Daphnia magna, we studied if the host performs better when receiving a microbiome from their source region than from another region when facing a stressful condition, more in particular exposure to the toxic cyanobacteria Microcystis aeruginosa. Therefore, a reciprocal transplant experiment was performed in which recipient, germ-free D. magna, isolated from different ponds, received a donor microbiome from sympatric or allopatric D. magna that were pre-exposed to toxic cyanobacteria or not. We tested for effects on host life history traits and gut microbiome composition. Our data indicate that Daphnia interact with particular microbial strains mediating local adaptation in host stress tolerance. Most recipient D. magna individuals performed better when inoculated with sympatric than with allopatric microbiomes. This effect was most pronounced when the donors were pre-exposed to the toxic cyanobacteria, but this effect was also pond and genotype dependent. We discuss how this host fitness benefit is associated with microbiome diversity patterns.While evidence for the role of the microbiome in shaping host stress tolerance is becoming well-established, to what extent this depends on the interaction between the host and its local microbiome is less clear. Therefore, we investigated whether locally adapted gut microbiomes affect host stress tolerance. In the water flea  Many organisms face different environmental conditions across their species’ range and thereby evolved local adaptation with organisms being relatively fit in their local conditions and relatively unfit in others , 2. LocaInsights in the genomic targets and the molecular mechanisms underpinning local adaptation are starting to accumulate thanks to the increased affordability of high-throughput sequencing –22. HoweDaphnia magna selects beneficial bacteria from the environment. We expect stronger host-mediated selection for beneficial bacterial strains in sympatric than in allopatric microbiomes. If so, the gut will be occupied by locally selected bacteria leaving less opportunity for other bacteria to establish, which may result in more convergent microbiomes over the host genotypes  Is fitness higher in D. magna receiving a sympatric than an allopatric microbiome and does this depend on the donor diet? (2) Do D. magna have a lower or higher microbial diversity when they receive a sympatric vs. an allopatric microbiome? and (3) how is this related to their fitness and genotype?To unravel whether a local microbiome could play a role in increased tolerance, bacteria –48. A conotoxins –51. Becagna host , 43, we D. magna genotypes from each of two regions in Belgium separated by circa 100 km (Kortrijk and Leuven) were collected (Table SID. magna genotypes from the Blauwe Hoeve (K_BH), Kennedypark (K_KP) and Morinnestraat (K_MS), by sampling one individual from the active population in each pond. In addition, two genotypes were obtained from a recent (top layer) resting eggs in Zwevegem (K_ZWE1 and K_ZWE2) as at the time of sampling no active population of D. magna was present. In Leuven, all genotypes were obtained from recent (T = top layer sediments) resting eggs of two ponds: Heverlee (L_OM2) and Oud-Heverlee . The ponds from Kortrijk out of which the genotypes were sampled did not have a Microcystis bloom at sampling , but in some ponds of Leuven  Microcystis blooms have been observed . It could be possible for the Leuven genotypes to be locally adapted to cyanobacteria. However, Daphnia adaptation to Microcystis strains is assumed to be locally structured [Daphnia. Maternal lines of the D. magna genotypes were cultured in filtered tap water at a temperature of 19 ± 1 °C and under a 16:8 h light:dark cycle in 2 L glass jars . They were fed three times per week with saturating amounts of the green algae Chlorella vulgaris. The medium of these cultures was refreshed once per week. Three months before the start of the experiment 10 L pond water was collected from each pond and stored at 4 °C to maintain the bacterioplankton community. At the time of pond water sampling, there was no visible presence of cyanobacteria in any pond. After sieving the pond water over a 100 µm sieve, two mixtures  were made, one from the Kortrijk ponds, and one mixture from the Leuven ponds to be inoculated in the experimental jars during the transplant experiment.Five n et al. ). After ructured , and we D. magna individuals were fed with the unicellular green algae Chlorella vulgaris  and part of the Culture Collections at Institute Pasteur . C. vulgaris and M. aeruginosa were grown in Wright’s Cryptophyte (WC) medium [D. magna ) or a coD. magna ). We use) medium  and modi) medium  agar pla) medium .D. magna shows a better performance upon exposure to toxic cyanobacteria in the presence of a sympatric vs. an allopatric gut microbiome, a reciprocal gut microbiome transplant experiment was conducted , per algal treatment  and per replicate and were kept at a density of 50  individuals in 2 L experimental jars with filtered local pond water. Donor diets had a final carbon concentration of 1 mg C/L and were given to the D. magna three times per week. The pooled donor D. magna genotypes stayed in these conditions for 10 days to adjust the gut microbiome when exposed to M. aeruginosa [D. magna individuals per donor region × diet combination were randomly isolated and kept in sterile-filtered tap water for 24 h to remove food particles from the gut, as well as environmental bacteria on the carapace and filter apparatus. Then, guts were extracted with dissection needles under a stereo microscope and placed in an Eppendorf tube containing 1 mL sterile milliQ water. These dissected guts were then stored at 4 °C for 7 days. Each donor condition was set up in triplicate, which resulted in 12 pooled donor inocula (two donor regions × two donor diets × three replicates). After 7 days, the stored donor inocula were crushed and given to sterile D. magna juveniles. In the recipient phase, each genotype received a pooled and crushed gut microbiome of one of the four donor treatment combinations. This resulted in a fully crossed design: donor region (two levels) × donor diet (two levels) × D. magna genotype  with three replicates per genotype . In sympatric combinations D. magna received a local donor inoculum, in allopatric combinations they received a foreign donor inoculum. Sterile D. magna juveniles were obtained following the protocols of Callens et al. [D. magna were incubated with the pooled donor inocula for 48 h, after which D. magna were individually transferred to new 50 mL falcon tubes filled with filtered local pond water. In these 48 h, the recipient D. magna will have taken up a substantial part of the bacteria from the pooled donor inoculum. After the transfer to (unsterile) filtered local pond water (filtered over a 100 µm sieve), all recipient D. magna were subjected to a cyanobacterial diet , which was administered every 48 h to test if there was an effect from the donor gut microbiome on Daphnia tolerance to toxic cyanobacteria in terms of host fitness, survival and reproduction, monitored every 48 h during 21 days. Body size was measured at days 3, 6, 10 and 20. After 21 days, the guts from the surviving D. magna were dissected for amplicon sequencing. The D. magna were placed in sterile-filtered tap water for 24 h to remove food particles from the gut, as well as environmental bacteria on the carapace and filter apparatus. After these 24 h, they were dissected under a stereo microscope using dissection needles and collected in 10 µL sterile milliQ water, after which they were stored in −20 °C.To determine if ted Fig. . We firsruginosa . Afterwas et al. , 59. TheD. magna at the end of the 21-day monitoring period, DNA was extracted using the PowerSoil DNA isolation kit (Qiagen). DNA was dissolved in 20 µL milliQ water. The total DNA yield was determined using a Qubit dsDNA HS assay (Invitrogen) on 1 µL of sample. A nested PCR was applied to increase specificity and amplicon yield. The full-length 16S rRNA gene was first amplified with EUB8F and 1492R primers on 10 ng of template using a high-fidelity SuperFi polymerase (Life Technologies) for 30 cycles: 98 °C—10 s; 50 °C—45 s; 72 °C—30 s. PCR products were subsequently purified using the QIAquick PCR purification kit. To obtain dual-index amplicons of the V4 region, a second amplification was performed on 5 µL (=20–50 ng) of PCR product using 515F and 806R primers for 30 cycles: 98 °C—10 s; 50 °C—5 s; 72 °C—30 s. Both primers contained an Illumina adapter and an 8-nucleotide (nt) barcode at the 5′-end. For each sample, PCRs were performed in triplicate, pooled and gel-purified using the QIAquick gel extraction kit. An equimolar library was prepared by normalizing amplicon concentrations with a SequalPrep Normalization Plate (Applied Biosystems) and subsequent pooling. Amplicons were sequenced using a v2 PE500 kit with custom primers on the Illumina Miseq, producing two ×250-nt paired-end reads. This way, 96 recipient samples were generated representing eight genotypes  × two donor regions × two donor diets × three replicates, and the 12 donor samples. The number of sequenced D. magna individuals per treatment is represented in the Table SITo characterize the gut microbial communities from the donor sets and from the recipient −5 were removed from the analysis. To visualize the bacterial families that differed between the treatments, OTUs were grouped at the family level, and families representing <1% of the reads were discarded . To test for differences in α- and β- diversity, all samples were rarefied to a depth of 1000 reads. One gut sample  had a lower number of reads, and was removed from the analysis.DNA sequences were processed following Callahan et al. . SequencF-tests and p values were created (Anova function of the car package [D. magna genotype (=clone) as fixed factors, with genotype nested within pond  as repeated measures and also controlled for unbalanced design with a restricted maximum likelihood estimation. To specifically analyze relationships in the response patterns to M. aeruginosa pre-exposure between traits, we calculated delta values as the clonal average trait value when pre-exposed to M. aeruginosa—the clonal average trait value when not pre-exposed. We did so for survival, total fecundity and body size . We then tested with a Pearson correlation test, with bonferroni correction for multiple testing . We used the Akaike information criterion (AIC) to select the best subset of variables to represent the best model (Table SI package ). In theage in R ), controage in R ), for paF-tests and p values were created  were calculated using the vegan package in R . Shannon package ). In the package . The eff package  on the t package . A UnionGiven the complexity of the results, we only focus on significant effects in the text below. For full results, see Tables SI M. aeruginosa or not), donor microbiome type  and recipient pond, was highly significant , but not when the microbiome was not pre-exposed to M. aeruginosa . When the microbiome was pre-exposed to the toxic M. aeruginosa strain, seven out of the nine genotypes  had a higher survival percentage when they received a sympatric than an allopatric microbiome, for the other two genotypes  the survival percentage was higher when they received an allopatric than a sympatric microbiome  reflecting that when D. magna individuals received a microbiome that was pre-exposed to the toxic M. aeruginosa strain more juveniles were produced when they received a sympatric than an allopatric microbiome .For D. magna body size, there was a three-way interaction between donor diet, microbiome type, and genotype . However, when the D. magna received a microbiome not pre-exposed to M. aeruginosa there was a marginally significant