contrasts with our expectations, which were based on the findings of most previous research that hyperactivity/impulsivity would be a stronger predictor ,13,55. I in ADHD ,57. The he PICTS ,33,58, aComparing the results of the two regressions, DSM-IV inattention was more closely related to criminogenic cognitions than was inattention/memory problems, which suggests that memory problems are not (or are only weakly) associated with criminogenic cognitions. Similarly, since impulsivity/emotional lability was more closely related to criminogenic cognitions than hyperactivity/impulsivity, and hyperactivity/restlessness did not predict criminogenic cognitions, it is likely that impulsivity is related to criminogenic cognitions, but that hyperactivity is not (or is only weakly) associated with criminogenic cognitions.T-scores > 60, although the analysis was slightly underpowered, as with the full sample inattention and impulsivity were significant predictors of criminogenic cognitions, and hyperactivity was not. Thus, our data showed no association between hyperactivity and criminogenic cognitions.The strength of the relationship between inattention and criminogenic cognitions, and the absence of a clear relationship between hyperactive symptoms and criminogenic cognitions, are both surprising and somewhat counter-intuitive, because at least in children, problematic externalizing behavior is primarily due to the hyperactive/impulsive symptom domain. However, it is important to bear in mind that hyperactivity was not independently tested as a variable in this study. Thus, our conclusions regarding hyperactivity, at this point, should be interpreted with caution. The reason for these contrasting results might be that we tested adults, and there is some debate about how symptom patterns and subtypes remit over the course of development, particularly in adulthood . In addiThe divergent findings of this and previous research are likely to reflect the differing levels of criminal behavior shown by the samples. In contrast to participants in most previous studies, the community-recruited participants in the current study reported lower levels of criminality, and of course, were not incarcerated. This suggests that the combination of criminogenic cognitions and hyperactive symptoms (that is characteristic of previous studies’ samples) predicts criminality, whereas criminogenic cognitions in combination with inattention  does not. If correct, such an inference would have profound implications for our understanding of ADHD, criminogenic cognitions, and criminal behavior. There would also be important implications for policy and practice, primarily in the identification of individuals with ADHD symptoms who are at risk for criminality (because they show high levels of both criminogenic cognitions and hyperactivity), and of those who are not . In addition, interventions aimed at preventing or reducing criminal behavior by these at risk individuals should focus on addressing their criminogenic cognitions, ADHD symptoms, or both.One approach to testing this possibility would be to compare rates of criminality among two groups of people with high levels of both criminogenic cognitions and ADHD. Those whose symptoms were of hyperactivity would be expected to engage in considerably more criminal behavior than those with symptoms of inattention. Another approach would be to conduct interventions designed to reduce criminal behavior by reducing criminogenic cognitions. We would predict that these interventions would be more successful when the primary diagnosis was of hyperactivity/impulsivity rather than inattention. Previous research has indicated that ADHD symptoms are a unique predictor over and above conduct problems. Unfortunately, in this study we were unable to collect assessments of conduct problems, and so we are not in a position to comment on how much of the variance in our ADHD-on-criminogenic cognition results may be shared with conduct problems, and how much variance is unique to ADHD . A seconThese findings indicate that, as well as age and gender, criminogenic cognitions in community-recruited adults are strongly related to inattention, moderately related to impulsivity and impulsivity/emotional lability, and not related to hyperactivity. Given that ADHD symptoms tend to remit over the course of development, we feel that these results are particularly important to understanding the relationship between adult symptoms of ADHD and criminogenic cognitions, and how the understanding of that relationship is important for understanding the relationship between criminogenic cognitions, ADHD, and criminal behavior.


It is known that cationic surfactants have an antimicrobial effect and act as enhancers. This paper studies three cationic surfactants from the group of alkyldimethylbenzylammonium chlorides . Interest is focused on the association of the surfactants with respect to temperature, partition balances and their influence on drug release, rheological properties, and the pH of hydrogels. The critical micelle concentrations (CMC) of the surfactants were estimated from dependencies of conductivity, density, spectrofluorimetry, and UV–VIS spectrophotometry on molarity in the temperature range of 25–50 °C. It was found that the temperature dependence of a CMC is U-shaped, with its minimum at 30 °C, and the CMC value decreases as the length of the chain increases. The pseudo-phase separation model was used for the calculation of various thermodynamic parameters, such as the Gibbs free energies (spontaneous process), enthalpies (exothermic process), and entropies of the micelles’ formation, CMCs, and the degree of counterion binding. All thermodynamic parameters, as functions of the temperature, were estimated. It was found that partition coefficients increase as the length of the alkyl chain and the pH = (5.0–7.0) increase. The influences of surfactants, below and above the CMC, on drug (chlorhexidine dihydrochloride) release from hydrogels, rheological properties, and pH at 30 °C were studied. Also, the amounts of the released drug increase as the alkyl chains of the surfactants prolongate. The amounts of the released drug with the surfactant below the CMC are greater than that above the CMC. All hydrogels  exhibit a non-Newtonian pseudo-plastic flow. The results obtained will be used in the formulation of the drug and surfactants into dosage forms. The optimization of drugs to reach their maximum therapeutic effect is a primordial problem of pharmaceutical technology, and it requires deep knowledge about the influences of auxiliary substances. Appropriate choices of auxiliary substances determine the first process of LADME, namely drug liberation, which in turn influences the subsequent