Multitier regulation of the E. coli extreme acid stress response by CsrA.

CsrA is an RNA-binding protein that regulates processes critical for growth and survival, including central carbon metabolism, motility, biofilm formation, stress responses, and expression of virulence factors in pathogens. Transcriptomics studies in Escherichia coli suggested that CsrA repressed genes involved in surviving extremely acidic conditions. Here, we examine the effects of disrupting CsrA-dependent regulation on the expression of genes and circuitry for acid stress survival and demonstrate CsrA-mediated repression at multiple levels. We show that this repression is critical for managing the trade-off between growth and survival; overexpression of acid stress genes caused by csrA disruption enhances survival under extreme acidity but is detrimental for growth under mildly acidic conditions. In vitro studies confirmed that CsrA binds specifically to mRNAs of structural and regulatory genes for acid stress survival, causing translational repression. We also found that translation of the top-tier acid stress regulator, evgA, is coupled to that of a small leader peptide, evgL, which is repressed by CsrA. Unlike dedicated acid stress response genes, csrA and its sRNA antagonists, csrB and csrC, did not exhibit a substantial response to acid shock. Furthermore, disruption of CsrA regulation of acid stress genes impacted host-microbe interactions in Caenorhabditis elegans, alleviating GABA deficiencies. This study expands the known regulon of CsrA to genes of the extreme acid stress response of E. coli and highlights a new facet of the global role played by CsrA in balancing the opposing physiological demands of stress resistance with the capacity for growth and modulating host interactions.IMPORTANCETo colonize/infect the mammalian intestinal tract, bacteria must survive exposure to the extreme acidity of the stomach. E. coli does this by expressing proteins that neutralize cytoplasmic acidity and cope with molecular damage caused by low pH. Because of the metabolic cost of these processes, genes for surviving acid stress are tightly regulated. Here, we show that CsrA negatively regulates the cascade of expression responsible for the acid stress response. Increased expression of acid response genes due to csrA disruption improved survival at extremely low pH but inhibited growth under mildly acidic conditions. Our findings define a new layer of regulation in the acid stress response of E. coli and a novel physiological function for CsrA.

Oh my gut! Is the microbial origin of neurodegenerative diseases real?

There is no cure or effective treatment for neurodegenerative protein conformational diseases (PCDs), such as Alzheimer's or Parkinson's diseases, mainly because the etiology of these diseases remains elusive. Recent data suggest that unique changes in the gut microbial composition are associated with these ailments; however, our current understanding of the bacterial role in the pathogenesis of PCDs is hindered by the complexity of the microbial communities associated with specific microbiomes, such as the gut, oral, or vaginal microbiota. The composition of these specific microbiomes is regarded as a unique fingerprint affected by factors such as infections, diet, lifestyle, and antibiotics. All of these factors also affect the severity of neurodegenerative diseases. The majority of studies that reveal microbial contribution are correlational, and various models, including worm, fly, and mouse, are being utilized to decipher the role of individual microbes that may affect disease onset and progression. Recent evidence from across model organisms and humans shows a positive correlation between the presence of gram-negative enteropathogenic bacteria and the pathogenesis of PCDs. While these correlational studies do not provide a mechanistic explanation, they do reveal contributing bacterial species and provide an important basis for further investigation. One of the lurking concerns related to the microbial contribution to PCDs is the increasing prevalence of antibiotic resistance and poor antibiotic stewardship, which ultimately select for proteotoxic bacteria, especially the gram-negative species that are known for intrinsic resistance. In this review, we summarize what is known about individual microbial contribution to PCDs and the potential impact of increasing antimicrobial resistance.

Colonization of the Caenorhabditis elegans gut with human enteric bacterial pathogens leads to proteostasis disruption that is rescued by butyrate.

