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E-cadherin is a calcium dependent cell adhesion molecule that is key to the organization of cells in the epithelial tissue. It is a multidomain, trans-membrane protein in which the extracellular domain forms the homotypic, adhesive interaction while the intracellular domain interacts with the actin cytoskeleton through the catenin family of adaptor proteins. A number of recent studies have provided novel insights into the mechanism of adhesion formation by this class of adhesion proteins. Here, we describe an updated view of the process of E-cadherin adhesion formation with an emphasis on the role of molecular mobility, clustering, and active cellular processes.

The amounts of annual rainfall in the southwestern part of Bangladesh are almost equal having very little spatial variation. Significant decreasing trend (Mann-Kendal) of annual rainfall over the region is found (-4.5 mm/year at Mongla, -9.14 mm/year at Jessore, -15.71 mm/year at Madaripur) except Khulna, Satkhira and Khepupara, where the trend is positive over the long period (1948-2014) but it exhibits a decreasing trend during the recent period i.e., after 1980s. It clearly indicates a gradual decrease of the amount of rainfall over southwestern part, which has become most prominent in the recent climatic period of 1981-2014. The trend is more intense at the upper southwestern part, i.e., places like Jessore and Madaripur. Annual rainfall deviates within the range -42.6% to +48.6% over the region. Most wide annual deviation is observed in Khulna from 48.6% to -35.5%. Like annual rainfall, seasonal rainfall also has anomalous behaviour over the region. Monsoon rainfall at maximum locations are syncline with annual trend, but at Khulna, Satkhira and Mongla though the annual trend is negative but the monsoon trend is positive, it clearly indicates, intensification of rainfall in monsoon period over the fore southwestern part. In pre-monsoon season, overall rainfall trend is significantly negative at maximum places (-8.49 mm/year at Jessore, -2.02 mm/year at Barisal, -7.80 mm/year at Madaripur) over the region except Khulna, satkhira and Khepupara. But in Khulna and Satkhira it is also significantly negative over recent climatic period. Rainfall deviation is higher in seasonal scale than the annual scale. Among the seasons wider deviation is observed in winter (from -100.0% to +586%) and narrower in monsoon (from -43.0% to +62.1%). The coastal region of southwest Bangladesh has been facing various natural extremes like salinity intensification, drainage congestion, inundation, water logging etc. Anomalous behavior of rainfall in combination with these problems, affecting agricultural crop production in the upazilas under study. The problem is serious in Rabi season, where groundwater irrigation facility is limited for salinity problem. In these areas agriculture is totally dependent on surface water (irrigation canal), which becomes scarce during the month of January, February, March and rainfall is also becoming low in this time (supports by public opinion and rainfall trend analysis) imposing water shortage in crop field and as a result farmers have to incur yield reduction. In monsoon season problem is different. Drainage congestion is a major problem in this time. A little heavy rainfall causes inundation and water logged condition. Besides this problem, increasing trend of rainfall in monsoon is very likely to intensify the risk of inundation. And the farmers of the study area have already faced total damage of Aus crop due to flooding in some years with comparatively high rainfall or fallow due to water logged condition. Moreover, overall decreasing trend of rainfall is more likely to intensify the risks of salinization due to decreasing upstream flow as well as sedimentation on river bed and consequent poor drainage and water logging. This intensified environmental problem is further likely to intensify the detrimental effects on crop production. So, changing pattern of rainfall in combination with the local environmental stress is being imposed on risk of agricultural crop production over the study area. It is also occasionally responsible for crop failure over the study area. It is also likely to further intensify the risk for future time.

