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is primarily a respiratory pathogen. However, 15% of infections worldwide occur at extrapulmonary sites causing additional complications for diagnosis and treatment of the disease. In addition, dissemination of out of the lungs is thought to be more than just a rare event leading to extrapulmonary tuberculosis, but rather a prerequisite step that occurs during all infections, producing secondary lesions that can become latent or productive. In this review we will cover the clinical range of extrapulmonary infections and the process of dissemination including evidence from both historical medical literature and animal experiments for dissemination and subsequent reseeding of the lungs through the lymphatic and circulatory systems. While the mechanisms of dissemination are not fully understood, we will discuss the various models that have been proposed to address how this process may occur and summarize the bacterial virulence factors that facilitate dissemination.

Background Diseases such as tuberculosis (TB) have always had a large impact on human health. Bacillus Calmette-Guérin (BCG) is used as a surrogate for TB during the development of anti-TB drugs. Nanoparticles (NPs) have attracted great interest in drug development. The purpose of this study was to examine the potential of NPs as anti-TB compounds by studying the interacting mechanisms between NPs and bacteria. Results We investigated effects of gold and silver NPs on BCG and Escherichia coli . Experimentally, particle size and shape were characterized using transmission electron microscopy (TEM). Different concentrations of NPs were applied in bacterial culture. The growth of E. coli was monitored through colony forming units (CFU). The mechanism of interaction between NPs and bacteria was analyzed through bacterial thin sections followed by TEM and scanning electron microscopy. Antibacterial effects on BCG were observed by recording fluorescent protein expression levels. Conclusions The results suggest NPs have potential applications as anti-TB compounds. The antibacterial effects and mechanism of action for NPs were dependent upon composition and surface modifications.

The slow growth of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), hinders development of new diagnostics, therapeutics and vaccines. Using non-invasive real-time imaging technologies to monitor the disease process in live animals would facilitate TB research in all areas. We developed fluorescent protein (FP) expressing Mycobacterium bovis BCG strains for in vivo imaging, which can be used to track bacterial location, and to quantify bacterial load in live animals. We selected an optimal FP for in vivo imaging, by first cloning six FPs: tdTomato, mCherry, mPlum, mKate, Katushka and mKeima, into mycobacteria under either a mycobacterial Hsp60 or L5 promoter, and compared their fluorescent signals in vitro and in vivo. Fluorescence from each FP-expressing strain was measured with a multimode reader using the optimal excitation and emission wavelengths for the FP. After normalizing bacterial numbers with optical density, the strain expressing L5-tdTomato displayed the highest fluorescence. We used the tdTomato-labeled M. bovis BCG to obtain real-time images of pulmonary infections in living mice and rapidly determined the number of bacteria present. Further comparison between L5-tdTomato and Hsp60-tdTomato revealed that L5-tdTomato carried four-fold more tdTomato gene copies than Hsp60-tdTomato, which eventually led to higher protein expression of tdTomato. Evaluating anti-TB efficacy of rifampicin and isoniazid therapy in vitro and in vivo using the L5-tdTomato strain demonstrated that this strain can be used to identify anti-TB therapeutic efficacy as quickly as 24 h post-treatment. These M. bovis BCG reporter strains represent a valuable new tool for evaluation of therapeutics, vaccines and virulence.

The slow growth rate and genetic intractability of tubercle bacilli has hindered progress toward understanding tuberculosis, one of the most frequent causes of death worldwide. We overcame this roadblock through development of near-infrared (NIR) fluorogenic substrates for β-lactamase, an enzyme expressed by tubercle bacilli, but not by their eukaryotic hosts, to allow real-time imaging of pulmonary infections and rapid quantification of bacteria in living animals by a strategy called reporter enzyme fluorescence (REF). This strategy has a detection limit of 6 ± 2 x 10² colony-forming units (CFU) of bacteria with the NIR substrate CNIR5 in only 24 h of incubation in vitro, and as few as 10⁴ CFU in the lungs of live mice. REF can also be used to differentiate infected from uninfected macrophages by using confocal microscopy and fluorescence activated cell sorting. Mycobacterium tuberculosis and the bacillus Calmette-Guérin can be tracked directly in the lungs of living mice without sacrificing the animals. Therapeutic efficacy can also be evaluated through loss of REF signal within 24 h posttreatment by using in vitro whole-bacteria assays directly in living mice. We expect that rapid quantification of bacteria within tissues of a living host and in the laboratory is potentially transformative for tuberculosis virulence studies, evaluation of therapeutics, and efficacy of vaccine candidates. This is a unique use of an endogenous bacterial enzyme probe to detect and image tubercle bacilli that demonstrates REF is likely to be useful for the study of many bacterial infections.