interaction between donor microbiome and genotype . In addition, there was a significant interaction between donor diet and microbiome type across the repeated size measurements . D. magna receiving a donor microbiome pre-exposed to M. aeruginosa had a smaller body size than when not pre-exposed to M. aeruginosa, and this effect was most pronounced when they received a sympatric microbiome . There was also a marginally significant interaction between microbiome type and genotype  for D. magna body size, and a significant main effect of genotype  and time .For  Table SI. SeparatD. magna survival and body size (at day 10) of the recipients . The recipient gut microbiomes also harbored Gammaproteobacteria and Bacteroida, but were more diverse than the donor microbiomes . There was a significant distinction between the gut microbiome of the four donor sets and the recipient D. magna measured by α- and β-diversity . In the population from Kortrijk the Shannon entropy was higher when they were fed with M. aeruginosa than when they were not fed with M. aeruginosa. In Leuven the opposite was true, there the Shannon entropy was higher when they were not fed with M. aeruginosa than when they were fed with M. aeruginosa . We observed inconsistent patterns on the genotype level. When the recipient D. magna received a donor microbiome in which the D. magna were pre-exposed to M. aeruginosa, three out of the eight genotypes  had a lower Shannon entropy when they received a sympatric than an allopatric microbiome. For two genotypes (K_BH and K_KP) the sequencing partly failed making it impossible to compare the sympatric and allopatric treatments when the donor diet was pre-exposed to M. aeruginosa, and for the other three genotypes  the Shannon entropy was higher when they received a sympatric than an allopatric microbiome. When the donor diet was not pre-exposed to M. aeruginosa, four out of the eight genotypes  had a lower Shannon entropy when they received a sympatric than an allopatric microbiome, for one genotype (K_KP) there was no data and for the other three genotypes  the Shannon entropy was higher when they received a sympatric than an allopatric microbiome , reflecting strong variation in the microbial community between the recipient genotypes . In the sympatric treatments, there was a main genotype effect in D. magna that received a donor diet not pre-exposed to M. aeruginosa . However, when they received a donor diet pre-exposed to M. aeruginosa, this genotype was no longer present  × donor microbiome type  × recipient genotype interactions for the life history traits survival and fecundity. Independent of the tested region, more than half of the genotypes showed a higher survival (seven out of nine) or fecundity (five out of eight) when they received a sympatric vs. an allopatric microbiome, especially if the donor microbiomes were pre-exposed to M. aeruginosa. This pattern where most D. magna genotypes had a higher fitness, in terms of survival and fecundity, when they received a local (sympatric) than a foreign  microbiome, is in agreement with what we expected. It has earlier been shown that in stressful environments, hosts may benefit from locally adapted microbes, as hosts may use specific microbial communities with large phenotypic effects to specialize and persist in novel niches [sensu lato, given that the adaptation is not at the level of a particular population, but rather on the level of the region, reflecting regional adaptation. In future work it would be very interesting to investigate these effects further on a microscale within a region.We here performed a reciprocal gut transplant experiment, in which germ-free l niches –41. HoweD. magna with a donor microbiome that had been pre-exposed to M. aeruginosa than to the control diet and this was especially so for the sympatric microbiomes. We found a marginally significant negative correlation between the delta values of the donor microbiomes (M. aeruginosa—control treatment) for survival and body size in the recipient D. magna genotypes. This indicates that D. magna genotypes that had a higher survival with a microbiome pre-exposed to M. aeruginosa were smaller. This suggests that carrying a microbiome that is associated with a higher tolerance upon M. aeruginosa exposure comes with a cost in terms of body size. A trade-off between growth and survival is shown in many organisms [Daphnia may be mediated through the microbiome, as also suggested by Callens et al. [For body size there was also a significant donor diet × donor microbiome type × recipient genotype interaction, and the interactions between donor diet and donor microbiome type, and between donor microbiome type and recipient genotype were significant. Body size was smaller in rganisms –74. Heres et al. . Hosts wD. magna genotypes that were tolerant to Microcystis, contained more Flavobacteriia. Gammaproteobacteria and Flavobacteriia have been described to be part of the mainly aerobic D. magna gut bacterial community [Microcystis cells and degrade dissolved organic matter derived from intracellular products of Microcystis [Daphnia may therefore provide the host individual access to otherwise inaccessible nutrients in addition to detoxification of cyanotoxins. The recipient gut microbiomes were more diverse than the donor gut microbiomes. Next to Gammaproteobacteria and Bacteroida, the recipient gut microbiomes also contained bacteria from the following classes: Actinobacteria, Alphaproteobacteria, Bacilli, Planctomycetacia and Verrucomicrobiae. The recipient microbiomes were inoculated with the donor microbiomes for 48 h in sterile-filtered tap water in our transplant experiment. After these 48 h, the recipient D. magna were placed in a mixture of pond water from the region of origin, which may explain the relatively higher diversity and the presence of environmental bacteria in the gut microbiome of the recipient [D. magna. According to the Unionplot, 71.2% of the OTUs were shared between donors and recipients, showing that in the 48 h the recipients took up a substantial part of the bacteria from the pooled donor inoculum. The analysis of the Bray Curtis dissimilarity matrix, showed that D. magna that received a sympatric microbiome that was pre-exposed to M. aeruginosa had a more convergent microbiome across the genotypes. This was demonstrated by the distance between donors and recipients varying less across the genotypes in the sympatric treatments that were pre-exposed to M. aeruginosa, than across the genotypes in allopatric and sympatric treatments not pre-exposed to M. aeruginosa . In the study of Macke et al. , D. magnommunity . Gammaprommunity , 76. Memrocystis , 78. Theecipient  D. magnaosa Fig. .D. magna may follow two patterns with respect to the selective uptake of microbial strains to obtain an increased tolerance upon toxic cyanobacterial exposure: (1) selection of a few adapted strains, or (2) selection for a high diversity of strains with complementary gene functions  followed the second pattern, where both D. magna fitness  and bacterial diversity were higher in the sympatric than in the allopatric treatment. Hosts can also benefit from a more diverse microbiome. For example, it has been shown that mice with a natural, more diverse, microbiome had a higher fitness and a lower inflammation response to two diseases than mice with a laboratory, less diverse, microbiome [Daphnia genotypes being non-selective and picking up randomly a high diversity of  strains in stressful environments.The host xposure:  selectio attacks –82. Selecrobiome . It is pcrobiome . AlternaD. magna genotypes had a higher fitness in terms of survival and fecundity, when they received a sympatric than an allopatric microbiome. These fitness benefits from a local microbiome were, however, genotype dependent, which conforms the hypothesis that different Daphnia genotypes differ in their tolerance to M. aeruginosa [D. magna genotypes fitness was higher while bacterial diversity was lower for sympatric than for allopatric microbiomes, suggesting selection of certain microbiome strains. While in others both the fitness and bacterial diversity were higher in sympatric than allopatric treatments, suggesting selection of a high-bacterial diversity. Future research should confirm if particular adaptive strains interact with the host genotype to structure the microbial community in the Daphnia gut in stressful cyanobacterial environments.We conclude that most ruginosa  and diffruginosa . As suchruginosa , 84. In Table SI1Table SI2Table SI3Table SI4Table SI5Table SI6Table SI7Table SI8Table SI9Table SI10Table SI11Table SI12Figure SI1Figure SI2Figure SI3Figure SI4Figure SI5figure SI6Figure SI7Figure SI8


CSF and serum albumin and IgG levels positively correlated to age in both sexes. CSF total protein content did not correlate with CSF leukocyte numbers but was higher in patients with marked pleocytosis. Conclusions: In neurological patients, men have higher levels of CSF serum-derived proteins, such as albumin and IgG.Background and aims: Cerebrospinal fluid (CSF) protein content presents a sexual dimorphism in humans. We investigated sex-related differences in CSF IgG levels and in the quantification of intrathecal IgG synthesis (IIS). Methods: CSF, serum albumin and IgG were measured in 1519 neurological patients and both linear and hyperbolic formulas were used for the quantification of IIS. CSF-restricted oligoclonal IgG bands (OCBs) were used as “gold standard”. Results: The linear IgG Index showed a weak agreement with OCBs in males and females , while the hyperbolic Reiber’s formulas had a moderate agreement with OCBs in females (k = 0.635) and a weak agreement in males (k = 0.565). Higher CSF albumin and IgG levels were found in men than in women in the whole population and in subjects without IIS after adjusting for age and for serum concentrations of albumin and IgG, respectively (Quade statistics,  Cerebrospinal fluid (CSF) is a watery body fluid that is continuously produced by the choroid plexuses of the cerebral ventricles and circulates around the surface of the brain and spinal cord. CSF typically contains low protein concentrations and few circulating cells . In addiCSF studies open “windows on the brain” . The diaThe most abundant proteins in blood serum are albumin and immunoglobulin G (IgG), approximately 40 and 10 mg/mL, respectively . AlbuminSexual dimorphisms have been reported related to brain development, structure and neurotransmission , and whiWhile it is clear that the concentration of plasma-derived proteins, such as albumin, is higher in the CSF of males than in females, the sex-specific concentration of immunoglobulins, another main component of plasma proteins, still remains unknown.We therefore aimed to search for possible sex differences in the CSF content of plasma proteins, with a focus on albumin and IgG, and to analyze the impact that these differences may have on the application of the most widely used mathematical formulas for measuring B-CSF-B damage and the intrathecal synthesis of immunoglobulins.Clinical and laboratory anonymized data were retrospectively collected from patients hospitalized from 2000 to 2018 at the “S. Anna” University Hospital , in Ferrara, northern Italy. The study was approved by the local Committee for Medical Ethics in Research, “Comitato Etico di Area Vasta Emilia Centro della Regione Emilia-Romagna”  and written informed consent was obtained.Two different cohorts of patients were included in this study. Cohort 1 were used for the analysis of CSF and serum albumin and IgG levels and for the determination of an intrathecal IgG synthesis. This group included neurological patients of which almost a quarter were multiple sclerosis-affected people, a quarter of the patients with other inflammatory neurological