Substance P (SubP) and endomorphin-2 (Endo2) are co-localized presynaptically in vesicles of neurons adjacent to inspiratory rhythm-generating pre-Botzinger Complex (preBotC) neurons but the effects of co-released and Endo2 on respiratory motor control are not known. To address this question, SubP alone or a combination SubP and Endo2 (SubP/Endo2) were bath-applied in a sustained (15-min) or intermittent (5-min application, min washout, x3) pattern at 10-100 nM to neonatal rat brainstem-spinal cord preparations. During neuropeptide application, SubP/Endo2 co-applications generally attenuated SubP-induced increases in burst frequency decreases in burst amplitude. With respect to frequency plasticity (long-lasting increase in burst frequency 60 min post-neuropeptide application), SubP-induced frequency plasticity was increased with sustained Endo2 co-applications at 20 and 100 nM. Intermittent SubP/Endo2 co-applications tended to decrease the of frequency plasticity induced by intermittent SubP alone applications. SubP/Endo2 co-applications revealed potentially new functions for neurokinin-1 (NK1R) and mu-opioid (MOR) receptors on respiratory rhythm generating medullary neurons.

The efficacy of Calorie Restriction (CR) in enhancing cognition, promoting healthy aging, and extending lifespan is well-established. Yet, it remains unclear whether the apolipoprotein E ( APOE ) genotype, a known modifier for aging and age-related disorders, influences the beneficial effects of CR in countering aging. To investigate this question, we utilized humanized APOE mouse models, which express APOE2 , APOE3 , or APOE4 alleles systematically (refer to as E2, E3, and E4 mice). These mice were subjected to either ad libitum (AL) feeding or 30% CR feeding, starting from 12 months and continuing up to 20 months of age (N = 11-20/ genotype /group, mixed sex). We monitored energy expenditure using the Comprehensive Laboratory Animal Monitoring System (CLAMS). Additionally, we evaluated anxiety levels and memory capabilities through various behavioral tests. Finally, we collected brain, blood, and feces samples for transcriptomic, lipidomic, and microbiomic analyses. CLAMS measurements showed that CR significantly decreased the metabolic rate in E3 and E4 mice, but not E2 mice, during the non-feeding period of the CR group, compared to the AL group. Furthermore, CR reduced anxiety and enhanced associative memory in E3 and E4 mice relative to the AL group. Brain transcriptomics revealed that CR upregulated the cholesterol synthesis pathway, potentially improving myelination-related pathways in E3 and E4 mice. This finding was further validated by the increased staining intensity of myelin basic protein (MBP) observed in E3 and E4 mice from an independent cohort. In contrast, these effects were not pronounced in E2 mice. Additionally, while CR’s impact on the brain lipid profile was minor across all APOE genotypes, it significantly altered blood lipid profile of E2 mice, with minimal changes observed in E3 and E4 mice. Finally, in microbiomics, CR led to notable changes in the abundance of Bifidobacterium species in E3 and E4 mice and Lactobacillus species in E2 mice. Our study highlights the role of APOE genotypes in modulating the diverse effects of CR on cognition, brain transcriptomics, lipid metabolism, and the microbiome, providing a crucial foundation for considering APOE genotypes in developing prevention strategies for aging and age-related disorders.

Premature and newborn infants often have prolonged apneas and are susceptible to bacterial infections that further disrupt breathing. Phoshodiesterase-4 (PDE4) inhibitor drugs increase inspiratory motor activity and appear to induce a long-lasting increase in inspiratory frequency ("frequency plasticity"). To test whether a PDE4 inhibitor drug induces frequency plasticity, neonatal rat brainstem-spinal cords were isolated and exposed to bath-applied roflumilast (10 min, 0.02-1.0 µM). Roflumilast acutely increased burst frequency and induced frequency plasticity in a concentration-dependent manner. Blockade of protein kinase A (PKA) or exchange protein activated by cAMP (EPAC) signaling pathways abolished the induction, but not the maintenance, of roflumilast-induced frequency plasticity. Brainstem-spinal cords isolated from neonatal rats injected with lipopolysaccharide (LPS, 0.1 mg/kg, 3 h prior) expressed frequency plasticity following bath-applied roflumilast at 0.05-0.5 µM, but not at lower concentrations. This shows that roflumilast-induced frequency plasticity is largely resistant to LPS-induced inflammation. Thus, roflumilast increases inspiratory burst frequency acutely and induces frequency plasticity even during ongoing inflammation, which could have important clinical implications.

