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Rationale Myocardial infarction (MI) triggers a dynamic microRNA response with the potential of yielding therapeutic targets. Objective We aimed to identify novel aberrantly expressed cardiac microRNAs post-MI with potential roles in adverse remodeling in a rat model, and to provide post-ischemic therapeutic inhibition of a candidate pathological microRNA in vivo. Methods and results Following microRNA array profiling in rat hearts 2 and 14 days post-MI, we identified a time-dependent up-regulation of miR-31 compared to sham-operated rats. A progressive increase of miR-31 (up to 91.4 ± 11.3 fold) was detected in the infarcted myocardium by quantitative real-time PCR. Following target prediction analysis, reporter gene assays confirmed that miR-31 targets the 3´UTR of cardiac troponin-T (Tnnt2), E2F transcription factor 6 (E2f6), mineralocorticoid receptor (Nr3c2) and metalloproteinase inhibitor 4 (Timp4) mRNAs. In vitro, hypoxia and oxidative stress up-regulated miR-31 and suppressed target genes in cardiac cell cultures, whereas LNA-based oligonucleotide inhibition of miR-31 (miR-31i) reversed its repressive effect on target mRNAs. Therapeutic post-ischemic administration of miR-31i in rats silenced cardiac miR-31 and enhanced expression of target genes, while preserving cardiac structure and function at 2 and 4 weeks post-MI. Left ventricular ejection fraction (EF) improved by 10% (from day 2 to 30 post-MI) in miR-31i-treated rats, whereas controls receiving scrambled LNA inhibitor or placebo incurred a 17% deterioration in EF. miR-31i decreased end-diastolic pressure and infarct size; attenuated interstitial fibrosis in the remote myocardium and enhanced cardiac output. Conclusion miR-31 induction after MI is deleterious to cardiac function while its therapeutic inhibition in vivo ameliorates cardiac dysfunction and prevents the development of post-ischemic adverse remodeling.

Long non-coding RNAs and microRNAs control gene expression to determine central nervous system development and function. Neuronal growth regulator 1 (NEGR1) is a cell adhesion molecule that plays an important role in neurite outgrowth during neuronal development and its precise expression is crucial for correct brain development. The data described here is related to the research article titled “A long non-coding RNA, BC048612 and a microRNA, miR-203 coordinate the gene expression of Neuronal growth regulator 1 (NEGR1) adhesion protein” [1]. This data article contains detailed bioinformatics analysis of genetic signatures at the Negr1 gene locus retrieved from the UCSC genome browser. This approach could be adopted to identify putative regulatory non-coding RNAs in other tissues and diseases. Keywords: Bioinformatics, Long non-coding RNA, MicroRNA, Negr1, Regulation

The regulatory roles for non-coding RNAs, the long non-coding RNAs and microRNAs, are emerging as crucial determinants of central nervous system development and function. Neuronal growth regulator 1 (NEGR1) is a cell adhesion molecule that has been shown to play an important role in neurite outgrowth during neuronal development. Precise expression of the Negr1 gene is crucial for proper brain development and is dysregulated during brain injury. Hence, we attempted to elucidate the non-coding RNAs that control Negr1 gene expression. A long non-coding RNA, BC048612, transcribed from the bidirectional GC-rich Negr1 gene promoter was found to influence Negr1 mRNA expression. In vitro knockdown of the long non-coding RNA resulted in significant down-regulation of Negr1 mRNA expression, NEGR1 protein levels and neurite length whereas over-expression enhanced Negr1 mRNA expression, NEGR1 protein levels and increased neurite length. Meanwhile, another non-coding RNA, microRNA-203, was found to target the 3' untranslated region of the Negr1 mRNA. Inhibition of microRNA-203 led to increased expression of Negr1 mRNA, elevated NEGR1 protein levels and increased neurite length. Conversely, microRNA-203 over-expression decreased the level of Negr1 mRNA, NEGR1 protein and neurite length. Neither microRNA-203 nor the long non-coding RNA, BC048612 could influence each other's expression. Hence, the long non-coding RNA, BC048612, and microRNA-203 were determined to be positive and negative regulators of Negr1 gene expression respectively. These processes have a direct effect on NEGR1 protein levels and neurite length, thus highlighting the importance of the regulatory non-coding RNAs in modulating Negr1 gene expression for precise neuronal development.

