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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.

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.

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.

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.

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.

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.

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.

The in vivo interactions between alpha-neurotoxin, cardiotoxin and two phospholipases A2 (sputa-phospholipase A2-1 and 3) isolated from Malayan cobra venom were assessed by examining the effects of simultaneous injection of sub-LD50 dose of one toxin on (i) i.v. LD50 S of the other toxins in mice; and (ii) mean survival times of mice injected with lethal doses of the other toxins. While LD50 measurements did not reveal any interaction between the toxins in vivo, survival time measurements suggest a synergy between the neurotoxin and sputa-phospholipase A2-1 and between sputa-phospholipase A2-1 and sputa-phospholipase A2-3. Our results also suggest that both sputa-phospholipases A2 interfere with the lethal action of the cardiotoxin, resulting in prolongation of the mean survival time of mice injected with a lethal dose of cardiotoxin. The patterns of in vivo interactions between phospholipase A2 and other venom toxins appear to depend on the nature and mode of pharmacological action of the phospholipase A2.

1. 1. The lethalities, anticoagulant effects, hemorrhagic, thrombin-like enzyme, hyaluronidase, protease, arginine ester hydrolase, 5′-nucleotidase, l -amino acid oxidase, alkaline phosphomonoesterase, phosphodiesterase and phospholipase A activities of twenty-three samples of venoms from twelve species of Asian lance-headed pit vipers (genus Trimeresurus) were examined. 2. 2. The results indicate that notwithstanding individual variations in venom properties, the differences in biological properties of the Trimeresurus venoms can be used for the differentiation of venoms from different species of Trimeresurus. 3. 3. The results also suggest that differences in the biological properties of snake venoms are useful parameters in the classification of snake species. 4. 4. Our results indicate that venoms from the species T. okinavensis exhibited biological properties markedly different from other Trimeresurus venoms examined. This observation supports the recently proposed reclassification of T. okinavensis as a member of the genus Ovophis, rather than the genus Trimeresurus.