processes, i.e., absorption, distribution, metabolism, and excretion. Therefore, the LADME process should be modelled and analyzed as a whole, which has been a long-lasting research subject of the authors ,2. ThereNumerous papers dealing with the phenomenon of micelle formation have been published. See for instance references ,7. The pThe temperature dependencies of ionic surfactants are not linear. That is because the changing temperature initiates a change of interactions between the hydrophilic and hydrophobic parts of a molecule. The values of the CMC first decrease as the temperature increases, and on reaching the minimum they start to increase. Hence, the dependencies are U-shaped curves. That is because two influences act against each other. The increased temperature causes a decrease of the hydrophilic group, what supports the micellization of the surfactant. At the same time, it develops disruptions in the structuralized water around the hydrophilic group, which in turn suppresses micellization. The ratio of the strengths of these effects decides whether the value of the CMC increases or decreases in a given temperature interval .Surfactants are able to increase the solubility of poorly soluble drugs in water, for instance chlorhexidine dihydrochloride  .Among the physicochemical properties of surfactants, which are influenced by a change in chemical structure, the partition coefficient characterizes lipophility, as it determines the behavior of the substance on the phase interface between the organic solvent and water ,11.The aims of the study were as follows.Association Balances(1) The aim of this part was to determine and compare the CMC values, the degree of counterion bindings, and the thermodynamic parameters of micellization  of three cationic surfactants from the group of alkyldimethylbenzylammonium chlorides where the alkyl was dodecyl: C12, tetradecyl: C14, and hexadecyl: C16. For measurements, four experimental methods were used . The results obtained were mutually compared and their suitability for the solution of the association balances of the surfactants were evaluated.Partition Balances(2) The aim of this part was to determine the dependence of the partition coefficient of the studied surfactants on the length of the alkyl chain and the change of pH of the water phase. The partition balance was measured in solution within the pH range of 5.0–7.5 in the system octanol/pH. This interval of pH is interesting and also important for the preparation of hydrogels.Liberation Balances(3) w/w) with the surfactant, both above and below the CMC.The aim of this part was to design hydrogels with chlorhexidine as a drug. The permeation characteristics of the drug and rheological properties of the hydrogels were investigated. The drug release profiles of the hydrogels without and with the surfactant were measured, analyzed, and finally compared. Experiments were aimed at the evaluation of the release kinetics contained in the same amounts of the drug  The rheological studies confirmed the influence of formulation characteristics on pharmaceutical availability.κ) or molar conductivity (Λ) versus the concentration of surfactants: molarity (c), molality (b), or molar fraction (x). For example, the conductivity measurement of the substance C12 in the solution . Four experimental methods—conductometry, UV–VIS spectrophotometry, spectrofluorimetry, and densitometry—were used. The critical micelle concentration of ionic surfactants is usually determined by the intersection of two straight lines on plots of the conductivity (κ/dc) or (d2κ/dc2) versus molarity. The inflection point of the sigmoid curve , The micellization and CMC of ionic surfactants are affected by various factors, including temperature, as the hydrophobic and hydrophilic interactions depend on the temperature. Studies of the CMC versus temperature have been performed to obtain information on these interactions. For the sake of better visibility, shown are various CMC values which were determined at different temperatures for all studied surfactants, see The CMC values of all studied surfactants pass at the temperature 30 °C through a shallow minimum. Their CMC values with respect to the length of the alkyl chain are ordered as follows:It is known that at a higher temperature the thermal motion increases, which results in de-micellization owing to the distraction of the palisade layer of the micelle, which subsequently enhances the CMC of surfactants . This isλ = 262 nm (f(csurf) show an abrupt change of the line slopes above the CMC (f(csurf)  b.β) were calculated from conductivity measurements according to Equation (1); see The degrees of counterion bindings  is essential for observations of the effect of structural and environmental factors on CMC values. Experimental values of CMC and degrees of counterion bindings were used for the calculation of thermodynamical parameters describing the micellization process according to Equations (2)–(5). The calculated values are summarized in The thermodynamic parameters of micellization play a vital role in understanding the process of micellization. The elucidation of these parameters  release from the hydrogels with and without surfactants, and also their concentration above and below the CMC. The aim was to find the influence of the length of alkyl chain on the drug release from the hydrogels. The average values of the released drug are summarized in From It can be seen that all drug release rate constants from the hydrogels with content of surfactant below the CMC are higher, which can be explained by the fact that in these systems the surfactants are present in a monomer form only; hence, they do not influence the release process to such an extent as if they were above the CMC.From Mr = 578.4) was obtained from Imperial Chemical Industries, Geshire, UK.Chlorhexidine dihydrochloride (Ph.Eur.9)—(abreviation CHX)—1,1′-bis [5-(4-chlorophenyl)biguanide]dihydrochloride, .Pyrene (abbreviation Pyr)—fenantrene—−3 H3PO4 (4.9 g 80% dm−3 H3PO4), 0.04 mol dm−3 CH3COOH (2.4 g CH3COOH), 0.04 mol dm−3 H3BO3 (2.474 g H3BO3), was mixed with a mL 0.2 mol dm−3 of solution of NaOH. The value of pH was within the interval 11.98–2.09. All used chemicals were of purity p.a.Universal buffer, 1000 mL of it contains 0.04 mol dmMr = 190.000–375.000) was obtained from Sigma Aldrich Chemie GmbH, Steinheim, Germany.Chitosan—medium (abbreviation CHIT)—Lactic acid solution (abbreviation LA) was supplied by Merck Chemical Company, (Germany).The 1-octanol for UV–VIS was supplied by Merck Chemical Company, (Germany).