RESUMEN
Risk factors contributing to dementia are multifactorial. Accumulating evidence suggests a role for pathogens as risk factors, but data is largely correlative with few causal relationships. Here, we demonstrate that intermittent murine cytomegalovirus (MCMV) infection of mice, alters blood brain barrier (BBB) permeability and metabolic pathways. Increased basal mitochondrial function is observed in brain microvessels cells (BMV) exposed to intermittent MCMV infection and is accompanied by elevated levels of superoxide. Further, mice score lower in cognitive assays compared to age-matched controls who were never administered MCMV. Our data show that repeated systemic infection with MCMV, increases markers of neuroinflammation, alters mitochondrial function, increases markers of oxidative stress and impacts cognition. Together, this suggests that viral burden may be a risk factor for dementia. These observations provide possible mechanistic insights through which pathogens may contribute to the progression or exacerbation of dementia.
Asunto(s)
Trastornos del Conocimiento , Disfunción Cognitiva , Infecciones por Citomegalovirus , Demencia , Animales , Ratones , Infecciones por Citomegalovirus/complicaciones , CogniciónRESUMEN
Treatment of Clostridioides difficile infection (CDI) is expensive and complex, with a high proportion of patients suffering infection relapse (20-35%), and some having multiple relapses. A healthy, unperturbed gut microbiome provides colonisation resistance against CDI through competition for nutrients and space. However, antibiotic consumption can disturb the gut microbiota (dysbiosis) resulting in the loss of colonisation resistance allowing C. difficile to colonise and establish infection. A unique feature of C. difficile is the production of high concentrations of the antimicrobial compound para-cresol, which provides the bacterium with a competitive advantage over other bacteria found in the gut. p-cresol is produced by the conversion of para-Hydroxyphenylacetic acid (p-HPA) by the HpdBCA enzyme complex. In this study, we have identified several promising inhibitors of HpdBCA decarboxylase, which reduce p-cresol production and render C. difficile less able to compete with a gut dwelling Escherichia coli strain. We demonstrate that the lead compound, 4-Hydroxyphenylacetonitrile, reduced p-cresol production by 99.0 ± 0.4%, whereas 4-Hydroxyphenylacetamide, a previously identified inhibitor of HpdBCA decarboxylase, only reduced p-cresol production by 54.9 ± 13.5%. To interpret efficacy of these first-generation inhibitors, we undertook molecular docking studies that predict the binding mode for these compounds. Notably, the predicted binding energy correlated well with the experimentally determined level of inhibition, providing a molecular basis for the differences in efficacy between the compounds. This study has identified promising p-cresol production inhibitors whose development could lead to beneficial therapeutics that help to restore colonisation resistance and therefore reduce the likelihood of CDI relapse.
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Carboxiliasas , Clostridioides difficile , Microbioma Gastrointestinal , Humanos , Simulación del Acoplamiento Molecular , Clostridioides , Escherichia coliRESUMEN
Glycoengineering of recombinant glycans and glycoconjugates is a rapidly evolving field. However, the production and exploitation of glycans has lagged behind that of proteins and nucleic acids. Biosynthetic glycoconjugate production requires the coordinated cooperation of three key components within a bacterial cell: a substrate protein, a coupling oligosaccharyltransferase, and a glycan biosynthesis locus. While the acceptor protein and oligosaccharyltransferase are the products of single genes, the glycan is a product of a multigene metabolic pathway. Typically, the glycan biosynthesis locus is cloned and transferred en bloc from the native organism to a suitable Escherichia coli strain. However, gene expression within these pathways has been optimized by natural selection in the native host and is unlikely to be optimal for heterologous production in an unrelated organism. In recent years, synthetic biology has addressed the challenges in heterologous expression of multigene systems by deconstructing these pathways and rebuilding them from the bottom up. The use of DNA assembly methods allows the convenient assembly of such pathways by combining defined parts with the requisite coding sequences in a single step. In this study, we apply combinatorial assembly to the heterologous biosynthesis of the Campylobacter jejuni N-glycosylation (pgl) pathway in E. coli. We engineered reconstructed biosynthesis clusters that faithfully reproduced the C. jejuni heptasaccharide glycan. Furthermore, following a single round of combinatorial assembly and screening, we identified pathway clones that outperform glycan and glycoconjugate production of the native unmodified pgl cluster. This platform offers a flexible method for optimal engineering of glycan structures in E. coli.
