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.

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.

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.

Insights into the key roles of proteoglycans in breast cancer biology and translational medicine.

Proteoglycans control numerous normal and pathological processes, among which are morphogenesis, tissue repair, inflammation, vascularization and cancer metastasis. During tumor development and growth, proteoglycan expression is markedly modified in the tumor microenvironment. Altered expression of proteoglycans on tumor and stromal cell membranes affects cancer cell signaling, growth and survival, cell adhesion, migration and angiogenesis. Despite the high complexity and heterogeneity of breast cancer, the rapid evolution in our knowledge that proteoglycans are among the key players in the breast tumor microenvironment suggests their potential as pharmacological targets in this type of cancer. It has been recently suggested that pharmacological treatment may target proteoglycan metabolism, their utilization as targets for immunotherapy or their direct use as therapeutic agents. The diversity inherent in the proteoglycans that will be presented herein provides the potential for multiple layers of regulation of breast tumor behavior. This review summarizes recent developments concerning the biology of selected proteoglycans in breast cancer, and presents potential targeted therapeutic approaches based on their novel key roles in breast cancer.

Fell-Muir Lecture: Syndecans: from peripheral coreceptors to mainstream regulators of cell behaviour.

In the 25 years, as the first of the syndecan family was cloned, interest in these transmembrane proteoglycans has steadily increased. While four distinct members are present in mammals, one is present in invertebrates, including C. elegans that is such a powerful genetic model. The syndecans, therefore, have a long evolutionary history, indicative of important roles. However, these roles have been elusive. The knockout in the worm has a developmental neuronal phenotype, while knockouts of the syndecans in the mouse are mild and mostly limited to post-natal rather than developmental effects. Moreover, their association with high-affinity receptors, such as integrins, growth factor receptors, frizzled and slit/robo, have led to the notion that syndecans are coreceptors, with minor roles. Given that their heparan sulphate chains can gather many different protein ligands, this gave credence to views that the importance of syndecans lay with their ability to concentrate ligands and that only the extracellular polysaccharide was of significance. Syndecans are increasingly identified with roles in the pathogenesis of many diseases, including tumour progression, vascular disease, arthritis and inflammation. This has provided impetus to understanding syndecan roles in more detail. It emerges that while the cytoplasmic domains of syndecans are small, they have clear interactive capabilities, most notably with the actin cytoskeleton. Moreover, through the binding and activation of signalling molecules, it is likely that syndecans are important receptors in their own right. Here, an overview of syndecan structure and function is provided, with some prospects for the future.

Alterations in anatomic and functional imaging parameters with repeated FDG PET-CT and MRI during radiotherapy for head and neck cancer: a pilot study.

BACKGROUND: The use of imaging to implement on-treatment adaptation of radiotherapy is a promising paradigm but current data on imaging changes during radiotherapy is limited. This is a hypothesis-generating pilot study to examine the changes on multi-modality anatomic and functional imaging during (chemo)radiotherapy treatment for head and neck squamous cell carcinoma (HNSCC). METHODS: Eight patients with locally advanced HNSCC underwent imaging including computed tomography (CT), Fluorine-18 fluorodeoxyglucose (FDG) positron emission tomography (PET)-CT and magnetic resonance imaging (MRI) (including diffusion weighted (DW) and dynamic contrast enhanced (DCE)) at baseline and during (chemo)radiotherapy treatment (after fractions 11 and 21). Regions of interest (ROI) were drawn around the primary tumour at baseline and during treatment. Imaging parameters included gross tumour volume (GTV) assessment, SUVmax, mean ADC value and DCE-MRI parameters including Plasma Flow (PF). On treatment changes and correlations between these parameters were analysed using a Wilcoxon rank sum test and Pearson's linear correlation coefficient respectively. A p-value <0.05 was considered statistically significant. RESULTS: Statistically significant reductions in GTV-CT, GTV-MRI and GTV-DW were observed between all imaging timepoints during radiotherapy. Changes in GTV-PET during radiotherapy were heterogeneous and non-significant. Significant changes in SUVmax, mean ADC value, Plasma Flow and Plasma Volume were observed between the baseline and the fraction 11 timepoint, whilst only changes in SUVmax between baseline and the fraction 21 timepoint were statistically significant. Significant correlations were observed between multiple imaging parameters, both anatomical and functional; 20 correlations between baseline to the fraction 11 timepoint; 12 correlations between baseline and the fraction 21 timepoints; and 4 correlations between the fraction 11 and fraction 21 timepoints. CONCLUSIONS: Multi-modality imaging during radiotherapy treatment demonstrates early changes (by fraction 11) in both anatomic and functional imaging parameters. All functional imaging modalities are potentially complementary and should be considered in combination to provide multi-parametric tumour assessment, to guide potential treatment adaptation strategies. TRIAL REGISTRATION: ISRCTN Registry: ISRCTN34165059 . Registered 2nd February 2015.