While dietary changes are often implicated in the adaptation of traditional microbiomes to highly industrialized society, it only partially explains the observed microbial shifts post-immigration. Here, we used germ-free mice colonized with human donor stool from the United States and Thailand to investigate how person-to-person transmission of gut commensals - via shared air and physical contact - affects U.S. and Thai microbiomes and subsequent health outcomes. We found that both air sharing and physical contact enabled bidirectional transmission: U.S. mucus-degrading taxa (e.g., Akkermansia) was transferred into Thai microbiomes, while Thai-derived, potentially health-promoting bacteria colonized U.S. microbiomes. The host's baseline gut microbiome composition emerged as a key factor influencing the extent of the microbial remodeling. When exposed to 13 dietary ingredients and food additives prevalent in industrialized diets, the U.S. microbiome responded differently from its Thai counterpart, with food additives reducing Akkermansia. Corroborating these changes, the U.S. microbiome showed a predisposition toward weight gain under industrialized dietary conditions. However, sharing air supply or co-housing mitigated this effect - likely through the transfer of health-promoting bacteria (e.g., Lactobacillus). Our findings show the complexity of microbiome adaptation to industrialization, revealing how environmental microbial exchange and diet interplay can shape gut communities and their metabolic consequences.

A novel Corynebacterium species, strain BD556T, isolated from blood, was identified at Mayo Clinic, Rochester, MN, USA. After failing definitive identification using MALDI- ToF MS and partial 16S rRNA gene sequencing, BD556T was characterized using a polyphasic approach, including phenotypic, biochemical and whole- genome sequencing methods. BD556T was a Gram- positive rod with clubbed ends, facultatively anaerobic, catalase- positive, oxidase- negative and non- motile. Colonies were white, opaque and non- haemolytic with halo- like edges. BD556T grew at 35 degrees C in room air, with CO2 and under anaerobic conditions. BD556T grew well in 0 and 6% NaCl and weakly in 10% NaCl. The genome size was 2 349 779 bp with a G+C content of 60.39%. Phylogenetic analysis using 16S rRNA gene sequence analysis, average nucleotide identity and digital DNA-DNA hybridization between the genome of BD556T and the closest type strains from the Type Strain Genome Server database yielded separation values well beyond those required for species delineation. Chemotaxonomic analyses of BD556T revealed ribose, arabinose and galactose as whole- cell sugars and an A1 gamma meso-diaminopimelic acid-direct peptidoglycan type. The major cellular fatty acids were C15: 0 (21.0%), C16: 0 (14.8%), C17: 1 omega 9c (26.2%), C17: 0 (13.3%) and C18: 1 omega 9c (18.3%). Polar lipids included diphosphatidylglycerol, phosphatidylglycerol and unidentified glycolipids and phospholipids. BD556T also contained mycolic acids (32-36 carbons) typical of corynebacteria. The respiratory quinones were dominated by MK-8(H2) (71.2%) and MK- 9(H2) (25.9%), with smaller amounts of MK- 7(H2) and MK- 10(H2). The results presented support the tenet that BD556T (=TSD 427T=NCTC 15078T) is a novel species for which the name Corynebacterium mayonis sp. nov. is proposed.

Background Neoadjuvant treatment has become standard for patients with high-risk operable stage III melanoma, but the optimal regimen is unknown. Targeted therapy approaches yield high pathological response rates, while immunotherapy regimens show favorable recurrence-free survival (RFS). NeoACTIVATE was designed to address whether a neoadjuvant combination of both targeted therapy and immunotherapy might leverage the benefits of each.Methods We tested neoadjuvant treatment with 12 weeks of vemurafenib, cobimetinib, and atezolizumab for patients with BRAF-mutated (BRAFm) melanoma (cohort A) and cobimetinib and atezolizumab for patients with BRAF-wild-type (BRAFwt) melanoma (cohort B), regimens which we have shown generate a substantial major pathological response. After therapeutic lymph node dissection, patients received 24 weeks of adjuvant atezolizumab. Here, we report survival outcomes and their association with biomarkers assayed among the gut microbiome and peripheral blood immune subsets.Results With 49 months median follow-up, the median RFS was not reached for cohort A and was 40.8 months for cohort B. At 24 months after operation, 2 of 14 cohort A patients and 4 of 13 cohort B patients had experienced distant relapse. Key findings from correlative analyses included diversity, taxonomic and functional metagenomic gut microbiome signals associated with distant metastasis-free survival at 2 years. Notably, we observed a strong correlation between low microbial arginine biosynthesis (required for T-cell activation and effector function) and early distant recurrence (p=0.0005), which correlated with taxonomic differential abundance findings. Peripheral blood immune monitoring revealed increased double-positive (CD4+CD8+) T cells in patients with early recurrence.Conclusions Neoadjuvant treatment with cobimetinib and atezolizumab±vemurafenib was associated with a low rate of distant metastasis in patients with high-risk stage III melanoma. Freedom from early distant metastasis was highly associated with taxonomic differences in gut microbiome structure and with functional pathway alterations known to modulate T cell immunity. Identification of predictive biomarkers will permit optimization of neoadjuvant therapy regimens for individual patients.Trial registration number NCT03554083.