Asunto(s)
Campylobacter jejuni , Escherichia coli , Escherichia coli/genética , ADN , Glicosilación , Campylobacter jejuni/genética , PolisacáridosRESUMEN
Human adipose-derived stem cells (hASCs) are potent modulators of inflammation and promising candidates for the treatment of inflammatory and autoimmune diseases. Strategies to improve hASC survival and immunoregulation are active areas of investigation. Autophagy, a homeostatic and stress-induced degradative pathway, plays a crucial role in hASC paracrine signaling-a primary mechanism of therapeutic action. Therefore, induction of autophagy with rapamycin (Rapa), or inhibition with 3-methyladenine (3-MA), was examined as a preconditioning strategy to enhance therapeutic efficacy. Following preconditioning, both Rapa and 3-MA-treated hASCs demonstrated preservation of stemness, as well as upregulated transcription of cyclooxygenase-2 (COX2) and interleukin-6 (IL-6). Rapa-ASCs further upregulated TNFα-stimulated gene-6 (TSG-6) and interleukin-1 beta (IL-1ß), indicating additional enhancement of immunomodulatory potential. Preconditioned cells were then stimulated with the inflammatory cytokine interferon-gamma (IFNγ) and assessed for immunomodulatory factor production. Rapa-pretreated cells, but not 3-MA-pretreated cells, further amplified COX2 and IL-6 transcripts following IFNγ exposure, and both groups upregulated secretion of prostaglandin-E2 (PGE2), the enzymatic product of COX2. These findings suggest that a 4-h Rapa preconditioning strategy may bestow the greatest improvement to hASC expression of cytokines known to promote tissue repair and regeneration and may hold promise for augmenting the therapeutic potential of hASCs for inflammation-driven pathological conditions.
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Autofagia , Ciclooxigenasa 2 , Dinoprostona , Células Madre Mesenquimatosas , Tejido Adiposo , Ciclooxigenasa 2/metabolismo , Citocinas/metabolismo , Dinoprostona/metabolismo , Humanos , Inflamación/metabolismo , Interferón gamma/metabolismo , Interleucina-6/metabolismo , Células Madre Mesenquimatosas/metabolismo , Fenotipo , SirolimusRESUMEN
Glioblastoma (GBM) is an aggressive primary central nervous system neoplasia with limited therapeutic options and poor prognosis. Following reports of cytomegalovirus (HCMV) in GBM tumors, the anti-viral drug Valganciclovir was administered and found to significantly increase the longevity of GBM patients. While these findings suggest a role for HCMV in GBM, the relationship between them is not clear and remains controversial. Treatment with anti-viral drugs may prove clinically useful; however, their results do not explain the underlying mechanism between HCMV infection and GBM progression. We hypothesized that HCMV infection would metabolically reprogram GBM cells and that these changes would allow for increased tumor progression. We infected LN-18 GBM cells and employed a Seahorse Bioanalyzer to characterize cellular metabolism. Increased mitochondrial respiration and glycolytic rates were observed following infection. These changes were accompanied by elevated production of reactive oxygen species and lactate. Due to lactate's numerous tumor-promoting effects, we examined the impact of paracrine signaling of HCMV-infected GBM cells on uninfected stromal cells. Our results indicated that, independent of viral transmission, the secretome of HCMV-infected GBM cells was able to alter the expression of key metabolic proteins and epigenetic markers. This suggests a mechanism of action where reprogramming of GBM cells alters the surrounding tumor microenvironment to be permissive to tumor progression in a manner akin to the Reverse-Warburg Effect. Overall, this suggests a potential oncomodulatory role for HCMV in the context of GBM.
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Infecciones por Citomegalovirus/fisiopatología , Citomegalovirus/fisiología , Glioblastoma/metabolismo , Glioblastoma/virología , Comunicación Paracrina , Secretoma , Línea Celular Tumoral , Citomegalovirus/genética , Infecciones por Citomegalovirus/virología , Glucólisis , Humanos , Ácido Láctico/metabolismo , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Fosforilación Oxidativa , Especies Reactivas de Oxígeno/metabolismo , Microambiente Tumoral , Replicación ViralRESUMEN
The human pathogen Clostridioides difficile colonises the gastrointestinal tract following antibiotic exposure, which causes perturbations in the beneficial microbiome. An unusual feature of C. difficile among the gut microbiota is its ability to produce high concentrations of the antimicrobial compound para-cresol, which selectively targets Gram-negative bacteria. Production of p-cresol occurs either by: (a) tyrosine fermentation via the intermediate para-hydroxyphenylacetate (p-HPA), or (b) direct turnover of exogenous p-HPA in the human gut. p-HPA is decarboxylated to produce p-cresol, by the action of HpdBCA decarboxylase encoded by the hpdBCA operon. HpdBCA decarboxylase production is induced at the transcriptional level by elevated p-HPA, which causes elevated p-cresol production, that significantly reduces microbiome diversity and richness. This deleterious effect of p-cresol on the beneficial gut microbiome is advantageous for C. difficile pathogenesis and infection relapse. Inhibiting this pathway would provide a highly specific therapeutic.