Protein conformational diseases are characterized by misfolding and toxic aggregation of metastable proteins, often culminating in neurodegeneration. Enteric bacteria influence the pathogenesis of neurodegenerative diseases; however, the complexity of the human microbiome hinders our understanding of how individual microbes influence these diseases. Disruption of host protein homeostasis, or proteostasis, affects the onset and progression of these diseases. To investigate the effect of bacteria on host proteostasis, we used Caenorhabditis elegans expressing tissue-specific polyglutamine reporters that detect changes in the protein folding environment. We found that colonization of the C. elegans gut with enteric bacterial pathogens disrupted proteostasis in the intestine, muscle, neurons, and the gonad, while the presence of bacteria that conditionally synthesize butyrate, a molecule previously shown to be beneficial in neurodegenerative disease models, suppressed aggregation and the associated proteotoxicity. Co-colonization with this butyrogenic strain suppressed bacteria-induced protein aggregation, emphasizing the importance of microbial interaction and its impact on host proteostasis. Further experiments demonstrated that the beneficial effect of butyrate depended on the bacteria that colonized the gut and that this protective effect required SKN-1/Nrf2 and DAF-16/FOXO transcription factors. We also found that bacteria-derived protein aggregates contribute to the observed disruption of host proteostasis. Together, these results reveal the significance of enteric infection and gut dysbiosis on the pathogenesis of protein conformational diseases and demonstrate the potential of using butyrate-producing microbes as a preventative and treatment strategy for neurodegenerative disease.

Bacteria-Derived Protein Aggregates Contribute to the Disruption of Host Proteostasis.

Neurodegenerative protein conformational diseases are characterized by the misfolding and aggregation of metastable proteins encoded within the host genome. The host is also home to thousands of proteins encoded within exogenous genomes harbored by bacteria, fungi, and viruses. Yet, their contributions to host protein-folding homeostasis, or proteostasis, remain elusive. Recent studies, including our previous work, suggest that bacterial products contribute to the toxic aggregation of endogenous host proteins. We refer to these products as bacteria-derived protein aggregates (BDPAs). Furthermore, antibiotics were recently associated with an increased risk for neurodegenerative diseases, including Parkinson's disease and amyotrophic lateral sclerosis-possibly by virtue of altering the composition of the human gut microbiota. Other studies have shown a negative correlation between disease progression and antibiotic administration, supporting their protective effect against neurodegenerative diseases. These contradicting studies emphasize the complexity of the human gut microbiota, the gut-brain axis, and the effect of antibiotics. Here, we further our understanding of bacteria's effect on host protein folding using the model Caenorhabditis elegans. We employed genetic and chemical methods to demonstrate that the proteotoxic effect of bacteria on host protein folding correlates with the presence of BDPAs. Furthermore, the abundance and proteotoxicity of BDPAs are influenced by gentamicin, an aminoglycoside antibiotic that induces protein misfolding, and by butyrate, a short-chain fatty acid that we previously found to affect host protein aggregation and the associated toxicity. Collectively, these results increase our understanding of host-bacteria interactions in the context of protein conformational diseases.

Neuropsychological Assessment and Screening in Heart Failure: a Meta-Analysis and Systematic Review.

A variety of neuropsychological changes secondary to heart failure have been documented in the literature. However, what remains unclear are which neuropsychological abilities are the most impacted by heart failure and what tests have the sensitivity to measure that impact. Eight databases were searched for articles that examined the neuropsychological functioning of patients with heart failure. Some of the inclusion criteria were articles had to have a heart failure group with a demographically comparable control group and standardized neuropsychological testing. Exclusion criteria included articles with a heart failure group with any other type of major organ failure, or comparisons that were between different classes of heart failure rather than between a heart failure and non-heart failure group. A total of 33 articles met the inclusion criteria (total heart failure sample n = 8900) and provided effect size data for 20 neuropsychological domains. All observed domain-level differences between heart failure and non-heart failure groups were statistically significant, except for simple motor functioning and confrontation naming. The greatest differences in performance were in executive functioning, global cognition, complex psychomotor speed, and verbal memory. The highest effect sizes came from Trail-Making Test-Part B, CAMCOG, Symbol Digit Modality Test, and California Verbal Learning Test. The neuropsychological patterns of heart failure suggested diffuse cognitive involvement, with higher-level processes being most affected. It is important to track neurocognition in this clinical population since neuropsychological impairment is prevalent, and screening measures appear to be reliable. Such screening and further assessment would inform future medical treatment and may improve patient care management.

Ovarian cancer cells direct monocyte differentiation through a non-canonical pathway.

