A dipyrrole derivative from Aloe vera inhibits an anti-diabetic drug target Dipeptidyl Peptidase (DPP)-IV in vitro.

Aloe vera, a succulent herb, has a long history of use in traditional medicine, including diabetes. Earlier studies from our laboratory demonstrated that the Aloe vera extract has the ability to inhibit the diabetic drug target dipeptidyl peptidase (DPP) IV in vitro. This current study focuses on the isolation of small water soluble active molecule(s) involved in DPP-IV inhibition from Aloe vera extract, and further to characterize its structure and to elucidate the mode of inhibition of the DPP-IV enzyme. Aloe vera gel ethanolic extract was subjected to preparative reverse-phase high-pressure liquid chromatography (RP-HPLC), LH-20 Sephadex gel filtration chromatography, followed by analytical RP-HPLC, to isolate the active molecule involved in DPP-IV inhibition. Based on the spectroscopic studies, the structure of the isolated DPP-IV inhibitor was predicted to be 3, 6-dioxo-3, 3a, 6, 6 a-tetrahydropyrrolo [3, 4-c] pyrrole-1, 4-dicarboxamide with the chemical formula C8H6N4O4, having the molecular weight of 225.175 Da. This molecule inhibited the DPP-IV enzyme in a noncompetitive manner with an IC50 value of 8.59 ± 2.61 µM, with a Ki of 4.7 ± 0.038 µM. Thus, the mechanism of DPP-IV inhibition and the inhibitory constants were determined. The results of our studies suggested that the inhibition of the DPP-IV enzyme as one of the pathways by which the Aloe vera extract may restore the pancreatic islets cell mass in diabetic animal model.

Recombinant irisin induces weight loss in high fat DIO mice through increase in energy consumption and thermogenesis.

OBJECTIVE: Irisin is known to be an important metabolic regulator of glucose and lipid metabolism. The aims of the present study are to assess the role of mouse Irisin in obesity and energy metabolism and its glucose and lipid-lowering effects in a high-fat diet-induced obesity (DIO) mice model. METHODS: DIO mice were treated with recombinant murine Irisin or vehicle, and parameters such as body weight, feed intake, glucose, and lipid levels, obesity, energy consumption, and insulin sensitivity were assessed. mRNA and protein levels of UCP1 and different thermogenesis biomarker were evaluated by quantitative real-time PCR and Western blot, respectively, in tissues and major metabolic organs. RESULTS: Irisin decreased body weight and whole-body fat mass in DIO mice in a dose dependent manner due to marked increases in total energy expenditure. It also lowered blood glucose, insulin, and lipid levels and possibly reversed hepatic steatosis. Irisin improved hepatic and peripheral insulin sensitivity in DIO mice along with body weight reduction and adiposity. Gene expression of UCP1 in different organs (adipose tissue and major organs, i.e., liver, kidney, heart, brain, and spleen) have suggested the role of irisin is global. Gene expression profile of different biomarkers in spleen suggest a profound role of Irisin in inflammation. Liver tissue have also shown significant increase of UCP1 expression in dose dependent manner which suggest a role of irisin in liver.

Expression of anti-tumor necrosis factor alpha (TNFα) single-chain variable fragment (scFv) in Spirodela punctata plants transformed with Agrobacterium tumefaciens.

Therapeutic antibodies against tumor necrosis factor alpha (TNFα) have been considered effective for some of the autoimmune diseases such as rheumatoid arthritis, Crohn's diseases, and so on. But associated limitations of the current therapeutics in terms of cost, availability, and immunogenicity have necessitated the need for alternative candidates. Single-chain variable fragment (scFv) can negate the limitations tagged with the anti-TNFα therapeutics to a greater extent. In the present study, Spirodela punctata plants were transformed with anti-TNFα through in planta transformation using Agrobacterium tumefaciens strain, EHA105. Instead of cefotaxime, garlic extract (1 mg/mL) was used to remove the agrobacterial cells after cocultivation. To the best of our knowledge, this report shows for the first time the application of plant extracts in transgenic plant development. 95% of the plants survived screening under hygromycin. ScFv cDNA integration in the plant genomic DNA was confirmed at the molecular level by PCR. The transgenic protein expression was followed up to 10 months. Expression of scFv was confirmed by immunodot blot. Protein expression levels of up to 6.3% of total soluble protein were observed. ß-Glucuronidase and green fluorescent protein expressions were also detected in the antibiotic resistant plants. The paper shows the generation of transgenic Spirodela punctuata plants through in planta transformation.

