Activation of protein kinase Cδ leads to increased pancreatic acinar cell dedifferentiation in the absence of MIST1

Pancreatic ductal adenocarcinoma (PDAC) has a 5 year survival rate post-diagnosis of \u3c 5%. Individuals with chronic pancreatitis (CP) are 20-fold more likely to develop PDAC, making it a significant risk factor for PDAC. While the relationship for the increased susceptibility to PDAC is unknown, loss of the acinar cell phenotype is common to both pathologies. Pancreatic acinar cells can dedifferentiate or trans-differentiate into a number of cell types including duct cells, β cells, hepatocytes and adipocytes. Knowledge of the molecular pathways that regulate this plasticity should provide insight into PDAC and CP. MIST1 (encoded by Bhlha15 in mice) is a transcription factor required for complete acinar cell maturation. The goal of this study was to examine the plasticity of acinar cells that do not express MIST1 (Mist1 -/-). The fate of acinar cells from C57Bl6 or congenic Mist1 -/- mice expressing an acinar specific, tamoxifen-inducible Cre recombinase mated to Rosa26 reporter LacZ mice (Mist1CreERT/- R26r) was determined following culture in a three-dimensional collagen matrix. Mist1CreERT/- R26r acini showed increased acinar dedifferentiation, formation of ductal cysts and transient increases in PDX1 expression compared to wild-type acinar cells. Other progenitor cell markers, including Foxa1, Sox9, Sca1 and Hes1, were elevated only in Mist1-/- cultures. Analysis of protein kinase C (PKC) isoforms by western blot and immunofluorescence identified increased PKCε accumulation and nuclear localization of PKCδ that correlated with increased duct formation. Treatment with rottlerin, a PKCδ-specific inhibitor, but not the PKCε-specific antagonist εV1-2, reduced acinar dedifferentiation, progenitor gene expression and ductal cyst formation. Immunocytochemistry on CP or PDAC tissue samples showed reduced MIST1 expression combined with increased nuclear PKCδ accumulation. These results suggest that the loss of MIST1 is a common event during PDAC and CP and events that affect MIST1 function and expression may increase susceptibility to these pathologies. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

Immunochemical characterization of polysaccharide antigens from six clinical strains of Enterococci

BACKGROUND: Enterococci have become major nosocomial pathogens due to their intrinsic and acquired resistance to a broad spectrum of antibiotics. Their increasing drug resistance prompts us to search for prominent antigens to develop vaccines against enterococci. Given the success of polysaccharide-based vaccines against various bacterial pathogens, we isolated and characterized the immunochemical properties of polysaccharide antigens from five strains of Enterococcus faecalis and one strain of vancomycin-resistant E. faecium. RESULTS: We cultured large batches of each strain, isolated sufficient quantities of polysaccharides, analyzed their chemical structures, and compared their antigenic specificity. Three classes of polysaccharides were isolated from each strain, including a polyglucan, a teichoic acid, and a heteroglycan composed of rhamnose, glucose, galactose, mannosamine, and glucosamine. The polyglucans from all six strains are identical and appear to be dextran. Yields of the teichoic acids were generally low. The most abundant polysaccharides are the heteroglycans. The six heteroglycans are structurally different as evidenced by NMR spectroscopy. They also differ in their antigenic specificities as revealed by competitive ELISA. The heteroglycans are not immunogenic by themselves but conjugation to protein carriers significantly enhanced their ability to induce antibodies. CONCLUSION: The six clinical strains of enterococci express abundant, strain-specific cell-surface heteroglycans. These polysaccharides may provide a molecular basis for serological typing of enterococcal strains and antigens for the development of vaccines against multi-drug resistant enterococci

Can Extreme Bacteria Teach Us About Extraterrestrial Life?

Have you ever wondered if there is life beyond Earth? Scientists have been studying this topic for a long time and believe the answer might lie in extremophilic microbes, small organisms that thrive in extreme environments. In a 2022 study, scientists took extremophilic microbes from an analogue environment, or place on Earth similar to Mars, and put them in simulated Martian conditions. After exposing them to higher ultraviolet radiation levels, low oxygen levels, a dry atmosphere, and moisture-free Mars-like soil, these microbes still were able to survive. This research is important in helping us understand if Mars can house life and give us clues into what that life might look like beyond Earth

Identifying nonalcoholic fatty liver disease patients with active fibrosis by measuring extracellular matrix remodeling rates in tissue and blood.

