Structure-based identification of naphthoquinones and derivatives as novel inhibitors of main protease Mpro and papain-like protease PLpro of SARS-CoV-2

The worldwide COVID-19 pandemic caused by the coronavirus SARS-CoV-2 urgently demands novel direct antiviral treatments. The main protease (Mpro) and papain-like protease (PLpro) are attractive drug targets among coronaviruses due to their essential role in processing the polyproteins translated from the viral RNA. In the present work, we virtually screened 688 naphthoquinoidal compounds and derivatives against Mpro of SARS-CoV-2. Twenty-four derivatives were selected and evaluated in biochemical assays against Mpro using a novel fluorogenic substrate. In parallel, these compounds were also assayed with SARS-CoV-2 PLpro. Four compounds inhibited Mpro with half-maximal inhibitory concentration (IC50) values between 0.41 μM and 66 μM. In addition, eight compounds inhibited PLpro with IC50 ranging from 1.7 μM to 46 μM. Molecular dynamics simulations suggest stable binding modes for Mpro inhibitors with frequent interactions with residues in the S1 and S2 pockets of the active site. For two PLpro inhibitors, interactions occur in the S3 and S4 pockets. In summary, our structure-based computational and biochemical approach identified novel naphthoquinonal scaffolds that can be further explored as SARS-CoV-2 antivirals.

Functional analysis of the polar amino acid in TatA transmembrane helix

The twin-arginine translocation (Tat) pathway utilizes the proton-motive force (PMF) to transport folded proteins across cytoplasmic membranes in bacteria and archaea, as well as across the thylakoid membrane in plants and the inner membrane in mitochondria. In most species, the minimal components required for Tat activity consist of three subunits, TatA, TatB, and TatC. Previous studies have shown that a polar amino acid is present at the N-terminus of the TatA transmembrane helix (TMH) across many different species. In order to systematically assess the functional importance of this polar amino acid in the TatA TMH in Escherichia coli, a complete set of 19-amino-acid substitutions was examined. Unexpectedly, although being preferred overall, our experiments suggest that the polar amino acid is not necessary for a functional TatA. Hydrophobicity and helix stabilizing properties of this polar amino acid were found to be highly correlated with the Tat activity. Specifically, change in charge status of the amino acid side chain due to pH resulted in a shift in hydrophobicity, which was demonstrated to impact the Tat transport activity. Furthermore, a four-residue motif at the N-terminus of the TatA TMH was identified by sequence alignment. Using a biochemical approach, the N-terminal motif was found to be functionally significant, with evidence indicating a potential role in the preference for utilizing different PMF components. Taken together, these findings yield new insights into the functionality of TatA and its potential role in the Tat transport mechanism.

Assessment of Genetic Diversity of Sugarcane (Saccharum spp.) Genotypes through Biochemical Approach

Background: Sugarcane is very important industrial and cash crop in Pakistan and in many countries of the world. It is worldwide an essential source of commercial sugar accounting for nearly 75 per cent of the world sugar production. Biochemical screening is first step in breeding to select superior genotypes in further breeding program. Methods: The experimental investigations were carried out on 10 different genotypes of sugarcane during consecutive years i.e., 2010-2012 to determine their genetic diversity by biochemical approach and SDS-PAGE analysis. The data on biochemical parameters i.e. reducing and non-reducing sugar, protein content, vitamin C, iron content, phosphorus content, calcium content and magnesium content in juice samples of various genotypes was recorded to assess biochemical composition. Result: The genotypes SPF-213, LHO83-153, CP-72-2086 performed better than other ones for reducing and non-reducing sugar, protein content, vitamin C and minerals content. However, on the basis of overall biochemical constituents CP-72-2086 was most efficient genotype. Electrophoretic mobility through SDS-PAGE was revealed clear genetic diversity among genotypes. As genotype S-06-US-469 and S-06-US-312 which showed unique pattern from common ancestors. These diverse genotypes could be successfully utilized in hybridization for improvement of crop.

