Extracellular and intracellular antiviral effects of ultraviolet A against severe acute respiratory syndrome coronavirus-2 are variant-independent.

Under controlled settings, narrow-band ultraviolet A (UVA) exposure exerts antiviral effects both in vivo and in vitro. The effect is thought to be mediated via direct effect on viral particles and indirectly, by modulation of metabolic pathways of host cells. We aimed to explore the extracellular and intracellular antiviral effects of UVA exposure against Alpha, Beta, and Delta variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: Vero E6 kidney normal epithelial cells and human tracheal epithelial cells were infected with Alpha, Beta, and Delta variants in a BSL-3 laboratory. To assess extracellular effects, SARS-CoV-2 variants were directly exposed to a single dose of UVA prior to infection of the host cells (Vero E6 kidney normal epithelial cells and human tracheal epithelial cells) The intracellular effects of UVA were assessed by first infecting the cells with SARS-CoV-2 variants followed by UVA treatment of infected cell monolayers. Efficacy was quantified by both plaque reduction assay and quantitative real-time polymerase chain reaction. Additionally, transcriptomic analysis was performed on exposed Vero E6 cells to assess differentially expressed genes and canonical pathways as compared to controls. RESULTS: SARS-CoV-2 Alpha, Beta and Delta variants are susceptible to UVA exposure prior to infection of Vero E6 cells. Importantly, the UVA-driven reduction in Delta variant load could be reproduced in human primary tracheal cells. Beta and Delta variants load also significantly decreased during Vero E6 cells intracellular experiments. UVA-driven reductions in viral loads ameliorate several host metabolic pathways, including canonical pathways related to viral infection and interferon signaling. CONCLUSION: Narrow-band UVA exhibits both extracellular effects on SARS-CoV-2 viral particles and intracellular effects on infected cells with SARS-CoV-2. Efficacy appears to be variant independent.

Defining Small Intestinal Bacterial Overgrowth by Culture and High Throughput Sequencing.

BACKGROUND& AIMS: Despite accelerated research in small intestinal bacterial overgrowth (SIBO), questions remain regarding optimal diagnostic approaches and definitions. Here, we aim to define SIBO using small bowel culture and sequencing, identifying specific contributory microbes, in the context of gastrointestinal symptoms. METHODS: Subjects undergoing esophagogastroduodenoscopy (without colonoscopy) were recruited and completed symptom severity questionnaires. Duodenal aspirates were plated on MacConkey and blood agar. Aspirate DNA was analyzed by 16S ribosomal RNA and shotgun sequencing. Microbial network connectivity for different SIBO thresholds and predicted microbial metabolic functions were also assessed. RESULTS: A total of 385 subjects with <103 colony forming units (CFU)/mL on MacConkey agar and 98 subjects with ≥103 CFU/mL, including ≥103 to <105 CFU/mL (N = 66) and ≥105 CFU/mL (N = 32), were identified. Duodenal microbial α-diversity progressively decreased, and relative abundance of Escherichia/Shigella and Klebsiella increased, in subjects with ≥103 to <105 CFU/mL and ≥105 CFU/mL. Microbial network connectivity also progressively decreased in these subjects, driven by the increased relative abundance of Escherichia (P < .0001) and Klebsiella (P = .0018). Microbial metabolic pathways for carbohydrate fermentation, hydrogen production, and hydrogen sulfide production were enhanced in subjects with ≥103 CFU/mL and correlated with symptoms. Shotgun sequencing (N = 38) identified 2 main Escherichia coli strains and 2 Klebsiella species representing 40.24% of all duodenal bacteria in subjects with ≥103 CFU/mL. CONCLUSIONS: Our findings confirm ≥103 CFU/mL is the optimal SIBO threshold, associated with gastrointestinal symptoms, significantly decreased microbial diversity, and network disruption. Microbial hydrogen- and hydrogen sulfide-related pathways were enhanced in SIBO subjects, supporting past studies. Remarkably few specific E coli and Klebsiella strains/species appear to dominate the microbiome in SIBO, and correlate with abdominal pain, diarrhea, and bloating severities.

Coronavirus Disease 2019 (COVID-19) Pandemic Lifestyle Changes May Have Influenced Small Bowel Microbial Composition and Microbial Resistance.

BACKGROUND: The coronavirus disease 2019 (COVID-19) global pandemic necessitated many severe lifestyle changes, including lockdowns, social distancing, altered food consumption and exercise patterns, and extensive hygiene practices. These extensive changes may have affected the human gut microbiome, which is highly influenced by lifestyle. AIMS: To examine the potential effects of pandemic-related lifestyle changes on the metabolically relevant small bowel microbiome. METHODS: Adult subjects presenting for upper endoscopy without colonoscopy were identified and divided into two matched groups: pre-pandemic (February 2019-March 2020) and intra-pandemic (April 2021-September 2021, all COVID-19 negative). Duodenal aspirates and blood samples were collected. Duodenal microbiomes were analyzed by 16S rRNA sequencing. Serum cytokine levels were analyzed by Luminex FlexMap3D. RESULTS: Fifty-six pre-pandemic and 38 COVID-negative intra-pandemic subjects were included. There were no significant changes in duodenal microbial alpha diversity in the intra-pandemic vs. pre-pandemic group, but beta diversity was significantly different. The relative abundance (RA) of phylum Deinococcus-Thermus and family Thermaceae, which are resistant extremophiles, was significantly higher in the intra-pandemic vs. pre-pandemic group. The RA of several Gram-negative taxa including Bacteroidaceae (phylum Bacteroidetes) and the Proteobacteria families Enterobacteriaceae and Pseudomonadaceae, and the RA of potential disruptor genera Escherichia-Shigella and Rothia, were significantly lower in the intra-pandemic vs. pre-pandemic group. Circulating levels of interleukin-18 were also lower in the intra-pandemic group. CONCLUSIONS: These findings suggest the small bowel microbiome underwent significant changes during the pandemic, in COVID-19-negative individuals. Given the key roles of the small bowel microbiota in host physiology, this may have implications for human health.

