CCADD: An online webserver for Alzheimer's disease detection from brain MRI.

Alzheimer's disease (AD) imposes a growing burden on public health due to its impact on memory, cognition, behavior, and social skills. Early detection using non-invasive brain magnetic resonance images (MRI) is vital for disease management. We introduce CCADD (Corpus Callosum-based Alzheimer's Disease Detection), a user-friendly webserver that automatically identifies and segments the corpus callosum (CC) region from brain MRI slices. Extracted shape and size-based features of CC are fed into Support Vector Machines (SVM), Random Forest (RF), eXtreme Gradient Boosting (XGBoost), K-Nearest Neighbor (KNN), and Artificial Neural Network (ANN) classifiers to predict AD or Mild Cognitive Impairment (MCI). Exhaustive benchmarking on ADNI data reveals high prediction accuracies for different AD severity levels. CCADD empowers clinicians and researchers for AD detection. This server is available at: http://www.hpppi.iicb.res.in/add.

EIF4A3 targeted therapeutic intervention in glioblastoma multiforme using phytochemicals from Indian medicinal plants - an integration of phytotherapy into precision onco-medicine.

Glioblastoma Multiforme (GBM), an aggressive brain tumor (grade-IV astrocytoma), poses treatment challenges. Poor prognosis results from the rapid growth, highlighting the role of EIF4A3 in regulating non-coding RNAs. EIF4A3 promotes the expression of several non-coding RNAs, viz, Circ matrix metallopeptidase 9 (MMP9), a prominent oncogene, by interacting with the upstream region of the circMMP9 mRNA transcript and acts on cell proliferation, migration, and invasion of GBM. However, research shows that EIF4A3 knockdown inhibits glioblastoma progression and increases apoptosis. In this study, we explored the efficiency of the phytochemicals from plants like Withania somnifera and Castanea sativa with potential anti-glioblastoma effects as obtained from the Indian Medicinal Plants, Phytochemistry and Therapeutics (IMPPAT) database. Consequently, we have performed a virtual screening of the compounds against the protein EIF4A3. We further investigated the efficiency of the shortlisted compounds based on docking scores evaluated using GOLD, AutoDock4.2, LeDock, and binding free energy analyses using Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA). Among the phytochemicals studied so far, several Withania-specific compounds from Withania somnifera and a single dietary compound, viz., Thiamine from Castanea sativa, have exhibited comparatively good blood-brain barrier permeability, significant binding affinity towards the EIF4A3, and good ADMET properties. Furthermore, we have verified the interaction stability of the lead molecules with EIF4A3 using MD simulations. Thus, the present study offers an opportunity to develop drug candidates targeting glioblastoma caused by EIF4A3 over-expression, integrating phytotherapy into precision oncology to create tailored and precise natural treatment strategies for cancer.Communicated by Ramaswamy H. Sarma.

CMT2A-linked MFN2 mutation, T206I promotes mitochondrial hyperfusion and predisposes cells towards mitophagy.

Mutations in Mitofusin2 (MFN2) associated with the pathology of the debilitating neuropathy Charcot-Marie-Tooth type 2A (CMT2A) are known to alter mitochondrial morphology. Previously, such mutations have been shown to elicit two diametrically opposite phenotypes - while some mutations have been causally linked to enhanced mitochondrial fragmentation, others have been shown to induce hyperfusion. Our study identifies one such MFN2 mutant, T206I that causes mitochondrial hyperfusion. Cells expressing this MFN2 mutant have elongated and interconnected mitochondria. T206I-MFN2 mutation in the GTPase domain increases MFN2 stability and renders cells susceptible to stress. We show that cells expressing T206I-MFN2 have a higher predisposition towards mitophagy under conditions of serum starvation. We also detect increased DRP1 recruitment onto the outer mitochondrial membrane, though the total DRP1 protein level remains unchanged. Here we have characterized a lesser studied CMT2A-linked MFN2 mutant to show that its presence affects mitochondrial morphology and homeostasis.