BACKGROUND Familial diarrhea disorders are, in most cases, severe and caused by recessive mutations. We describe the cause of a novel dominant disease in 32 members of a Norwegian family. The affected members have chronic diarrhea that is of early onset, is relatively mild, and is associated with increased susceptibility to inflammatory bowel disease, small-bowel obstruction, and esophagitis. METHODS We used linkage analysis, based on arrays with single-nucleotide polymorphisms, to identify a candidate region on chromosome 12 and then sequenced GUCY2C, encoding guanylate cyclase C (GC-C), an intestinal receptor for bacterial heat-stable enterotoxins. We performed exome sequencing of the entire candidate region from three affected family members, to exclude the possibility that mutations in genes other than GUCY2C could cause or contribute to susceptibility to the disease. We carried out functional studies of mutant GC-C using HEK293T cells. RESULTS We identified a heterozygous missense mutation (c.2519G -> T) in GUCY2C in all affected family members and observed no other rare variants in the exons of genes in the candidate region. Exposure of the mutant receptor to its ligands resulted in markedly increased production of cyclic guanosine monophosphate (cGMP). This may cause hyperactivation of the cystic fibrosis transmembrane regulator (CFTR), leading to increased chloride and water secretion from the enterocytes, and may thus explain the chronic diarrhea in the affected family members. CONCLUSIONS Increased GC-C signaling disturbs normal bowel function and appears to have a proinflammatory effect, either through increased chloride secretion or additional effects of elevated cellular cGMP. Further investigation of the relevance of genetic variants affecting the GC-C-CFTR pathway to conditions such as Crohn's disease is warranted. (Funded by Helse Vest Western Norway Regional Health Authority] and the Department of Science and Technology, Government of India.)

Deoxyribonucleic acid (DNA) has been used as a material for a variety of applications, including surface functionalization for cell biological or in vitro reconstitution studies. Use of DNA-based surface functionalization eliminates limitations of multiplexing posed by traditionally used methods in applications requiring spatially segregated surface functionalization. Recently, we have reported a stochastic, membrane fusion-based strategy to fabricate multicomponent membrane array substrates displaying spatially segregated protein ligands using biotin-streptavidin and Ni-NTA-polyhistidine interactions. Here, we report the delivery of DNA oligonucleotide-conjugated lipid molecules to membrane corrals, allowing spatially segregated membrane corral functionalization in a membrane microarray. Incubation of microbeads coated with the supported membrane resulted in an exchange of lipid contents with planar membrane corrals present on a micropatterned substrate. Increases in the system temperature and membrane corral size resulted in alterations in the rate constant of lipid exchange, which are in agreement with our previously developed analytical model and further confirm that lipid exchange is a diffusion-based process that takes place after the formation of a long "fusion-stalk" between the two membranes. We take advantage of the physical dimensions of the fusion-stalk with a large aspect ratio to deliver DNA oligonucleotide-conjugated lipid molecules to membrane corrals. We believe that the ability to functionalize membrane corrals with DNA oligonucleotides significantly increases the utility of the stochastic fusion-mediated lipid delivery strategy in the functionalization of biomolecules such as DNA or DNA-conjugated protein ligands. NRF (Natl Research Foundation, S’pore)

Receptor guanylyl cyclases are multidomain proteins, and ligand binding to the extracellular domain increases the levels of intracellular cGMP. The intracellular domain of these receptors is composed of a kinase homology domain (KHD), a linker of ∼70 amino acids, followed by the C-terminal guanylyl cyclase domain. Mechanisms by which these receptors are allosterically regulated by ligand binding to the extracellular domain and ATP binding to the KHD are not completely understood. Here we examine the role of the linker region in receptor guanylyl cyclases by a series of point mutations in receptor guanylyl cyclase C. The linker region is predicted to adopt a coiled coil structure and aid in dimerization, but we find that the effects of mutations neither follow a pattern predicted for a coiled coil peptide nor abrogate dimerization. Importantly, this region is critical for repressing the guanylyl cyclase activity of the receptor in the absence of ligand and permitting ligand-mediated activation of the cyclase domain. Mutant receptors with high basal guanylyl cyclase activity show no further activation in the presence of non-ionic detergents, suggesting that hydrophobic interactions in the basal and inactive conformation of the guanylyl cyclase domain are disrupted by mutation. Equivalent mutations in the linker region of guanylyl cyclase A also elevated the basal activity and abolished ligand- and detergent-mediated activation. We, therefore, have defined a key regulatory role for the linker region of receptor guanylyl cyclases which serves as a transducer of information from the extracellular domain via the KHD to the catalytic domain.