Excess collagen synthesis (fibrogenesis) in the liver plays a causal role in the progression of nonalcoholic fatty liver disease (NAFLD). Methods are needed to identify patients with more rapidly progressing disease and to demonstrate early response to treatment. We describe here a novel method to quantify hepatic fibrogenesis flux rates both directly in liver tissue and noninvasively in blood. Twenty-one patients with suspected NAFLD ingested heavy water (2 H2 O, 50-mL aliquots) two to three times daily for 3-5 weeks prior to a clinically indicated liver biopsy. Liver collagen fractional synthesis rate (FSR) and plasma lumican FSR were measured based on 2 H labeling using tandem mass spectrometry. Patients were classified by histology for fibrosis stage (F0-F4) and as having nonalcoholic fatty liver or nonalcoholic steatohepatitis (NASH). Magnetic resonance elastography measurements of liver stiffness were also performed. Hepatic collagen FSR in NAFLD increased with advancing disease stage (e.g., higher in NASH than nonalcoholic fatty liver, positive correlation with fibrosis score and liver stiffness) and correlated with hemoglobin A1C. In addition, plasma lumican FSR demonstrated a significant correlation with hepatic collagen FSR.ConclusionUsing a well-characterized cohort of patients with biopsy-proven NAFLD, this study demonstrates that hepatic scar in NASH is actively remodeled even in advanced fibrosis, a disease that is generally regarded as static and slowly progressive. Moreover, hepatic collagen FSR correlates with established risks for fibrotic disease progression in NASH, and plasma lumican FSR correlates with hepatic collagen FSR, suggesting applications as direct or surrogate markers, respectively, of hepatic fibrogenesis in humans. (Hepatology 2017;65:78-88)

Large scale real-time PCR validation on gene expression measurements from two commercial long-oligonucleotide microarrays

BACKGROUND: DNA microarrays are rapidly becoming a fundamental tool in discovery-based genomic and biomedical research. However, the reliability of the microarray results is being challenged due to the existence of different technologies and non-standard methods of data analysis and interpretation. In the absence of a "gold standard"/"reference method" for the gene expression measurements, studies evaluating and comparing the performance of various microarray platforms have often yielded subjective and conflicting conclusions. To address this issue we have conducted a large scale TaqMan(® )Gene Expression Assay based real-time PCR experiment and used this data set as the reference to evaluate the performance of two representative commercial microarray platforms. RESULTS: In this study, we analyzed the gene expression profiles of three human tissues: brain, lung, liver and one universal human reference sample (UHR) using two representative commercial long-oligonucleotide microarray platforms: (1) Applied Biosystems Human Genome Survey Microarrays (based on single-color detection); (2) Agilent Whole Human Genome Oligo Microarrays (based on two-color detection). 1,375 genes represented by both microarray platforms and spanning a wide dynamic range in gene expression levels, were selected for TaqMan(® )Gene Expression Assay based real-time PCR validation. For each platform, four technical replicates were performed on the same total RNA samples according to each manufacturer's standard protocols. For Agilent arrays, comparative hybridization was performed using incorporation of Cy5 for brain/lung/liver RNA and Cy3 for UHR RNA (common reference). Using the TaqMan(® )Gene Expression Assay based real-time PCR data set as the reference set, the performance of the two microarray platforms was evaluated focusing on the following criteria: (1) Sensitivity and accuracy in detection of expression; (2) Fold change correlation with real-time PCR data in pair-wise tissues as well as in gene expression profiles determined across all tissues; (3) Sensitivity and accuracy in detection of differential expression. CONCLUSION: Our study provides one of the largest "reference" data set of gene expression measurements using TaqMan(® )Gene Expression Assay based real-time PCR technology. This data set allowed us to use an alternative gene expression technology to evaluate the performance of different microarray platforms. We conclude that microarrays are indeed invaluable discovery tools with acceptable reliability for genome-wide gene expression screening, though validation of putative changes in gene expression remains advisable. Our study also characterizes the limitations of microarrays; understanding these limitations will enable researchers to more effectively evaluate microarray results in a more cautious and appropriate manner

Influenza Vaccination Generates Cytokine-Induced Memory-like NK Cells:Impact of Human Cytomegalovirus Infection

