A nucleic acid binding protein map of germline regulation in Caenorhabditis elegans.

Fertility requires the faithful proliferation of germ cells and their differentiation into gametes. Controlling these cellular states demands precise timing and expression of gene networks. Nucleic acid binding proteins (NBPs) play critical roles in gene expression networks that influence germ cell development. There has, however, been no functional analysis of the entire NBP repertoire in controlling in vivo germ cell development. Here, we analyzed germ cell states and germline architecture to systematically investigate the function of 364 germline-expressed NBPs in the Caenorhabditis elegans germ line. Using germline-specific knockdown, automated germ cell counting, and high-content analysis of germ cell nuclei and plasma membrane organization, we identify 156 NBPs with discrete autonomous germline functions. By identifying NBPs that control the germ cell cycle, proliferation, differentiation, germline structure and fertility, we have created an atlas for mechanistic dissection of germ cell behavior and gamete production.

The matrisome landscape controlling in vivo germ cell fates.

The developmental fate of cells is regulated by intrinsic factors and the extracellular environment. The extracellular matrix (matrisome) delivers chemical and mechanical cues that can modify cellular development. However, comprehensive understanding of how matrisome factors control cells in vivo is lacking. Here we show that specific matrisome factors act individually and collectively to control germ cell development. Surveying development of undifferentiated germline stem cells through to mature oocytes in the Caenorhabditis elegans germ line enabled holistic functional analysis of 443 conserved matrisome-coding genes. Using high-content imaging, 3D reconstruction, and cell behavior analysis, we identify 321 matrisome genes that impact germ cell development, the majority of which (>80%) are undescribed. Our analysis identifies key matrisome networks acting autonomously and non-autonomously to coordinate germ cell behavior. Further, our results demonstrate that germ cell development requires continual remodeling of the matrisome landscape. Together, this study provides a comprehensive platform for deciphering how extracellular signaling controls cellular development and anticipate this will establish new opportunities for manipulating cell fates.

Assessment of the comparative agreement between chest radiographs and CT scans in intensive care units.

PURPOSE: Chest radiographs in critically ill patients can be difficult to interpret due to technical and clinical factors. We sought to determine the agreement of chest radiographs and CT scans, and the inter-observer variation of chest radiograph interpretation, in intensive care units (ICUs). METHODS: Chest radiographs and corresponding thoracic computerised tomography (CT) scans (as reference standard) were collected from 45 ICU patients. All radiographs were analysed by 20 doctors (radiology consultants, radiology trainees, ICU consultants, ICU trainees) from 4 different centres, blinded to CT results. Specificity/sensitivity were determined for pleural effusion, lobar collapse and consolidation/atelectasis. Separately, Fleiss' kappa for multiple raters was used to determine inter-observer variation for chest radiographs. RESULTS: The median sensitivity and specificity of chest radiographs for detecting abnormalities seen on CTs scans were 43.2% and 85.9% respectively. Diagnostic sensitivity for pleural effusion was significantly higher among radiology consultants but no specialty/experience distinctions were observed for specificity. Median inter-observer kappa coefficient among assessors was 0.295 ("fair"). CONCLUSIONS: Chest radiographs commonly miss important radiological features in critically ill patients. Inter-observer agreement in chest radiograph interpretation is only "fair". Consultant radiologists are least likely to miss thoracic radiological abnormalities. The consequences of misdiagnosis by chest radiographs remain to be determined.

Nuclear factor Y is a pervasive regulator of neuronal gene expression.

Nervous system function relies on the establishment of complex gene expression programs that provide neuron-type-specific and core pan-neuronal features. These complementary regulatory paradigms are controlled by terminal selector and parallel-acting transcription factors (TFs), respectively. Here, we identify the nuclear factor Y (NF-Y) TF as a pervasive direct and indirect regulator of both neuron-type-specific and pan-neuronal gene expression. Mapping global NF-Y targets reveals direct binding to the cis-regulatory regions of pan-neuronal genes and terminal selector TFs. We show that NFYA-1 controls pan-neuronal gene expression directly through binding to CCAAT boxes in target gene promoters and indirectly by regulating the expression of terminal selector TFs. Further, we find that NFYA-1 regulation of neuronal gene expression is important for neuronal activity and motor function. Thus, our research sheds light on how global neuronal gene expression programs are buffered through direct and indirect regulatory mechanisms.