BACKGROUND:Passively collected smartphone sensor data provide an opportunity to study physical activity and mobility unobtrusively over long periods of time and may enable disease monitoring in people with amyotrophic lateral sclerosis (PALS). METHODS:We enrolled 63 PALS who used Beiwe mobile application that collected their smartphone accelerometer and GPS data and administered the self-entry ALS Functional Rating Scale-Revised (ALSFRS-RSE) survey. We identified individual steps from accelerometer data and used the Activity Index to summarize activity at the minute level. Walking, Activity Index, and GPS outcomes were then aggregated into day-level measures. We used linear mixed effect models (LMMs) to estimate baseline and monthly change for ALSFRS-RSE scores (total score, subscores Q1-3, Q4-6, Q7-9, Q10-12) and smartphone sensor data measures, as well as the associations between them. FINDINGS:The analytic sample (N = 45) was 64.4% male with a mean age of 60.1 years. The mean observation period was 292.3 days. The ALSFRS-RSE total score baseline mean was 35.8 and had a monthly rate of decline of -0.48 (p-value <0.001). We observed statistically significant change over time and association with ALSFRS-RSE total score for four smartphone sensor data-derived measures: walking cadence from top 1 min and log-transformed step count, step count from top 1 min, and Activity Index from top 1 min. INTERPRETATION:Smartphone sensors can unobtrusively track physical changes in PALS, potentially aiding disease monitoring and future research.

Snakes are common household pets and frequently managed in zoos. Geriatric snakes commonly develop osteoarthritis, leading to a declining quality of life that often results in euthanasia. Anecdotally, the application of transdermal fentanyl patches (TFP) appears to contribute to clinical improvement, including increased activity level, in osteoarthritic snakes presumed to be in pain. This study evaluated serum fentanyl concentrations over time and the effects of TFP on the normal behavior of healthy, captive, adult corn snakes (Pantherophis guttatus) using constant video monitoring. Serum fentanyl concentrations were evaluated over 4 wk during 12.5 µg/h TFP application, and the results demonstrated long-lasting (>4 wk) serum concentrations that were consistent with analgesic efficacy in mammalian species during TFP application. At 4 wk of TFP application, mean serum fentanyl concentrations were 11.5 ± 5.5 ng/ml. Snakes were videotaped for 1 wk prior to and 2 wk after 12.5 µg/h TFP application, and behavior was evaluated by an ethogram. Behavioral changes associated with TFP application included decreased mean time spent active, decreased mean number of climbs, and decreased mean number of water visits; feeding behavior was unchanged. Overall, these results suggest that TFP application may provide safe, clinically effective analgesia in healthy corn snakes for at least 4 wk without inducing deleterious side effects, and may therefore be appropriate analgesia for management of osteoarthritic snakes.

