Mapping the geographical distribution of the mucosa-associated gut microbiome in GI-symptomatic children with autism spectrum disorder.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by cognitive, behavioral, and communication impairments. In the past few years, it has been proposed that alterations in the gut microbiota may contribute to an aberrant communication between the gut and brain in children with ASD. Consistent with this notion, several studies have demonstrated that children with ASD have an altered fecal microbiota compared with typically developing (TD) children. However, it is unclear where along the length of the gastrointestinal (GI) tract these alterations in microbial communities occur. In addition, the variation between specific mucosa-associated communities remains unknown. To address this gap in knowledge of the microbiome associated with ASD, biopsies from the antrum, duodenum, ileum, right colon, and rectum of children with ASD and age- and sex-matched TD children were examined by 16S rRNA sequencing. We observed an overall elevated abundance of Bacillota and Bacteroidota and a decreased abundance of Pseudomonadota in all GI tract regions of both male and female children with ASD compared with TD children. Further analysis at the genera level revealed unique differences in the microbiome in the different regions of the GI tract in children with ASD compared with TD children. We also observed sex-specific differences in the gut microbiota composition in children with ASD. These data indicate that the microbiota of children with ASD is altered in multiple regions of the GI tract and that different anatomic locations have unique alterations in mucosa-associated bacterial genera.NEW & NOTEWORTHY Analysis in stool samples has shown gut microbiota alterations in children with autism spectrum disorder (ASD) compared with typically developing (TD) children. However, it is unclear which segment(s) of the gut exhibit alterations in microbiome composition. In this study, we examined microbiota composition along the gastrointestinal (GI) tract in the stomach, duodenum, ileum, right colon, and rectum. We found site-specific and sex-specific differences in the gut microbiota of children with ASD, compared with controls.

Blood pressure and the kidney cortex transcriptome response to high-sodium diet challenge in female nonhuman primates.

Blood pressure (BP) is influenced by genetic variation and sodium intake with sex-specific differences; however, studies to identify renal molecular mechanisms underlying the influence of sodium intake on BP in nonhuman primates (NHP) have focused on males. To address the gap in our understanding of molecular mechanisms regulating BP in female primates, we studied sodium-naïve female baboons (n = 7) fed a high-sodium (HS) diet for 6 wk. We hypothesized that in female baboons variation in renal transcriptional networks correlates with variation in BP response to a high-sodium diet. BP was continuously measured for 64-h periods throughout the study by implantable telemetry devices. Sodium intake, blood samples for clinical chemistries, and ultrasound-guided kidney biopsies were collected before and after the HS diet for RNA-Seq and bioinformatic analyses. We found that on the LS diet but not the HS diet, sodium intake and serum 17 ß-estradiol concentration correlated with BP. Furthermore, kidney transcriptomes differed by diet-unbiased weighted gene coexpression network analysis revealed modules of genes correlated with BP on the HS diet but not the LS diet. Our results showed variation in BP on the HS diet correlated with variation in novel kidney gene networks regulated by ESR1 and MYC; i.e., these regulators have not been associated with BP regulation in male humans or rodents. Validation of the mechanisms underlying regulation of BP-associated gene networks in female NHP will inform better therapies toward greater precision medicine for women.

Postnatal persistence of nonhuman primate sex-dependent renal structural and molecular changes programmed by intrauterine growth restriction.

BACKGROUND: Poor nutrition during fetal development programs postnatal kidney function. Understanding postnatal consequences in nonhuman primates (NHP) is important for translation to our understanding the impact on human kidney function and disease risk. We hypothesized that intrauterine growth restriction (IUGR) in NHP persists postnatally, with potential molecular mechanisms revealed by Western-type diet challenge. METHODS: IUGR juvenile baboons were fed a 7-week Western diet, with kidney biopsies, blood, and urine collected before and after challenge. Transcriptomics and metabolomics were used to analyze biosamples. RESULTS: Pre-challenge IUGR kidney transcriptome and urine metabolome differed from controls. Post-challenge, sex and diet-specific responses in urine metabolite and renal signaling pathways were observed. Dysregulated mTOR signaling persisted postnatally in female pre-challenge. Post-challenge IUGR male response showed uncoordinated signaling suggesting proximal tubule injury. CONCLUSION: Fetal undernutrition impacts juvenile offspring kidneys at the molecular level suggesting early-onset blood pressure dysregulation.

The non-human primate kidney transcriptome in fetal development.

