ESRRG-controlled downregulation of KCNN1 in primary sensory neurons is required for neuropathic pain.

Peripheral nerve injury-induced neuronal hyperactivity in the dorsal root ganglion (DRG) participates in neuropathic pain. The calcium-activated potassium channel subfamily N member 1 (KCNN1) mediates action potential afterhyperpolarization (AHP) and gates neuronal excitability. However, the specific contribution of DRG KCNN1 to neuropathic pain is not yet clear. We report that chronic constriction injury (CCI) of the unilateral sciatic nerve or unilateral ligation of the fourth lumbar nerve produced the downregulation of Kcnn1 mRNA and KCNN1 protein in the injured DRG. This downregulation was partially attributed to a decrease in DRG estrogen-related receptor gamma (ESRRG), a transcription factor, which led to reduced binding to the Kcnn1 promoter. Rescuing this downregulation prevented CCI-induced decreases in total potassium voltage currents and AHP currents, reduced excitability in the injured DRG neurons, and alleviated CCI-induced development and maintenance of nociceptive hypersensitivities, without affecting locomotor function and acute pain. Mimicking the CCI-induced DRG KCNN1 downregulation resulted in augmented responses to mechanical, heat, and cold stimuli in naive mice. Our findings indicate that ESRRG-controlled downregulation of DRG KCNN1 is likely essential for the development and maintenance of neuropathic pain. Thus, KCNN1 may serve as a potential target for managing this disorder.

NT-3 contributes to chemotherapy-induced neuropathic pain through TrkC-mediated CCL2 elevation in DRG neurons.

Cancer patients undergoing treatment with antineoplastic drugs often experience chemotherapy-induced neuropathic pain (CINP), and the therapeutic options for managing CINP are limited. Here, we show that systemic paclitaxel administration upregulates the expression of neurotrophin-3 (Nt3) mRNA and NT3 protein in the neurons of dorsal root ganglia (DRG), but not in the spinal cord. Blocking NT3 upregulation attenuates paclitaxel-induced mechanical, heat, and cold nociceptive hypersensitivities and spontaneous pain without altering acute pain and locomotor activity in male and female mice. Conversely, mimicking this increase produces enhanced responses to mechanical, heat, and cold stimuli and spontaneous pain in naive male and female mice. Mechanistically, NT3 triggers tropomyosin receptor kinase C (TrkC) activation and participates in the paclitaxel-induced increases of C-C chemokine ligand 2 (Ccl2) mRNA and CCL2 protein in the DRG. Given that CCL2 is an endogenous initiator of CINP and that Nt3 mRNA co-expresses with TrkC and Ccl2 mRNAs in DRG neurons, NT3 likely contributes to CINP through TrkC-mediated activation of the Ccl2 gene in DRG neurons. NT3 may be thus a potential target for CINP treatment.

Transcription factor EBF1 mitigates neuropathic pain by rescuing Kv1.2 expression in primary sensory neurons.

Nerve injury-induced alternations of gene expression in primary sensory neurons of the dorsal root ganglion (DRG) are molecular basis of neuropathic pain genesis. Transcription factors regulate gene expression. In this study, we examined whether early B cell factor 1 (EBF1), a transcription factor, in the DRG, participated in neuropathic pain caused by chronic constriction injury (CCI) of the sciatic nerve. EBF1 was distributed exclusively in the neuronal nucleus and coexpressed with cytoplasmic/membrane Kv1.2 in individual DRG neurons. The expression of Ebf1 mRNA and protein was time-dependently downregulated in the ipsilateral lumbar (L) 3/4 DRGs after unilateral CCI. Rescuing this downregulation through microinjection of the adeno-associated virus 5 expressing full-length Ebf1 mRNA into the ipsilateral L3/4 DRGs reversed the CCI-induced decrease of DRG Kv1.2 expression and alleviated the development and maintenance of mechanical, heat and cold hypersensitivities. Conversely, mimicking the downregulation of DRG EBF1 through microinjection of AAV5-expressing Ebf1 shRNA into unilateral L3/4 DRGs produced a reduction of Kv1.2 expression in the ipsilateral L3/4 DRGs, spontaneous pain, and the enhanced responses to mechanical, heat and cold stimuli in naive mice. Mechanistically, EBF1 not only bound to the Kcna2 gene (encoding Kv1.2) promoter but also directly activated its activity. CCI decreased the EBF1 binding to the Kcna2 promoter in the ipsilateral L3/4 DRGs. Our findings suggest that DRG EBF1 downregulation contributes to neuropathic pain likely by losing its binding to Kcna2 promoter and subsequently silencing Kv1.2 expression in primary sensory neurons. Exogenous EBF1 administration may mitigate neuropathic pain by rescuing DRG Kv1.2 expression.

