Publicly available genome data provides valuable information on the genetic variation patterns across different modern human populations. Neuropeptide genes are crucial to the nervous, immune, endocrine system, and physiological homeostasis as they play an essential role in communicating information in neuronal functions. It remains unclear how evolutionary forces, such as natural selection and random genetic drift, have affected neuropeptide genes among human populations. To date, there are over 100 known human neuropeptides from the over 1000 predicted peptides encoded in the genome. The purpose of this study is to analyze and explore the genetic variation in continental human populations across all known neuropeptide genes by examining highly differentiated SNPs between African and non-African populations.NPPA (rs5065) with high impact stop lost variant; CHGB  with multiple moderate impact missense variants; IGF2 (rs10770125) and INS (rs3842753) with moderate impact missense variants that are in linkage disequilibrium. Phenotype and disease associations of these differentiated SNPs indicated their association with hypertension and diabetes and highlighted the pleiotropic effects of these neuropeptides and their role in maintaining physiological homeostasis in humans.We identified a total of 644,225 SNPs in 131 neuropeptide genes in 6 worldwide population groups from a public database. Of these, 5163 SNPs that had ΔDAF |(African - non-African)| ≥ 0.20 were identified and fully annotated. A total of 20 outlier SNPs that included 19 missense SNPs with a moderate impact and one stop lost SNP with high impact, were identified in 16 neuropeptide genes. Our results indicate that an overall strong population differentiation was observed in the non-African populations that had a higher derived allele frequency for 15/20 of those SNPs. Highly differentiated SNPs in four genes were particularly striking: We compiled a list of 131 human neuropeptide genes from multiple databases and literature survey. We detect significant population differentiation in the derived allele frequencies of variants in several neuropeptide genes in African and non-African populations. The results highlights SNPs in these genes that may also contribute to population disparities in prevalence of diseases such as hypertension and diabetes. Neuropeptide genes , 2 are nNeuropeptides are also expressed in the endocrine and immune system and play a major role in physiological homeostasis. They intersect the immune, nervous and endocrine systems through autocrine, neurocrine, paracrine and endocrine manners, thus playing a core role in influencing postsynaptic cells in a large target area . In physrs2478523 in the AGT gene shows an increase in the risk of high altitude polycythemia (HAPC) in the Tibetan population while in the Han population, rs699, rs4762 and rs5051 are associated with reduced HAPC susceptibility [rs5065 in NPPA was identified as a marker of increased cardiovascular risk [rs1042571 in POMC was shown to increase the risk of obesity [As humans migrated into new frontiers outside Africa their populations became fragmented and genetically differentiated. This genetic diversity can also be a source of differences in genetic risk for particular ailments between different populations. For example, variant tibility . Also, ilar risk , and in  obesity . Due to  obesity –15 so fa obesity , 16–19.The rapid development in sequencing technology and decreasing costs of genome sequencing now proffer an unbiased examination of human genetic variation and have led to the development of several large scale human whole exome and whole genome databases, such as the 1000 Genomes Project , the TraIn this study, we analyzed the genetic variation in continental human populations across known neuropeptide genes. In particular, we examined single nucleotide polymorphisms (SNP) that were highly differentiated between African and non-African populations in publicly available datasets, to gain insights about the patterns of genetic variations in genes that code for neuropeptides and examine whether any are undergoing any adaptive selection in these populations.Homo sapiens”. An additional 26 neuropeptide genes were identified by Ensembl [A total of 105 neuropeptide genes were identified from four neuropeptide databases; StraPep , neurope Ensembl . TherefoUsing the whole genome sequence data, we extracted variants for the 131 neuropeptide genes in 15,164 individuals belonging to 6 different populations, Africans, Latino, Ashkenazi Jewish, East Asian, Finnish and Non-Finnish Europeans. A total of 769,597 variable sites were identified in the 131 neuropeptide genes  between African and non-African populations equal to or more than 0.20, were identified and functionally annotated Figs. . A cutofTwenty SNPs that were highly differentiated (ΔDAF ≥0.20) between Africans and non-Africans occurred in 16 of 131 neuropeptide genes. Their functional consequences were analyzed using available phenotype data in Genome Wide Association Studies (GWAS) catalogue , Online NPPA, CHGB, IGF2 and INS) were especially striking because of the following salient features: NPPA with a high impact stop lost variant (rs5065); CHGB with multiple moderate impact missense variants ; IGF2 (rs10770125) and INS (rs3842753) with moderate impact missense variants that are in linkage disequilibrium. These variants are further examined in the following sections.Variants in four of these genes (rs5065) in NPPA has been associated with cardiovascular disease risk [NPPA . Three of these SNPs  had a high derived allele frequency in African populations and two SNPs (rs6085324 and rs742711) in non-Africans (Table rs6085324 and rs742711) have been associated with schizophrenia in the Korean population [rs236152 has also been associated with schizophrenia in the Japanese population [Five highly differentiated SNPs occurred in the pulation  and SNP pulation .Table 2CHGB 14 kb genomic region , separate the two major haplotypes, whereas the remaining 2 SNPs (rs236150 and rs742710) mainly separate other Africans and minor non-Africans haplotypes from one another.The relationship between these 5 SNPs was further explored by using Africans and non-Africans allele linkage disequilibrium (LD) Table . A mediars10770125) in IGF2 and (rs3842753) in INS are located close together on chromosome 11. GTEx data shows that IGF2 is highly expressed in the Adipose – Visceral (Omentum) and INS is highly expressed in the pancreas. The derived allele frequencies of both SNPs are higher in non-Africans as compared to Africans (Table 2 = 0.336) than in non-Africans (r2 = 0.056). A haplotype network based upon 65 SNPs in a 1 kb genomic region of IGF2  patients in the Uruguayan population of predominantly European ancestry. In a separate study of European American descents in the GoKinD project [IGF2 rs10770125 has been associated with diabetic nephropathy in male patients with T1D, but not in female patients [The SNP  to extract variants for 131 neuropeptide genes. Using differences in derived allele frequencies we identified 20 highly differentiated SNPs between Africans and non-African populations in 16 neuropeptide genes (Table (rs5065) in NPPA has been associated with increased acute coronary syndrome [NPPA encodes a protein implicated in the control of extracellular fluid volume and electrolyte homeostasis and is highly expressed by the heart muscle. Furthermore, the ventricular expression of this gene is strongly increased in the cardiac muscle cells of the mice during stress [The high impact stop lost variant syndrome  and cardsyndrome , 31. NPPg stress .CHGB [CHGB locus, especially in the proximal promoter, influences CHGB expression, catecholamine secretion and the early heritable responses to environmental stress and is associated with changes in blood pressure in the sub-Saharan African and European ancestry groups [ that lie in a single exon have high ΔDAF between Africans and non-Africans and three  of these (Table CHGB expression in the GTEx dataset [(rs236152) has a higher derived allele frequency (63%) in Africans. Another close SNP (rs236150) that also has a higher derived allele frequency (21%) in Africans is also predicted to be differentially O-glycosylated. Another calcium binding protein with a highly differentiated SNP (rs757081) was NUCB2. NUCB2 shares a 60% sequence homology with NUCB1 in the human and mouse genome [AGT, another gene with a highly differentiated SNP, rs699, with a derived allele frequency of 17% in Africans, has also been associated with hypertension in African populations [NUCB2 rs757081 and AGT rs699 decreases their gene expression levels in several tissues and both SNPs have been associated with hypertension in the GWAS catalogue [A number of these highly differentiated SNPs were in genes that help regulate the amount of intracellular calcium that is known to play a crucial role in the regulation of cardiovascular functions. An increase in calcium in vascular smooth muscle cells leads to an augmented muscular tone which further increases vascular resistance that eventually raises the blood pressure . One sucCHGB  that stiCHGB . Common y groups . Five mi dataset . Of these genome  and playe genome . This SNe genome , and in e genome . AGT, anulations . Based oatalogue .INS, GIP and IGF2 with highly differentiated SNPs are involved in regulating glucose homeostasis. Evidence from epidemiological studies suggests that African Americans are also more insulin resistant and have higher insulin responses to glucose than European Americans [INS rs3842753 with a derived allele frequency of more than 75% in non-African populations has been identified as a marker for atypical T2D in the Uruguayan population [IGF2 rs10770125 has been associated with diabetic nephropathy in people with European American ancestry [GIP is secreted from K cells and acts on pancreatic beta cells to stimulate the release of insulin. Using the HGDP-CEPH project and the Human Genome Center at the University of Tokyo datasets, a previous study [rs2291725 is significantly higher (> 60%) in the majority of East Asian populations while varying widely in other populations, ranging between 0.0–9.5% in sub-Saharan Africans and increasing to > 40% in European and Middle Eastern populations. We also noted a low derived allele frequency of 14% for this SNP in the Africans and a significantly higher derived allele frequency of 52% for non-Africans. The highest derived allele frequency was also seen in East