−1). The precision of the measurements was ± 0.01 μS cm−1. The solutions were continually stirred and thermostated (thermostat JULABO 5E: Swiss) of the precision ± 0.1 °C. Values of CMC were estimated from measured dependencies (κ = f(csurf), Λ = f(csurf), dκ/dc = f(csurf), or d2κ/dc = f(csurf)).Conductivity dependencies were measured by conductometric titration (by dilution of more concentrated solution with redistilled water) at the temperature range of 20–50 °C using a digital conductomer ino Lab (Germany), with the double conductivity cell and platinum electrode Tetra Con 325 (cell constant K = 0.474 μS cmβ were determined from the slopes of two linear parts of the conductivity curves (κ = f(csurf)) and calculated according Equation (1):S1 is the slope of the linear parts of the conductivity curve below the CMC, and S2 is the slope above the CMC. Units of S1 and S2 are S m2 mol−1 [Values of m2 mol−1 .The pseudo-phase model was applied to estimate thermodynamic parameters because it is of wide acceptance for the interpretation of the energetic state of micelle formation.β values were used in the calculation of the thermodynamic parameters of micellization according to the equations [The CMC and quations :mGΔ):Molar Gibbs energy of micellization (mHΔ):B and C are the parameters of the second-order polynomial:Molar enthalpy of micellization (mSΔ):Molar entropy of micellization (A) of the solutions for association and partition were measured with a UV–VIS spectrophotometer Hewlett Packard 8452A (diode array) in 0.5 or 1.0 cm cuvettes at λmax = 262 nm and a temperature of 25 °C. As a blank was used distilled water. The absorbance (A) of the solutions of hydrogels was measured in 1.0 cm cuvettes at λmax = 254 nm and a temperature of 30 °C.The absorbance (ρ = f(csurf)) at the different temperatures.The samples were prepared by an accurate weighting of the surfactants and redistilled water. All prepared solutions were degassed (10 min) before the densitometry measurement. Density was measured by the densitometer M 4500, Anton Paar  at the temperature range of 20–50 °C. The CMC values of the surfactants were calculated from the dependence (cpyr = 5 × 10−4 mol dm−3) used in the role of a fluorimetric probe. The peaks of intensities I1 and I3 were measured with the spectrofluorimeter Fluoromax-4 Horiba Jobin Yvon  under the following conditions: excitation wave length = 332 nm, excitation slot = 2 nm, emission spectrum = 340–450 nm, step size = 1 nm, and emission slot = 2 nm. From the dependence I1/I3 = f(csurf) sigmoidal curve was calculated the CMC values at the temperatures 25–50 °C.Into the prepared solutions of surfactants was (before the measurement) added 6 μL of pyrene (w) was used solutions of different pH  and as a lipophilic phase was used 1-octanol. Masses of surfactants (cca 5 mg) were dissolved in water phase (10 mL) and octanol (5 mL). Solutions were intensively shacked for 1 h, and after 24 h of staying the absorbance was measured at the wavelength 262 nm. The absorbance was recalculated by calibration curves at concentrations and the values of partition coefficients were calculated in accordance with the equation:m0: mass of sample (surfactants) (kg).cW: molarity of surfactant in water phase (mol m−3).M: molar mass of surfactant (kg mol−1).WV: volume of water phase (m3).OV: volume of octanol phase (m3).The experimental partition coefficient was determined by the method “shake-flask” between phase o/w. In the role of the water phase (w/w) were prepared in 1% (w/w) lactic acid solution with and without surfactants. The concentration of chlorhexidine was 0.1% (w/w). Gels of chlorhexidine were prepared with the three surfactants C12–C16 (Chitosan hydrogels of concentration 2.5% ( C12–C16 . The conw/w) solution of lactic acid was added (up to 50.0 g including other substances). The system was slowly stirred while all particles swelled and dissolved. In this way, the homogenous hydrogel was prepared. The blanks (hydrogels without the drug but with surfactants) were prepared by the same procedure.The hydrogels were prepared in two parallel samples. Accurate amounts of the gelling agent: chitosan, and the permeation enhancer: surfactant were given into the balanced flask. Then, the appropriate amount of the 1.0% −lnWsThe pH values of hydrogels were measured by a combined glass electrode of pH-meter Metrohm (WTW series Inolab at 30 °C after 48 h since the preparation).The association and partition balances of three cationic surfactants from the group of benzyldimethylalkylammonium chlorides  were studied.β, and thermodynamic parameters of micellization.In particular, association balances were studied in the temperature range of 20–50 °C. From the obtained conductivity curves were calculated the values of CMC, f(t) reach a shallow minimum at a temperature of about 30 °C. The values of CMC of the studied surfactants are decrease in the order C12 > C14 > C16, which is caused by increasing lipophility, i.e., the prolongation of the alkyl chain in the molecule of the surfactant.The values of CMC increase with an increasing temperature of the micellization process. The curves of CMC = For verification of the determined CMC from conductivity measurements were used another three experimental methods: densitometry, spectrofluorimetry, and UV–VIS spectrophotometry. All methods are suitable for the study of the formation of micelles.β) of the studied water systems linearly decrease with both increasing temperature and prolongation of the alkyl chain of the surfactant.The degrees of counterion bindings  of the water phase in the order C12 < C14 < C16, which is in correspondence with increasing their lipophility.Liberation balancesThe amount of the released chlorhexidine dihydrochloride (CHX) increased with the prolongation of the alkyl chain of the surfactants. If the surfactant was below the CMC, a greater amount of the drug permeated through the semipermeable membrane in comparison with the case when the surfactant was above the CMC. That was because, in the case where the surfactant was above the CMC, the drug was closed in the micelles. The release rate constants of the drug from hydrogels are higher in the systems with surfactant concentration below the CMC. All evaluated hydrogels  exhibit a non-Newtonian pseudo-plastic flow. The hydrogels’ pH was not statistically significantly influenced by changing the length of the alkyl chain .The results obtained are significant for pharmaceutical technology. They will be effectively utilized in the process of drug formulation, as the surfactants are important pharmaceutical auxiliary substances used as enhancers or antimicrobial agents. The found behaviors and relations between parameters such as association, distribution, liberation, and rheological balance provide a drug designer with a serious knowledge base. The obtained knowledge is inevitable for drug optimization because obtaining good release kinetics is necessary for reaching a maximum therapeutic effect. Therefore, the obtained results will be used in ongoing research activities at the workplace of the authors. The aim of this research is to develop optimal control of drug liberation from semisolid dosage forms and computer simulation of the LADME process with possible prediction of the therapeutic effect ,18,19,20