Glucose regulation by newly synthesized boronic acid functionalized molecules as dipeptidyl peptidase IV inhibitor: a potential compound for therapeutic intervention in hyperglycaemia.

Management of type 2 diabetes mellitus (T2DM) using dipeptidyl peptidase IV (DPP IV) inhibitors is gaining precedence as this enzyme plays an indispensable role in cleaving and inactivating peptides, such as glucagon-like peptide-1 (GLP-1), incretin hormones, and glucose-dependent insulinotropic polypeptide (GIP). There are several DPP IV inhibitors used to treat T2DM, but limited by side effects such as disturbed GIT, flu-like symptoms, etc. Thus, there is an urgent need for the development of novel and better DPP IV inhibitors for the management of the same. In the present study, we investigated the effect of new boronic acid-based thiazole compounds as DPP IV inhibitors. We used substituted anilines that were progressively modified through a multi-step synthesis and then chemically characterised. These molecules have good binding affinity and molecular interactions at the active site of the DPP IV enzyme. Two boronic acid-based molecules, i.e. PC06R58 and PC06R108, were used for the assessment of their in-vitro enzymatic activities. Both molecules (PC06108 and PC06R58) exhibited potent uncompetitive DPP IV enzyme inhibition at two different concentrations of 90.9 and 15.6 nM, respectively, compared to sitagliptin having an IC50 of 17.3 nM. Furthermore, the oral glucose tolerance test suggested significantly reduced blood glucose levels at 20 mg/kg of the body weight upon administration of PC06R58 and PC06R108 molecules in rats after glucose ingestion (2 g/kg of the body weight). The compounds showed satisfactory DPP IV inhibition. Furthermore, DPP IV inhibitory activity and acceptable pre-ADME/Tox profile indicate it is a lead compound in this novel class of DPP IV inhibitors.Communicated by Ramaswamy H. Sarma.

RALY participates in nerve trauma-induced nociceptive hypersensitivity through triggering Eif4g2 gene expression in primary sensory neurons.

BACKGROUND AND PURPOSE: Peripheral nerve trauma-induced dysregulation of pain-associated genes in the primary sensory neurons of dorsal root ganglion (DRG) contributes to neuropathic pain genesis. RNA-binding proteins participate in gene transcription. We hypothesized that RALY, an RNA-binding protein, participated in nerve trauma-induced dysregulation of DRG pain-associated genes and nociceptive hypersensitivity. METHODS AND RESULTS: Immunohistochemistry staining showed that RALY was expressed exclusively in the nuclei of DRG neurons. Peripheral nerve trauma caused by chronic constriction injury (CCI) of unilateral sciatic nerve produced time-dependent increases in the levels of Raly mRNA and RALY protein in injured DRG. Blocking this increase through DRG microinjection of adeno-associated virus 5 (AAV5)-expressing Raly shRNA reduced the CCI-induced elevation in the amount of eukaryotic initiation factor 4 gamma 2 (Eif4g2) mRNA and Eif4g2 protein in injured DRG and mitigated the development and maintenance of CCI-induced nociceptive hypersensitivity, without altering basal (acute) response to noxious stimuli and locomotor activity. Mimicking DRG increased RALY through DRG microinjection of AAV5 expressing Raly mRNA up-regulated the expression of Eif4g2 mRNA and Eif4g2 protein in the DRG and led to hypersensitive responses to noxious stimuli in the absence of nerve trauma. Mechanistically, CCI promoted the binding of RALY to the promoter of Eif4g2 gene and triggered its transcriptional activity. CONCLUSION AND IMPLICATIONS: Our findings indicate that RALY participates in nerve trauma-induced nociceptive hypersensitivity likely through transcriptionally triggering Eif4g2 expression in the DRG. RALY may be a potential target in neuropathic pain management.

Characterization of homogenous acid catalyzed biodiesel production from palm oil: experimental investigation and numerical simulation.

Biodiesel is a biological renewable source produced from the conversion of triglycerides to alkyl esters. Palm oil is one of the most used lipid feedstocks for biodiesel production. It becomes necessary to optimize the transesterification reaction parameters to reduce the cost and enhance the quality of biodiesel. This study focuses on the use of homogenous sulfuric acid as a catalyst for the transesterification of palm fatty acids to methyl esters in a batch-scale reactor. A novel examination of transesterification reaction input parameters using the technique for order performance by similarity to ideal solution optimization technique and the effect of these parameters on yield, viscosity, and density of palm biodiesel using 3D surface graphs is investigated in this research. The present optimization approach is implemented to find out the optimum ranking of biodiesel production. From the experimental and numerical simulation, optimum results were observed at the catalyst concentration of 6% (w/w), reaction temperature of 70 °C, the reaction time of 120 min, and alcohol to oil molar ratio of 30:1 at which yield of 95.35%, viscosity of 5.0 cSt, and density of 880 kg/m3 of palm biodiesel were obtained. The different physicochemical properties of produced palm methyl esters are obtained within standards set by international authorities. Selected optimized process parameters can be used for commercial-scale biodiesel production.