Dual-specific autophosphorylation of kinase IKK2 enables phosphorylation of substrate IκBα through a phosphoenzyme intermediate.

Rapid and high-fidelity phosphorylation of two serines (S32 and S36) of IκBα by a prototype Ser/Thr kinase IKK2 is critical for fruitful canonical NF-κB activation. Here, we report that IKK2 is a dual specificity Ser/Thr kinase that autophosphorylates itself at tyrosine residues in addition to its activation loop serines. Mutation of one such tyrosine, Y169, located in proximity to the active site, to phenylalanine, renders IKK2 inactive for phosphorylation of S32 of IκBα. Surprisingly, auto-phosphorylated IKK2 relayed phosphate group(s) to IκBα without ATP when ADP is present. We also observed that mutation of K44, an ATP-binding lysine conserved in all protein kinases, to methionine renders IKK2 inactive towards specific phosphorylation of S32 or S36 of IκBα, but not non-specific substrates. These observations highlight an unusual evolution of IKK2, in which autophosphorylation of tyrosine(s) in the activation loop and the invariant ATP-binding K44 residue define its signal-responsive substrate specificity ensuring the fidelity of NF-κB activation.

A Novel spice-antioxidant-based nano-vehicle as a putative green alternative of synthetic AChE inhibitor drugs.

The present treatment for Alzheimer's disease (AD) involves well known synthetic acetylcholine esterase (AChE) inhibitor drugs which besides having short duration of action also have deleterious impact on human health. Therefore, there is a need for natural plant-based biomolecule(s) with potential AChE inhibition activity (ies). The aim of the work is to design a spice-based nano-vehicle as a novel green alternative of synthetic AD drugs by nanoencapsulating a solvent-less supercritical CO2 extract of small cardamom seeds (SCE) having a synergistic consortium of five antioxidant molecules, using polyethylene glycol and emulsifiers, selected based on Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) analyses. Ellman's assay and enzyme inhibition kinetics of the antioxidant molecules as well as the extract and its nanoliposomal formulation (SCE-NL) were performed, followed by rigorous molecular docking and dynamics studies using MM-PBSA and umbrella sampling. The antioxidants exhibited significant AChE inhibition in vitro, individually with 1, 8-cineole having the least IC50 value of 65.53 ± 0.05 µg/mL. . Although SCE-NL had higher IC50 value (575.67 ± 0.5 µg/mL) vis-à-vis that of rivastigmine (67.52 ± 0.02 µg/mL), it is safer for usage being 'green'.The Lineweaver-Burk plots (Vmax ∼1.04 mM/min) revealed competitive mode(s) of inhibition of AChE with each of these antioxidants. Binding energy analyses suggested very good binding free energies and stable docking/binding complexes (between the antioxidants and AChE). This study has delivered a nanoliposomal vehicle of food antioxidants as a putative 'green' alternative of synthetic AChE inhibitor drugs.Communicated by Ramaswamy H. Sarma.

Accelerated export of Dicer1 from lipid-challenged hepatocytes buffers cellular miRNA-122 levels and prevents cell death.

Hepatocytes on exposure to high levels of lipids reorganize the metabolic program while fighting against the toxicity associated with elevated cellular lipids. The mechanism of this metabolic reorientation and stress management in lipid-challenged hepatocytes has not been well explored. We have noted the lowering of miR-122, a liver-specific miRNA, in the liver of mice fed with either a high-fat diet or a methionine-choline-deficient diet that is associated with increased fat accumulation in mice liver. Interestingly, low miR-122 levels are attributed to the enhanced extracellular export of miRNA processor enzyme Dicer1 from hepatocytes in the presence of high lipids. Export of Dicer1 can also account for the increased cellular levels of pre-miR-122-the substrate of Dicer1. Interestingly, restoration of Dicer1 levels in the mouse liver resulted in a strong inflammatory response and cell death in the presence of high lipids. Increasing death of hepatocytes was found to be caused by increased miR-122 levels in hepatocytes restored for Dicer1. Thus, the Dicer1 export by hepatocytes seems to be a key mechanism to combat lipotoxic stress by shunting out miR-122 from stressed hepatocytes. Finally, as part of this stress management, we determined that the Ago2-interacting pool of Dicer1, responsible for mature microribonucleoprotein formation in mammalian cells, gets depleted. miRNA-binder and exporter protein HuR is found to accelerate Ago2-Dicer1 uncoupling to ensure export of Dicer1 via extracellular vesicles in lipid-loaded hepatocytes.

