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Background In addition to the widespread use of antibiotics in healthcare settings, the current COVID-19 pandemic has escalated the emergence of antibiotic resistance. Nosocomial infections among hospitalized patients is a leading site for such resistant microbial colonization due to prolonged use of invasive devices and antibiotics in therapies. Invasive medical devices, especially catheters, are prone to infections that could accelerate the development of resistant microbes. Often, catheters - particularly urinary catheters - are prone to high infection rates. Antibiotic-coated catheters can reduce infection rates and although commercially available, are limited in efficacy and choices. Methods Herein, a novel and facile method to fabricate PMDS-based biomaterial for the development of antimicrobial eluting catheters is presented. Silicone based organic polymer polydimethylsiloxane (PDMS) was used to prepare a biomaterial containing novel polymeric imidazolium antimicrobial compound. Results It was found that the PDMS-based biomaterials could eradicate microbial colonization even after 60 days in culture with continuous microbial challenge, be recycled over multiple uses, stored at room temperature for long-term usage and importantly is biocompatible. Conclusion The PDMS-based biomaterial displayed biocidal functionality on microbes of clinical origin, which form major threats in hospital acquired infections. Graphical Abstract

In the search for a fast contact-killing antimicrobial surface to break the transmission pathway of lethal pathogens, nanostructured copper surfaces were found to exhibit the desired antimicrobial properties. Compared with plain copper, these nanostructured copper surfaces with Cu(OH)2 nano-sword or CuO nano-foam were found to completely eliminate pathogens at a fast rate, including clinically isolated drug resistant species. Additionally these nanostructured copper surfaces demonstrated potential antiviral properties when assessed against bacteriophages, as a viral surrogate, and murine hepatitis virus, a surrogate for SARS-CoV-2. The multiple modes of killing, physical killing and copper ion mediated killing contribute to the superior and fast kinetics of antimicrobial action against common microbes, and ESKAPE pathogens. Prototypes for air and water cleaning with current nanostructured copper surface have also been demonstrated.

Antibiotic resistance has become one of the major, deadly threats to public health worldwide. This paper highlights several recent works, which may initiate the development of comprehensive approaches to mitigate antibiotic resistance. The new strategies demonstrate efficiency and efficacy, with very little probability of inducing drug resistance, paving the way for further breakthroughs in drug discovery for infection control.

Antimicrobial resistance poses an increasingly serious global health threat. Hence, new antimicrobials with low propensity toward inducing resistance in bacteria are being developed to combat this threat. In this work, a series of imidazolium tetramers have been synthesized by modulating the linkers between imidazoliums or the length of the end groups within the structures of oligomers in order to optimize the activity, selectivity, and biocompatibility of the compounds. These new materials possess high biocompatibility, Gram selectivity, and high efficacy against the selected bacterium as well as clinically isolated methicillin-resistant Staphylococcus aureus species without inducing drug resistance. Therefore, we believe that these compounds can potentially be used to mitigate resistance as highly effective disinfectants in healthcare products or as antimicrobial therapies specifically for Gram-positive bacterial infections.

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.

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.

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.

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.

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.

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.

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.

Mucuna pruriens is widely used in traditional medicine for treatments of various diseases. In certain region of Nigeria, the seed is used as oral prophylactics for snakebite. Rats pretreated with the aqueous extract from M. pruriens seed (MPE) were protected against the lethal effects of Naja sputatrix (Javan spitting cobra) venom [Tan et al., J Ethnopharmacol, 123 (2009) 356]. The pretreatment also protected against venom-induced histopathological changes in rat heart. To contribute to the understanding of the mechanism of cardio-protective action, the present study examined the effects of MPE-pretreatment on gene expression profile of rat heart as well as effect of MPE-pretreatment on N. sputatrix venom-induced gene expression alterations in rat heart. The gene expression profiles were examined by microarray analysis and verified by real time PCR. The results showed that pretreatment with MPE caused 50 genes in the rat heart substantially up-regulated of which 19 were related to immune responses, 7 were related to energy production and metabolism. The up-regulation of genes related to energy metabolism probably plays a role in maintaining the viability of the heart. Four other genes that were up-regulated (alpha synuclein, natriuretic peptide precursor, calsequestrin and triadin) were involved in the maintenance of homeostasis of the heart or maintaining its viability, thereby contributing to the direct protective action. The results demonstrated that protective effect of MPE pretreatment against snake venom poisoning may involve a direct action on the heart.