BACKGROUND: Little is known about the repertoire of non-human primate kidney genes expressed throughout development. The present work establishes an understanding of the primate renal transcriptome during fetal development in the context of renal maturation. METHODS: The baboon kidney transcriptome was characterized at 60-day gestation (DG), 90 DG, 125 DG, 140 DG, 160 DG and adulthood (6-12 years) using gene arrays and validated by QRT-PCR. Pathway and cluster analyses were used to characterize gene expression in the context of biological pathways. RESULTS: Pathway analysis indicated activation of pathways not previously reported as relevant to kidney development. Cluster analysis also revealed gene splice variants with discordant expression profiles during development. CONCLUSIONS: This study provides the first detailed genetic analysis of the developing primate kidney, and our findings of discordant expression of gene splice variants suggest that gene arrays likely provide a simplified view and demonstrate the need to study the fetal renal proteome.

Simulated microgravity impairs human NK cell cytotoxic activity against space radiation-relevant leukemic cells.

Natural killer (NK) cells are an important first-line of defense against malignant cells. Because of the potential for increased cancer risk from astronaut exposure to space radiation, we determined whether microgravity present during spaceflight affects the body's defenses against leukemogenesis. Human NK cells were cultured for 48 h under normal gravity and simulated microgravity (sµG), and cytotoxicity against K-562 (CML) and MOLT-4 (T-ALL) cells was measured using standard methodology or under continuous sµG. This brief exposure to sµG markedly reduced NK cytotoxicity against both leukemias, and these deleterious effects were more pronounced in continuous sµG. RNA-seq performed on NK cells from two additional healthy donors provided insight into the mechanism(s) by which sµG reduced cytotoxicity. Given our prior report of space radiation-induced human T-ALL in vivo, the reduced cytotoxicity against MOLT-4 is striking and raises the possibility that µG may increase astronaut risk of leukemogenesis during prolonged missions beyond LEO.

Cell-type deconvolution of bulk RNA-Seq from kidney using opensource bioinformatic tools.

Traditional bulk RNA-Seq pipelines do not assess cell-type composition within heterogeneous tissues. Therefore, it is difficult to determine whether conflicting findings among samples or datasets are the result of biological differences or technical differences due to variation in sample collections. This report provides a user-friendly, open source method to assess cell-type composition in bulk RNA-Seq datasets for heterogeneous tissues using published single cell (sc)RNA-Seq data as a reference. As an example, we apply the method to analysis of kidney cortex bulk RNA-Seq data from female (N=8) and male (N=9) baboons to assess whether observed transcriptome sex differences are biological or technical, i.e., variation due to ultrasound guided biopsy collections. We found cell-type composition was not statistically different in female versus male transcriptomes based on expression of 274 kidney cell-type specific transcripts, indicating differences in gene expression are not due to sampling differences. This method of cell-type composition analysis is recommended for providing rigor in analysis of bulk RNA-Seq datasets from complex tissues. It is clear that with reduced costs, more analyses will be done using scRNA-Seq; however, the approach described here is relevant for data mining and meta analyses of the thousands of bulk RNA-Seq data archived in the NCBI GEO public database.

Development of a novel air-liquid interface airway tissue equivalent model for in vitro respiratory modeling studies.

The human airways are complex structures with important interactions between cells, extracellular matrix (ECM) proteins and the biomechanical microenvironment. A robust, well-differentiated in vitro culture system that accurately models these interactions would provide a useful tool for studying normal and pathological airway biology. Here, we report the development and characterization of a physiologically relevant air-liquid interface (ALI) 3D airway 'organ tissue equivalent' (OTE) model with three novel features: native pulmonary fibroblasts, solubilized lung ECM, and hydrogel substrate with tunable stiffness and porosity. We demonstrate the versatility of the OTE model by evaluating the impact of these features on human bronchial epithelial (HBE) cell phenotype. Variations of this model were analyzed during 28 days of ALI culture by evaluating epithelial confluence, trans-epithelial electrical resistance, and epithelial phenotype via multispectral immuno-histochemistry and next-generation sequencing. Cultures that included both solubilized lung ECM and native pulmonary fibroblasts within the hydrogel substrate formed well-differentiated ALI cultures that maintained a barrier function and expressed mature epithelial markers relating to goblet, club, and ciliated cells. Modulation of hydrogel stiffness did not negatively impact HBE differentiation and could be a valuable variable to alter epithelial phenotype. This study highlights the feasibility and versatility of a 3D airway OTE model to model the multiple components of the human airway 3D microenvironment.

Severe acute respiratory coronavirus virus 2 (SARS-CoV-2) infections occurring in healthcare workers after booster vaccination: A comparison of delta versus omicron variants.

In this study, we used genomic sequencing to identify variants of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) in healthcare workers with coronavirus disease 2019 (COVID-19) after receiving a booster vaccination. We compared symptoms, comorbidities, exposure risks, and vaccine history between the variants. Postbooster COVID-19 cases increased as the SARS-CoV-2 omicron variant predominated.

Hepatic transcript signatures predict atherosclerotic lesion burden prior to a 2-year high cholesterol, high fat diet challenge.

