Iron-Gold Composites for Biodegradable Implants: In Vitro Investigation on Biodegradation and Biomineralization.

The biocompatibility and biodegradation of iron (Fe) make it a suitable candidate for developing biodegradable metallic implants. However, the degradation rate of Fe in a physiological environment is extremely slow and needs to be enhanced to a rate compatible with tissue growth. Incorporating noble metals improves the Fe degradation rate by forming galvanic couples. This study incorporated gold (Au) into Fe at very low concentrations of 1.25 and 2.37 µg/g to improve the degradation rate. The electrochemical corrosion test of the samples revealed that the Au-containing samples showed a four-time and nine-time faster degradation rate than pure Fe. Furthermore, the immersion test and long-term electrochemical impedance spectroscopy conducted in simulated body fluid (SBF) revealed that the Au-incorporated samples exhibited increased bioactivity and degraded faster than pure Fe. Integrating nanogold into a Fe matrix increased the in situ formation of hydroxyapatite on the sample's surface and did not cause toxicity to L929-murine fibroblast cells. It is suggested that Fe-Au composites with low concentrations of Au can be used to tailor the biodegradation rate and promote the biomineralization of Fe-based implants in the physiological environment.

High doses of clethodim-based herbicide GrassOut Max poses reproductive hazard by affecting male reproductive function and early embryogenesis in Swiss albino mice.

Clethodim is a widely used and approved class II herbicide, with little information about its impact on the reproductive system. Herein, we investigated the male reproductive toxicity of clethodim using a mouse model. GrassOut Max (26% clethodim-equivalent) or analytical grade clethodim (≥90%) were given orally to male mice for 10 d in varying doses. All parameters were assessed at 35 d post-treatment. Significant decrease in testicular weight, decreased germ cell population, elevated DNA damage in testicular cells and lower serum testosterone level was observed post clethodim based herbicide exposure. Epididymal spermatozoa were characterized with significant decrease in motility, elevated DNA damage, abnormal morphology, chromatin immaturity and, decreased acetylated-lysine of sperm proteins. In the testicular cells of clethodim-based herbicide treated mice, the expression of Erß and Gper was significantly higher. Proteomic analysis revealed lower metabolic activity, poor sperm-oocyte binding potential and defective mitochondrial electron transport in spermatozoa of clethodim-based herbicide treated mice. Further, fertilizing ability of spermatozoa was compromised and resulted in defective preimplantation embryo development. Together, our data suggest that clethodim exposure risks male reproductive function and early embryogenesis in Swiss albino mice via endocrine disrupting function.

Carnosine Protects against Cerebral Ischemic Injury by Inhibiting Matrix-Metalloproteinases.

Stroke is one of the leading causes of death and disability worldwide. However, treatment options for ischemic stroke remain limited. Matrix-metalloproteinases (MMPs) contribute to brain damage during ischemic strokes by disrupting the blood-brain barrier (BBB) and causing brain edemas. Carnosine, an endogenous dipeptide, was found by us and others to be protective against ischemic brain injury. In this study, we investigated whether carnosine influences MMP activity. Brain MMP levels and activity were measured by gelatin zymography after permanent occlusion of the middle cerebral artery (pMCAO) in rats and in vitro enzyme assays. Carnosine significantly reduced infarct volume and edema. Gelatin zymography and in vitro enzyme assays showed that carnosine inhibited brain MMPs. We showed that carnosine inhibited both MMP-2 and MMP-9 activity by chelating zinc. Carnosine also reduced the ischemia-mediated degradation of the tight junction proteins that comprise the BBB. In summary, our findings show that carnosine inhibits MMP activity by chelating zinc, an essential MMP co-factor, resulting in the reduction of edema and brain injury. We believe that our findings shed new light on the neuroprotective mechanism of carnosine against ischemic brain damage.

Pseudokinases: Prospects for expanding the therapeutic targets armamentarium.

Pseudokinases are pseudoenzyme variants of the protein kinase superfamily that primarily signal through non-catalytic mechanisms. The aberrant expression of pseudokinases correlates with the pathogenesis of many human diseases. However, pseudokinases remain relatively untapped as therapeutic targets due to difficulties associated with regulating their biological functions. Many protein kinase- and few pseudokinase-specific inhibitors have been reported to influence the non-catalytic functions of active kinases, giving the hope that pseudokinases can also be exploited for therapeutic purposes. This chapter presents the structural characteristics of selected pseudokinases, their known roles in human diseases, and the progress made toward developing pseudokinase-centric therapies.