MicroRNAs (miRNA), small sequences of non-coding RNA which interact with complementary sequences on the 3’untranslated region of target messenger RNAs to modulate translation, have a pivotal role in the development of the heart and its response to injury. Myocardial infarction (MI) triggers a dynamic miRNA response with the potential of yielding therapeutic targets. Following miRNA array profiling in rat hearts 2, 7 and 14 days after MI induced by coronary ligation, we identified a progressive time-dependent up-regulation of miR-31 compared to sham rats. Increase of miR-31 in heart tissue in the acute and subacute phases after MI (up to 90-fold) was also detected by Real-Time PCR (P=0.02 at day 2; P

Natriuretic peptide receptor 3 (NPR3) is the clearance receptor for the cardiac natriuretic peptides (NPs). By modulating the level of NPs, NPR3 plays an important role in cardiovascular homeostasis. Although the physiological functions of NPR3 have been explored, little is known about its regulation in health or disease. MicroRNAs play an essential role in the post-transcriptional expression of many genes. Our aim was to investigate potential microRNA-based regulation of NPR3 in multiple models. Hypoxic challenge elevated levels of NPPB and ADM mRNA, as well as NT-proBNP and MR-proADM in human left ventricle derived cardiac cells (HCMa), and in the corresponding conditioned medium, as revealed by qRT-PCR and ELISA. NPR3 was decreased while NPR1 was increased by hypoxia at mRNA and protein levels in HCMa. Down-regulation of NPR3 mRNA was also observed in infarct and peri-infarct cardiac tissue from rats undergoing myocardial infarction. From microRNA microarray analyses and microRNA target predictive databases, miR-100 was selected as a candidate regulator of NPR3 expression. Further analyses confirmed up-regulation of miR-100 in hypoxic cells and associated conditioned media. Antagomir-based silencing of miR-100 enhanced NPR3 expression in HCMa. Furthermore, miR-100 levels were markedly up-regulated in rat hearts and in peripheral blood after myocardial infarction and in the blood from heart failure patients. Results from this study point to a role for miR-100 in the regulation of NPR3 expression, and suggest a possible therapeutic target for modulation of NP bioactivity in heart disease.

An Australian common brown snake, Pseudonaja textilis, is known to contain highly lethal neurotoxins. Among them, a long-chain alpha-neurotoxin, pseudonajatoxin b, has been identified. In this report, while presenting evidence for the presence of at least four such long-chain alpha-neurotoxins in the venom of P. textilis, we describe the characteristics of both the mRNA and the gene responsible for the synthesis of these neurotoxins. A precursor toxin synthesized from the gene has been identified as being capable of producing the isoforms possibly by post-translational modifications at its C-terminal end. Recombinant toxins corresponding to the precursor and its product have been found to possess similar binding affinities for muscular acetylcholine receptors (IC(50)=3x10(-8) M) and a lethality, LD(50), of 0.15 microg/g in mice.

This report is the first study of the regulation of expression of a toxin gene and it also demonstrates the novel finding that the cardiotoxin (CTX)-2 gene from Naja sputatrix is expressed in the venom gland as well as in other tissues in the snake, such as liver, heart and muscle. The venom gland produces a 500-bp (spliced) CTX-2 mRNA as the final transcript. However, the liver produces two types of CTX-2 mRNA, of which the unspliced transcript (1 kb) is predominant; the 500 bp spliced transcript is the minor species. This differential expression of the CTX gene has been attributed to the usage of alternative promoter consisting of independent TATA boxes and corresponding transcription initiation sites. Among the several transcription factors that have been identified by a search of the TFIID database, the participation of two glucocorticoid elements in the expression of the CTX gene has been demonstrated by promoter deletion analysis. Putative binding sites for SP-1, C/EBP, CACCC-binding factor and at least two unknown binding factors have also been identified by DNase I footprinting of the promoter.