diseases, a quarter of the patients with non-inflammatory neurological diseases and the last quarter were people with undefined diagnosis as published before . Cohort 2 were used as the control group to study the relationship between CSF total protein content and pleocytosis and included patients who underwent lumbar puncture for diagnostic purposes which had (i) normal (up to 5 cells/μL), (ii) mild (6–25 cells/μL), (iii) moderate (26–100 cells/μL) and (iv) marked pleocytosis (more than 100 cells/μL).All patients in both cohorts were analyzed blindly with respect to diagnostic suspicion and definite clinical diagnosis.Pairs of CSF and blood samples were withdrawn from all patients included in the study for diagnostic purposes. CSF and serum samples were analyzed at room temperature immediately after centrifugation or stored in aliquots at −80 °C until assay. Exclusion criteria were: age less than 16 years, lack of demographic information (sex and/or age) and the presence of xanthochromia and/or high levels red blood cells (>2000/µL) in CSF, as suggestive for traumatic lumbar puncture or subarachnoid hemorrhage.All analyses were performed on the 2nd–4th mL of CSF after lumbar puncture .Albumin and IgG levels were measured as part of the diagnostic work-up in cell-free CSF and paired serum samples by immunochemical nephelometry with the Beckman Array Protein System or IMMAGE 800 Immunochemistry System  following the procedure of Salden . AlbuminNormal QAlb values were considered as <6.5 for patients aged 15–40 years, <8.0 for patients aged 41–60 years and <9.0 for patients over 60 years ,21. Acco(1) linear IgG Index = QIgG/QAlbCSF/[IgG]serum × 1000where QIgG = [IgG](2) the hyperbolic Reiber’s formula, IgGLoc = (QIgG − QLim) × [IgG]serumwhere QLim Quantification of the IIS was done with both linear and hyperbolic mathematical formulas as follow:The normal limits were considered 0.7 and 0 for IgG Index and for Reiber’s formula, respectively.Qualitative demonstration of the IIS was done on paired CSF and serum samples with the “gold standard” of isoelectric focusing on agarose gel followed by IgG-specific immunoblotting using a Helena Biosciences IgG IEF kit . The immunoblotting patterns were first independently assessed by two experienced operators blinded each other and for clinical information, and finally the presence/absence of IgG oligoclonal bands (OCBs) were established through an interobserver agreement. The following CSF patterns were considered: pattern 1 = absence of IgG OCBs; pattern 2 = 2 or more CSF-restricted IgG OCBs; pattern 3 = 2 or more CSF-restricted IgG OCBs with additional identical IgG OCBs in CSF and serum; pattern 4 = 2 or more identical IgG OCBs in CSF and serum; pattern 5 = some identical IgG OCBs in CSF and serum in a restricted pH range. Only patterns 2 and 3 indicated an intrathecal IgG synthesis .CSF total protein content was measured in all samples as a part of the diagnostic work-up. As published before  analysisCSF leukocytes were counted in a Fuchs-Rosenthal chamber and counts were reported as cells to a standard volume of 1 μL. A cell count less than or equal to 5 cells/μL was considered normal, higher values were considered suggestive of pleocytosis.Continuous variables presenting with a non-normal distribution (Kolmogorov–Smirnov test) were reported as median and interquartile range (IQR) and Mann–Whitney test was used for all comparisons. Categorical variables were reported as counts (percentages) and the Fisher’s exact test was used to test significance. Correlations between age and biological variables were investigated with the Spearman test. In regression analysis F-test was used to compare the fits of linear models. The agreement with quantitative and qualitative methods for the determination of an IIS was calculated through the Cohen’s kappa coefficient  with a fhttps://www.graphpad.com/quickcalcs/kappa1/. p-values < 0.05 were considered statistically significant. The Statistical Package for the Social Sciences  version 21.0 for Windows and Prism 9 for MacOS  were used for the statistical analysis. Kappa values were interpreted as follows: 0.00–0.20, slight agreement; 0.21–0.40, fair agreement; 0.41–0.60, moderate agreement; 0.61–0.80, substantial agreement; 0.81–1.00, almost perfect agreement. The sensitivity and specificity of the mathematical formulas in identifying the presence of IIS were calculated with respect to the “gold standard” of OCBs using 2 × 2 contingency tables. The robust regression and outlier removal (ROUT) method was used to determine outliers, assuming a maximum desired false discovery rate (Q) equal to 1%. Quade statistics was used for multivariate analysis. Two tailed Cohort 1 included 1519 patients  aged between 15 and 93 . The womThe linear “IgG Index” showed a moderate agreement both in females (k = 0.587) and males (k = 0.559) while the hyperbolic Reiber’s formula demonstrated a good agreement in females (k = 0.635) and a moderate agreement in males (k = 0.565) .Sensitivity and specificity of IgG index and hyperbolic Reiber’s formula were calculated and compared in the two sexes . While tCSF and serum concentrations of albumin and IgG and their CSF/serum quotients were investigated by age and sex. Patients with IgG intrathecal synthesis (OCBs patterns 2 and 3) were excluded from this analysis to avoid the influence of the intrathecal IgG fraction on the blood-derived CSF IgG concentration and consequently on the CSF/serum IgG quotient. Moreover, to reduce potential biases related to abnormal CSF or serum concentrations, outliers were excluded from any single analysis.p = 0.031; the difference between Y-intercepts was undeterminable for too large difference of slopes) with a positive correlation (Spearman: r = 0.2218 and p < 0.0001) in females and with no correlation in males   and a positive correlation in females  and in males   in males  than in females (Spearman: r = −0.2614 and p < 0.0001)  despite a slightly higher negative correlation in females  than in males   and males  and the regression line was constantly higher in males than in females  (p < 0.0001) and males  with a higher regression line in the male group   , panel a 0.0368) , panel b 0.0001) , panel c 0.3782) , panel d 0.0001) , panel e 0.0001) , panel fAs for the previous analysis, patients positive for an IgG intrathecal synthesis (OCBs patterns 2 and 3) were excluded to avoid the influence of the intrathecal IgG fraction. CSF albumin and IgG levels were compared in men and women grouped by age. As reported in p < 0.000001) and IgG  median levels were significantly higher in men than in women even after adjusting for age at the time of lumbar puncture and for serum levels of both albumin and IgG.A multivariate model was applied to patients negative for the presence of an intrathecal IgG synthesis. As reported in Cohort 2 included 577 subjects, 299 women and 278 men, grouped by pleocytosis severity in four groups: (i) normal (no more than 5 cells/μL), (ii) mild (6–25 cells/μL), (iii) moderate (26–100 cells/μL) and (iv) marked (more than 100 cells/μL). All groups had a similar M/F ratio with no differences in age between sexes (data not shown). As reported in CSF protein levels were further correlated to the number of CSF infiltrating cells in both men and women with mild, moderate and marked pleocytosis but no associations were found .In this study, we highlight for the first time that CSF concentrations of serum-derived IgG, similar to that already described for albumin, are higher in male than in female neurological patients and that these concentrations increase with age in a parallel manner in the two sexes. These differences in CSF composition have an impact on the applicability of the quantitative indices of IIS as shown with the differences between sensitivities, specificities and agreement with the “gold standard” of OCBs by the Reiber’s hyperbolic formula.The CSF analysis is a widely used laboratory tool supporting the diagnosis of neurological disorders. In particular, quantitative CSF analysis, through the measurement of albumin and IgG in paired serum and CSF, together with the subsequent calculation of mathematical indices, can give information regarding the B-CSF-B functionality and the presence of inflammation within the CNS ,24. The In the present study we also demonstrated that the different CSF concentrations of plasma-derived proteins not only concerned albumin, but also IgG, the second most abundant plasma protein. A consequence of this sex-specific distribution of albumin and IgG was detected in the different performance of the quantitative indices of IIS in females compared to males. In particular, the hyperbolic Reiber’s formula showed sex-related differences in sensitivity, specificity and concordance towards the “gold standard” of OCBs. To exclude the effect of the intrathecally produced IgG fraction on the CSF, total IgG concentration in all patients with an IIS by means of IgG OCBs were excluded from subsequent analyses. Surprisingly, serum IgG levels remained constant and were similar with age in the two sexes, while CSF IgG levels were higher in males and increased with age in a similar way in both sexes. The consequence of this sexual dimorphism in CSF protein content was that the CSF/serum IgG quotient (QIgG) increased in parallel in the two sexes, maintaining higher levels in males than in females at any age, similar to what has been reported about QAlb. Taken together, this evidence highlights a higher concentration of plasma-derived proteins, i.e., albumin and IgG, in males due to a greater permeability of B-CSF-B, as shown by the differences between the respective CSF/serum quotients.The higher CSF concentration of plasma-derived proteins in males compared to females is reflected in that males tend to have a higher CSF total protein content than females . DiffereInterestingly, in both sexes a marked pleocytosis is accompanied by a high CSF protein content while this association is not present for less severe pleocytosis. Furthermore, the CNS infiltrating lymphocyte count does not appear to correlate directly with CSF protein concentration values in our neurological patients.Due to the observational nature of our study, we are unable to draw conclusions on the causes of this different CSF biochemical composition. Sex-related differences in CSF composition may be secondary to multiple mechanisms. The female hormone 17β-estradiol  could drIn light of these results, it is evident that the study design of any CSF biomarker, especially if of possible  plasma derivation, must necessarily take into account the effects of sex and age.Our study has some limitations. Our analyses did not include a population of controls from the general populations, in relation to the lumbar puncture practice only performed for diagnostic or therapeutic purposes. Moreover, the lack of clinical diagnoses did not allow us to verify the impact of this sex-related discrepancy in those conditions such as MS in which the presence of IIS is one of the diagnostic criteria. Finally, due to the retrospective nature of the study, it was not possible to include some physical variables such as weight, height and body mass index in our analyses.In conclusion, our study confirms the presence of a sexual dimorphism with regard to CSF protein content and suggests that this is mainly due to higher levels of plasma-derived proteins in male CSF. The effects of this gap between the two sexes fall on the quantitative indices of IIS and evaluation of the functionality of B-CSF-B. Future studies are desirable, and especially on populations of subjects with no neurological disorders, to provide indications on possible corrections to apply to the quantitative parameters of CSF analysis in order to standardize the physiological gap between the two sexes.