Asian populations with frequency of 0.75. NUCB2 rs757081 variant was also associated with the decreased risk of developing T2D in Chinese Han population [CHGB gene is also essential for adequate secretion of islet hormones in mice, where its deficiency led to a phenotype with some hallmarks of human T2D including loss of initial rapid insulin secretion [ have been associated with schizophrenia and increased risk for T2D [Several of the other genes, including mericans . The balmericans . INS rs3pulation , while Iancestry . GIP is us study  showed tpulation . The CHGecretion . Three m for T2D .ST for the highly differentiated SNPs. As expected haplotype networks for the highly differentiated genes show population sub-structure with high frequency population specific haplotypes. However, this could not be considered an unusual feature, because it is dependent upon the underlying linkage disequilibrium between SNPs in these populations and confounded by selection and demography.A major limitation of the study was the non-availability of individual sequences in the gnomAD dataset. Therefore, selected sequences from the 1000 Genomes Project continental populations representing Africans and non-Africans were used to construct the haplotype networks, compute LD and FOur study shows substantial population differentiation between African and non-African, as measured by differences in derived allele frequencies, in variants located in 131 neuropeptide genes. Twenty outlier SNPs with ΔDAF |(African – non-African)| ≥ 0.20 were identified in 16 neuropeptide genes and their functional significance was evaluated. The product of these genes appeared to affect multiple systems and some were associated with ethnic differences in incidence of common human diseases such as high blood pressure and type 2 diabetes. Significantly, our analysis adds to our knowledge of the genetic variation in continental human populations across all known neuropeptide genes. It also highlights the pleiotropic nature of these neuropeptides, their functional significance in extra neuronal tissues and their association with cardiovascular and metabolic diseases.Homo sapiens”. The list was further refined by adding more neuropeptide genes using the search term “Neuropeptide” in Homo sapiens in Ensembl (Ensembl GRCh37.p13) [GO:0005184 neuropeptide hormone activityGO:0051428 peptide hormone receptor bindingGO:0071855 neuropeptide receptor bindingGene ontology - Molecular functionGO:0007218 neuropeptide signaling pathwayGene ontology - Biological processA list of human neuropeptide genes was manually generated by integrating information from neuropeptide databases, Ensembl, and AmiGO Gene Ontology. Four neuropeptide databases were used for obtaining the gene list and included: StraPep , neuropeh37.p13)  and AmiGh37.p13) . In addiThe final list comprised a total of 131 human neuropeptide genes | ≥ 0.20, as this was above the 99VEP tool  to deterVEP tool  databaseVEP tool  and GWASVEP tool  to underVEP tool  was also2 ≤ 0.2 between the most differentiated and other SNPs in the region  package , to inveAdditional file 1 : Table S1. List of 131 neuropeptide genes in human.Additional file 2 : Table S2. General information on the variants data for 131 neuropeptide genes.Additional file 3 : Figure S1. Distribution of ΔDAF values between Africans and non-Africans.Additional file 4 : Table S3. SNPs with absolute differences in derived allele frequencies between African and non-African population equal to or more than 0.20. The data are sorted by differences of derived allele frequency.Additional file 5 : Figure S2. Annotating 5163 SNPs consequences using the Ensembl Variant Effect Predictor (VEP) tool.Additional file 6 : Figure S3. Median-joining haplotype networks for SNPs listed in Table Additional file 7 : Figure S4. Median-joining haplotype networks for selected genomic regions in Africans (YRI) and non-Africans (CEU + CHB). A). Five kb region encompassing INS rs3842753 . B). Another 5 kb region on the same chromosome 11 where ΔDAF = 0.012 between Africans and non-Africans. Both these genomic regions do not have any SNPs with ΔDAF ≥0.20.Additional file 8 : Figure S5. Haplotype network of a 14 kb region encompassing CHGB in Africans (YRI), East Asians (CHB) and Europeans (CEU).Additional file 9 : Figure S6. Haplotype network of a 1 kb region encompassing IGF2 in Africans (YRI), East Asians (CHB) and Europeans (CEU).Additional file 10 : Figure S7. Haplotype network of a 1 kb region encompassing INS in Africans (YRI), East Asians (CHB) and Europeans (CEU).Additional file 11 : Table S4. Analyzed populations and samples. Populations were split into two categories. Sample size indicates the number of individuals for each population.Additional file 12 : Table S5. LD patterns in Africans and non-Africans for highly differentiated SNPs listed in Table


Fecal microbial transplantation (FMT), a treatment for certain gastrointestinal conditions associated with dysbiosis in people, is also empirically employed in horses with colitis. This study used microbiota high-throughput sequencing to compare the fecal microbial profile of healthy horses to that of geriatric microbial transplant recipients experiencing diarrhea and tested whether FMT restores microbiota diversity.To evaluate the effect of environment and donor characteristics on the intestinal microbiota, fecal samples were collected per rectum from 15 healthy young-adult (2–12 years) and 15 geriatric (≥20 years) horses. Additionally, FMT was performed for 3 consecutive days in 5 geriatric horses with diarrhea using feces from the same healthy donor. Fecal samples were collected from both donor and recipient prior to each FMT and from recipients 24 hours following the last FMT. The profile of the fecal bacterial microbiota was compared using 16S amplicon sequencing.In contrast to diet and farm location, age did not significantly affect the healthy equine fecal microbiota, indicating that both healthy geriatric and young-adult horses may serve as FMT donors. The fecal microbiota of horses with diarrhea was significantly more variable in terms of β-diversity than that of healthy horses. An inverse correlation between diarrhea score and relative abundance of Verrucomicrobia was identified in surviving FMT recipients. At study completion, the fecal microbiota of horses which responded to FMT had a higher α-diversity than prior to treatment and was phylogenetically more similar to that of the donor. Intestinal microbiome imbalance, or dysbiosis, is a key factor in the development of diarrhea and equine colitis, which remains a leading cause of critical illness in horses, with an estimated disease fatality of 25.4% to 35%., 2 AntibClostridium difficile. A well-described clinical trial in humans with refractory, chronic C. difficile diarrhea showed over 85% effectiveness for one FMT, and over 90% effectiveness for two FMTs in comparison to standard treatment with vancomycin. A recent study in mice further demonstrated that autologous FMT significantly enhanced fecal and gastrointestinal microbiota reconstitution in mice following antimicrobial treatment.[It has recently been shown that the fecal bacterial microbiota of horses with colitis of various etiologies is less diverse than that of unaffected animals., 6 As wireatment. SimilarlA recent study showed that the outcome of colitis is less favorable in geriatric compared to younger horses. At the sAlthough recent studies have improved our understanding of the equine gut microbiota,, 16 littThis study was approved by the Cummings School of Veterinary Medicine Institutional Animal Care and Use Committee (#G2016-179) and Clinical Studies Review Committee (#161215) and met all requirements for ethical care and treatment of animals.Thirty clinically healthy horses of two age groups  were selected from 5 housing facilities located in Massachusetts and Connecticut, based on the availability of phenotypically matched young and aged horses at each location, exposure to a comparable diet, and similar management. The latter selection criteria were used to determine which management and phenotypic features may contribute to differences in the fecal microbiota of healthy horses and should thus be considered in selecting a donor for fecal microbial transplantation. Prior to fecal collection, a complete diet history, medical history, and physical examination were obtained to ensure clinical health. Exclusion criteria included any recent gastrointestinal illness , transport, medical treatment, or dietary supplementation with probiotics. Breed, age, body condition score, heart rate, respiratory rate, rectal temperature, attitude, and borborygmi were recorded prior to manure collection. Up to 10-mL of feces were collected per rectum using a clean gloved hand at two separate time-points 2 weeks apart and stored at -80°C for subsequent analysis. The experimental design and methods were approved by the Clinical Studies Review Committee and informed client consent was obtained for all horses.Clostridium difficile toxins A and B, Clostridium perfringens antigens, Lawsonia intracellularis, Neorickettsia risticii and Salmonella sp.  were confirmed to be negative for 3 control horses that served as FMT donors to clinically ill geriatric patients with diarrhea. Prior to each FMT on 3 consecutive days, a 10-mL fecal sample was obtained from the donor manure pile that was subsequently processed and used as transfaunate for a single patient. All donor samples obtained on days 1–3 of transfaunation were stored and sequenced individually, thus analyzing a total of 3 FMT samples per donor obtained on consecutive days and administered to a single recipient.A quantitative fecal egg count and Equine Diarrhea PCR panel for Coronavirus, Five geriatric horses hospitalized at Tufts Cummings School of Veterinary Medicine with diarrhea (pudding to watery consistency) served as fecal transplant recipients. Exclusion criteria included a history of gastrointestinal reflux within 3 days prior to enrollment in the study. The following clinical data were obtained: signalment; presenting complaint; predisposing factors such as antibiotic or non-steroidal anti-inflammatory therapy, recent long-distance travel, anesthesia, feed changes, or prior enteral treatment with mineral oil, surfactants, or cathartics; duration of diarrhea; development of complications such as laminitis or thrombophlebitis; length of hospitalization; and outcome. Frequency of diarrhea during hospitalization was recorded