Predictors of Short-term Mortality in Patients of Cirrhosis of Liver Presenting as Acute Kidney Injury: An In-hospital Prospective Observational Study.

Background: Acute kidney injury (AKI) is known to be associated with increased short-term mortality among cirrhotic patients. On this background, we designed this study to evaluate various causes of AKI among admitted patients with cirrhosis of liver and predictors of 90-day mortality. Methods: One hundred and two consecutive adult patients with cirrhosis of liver with AKI hospitalized between November 2016 and March 2018 were enrolled in this prospective study. Their detailed clinical profile, including biochemical parameters, the etiology of AKI, and their clinical outcome of survival or mortality at 90-days, were recorded. Results: The most common causes of AKI were infections, followed by hypovolemia, seen in 55.88% and 31.37% of the patients, respectively. Hepatorenal syndrome (HRS) was seen in 10.78%, while parenchymal renal disease was the least common (1.9%). The in-hospital mortality rate was 28.4%, while 90-day mortality was 39.21%. The HRS group had a high 90-day mortality rate of 54.54%. ROC analysis of various biochemical parameters revealed that serum creatinine (sCr), Model for End-Stage Liver Disease (MELD), International Normalized Ratio (INR), and Neutrophil-Lymphocyte ratio (NLR), followed by Child Turcotte Pugh (CTP), had high area under the curves of 0.785, 0.773, 0.747, 0.740, and 0.718, respectively, for the prediction of 90-day mortality. Conclusion: Infection is the commonest cause of AKI in cirrhosis; however, mortality in patients with HRS-AKI is higher than that in those with infection-related AKI. Serum creatinine at admission, INR, NLR, and CTP scores predict short-term mortality among patients with AKI in cirrhosis. Further, large prospective studies are needed to confirm these findings.

Treatment of Hospital-Acquired Infections in Patients with Cirrhosis - New Challenges.

Background: Hospital acquired infections (HAI) in the cirrhotic patients contribute to hepatic decompensation. With emergence of bacterial drug resistance, designing the treatment protocol of HA infection has become the foremost challenge. Purpose: To analyze the resistance pattern of organisms isolated from hospital-acquired (HA) infections and determine appropriate antibiotics treatment protocols for these infections. Study Design: A prospective hospital based observational study was undertaken. Patients and Methods: The present study was conducted over 18 months at Kasturba Medical College, Mangalore, Karnataka, India. Patients with suspected HA infections were subjected to clinical, hematological and microbiological evaluation. Antibiotic sensitivity evaluation was undertaken for the bacteria isolated from these patients. Results: During the study period, 398 patients with cirrhosis were 472 times admitted to the hospital for treatment. Out of these patients, 40 patients were diagnosed with 50 HA infections. Fifty five different organisms were isolated from these infections. It was found that these 55 bacteria isolates comprised 30 (54.54%) gram-negative (GN) and 25 (45.45%) gram-positive (GP) bacteria. Quite seriously, extended-spectrum beta-lactamase (ESBL) producers and methicillin-resistant Staphylococcus aureus (MRSA) were detected in 40% and 58% of GN and GP infections respectively. A total of 36 (65.4%) and (14.5%) 8 out of 55 isolated organisms exhibited multi-drug resistance (MDR) and extensive drug resistance (XDR) behavior, respectively. Conclusion: Cirrhosis patients with HA infection possess higher prevalence of MDR and XDR infections. In such sick patients, cephalosporin and quinolones are not the appropriate empirical antibiotics. Herein, we propose a tigecycline with carbapenem like meropenem and vancomycin based empirical antibiotics protocol to be prescribed for such patients. De-escalation is advised after the culture sensitivity report is obtained.

Functional recovery of the germ line following splicing collapse.

Splicing introns from precursor-messenger RNA (pre-mRNA) transcripts is essential for translating functional proteins. Here, we report that the previously uncharacterized Caenorhabditis elegans protein MOG-7 acts as a pre-mRNA splicing factor. Depleting MOG-7 from the C. elegans germ line causes intron retention in most germline-expressed genes, impeding the germ cell cycle, and causing defects in nuclear morphology, germ cell identity and sterility. Despite the deleterious consequences caused by MOG-7 loss, the adult germ line can functionally recover to produce viable and fertile progeny when MOG-7 is restored. Germline recovery is dependent on a burst of apoptosis that likely clears defective germ cells, and viable gametes generated from the proliferation of germ cells in the progenitor zone. Together, these findings reveal that MOG-7 is essential for germ cell development, and that the germ line can functionally recover after a collapse in RNA splicing.