Integrating Multi-Omics Data to Construct Reliable Interconnected Models of Signaling, Gene Regulatory, and Metabolic Pathways.

Alteration of the status of the metabolic enzymes could be a probable way to regulate metabolic reprogramming, which is a critical cellular adaptation mechanism especially for cancer cells. Coordination among biological pathways, such as gene-regulatory, signaling, and metabolic pathways is crucial for regulating metabolic adaptation. Also, incorporation of resident microbial metabolic potential in human body can influence the interplay between the microbiome and the systemic or tissue metabolic environments. Systemic framework for model-based integration of multi-omics data can ultimately improve our understanding of metabolic reprogramming at holistic level. However, the interconnectivity and novel meta-pathway regulatory mechanisms are relatively lesser explored and understood. Hence, we propose a computational protocol that utilizes multi-omics data to identify probable cross-pathway regulatory and protein-protein interaction (PPI) links connecting signaling proteins or transcription factors or miRNAs to metabolic enzymes and their metabolites using network analysis and mathematical modeling. These cross-pathway links were shown to play important roles in metabolic reprogramming in cancer scenarios.

Oncogene-mediated nuclear accumulation of lactate promotes epigenetic alterations to induce cancer cell proliferation.

Homeobox gene families are associated with embryonic development and organogenesis. Pieces of evidence suggest that these Homeobox genes are also crucial in facilitating oncogenesis when mutated or overexpressed. Paired homeodomain transcription factor-2 (PITX2), one of the members of this family, is involved in oncogenic regulation apart from its different development regulatory functions. PITX2 has been earlier shown to induce ovarian cancer cell proliferation through the activation of different signaling cascades. Increased cancer cell proliferation requires a constant supply of nutrients for both adenosine triphosphate and biomass synthesis, which is facilitated by altered cancer cell metabolism that includes enhanced glucose uptake and increased glycolytic rate. This present study highlights the involvement of PITX2 in enhancing the cellular glycolysis pathway in ovarian cancer cells through protein kinase B-phosphorylation (phospho-AKT). PITX2 expression correlates positively with that of the glycolytic rate-determining enzyme, lactate dehydrogenase-A (LDHA), in both high-grade serous ovarian cancer tissues and common ovarian cancer cell lines. Interestingly, transient localization of enzymatically active LDHA in the nucleus was observed in PITX2-overexpressed ovarian cancer cells. This nuclear LDHA produces higher concentrations of the glycolytic end product, lactate, which accumulates in the nuclear compartment resulting in decreased histone deacetylase (HDAC1/2) expression and increased histone acetylation at H3/H4. However, the mechanistic details of lactate-HDAC interaction are still elusive in the earlier reports. Our in silico studies elaborated on the interaction dynamics of lactate in the HDAC catalytic core through ligand-binding studies and molecular dynamics simulation approaches. Blocking lactate production by silencing LDHA reduced cancer cell proliferation. Thus, PITX2-induced epigenetic changes can lead to high cellular proliferation and increase the size of tumors in syngeneic mice as well. Taken together, this is the first report of its kind to show that the developmental regulatory homeobox gene PITX2 could enhance oncogenesis through enhanced glycolysis of tumor cells followed by epigenetic modifications.

MetaDOCK: A Combinatorial Molecular Docking Approach.