BACKGROUND: Alternatively-activated macrophages (AAMs), an anti-inflammatory macrophage subpopulation, have been implicated in the progression of high grade serous ovarian carcinoma (HGSOC). Increased levels of AAMs are correlated with poor HGSOC survival rates, and AAMs increase the attachment and spread of HGSOC cells in vitro. However, the mechanism by which monocytes in the HGSOC tumor microenvironment are differentiated and polarized to AAMs remains unknown. METHODS: Using an in vitro co-culture device, we cultured naïve, primary human monocytes with a panel of five HGSOC cell lines over the course of 7 days. An empirical Bayesian statistical method, EBSeq, was used to couple RNA-seq with observed monocyte-derived cell phenotype to explore which HGSOC-derived soluble factors supported differentiation to CD68+ macrophages and subsequent polarization towards CD163+ AAMs. Pathways of interest were interrogated using small molecule inhibitors, neutralizing antibodies, and CRISPR knockout cell lines. RESULTS: HGSOC cell lines displayed a wide range of abilities to generate AAMs from naïve monocytes. Much of this variation appeared to result from differential ability to generate CD68+ macrophages, as most CD68+ cells were also CD163+. Differences in tumor cell potential to generate macrophages was not due to a MCSF-dependent mechanism, nor variance in established pro-AAM factors. TGFα was implicated as a potential signaling molecule produced by tumor cells that could induce macrophage differentiation, which was validated using a CRISPR knockout of TGFA in the OVCAR5 cell line. CONCLUSIONS: HGSOC production of TGFα drives monocytes to differentiate into macrophages, representing a central arm of the mechanism by which AAMs are generated in the tumor microenvironment.

Withaferin A inhibits lysosomal activity to block autophagic flux and induces apoptosis via energetic impairment in breast cancer cells.

Withaferin A (WFA), a steroidal lactone, negatively regulates breast cancer growth however, its mechanisms of action remain largely elusive. We found that WFA blocks autophagy flux and lysosomal proteolytic activity in breast cancer cells. WFA increases accumulation of autophagosomes, LC3B-II conversion, expression of autophagy-related proteins and autophagosome/lysosome fusion. Autolysosomes display the characteristics of acidic compartments in WFA-treated cells; however, the protein degradation activity of lysosomes is inhibited. Blockade of autophagic flux reduces the recycling of cellular fuels leading to insufficient substrates for tricarboxylic acid (TCA) cycle and impaired oxidative phosphorylation. WFA decreases expression and phosphorylation of lactate dehydrogenase, the key enzyme that catalyzes pyruvate-to-lactate conversion, reduces adenosine triphosphate levels and increases AMP-activated protein kinase (AMPK) activation. AMPK inhibition abrogates while AMPK activation potentiates WFA's effect. WFA and 2-deoxy-d-glucose combination elicits synergistic inhibition of breast cancer cells. Genetic knockout of BECN1 and ATG7 fails to rescue cells from WFA treatment; in contrast, addition of methyl pyruvate to supplement TCA cycle protects WFA-treated cells. Together, these results implicate that WFA is a potent lysosomal inhibitor; energetic impairment is required for WFA-induced apoptosis and growth inhibition and combining WFA and 2-DG is a promising therapeutic strategy for breast cancer.

Breaking Boundaries in Pneumonia Diagnostics: Transitioning from Tradition to Molecular Frontiers with Multiplex PCR.

The advent of rapid molecular microbiology testing has revolutionized infectious disease diagnostics and is now impacting pneumonia diagnosis and management. Molecular platforms offer highly multiplexed assays for diverse viral and bacterial detection, alongside antimicrobial resistance markers, providing the potential to significantly shape patient care. Despite the superiority in sensitivity and speed, debates continue regarding the clinical role of multiplex molecular testing, notably in comparison to standard methods and distinguishing colonization from infection. Recent guidelines endorse molecular pneumonia panels for enhanced sensitivity and rapidity, but implementation requires addressing methodological differences and ensuring clinical relevance. Diagnostic stewardship should be leveraged to optimize pneumonia testing, emphasizing pre- and post-analytical strategies. Collaboration between clinical microbiologists and bedside providers is essential in developing implementation strategies to maximize the clinical utility of multiplex molecular diagnostics in pneumonia. This narrative review explores these multifaceted issues, examining the current evidence on the clinical performance of multiplex molecular assays in pneumonia, and reflects on lessons learned from previous microbiological advances. Additionally, given the complexity of pneumonia and the sensitivity of molecular diagnostics, diagnostic stewardship is discussed within the context of current literature, including implementation strategies that consider pre-analytical and post-analytical modifications to optimize the clinical utility of advanced technologies like multiplex PCR.

Divergent iron-regulatory states contribute to heterogeneity in breast cancer aggressiveness.