There are well known behavioral complications of ADHD at adulthood such as learning difficulties resulting in lower education attainments; increased rate of car and other accidents; substance abuse; misconduct and imprisonment. These complications can be prevented or alleviated by effective treatment. In this study we calculated the economic burden of ADHD among adults in Israel and the cost of diagnosing and treating ADHD from childhood to adulthood. We then obtained the cost-benefit ratio of the treatment.The data were calculated using accepted estimations of prevalence and cost for the Israeli population assuming a prevalence of 4% among adults which is based on the ADHD prevalence among school age children.The estimated cost per person with ADHD due to lower education attainment, higher involvement in crime and car accidents and more drug abuse is 289,969 USD and the estimated cost for optimal treatment is 41,667 USD. Hence, the benefit cost ratio is 7.02 and, assuming only 50% success of treatment, it is 3.51, still a very high cost benefit ratio.Since early diagnosis and appropriate treatment of ADHD is very effective in reducing the various symptoms and complications at adulthood thus enabling a better education and higher income, it seems important to diagnose and offer comprehensive treatment to children with ADHD. Moreover, it seems equally important to continue treatment at adulthood. In the recent decades it has become clear that the sound development of adults depends much on the interaction of genetics and individual experience at childhood. In the words of Nobel Prize winner, the economist James Heckman: ‘Life cycle skill formation is a dynamic process in which early inputs strongly affect the productivity of later inputs… Four core concepts important to devising sound social policy toward early childhood have emerged from decades of independent research in economics, neuroscience, and developmental psychology… cognitive, linguistic, social, and emotional competencies are interdependent; all are shaped powerfully by the experiences of the developing child; and all contribute to success in the society at large .The public policy implications of this insight are very costly. For example, if the vocabulary of children is determined at a very early age, high quality nursery schools are needed at a very young age . The reqWe propose to have national screening in Israel for ADHD at the elementary school system, and then treat the children with the disorder. Below we explain that this neurobehavioral disorder can harm the development of children thus impeding their full development. This may lead to lower incomes as adults, and to the pursuit of criminal careers for the misfits, as well as other harmful behaviors. We then proceed to estimate the benefits and the costs of this national program, and show its economic effectiveness with the tool of benefit-cost analysis.Attention deficit hyperactivity disorder (ADHD) is a neurobehavioral disorder defined by the Diagnostic Statistical Manual 5 (DSM 5) as a “persistent disorder of inattention and/or hyperactivity –impulsivity that interferes with functioning or development” . At chilIn a study that compared the number of children living in the central regions of Israel aged 6–18 years and were prescribed methylphenidate in 2007 and 2011, the prevalence increased from 4.2% to 7.5% with 4 times more prescriptions among Jewish children as compared to Arab children [We recently carried out a study on 971 Jewish children of grades 1 and 2 from 35 elementary schools in Jerusalem [.Although ADHD is a neurobehavioral disorder of childhood, in most cases it extends into- adolescence and in 40%-50% the symptoms also continue into adulthood . ChildreMany adults with ADHD suffer also from other long – term complications. The rate of abuse of alcohol and illicit drugs among adolescents or adults with ADHD is higher than in the general population tending to start earlier. ADHD persons are also more often involved in criminal acts, do not obey the laws and are more involved in accidents, especially in car and road accidents –14. The Dalsgaard et al.  found inThere are apparently no studies showing the benefit of early diagnosis and treatment of ADHD in direct reduction of crime and imprisonment, mainly because of the difficulties is planning such studies. However, several studies and reviews have demonstrated that early treatment of persons with ADHD significantly improved the social function and the antisocial behavior.  is defined as a condition where the use of illicit drugs causes physiological and behavioral symptoms. Substance abuse defines the repeated use of illicit drugs without dependence, but with social consequences.Lee et al., found  that chiDalsgaard et al.  found amIt can be summarized that the danger of substance abuse and substance use disorder among adolescents and adults with ADHD is 3–4 times higher and early diagnosis and treatment significantly reduces this danger.More car accidents in persons with ADHD probably stem from the decreased concentration and the tendency not to follow rules and directions. Groom et al.  used theIt can be summarized that the rate of serious car accidents is at least doubled in drivers with ADHD compared to non ADHD drivers; early diagnosis and effective treatment almost normalizes that rate.. In a study on 370 children with ADHD compared to 740 non-ADHD children, Barbaresi et al. [A high rate of learning difficulties (30%-50%) is observed in children with ADHD and a high rate of ADHD is present among children with learning difficulties and their scores were 10–15 points lower compared to their peers even when controlling for IQ. –35. Schoi et al.  found thIn a study by Mannuza et al.  on 91 adBarkley (34?) found that, 42% of children with ADHD are held back compared to only 13% in non-ADHD; 32–36% of adolescents do not complete high school compared to 5% in non-ADHD; only 22% enter college and 5% graduate. We found  that amoOptimal treatment of ADHD children often alleviates the learning difficulties, mainly due to improved attention and short-term memory . Ornoy eIt can be summarized that the ability for high educational achievements of people with ADHD is reduced more than 50% compared to non – ADHD persons; early diagnosis and treatment increase these achievements.All existing data are a description of relatively small cohorts of adults