Novel monoclonal antibody against truncated C terminal region of Histidine Rich Protein2 (PfHRP2) and its utility for the specific diagnosis of malaria caused by Plasmodium falciparum.

An accurate diagnosis of malarial infection is an important element in combating this deadly disease. Malaria diagnostic test including, microscopy and other molecular tests are highly sensitive but too complex for field conditions. Rapid detection tests for P. falciparum infection using monoclonal antibodies (mAbs) against highly polymorphic PfHRP2 (Histidine Rich Protein2) are still most preferred test in field conditions, but with limitations such as specificity, and sensitivity leading to false positive and false negative results. To overcome these limitations, we carried out bioinformatics analysis PfHRP2 and PfHRP3 and found that the C-terminal region of PfHRP2 (~105 amino acids) displayed relatively lower sequence identity with PfHRP3. This C-terminal region of PfHRP2 contained unique peptide repeats and was found to be conserved in various isolates of P. falciparum. Moreover, this region was also found to be highly antigenic as predicted by antigenicity propensity scores. Thus we constructed a cDNA clone of the truncated PfHRP2 (recPfHRP2-T3) coding for C-terminal 105 amino acids and expressed it in E. coli and purified the polypeptide to homogeneity. The purified recPfHRP2-T3 was used as an antigen for development of both polyclonal and monoclonal antibody (mAb). The mAbs b10c1 and Aa3c10 developed against recPfHRP2-T3 was found to efficiently recognize recombinant PfHRP2 but not PfHRP3. In addition, the above mAbs reacted positively with spent media and serum sample of P. falciparum infection recognizing the native PfHRP2. The affinity constant of both the clones were found to be 10(9) M(-1). Quantitatively, both these clones showed ~4.4 fold higher reactivity with P. falciparum infected serum compared to serum from healthy volunteers or P. vivax infected patient samples. Thus these anti-C-terminal PfHRP2 mAbs (Aa3c10 and b10c1) display a very high potential for improvising the existing malarial diagnostic tools for detection of P. falciparum infection especially in areas where PfHRP2 polymorphism is highly prevalent.

The use of Bacillus subtilis as a cost-effective expression system for production of Cholera Toxin B fused factor VIII epitope regions applicable for inducing oral immune tolerance.

Coagulation factor replacement therapy for the X-linked bleeding disorder Haemophilia, characterized by a deficiency of coagulation protein factor VIII (FVIII), is severely complicated by antibody (inhibitors) formation. The development of FVIII inhibitors drastically alters the quality of life of the patients and is associated with a tremendous increase in morbidity as well as treatment costs. The ultimate goal of inhibitor control is antibody elimination. Immune tolerance induction (ITI) is the only clinically established approach for developing antigen-specific tolerance to FVIII. This work aims to establish a novel cost-effective strategy to produce FVIII molecules in fusion with cholera toxin B (CTB) subunit at the N terminus using the Bacillus subtilis expression system for oral tolerance, as the current clinical immune tolerance protocols are expensive. Regions of B-Domain Deleted (BDD)-FVIII that have potential epitopes were identified by employing Bepipred linear epitope prediction; 2 or more epitopes in each domain were combined and cDNA encoding these regions were fused with CTB and cloned in the Bacillus subtilis expression vector pHT43 and expression analysis was carried out. The expressed CTB-fused FVIII epitope domains showed strong binding affinity towards the CTB-receptor GM1 ganglioside. To conclude, Bacillus subtilis expressing FVIII molecules might be a promising candidate for exploring for the induction of oral immune tolerance.

Binding Promiscuity of Therapeutic Factor VIII.