From the serum of the nonvenomous snake Python reticulatus, a new phospholipase A(2) (PLA(2)) inhibitor termed phospholipase inhibitor from python (PIP) was purified by sequential chromatography and cloned to elucidate its primary structure and fundamental biochemical characteristics. A cDNA clone encoding PIP was isolated from the liver total RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). It contained a 603 bp open reading frame that encoded a 19-residue signal sequence and a 182-residue protein. PIP showed about 60% sequence homology with those PLA(2) inhibitors having a urokinase-type plasminogen activator receptor-like domain structure. PIP was also functionally expressed as a fusion protein in Escherichia coli to explore its potential therapeutic significance. The recombinant PIP was shown to be identical to the native form in chromatographic behavior and biochemical characteristics. Both the native and recombinant PIP appear to exist as a hexamer of 23-kDa subunits having an apparent molecular mass of approximately 140 kDa. PIP showed ability to bind to the major PLA(2) toxin (daboiatoxin, DbTx) of Daboia russelli siamensis at 1-2-fold molar excess of inhibitor to toxin. It exhibited broad spectra in neutralizing the toxicity of various snake venoms and toxins and inhibited the formation of edema in mice. Our data demonstrate the venom neutralizing potential of the recombinant PIP and suggest that the proline-rich hydrophobic core region may play a role in binding to PLA(2).

The structure and organization of five genes responsible for the synthesis of six isoforms of short-chain alpha-neurotoxins in Pseudonaja textilis venom are reported in this paper. This also forms the first report which describes the synthesis of two neurotoxin mRNA variants from one of these genes (Pt-sntx1) as a result of alternative splicing. Each gene consists of three exons which are separated by two introns and each has a functional promoter. The promoter activity was confirmed by both CAT assay and Real-Time PCR. A transcription initiation site, two putative TATA boxes, one CCAAT box and the transcription factor binding consensus sites for AP-1, GATA-2, c/EBPb were identified in the 5' non-coding region of each gene. Phylogenetic analysis showed that these five genes from P. textilis constituted a distinct group which has evolved by gene duplication followed by accelerated evolution from an ancestral gene.

Four phospholipase A 2 (PLA 2 ) enzymes (Superbins a, b, c, and d) with varying platelet aggregation inhibitor activities have been purified from Austrelaps superbus by a combination of gel filtration, ion-exchange, and reversed-phase high-pressure liquid chromatography. Purity and homogeneity of the superbins have been confirmed by high-performance capillary zone electrophoresis and mass spectrometry. The electron spray ionization mass spectrometry data showed that their molecular masses range from 13,140 to 13,236 Da. Each of the proteins has been found to be basic and exhibit varying degrees of PLA 2 activity. They also displayed different platelet aggregation inhibitory activities. Superbin a was found to possess the most potent inhibitory activity with an IC 50 of 9.0 nM, whereas Superbin d was found to be least effective with an IC 50 of 3.0 μM. Superbins b and c were moderately effective with IC 50 values of 0.05 and 0.5 μM, respectively. The amino-terminal sequencing confirmed the identity of these superbins. cDNA cloning resulted in the identification of 17 more PLA 2 isoforms in A. superbus venom. It has also provided complete information on the precursor PLA 2 . The precursor PLA 2 contained a 27-amino-acid signal peptide and 117- to 125-amino-acid PLA 2 (molecular mass ranging from 13,000 to 14,000 Da). Two of these PLA 2 enzymes resembled more closely (87%) Superbin a in structure. Two unique PLA 2 enzymes containing an extra pancreatic loop also have been identified among the isoforms.

A sensitive and highly reproducible method has been used to show that Aquaporin 3 (AQP 3 ) mRNA is present in the ovine placenta and chorion from at least 60 days of gestation (term=145–150d) with levels increasing substantially (>16 fold) at 100 days, and remaining constant thereafter. By immuno- and hybridization histochemistry, the epithelial cells expressing AQP 3 were found to be the trophoblast cells. Some AQP 3 was expressed in fibroblasts of the amnion and allantois but none was expressed in the epithelia of these membranes. AQP 1 was expressed in endothelial cells of fetal and maternal blood vessels but not in any epithelial cell of the ovine placenta and fetal membranes. The level of AQP 3 expression is consistent with known ovine placental permeabilities to water, glycerol and urea.