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Carboxiliasas , Clostridioides difficile , Infecciones por Clostridium , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Carboxiliasas/metabolismo , Carboxiliasas/uso terapéutico , Clostridioides , Clostridioides difficile/genética , Infecciones por Clostridium/microbiología , Cresoles , Bacterias Gramnegativas/metabolismo , HumanosRESUMEN
Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and is capable of causing severe symptoms, such as pseudomembranous colitis and toxic megacolon. An unusual feature of C. difficile is the distinctive production of high levels of the antimicrobial compound para-cresol. p-Cresol production provides C. difficile with a competitive colonization advantage over gut commensal species, in particular, Gram-negative species. p-Cresol is produced by the conversion of para-hydroxyphenylacetic acid (p-HPA) via the actions of HpdBCA decarboxylase coded by the hpdBCA operon. Host cells and certain bacterial species produce p-HPA; however, the effects of p-HPA on the viability of C. difficile and other gut microbiota are unknown. Here we show that representative strains from all five C. difficile clades are able to produce p-cresol by two distinct mechanisms: (i) via fermentation of p-tyrosine and (ii) via uptake and turnover of exogenous p-HPA. We observed strain-specific differences in p-cresol production, resulting from differential efficiency of p-tyrosine fermentation; representatives of clade 3 (CD305) and clade 5 (M120) produced the highest levels of p-cresol via tyrosine metabolism, whereas the toxin A-/B+ isolate from clade 4 (M68) produced the lowest level of p-cresol. All five lineages share at least 97.3% homology across the hpdBCA operon, responsible for decarboxylation of p-HPA to p-cresol, suggesting that the limiting step in p-cresol production may result from tyrosine to p-HPA conversion. We identified that elevated intracellular p-HPA, modulated indirectly via CodY, controls p-cresol production via inducing the expression of HpdBCA decarboxylase ubiquitously in C. difficile populations. Efficient turnover of p-HPA is advantageous to C. difficile as p-HPA has a deleterious effect on the growth of C. difficile and other representative Gram-negative gut bacteria, transduced potentially by the disruption of membrane permeability and release of intracellular phosphate. This study provides insights into the importance of HpdBCA decarboxylase in C. difficile pathogenesis, both in terms of p-cresol production and detoxification of p-HPA, highlighting its importance to cell survival and as a highly specific therapeutic target for the inhibition of p-cresol production across C. difficile species.
Asunto(s)
Clostridioides difficile , Cresoles/metabolismo , Clostridioides difficile/crecimiento & desarrollo , Clostridioides difficile/metabolismo , Descarboxilación , Fenilacetatos/metabolismoRESUMEN
Human cytomegalovirus (HCMV) is a near ubiquitous herpesvirus that relies on host cell metabolism for efficient replication. Although it has been shown that HCMV requires functional host cell mitochondria for efficient replication, it is unknown whether mitochondrial targeted pharmacological agents can be repurposed as antivirals. Here we report that treatment with drugs targeting the electron transport chain (ETC) complexes inhibit HCMV replication. Addition of rotenone, oligomycin, antimycin and metformin resulted in decreased HCMV titers in vitro, independent of HCMV strain. This further illustrates the dependence of HCMV replication on functional mitochondria. Metformin, an FDA approved drug, delays HCMV replication kinetics resulting in a reduction of viral titers. Repurposing metformin as an antiviral is advantageous as its safety profile and epidemiological data are well accepted. Our findings provide new insight into the potential for targeting HCMV infection through host cell metabolism and how these pharmacological interventions function.