The binding promiscuity of proteins defines their ability to indiscriminately bind multiple unrelated molecules. Binding promiscuity is implicated, at least in part, in the off-target reactivity, nonspecific biodistribution, immunogenicity, and/or short half-life of potentially efficacious protein drugs, thus affecting their clinical use. In this review, we discuss the current evidence for the binding promiscuity of factor VIII (FVIII), a protein used for the treatment of hemophilia A, which displays poor pharmacokinetics, and elevated immunogenicity. We summarize the different canonical and noncanonical interactions that FVIII may establish in the circulation and that could be responsible for its therapeutic liabilities. We also provide information suggesting that the FVIII light chain, and especially its C1 and C2 domains, could play an important role in the binding promiscuity. We believe that the knowledge accumulated over years of FVIII usage could be exploited for the development of strategies to predict protein binding promiscuity and therefore anticipate drug efficacy and toxicity. This would open a mutational space to reduce the binding promiscuity of emerging protein drugs while conserving their therapeutic potency.

Cholesterol transport and beyond: Illuminating the versatile functions of HDL apolipoproteins through structural insights and functional implications.

High-density lipoproteins (HDLs) play a vital role in lipid metabolism and cardiovascular health, as they are intricately involved in cholesterol transport and inflammation modulation. The proteome of HDL particles is indeed complex and distinct from other components in the bloodstream. Proteomics studies have identified nearly 285 different proteins associated with HDL; however, this review focuses more on the 15 or so traditionally named "apo" lipoproteins. Important lipid metabolizing enzymes closely working with the apolipoproteins are also discussed. Apolipoproteins stand out for their integral role in HDL stability, structure, function, and metabolism. The unique structure and functions of each apolipoprotein influence important processes such as inflammation regulation and lipid metabolism. These interactions also shape the stability and performance of HDL particles. HDLs apolipoproteins have multifaceted roles beyond cardiovascular diseases (CVDs) and are involved in various physiological processes and disease states. Therefore, a detailed exploration of these apolipoproteins can offer valuable insights into potential diagnostic markers and therapeutic targets. This comprehensive review article aims to provide an in-depth understanding of HDL apolipoproteins, highlighting their distinct structures, functions, and contributions to various physiological processes. Exploiting this knowledge holds great potential for improving HDL function, enhancing cholesterol efflux, and modulating inflammatory processes, ultimately benefiting individuals by limiting the risks associated with CVDs and other inflammation-based pathologies. Understanding the nature of all 15 apolipoproteins expands our knowledge of HDL metabolism, sheds light on their pathological implications, and paves the way for advancements in the diagnosis, prevention, and treatment of lipid and inflammatory-related disorders.

High factor VIII concentrations interfere with glycoprotein VI-mediated platelet activation in vitro.

BACKGROUND: The recruitment of activated factor VIII (FVIII) at the surface of activated platelets is a key step toward the burst of thrombin and fibrin generation during thrombus formation at the site of vascular injury. It involves binding to phosphatidylserine and, possibly, to fibrin-bound αIIbß3. Seminal work had shown the binding of FVIII to resting platelets, yet without a clear understanding of a putative physiological relevance. OBJECTIVES: To characterize the effects of FVIII-platelet interaction and its potential modulation of platelet function. METHODS: FVIII was incubated with washed platelets. The effects on platelet activation (spontaneously or triggered by collagen and thrombin) were studied by flow cytometry and light transmission aggregometry. We explored the involvement of downstream pathways by studying phosphorylation profiles (Western blot). The FVIII-glycoprotein (GP) VI interaction was investigated by ELISA, confocal microscopy, and proximity ligation assay. RESULTS: FVIII bound to the surface of resting and activated platelets in a dose-dependent manner. FVIII at supraphysiological concentrations did not induce platelet activation but rather specifically inhibited collagen-induced platelet aggregation and altered glycoprotein VI (GPVI)-dependent phosphorylation. FVIII, freed of its chaperone protein von Willebrand factor (VWF), interacted in close proximity with GPVI at the platelet surface. CONCLUSION: We showed that VWF-free FVIII binding to, or close to, GPVI modulates platelet activation in vitro. This may represent an uncharacterized negative feedback loop to control overt platelet activation. Whether locally activated FVIII concentrations achieved during platelet accumulation and thrombus formation at the site of vascular injury in vivo are compatible with such a function remains to be determined.