every 6 hours in clinical patients (colitis horses), and diarrhea quality described every 6 hours on a scale of 0–5 according to the following guidelines: 0—Normal, firm but moist balls of manure; 1 –Soft-formed balls of manure that lose their form upon reaching the ground; 2—Pudding-consistency manure that still holds some shape; 3—Pudding-consistency manure that spreads out upon reaching the ground; 4—Watery manure with some formed pieces; 5—Watery manure without formed pieces.Five client-owned geriatric horses (≥ 20 years old) that presented for colitis, or developed diarrhea in-hospital for any reason, received fecal microbial transplants from a single donor horse on 3 consecutive days. FMT was performed using standard clinical techniques. In short, 2.5 pounds of fresh manure was collected and mixed in 4 liters of lukewarm water within a bouffant cap  for 10 minutes. The mixture was subsequently strained and administered within 15 minutes of processing via nasogastric tube to the recipient horse. Horses were checked for reflux using no more than 2 L of water prior to administration of FMT, with <2 L net reflux considered acceptable. A 10 mL fecal sample was collected per rectum from each recipient prior to each FMT (days 1–3) and 24 hours following the last FMT (day 4) . All samClinical data are presented descriptively as mean +/- standard deviation (SD) or median +/- interquartile range (IQR) or range. Univariate statistical analyses were based on the normality of data distribution (Shapiro-Wilk test), employing independent samples T-test, Mann-Whitney U and Chi-Square analyses to compare data between geriatric and young-adult donor horses, with an accepted significance level of P<0.05. These numerical analyses were performed with the IBM SPSS Statistics 22 package.tucf.org) using the single-end 300-nucleotide strategy. To control for technical variation introduced during PCR, library preparation and sequencing, the 16S library included two replicates of a randomly selected sample. Replication involved the separate processing of 2 subsamples of a sample and tagging each amplicon with a different 6-nucleotide barcode.The procedures for DNA extraction, amplicon library construction and bioinformatics were previously described –19. Briemothur [mothur using program unifrac.weighted. The UniFrac distance ranges from 0 for identical samples to 1 for samples that are completely different and share no sequences or no taxa. GenAlEx [anosim was run in mothur using weighted UniFrac distance matrices as input. Operational Taxonomic Units (OTUs) were obtained using program cluster and the OptiClust clustering method [mothur. Constrained ordination analysis was performed in CANOCO [FASTQ formatted sequences were processed using programs found in mothur  essentiamothur . Brieflymothur  between  GenAlEx  was used GenAlEx  was usedg method . A distag method  was usedn CANOCO .Sequence data accession numbers. 16S sequence data were deposited in the Sequence Read Archive under study accession number PRJEB32490.Fecal samples were obtained from 15 paired young-adult (median age: 6.7 ± 3.7 years) and geriatric horses (median age: 22.6 ± 1.8 years) exposed to comparable management conditions, including 21 mares and 9 geldings. Aged and young-adult horses were matched based on phenotype, body condition score, sex and diet, except for one horse pair where a young gelding was matched with an aged mare . The mosClostridium perfringens antigen, all horses likely experienced undifferentiated colitis. Horse H did not have a diarrhea PCR panel performed but showed evidence of sand enteropathy based on abdominal radiographs and removal of enteric sand on rectal palpation.Five geriatric (≥ 20 years of age) client-owned horses, including 3 geldings and 2 mares, presented for colitis or developed diarrhea while hospitalized, prior to receiving 3 consecutive daily FMT from a single donor. Duration of hospitalization ranged between 5 to 8 days, with 3/5 horses surviving to discharge. The horses’ historical, clinical and hematologic characteristics are specified in The fecal microbiota of horses treated with FMT was highly heterogeneous. For 190 pairwise weighted UniFrac distance values between 20 samples (4 samples x 5 recipients), the mean was 0.73 (n = 20x19/2 = 190). This value compares to a mean pairwise distance between healthy horses of 0.44 (n = 2926). The comparison between healthy and diarrheic horses was statistically highly significant . As apparent in Due to the relatively large phylogenetic distances between the microbiota of healthy and of diarrheic horses, the datapoints from healthy horses in Trabulsiella. In contrast, W's microbiota was dominated by Bacteroidetes, mostly from the order Bacteroidales. Horse W was ultimately diagnosed with enterolithiasis and Horse F was treated with broad-spectrum antimicrobials throughout the study period  beginning 4 hours prior to first sampling and FMT. This horse also received one injection of oxytetracycline (7.7 mg/kg intravenously once) from the referring veterinarian the day prior to hospital admission. No other FMT recipients were exposed to systemic or enteral antimicrobials.Classified at phylum level, the microbiota taxonomies  revealedIn addition to the increasing Verrucomicrobia abundance mentioned above, two other changes were observed in the 3 horses  that experienced improving diarrhea scores during treatment and survived to discharge: increasing α-diversity and decreasing β-diversity to the donor’s microbiota. The changes in microbiota α-diversity during the 4 FMT days are illustrated in Horses which received FMT from donor horse To (F and W) were transplanted with a very similar microbiome . The relThe relative effect of donor and of day of transplant was further investigated by Canonical Correspondence Analysis using recipient and day, respectively, as independent variables. As apparent in Fecal Microbial Transplantation (FMT) was associated with improving diarrhea scores in a small group of surviving horses that presented for acute or progressive diarrhea to a referral center. More specifically, increasing abundance of Verrucomicrobia and increasing α-diversity was observed in treatment responders. The latter group established a microbiome that more closely resembled the donor horse following 3 consecutive daily fecal transplants. In this study, diminishing UniFrac distance over time between donor and FMT recipient was suggestive of transplanted bacteria colonizing the gastro-intestinal tract of the recipient horse and normalizing the intestinal microbiome. Re-establishing a healthy gut microbiome in horses with colitis is considered essential to digestion, is expected to act as a bulwark against pathogenic bacteria and may stimulate the development of a robust and effective immune system to counteract infection. Additionally, normal microbial inhabitants of the gut are thought to help maintain a balance between inflammatory and anti-inflammatory mediators in the intestinal tract, and may C. difficile infection (CDI) in humans.[Clostridium difficile infections in people,[The fecal microbiome of horses that survived to discharge (3/5) showed a significantly higher α-diversity (higher species richness) after FMT treatment and were considered treatment responders. Bacterial species richness and diversity are important elements of a healthy intestinal microbiome, and a reduction in richness and diversity has been associated with conditions such as chronic diarrhea and recurrent n humans., 33 Moren people,. While sn people, Thereforn people, and thatn people,. A recenn people, In contrn people, EmergingTrabulsiella), a feature that was not observed in the second non-responder (Horse W). Firmicutes were under-represented in W's microbiome and no increase in Verrucomicrobia abundance was observed in this horse following FMT  did not significantly affect the equine fecal microbiome in the population of healthy horses screened in this study. In contrast, previous reports have documented inconsistent effects of advancing age on the equine intestinal microbiome, with both inverse and direAs expected, we documented that diet affected the fecal microbiome of healthy donor horses. Healthy fecal donor samples were collected from 5 study locations in close geographical proximity. Since different locations also differed by diet, it is not feasible to fully assess the impact of diet by itself on the fecal microbiome. Study location “Uc” was the only site where horses did not have access to pasture. Consistent with a strong effect of diet on the equine fecal microbiome observed by others , microbiOverall, this preliminary work supports FMT as a mechanism for establishing a more diverse microbiome in horses with colitis and may thus represent a cost-effective therapy to facilitate restoration of gut function in horses with dysbiosis or colitis. As such, surviving horses that responded to FMT with diminishing diarrhea scores in this study were more likely to have improved alpha diversity with increasing abundance of Verrucomicrobia, and acquired a microbiome that began to resemble that of their donor horse. However, larger case-controlled studies are needed to ensure reliability of these results. The fecal microbiome assessment of both healthy geriatric and young-adult horses suggests that age-matching between donor and FMT recipients is not necessary to optimize microbial transplantation in adult horses, while diet-matching may be prudent.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S1 FigBlack, Location Bo; grey, Location RHF; brown, Location Uc; turquoise, Location Um. Circles, aged healthy horses; diamonds, young-adults.(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 FigColor indicates recipient as used in Figs (TIFF)Click here for additional data file.S4 FigLeft. FMT recipients are represented with red triangles, labelled as in Figs Right. FMT days are represented with red triangles. Each pie symbol represents an OTU. Pie segmentation represents the OTU’s relative abundance in samples collected on each FMT day, as indicated by white-to-black shading, where white is day 1, light grey day 2, dark grey day 3, and black day 4. The distances between a pie symbol and each triangle shows the relative abundance of that OTU on individual FMT days. In other words, the OTU is predicted to be most abundant in the FMT day located at the shortest distance on the plot. For additional explanations, see left panel legend.(TIFF)Click here for additional data file.