Aquaporins are transmembrane water channel proteins present in biological plasma membranes that aid in biological water filtration processes by transporting water molecules through at high speeds, while selectively blocking out other kinds of solutes. Aquaporin Z incorporated biomimetic membranes are envisaged to overcome the problem of high pressure needed, and holds great potential for use in water purification processes, giving high flux while keeping energy consumption low. The functionality of aquaporin Z in terms of osmotic permeability might be regulated by factors such as pH, temperature, crosslinking and hydrophobic thickness of the reconstituted bilayers. Hence, we reconstituted aquaporin Z into vesicles that are made from a series of amphiphilic block copolymers PMOXA-PDMS-PMOXAs with various hydrophobic molecular weights. The osmotic permeability of aquaporin Z in these vesicles was determined through a stopped-flow spectroscopy. In addition, the temperature and pH value of the vesicle solutions were adjusted within wide ranges to investigate the regulation of osmotic permeability of aquaporin Z through external conditions. Our results show that aquaporin Z permeability was enhanced by hydrophobic mismatch. In addition, the water filtration mechanism of aquaporin Z is significantly affected by the concentration of H+ and OH- ions.

Aim The potential diagnostic utility of circulating microRNAs in heart failure (HF) or in distinguishing HF with reduced vs. preserved left ventricular ejection fraction (HFREF and HFPEF, respectively) is unclear. We sought to identify microRNAs suitable for diagnosis of HF and for distinguishing both HFREF and HFPEF from non-HF controls and HFREF from HFPEF. Methods and results MicroRNA profiling performed on whole blood and corresponding plasma samples of 28 controls, 39 HFREF and 19 HFPEF identified 344 microRNAs to be dysregulated among the three groups. Further analysis using an independent cohort of 30 controls, 30 HFREF and 30 HFPEF, presented 12 microRNAs with diagnostic potential for one or both HF phenotypes. Of these, miR-1233, -183-3p, -190a, -193b-3p, -193b-5p, -211-5p, -494, and -671-5p distinguished HF from controls. Altered levels of miR-125a-5p, -183-3p, -193b-3p, -211-5p, -494, -638, and -671-5p were found in HFREF while levels of miR-1233, -183-3p, -190a, -193b-3p, -193b-5p, and -545-5p distinguished HFPEF from controls. Four microRNAs (miR-125a-5p, -190a, -550a-5p, and -638) distinguished HFREF from HFPEF. Selective microRNA panels showed stronger discriminative power than N-terminal pro-brain natriuretic peptide (NT-proBNP). In addition, individual or multiple microRNAs used in combination with NT-proBNP increased NT-proBNP's discriminative performance, achieving perfect intergroup distinction. Pathway analysis revealed that the altered microRNAs expression was associated with several mechanisms of potential significance in HF. Conclusions We report specific microRNAs as potential biomarkers in distinguishing HF from non-HF controls and in differentiating between HFREF and HFPEF.

Ischemic stroke is a major cause of mortality and morbidity globally. Among the ischemic stroke subtypes, cardioembolic stroke is with poor functional outcome (Modified Rankin score ≥ 2). Early diagnosis of cardioembolic stroke will prove beneficial. This study examined the microRNAs targeting cluster of differentiation 46 (CD46), a potential biomarker for cardioembolic stroke. CD46 mRNA level was shown to be differentially expressed (p < 0.001) between cardioembolic stroke (median = 1.32) and non-cardioembolic stroke subtypes (large artery stroke median = 5.05; small vessel stroke median = 6.45). Bioinformatic search showed that miR-19a, -20a, -185 and -374b were found to target CD46 mRNA and further verified by luciferase reporter assay. The levels of miRNAs targeting CD46 were significantly reduced (p < 0.05) in non-cardioembolic stroke patients (large artery stroke median: miR-19a = 0.63, miR-20a = 0.42, miR-185 = 0.32, miR-374b = 0.27; small artery stroke median: miR-19a = 0.07, miR-20a = 0.06, miR-185 = 0.07, miR-374b = 0.05) as compared to cardioembolic stroke patients (median: miR-19a = 2.69, miR-20a = 1.36, miR-185 = 1.05, miR-374b = 1.23). ROC curve showed that the miRNAs could distinguish cardioembolic stroke from non-cardioembolic stroke with better AUC value as compared to CD46. Endogenous expression of CD46 in Human Umbilical Vein Endothelial Cells (HUVECs) were found to be regulated by miR-19a and miR-20a. Thus implicating that miR-19a and -20a may play a role in pathogenesis of cardioembolic stroke, possibly via the endothelial cells.