with ADHD. Large population studies, that seem to be lacking, might have changed these findings. We hope that studies like ours will raise the interest of decision makers and will result in detailed population studies. Such studies may fine tune our results.The purpose of this study was to estimate the average cost of a non-treated person with ADHD taking into account the interference at adulthood in daily activities, their lower education, higher rate of substance abuse and involvement in accidents and crime. In addition we were interested to calculate the cost of effective treatment and the cost benefit ration.The cost of non-treated persons with ADHD is calculated in order to assess the economic benefits of early diagnosis at childhood and effective treatment throughout life. We presumed that comprehensive treatment is effective only in 40%-60% of ADHD cases, with an average of 50%. Calculation of economic gain from treatment was assigned both individually and also for the whole Israeli economy. We assumed, based on the prevalence of ADHD in school age children in Israel, that about 4% of adults have ADHD. The cost was calculated for each one of the complications individually. Medical expenses were calculated for 55 years, taking into account the average age at diagnosis (10 years) and the working years until retirement at 65. The loss of income was calculated for the ages of 25–65, which is the average age that people work and have an income.Israel is one of the OECD countries and many of its economic features are common to the other OECD countries. Thus, the cost benefit of ADHD treatment in Israel may be relevant to many other countries.We use in our calculations on the cost-benefit of successful treatment of ADHD the data on the different comorbidities assuming that their rate in the Israeli population of children and adults with ADHD are similar to those described in the literature. According to the Israeli Central Bureau of Statistics, there were in 2015 about 3,830,000 adults  aged 25–65, hence, 4% are about 153,000 people with ADHD. Our calculations are presented for an individual person, and the total cost for Israel is that received after multiplication with the presumed number of adults with ADHD.Quantitative evaluation of the cost effectiveness was carried out using the method of Cost-Benefit analysis described by Mishan , in his The validity and usefulness of Cost-Benefit analysis is exhibited in the fact that governments in all parts of the world use it to evaluate public policies. In the US it began with a law from 1936 stipulating that all public projects must meet the criterion for surplus of benefits over costs. Since then, more areas have used Cost-Benefit analysis to evaluate public policy, to water management, environmental policies etc. In the academic world, Weisbord  has applBased on the studies mentioned above we estimate conservatively the usefulness of comprehensive treatment to be 50%. Hence, a benefit of treatment of only 50% was used for all our calculations, reducing the benefit/cost to half. The cost of treatment is an estimate, where some costs  are based on real cost and the other  are based on realistic assumptions.The educational data Table  show the148,752 USD  is 1.109 billion USD. The total annual cost would be about 112,819 million USD, gaining 996 million USD/year. Assuming a 50% success of treatment, it would be 498 million USD/year. A net profit of about half a billion dollars for a country with budgetary hardships and only 8 million people, is rather significant, not to add the social behavioral profits of effective treatment of ADHD.The total benefit for Israel, assuming 153,000 adults with ADHD is It seems obvious that early diagnosis and successful treatment of ADHD in Israel are highly cost-effective as the economic benefit from treatment with 100% success is 7.02 and with 50% success it is 3.51. These ratios are robust to our assumptions, since we used conservative estimates throughout the study. The advantage of this study is the fact that the calculated figures were calculated bringing into consideration most daily life problems of adults with untreated ADHD. To our knowledge, this is the first study that considered most possible effects of untreated ADHD on daily life at adulthood. Note, however, that we did not consider the enormous social impact of untreated ADHD on the individual family and society, which have significant additional disadvantages and costs. This enhances the fact that we chose to be on the conservative side in our estimates of the economic advantage of treatment.There are many studies that calculated the cost-effectiveness of ADHD treatment. Most of them, however, calculated the actual cost of different treatment modalities, concluding that the long acting stimulants are apparently more cost-effective than the short acting ones . For exa289,969 USD. However, we also calculated the cost of optimal treatment, thus calculating the cost benefit ratio. It is obvious from our data that the highest contribution to the cost is the lower income that stems from the fact that children with ADHD have a lower education. We presume that good medical, educational and psychosocial supportive treatment, especially during childhood, can significantly improve school performance of the ADHD children, thus increasing their future level of education and income.Several investigators have calculated the loss in income of an untreated person with ADHD. Cohen  estimateThe lower education among children with ADHD which is the main contributor to the high cost, deserves some additional comments. Based on the literature and our data , we presLearning difficulties are influenced by genetic factors and a variety of other factors such as parental SES and behavior and cognitive level of the child and his parents. Moreover, the social difficulties that children with ADHD have in the regular school settings are generally not dealt with, increasing the learning difficulties. It is to be hoped that with increased awareness, the school environment will become more appropriate for all children with learning difficulties, including those with ADHD.The high benefit cost ratio, and the fact that early diagnosis of ADHD and treatment improves the outcome at adulthood, point to the importance of screening early school age children for ADHD. Furthermore, our estimates were carried out using conservative assumptions throughout the study. This screening can easily be done by the use of DSM 5 questionnaire or by other screening/diagnostic questionnaires filled out by the teachers, and if ADHD is suspected, also by the parents. This will enable the treating physicians to carry out proper diagnosis and offer appropriate treatment as early as possible.


