FBXL4: safeguarding against mitochondrial depletion through suppression of mitophagy.

Mitophagy is a critical mitochondrial quality control process that selectively removes dysfunctional or excess mitochondria through the autophagy-lysosome system. The process is tightly controlled to ensure cellular and physiological homeostasis. Insufficient mitophagy can result in failure to remove damaged mitochondria and consequent cellular degeneration, but it is equally important to appropriately restrain mitophagy to prevent excessive mitochondrial depletion. Here, we discuss our recent discovery that the SKP1-CUL1-F-box (SCF)-FBXL4 (F-box and leucine-rich repeat protein 4) E3 ubiquitin ligase localizes to the mitochondrial outer membrane, where it constitutively mediates the ubiquitination and degradation of BNIP3L/NIX and BNIP3 mitophagy receptors to suppress mitophagy. The post-translational regulation of BNIP3L and BNIP3 is disrupted in mitochondrial DNA depletion syndrome 13 (MTDPS13), a multi-systemic disorder caused by mutations in the FBXL4 gene and characterized by elevated mitophagy and mitochondrial DNA/mtDNA depletion in patient fibroblasts. Our results demonstrate that mitophagy is not solely stimulated in response to specific conditions but is instead also actively suppressed through the continuous degradation of BNIP3L and BNIP3 mediated by the SCF-FBXL4 ubiquitin ligase. Thus, cellular conditions or signaling events that prevent the FBXL4-mediated turnover of BNIP3L and BNIP3 on specific mitochondria are expected to facilitate their selective removal.

Prospective study to compare results of FIA (fluorescent immunoassay) test with gold standard ELISA test in Dengue NS1 patients admitted in a tertiary care hospital.

PURPOSE: The objective of this study was to compare the performance of STANDARD F Dengue NS1 Ag FIA SD Biosensor and ELISA for diagnosis of dengue in patients admitted in a tertiary care hospital. The results of the FIA test were compared with ELISA test, the gold standard in terms of sensitivity, specificity, positive predictive value, negative predictive value. METHODS: Total 19,890 patients suffering from acute febrile illness suspecting dengue were screened for dengue NS1 antigen by rapid immunochromatography test during the period of one year and six months from June 2020 to November 2021. Out of them, a total of 300 blood samples were selected by simple random sampling method for the study. The blood samples were collected and subjected to dengue NS1 ELISA and FIA test according to the kit literature. RESULTS: Out of total 300 samples, 67.67% (203) patients were positive for dengue NS1 antigen by FIA test. Total 200 patients (66.67%) were positive for dengue NS1 antigen by ELISA and (190) 63.33% were positive for dengue NS1 antigen by rapid ICT. It was also seen that there was an inverse relation between the Cutoff Index (COI) value of FIA and the platelet count of the dengue NS1 positive patients. CONCLUSION: We conclude that the results of Dengue NS1 Microwell ELISA test and Dengue NS1 FIA test are almost equivalent in relation to sensitivity, specificity, positive predictive value, and negative predictive value. FIA gives rapid test results, thus is time saving and can be routinely used. The COI value can also give a relative idea about the severity of the disease.

Polytetrafluoroethylene tape to combat tooth sensitivity during complete mouth rehabilitation.

Dentin sensitivity is a common complaint from patients during and after tooth preparation for complete coverage restorations. Techniques to reduce sensitivity during tooth preparation include immediate dentin sealing and application of desensitizers. However, managing dentin sensitivity during complete mouth rehabilitation on natural teeth can be challenging, especially for patients with dentin hypersensitivity. A technique to protect the prepared teeth during complete mouth rehabilitation using polytetrafluoroethylene (PTFE) tape is described.

FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors.

