Genetic comparison among various coronavirus strains for the identification of potential vaccine targets of SARS-CoV2.

On-going pandemic pneumonia outbreak COVID-19 has raised an urgent public health issue worldwide impacting millions of people with a continuous increase in both morbidity and mortality. The causative agent of this disease is identified and named as SARS-CoV2 because of its genetic relatedness to SARS-CoV species that was responsible for the 2003 coronavirus outbreak. The immense spread of the disease in a very small period demands urgent development of therapeutic and prophylactic interventions for the treatment of SARS-CoV2 infected patients. A plethora of research is being conducted globally on this novel coronavirus strain to gain knowledge about its origin, evolutionary history, and phylogeny. This review is an effort to compare genetic similarities and diversifications among coronavirus strains, which can hint towards the susceptible antigen targets of SARS-CoV2 to come up with the potential therapeutic and prophylactic interventions for the prevention of this public threat.

Design and Characterization of Apocynin Loaded PLGA Nanoparticles and their In vivo Efficacy in Hyperoxaluric Rats.

BACKGROUND: Apocynin has become a drug of choice in NADPH oxidase induced pathological conditions. Hyperoxaluria is one such pathological condition where NADPH oxidase is involved in eliciting renal injury. OBJECTIVE: Recently apocynin has shown to reverse the transcriptome profile of the NADPH oxidaseassociated genes and reduced oxidative burden in hyperoxaluric animals. The poor solubility of this drug creates certain apprehensions about its bioavailability. PLGA (Poly Lactic co-Glycolic Acid) encapsulation of drug nanoparticles have showed to induce sustain release and henceforth enhance the efficiency and bioavailability of drugs. Therefore, the present study is aimed to envisage a novel approach of synthesizing apocynin doped PLGA nanoparticles. METHODS: The PLGA nanoparticles (both unloaded and loaded) were prepared using solvent extraction method and analyzed for size and stability by Dynamic Light Scattering (DLS), TEM (transmission electron microscopy) and zeta potential. Furthermore, the drug release and encapsulation efficiency of the drug was calculated in vitro. RESULTS: The nanoencapsulation formed was stable with desired size (217-259 nm) and posses a controlled drug release of 20%. Further this nanoencapsulation was explored for its potential to reduce hyperoxaluric manifestations in rats given ethylene glycol with ammonium chloride for 9 days. CONCLUSION: In comparison to free apocynin, it was found that nanoparticles containing apocynin showed moderately better results in vivo by maintaining serum urea and createnine levels. These nanoparticles can be used in diseases where a sustained release of apocynin is required.

Implication of hyperoxaluria on osteopontin and ER stress mediated apoptosis in renal tissue of rats.

Hyperoxaluria is a stress that leads to calcium oxalate crystal deposition which further causes inflammation and renal cell necroptosis. Many studies have linked osteopontin expression with apoptosis and inflammation but so far its association with apoptosis with regard to hyperoxaluria is undiscovered. Moreover, a recent report has suggested that osteopontin induces endoplasmic reticulum stress and subsequently apoptosis in myocytes. In this study, the impact of hyperoxaluria on the modulation of osteopontin expression and endoplasmic reticulum (ER) stress mediated apoptosis in rats is explored. Hyperoxaluria was induced in rats by three different doses viz. ethylene glycol alone, ethylene glycol and ammonium chloride together and third group were fed with hydroxyl-l-proline. After hyperoxaluria induction rats were sacrificed and renal tissue was analysed for crystal depositions, osteopontin expression, inflammation, ER stress and subsequent unfolded protein response intermediates (UPR). Altered histoarchitecture of renal tissue and elevated levels of reactive oxygen species (ROS) along with the presence of calcium oxalate crystals were observed in the hyperoxaluric groups. As expected, inflammation and apoptosis was significantly high in all hyperoxaluria groups. Osteopontin expression showed significant up-regulation following hyperoxaluria. Further, a similar trend between expression of osteopontin and elevated ER stress level was observed. Moreover, UPR intermediates expression was also concurrent with osteopontin levels. It is observed that the extent of calcium oxalate crystal deposition is directly associated with the expression of osteopontin, inflammation and ER stress. The results advocate possible association of osteopontin with ER stress, thus suggesting that the ER could be a new target for developing therapeutic regimes for kidney stones.