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.

On the roles and regulation of chondroitin sulfate and heparan sulfate in zebrafish pharyngeal cartilage morphogenesis.

The present study addresses the roles of heparan sulfate (HS) proteoglycans and chondroitin sulfate (CS) proteoglycans in the development of zebrafish pharyngeal cartilage structures. uxs1 and b3gat3 mutants, predicted to have impaired biosynthesis of both HS and CS because of defective formation of the common proteoglycan linkage tetrasaccharide were analyzed along with ext2 and extl3 mutants, predicted to have defective HS polymerization. Notably, the effects on HS and CS biosynthesis in the respective mutant strains were shown to differ from what had been hypothesized. In uxs1 and b3gat3 mutant larvae, biosynthesis of CS was shown to be virtually abolished, whereas these mutants still were capable of synthesizing 50% of the HS produced in control larvae. extl3 and ext2 mutants on the other hand were shown to synthesize reduced amounts of hypersulfated HS. Further, extl3 mutants produced higher levels of CS than control larvae, whereas morpholino-mediated suppression of csgalnact1/csgalnact2 resulted in increased HS biosynthesis. Thus, the balance of the Extl3 and Csgalnact1/Csgalnact2 proteins influences the HS/CS ratio. A characterization of the pharyngeal cartilage element morphologies in the single mutant strains, as well as in ext2;uxs1 double mutants, was conducted. A correlation between HS and CS production and phenotypes was found, such that impaired HS biosynthesis was shown to affect chondrocyte intercalation, whereas impaired CS biosynthesis inhibited formation of the extracellular matrix surrounding chondrocytes.

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.

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.

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.

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.

Heparan sulfate chain valency controls syndecan-4 function in cell adhesion.

Fibroblasts null for the transmembrane proteoglycan, syndecan-4, have an altered actin cytoskeleton, compared with matching wild-type cells. They do not organize alpha-smooth muscle actin into bundles, but will do so when full-length syndecan-4 is re-expressed. This requires the central V region of the core protein cytoplasmic domain, though not interactions with PDZ proteins. A second key requirement is multiple heparan sulfate chains. Mutant syndecan-4 with no chains, or only one chain, failed to restore the wild-type phenotype, whereas those expressing two or three were competent. However, clustering of one-chain syndecan-4 forms with antibodies overcame the block, indicating that valency of interactions with ligands is a key component of syndecan-4 function. Measurements of focal contact/adhesion size and focal adhesion kinase phosphorylation correlated with syndecan-4 status and alpha-smooth muscle actin organization, being reduced where syndecan-4 function was compromised by a lack of multiple heparan sulfate chains.

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.

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.

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.

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.

Syndecans as receptors and organizers of the extracellular matrix.

Syndecans are type I transmembrane proteins having a core protein modified with glycosaminoglycan chains, most commonly heparan sulphate. They are an ancient group of molecules, present in invertebrates and vertebrates. Among the plethora of molecules that can interact with heparan sulphate, the collagens and glycoproteins of the extracellular matrix are prominent. Frequently, they do so in conjunction with other receptors, most notably the integrins. For this reason, they are often referred to as "co-receptors". However, just as with integrins, syndecans can interact with actin-associated proteins and signalling molecules, such as protein kinases. Some aspects of syndecan signalling are understood but much remains to be learned. The functions of syndecans in regulating cell adhesion and extracellular matrix assembly are described here. Evidence from null mice suggests that syndecans have roles in postnatal tissue repair, inflammation and tumour progression. Developmental deficits in lower vertebrates in which syndecans are eliminated are also informative and suggest that, in mammals, redundancy is a key issue.

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.