Prevalence of Small Intestinal Bacterial Overgrowth Syndrome in Patients with Non-Alcoholic Fatty Liver Disease/Non-Alcoholic Steatohepatitis: A Cross-Sectional Study.

INTRODUCTION: Non-alcoholic fatty liver disease (NAFLD) is a multifactorial, wide-spectrum liver disorder. Small intestinal bacterial overgrowth (SIBO) is characterized by an increase in the number and/or type of colonic bacteria in the upper gastrointestinal tract. SIBO, through energy salvage and induction of inflammation, may be a pathophysiological factor for NAFLD development and progression. AIM/METHODS: Consecutive patients with histological, biochemical, or radiological diagnosis of any stage of NAFLD (non-alcoholic fatty liver [NAFL], non-alcoholic steatohepatitis [NASH], cirrhosis) underwent upper gastrointestinal endoscopy. Duodenal fluid (2cc) was aspirated from the 3rd-4th part of duodenum into sterile containers. SIBO was defined as ≥103 aerobic colony-forming units (CFU)/mL of duodenal aspirate and/or the presence of colonic-type bacteria. Patients without any liver disease undergoing gastroscopy due to gastroesophageal reflux disease (GERD) comprised the healthy control (HC) group. Concentrations (pg/mL) of tumor necrosis factor alpha (TNFα), interleukin (IL)-1ß, and IL-6 were also measured in the duodenal fluid. The primary endpoint was to evaluate the prevalence of SIBO in NAFLD patients, while the comparison of SIBO prevalence among NAFLD patients and healthy controls was a secondary endpoint. RESULTS: We enrolled 125 patients (51 NAFL, 27 NASH, 17 cirrhosis, and 30 HC) aged 54 ± 11.9 years and with a weight of 88.3 ± 19.6 kg (NAFLD vs. HC 90.7 ± 19.1 vs. 80.8 ± 19.6 kg, p = 0.02). Overall, SIBO was diagnosed in 23/125 (18.4%) patients, with Gram-negative bacteria being the predominant species (19/23; 82.6%). SIBO prevalence was higher in the NAFLD cohort compared to HC (22/95; 23.2% vs. 1/30; 3.3%, p = 0.014). Patients with NASH had higher SIBO prevalence (6/27; 22.2%) compared to NAFL individuals (8/51; 15.7%), but this difference did not reach statistical significance (p = 0.11). Patients with NASH-associated cirrhosis had a higher SIBO prevalence compared to patients with NAFL (8/17; 47.1% vs. 8/51; 15.7%, p = 0.02), while SIBO prevalence between patients with NASH-associated cirrhosis and NASH was not statistically different (8/17; 47.1% vs. 6/27; 22.2%, p = 0.11). Mean concentration of TNF-α, IL-1ß, and IL-6 did not differ among the different groups. CONCLUSION: The prevalence of SIBO is significantly higher in a cohort of patients with NAFLD compared to healthy controls. Moreover, SIBO is more prevalent in patients with NASH-associated cirrhosis compared to patients with NAFL.

The Impact of the CTHRSSVVC Peptide Upon Experimental Models of Trypanosoma cruzi Infection.

Chagas disease (CD), caused by the hemoflagellate protozoan Trypanosoma cruzi, affects more than six million people worldwide and presents an unsatisfactory therapy, based on two nitroderivatives, introduced in clinical medicine for decades. The synthetic peptide, with CTHRSSVVC sequence (PepA), mimics the CD163 and TNF-α tripeptide "RSS" motif and binds to atheromatous plaques in carotid biopsies of human patients, spleen tissues, and a low-density lipoprotein receptor knockout (LDLr-/-) mouse model of atherosclerosis. CD163 receptor is present on monocytes, macrophages, and neutrophils, acting as a regulator of acute-phase processes and modulating aspects of the inflammatory response and the establishment of infections. Due to the potential theranostic role of PepA, our aim was to investigate its effect upon T. cruzi infection in vitro and in vivo. PepA and two other peptides with shuffled sequences were assayed upon different binomials of host cell/parasite, including professional [as peritoneal mouse macrophages (PMM)] and non-professional phagocytes [primary cultures of cardiac cells (CM)], under different protocols. Also, their impact was further addressed in vivo using a mouse model of acute experimental Chagas disease. Our in-vitro findings demonstrate that PepA and PepB (the peptide with random sequence retaining the "RS" sequence) reduced the intracellular parasitism of the PMM but were inactive during the infection of cardiac cells. Another set of in-vitro and in-vivo studies showed that they do not display a trypanocidal effect on bloodstream trypomastigotes nor exhibit in-vivo efficacy when administered after the parasite inoculation. Our data report the in-vitro activity of PepA and PepB upon the infection of PMM by T. cruzi, possibly triggering the microbicidal arsenal of the host professional phagocytes, capable of controlling parasitic invasion and proliferation.

Smoking has disruptive effects on the small bowel luminal microbiome.