A somatic proteoglycan controls Notch-directed germ cell fate.

Communication between the soma and germline optimizes germ cell fate programs. Notch receptors are key determinants of germ cell fate but how somatic signals direct Notch-dependent germ cell behavior is undefined. Here we demonstrate that SDN-1 (syndecan-1), a somatic transmembrane proteoglycan, controls expression of the GLP-1 (germline proliferation-1) Notch receptor in the Caenorhabditis elegans germline. We find that SDN-1 control of a somatic TRP calcium channel governs calcium-dependent binding of an AP-2 transcription factor (APTF-2) to the glp-1 promoter. Hence, SDN-1 signaling promotes GLP-1 expression and mitotic germ cell fate. Together, these data reveal SDN-1 as a putative communication nexus between the germline and its somatic environment to control germ cell fate decisions.

Efficacy and Validity of Image-Guided Percutaneous Fine Needle Aspiration and Core Biopsy of Liver Pathologies: Saga of Focal Hepatic Lesions from the Nodule to the Needle to the Slide.

CONTEXT: Radiology and pathology are pivotal tools in the investigational artillery for management of wide spectrum of hepatic lesions and early detection is of a paramount importance. AIMS: The study aimed at analyzing the efficacy, comparative yield and validity of image-guided aspiration cytology (FNA)/core biopsy (CB) in focal hepatic lesions. SETTINGS AND DESIGN: A retrospective hospital-based study was conducted in departments of Pathology and Radiology and Imaging of a tertiary care center. MATERIALS AND METHODS: Cases of focal hepatic lesions that underwent percutaneous image guided-FNA reported (2011-2018) were analyzed. Cytological-histopathological correlation was performed where available. FNA diagnoses were divided into four categories-positive for malignancy (group 1), atypical (group 2), negative for malignancy (group 3), and non-diagnostic (group 4). STATISTICAL ANALYSIS USED: Categorical data was depicted in the form of frequencies and proportions. Validity of percutaneous image-guided FNA diagnosis was collated with the final diagnosis and results were analyzed. RESULTS: A total of 338 FNA of focal hepatic lesions were reported in which 217 (68.2%) cases in group 1; 21 (6.2%) in group 2; 58 (17.2%) in group 3 and 42 (12.4%) in group 4. CB correlation was available in 123 cases. Based on clinical, radiological and pathological findings, conclusive final diagnoses were obtained and the cases were regrouped [malignant cases-245, benign lesions-57 and uncertain lesions-36]. Metastasis was the most common malignancy (175/245; 71.4%). Sensitivity, specificity, and overall diagnostic accuracy of FNA to categorize the lesion as benign or malignant were 96.94%, 100% and 97.51%, respectively. However, the cytology-histopathology correlation revealed discordance of subtyping the lesion in 20% of cases and sensitivity and specificity reduced to 80% and 50% respectively in rendering the specific diagnosis. CONCLUSIONS: Percutaneous image-guided FNA is a sensitive and specific tool with high diagnostic accuracy in evaluating focal hepatic lesions. The study highlights the pre-eminence of interventional radiology and cytology in the care of patients with liver lesions.

Functions of the extracellular matrix in development: Lessons from Caenorhabditis elegans.

Cell-extracellular matrix interactions are crucial for the development of an organism from the earliest stages of embryogenesis. The main constituents of the extracellular matrix are collagens, laminins, proteoglycans and glycosaminoglycans that form a network of interactions. The extracellular matrix and its associated molecules provide developmental cues and structural support from the outside of cells during development. The complex nature of the extracellular matrix and its ability for continuous remodeling poses challenges when investigating extracellular matrix-based signaling during development. One way to address these challenges is to employ invertebrate models such as Caenorhabditis elegans, which are easy to genetically manipulate and have an invariant developmental program. C. elegans also expresses fewer extracellular matrix protein isoforms and exhibits reduced redundancy compared to mammalian models, thus providing a simpler platform for exploring development. This review summarizes our current understanding of how the extracellular matrix controls the development of neurons, muscles and the germline in C. elegans.