Primary tumors with similar mutational profiles can progress to vastly different outcomes where transcriptional state, rather than mutational profile, predicts prognosis. A key challenge is to understand how distinct tumor cell states are induced and maintained. In triple negative breast cancer cells, invasive behaviors and aggressive transcriptional signatures linked to poor patient prognosis can emerge in response to contact with collagen type I. Herein, collagen-induced migration heterogeneity within a TNBC cell line was leveraged to identify transcriptional programs associated with invasive versus non-invasive phenotypes and implicate molecular switches. Phenotype-guided sequencing revealed that invasive cells upregulate iron uptake and utilization machinery, anapleurotic TCA cycle genes, actin polymerization promoters, and a distinct signature of Rho GTPase activity and contractility regulating genes. The non-invasive cell state is characterized by actin and iron sequestration modules along with glycolysis gene expression. These unique tumor cell states are evident in patient tumors and predict divergent outcomes for TNBC patients. Glucose tracing confirmed that non-invasive cells are more glycolytic than invasive cells, and functional studies in cell lines and PDO models demonstrated a causal relationship between phenotype and metabolic state. Mechanistically, the OXPHOS dependent invasive state resulted from transient HO-1 upregulation triggered by contact with dense collagen that reduced heme levels and mitochondrial chelatable iron levels. This induced expression of low cytoplasmic iron response genes regulated by ACO1/IRP1. Knockdown or inhibition of HO-1, ACO1/IRP1, MRCK, or OXPHOS abrogated invasion. These findings support an emerging theory that heme and iron flux serve as important regulators of TNBC aggressiveness.

Identification of proteotoxic and proteoprotective bacteria that non-specifically affect proteins associated with neurodegenerative diseases.

Neurodegenerative protein conformational diseases (PCDs), such as Alzheimer's, Parkinson's, and Huntington's, are a leading cause of death and disability worldwide and have no known cures or effective treatments. Emerging evidence suggests a role for the gut microbiota in the pathogenesis of neurodegenerative PCDs; however, the influence of specific bacteria on the culprit proteins associated with each of these diseases remains elusive, primarily due to the complexity of the microbiota. In the present study, we employed a single-strain screening approach to identify human bacterial isolates that enhance or suppress the aggregation of culprit proteins and the associated toxicity in Caenorhabditis elegans expressing Aß1-42, α-synuclein, and polyglutamine tracts. Here, we reveal the first comprehensive analysis of the human microbiome for its effect on proteins associated with neurodegenerative diseases. Our results suggest that bacteria affect the aggregation of metastable proteins by modulating host proteostasis rather than selectively targeting specific disease-associated proteins. These results reveal bacteria that potentially influence the pathogenesis of PCDs and open new promising prevention and treatment opportunities by altering the abundance of beneficial and detrimental microbes.

Neuropsychological Performance in Alzheimer's Disease versus Late-Life Depression: A Systematic Review and Meta-Analysis.

OBJECTIVE: Despite decades of research, neuropsychological tests (NPTs) that clearly differentiate between Alzheimer's disease (AD) and late-life depression (LLD) have yet to be agreed upon. Given this gap in knowledge and the rapid deployment of disease-modifying drugs for the two disorders, accurate clinical diagnosis using evidence-based assessment is essential. This study aims to systematically examine the literature to identify NPTs that would be able to differentiate AD and LLD. METHOD: Databases and bibliographies were searched to identify articles for analysis. Two major inclusion criteria were that the studies compared neuropsychological functioning of AD versus LLD using normed NPTs and provided data for effect size calculation. Risk of bias was minimized by having independent coders for all steps in the review. RESULTS: Forty-one studies met inclusion criteria (N = 2,797) and provided effect sizes for tests that were classified as belonging to 15 domains of functioning. The two groups were well differentiated by tasks of delayed contextual verbal memory as compared to immediate or non-contextual memory, recognition cueing, confrontation naming, visuospatial construction, and conceptualization. Specific NPTs that appear to be useful for differential diagnosis include the Rey Auditory Verbal Learning Test-Delayed Recognition; Boston Naming Test; the Dementia Rating Scale's memory, conceptualization, and construction subscales; and the CERAD Constructional Praxis. CONCLUSIONS: The NPTs highlighted in this systematic review could be used as a relatively simple and cost-effective method to differentiate between patients with cognitive dysfunction due to AD versus LLD.

Time-off-pick Assay to Measure Caenorhabditis elegans Motility.