Guanylyl cyclases (GCs) are enzymes that generate cyclic GMP and regulate different physiologic and developmental processes in a number of organisms. GCs possess sequence similarity to class III adenylyl cyclases (ACs) and are present as either membrane-bound receptor GCs or cytosolic soluble GCs. We sought to determine the evolution of GCs using a large-scale bioinformatic analysis and found multiple lineage-specific expansions of GC genes in the genomes of many eukaryotes. Moreover, a few GC-like proteins were identified in prokaryotes, which come fused to a number of different domains, suggesting allosteric regulation of nucleotide cyclase activity. Eukaryotic receptor GCs are associated with a kinase homology domain (KHD), and phylogenetic analysis of these proteins suggest coevolution of the KHD and the associated cyclase domain as well as a conservation of the sequence and the size of the linker region between the KHD and the associated cyclase domain. Finally, we also report the existence of mimiviral proteins that contain putative active kinase domains associated with a cyclase domain, which could suggest early evolution of the fusion of these two important domains involved in signal transduction.

We describe here a novel sensor for cGMP based on the GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase 5 (PDE5) using bioluminescence resonance energy transfer (BRET). The wild type GAFa domain, capable of binding cGMP with high affinity, and a mutant (GAFa F163A) unable to bind cGMP were cloned as fusions between GFP and Rluc for BRET (2) assays. BRET (2) ratios of the wild type GAFa fusion protein, but not GAFa F163A, increased in the presence of cGMP but not cAMP. Higher basal BRET (2) ratios were observed in cells expressing the wild type GAFa domain than in cells expressing GAFa F163A. This was correlated with elevated basal intracellular levels of cGMP, indicating that the GAF domain could act as a sink for cGMP. The tandem GAF domains in full length PDE5 could also sequester cGMP when the catalytic activity of PDE5 was inhibited. Therefore, these results describe a cGMP sensor utilizing BRET (2) technology and experimentally demonstrate the reservoir of cGMP that can be present in cells that express cGMP-binding GAF domain-containing proteins. PDE5 is the target for the anti-impotence drug sildenafil citrate; therefore, this GAF-BRET (2) sensor could be used for the identification of novel compounds that inhibit cGMP binding to the GAF domain, thereby regulating PDE5 catalytic activity.

The turnover of integrin receptors is critical for cell migration and adhesion dynamics. Here we find that force development at integrins regulates adaptor protein recruitment and endocytosis. Using mobile RGD (Arg-Gly-Asp) ligands on supported lipid membranes (RGD membranes) and rigid RGD ligands on glass (RGD-glass), we find that matrix force-dependent integrin signals block endocytosis. Dab2, an adaptor protein of clathrin-mediated endocytosis, is not recruited to activated integrin-beta3 clusters on RGD-glass; however, it is recruited to integrin-mediated adhesions on RGD membranes. Further, when force generation is inhibited on RGD-glass, Dab2 binds to integrin-beta3 clusters. Dab2 binding to integrin-beta3 excludes other adhesion-related adaptor proteins, such as talin. The clathrin-mediated endocytic machinery combines with Dab2 to facilitate the endocytosis of RGD-integrin-beta3 clusters. From these observations, we propose that loss of traction force on ligand-bound integrin-beta3 causes recruitment of Dab2/clathrin, resulting in endocytosis of integrins., Force is known to recruit adaptor proteins to the intracellular tails of integrin extracellular matrix receptors. Here the authors show that matrix force-dependent β3 integrin signals block endocytosis by preventing the recruitment of the clathrin adaptor Dab2.

GAF domains are a large family of regulatory domains, and a subset are found associated with enzymes involved in cyclic nucleotide (cNMP) metabolism such as adenylyl cyclases and phosphodiesterases. CyaB2, an adenylyl cyclase from Anabaena, contains two GAF domains in tandem at the N-terminus and an adenylyl cyclase domain at the C-terminus. Cyclic AMP, but not cGMP, binding to the GAF domains of CyaB2 increases the activity of the cyclase domain leading to enhanced synthesis of cAMP. Here we show that the isolated GAFb domain of CyaB2 can bind both cAMP and cGMP, and enhanced specificity for cAMP is observed only when both the GAFa and the GAFb domains are present in tandem (GAFab domain). In silico docking and mutational analysis identified distinct residues important for interaction with either cAMP or cGMP in the GAFb domain. Structural changes associated with ligand binding to the GAF domains could not be detected by bioluminescence resonance energy transfer (BRET) experiments. However, amide hydrogen-deuterium exchange mass spectrometry (HDXMS) experiments provided insights into the structural basis for cAMP-induced allosteric regulation of the GAF domains, and differences in the changes induced by cAMP and cGMP binding to the GAF domain. Thus, our findings could allow the development of molecules that modulate the allosteric regulation by GAF domains present in pharmacologically relevant proteins.