Syndecan receptors: pericellular regulators in development and inflammatory disease.

The syndecans are the major family of transmembrane proteoglycans, usually bearing multiple heparan sulfate chains. They are present on virtually all nucleated cells of vertebrates and are also present in invertebrates, indicative of a long evolutionary history. Genetic models in both vertebrates and invertebrates have shown that syndecans link to the actin cytoskeleton and can fine-tune cell adhesion, migration, junction formation, polarity and differentiation. Although often associated as co-receptors with other classes of receptors (e.g. integrins, growth factor and morphogen receptors), syndecans can nonetheless signal to the cytoplasm in discrete ways. Syndecan expression levels are upregulated in development, tissue repair and an array of human diseases, which has led to the increased appreciation that they may be important in pathogenesis not only as diagnostic or prognostic agents, but also as potential targets. Here, their functions in development and inflammatory diseases are summarized, including their potential roles as conduits for viral pathogen entry into cells.

Syndecans in Inflammation at a Glance.

Syndecans are transmembrane proteoglycans with heparan and chondroitin sulfate chains attached to their extracellular domain. Like many proteoglycans, they interact with a large number of ligands, such as growth factors, adhesion receptors, soluble small molecules, proteinases, and other extracellular matrix proteins to initiate downstream signaling pathways. Syndecans play a major role in inflammation, mainly by regulating leukocyte extravasation and cytokine function. At the same time, syndecans can undergo cytokine mediated changes in their expression levels during inflammation. The function of syndecans during inflammation appears to depend on the stage of inflammation, sulfation of heparan/chondroitin sulfate chains, the rate of ectodomain shedding and the solubility of the ectodomains. From the current literature, it is clear that syndecans are not only involved in the initial recruitment of pro-inflammatory molecules but also in establishing a balanced progression of inflammation. This review will summarize how cell surface and soluble syndecans regulate multiple aspects of inflammation.

Calcium in Cell-Extracellular Matrix Interactions.

In multicellular organisms, the cells are surrounded by persistent, dynamic extracellular matrix (ECM), the largest calcium reservoir in animals. ECM regulates several aspects of cell behavior including cell migration and adhesion, survival, gene expression and differentiation, thus playing a significant role in health and disease. Calcium is reported to be important in the assembly of ECM, where it binds to many ECM proteins. While serving as a calcium reservoir, ECM macromolecules can directly interact with cell surface receptors resulting in calcium transport across the membrane. This chapter mainly focusses on the role of cell-ECM interactions in cellular calcium regulation and how calcium itself mediates these interactions.

Interferon-ß-induced miR-1 alleviates toxic protein accumulation by controlling autophagy.

Appropriate regulation of autophagy is crucial for clearing toxic proteins from cells. Defective autophagy results in accumulation of toxic protein aggregates that detrimentally affect cellular function and organismal survival. Here, we report that the microRNA miR-1 regulates the autophagy pathway through conserved targeting of the orthologous Tre-2/Bub2/CDC16 (TBC) Rab GTPase-activating proteins TBC-7 and TBC1D15 in Caenorhabditis elegans and mammalian cells, respectively. Loss of miR-1 causes TBC-7/TBC1D15 overexpression, leading to a block on autophagy. Further, we found that the cytokine interferon-ß (IFN-ß) can induce miR-1 expression in mammalian cells, reducing TBC1D15 levels, and safeguarding against proteotoxic challenges. Therefore, this work provides a potential therapeutic strategy for protein aggregation disorders.

A Protein Disulfide Isomerase Controls Neuronal Migration through Regulation of Wnt Secretion.

Appropriate Wnt morphogen secretion is required to control animal development and homeostasis. Although correct Wnt globular structure is essential for secretion, proteins that directly mediate Wnt folding and maturation remain uncharacterized. Here, we report that protein disulfide isomerase-1 (PDI-1), a protein-folding catalyst and chaperone, controls secretion of the Caenorhabditis elegans Wnt ortholog EGL-20. We find that PDI-1 function is required to correctly form an anteroposterior EGL-20/Wnt gradient during embryonic development. Furthermore, PDI-1 performs this role in EGL-20/Wnt-producing epidermal cells to cell-non-autonomously control EGL-20/Wnt-dependent neuronal migration. Using pharmacological inhibition, we further show that PDI function is required in human cells for Wnt3a secretion, revealing a conserved role for disulfide isomerases. Together, these results demonstrate a critical role for PDIs within Wnt-producing cells to control long-range developmental events that are dependent on Wnt secretion.