MicroRNAs have been identified as key regulators of gene expression and thus their potential in disease diagnostics, prognosis and therapy is being actively pursued. Deregulation of microRNAs in cerebral pathogenesis has been reported to a limited extent in both animal models and human. Due to the complexity of the pathology, identifying stroke specific microRNAs has been a challenge. This study shows that microRNA profiles reflect not only the temporal progression of stroke but also the specific etiologies. A panel of 32 microRNAs, which could differentiate stroke etiologies during acute phase was identified and verified using a customized TaqMan Low Density Array (TLDA). Furthermore we also found 5 microRNAs, miR-125b-2*, -27a*, -422a, -488 and -627 to be consistently altered in acute stroke irrespective of age or severity or confounding metabolic complications. Differential expression of these 5 microRNAs was also observed in rat stroke models. Hence, their specificity to the stroke pathology emphasizes the possibility of developing these microRNAs into accurate and useful tools for diagnosis of stroke.

Wound healing is a major problem in diabetic patients and current treatments have met with limited success. We evaluated the treatment of excisional and diabetic wounds using a stem cell isolated from the human umbilical cord Wharton's jelly (hWJSC) that shares unique properties with embryonic and adult mesenchymal stem cells. hWJSCs are non-controversial, available in abundance, hypo-immunogenic, non-tumorigenic, differentiate into keratinocytes, and secrete important molecules for tissue repair. When human skin fibroblasts (CCD) in conventional scratch-wound assays were exposed to hWJSC-conditioned medium (hWJSC-CM) the fibroblasts at the wound edges migrated and completely covered the spaces by day 2 compared to controls. The number of invaded cells, cell viability, total collagen, elastin, and fibronectin levels were significantly greater in the hWJSC-CM treatment arm compared to controls (P

Seawater desalination and water reuse using membrane technology can provide a sustainable water supply to the world if such processes can be more energy-efficient. To harness the highly efficient water transport used by nature, it is proposed to incorporate trans-membrane water channel, AquaporinZ (AqpZ), into biomimetic membranes. However, the biomimetic membranes are intrinsically too fragile to be used in water purification. Here, we report a robust vesicular biomimetic membrane design and the synthesis route. The membrane is formed by cross-linking AqpZ-embedded block copolymer vesicles, followed by immobilizing vesicles on the membrane support via covalent binding, and then stabilizing through an optimized layer-by-layer polydopamine (PDA)–histidine (His) coating process. As compared with commercially available HTI membranes, the AqpZ-embedded vesicular membrane shows an order-of-magnitude increment in water flux (17.6 L/m 2 /h) with high salt retention (91.8%) when using 6000 ppm NaCl as the feed and 0.8 M sucrose as the draw solute in the forward osmosis operation. Thus, the vesicular membrane design may provide new insights into the design and fabrication of Aqp-embedded biomimetic membranes.

Ischemic stroke is a multi-factorial disease where some patients present themselves with little or no risk factors. Blood microRNA expression profiles are becoming useful in the diagnosis and prognosis of human diseases. We therefore investigated the blood microRNA profiles in young stroke patients who presented with minimal or absence of risk factors for stroke such as type 2 diabetes, dyslipidemia and hypertension. Blood microRNA profiles from these patients varied with stroke subtypes as well as different functional outcomes (based on modified Rankin Score). These microRNAs have been shown to target genes that are involved in stroke pathogenesis. The findings from our study suggest that molecular mechanisms in stroke pathogenesis involving low or no risk ischemic stroke patients could differ substantially from those with pre-existing risk factors.

Aquaporin (AQP) is a water channel protein in biological membranes that possesses exceptional water selectivity and permeability. The AQP-embedded biomimetic membrane can be potentially used in water purification and pharmaceutical separation. A practical and stable design of AQP-embedded mixed matrix membrane has been revealed in this work using the multilayer polyelectrolyte adsorption method. By encapsulating AQP-incorporated vesicles with positively charged poly-L-lysine molecules, the vesicles could be adsorbed onto polyanion films. AFM and FESEM studies showed that the amount of adsorbed vesicles was proportional to the percentage of charged lipids that was present in the liposomes. The AQP embedded layer-by-layer (LbL) membrane demonstrated a water permeability of 6 L m−2 h−1 bar−1 and MgCl2 rejection greater than 95%. In comparison with the control LbL membrane without embedding any liposomes, the newly designed mixed matrix membrane improved the water permeability by 60% because of the presence of AQPs. The AQPZ-incorporated membrane was also tested for glutathione separation at various pH levels. The rejection rate of glutathione was greater than 93% at pH values ranging from 4 to 9. Since the LbL approach provides the AQP embedded biomimetic membrane with satisfactory stability and separation performance, it makes the large scale fabrication of AQPZ-incorporated membranes feasible and practical.

