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The syndrome of spontaneous preterm birth (sPTB) presents a challenge to mechanistic understanding, effective risk stratification, and clinical management. Individual associations between sPTB, self-reported ethnic ancestry, vaginal microbiota, metabolome, and innate immune response are known but not fully understood, and knowledge has yet to impact clinical practice. Here, we used multi-data type integration and composite statistical models to gain insight into sPTB risk by exploring the cervicovaginal environment of an ethnically heterogenous pregnant population (n = 346 women; n = 60 sPTB < 37 weeks' gestation, including n = 27 sPTB < 34 weeks). Analysis of cervicovaginal samples (10-15+6 weeks) identified potentially novel interactions between risk of sPTB and microbiota, metabolite, and maternal host defense molecules. Statistical modeling identified a composite of metabolites (leucine, tyrosine, aspartate, lactate, betaine, acetate, and Ca2+) associated with risk of sPTB < 37 weeks (AUC 0.752). A combination of glucose, aspartate, Ca2+, Lactobacillus crispatus, and L. acidophilus relative abundance identified risk of early sPTB < 34 weeks (AUC 0.758), improved by stratification by ethnicity (AUC 0.835). Increased relative abundance of L. acidophilus appeared protective against sPTB < 34 weeks. By using cervicovaginal fluid samples, we demonstrate the potential of multi-data type integration for developing composite models toward understanding the contribution of the vaginal environment to risk of sPTB.

IBD patients can have reduced gut commensal abundance, relative expansion of pro-inflammatory taxa, and reduced microbial diversity and short-chain fatty acid production. FMT leads to clinical remission in some UC patients, but is a complex, nonscalable process with inconsistent composition/quality that has led to death from transmission of pathogens undetected in screening. VE202 is a rationally defined LBP of 16 beneficial Clostridia from clusters IV, XIVa, and XVIII, with properties including association with healthy human microbiomes, promotion of colonic Treg cells, production of beneficial and immunoregulatory metabolites, efficacy in mouse colitis models, and lack of overt pathogenic or toxigenic features. These properties could help restore a healthy microbiome in UC patients. Another rationally defined LBP, VE303, showed activity in a Phase 2 study for prevention of recurrentC. difficile. The goal of the current Phase 1 study was to evaluate safety and colonization of VE202, to inform planned Phase 2 trials in UC patients. METHODS: After 5 days of vancomycin pretreatment at 125mg po QID to decrease gut microbial burden, 31 healthy adults received 1 dose at 2 dosage levels (6 VE202, 3 PBO per dosage level) or a 14-day course (9 VE202, 4 PBO) of the lower dose. Each VE202 delayed-release enteric capsule contained ∼1x109 CFU of bacteria. Safety was monitored through Week 12, with phone follow-up at Week 24. Stool was collected for LBP strain detection and exploratory microbiome, metabolomic, and immune markers. RESULTS: VE202 was well tolerated with no dose-dependent pattern of AEs. The only MedDRA organ classes with ≥20% reported AEs in VE202 recipients were: —Single-dose cohorts (N=12): GI (n=6; 50%), nervous system (n=4; 33.3%), investigations and infections/infestations (n=3 each; 25%) —14-day cohort (N=9): GI (n=5; 55.6%), general/administration site, musculoskeletal/connective tissue and nervous system (n=3 each; 33.3%), and skin/subcutaneous tissue (n=2; 22.2%) In VE202 recipients (N=21), the only AEs seen in ≥10% of subjects were abdominal discomfort, diarrhea, headache (n=4 each; 19.0%), and fatigue (n=3; 14.3%). No severe AEs or clinically significant trends in lab values were seen. Two SAEs, neither treatment-related, occurred several weeks after completion of VE202 dosing: abdominal pain caused by a suspected passed bile duct stone (Day 71) and hip pain leading to hip replacement (Day 120). Sustained VE202 strain detection (Figure 1) and relative abundance (Figure 2) in the vancomycin-perturbed gut was seen as soon as Day 2, sustained through 2 weeks after dosing completion, then declined slowly but remained substantially above baseline through Week 24. A dose- and duration-dependent trend in colonization was seen. CONCLUSION: VE202 is well tolerated in healthy adults and provides robust, durable, abundant colonization of LBP strains.