Calcification of vascular smooth muscle cells is induced by secondary calciprotein particles and enhanced by tumor necrosis factor-α.

BACKGROUND AND AIMS: Vascular calcification is prevalent in clinical states characterized by low-grade chronic inflammation, such as chronic kidney disease (CKD). Calciprotein particles (CPP) are calcium phosphate-containing nano-aggregates, which have been found in the blood of CKD patients and appear pro-inflammatory in vitro. The interplay of CPPs and inflammatory cytokines with regard to the calcification of vascular smooth muscle cells (VSMC), in vitro, has not been investigated yet. METHODS: Primary or secondary CPP were generated using phosphate-enriched culture medium (DMEM/10% FBS) incubated at 37 °C. Human VSMC were cultured with these media and mineralization was measured. Expression of TNF-α was detected by qPCR, ELISA and Western blot in calcified VSMC. To further characterize the significance of TNF-α and its receptors for the calcification of VSMC, RNA interference experiments using siTNF-α, siTNFR1 and siTNFR2 were performed. RESULTS: The addition of phosphate to cell culture medium containing DMEM/10% FBS led to the rapid formation of primary CPP, which underwent spontaneous transformation to secondary CPP. Exposure of VSMC towards secondary CPP led to pronounced and concentration-dependent calcification, whereas exposure towards primary CPP did not. Importantly, secondary CPP induced oxidative stress, and led to the up-regulation and release of TNF-α. Addition of TNF-α to the cell culture medium enhanced, whereas the suppression of endogenous TNF-α or TNF receptor type 1 (TNFR1) expression by siRNA, ameliorated calcification. CONCLUSIONS: Secondary, but not primary CPP, induce VSMC calcification. Secondary CPP induce the expression and release of TNF-α, which enhances calcification via its receptor TNFR1.

Sodium thiosulfate ameliorates oxidative stress and preserves renal function in hyperoxaluric rats.

BACKGROUND: Hyperoxaluria causes crystal deposition in the kidney, which leads to oxidative stress and to injury and damage of the renal epithelium. Sodium thiosulfate (STS, Na2S2O3) is an anti-oxidant, which has been used in human medicine for decades. The effect of STS on hyperoxaluria-induced renal damage is not known. METHODS: Hyperoxaluria and renal injury were induced in healthy male Wistar rats by chronic exposure to ethylene glycol (EG, 0.75%) in the drinking water for 4 weeks. The treatment effects of STS, NaCl or Na2SO4 were compared. Furthermore, the effects of STS on oxalate-induced oxidative stress were investigated in vitro in renal LLC-PK1 cells. RESULTS: Chronic EG exposure led to hyperoxaluria, oxidative stress, calcium oxalate crystalluria and crystal deposition in the kidneys. Whereas all tested compounds significantly reduced crystal load, only STS-treatment maintained tissue superoxide dismutase activity and urine 8-isoprostaglandin levels in vivo and preserved renal function. In in vitro studies, STS showed the ability to scavenge oxalate-induced ROS accumulation dose dependently, reduced cell-released hydrogen peroxide and preserved superoxide dismutase activity. As a mechanism explaining this finding, STS was able to directly inactivate hydrogen peroxide in cell-free experiments. CONCLUSIONS: STS is an antioxidant, which preserves renal function in a chronic EG rat model. Its therapeutic use in oxidative-stress induced renal-failure should be considered.

The most potent antilithiatic agent ameliorating renal dysfunction and oxidative stress from Bergenia ligulata rhizome.