Acute liver failure (ALF) is a complicated clinical syndrome associated with high mortality, with liver transplantation as the only treatment option. Treatment of mesenchymal stem cells has shown a potential therapeutic option for acute liver failure. However, the lack of random clinical trials and large non-human primate studies makes it necessary to assess the efficacy and safety in the clinic.We treated the monkeys with peripheral delivery of human umbilical MSCs (hUC-MSCs) and investigated the role of hUC-MSCs in modulating the progress of acute liver failure.The use of early peripheral infusion of human umbilical cord MSC infusion did not improve liver regeneration or modulate adaptive immunity. However, it significantly suppressed the hepatic aggregation and maturation of circulating monocytes and their IL-6 secretion, greatly improving liver histology, systemic homeostasis, and survival.Our study reveals the critical role of monocyte-derived IL-6 in initiating and accelerating acute liver failure and hUC-MSC treatment can disrupt the development of the inflammatory cascade by inhibiting monocyte activation. Early hUC-MSC treatment disrupts the development of the inflammatory cascade, indicating a potential clinical solution for acute liver failure.The online version of this article (10.1186/s13287-019-1184-2) contains supplementary material, which is available to authorized users. Acute liver failure (ALF), caused by sudden and severe hepatic injury, is a complicated clinical syndrome composed of metabolic and immunological dysfunction, hepatic encephalopathy, coagulopathy, sepsis, and multi-organ failure. It causes over 60% mortality in western countries if liver transplantation is not provided. However, the shortage of donor livers and life-long immunosuppression have restricted the use of this intervention. The alternative strategies for liver transplantation, such as hepatocyte transplantation, bio-artificial liver support systems, and tissue-engineering livers, have been shown as a potential treatment option for ALF , 2. NoneThe pathophysiological process underlying ALF is still far from clear –4. GivenMacaca mulatta) model of ALF, in which we induced ALF with a single intraperitoneal injection of low-dose α-amatoxin and lipopolysaccharide (LPS) [We previously established a non-human primate (de (LPS) , 10. Folde (LPS) . Moreovede (LPS) . TherefoMesenchymal stem cells are a type of somatic stem cells derived from mesodermal tissues, including bone marrow, fat, cord blood, umbilical cord, placenta, etc. MSC-based therapies have been widely used for many diseases –13. MoreIt has been reported that Wharton’s jelly is one of the main sources of extracting human MSCs . In receHere, using a toxin-induced monkey model of ALF, we explored the efficacy and safety of hUC-MSC-based therapy for ALF. Our work demonstrates that peripheral infusions of hUC-MSCs profoundly suppressed the activation of c-Mos and their IL-6 secretion, which resulted in the prevention of the development of lethal ALF in monkeys. These results, in combination with those demonstrating the efficacy and safety approach, indicate that hUC-MSC-based therapies are promising evidence which need further investigations and validation before being applied in clinical practice.The animal protocols used in this study were approved by the Institutional Animal Care and Use Committee of the Traditional Chinese Medicine National Center  (Protocol: IACUC-2012001C). Adult healthy experimental rhesus monkeys were provided by Chengdu Ping’an Experimental Animal Reproduction Center (license no.: SCXK (CHUAN) 2014-013, Chengdu, China). For a detailed information of the monkeys and their treatments, refer to Supplemental materials and methods Additional file Human umbilical cords were donated by women who underwent cesarean sections. Informed consent was obtained from the subjects’ families. hUC-MSCs were collected at the Sichuan Stem Cell Bank, Chengdu, China and cultured with serum-free medium . For hUC-MSC quality control, cultured cells at the third passage (P3) were tested to determine the expression levels of surface markers by flow cytometry . Statistical analysis was performed using GraphPad 5.0 or SPSS 13.0 software. Survival was analyzed using a Kaplan–Meier plot and log-rank analysis. Statistical evaluation of two groups was performed using Student Other materials and methods are available in the Additional file 7 cells/monkey/day)  was repeatedly peripherally infused with 1 × 107 hUC-MSCs and another group (n = 7) received an equal volume of saline at 2 h, 24 h (day 1), and 48 h (day 2) after challenge with the toxins  acts a critical role in initiating and accelerating ALF , 27, we As a pro-inflammatory cytokine, IL-6 targets numerous genes that are known to regulate liver repair following a diverse array of injuries . HoweverOur data suggested that early infusion of hUC-MSCs disrupts the inflammatory cascade by inhibiting the overproduction of monocyte-derived IL-6. However, in humans, patients seek medications only when their symptoms become apparent, with their systemic homeostasis already severely impaired. We therefore assessed whether treatment with hUC-MSCs improves prognoses in monkeys with fully developed ALF. We began to infuse the monkeys with hUC-MSCs at 24 h after injection of the toxins Fig. . AlthougTo further confirm that hUC-MSCs suppress the activation of c-Mos, we isolated quiescent monocytes from healthy monkeys and stimulated them with LPS for activation . After 2https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-4750/). We further analyzed the profiles of the upregulated genes to identify the networks and biological processes involved in the changes. We found that components of molecular networks associated with inflammatory responses, defenses, monocyte differentiation, and myeloid cell differentiation were upregulated  or inactive serum . After a 30-min stimulation, the cells were collected for microarray gene expression profiling. The results revealed that a total of 188 genes were upregulated while 433 genes were downregulated in hUC-MSCs  Flow cytometric analysis of the cell markers of mesenchymal stem cells. (B) Adipogenic differentiation and osteogenic differentiation of hUC-MSCs were determined by Oil red O staining and alizarin red staining. Scale bar = 50 μm. Figure S2. The rhesus monkeys completely tolerated the xenogeneic hUC-MSCs. (A) Schematic representations of the experimental designs. (B) Sum and sort counting of leukocytes.  Flow cytometric analysis and quantitation of the ratio of the CD4+/CD8+ cells.  Flow cytometric analysis and quantitation of the proportion of CD4+CD25+FoxP3+ regulatory T cells. (G) Serum levels of IgA, IgG, and IgM over time. Each bar represents the mean ± s.e.m., n ≥ 3. Figure S3. hUC-MSC infusion does not change peripheral leukocytes, ratio of CD4+/CD8+ T cell, regulatory T cells and DCs. (A) Flow cytometric analysis and quantitation of the ratio of CD4+/CD8+ T cells. (B) Flow cytometric analysis of CD4+/CD25+FoxP3+ regulatory T cells. (C) Flow cytometric analysis of CD1a+/CD80+/CD86+ dendritic cells. Each bar represents the mean ± s.e.m., n ≥ 5/group. Figure S4. hUC-MSC infusion does not disturb antibodies and complements. Student’s t test, the data are presented as the means±s.e.m., n ≥ 5. Figure S5. Heat map of altered genes from microarray analysis. ArrayExpress accession number: E-MTAB-4750, https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-4750/. (DOCX 3772 kb)