Parathyroid allotransplantation is a burgeoning treatment for severe hypoparathyroidism. Deceased donor parathyroid gland (PTG) procurement can be technically challenging due to lack of normal intraoperative landmarks and exposure constraints in the neck of organ donors. In this study, we assessed standard 4-gland exposure in situ and en bloc surgical techniques for PTG procurement and ex vivo near-infrared autofluorescence (NIRAF) imaging for identification of PTGs during organ recovery.Research tissue consent was obtained from organ donors or donor families for PTG procurement. All donors were normocalcemic, brain-dead, solid organ donors between 18 and 65 y of age. PTGs were procured initially using a standard 4-gland exposure technique in situ and subsequently using a novel en bloc resection technique after systemic organ preservation flushing. Parathyroid tissue was stored at 4 °C in the University of Wisconsin solution up to 48 h post-procurement. Fluoptics Fluobeam NIRAF camera and Image J software were utilized for quantification of NIRAF signal.P < 0.0001). Quantification of NIRAF integrated density signal demonstrated >1-fold log difference in PTG (2.13 × 105 pixels) versus surrounding anterior neck structures . PTGs maintain distinct NIRAF signal from the time of recovery (1.88 × 105 pixels) up to 48 h post-procurement (1.55 × 105 pixels) in organ preservation cold storage (P = 0.34).Thirty-one brain-dead deceased donor PTG procurements were performed by abdominal transplant surgeons. In the initial 8 deceased donors, a mean of 1.75 glands (±1.48 glands SD) per donor were recovered using the 4-gland in situ technique. Implementation of combined en bloc resection with ex vivo NIRAF imaging in 23 consecutive donors yielded a mean of 3.60 glands (±0.4 SD) recovered per donor (The use of an en bloc surgical technique with ex vivo NIRAF imaging significantly enhances the identification and recovery of PTG from deceased donors. A recent Scandinavian registry study found that permanent hypoPT patients had an increased risk for renal insufficiency , for any malignancy , for cardiovascular events , and for death .5Hypoparathyroidism (hypoPT), defined by hypocalcemia from inappropriately low parathyroid hormone (PTH) levels, is currently one of the most common and debilitating complications of neck surgery worldwide. Transient postsurgical hypoPT lasting <6 mo is estimated to occur in 25.4% to 83% of patients worldwide after neck surgery,6 Furthermore, a recombinant PTH analogue has been shown to have limited clinical success, and the drug can cost up to $60 000 to $ 100 000 annually.7In many patients, hypoPT is managed with regular use of calcium supplements and active vitamin D analogues, but a significant proportion of patients continue to experience severe symptoms requiring repeated emergency admissions and ultimately fail supplementation therapy alone.9 The most common source of viable parathyroid tissue for allotransplantation has come from hyperplastic glands of living donor patients with secondary hyperparathyroidism triggered by chronic renal failure.9 These glands were most often cryopreserved or cultured before transplantation, leading to a large degree of variance in allotransplantation strategies and outcomes.9 With respect to deceased donor sources of parathyroid tissue for allotransplantation, there are 3 case reports in the literature to date. In 1983, Sollinger et al described the outcomes of 2 patients transplanted with deceased donor parathyroid tissue from a cadaveric donor into the brachioradialis muscle with allograft function out to 16 mo.10 Chapelle et al described a combined deceased donor kidney/parathyroid transplantation for a patient with terminal renal failure and a previous total parathyroidectomy with excellent parathyroid allograft function.11 Most recently, Aysan et al reported a deceased donor parathyroid allotransplant with methylprednisolone induction and no maintenance immunosuppression with continued allograft function out to 36 mo.12A promising approach to treating permanent hypoPT is parathyroid allotransplantation. A wide range of case reports and methodologies have been described dating back to the 1950s with relative success.Because of the paucity of literature surrounding deceased donor parathyroid gland (PTG) procurement, this study’s aim was to describe our group’s initial learning curve with standard 4-gland exploration in situ and evolution toward en bloc cadaveric dissection ex vivo. Importantly, because of timing constraints related to concurrent recipient operations , the donor PTG procurement was performed after organ flushing, and all transplantable organs were procured. The evolution toward en bloc cadaveric dissection specifically addressed 3 major concerns related to cadaveric parathyroid procurement: (1) operative exposure constraints in the neck of organ donors due to postmortem concerns around funeral viewings; (2) significant change in tissue color and anatomical relationships between the PTG, thyroid gland, and lymph nodes in the cold phase of the donor operation; (3) any potential donation after circulatory death donor PTG procurements would be performed in the cold phase. These concerns are compounded by the fact that the recovering team consists of non-endocrine trained surgeons but rather abdominal transplant trained surgeons and organ/tissue procurement coordinators.14 Furthermore, we highlight the novel finding of parathyroid NIRAF signal stability for up to 48 h in cold storage.Additionally, we describe a new indication for PTG near-infrared autofluorescence (NIRAF) imaging in identifying normal parathyroid tissue in cadaveric donors. NIRAF imaging was adopted ex vivo because its use has decreased inadvertent resection of PTGs during thyroidectomy and lowered risk of postoperative hypocalcemia in randomized trials.Research tissue consent was obtained from organ donors and donor families for PTG procurement. Donor selection included normocalcemic, brain-dead, solid organ donors between 18 and 65 y of age. The PTGs’ vascular supply was not included in cold perfusion circuit, and the glands were recovered after procurement of all intra-abdominal and intrathoracic organs to be used for clinical transplantation.Standard deceased donor procedures would occur, including warm dissection of thoracic and abdominal organs, administration of heparin, cannulation, aortic cross clamping, venting, ice cooling and cold dissection, and multiorgan sequential extraction. Vessel recovery would then be conducted. Once all standard organs and vessels had been extracted, the abdominal transplant procurement team would recover the PTGs. Our initial operative protocol involved replicating a standard 4-gland parathyroid exploration in situ in the first 8 donors. A standard collar incision was not utilized to avoid a visible incision during funeral viewing. Instead, due to these constraints, the standard donor sternotomy incision was only extended cephalad to the level of the cricoid cartilage. The overlying platysma and strap muscles were divided exposing the anterior portion of the thyroid. The superior pole of the thyroid was then fully mobilized bilaterally. The lateral aspects of the thyroid gland were then mobilized to allow medial and lateral rotation of the thyroid to expose the PTGs. Identified PTGs were then placed in cold University of Wisconsin (UW) solution for secondary confirmatory identification via histological examination. Additionally, the Fluobeam system was used ex vivo to confirm identification of candidate PTGs.Fluoptics Fluobeam camera and software were utilized for all PTG NIRAF imaging using high-sensitivity factory settings. NIRAF imaging was performed within 6 h of procurement and after 48 h of cold storage in the UW solution. PTGs identified by Fluobeam were confirmed by frozen section histology. After frozen sections were cut, hematoxylin and eosin staining was performed per the University of California San Francisco Department of Pathology protocols.http://links.lww.com/TXD/A407.NIRAF images of en bloc specimens obtained on the Fluobeam platform were uploaded directly to the Image J software  for quantification of the integrated density . Equivalently sized regions of interest in the thyroid gland and anterior neck structures (5-mm diameter) were compared. Example images of quantification methodology are illustrated in Figure S1, SDC, t test and Mann-Whitney U test were performed using the Prism GraphPad Pro  software.Two-tailed unpaired From January 2018 to February 2019, 31 brain-dead deceased donor PTG procurements were performed by abdominal transplant recovery teams. In our initial experience in 8 donors, we replicated a standard 4-gland exploratory technique in situ through the sternotomy incision during cold phase of donor procurement , and a significant change in the distinctive color differences between the PTG, thyroid gland, and lymph node tissue in the cold phase of the operation. An evolution to en bloc resection of the thyroid, parathyroids, and surrounding anterior cervical structures was adopted in the following 23 consecutive donors. The anatomic borders of the en bloc resection can be found in Table En bloc tissue was procured after systemic organ preservation flushing and placed in cold UW preservation solution Figure A and B. P < 0.0001) were identified and confirmed by histological confirmation.NIRAF imaging was then implemented ex vivo using the Fluobeam platform to identify candidate PTGs Figure C and D. 5 pixels; SEM, ±2.8 × 104) compared with thyroid tissue  in the thyroid gland and showed there is a significant increase in the integrated density signal in PTGs  versus other anterior neck structures  surrounding anterior neck structures . Using 4 different donors, we compared 14 PTGs versus 9 anterior neck structures and showed there is a significantly >1-fold log difference in integrated density signal in PTG  compared with after 48 h in cold storage UW preservation solution . False positive signals can still occur in some tissues such as brown fat, colloid nodules, and thyroid cancer. Although this study utilized only a NIRAF imaging platform , we believe a NIRAF probe platform  could be used interchangeably or synergistically to identify parathyroid candidates ex vivo. NIRAF probe has the advantage of quantitative measurement, meaning it could be used to pinpoint (instead of searching for) parathyroid tissue.Limitations of this study include that due to the institutional review board and organ procurement organization (OPO) constraints, a standard collar incision utilized for most standard thyroid operations was not allowed due to potential cosmetic reasons postmortem. Additionally, PTGs were recovered in the cold phase after all transplantable organs were retrieved in conjunction with local OPO policies. The potential recovery of PTG during the warm phase of the donor operation with the presence of formally trained endocrine surgeons using a standard collar incision in cooperation with local OPO policies may lead to more consistent yield of deceased donor PTGs. This would require future study and policy changes for full practical implementation with current donor operation logistics.https://www.clinicaltrials.gov; unique identifier: NCT03977662; registered in 2019), we believe the renewed and emerging interest in exploring parathyroid allotransplantation as a treatment for permanent hypoPT will require a thorough understanding of PTG procurement techniques and storage and viability practices for potential allotransplantation protocols.21Along with our group’s recently described novel application of whole PTG cotransplantation to improve pancreatic islet engraftment and diabetes reversal (Pancreatic Islets and Parathyroid Gland Co-Transplantation for Treatment of Type 1 Diabetes; In summary, this study describes our group’s learning curve and surgical technique for enhanced identification of normal cadaveric PTGs via en bloc resection and ex vivo NIRAF imaging. We believe the standardization of a deceased donor PTG procurement technique with the aid of NIRAF imaging will have vast implications for future basic research of normal PTGs and potential allogeneic transplantation trials of PTG for hypoPT and cotransplantation with pancreatic islets for type 1 diabetes.


