Analysis of the Biomarkers for Neurodegenerative Diseases in Aged Progranulin Deficient Mice.

Neurodegenerative diseases are debilitating impairments that affect millions of people worldwide and are characterized by progressive degeneration of structure and function of the central or peripheral nervous system. Effective biomarkers for neurodegenerative diseases can be used to improve the diagnostic workup in the clinic as well as facilitate the development of effective disease-modifying therapies. Progranulin (PGRN) has been reported to be involved in various neurodegenerative disorders. Hence, in the current study we systematically compared the inflammation and accumulation of typical neurodegenerative disease markers in the brain tissue between PGRN knockout (PGRN KO) and wildtype (WT) mice. We found that PGRN deficiency led to significant neuron loss as well as activation of microglia and astrocytes in aged mice. Several characteristic neurodegenerative markers, including α-synuclein, TAR DNA-binding protein 43 (TDP-43), Tau, and ß-amyloid, were all accumulated in the brain of PGRN-deficient mice as compared to WT mice. Moreover, higher aggregation of lipofuscin was observed in the brain tissue of PGRN-deficient mice compared with WT mice. In addition, the autophagy was also defective in the brain of PGRN-deficient mice, indicated by the abnormal expression level of autophagy marker LC3-II. Collectively, comprehensive assays support the idea that PGRN plays an important role during the development of neurodegenerative disease, indicating that PGRN might be a useful biomarker for neurodegenerative diseases in clinical settings.

Calcium fluoride nanoparticles induced suppression of Streptococcus mutans biofilm: an in vitro and in vivo approach.

Biofilm formation on the tooth surface is the root cause of dental caries and periodontal diseases. Streptococcus mutans is known to produce biofilm which is one of the primary causes of dental caries. Acid production and acid tolerance along with exopolysaccharide (EPS) formation are major virulence factors of S. mutans biofilm. In the current study, calcium fluoride nanoparticles (CaF2-NPs) were evaluated for their effect on the biofilm forming ability of S. mutans in vivo and in vitro. The in vitro studies revealed 89 % and 90 % reduction in biofilm formation and EPS production, respectively. Moreover, acid production and acid tolerance abilities of S. mutans were also reduced considerably in the presence of CaF2-NPs. Confocal laser scanning microscopy and transmission electron microscopy images were in accordance with the other results indicating inhibition of biofilm without affecting bacterial viability. The qRT-PCR gene expression analysis showed significant downregulation of various virulence genes (vicR, gtfC, ftf, spaP, comDE) associated with biofilm formation. Furthermore, CaF2-NPs were found to substantially decrease the caries in treated rat groups as compared to the untreated groups in in vivo studies. Scanning electron micrographs of rat's teeth further validated our results. These findings suggest that the CaF2-NPs may be used as a potential antibiofilm applicant against S. mutans and may be applied as a topical agent to reduce dental caries.

Inhibitory effect of zingiber officinale towards Streptococcus mutans virulence and caries development: in vitro and in vivo studies.

BACKGROUND: Streptococcus mutans is known as a key causative agent of dental caries. It metabolizes dietary carbohydrate to produce acids which reduce the environmental pH leading to tooth demineralization. The ability of this bacterium to tolerate acids coupled with acid production, allows its effective colonization in the oral cavity leading to the establishment of highly cariogenic plaque. For this reason, S. mutans is the only bacterium found in significantly higher numbers than other bacteria in the dental plaque. The aim of this study was to evaluate the effect of crude extract and methanolic fraction of Z. officinale against S. mutans virulence properties. RESULTS: We investigated in vitro and in vivo activity of crude extract and methanolic fraction at sub- MIC levels against cariogenic properties of S. mutans. We found that these extracts strongly inhibited a variety of virulence properties which are critical for its pathogenesis. The biofilm formation in S. mutans was found to be reduced during critical growth phases. Furthermore, the glucan synthesis and adherence was also found to be inhibited. Nevertheless, the insoluble glucan synthesis and sucrose dependent adherence were apparently more reduced as compared to soluble glucan synthesis and sucrose- independent adherence. Biofilm architecture inspected with the help of confocal and scanning electron microscopy, showed dispersion of cells in the treated group as compared to the control. The Quantitative Real Time PCR (qRT-PCR) data had shown the down regulation of the virulence genes, which is believed to be one of the major reasons responsible for the observed reduction in the virulence properties. The incredible reduction of caries development was found in treated group of rats as compared to the untreated group which further validate our in vitro data. CONCLUSION: The whole study concludes a prospective role of crude extract and methanolic fraction of Z. officinale in targeting complete array of cariogenic properties of S. mutans, thus reducing its pathogenesis. Hence, it may be strongly proposed as a putative anti- cariogenic agent.