Hyperglycemia is closely associated with prediabetes and Type 2 Diabetes Mellitus. Hyperglycemia increases the risk of vascular complications such as diabetic retinopathy, diabetic nephropathy, peripheral vascular disease and cerebro/cardiovascular diseases. Under hyperglycemic conditions, the endothelial cells become dysfunctional. In this study, we investigated the miRNA expression changes in human umbilical vein endothelial cells exposed to different glucose concentrations (5, 10, 25 and 40 mM glucose) and at various time intervals (6, 12, 24 and 48 h). miRNA microarray analyses showed that there is a correlation between hyperglycemia induced endothelial dysfunction and miRNA expression. In silico pathways analyses on the altered miRNA expression showed that the majority of the affected biological pathways appeared to be associated to endothelial cell dysfunction and apoptosis. We found the expression of ten miRNAs (miR-26a-5p, -26b-5p, 29b-3p, -29c-3p, -125b-1-3p, -130b-3p, -140-5p, -192-5p, -221-3p and -320a) to increase gradually with increasing concentration of glucose. These miRNAs were also found to be involved in endothelial dysfunction. At least seven of them, miR-29b-3p, -29c-3p, -125b-1-3p, -130b-3p, -221-3p, -320a and -192-5p, can be correlated to endothelial cell apoptosis.

Coordinated interplay of dysregulated microRNAs in isolated metabolic disorder is implicated in the pathogenesis of metabolic syndrome.The objective of the study was to characterize microRNA expression in the blood and exosomes of individuals with metabolic syndrome and compare them with those manifesting one of the metabolic vascular risk factors (type 2 diabetes, hypercholesterolemia, or hypertension). RESEARCH DESIGN/SETTING/PARTICIPANTS: A total of 265 participants were recruited in a health screening and characterized into distinct groups as follows: 1) healthy controls (n = 46); 2) metabolic syndrome (n = 50); 3) type 2 diabetes (n = 50); 4) hypercholesterolemia (n = 89); and 5) hypertension (n = 30). Total RNA was subjected to microRNA profiling, and a panel of significantly dysregulated microRNAs was validated using quantitative PCR.Analysis of profiling data characterized unique pools of miRNAs that could categorize the different risk factors of metabolic syndrome.We have identified miR-197, miR-23a, and miR-509-5p as potential contributors of dyslipidemia in metabolic syndrome (correlation with body mass index; P = 0.029, 0.021, and 0.042, respectively) and miR-130a and miR-195 as contributors of hypertension (correlation with blood pressure; P = 0.019 and 0.045, respectively). A plausible association of miR-27a and miR-320a with metabolic syndrome and type 2 diabetes patients has also been found because these miRNAs remained dysregulated in both cases (correlation with fasting glucose; P = 0.010 and 0.016, respectively).Significant dysregulation of seven candidate microRNAs has been found to be associated with risks involved in the manifestation of metabolic syndrome.

The aim of the present study was to investigate and demonstrate the preparation and characterization of planar biomimetic water-channel protein Aquaporin Z (AqpZ)-incorporated polymer membranes on different porous alumina substrates by vesicle spreading. AqpZ-incorporated polymer vesicles were prepared from disulfide-functionalized poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyloxazoline) (disulfide-functionalized PMOXA 20 -PDMS 75 -PMOXA 20 ) tri-block copolymer which was previously proven to be able to maintain protein activity after the incorporation as found in lipid vesicles. AqpZ-incorporated vesicle permeability was evaluated using stopped-flow spectroscopy. The permeability of AqpZ-vesicles measured by stopped-flow spectroscopy was 4680 μm/s at room temperature when the molar ratio of AqpZ/polymer used to prepare the vesicles was 1/50. The result demonstrates that AqpZ could maintain its performance after inserting into polymer vesicles. In addition, the vesicles were designed with disulfide functional end groups to enhance vesicle spreading on gold-coated solid substrates by covalent interactions between polymer membranes and substrates. The morphology of polymer membranes on different types of porous alumina substrates was elucidated by using atomic force microscopy (AFM) and field-emission scanning electron microscopy (FESEM). AqpZ-incorporated polymer vesicles were demonstrated to be able to form planar AqpZ-incorporated polymer membranes on the porous alumina substrates. Preliminary studies of membrane's permeability showed that AqpZ were still in the active form on the planar membranes and the resultant membranes may be useful tools for nanofiltration process for water reuse.