Oxidative stress and cardiovascular health: therapeutic potential of polyphenols.

Reactive oxygen species (ROS) are important in normal cellular function and physiology. However, oxidative stress resulting from an accumulation of ROS has a detrimental impact on cellular function, and ROS has been implicated in the pathogenesis of a number of diseases, including cardiovascular diseases. This review provides a summary of the impact of ROS on cardiovascular health and diseases, highlighting the therapeutic use of antioxidants. In addition, this review summarizes the health benefits of polyphenols, and the recent progress on understanding the cellular and physiological actions by which polyphenols may impart their beneficial properties on cardiovascular health.

Genes involved in pancreatic islet cell rejuvenation.

Pancreas plays an important role in maintaining the glucose homeostasis. The deterioration of ß-cells in the pancreas is a crucial factor in the progression of diabetes mellitus; therefore, the restoration of ß-cell mass and its function is of vital importance for effective therapeutic strategies. The precise mechanism for increase in functional ß-cell mass is still unknown. This review focuses on the importance of certain genes which are involved in the rejuvenation of pancreas. These genes are divided according to their functions into three categories: participate either in proliferation (mitotic division of differentiated ß-cells), neogenesis/transdifferentiation (development from precursor cells) or inhibition of ß-cell apoptosis (programmed cell death). The rate of ß-cell rejuvenation is the balance among the rates of ß-cell proliferation, neogenesis and apoptosis. Understanding these genes and their pathways may lead to the discovery of new drugs, target based gene delivery and development of safer antidiabetic drugs.

Oxidative stress in aging--matters of the heart and mind.

Oxidative damage is considered to be the primary cause of several aging associated disease pathologies. Cumulative oxidative damage tends to be pervasive among cellular macromolecules, impacting proteins, lipids, RNA and DNA of cells. At a systemic level, events subsequent to oxidative damage induce an inflammatory response to sites of oxidative damage, often contributing to additional oxidative stress. At a cellular level, oxidative damage to mitochondria results in acidification of the cytoplasm and release of cytochrome c, causing apoptosis. This review summarizes findings in the literature on oxidative stress and consequent damage on cells and tissues of the cardiovascular system and the central nervous system, with a focus on aging-related diseases that have well-documented evidence of oxidative damage in initiation and/or progression of the disease. The current understanding of the cellular mechanisms with a focus on macromolecular damage, impacted cellular pathways and gross morphological changes associated with oxidative damage is also reviewed. Additionally, the impact of calorific restriction with its profound impact on cardiovascular and neuronal aging is addressed.

Delineating the impact of pathogenic mutations on the conformational dynamics of HDL's vital protein ApoA1: a combined computational and molecular dynamic simulation approach.

Apolipoprotein A1 (ApoA1), is the important component of high-density lipoproteins (HDL), that has key role in HDL biogenesis, cholesterol trafficking, and reverse cholesterol transport (RCT). Non-synonymous Single Nucleotide Polymorphisms (nsSNPs) in ApoA1 have been linked to cardiovascular diseases and amyloidosis as they alter the protein's native structure and function. Therefore in this study, we attempted to understand the molecular pathogenicity profile of nsSNPs of ApoA1 using various computational approaches. We used state-of-the-art computational methods to thoroughly investigate the 295 ApoA1 nsSNPs at sequence and structural levels. Seven nsSNPs (L13R, L84R, L84P, L99P, R173P, L187P, and L238P) out of 295 were classified as the most deleterious and destabilizing. In order to estimate the effect of such destabilizing mutations on the protein conformation, all-atom molecular dynamics simulations (MDS) of ApoA1 wild-type (WT), L99P and R173P for 100 ns, was carried out using GROMACS 5.0.1 package. The MD simulation investigation revealed significant structural alterations in L99P and R173P. In addition, they had changed principal component analysis and electrostatic surface potential, decreased structural compactness, and intramolecular hydrogen bonds, which supported the rationale underpinning ApoA1 dysfunction with such mutations. This work sheds light on ApoA1 dysfunction due to single amino acid alterations, and offers new insight into the molecular basis of ApoA1-related diseases progression.Communicated by Ramaswamy H. Sarma.