Cells sense biochemical as well as mechanical signals from their microenvironment by engaging multiple receptors. In many cases, multiple receptors operate in concert, and it can be misleading to attempt to isolate a single ligand–receptor interaction. Supported lipid membranes are employed to reconstitute a number of cell receptor systems. Efforts are also made to fabricate membrane microarrays presenting multiple ligands in a spatially segregated manner. However, such membrane multiplexing methods are generally limited by complex instrumentation and scalability. Here, a straightforward method is presented to produce centimeter‐scale membrane microarrays displaying multiple, spatially segregated membrane‐anchored protein ligands suitable for live single‐cell studies. The method is based on stochastic membrane fusion and subsequent diffusion‐mediated mixing of their lipid content. The result is the delivery of membrane contents into spatially segregated membrane corrals. Utilizing the technology developed here, this work probes the recruitment of an adaptor protein, Shc1, to epidermal growth factor receptor (EGFR) and EphA2 receptors and shows that activation of EGFR results in a decrease in the recruitment of protein to activate EphA2 same cell. National Research Foundation (NRF)

Infants and children are at higher risk of heat stroke for several reasons. We report these cases to highlight the danger of leaving children unsupervised in vehicles, aid prompt diagnosis, and management of heat stroke. Two Nigerian siblings aged ranges 5 and 3 years old, were trapped inside an unlocked vehicle and subsequently developed heat stroke. Both children presented with hyperthermia, severe dehydration, convulsions, and loss of consciousness. One of them also had hematuria. They were treated by spraying water onto their bodies to bring down the temperature, intravenous fluid resuscitation, oxygen therapy, and anticonvulsants. Both eventually recovered and were discharged with no obvious neurologic sequalae, but are being followed.up. Key words: Childhood, heatstroke, hyperthermia, vehicular entrapment

The main objective of this study is to estimate the impact of bioslurry to Boro rice production in Bangladesh. Translog production function through Stochastic Frontier Apoproach (SFA) was applied for estimating the efficiency of Boro production. Data were collected from biogas users in the four district of Bangladesh: Mymensingh, Pabna, Thakurgaon and Dinajpur. Biogas users have received significant impact from bio-slurry to Boro rice production while chemical fertilizers have no significant impact to same production. The production efficiency of biogas users is notably different from traditional farms. The efficiency differences are explained mostly by farm size, year of education, family size and off-farm income. Bio-slurry could be applied for reducing application of chemical fertilizers, pesticides and labour requirement that lead to more organic agriculture practices with producing more output, earn more income and save foreign currency.

The behavior of cells in a tissue is regulated by chemical as well as physical signals arising from their microenvironment. While gel-based substrates have been widely used for mimicking a range of substrate rigidities, there is a need for the development of low rigidity substrates for mimicking the physical properties of soft tissues. In this study, the authors report the development of a supported lipid bilayer (SLB)-based low rigidity substrate for cell adhesion studies. SLBs are functionalized with either collagen I or fibronectin via covalent, amine coupling to a carboxyl group-modified lipid molecule. While the lipid molecules in the bilayer show long-range lateral mobility, the covalently functionalized extracellular matrix (ECM) proteins are immobile on the bilayer surface. Specific adhesion of cells results in an enrichment of the protein on the bilayer and the appearance of a zone of depletion around the cells. Further, the lateral reorganization of the ECM proteins is controlled by altering the fluidity of lipid molecules in the substrate. Thus, the experimental platform developed in this study can be utilized for addressing basic questions related to cell adhesion on low rigidity substrates as well as biomedical applications requiring adhesion of cells to low rigidity substrates.