Tobacco use is the leading preventable cause of cancer, and affects the respiratory, oral, fecal, and duodenal mucosa-associated microbiota. However, the effects of smoking on the duodenal luminal microbiome have not been studied directly. We aimed to compare the duodenal luminal microbiome in never-smokers, current smokers, and ex-smokers who quit ≥ 10 years ago. In a cross-sectional study, current smokers (CS, n = 24) were identified and matched to never-smokers (NS, n = 27) and ex-smokers (XS, n = 27) by age (± 5 years), body mass index (BMI, ± 3 kg/m2), and sex. Current antibiotic users were excluded. The duodenal luminal microbiome was analysed in 1 aspirate sample per subject by 16S rRNA gene sequencing. Relative abundances (RA) of families associated with increased duodenal microbial diversity, Prevotellaceae, Neisseriaceae, and Porphyromonadaceae, were significantly lower in CS vs. NS. This was driven by lower RA of unknown Prevotella and Porphyromonas species, and Neisseria subflava and N. cinerea, in CS. In contrast, RA of Enterobacteriaceae and Lactobacillaceae (associated with decreased diversity), were significantly higher in CS, due to higher RA of Escherichia-Shigella, Klebsiella and Lactobacillus species. Many of these changes were absent or less pronounced in XS, who exhibited a duodenal luminal microbiome more similar to NS. RA of taxa previously found to be increased in the oral and respiratory microbiota of smokers were also higher in the duodenal luminal microbiome, including Bulledia extructa and an unknown Filifactor species. In conclusion, smoking is associated with an altered duodenal luminal microbiome. However, ex-smokers have a duodenal luminal microbiome that is similar to never-smokers.

Duodenal microbiome changes in postmenopausal women: effects of hormone therapy and implications for cardiovascular risk.

OBJECTIVE: Hormone therapy (HT) is used to treat menopause-related conditions and symptoms. The small intestine plays key roles in metabolic and endocrine function, but the effects of HT on the small intestinal microbiome are unknown. Here, we characterize duodenal microbiome differences, and the effects of HT, in postmenopausal women. METHODS: Female participants undergoing esophagogastroduodenoscopy who were postmenopausal and taking HT (HT+), postmenopausal but not taking HT (HT-), or of reproductive age and not taking exogenous hormones (RA), were identified and matched for body mass index (±3 kg/m2). DNAs were isolated from duodenal aspirates obtained during upper endoscopy. V3 and V4 libraries were used for 16S rRNA sequencing. Serum hormone levels were analyzed by Luminex FlexMap. RESULTS: The core duodenal microbiome was different in HT- participants (n = 12) when compared with RA participants (n = 10), but more similar in HT+ (n = 13) and RA participants. HT- participants had increased Proteobacteria taxa, leading to greater microbial dysbiosis compared with HT+ participants, and had decreased prevalence of Bacteroidetes, which was associated with higher fasting glucose levels, lower duodenal microbial diversity, and lower testosterone levels. HT+ participants had significantly higher estradiol (P = 0.04) and progesterone (P = 0.04), and lower fasting glucose (P = 0.03), than HT- participants, and had increased relative abundance of Prevotella (P = 0.01), and decreased Escherichia (P = 1.12E-7), Klebsiella (P = 5.93E-7), and Lactobacillus (P = 0.02), all associated with lower cardiovascular disease risks. CONCLUSIONS: These findings support previous studies suggesting that HT may have beneficial effects following menopause, and although preliminary, may also support a beneficial effect of HT on the duodenal microbiome.

Effects of Proton Pump Inhibitors on the Small Bowel and Stool Microbiomes.

BACKGROUND: Proton pump inhibitor (PPI) use is extremely common. PPIs have been suggested to affect the gut microbiome, and increase risks of Clostridium difficile infection and small intestinal bacterial overgrowth (SIBO). However, existing data are based on stool analyses and PPIs act on the foregut. AIMS: To compare the duodenal and stool microbiomes in PPI and non-PPI users. METHODS: Consecutive subjects presenting for upper endoscopy without colonoscopy were recruited. Current antibiotic users were excluded. Subjects taking PPI were age- and gender-matched 1:2 to non-PPI controls. Subjects completed medical history questionnaires, and duodenal aspirates were collected using a validated protected catheter. A subset also provided stool samples. Duodenal and stool microbiomes were analyzed by 16S rRNA sequencing. RESULTS: The duodenal microbiome exhibited no phylum-level differences between PPI (N = 59) and non-PPI subjects (N = 118), but demonstrated significantly higher relative abundances of families Campylobacteraceae (3.13-fold, FDR P value < 0.01) and Bifidobacteriaceae (2.9-fold, FDR P value < 0.01), and lower relative abundance of Clostridiaceae (88.24-fold, FDR P value < 0.0001), in PPI subjects. SIBO rates were not significantly different between groups, whether defined by culture (> 103 CFU/ml) or 16S sequencing, nor between subjects taking different PPIs. The stool microbiome exhibited significantly higher abundance of family Streptococcaceae (2.14-fold, P = 0.003), and lower Clostridiaceae (2.60-fold, FDR P value = 8.61E-13), in PPI (N = 22) versus non-PPI (N = 47) subjects. CONCLUSIONS: These findings suggest that PPI use is not associated with higher rates of SIBO. Relative abundance of Clostridiaceae was reduced in both the duodenal and stool microbiomes, and Streptococcaceae was increased in stool. The clinical implications of these findings are unknown.

Ultraviolet-A light increases mitochondrial anti-viral signaling protein in confluent human tracheal cells via cell-cell signaling.