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Antivirales/farmacología , Citomegalovirus/efectos de los fármacos , Transporte de Electrón/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/virología , Prepucio/citología , Humanos , Masculino , Metformina/farmacología , Oligomicinas/farmacologíaRESUMEN
ABSTRACT: The ubiquity of Listeria monocytogenes in the environment affects the food industry and presents concerns for frozen food facilities. This study determined the prevalence and numbers of Listeria species and L. monocytogenes on raw produce arriving at frozen food facilities. Raw produce was collected using multilevel blinding protocols to ensure anonymity of participants and avoid traceback. Five raw vegetables were selected: corn, carrots, green beans, peas, and spinach. Raw products were collected after arrival at the facilities but before cleaning or other preprocessing steps that are typically performed inside the facility. The U.S. Food and Drug Administration's Bacteriological Analytical Manual method for detection of Listeria spp. and L. monocytogenes was followed, with PCR screening followed by selective plating methods. Listeria numbers were estimated from positive samples using the most-probable-number (MPN) methodology. A total of 290 samples were collected, with 96 and 17 samples positive for Listeria spp. (33.1%) and L. monocytogenes (5.9%), respectively. Enumeration data for the 96 Listeria spp. samples indicated 82 samples had greater than 100 MPN of Listeria spp. per g and 14 samples had less than 100 MPN Listeria spp. per g. The prevalence of Listeria spp. varied by commodity: spinach (66.7%), peas (50%), corn (32.2%), green beans (22.2%), and carrots (13%). L. monocytogenes prevalence was determined in corn (13.6%), peas (6.3%), and green beans (4.2%) arriving at processing facilities. Such data were previously unavailable to frozen vegetable processors and are valuable in implementing process control standards. The prevalence and pathogen concentration data from raw commodities found in this study can provide the industry with information to conduct more accurate quantitative risk assessments and a baseline to model and target appropriate pathogen reduction steps during processing.
Asunto(s)
Listeria monocytogenes , Listeria , Contaminación de Alimentos/análisis , Microbiología de Alimentos , Alimentos Congelados , Humanos , Instalaciones Industriales y de Fabricación , PrevalenciaRESUMEN
Clostridioides difficile is the leading cause of nosocomial antibiotic-associated diarrhoea worldwide, yet there is little insight into intestinal tract colonisation and relapse. In many bacterial species, the secondary messenger cyclic-di-GMP mediates switching between planktonic phase, sessile growth and biofilm formation. We demonstrate that c-di-GMP promotes early biofilm formation in C. difficile and that four cell surface proteins contribute to biofilm formation, including two c-di-GMP regulated; CD2831 and CD3246, and two c-di-GMP-independent; CD3392 and CD0183. We demonstrate that C. difficile biofilms are composed of extracellular DNA (eDNA), cell surface and intracellular proteins, which form a protective matrix around C. difficile vegetative cells and spores, as shown by a protective effect against the antibiotic vancomycin. We demonstrate a positive correlation between biofilm biomass, sporulation frequency and eDNA abundance in all five C. difficile lineages. Strains 630 (RT012), CD305 (RT023) and M120 (RT078) contain significantly more eDNA in their biofilm matrix than strains R20291 (RT027) and M68 (RT017). DNase has a profound effect on biofilm integrity, resulting in complete disassembly of the biofilm matrix, inhibition of biofilm formation and reduced spore germination. The addition of exogenous DNase could be exploited in treatment of C. difficile infection and relapse, to improve antibiotic efficacy.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas , Clostridioides difficile/fisiología , GMP Cíclico/análogos & derivados , ADN Bacteriano/metabolismo , Biopelículas/crecimiento & desarrollo , Clostridioides difficile/ultraestructura , Infecciones por Clostridium/microbiología , GMP Cíclico/metabolismo , HumanosRESUMEN
OBJECTIVES: Azithromycin treatment of Chlamydia trachomatis (CT) may not be adequate to treat concomitant Mycoplasma genitalium (MG) infection, and particularly if MG has macrolide resistance-associated mutations (MG-MRAMs). We estimated prevalence of coinfections of CT with MG carrying MRAM, and risk factors for MG-MRAM among a sexual health clinic population. STUDY DESIGN AND SETTING: Among symptomatic and STI-contact clinic attendees in London, prevalence of CT-MG coinfection and MG-MRAM were estimated using nucleic acid amplification testing and Sanger sequencing, respectively, and their associated risk factors analysed using logistic regression. RESULTS: MG prevalence was 7.5% (23/307), 17.3% (30/173), and 11.4% (8/70) in females, men who have sex with women (MSW) and men who have sex with men (MSM), respectively; MG coinfection in CT-infected participants represented 28.0% (7/25), 13.5% (5/37), 0.0% (0/0), respectively. Presence of MG-MRAM was 39.1% (9/23) in female swabs, 70.0% (21/30) in MSW urine and 83.3% (5/6) in MSM rectal swabs. In multivariate analyses, coinfection with another STI was strongly associated with MG-MRAM (OR: 7.19; 95% CI: 2.4 to 21.5). CONCLUSION: A significant proportion of participants in our study of symptomatic patients and STI contacts were infected with macrolide-resistant MG, suggesting that testing for MG and MRAM, for MG positives, might be clinically useful. The findings also suggest services explore potential benefits of testing CT positive samples for MG in these patient groups. Where MG testing is not available, potential high rates of MG coinfection should be borne in mind when considering azithromycin in the treatment of CT among STI contacts and symptomatic patients.