6 GAF domains are a large family of regulatory domains, and a subset are found associated with 7 enzymes involved in cyclic nucleotide (cNMP) metabolism such as adenylyl cyclases and 8 phosphodiesterases. CyaB2, an adenylyl cyclase from Anabaena, contains two GAF domains in 9 tandem at the N-terminus and an adenylyl cyclase domain at the C-terminus. Cyclic AMP, but 10 not cGMP, binding to the GAF domains of CyaB2 increases the activity of the cyclase domain 11 leading to enhanced synthesis of cAMP. Here we show that the isolated GAFb domain of CyaB2 12 can bind both cAMP and cGMP, and enhanced specificity for cAMP is observed only when both 13 the GAFa and the GAFb domains are present in tandem (GAFab domain). In silico docking and 14 mutational analysis indicated distinct modes of binding of cAMP and cGMP to the GAFb 15 domain. Structural changes associated with ligand binding to the GAF domains could not be 16 detected by the highly sensitive Bioluminescence Resonance Energy Transfer (BRET) 17 experiments. Amide hydrogen-deuterium exchange mass spectrometry (HDXMS) experiments, 18 however, revealed the structural basis for cAMP-induced allosteric regulation of the GAF 19 domains, and differences in the structural changes induced by cAMP and cGMP binding to the 20 GAF domain. Thus, our results provide an insight into structural mechanisms of ligand binding 21 to GAF domains in general, which can be utilized in developing molecules that modulate the 22 allosteric regulation by GAF domains in pharmacologically relevant proteins. 23 PeerJ PrePrints | http://dx.doi.org/10.7287/peerj.preprints.614v1 | CC-BY 4.0 Open Access | rec: 15 Nov 2014, publ: 15 Nov 2014 P re P rin ts

The Eph receptors comprise the largest family of receptor tyrosine kinases. Their activation depends on juxtacrine signalling, triggered by ephrin ligands on an apposed cell membrane. Eph and ephrin are key regulators in many physiological developmental and pathological processes. For example, EphA2 is overexpressed in over 40% of all breast cancers, and their overexpression is highly correlated with metastasis and poor prognosis. However, the underlying mechanism of how EphA2 is triggered and related with metastasis is largely unknown.Using a reconstituted system made of supported lipid bilayers displaying ephrinA1 ligands and live cells expressing EphA2 receptors, we have previously observed the binding, clustering and centripetal movement of ligand-receptor complexes, followed by the activation of downstream signalling, including phosphorylation of EphA2 kinase and recruitment of ADAM10 metalloproteinase. Importantly, when ephrinA1-EphA2 lateral movement was inhibited by surface diffusion barriers, ADAM10 recruitment was greatly reduced, indicating the spatial sensing ability of EphA2 receptors.Here, we developed a subcellular-scale hybrid pattern of fluid ephrinA1 and immobilized RGD peptide to simultaneously mimic both cell membrane and extracellular matrix environments, with controllable shape and size. Fluorescence recovery after photobleaching showed that the lateral diffusion of ephrinA1 was not affected by the presence of RGD patterns. Live cell experiments revealed that cells formed integrin adhesions as well as ephrinA1-EphA2 complexes, and they displayed a more spread geometry. Remarkably, instead of centripetal movement of ephrinA1-EphA2 clusters, as seen on lipid bilayers alone, the ephrinA1-EphA2 clusters in this hybrid pattern are smaller and more dispersed. Ongoing work is aimed at determining the differential responses of downstream signalling such as ADAM10 recruitment and EphA2 endocytosis. This method can be extended to the study of other cell-cell and cell-matrix interactions.

E-cadherin-based cell-cell adhesions are key to development and maintenance of the epithelial tissue, and a loss of these adhesions may contribute to cancer development. These are mechanosensitive structures in that they are strengthened under tension. Mechanotransduction in these adhesions has been postulated to be mediated, in part, by a force-dependent conformational activation of α-catenin, which allows it to interact with vinculin, in addition to F-actin, resulting in strengthening of junctions. Here, using E-cadherin adhesions reconstituted on synthetic, nanopatterned membranes, we show that activation of α-catenin is dependent on E-cadherin clustering, and is sustained in the absence of mechanical force or association with F-actin or vinculin. Adhesions are formed by filopodia-mediated nucleation and micron-scale assembly of E-cadherin clusters, which could be distinguished as either peripheral or central depending on their relative location at the cell-bilayer adhesion. While F-actin, vinculin and phosphorylated myosin light chain associate only with the peripheral assemblies, activated α-catenin is present in both peripheral and central assemblies, and persisted in the central assemblies in the absence of actomyosin tension. Impeding filopodia-mediated nucleation and micron-scale assembly of E-cadherin adhesion complexes, by confining bilayer bound E-cadherin extracellular domain movement on nanopatterned substrates, reduced levels of activated α-catenin. Taken together, although the initial activation of α-catenin requires micron-scale clustering that may allow development of mechanical forces, sustained force is not required for maintaining α-catenin in the active state.