P = 0.041, P = 0.032, and P = 0.014, respectively). The microbiota community structure of LBW infants were significantly different from NBW infants throughout the 3 months  but not at stage 2 (P = 0.054). There was a significant increase in abundance in Firmicutes while a decrease in Proteobacteria, and at genus level the abundance of Enterococcus, Klebsiella, and Streptococcus increased while it decreased for Haemophilus in LBW group. Birth weight was the main factor explaining the observed variation at all stages, except at stage 2. Delivery mode (4.78%) and antibiotic usage (3.50%) contributed to explain the observed variation at stage 3, and pregestational BMI (4.61%) partially explained the observed variation at stage 4. In conclusion, gut microbial communities differed in NBW and LBW infants from birth to 3 months of life, and were affected by birth weight, delivery mode, antibiotic treatment, and pregestational BMI.Establishment of low birth weight (LBW) infant gut microbiota may have lifelong implications for the health of individuals. However, no longitudinal cohort studies have been conducted to characterize the gut microbial profiles of LBW infants and their influencing factors. Our objective was to understand how the gut bacterial community structure of LBW and normal birth weight (NBW) infants varies across the first 3 months of life and assess the influencing factors. In this observational cohort study, gut bacterial composition was identified with sequencing of the 16S rRNA gene in fecal samples of 69 LBW infants and 65 NBW controls at 0 day, 3 days, 2 weeks, 6 weeks, and 3 months (defined as stages 1–5) after birth. Alpha-diversity of both groups displayed a decreasing trend followed by slight variations. There were significant differences on the Shannon index of the two groups at stages 1 to 3 ( The fecal samples were delivered immediately to the laboratory and then stored at −80°C until DNA extraction. Fecal samples were collected at 0 day, 3 days, 2 weeks, 6 weeks, and 3 months post-birth, periods that were defined, respectively, as Stage 1, Stage 2, Stage 3, Stage 4, and Stage 5. NBW infants in the newborn nursery were accompanied by their mothers, fed with breast milk during the first 2–3 days after birth, and then discharged. In contrast, the LBW infants were immediately hospitalized in the NICU after their birth to receive observation and treatment for at least 1–2 weeks, during which time parental skin-to-skin contact was impossible. However, breast milk from their mother was collected and provided to the LBW infants or supplemented by infant formula as a nutritional alternative. In our study, 40.6% LBW infants (28/69) were hospitalized over 2 weeks after their birth. Information about their mothers was collected before delivery including age, height, pre-pregnancy weight, and history of GDM and gestational hypertension. Additionally, information on delivery mode, gestational weeks, gender and birth weight of infants was recorded at delivery. Information on antibiotic usage and length of hospital stay were recorded from medical records and type of feeding was collected by telephone follow-up at each of the stages following discharge.The study was approved by the Ethics Committee of the Beijing Obstetrics and Gynecology Hospital (No. 2017-KY-027-01), and informed written consent was obtained from parents of each infant. The study was registered in Chinese Clinical Trials Register (registration number ChiCTR1800014390) on January 10, 2018. Sixty-nine LBW infants  and 65 NBW  infants were enrolled in this prospective observational cohort study and the inclusion criteria were as follows: All the pregnant women were admitted to the Beijing Obstetrics and Gynecology Hospital before delivery, the LBW infants were immediately hospitalized in the Neonatal Intensive Care Unit (NICU) of the Beijing Obstetrics and Gynecology Hospital after delivery, and the NBW infants were immediately placed in the newborn nursery with their mother after delivery. Infants were excluded if they had neonatal asphyxia, congenital heart disease, neural tube defects, genetic defects, or had diseases requiring surgery during the 3-month follow-up period. Fecal samples were collected by using a collection tube pre-filled with 8 mL of DNA stabilizer solution , following the manufacturer’s instructions, which included a bead-beater step. The V4 hypervariable region of the 16S rRNA gene were amplified, sequenced and analyzed to define composition of the bacterial community. Amplification primers were generated with the following PCR primers: Forward, 515F (5′-GTGCCAGCMGCCGCGGTAA-3′); reverse, 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The first PCR conditions were as follows: initial denaturation at 95°C for 3 min, followed by 25 cycles of 95°C for 30 s, 55°C for 30 s and 72°C for 30 s, followed by a final extension at 72°C for 5 min and then holding at 4°C. The first PCR products were excised from 1% agarose gel electrophoresis and purified using AMPure XP beads, followed by index PCR. Each index PCR reaction mixture contained the purified products of the first PCR experiment (5 μl), 5μl of both Nextera XT index primer 1 and primer 2, and 1X KAPA HiFi Hotstart Ready Mix. The second PCR conditions were as follows: 95°C for 3 min, followed by eight cycles of 95°C for 30 s, 55°C for 30 s and 72°C for 30 s, and finally, 72°C for 5 min. The index PCR products were purified with AMPure XP beads and the final amplicon libraries were approximately 630 bp. After pooling equally with unique indices, the multiplex amplified libraries were denatured with NaOH and sequenced by Illumina MiSeq with 2 × 300 paired-end V4 sequencing reagents . Sequence data were