The impact of cardiovascular disease (CVD) comorbidity on resection rates and survival for patients with early-stage non-small-cell lung cancer (NSCLC) is unclear. We explored if CVD comorbidity explained surgical resection rate variation and the impact on survival if resection rates increased.Cancer registry data consisted of English patients diagnosed with NSCLC from 2012 to 2016. Linked hospital records identified CVD comorbidities. We investigated resection rate variation by geographical region using funnel plots; resection and death rates using time-to-event analysis. We modelled an increased propensity for resection in regions with the lowest resection rates and estimated survival change.Among 57,373 patients with Stage 1−3A NSCLC, resection rates varied considerably between regions. Patients with CVD comorbidity had lower resection rates and higher mortality rates. CVD comorbidity explained only 1.9% of the variation in resection rates. For every 100 CVD comorbid patients, increasing resection in regions with the lowest rates from 24 to 44% would result in 16 more patients resected and alive after 1 year and two fewer deaths overall.Variation in regional resection rate is not explained by CVD comorbidities. Increasing resection in patients with CVD comorbidity to the levels of the highest resecting region would increase 1-year survival. Curative surgical resection has the potential to lead to long-term cancer-free survival in patients with NSCLC3 leading to recent recommendations aiming to increase the proportion of the resectable patient population treated with surgery. However, lung resection is a major surgical procedure with significant peri-operative risks, which increase in patients with serious comorbidities, the most important of which are cardiovascular and lung disease.4 Clinical decision-making in NSCLC patients with cardiovascular disease (CVD) aims to find a balance between the risk of adverse operative outcomes and the potential prognostic gains of successful surgery. It is not known if current practice strikes the right operative balance and whether increasing lung resection in NSCLC patients with CVD has significant potential to improve survival rates.The impact of cardiovascular comorbidity on outcomes in patients with non-small-cell lung cancer (NSCLC) is critical to clinical decision-making but is complex and relatively poorly understood in a real-world context. Patients with NSCLC have a disproportionally high rate of cardiovascular comorbidities5 Data from the National Lung Cancer Audit and the English Cancer Registries showed wide variation in the proportion of patients undergoing surgical resection between hospital trusts and commissioning areas in England.7 Resection rates have increased significantly in England in recent years as has 1- and 5-year survival9 but variation still exists. Existing literature suggests further improvements in longer term survival if the highest current resection rates could be applied to all NSCLC patients in England.7 Very little is known about the extent these low and variable resection rates can be explained by differences in the rates of significant comorbidities. Two studies comparing regional populations in the North East of England and Italy have demonstrated that lower resection rates in England can be explained, at least in part, by higher rates of comorbidities.11Historically, survival rates from lung cancer have been poor in the UK and surgical resection rates have been low in comparison to many other western countries.As part of the Virtual Cardio-Oncology Research Institute (VICORI), we used data from the Public Health England National Cancer Registration and Analysis Service (NCRAS) linked with Hospital Episode Statistics (HES) to identify CVD comorbidity recorded in hospital records before a diagnosis of NSCLC. We aimed first to investigate to what extent CVD comorbidities explain geographical variation in surgical resection rates; second, how CVD comorbidity affects operative outcome and third, how CVD comorbidity affects survival in both the operated and non-operated groups using a multi-state model. This sophisticated statistical framework allows us to accurately model the whole patient care pathway and has not previously been applied in this context. Using this model, we can control for case-mix factors between geographical regions  and model the effect of standardising regional resection rates to the equivalent of the highest fifth of current practice.n = 57,373).We extracted data from 187,432 patients with 189,310 lung cancer tumours (ICD10 C33-C34) diagnosed between 2012 and 2016 from NCRAS linked to HES. Following exclusions  were not considered as resections in the analysis. Due to imprecision of some cancer diagnosis recording, any resections up to 30 days before the recorded diagnosis date were recoded to occur at the cancer diagnosis date . Length of stay in hospital post-resection and re-admission within 30 days of resection were identified in relation to admission and discharge dates for the hospital admission associated with resection. The length of time from NSCLC diagnosis to death or censoring was calculated using vital status information from NCRAS, with mortality follow-up until 28 January 2018.Patients who underwent surgical resection with curative intent were identified from HES OPCS-4  codes recorded between 30 days before and 365 days after cancer diagnosis (full list of codes in Supplementary Table CVD procedures prior to cancer diagnosis were identified from HES records (available from 1 January 2000) based on OPCS-4 procedure “K” codes and further subdivided into coronary artery bypass graft (CABG), percutaneous coronary interventions (PCI), valve surgery, diagnostic procedures and other CVD procedures , which were categorised into eight groups  any CABG, (2) any PCI, (3) any valve surgery, (4) only diagnostic procedures, (5) only other CVD procedures, (6) CABG and PCI  patients only), (7) CABG and PCI (in non-AMI patients only), (8) CABG (in AMI patients) and (9) PCI (in AMI patients). We also investigated the effect of type of CVD diagnosis by defining the following exposure variables: (1) AMI, (2) ischaemic heart disease (not AMI), (3) congestive heart failure, (4) peripheral artery disease, (5) cerebrovascular disease, (6) stroke, (7) valvular heart disease, (8) any other CVD diagnosis and (9) hypertension only. Each exposure category was compared against no CVD procedure or diagnosis codes.https://www.gov.uk/government/statistics/english-indices-of-deprivation-2015), which measures area-level deprivation. We identified geographical regions each patient was resident in at the time of NSCLC diagnosis using Clinical Commissioning Groups, which are clinically led statutory NHS bodies responsible for the planning and commissioning of healthcare services for their local area. We calculated the proportion of Stage 1−3A lung cancer patients resident in each region who underwent resection and obtained resection rate quintiles. Each region, and hence each patient within that region, was assigned one of these fifths. Regional resection quintiles were re-calculated based on the subgroup of patients with CVD comorbidity (prior procedure or diagnosis). We extracted cancer stage, morphology and the Charlson Comorbidity Index (CCI)12 derived using comorbidities recorded in HES 27 to 3 months prior to NSCLC diagnosis. We derived a modified CCI, which excluded CVD diagnosis codes, to avoid counting them in both the CVD exposure and the CCI (Supplementary Table Socio-economic status was measured using the full Index of Multiple Deprivation (IMD) 2015 score , year of diagnosis , IMD and the modified CCI. Non-linear effects of age-at-diagnosis were modelled using a restricted cubic spline function with three knots.Funnel plots were used to investigate variations in regional resection rates by plotting standardised resection ratios (SRRs), calculated by dividing the observed number of resections in each region by the predicted number of resections, obtained from a logistic regression model.14) to separately investigate the association between CVD comorbidity and three outcomes: (1) time from NSCLC diagnosis to resection, (2) time from NSCLC diagnosis to death prior to or without resection, and (3) time from resection to post-resection death. Time-to-event analyses were used to account for competing risks of the three outcomes  and compared to marginal estimates if they had resection rates of regions with the highest rates of resection (i.e. the highest fifth), given the patient mix (including CVD comorbidities) of these patients remained unchanged. We repeated this analysis restricted to patients with a CVD procedure or diagnosis, recalculating the regional resection quintiles for this population. Further details are given in Supplementary Appendix The fitted flexible parametric models were used within a multi-state framework  only including NSCLC patients with cancer stages 1 or 2.n = 77,200; 47.9%), followed by ischaemic heart disease and peripheral artery disease  had a CVD procedure  prior to diagnosis, 70,224 (43.6%) had no CVD procedure but had a CVD diagnosis (excluding patients with only hypertension), 23,201 (14.4%) had only hypertension or a non-interventional procedure code and 56,776 (35.2%) had no CVD procedure or diagnosis codes recorded. The most prevalent CVD diagnosis code was hypertension (p < 0.001).Amongst the 57,373 patients with cancer stages 1, 2 or 3A, patients with CVD comorbidity were on average older compared to those without CVD and were more likely to be male, have a more recent NSCLC diagnosis, belong to a higher category of multiple deprivation and have a clinical diagnosis of lung cancer  (Supplementary Table p < 0.001 for each cancer stage). In the group of patients with stage 1 disease and no recorded CVD or procedures (n = 7565), the resection rate was 62.8% compared with only 44.4% in those patients with stage 1 disease who had a CVD procedure (n = 2194). Patients with a CVD procedure were, however, more likely to have stage 1 lung cancer compared with patients with no CVD; 2194/11,030 (21.7%) of patients with a CVD procedure compared with 7565/56,776 (14.4%) of patients with no CVD.The resection rate was 15.2% across all patients and 39.0% amongst patients with clinical stages 1, 2 or 3A. The majority of patients presented with late stage disease (55.1% stage 3B or 4 at diagnosis and 9.3% missing stage information) (Table R2 = 1.9%).17 In comparison, age explained 10.2% of the variation, cancer stage 9.1%, sex 0.1%, and CCI < 0.1%.Resection rates for cancer stages 1−3A varied considerably between geographical regions (from 20.6 to 64.6%) with variation between regions greater than that expected due to chance (10/209 (4.8%) regions had observed resection rates outside of the 99.8% confidence bands). Adjusting for case-mix, including CVD comorbidity, did not substantially reduce the variation . We did not find a significant difference between resection rates for NSCLC patients who had CABG and those who had valve surgery . Patients diagnosed with congestive cardiac failure had the lowest rate of resection compared to other CVD diagnoses  and 0.66 , respectively). After adjusting for potential case-mix , the hazard ratio for CVD procedure attenuated to 0.82  and for CVD diagnosis to 0.82  Fig. . ResectiThe mortality rate was higher in patients with CVD procedures or diagnoses compared to patients with no CVD, both prior to or without resection  , due to a more favourable case-mix, with a mean restricted survival (at 5 years) of 2.65 years  increase early diagnosis of NSCLC; secondly, that CVD comorbidities reduce the likelihood of lung cancer resection; thirdly, CVD comorbidities worsen survival prognosis in both resected and non-resected populations and finally that increasing resection rates in patients with CVD comorbidities may offer small but potentially important improvements in overall survival with a sizeable