MicroMPN: methods and software for high-throughput screening of microbe suppression in mixed populations.

Screening assays are used to test if one or more microbes suppress a pathogen of interest. In the presence of more than one microbe, the screening method must be able to accurately distinguish viable pathogen cells from non-viable and non-target microbes in a sample. Current screening methods are time-consuming and require special reagents to detect viability in mixed microbial communities. Screening assays performed using soil or other complex matrices present additional challenges for screening. Here, we develop an experimental workflow based on the most probable number (MPN) assay for testing the ability of synthetic microbial communities to suppress a soil-borne pathogen. Our approach, fluorMPN, uses a fluorescently labeled pathogen and microplate format to enable high-throughput comparative screening. In parallel, we developed a command-line tool, MicroMPN, which significantly reduces the complexity of calculating MPN values from microplates. We compared the performance of the fluorMPN assay with spotting on agar and found that both methods produced strongly correlated counts of equal precision. The suppressive effect of synthetic communities on the pathogen was equally recoverable by both methods. The application of this workflow for discriminating which communities lead to pathogen reduction helps narrow down candidates for additional characterization. Together, the resources offered here are meant to facilitate and simplify the application of MPN-based assays for comparative screening projects.IMPORTANCEWe created a unified set of software and laboratory protocols for screening microbe libraries to assess the suppression of a pathogen in a mixed microbial community. Existing methods of fluorescent labeling were combined with the most probable number (MPN) assay in a microplate format to enumerate the reduction of a pathogenic soil microbe from complex soil matrices. This work provides a fluorescent expression vector available from Addgene, step-by-step laboratory protocols hosted by protocols.io, and MicroMPN, a command-line software for processing plate reader outputs. MicroMPN simplifies MPN estimation from 96- and 384-well microplates. The microplate screening assay is amenable to robotic automation with standard liquid handling robots, further reducing the hands-on processing time. This tool was designed to evaluate synthetic microbial communities for use as microbial inoculates or probiotics. The fluorMPN method is also useful for screening chemical and antimicrobial libraries for pathogen suppression in complex bacterial communities like soil.

Role of RpoS in Regulating Stationary Phase Salmonella Typhimurium Pathogenesis-Related Stress Responses under Physiological Low Fluid Shear Force Conditions.

The discovery that biomechanical forces regulate microbial virulence was established with the finding that physiological low fluid shear (LFS) forces altered gene expression, stress responses, and virulence of the enteric pathogen Salmonella enterica serovar Typhimurium during the log phase. These log phase LFS-induced phenotypes were independent of the master stress response regulator, RpoS (σS). Given the central importance of RpoS in regulating stationary-phase stress responses of S. Typhimurium cultured under conventional shake flask and static conditions, we examined its role in stationary-phase cultures grown under physiological LFS. We constructed an isogenic rpoS mutant derivative of wild-type S. Typhimurium and compared the ability of these strains to survive in vitro pathogenesis-related stresses that mimic those encountered in the infected host and environment. We also compared the ability of these strains to colonize (adhere, invade, and survive within) human intestinal epithelial cell cultures. Unexpectedly, LFS-induced resistance of stationary-phase S. Typhimurium cultures to acid and bile salts stresses did not rely on RpoS. Likewise, RpoS was dispensable for stationary-phase LFS cultures to adhere to and survive within intestinal epithelial cells. In contrast, the resistance of these cultures to challenges of oxidative and thermal stresses, and their invasion into intestinal epithelial cells was influenced by RpoS. These findings expand our mechanistic understanding of how physiological fluid shear forces modulate stationary-phase S. Typhimurium physiology in unexpected ways and provide clues into microbial mechanobiology and nuances of Salmonella responses to microenvironmental niches in the infected host. IMPORTANCE Bacterial pathogens respond dynamically to a variety of stresses in the infected host, including physical forces of fluid flow (fluid shear) across their surfaces. While pathogens experience wide fluctuations in fluid shear during infection, little is known about how these forces regulate microbial pathogenesis. This is especially important for stationary-phase bacterial growth, which is a critical period to understand microbial resistance, survival, and infection potential, and is regulated in many bacteria by the general stationary-phase stress response protein RpoS. Here, we showed that, unlike conventional culture conditions, several stationary-phase Salmonella pathogenic stress responses were not impacted by RpoS when bacteria were cultured under fluid shear conditions relevant to those encountered in the intestine of the infected host. These findings offer new insight into how physiological fluid shear forces encountered by Salmonella during infection might impact pathogenic responses in unexpected ways that are relevant to their disease-causing ability.