Molecular docking plays a major role in academic and industrial drug screening and discovery processes. Despite the availability of numerous docking software packages, there is a lot of scope for improvement for the docking algorithms in terms of becoming more reliable to replicate the experimental binding results. Here, we propose a combinatorial or consensus docking approach where complementary powers of the existing methods are captured. We created a meta-docking protocol by combining the results of AutoDock4.2, LeDock, and rDOCK programs as these are freely available, easy to use, and suitable for large-scale analysis and produced better performance on benchmarking studies. Rigorous benchmarking analyses were undertaken to evaluate the scoring, posing, and screening capability of our approach. Further, the performance measures were compared against one standard state-of-the-art commercial docking software, GOLD, and one freely available software, PLANTS. Performances of MetaDOCK for scoring, posing, and screening the protein-ligand complexes were found to be quite superior compared to the reference programs. Exhaustive molecular dynamics simulation and molecular mechanics Poisson-Boltzmann and surface area-based free energy estimation also suggest better energetic stability of the docking solutions produced by our meta-approach. We believe that the MetaDOCK approach is a useful packaging of the freely available software and provides a better alternative to the scientific community who are unable to afford costly commercial packages.

Rodent Model Preclinical Assessment of PEGylated Block Copolymer Targeting Cognition and Oxidative Stress Insults of Alzheimer's Disease.

Misfolded peptide amyloid beta (Aß42), neurofibrillary tangles of hyper-phosphorylated tau, oxidative damage to the brain, and neuroinflammation are distinguished determinants of Alzheimer's disease (AD) responsible for disease progression. This multifaceted neurodegenerative disease is challenging to cure under a single treatment regime until the key disease determinants are traced for their sequential occurrence in disease progression. In an early report, a novel side-chain tripeptide containing PEGylated block copolymer has been tested thoroughly in vitro and in silico for the early inhibition of Aß42 aggregation as well as degradation of preformed Aß42 fibril deposits. The present study demonstrates a preclinical assessment of the PEGylated block copolymer in colchicine-induced AD-mimicking rodent model. The colchicine-induced Wistar rats receiving an intranasal delivery of the block copolymer at a daily dosage of 100 µg/kg and 200 µg/kg body weights, respectively, for 14 days manifested a notable attenuation of behavioral deficit pattern, oxidative stress, and neurotransmitters' deficiency as compared to the untreated ones. The current study also reports the ameliorative property of the PEGylated compound for progressive neuroinflammation and decreased mitochondrial bioenergetics in astrocytoma cell line, viz., U87. A closer look into the drug mechanism of action of a compact 3D PEGylated block copolymer confirmed its disintegrative interaction with Aß42 fibril via in silico simulation. The results obtained from this study signify the potential of the novel PEGylated block copolymer to ameliorate the cognitive decline and progressive oxidative insults in AD and may envision a successful clinical phase trial. The amelioration of disease condition of colchicine-induced AD rat. Initially the rat has given colchicine via stereotaxic surgery which led to a mimicking condition of AD including neuronal death in hippocampal CA1 region. After recovery from the surgery, the rat was treated with the PEGylated block copolymer through intranasal delivery, and this has led to the decrease in neuronal death in hippocampal CA1 region. The mechanism of drug action has shown by the separation of monomer chains of Aß42.

Genome characterization, phylogenomic assessment and spatio-temporal dynamics study of highly mutated BA variants from India.