Identification of natural compounds (proanthocyanidin and rhapontin) as high-affinity inhibitors of SARS-CoV-2 Mpro and PLpro using computational strategies.

Introduction: The emergence of a new and highly pathogenic coronavirus (SARS-CoV-2) in Wuhan (China) and its spread worldwide has resulted in enormous social and economic losses. Amongst many proteins encoded by the SARS-CoV-2 genome, the main protease (Mpro) or chymotrypsin-like cysteine protease (3CLpro) and papain-like protease (PLpro) serve as attractive drug targets. Material and methods: We screened a library of 2267 natural compounds against Mpro and PLpro using high throughput virtual screening (HTVS). Fifty top-scoring compounds against each protein in HTVS were further evaluated by standard-precision (SP) docking. Compounds with SP docking energy of ≤ -8.0 kcal/mol against Mpro and ≤ -5.0 kcal/mol against PLpro were subjected to extra-precision (XP) docking. Finally, six compounds against each target proteins were identified and subjected to Prime/MM-GBSA free energy calculations. Compounds with the lowest Prime/MM-GBSA energy were subjected to molecular dynamics simulation to evaluate the stability of protein-ligand complexes. Results: Proanthocyanidin and rhapontin were identified as the most potent inhibitors of Mpro and PLpro, respectively. Analysis of protein-inhibitor interaction revealed that both protein-inhibitor complexes were stabilized by hydrogen bonding and hydrophobic interactions. Proanthocyanidin interacted with the catalytic residues (His41 and Cys145) of Mpro, while rhapontin contacted the active site residues (Trp106, His272, Asp286) of PLpro. The docking energies of proanthocyanidin and rhapontin towards their respective targets were -10.566 and -10.022 kcal/mol. Conclusions: This study's outcome may support application of proanthocyanidin and rhapontin as a scaffold to build more potent inhibitors with desirable drug-like properties. However, it requires further validation by in vitro and in vivo studies.

Dietary pyruvate targets cytosolic phospholipase A2 to mitigate inflammation and obesity in mice.

Obesity has a multifactorial etiology and is known to be a state of chronic low-grade inflammation, known as meta-inflammation. This state is associated with the development of metabolic disorders such as glucose intolerance and nonalcoholic fatty liver disease. Pyruvate is a glycolytic metabolite and a crucial node in various metabolic pathways. However, its role and molecular mechanism in obesity and associated complications are obscure. In this study, we reported that pyruvate substantially inhibited adipogenic differentiation in vitro and its administration significantly prevented HFD-induced weight gain, white adipose tissue inflammation, and metabolic dysregulation. To identify the target proteins of pyruvate, drug affinity responsive target stability was employed with proteomics, cellular thermal shift assay, and isothermal drug response to detect the interactions between pyruvate and its molecular targets. Consequently, we identified cytosolic phospholipase A2 (cPLA2) as a novel molecular target of pyruvate and demonstrated that pyruvate restrained diet-induced obesity, white adipose tissue inflammation, and hepatic steatosis in a cPLA2-dependent manner. Studies with global ablation of cPLA2 in mice showed that the protective effects of pyruvate were largely abrogated, confirming the importance of pyruvate/cPLA2 interaction in pyruvate attenuation of inflammation and obesity. Overall, our study not only establishes pyruvate as an antagonist of cPLA2 signaling and a potential therapeutic option for obesity, but it also sheds light on the mechanism of its action. Pyruvate's prior clinical use indicates that it can be considered a safe and viable alternative for obesity, whether consumed as a dietary supplement or as part of a regular diet.