Caenorhabditis elegans is a simple metazoan that is often used as a model organism to study various human ailments with impaired motility phenotypes, including protein conformational diseases. Numerous motility assays that measure neuro-muscular function have been employed using C. elegans . Here, we describe "time-off-pick" (TOP), a novel assay for assessing motility in C. elegans . TOP is conducted by sliding an eyebrow hair under the mid-section of the worm and counting the number of seconds it takes for the worm to crawl completely off. The time it takes for the worm to crawl off the eyebrow hair is proportional to the severity of its motility defect. Other readouts of motility include crawling or swimming phenotypes, and although widely established, have some limitations. For example, worms that are roller mutants are less suitable for crawling or swimming assays. We demonstrated that our novel TOP assay is sensitive to age-dependent changes in motility, thus, providing another more inclusive method to assess motor function in C. elegans . Graphical abstract: Conceptual overview of the "time-off-pick" (TOP) assay. Various C. elegans models exhibit age-dependent defects in motility. The time it takes for a worm to crawl off of an eyebrow pick that is slid under its mid-section is measured in TOP seconds. A greater TOP is indicative of a greater motility defect. Eventually, worms with phenotypes that lead to paralysis will not be able to leave the pick.

Quantification of Bacterial Loads in Caenorhabditis elegans.

Caenorhabditis elegans is a ubiquitous free-living nematode that feeds on bacteria. The organism was introduced into a laboratory setting in the 1970s and has since gained popularity as a model to study host-bacteria interactions. One advantage of using C. elegans is that its intestine can be colonized by the bacteria on which it feeds. Quantifying the bacterial load within C. elegans is an important and easily obtainable metric when investigating host-bacteria interactions. Although quantification of bacteria harbored in C. elegans via whole-worm lysis is not a novel assay, there is great variation between existing methods. To lyse C. elegans, many protocols rely on the use of a hand-held homogenizer, which could introduce systematic error and subsequent variation between researchers performing the same experiment. Here, we describe a method of lysing the intestines of C. elegans to quantify the bacterial load within the intestine. Our method has been optimized for removing exogenous bacteria while maintaining worm paralysis, to ensure no bactericidal agents are swallowed, which could kill bacteria within the intestine and affect results. We utilize and compare the efficiency of two different homogenization tools: a battery-powered hand-held homogenizer, and a benchtop electric homogenizer, where the latter minimizes variability. Thus, our protocol has been optimized to reduce systematic error and decrease the potential for variability among experimenters. Graphic abstract: Simplified overview of the procedure used to quantify the bacterial load within C. elegans. The two different methods are herein described for worm lysis: "Option 1" is a hand-held homogenizer, and "Option 2" is a benchtop homogenizer.

Automating Aggregate Quantification in Caenorhabditis elegans.

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Ovarian Cells Have Increased Proliferation in Response to Heparin-Binding Epidermal Growth Factor as Collagen Density Increases.

It is well known that during ovarian cancer progression, the omentum transforms from a thin lacy organ to a thick tougher tissue. However, the mechanisms regulating this transformation and the implications of the altered microenvironment on ovarian cancer progression remain unclear. To address these questions, the global and local concentrations of collagen I were determined for normal and metastatic human omentum. Collagen I was increased 5.3-fold in omenta from ovarian cancer patients and localized to areas of activated fibroblasts rather than regions with a high density of cancer cells. Transforming growth factor beta 1 (TGFß1) was detected in ascites from ovarian cancer patients (4 ng/mL), suggesting a potential role for TGFß1 in the observed increase in collagen. Treatment with TGFß1 induced fibroblast activation, proliferation, and collagen deposition in mouse omental explants and an in vitro model with human omental fibroblasts. Finally, the impact of increased collagen I on ovarian cancer cells was determined by examining proliferation on collagen I gels formulated to mimic normal and cancerous omenta. While collagen density alone had no impact on proliferation, a synergistic effect was observed with collagen density and heparin-binding epidermal growth factor treatment. These results suggest that TGFß1 induces collagen deposition from the resident fibroblasts in the omentum and that this altered microenvironment impacts cancer cell response to growth factors found in ascites. Impact statement Using quantitative analysis of patient samples, in vitro models of the metastatic ovarian cancer microenvironment were designed with pathologically relevant collagen densities and growth factor concentrations. Studies in these models support a mechanism where transforming growth factor ß1 in the ascites fluid induces omental fibroblast proliferation, activation, and deposition of collagen I, which then impacts tumor cell proliferation in response to additional ascites growth factors such as heparin-binding epidermal growth factor. This approach can be used to dissect mechanisms involved in microenvironmental modeling in multiple disease applications.