PURPOSE: The emergence of highly mutated and transmissible BA variants has caused an unprecedented surge in COVID-19 infections worldwide. Thorough analysis of its genome structure and phylogenomic evolutionary details will serve as scientific reference for future research. METHOD: Here, we have analyzed the BA variants from India using whole-genome sequencing, spike protein mutation study, spatio-temporal surveillance, phylogenomic assessment and epitope mapping. RESULTS: The predominance of BA.2/BA.2-like was observed in India during COVID-19 third wave. Genome analysis and mutation study highlighted the existence of 2128 amino acid changes within BA as compared to NC_045512.2. Presence of 23 unknown mutation sites (spanning region 61-831) were observed among the Indian BA variants as compared to the global BA strains. Unassigned probable Omicron showed the highest number of mutations (370) followed by BA.1 (104), BA.2.3 (56), and BA.2 (27). Presence of mutations 'Q493R â€‹+ â€‹Q498R â€‹+ â€‹N501Y', and 'K417 â€‹N â€‹+ â€‹E484A â€‹+ â€‹N501Y' remained exclusive to BA.2 as well as unassigned probable Omicron. The time-tree and phylogenomic network assessed the evolutionary relationship of the BA variants. Existence of 424 segregating sites and 113 parsimony informative sites within BA genomes were observed through haplotype network analysis. Epitope mapping depicted the presence of unique antigenic sites within the receptor binding domain of the BA variants that could be exploited for robust vaccine development. CONCLUSION: These findings provide important scientific insights about the nature, diversity, and evolution of Indian BA variants. The study further divulges in the avenues of therapeutic upgradation for better management and eventual eradication of COVID-19.

Virtual screening of natural products inspired in-house library to discover potential lead molecules against the SARS-CoV-2 main protease.

SARS-CoV-2, a new coronavirus emerged in 2019, causing a global healthcare epidemic. Although a variety of drug targets have been identified as potential antiviral therapies, and effective candidate against SARS-CoV-2 remains elusive. One of the most promising targets for combating COVID-19 is SARS-CoV-2 Main protease (Mpro, a protein responsible for viral replication. In this work, an in-house curated library was thoroughly evaluated for druggability against Mpro. We identified four ligands (FG, Q5, P5, and PJ4) as potential inhibitors based on docking scores, predicted binding energies (MMGBSA), in silico ADME, and RMSD trajectory analysis. Among the selected ligands, FG, a natural product from Andrographis nallamalayana, exhibited the highest binding energy of -10.31 kcal/mol close to the docking score of clinical candidates Boceprevir and GC376. Other ligands (P5, natural product from cardiospermum halicacabum and two synthetic molecules Q5 and PJ4) have shown comparable docking scores ranging -7.65 kcal/mol to -7.18 kcal/mol. Interestingly, we found all four top ligands had Pi bond interaction with the main amino acid residues HIS41 and CYS145 (catalytic dyad), H-bonding interactions with GLU166, ARG188, and GLN189, and hydrophobic interactions with MET49 and MET165 in the binding site of Mpro. According to the ADME analysis, Q5 and P5 are within the acceptable range of drug likeliness, compared to FG and PJ4. The interaction stability of the lead molecules with viral protease was verified using replicated MD simulations. Thus, the present study opens up the opportunity of developing drug candidates targeting SARS-CoV-2 main protease (Mpro) to mitigate the disease.Communicated by Ramaswamy H. Sarma.

USP7 targets XIAP for cancer progression: Establishment of a p53-independent therapeutic avenue for glioma.

Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific target protein substrates in order to alter their degradation rate, sub-cellular localization, interaction, and activity. The induction of apoptosis upon USP7 inhibition is well established in cancer containing wild type p53, which operates through the 'USP7-Mdm2-p53' axis. However, in cancers without functional p53, USP7-dependent apoptosis is induced through many other alternative pathways. Here, we have identified another critical p53 independent path active under USP7 to regulate apoptosis. Proteomics analysis identifies XIAP as a potential target of USP7-dependent deubiquitination. GSEA analysis revealed up-regulation of apoptosis signalling upon USP7 inhibition associated with XIAP down-regulation. Modulation of USP7 expression and activity in multiple cancer cell lines showed that USP7 deubiquitinates XIAP to inhibit apoptosis in a caspase-dependent pathway, and the combinatorial inhibition of USP7 and XIAP induces apoptosis in vitro and in vivo. Immunohistochemical staining revealed that grade-wise accumulation of USP7 correlated with an elevated level of XIAP in glioma tissue. This is the first report on the identification and validation of XIAP as a novel substrate of USP7 and together, they involve in the empowerment of the tumorigenic potential of cancer cells by inhibiting apoptosis.