A. cursor, A. montensis, N. lasiurus, and O. nigripes). These species belong to the Cricetidae family, which is the most diverse family of rodents in South America, with a total of ~120 species in Brazil. The results found for the gonadosomatic index and the sickled sperm head shape observed strongly suggest that the species here evaluated are promiscuous, prolific, and short-lived. The duration of spermatogenesis was relatively short and varied from ~35–40 days. Both the percentage of seminiferous tubules (ST) in the testis parenchyma (~95–97%) and the number of Sertoli cells (SC) (~48–70 million) per testis gram were very high, whereas a fairly good SC efficiency (~8–13 round spermatids per SC) was observed. In comparison to other mammalian species studied, particularly the rodents of the suborder Myomorpha , the rodents herein investigated exhibited very high (~62–80 million) daily sperm production per testis gram. This impressive spermatogenic efficiency resulted mainly from the short duration of spermatogenesis and quite high values found for the ST percentage in the testis and the SC number per testis gram. We expect that the knowledge here obtained will help conservation programs and the proper management of wildlife.Although rodents represent approximately 40% of all living mammalian species, our knowledge regarding their reproductive biology is still scarce. Due to their high vulnerability to environmental changes, wild rodents have become beneficial models for ecological studies. Thus, we aimed to comparatively investigate key functional testis parameters in four sexually mature wild rodent species ( Akodon cursor, Akodon montensis, Necromys lasiurus, and Oligoryzomys nigripes) investigated in the present study belong to the Cricetidae family, which is the most diverse family of rodents in South America and particularly in Brazil where they comprise a single neotropical Sigmodintinae subfamily with a total of 117 species and 36 genera . Subsequently, testes were perfused-fixed by gravity-fed perfusion through the left ventricle with 0.9% saline and 4% buffered glutaraldehyde for 25-30min . Followin = 2 per each species) of tritiated thymidine  were given near the cauda of the epididymis. Two-time intervals (1 hour and approximately three weeks) were considered after thymidine injections for A. cursor, A. montensis, and N. Lasiurus, whereas for O. nigripes these intervals were 1 hour and approximately two weeks.In order to estimate the duration of spermatogenesis and before orchiectomy, intratesticular injections  were dipped in autoradiographic emulsion  at 43–45°C. After drying for approximately 1h at 25°C, testis sections were placed in sealed black boxes and stored in a refrigerator at 4°C for approximately four weeks. Subsequently, they were developed in Kodak D-19  solution at 15°C  and staiThe volume densities of testicular tissue components were determined by light microscopy using a 441-intersection grid placed in the ocular. Fifteen randomly chosen fields  were scored for each animal at 400X magnification. The tubular diameter and seminiferous tubule epithelium height were measured at 200X magnification using an ocular micrometer. Thirty tubular profiles (round or nearly round) were chosen randomly and measured for each animal. The epithelium height was obtained in the same tubules used to determine tubular diameter. The total length of seminiferous tubules (meters) was obtained by dividing seminiferous tubule volume by the squared radius of the tubule multiplied by π .Seminiferous epithelium cycle (SEC) stages were characterized according to the development of the acrosome system and morphology of the developing spermatid nucleus . The relThe spermatogenic cycle length was estimated based on the stage frequencies and the most advanced germ cell type labeled at different periods following thymidine injections. The total duration of spermatogenesis took into account that approximately 4.5 cycles are necessary to complete the spermatogenic process , 54. SinThe cells present in stage VIII of the cycle were counted in ten seminiferous tubule cross-sections per animal. These counts were corrected based on the method described by Abercrombie (1946) , as modiThe Sertoli cell numbers per testis and testis gram were estimated from the Sertoli cell nucleoli number per seminiferous tubule cross-section and the obtained seminiferous tubule total length , 59. Thi3, in which R = nuclear diameter/2). The proportion between the nucleus and cytoplasm was estimated, scoring 1,000 points over Leydig cells per animal using a grid placed in the ocular at 400X magnification. The number of Leydig cells per testis was estimated from the individual Leydig cell volume and the total volume occupied by these cells in the testis parenchyma.Leydig cell volume was obtained using the nuclear volume as well as the proportion between the nucleus and cytoplasm. For this purpose, 30 nuclei were measured per animal. Leydig cell nuclear volume was obtained using the sphere formula  were in the range of ~4–9% in all four species studied. The frequencies of pre-meiotic and post-meiotic phases were similar among A. cursor, A. montensis, N. lasiurus, and O. nigripes, and in the range of respectively 28–33% and 61–64%.The mean percentages of each of the twelve stages characterized, as well as the frequencies of pre-meiotic (stages VIII-XI), meiotic (stage XII) and post-meiotic (stages I-VII) phases, are displayed in A. cursor and stage IX in the other species , 40 ± 0.9 (A. montensis), 35.1 ± 0.3 (N. lasiurus), and 35.6 ± 0.1 (O. nigripes). For each species, a schematic illustration of the stage frequencies and their duration is depicted in Based on the labeled germ cells observed in each period following thymidine injection and the stage frequencies Tables , the mea0.1 days . Since aO. nigripes, which presented a lower value, the meiotic index (number of round spermatids per primary spermatocyte) was approximately 3, meaning that the germ cell loss during meiosis was around 25%. Regarding the Sertoli cell efficiency (number of spermatids per Sertoli cell), N. lasiurus exhibited the highest value (~13:1), while O. nigripes showed the lowest efficiency (~8:1). The number of Sertoli cells per gram of testis in O. nigripes was almost 70 million, whereas in the other three rodent species the values found were in the range of ~48–55 million. Due to testis size, the total number of Sertoli cells per testis was ~4 million in O. nigripes and ~11–14 million in the other three species. In relation to the spermatogenic efficiency DSP/gram/testis, the values observed were ~62 million and ~66 million in A. montensis and A. cursor, respectively. This efficiency reached 74 million in O. nigripes and almost 80 million in N. lasiurus. Finally, the sperm production per testis was around 18 million in A. cursor and N. lasiurus, ~13 million in A. montensis, and less than 5 million in O. nigripes . Because the Leydig cell individual volume is small in O. nigripes, overall, its number per testis gram (~33 million) was two-fold higher (~15–19 million) than in the three other species. However, as expected, its number per testis is much lower in O. nigripes . An interesting aspect suggested in the literature, particularly in studies conducted in our laboratory investigating about twenty different species [In mammals, although under the control of the germ cell genotype , the len species , 75, 76,A. cursor and A. montensis. According to the literature [Similar to the mice strains usually utilized in laboratory studies , the speterature , relativterature . In geneterature , 89. In terature . Moreoveterature , 91, theIn the present study, the percentage of the seminiferous tubules in the testis parenchyma was very high and similar to most rodents from the Myomorpha and Hystrichomorpha suborders investigated see . It meanP. guyannensis and T. moojeni (~78–82 million) that belong to the Hystricomorpha suborder  in the seminiferous epithelium in the present investigation. However, based on the Sertoli cell results herein obtained, such as their comparatively high number per testis gram and their good support capacity for germ cells , accordisuborder . Also copressive .In summary, in comparison to the other mammalian species already investigated and, in particular, with other rodents from the suborder Myomorpha, the four cricetid rodents herein evaluated exhibited very high spermatogenic efficiency. This efficiency is mainly related to the short duration of spermatogenesis, the high values found for the percentage of seminiferous tubules in the testis parenchyma and the number of Sertoli cells per testis gram. We expect that, besides contributing to comparative and evolutionary reproductive biology studies, the knowledge herein generated will help to advance the conservation programs and the proper management of wildlife.S1 Table(DOCX)Click here for additional data file.