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Antibacterianos/uso terapéutico , Azitromicina/uso terapéutico , Coinfección/epidemiología , Farmacorresistencia Bacteriana , Infecciones por Mycoplasma/epidemiología , Mycoplasma genitalium/efectos de los fármacos , Infecciones por Chlamydia/epidemiología , Chlamydia trachomatis/efectos de los fármacos , Femenino , Gonorrea/epidemiología , Humanos , Londres , Masculino , Neisseria gonorrhoeae/efectos de los fármacos , Prevalencia , Estudios ProspectivosRESUMEN
ABSTRACT: Difficulties associated with addressing research problems can revolve around the collection of data from private entities. Potential issues can arise when collecting food samples or food safety data from industry or third-party sources because of concerns about distribution or exposure of potentially sensitive information. Industry is cautious of its involvement in research projects because issues associated with production levels, capital investment, regulatory inquiries, unwarranted publicity, or other legal aspects can arise depending on the nature of information gathered, and information may be inadvertently released into the public domain. Well-designed clinical trials with animals or humans use blinding methods to reduce bias. In this study, a similar strategy was applied to acquisition of sensitive data to gather meaningful food safety related data while assuring that information provided was not at risk. Blinding methods for collecting electronic data and material samples were created to obtain materials and records directly from participating frozen food companies. This approach provided insight into current industry practices without potential downsides for participating companies. Analysis of food safety concerns using industry data and the distribution of findings can be of assistance industry-wide for conducting risk assessments and developing improved research-based food safety plans. The method described was designed to collect information using blinding protocols to reduce bias and prevent traceback to the original source. The use of blinding protocols promotes industry participation and creates data collection with anonymity for the original source, which can improve reliability of the research and applicability for industry. These blinding protocols are suitable for use in future food safety research projects involving data within and between various segments of the food industry and could be used to encourage collection of valuable industry samples and data.
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Inocuidad de los Alimentos , Industria de Procesamiento de Alimentos , Animales , Industria de Alimentos , Alimentos Congelados , Humanos , Reproducibilidad de los ResultadosRESUMEN
Macrophages and microglia represent the primary phagocytes and first line of defense in the peripheral and central immune systems. They activate and polarize into a spectrum of pro- and anti-inflammatory phenotypes in response to various stimuli. This activation is tightly regulated to balance the appropriate immune response with tissue repair and homeostasis. Disruption of this balance results in inflammatory disease states and tissue damage. Adipose stem cells (ASCs) have great therapeutic potential because of the potent immunomodulatory capabilities which induce the polarization of microglia and macrophages to the anti-inflammatory, M2, phenotype. In this study, we examined the effects of donor heterogeneity on ASC function. Specifically, we investigated the impact of donor obesity on ASC stemness and immunomodulatory abilities. Our findings revealed that ASCs from obese donors (ObASCs) exhibited reduced stem cell characteristics when compared to ASCs from lean donors (LnASCs). We also found that ObASCs promote a pro-inflammatory phenotype in murine macrophage and microglial cells, as indicated by the upregulated expression of pro-inflammatory genes, increased nitric oxide pathway activity, and impaired phagocytosis and migration. These findings highlight the importance of considering individual donor characteristics such as obesity when selecting donors and cells for use in ASC therapeutic applications and regenerative medicine.