Fospropofol: a new sedative-hypnotic agent for monitored anesthesia care.

OBJECTIVE: To summarize the published clinical data on fospropofol, critically review the safety and efficacy information, and provide pertinent information for formulary review. DATA SOURCES: Data were collected from searches of MEDLINE (1966-June 30, 2009), EMBASE (1974-June 30, 2009), bibliographies of manuscripts, and www.fda.gov. Key search terms included fospropofol, Lusedra, Aquavan, sedative-hypnotic, and monitored anesthesia care. STUDY SELECTION AND DATA EXTRACTION: All Phase 1, Phase 2, and Phase 3 clinical trials studying the safety and efficacy of fospropofol were reviewed. DATA SYNTHESIS: Fospropofol is a water-soluble prodrug of propofol, a potent sedative-hypnotic agent. Propofol is highly lipophilic and is formulated in lipid-containing solvents, which have known disadvantages, including pain on injection, narrow therapeutic window with the potential to cause deep sedation, high lipid intake during long-term sedation, and risk of infection resulting from bacterial contamination. Due to its water solubility, fospropofol eliminates some of the known lipid emulsion-associated disadvantages of propofol and provides a more predictable peak onset of activity and more gradual recovery to a full state of consciousness. The pharmacokinetic and pharmacodynamic profiles of fospropofol make it an attractive agent for sedation for procedures of short duration. Unfortunately, the number of patients studied has been relatively small and the amount of safety data is limited. Of concern are reports of hypoxemia and hypotension; these reports are limited in number, but the episodes are serious and may require acute intervention. Although fospropofol holds promise for procedural sedation, due to limited safety data, the Food and Drug Administration has limited approval of fospropofol to monitored anesthesia care in patients undergoing diagnostic or therapeutic procedures. CONCLUSIONS: Fospropofol is a viable addition to the class of sedative-hypnotic agents due to the minimization of unwanted adverse effects of propofol and maintenance of a favorable pharmacokinetic profile facilitating sedation, anxiolysis, and rapid recovery. However, there are limited safety data to justify its use without the presence of dedicated anesthesia personnel.

Substrate curvature induces fallopian tube epithelial cell invasion via cell-cell tension in a model of ovarian cortical inclusion cysts.

Throughout the body, epithelial tissues contain curved features (e.g. cysts, ducts and crypts) that influence cell behaviors. These structures have varied curvature, with flat structures having zero curvature and structures such as crypts having large curvature. In the ovary, cortical inclusion cysts (CICs) of varying curvatures are found, and fallopian tube epithelial (FTE) cells have been found trapped within these cysts. FTE are the precursor for ovarian cancer, and the CIC niche has been proposed to play a role in ovarian cancer progression. We hypothesized that variations in ovarian CIC curvature that occur during cyst resolution impact the ability of trapped FTE cells to invade into the surrounding stroma. Using a lumen model in collagen gels, we determined that increased curvature resulted in more invasions of mouse FTE cells. To isolate curvature as a system parameter, we developed a novel technique to pattern concave curvatures into collagen gels. When FTE cells were seeded to confluency on curved substrates, increases in curvature increased the number of invading FTE cells and the invasion distance. FTE invasion into collagen substrates with higher curvature depended on matrix metalloproteinases (MMPs), but expression of collagen I degrading Mmps was not different on curved and flat regions. A finite-element model predicted that contractility and cell-cell connections were essential for increased invasion on substrates with higher curvature, while cell-substrate interactions had minimal effect. Experiments supported these predictions, with invasion decreased by blebbistatin, ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or N-cadherin-blocking antibody, but with no effect from a focal adhesion kinase inhibitor. Finally, experimental evidence supports that cell invasion on curved substrates occurs in two phases-a cell-cell-dependent initiation phase where individual cells break away from the monolayer and an MMP-dependent phase as cells migrate further into the collagen matrix.

Nrf2 signaling and autophagy are complementary in protecting breast cancer cells during glucose deprivation.