Mitochondrial antiviral signaling (MAVS) protein mediates innate antiviral responses, including responses to certain coronaviruses such as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We have previously shown that ultraviolet-A (UVA) therapy can prevent virus-induced cell death in human ciliated tracheal epithelial cells (HTEpC) infected with coronavirus-229E (CoV-229E), and results in increased intracellular levels of MAVS. In this study, we explored the mechanisms by which UVA light can activate MAVS, and whether local UVA light application can activate MAVS at locations distant from the light source (e.g. via cell-to-cell communication). MAVS levels were compared in HTEpC exposed to 2 mW/cm2 narrow band (NB)-UVA for 20 min and in unexposed controls at 30-40% and at 100% confluency, and in unexposed HTEpC treated with supernatants or lysates from UVA-exposed cells or from unexposed controls. MAVS was also assessed in different sections of confluent monolayer plates where only one section was exposed to NB-UVA. Our results showed that UVA increases the expression of MAVS protein. Further, cells in a confluent monolayer exposed to UVA conferred an elevation in MAVS in cells adjacent to the exposed section, and also in cells in the most distant sections which were not exposed to UVA. In this study, human ciliated tracheal epithelial cells exposed to UVA demonstrate increased MAVS protein, and also appear to transmit this influence to confluent cells not exposed to UVA, likely via cell-cell signaling.

Ultraviolet-A light reduces cellular cytokine release from human endotracheal cells infected with Coronavirus.

BACKGROUND: An important clinical feature of coronavirus disease 2019 (COVID-19) is hypercytokinemia (cytokine storm). We previously showed that narrow band ultraviolet-A (NB-UVA) treatment salvages coronavirus (CoV)-229E-infected human tracheal cells, and that daily endotracheal NB-UVA therapy reduced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) levels in human subjects, with improved clinical outcomes. Here, we examined NB-UVA effects on cytokine release during CoV-229E infection. METHODS: Primary human tracheal epithelial cells were transfected with CoV-229E, then exposed to 2 mW/cm2 NB-UVA for 20 minutes every 24h, either 3 or 4 times. Secreted cytokine/chemokine levels were analyzed in supernatants collected from CoV-229E-infected/UVA-exposed cells 24h after the last UVA treatment, and from matched non-infected/UVA-exposed controls, CoV-229E-infected/non-exposed controls, and non-infected/non-exposed (naïve) controls. Metabolic pathway/downstream prediction analyses were also performed. RESULTS: Pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF), and chemokines IL-8, monocyte chemoattractant protein-1 (MCP1), and interferon gamma-induced protein 10 (IP-10), were significantly increased in CoV-229E-infected cells, and significantly decreased following NB-UVA treatment. Interferon (IFN)-α2, IFN-γ, and IL-10 were not upregulated in response to CoV-229E. Metabolic pathway predictions indicated hypercytokinemia as the top inflammatory response in CoV-229E-infected cells, whereas the top predicted pathway in CoV-229E-infected/UVA-exposed cells was the recovery stage of severe acute respiratory syndrome. CONCLUSIONS: Human tracheal epithelial cells infected with CoV-229E showed reduced cytokine secretions including IL-6, TNF, IL-8, and MCP-1, following NB-UVA exposure. This reduction of cytokine levels in vitro, coupled with previously identified reduced cell death in CoV-229E-infected/UVA-exposed cells, suggests that determining UVA effects on cytokine storm in human SARS-Co-V2 patients is warranted.

The duodenal microbiome is altered in small intestinal bacterial overgrowth.

Small intestinal bacterial overgrowth (SIBO) is highly prevalent and is associated with numerous gastrointestinal disorders, but the microbes involved remain poorly defined. Moreover, existing studies of microbiome alterations in SIBO have utilized stool samples, which are not representative of the entire gastrointestinal tract. Therefore, we aimed to determine and compare the duodenal microbiome composition in SIBO and non-SIBO subjects, using duodenal aspirates from subjects undergoing standard-of-care esophagogastroduodenoscopy without colon preparation. Using the recently-redefined cutoff for SIBO of >103 colony forming units per milliliter (CFU/mL), 42 SIBO and 98 non-SIBO subjects were identified. Duodenal samples from SIBO subjects had 4x103-fold higher counts than non-SIBO subjects when plated on MacConkey agar (P<0.0001), and 3.8-fold higher counts when plated on blood agar (P<0.0001). Twenty subjects had also undergone lactulose hydrogen breath tests (LHBTs), of whom 7/20 had SIBO. At the 90-minute timepoint, 4/7 SIBO subjects had positive LHBTs (rise in hydrogen (H2) ≥ 20 ppm above baseline), as compared to 2/13 non-SIBO subjects. 16S ribosomal RNA (rRNA) sequencing revealed that SIBO subjects had 4.31-fold higher relative abundance of Proteobacteria (FDR P<0.0001) and 1.64-fold lower Firmicutes (P<0.0003) than non-SIBO subjects. This increased relative abundance of Proteobacteria correlated with decreased α-diversity in SIBO subjects (Spearman R = 0.4866, P<0.0001) Specific increases in class Gammaproteobacteria correlated with the area-under-the-curve for H2 for 0-90 mins during LHBT (R = 0.630, P = 0.002). Increases in Gammaproteobacteria resulted primarily from higher relative abundances of the family Enterobacteriaceae (FDR P<0.0001), which correlated with the symptom of bloating (Spearman R = 0.185, 2-tailed P = 0.028). Increases in family Aeromonadaceae correlated with urgency with bowel movement (Spearman R = 0.186, 2-tailed P = 0.028). These results validate the >103 CFU/mL cutoff for the definition of SIBO, and also reveal specific overgrowth of Proteobacteria in SIBO vs. non-SIBO subjects, coupled with an altered Proteobacterial profile that correlates with symptom severity. Future research may elucidate host-microbiome interactions underlying these symptoms in SIBO patients.