To maintain both mitochondrial quality and quantity, cells selectively remove damaged or excessive mitochondria through mitophagy, which is a specialised form of autophagy. Mitophagy is induced in response to diverse conditions, including hypoxia, cellular differentiation and mitochondrial damage. However, the mechanisms that govern the removal of specific dysfunctional mitochondria under steady-state conditions to fine-tune mitochondrial content are not well understood. Here, we report that SCFFBXL4 , an SKP1/CUL1/F-box protein ubiquitin ligase complex, localises to the mitochondrial outer membrane in unstressed cells and mediates the constitutive ubiquitylation and degradation of the mitophagy receptors NIX and BNIP3 to suppress basal levels of mitophagy. We demonstrate that the pathogenic variants of FBXL4 that cause encephalopathic mtDNA depletion syndrome (MTDPS13) do not efficiently interact with the core SCF ubiquitin ligase machinery or mediate the degradation of NIX and BNIP3. Thus, we reveal a molecular mechanism whereby FBXL4 actively suppresses mitophagy by preventing NIX and BNIP3 accumulation. We propose that the dysregulation of NIX and BNIP3 turnover causes excessive basal mitophagy in FBXL4-associated mtDNA depletion syndrome.

A novel property of hexokinase inhibition by Favipiravir and proposed advantages over Molnupiravir and 2 Deoxy D glucose in treating COVID-19.

PURPOSE: In the wake of SARS-CoV-2's global spread, human activities from health to social life to education have been affected. Favipiravir and Molnupiravir exhibited novel hexokinase inhibition and we discuss advantages of this property in their COVID-19 inhibition potential. METHODS: This paper describes molecular docking data of human hexokinase II with Favipiravir, Cyan 20, Remdesivir, 2DG, and Molnupiravir along with hexokinase inhibition assays. RESULTS: Favipiravir, an antiviral drug previously cleared for treating the flu and ebola, has shown some promise in early trials to treat COVID-19. We observed potent human hexokinase inhibiting potential of Favipiravir (50%) as against 4% and merely 0.3% hexokinase inhibition with Molnupiravir and 2 Deoxy D glucose at 0.1 mM concentration supported by molecular docking studies. CONCLUSION: Favipiravir could continue to be part of the COVID-19 treatment regimen due to its resistance to host esterases, hexokinase inhibition potential and proven safety through human trials.

Hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) Simulation: A Tool for Structure-Based Drug Design and Discovery.

Quantum Mechanics (QM) is the physics-based theory that explains the physical properties of nature at the level of atoms and sub-atoms. Molecular mechanics (MM) construct molecular systems through the use of classical mechanics. So, when combined, hybrid quantum mechanics and molecular mechanics (QM/MM) can act as computer-based methods that can be used to calculate the structure and property data of molecular structures. Hybrid QM/MM combines the strengths of QM with accuracy and MM with speed. QM/MM simulation can also be applied for the study of chemical processes in solutions, as well as in the proteins, and has a great scope in structure-based drug design (SBDD) and discovery. Hybrid QM/MM can also be applied to HTS to derive QSAR models. Due to the availability of many protein crystal structures, it has a great role in computational chemistry, especially in structure- and fragment-based drug design. Fused QM/MM simulations have been developed as a widespread method to explore chemical reactions in condensed phases. In QM/MM simulations, the quantum chemistry theory is used to treat the space in which the chemical reactions occur; however, the rest is defined through the molecular mechanics force field (MMFF). In this review, we have extensively reviewed recent literature pertaining to the use and applications of hybrid QM/MM simulations for ligand and structure-based computational methods for the design and discovery of therapeutic agents.

An Autonomous Molecular Bioluminescent Reporter (AMBER) for Voltage Imaging in Freely Moving Animals.