increase in patients treated with surgery with curative intent. In absolute terms, for CVD comorbid patients with NSCLC, for every ten additional resections, one death could be avoided by 5 years.18 Studies of NSCLC in England report variable lung cancer surgical resection rates by primary care trust (PCT)7 and treating surgical unit.6 Our study confirmed wide variation by area of residence. Whilst previous work showed that this variation is not fully explained by overall rates of comorbidity , this study extends these findings to investigate the specific impact of rates of CVD comorbidity. We have demonstrated that CVD comorbidity explains 1.9% of the overall variation in resection rate and geographical variation is not well explained by variation in incidence of CVD comorbidities. We observed that age was the biggest contributor to geographical variation in resection rates. Age is a non-modifiable risk factor, but we know that high volume thoracic surgical centres treat more older patients than the low volume ones,7 so the clinical management of older patients is certainly modifiable.Variation in other aspects of the treatment of lung cancer patients has been well documented.19 60% in the Netherlands20 and 67% in Italy.10 These studies suggest possible reasons for lower resection rates in the UK may be due to fewer multidisciplinary teams, less frequent imaging and lower diagnosis volume. These suggestions agree with recommendations from a report to improve care for lung cancer patients in the UK.21 However, even though the study from the Netherlands found higher resection rates for hospitals with a teaching status for thoracic surgeons and high diagnostic volume, hospital teaching status and volume did not explain variation in resection rates between hospitals.20We found resection rate of 39% in England for patients with stages 1, 2 or 3A NSCLC from 2012 to 2016, which is an increase in resection rates compared to studies using earlier data, but still low compared to some other European studies. For stages 1 and 2 NSCLC, resection rates were 27% in Sweden,10We observed an inverse association between CVD comorbidity and cancer stage. Patients with CVD comorbidity were more likely to have a lower cancer stage compared to patients with no CVD comorbidity. Diagnosis earlier in the natural history of the cancer is likely to be a result of more intensive hospital investigations relating to the CVD comorbidity, resulting in earlier, incidental detection of the tumour, which may increase survival.Patients who had received a CABG had a lower rate of resection compared to PCI patients, possibly because CABG patients generally have more advanced/complex coronary artery disease than PCI patients. Another concern might be the technical challenges of lung resection post CABG .22 which agrees with our study. However, using the multi-state framework to model the patient care pathway allowed us not only to report survival for resected and non-resected patients, but also investigate how increasing resection rates affects survival. A strength of this study is we were able to analyse large-scale national linked cancer and hospital episode data within a multi-state modelling framework to estimate outcomes in the presence of competing risks. Marginal effects associated with increasing resection rates in different populations were estimated by “moving” patients from one regional fifth to another and using standardisation to control for other modelled patient factors. An assumption of this approach is that effects are exchangeable when moving patients from one regional fifth to another, conditional on their covariate pattern.A recent study investigated the association between age, deprivation and CVD comorbid conditions and NSCLC resection in England and found strong evidence that comorbidities reduced the receipt of surgery in early-stage patients,22 Second, smoking status is unavailable, which is likely to be associated with CVD comorbidity (particularly COPD) and resection rate, but IMD may be a suitable proxy for some health and lifestyle choices. Third, we could only identify CVD comorbidity for NSCLC patients with a previous hospital admission because we used the in-patient HES dataset, extracted directly from individual hospital patient administration systems, and missed patients who never had a hospital admission over the time period of our study. The results from previous work, validating the use of HES to identify patients with myocardial infarction,23 suggest we potentially missed some CVD events. The only other potential source to identify these missing patients would be the out-patient HES dataset, which we did not use because it is known to have limited diagnoses data. However, we did use all the in-patient HES records available to us to maximise the chance of capturing every CVD event. We excluded CVD events at the time of cancer diagnosis to ensure CVD was before cancer diagnosis and not a CVD event caused by cancer treatment (this approach was used by other studies22). Another limitation of using HES to identify CVD comorbidity is we were not able to obtain details of severity. Fourth, we were not able to consider other potentially curative treatments , which may be more suitable for patients with early-stage NSCLC than resection. However, a recent study24 examined variation in resection rates and radical radiotherapy rates for English NSCLC patients using NCRAS did not find an association between resection rates and radical radiotherapy rates, suggesting areas with low resection rates do not necessarily have high radical radiotherapy rates. Finally, NSCLC patients with cancer stage 3A may be a heterogeneous group because the standard of care is not consistent in England, but repeating the analysis excluding patients with cancer stage 3A did not alter the interpretation of the results.There are some limitations of this study. First, we were unable to include WHO performance status classification due to high proportion of patients with missing values, which has been shown to be associated with resection rates.In conclusion, this study found CVD comorbidity did not substantially explain resection rate variation and a small but significant increase in survival was predicted when we modelled an increased propensity for resection in CVD comorbid patients in regions with lowest resection rates. Our findings suggest that decision-making about resection in CVD comorbid patients is overly conservative and that increasing resection rates in CVD comorbid patients, at least to the level currently seen in those regions in the top fifth for resections in this population, will improve population-level survival. Further work is required to understand the reasons for variation in cancer treatment and how best to ensure that all patients have access to optimal care, which may be treatments other than resection.Supplement


The adjusted regression analyses evidenced in AD patients an association between plasma and CSF sTREM2 levels , as well as β-amyloid concentrations  and an association between CSF sTREM2 and phospho-Tau concentrations . No significant relation was found in iNPH patients. In conclusion, these differences in sTREM2 profiles between AD and iNPH reinforce the notion that this receptor has a role in neurodegeneration.Microglia performs a variety of functions during brain development designed to maintain brain homeostasis. Triggering receptor expressed on myeloid cells 2 (TREM2) is expressed in microglial cells modulating phagocytosis, cytokine production, cell proliferation, and cell survival. Interestingly, the levels of soluble TREM2  were higher in cerebrospinal fluid (CSF) from Alzheimer's disease (AD) patients than subjects without cognitive decline. It is noteworthy that, while CSF sTREM2 levels have been extensively studied, few studies have investigated sTREM2 in blood producing conflicting results. We aimed to investigate the levels of sTREM2 in CSF and blood from a cohort of well-characterized AD comparing the results to those obtained in patients suffering from idiopathic normal pressure hydrocephalus (iNPH), a potentially reversible cognitive impairment. Our findings underlined a significantly lower plasma sTREM2 concentration in AD patients compared to iNPH subjects [39.1 ng/mL (standard deviation (SD), 15.0) and 47.2 ng/mL , respectively;  Microglial cells are the resident immune cellular population of the central nervous system (CNS) , neuroinflammation exerts a key role in the disease progression. It has been demonstrated that β-amyloid (Aβ) can activate microglia cells and initiate the chronic inflammatory response  responsible for neurodegeneration  to inflammation, leading to an increased release of proinflammatory cytokines. In other words, the progressive decline of brain TREM2 expression can contribute to switching the microglia phenotype into a neurodegenerative pattern  has been found in cerebrospinal fluid (CSF) from AD patients, where it exerts a proinflammatory action  study the concentrations of sTREM2 in CSF and blood from a cohort of well-characterized persons with AD, (ii) correlate the concentrations with CSF markers of AD progression, and (iii) compare the results shown in the AD group to those obtained in a cohort of patients suffering from a potentially reversible cognitive impairment  . Thirty-three individuals without cognitive decline (CT), age-, and sex-matched with the AD group, were selected to compare the plasmatic concentrations of sTREM2.The diagnosis of AD was made according to the criteria proposed by Dubois et al. . In contThe study protocol received approval from the local ethical committee. All subjects gave their informed consent to participate in the study.1−42, Tau, and phospho-Tau (pTau) were assessed using the commercially available enzyme-linked immunosorbent assay (ELISA) kits . The CSF and plasma concentrations of sTREM2 were measured using the Human TREM2 ELISA kit  according to the manufacturer's instructions. The apolipoprotein E (APOE) genotype was determined as previously described . A p < 0.05 was considered as the threshold for statistical significance.SPSS statistical package was used to conduct the statistical analyses . All the variables were normally distributed. Results are expressed as mean [standard deviation (SD)] and/or percentage. Student's p = 0.09) (The AD patients showed significantly different CSF biomarkers concentrations compared to iNPH subjects . Interes = 0.09) .AD patients showed negative correlations of age with CSF Tau and pTau, whereas a positive correlation was reported between age and plasma sTREM2 concentrations . In the 1−42 concentrations, whereas no significant correlation was highlighted in iNPH subjects , as well as Aβ1−42 concentrations . Moreover, regression analysis highlighted an association between CSF sTREM2 and pTau concentrations . No significant relation was found in iNPH patients.The regression analyses adjusted by age, sex, and presence of the ε4 allele of the ApoE gene confirmed in AD patients the association between plasma sTREM2 levels and CSF levels (1−42 concentrations (differently from previous reports)  (Ma et al., TREM2 has a role in protecting against neuroinflammation by the inhibition of the persistent activation of microglia, promoting phagocytosis, and clearing apoptotic neurons (Colonna and Wang, An exciting aspect of TREM2 resides in releasing its soluble variant in various human biofluids, such as CSF and blood (Bekris et al., Existing studies report no significant difference in the plasma levels of sTREM2 between AD cases and healthy controls (Piccio et al., Evidence suggests that sTREM2 is able to prevent apoptosis in macrophages (Wu et al., Under these premises, in this study, we compared two neurological diseases, both characterized by cognitive impairment and difficult to differentiate from each other without diagnostic insights (Jingami et al., The results of our study highlighted lower plasmatic levels of sTREM2 in patients