Spaceflight Analogue Culture Enhances the Host-Pathogen Interaction Between Salmonella and a 3-D Biomimetic Intestinal Co-Culture Model.

Physical forces associated with spaceflight and spaceflight analogue culture regulate a wide range of physiological responses by both bacterial and mammalian cells that can impact infection. However, our mechanistic understanding of how these environments regulate host-pathogen interactions in humans is poorly understood. Using a spaceflight analogue low fluid shear culture system, we investigated the effect of Low Shear Modeled Microgravity (LSMMG) culture on the colonization of Salmonella Typhimurium in a 3-D biomimetic model of human colonic epithelium containing macrophages. RNA-seq profiling of stationary phase wild type and Δhfq mutant bacteria alone indicated that LSMMG culture induced global changes in gene expression in both strains and that the RNA binding protein Hfq played a significant role in regulating the transcriptional response of the pathogen to LSMMG culture. However, a core set of genes important for adhesion, invasion, and motility were commonly induced in both strains. LSMMG culture enhanced the colonization (adherence, invasion and intracellular survival) of Salmonella in this advanced model of intestinal epithelium using a mechanism that was independent of Hfq. Although S. Typhimurium Δhfq mutants are normally defective for invasion when grown as conventional shaking cultures, LSMMG conditions unexpectedly enabled high levels of colonization by an isogenic Δhfq mutant. In response to infection with either the wild type or mutant, host cells upregulated transcripts involved in inflammation, tissue remodeling, and wound healing during intracellular survival. Interestingly, infection by the Δhfq mutant led to fewer transcriptional differences between LSMMG- and control-infected host cells relative to infection with the wild type strain. This is the first study to investigate the effect of LSMMG culture on the interaction between S. Typhimurium and a 3-D model of human intestinal tissue. These findings advance our understanding of how physical forces can impact the early stages of human enteric salmonellosis.

Advancing Equity and Inclusion in Microbiome Research and Training.

This article proposes ways to improve inclusion and training in microbiome science and advocates for resource expansion to improve scientific capacity across institutions and countries. Specifically, we urge mentors, collaborators, and decision-makers to commit to inclusive and accessible research and training that improves the quality of microbiome science and begins to rectify long-standing inequities imposed by wealth disparities and racism that stall scientific progress.

Communication calls of little brown bats display individual-specific characteristics.

Bats' echolocation signals have been shown to be situation-, colony-, and individual-specific, but whether or not these findings apply to bats' communication signals is not fully understood. The primary goal of this study was to test the hypothesis that the communication calls of adult little brown bats (Myotis lucifugus) are individual specific. Bats were paired to form focal pairs from June 2007 to August 2008. Each bat's vocalizations were recorded on a PC-based digital recorder with a custom made ultrasonic microphone. The vocal signals were first classified using a previously established classification scheme. Three acoustic parameters (the minimum and maximum frequencies, and the call duration) of two of the dominant call-types, the steep-FM and broadband noise bursts, of individual bats were further analyzed. Discriminant function analysis, and multi- and univariate analyses of variance of these parameters revealed that these vocal signals were individually distinct and likely contain individual signatures to allow bats to identify individuals acoustically.