We report the genomic structure, organization and the presence of multiple isoforms of the gene encoding cardiotoxins (CTX) of Naja naja sputatrix . The cardiotoxin gene consists of six CTX isoforms, each (2.2 kb) having three exons and two introns. Two possible transcription initiation sites as well as consensus TATA boxes and transcription factor binding motifs, AP-2, NFIL-6/C/EBP, NF-κB and PuF have been identified in the 5′-region of the gene. The CTX gene isoforms show nucleotide variations at specific segments in exon 2 and exon 3, which correspond to the functional domains in the three-finger loop structure of the cardiotoxin molecule. The diverse functions of cardiotoxins together with our findings suggest that the cardiotoxin gene isoforms may have evolved under adaptive pressure through a positive Darwinian selection process.

In obese individuals, a high efflux of glycerol from accumulated fat in adipose tissue into the liver is known to be associated with the development of type 2 diabetes. Aquaporin 9 (AQP9) is an aquaglyceroporin which serves as the primary route of hepatic glycerol uptake for gluconeogenesis. Hence, development of AQP9 blockers/regulators may be of potential benefit in controlling hyperglycaemia especially in obese and diabetic individuals. HTS13286 was recently identified as a specific AQP9 inhibitor, even so its limited solubility renders the molecule unsuitable for in vivo application. microRNAs are naturally occurring gene regulators that are often associated with various diseases. The feasibility in selective modulation of microRNAs has introduced a new paradigm for therapeutic applications. In this study, we explore the possibility of using microRNAs to regulate AQP9 expression and eventually glycerolbased gluconeogenesis. In silico prediction of microRNAs targeting the 3’ untranslated region of AQP9 was conducted using miRWalk database. Among the list of potential microRNAs, miR-22 and miR-23a were shortlisted for their high expression in liver and further confirmed to interact with AQP9 via luciferase assay. Over-expression of miR-22 or miR- 23a was able to reduce AQP9 expression (mRNA and protein) and ultimately inhibit glycerol-based gluconeogenesis in HepG2 cells. Livers of diabetic rats were also observed to exhibit an inverse correlation between miR-22, miR-23a and AQP9 expression. As negative modulators of AQP9 expression, miR-22 and miR-23a suggest a potential role in regulating glycerol entry into hepatocytes which could be beneficial in managing glycerol-dependent hyperglycaemic conditions.

cDNAs encoding three phospholipase A2 (PLA2) isoforms in Naja naja sputatrix were cloned and characterized. One of them encoded an acidic PLA2 (APLA) while the others encoded neutral PLA2 (NPLA-1 and NPLA-2). The specific characteristics of APLA and NPLA were attributed to mutations at nt139 and nt328 from G to C and G to A, respectively, resulting in amino acid substitutions from Asp20 and 83 in APLA to His20 and Asn83 in NPLA. Amino acid sequencing of purified protein also showed the presence of this Asp20 and His20 in APLA and NPLA, respectively. The cDNA encoding one of the PLA2 (NAJPLA-2A), when expressed in Escherichia coli, yielded a protein that exhibited PLA2 activity.

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 0.05). When a single application of green fluorescent protein (GFP)-labeled hWJSCs (GFP-hWJSCs) or hWJSC-CM was administered to full-thickness murine excisional and diabetic wounds, healing rates were significantly greater compared to controls (P 0.05). Wound biopsies collected at various time points showed the presence of green GFP-labeled hWJSCs, positive human keratinocyte markers (cytokeratin, involucrin, filaggrin) and expression of ICAM-1, TIMP-1, and VEGF-A. On histology, the GFP-hWJSCs and hWJSC-CM treated wounds showed reepithelialization, increased vascularity and cellular density and increased sebaceous gland and hair follicle numbers compared to controls. hWJSCs showed increased expression of several miRNAs associated with wound healing compared to CCDs. Our studies demonstrated that hWJSCs enhance healing of excisional and diabetic wounds via differentiation into keratinocytes and release of important molecules.

Over the past decade, scientific discoveries have highlighted new roles for a unique class of non-coding RNAs. Transcribed from the genome, these non-coding RNAs have been implicated in determining the biological complexity seen in mammals by acting as transcriptional and translational regulators. Non-coding RNAs, which can be sub-classified into long non-coding RNAs, microRNAs, PIWI-interacting RNAs and several others, are widely expressed in the nervous system with roles in neurogenesis, development and maintenance of the neuronal phenotype. Perturbations of these non-coding transcripts have been observed in ischemic preconditioning as well as ischemic brain injury with characterization of the mechanisms by which they confer toxicity. Their dysregulation may also confer pathogenic conditions in neurovascular diseases. A better understanding of their expression patterns and functions has uncovered the potential use of these riboregulators as neuroprotectants to antagonize the detrimental molecular events taking place upon ischemic-reperfusion injury. In this review, we discuss the various roles of non-coding RNAs in brain development and their mechanisms of gene regulation in relation to ischemic brain injury. We will also address the future directions and open questions for identifying promising non-coding RNAs that could eventually serve as potential neuroprotectants against ischemic brain injury.