Genetically encoded reporters have greatly increased our understanding of biology. While fluorescent reporters have been widely used, photostability and phototoxicity have hindered their use in long-term experiments. Bioluminescence overcomes some of these challenges but requires the addition of an exogenous luciferin limiting its use. Using a modular approach, Autonomous Molecular BioluminEscent Reporter (AMBER), an indicator of membrane potential is engineered. Unlike other bioluminescent systems, AMBER is a voltage-gated luciferase coupling the functionalities of the Ciona voltage-sensing domain (VSD) and bacterial luciferase, luxAB. When co-expressed with the luciferin-producing genes, AMBER reversibly switches the bioluminescent intensity as a function of membrane potential. Using biophysical and biochemical methods, it is shown that AMBER switches its enzymatic activity from an OFF to an ON state as a function of the membrane potential. Upon depolarization, AMBER switches from a low to a high enzymatic activity state, showing a several-fold increase in the bioluminescence output (ΔL/L). AMBER in the pharyngeal muscles and mechanosensory touch neurons of Caenorhabditis elegans is expressed. Using the compressed sensing approach, the electropharingeogram of the C. elegans pharynx is reconstructed, validating the sensor in vivo. Thus, AMBER represents the first fully genetically encoded bioluminescent reporter without requiring exogenous luciferin addition.

Bioanalytical method development for momordicinin and its application to long-term pharmacokinetics in diabetic rats.

OBJECTIVE: To develop and validate bioanalytical RP-HPLC method to evaluate pharmacokinetics and tissue distribution pattern of momordicinin (MRN). SIGNIFICANCE: MRN is one of the major cucurbitane triterpenoid found in Momordica charantia Linn (MC). However, MRN has not been explored for its pharmacokinetic profile, tissue distribution, and stability in order to establish it as an antidiabetic agent. METHODS: In 28 days pharmacokinetic study, 54 diabetic male wistar rats were divided into three different groups and administered with 25, 50, and 100 mg/kg MRN orally. The blood samples were collected at 1, 7, 14, 21, and 28th day of the treatment and plasma quantification of MRN was done by validated RP-HPLC method. The rats were sacrificed at end of the study for tissue distribution. RESULTS: The developed method was successfully applied to investigate pharmacokinetic profile of MRN. In pharmacokinetic analysis, the Cmax for 25, 50, and 100 mg/kg was found to be 8.412, 10.443, and 11.829 µg/mL respectively suggesting the dose dependent activity. The maximum plasma concentration was achieved at 2 h for all doses. MRN showed major distribution in liver followed by kidney, spleen, and pancreas. CONCLUSION: The newly developed and validated method was used to assay MRN in plasma as well as in tissues to evaluate pharmacokinetics of the drug for the first time. Undoubtedly, these findings can be taken into consideration while concluding its therapeutic effects after oral administration.

Isolation, characterisation and investigation of in vitro antidiabetic and antioxidant activity of phytoconstituents from fruit of Momordica charantia Linn.

The dry powder of MC fruits was extracted by maceration, ultrasonication, liquid-liquid partition and soxhlation. The in vitro antidiabetic and antioxidant assays were used to screen extracts and fractions. Refluxed and liquid partitioned extracts were fractionated using petroleum ether and ethyl acetate and purified with the help of preparative HPLC to give 2 phytoconstituents M1 and M2 respectively. Compound M1 (1) was identified as charantin and Compound M2 (2) was identified as momordicinin using spectral studies. Momordicinin showed potent α-amylase inhibitory activity with IC50 15.86µg/ml which was reported for the first time.

Tissue-Penetrating, Hypoxia-Responsive Echogenic Polymersomes For Drug Delivery To Solid Tumors.

Hypoxia in solid tumors facilitates the progression of the disease, develops resistance to chemo and radiotherapy, and contributes to relapse. Due to the lack of tumor penetration, most of the reported drug carriers are unable to reach the hypoxic niches of the solid tumors. We have developed tissue-penetrating, hypoxia-responsive echogenic polymersomes to deliver anticancer drugs to solid tumors. The polymersomes are composed of a hypoxia-responsive azobenzene conjugated and a tissue penetrating peptide functionalized polylactic acid-polyethylene glycol polymer. The drug-encapsulated, hypoxia-responsive polymersomes substantially decreased the viability of pancreatic cancer cells in spheroidal cultures. Under normoxic conditions, polymersomes were echogenic at diagnostic ultrasound frequencies but lose the echogenicity under hypoxia. In-vivo imaging studies with xenograft mouse model further confirmed the ability of the polymersomes to target, penetrate, and deliver the encapsulated contents in hypoxic pancreatic tumor tissues.