A Systematic Review of the Potential of Pichia pastoris (Komagataella phaffii) as an Alternative Host for Biologics Production.

The methylotrophic yeast Pichia pastoris is garnering interest as a chassis cell factory for the manufacture of recombinant proteins because it effectively satisfies the requirements of both laboratory and industrial set up. The optimisation of P. pastoris cultivation is still necessary due to strain- and product-specific problems such as promoter strength, methanol utilisation type, and culturing conditions to realize the high yields of heterologous protein(s) of interest. Techniques integrating genetic and process engineering have been used to overcome these problems. Insight into the Pichia as an expression system utilizing MUT pathway and the development of methanol free systems are highlighted in this systematic review. Recent developments in the improved production of proteins in P. pastoris by (i) diverse genetic engineering such as codon optimization and gene dosage; (ii) cultivating tactics including co-expression of chaperones; (iii) advances in the use of the 2A peptide system, and (iv) CRISPR/Cas technologies are widely discussed. We believe that by combining these strategies, P. pastoris will become a formidable platform for the production of high value therapeutic proteins.

A commentary on the paper, 'Development and validation of a novel automatable assay for cholesterol efflux capacity'.

The determination of functionality or quality of high-density lipoproteins (HDL) is assuming a central stage in the prediction of cardiovascular diseases (CVD). To assess HDL quality, several attempts have been made to develop an automated, cost-effective cholesterol efflux capacity (CEC) system with few operational steps that might be used in clinical settings for large throughput testing. The work of Dr. Ohkawa and co-workers seems to address this issue and provide a solution for the same (Bioscience Reports (2023), 43 BSR20221519, https://doi.org/10.1042/BSR20221519). Earlier work from the author's lab utilized a radioisotope and cell-free CEC assay known as the immobilized liposome-bound gel beads (ILGs) method. However, this assay required a centrifugation step to separate the cells and was not suitable for automation. To overcome these limitations, two very important changes were made: (i) magnetic beads were used instead of gel beads that allowed them to avoid the centrifugation process that would allow ease of setting up an autonomous analyzer; (ii) porous magnetic beads were coated with liposomes containing fluorescently tagged cholesterol instead radiolabeled cholesterol. These two changes can be considered not only significant but also novel as they were highly suitable for CEC testing. The authors reported the successful development of a simple immobilized liposome-based magnetic beads (ILMs) automated system to measure CEC, which provided both consistent performance and satisfactory correlation with the other methods. Thus, we feel the present study will open newer avenues for measuring the quality of HDL in addition to the quantity of HDL-cholesterol in clinical settings in a more robust way.

GPR56 signaling pathway network and its dynamics in the mesenchymal transition of glioblastoma.

G protein-coupled receptor 56 (GPR56/ADGRG1) is a multifunctional adhesion GPCR involved in diverse biological processes ranging from development to cancer. In our earlier study, we reported that GPR56 is expressed heterogeneously in glioblastoma (GBM) and is involved in the mesenchymal transition, making it a promising therapeutic target (Ganesh et al., 2022). Despite its important role in cancer, its mechanism of action or signaling is not completely understood. Thus, based on transcriptomic, proteomic, and phosphoproteomic differential expression data of GPR56 knockdown U373-GBM cells included in our above study along with detailed literature mining of the molecular events plausibly associated with GPR56 activity, we have constructed a signaling pathway map of GPR56 as may be applicable in mesenchymal transition in GBM. The map incorporates more than 100 molecular entities including kinases, receptors, ion channels, and others associated with Wnt, integrin, calcium signaling, growth factors, and inflammation signaling pathways. We also considered intracellular and extracellular factors that may influence the activity of the pathway entities. Here we present a curated signaling map of GPR56 in the context of GBM and discuss the relevance and plausible cross-connectivity across different axes attributable to GPR56 function. GPR56 signaling and mesenchymal transition.

The Effects of Oral Probiotics on Type 2 Diabetes Mellitus (T2DM): A Clinical Trial Systematic Literature Review.