G-CSF concentrations were lower in patients with SUD than in controls (p < 0.05), specifically in the cocaine subgroup (p < 0.05). Patients with SUD and comorbid MDD had lower G-CSF concentrations than patients with SUD but not comorbid MDD or controls (p < 0.05). In contrast, patients with MDD but not SUD showed no differences compared with their controls. The negative association between G-CSF concentrations and age in the sample was not observed in patients with MDD. G-CSF concentrations were decreased in patients with SUD and comorbid MDD but not in patients with MDD. Therefore, G-CSF may be useful to improve the stratification of patients with dual diagnosis seeking treatment. Further investigation is needed to explore the impact of sex and type of drug on the expression of G-CSF.Granulocyte colony–stimulating factor (G-CSF) has raised much interest because of its role in cocaine addiction in preclinical models. We explored the plasma concentrations of G-CSF in patients diagnosed with substance use disorder (SUD) and highly comorbid psychiatric disorders. In particular, we investigated the association between G-CSF concentrations and comorbid major depressive disorder (MDD) in patients with cocaine and alcohol use disorders . Additionally, patients with MDD but not SUD were included in the study. Three hundred and eleven participants were enrolled in this exploratory study: 136 control subjects, 125 patients with SUD (SUD group) from outpatient treatment programs for cocaine  and alcohol , and 50 patients with MDD but not SUD (MDD group) from primary-care settings. Participants were assessed based on DSM-IV-TR criteria, and a blood sample was collected to examine the plasma concentrations of G-CSF. G-CSF concentrations were negatively correlated with age in the entire sample (r = − 0.233,  In recent years, it has been confirmed that SUDs are associated with neuroinflammation, a process that can cause long-term behavioral alterations by activating glial cells, mainly microglia and astrocytes, and modifying the plasticity of the central nervous system (CNS)3. Alcohol and cocaine are two of the most commonly used drugs, both of which are well known to cause neuroinflammation; in fact, several studies have explored how these inflammatory effects contribute to the clinical course of SUDs and comorbid pathologies6.Substance use disorder (SUD) is a chronic neurobiological-based medical illness contributing to almost 6% of all deaths worldwide4, modifications of the lipid membrane causing alteration of the cell membrane permeability, changes in proliferation and maturation of neurons, and eventually neuronal apoptosis7. Interestingly, alcohol directly induces the activation of proinflammatory immune signaling, as revealed by the characterization of Toll-like receptor 4 (TLR4)-dependent glial activation responses associated with alcohol exposure8. This finding supported the inflammatory view of alcohol-induced toxic effects: preclinical studies based on reward and alcohol withdrawal behavioral responses have revealed that there are several proinflammatory mediators involved in the regulation of alcohol effects11. For example, the chemokine monocyte chemoattractant protein-1 (MCP-1) secreted by glial cells was found to be associated with alcohol consumption and alcohol-related neurodegeneration13. Other studies monitoring plasma concentrations of immune mediators have shown alcohol-induced dysregulation of certain chemokines related to immune responses such as SDF-1 [stromal derived factor (CXCL12)] and fractalkine (CX3CL1), which were found to be decreased in patients with alcohol use disorder (AUD)14. Finally, important neurotrophic factors such as BDNF and insulin-like growth factor-1 (IGF-1) were found to be profoundly affected in abstinent patients with AUD15.Regarding alcohol, its pharmacological actions in the CNS include multiple neurotoxic effects, such as activation of microglial cells, alterations in the release of mediators 16. Cocaine consumption is associated with neuronal cytotoxicity and altered neurotrophic responses through a nitric oxide-mediated signaling pathway, among other mechanisms17. However, although cocaine use disorder (CUD) is mostly characterized by dysfunctions in reward-related brain circuits and dysregulation of dopaminergic reward pathways and glutamate receptor-dependent signaling cascades19, multiple studies have also described immune signaling alterations associated with cocaine use8. Regarding circulating immunological mediators related to cocaine consumption, proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) were found to be elevated in plasma while the anti-inflammatory interleukin 10 (IL-10) was found to be decreased and linked to cocaine-related chronic stress20. Moreover, plasma concentrations of interleukin 1β (IL1β), fractalkine and SDF-1 were found to predict the severity of CUD symptoms, which suggests a relevant contribution of the immune system to cocaine addiction21.In contrast, cocaine is a stimulant substance in the CNS with a high potential for abuse23, which affects the course of each psychiatric condition24. Mood, anxiety, psychotic, and personality disorders are the most prevalent diagnoses found in patients with SUD27. The dysregulation of inflammatory and immune signaling has been found to be associated with several psychiatric disorders, mainly mood disorders such as major depressive disorder (MDD)29. Immunological and affective regulatory interactions are an important aspect of MDD development30. Previously, it has been reported that the gene expression of IL-17, IL-21, IL-23, and IL-35 was substantially higher in patients with MDD than in controls, which suggests that the expression of specific inflammatory genes may be a factor in the etiopathogenesis of depressive disorders. Additionally, these cytokines may affect the metabolism of neurotransmitters and neuroendocrine functions in the brain and may be markers and new potential therapeutic targets for recurrent depressive disorders32.The contribution of the immune system in addiction not only has been associated with the severity of SUD or the toxic effects of substances but also it might contribute to the development of comorbid disorders. Patients with SUD are more likely to suffer comorbid psychiatric disorders throughout their lives than the general population33. G-CSF participates in the proliferation and differentiation of myeloid progenitors33, promotes the generation of granulocyte precursors and exerts antiapoptotic actions35. In the CNS, G-CSF receptors have been studied because of their neuroprotective properties36, including antiinflammatory effects on dopaminergic neurons in neurodegenerative disorders such as Parkinson’s disease37 and Alzheimer’s disease38. Unlike other inflammatory signals, few studies examining G-CSF have been reported in the context of mood disorders, and the only study in patients suffering MDD with long-term symptomatology found no differences in serum G-CSF relative to controls39. Regarding addiction, G-CSF was found to be a relevant signal that promoted cocaine self-administration and consolidates cocaine seeking behavior40. However, alterations in this immunomodulatory growth factor in patients with SUD have been inconclusive, evidenced by a recent meta-analysis of 74 studies has recently reported41.The search for immune mediators participating in the natural history of addiction has brought attention to new molecules such as the granulocyte colony-stimulating factor (G-CSF), a glycoprotein that acts as a growth factor, cytokine and hormone in the bone marrow, blood and CNS42. Because several studies have reported a systemic inflammatory state in patients with SUD and common psychiatric disorders such as MDD, we explored whether circulating concentrations of G-CSF in the plasma were altered in patients with SUD (CUD and AUD) and psychiatric comorbidity from outpatient treatment programs, as well as in patients with MDD but not SUD from primary-care settings in Spain.The impact of addictive disorders in the health system emphasizes the importance of effective prevention programs and treatmentsThree hundred thirty-five volunteers were originally enrolled in this exploratory study: 140 healthy control subjects, 140 abstinent patients diagnosed with SUD and 55 patients diagnosed with MDD. However, 24 participants were excluded because they did not meet the participation criteria .Hospital Regional Universitario de Málaga, Málaga, Spain) and a second cohort obtained from volunteers donating data and plasma to the National Biobank of DNA . Control subjects were divided into two groups based on the age, sex, and body mass index (BMI) of patients with SUD  and MDD . Notably, the control group for SUD was sex-balanced and showed no statistically significant differences in age and BMI with the SUD group, and the control group for MDD showed no statistically significant differences in sex, age and BMI with the MDD group.Control subjects (N = 136) were recruited from two different sources, a multidisciplinary staff cohort of volunteers working at the Spanish National Public Health System .Patients with SUD  were recruited from outpatient treatment programs for alcohol  and cocaine , and patients with MDD  were recruited from primary-care settings at the Spanish National Public Health System  for all patients, diagnosis of AUD or CUD (a minimum of 4 weeks of abstinence) for the SUD group and diagnosis of MDD (last two months of depressive symptoms) for the MDD group. Lifetime SUD and MDD were diagnosed based on the DSM-IV-TR criteria. The exclusion criteria included personal history of chronic inflammatory diseases or cancer, infectious diseases, severe mental disorders precluding evaluation, pregnancy or breastfeeding and less than 4 weeks of abstinence from any drug, except for nicotine and caffeine. Psychiatric comorbidity was accepted for the SUD and MDD groups . Regarding healthy control subjects, the exclusion criteria were identical to those for the groups of patients but also included the use of psychotropic medication during the last year and the diagnosis of psychiatric disorders based on the DSM-IV-TR criteria.Hospital Regional Universitario de Malaga in accordance with the Ethical Principles for Medical Research Involving Human Subjects adopted in the Declaration of Helsinki by the World Medical Association  and Recommendation No. R (97) 5 of the Committee of Ministers to Member States on the Protection of Medical Data (1997), and Spanish data protection act  on plasma concentrations of G-CSF while controlling for “age” as a covariate. Because plasma concentrations of G-CSF showed a positively skewed distribution and did not pass the D'Agostino-Pearson normality test, raw data were log10-transformed data) and relevant SUD-related variables  were performed using the correlation coefficients of Pearson (r) and Spearman (rho) with continuous and categorical variables, respectively.Correlation analyses between plasma concentrations of G-CSF  and IBM SPSS Statistics version 22 . A p-value < 0.05 was considered statistically significant.This study included 311 participants grouped into four experimental groups to explore plasma concentrations of G-CSF: the SUD group, the MDD group and their control groups. Table 2), there were differences in the sex composition because the control group was originally sex-balanced in men and women and the SUD group was mainly composed of men (67%). The SUD group was divided into the cocaine subgroup and the alcohol subgroup. In this case, the comparison between both subgroups revealed significant differences in age and education level. Thus, the patients with CUD were younger than the patients with AUD, and the percentage of participants with elementary education was greater in the alcohol subgroup.Although the control group for patients with SUD showed comparable age and BMI to the SUD group .10-transformed G-CSF concentrations and BMI were not correlated, log10-transformed G-CSF concentrations and age were significantly correlated  in the total sample. Interestingly, there were differences in this association based on the group .An initial analysis was performed to explore the correlations between plasma concentrations of G-CSF and variables that could be included in the ANCOVA models as covariates or confounding variables (age and BMI). While log10-transformed for the analysis with “SUD diagnosis” [SUD (N = 125) and control (N = 92)] and “sex” as factors and “age” as an important covariate. ANCOVA revealed a significant main effect of “SUD diagnosis” on plasma concentrations of G-CSF  , alcohol (N = 65) and control (N = 92)] as a factor. There was a significant main effect of “type of SUD diagnosis” on plasma concentrations of G-CSF   = 3.393; p = 0.035]  . However, significantly lower G-CSF plasma concentrations were observed in the alcohol subgroup than in the control group in women (p < 0.05) .To elucidate the contribution of each SUD subgroup, we performed a second two-way ANCOVA on log] Figure A. The mu10-transformed) and these variables (Table p < 0.05).Because there was a significant association between lifetime SUD diagnosis and plasma concentrations of G-CSF, we explored relevant variables related to cocaine and alcohol addiction: the severity of SUD (based on DSM-IV-TR criteria for AUD and CUD), duration of the last abstinence and problematic substance use. To this end, the SUD group was divided into alcohol and cocaine subgroups, and correlation analyses were performed between plasma concentrations of G-CSF . We assessed all patients with SUD, and the high prevalence of psychiatric comorbidity was confirmed as a primary characteristic of the sample: 64 patients with SUD (51.6%), including 19 patients with