Ultraviolet A light effectively reduces bacteria and viruses including coronavirus.

Antimicrobial-resistant and novel pathogens continue to emerge, outpacing efforts to contain and treat them. Therefore, there is a crucial need for safe and effective therapies. Ultraviolet-A (UVA) phototherapy is FDA-approved for several dermatological diseases but not for internal applications. We investigated UVA effects on human cells in vitro, mouse colonic tissue in vivo, and UVA efficacy against bacteria, yeast, coxsackievirus group B and coronavirus-229E. Several pathogens and virally transfected human cells were exposed to a series of specific UVA exposure regimens. HeLa, alveolar and primary human tracheal epithelial cell viability was assessed after UVA exposure, and 8-Oxo-2'-deoxyguanosine was measured as an oxidative DNA damage marker. Furthermore, wild-type mice were exposed to intracolonic UVA as an in vivo model to assess safety of internal UVA exposure. Controlled UVA exposure yielded significant reductions in Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Enterococcus faecalis, Clostridioides difficile, Streptococcus pyogenes, Staphylococcus epidermidis, Proteus mirabilis and Candida albicans. UVA-treated coxsackievirus-transfected HeLa cells exhibited significantly increased cell survival compared to controls. UVA-treated coronavirus-229E-transfected tracheal cells exhibited significant coronavirus spike protein reduction, increased mitochondrial antiviral-signaling protein and decreased coronavirus-229E-induced cell death. Specific controlled UVA exposure had no significant effect on growth or 8-Oxo-2'-deoxyguanosine levels in three types of human cells. Single or repeated in vivo intraluminal UVA exposure produced no discernible endoscopic, histologic or dysplastic changes in mice. These findings suggest that, under specific conditions, UVA reduces various pathogens including coronavirus-229E, and may provide a safe and effective treatment for infectious diseases of internal viscera. Clinical studies are warranted to further elucidate the safety and efficacy of UVA in humans.

Acute appendicitis is associated with appendiceal microbiome changes including elevated Campylobacter jejuni levels.

OBJECTIVES: To compare the appendiceal microbiomes and examine the prevalence of Campylobacter species in the appendices of adult subjects with confirmed acute non-perforated appendicitis and controls with healthy appendices. DESIGN: Archived samples of formalin-fixed paraffin-embedded appendiceal tissues were obtained from 50 consecutive female subjects who underwent appendectomy for acute, non-perforated appendicitis, and 35 consecutive female controls who underwent incidental appendectomy during gynaecological surgery. RESULTS: 16S rRNA gene sequencing revealed that the relative abundances (RAs) of the major phyla in appendiceal tissues (Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria) were similar in both groups. Beta diversity was significantly different due to differences in Bacteroidetes and Proteobacteria (p<0.0001). Within Proteobacteria, RAs of classes Alphaproteobacteria (~21%, fold change (FC)=1.31, false discovery rate (FDR) p value=0.03) and Epsilonproteobacteria (~1%, FC=0.25, FDR p value>0.05) were increased in acute appendicitis samples. RAs of unknown genera from families Burkholderiaceae and Enterobacteriaceae were decreased in appendicitis samples, and 14 genera were increased, including Neisseria, Acinetobacter and Campylobacter. Quantitative PCR revealed that levels of Campylobacter jejuni DNA, but not other Campylobacter species or pathogens tested, were significantly higher in appendicitis samples than in controls (p=0.013). Using a cut-off of 0.31 pg/µL, 40% of appendicitis cases and 6% of controls were positive for C. jejuni, indicating specificity of 93.7% (95% Cl 79.2 to 99.2), sensitivity of 40.9% (95% Cl 24.7 to 54.5), and OR of 10.38 (Fisher's p value=0.0006, 95% Cl 2.3 to 47.4). CONCLUSIONS: Our findings indicate that Campylobacter jejuni may be a significant cause of acute appendicitis. This supports earlier studies and suggests that targeted antibiotic therapies could be an alternative treatment for a subset of non-complicated acute appendicitis cases.

Optimizing microbiome sequencing for small intestinal aspirates: validation of novel techniques through the REIMAGINE study.