Type 2 diabetes mellitus (T2DM) remains a global health concern. Emerging clinical trial (CT) evidence suggests that probiotic intervention may promote a healthy gut microbiome in individuals with T2DM, thereby improving management of the disease. This systematic literature review summarizes thirty-three CTs investigating the use of oral probiotics for the management of T2DM. Here, twenty-one studies (64%) demonstrated an improvement in at least one glycemic parameter, while fifteen studies (45%) showed an improvement in at least one lipid parameter. However, no article in this review was able to establish a uniform decrease in glycemic, lipid, or blood pressure profiles. The lack of consistency across the studies may be attributed to differences in probiotic composition, duration of probiotic consumption, and probiotic dose. An interesting finding of this literature review was the beneficial trend of metformin and probiotic co-administration. Here, patients with T2DM taking metformin demonstrated enhanced glycemic control via the co-administration of probiotics. Taken together, the overall positive findings reported across the studies in combination with minimal adverse effects constitute ground for further quality CTs. This review provides recommendations for future CTs that may address the shortcomings of the current studies and help to extract useful data from future investigations of the use of probiotics in T2DM management.

Molecular Mechanisms Associated with Antifungal Resistance in Pathogenic Candida Species.

Candidiasis is a highly pervasive infection posing major health risks, especially for immunocompromised populations. Pathogenic Candida species have evolved intrinsic and acquired resistance to a variety of antifungal medications. The primary goal of this literature review is to summarize the molecular mechanisms associated with antifungal resistance in Candida species. Resistance can be conferred via gain-of-function mutations in target pathway genes or their transcriptional regulators. Therefore, an overview of the known gene mutations is presented for the following antifungals: azoles (fluconazole, voriconazole, posaconazole and itraconazole), echinocandins (caspofungin, anidulafungin and micafungin), polyenes (amphotericin B and nystatin) and 5-fluorocytosine (5-FC). The following mutation hot spots were identified: (1) ergosterol biosynthesis pathway mutations (ERG11 and UPC2), resulting in azole resistance; (2) overexpression of the efflux pumps, promoting azole resistance (transcription factor genes: tac1 and mrr1; transporter genes: CDR1, CDR2, MDR1, PDR16 and SNQ2); (3) cell wall biosynthesis mutations (FKS1, FKS2 and PDR1), conferring resistance to echinocandins; (4) mutations of nucleic acid synthesis/repair genes (FCY1, FCY2 and FUR1), resulting in 5-FC resistance; and (5) biofilm production, promoting general antifungal resistance. This review also provides a summary of standardized inhibitory breakpoints obtained from international guidelines for prominent Candida species. Notably, N. glabrata, P. kudriavzevii and C. auris demonstrate fluconazole resistance.

Leveraging knowledge of HDLs major protein ApoA1: Structure, function, mutations, and potential therapeutics.

Apolipoprotein A1 (ApoA1) is a member of the Apolipoprotein family of proteins. It's a vital protein that helps in the production of high-density lipoprotein (HDL) particles, which are crucial for reverse cholesterol transport (RCT). It also has anti-inflammatory, anti-atherogenic, anti-apoptotic, and anti-thrombotic properties. These functions interact to give HDL particles their cardioprotective characteristics. ApoA1 has recently been investigated for its potential role in atherosclerosis, diabetes, neurological diseases, cancer, and certain infectious diseases. Since ApoA1's discovery, numerous mutations have been reported that affect its structural integrity and alter its function. Hence these insights have led to the development of clinically relevant peptides and synthetic reconstituted HDL (rHDL) that mimics the function of ApoA1. As a result, this review has aimed to provide an organized explanation of our understanding of the ApoA1 protein structure and its role in various essential pathways. Furthermore, we have comprehensively reviewed the important ApoA1 mutations (24 mutations) that are reported to be involved in various diseases. Finally, we've focused on the therapeutic potentials of some of the beneficial mutations, small peptides, and synthetic rHDL that are currently being researched or developed, since these will aid in the development of novel therapeutics in the future.

Expression of a Tagless Single-Chain Variable Fragment (scFv) of Anti-TNF-α by a Salt Inducible System and its Purification and Characterization.