Tumor microenvironment targeted nanotherapeutics for cancer therapy and diagnosis: A review.

Recent findings suggest that the cellular and extracellular materials surrounding the cancerous cells from an atypical tumor microenvironment (TM) play a pivotal role in the process of tumor initiation and progression. TM comprises an intricate system involving diverse cell types including endothelial cells, pericytes, smooth muscle cells, fibroblasts, various inflammatory cells, dendritic cells, and cancer stem cells (CSCs). The TM-forming cells dynamically interact with the cancerous cells through various signaling mechanisms and pathways. The existence of this dynamic cellular communication is responsible for creating an environment suitable for sustaining a reasonably high cellular proliferation. Presently, researchers are showing interest to use these TM conditions to mediate effective targeting measures for cancer therapy. The use of nanotherapeutics-based combination therapy; stimuli-responsive nanotherapeutics targeting acidic pH, hypoxic environment; and nanoparticle-induced hyperthermia are some of the approaches that are under intense investigation for cancer therapy. This review discusses TM and its role in cancer progression and crosstalk understanding, opportunities, and epigenetic modifications involved therein to materialize the capability of nanotherapeutics to target cancer by availing TM. STATEMENT OF SIGNIFICANCE: This article presents various recent reports, proof-of-concept studies, patents, and clinical trials on the concept of tumor microenvironment for mediating the cancer-specific delivery of nanotechnology-based systems bearing anticancer drug and diagnostics. We highlight the potential of tumor microenvironment; its role in disease progression, opportunities, challenges, and allied treatment strategies for effective cancer therapy by conceptual understanding of tumor microenvironment and epigenetic modifications involved. Specifically, nanoparticle-based approaches to target various processes related to tumor microenvironment (pH responsive, hypoxic environment responsive, targeting of specific cells involved in tumor microenvironment, etc.) are dealt in detail.

Cardiac Dysfunction in Patients with Liver Cirrhosis.

BACKGROUND: The clinical picture in cirrhosis is dominated by the classical complications such as ascites, bleeding varices, portal hypertension and encephalopathy. Cardiac dysfunction in patients with cirrhosis, which contributes significantly to the morbidity and, mortality though prevalent, is less studied and not widely recognized entity since it is largely asymptomatic at rest, with overt heart failure seen mainly during pharmacological stress, transjugular intrahepatic portosystemic shunt, liver transplantation. METHODS: It is a cross sectional study done on patients admitted in wards or attending to outpatient department of Liver unit, Bir Hospital, between May 2015 to May 2016. Diagnosis of cirrhosis was based on clinical examination, lab parameters, ultrasound examination, endoscopy and/or liver biopsy. Cirrhotic patients after assessing the exclusion criteria were recruited for the study. Child Pugh and model for end stage liver disease scores were calculated to assess the liver function. Cardiac function was evaluated by resting pulse, mean arterial pressure, electrocardiography, and 2 dimensional echocardiography. RESULTS: Diastolic dysfunction was seen in 61.9%(48) and was more common in alcoholic group (63.2% Vs 58.6%). Systolic dysfunction was seen in 6.6% of alcoholic patients only. 51.4% had cirrhotic cardiomyopathy according to the criteria (proposed by World congress of gastroenterology in 2005). Prolonged QTc of >0.44 seconds was noted in 79%, mainly in child pugh C, with model for end stage liver disease score >10. CONCLUSIONS: Cardiac dysfunction is prevalent with sizeable number of patients with cirrhosis especially in the form of diastolic dysfunction independent of etiology. QTc prolongation might be an early indicator of cardiac dysfunction and is directly correlated with child pugh and model for end stage liver disease scores.

Fast dissolving electrospun polymeric films of anti-diabetic drug repaglinide: formulation and evaluation.