Quinoline Derivative Enhances Human Sperm Motility and Improves the Functional Competence.

In this study, we aimed to explore the beneficial properties of novel quinoline derivatives on human sperm motility and its functional competence. Nine novel quinoline derivatives were screened for their effect on motility in human spermatozoa from normozoospermic ejaculates. Compounds with impressive sperm motility enhancement properties were further assessed for their effect on functional competence of human spermatozoa. To determine the effect on the fertilizing ability of spermatozoa processed with quinoline derivatives and to assess developmental competence of embryos derived, in vitro fertilization (IVF) was performed using mouse model. Among the nine quinoline derivatives, 2 compounds (6MQT and 2,6DQT) exhibited significant enhancement in sperm progressive motility and survival at 24 h. Further, non-significant increase in curvilinear velocity (VCL), straight line velocity (VSL), and amplitude of lateral head displacement (ALH) was observed. Capacitation, intracellular cAMP level and tyrosine phosphorylated sperm proteins were significantly higher in 6MQT (P < 0.05) and 2,6DQT (P < 0.001) compared to control. In vitro fertilization (IVF) experiments using Swiss albino mice revealed that spermatozoa processed with 6MQT had non-significantly higher blastocyst rate and a superior blastocyst quality, while, 2,6DQT resulted in significantly lower blastocyst rate (P < 0.05) compared to control. Quinoline derivative 6MQT has significant motility enhancement property under in vitro conditions. Graphical abstract.

Hydrogel Scaffolds: Towards Restitution of Ischemic Stroke-Injured Brain.

Chronic brain injury following cerebral ischemia is a severe debilitating neurological condition, where clinical intervention is well known to decrease morbidity and mortality. Despite the development of several therapeutic strategies, clinical outcome in the majority of patients could be better improved, since many still face life-long neurological deficits. Among the several strategic options that are currently being pursued, tissue engineering provides much promise for neural tissue salvage and regeneration in brain ischemia. Specifically, hydrogel biomaterials have been utilized to docket biomolecules, adhesion motifs, growth factors, and other proneural cues for stable stem cell encapsulation. Here, we provide an overview of therapeutic applications of hydrogels in stroke treatment. Special focus is given to design considerations for generation of efficient hydrogel systems for neurological applications. Therapeutic applications of hydrogels in stroke as conducive microenvironments for stem cell transplantation and drug delivery have been discussed. Finally, we present our perspectives on clinical translation of hydrogels for neural tissue regeneration.

miR-9 Upregulation Integrates Post-ischemic Neuronal Survival and Regeneration In Vitro.

The irrefutable change in the expression of brain-enriched microRNAs (miRNAs) following ischemic stroke has promoted the development of radical miRNA-based therapeutics encompassing neuroprotection and neuronal restoration. Our previous report on the systems-level prediction of miR-9 in post-stroke-induced neurogenesis served as a premise to experimentally uncover the functional role of miR-9 in post-ischemic neuronal survival and regeneration. The oxygen-glucose deprivation (OGD) in SH-SY5Y cells significantly reduced miR-9 expression, while miR-9 mimic transfection enhanced post-ischemic neuronal cell viability. The next major objective involved the execution of a drug repositioning strategy to augment miR-9 expression via structure-based screening of Food and Drug Administration (FDA)-approved drugs that bind to Histone Deacetylase 4 (HDAC4), a known miR-9 target. Glucosamine emerged as the top hit and its binding potential to HDAC4 was verified by Molecular Dynamics (MD) Simulation, Drug Affinity Responsive Target Stability (DARTS) assay, and MALDI-TOF MS. It was intriguing that the glucosamine treatment 1-h post-OGD was associated with the increased miR-9 level as well as enhanced neuronal viability. miR-9 mimic or post-OGD glucosamine treatment significantly increased the cellular proliferation (BrdU assay), while the neurite outgrowth assay displayed elongated neurites. The enhanced BCL2 and VEGF parallel with the reduced NFκB1, TNF-α, IL-1ß, and iNOS mRNA levels in miR-9 mimic or glucosamine-treated cells further substantiated their post-ischemic neuroprotective and regenerative efficacy. Hence, this study unleashes a potential therapeutic approach that integrates neuronal survival and regeneration via small-molecule-based regulation of miR-9 favoring long-term recovery against ischemic stroke.

Circular RNAs in Brain Physiology and Disease.