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Tejido Adiposo , Macrófagos/metabolismo , Células Madre Mesenquimatosas , Obesidad/metabolismo , Tejido Adiposo/citología , Tejido Adiposo/inmunología , Animales , Diferenciación Celular , Proliferación Celular , Técnicas de Cocultivo , Femenino , Humanos , Inmunomodulación , Macrófagos/citología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/inmunología , Ratones , Fenotipo , Cultivo Primario de Células , Células RAW 264.7RESUMEN
Human adipose-derived stem cells (ASCs) show immense promise for treating inflammatory diseases, attributed primarily to their potent paracrine signaling. Previous investigations demonstrated that short-term Rapamycin preconditioning of bone marrow-derived stem cells (BMSCs) elevated secretion of prostaglandin E2, a pleiotropic molecule with therapeutic effects in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), and enhanced immunosuppressive capacity in vitro. However, this has yet to be examined in ASCs. The present study examined the therapeutic potential of short-term Rapamycin-preconditioned ASCs in the EAE model. Animals were treated at peak disease with control ASCs (EAE-ASCs), Rapa-preconditioned ASCs (EAE-Rapa-ASCs), or vehicle control (EAE). Results show that EAE-ASCs improved clinical disease scores and elevated intact myelin compared to both EAE and EAE-Rapa-ASC animals. These results correlated with augmented CD4+ T helper (Th) and T regulatory (Treg) cell populations in the spinal cord, and increased gene expression of interleukin-10 (IL-10), an anti-inflammatory cytokine. Conversely, EAE-Rapa-ASC mice showed no improvement in clinical disease scores, reduced myelin levels, and significantly less Th and Treg cells in the spinal cord. These findings suggest that short-term Rapamycin preconditioning reduces the therapeutic efficacy of ASCs when applied to late-stage EAE.
Asunto(s)
Antibacterianos/uso terapéutico , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/metabolismo , Esclerosis Múltiple/tratamiento farmacológico , Sirolimus/efectos adversos , Animales , Antibacterianos/farmacología , Modelos Animales de Enfermedad , Humanos , Ratones , Sirolimus/farmacologíaRESUMEN
Clostridioides difficile is an etiological agent for antibiotic-associated diarrheal disease. C. difficile produces a phenolic compound, para-cresol, which selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. C. difficile decarboxylates para-hydroxyphenylacetate (p-HPA) to produce p-cresol by the action of the HpdBCA decarboxylase encoded by the hpdBCA operon. Here, we investigate regulation of the hpdBCA operon and directly compare three independent reporter systems; SNAP-tag, glucuronidase gusA, and alkaline phosphatase phoZ reporters to detect basal and inducible expression. We show that expression of hpdBCA is upregulated in response to elevated p-HPA. In silico analysis identified three putative promoters upstream of hpdBCA operon-P1, P2, and Pσ54; only the P1 promoter was responsible for both basal and p-HPA-inducible expression of hpdBCA We demonstrated that turnover of tyrosine, a precursor for p-HPA, is insufficient to induce expression of the hpdBCA operon above basal levels because it is inefficiently converted to p-HPA in minimal media. We show that induction of the hpdBCA operon in response to p-HPA occurs in a dose-dependent manner. We also identified an inverted palindromic repeat (AAAAAG-N13-CTTTTT) upstream of the hpdBCA start codon (ATG) that is essential for inducing transcription of the hpdBCA operon in response to p-HPA, which drives the production of p-cresol. This provides insights into the regulatory control of p-cresol production, which affords a competitive advantage for C. difficile over other intestinal bacteria, promoting dysbiosis.IMPORTANCEClostridioides difficile infection results from antibiotic-associated dysbiosis. para-Cresol, a phenolic compound produced by C. difficile, selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. Here, we demonstrate that expression of the hpdBCA operon, encoding the HpdBCA decarboxylase which converts p-HPA to p-cresol, is upregulated in response to elevated exogenous p-HPA, with induction occurring between >0.1 and ≤0.25 mg/ml. We determined a single promoter and an inverted palindromic repeat responsible for basal and p-HPA-inducible hpdBCA expression. We identified turnover of tyrosine, a p-HPA precursor, does not induce hpdBCA expression above basal level, indicating that exogenous p-HPA was required for p-cresol production. Identifying regulatory controls of p-cresol production will provide novel therapeutic targets to prevent p-cresol production, reducing C. difficile's competitive advantage.