Novel peptide inhibitor of human tumor necrosis factor-α has antiarthritic activity.

The inhibition of tumor necrosis factor (TNF)-α trimer formation renders it inactive for binding to its receptors, thus mitigating the vicious cycle of inflammation. We designed a peptide (PIYLGGVFQ) that simulates a sequence strand of human TNFα monomer using a series of in silico methods, such as active site finding (Acsite), protein-protein interaction (PPI), docking studies (GOLD and Flex-X) followed by molecular dynamics (MD) simulation studies. The MD studies confirmed the intermolecular interaction of the peptide with the TNFα. Fluorescence-activated cell sorting and fluorescence microscopy revealed that the peptide effectively inhibited the binding of TNF to the cell surface receptors. The cell culture assays showed that the peptide significantly inhibited the TNFα-mediated cell death. In addition, the nuclear translocation of the nuclear factor kappa B (NFκB) was significantly suppressed in the peptide-treated A549 cells, as observed in immunofluorescence and gel mobility-shift assays. Furthermore, the peptide protected against joint damage in the collagen-induced arthritis (CIA) mouse model, as revealed in the micro focal-CT scans. In conclusion, this TNFα antagonist would be helpful for the prevention and repair of inflammatory bone destruction and subsequent loss in the mouse model of CIA as well as human rheumatoid arthritis (RA) patients. This calls upon further clinical investigation to utilize its potential effect as an antiarthritic drug.

Interaction with ERp57 is required for progranulin protection against Type 2 Gaucher disease.

Gaucher disease (GD), one of the most common lysosomal storage diseases, is caused by GBA1 mutations resulting in defective glucocerebrosidase (GCase) and consequent accumulation of its substrates ß-glucosylceramide (ß-GlcCer). We reported progranulin (PGRN), a secretary growth factor-like molecule and an intracellular lysosomal protein was a crucial co-factor of GCase. PGRN binds to GCase and recruits Heat Shock Protein 70 (Hsp70) to GCase through its C-terminal Granulin (Grn) E domain, termed as ND7. In addition, both PGRN and ND7 are therapeutic against GD. Herein we found that both PGRN and its derived ND7 still displayed significant protective effects against GD in Hsp70 deficient cells. To delineate the molecular mechanisms underlying PGRN's Hsp70-independent regulation of GD, we performed a biochemical co-purification and mass spectrometry with His-tagged PGRN and His-tagged ND7 in Hsp70 deficient cells, which led to the identification of ERp57, also referred to as protein disulfide isomerase A3 (PDIA3), as a protein that binds to both PGRN and ND7. Within type 2 neuropathic GD patient fibroblasts L444P, bearing GBA1 L444P mutation, deletion of ERp57 largely abolished the therapeutic effects of PGRN and ND7, as manifested by loss of effects on lysosomal storage, GCase activity, and ß-GlcCer accumulation. Additionally, recombinant ERp57 effectively restored the therapeutic effects of PGRN and ND7 in ERp57 knockout L444P fibroblasts. Collectively, this study reports ERp57 as a previously unrecognized binding partner of PGRN that contributes to PGRN regulation of GD.

Injectable recombinant block polymer gel for sustained delivery of therapeutic protein in post traumatic osteoarthritis.

Protein-based biomaterials offer several advantages over synthetic materials, owing to their unique stimuli-responsive properties, biocompatibility and modular nature. Here, we demonstrate that E5C, a recombinant protein block polymer, consisting of five repeats of elastin like polypeptide (E) and a coiled-coil domain of cartilage oligomeric matrix protein (C), is capable of forming a porous networked gel at physiological temperature, making it an excellent candidate for injectable biomaterials. Combination of E5C with Atsttrin, a chondroprotective engineered derivative of anti-inflammatory growth factor progranulin, provides a unique biochemical and biomechanical environment to protect against post-traumatic osteoarthritis (PTOA) onset and progression. E5C gel was demonstrated to provide prolonged release of Atsttrin and inhibit chondrocyte catabolism while facilitating anabolic signaling in vitro. We also provide in vivo evidence that prophylactic and therapeutic application of Atsttrin-loaded E5C gels protected against PTOA onset and progression in a rabbit anterior cruciate ligament transection model. Collectively, we have developed a unique protein-based gel capable of minimally invasive, sustained delivery of prospective therapeutics, particularly the progranulin-derivative Atsttrin, for therapeutic application in OA.