Supplemental Digital Content is available in the text., Objective: Perivascular adipose tissue (PVAT) plays a vital role in maintaining vascular homeostasis. However, most studies ascribed the function of PVAT in vascular remodeling to adipokines secreted by the perivascular adipocytes. Whether mesenchymal stem cells exist in PVAT and play a role in vascular regeneration remain unknown. Approach and Results: Single-cell RNA-sequencing allowed direct visualization of the heterogeneous PVAT-derived mesenchymal stem cells (PV-ADSCs) at a high resolution and revealed 2 distinct subpopulations, among which one featured signaling pathways crucial for smooth muscle differentiation. Pseudotime analysis of cultured PV-ADSCs unraveled their smooth muscle differentiation trajectory. Transplantation of cultured PV-ADSCs in mouse vein graft model suggested the contribution of PV-ADSCs to vascular remodeling through smooth muscle differentiation. Mechanistically, treatment with TGF-β1 (transforming growth factor β1) and transfection of microRNA (miR)-378a-3p mimics induced a similar metabolic reprogramming of PV-ADSCs, including upregulated mitochondrial potential and altered lipid levels, such as increased cholesterol and promoted smooth muscle differentiation. Conclusions: Single-cell RNA-sequencing allows direct visualization of PV-ADSC heterogeneity at a single-cell level and uncovers 2 subpopulations with distinct signature genes and signaling pathways. The function of PVAT in vascular regeneration is partly attributed to PV-ADSCs and their differentiation towards smooth muscle lineage. Mechanistic study presents miR-378a-3p which is a potent regulator of metabolic reprogramming as a potential therapeutic target for vascular regeneration.

In the phase 2 GALAHAD study (NCT02854436), the PARP inhibitor (PARPi) niraparib, was evaluated in heavily pre-treated patients with mCRPC and DNA-repair gene defects (DRD) who progressed after androgen-receptor (AR) targeted therapy and taxane-based chemotherapy. The results showed that objective response rate (ORR) was 34.2% for patients with measurable disease having biallelic BRCA1/2 alterations (n=76). The development of BRCA reversion mutations, a type of secondary mutation that restores protein function, has been proposed as a key resistance mechanism to PARP inhibition. We aimed to evaluate the relationship between reversion mutations and treatment response in BRCA1/2 altered patients treated with niraparib in the GALAHAD study. Thirty-three patients with biallelic BRCA alterations (excluding patients with homozygous deletions) had donated an end-of-treatment (EOT) ctDNA sample. We performed sequencing to detect reversion mutations using the Resolution Bioscience ctDx-HRD assay. Mutation patterns at baseline included splice site, nonsense, missense, and frameshift mutations, with the latter being the most common (24/33; 73%). No reversions were detected at baseline and the baseline BRCA alterations were detected at EOT for every patient. Most BRCA patients (28/33; 85%) had at least 1 reversion mutation (range: 1-38 different BRCA alterations) at EOT. Of the 28 patients with reversions, 5 were classified as low reversions: 3 patients had only one reversion mutation, 1 patient had 2 reversions at low allele frequency and 1 patient had 4 reversions at low allele frequency. Patients with reversion mutations showed better composite response (defined as ORR by RECIST 1.1, or CTC conversion to Citation Format: Karen Urtishak, Gerhardt Attard, Tokuwa Kanno, Shibu Thomas, Gary E. Mason, Byron Espina, Eugene Zhu, Natalie Hutnick, Mary Guckert, Adam del Corral, Mark Li, Angela Lopez-Gitlitz, Kim Chi, William Kevin Kelly, Evan Y. Yu, Karim Fizazi, Matthew Smith. High prevalence and heterogeneity of emergence of BRCA reversion mutations at progression on niraparib treatment in BRCA-mutant metastatic castration-resistant prostate cancer (mCRPC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4133.