Glycan Mimetics from Natural Products: New Therapeutic Opportunities for Neurodegenerative Disease.

Neurodegenerative diseases (NDs) affect millions of people worldwide. Characterized by the functional loss and death of neurons, NDs lead to symptoms (dementia and seizures) that affect the daily lives of patients. In spite of extensive research into NDs, the number of approved drugs for their treatment remains limited. There is therefore an urgent need to develop new approaches for the prevention and treatment of NDs. Glycans (carbohydrate chains) are ubiquitous, abundant, and structural complex natural biopolymers. Glycans often covalently attach to proteins and lipids to regulate cellular recognition, adhesion, and signaling. The importance of glycans in both the developing and mature nervous system is well characterized. Moreover, glycan dysregulation has been observed in NDs such as Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS). Therefore, glycans are promising but underexploited therapeutic targets. In this review, we summarize the current understanding of glycans in NDs. We also discuss a number of natural products that functionally mimic glycans to protect neurons, which therefore represent promising new therapeutic approaches for patients with NDs.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Automated three-dimensional reconstruction of the Caenorhabditis elegans germline.

The Caenorhabditis elegans germline is widely used as a model to study stem cell development, chromosome dynamics and apoptosis. Major readouts of germline phenotypes such as cell counting and protein expression profiling are routinely analyzed manually and in a two-dimensional manner. The major disadvantages of the existing approaches are 1) they are time-consuming and laborious and 2) there is an inability to study the effects of genetic mutations in three dimensions. Here, we demonstrate a rapid, automated method for analyzing the three-dimensional distribution of proteins, germline nuclei and cytoskeletal structures in the C. elegans germline. Using this method, we have revealed previously unappreciated germline organization and cytoskeletal structures that will have a major impact on the characterization of germline phenotypes. To conclude, our new method dramatically enhances the efficiency and resolution of C. elegans germline analysis and may be applied to other cellular structures.

Fibronectin-guided migration of carcinoma collectives.

Functional interplay between tumour cells and their neoplastic extracellular matrix plays a decisive role in malignant progression of carcinomas. Here we provide a comprehensive data set of the human HNSCC-associated fibroblast matrisome. Although much attention has been paid to the deposit of collagen, we identify oncofetal fibronectin (FN) as a major and obligate component of the matrix assembled by stromal fibroblasts from head and neck squamous cell carcinomas (HNSCC). FN overexpression in tumours from 435 patients corresponds to an independent unfavourable prognostic indicator. We show that migration of carcinoma collectives on fibrillar FN-rich matrices is achieved through αvß6 and α9ß1 engagement, rather than α5ß1. Moreover, αvß6-driven migration occurs independently of latent TGF-ß activation and Smad-dependent signalling in tumour epithelial cells. These results provide insights into the adhesion-dependent events at the tumour-stroma interface that govern the collective mode of migration adopted by carcinoma cells to invade surrounding stroma in HNSCC.

Cell-extracellular matrix and cell-cell adhesion are linked by syndecan-4.

Cell-extracellular matrix (ECM) and cell-cell junctions that employ microfilaments are sites of tension. They are important for tissue repair, morphogenetic movements and can be emblematic of matrix contraction in fibrotic disease and the stroma of solid tumors. One cell surface receptor, syndecan-4, has been shown to regulate focal adhesions, junctions that form at the ends of microfilament bundles in response to matrix components such as fibronectin. Recently it has been shown that signaling emanating from this proteoglycan receptor includes regulation of Rho family GTPases and cytosolic calcium. While it is known that cell-ECM and cell-cell junctions may be linked, possible roles for syndecans in this process are not understood. Here we show that wild type primary fibroblasts and those lacking syndecan-4 utilize different cadherins in their adherens junctions and that tension is a major factor in this differential response. This corresponds to the reduced ability of fibroblasts lacking syndecan-4 to exert tension on the ECM and we now show that this may extend to reduced tension in cell-cell adhesion.