CUD (31.7%) and 45 patients with AUD (69.2%), were diagnosed with other mental disorders. The most prevalent mental disorders in the SUD group were as follows: 31.7%, mood disorders; 27%, anxiety disorders; 15%, cluster B personality disorders; and 4.8%, psychotic disorders. No significant differences were found in the sex proportions among these comorbid mental disorders.Table disulfiram (39%). To investigate the potential effects of psychiatric medication on G-CSF concentrations, we performed two-way ANCOVA with “psychiatric medication” (nonmedicated and medicated patients with SUD) and “sex” as factors and “age” as a covariate. However, the analysis revealed no main effects of “psychiatric medication” on log10-transformed G-CSF concentrations , anxiolytics (40.7%), anticonvulsants (22.8%), antipsychotics (8.9%) and 0.7%, anx10-transformed G-CSF concentrations and “comorbid MDD diagnosis” but not with other comorbid mental disorders (data not shown).Because we observed no significant effect of “psychiatric medication”, we analyzed G-CSF concentrations in the SUD group based on the most prevalent comorbid mental disorders using two-way ANCOVA with “sex” as a factor while controlling for “age” but not for “psychiatric medication”. The analyses revealed a significant association between logF = 5.270; p = 0.006]  . Regarding the “sex” factor, there was no main effect or interaction on plasma concentrations of G-CSF.Therefore, there was a significant main effect of “comorbid MDD diagnosis” on plasma concentrations of G-CSF   = 4.384; p = 0.038]  in patients with MDD and antidepressant treatment48, we investigated the effects of antidepressants and anxiolytics on G-CSF concentrations. However, the analyses revealed no significant main effects of “antidepressants” on log10-transformed G-CSF concentrations  and anxiolytics (40%)] and recent research in our group has reported high plasma concentrations of antiinflammatory molecules  Plasma concentrations of G-CSF were significantly and inversely correlated with age in the control group and the SUD group, but the correlation was nonsignificant in the MDD group; (2) G-CSF concentrations were significantly lower in the patients with SUD than in the control subjects, specifically in the cocaine subgroup; (3) The patients with SUD and comorbid MDD had significantly lower G-CSF concentrations than the patients with SUD but not comorbid MDD or control subjects; (4) G-CSF concentrations were significantly lower in the cocaine subgroup with comorbid MDD but not in the alcohol subgroup with comorbid MDD; and (5) The patients with MDD showed no differences in plasma concentrations of G-CSF compared with their control subjects. The results suggest a potential role of this immunomodulatory factor in MDD disorders associated with SUDs rather than in primary MDD disorders.50. Unlike the control group, our data showed fluctuations in the inverse association between G-CSF concentrations and age across the different groups of patients. Thus, the inverse association was attenuated in the patients with SUD and nonsignificant in the patients with MDD. This suggests that a history of MDD could be associated with disturbances in the immune system through immunomodulatory growth factors.Consistent with our findings, it is known that G-CSF is close and negatively correlated with age as a typical growth factor. Previous studies have reported G-CSF as a risk factor for cognitive impairment in preclinical models and as one of the 18 plasma signaling proteins that are collectively predictive of conversion from mild cognitive impairment to Alzheimer’s disease51, whether G-CSF treatment in preclinical models of cocaine reward is associated with boosting or sustaining cocaine-seeking behavior40, it is reasonable to consider that G-CSF might be downregulated during a period of abstinence after chronic cocaine use in severe cocaine-addicted patients. However, these results regarding cocaine need to be conclusively determined. Regarding alcohol, G-CSF has been linked to alcohol-related liver disease, and a study is currently validating the efficacy of liver regeneration through G-CSF therapy52. Unfortunately, we have no data regarding accessory digestive organ diseases in the present sample to test this association. However, previous studies in patients with AUD from the same source as our group reported a high prevalence of alcohol-related liver and pancreas diseases53. In contrast, our study revealed a positive correlation between plasma concentrations of G-CSF and the duration of the last abstinence from alcohol. Similar to G-CSF, other inflammation-related signals in the plasma [such as the lipid messenger N-oleoilethanolamine (OEA)] were found to be related to the duration of alcohol abstinence. Thus, OEA decreases as the duration of alcohol abstinence increases in patients with AUD, and it has been proposed as a marker of abstinence54.However, our results confirm that changes in the plasma concentrations of G-CSF might be attributed to the history of addiction in patients with AUD and CUD, although the nature of this relationship demands further research. While in preclinical studies this immunomodulatory growth factor helps to consolidate cocaine reward/cocaine seeking behavior, in abstinent humans we observed that G-CSF concentrations are lowered in patients with CUD and with a nonsignificant trend toward a negative correlation with the severity of CUD. Because there is a lack of data on the acute effects of cocaine on circulating concentrations of G-CSF, we can only speculate on the nature of this finding. In accordance with the allostatic model set in place for most biological modulatory transmitters related to addiction55, with several forms of dysregulation observed in relation to proinflammatory status56. Recent studies from our group have described differences in other peripheral inflammatory mediators relative to the presence of comorbid psychiatric disorders in patients with AUD. In particular, decreased concentrations of the chemokine eotaxin-1 (CCL11) in the plasma of abstinent patients with AUD have been associated with comorbid mood and anxiety disorders14. Another study reported lower concentrations of IL-1β, CXCL12 and CCL11 in cocaine-induced MDD57. Although we observed lower concentrations of G-CSF in patients with CUD and comorbid MDD, these changes in the concentrations were not linked to the type of MDD . How the interaction of SUD and MDD affects G-CSF must be elucidated with further research.Our results also showed that plasma concentrations of G-CSF were lower in the patients with SUD and comorbid MDD but not in the patients with primary MDD without SUD. Long-lasting stress effects have been studied in the development of mood disorders58. Therefore, we consider that it would be interesting to measure the role of G-CSF in a neuropsychological cohort of patients with SUD and substance-induced memory deficits.Furthermore, this immunomodulatory growth factor has also been studied in relation to memory functions in a preclinical model, showing that deficiencies in G-CSF concentrations in the hippocampus decreased spatial learning performance and memory formation10-transformation), calculation of sample size, number of independent variables/covariates in the linear models, etc. Finally, it is important to replicate these findings with larger samples in cohorts from different geographical and cultural backgrounds, as well as to characterize G-CSF concentrations in longitudinal studies during critical stages in the course of MDD and SUD, such as substance abstinence and active substance use.These findings support the importance of monitoring G-CSF in the context of SUD and psychiatric comorbidity, but we are aware of the limitations of this exploratory study: (1) The high prevalence of psychiatric comorbidity in patients with SUD increased the difficulty in the characterization of a specific comorbid disorder such as MDD; (2) There are additional uncontrolled biological and sociodemographic variables that were not included in the present study but could be a source of variability in G-CSF concentrations; (3) The findings were restricted based on statistical limitations such as compliance of parametric assumptions and/or appropriate transformations of raw data . Further research is necessary to elucidate the role of G-CSF as a potential biological marker of immunomodulatory and inflammatory states in addiction, mood disorders and dual diagnosis.Supplementary Information.


Many scholars have taken measures to improve the accuracy of gait recognition, but several real-time limitations affect its applicability, of which variation in the load styles is obvious. The purposes of this study are to (1) investigate the impact of different load styles on gait recognition; (2) study whether good gait recognition performance can be obtained when a convolutional neural network (CNN) is used to deal with the sEMG image from sparse multichannel sEMG (SMC-sEMG); and (3) explore whether the control system of the lower-limb exoskeleton trained by sEMG from part of the load styles still works efficiently in a real-time environment where multiload styles are required. In addition, we discuss an effective method to improve gait recognition at the levels of the load styles. In our experiment, fifteen able-bodied male graduate students with load (20% of body weight) and using three load styles  were asked to walk uniformly on a treadmill. Each subject performed 50 continuous gait cycles under three speeds . A CNN was employed to deal with sEMG images from sEMG signals for gait recognition, and back propagation neural networks (BPNNs) and support vector machines (SVMs) were used for comparison by dealing with the same sEMG signal. The results indicated that (1) different load styles had remarkable impact on the gait recognition at three speeds under three load styles ( The surface electromyogram (sEMG) signal is employed widely as a neural control source for lower-limb exoskeleton and prosthesis devices ,2; theseThe above literature focuses mainly on increasing the gait recognition performance based on offline sEMG signals by multisensor cooperation , algoritAlgorithms, such as BPNN , Long ShRecently, CNN have achieved great success in computer vision , image pInspired by the success of EMG maps ,27 and mTo clarify these issues thoroughly, fifteen adult males took part in our experiment. Every subject walked on a uniform running treadmill with three load styles under three speeds. The CNN was employed to deal with the SMC-sEMG image from the SMC-sEMG signal to investigate (1) whether the different load styles have a significant impact on gait recognition; (2) whether the CNN based on SMC-sEMG images performs well in gait recognition; and (3) whether a lower-limb exoskeleton (or prosthesis) control system trained by sEMG signals from single or parts of load styles still functions well in the face of multiload style conditions.2) from Zhejiang University who were skilled in using a treadmill participated in our study. Before the experiment, all subjects underwent anthropometric measurements, including age, height, and weight. Then, they were given a complete physical examination to ensure that they were free from neurological and musculoskeletal diseases. They were not allowed to participate in any physical strenuous exercise that might introduce fatigue. The purpose, details, and procedure of the experiment were provided to the local human ethical clearance committee of Zhejiang University for review and were approved. All subjects provided informed consent after they completely understood the experiment.Fifteen able-bodied (intact) male graduate students  = 21.6 ± 0.7 kg/mAccording to the different roles the lower-limb muscles play during the gait cycle, four thigh muscles  were selected for the acquisition of the surface EMG signal; of these, the tensor fasciae latae and the semitendinosus are closely related to the stance phase, while the adductor longus and the vastus medialis mainly affect the swing phase ,34, as sIn the experiment, all the subjects were asked to wear T-shirts and white shoes to strengthen the colour contrast between their shoes and the treadmill belt, which is good for the division of the gait phase by identifying the demarcation points of the heel strike and toe off of both feet. Then, the subjects were familiarized with the entire experimental process and the equipment.Four electrode groups were used to acquire sEMG signals, every group contained 3 Ag/AgCl electrodes. Each electrode group was adhered to a target muscle, and the line through the two electrodes (non-reference electrode) aligned with the direction of target muscle fibres on the midline of the muscle belly . Then, tBP, SCS, and SSS; After all instruments were configured correctly and the signal was checked to be in a very good steady state, the experiment continued to the next step. The subjects were required to walk on the treadmill at three uniform speeds   under thIn the experiment, we chose a traditional one-shoulder bag and backpack with no sternum strap or hip-loading belt to carry the load. The adoption of this bag and backpack makes the experiment more convincing for their universal applicability. Then, the shoulder straps of the backpack were adjusted to ensure that the load was placed on the level of pelvis , and theThe subjects were instructed that data would be collected when they walked steadily on the treadmill. The surface electromyographic (sEMG) signals of the target muscles of subjects were collected by MyoScan-Pro sensors, which have the ability to record up to 1600-microvolt (μV) sEMG signals. The collected signal was filtered in a range of 10 Hz to 400 Hz, and a 50 Hz notch filter was employed to remove line interference. Then, the signals were input into a PC for data analysis using a digital sEMG system , which contains ten channels and a video collection system , which cThe video data were collected synchronously by a high-speed, high-resolution camera at 60 frames per second. The camera was fixed perpendicular to the direction of the running belt of the treadmill, and the vertical distance to the treadmill was adjusted to ensure that the entire running belt was in the camera’s frame .All sEMG and video signals were processed offline. The synchronized video of each experiment was segmented into independent gait cycles one by one, and then each gait cycle was subdivided into 5 separate gait subphases  by a trained and experienced observer by identifying the demarcation point of the heel strike and toe off of both feet . Then, t1. sEMG image.The sample size of the sEMG signal after pre-treatment was 240 . The sEMG signal of each analysis window was recombined and arranged into a 4 × 60 greyscale image to take advantage of the CNN in image processing for feature extraction and classification.2. CNN structure.We structured the CNN as shown in l, m, r, and f), in which l was the layer, m was the feature image, r was the specific position of neurons in the feature image, and f was the activation function. The input and out of a neuron were recorded as f (u) =tanh (u) as the activation function . In F4 C,TH , which can be expressed as 4 × 60.