For the first time, planar biomimetic membranes consisting of Aquaporin Z (AqpZ) were fabricated upon cellulose acetate membrane substrate functionalized with methacrylate end groups. By vesicle rupture of triblock copolymer (ABA) vesicles and UV polymerization, a selective layer upon the substrate for nano-filtration (NF) was formed. The AqpZ:ABA ratio was varied from 1:200 to 1:50 and its effects on nanofiltration performance were elucidated. It is found that the NF membranes comprising AqpZ:ABA ratio of 1:50 can give an impressive water permeability of 34 LMH bar −1 and NaCl rejection of more than 30%. This study opens up new possibilities of using AqpZ embedded biomimetic membranes for water purification with advantages that include high throughput with lesser energy consumption.

Nerve growth factor (NGF) is synthesized as a precursor, proNGF that undergoes post-translational processing to generate the biologically active mature NGF. While the neurotrophic function of NGF is well established, the activity of the proNGF precursor is still unclear. In this study, we have cloned the pro-domain of the precursor NGF molecule and have elucidated its function. We have used both mature and the furin resistant pro (R/G) NGF as controls in our experiments. Both pro (R/G) NGF and mature NGF (NGF) exhibited neurotrophic activity on PC12 cells while the pro-domain itself promoted cell death. The pro-domain, has been found to mediate apoptosis possibly by promoting the formation of a signaling complex comprising of endogenous p75 NTR receptor, Bim/Bcl2 group of proteins and JNK and MEK1/2 signaling pathways.

In this work, a new approach of incorporating transmembrane protein AquaporinZ (AqpZ) into a lipid bilayer has been developed with the aid of the Langmuir–Blodgett technique. The binary-lipid monolayer for AqpZ incorporation is composed of (1) gel-phase lipids resistant to detergent dissolution and (2) nickel-chelating lipids that can attach the histidine-tagged AqpZ from the subphase. Upon removal of subphase detergent with BioBeads, the incorporation is achieved by transferring the AqpZ-associated binary-lipid monolayer onto a preformed pure binary-lipid monolayer using the Langmuir–Schaefer deposition method. AFM images show an indication of AqpZ incorporation in the bilayer. Furthermore, it is also shown that BioBeads can remove a significant amount of detergent in the subphase and lipid film integrity is restored after detergent removal. The detergent removal rate is correlated to BioBeads amount and subphase circulation. The new approach of AqpZ reconstitution revealed in this work could potentially be applied in biomimetic membrane formation for water purification applications.

Aquaporins facilitate efficient diffusion of water across cellular membranes, and water homeostasis is critically important in conditions such as cerebral edema. Changes in aquaporin 1 and 4 expression in the brain are associated with cerebral edema, and the lack of water channel modulators is often highlighted. Here we present evidence of an endogenous modulator of aquaporin 1 and 4. We identify miR-320a as a potential modulator of aquaporin 1 and 4 and explore the possibility of using miR-320a to alter the expression of aquaporin 1 and 4 in normal and ischemic conditions. We show that precursor miR-320a can function as an inhibitor, whereas anti-miR-320a can act as an activator of aquaporin 1 and 4 expressions. We have also shown that anti-miR-320a could bring about a reduction of infarct volume in cerebral ischemia with a concomitant increase in aquaporins 1 and 4 mRNA and protein expression.

Pathogenesis of cerebral ischemia has so far been described in the context of proteins and the pathways that they regulate. The discovery of biomarkers has also been focussed mainly on proteins and to some extent on the mRNAs that encode them. The knowledge on the role of microRNAs in understanding the pathogenesis of cerebral ischemia is still at its infancy. In this study, using rat models subjected to middle cerebral artery occlusion, we have profiled the microRNAs at different reperfusion times (0 to 48 h) to understand the progression of cerebral ischemia. We have also attempted to correlate the expression of microRNAs to treatment with an NMDA antagonist (MK801) and to protein expression with the hope of demonstrating the potential use of microRNAs as early biomarkers of stroke.

Background: Over the past 10 years clinical trials aimed at finding suitable neuroprotectants against the debilitating effects of stroke have met with no success. Identifying novel neuroprotectants that can reverse the effects of stroke is becoming a challenge to both clinicians and scientists. Objectives: This review focused on the current status on the topic and highlights some of the neuroprotectants that are worth examining or re-examining further. Methods: Recent findings on the subject have been included. Conclusion: Many neuroprotectants that have worked in preclinical evaluations have been found to be ineffective in clinical trials. Nevertheless, some of them are still considered to be worth re-examining. Non-coding small RNAs (riboregulators) as novel therapeutic leads have also been introduced in this review.

