Akt may associate with insulin-responsive vesicles via interaction with sortilin.

Insulin-responsive vesicles (IRVs) deliver the glucose transporter Glut4 to the plasma membrane in response to activation of the insulin signaling cascade: insulin receptor-IRS-PI3 kinase-Akt-TBC1D4-Rab10. Previous studies have shown that Akt, TBC1D4, and Rab10 are compartmentalized on the IRVs. Although functionally significant, the mechanism of Akt association with the IRVs remains unknown. Using pull-down assays, immunofluorescence microscopy, and cross-linking, we have found that Akt may be recruited to the IRVs via the interaction with the juxtamembrane domain of the cytoplasmic C terminus of sortilin, a major IRV protein. Overexpression of full-length sortilin increases insulin-stimulated phosphorylation of TBC1D4 and glucose uptake in adipocytes, while overexpression of the cytoplasmic tail of sortilin has the opposite effect. Our findings demonstrate that the IRVs represent both a scaffold and a target of insulin signaling.

The BACH1 inhibitor ASP8731 inhibits inflammation and vaso-occlusion and induces fetal hemoglobin in sickle cell disease.

In sickle cell disease (SCD), heme released during intravascular hemolysis promotes oxidative stress, inflammation, and vaso-occlusion. Conversely, free heme can also activate expression of antioxidant and globin genes. Heme binds to the transcription factor BACH1, which represses NRF2-mediated gene transcription. ASP8731, is a selective small molecule inhibitor of BACH1. We investigated the ability of ASP8731 to modulate pathways involved in SCD pathophysiology. In HepG2 liver cells, ASP8731 increased HMOX1 and FTH1 mRNA. In pulmonary endothelial cells, ASP8731 decreased VCAM1 mRNA in response to TNF-α and blocked a decrease in glutathione in response to hemin. Townes-SS mice were gavaged once per day for 4 weeks with ASP8731, hydroxyurea (HU) or vehicle. Both ASP8731 and HU inhibited heme-mediated microvascular stasis and in combination, ASP8731 significantly reduced microvascular stasis compared to HU alone. In Townes-SS mice, ASP8731 and HU markedly increased heme oxygenase-1 and decreased hepatic ICAM-1, NF-kB phospho-p65 protein expression in the liver, and white blood cell counts. In addition, ASP8731 increased gamma-globin expression and HbF+ cells (F-cells) as compared to vehicle-treated mice. In human erythroid differentiated CD34+ cells, ASP8731 increased HGB mRNA and increased the percentage of F-cells 2-fold in manner similar to HU. ASP8731 and HU when given together induced more HbF+ cells compared to either drug alone. In CD34+ cells from one donor that was non-responsive to HU, ASP8731 induced HbF+ cells ~2-fold. ASP8731 and HU also increased HBG and HBA, but not HBB mRNA in erythroid differentiated CD34+ cells derived from SCD patients. These data indicate that BACH1 may offer a new therapeutic target to treat SCD.

Twitter sentiment analysis for COVID-19 associated mucormycosis.

Purpose: COVID-19-associated mucormycosis (CAM) was a serious public health problem during the second wave of COVID-19 in India. We planned to analyze public perceptions by sentiment analysis of Twitter data regarding CAM. Methods: In this observational study, the application programming interface (API) provided by the Twitter platform was used for extracting real-time conversations by using keywords related to mucormycosis (colloquially known as "black fungus"), from May 3 to August 29, 2021. Lexicon-based sentiment analysis of the tweets was done using the Vader sentiment analysis tool. To identify the overall sentiment of a user on any given topic, an algorithm to label a user "k" based on their sentiments was used. Results: A total of 4,01,037 tweets were collected between May 3 and August 29, 2021, and the peak frequency of 1,60,000 tweets was observed from May 17 to May 23, 2021. Positive sentiment tweets constituted a larger share as compared to negative sentiment tweets, with weekly variations. A temporal analysis of the demand for utilities showed that the demand was high in the initial period but decreased with time, which was associated with the availability of resources. Conclusion: Sentiment analysis using Twitter data revealed that social media platforms are gaining popularity to express one's emotions during the ongoing COVID-19 pandemic. In our study, time-based assessment of tweets showed a reduction over time in the frequency of negative sentiment tweets. The polarization in the retweet network of users, based on sentiment polarity, showed that the users were well connected, highlighting the fact that such issues bond our society rather than segregating it.