deposited in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA 503135.Microbial DNA was isolated from fecal samples using the PSP Spin Stool DNA Plus kit (PSPQ-score ≥ 25). Chimera removal and dereplication of the reads was performed by using USEARCH8  of effect size (LEfSe) was applied to determine the most discriminant taxa between NBW and LBW groups at week 2 and 6 after birth , and then the canonical RDA was conducted with the Vegan package to better understand the relationship between gut microbial composition and characteristics of mothers and infants. R software was used for plotting and statistical analysis.All paired-end reads from the original DNA fragment were assigned to each sample according to the unique barcodes and then merged using FLASH . Merged USEARCH8 . The totUSEARCH8  in the RUSEARCH8 . Alpha-dA total of 404 samples from 0 day (meconium), 3 days, 2 weeks, 6 weeks, and 3 months after birth were obtained from 134 infants  selected in this cohort study . MothersP = 0.032). Additionally, there were no significant differences of the Chao1 index between the two groups at corresponding stages (P < 0.001). Interestingly, the Shannon index of the NBW group was significantly higher than it was in the LBW group at stage 1 (P = 0.041) and lower in stages 2 and 3 .Our results showed that alpha-diversity (Shannon and Chao1 index) of gut microbiota , firstlyg stages , except P < 0.01). Along the second axis, sample distribution of the two groups showed a similar change trend over time in PC2. We compared the β-diversity distances within and between the groups to see whether there are distinctive associations. Microbiota community structure of NBW infants differed over time in the first 6 weeks of early life, when comparing different time points across all samples in the same group . Though similar differences were also found in LBW infants, there was no significant difference in microbiota community structure between stages 1 and 2 (adonis P = 0.204). Furthermore, no significant differences of microbiota community structure between stages 4 and 5 were found in NBW and LBW infants . We also found the microbiota community structure of NBW infants was significantly different from that of LBW infants (P < 0.05) but not at stage 2 (adonis P = 0.054). In our cohort, 4.6% infants (3/65) in the NBW group was preterm infants, and 27.5% infants (19/69) in the LBW group was term infants. To address the possible influence of gestational age, we added a subgroup analysis of the difference of the microbiota community structure between term NBW and preterm LBW infants (P < 0.05) but not at stage 2 (adonis P = 0.176). Besides, no significant difference was observed in the microbiota community structure between the samples of preterm LBW and term LBW infants (adonis P > 0.05).We used PCoA based on Bray–Curtis dissimilarity to examine the relationship of microbial communities across different groups and stages . Bacteri infants  at stage infants . The micP < 0.001). Similarly, differences in the bacterial community structure between two groups was also found at stage 4 (adonis P = 0.006), although it was not obvious in PCoA plot  and higher abundance of Firmicutes  in the LBW group compared to NBW group. At the phylum level , while the LBW group had a greater abundance of Firmicutes at stages 1, 3, and 4 . At the family level, the gut microbiota of the LBW group had a greater abundance of Ruminococcaceae at stages 3 to 5 . At the genus level, there were a large number of genera with significant different abundances between the two groups  except stage 2, and an unknown Enterobacteriaceae genus at stages 3 to 5 , while the LBW group had a greater abundance of Enterococcus throughout the first 3 months , and Streptococcus at stages 2 and 3 (both P < 0.05), as well as Klebsiella at stages 4 and 5 (both P < 0.01)  .P < 0.001) phylum at stage 3, an unknown Enterobacteriaceae genus , and Haemophilus  genus at stages 3 and 4. It was noteworthy that Bifidobacterium  was enriched in NBW infants at stage 4. Conversely, taxa enriched in the LBW infants included Firmicutes  phylum, Ruminococcaceae  family, Enterococcus  genus at stages 3 and 4, Streptococcus  genus at stage 3, Lachnospiraceae ND3007 group  and Klebsiella  genus at stage 4.To explore the variation of the microbial community composition between the NBW and LBW infants, we performed LEfSe tests to detect differences in relative abundance of bacterial taxa across the two stages . Taxa whP = 0.026) (P = 0.026). At stage 3, it still was the main factor explaining the observed variation , while other factors were delivery mode  and antibiotic usage  (P = 0.004) and pre-gestational BMI  at stage 4 (P = 0.01), as well as compared with the NBW infants, respectively (both adonis P < 0.001)  was enriched in LBW infants who received no antibiotics, while Enterococcus  was enriched in LBW infants who received antibiotics treatment at stage 3   and 5 (3= 0.003) . The obs stage 4 . Based o< 0.001) . LEfSe t stage 3 .In our study, we characterized gut microbiota profiles of the LBW and NBW infants, described their change trend across different stages within each group, identified the differences between the two groups, and explored the underlying factors responsible for microbiota community in the first 3 months of early life, using data from a prospective cohort study on Chinese infants.Low birth weight infants have an underdeveloped immune system and a slower progression of bacterial colonization , possiblEnterococcus throughout all the stages, which is consistent with previous research  because it was difficult to recruit infants with formula feeding and randomization on feeding practices was not ethically acceptable. In addition, the amount of formula intake of mixed feeding practice was hard to record as it varied with the mother’s milk supply and the LBW infant’s demand. Therefore, the effect of formula and mix feeding practices on the development of gut microbiota could not be measured precisely. A multi-center study should be considered as the gut microbial community differed in the study locations such as different hospitals , and informed written consent was obtained from parents of each infant.CY, BZ, and YX designed the study. CC, RL, YW, XD, HZ, GL, QS, XH, LC, TJ, and JZ recruited the infants and collected the data and samples. YX, NL, and YH performed the microbiological analyses. YX and YH analyzed the data. CC and YX generated the figures and wrote the manuscript. JS and NB critically reviewed and edited the manuscript. All authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.