Venoms and toxins are of significant interest due to their ability to cause a wide range of pathophysiological conditions that can potentially result in death. Despite their wide distribution among plants and animals, the biochemical pathways associated with these pathogenic agents remain largely unexplored. Impoverished and underdeveloped regions appear especially susceptible to increased incidence and severity due to poor socioeconomic conditions and lack of appropriate medical treatment infrastructure. To facilitate better management and treatment of envenomation victims, it is essential that the biochemical mechanisms of their action be elucidated. This review aims to characterize downstream envenomation mechanisms by addressing the major neuro-, cardio-, and hemotoxins as well as ion-channel toxins. Because of their use in folk and traditional medicine, the biochemistry behind venom therapy and possible implications on conventional medicine will also be addressed.

Scorpion venoms are rich sources of bioactive peptides and are widely known for their ion channel inhibiting properties. We have isolated, cloned and characterized a venom protein (Bumarsin) from the Chinese scorpion, Mesobuthus martensii Karsch. Bumarsin cDNA encodes a 8132 Da, 72 amino acid mature protein that most probably exists in its native form as a Cys-bridged homodimer. We have identified this novel protein to be an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity. 0.6 μM of Bumarsin inhibits 32% of the HMG-CoA reductase activity, in comparison to 10 μM simvastatin which only inhibits 35% of the activity. RT-PCR and SELDI-TOF mass spectrometric studies demonstrate that bumarsin regulates the expression of both genes and proteins involved in cholesterol homeostasis. Our results suggest that bumarsin may provide a model for the design of novel drugs that can be used to modulate cholesterol homeostasis.

Aquaporins (AQPs) are transmembrane water channels ubiquitously expressed in mammalian tissues. They play prominent roles in maintaining cellular fluid balance. Although expression of AQP1, -3, -4, -5, -8, -9, and -11 has been reported in the central nervous system, it is AQP4 that is predominately expressed. Its importance in fluid regulation in cerebral edema conditions has been highlighted in several studies, and we have also shown that translational regulation of AQP4 by miR-320a could prove to be useful in infarct volume reduction in middle cerebral artery occluded rat brain. There is evidence for the existence of two AQP4 transcripts (M1 and M23) in the brain arising from two alternative promoters. Because the AQP4 M1 isoform exhibits greater water permeability, in this study, we explored the possibility of microRNA-based transcriptional regulation of the AQP4 M1 promoter. Using RegRNA software, we identified 34 microRNAs predicted to target the AQP4 M1 promoter region. MicroRNA profiling, quantitative stem-loop PCR, and luciferase reporter assays revealed that miR-130a, -152, -668, -939, and -1280, which were highly expressed in astrocytes, could regulate the promoter activity. Of these, miR-130a was identified as a strong transcriptional repressor of the AQP4 M1 isoform. In vivo studies revealed that LNA(TM) anti-miR-130a could up-regulate the AQP4 M1 transcript and its protein to bring about a reduction in cerebral infarct and promote recovery.

MicroRNAs (miRNAs) are endogenous short (20–22 nucleotides) non-coding RNA molecules that mediate gene expression. This is an important regulatory mechanism to modulate fundamental cellular processes such as differentiation, proliferation, death, metabolism, and pathophysiology of many diseases. The miRNA expression profile of the kidney differs greatly from that of other organs, as well as between the different regions in the kidney. In kidneys, miRNAs are indispensable for development and homeostasis. In this review, we explore the involvement of miRNAs in the regulation of blood pressure, hormone, water, and ion balance pertaining to kidney homeostasis. We also highlight their importance in renal pathophysiology, such as in polycystic disease, diabetic nephropathy, nephrogenic diabetes insipidus, hypertension, renal cancer, and kidney fibrosis (epithelial–mesenchymal transition). In addition, we highlight the need for further investigations on miRNA-based studies in the development of diagnostic, prognostic, and therapeutic tools for renal diseases.