with AD compared to those with iNPH. Interestingly, we did not find substantial differences comparing the sTREM2 plasma levels between iNPH and control subjects characterized by normal cognitive functions. These results suggest that plasmatic sTREM2 may be a useful marker to identify cognitive impairment due to neurodegenerative processes. Moreover, the comparable results obtained in iNPH and CT subjects confirmed our previous findings showing blood and CSF protein profiles similar in these two groups (Fania et al., Regarding CSF, our results did not show a difference in sTREM2 concentrations between overt AD and iNPH patients, as already reported in studies that compare AD and healthy controls (Henjum et al., Moreover, our study results confirmed the positive association between CSF sTREM2 and plasma sTREM2 in AD patients (Bekris et al., 1−42 concentrations in AD patients, suggesting that sTREM2 might play a role in the pathological processes occurring after Aβ1−42 accumulation (Zhong and Chen, The regression analyses highlighted an association between CSF sTREM2 with pTau, but not with Aβ1−42 in AD. Although the function of TREM2 in AD pathology is partly unclear, it is hypothesized a role of TREM2 in clearing the soluble Aβ aggregates and other toxic debris and controlling the inflammatory reactions elicited by the AD pathology (Hsieh et al., 1−42 CSF concentrations.Interestingly, despite the fact that a recent study stated that plasma sTREM2 did not significantly correlate with CSF AD-related biomarkers (Bekris et al., However, we could not exclude the fact that aging, the most significant risk factor for AD, may be in part responsible for the sTREM2 alterations that emerged from our results. Indeed, aging is associated with gliosis, increased microglial activity (Heneka et al., In our cohort of subjects, plasma sTREM2 concentrations strongly correlated with age in AD patients and slightly in the CT group, whereas there was no correlation in iNPH subjects. Despite these assumptions, the association between CSF sTREM2 and plasma sTREM2 levels was confirmed in the AD group even after adjusting for age and sex, further supporting a role of TREM2 in the pathobiology of AD.There are limitations to this study. First, the number of the study participants is limited, and thus these findings are preliminary and need to be further investigated in large cohorts of patients. Another limitation is the CSF unavailability for subjects without cognitive decline. Indeed, we were not able to collect CSF from these subjects for ethical reasons, contrarily to AD and iNPH subjects for which CSF has been collected for diagnostic purposes (Dubois et al., In conclusion, we demonstrate that plasma levels of sTREM2 are decreased in AD patients, but not in iNPH and subjects without cognitive decline. Moreover, only in the AD group, we observe a significant association between CSF sTREM2 and plasma sTREM2 levels, not evidenced in iNPH subjects. These significant differences in sTREM2 profiles between AD and iNPH reinforce the notion that this receptor has a role in neurodegeneration.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico. The patients/participants provided their written informed consent to participate in this study.BA conceived and designed the study. BA and EF analyzed the data, interpreted the data, and results. BA, EF, and MC drafted the manuscript. MC provided critical revision. PDR and SC performed clinical assessment. AG performed biochemical analyses. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Zea mays (L.) (Poaceae). We conducted a genome wide association study (GWAS) of 146 maize genotypes comprising of landraces, inbred lines and commercial hybrids. Plants were phenotyped in bioassays measuring parasitic wasp Cotesia sesamiae (Cameron) (Hymenoptera: Braconidae) attraction to volatiles collected from plants exposed to stemborer Chilo partellus (Swinhoe) (Lepidoptera: Crambidae) eggs. Genotyping-by-sequencing was used to generate maize germplasm SNP data for GWAS. The egg-induced parasitoid attraction trait was more common in landraces than in improved inbred lines and hybrids. GWAS identified 101 marker-trait associations (MTAs), some of which were adjacent to genes involved in the JA-defence pathway , terpene biosynthesis , benzoxazinone synthesis  and known resistance genes . Intriguingly, there was also association with a transmembrane protein kinase that may function as a receptor for the egg elicitor and other genes implicated in early plant defence signalling. We report maize genomic regions associated with indirect defence and provide a valuable resource for future studies of tritrophic interactions in maize. The markers identified may facilitate selection of indirect defence by maize breeders.Tritrophic interactions allow plants to recruit natural enemies for protection against herbivory. Here we investigated genetic variability in induced responses to stemborer egg-laying in maize  These defences are wide ranging and can include physical barriers such as lignin, anti-nutritive substances like tannin, production of antibiotics and interactions with associated organisms, which function as natural enemies of the herbivores4. Some defences are constitutively expressed, whereas others are induced after exposure to herbivore attack, when elicitors from the insect saliva or insect eggs trigger defence responses7. When plants are exposed to herbivory, they emit herbivore induced plant volatiles (HIPVs)10. Plants tend to emit larger amounts of volatiles than the insect that is feeding on it and parasitoids and predators have evolved to use HIPVs as cues to locate the herbivores they use as hosts or prey12. Plant defence involving interaction with the third trophic level is referred to as “indirect defence”, in contrast to direct defences that make the plant less suitable for the herbivore10.Plants, being rooted to the ground and unable to flee from attack, have evolved highly sophisticated ways of defending themselves from insect herbivores, over a 400-million-year period of plant-insect interaction and coevolution15. These crop plants are vulnerable to attack by adapted insect herbivores, which become pests in this context16. Modern maize (corn), Zea mays (L.) (Poaceae), was domesticated approximately 9,000 years ago from wild teosinte species17. Maize is an essential staple and cash crop for millions of people, particularly in sub-Saharan Africa (SSA). Maize production remains severely constrained by Lepidopteran stemborers, devastating pests of staple cereals in SSA, which reduce yields by up to 80%, depending on the pest population density and the phenological stage of the crop at infestation18. Stemborers infest about 50% of the agricultural land in the SSA region, affect the lives of nearly 300 million people and cause yield losses of approximately US$ 1.5 billion per annum18.While wild plants in natural ecosystems are under natural selection for the ability to defend themselves, domesticated crops have been subjected to artificial selection and are often grown as monocrops19. Conventional breeding for host plant resistance against attacking insect pests has largely been done by trial and error or by exposing different genetic lines of crops to the herbivore pests. Potential interactions between different crop genotypes and the natural enemies of the pests have received less attention. We hypothesised that locally adapted varieties (landraces) preferred by smallholder farmers who cannot afford pesticides might have better indirect defence than commercially bred genotypes. Evidence in support of this hypothesis was provided when we discovered that three farmer-selected landraces of maize emitted HIPVs in response to stemborer, Chilo partellus (Swinhoe) (Lepidoptera: Crambidae), egg laying whereas the two commercial varieties initially tested did not7. The HIPVs emitted by the landraces attracted Cotesia sesamiae (Cameron) (Hymenoptera: Braconidae) parasitoid wasps, which are a key natural enemy of the stemborer. These landraces were of South American origin and further studies provided evidence that the egg induced indirect defence trait also exists in some African maize landraces20 and in wild teosinte species21.Nearly all recent commercial maize breeding  has been undertaken in a pesticide treated backgroundC. sesamiae parasitic wasp bodyguards after egg deposition by C. partellus moths. Our objectives were 1. to determine how widespread this trait was in a wider germplasm collection comprising locally adapted landraces, improved breeding lines and higher yielding commercial varieties, and 2. to develop molecular markers for the indirect plant defence trait. We used GWAS to discover genomic regions and molecular markers associated with it. To the best of our knowledge, this is the first GWAS of parasitoid response to plants induced with insect eggs.Given these promising initial findings, we embarked on a much larger genome wide association study (GWAS), involving 146 maize genotypes, reported here. The plant trait (phenotype) studied was the ability to “cry for help” by emitting HIPVs to attract C. sesamiae parasitoid wasps to these volatiles were measured in an olfactometer bioassay, with at least 9 parasitoid wasp bioassays per genotype. DNA samples were collected from the same plants (see below). Olfactometer bioassay data are shown in Supplementary Table A diverse collection of 146 genotypes was tested  revealed significant differences in parasitoid wasp responses (mentclass2pt{minimmentclass2pt{minimGenotyping-by-sequencing (GBS) data were generated from 1018 individual maize plants (4-6 replicates per genotype), which were representative of 146 diverse accessions  is shown in Fig. A total of 101 significant SNP-trait associations were identified (Supplementary Table https://www.maizegdb.org/gbrowse). These genes have previously been annotated with a plant defence function. The phenotypic variation (cdpk13 gene) to 9.8 mb (bx7 gene). Detailed information about candidate genes and their roles in plant defence is given in Supplementary Table We retrieved 33 candidate genes .Multitrophic interactions with natural enemies of herbivores allow plants to increase herbivore mortality by recruitment of “bodyguards” after changing their volatile emission profile to become more attractive to the natural enemies20revealed the suite of plant volatiles induced by C. partellus eggs in maize. Thus, identification of the HIPVs was not the focus of the current study. Volatile samples in the current study were analysed by gas chromatography (data not shown) and similar key compounds, in particular (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), were induced. Here we aimed to identify molecular markers for parasitoid attraction and obtain insight into adjacent potential candidate genes underpinning this indirect defence trait. Availability of molecular markers, provided in the current study, could facilitate accelerated breeding for improved maize cultivars with the indirect defence trait through marker assisted selection (MAS). We used the parasitoid bioassay response itself to directly measure parasitoid attraction, rather than use a proxy in terms of HIPVs. Our study used a biodiverse collection of maize genotypes, which were exposed to C. partellus eggs prior to volatile collection. Volatile samples were then used in large scale parasitoid bioassays, for all 146 genotypes, in a choice test, testing if volatiles from egg exposed plants were significantly preferred to volatiles from unexposed control plants. We found the indirect defence trait was more widespread in landrace germplasm (40% of genotypes) but, because these were not genetically uniform, considerable variation between individual plants was observed. The trait was found in 25% of inbred lines and in 13% of hybrids. These lines were more consistent because they were genetically uniform. Furthermore, discovering the indirect defence trait in improved lines opens up