CMT2A-linked mitochondrial hyperfusion-driving mutant MFN2 perturbs ER-mitochondrial associations and Ca2+ homeostasis.

BACKGROUND INFORMATION: Mitofusin2 (MFN2), an important molecular player that regulates mitochondrial fusion, also helps maintain the inter-organellar contact sites, referred as mitochondria associated membranes (MAMs) that exist between the ER and mitochondria. The study deals with a mutant of MFN2, R364W-MFN2, linked with the neuropathy, Charcot Marie Tooth (CMT) disease. Previous studies show that this mutant promotes mitochondrial hyperfusion. Here, we try to decipher the role of R364W-MFN2 in affecting the ER mitochondrial associations at the MAM junctions and inter-organellar calcium signalling between the ER and the mitochondria. RESULTS: Our results show that R364W-MFN2 altered ER-mitochondria association at the MAM junctions, predisposed mitochondria towards cellular stress with the mitochondria undergoing rapid fission upon induction of mild stress and perturbs inter-organellar calcium homeostasis. CONCLUSION: The results indicate that R364W-MFN2 not only affects mitochondrial morphology and dynamics but also modulate its interaction with the ER and Ca2+ signalling between the two organelles. SIGNIFICANCE: This study provides significant insight that presence of the R364W-MFN2 mutation makes cells susceptible towards stress, thus negatively affecting cellular health which altogether might culminate in the form of the CMT neuropathy.

Target-Dependent Coordinated Biogenesis of Secondary MicroRNAs by miR-146a Balances Macrophage Activation Processes.

MicroRNAs (miRNAs) repress protein expression by binding to the target mRNAs. Exploring whether the expression of one miRNA can regulate the abundance and activity of other miRNAs, we noted the coordinated biogenesis of miRNAs in activated macrophages. miRNAs with higher numbers of binding sites (the "primary" miRNAs) induce expression of other miRNAs ("secondary" miRNAs) having binding sites on the 3' untranslated region (UTR) of common target mRNAs. miR-146a-5p, in activated macrophages, acts as a "primary" miRNA that coordinates biogenesis of "secondary" miR-125b, miR-21, or miR-142-3p to target new sets of mRNAs to balance the immune responses. During coordinated biogenesis, primary miRNA drives the biogenesis of secondary miRNA in a target mRNA- and Dicer1 activity-dependent manner. The coordinated biogenesis of miRNAs was observed across different cell types. The target-dependent coordinated miRNA biogenesis also ensures a cumulative mode of action of primary and secondary miRNAs on the secondary target mRNAs. Interestingly, using the "primary" miR-146a-5p-specific inhibitor, we could inhibit the target-dependent biogenesis of secondary miRNAs that can stop the miRNA-mediated buffering of cytokine expression and inflammatory response occurring in activated macrophages. Computational analysis suggests the prevalence of coordinated biogenesis of miRNAs also in other contexts in human and in mouse.

Role of systems biology and multi-omics analyses in delineating spatial interconnectivity and temporal dynamicity of ER stress mediated cellular responses.

The endoplasmic reticulum (ER) is a membranous organelle involved in calcium storage, lipid biosynthesis, protein folding and processing. Many patho-physiological conditions and pharmacological agents are known to perturb normal ER function and can lead to ER stress, which severely compromise protein folding mechanism and hence poses high risk of proteotoxicity. Upon sensing ER stress, the different stress signaling pathways interconnect with each other and work together to preserve cellular homeostasis. ER stress response is a part of the integrative stress response (ISR) and might play an important role in the pathogenesis of chronic neurodegenerative diseases, where misfolded protein accumulation and cell death are common. The initiation, manifestation and progression of ER stress mediated unfolded protein response (UPR) is a complex procedure involving multiple proteins, pathways and cellular organelles. To understand the cause and consequences of such complex processes, implementation of an integrative holistic approach is required to identify novel players and regulators of ER stress. As multi-omics data-based systems analyses have shown potential to unravel the underneath molecular mechanism of complex biological systems, it is important to emphasize the utility of this approach in understanding the ER stress biology. In this review we first discuss the ER stress signaling pathways and regulatory players, along with their inter-connectivity. We next highlight the importance of systems and network biology approaches using multi-omics data in understanding ER stress mediated cellular responses. This report would help advance our current understanding of the multivariate spatial interconnectivity and temporal dynamicity of ER stress.