The purpose of this study was to identify molecular mechanisms by which the liver influences total lesion burden in a nonhuman primate model (NHP) of cardiovascular disease with acute and chronic feeding of a high cholesterol, high fat (HCHF) diet. Baboons (47 females, 64 males) were fed a HCHF diet for 2 years (y); liver biopsies were collected at baseline, 7 weeks (w) and 2y, and lesions were quantified in aortic arch, descending aorta, and common iliac at 2y. Unbiased weighted gene co-expression network analysis (WGCNA) revealed several modules of hepatic genes correlated with lesions at different time points of dietary challenge. Pathway and network analyses were performed to study the roles of hepatic module genes. More significant pathways were observed in males than females. In males, we found modules enriched for genes in oxidative phosphorylation at baseline, opioid signaling at 7w, and EIF2 signaling and HNF1A and HNF4A networks at baseline and 2y. One module enriched for fatty acid ß oxidation pathway genes was found in males and females at 2y. To our knowledge, this is the first study of a large NHP cohort to identify hepatic genes that correlate with lesion burden. Correlations of baseline and 7w module genes with lesions at 2y were observed in males but not in females. Pathway analyses of baseline and 7w module genes indicate EIF2 signaling, oxidative phosphorylation, and µ-opioid signaling are possible mechanisms that predict lesion formation induced by HCHF diet consumption in males. Our findings of coordinated hepatic transcriptional response in male baboons but not female baboons indicate underlying molecular mechanisms differ between female and male primate atherosclerosis.

Diet, obesity, and the gut microbiome as determinants modulating metabolic outcomes in a non-human primate model.

BACKGROUND: The objective of this study was to increase understanding of the complex interactions between diet, obesity, and the gut microbiome of adult female non-human primates (NHPs). Subjects consumed either a Western (n=15) or Mediterranean (n=14) diet designed to represent human dietary patterns for 31 months. Body composition was determined using CT, fecal samples were collected, and shotgun metagenomic sequencing was performed. Gut microbiome results were grouped by diet and adiposity. RESULTS: Diet was the main contributor to gut microbiome bacterial diversity. Adiposity within each diet was associated with subtle shifts in the proportional abundance of several taxa. Mediterranean diet-fed NHPs with lower body fat had a greater proportion of Lactobacillus animalis than their higher body fat counterparts. Higher body fat Western diet-fed NHPs had more Ruminococcus champaneliensis and less Bacteroides uniformis than their low body fat counterparts. Western diet-fed NHPs had significantly higher levels of Prevotella copri than Mediterranean diet NHPs. Western diet-fed subjects were stratified by P. copri abundance (P. copriHIGH versus P. copriLOW), which was not associated with adiposity. Overall, Western diet-fed animals in the P. copriHIGH group showed greater proportional abundance of B. ovatus, B. faecis, P. stercorea, P. brevis, and Faecalibacterium prausnitzii than those in the Western P. copriLOW group. Western diet P. copriLOW subjects had a greater proportion of Eubacterium siraeum. E. siraeum negatively correlated with P. copri proportional abundance regardless of dietary consumption. In the Western diet group, Shannon diversity was significantly higher in P. copriLOW when compared to P. copriHIGH subjects. Furthermore, gut E. siraeum abundance positively correlated with HDL plasma cholesterol indicating that those in the P. copriLOW population may represent a more metabolically healthy population. Untargeted metabolomics on urine and plasma from Western diet-fed P. copriHIGH and P. copriLOW subjects suggest early kidney dysfunction in Western diet-fed P. copriHIGH subjects. CONCLUSIONS: In summary, the data indicate diet to be the major influencer of gut bacterial diversity. However, diet and adiposity must be considered together when analyzing changes in abundance of specific bacterial taxa. Interestingly, P. copri appears to mediate metabolic dysfunction in Western diet-fed NHPs. Video abstract.

Shotgun Metagenomic Sequencing Identifies Dysbiosis in Triplet Sibling with Gastrointestinal Symptoms and ASD.

The gut microbiome profile of a child with autism spectrum disorder (ASD) and co-occurring gastrointestinal (GI) symptoms was compared to that of her healthy triplet siblings to determine if she exhibited intestinal dysbiosis. Shotgun metagenomic sequencing was performed in individual fecal samples, and relative microbial abundance and diversity was determined. Microbial diversity was lower in sibling #3, coupled with a higher Bacteroidetes/Firmicutes ratio, a lower relative abundance of Actinobacteria, and an increased relative abundance of Proteobacteria. Our findings are suggestive of gut dysbiosis in a child with ASD and co-occurring GI symptoms, compared to her two healthy triplet siblings.

Mammalian Glutamyl Aminopeptidase Genes (ENPEP) and Proteins: Comparative Studies of a Major Contributor to Arterial Hypertension.