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Proteínas Bacterianas/metabolismo , Carboxiliasas/metabolismo , Clostridioides difficile/metabolismo , Cresoles/metabolismo , Fenilacetatos/metabolismo , Regulación Bacteriana de la Expresión Génica , Operón , Regiones Promotoras GenéticasRESUMEN
Obesity is associated with poorer responses to chemo- and radiation therapy for breast cancer, which leads to higher mortality rates for obese women who develop breast cancer. Adipose stem cells (ASCs) are an integral stromal component of the tumor microenvironment (TME). In this study, the effects of obesity-altered ASCs (obASCs) on estrogen receptor positive breast cancer cell's (ER+BCCs) response to radiotherapy (RT) were evaluated. We determined that BCCs had a decreased apoptotic index and increased surviving fraction following RT when co-cultured with obASCs compared to lnASCs or non-co-cultured cells. Further, obASCs reduced oxidative stress and induced IL-6 expression in co-cultured BCCs after radiation. obASCs produce increased levels of leptin relative to ASCs from normal-weight individuals (lnASCs). obASCs upregulate the expression of IL-6 compared to non-co-cultured BCCs, but BCCs co-cultured with leptin knockdown obASCs did not upregulate IL-6. The impact of shLeptin obASCs on radiation resistance of ER+BCCs demonstrate a decreased radioprotective ability compared to shControl obASCs. Key NOTCH signaling players were enhanced in ER+BBCs following co-culture with shCtrl obASCs but not shLep obASCs. This work demonstrates that obesity-altered ASCs, via enhanced secretion of leptin, promote IL-6 and NOTCH signaling pathways in ER+BCCs leading to radiation resistance.
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Tejido Adiposo/citología , Neoplasias de la Mama/radioterapia , Leptina/metabolismo , Células Madre Mesenquimatosas/metabolismo , Obesidad/metabolismo , Comunicación Paracrina/efectos de la radiación , Receptores de Estrógenos/metabolismo , Tejido Adiposo/metabolismo , Animales , Apoptosis/efectos de la radiación , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Técnicas de Cocultivo , Daño del ADN/efectos de la radiación , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Interleucina-6/metabolismo , Leptina/genética , Células MCF-7 , Ratones , Estrés Oxidativo/efectos de la radiación , ARN Interferente Pequeño , Radiación , Receptores Notch/metabolismo , Puntos de Control de la Fase S del Ciclo Celular/efectos de la radiación , Transducción de Señal/efectos de la radiación , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Food processors face serious challenges due to Listeria monocytogenes contamination. Environmental monitoring is used to control L. monocytogenes from the processing environment. Although frozen foods do not support the growth of L. monocytogenes, the moist and cold conditions in frozen food production environments are favorable for growth of L. monocytogenes. The purpose of the study was to determine the current state of awareness and practices applied across a variety of frozen food facilities related to environmental monitoring for Listeria. A survey tool was created to elicit information on existing environmental monitoring programs within the frozen food industry. The topics included cleaning and sanitizing applications and frequency, microbiological testing, and environmental areas of concern. The survey was reviewed by academic and industry experts with knowledge of microbiology and frozen food processing and was field tested by industry personnel with extensive knowledge of environmental monitoring. The survey was distributed and analyzed electronically via Qualtrics among 150 frozen food contacts. Data were gathered anonymously with a response rate of 31% (n = 46). The survey indicated that facilities are more likely to test for Listeria spp. in environmental monitoring zones 2 to 4 (nonfood contact areas) on a weekly basis. The major areas of concern in facilities for finding Listeria-positive results are floors, walls, and drains. At the time of the survey, few facilities incorporated active raw material and finished product testing for Listeria; instead, programs emphasized the need to identify presence of Listeria in the processing environment and mitigate potential for product contamination. Recognition of environmental monitoring as a key component of a comprehensive food safety plan was evident, along with an industry focus to further improve and develop verification programs to reduce prevalence of L. monocytogenes in frozen food processing environments.