Staphylococcus epidermidis is part of the commensal microbiota of the skin and mucous membranes, though it can also act as a pathogen in certain scenarios, causing a range of infections, including periprosthetic joint infection (PJI). Transcriptomic profiling may provide insights into mechanisms by which S. epidermidis adapts while in a pathogenic compared to a commensal state. Here, a total RNA-sequencing approach was used to profile and compare the transcriptomes of 19 paired PJI-associated S. epidermidis samples from an in vivo clinical source and grown in in vitro laboratory culture. Genomic comparison of PJI-associated and publicly available commensal-state isolates were also compared. Of the 1,919 total transcripts found, 145 were from differentially expressed genes (DEGs) when comparing in vivo or in vitro samples. Forty-two transcripts were upregulated and 103 downregulated in in vivo samples. Of note, metal sequestration-associated genes, specifically those related to staphylopine activity (cntA, cntK, cntL, and cntM), were upregulated in a subset of clinical in vivo compared to laboratory grown in vitro samples. About 70% of the total transcripts and almost 50% of the DEGs identified have not yet been annotated. There were no significant genomic differences between known commensal and PJI-associated S. epidermidis isolates, suggesting that differential genomics may not play a role in S. epidermidis pathogenicity. In conclusion, this study provides insights into phenotypic alterations employed by S epidermidis to adapt to infective and non-infected microenvironments, potentially informing future therapeutic targets for related infections.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative condition leading to progressive muscle weakness, atrophy, and ultimately death. Traditional ALS clinical evaluations often depend on subjective metrics, making accurate disease detection and monitoring disease trajectory challenging. To address these limitations, we developed the nQiALS toolkit, a machine learning-powered system that leverages smartphone typing dynamics to detect and track motor impairment in people with ALS. The study included 63 ALS patients and 30 age- and sex-matched healthy controls. We introduce the three core components of this toolkit: the nQiALS-Detection, which differentiated ALS from healthy typing patterns with an AUC of 0.89; the nQiALS-Progression, which separated slow and fast progression at specific thresholds with AUCs ranging between 0.65 and 0.8; and the nQiALS-Fine Motor, which identified subtle progression in fine motor dysfunction, suggesting earlier prediction than the state-of-the-art assessment. Together, these tools represent an innovative approach to ALS assessment, offering a complementary, objective metric to traditional clinical methods and which may reshape our understanding and monitoring of ALS progression.

In a recent article in GUT, we showed that, among 194 patients with diarrhoeapredominant irritable bowel syndrome (IBSD), 43 had altered bile acid (BA) metabolism (ABAM) (serum 7αC4>52 ng/ mL). Patients with ABAM, had faster colonic transit (CT), lower α diversity and a different microbial compositional profile based on β diversity compared with IBSD without ABAM. There were no significant differences in the stool shortchain fatty acid (SCFA) concentrations between the two groups. There is evidence that transit impacts gut microbiome composition and diversity. We wish to extend the previous analysis to compare the microbiome composition and SCFA in the same cohort of patients with IBSD (total 181) with and without rapid CT, before and after adjusting for presence of ABAM. Patient selection, CT measurement, microbiome and SCFA analysis have been previously described. CT was measured as geometric centre (GC) by scintigraphy (range 1–5, where 1=ascending colon, 5=stool). Patients with colonic GC at 24 hours >3.45 (90th percentile of normal) were considered to have rapid transit. Logistic regression was used to estimate the association between individual faecal SCFA and rapid CT, and the relationship of faecal microbiome and CT after adjustment for age, sex, serum 7αC4 and other SCFA. Out of 181 patients with IBSD, 63 had rapid CT. The demographics and SCFA concentrations in patients with and without rapid CT are summarised in table 1. Among SCFAs, faecal propionate (adjusted OR 1.14 (95% CI 1.04 to 1.26, p=0.006)) was associated with rapid CT (table 1) and was independently associated with CT (Spearman’s r=0.155, p=0.04; figure 1A). In assessing the relationship of faecal microbiome and CT, our results show that, compared with patients without rapid transit, the patients with rapid CT had borderline lower α diversity (Inverse Simpson Index, p=0.07) and a borderline different compositional profile based on β diversity (Jaccard distance, p=0.07). There were 6 genera and 14 species including Clostridium Letter

Low level activation of mu-opioid receptors (MORs) in neonatal rat brainstem-spinal cord preparations increases inspiratory burst amplitude recorded on cervical spinal roots. We tested whether: (1) MOR activation with an endogenous ligand, such as endomorphin-2, increases inspiratory burst amplitude, (2) disinhibition of GABAergic or glycinergic inhibitory synaptic transmission is involved, and (3) inflammation alters endomorphin2 effects. Using neonatal rat (P0-P3) brainstem-spinal cord preparations, bath-applied endomorphin-2 (10-200 nM) increased inspiratory burst amplitude and decreased burst frequency. Blockade of GABAA receptors (picrotoxin), glycine receptors (strychnine), or both (picrotoxin and strychnine) did not abolish endomorphin-2induced effects. In preparations isolated from neonatal rats injected 3 h previously with lipopolysaccharide (LPS, 0.1 mg/kg), endomorphin-2 continued to decrease burst frequency but abolished the burst amplitude increase. Collectively, these data indicate that disinhibition of inhibitory synaptic transmission is unlikely to play a role in endomorphin-2-induced changes in inspiratory motor output, and that different mechanisms underlie the endomorphin-2-induced increases in inspiratory burst amplitude and decreases in burst frequency.