DeriveNet for (Very) Low Resolution Image Classification.

Images captured from a distance often result in (very) low resolution (VLR/LR) region of interest, requiring automated identification. VLR/LR images (or regions of interest) often contain less information content, rendering ineffective feature extraction and classification. To this effect, this research proposes a novel DeriveNet model for VLR/LR classification, which focuses on learning effective class boundaries by utilizing the class-specific domain knowledge. DeriveNet model is jointly trained via two losses: (i) proposed Derived-Margin softmax loss and (ii) the proposed Reconstruction-Center (ReCent) loss. The Derived-Margin softmax loss focuses on learning an effective VLR classifier while explicitly modeling the inter-class variations. The ReCent loss incorporates domain information by learning a HR reconstruction space for approximating the class variations for the VLR/LR samples. It is utilized to derive inter-class margins for the Derived-Margin softmax loss. The DeriveNet model has been trained with a novel Multi-resolution Pyramid based data augmentation which enables the model to learn from varying resolutions during training. Experiments and analysis have been performed on multiple datasets for (i) VLR/LR face recognition, (ii) VLR digit classification, and (iii) VLR/LR face recognition from drone-shot videos. The DeriveNet model achieves state-of-the-art performance across different datasets, thus promoting its utility for several VLR/LR classification tasks.

Morality-Based Assertion and Homophily on Social Media: A Cultural Comparison Between English and Japanese Languages.

Moral psychology is a domain that deals with moral identity, appraisals and emotions. Previous work has primarily focused on moral development and the associated role of culture. Knowing that language is an inherent element of a culture, we used the social media platform Twitter to compare moral behaviors of Japanese tweets with English tweets. The five basic moral foundations, i.e., Care, Fairness, Ingroup, Authority, and Purity, along with the associated emotional valence were compared between English and Japanese tweets. The tweets from Japanese users depicted relatively higher Fairness, Ingroup, and Purity, whereas English tweets expressed more positive emotions for all moral dimensions. Considering moral similarities in connecting users on social media, we quantified homophily concerning different moral dimensions using our proposed method. The moral dimensions Care, Authority, and Purity for English and Ingroup, Authority and Purity for Japanese depicted homophily on Twitter. Overall, our study uncovers the underlying cultural differences with respect to moral behavior in English- and Japanese-speaking users.

Author Correction: Mesenchymal Stromal Cell Bioreactor for Ex Vivo Reprogramming of Human Immune Cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mesenchymal Stromal Cell Bioreactor for Ex Vivo Reprogramming of Human Immune Cells.

Bone marrow mesenchymal stromal cells (MSCs) have been studied for decades as potent immunomodulators. Clinically, they have shown some promise but with limited success. Here, we report the ability of a scalable hollow fiber bioreactor to effectively maintain ideal MSC function as a single population while also being able to impart an immunoregulatory effect when cultured in tandem with an inflamed lymphocyte population. MSCs were seeded on the extraluminal side of hollow fibers within a bioreactor where they indirectly interact with immune cells flowing within the lumen of the fibers. MSCs showed a stable and predictable metabolite and secreted factor profile during several days of perfusion culture. Exposure of bioreactor-seeded MSCs to inflammatory stimuli reproducibly switched MSC secreted factor profiles and altered microvesicle composition. Furthermore, circulating, activated human peripheral blood mononuclear cells (PBMCs) were suppressed by MSC bioreactor culture confirmed by a durable change in their immunophenotype and function. This platform was useful to study a model of immobilized MSCs and circulating immune cells and showed that monocytes play an important role in MSC driven immunomodulation. This coculture technology can have broad implications for use in studying MSC-immune interactions under flow conditions as well as in the generation of ex vivo derived immune cellular therapeutics.

Identification of genomic regions associated with early plant vigour in lentil (Lens culinaris).