Programmed cell death (PCD), is a highly regulated and sophisticated cellular mechanism that commits cell to isolated death fate. PCD has been implicated in the pathogenesis of numerous neurodegenerative disorders. Countless molecular events underlie this phenomenon, with each playing a crucial role in death commitment. A precedent event, apoptotic volume decrease (AVD), is ubiquitously observed in various forms of PCD induced by different cellular insults. Under physiological conditions, cells when subjected to osmotic fluctuations will undergo regulatory volume increase/decrease (RVI/RVD) to achieve homeostatic balance with neurons in the brain being additionally protected by the blood-brain-barrier. However, during AVD following apoptotic trigger, cell undergoes anistonic shrinkage that involves the loss of water and ions, particularly monovalent ions e.g. K(+), Na(+) and Cl(-). It is worthwhile to concentrate on the molecular implications underlying the loss of these cellular components which posed to be significant and crucial in the successful propagation of the apoptotic signals. Microarray and real-time PCR analyses demonstrated several ion and water channel genes are regulated upon the onset of lactacystin (a proteosomal inhibitor)-mediated apoptosis. A time course study revealed that gene expressions of water and ion channels are being modulated just prior to apoptosis, some of which are aquaporin 4 and 9, potassium channels and chloride channels. In this review, we shall looked into the molecular protein machineries involved in the execution of AVD in the central nervous system (CNS), and focus on the significance of movements of each cellular component in affecting PCD commitment, thus provide some pharmacological advantages in the global apoptotic cell death.

The red scorpion, Mesobuthus tamulus, is found in two distinct biotopes within the Indian state of Maharastra - a tropical, sea-level biotope and a semi-arid biotope, up to 600 m. Scorpions from these two geographical areas show marked differences in toxicity. Using mass spectrometry, we have shown biotope-specific variation in the expression of peptides from scorpions collected from these two distinct areas. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) and reversed-phase liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) were assessed as techniques for obtaining mass fingerprint data. On line LC/ESI-MS was judged to be the method of choice and unique biotope-specific mass fingerprints, with key diagnostic markers, were obtained.

Background Dengue is the most common arboviral illness worldwide. While most infected patients recover, a proportion of them develop severe complications or fatality. Nevertheless, the pathophysiological mechanisms which distinguish the disease severity and associated complications are not clearly understood. We studied blood profiles of dengue patients in order to identify microRNAs that could play a role in these pathophysiological mechanisms. Methods Blood samples from 26 dengue-infected patients were collected within 0–14 days of infection. Together with samples obtained from six healthy individuals, microRNA profiles were generated to identify significantly altered microRNAs upon dengue infection. Profiles of patients with influenza were also used to determine the disease specificity of these altered microRNAs. Their discriminative power to distinguish dengue from influenza was then tested statistically. Results Several significantly altered microRNAs were identified in patients with dengue. Twelve microRNAs were specifically altered upon acute dengue whereas 14 microRNAs exhibited similar expression between dengue and influenza. Seventeen microRNAs which could potentially distinguish dengue-related complications were also identified. Expression of miR-24-1-5p, miR-512-5p and miR-4640-3p distinguished mild dengue from those exhibiting liver complications whereas miR-383 was significantly upregulated in mild dengue compared to those diagnosed as severe dengue with fluid accumulation. Conclusions We identified two panels of microRNAs – one specific for dengue and the other common to dengue and influenza. We also report on the differentially expressed microRNAs in patients with mild versus severe dengue, which could be the basis for the complications seen in them.

Various clinical manifestations leading to death have been documented in most cases of bites caused by venomous snakes. Cobra envenomation is an extremely variable process and known to cause profound neurological abnormalities. The complexity of cobra venom can induce multiple-organ failure, leading to death in case of severe envenomation. Intramuscular administration of Malayan spitting cobra (Naja sputatrix) crude venom at 1 microg/g dose caused death in mice in approximately 3 h. Analysis of gene expression profiles in the heart, brain, kidney, liver and lung revealed 203 genes whose expression was altered by at least 3-fold in response to venom treatment. Of these, 50% were differentially expressed in the heart and included genes involved in inflammation, apoptosis, ion transport and energy metabolism. Electrocardiogram recordings and serum troponin T measurements indicated declining cardiac function and myocardial damage. This not only sheds light on the cardiotoxicity of cobra venom but also reveals the molecular networks affected during envenomation.