Circular RNAs (circRNAs) are endogenously expressed non-coding RNAs discovered in the early 1990s as a transcriptional by-product of little importance. It was only recently that they were identified as a key player in regulating the gene expression by targeting and modulating the functions of microRNA, a process known as microRNA sponging. They are distributed throughout the system in a tissue-specific manner showing abundant enrichment in neuronal tissue. Their physiological functions in the brain such as neuronal maturation, differentiation, etc. as well as their implications in numerous brain-related disorders have made its entry into the spotlight. Yet the wider scope and molecular mechanism of circRNAs still remain elusive. In this chapter, we describe in detail the functional aspects and importance of circRNAs in the human brain and how it is associated with various neurological diseases.

The Synergistic Combination of Everolimus and Paroxetine Exerts Post-ischemic Neuroprotection In Vitro.

Ischemic stroke is a debilitating multi-factorial cerebrovascular disorder, representing an area of tremendous unmet medical need. Combination treatment has been proposed as a promising therapeutic approach towards combating ischemic stroke. The present study employs in vitro oxygen glucose deprivation (OGD) model to evaluate the post-ischemic neuroprotective efficacy of Everolimus and Paroxetine, alone and in combination. Post-OGD treatment with Everolimus and Paroxetine, alone or in combination, significantly improved the cell survival (~ 80%) when compared to the cells subjected to ischemic injury alone. The individual neuroprotective doses of Everolimus and Paroxetine were found to be at 6.25 and 25 nM, respectively. Whereas, the synergistic neuroprotective dose for Everolimus:Paroxetine was 2:10 nM, calculated using the Chou-Talalay combination index and other four mathematical models. The synergistic combination dose downregulated neuroinflammatory genes (Tnf-α, Il1b, Nf-κB, and iNos) and upregulated the neuroprotective genes (Bcl-2, Bcl-xl, Hif-1, and Epo). The mitochondrial functioning and ROS neutralizing ability increased with combination treatment. Further, the active role of nitric oxide synthase and calmodulin were revealed while exploring the bio-activity of Everolimus and Paroxetine through network pharmacology. The present study for the first time demonstrates the synergistic post-ischemic neuroprotective efficacy of combination treatment with Everolimus and Paroxetine in vitro. Taken together, these findings clearly suggest that Everolimus in combination with Paroxetine may represent a promising therapeutic strategy for the treatment of ischemic stroke, further supporting the combination treatment strategy for this debilitating disorder.

A Novel Five-Node Feed-Forward Loop Unravels miRNA-Gene-TF Regulatory Relationships in Ischemic Stroke.

The complex and interlinked cascade of events regulated by microRNAs (miRNAs), transcription factors (TF), and target genes highlight the multifactorial nature of ischemic stroke pathology. The complexity of ischemic stroke requires a wider assessment than the existing experimental research that deals with only a few regulatory components. Here, we assessed a massive set of genes, miRNAs, and transcription factors to build a miRNA-gene-transcription factor regulatory network to elucidate the underlying post-transcriptional mechanisms in ischemic stroke. Feed-forward loops (three-node, four-node, and novel five-node) were converged to establish regulatory relationships between miRNAs, TFs, and genes. The synergistic function of miRNAs in ischemic stroke was predicted and incorporated into a novel five-node feed-forward loop. Significant miRNA-TF pairs were identified using cumulative hypergeometric distribution. Two subnetworks were derived from the extensive miRNA-TF regulatory network and analyzed to predict the molecular mechanism relating the regulatory components. NFKB and STAT were identified to be the chief regulators of innate inflammatory and neuronal survival mechanisms, respectively. Exclusive novel interactions between miR-9 and miR-124 with TLX, BCL2, and HDAC4 were identified to explain the post-stroke induced neurogenesis mechanism. Therefore, this network-based approach to delineate miRNA, TF, and gene interactions might promote the development of effective therapeutics against ischemic stroke.

Folic Acid Exerts Post-Ischemic Neuroprotection In Vitro Through HIF-1α Stabilization.