BACKGROUND: The human small intestine plays a central role in the processes of digestion and nutrient absorption. However, characterizations of the human gut microbiome have largely relied on stool samples, and the associated methodologies are ill-suited for the viscosity and low microbial biomass of small intestine samples. As part of the REIMAGINE study to examine the specific roles of the small bowel microbiome in human health and disease, this study aimed to develop and validate methodologies to optimize microbial analysis of the small intestine. RESULTS: Subjects undergoing esophagogastroduodenoscopy without colon preparation for standard of care were prospectively recruited, and ~ 2 ml samples of luminal fluid were obtained from the duodenum using a custom sterile aspiration catheter. Samples of duodenal aspirates were either untreated (DA-U, N = 127) or pretreated with dithiothreitol (DA-DTT, N = 101), then cultured on MacConkey agar for quantitation of aerobic gram-negative bacteria, typically from the class Gammaproteobacteria, and on blood agar for quantitation of anaerobic microorganisms. DA-DTT exhibited 2.86-fold greater anaerobic bacterial counts compared to DA-U (P = 0.0101), but were not statistically different on MacConkey agar. DNA isolation from DA-U (N = 112) and DA-DTT (N = 43) samples and library preparation for 16S rRNA gene sequencing were also performed using modified protocols. DA-DTT samples exhibited 3.81-fold higher DNA concentrations (P = 0.0014) and 4.18-fold higher 16S library concentrations (P < 0.0001) then DA-U samples. 16S rRNA gene sequencing revealed increases in the detected relative abundances of obligate and facultative anaerobes in DA-DTT samples, including increases in the genera Clostridium (false discovery rate (FDR) P = 4.38E-6), Enterococcus (FDR P = 2.57E-8), Fusobacterium (FDR P = 0.02) and Bacteroides (FDR P = 5.43E-9). Detected levels of Gram-negative enteropathogens from the phylum Proteobacteria, such as Klebsiella (FDR P = 2.73E-6) and Providencia (FDR P < 0.0001) (family Enterobacteriaceae) and Pseudomonas (family Pseudomonadaceae) (FDR P = 0.04), were also increased in DA-DTT samples. CONCLUSIONS: This study validates novel DTT-based methodology which optimizes microbial culture and 16S rRNA gene sequencing for the study of the small bowel microbiome. The microbial analyses indicate increased isolation of facultative and obligate anaerobes from the mucus layer using these novel techniques.

Effect of High Voltage Pulsed Current on the integration of total skin grafts in rats submitted to nicotine action.

OBJECTIVE: The aim of this study was to evaluate the impact of High Voltage Pulsed Current (HVPC) on the integration of total skin grafts in rats submitted to nicotine action. MATERIALS AND METHODS: For this purpose, 60 adult Wistar rats randomly distributed in 6 groups of 10 animals were analyzed. The electrical stimulation (anodic and cathodic stimulation, motor level, 30 min at 10 Hz; minimum voltage 20 µs and 100 µs pulse interval) was applied for seven days, starting on the third day after surgery and after the dressing was removed from the graft. RESULTS: Anodic HVPC promoted greater graft integration, demonstrating a lower percentage of tissue contraction, a lower number of inflammatory infiltrates and a greater amount of vascular endothelial growth factor (VEGF), as well as a higher number of newly formed blood vessels. CONCLUSIONS: HVPC can positively influence the integration of skin grafts in nicotine-treated rats. anodic HVPC is shown to promote greater integration in relation to a lower percentage of tissue contraction, a lower number of inflammatory infiltrates and a greater amount of vascular endothelial growth factor and newformed blood vessels. Whereas, the cathodic polarity has presented smaller amount of tissue gap.

Extent of rescue of F508del-CFTR function by VX-809 and VX-770 in human nasal epithelial cells correlates with SNP rs7512462 in SLC26A9 gene in F508del/F508del Cystic Fibrosis patients.

BACKGROUND: We analyzed the CFTR response to VX-809/VX-770 drugs in conditionally reprogrammed cells (CRC) of human nasal epithelium (HNE) from F508del/F508del patients based on SNP rs7512462 in the Solute Carrier Family 26, Member 9 (SLC26A9; MIM: 608481) gene. METHODS: The Isc-eq measurements of primary nasal epithelial cells from F508del/F508del patients (n = 12) for CFTR function were performed in micro Ussing chambers and compared with non-CF controls (n = 2). Data were analyzed according to the rs7512462 genotype which were determined by real-time PCR. RESULTS: The CRC-HNE cells from F508del/F508del patients evidenced high variability in the basal levels of CFTR function. Also, the rs7512462*C allele showed an increased basal CFTR function and higher responses to VX-809 + VX-770. The rs7512462*CC + CT genotypes together evidenced CFTR function levels of 14.89% relatively to wt/wt (rs7512462*CT alone-15.29%) i.e., almost double of rs7512462*TT (7.13%). Furthermore, sweat [Cl-] and body mass index of patients also evidenced an association with the rs7512462 genotype. CONCLUSION: The CFTR function can be performed in F508del/F508del patient-derived CRC-HNEs and its function and responses to VX-809 + VX-770 combination as well as clinical data, are all associated with the rs7512462 variant, which partially sheds light on the generally inter-individual phenotypic variability and in personalized responses to CFTR modulator drugs.

Analysis of Circulating miR-1, miR-23a, and miR-26a in Atrial Fibrillation Patients Undergoing Coronary Bypass Artery Grafting Surgery.

Atrial fibrillation (AF) is the most common arrhythmia after cardiac surgery. From a pathophysiological point of view, a myriad of factors such as trauma, atrial dilation, ischemia, mechanical myopericarditis, autonomic imbalance, loss of connexins, AF nest remodeling, inflammation, sutures, and dysfunction caused by postextracorporeal circulation can contribute to postoperative atrial fibrillation (POAF) resulting in a longer hospital stay and consequently higher cost. Recent studies showed that short fragments of RNA, called microRNA (miRNA), can contribute to the development of several cardiovascular diseases, including AF. The aim of this study was to evaluate the levels of circulating miRNAs (miR-1, -23a, and -26a) that can be involved in POAF. Patients submitted to coronary artery bypass graft surgery were grouped in POAF (24 patients) and without POAF (24 patients). Results showed older age, longer clamp-time, and more days in the intensive care unit as well as a longer total hospital stay in the POAF group. Preoperative levels of circulating miRNAs were similar. Analysis of miRNAs revealed significantly lower circulating levels of miRNA-23a (P = 0.02) and -26a (P = 0.01) in the POAF group during the postoperative period. Receiver operating characteristic (ROC) analysis showed the area under the ROC curve of miR-23a and miR-26a for predicting FA was 0.63 (95% confidence interval [CI]: 0.51-0.74; P = 0.02) and 0.66 (95% CI: 0.55-0.77; P = 0.01), respectively. Our data suggests that circulating miRNA-23a and -26a may be involved in the underlying biology of postoperative AF development.