Variability in the Responses of Hepatitis B Virus D-Subgenotypes to Antiviral Therapy: Designing Pan-D-Subgenotypic Reverse Transcriptase Inhibitors.

Nucleos(t)ide analogues entecavir (ETV) and tenofovir disoproxil fumarate (TDF) are recommended as first-line monotherapies for chronic hepatitis B (CHB). Multiple HBV genotypes/subgenotypes have been described, but their impact on treatment response remains largely elusive. We investigated the effectiveness of ETV/TDF on HBV/D-subgenotypes, D1/D2/D3/D5, studied the structural/functional differences in subgenotype-specific reverse transcriptase (RT) domains of viral polymerase, and identified novel molecules with robust inhibitory activity on various D-subgenotypes. Transfection of Huh7 cells with full-length D1/D2/D3/D5 and in vitro TDF/ETV susceptibility assays demonstrated that D1/D2 had greater susceptibility to TDF/ETV while D3/D5 exhibited poorer response. Additionally, HBV load was substantially reduced in TDF-treated CHB patients carrying D1/D2 but minimally reduced in D3/D5-infected patients. Comparison of RT sequences of D-subgenotypes led to identification of unique subgenotype-specific residues, and molecular modeling/docking/simulation studies depicted differential bindings of TDF/ETV to the active site of their respective RTs. Replacement of signature residues in D3/D5 HBV clones with corresponding amino acids seen in D1/D2 improved their susceptibility to TDF/ETV. Using high throughput virtual screening, we identified N(9)-[3-fluoro-2-(phosphonomethoxy)propyl] (FPMP) derivatives of purine bases, including N6-substituted (S)-FPMP derivative of 2,6-diaminopurine (DAP) (OB-123-VK), as potential binders of RT of different D-subgenotypes. We synthesized (S)-FPMPG prodrugs (FK-381-FEE/FK-381-SEE/FK-382) and tested their effectiveness along with OB-123-VK. Both OB-123-VK and FK-381-FEE exerted similar antiviral activities against all D-subgenotypes, although FK-381-FEE was more potent. Our study highlighted the natural variation in therapeutic response of D1/D2/D3/D5 and emphasized the need for HBV subgenotype determination before treatment. Novel molecules described here could benefit future design/discovery of pan-D-subgenotypic inhibitors. IMPORTANCE Current treatment of chronic hepatitis B relies heavily on nucleotide/nucleoside analogs in particular, tenofovir disoproxil fumarate (TDF) and entecavir (ETV) to keep HBV replication under control and prevent end-stage liver diseases. However, it was unclear whether the therapeutic effects of TDF/ETV differ among patients infected with different HBV genotypes and subgenotypes. HBV genotype D is the most widespread of all HBV genotypes and multiple D-subgenotypes have been described. We here report that different subgenotypes of HBV genotype-D exhibit variable response toward TDF and ETV and this could be attributed to naturally occurring amino acid changes in the reverse transcriptase domain of the subgenotype-specific polymerase. Further, we identified novel molecules and also synthesized prodrugs that are equally effective on different D-subgenotypes and could facilitate management of HBV/D-infected patients irrespective of D-subgenotype.

MITOL-mediated DRP1 ubiquitylation and degradation promotes mitochondrial hyperfusion in a CMT2A-linked MFN2 mutant.