Antiviral potential of some novel structural analogs of standard drugs repurposed for the treatment of COVID-19.

SARS-CoV-2 pandemic has claimed millions of lives across the world. As of June 2020, there is no FDA approved antiviral therapy to eradicate this dreadful virus. In this study, drug re-purposing and computational approaches were employed to identify high affinity inhibitors of SARS-CoV-2 Main protease (3CLpro), Papain-like protease (PLpro) and the receptor domain of Spike protein. Molecular docking on 40 derivatives of standard drugs (Remdesivir, Lopinavir and Theophylline) led to the identification of R10, R2 and L9 as potential inhibitors of 3CLpro, PLpro and Spike protein, respectively. The binding affinity of R10, R2 and L9 towards 3CLpro, PLpro and Spike protein were 4.03 × 106, 3.72 × 104 and 1.31 × 104M-1, respectively. These inhibitors interact with the active site or catalytic amino acid residues of 3CLpro, PLpro and Spike protein. We also examined the stability and dynamic behavior of protein-inhibitor complex by employing molecular dynamics simulation. RMSDs, RMSFs and variation in secondary structure of target proteins alone or in complex with their respective inhibitors were used to ascertain the integrity of proteins' structure during simulation. Moreover, physicochemical and ADMET properties of R10, R2 and L9 along with Remdesivir, Lopinavir and Theophylline were determined. In vitro and In vivo studies are needed to further validate the potential of these derivatives before they can be developed into potential drug molecules.Communicated by Ramaswamy H. Sarma.

Genotoxic assessment of chlorhexidine mouthwash on exfoliated buccal epithelial cells in chronic gingivitis patients.

BACKGROUND: Chlorhexidine (CHX) is the gold standard of all chemical plaque control agents and the most commonly prescribed mouthwash. However, several studies have shown cytotoxic and genotoxic effects of CHX on various eukaryotic cells. In this study, we have used micronuclei as a biomarker of DNA damage in buccal epithelial cells of chronic gingivitis patients who were given adjunct 0.2% CHX for plaque control. MATERIALS AND METHODS: Chronic gingivitis patients who were exclusively on mechanical plaque control methods were taken as control (Group A) (n = 101), and chronic gingivitis patients who along with mechanical plaque control measures were taking 0.2% chlorhexidine mouthwash as adjunct were taken as cases (Group B) (n = 255). The Group B was further divided into 5 subgroups (B1, B2, B3, B4, B5) (n = 51) on increasing duration of usage of CHX from ≤1 week to 24 weeks. Buccal epithelial cells were gently scrapped from the buccal mucosa using soft toothbrush. The epithelial cells were collected in buffer solution and centrifuged at 8000 rpm for 5 min. The buccal epithelial cells were air dried, fixed, and stained with 5% Giemsa stain on preheated glass microscopic slides and observed under microscope to screen 2000 nucleated cells per individual for number of micronucleated cells and micronuclei as genotoxic measure. RESULTS: The mean number of micronucleated cells was found to be 0.41 ± 0.71 for Group A as compared values ranging from 1.65 ± 2.09 (Group B1) to 11.7 ± 1.87 (Group B5) in different subgroups of Group B, and similarly, the mean number of micronuclei was found to be 0.48 ± 0.80 for Group A as compared to values ranging from 2.57 ± 1.64 (Group B1) to 14.5 ± 2.49 (Group B5) in different subgroups of Group B using analysis of variance (P < 0.001). CONCLUSION: We conclude that CHX mouthwash is genotoxic to buccal epithelial cells and there is incremental trend in genotoxicity as the duration of usage is increased.

Inhibition of major virulence pathways of Streptococcus mutans by quercitrin and deoxynojirimycin: a synergistic approach of infection control.