The Peer and Non-peer: the potential of risk management for HIV prevention in contexts of prostitution / O Par e o Ímpar: o potencial de gestão de risco para a prevenção de DST/HIV/AIDS em contextos de prostituição

ABSTRACT Sex workers have been the protagonists and focus of HIV prevention campaigns and research since the late 1980s in Brazil. Through a review of national and international literature, combined with a history of sex workers' involvement in the construction of the Brazilian response, this article explores the overlaps and disconnects between research and practice in contexts of prostitution over the past three decades. We review the scientific literature on the epidemiology of HIV among sex workers and prevention methodologies. We conclude that although research focus and designs often reinforce the idea that sex workers' vulnerability is due to their sexual relationships with clients, their greatest vulnerability has been found to be with their nonpaying intimate partners. Few studies explore their work contexts and structural factors that influence safe sex practices with both types of partners. The negative effects of criminalization, stigma, and exclusively biomedical and peer education-based approaches are well documented in the scientific literature and experiences of sex worker activists, as is the importance of prevention programs that combine empowerment and human rightsbased approach to reduce HIV infection rates. We conclude that there is a need for actions, policies, and research that encompass the environment and context of sex workers' lives and reincorporate the human rights and citizenship frame that dominated the Brazilian response until the end of the 2000s. As part of HIV prevention efforts, female sex workers need to be considered above all as women, equal to all others.

RESUMO Prostitutas têm sido protagonistas e foco de campanhas de prevenção de HIV desde o final da década de 1980 no Brasil. Com base em um levantamento da literatura nacional e internacional, combinada com a trajetória do movimento de prostitutas na construção da resposta brasileira à epidemia, este artigo explora as sobreposições e incoerências entre pesquisa e prática em contextos de prostituição nas últimas três décadas. Na revisão da literatura científica, verificamos que a maior vulnerabilidade desse grupo social ocorre com os parceiros íntimos, não-comerciais; entretanto, o foco das pesquisas e a forma que são feitas geralmente reforçam a ideia de que a vulnerabilidade decorre de seus clientes. Ao mesmo tempo, há poucos estudos sobre seus contextos de trabalho e fatores estruturais que influenciam práticas sexuais mais seguras com ambos os tipos de parceiros. Os efeitos negativos da criminalização, do estigma, e de abordagens exclusivamente biomédicas e baseadas de uma forma isolada na metodologia de educação pelos pares estão bem documentados na literatura científica e nas experiências de ativistas, assim como a importância de programas de prevenção baseados em direitos humanos e sexuais. Concluímos que há necessidade de ações, políticas e pesquisas que incluam o ambiente e contexto nos quais profissionais do sexo trabalham, que reincorporem o arcabouço de direitos humanos e cidadania que dominou a resposta brasileira até o final da década de 2000, e que prostitutas devem ser consideradas e tratadas como mulheres, iguais a todas as outras.

Tradução e adaptação cultural do instrumento Supportive Care Needs Survey Short Form 34 / Translation and cultural adaptation of the Supportive Care Needs Survey Short Form 34

O presente estudo tem como objetivo traduzir para a língua portuguesa e adaptar culturalmente o instrumento Supportive Care Needs Survey ­ Short Form (SCNS-SF34), o qual é composto por 34 questões, desenvolvido para avaliar as diferentes áreas de necessidades para o paciente com câncer. É estruturado em cinco domínios: Psicológico; Sistema de Saúde e Informação; Físico e Vida Diária; Suporte e Cuidado; e Sexualidade. A pesquisa foi realizada na Central de Quimioterapia do A.C. Camargo Cancer Center. Foi um estudo descritivo, caracterizado por duas etapas: a adaptação cultural do instrumento SCNS-SF34; e a análise das propriedades psicométricas (validade de construto e confiabilidade) da versão adaptada. A população é constituída de pacientes portadores de câncer com diagnóstico realizado há, pelo menos, três meses, que estejam em quimioterapia, atendidos na Central de Quimioterapia. Foi utilizada uma amostra não probabilística. Para a avaliação da validade de construto foi realizada a análise fatorial, seguida da análise de confiabilidade com avaliação do coeficiente alfa de Cronbach. Em seguida, foi realizada a avaliação da validade convergente, considerando o instrumento EORTC QLQ-C-30, e avaliação pelo coeficiente de Correlação de Pearson. Para avaliação da estabilidade, foi realizado teste-reteste com teste de médias t-student. Foram entrevistados 115 pacientes. A maioria do sexo feminino (71,3%); com ensino superior (47,7%); casada (73,9%). O tumor mais frequente foi o tumor na mama (54,8), e o estádio III (47,8%). A solução da análise fatorial foi com sete fatores. O coeficiente alfa de Cronbach foi superior a 0,80 em todos os domínios avaliados. Optou-se por duas questões permanecerem separadamente na escala em razão das avaliações da análise de construto. A validade convergente mostrou que a escala apresenta correlação com sintomas do EORTC QLQ-C30. O instrumento mostrou estabilidade, não apresentando diferenças significativas entre as duas medidas. Com base nos dados apresentados, foi possível concluir que o SNCS-SF34 é um instrumento que está validado para o idioma Português e apresenta confiabilidade adequada.