ETHNOPHARMACOLOGICAL RELEVANCE: The rhizome of Bergenia ligulata is referred by the Ayurvedic system for the treatment of kidney stone since decades and a few, in vitro and in vivo studies also support it. To identify the main phytochemical constituent(s) responsible for antilithiatic activity of its rhizome. MATERIALS AND METHODS: In order to identify the most potent antilithiatic metabolite, the crude extract of rhizome was fractionated using in vitro Calcium oxalate (CaOx) crystal growth inhibitory activity guided fractionation followed by its characterization via LC-MS, FTIR and NMR. Further, the antioxidant potential of purified molecule was assessed using in vitro assays (FRAP and H2O2 scavenging). In vivo activity of the metabolite was evaluated in hyperoxaluric rats given 0.4% ethylene glycol (EG) and 1.0% ammonium chloride (NH4Cl) for 9 days. RESULTS: Activity guided fractionation led to the isolation of most potent antilithiatic metabolite from the rhizome of Bergenia ligulata and spectroscopic analysis revealed it as bergenin. Bergenin showed reducing ability and H2O2 scavenging activity comparable with commercially available anitioxidant, α-tocopherol. At a dose of 10mg/kg body weight of the treated rat, it protected against deleterious effects of lithogenic treatment including weight loss, impaired renal function and oxidative stress, manifested as increased malondialdehyde, reduced redox ratio and decreased antioxidant enzyme activities in the kidneys of hyperoxaluric rats. The creatinine clearance and kidney damage were more improved by bergenin as compared to crude extract of rhizome. CONCLUSIONS: Since, bergenin maintained oxidant/antioxidant balance in hyperoxaluric rats, thus mechanistic insight of its antilithiatic activity was attributed to the antioxidant capability of bergenin. The results of the present study provide significant evidence that bergenin is an active component present in the rhizome of Bergenia ligulata for managing CaOx calculi.

In vivo efficacy of Trachyspermum ammi anticalcifying protein in urolithiatic rat model.

ETHNOPHARMACOLOGICAL RELEVANCE: Many medicinal plants have been employed during ages to treat urinary stones though the rationale behind their use is not well established. Recently, we have successfully purified an anticalcifying protein from the seeds of Trachyspermum ammi (L.) Sprague ex Turril (Umbelliferae) using oxalate depletion assay and deciphered its inhibitory activity against calcium oxalate crystal growth. AIM: In this report, the antilithiatic activity of Trachyspermum ammi anticalcifying protein (TAP) was studied in urolithiatic rat model. METHODOLOGY: Urolithiasis was induced by exposure of 0.4% ethylene glycol (EG) and 1.0% ammonium chloride (NH(4)Cl) for 9 days. The efficacy of TAP was studied in another group given same dose of EG and NH(4)Cl in addition to 2mg/kg body weight of TAP. Further, we evaluated ability of TAP to inhibit the attachment of calcium oxalate (CaO(x)) crystal in kidney tissue and studied the consequences of CaO(x) adhesion on renal functioning and tissue integrity. RESULTS: The antilithiatic potential of TAP was confirmed by its ability to maintain renal functioning, reduce renal injury and decrease crystal excretion in urine and retention in renal tissues. CONCLUSIONS: Thus, the present investigation suggests the potential of TAP in preventing calcium oxalate deposition and forms the basis for the development of antilithiatic drug interventions against urolithiasis.

A novel calcium oxalate crystal growth inhibitory protein from the seeds of Dolichos biflorus (L.).

Recurrence and persistent side effects of present day treatment for urolithiasis restrict their use, so an alternate, using phytotherapy is being sought. Dolichos biflorus seeds, which are used as dietary food in India, possess antilithiatic properties. In the present study, a novel dimeric antilithiatic protein (98 kDa) from its seeds was purified based on its ability to inhibit calcium oxalate crystallization in vitro. Amino acid analysis of Dolichos biflorus antilithiatic protein showed abundant acidic amino acids. The mascot search engine presented sequence similarity with a calcium binding protein, calnexin of Pisum sativum from the m/z data obtained by MALDI TOF mass spectrometer. Above results demonstrate the anticalcifying/antilithiatic nature of a novel protein from the seeds of Dolichos biflorus and thus open new vistas for using plant proteins as therapeutic agents to treat urolithiasis.