INTRODUCTION The low FODMAP diet (LFD) reduces symptoms and bifidobacteria in irritable bowel syndrome (IBS). β-galactooligosaccharides (B-GOS) may reduce the symptoms and increase bifidobacteria in IBS. We investigated whether B-GOS supplementation alongside the LFD improves IBS symptoms while preventing the decline in bifidobacteria. METHODS We performed a randomized, placebo-controlled, 3-arm trial of 69 Rome III adult patients with IBS from secondary care in the United Kingdom. Patients were randomized to a sham diet with placebo supplement (control) or LFD supplemented with either placebo (LFD) or 1.4 g/d B-GOS (LFD/B-GOS) for 4 weeks. Gastrointestinal symptoms, fecal microbiota (fluorescent in situ hybridization and 16S rRNA sequencing), fecal short-chain fatty acids (gas-liquid chromatography) and pH (probe), and urine metabolites (H NMR) were analyzed. RESULTS At 4 weeks, adequate symptom relief was higher in the LFD/B-GOS group (16/24, 67%) than in the control group (7/23, 30%) (odds ratio 4.6, 95% confidence interval: 1.3-15.6; P = 0.015); Bifidobacterium concentrations (log10 cells/g dry weight) were not different between LFD and LFD/B-GOS but were lower in the LFD/B-GOS (9.49 [0.73]) than in the control (9.77 [0.41], P = 0.018). A proportion of Actinobacteria was lower in LFD (1.9%, P = 0.003) and LFD/B-GOS (1.8%, P < 0.001) groups than in the control group (4.2%). Fecal butyrate was lower in the LFD (387.3, P = 0.028) and LFD/B-GOS (346.0, P = 0.007) groups than in the control group (609.2). DISCUSSION The LFD combined with B-GOS prebiotic produced a greater symptom response than the sham diet plus placebo, but addition of 1.4 g/d B-GOS did not prevent the reduction of bifidobacteria. The LFD reduces fecal Actinobacteria and butyrate thus strict long-term use should not be advised.

Chronic disruption of the intestinal microbiota in adult cystic fibrosis (CF) patients is associated with local and systemic inflammation, and has been linked to the risk of serious comorbidities. Supplementation with high amylose maize starch (HAMS) might provide clinical benefit by promoting commensal bacteria and the biosynthesis of immunomodulatory metabolites. However, whether the disrupted CF gut microbiota has the capacity to utilise these substrates is not known. We combined metagenomic sequencing, in vitro fermentation, amplicon sequencing, and metabolomics to define the characteristics of the faecal microbiota in adult CF patients and assess HAMS fermentation capacity. Compared to healthy controls, the faecal metagenome of adult CF patients had reduced bacterial diversity and prevalence of commensal fermentative clades. In vitro fermentation models seeded with CF faecal slurries exhibited reduced acetate levels compared to healthy control reactions, but comparable levels of butyrate and propionate. While the commensal genus Faecalibacterium was strongly associated with short chain fatty acid (SCFA) production by healthy microbiota, it was displaced in this role by Clostridium sensu stricto 1 in the microbiota of CF patients. A subset of CF reactions exhibited enterococcal overgrowth, resulting in lactate accumulation and reduced SCFA biosynthesis. The addition of healthy microbiota to CF faecal slurries failed to displace predominant CF taxa, or substantially influence metabolite biosynthesis. Despite significant microbiota disruption, the adult CF gut microbiota retains the capacity to exploit HAMS. Our findings highlight the potential for taxa associated with the altered CF gut microbiotato mediate prebiotic effects in microbial systems subject to ongoing perturbation, irrespective of the depletion of common commensal clades.

The mycobacterial cell wall affords natural resistance to antibiotics. Antimicrobial peptides (AMPs) modify the surface properties of mycobacteria and can act synergistically with antibiotics from differing classes. Here we investigate the response ofMycobacterium smegmatisto the presence of rifampicin or capreomycin, either alone or in combination with two synthetic, cationic, α-helical AMPs; distinguished by the presence (D-LAK120-HP13) or absence (D-LAK120-A) of a kink-inducing proline. Using a combination of high-resolution magic angle spinning (HR-MAS) NMR of bacteria, diphenylhexatriene (DPH) fluorescence anisotropy and laurdan emission spectroscopy we show thatM. smegmatisresponds to challenge with rifampicin or capreomycin by substantially altering its metabolism and, in particular, by remodelling the cell envelope. In NMR spectra of bacteria, reductions in intensity for mycolic acid lipid −(CH2)-, -CH3, R2CH-COOH, R2CH-OH and also -CH2-(CH==CH)- and -CH=CH- resonances were observed following challenge with rifampicin and capreomycin, while the latter also caused an increase in trehalose. These changes are consistent with a reduction of trehalose dimycolate and increase of trehalose monomycolate and are associated with an increase in rigidity of the mycolic acid layer observed following challenge by capreomycin but not rifampicin. Challenge with D-LAK120-A or D-LAK120-HP13 induced no or modest changes respectively in these metabolites and did not induce a significant increase in rigidity of the mycolic acid layer. Further, the response to rifampicin or capreomycin was significantly reduced when these were combined respectively with D-LAK120-HP13 and D-LAK120-A, suggesting a possible mechanism for the synergy of these combinations. The remodelling of the mycomembrane inM. smegmatisis therefore identified as an important countermeasure deployed against rifampicin or capreomycin, but this can be mitigated, and rifampicin or capreomycin efficacy potentiated, by combining with AMPs.