Autophagy can serve as a mechanism for survival of cells during nutrient deprivation by recycling cellular macromolecules and organelles transiently to provide essential metabolic substrates. However, autophagy itself causes metabolic stress to cells, and other cellular protective mechanisms likely cooperate with autophagy to promote cell survival during nutrient deprivation. In this study, we explored protective mechanisms in breast cancer cells in the setting of glucose deprivation. While breast cancer cells (MCF7 and T47D) survive in glucose-free medium for three days or more, autophagy is induced in this setting. Blocking autophagy pharmacologically with chloroquine or by knock-out of an essential autophagy gene, such as Beclin 1 or ATG7, markedly reduces the ability of cells to survive during glucose deprivation. Autophagy previously was shown to degrade p62, a protein that sequesters KEAP1, and KEAP1 in turn sequesters Nrf2, a master regulator of the antioxidant response. Hence, we investigated how the Nrf2 signaling pathway might be affected by glucose deprivation and autophagy. We found that while glucose deprivation does cause decreased cellular levels of p62, Nrf2 protein levels and activity unexpectedly increase in this setting. Moreover, this increase in Nrf2 activity provides important protection to breast cancer cells during glucose deprivation, since siRNA knockdown of Nrf2 markedly impairs survival during glucose deprivation. Antioxidants, N-acetyl cysteine and glutathione also protect these cells during glucose deprivation, leading us to conclude that Nrf2 signaling via its antioxidant activity has a critical and previously undescribed role of protecting cells during glucose deprivation-induced autophagy.

The Extracellular Matrix of Ovarian Cortical Inclusion Cysts Modulates Invasion of Fallopian Tube Epithelial Cells.

A growing body of research supports the idea that the fallopian tube epithelium (FTE) is the precursor for most high-grade serous ovarian canacers (HGSOC) but that the ovary plays a critical role in tumor metastasis. Cortical inclusion cysts (CICs) in the ovarian cortex have been hypothesized to create a niche environment that plays a role in HGSOC progression. Through histological analysis of pathology samples from human ovaries, we determined that collagen I and III were elevated near CICs and that the collagen fibers in this dense region were oriented parallel to the cyst boundary. Using this information from human samples as design parameters, we engineered an in vitro model that recreates the size, shape, and extracellular matrix (ECM) properties of CICs. We found that FTE cells within our model underwent robust invasion that was responsive to stimulation with follicular fluid, while ovarian surface epithelial (OSE) cells, the native cells of the ovary, were not invasive. We provide experimental evidence to support a role of the extracellular matrix in modulating FTE cell invasion, as decreased collagen I concentration or the addition of collagen III to the matrix surrounding FTE cells increased FTE cell invasion. Taken together, we show that an in vitro model of CICs informed by the analysis of human tissue can act as an important tool for understanding FTE cell interactions with their environment.

PGE2 Augments Inflammasome Activation and M1 Polarization in Macrophages Infected With Salmonella Typhimurium and Yersinia enterocolitica.

Eicosanoids are cellular metabolites, which shape the immune response, including inflammatory processes in macrophages. The effects of these lipid mediators on inflammation and bacterial pathogenesis are not clearly understood. Certain eicosanoids are suspected to act as molecular sensors for the recruitment of neutrophils, while others regulate bacterial uptake. In this study, gene expression analyses indicated that genes involved in eicosanoid biosynthesis including COX-1, COX-2, DAGL, and PLA-2 are differentially regulated in THP-1 human macrophages infected with Salmonella enterica Typhimurium or Yersinia enterocolitica. By using targeted metabolomics approach, we found that the eicosanoid precursor, arachidonic acid (AA) as well as its derivatives, including prostaglandins (PGs) PGF2α or PGE2/PGD2, and thromboxane TxB2, are rapidly secreted from macrophages infected with these Gram-negative pathogenic bacteria. The magnitude of eicosanoid biosynthesis in infected host cells depends on the presence of virulence factors of Y. enterocolitica and S. Typhimurium strains, albeit in an opposite way in Y. enterocolitica compared to S. Typhimurium infection. Trials with combinations of EP2/EP4 PGE2 receptor agonists and antagonists suggest that PGE2 signaling in these infection models works primarily through the EP4 receptor. Downstream of EP4 activation, PGE2 enhances inflammasome activation and represses M2 macrophage polarization while inducing key M1-type markers. PGE2 also led to a decreased numbers of Y. enterocolitica within macrophages. To summarize, PGE2 is a potent autocrine/paracrine activator of inflammation during infection in Gram-negative bacteria, and it affects macrophage polarization, likely controlling bacterial clearance by macrophages.