BACKGROUND: Anti-TNF-α scFv is gaining acceptance as an effective drug for various diseases, such as rheumatoid arthritis and Crohn's disease that involve elevated levels of TNF-α. The single-chain variable fragment (scFv) consists of variable regions of heavy and light chains of monoclonal antibodies (mAb). Due to its smaller size, it curbs the mAb's auto-antibody effects and their limitation of penetration into the tissues during the neutralization of TNF-α. OBJECTIVE: In this work, a cDNA coding for anti-TNF-α scFv was successfully cloned into a pRSET-B vector and efficiently expressed in an E. coli strain GJ1158, a salt inducible system that uses sodium chloride instead of IPTG as an inducer. METHODS: The protein was expressed in the form of inclusion bodies (IB), solubilized using urea, and refolded by pulse dilution. Further, the amino acid sequence coverage of scFv was confirmed by ESI-Q-TOF MS/MS and MALDI-TOF. Further studies on scaling up the production of scFv and its application of scFv are being carried out. RESULTS: The soluble fraction of anti-TNF-α scFv was then purified in a single chromatographic step using CM-Sephadex chromatography, a weak cation exchanger with a yield of 10.3 mg/L. The molecular weight of the scFv was found to be ~ 28 kDa by SDS PAGE, and its presence was confirmed by western blot analysis and mass spectrometry. CONCLUSION: Anti-TNF-α scFv has been successfully purified in a salt inducible system GJ1158. As per the best of our knowledge, this is the first report of purification of Anti-TNF-α scFv in a salt inducible system from soluble fractions as well as inclusion bodies.

Alleviation of diabetes mellitus through the restoration of ß-cell function and lipid metabolism by Aloe vera (L.) Burm. f. extract in obesogenic WNIN/GR-Ob rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Aloe vera (L.) Burm. f. extract has been medicinally used for over 5000 years in different cultures for its curative and therapeutic properties ranging from dermatitis to diabetes. It has been demonstrated to alleviate diabetes through its protective effects on pancreatic islets and by improving insulin secretion. AIM OF THE STUDY: To investigate the simultaneous effect of ethanolic A. vera gel extract on diabetes and obesogenic milieu in Streptozotocin-induced WNIN/GR-Ob mutant obese rats. MATERIALS AND METHODS: A total of 30 rats were grouped equally into WNIN/GR-Ob control (received water as a vehicle), WNIN/GR-Ob Diabetic rats (Streptozotocin-35 mg/kg bw), WNIN/GR-Ob Diabetic rats + Sitagliptin (10 mg/kg bw), WNIN/GR-Ob Diabetic rats + A. vera (300 mg/kg bw) and GR-Ob control + A. vera (300 mg/kg bw). After 4 weeks of treatment, fasting blood glucose, serum insulin, Homeostatic Model Assessment - Insulin Resistance and ß-cell function, glucose-stimulated insulin secretion, Dipeptidyl peptidase-IV activity, and lipid profiles were studied. In addition, ultrastructural analysis of isolated islets and dual-energy X-ray absorptiometry analysis for body composition were also carried out. RESULTS: The A. vera treated group showed a significant reduction (p < 0.05) in triglyceride, Very low-density lipoprotein levels, Triglyceride to High-density lipoprotein ratio as well as fasting blood glucose levels and DPP-IV activity with a concomitant increase in the serum insulin levels. The increase in IR was observed in both WNIN/GR-Ob control and diabetic rats with a significant decrease in ß-cell function in the diabetic rats as per Homeostatic Model Assessment values. Oral administration of A. vera was effective in both reducing Homeostatic Model Assessment-Insulin Resistance and increasing Homeostatic Model Assessment-ß values. Also, the treated group demonstrated preservation of islets and a significant increase (p < 0.05) in the diameter of ß-cell as evident through Scanning electron microscope analysis. The increase in lean body mass was manifested in the treated group with a reduction in Fat percent in comparison with other groups. CONCLUSION: The beneficial effects of A. vera in WNIN/GR-Ob strain may be attributed to its ability to lower lipid profile thus improve insulin sensitivity and/or modulating ß-cell function. Thus, it has great therapeutic potential as an herbal remedy for the treatment of diabetes and associated adverse effects such as obesity. The exact mechanism underlying the observation needs to be investigated further to explore the anti-obesity and anti-diabetic properties of A. vera and advocate its potential application as alternative medicine.