EGF-receptor phosphorylation and downstream signaling are activated by benzo[a]pyrene 3,6-quinone and benzo[a]pyrene 1,6-quinone in human mammary epithelial cells.

Benzo[a]pyrene (BaP) is activated by xenobiotic-metabolizing enzymes to highly mutagenic and carcinogenic metabolites. Previous studies in this laboratory have shown that benzo[a]pyrene quinones (BPQs), 1,6-BPQ and 3,6-BPQ, are able to induce epidermal growth factor receptor (EGFR) cell signaling through the production of reactive oxygen species. Recently, we have reported that BPQs have the potential to induce the expression of genes involved in numerous pathways associated with cell proliferation and survival in human mammary epithelial cells. In the present study we demonstrated that BPQs not only induced EGFR tyrosine autophosphorylation, but also induced EGFR-dependent tyrosine phosphorylation of phospholipase C-gamma1 and several signal transducers and activators of transcription (STATs). The effects of BPQs were evaluated in a model of EGF withdrawal in MCF10-A cells. We found that BPQs (1 muM), induced EGFR tyrosine phosphorylation at positions Y845, Y992, Y1068, and Y1086. PLC-gamma1 phosphorylation correlated with the phosphorylation of tyrosine-Y992, a proposed docking site for PLC-gamma1 on the EGFR. Additionally, we found that BPQs induced the activation of STAT-1, STAT-3, STAT-5a and STAT-5b. STAT5 was shown to translocate to the nucleus following 3,6-BPQ and 1,6-BPQ exposures. Although the patterns of phosphorylation at EGFR, PLC-gamma1 and STATs were quite similar to those induced by EGF, an important difference between BPQ-mediated signaling of the EGFR was observed. Signaling produced by EGF ligand produced a rapid disappearance of EGFR from the cell surface, whereas BPQ signaling maintained EGFR receptors on the cell membrane. Thus, the results of these studies show that 1,6-BPQ and 3,6-BPQ can produce early events as evidenced by EGFR expression, and a prolonged transactivation of EGFR leading to downstream cell signaling pathways.

[Familial adenomatous polyposis: case report of male twins]. / Poliposis adenomatosa familiar: presentación de dos casos en gemelos masculinos idénticos.

BACKGROUND: One hundred percent of the cases of familial adenomatous polyposis (FAP) will develop carcinoma; therefore, the necessity of diagnosis at an early age with immediate therapy is essential. In the presence of identical twins, it is mandatory for both to undergo comprehensive colonic examination as early as possible. The study took place at a third-level general hospital with the objective of explaining in detail the importance of early diagnosis of FAP. CLINICAL CASES: We report on FAP in identical male twins who were operated on at different times with different outcomes and prognosis. The first twin was treated 20 years previously at an early age and underwent subtotal colectomy with ileoproctostomy. This patient is currently asymptomatic with no evidence of malignancy. The second twin was operated on at the age of 33 years and was already a carrier of a well differentiated rectal adenocarcinoma. CONCLUSIONS: Opportune therapy carried out on the first twin has resulted in a disease-free status, in contrast with the delay in treatment of the second twin who developed carcinoma.

Poliposis adenomatosa familiar: presentación de dos casos en gemelos masculinos idénticos / Familial adenomatous polyposis: case report of male twins

BACKGROUND: One hundred percent of the cases of familial adenomatous polyposis (FAP) will develop carcinoma; therefore, the necessity of diagnosis at an early age with immediate therapy is essential. In the presence of identical twins, it is mandatory for both to undergo comprehensive colonic examination as early as possible. The study took place at a third-level general hospital with the objective of explaining in detail the importance of early diagnosis of FAP. CLINICAL CASES: We report on FAP in identical male twins who were operated on at different times with different outcomes and prognosis. The first twin was treated 20 years previously at an early age and underwent subtotal colectomy with ileoproctostomy. This patient is currently asymptomatic with no evidence of malignancy. The second twin was operated on at the age of 33 years and was already a carrier of a well differentiated rectal adenocarcinoma. CONCLUSIONS: Opportune therapy carried out on the first twin has resulted in a disease-free status, in contrast with the delay in treatment of the second twin who developed carcinoma.