The constant failure of single-target drug therapies for ischemic stroke necessitates the development of novel pleiotropic pharmacological treatment approaches, to effectively combat the aftermath of this devastating disorder. The major objective of our study involves a multi-target drug repurposing strategy to stabilize hypoxia-inducible factor-1 α (HIF-1α) via a structure-based screening approach to simultaneously inhibit its regulatory proteins, PHD2, FIH, and pVHL. Out of 1424 Food and Drug Administration (FDA)-approved drugs that were screened, folic acid (FA) emerged as the top hit and its binding potential to PHD2, FIH, and pVHL was further verified by re-docking, molecular dynamics (MD) simulation and by Drug Affinity Responsive Target Stability (DARTS) assay. HIF-1α stabilization by FA was demonstrated by the nuclear translocation and increased green fluorescence emission of HIF-1α using HIF1α-GFPSpark tag vector. Further, FA treatment enhanced the cell survival following oxygen glucose deprivation and its neuroprotective mechanism was elucidated by measuring the expression of BAX, NFE2L2, VEGF, and EPO genes in a time-dependent manner (5 and 11 h following FA treatment). VEGF and EPO expressions were significantly increased by 5.41- and 1.35-folds, respectively, whereas BAX expression reduced by 4-fold at 11 h post-FA treatment. NFE2L2 expression was elevated (1.65-fold) at 5 h with no major difference at 11 h post-FA treatment. The chicken chorioallantoic membrane (CAM) assay demonstrated the pro-angiogenic potential of FA as evidenced by an increased blood vessel density and branching. The present study elucidates for the first time that the post-ischemic neuroprotection exerted by FA may be attributed to its HIF-1α stabilization and pro-angiogenic properties.

Taxifolin as dual inhibitor of Mtb DNA gyrase and isoleucyl-tRNA synthetase: in silico molecular docking, dynamics simulation and in vitro assays.

DNA gyrase and aminoacyl-tRNA synthetases are two essential bacterial enzymes involved in DNA replication, transcription and translation. Flavonoids are plant secondary metabolites with variable phenolic structures. In this study, eight flavonoids structurally similar to quercetin were selected and their ADMET properties were evaluated. Molecular docking and free energy calculations were carried out to examine the binding of these flavonoids to the ATP-binding site and editing domain of DNA gyrase and Isoleucyl-tRNA synthetase, respectively. Taxifolin was found out to be the top lead molecule in both the docking studies with a good number of interactions with the active site amino acids. Further, binding of taxifolin to the proteins was extensively studied using 50 ns molecular dynamics simulation. In vitro anti-tuberculosis activity of taxifolin was evaluated and compared with the standard drugs. Minimal inhibition concentration of taxifolin was found to be ≤ 12.5 µg/ml.

Hypoxia Mimetic Agents for Ischemic Stroke.

Every year stroke claims more than 6 million lives worldwide. The majority of them are ischemic stroke. Small molecule-based therapeutics for ischemic stroke has attracted a lot of attention, but none has been shown to be clinically useful so far. Hypoxia-inducible factor-1 (HIF-1) plays a crucial role in the transcriptional adaptation of cells to hypoxia. Small molecule-based hypoxia-mimetic agents either stabilize HIF-1α via HIF-prolyl hydroxylases (PHDs) inhibition or through other mechanisms. In both the cases, these agents have been shown to confer ischemic neuroprotection in vitro and in vivo. The agents which act via PHD inhibition are mainly classified into iron chelators, iron competitors, and 2 oxoglutarate (2OG) analogs. This review discusses HIF structure and key players in the HIF-1 degradation pathway as well as the genes, proteins and chemical molecules that are connected to HIF-1 and how they affect cell survival following ischemic injury. Furthermore, this review gives a summary of studies that used PHD inhibitors and other HIF-1α stabilizers as hypoxia-mimetic agents for the treatment of ischemic injury.

Decoding the ubiquitous role of microRNAs in neurogenesis.

Neurogenesis generates fledgling neurons that mature to form an intricate neuronal circuitry. The delusion on adult neurogenesis was far resolved in the past decade and became one of the largely explored domains to identify multifaceted mechanisms bridging neurodevelopment and neuropathology. Neurogenesis encompasses multiple processes including neural stem cell proliferation, neuronal differentiation, and cell fate determination. Each neurogenic process is specifically governed by manifold signaling pathways, several growth factors, coding, and non-coding RNAs. A class of small non-coding RNAs, microRNAs (miRNAs), is ubiquitously expressed in the brain and has emerged to be potent regulators of neurogenesis. It functions by fine-tuning the expression of specific neurogenic gene targets at the post-transcriptional level and modulates the development of mature neurons from neural progenitor cells. Besides the commonly discussed intrinsic factors, the neuronal morphogenesis is also under the control of several extrinsic temporal cues, which in turn are regulated by miRNAs. This review enlightens on dicer controlled switch from neurogenesis to gliogenesis, miRNA regulation of neuronal maturation and the differential expression of miRNAs in response to various extrinsic cues affecting neurogenesis.