Retention of CAD/CAM resin composite crowns following different bonding protocols.

PURPOSE: To evaluate the effect of different surface treatments and primers with a CAD/CAM resin composite block on its crown retention. METHODS: 120 human molars were prepared with a 24° total convergence angle, 1.5 mm height, and axial walls in dentin. Surface area was measured by digital microscopy. Crowns were machined from CAD/CAM resin composite blocks. Teeth were randomly allocated to 12 groups (n= 10) based on possible combinations of three surface treatments: [Control, Alumina air abrasion (50-µm Al2O3 at 0.28 MPa) ]; 5% hydrofluoric acid etch (20-second scrub); silane application (with or without Kerr Silane primer); and adhesive application (with or without Optibond XTR Adhesive). Optibond XTR Adhesive was applied to the tooth preparations and crowns were bonded with MaxCem Elite cement. Crowns were fatigued for 100,000 cycles at 100 N in water and debonded in tension (1 mm/minute). Crown retention strength (maximum load/surface area) values were analyzed using a three-way ANOVA with Tukey's post-hoc tests (α= 0.05). RESULTS: Surface treatment, silane and adhesive applications independently affect retention force (P< 0.05). All interactions were not significant (P> 0.05). Alumina airborne abrasion surface treatment, silane and adhesive applications all improve retention strength. Therefore, CAD/CAM resin composite crowns can withstand debonding while undergoing mechanical fatigue. Although all forms of surface treatment and primer application improve bond strength, the highest mean retention strength values were recorded when the crowns were alumina particle abraded and coated with adhesive (with or without silane). CLINICAL SIGNIFICANCE: In order to improve the bonding of resin composite crowns, application of alumina airborne particle abrasion and a coat of adhesive (proceeded by an optional coat of silane) is recommended. If hydrofluoric acid is utilized, the crowns should be treated with a coat of silane followed by adhesive application.

Acoustic Characterization of Echogenic Polymersomes Prepared From Amphiphilic Block Copolymers.

Polymersomes are a class of artificial vesicles prepared from amphiphilic polymers. Like lipid vesicles (liposomes), they too can encapsulate hydrophilic and hydrophobic drug molecules in the aqueous core and the hydrophobic bilayer respectively, but are more stable than liposomes. Although echogenic liposomes have been widely investigated for simultaneous ultrasound imaging and controlled drug delivery, the potential of the polymersomes remains unexplored. We prepared two different echogenic polymersomes from the amphiphilic copolymers polyethylene glycol-poly-DL-lactic acid (PEG-PLA) and polyethylene glycol-poly-L-lactic acid (PEG-PLLA), incorporating multiple freeze-dry cycles in the synthesis protocol to ensure their echogenicity. We investigated acoustic behavior with potential applications in biomedical imaging. We characterized the polymeric vesicles acoustically with three different excitation frequencies of 2.25, 5 and 10 MHz at 500 kPa. The polymersomes exhibited strong echogenicity at all three excitation frequencies (about 50- and 25-dB enhancements in fundamental and subharmonic, respectively, at 5-MHz excitation from 20 µg/mL polymers in solution). Unlike echogenic liposomes, they emitted strong subharmonic responses. The scattering results indicated their potential as contrast agents, which was also confirmed by clinical ultrasound imaging.

Hypoxia Responsive, Tumor Penetrating Lipid Nanoparticles for Delivery of Chemotherapeutics to Pancreatic Cancer Cell Spheroids.