Human NK cells are activated by cytokines, immune complexes, and signals transduced via activating ligands on other host cells. After vaccination, or during secondary infection, adaptive immune responses can enhance both cytokine-driven and Ab-dependent NK cell responses. However, induction of NK cells for enhanced function after in vitro exposure to innate inflammatory cytokines has also been reported and may synergize with adaptive signals to potentiate NK cell activity during infection or vaccination. To test this hypothesis, we examined the effect of seasonal influenza vaccination on NK cell function and phenotype in 52 previously unvaccinated individuals. Enhanced, IL-2-dependent, NK cell IFN-γ responses to Influenza A/California/7/2009 virus were detected up to 4 wk postvaccination and higher in human CMV (HCMV)-seronegative (HCMV(-)) individuals than in HCMV-seropositive (HCMV(+)) individuals. By comparison, robust NK cell degranulation responses were observed both before and after vaccination, due to high titers of naturally occurring anti-influenza Abs in human plasma, and did not differ between HCMV(+) and HCMV(-) subjects. In addition to these IL-2-dependent and Ab-dependent responses, NK cell responses to innate cytokines were also enhanced after influenza vaccination; this was associated with proliferation of CD57(-) NK cells and was most evident in HCMV(+) subjects. Similar enhancement of cytokine responsiveness was observed when NK cells were cocultured in vitro with Influenza A/California/7/2009 virus, and this was at least partially dependent upon IFN-αβR2. In summary, our data indicate that attenuated or live viral vaccines promote cytokine-induced memory-like NK cells and that this process is influenced by HCMV infection

Fibroblast growth factor receptor 1 signaling in adult cardiomyocytes increases contractility and results in a hypertrophic cardiomyopathy

Fibroblast growth factors (FGFs) and their receptors are highly conserved signaling molecules that have been implicated in postnatal cardiac remodeling. However, it is not known whether cardiomyocyte-expressed FGF receptors are necessary or sufficient for ventricular remodeling in the adult heart. To determine whether cardiomyocytes were competent to respond to an activated FGF receptor, and to determine if this signal would result in the development of hypertrophy, we engineered a doxycycline (DOX)-inducible, cardiomyocyte-specific, constitutively active FGF receptor mouse model (αMHC-rtTA, TRE-caFgfr1-myc). Echocardiographic and hemodynamic analysis indicated that acute expression of caFGFR1 rapidly and directly increased cardiac contractility, while chronic expression resulted in significant hypertrophy with preservation of systolic function. Subsequent histologic analysis showed increased cardiomyocyte cross-sectional area and regions of myocyte disarray and fibrosis, classic features of hypertrophic cardiomyopathy (HCM). Analysis of downstream pathways revealed a lack of clear activation of classical FGF-mediated signaling pathways, but did demonstrate a reduction in Serca2 expression and troponin I phosphorylation. Isolated ventricular myocytes showed enhanced contractility and reduced relaxation, an effect that was partially reversed by inhibition of actin-myosin interactions. We conclude that adult cardiomyocytes are competent to transduce FGF signaling and that FGF signaling is sufficient to promote increased cardiomyocyte contractility in vitro and in vivo through enhanced intrinsic actin-myosin interactions. Long-term, FGFR overexpression results in HCM with a dynamic outflow tract obstruction, and may serve as a unique model of HCM

NGF-p75 signaling coordinates skeletal cell migration during bone repair

: Bone regeneration following injury is initiated by inflammatory signals and occurs in association with infiltration by sensory nerve fibers. Together, these events are believed to coordinate angiogenesis and tissue reprogramming, but the mechanism of coupling immune signals to reinnervation and osteogenesis is unknown. Here, we found that nerve growth factor (NGF) is expressed following cranial bone injury and signals via p75 in resident mesenchymal osteogenic precursors to affect their migration into the damaged tissue. Mice lacking Ngf in myeloid cells demonstrated reduced migration of osteogenic precursors to the injury site with consequently delayed bone healing. These features were phenocopied by mice lacking p75 in Pdgfra+ osteoblast precursors. Single-cell transcriptomics identified mesenchymal subpopulations with potential roles in cell migration and immune response, altered in the context of p75 deletion. Together, these results identify the role of p75 signaling pathway in coordinating skeletal cell migration during early bone repair

A Neurotrophic Mechanism Directs Sensory Nerve Transit in Cranial Bone

The flat bones of the skull are densely innervated during development, but little is known regarding their role during repair. We describe a neurotrophic mechanism that directs sensory nerve transit in the mouse calvaria. Patent cranial suture mesenchyme represents an NGF (nerve growth factor)-rich domain, in which sensory nerves transit. Experimental calvarial injury upregulates Ngf in an IL-1 beta/TNF-alpha-rich defect niche, with consequent axonal ingrowth. In calvarial osteoblasts, IL-1 beta and TNF-alpha stimulate Ngf and downstream NF-kappa B signaling. Locoregional deletion of Ngf delays defect site re-innervation and blunted repair. Genetic disruption of Ngf among LysM-expressing macrophages phenocopies these observations, whereas conditional knockout of Ngf among Pdgfra-expressing cells does not. Finally, inhibition of TrkA catalytic activity similarly delays re-innervation and repair. These results demonstrate an essential role of NGF-TrkA signaling in bone healing and implicate macrophage-derived NGF-induced ingrowth of skeletal sensory nerves as an important mediator of this repair