Glutamyl aminopeptidase (ENPEP) is a member of the M1 family of endopeptidases which are mammalian type II integral membrane zinc-containing endopeptidases. ENPEP is involved in the catabolic pathway of the renin-angiotensin system forming angiotensin III, which participates in blood pressure regulation and blood vessel formation. Comparative ENPEP amino acid sequences and structures and ENPEP gene locations were examined using data from several mammalian genome projects. Mammalian ENPEP sequences shared 71-98% identities. Five N-glycosylation sites were conserved for all mammalian ENPEP proteins examined although 9-18 sites were observed, in each case. Sequence alignments, key amino acid residues and predicted secondary and tertiary structures were also studied, including transmembrane and cytoplasmic sequences and active site residues. Highest levels of human ENPEP expression were observed in the terminal ileum of the small intestine and in the kidney cortex. Mammalian ENPEP genes contained 20 coding exons. The human ENPEP gene promoter and first coding exon contained a CpG island (CpG27) and at least 6 transcription factor binding sites, whereas the 3'-UTR region contained 7 miRNA target sites, which may contribute to the regulation of ENPEP gene expression in tissues of the body. Phylogenetic analyses examined the relationships of mammalian ENPEP genes and proteins, including primate, other eutherian, marsupial and monotreme sources, using chicken ENPEP as a primordial sequence for comparative purposes.

Primate response to angiotensin infusion and high sodium intake differ by sodium lithium countertransport phenotype.

An increased level of sodium-lithium countertransport (SLC) activity has been associated with salt-sensitive hypertension. Previous findings have suggested that dysregulation of the renin-angiotensin-aldosterone system (RAAS) may be involved in the mechanism linking elevated SLC activity and hypertension. Therefore, baboons with different levels of SLC activity were given two diets differing in sodium content, with and without an angiotensin II (ANG II) infusion, to investigate the relationship between SLC activity, the RAAS, and physiological regulation by sodium. Although we anticipated that high SLC activity would be associated with inappropriate function of the RAAS and greater arterial pressure sensitivity to dietary sodium and ANG II and that low SLC activity would be associated with the least BP sensitivity, we found that the low SLC phenotype correlated with BP sensitivity similar to the high SLC phenotype, and the normal SLC phenotype showed the least BP sensitivity to dietary sodium and ANG II.

Evolution of Vertebrate Solute Carrier Family 9B Genes and Proteins (SLC9B): Evidence for a Marsupial Origin for Testis Specific SLC9B1 from an Ancestral Vertebrate SLC9B2 Gene.

SLC9B genes and proteins are members of the sodium/lithium hydrogen antiporter family which function as solute exchangers within cellular membranes of mammalian tissues. SLC9B2 and SLC9B1 amino acid sequences and structures and SLC9B-like gene locations were examined using bioinformatic data from several vertebrate genome projects. Vertebrate SLC9B2 sequences shared 56-98% identity as compared with ∼50% identities with mammalian SLC9B1 sequences. Sequence alignments, key amino acid residues and conserved predicted transmembrane structures were also studied. Mammalian SLC9B2 and SLC9B1 genes usually contained 11 or 12 coding exons with differential tissue expression patterns: SLC9B2, broad tissue distribution; and SLC9B1, being testis specific. Transcription factor binding sites and CpG islands within the human SLC9B2 and SLC9B1 gene promoters were identified. Phylogenetic analyses suggested that SLC9B1 originated in an ancestral marsupial genome from a SLC9B2 gene duplication event.

Neuroprotective mechanisms activated in non-seizing rats exposed to sarin.

Exposure to organophosphate (OP) nerve agents, such as sarin, may lead to uncontrolled seizures and irreversible brain injury and neuropathology. In rat studies, a median lethal dose of sarin leads to approximately half of the animals developing seizures. Whereas previous studies analyzed transcriptomic effects associated with seizing sarin-exposed rats, our study focused on the cohort of sarin-exposed rats that did not develop seizures. We analyzed the genomic changes occurring in sarin-exposed, non-seizing rats and compared differentially expressed genes and pathway activation to those of seizing rats. At the earliest time point (0.25 h) and in multiple sarin-sensitive brain regions, defense response genes were commonly expressed in both groups of animals as compared to the control groups. All sarin-exposed animals activated the MAPK signaling pathway, but only the seizing rats activated the apoptotic-associated JNK and p38 MAPK signaling sub-pathway. A unique phenotype of the non-seizing rats was the altered expression levels of genes that generally suppress inflammation or apoptosis. Importantly, the early transcriptional response for inflammation- and apoptosis-related genes in the thalamus showed opposite trends, with significantly down-regulated genes being up-regulated, and vice versa, between the seizing and non-seizing rats. These observations lend support to the hypothesis that regulation of anti-inflammatory genes might be part of an active and sufficient response in the non-seizing group to protect against the onset of seizures. As such, stimulating or activating these responses via pretreatment strategies could boost resilience against nerve agent exposures.