OBJECTIVES: To evaluate the synergistic effect of Quercitrin and Deoxynojirimycin (DNJ) together with their individual inhibitory effect against virulence pathways of Streptococcus mutans. METHODOLOGY: MICs of both the compounds were determined by the microdilution method, followed by their in vitrosynergy using checkerboard and time kill assay. The nature of interaction was classified as synergistic on the basis of fractional inhibitory concentration index (FICI) value of ≤0.5. Furthermore, the activity of Quercitrin and DNJ was evaluated individually and in combination against various cariogenic properties of S. mutans UA159 such as acidogenesis, aciduracity, glucan production, hydrophobicity, biofilm and adherence. Moreover, expression of virulent genes in S. mutans was analysed by quantitative RT- PCR (qRT-PCR) and inhibition of F1F0-ATPase, lactate dehydrogenase and enolase was also evaluated. Finally, scanning electron microscopy (SEM) was used to investigate structural obliteration of biofilm. RESULTS: The in vitro synergism between Quercitrin and DNJ was observed, with a FICI of 0.313. Their MIC values were found to be 64 µg/ml and 16 µg/ml respectively. The synergistic combination consistently showed best activity against all the virulence factors as compared to Quercitrin and DNJ individually. A reduction in glucan synthesis and biofilm formation was observed at different phases of growth. The qRT-PCR revealed significant downregulation of various virulent genes. Electron micrographs depicted the obliteration of biofilm as compared to control and the activity of cariogenic enzymes was also inhibited. CONCLUSIONS: The whole study reflects a prospective role of Quercitrin and DNJ in combination as a potent anticariogenic agent against S. mutans.

Novel combinations of antibiotics to inhibit extended-spectrum ß-lactamase and metallo-ß-lactamase producers in vitro: a synergistic approach.

AIM: To determine the synergistic effect of different antibiotics, alone and in combination, against extended-spectrum ß-lactamase and metallo-ß-lactamase producers. MATERIALS & METHODS: MIC values of different antibiotics were determined for a total of 12 strains, of which nine strains were of clinical origin and the remainder were reference strains. In vitro synergy testing was performed for all the isolates using microdilution, chequerboard techniques and time-kill assays. RESULTS: Out of the all the combinations, cefoxitin gave the highest rate of synergy by pairing up with streptomycin and cefotaxime to form effective combinations against multidrug-resistant strains of bacteria. However, the combination of cefotaxime and cefoxitin showed synergistic activity against both NDM-1-producing strains. CONCLUSION: Novel synergistic combinations were observed against different resistant strains, including NDM-1 producers.

Efficacy of E. officinalis on the cariogenic properties of Streptococcus mutans: a novel and alternative approach to suppress quorum-sensing mechanism.

The present study was focused on evaluating the potential of Emblica officinalis against cariogenic properties of Streptococcus mutans, a causative microorganism for caries. The effect of crude extract and ethanolic fraction from Emblica officinalis fruit was analysed against S. mutans. The sub-MIC concentrations of crude and ethanolic fraction of E. officinalis were evaluated for its cariogenic properties such as acid production, biofilm formation, cell-surface hydrophobicity, glucan production, sucrose-dependent and independent adherence. Its effect on biofilm architecture was also investigated with the help of confocal and scanning electron microscopy (SEM). Moreover, expression of genes involved in biofilm formation was also studied by quantitative RT- PCR. This study showed 50% reduction in adherence at concentrations 156 µg/ and 312.5 µg/ml of crude extract and ethanolic fraction respectively. However, the biofilm was reduced to 50% in the presence of crude extract (39.04 µg/ml) and ethanolic fraction (78.08 µg/ml). Furthermore, effective reduction was observed in the glucan synthesis and cell surface hydrophobicity. The qRT-PCR revealed significant suppression of the genes involved in its virulence. Confocal and scanning electron microscopy clearly depicted the obliteration of biofilm structure with reference to control. Hence, this study reveals the potential of E. officinalis fruit extracts as an alternative and complementary medicine for dental caries by inhibiting the virulence factors of Streptococcus mutans.