Solid tumors are often poorly irrigated due to structurally compromised microcirculation. Uncontrolled multiplication of cancer cells, insufficient blood flow, and the lack of enough oxygen and nutrients lead to the development of hypoxic regions in the tumor tissues. As the partial pressure of oxygen drops below the necessary level (10 psi), the cancer cells modulate their genetic makeup to survive. Hypoxia triggers tumor progression by enhancing angiogenesis, cancer stem cell production, remodeling of the extracellular matrix, and epigenetic changes in the cancer cells. However, the hypoxic regions are usually located deep in the tumors and are usually inaccessible to the intravenously injected drug carrier or the drug. Considering the designs of the reported nanoparticles, it is likely that the drug is delivered to the peripheral tumor tissues, close to the blood vessels. In this study, we prepared lipid nanoparticles (LNs) comprising the synthesized hypoxia-responsive lipid and a peptide-lipid conjugate. We observed that the resultant LNs penetrated to the hypoxic regions of the tumors. Under low oxygen partial pressure, the hypoxia-responsive lipid undergoes reduction, destabilizing the lipid membrane, and releasing encapsulated drugs from the nanoparticles. We demonstrated the results employing spheroidal cultures of the pancreatic cancer cells BxPC-3. We observed that the peptide-decorated, drug encapsulated LNs reduced the viability of pancreatic cancer cells of the spheroids to 35% under hypoxic conditions.

Hypoxia-Responsive Polymersomes for Drug Delivery to Hypoxic Pancreatic Cancer Cells.

Hypoxia in tumors contributes to overall tumor progression by assisting in epithelial-to-mesenchymal transition, angiogenesis, and metastasis of cancer. In this study, we have synthesized a hypoxia-responsive, diblock copolymer poly(lactic acid)-azobenzene-poly(ethylene glycol), which self-assembles to form polymersomes in an aqueous medium. The polymersomes did not release any encapsulated contents for 50 min under normoxic conditions. However, under hypoxia, 90% of the encapsulated dye was released in 50 min. The polymersomes encapsulated the combination of anticancer drugs gemcitabine and erlotinib with entrapment efficiency of 40% and 28%, respectively. We used three-dimensional spheroid cultures of pancreatic cancer cells BxPC-3 to demonstrate hypoxia-mediated release of the drugs from the polymersomes. The vesicles were nontoxic. However, a significant decrease in cell viability was observed in hypoxic spheroidal cultures of BxPC-3 cells in the presence of drug encapsulated polymersomes. These polymersomes have potential for future applications in imaging and treatment of hypoxic tumors.

Quantitative proteomic analysis of global effect of LLL12 on U87 cell's proteome: An insight into the molecular mechanism of LLL12.

Glioblastoma multiforme (GBM) is one of the most devastating and dreadful WHO grade IV brain tumors associated with poor survival rate and limited therapeutics. Signal transducer and activator of transcription factor 3 (STAT3) is persistently active in several cancers, including gliomas, and STAT3 inhibitors hold great promise for treatment of glioma. LLL12, a curcumin derivative, inhibits STAT3 functions, thereby reduces growth of GBM. However, the global effects of targeting STAT3 using LLL12 have not been studied well. To shed light on this aspect, we performed quantitative proteomic analyses using differential in-gel electrophoresis (2D-DIGE) and isobaric tags for relative and absolute quantitation (iTRAQ) as well as label-free mass spectrometric analysis with 0.5µM (IC50) concentration of LLL12. Through this approach, we identified a total dataset of 1012 proteins with 1% FDR, of which 143 proteins were differentially expressed associated with various cellular functions. Results suggest that LLL12 influences central cellular metabolism and cytoskeletal proteins, in addition to its apoptosis inducing and anti-angiogenic activities, which altogether contribute to its anti-tumorigenic function. Interestingly, triose phosphate isomerase (TPI), phosphoglycerate mutase 1 (PGAM1), adaptor molecule (CRK2), protein DJ-1 (PARK7) and basic transcription factor 3 (BTF3) were found to be down-regulated and can be studied further to understand their therapeutic potential in gliomas. TPI1 and PGAM1 protein expressions were validated using immunoblot. Conclusively, our results suggest the therapeutic potential of LLL12 and it can be investigated further for a significant role in glioma treatment. BIOLOGICAL SIGNIFICANCE: LLL12 holds great promise for therapeutic development in gliomas with constitutive expression of STAT3. This study investigated the global effect of LLL12 on the proteome of U87 glioma cells using complementary proteomic approaches, and our findings suggest that LLL12 influences central metabolism, translation, transport processes, and cytoskeleton of a cell in addition to its anti-angiogenic and apoptosis inducing functions which altogether contributes to anti-tumorigenic activity of LLL12. This study leads to the identification of several proteins which may serve as prognostic or predictive markers in GBM. We identified TPI1, PGAM1, CRK and BTF3 as potential therapeutic targets and further investigations on these candidates may facilitate therapeutic development.