Asunto(s)
Contaminación de Alimentos/análisis , Alimentos Congelados/microbiología , Listeria monocytogenes/aislamiento & purificación , Instalaciones Industriales y de Fabricación , Microbiología Ambiental , Monitoreo del Ambiente , Microbiología de Alimentos , Industria de Procesamiento de AlimentosRESUMEN
Critical-sized bone defects fail to heal and often cause non-union. Standard treatments employ autologous bone grafting, which can cause donor tissue loss/pain. Although several scaffold types can enhance bone regeneration, multiple factors limit their level of success. To address this issue, this study evaluated a novel decellularized human adipose tissue (DAT) hydrogel as an alternative. In this study, DAT hydrogel alone, or in combination with adipose-derived stromal/stem cells (ASC), osteo-induced ASCs (OIASC), and hydroxyapatite were tested for their ability to mediate repair of a critical-sized (3 mm) femoral defect created in C57BL/6 mice. Micro-computed tomography results showed that all DAT hydrogel treated groups significantly enhanced bone regeneration, with OIASC + hydroxyapatite treated group displaying the most robust bone regeneration. Histological analyses revealed that all treatments resulted in significantly higher tissue areas with the relative mineralized tissue area significantly increased at 12 weeks; however, cartilaginous content was lowest among treatment groups with OIASC. Immunohistochemical analyses showed that DAT hydrogel enhanced collagen I and osteopontin expression, while the addition of OIASCs to the hydrogel reduced collagen II levels. Thus, DAT hydrogel promotes bone regeneration in a critical-sized femoral defect model that is further enhanced in the presence of OIASCs and hydroxyapatite.
RESUMEN
Background High rates of antimicrobial resistance (AMR) in Neisseria gonorrhoeae hinder effective treatment, but molecular AMR diagnostics may help address the challenge. This study aimed to appraise the literature for resistance-associated genotypic markers linked to fluoroquinolones and macrolides, to identify and review their use in diagnostics. METHODS: Medline and EMBASE databases were searched and data pooled to evaluate associations between genotype and phenotypic resistance. The minimum inhibitory concentration (MIC) cut-offs were ≤ 0.06 mg L-1 for non-resistance to ciprofloxacin and ≤ 0.5 mg L-1 for non-resistance to azithromycin. RESULTS: Diagnostic accuracy estimates were limited by data availability and reporting. It was found that: 1) S91 and D95 mutations in the GyrA protein independently predicted ciprofloxacin resistance and, used together, gave 98.6% (95% confidence interval (CI) 98.0-99.0%) sensitivity and 91.4% (95%CI 88.6-93.7%) specificity; 2) the number of 23S rRNA gene alleles with C2611T or A2059G mutations was highly correlated with azithromycin resistance, with mutation in any allele giving a sensitivity and specificity of 66.1% (95%CI 62.1-70.0%) and 98.9% (95%CI 97.5-99.5%) respectively. Estimated negative (NPV) and positive predictive values (PPV) for a 23S rRNA diagnostic were 98.6% (95%CI 96.8-99.4%) and 71.5% (95%CI 68.0-74.8%) respectively; 3) mutation at amino acid position G45 in the MtrR protein independently predicted azithromycin resistance; however, when combined with 23S rRNA, did not improve the PPV or NPV. CONCLUSIONS: Viable candidates for markers of resistance detection for incorporation into diagnostics were demonstrated. Such tests may enhance antibiotic stewardship and treatment options.
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Fluoroquinolonas/farmacología , Macrólidos/farmacología , Neisseria gonorrhoeae/genética , Genes Bacterianos/genética , Estudios de Asociación Genética , Gonorrea/tratamiento farmacológico , Humanos , Neisseria gonorrhoeae/efectos de los fármacos , ARN Ribosómico 23S/genéticaRESUMEN
Adipose stem cells (ASCs) play an essential role in tumor microenvironments. These cells are altered by obesity (obASCs) and previous studies have shown that obASCs secrete higher levels of leptin. Increased leptin, which upregulates estrogen receptor alpha (ERα) and aromatase, enhances estrogen bioavailability and signaling in estrogen receptor positive (ERâº) breast cancer (BC) tumor growth and metastasis. In this study, we evaluate the effect of obASCs on ERâºBC outside of the ERα signaling axis using breast cancer models with constitutively active ERα resulting from clinically relevant mutations (Y537S and D538G). We found that while obASCs promote tumor growth and proliferation, it occurs mostly through abrogated estrogen signaling when BC has constitutive ER activity. However, obASCs have a similar promotion of metastasis irrespective of ER status, demonstrating that obASC promotion of metastasis may not be completely estrogen dependent. We found that obASCs upregulate two genes in both ER wild type (WT) and ER mutant (MUT) BC: SERPINE1 and ABCB1. This study demonstrates that obASCs promote metastasis in ER WT and MUT xenografts and an ER MUT patient derived xenograft (PDX) model. However, obASCs promote tumor growth only in ER WT xenografts.