Bacillus Calmette–Guérin (BCG) is the most widely used vaccine worldwide and has been used to prevent tuberculosis for a century. BCG also stimulates an anti-tumour immune response, which urologists have harnessed for the treatment of non-muscle-invasive bladder cancer. A growing body of evidence indicates that BCG offers protection against various non-mycobacterial and viral infections. The non-specific effects of BCG occur via the induction of trained immunity and form the basis for the hypothesis that BCG vaccination could be used to protect against the severity of coronavirus disease 2019 (COVID-19). This Perspective article highlights key milestones in the 100-year history of BCG and projects its potential role in the COVID-19 pandemic.Bacillus Calmette–Guérin (BCG) has been used to prevent tuberculosis for a century and is also a standard approach for the treatment of non-muscle-invasive bladder cancer. However, BCG also has a plethora of non-specific effects that occur via the induction of trained immunity and have raised the hypothesis that BCG vaccination could be used to protect against coronavirus disease 2019 (COVID-19). In this Perspective, the authors describe the history of BCG, discuss the mechanisms of its effects, and consider its potential role during the COVID-19 pandemic.

Advanced ovarian cancer is one of the most lethal gynecological tumor, mainly due to late diagnoses and acquired drug resistance. MicroRNAs (miRNAs) are small-non coding RNA acting as tumor suppressor/oncogenes differentially expressed in normal and epithelial ovarian cancer and has been recognized as a new class of tumor early detection biomarkers as they are released in blood fluids since tumor initiation process. Here, we evaluated by droplet digital PCR (ddPCR) circulating miRNAs in serum samples from healthy (N = 105) and untreated ovarian cancer patients (stages I to IV) (N = 72), grouped into a discovery/training and clinical validation set with the goal to identify the best classifier allowing the discrimination between earlier ovarian tumors from health controls women. The selection of 45 candidate miRNAs to be evaluated in the discovery set was based on miRNAs represented in ovarian cancer explorative commercial panels. We found six miRNAs showing increased levels in the blood of early or late-stage ovarian cancer groups compared to healthy controls. The serum levels of miR-320b and miR-141-3p were considered independent markers of malignancy in a multivariate logistic regression analysis. These markers were used to train diagnostic classifiers comprising miRNAs (miR-320b and miR-141-3p) and miRNAs combined with well-established ovarian cancer protein markers (miR-320b, miR-141-3p, CA-125 and HE4). The miRNA-based classifier was able to accurately discriminate early-stage ovarian cancer patients from health-controls in an independent sample set (Sensitivity = 80.0%, Specificity = 70.3%, AUC = 0.789). In addition, the integration of the serum proteins in the model markedly improved the performance (Sensitivity = 88.9%, Specificity = 100%, AUC = 1.000). A cross-study validation was carried out using four data series obtained from Gene Expression Omnibus (GEO), corroborating the performance of the miRNA-based classifier (AUCs ranging from 0.637 to 0.979). The clinical utility of the miRNA model should be validated in a prospective cohort in order to investigate their feasibility as an ovarian cancer early detection tool.

Objective Circulating cell-free microRNAs (miRNAs) which consist of short-sequence RNAs are released from cells into the blood stream and has emerged as new biomarkers in the clinical cancer diagnosis and treatment. For instance, ovarian cancer comprises one of the three major malignant tumor types in the female reproductive system. The mortality rate of this cancer is the highest among all gynecological tumors, with ovarian cancer metastasis constituting an important cause of death. Therefore, development of a diagnostic tool that enables the ovarian cancer diagnosis in earlier stages is urgent. Results We have described an efficient protocol for an accurate absolute quantification of circulating miRNAs in healthy and ovarian cancer serum samples. Our data showed that ddPCR methodology can accurately measure circulating miRNAs levels and that can be a useful tool in biomarkers discovery for ovarian cancer detection.