This study aims to translate into Portuguese and also to culturally adapt the Supportive Care Needs Survey Instrument - Short Form (SCNS-SF34), which consists of 34 questions designed to evaluate the different areas of needs of cancer patients. This instrument is structured in five domains: Psychological; Health System and Information; Physical and Daily Life; Support and Care; and Sexuality. The survey was conducted at the Chemotherapy Center of AC Camargo Cancer Center. It was a descriptive study, characterized by two stages: the cultural adaptation of SCNS-SF34 instrument; and the analysis of the psychometric properties (construct validity and reliability) of the adapted version. The population consists of cancer patients diagnosed at least three months prior to the survey, undergoing chemotherapy treatment at the Chemotherapy Center. A non-probabilistic sample was used. To evaluate the construct validity we performed a factorial analysis followed by the reliability analysis based on the evaluation of Cronbach's alpha coefficient. Next, we conducted the evaluation of the convergent validity, considering the EORTC QLQ- C-30 instrument, and we also performed a Pearson correlation coefficient evaluation. To evaluate the stability, we performed test-retest with Student's t-test. We interrogated 115 patients. The majority was female (71.3%); with university degree (47.7%); married (73.9%). The most common tumor was breast cancer (54.8) stage III (47.8%). The factorial analysis solution presented seven factors. The Cronbach alpha coefficient was higher than 0.80 in all domains assessed. We decided to separate two questions in the scale due to the construct analysis assessments. The convergent validity showed that the scale correlates with the EORTC QLQ-C30 symptoms. The instrument showed to be stable, with no significant differences between the two measures. Based on these data, we concluded that the SCNS-SF34 instrument is validated for Portuguese language and has adequate reliability.

Tradução e adaptação cultural do instrumento Supportive Care Needs Survey Short Form 34 / Translation and cultural adaptation of the Supportive Care Needs Survey Short Form 34

O presente estudo tem como objetivo traduzir para a língua portuguesa eadaptar culturalmente o instrumento Supportive Care Needs Survey – ShortForm (SCNS-SF34), o qual é composto por 34 questões, desenvolvido paraavaliar as diferentes áreas de necessidades para o paciente com câncer. É estruturado em cinco domínios: Psicológico; Sistema de Saúde e Informação; Físico e Vida Diária; Suporte e Cuidado; e Sexualidade. A pesquisa foi realizada na Central de Quimioterapia do A.C. Camargo Cancer Center. Foi um estudo descritivo, caracterizado por duas etapas: a adaptação cultural do instrumento SCNS-SF34; e a análise das propriedades psicométricas (validade de construto e confiabilidade) da versão adaptada. A população é constituída de pacientes portadores de câncer com diagnóstico realizado há, pelo menos, três meses, que estejam em quimioterapia,atendidos na Central de Quimioterapia. Foi utilizada uma amostra não probabilística. Para a avaliação da validade de construto foi realizada aanálise fatorial, seguida da análise de confiabilidade com avaliação do coeficiente alfa de Cronbach. Em seguida, foi realizada a avaliação da validade convergente, considerando o instrumento EORTC QLQ-C-30, e avaliação pelo coeficiente de Correlação de Pearson. Para avaliação da estabilidade, foi realizado teste-reteste com teste de médias t-student. Foram entrevistados 115 pacientes. A maioria do sexo feminino (71,3%); com ensino superior (47,7%); casada (73,9%). O tumor mais frequente foi o tumor na mama (54,8), e o estádio III (47,8%). A solução da análise fatorial foi com sete fatores. O coeficiente alfa de Cronbach foi superior a 0,80 em todos os domínios avaliados. Optou-se por duas questões permanecerem separadamente na escala em razão das avaliações da análise de construto A validade convergente mostrou que a escala apresenta correlação comsintomas do EORTC QLQ-C30. O instrumento mostrou estabilidade, nãoapresentando diferenças significativas entre as duas...

This study aims to translate into Portuguese and also to culturally adapt theSupportive Care Needs Survey Instrument - Short Form (SCNS-SF34), which consists of 34 questions designed to evaluate the different areas of needs ofcancer patients. This instrument is structured in five domains: Psychological;Health System and Information; Physical and Daily Life; Support and Care; and Sexuality. The survey was conducted at the Chemotherapy Center of AC Camargo Cancer Center. It was a descriptive study, characterized by two stages: the cultural adaptation of SCNS-SF34 instrument; and the analysis of the psychometric properties (construct validity and reliability) of the adapted version. The population consists of cancer patients diagnosed at least three months prior to the survey, undergoing chemotherapy treatment at the Chemotherapy Center. A non-probabilistic sample was used. To evaluate the construct validity we performed a factorial analysis followed by the reliability analysis based on the evaluation of Cronbach's alpha coefficient. Next, we conducted the evaluation of the convergent validity, considering the EORTC QLQ- C-30 instrument, and we also performed a Pearson correlationcoefficient evaluation. To evaluate the stability, we performed test-retest withStudent's t-test. We interrogated 115 patients. The majority was female(71.3%); with university degree (47.7%); married (73.9%). The most common tumor was breast cancer (54.8) stage III (47.8%). The factorial analysissolution presented seven factors. The Cronbach alpha coefficient was higherthan 0.80 in all domains assessed. We decided to separate two questions inthe scale due to the construct analysis assessments. The convergent validityshowed that the scale correlates with the EORTC QLQ-C30 symptoms. Theinstrument showed to be stable, with no significant differences between thetwo measures. Based on these data, we concluded that the SCNS-SF34 instrument is validated for...