Mmp-9 responsive PEG cleavable nanovesicles for efficient delivery of chemotherapeutics to pancreatic cancer.

Significant differences in biochemical parameters between normal and tumor tissues offer an opportunity to chemically design drug carriers which respond to these changes and deliver the drugs at the desired site. For example, overexpression of the matrix metalloproteinase-9 (MMP-9) enzyme in the extracellular matrix of tumor tissues can act as a trigger to chemically modulate the drug delivery from the carriers. In this study, we have synthesized an MMP-9-cleavable, collagen mimetic lipopeptide which forms nanosized vesicles with the POPC, POPE-SS-PEG, and cholesteryl-hemisuccinate lipids. The lipopeptide retains the triple-helical conformation when incorporated into these nanovesicles. The PEG groups shield the substrate lipopeptides from hydrolysis by MMP-9. However, in the presence of elevated glutathione levels, the PEG groups are reductively removed, exposing the lipopeptides to MMP-9. The resultant peptide-bond cleavage disturbs the vesicles' lipid bilayer, leading to the release of encapsulated contents. These PEGylated nanovesicles are capable of encapsulating the anticancer drug gemcitabine with 50% efficiency. They were stable in physiological conditions and in human serum. Effective drug release was demonstrated using the pancreatic ductal carcinoma cells (PANC-1 and MIAPaCa-2) in two-dimensional and three-dimensional "tumor-like" spheroid cultures. A reduction in tumor growth was observed after intravenous administration of the gemcitabine-encapsulated nanovesicles in the xenograft model of athymic, female nude mice.

High-performance liquid chromatographic fingerprint for quality control of Terminalia arjuna containing Ayurvedic churna formulation.

Because Ayurvedic herbal preparations contain a myriad of compounds in complex matrixes, it is difficult to establish quality control standards for raw materials and to standardize finished Ayurvedic drugs. A novel, accurate, and valid fingerprint method was developed using HPLC for quality control of a traditional Ayurvedic Arjuna churna formulation, which is used as a cardiotonic drug. Comprehensive comparison of chromatograms of standardized formulation of Arjuna churna and marketed formulations revealed eight characteristic peaks in chromatograms, which unambiguously confirmed the presence of authentic raw material used in the formulation on the basis of their retention time values and UV data. An HPLC fingerprint was also developed for total sapogenins present in Terminalia arjuna. The six common peaks observed in chromatograms of isolated sapogenins, standardized formulations, and marketed formulations can serve as a quality control tool for qualitative estimation of total saponin glycosides present in an Arjuna churna formulation.

Ocular defects in cerebral palsy.

There is a high prevalence of ocular defects in children with developmental disabilities. This study evaluated visual disability in a group of 200 cerebral palsy (CP) patients and found that 68% of the children had significant visual morbidity. These findings emphasize the need for an early ocular examination in patients with CP.