Mycobacterium tuberculosis ( Mtb ) has complex and dynamic interactions with the human host, and subpopulations of Mtb that emerge during infection can influence disease outcomes. This study implicates zinc ion (Zn 2+ ) availability as a likely driver of bacterial phenotypic heterogeneity in vivo . Zn 2+ sequestration is part of “nutritional immunity”, where the immune system limits micronutrients to control pathogen growth, but this defense mechanism seems to be ineffective in controlling Mtb infection. Nonetheless, Zn 2+ -limitation is an environmental cue sensed by Mtb , as calprotectin triggers the zinc uptake regulator (Zur) regulon response in vitro and co-localizes with Zn 2+ -limited Mtb in vivo . Prolonged Zn 2+ limitation leads to numerous physiological changes in vitro , including differential expression of certain antigens, alterations in lipid metabolism and distinct cell surface morphology. Furthermore, Mtb enduring limited Zn 2+ employ defensive measures to fight oxidative stress, by increasing expression of proteins involved in DNA repair and antioxidant activity, including well described virulence factors KatG and AhpC, along with altered utilization of redox cofactors. Here, we propose a model in which prolonged Zn 2+ limitation defines a population of Mtb with anticipatory adaptations against impending immune attack, based on the evidence that Zn 2+ -limited Mtb are more resistant to oxidative stress and exhibit increased survival and induce more severe pulmonary granulomas in mice. Considering that extracellular Mtb may transit through the Zn 2+ -limited caseum before infecting naïve immune cells or upon host-to-host transmission, the resulting phenotypic heterogeneity driven by varied Zn 2+ availability likely plays a key role during early interactions with host cells.

Soon after commencement of the SARS-CoV-2 disease outbreak of 2019 (COVID-19), it became evident that the receptor-binding domain of the viral spike protein is the target of neutralizing antibodies that comprise a critical element of protective immunity to the virus. This study addresses the relative lack of information regarding actual antibody concentrations and binding affinities in convalescent plasma (CP) samples from COVID-19 patients and extends these analyses to post-vaccination (PV) samples to estimate protective IgG antibody (Ab) levels. A direct enzyme-linked immunosorbent assay (ELISA) was used to measure IgG anti-spike protein (SP) antibodies (Abs) relative to human chimeric spike S1 Ab standards. Microplate wells were coated with recombinant SP. Affinities of Ab binding to SP were determined by previously described methods. Binding affinities were also determined in an RBD-specific sandwich ELISA. Two indices of protective immunity were determined as permutations of Ab molar concentration divided by affinity as dissociation constant (K D ). The range and geometric means of Ab concentrations in 21 CP and 21 PV samples were similar and a protective Ab level of 7.5 μg/ml was determined for the latter population, based on 95% of the normal distribution of the PV population. A population (n = 21) of plasma samples from individuals receiving only one vaccination with the BNT162b2 or mRNA-1273 vaccines (PtV) exhibited a geometric mean Ab concentration significantly (p < 0.03) lower than the PV population. The results of this study have implications for future vaccine development, projection of protective efficacy duration, and understanding of the immune response to SARS-CoV-2 infection.

Summary Indole is an extracellular biofilm signal for Escherichia coli, and many bacterial oxygenases readily convert indole to various oxidized compounds including 7‐hydroxyindole (7HI). Here we investigate the impact of indole and 7HI on Pseudomonas aeruginosa PAO1 virulence and quorum sensing (QS)‐regulated phenotypes; this strain does not synthesize these compounds but degrades them rapidly. Indole and 7HI both altered extensively gene expression in a manner opposite that of acylhomoserine lactones; the most repressed genes encode the mexGHI‐opmD multidrug efflux pump and genes involved in the synthesis of QS‐regulated virulence factors including pyocyanin (phz operon), 2‐heptyl‐3‐hydroxy‐4(1H)‐quinolone (PQS) signal (pqs operon), pyochelin (pch operon) and pyoverdine (pvd operon). Corroborating these microarray results, indole and 7HI decreased production of pyocyanin, rhamnolipid, PQS and pyoverdine and enhanced antibiotic resistance. In addition, indole affected the utilization of carbon, nitrogen and phosphorus, and 7HI abolished swarming motility. Furthermore, 7HI reduced pulmonary colonization of P. aeruginosa in guinea pigs and increased clearance in lungs. Hence, indole‐related compounds have potential as a novel antivirulence approach for the recalcitrant pathogen P. aeruginosa.