The NGF (nerve growth factor) from Naja sputatrix has been purified by gel filtration followed by reversed-phase HPLC. The protein showed a very high ability to induce neurite formation in PC12 cells relative to the mouse NGF. Two cDNAs encoding isoforms of NGF have been cloned and an active recombinant NGF, sputa NGF, has been produced in Escherichia coli as a His-tagged fusion protein. Sputa NGF has been found to be non-toxic under both in vivo and in vitro conditions. The induction of neurite outgrowth by this NGF has been found to involve the high-affinity trkA–p75NTR complex of receptors. The pro-survival mechanism of p75NTR has been mediated by the activation of nuclear factor κB gene by a corresponding down-regulation of inhibitory κB gene. Real-time PCR and protein profiling (by surface-enhanced laser-desorption–ionization time-of-flight) have confirmed that sputa NGF up-regulates the expression of the endogenous NGF in PC12 cells. Preliminary microarray analysis has also shown that sputa NGF is capable of promoting additional beneficial effects such as the up-regulation of arginine vasopressin receptor 1A, voltage-dependent T-type calcium channel. Hence, sputa NGF forms a new and useful NGF.

Motivation: Sequence annotations, functional and structural data on snake venom neurotoxins (svNTXs) are scattered across multiple databases and literature sources. Sequence annotations and structural data are available in the public molecular databases, while functional data are almost exclusively available in the published articles. There is a need for a specialized svNTXs database that contains NTX entries, which are organized, well annotated and classified in a systematic manner. Results: We have systematically analyzed svNTXs and classified them using structure–function groups based on their structural, functional and phylogenetic properties. Using conserved motifs in each phylogenetic group, we built an intelligent module for the prediction of structural and functional properties of unknown NTXs. We also developed an annotation tool to aid the functional prediction of newly identified NTXs as an additional resource for the venom research community. Availability: We created a searchable online database of NTX proteins sequences (http://research.i2r.a-star.edu.sg/Templar/DB/snake_neurotoxin). This database can also be found under Swiss-Prot Toxin Annotation Project website (http://www.expasy.org/sprot/)

The use of toxins as novel molecular probes to study the structure-function relationship of ion-channels and receptors as well as potential therapeutics in the treatment of wide variety of diseases is well documented. The high specificity and selectivity of these toxins have attracted a great deal of interest as candidates for drug development. This review highlights the involvement of the proteins and peptide toxins as well as non-proteinaceous compounds derived from both venomous and non-venomous animals, in anti-nociception and anti-inflammation. The possible mechanisms of these potential therapeutic agents and possible clinical applications in the treatment of pain and inflammation are also summarized.

Neurodegeneration induced by excitatory neurotransmitter glutamate is considered to be of particular relevance in several types of acute and chronic neurological impairments ranging from cerebral ischaemia to neuropathological conditions such as motor neuron disease, Alzheimer's, Parkinson's disease and epilepsy. The hyperexcitation of glutamate receptors coupled with calcium overload can be prevented or modulated by using well-established competitive and non-competitive antagonists targeting ion/receptor channels. The exponentially increasing body of pharmacological evidence over the years indicates potential applications of peptide toxins, due to their exquisite subtype selectivity on ion channels and receptors, as lead structures for the development of drugs for the treatment of wide variety of neurological disorders. This review comprehensively highlights the overview of the diversity in the molecular as well as neurobiological mechanisms of different peptide toxins derived from venomous animals with particular reference to neuroprotection. In addition, the potential applications of peptide toxins in the diagnosis and treatment of neurological disorders such as neuromuscular disorders, epilepsy, Alzheimer's and Parkinson's diseases, gliomas and ischaemic stroke and their future prospects in the diagnosis as well as in the therapy are addressed.

Snake venoms are complex mixtures of biologically active polypeptides that target a variety of vital physiological functions in mammals. alpha-Neurotoxins, toxins that cause paralysis by binding to the nicotinic receptors at the postsynaptic region of the neuromuscular junction have been widely studied in terms of their structure-function relationships as well as gene structure, organization and expression. In this review, we describe the structure of alpha-neurotoxin genes and discuss their evolutionary relationships. Almost all members of neurotoxins have been found to exhibit a common evolutionary origin. The importance of alpha-neurotoxins in therapy and research has also been discussed to highlight their potential applications especially in the area of drug discovery.