the prospect of introgressing the trait into other higher yielding maize cultivars with desirable agronomic characteristics.Our earlier studiestps2, tps3, tps4, tps5, tps7, tps9 and tps10). This is not surprising because the indirect defence trait operates by emission of volatiles. Previous studies have linked terpene synthases to indirect defence24. Genes implicated in DMNT emission, induced by a synthetic jasmonic acid (JA) analogue, were investigated in an earlier GWAS by Richter et al.24 who found a strong association with tps2. Farnesyl diphosphate synthase3 (fps3) is another candidate gene and catalyses biosynthesis of precursor molecules for terpene biosynthesis. Several of our other candidate genes are implicated in plant secondary metabolism. The most notable of these are 12-oxo-phytodienoic acid reductase7 (opr7), allene oxide synthesis1 (aos1) allene oxide synthesis2 (aos2) and allene oxide synthesis3 (aos3) which encode key enzymes in the JA-defence pathway26. Another candidate gene that potentially plays a role is methionine S-methyltransferase1 (mmt1) as methyltransferases can be involved in plant volatile biosysnthesis27.Data were subjected to a GWAS analysis which revealed 101 SNPs strongly associated with the trait. Within a 10mb region of the genome next to these SNPs, there were 33 candidate genes that may code for the trait. Of these, 7 are terpene synthase genes  which could play a role in interactions with eggs that contain chitin.To trigger the plant defence cascade culminating in release of herbivore induced volatiles, the plant needs to detect the presence of the insect eggs through molecular recognition of the egg elicitor. A putative receptor gene, GRMZM2G438840, is strongly associated with the trait. It is annotated as a leucine-rich repeat transmembrane protein kinase family protein and was identified bycalcium dependent protein kinase13 (cdpk13) has been shown to be a component of touch- and wound-induced pathways involved in early stages of local and systemic responses in maize30. Calcium-dependent protein kinases (CDPKs) play a vital role in stress signalling by detecting increases in 31. We also found a mitogen-activated protein kinase, MAP kinase15 (mpk15), associated with the indirect defence trait. Reducing the function of MAP kinases has been reported to impair the synthesis of secondary stress signals, including JA, and loss of MAPK function results in reduced resistance of plants to herbivorous insects32. It thus seems plausible that cdpk13 and/or mpk15 play a role in egg-induced signal transduction. We also found two pectin methylesterases (PMEs) - pectin methylesterase1 (pme1) and pectin methylesterase31 (pme31). These are noteworthy because PMEs are involved in cell wall modification and pectin catabolic processes33.Two of our candidate genes are implicated in early plant defence signalling: maize insect resistance1 (mir1) and maize insect resistance2 (mir2) which encode cysteine proteinases (key defensive proteins against chewing insect pests in maize)34; benzoxazinone synthesis7 (bx7) and benzoxazinone synthesis9 (bx9), genes for benzoxazinoid biosynthesis35, and brown midrib 2 (bm2) which is associated with lignin synthesis36, a physical defence. Although we used an unbiased approach in selecting SNPs via the GWAS procedure, the selection of candidate genes was limited by searching for genes annotated with defence functions and it is likely that more genes are known for direct defence than are currently known for indirect defence. Another explanation is that plants that can recognise insect eggs have a suite of defences that are triggered upon detection of eggs which include direct as well as indirect defences. It is possible that genes encoding direct and indirect defences could cluster together in the genome but this would require further study.Interestingly, some of our candidate genes are associated with direct defence. These include C. partellus egg elicitor is detected; secondly, the JA-defence pathway is triggered, and, thirdly, JA-associated defences, including HIPV emission are triggered. Thus, the egg sensitive genotypes elicit a suite of defences following stemborer oviposition that comprise both direct and indirect defences which will protect the plant against caterpillars emerging from the eggs.Given the above candidate genes, we would like to suggest a hypothetical model by which the egg sensitive maize genotypes respond: Firstly, there is a molecular recognition process by which the 37, and biosynthetically related to the egg elicitor . Thus, we hope our dataset will allow identification of novel genes involved in indirect defence signalling between maize plants provoked by herbivore (C. partellus) eggs and parasitoid wasp “bodyguards” that have not previously been annotated as having roles in plant defence. We provide information . For example, the CIMMYT ESA hybrid maize breeding program is mainly based on four parental lines 40. We found that CML312 and CML442 possess the egg-induced parasitoid attraction trait, whereas CML395 and CML444 do not. Therefore our study identifies germplasm that could be used to introgress the trait into improved crops. Such crops would be more resilient to insect attack, difficult for insect to develop resistance and less dependent on pesticide application. They would, however, require natural enemies of pests to be present in the agricultural ecosystem as an ecosystem service. A recent meta-analysis41 found that “top-down” control of herbivorous insect populations by natural enemies is at least as important as “bottom-up” control by the plant and, thus, breeding crops for increased tritophic interaction with natural enemies42 could be a promising approach. Future work should investigate if the genetics identified in the current study with C. partellus stemborers is also involved in indirect defence against a new threat to maize in Africa—the invasive fall armyworm, Spodoptera frugiperda.There are global pressures to reduce pesticide use in agriculture and in any case few African smallholder farmers in the study region have access to pesticides. The current findings will help develop improved maize varieties with indirect defence against stemborers because we have already identified improved lines and hybrids possessing the trait. Preliminary field trials indicate an increase in parasitism of maize stemborers in genotypes with the indirect defence trait. The indirect defence trait was rarer in improved lines than in landraces, perhaps because selection for yield and quality in commercial crop breeding environments could have compromised defence traits because the value of any defence traits would not be realised when plants were treated with insecticideicipe-Thomas Odhiambo campus (ITOC), Mbita Point , western Kenya. All plants were grown under natural conditions .A diverse collection of 146 maize genotypes comprising 9 landraces, 116 inbred lines and 21 commercial hybrid varieties were obtained from local farmers (farmer preferred landraces), Maseno University (Kenya), the International Maize and Wheat Improvement Center  and commercial seed suppliers (Table C. partellus were reared on a semi-synthetic diet containing sorghum (Sorghum bicolor) leaf powder43. The larval parasitoid C. sesamiae was reared on stemborer larvae using methodologies described previously44. Experimental insects were maintained at the insect mass rearing unit of icipe-Thomas Odhiambo campus  through the inlet port. Volatiles were collected on Porapak Q  filters inserted in the outlet port through which air was drawn at 300 mL min−1. After entrainment, volatiles were eluted with 0.5 mL dichloromethane (Sigma Aldrich) for use in subsequent bioassays. Volatiles were collected from 1,168 plants representing 146 genotypes.Volatile compounds from whole maize plants, with and without stemborer eggs, were collected by headspace sampling7. Air was drawn through the four arms towards the centre at 260 mL min−1. Headspace samples  were applied, using a micropipette , to a piece of filter paper (4 × 25 mm) subsequently placed in an inlet port at the end of each Olfactometer arm. Mated female parasitoids, without previous exposure to plants or hosts, were transferred individually into the central chamber of the Olfactometer using a custom-made piece of glass tubing. Time spent in each olfactometer arm was recorded with “Olfa” software  for 12 min.To phenotype the egg-induced indirect defence trait, behavioural responses of parasitoids to volatiles from different maize genotypes were tested in a Perspex four-arm olfactometer Fig.  describe7. The remaining two arms were solvent controls. For each plant, we calculated the average proportion of time spent by the parasitoid in each olfactometer arm across all replications and compared the means using analysis of variance (ANOVA). The means from the two arms representing the solvent controls were analysed together. Comparisons were made: 1. between time spent in arms containing volatiles from solvent control and from a plant with eggs, and 2. between time spent in arms containing volatiles from “with eggs” and “without eggs” plants. Significant observations were determined using P10 transforming the data. These attraction index values were used to draw normal distribution curves using the ggplot2 package in R studio (Version 1.1.383)  that had been collected from plants that had stemborer eggs and those without the eggs .Fresh leaf samples were collected from assayed plants, immersed in liquid nitrogen, and crushed into fine powder using mortar and pestle. DNA was extracted from 1018 plants   of the maize reference genome, ZmB73 RefGen v2 (https://www.maizegdb.org/gbrowse). The selection of these candidate genes was limited by searching for genes annotated with defence functions. In addition, we selected, regardless of any existing annotation, the top 16 SNPs that had an A filtered SNP dataset was used for all molecular analysis in this study. Genetic fidelity was confirmed with identity-by-state distance matrix in Tassel 5.0. We used the filtered SNP data set to generate a Neighbor-Joining cladogram and estimated principal component analysis (PCA) with covariance and five components. The population structure of the genotyped plants was determined using the admixture model with correlated allele frequencies. The estimated proportions of each individual’s genome originating from each of the K ancestral populations (q) was calculated for K ranging from 1 to 10 ancestral populations (or clusters), with 10 runs for each K value. The structure harvester program was used to estimate optimum K value from admixture analysis resultsSupplementary file1 (DOCX 2626 kb)Supplementary file2 (DOCX 137 kb)