Objective: Repaglinide is a well-known FDA approved drug from category of meglitinide; used for the treatment of diabetes. However, its use is limited because of its poor water solubility which leads to erratic drug absorption. Present work focuses on formulation and evaluation of polyvinyl alcohol (PVA)-polyvinyl pyrrolidone (PVP) nanofibers to counter this problem of poor water solubility.Significance: Prepared nanofibers with hydrophilic polymers were expected to tackle the problem of poor water solubility.Methods: Nanofibers were prepared by electrospinning technique with the optimization of parameters affecting final product. Further prepared formulation was characterized using various techniques.Results: Successful development of drug loaded nanofibers was commenced utilizing electrospinning technique. Further casted film of same polymeric blend was prepared and compared with nanofibers. Optimized nanofibers showed an average diameter of 600-800 nm with smooth surface morphology. Prepared nanofibers and casted film was analyzed in terms of surface morphology, mechanical strength, solid state of drug present, effects of hydrogen bond formation and drug release profile. Results from the glucose tolerance test suggested both the formulations to be having better control over glucose levels as compared to free drug.Conclusion: Overall developed nanofibers presented themselves to be potential drug delivery candidates for drugs having poor water solubility.

A durability study of a compression ignition engine operating with Thumba (Citrullus colocyntis) vegetable oil.

Vegetable oils are found suitable alternate of diesel fuel as per the results of short-run studies. Long-run studies with vegetable oil as a fuel pointed out the problems related to wear and maintenance of the engine. A single cylinder, variable compression ratio diesel engine was tested for 512 h (32 cycles of 16 h per day) to investigate longevity implications of fueling Thumba vegetable oil. Results of the study revealed that a very little damage was observed over the running surface of the cylinder liner, piston rings, valves, and valve seats. Wear in the piston outer diameter was observed to be 13 to 30 microns. Cylinder wear was about 80 microns. The closed gap in the oil piston ring increased up to 200 microns. Heavy carbon deposition was found on different internal parts of the engine, which indicates poor combustion of fuel. Amount of copper (66 mg/kg) and silicon (112 mg/kg) dissolved in the lubricating oil was found more than permissible limits (Cu 50 mg/kg, Si 25 mg/kg), after 450-h engine test run. But all the dissolve materials remain in allowable limits when the durability test conducted with diesel. Smoke, CO, HC, and NOX emissions were found to increase initially then decrease in the further engine running hours. But these emissions were found inferior to the engine emissions fueled with diesel in all the running hours. CO2 emissions were found superior throughout the test with the preheated T20 Thumba oil blend than diesel. The maximum reduction in the viscosity of the lubricating oil, during endurance testing, was found 60 centipoises but it was found 25 centipoises when the test conducted with diesel.

Kinesins: Motor Proteins as Novel Target for the Treatment of Chronic Pain.

Kinesins are one of the neoteric and efficacious targets recently reported to play an important role in the initiation and progression of chronic pain. Kinesins are anterograde microtubule-based motor proteins that are involved in trafficking of receptors including nociceptors and progression of pain. The specific kinesin and regulatory proteins interplay is crucial for the delivery of nociceptors to the synapse. If this complex and less understood interplay is inhibited, it may result in a decrease in central sensitization, and thus attenuation of pain. This review is focused on the transportation process of receptors/cargos, the role of regulatory proteins influencing the respective kinesin, and their relationship with chronic pain. The review also features specific strategies adopted by researchers for targeting kinesin and chronic pain. Considering the recent preclinical success of kinesin inhibition in pain, it is expected that inhibitors for kinesin or enzymes responsible for kinesin activation could be developed or repurposed as alternative, safe, and potential therapies for the treatment of chronic pain.

Neuroprotective effects of silibinin: an in silico and in vitro study.

AIM OF THE STUDY: Astrogliosis is a key contributor for many neurological disorders involving apoptosis, neuroinflammation and subsequent neuronal death. Silibinin, a polyphenol isolated from milk thistle (Silybum marianum), has been shown to suppress the astrocyte activation in various neurodegenerative disorders and also exhibit a neuroprotective role in neuroinflammation-driven oxidative damage. The present study was designed with an aim to investigate the neuroprotective effects of Silibinin against LPS induced oxido-inflammatory cascade and astrocyte activation. MATERIALS AND METHODS: We have used in-silico molecular modelling techniques to study the interaction and binding affinity of silibinin with chemokine receptors associated with neuroinflammation. We have also tested silibinin against LPS induced oxido-inflammatory cascade and astrocyte activation in C6 glia cell lines. RESULTS: In the present study, we found that treatment with silibinin significantly attenuates LPS-oxidative-nitrosative stress in C6 astrocytoma cells. We also observed the significant inhibition of induced astrocyte activity after treatment with silibinin. Moreover, molecular modelling studies have proposed a binding pose of silibinin with binding sites of p38 MAPK, CX3CR1 and P2X4 which is an important downstream cascade involved in glia cell activation and neuroinflammation. CONCLUSIONS: Overall, the findings from the current study suggests that silibinin exhibits neuroprotective activity by attenuating oxidative damage and astrocytes activation.