Introduction Prebiotic β-galactooligosaccharides (B-GOS) may counteract the microbiome modifying effect of the low FODMAP diet (LFD) in patients with irritable bowel syndrome (IBS). Faecal metabolites may predict why only some patients respond to the LFD paving the way towards more personalised treatment. The aim of this randomised controlled trial (RCT) was to investigate: a) the impact of the LFD and LFD+1.4 g/d B-GOS compared to Control on the gut microbiome in IBS, and b) if differences in faecal or urinary metabolites predict response to the LFD. Methods A 3-arm RCT was performed in 69 IBS patients randomised to: Sham diet +Placebo (Control), LFD +Placebo (LFD) or LFD +B GOS. This study investigated global symptom response (adequate relief), gut microbiome (16S rRNA sequencing), short-chain fatty acids (SCFA, gas liquid chromatography), volatile organic compounds (VOC) (gas chromatography/mass spectrometry) and urine metabolomics (NMR spectroscopy) at baseline (BL) and 4 weeks. Data were analysed by Chi2 for dichotomous outcomes and Kruskal-WallisMann-Whitney U tests for continuous variables. Predictive analysis was tested using receiver operator curves (ROC) and orthoganol partial least squared discriminant analysis (OPLS-DA). Results Response rate (adequate relief) at 4 weeks was greater in the LFD+GOS (67%) and LFD (50%) than the Control group (30%) (p=0.046). The phylum Actinobacteria was lower (p Conclusions The LFD +B GOS improves symptoms in IBS compared to Control but the LFD significantly reduces Actinobacteria and addition of 1.4 g/d of prebiotic B-GOS does not overcome this effect. Faecal VOCs and SCFAs and urine metabolomes may predict clinical response to the LFD, and specific bacterial groups correlate with predictive metabolites. Larger studies are required to validate these algorithms to develop personalised nutrition in IBS.

Background: Antimicrobial peptides (AMPs) have the potential to act against multiple pathogenic targets. Results: AMPs that maintain conformational flexibility are more potent against multiple pathogens and less hemolytic. Conclusion: Antimicrobial action and hemolysis proceed via differing mechanisms. Significance: The potency, selectivity, and ability of AMPs to reach intracellular pathogens can be modulated using general principles., We used a combination of fluorescence, circular dichroism (CD), and NMR spectroscopies in conjunction with size exclusion chromatography to help rationalize the relative antibacterial, antiplasmodial, and cytotoxic activities of a series of proline-free and proline-containing model antimicrobial peptides (AMPs) in terms of their structural properties. When compared with proline-free analogs, proline-containing peptides had greater activity against Gram-negative bacteria, two mammalian cancer cell lines, and intraerythrocytic Plasmodium falciparum, which they were capable of killing without causing hemolysis. In contrast, incorporation of proline did not have a consistent effect on peptide activity against Mycobacterium tuberculosis. In membrane-mimicking environments, structures with high α-helix content were adopted by both proline-free and proline-containing peptides. In solution, AMPs generally adopted disordered structures unless their sequences comprised more hydrophobic amino acids or until coordinating phosphate ions were added. Proline-containing peptides resisted ordering induced by either method. The roles of the angle subtended by positively charged amino acids and the positioning of the proline residues were also investigated. Careful positioning of proline residues in AMP sequences is required to enable the peptide to resist ordering and maintain optimal antibacterial activity, whereas varying the angle subtended by positively charged amino acids can attenuate hemolytic potential albeit with a modest reduction in potency. Maintaining conformational flexibility improves AMP potency and selectivity toward bacterial, plasmodial, and cancerous cells while enabling the targeting of intracellular pathogens.