Pseudonaja textilis , an Australian Elapid, is known to produce a highly toxic venom. Both protein profiling and N-terminal sequence analysis showed the presence of four new phospholipases A 2 in this venom. Besides being non-lethal, the phospholipase A 2 proteins were found to be moderately active enzymes and they showed procoagulant property. cDNA cloning and characterization indicated the presence of two isoforms of PLA 2 proteins in a single snake, each containing the “pancreatic loop,” characteristic of group IB phospholipase A 2 . The genomic cloning also confirmed the presence of two genes each containing four exons that are interrupted by three introns. Phylogenetic analysis showed that the venom group IB PLA 2 gene is primitive and could have evolved from the same ancestor as the mammalian and venom group IA PLA 2 genes. In the present study, we report that the Pt-PLA2 gene could be responsible for the production of PL1, 2, and 3 possibly via RNA editing process.

The structure and organization of the genes encoding the long-chain neurotoxins and four other isoforms of weak neurotoxins in the venom of Naja sputatrix are reported. The genes contained three exons interrupted by two introns, a structure similar to other members of the three-finger toxin family. The proteins encoded by these genes, however, show varied affinity towards nicotinic acetylcholine receptors. Phylogenetic analysis of these genes showed that the weak neurotoxin gene is confined to a distinct group. We also observe that specific mutations of the gene provide the diversity in function in these toxins while maintaining a common structural scaffold. This forms the first report where the molecular basis of evolution of postsynaptic neurotoxins from an ancestral gene can be demonstrated using the same species of snake.

Fetal lung development is dependent upon secretion of liquid into the future airways which must be cleared at birth to establish air-breathing. Aquaporins (AQP) 1, 3, 4 and 5 are membranous water channel proteins that are present in the lung after birth in rodents, with little expression before birth. Our aim was to describe the changes in AQP1, 3, 4 and 5 expression and protein levels in the fetal lung of a long-gestation species (sheep) and in response to physiological factors known to alter fetal lung liquid dynamics. Both mRNA and high protein levels were detected for AQP1, 3, 4 and 5 by day 100 (term is ~150 days in ovine fetuses). A cortisol infusion (120-131 days) significantly (P < 0.05) increased AQP1 (0.9 +/- 0.2 (n = 4) vs.1.8 +/- 0.3 (n = 5)) and AQP5 (8.8 +/- 0.6 vs. 14.1 +/- 1.2) mRNA levels in fetal lung (measured by real-time PCR). Ten days of tracheal obstruction significantly (P < 0.05) decreased AQP5 mRNA levels (6.1 +/- 0.9 (n = 5) vs. 2.7 +/- 0.3 (n = 5)). Immunohistochemistry was used to show that protein levels changed in parallel with the mRNA changes. These findings suggest that AQPs could be involved in lung liquid production and reabsorption during fetal development in long-gestation species.

Weak neurotoxins from snake venom are small proteins with five disulfide bonds, which have been shown to be poor binders of nicotinic acetylcholine receptors. We report on the cloning and sequencing of four cDNAs encoding weak neurotoxins from Naja sputatrix venom glands. The protein encoded by one of them, Wntx-5, has been synthesized by solid-phase synthesis and characterized. The physicochemical properties of the synthetic toxin (sWntx-5) agree with those anticipated for the natural toxin. We show that this toxin interacts with relatively low affinity (Kd = 180 nm) with the muscular-type acetylcholine receptor of the electric organ of T. marmorata, and with an even weaker affinity (90 µm) with the neuronal α7 receptor of chicken. Electrophysiological recordings using isolated mouse hemidiaphragm and frog cutaneous pectoris nerve–muscle preparations revealed no blocking activity of sWntx-5 at µm concentrations. Our data confirm previous observations that natural weak neurotoxins from cobras have poor affinity for nicotinic acetylcholine receptors.

The possible involvement of specific regions/loops of cardiotoxin from Naja sputatrix venom in mediating its cytolytic activity is evaluated using a new cytolytic assay. In this assay, the amount of chloramphenicol acetyltransferase (CAT) that is released upon lysis of the cellular membranes by the cytotoxin has been measured as an index of cytolysis. This newly developed CAT system is more sensitive than the traditional haemolysis method utilizing red blood cells or the lactate dehydrogenase assay for cytolysis. Series of chimaeric toxin molecules have been constructed by swapping the loops between highly hydrophilic neurotoxin and highly hydrophobic cardiotoxin molecules from Naja sputatrix, which are known to exhibit structural similarity (three-finger conformation) but to have different functional properties. Comparison of the cytolytic activities of the recombinant chimaeric toxins demonstrated the possible involvement of all three loops of cardiotoxin in its cytolytic potency. However, the first two loops of the protein appear to make the major contribution to its lytic activity. cDNAs encoding cardiotoxin and the chimaeric toxins, when expressed in transfected cultured Chinese hamster ovary cells, resulted in cell lysis, indicating that these cDNAs can be developed as useful cytolytic agents.