Comparative evaluation of electrospraying and lyophilization techniques on solid state properties of Erlotinib nanocrystals: Assessment of In-vitro cytotoxicity.

INTRODUCTION: Erlotinib is a well known FDA approved drug from category of tyrosine kinase inhibitors; used for the treatment of lung cancer. However its use is limited because of its poor water solubility. OBJECTIVE: The aim of present work was to improve solubility by developing a stable nanocrystal based drug delivery system of ERL with the aid of sodium lauryl sulfate as potential stabilizer and to carry out comparative evaluation of electrospraying and lyophilization as solidification techniques on its solid state properties. EXPERIMENTAL: Nanocrystal formulation was developed with antisolvent precipitation method having particle size, polydispersity index and zetapotential of 232.4±4.3nm, 0.162 and -9.82mV respectively. Further comparative evaluation of lyophilization and electrospraying was commenced as potential solidification techniques and solid powder matrix obtained from both the solidification techniques were compared in terms of size after re-dispersion (260±4.8 and 329±5.2nm respectively), particle morphology, surface area (0.984±0.11 and 0.341±0.05m2/g respectively), pore volume (0.0014 and 0.0009cc/g respectively), solid state of drug present and % drug release (~100% and ~78% respectively in 600min). In vitro cytotoxicity studies shared that obtained formulation was having reduced IC50 values in comparison to drug. Further intracellular reactive oxygen species production was found to be higher for formulation treated cells when compared to free drug. Overall developed formulation was found to be potential drug delivery system for lung cancer therapy.

Surface Engineered Dendrimers in siRNA Delivery and Gene Silencing.

BACKGROUND: Therapeutic efficacy of dreadful diseases like cancer, HIV (Human Immunodeficiency Virus) can be enhanced by delivering molecules which regulate function at gene level rather than at receptor level. Silencing RNA is one such approach recently used to silence target gene expressed diseases; and thereby reduce target protein levels. Many of the non-viral vectors are proved to act as carriers for silencing RNA. Dendrimers being one of them have less size, low poly dispersibility index, water solubility, multivalence, and easy surface modification. Many such surface modifications have been carried out to improve the delivery potential of small interfering RNA (siRNA) modified dendrimers compared to simple plain dendrimers. METHODS: Dendrimer was taken as a core whose surface was modified with fluorine, amino acids, phosphate, lipids, folate, specific antibody or RGD (Arg-Gly-Asp). The purpose of these modifications was to increase the therapeutic siRNA efficiency, lower the toxicity and improve the targeting potential of dendrimers. RESULTS: Fluorinated dendrimers have highest electronegativity and highest siRNA loading capacity. Amino acid functionalized dendrimers are made up of endogenous amino acids which improve biocompatibility of dendrimer and endosomal escape. Carbosilane dendrimers increase the gene transfection ability of tissues to be treated. Phosphate dendrimers having hydrophobic backbone and hydrophilic surface increase the permeability towards targeted tissue. Lipid based dendrimer causes endosomal escape and improves the permeability of dendrimers. Targeting of specific tissues is achieved by coupling dendrimer with folate, RGD and specific antibody, thereby reducing off target effect. CONCLUSION: Thus, surface modified dendrimers render a complete pack which offers increased siRNA loading, increased transfection and permeability, efficient targeting, endosomal escape and protecting siRNA from degradation by RNase and other such enzymes. The objective of this manuscript is to provide different approaches currently available for surface modifications of dendrimers and their overall effect on transfection ability of siRNA to target tissues.

Study of severe and rare complications of transarterial chemoembolization (TACE) for liver cancer.

OBJECTIVE: To study severe and rare complications of transarterial chemoembolization (TACE) for liver cancer. METHODS: Clinical records of severe and rare complications following TACE in 1348 cases of liver cancer from January 1997 to February 2004 were studied retrospectively. RESULTS: A total of 2012 TACE procedures were performed for 1348 patients. There were 3 cases of spontaneous rupture of liver cancer, 1 case of perforation of duodenum, 3 cases of liver abscess (1 of them was associated with sepsis), 1 case of pulmonary embolism, 1 case of spasm of the hepatic artery, 40 cases of hepatic artery occlusion, 3 cases of femoral nerve injury, 1 case of bilioma and 1 case of acute renal failure. CONCLUSION: Although the severe complications of TACE are rare, the procedure should be done cautiously including super selection of hepatic artery, slow infusion of lipiodol, careful postoperative observations and early detection and management of complications.