Chloroquine and cytosolic galectins affect endosomal escape of antisense oligonucleotides after Stabilin-mediated endocytosis

Non-DNA-binding Stabilin-2/HARE receptors expressed on liver sinusoidal endothelial cells specifically bind to and internalize several classes of phosphorothioate antisense oligonucleotides (PS-ASOs). After Stabilin-mediated uptake, PS-ASOs are trafficked within endosomes (\u3e97%–99%), ultimately resulting in destruction in the lysosome. The ASO entrapment in endosomes lowers therapeutic efficacy, thereby increasing the overall dose for patients. Here, we use confocal microscopy to characterize the intracellular route transverse by PS-ASOs after Stabilin receptor-mediated uptake in stable recombinant Stabilin-1 and -2 cell lines. We found that PS-ASOs as well as the Stabilin-2 receptor transverse the classic path: clathrincoated vesicle-early endosome-late endosome-lysosome. Chloroquine exposure facilitated endosomal escape of PS-ASOs leading to target knockdown by more than 50% as compared to untreated cells, resulting in increased PS-ASO efficacy. We also characterize cytosolic galectins as novel contributor for PS-ASO escape. Galectins knockdown enhances ASO efficacy by more than 60% by modulating EEA1, Rab5C, and Rab7A mRNA expression, leading to a delay in the endosomal vesicle maturation process. Collectively, our results provide additional insight for increasing PS-ASO efficacy by enhancing endosomal escape, which can further be utilized for other nucleic acid-based modalities

ON EXTENSION OF MITTAG-LEFFLER FUNCTION

In this paper, we study the extended Mittag -Leffler function by using generalized beta function and obtain various differential properties, integral representations. Further, we discuss Mellin transform of these functions in terms of generalized Wright hyper geometric function and evaluate Laplace transform, and Whittaker transform in terms of extended beta function. Finally, several interesting special cases of extended Mittag -Leffler functions have also be given

Impact of Janani SurakshaYojana (JSY): A study across two Delhi hospitals

This paperattemptsto understand the functioning of Janani Suraksha Yojana (JSY) in New Delhi. The study involved a primary survey of the JSY beneficiaries in two major districts of Delhi. The results of the survey suggest that the scheme has not been very successful in facilitating institutional delivery in Delhi.The scheme has failed to benefit the poor and the underprivileged groups and its limited benefits have only reached the better off households. The implementation of the scheme also has been uneven and faulty and many beneficiaries face delays in receiving benefits. However, the transition towards the direct benefit transfer (DBT) method of payment shows promise in improving implementation of the scheme

\u3ci\u3eAkkermansia muciniphila\u3c/i\u3e and its membrane protein ameliorates intestinal inflammatory stress and promotes epithelial wound healing via CREBH and miR‑143/145

Background The intestinal epithelial barrier is the interface for interaction between gut microbiota and host metabolic systems. Akkermansia muciniphila (A. muciniphila) is a key player in the colonic microbiota that resides in the mucus layer, whose abundance is selectively decreased in the faecal microbiota of inflammatory bowel disease (IBD) patients. This study aims to investigate the regulatory mechanism among A. muciniphila, a transcription factor cAMPresponsive element-binding protein H (CREBH), and microRNA-143/145 (miR-143/145) in intestinal inflammatory stress, gut barrier integrity and epithelial regeneration. Methods A novel mouse model with increased colonization of A muciniphila in the intestine of CREBH knockout mice, an epithelial wound healing assay and several molecular biological techniques were applied in this study. Results were analysed using a homoscedastic 2-tailed t-test. Results Increased colonization of A. muciniphila in mouse gut enhanced expression of intestinal CREBH, which was associated with the mitigation of intestinal endoplasmic reticulum (ER) stress, gut barrier leakage and blood endotoxemia induced by dextran sulfate sodium (DSS). Genetic depletion of CREBH (CREBH-KO) significantly inhibited the expression of tight junction proteins that are associated with gut barrier integrity, including Claudin5 and Claudin8, but upregulated Claudin2, a tight junction protein that enhances gut permeability, resulting in intestinal hyperpermeability and inflammation. Upregulation of CREBH by A. muciniphila further coupled with miR-143/145 promoted intestinal epithelial cell (IEC) regeneration and wound repair via insulin-like growth factor (IGF) and IGFBP5 signalling. Moreover, the gene expressing an outer membrane protein of A. muciniphila, Amuc_1100, was cloned into a mammalian cell-expression vector and successfully expressed in porcine and human IECs. Expression of Amuc_1100 in IECs could recapitulate the health beneficial effect of A. muciniphila on the gut by activating CREBH, inhibiting ER stress and enhancing the expression of genes involved in gut barrier integrity and IEC’s regeneration. Conclusions This study uncovers a novel mechanism that links A. muciniphila and its membrane protein with host CREBH, IGF signalling and miRNAs in mitigating intestinal inflammatory stress–gut barrier permeability and promoting intestinal wound healing. This novel finding may lend support to the development of therapeutic approaches for IBD by manipulating the interaction between host genes, gut bacteria and its bioactive components