Effect of concurrent chronic exposure of fluoride and aluminum on rat brain.

The present in vivo study was designed to investigate the toxic potential of fluoride alone and in conjugation with aluminum on the rat brain. The region-specific response of both elements was studied in different regions of brain, namely the cerebrum, cerebellum, and medulla oblongata. Following fluoride exposure, oxidative stress increased significantly, estimated by increased lipid peroxidation and a decrease in the activity of the antioxidant enzyme, superoxide dismutase. The neurotransmitter (e.g., dopamine, norepinephrine, and serotonin) content was also altered. However, these aspects were more pronounced in animals given fluoride and aluminum together. Histological evidence showed deprivation of neuronal integrity with higher magnitude in concurrent fluoride and aluminum exposure, as compared to fluoride alone. Thus, it can be concluded that aluminum appears to enhance the neurotoxic hazards caused by fluoride.

Purification and characterization of an anticalcifying protein from the seeds of Trachyspermum ammi (L.).

Till date various plants extract have been studied to reduce the incidence of urolithiasis but the identification of naturally occurring calcium oxalate (CaOx) inhibitory biomolecules from plants was hampered in past by limitation in identification method. The present study is aimed at examining the efficacy of Trachyspermum ammi on CaOx crystallization in vitro and further by combining conventional biochemical methods with recent advances in mass spectrometry, a novel calcium oxalate (CaOx) crystal growth inhibitor was purified from the seeds of Trachyspermum ammi. An anticalcifying protein from the seeds of Trachyspermum ammi was purified by three step purification scheme; ammonium sulphate fractionation, anion exchange chromatography and molecular sieve chromatography based on its ability to inhibit calcium oxalate crystallization in vitro. An anticalcifying protein having molecular weight 107 kDa and isolectric point 6.2 was isolated. Amino acid analysis of Trachyspermum ammi anticalcifying protein (TAP) showed abundant presence of acidic amino acids (Asp and Glu). Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry of TAP showed similarities with an unnamed protein product of Vitis vinifera (CAO23876) after matching peptide mass fingerprints in MASCOT search engine. Two EF hand domains were identified in unnamed protein product of Vitis vinifera (CAO23876) by SMART normal module. Due to a significant similarity of TAP with unnamed protein product of Vitis vinifera, presence of two EF hand domains in TAP was anticipated, signifying its calcium binding properties which is a feature of most kidney stone inhibitory proteins.

Modulatory effects of N-acetylcysteine on hyperoxaluric manifestations in rat kidney.

Hyperoxaluria is a condition where excessive oxalate is present in the urine. Many reports have documented free radical generation followed by hyperoxaluria as a consequence of which calcium oxalate deposition occurs in the kidney tissue. The present invivo study was designed to investigate the potential of N-acetylcysteine in modulating hyperoxaluric manifestation induced by sodium oxalate in the rat kidneys. Male wistar rats in one group were administered single dose of sodium oxalate (70mg/kg body weight) intraperitoneally to induce hyperoxaluric conditions and in the other group, rats were injected N-acetylcysteine (NAC) (200mg/kg body weight) intraperitoneally, half an hour after sodium oxalate dose. The treatment is for a period of 24h. N-acetylcysteine significantly reduced hyperoxaluria caused oxidative stress by reducing lipid peroxidation, restoring antioxidant enzymes activity in kidney tissue, followed by reduction in impairment of renal functioning. In addition, NAC administration reduced the number of calcium oxalate monohydrate (COM) crystals in the urine as observed under polarization microscope. Histological analysis depicted that NAC treatment decreased renal epithelial damage, inflammation and restored normal glomeruli morphology. Thus, it shows that use of an extraneous antioxidant may prove beneficial for combating the conditions of oxidative stress produced by hyperoxaluria.