Lentil is one of the most important food legume species, however its genetic and genomic resources remained largely uncharacterized and unexploited. In the past few years, a number of genetic maps have been constructed and marker resources have been developed in lentil. These resources could be exploited for understanding the extent and distribution of genetic variation in genus Lens and also for developing saturated and consensus genetic maps suitable for quantitative trait loci (QTL) mapping and marker-assisted selection. The present study aims to enrich polymerase chain reaction-based linkage map of F10 recombinant inbred lines (RILs) population of 94 individuals derived from cross WA8649090 9 Precoz and identification of QTLs linked to early plant vigour traits. Of the 268 polymorphic markers (93 simple sequence repeats (SSR), three inter-simple sequence repeats (ISSRs) and 172 random amplified polymorphic DNA (RAPDs)), 265 (90 SSRs, three ISSRs and 172 RAPDs) were mapped on seven linkage groups, varying in length between 25.6 and 210.3 cM, coverage of 809.4 cM with an average marker spacing of 3.05 cM. The study also reported assigning of 24 new cross-genera SSRs of Trifolium pratense on the present linkage map. The RILs along with the parents were screened for shoot length, root length, seedling length, dry weight, number of leaves and number of branches based on two replications under polyhouse conditions. A QTLhotspot consisting of six QTLs for shoot length (cm), root length (cm) and seedling length (cm) was observed between a map distances of56.61 and 86.81 cM on LG1.

Sortilin and retromer mediate retrograde transport of Glut4 in 3T3-L1 adipocytes.

Sortilin is a multiligand sorting receptor responsible for the anterograde transport of lysosomal enzymes and substrates. Here we demonstrate that sortilin is also involved in retrograde protein traffic. In cultured 3T3-L1 adipocytes, sortilin together with retromer rescues Glut4 from degradation in lysosomes and retrieves it to the TGN, where insulin--responsive vesicles are formed. Mechanistically, the luminal Vps10p domain of sortilin interacts with the first luminal loop of Glut4, and the cytoplasmic tail of sortilin binds to retromer. Ablation of the retromer does not affect insulin signaling but decreases the stability of sortilin and Glut4 and blocks their entry into the small vesicular carriers. As a result, Glut4 cannot reach the insulin-responsive compartment, and insulin-stimulated glucose uptake in adipocytes is suppressed. We suggest that sortilin- and retromer-mediated Glut4 retrieval from endosomes may represent a step in the Glut4 pathway vulnerable to the development of insulin resistance and diabetes.

4E-BPs Control Fat Storage by Regulating the Expression of Egr1 and ATGL.

Early growth response transcription factor Egr1 controls multiple aspects of cell physiology and metabolism. In particular, Egr1 suppresses lipolysis and promotes fat accumulation in adipocytes by inhibiting the expression of adipose triglyceride lipase. According to current dogma, regulation of the Egr1 expression takes place primarily at the level of transcription. Correspondingly, treatment of cultured adipocytes with insulin stimulates expression of Egr1 mRNA and protein. Unexpectedly, the MEK inhibitor PD98059 completely blocks insulin-stimulated increase in the Egr1 mRNA but has only a moderate effect on the Egr1 protein. At the same time, mTORC1 inhibitors rapamycin and PP242 suppress expression of the Egr1 protein and have an opposite effect on the Egr1 mRNA. Mouse embryonic fibroblasts with genetic ablations of TSC2 or 4E-BP1/2 express less Egr1 mRNA but more Egr1 protein than wild type controls. (35)S-labeling has confirmed that translation of the Egr1 mRNA is much more effective in 4E-BP1/2-null cells than in control. A selective agonist of the CB1 receptors, ACEA, up-regulates Egr1 mRNA, but does not activate mTORC1 and does not increase Egr1 protein in adipocytes. These data suggest that although insulin activates both the Erk and the mTORC1 signaling pathways in adipocytes, regulation of the Egr1 expression takes place predominantly via the mTORC1/4E-BP-mediated axis. In confirmation of this model, we show that 4E-BP1/2-null MEFs express less ATGL and accumulate more fat than control cells, while knock down of Egr1 in 4E-BP1/2-null MEFs increases ATGL expression and decreases fat storage.

Fat-specific protein 27 inhibits lipolysis by facilitating the inhibitory effect of transcription factor Egr1 on transcription of adipose triglyceride lipase.

Lipolysis in fat tissue represents a major source of circulating fatty acids. Previously, we have found that lipolysis in adipocytes is controlled by early growth response transcription factor Egr1 that directly inhibits transcription of adipose triglyceride lipase, ATGL (Chakrabarti, P., Kim, J. Y., Singh, M., Shin, Y. K., Kim, J., Kumbrink, J., Wu, Y., Lee, M. J., Kirsch, K. H., Fried, S. K., and Kandror, K. V. (2013) Mol. Cell. Biol. 33, 3659-3666). Here we demonstrate that knockdown of the lipid droplet protein FSP27 (a.k.a. CIDEC) in human adipocytes increases expression of ATGL at the level of transcription, whereas overexpression of FSP27 has the opposite effect. FSP27 suppresses the activity of the ATGL promoter in vitro, and the proximal Egr1 binding site is responsible for this effect. FSP27 co-immunoprecipitates with Egr1 and increases its association with and inhibition of the ATGL promoter. Knockdown of Egr1 attenuates the inhibitory effect of FSP27. These results provide a new model of transcriptional regulation of ATGL.