Stabilin receptors clear LPS and control systemic inflammation

Lipopolysaccharides (LPSs) cause lethal endotoxemia if not rapidly cleared from blood circulation. Liver sinusoidal endothelial cells (LSEC) systemically clear LPS by unknown mechanisms. We discovered that LPS clearance through LSEC involves endocytosis and lysosomal inactivation via Stabilin-1 and 2 (Stab1 and Stab2) but does not involve TLR4. Cytokine production was inversely related to clearance/endocytosis of LPS by LSEC. When exposed to LPS, Stabilin double knockout mice (Stab DK) and Stab1 KO, but not Stab2 KO, showed significantly enhanced systemic inflammatory cytokine production and early death compared with WT mice. Stab1 KO is not significantly different from Stab DK in circulatory LPS clearance, LPS uptake and endocytosis by LSEC, and cytokine production. These data indicate that (1) Stab1 receptor primarily facilitates the proactive clearance of LPS and limits TLR4-mediated inflammation and (2) TLR4 and Stab1 are functionally opposing LPS receptors. These findings suggest that endotoxemia can be controlled by optimizing LPS clearance by Stab1

Photosensitized INA-Labelled protein 1 (PhIL1) is novel component of the inner membrane complex and is required for Plasmodium parasite development.

Plasmodium parasites, the causative agents of malaria, possess a distinctive membranous structure of flattened alveolar vesicles supported by a proteinaceous network, and referred to as the inner membrane complex (IMC). The IMC has a role in actomyosin-mediated motility and host cell invasion. Here, we examine the location, protein interactome and function of PhIL1, an IMC-associated protein on the motile and invasive stages of both human and rodent parasites. We show that PhIL1 is located in the IMC in all three invasive (merozoite, ookinete-, and sporozoite) stages of development, as well as in the male gametocyte and locates both at the apical and basal ends of ookinete and sporozoite stages. Proteins interacting with PhIL1 were identified, showing that PhIL1 was bound to only some proteins present in the glideosome motor complex (GAP50, GAPM1-3) of both P. falciparum and P. berghei. Analysis of PhIL1 function using gene targeting approaches indicated that the protein is required for both asexual and sexual stages of development. In conclusion, we show that PhIL1 is required for development of all zoite stages of Plasmodium and it is part of a novel protein complex with an overall composition overlapping with but different to that of the glideosome

Investigating the Intracellular Trafficking of Antisense Oligonucleotide After Stabilin Receptor Mediated Endocytosis and Finding Novel Strategies to Enhance Its Endosomal Escape