Mutations in mitofusin 2 (MFN2) that are associated with the pathology of the debilitating neuropathy Charcot-Marie-Tooth type 2A (CMT2A) are known to alter mitochondrial morphology. One such abundant MFN2 mutation, R364W, results in the generation of elongated, interconnected mitochondria. However, the mechanism leading to this mitochondrial aberration remains poorly understood. Here, we show that mitochondrial hyperfusion in the presence of R364W-MFN2 is due to increased degradation of DRP1 (also known as DNM1L). The E3 ubiquitin ligase MITOL (also known as MARCHF5) is known to ubiquitylate both MFN2 and DRP1. Interaction with and subsequent ubiquitylation by MITOL is stronger in the presence of wild-type MFN2 than with R364W-MFN2. This differential interaction of MITOL with MFN2 in the presence of R364W-MFN2 renders the ligase more available for DRP1 ubiquitylation. Multi-monoubiquitylation and proteasomal degradation of DRP1 in R364W-MFN2 cells in the presence of MITOL eventually leads to mitochondrial hyperfusion. Here, we provide a mechanistic insight into mitochondrial hyperfusion, while also reporting that MFN2 can indirectly modulate DRP1 - an effect not shown previously. This article has an associated First Person interview with the first author of the paper.

Inhibition of extracellular vesicle-associated MMP2 abrogates intercellular hepatic miR-122 transfer to liver macrophages and curtails inflammation.

Hepatic miRNA, miR-122, plays an important role in controlling metabolic homeostasis in mammalian liver. Intercellular transfer of miR-122 was found to play a role in controlling tissue inflammation. miR-122, as part of extracellular vesicles released by lipid-exposed hepatic cells, are taken up by tissue macrophages to activate them and produce inflammatory cytokines. Matrix metalloprotease 2 or MMP2 was found to be essential for transfer of extracellular vesicles and their miRNA content from hepatic to non-hepatic cells. MMP2 was found to increase the movement of the extracellular vesicles along the extracellular matrix to enhance their uptake in recipient cells. Inhibition of MMP2 restricts functional transfer of hepatic miRNAs across the hepatic and non-hepatic cell boundaries, and by targeting MMP2, we could reduce the innate immune response in mammalian liver by preventing intra-tissue miR-122 transfer. MMP2 thus could be a useful target to restrict high-fat-diet-induced obesity-related metaflammation.

Corpus Callosum Atrophy in Detection of Mild and Moderate Alzheimer's Disease Using Brain Magnetic Resonance Image Processing and Machine Learning Techniques.

BACKGROUND: The total number of people with dementia is projected to reach 82 million in 2030 and 152 in 2050. Early and accurate identification of the underlying causes of dementia, such as Alzheimer's disease (AD) is of utmost importance. A large body of research has shown that imaging techniques are most promising technologies to improve subclinical and early diagnosis of dementia. Morphological changes, especially atrophy in various structures like cingulate gyri, caudate nucleus, hippocampus, frontotemporal lobe, etc., have been established as markers for AD. Being the largest white matter structure with a high demand of blood supply from several main arterial systems, anatomical alterations of the corpus callosum (CC) may serve as potential indication neurodegenerative disease. OBJECTIVE: To detect mild and moderate AD using brain magnetic resonance image (MRI) processing and machine learning techniques. METHODS: We have performed automatic detection and segmentation of the CC and calculated its morphological features to feed into a multivariate pattern analysis using support vector machine (SVM) learning techniques. RESULTS: Our results using large patients' cohort show CC atrophy-based features are capable of distinguishing healthy and mild/moderate AD patients. Our classifiers obtain more than 90%sensitivity and specificity in differentiating demented patients from healthy cohorts and importantly, achieved more than 90%sensitivity and > 80%specificity in detecting mild AD patients. CONCLUSION: Results from this analysis are encouraging and advocate development of an image analysis software package to detect dementia from brain MRI using morphological alterations of the CC.