STAT1 activation causes translocation of Bax to the endoplasmic reticulum during the resolution of airway mucous cell hyperplasia by IFN-gamma.

Disruption of the normal resolution process of inflammation-induced mucous cell hyperplasia may lead to sustained mucous hypersecretion in chronic diseases. During prolonged exposure of mice to allergen, IFN-gamma reduces mucous cell hyperplasia, but the signaling responsible for the cell death is largely unknown. A brief phosphorylation of STAT1 by IFN-gamma was required for cell death in airway epithelial cells (AEC), and during prolonged exposure to allergen, mucous cell hyperplasia remained elevated in STAT1(-/-) but was resolved in STAT1(+/+) mice. Although IFN-gamma treatment of primary human AECs and other airway cell lines left Bax protein levels unchanged, it caused translocation of Bax from the cytosol to the endoplasmic reticulum (ER) but not to the mitochondria. Localization of Bax to the ER was observed in IFN-gamma-treated primary AECs isolated from STAT1(+/+) mice but not in cells from STAT1(-/-) mice. In addition, ER Bax was detected in mucous cells of STAT1(+/+) but not STAT1(-/-) airways of mice exposed to allergen for prolonged periods. IFN-gamma did not release cytochrome c from mitochondria but reduced ER calcium stores and dilated the ER, confirming that the IFN-gamma-induced cell death is mediated through changes localized in the ER. Collectively, these observations suggest that STAT1-dependent translocation of Bax to the ER is crucial for IFN-gamma-induced cell death of AECs and the resolution of allergen-induced mucous cell hyperplasia.

Classification of communication signals of the little brown bat.

Little brown bats, Myotis lucifugus, are known for their ability to echolocate and utilize their echolocation system to navigate, and locate and identify prey. Their echolocation signals have been characterized in detail but their communication signals are less well understood despite their widespread use during social interactions. The goal of this study was to develop an automatic classification algorithm for characterizing the communication signals of little brown bats. Sound recordings were made overnight on five individual male bats (housed separately from a large group of captive bats) for 7 nights, using a bat detector and a digital recorder. The spectral and temporal characteristics of recorded sounds were first analyzed and classified by visual observation of a call's temporal pattern and spectral composition. Sounds were later classified using an automatic classification scheme based on multivariate statistical parameters in MATLAB. Human- and machine-based analysis revealed five discrete classes of bat's communication signals: downward frequency-modulated calls, steep frequency-modulated calls, constant frequency calls, broadband noise bursts, and broadband click trains.

Heat shock protein 70 and glycoprotein 96 are differentially expressed on the surface of malignant and nonmalignant breast cells.

Heat shock proteins (HSPs), which are important for a number of different intracellular functions, are occasionally found on the surface of cells. The function of heat shock protein on the cell surface is not understood, although it has been shown to be greater in some tumor cells and some virally infected cells. Surface expression of both glycoprotein 96 (gp96) and Hsp70 occurs on tumor cells, and this expression correlates with natural killer cell killing of the cells. We examined the surface expression of gp96 and Hsp70 on human breast cell lines MCF7, MCF10A, AU565, and HS578, and in primary human mammary epithelial cells by immunofluorescence microscopy and flow cytometry. The nonmalignant cell lines HS578, MCF10A, and HMEC showed no surface expression of gp96, whereas malignant cell lines MCF7 and AU565 were positive for gp96 surface expression. All of the breast cell lines examined showed Hsp70 surface expression. These results also confirm previous studies, demonstrating that Hsp70 is on the plasma membrane of tumor cell lines. Given the involvement of heat shock proteins, gp96 and Hsp70, in innate and adaptive immunity, these observations may be important in the immune response to tumor cells.