Vitamin D deficiency is prevalent in all ages regardless of climate or geographical location and evidence is emerging that the incidence of autoimmune diseases is increasing worldwide. Women make up a large proportion of autoimmune disease diagnoses, underscoring the importance of fully elucidating the complex synergistic relationships between estrogens and vitamin D. Vitamin D receptor-activating drugs appear to enhance remyelination in patients diagnosed with multiple sclerosis (MS) and other demyelinating diseases such as neuromyelitis optica (NMO). This review is intended to update health practitioners about the potential role of vitamin D deficiency demyelination and to motivate future research on dietary recommendations for vitamin D in preventing and treating demyel1nating diseases. The incidence of autoimmune diseases continues to rise globally  while poNMO and MS are both immune-mediated demyelinating diseases targeting the CNS. Since the CNS has a limited and questionable regenerative capacity, restoring lost function is not always guaranteed for patients and most treatments are aimed at preventing future episodes (prophylaxis) and symptom relief as opposed to curative measures. Vitamin D, a hormone that humans endogenously produce and also consume, has long been the focus of many MS studies, but may require an innovative approach to yield beneficial therapeutic results.Vitamin D is mostly known for its roles in calcium and bone homeostasis; however, it also contributes to nervous system development, blood-brain barrier maintenance, axon growth/regeneration, and myelination which are all important aspects to be considered in autoimmune demyelinating disease . FurtherResearch demonstrating correlations between vitamin D status, disability, and relapse rate for NMO has provided the rationale for vitamin D supplementation as a therapeutic and preventive strategy for this disease . On the Exposure to UVB radiation converts an endogenous precursor, 7-dehydrocholesterol in skin to the animal form of vitamin D, cholecalciferol, also known as vitamin D3 . Cutaneous penetration of ultraviolet radiation is a key step in the biochemical activation of human vitamin D. Thus, darker skin pigmentation increases the risk of vitamin D deficiency. Evidence is emerging that dark skin pigmentation is a risk factor for autoimmune disease; therefore, providing a logical linkage between these conditions and African ancestry . Specifiα-hydroxylases to become 1,25-dihydroxy vitamin D D), also known as calcitriol. According to the Food and Nutrition Board [Before vitamin D can perform its functions, it is activated first by liver 25-hydroxylases (to 25(OH)D) and then by kidney 1on Board , the beson Board . 25(OH)Don Board .cis retinoic acid, a form of vitamin A known for its functions in the Wnt stem cell signaling pathway (an important regulator of tissue development and carcinogenesis) [The actions of vitamin D3 are produced by a signaling cascade initiated by calcitriol binding to the nuclear vitamin D receptor (VDR) which acts as a transcription factor. Calcitriol forms a heterodimer with the retinoid-X-receptor which also binds 9-genesis) . The calgenesis) . The VDRgenesis) . VDRs argenesis)  calling genesis) . As a regenesis) . The folα-hydroxylase activity and express VDRs [Vitamin D is more popularly known for maintaining appropriate calcium levels to support bone mineralization and less recognized for its immunomodulatory function. Cells of both the innate and adaptive branches of the immune system display 1ess VDRs . Vitaminess VDRs . Vitaminess VDRs . In turness VDRs . Studiesess VDRs . For a sγ, whereas Th17 cells disrupt the blood brain barrier (an early event in MS) and attract other leukocytes to help in the attack against oligodendrocytes [Multiple sclerosis is the most common autoimmune demyelinating disease , yet thedrocytes . Oligodedrocytes . Remyelidrocytes  and eviddrocytes .Vitamin D may be a key mediator in NMO, an autoimmune disease which demyelinates the optic nerve, brain stem and spinal cord. Antibodies specifically directed against aquaporins (water channels) within astrocytes appear to be a key pathophysiological feature of NMO; however, the initiating factor remains unknown . IdentifThe majority of NMO spectrum cases are characterized by relapsing attacks of optic neuritis and/or myelitis happening sporadically . Fewer pFrom an immunological standpoint, the cerebrospinal fluid of patients with MS contains all mononuclear white blood cells while NMO relapsing patients have polymononuclear components . Unlike NMO spectrum disorders present with fatigue, pain, depression, and sleep problems, painting a very similar clinical picture to MS . RelapseTreatment for NMO spectrum disorders and MS are both aimed at immunosuppression and still very experimental. Both utilize corticosteroids for acute exacerbations , 25, butVitamin D plays a vital role in immunity, reflected in the low serum vitamin D levels in patients diagnosed with various autoimmune diseases  includinSignificantly lower vitamin D levels in NMO patients relative to healthy controls has been reported with an inverse correlation between vitamin D levels and disability status score . HoweverMyasthenia gravis (MG) is another autoimmune disease that may clinically present similar to MS and NMO (and may coexist with the latter), but antibodies target postsynaptic acetylcholine receptors of the neuromuscular junction. Askmark et al.  reportedIt is currently recommended that adults consume at least 600 IU of vitamin D daily to achieve adequate serum levels of 25-hydroxyvitamin D (25(OH)D) greater than 75 nmol/L . Fish oiα, IFN-γ, and IL-13) with vitamin D supplementation although slight reductions in IL-2 mRNA levels. Mosayebi et al. [β and IL-10, relative to the control group. The study was not able to identify a significant difference however between treatment and control in terms of expanded disability status score and gadolinium enhancing lesions. Soilu-Hänninen et al. [According to a study by Burton et al. , a normai et al.  administn et al.  raised tn et al. . Kampmann et al.  observedn et al. . The mixTo uncover the potential benefits vitamin D may have in treatment of NMO, MS and other autoimmune diseases, its mechanism of action as an immunomodulatory agent must be investigated more thoroughly. This review identifies epigenetic modifications, VDR polymorphisms, and estrogen interaction as potential avenues for further study.IL2RA) and IL-7 (IL7RA) α-chain receptor genes in MS [IL7RA∗C and IL2RA∗T downregulate the protective N-glycan branching pathway by inhibiting expression of MGAT1, a glucosaminyltransferase [Glycosylation is shown to be an important epigenetic modification of the immune response . A studyes in MS . The varnsferase . Grigorinsferase  reportednsferase .Vitamin D status and VDR polymorphisms appear to correlate with the incidence and severity of autoimmune diseases . Few polVDR mRNA [The human VDR gene spans approximately 75 kb on chromosome 12q13-14, composed of 11 exons with introns in between . The fouVDR mRNA . The resApaI polymorphism when codominant, and FokI polymorphism in both dominant and codominant models. Apa and FokII homozygote genotypes (AA and FF) were found to pose a high risk of MS but TaqI and BsmI polymorphisms were not implicated [A significant association is reported for MS and the FokI polymorphism in a region of Turkey . Howeverplicated . It seemplicated .Results from various studies are clearly contradictory and reasons may include but are not limited to, small sample size, low statistical power, difference in ethnicities, geographic variation, interaction with other genetic or environmental factors and clinical heterogeneity .ApaI, BsmI and TaqI polymorphisms result in different stability or translational efficiency of RNA, they do not appear to affect VDR protein structure directly. FokI polymorphism is an exception as it appears to affect both VDR protein structure and transcriptional activity [ApaI polymorphism and MS risk.The etiologies of autoimmune diseases such as NMO and MS are complex and polymorphisms may exert only a modest effect. Although activity , 46. Oveactivity  illustraactivity . This meactivity  meta-anaVDR gene polymorphism with MS might only be penetrant in a population with a sufficient vitamin D status [Some relationships between genotype and disease depend on exposure level to an environmental factor. In other words, some associations are only apparent if there is high exposure while others are observed with lower exposure. One study hypothesized that an association of a D status . This isD status  which waD status .GC (vitamin D-binding protein), DHCR7 , CYP2R1 (vitamin D-25-hydroxylase), and CYP24A1 [CYP24A1-linked SNP alone [CYP24A1 is purported to be associated with MS risk due to its increased transcription found along with frontal cortex inflammation. This finding suggests that increased CYP24A1-related vitamin D degradation may make the brain vulnerable for MS attack [Considered together, the aforementioned studies suggest that vitamin D polymorphisms are probably important to the etiology of NMO and MS, but should not overshadow the potentially bigger problem of vitamin D deficiency. The issue of vitamin D deficiency is, however, further complicated by findings that there are some gene polymorphisms important for vitamin D function which predispose individuals to developing vitamin D deficiency , and postamin D) . These Stamin D) . When sttamin D)  reportedNP alone . A variaS attack .Involvement of estrogen in vitamin D-related autoimmune disease seems to be logical in that women are at a higher risk than men for autoimmune diseases . Being tEstrogen appears to be a modulator of CYP24A1 expression  and the The role of estrogens in neuroprotective mechanisms has been illustrated in many studies. Precisely how the immunological effects of estrogens appear to be mediated specifically through vitamin D is of great interest, but not well studied. Both estrogens and vitamin D  are steroid hormones. These steroid hormones may work alongside each other in a synergistic relationship and it may be more helpful to view them as partners to better understand the mechanisms underlying autoimmune demyelinating diseases. Estrogen enhances VDR gene expression  and appeThe mechanisms by which estrogen increases the half-life of vitamin D and its responsiveness through VDR transcripts and CYP24A1 repression are unknown. A study investigating signaling pathways in breast and colon cancer suggest that E2 induces ERK 1/2 activation and transcriptional activity, which results in upregulation of expression of the VDR gene . SpanierCYP19 gene expression in VDR-null female rodents is associated with lower aromatase activity and lower circulating levels of E2 compared to wild-type rodents with intact VDR function [CYP19 and HSD17B4 (encodes for 17β-hydroxysteroid dehydrogenase which interconverts estrone (E1) and estradiol (E2)) in splenic T lymphocytes of rodents [Vitamin D also upregulates aromatase mRNA in bone tissue which increases E2 levels and increases osteoblast and fibroblast activity for promoting bone mineralization . Vitaminfunction . Another rodents . These fAs previously mentioned, the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, is increased by vitamin D deficiency and decreased by vitamin D supplementation, notably in a female-biased manner . These fEpidemiological evidence points to global vitamin D deficiency, prevalent in all ages regardless of climate or geographical location . AutoimmTaken together, the findings of this review suggest that vitamin D supplementation has potential as a treatment for demyelinating autoimmune diseases. Existing studies, however, continue to present conflicting data regarding clinical significance. Determining whether vitamin D may even serve as prophylaxis for NMO and MS or even reduce disability/relapse rate still requires intense investigation. The role of vitamin D is so complex and the intricacies of its role in immunity depend on many factors. This creates some difficulty in formulating an ideal way to utilize vitamin D for its immunological protection. Hence a more detailed mechanism is needed and this review pinpoints VDR polymorphisms and estrogen as potential gateways for understanding how vitamin D mediates protection against demyelinating autoimmune diseases such as NMO and MS. Environmental interactions dictate VDR function to an extent and UV levels cannot be neglected when comparing and assessing vitamin D sufficiency. Women make up a considerable proportion of autoimmune disease diagnoses and studies are currently exploiting the synergistic relationship estrogen has with vitamin D to observe for clinical improvement. Each autoimmune disease has different pathological origin so observations may not be applicable across the board , but thiWhile it may appear difficult, it should not be too long before we identify how to prevent the painful relapses and crippling disabilities that come about from immune systems gone haywire. A study is already hypothesizing VDR-activating drugs may be able to enhance remyelination in MS patients and other demyelinating diseases which may possibly include NMO . If the