Amikacin Inhibits miR-497 Maturation and Exerts Post-ischemic Neuroprotection.

MicroRNAs (miRNAs) are a group of small non-coding RNAs that regulate numerous signaling pathways involved in cerebral ischemia reperfusion injury. Recent finding demonstrated that miR-497 promotes ischemic neuronal death by negatively regulating anti-apoptotic proteins and therefore serves as a promising therapeutic target for cerebral ischemic injury. In this study, we present a systematic computational approach that includes 3D modeling, docking-based virtual screening, and molecular dynamics simulation to identify small-molecule inhibitors of pre-miR-497 maturation. The top hit, aminoglycosidic antibiotic, amikacin, formed a stable complex with pre-miR-497. Later, the protective efficacy of amikacin was evaluated against oxygen-glucose deprivation (OGD) and reoxygenation-induced neuronal cell death in SH-SY5Y cells and mouse organotypic hippocampal slice cultures. To confirm the inhibitory potential of amikacin on miR-497 maturation, quantitative real-time PCR was performed to check the expression of bcl-2, one of the primary anti-apoptotic targets of miR-497. Additionally, the expression level of mature miR-497 was quantified using TaqMan® MiRNA Assay Kit. Amikacin treatment effectively reduced OGD-induced cell death compared to control groups both in vitro and organotypic hippocampal slice cultures. Further, amikacin effectively increased the expression of bcl-2 in SH-SY5Y cells subjected to OGD. Interestingly, SH-SY5Y cells treated with amikacin displayed decreased expression of miR-497, probably due to inhibition of pre-miRic form. Our study provides strong evidence that amikacin inhibits miR-497 maturation and promotes ischemic neuronal survival by upregulating anti-apoptotic protein, bcl-2. Future studies directed at evaluating the neuroprotective efficacy and mechanism of amikacin animal models may lead to new therapeutic opportunities for preventing neuronal death after stroke.

Systematic review and stratified meta-analysis of the efficacy of carnosine in animal models of ischemic stroke.

Carnosine is a naturally occurring pleotropic dipeptide which influences multiple deleterious mechanisms that are activated during stroke. Numerous published studies have reported that carnosine has robust efficacy in ischemic stroke models. To further evaluate these data, we have conducted a systematic review and meta-analysis of published studies. We included publications describing in vivo models of ischemic stroke where the neuroprotective efficacy of carnosine was being evaluated through the reporting of infarct volume and/or neurological score as outcomes. Overall efficacy was evaluated using weighted mean difference random effects meta-analysis. We also evaluated for study quality and publication bias. We identified eight publications that met our inclusion criteria describing a total of 29 comparisons and 454 animals. Overall methodological quality of studies was moderate (median = 4/9). Carnosine reduced infarct volume by 29.4% (95% confidence interval (CI), 24.0% to 34.9%; 29 comparisons). A clear dose-response effect was observed, and efficacy was reduced when carnosine was administered more than 6 h after ischemia. Our findings suggest that carnosine administered before or after the onset of ischemia exhibits robust efficacy in experimental ischemic stroke. However, the methodological quality of some of the studies was low and testing occurred only in healthy young male animals.

ß-N-oxalyl-L-α, ß- diaminopropionic acid induces HRE expression by inhibiting HIF-prolyl hydroxylase-2 in normoxic conditions.

Hypoxia inducible factor (HIF)-1α, a subunit of HIF transcription factor, regulates cellular response to hypoxia. In normoxic conditions, it is hydroxylated by prolyl hydroxylase (PHD)-2 and targeted for proteosomal degradation. Drugs which inhibit PHD-2 have implications in conditions arising from insufficient blood supply. ß-ODAP (ß-N- oxalyl-L-α, ß- diaminopropionic acid), a non-protein excitatory amino acid present in Lathyrus sativus, is an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor agonist known to activate conventional protein kinase C and stabilize HIF-1α under normoxic conditions. However, the mechanism of HIF-1α stabilization by this compound is unknown. In silico approach was used to understand the mechanism of stabilization of HIF-1α which revealed ß-ODAP interacts with key amino acid residues and Fe2+ at the catalytic site of PHD-2. These results were further corroborated with luciferase HRE (hypoxia response element) reporter system in HeLa cells. Different chemical modulators of PHD-2 activity and HIF-1α levels were included in the study for comparison. Results obtained indicate that ß-ODAP inhibits PHD-2 and facilitates HIF dependent HRE expression and hence, might be helpful in conditions arising from hypoxia.