Insulin responsiveness of glucose transporter 4 in 3T3-L1 cells depends on the presence of sortilin.

Insulin-dependent translocation of glucose transporter 4 (Glut4) to the plasma membrane of fat and skeletal muscle cells plays the key role in postprandial clearance of blood glucose. Glut4 represents the major cell-specific component of the insulin-responsive vesicles (IRVs). It is not clear, however, whether the presence of Glut4 in the IRVs is essential for their ability to respond to insulin stimulation. We prepared two lines of 3T3-L1 cells with low and high expression of myc7-Glut4 and studied its translocation to the plasma membrane upon insulin stimulation, using fluorescence-assisted cell sorting and cell surface biotinylation. In undifferentiated 3T3-L1 preadipocytes, translocation of myc7-Glut4 was low regardless of its expression levels. Coexpression of sortilin increased targeting of myc7-Glut4 to the IRVs, and its insulin responsiveness rose to the maximal levels observed in fully differentiated adipocytes. Sortilin ectopically expressed in undifferentiated cells was translocated to the plasma membrane regardless of the presence or absence of myc7-Glut4. AS160/TBC1D4 is expressed at low levels in preadipocytes but is induced in differentiation and provides an additional mechanism for the intracellular retention and insulin-stimulated release of Glut4.

Insulin inhibits lipolysis in adipocytes via the evolutionarily conserved mTORC1-Egr1-ATGL-mediated pathway.

One of the basic functions of insulin in the body is to inhibit lipolysis in adipocytes. Recently, we have found that insulin inhibits lipolysis and promotes triglyceride storage by decreasing transcription of adipose triglyceride lipase via the mTORC1-mediated pathway (P. Chakrabarti et al., Diabetes 59:775-781, 2010), although the mechanism of this effect remained unknown. Here, we used a genetic screen in Saccharomyces cerevisiae in order to identify a transcription factor that mediates the effect of Tor1 on the expression of the ATGL ortholog in yeast. This factor, Msn4p, has homologues in mammalian cells that form a family of early growth response transcription factors. One member of the family, Egr1, is induced by insulin and nutrients and directly inhibits activity of the ATGL promoter in vitro and expression of ATGL in cultured adipocytes. Feeding animals a high-fat diet increases the activity of mTORC1 and the expression of Egr1 while decreasing ATGL levels in epididymal fat. We suggest that the evolutionarily conserved mTORC1-Egr1-ATGL regulatory pathway represents an important component of the antilipolytic effect of insulin in the mammalian organism.

In silico modeling of ligand molecule for non structural 3 (NS3) protein target of flaviviruses.

UNLABELLED: Flaviviruses are small, enveloped RNA viruses which cause a variety of diseases into animals and man. Despite the existence of licensed vaccines, yellow fever, Japanese encephalitis and tick-borne encephalitis also claim many thousands of victims each year across their vast endemic areas. A number of studies have already revealed that the non-structural NS3 serine protease is required for the maturation of the viral polyprotein and thus is a promising target for the development of antiviral inhibitors. Hence, the 3D structure of NS3 protein was modeled using homology modeling by MODELLER 9v7. Validation of the constructed NS3 protein models were done by PROCHECK, VERYFY3D and through ProSA calculations. Ligands for the catalytic triad (H51, D75, and S135) were designed using LIGBUILDER. The NS3 protein's catalytic triad was explored to find out the interactions pattern for inhibitor binding using molecular docking methodology using AUTODOCK Vina. The interactions of complex NS3protein-ligand conformations, including hydrogen bonds and the bond lengths were analyzed using Accelrys DS Visualizer software. Hence, from this observation, the novel molecule designed was observed to be the best ligand against the NS3 protein of flavivirus. This molecule may prove to be a potential identity in modulating disease manifestation for all the selected flavivirus members. ABBREVIATIONS: NCBI - National Centre for Biotechnological Information, BLAST - Basic Local Alignment Search Tool, DOPE - Discrete optimized protein energy, GROMOS96 - GROningen MOlecular Simulation package, SAVS - Structure Analysis and Validation Server.