(1) L1: input layer. the input matrix (2) C2: convolutional layer. The aim of this layer was to carry out spatial filtering for input signals. We used 7 spatial filters and set the convolution kernel [4 × 1] in the layer, so 7 feature maps (the size of each feature map was [1 × 60]) were obtained. The function can be expressed as (1) .(1)Pm2=(3) C3: which was similar to C2, which filtered input signals in the time domain. We used 6 time filters and set the convolution kernel size [1 × 6], and 42 feature maps (each size [1 × 10]) were obtained. The function can be expressed as (2) .(2)Pm3=(4) F4: fully connected layer, which is fully connected with C3, and 110 neurons were set in the layer.(5) O5: outer layer, which was fully connected to F4 and included 5 neurons . The expression is4 iterations. The range of input weight and bias was ith neuron in this layer. The learning rates In our CNN, gradient descent was employed to adjust the weight and bias to ensure the smallest error. The model’s loss convergence was determined to be optimal after 10 and (6) ,42.(5)σIn contrast to a CNN, an SVM and BPNN were employed to recognize the gait phases.A: Feature ExtractioniEMG) and the root mean square (RMS) were employed as the input features of SVM and BPNN, and they can be expressed as shown in Equations (7) and (8).ith sEMG data sampling. k = 1, 2…, N. N is the number of sampling points in each time window.After filtering, labelling, and segmentation, the sEMG data were extracted to reduce the sEMG signal dimension, which decreased the complexity and improved the computational efficiency of pattern recognition and classification. Based on the same thinking, it was important to choose features with clear distinction, less complexity, and substantial efficiency . In our P can be expressed asTherefore, the input vector kP = {RMSk1, iEMGk1, RMSk2, iEMGk2, RMSkr, iEMGkr}, where k is the number of time windows and r is the number of sEMG channels.In this experiment, the sEMG data were collected from four muscles, which means that each time window included eight features. The outputs were five gait phases, i.e., initial stance, midstance, terminal stance, initial swing, and terminal swing.B: Classification MethodsThe SVM is widely used to process EMG signals. With appropriate kernel functions, the SVM can obtain good adaptability and robustness. Therefore, in this study, we chose the Gaussian radial basis function (RBF) as the kernel function to construct the SVM model . Cross-vBPNNs have achieved great success in pattern recognition and classification based on sEMG signals. In our study, we designed a three-layer BPNN, which includes 20 nodes in the hidden layer. This means that there were eight nodes in the input layer and one node in the output layer, which was the gait phase corresponding to the time window.To study the impact of load styles on gait recognition, we converted the sEMG signal into an sEMG image from different load styles and speeds  and took the sEMG image as the input of the CNN model. In each experiment, 60% of the data were selected randomly for training CNN models, 20% of the data were set for selecting optimal parameters, and the remaining 20% of the data were set to test the performance. In each experiment, the recognition accuracies of the five gait phases were obtained separately, and the five values were averaged to acquire the average recognition accuracy of a single trial. For comparison, the SVM and BPNN were employed to process the same data.Univariate analysis of variance (UNIANOVA) was adopted to investigate the impact of load styles on gait recognition. The least significant difference (LSD) post hoc test was implemented to search for the full effect if the impact was significant. SPSS 24.0  was employed to analyse the experimental data, and a confidence level of 95% was selected.BP under V5, and the lowest value (80.73%) was from SCS under V3. When only the load style was considered, the highest accuracy of gait recognition at three different speeds came from SBP , followed by SSS , and the lowest value occurred at SCS . The gait recognition total average accuracies at the three load styles and three speeds were significantly different. V5 had the highest value (93.40%), and V3 had the lowest value (84.83%).Three different CNN models were constructed under each speed, as shown in p < 0.001); the same conclusion was also drawn from the speed data. In addition, the LSD post hoc test showed a significant difference among the three load styles (p < 0.05).UNIANOVA revealed that the different load styles had a substantial effect on gait recognition .For comparison, we adopted an SVM and BPNN to process the same data. The gait recognition performance of the three algorithms is presented in t-test indicated that there was a marked difference (p < 0.05) between the values on and off the main diagonal, which means that load styles had a significant effect on gait recognition. For example, when the testing data were from SBP and the training data were from SBP, SCS, and SSS under V5, the gait recognition performances  were markedly different. A similar phenomenon was also found in other experiments. Among all the experiments, the best and poorest performances at V3 appeared when the training data came from SBP and SCS and the testing data came from SBP . For V5 and V7, the best and poorest performances appeared when the training data came from SBP and the testing data came from SBP and SCS .Three different CNN models were constructed at each speed, which were trained by the data from one of the three load styles, and the test data were from all three load styles evaluated one by one at the same speed. All the performances are presented in Statistics were employed to analyse all the values in the three matrices, and the intra- and inter-load styles and overall accuracy, expressed as the mean ± SD, are presented in At each speed, all the data from the three load styles were mixed as the testing data group (group 3 (V3), group 5 (V5), and group 7 (V7)), and the training data were mixed as one or several load styles. This means that there were seven different combinations of load styles in the training data at each speed: one load style in the training data (three kinds), two load styles in the training data (three kinds), and three load styles in the training data (one kind). The performances are summarized in In this study, we first investigated the impact of load styles on gait recognition based on sEMG images from sEMG signals using a CNN. Then, we discussed whether a lower-limb exoskeleton control system with sEMG as the control signal and trained by parts of the load styles can still function efficiently in real-time conditions where a multiload style is required.p < 0.001), and the LSD post hoc test showed that a significant difference existed among the three load styles (p < 0.05). The load had a marked effect on the muscle activity, and the different load styles could influence the activity levels of muscles involved, which may influence the fatigue of the corresponding muscles and the gait stability [SS, SCS) had a markedly greater energy cost than a SBP [CS and SSS were much worse than that of SBP. Different load styles had significant effects on posture and gait balance [SS and SCS, the load was placed on one side of the body, which caused much greater lateral spinal bending and worse balance than with SBP [SS and SCS were much greater than that of SBP at the same speed, which leads to worse physical stability [CS, SSS in our study) than that with a SBP, which can explain to some extent why the gait recognition results of SSS and SCS were worse than that of SBP.tability , which wtability  revealedan a SBP , indicat balance . With SSwith SBP , and thewith SBP . Moreovetability . Yu-Chihtability  revealedp < 0.05); however, the performances of SVM and BPNN were generally worse than that of the CNN at the three tested speeds. A possible reason for this difference was that no information was lost in the feature exaction of the CNN, while the hand-crafted features  in the SVM and BPNN may drop inherent information [The SVM and BPNN algorithms were employed to deal with the same data separately to verify our conclusion, and the results shown in ormation , resultiTo explore whether lower-limb exoskeletons still work well in a real-time environment, a feasible method was to test whether the control system using sEMG as a control signal was sufficiently effective when the training and testing load styles were unequal  . Our expp < 0.001), and based on these results, we can conclude that increasing the kinds of load styles in the training set can clearly improve gait recognition and can subsequently improve the effectiveness of the exoskeleton using the EMG signal as a control signal in a real-time environment.We further studied the impact of load styles on gait recognition in real-time environments and explored ways to increase gait recognition at the level of load styles. All the data at each speed were collected as a group for the testing set to imitate a real-time environment, in which varied load styles are the norm. The three datasets from the three load styles were freely combined into seven datasets as a training set to imitate the possible load styles that people encounter in real environments. The performance of gait recognition at each speed revealed the same conclusion: if more load styles are included in the training set, better performance is achieved, as shown in In addition, we found that speed was not a factor that could be ignored in gait recognition, as shown in However, some limitations are present in our experiments. For instance, the subjects in our experiments were all able-bodied people who were not afflicted by stroke or hemiplegia . Second, we only explored whether the CNN based on SMC-sEMG images performs well in gait recognition and investigated the impact of different load styles on the accuracy of gait recognition. However, we did not validate the control system based on sEMG with the exoskeleton on. Therefore, the next study will focus on the inclusion of people with disabilities beside verification of sEMG control system with the exoskeleton on. With these factors taken into consideration in studies on the effects on gait recognition, we will attempt to improve the accuracy of gait recognition and the effectiveness of the control system for lower-limb exoskeletons based on sEMG in real-time environments.In this paper, we demonstrated that the different load styles had marked effects on gait recognition based on sEMG images using a CNN, and the same conclusion was obtained from a BPNN and SVM, which means that the marked effect was not mainly caused by algorithms but load styles. In our study, the gait recognition based on SMC-sEMG images using the CNN was much better than those using the SVM and BPNN, indicating that the CNN has good performance in processing SMC-sEMG images for gait cognition in addition to HD-sEMG images. We also found that the performance of gait recognition when the training and testing data of CNN were from intra-load styles was much better than the performance when they were from inter-load styles. Moreover, we freely combined the data from three load styles at each speed to imitate the possible load styles that the users of lower-limb exoskeletons may meet in real-time environments and pooled all data at each speed as a group to imitate a real-time environment at the level of the load styles. The results showed that if more load styles were included in the training data, better gait recognition performance was obtained, which suggests that a lower-limb exoskeleton control system trained by a single load style or by only some load styles does not work efficiently in real-time environments. We can also deduce that including more load styles in the training data was an effective method to improve gait recognition in a real-time environment at the level of the load styles. This study not only contributes to the EMG guidance of a lower-extremity exoskeleton but, at a certain level, also explains that the difference in load styles significantly impacts the posture and gait balance of the human body, providing good guidance for the posture correction of the human body, especially for teenagers.