Phosphorothioate antisense oligonucleotides (PS-ASOs) are internalized inside the cells via various receptor mediated endocytic pathways. Previous studies have reported that non-DNA binding Stabilin2/HARE receptor present on endothelial cells specifically binds to and internalize several classes of ASOs. However, once internalized by Stabilin receptor, ASOs are trafficked within endosomes (\u3e99%), ultimately resulting in destruction in the lysosome. The ASO entrapment in endosomes is considered a bottleneck in the oligonucleotide therapeutic advancement. As a first step towards solving endosomal entrapment problem, we first characterized the intracellular route transversed by ASOs after Stabilin receptor mediated uptake. We found that PS-ASO as well as Stabilin-2 receptor follow the classical path: Clathrin coated vesicle \u3e early endosome \u3e late endosome \u3e lysosome. However, Stabilin-1 traffick the PS-ASO via different route after clathrin mediated endocytosis which needs further investigation. Next, we investigated different strategies to enhance endosomal escape of PS-ASO after Stabilin-2 receptor mediated endocytosis. First, we evaluated a small molecule, Chloroquine, as an effective strategy for promoting the release of PS-ASO from endosomes. Chloroquine effectively enhanced the activity of PS-ASO in Stabilin-2 overexpressing cell lines. We also established its dominant role in enhancing PS-ASO escape as compared to other important endosome associated proteins. Next, we identified a novel role of galectin’s in enhancing endosomal escape of PS-ASO in Stabilin-2 cell line. Till date, galectins have been used as an imaging sensor for visualizing endosomal escape of siRNA. In our study, we showed that galectin knockdown efficiently enhances activity of PS-ASO by 50-70%. Galectin-1 modulated the expression of endosomal associated gene. This observation led us to hypothesize that galectin-1 affect endosomal vesicle maturation which could be a possible mechanism for how galectins are enhancing PS-ASO activity. This study brings us one step closer towards solving endosomal entrapment problem of PS-ASOs. Enhancing endosomal escape will result in lower drug administration, which will reduce the toxicity, thereby making PS-ASO drugs more cost-effective

Chloroquine and cytosolic galectins affect endosomal escape of antisense oligonucleotides after Stabilin-mediated endocytosis

Non-DNA-binding Stabilin-2/HARE receptors expressed on liver sinusoidal endothelial cells specifically bind to and internalize several classes of phosphorothioate antisense oligonucleotides (PS-ASOs). After Stabilin-mediated uptake, PS-ASOs are trafficked within endosomes (>97%–99%), ultimately resulting in destruction in the lysosome. The ASO entrapment in endosomes lowers therapeutic efficacy, thereby increasing the overall dose for patients. Here, we use confocal microscopy to characterize the intracellular route transverse by PS-ASOs after Stabilin receptor-mediated uptake in stable recombinant Stabilin-1 and -2 cell lines. We found that PS-ASOs as well as the Stabilin-2 receptor transverse the classic path: clathrin-coated vesicle-early endosome-late endosome-lysosome. Chloroquine exposure facilitated endosomal escape of PS-ASOs leading to target knockdown by more than 50% as compared to untreated cells, resulting in increased PS-ASO efficacy. We also characterize cytosolic galectins as novel contributor for PS-ASO escape. Galectins knockdown enhances ASO efficacy by more than 60% by modulating EEA1, Rab5C, and Rab7A mRNA expression, leading to a delay in the endosomal vesicle maturation process. Collectively, our results provide additional insight for increasing PS-ASO efficacy by enhancing endosomal escape, which can further be utilized for other nucleic acid-based modalities

Role of alcoholic extract of shoot of <i>Hypericum perforatum </i>Linn on lipid peroxidation and various species of free radicals in rats

567-571The alcoholic extract of the shoot of H. perforatum shows strong antioxidant property. It possesses the iron chelation property with more affinity to the ferrous form. It has scavenging property for both superoxide and for hydroxyl radicals but the response is more towards the superoxide radicals. Thus in addition to the anti-depressant property it has strong antioxidant property also

Aggregation in oxidation of aspartic and glutamic acids by chloramine-T in presence of surfactants: A kinetic study

1186-1192<span style="font-size:14.0pt;line-height:115%; font-family:" times="" new="" roman";mso-fareast-font-family:"times="" roman";="" mso-ansi-language:en-in;mso-fareast-language:en-in;mso-bidi-language:hi"="">The kinetics of oxidation of aspartic and glutamic acids by chloramine-Tin HClO4 medium have been investigated in the absence and presence of anionic (sodium lauryl sulphate), cationic (cetyltrimethylammonium bromide) and non-ionic (Brij 35) surfactants.</span