Homeodomain transcription factors regulate BMP-2-induced osteoactivin transcription in osteoblasts.

Osteoactivin (OA) is required for the differentiation of osteoblast cells. OA expression is stimulated by bone morphogenetic protein-2 (BMP-2). BMP-2 recruits homeodomain transcription factors Dlx3, Dlx5, and Msx2 to selectively activate or repress transcription of osteogenic genes and hence tightly regulate their transcription during osteoblast differentiation. Considering the key roles of Dlx3, Dlx5, and Msx2 in osteoblast differentiation, here we hypothesize that homeodomain proteins regulate BMP-2-induced OA transcription during osteoblast differentiation. Four classical homeodomain binding sites were identified in the proximal 0.96 kb region of rat OA promoter. Deletions and mutagenesis studies of the OA promoter region indicated that all four homeodomain binding sites are crucial for BMP-2-induced OA promoter activity. Simultaneous disruption of homeodomain binding sites at -852 and -843 of the transcription start site of OA gene significantly decreased the BMP-2-induced OA transcription and inhibited binding of Dlx3, Dlx5, and Msx2 proteins to the OA promoter. Dlx3 and Dlx5 proteins were found to activate the OA transcription, whereas, Msx2 suppressed BMP-2-induced OA transcription. Using chromatin immunoprecipitation assays, we demonstrated that the OA promoter is predominantly occupied by Dlx3 and Dlx5 during the proliferation and matrix maturation stages of osteoblast differentiation, respectively. During the matrix mineralization stage, BMP-2 robustly enhanced the recruitment of Dlx5 and to a lesser extent of Dlx3 and Msx2 to the OA promoter region. Collectively, our results show that the BMP-2-induced OA transcription is differentially regulated by Dlx3, Dlx5, and Msx2 during osteoblast differentiation.

A comprehensive molecular interaction map for Hepatitis B virus and drug designing of a novel inhibitor for Hepatitis B X protein.

Hepatitis B virus (HBV) infection is a leading source of liver diseases such as hepatitis, cirrhosis and hepatocellular carcinoma. In this study, we use computation methods in order to improve our understanding of the complex interactions that occur between molecules related to Hepatitis B virus (HBV). Due to the complexity of the disease and the numerous molecular players involved, we devised a method to construct a systemic network of interactions of the processes ongoing in patients affected by HBV. The network is based on high-throughput data, refined semi-automatically with carefully curated literature-based information. We find that some nodes in the network that prove to be topologically important, in particular HBx is also known to be important target protein used for the treatment of HBV. Therefore, HBx protein is the preferential choice for inhibition to stop the proteolytic processing. Hence, the 3D structure of HBx protein was downloaded from PDB. Ligands for the active site were designed using LIGBUILDER. The HBx protein's active site was explored to find out the critical interactions pattern for inhibitor binding using molecular docking methodology using AUTODOCK Vina. It should be noted that these predicted data should be validated using suitable assays for further consideration.

Annotation of hypothetical proteins orthologous in Pongo abelii and Sus scrofa.

UNLABELLED: A hypothetical protein is predicted to be expressed from an open reading frame without known experimental evidence of translation. They constitute a substantial fraction of proteomes. Domain extraction from these hypothetical sequences helps to search for protein coding genes for protein structural and functional annotation. We describe the analysis of prediction data in a sequence dataset of hypothetical protein orthologs of Pongo abelii (orangutan) and Sus scrofa (pig). It should be noted that these orangutan-pig orthologs are also non-homologous to human proteins. These predicted data find application in the genome wide annotation of proteins in poorly understood genomes. ABBREVIATIONS: PDB - Protein Data Bank, DEG - Database of Essential Genes, CDD - Conserved Domain Database, IUCN - International Union for Conservation of Nature.

Functional roles of osteoactivin in normal and disease processes.

Osteoactivin (OA) protein was discovered in bone cells a decade ago. Recent literature suggests that osteoactivin is crucial for the differentiation and functioning of different cell types, including bone-forming osteoblasts and bone-resorbing osteoclast cells. Here, we review the literature to date on various regulatory functions of osteoactivin, as well as its discovery, structure, expression, and function in different tissues and cells. The transcriptional regulation of osteoactivin and its mechanism of action in normal and diseased conditions with special emphasis on bone are also covered in this review. In addition, we touch on the therapeutic potential of osteoactivin in cancer and bone diseases.