Exploring the phytochemical and antioxidant potentialof Hylocereuspolyrhizus peel extract using biochemical approach

Red dragon fruit or Hylocereuspolyrhizus is one of the most popular fruits in Indonesia. Besides being consumed directly, H. Polyrhizus processed into various forms of processed food products such as jams, syrups, sweets, tea, and functional drinks. Unfortunately, massive quantities of solid waste, including H. polyrhizuspeel produced every year, continues to increase from year to year. Their disposal led to severe environmental issues. Whereas, H. polyrhizuspeels are abundant in beneficial secondary metabolites compoundespecially flavonoid and phenolic. The presence of flavonoid and phenolic content provides many benefits in the development of natural medicines, especially as antioxidants. However, the research related to exploring antioxidant potentials of H. polyrhizuspeel is still very limited. This study aimed to explore the phytochemical of H. polyrhizuspeel and their role as a natural-antioxidant agent. H. polyrhizuspeels were extracted through a maceration method using 96% of ethanol as their solvent. A total phenolic essay is determined by the method of Folin-Ciocalteu reagent using gallic acid as a reference. AlCl3 reagent is used to analyse the flavonoid content by comparing with quercetin. Antioxidant activity was done by DPPH and ABTS free radical scavenging methods. The total phenolic and flavonoid content of H. polyrhizuspeel extract (HPPE) at 107.35 ± 8.02 mg GAE/g and 108.82 ± 12.69 mg QE/g respectively. Furthermore, antioxidant activity of HPPE showed IC50 value at 136.20 ± 0.70 Lig/ml Lig/ml with DPPH methods and 390.70 ± 1.25 Lig/ml ug/ml with ABTS methods. Based on this recent study, HPPE has a moderate antioxidant activity by reducing free radicals in dose dependent manner.

Changes to the Collagen Structure using Vibrational Spectroscopy and Chemometrics: A Comparison between Chemical and Sulfide-Free Leather Process

Chemical and physical changes take place when hides and skins are processed to leather that affect the quality and strength of the material. Understanding the structure at each leather-making stage is the basis of this study but also intend to improve the process through a biochemical approach, employing a proteolytic enzyme for processing leather more cleanly with reduced environmental impact. Raman and ATR-FTIR spectroscopy in conjunction with chemometrics was used to investigate each leather-making stage from fresh green cattle hide to dry crust leather. The changes in proteins, lipids, nucleic acids and other biomolecules with leather processing was measured and reported using three novel Raman ratiometric markers, 920/1476, 1345/1259 and 1605/1476 cm-1, to discriminate the structural changes in collagen of hide using standard chemical and enzymatic method. Amide I band was deconvoluted to investigate thecollagen secondary structures using curve fitting by Gaussians function. The results of Principal Component Analysis are well-corroborated with the ratiometric markers of structural changes.

Replication Fork Uncoupling Causes Nascent Strand Degradation and Fork Reversal

Genotoxins cause nascent strand degradation (NSD) and fork reversal during DNA replication. NSD and fork reversal are crucial for genome stability and exploited by chemotherapeutic approaches. However, it is unclear how NSD and fork reversal are triggered. Additionally, the fate of the replicative helicase during these processes is unknown. We developed a biochemical approach to study synchronous, localized NSD and fork reversal using Xenopus egg extracts. We show that replication fork uncoupling stimulates NSD of both nascent strands and progressive conversion of uncoupled forks to reversed forks. The replicative helicase remains bound during NSD and fork reversal, indicating that both processes take place behind the helicase. Unexpectedly, NSD occurs before and after fork reversal, indicating that multiple degradation steps take place. Overall, our data show that uncoupling causes NSD and fork reversal and identify key steps involved in these processes.

17β-Estradiol Regulates miR-9-5p and miR-9-3p Stability and Function in the Aged Female Rat Brain

Clinical studies demonstrated that the ovarian hormone 17β-estradiol (E2) is neuroprotective within a narrow window of time following menopause, suggesting that there is a biological switch in E2 action that is temporally dependent. However, the molecular mechanisms mediating this temporal switch have not been determined. Our previous studies focused on microRNAs (miRNA) as one potential molecular mediator and showed that E2 differentially regulated a subset of mature miRNAs which was dependent on age and the length of time following E2 deprivation. Notably, E2 significantly increased both strands of the miR-9 duplex (miR-9-5p and miR-9-3p) in the hypothalamus, raising the possibility that E2 could regulate miRNA stability/degradation. We tested this hypothesis using a biochemical approach to measure miRNA decay in a hypothalamic neuronal cell line and in hypothalamic brain tissue from a rat model of surgical menopause. Notably, we found that E2 treatment stabilized both miRNAs in neuronal cells and in the rat hypothalamus. We also used polysome profiling as a proxy for miR-9-5p and miR-9-3p function and found that E2 was able to shift polysome loading of the miRNAs, which repressed the translation of a predicted miR-9-3p target. Moreover, miR-9-5p and miR-9-3p transcripts appeared to occupy different fractions of the polysome profile, indicating differential subcellular. localization. Together, these studies reveal a novel role for E2 in modulating mature miRNA behavior, independent of its effects at regulating the primary and/or precursor form of miRNAs.

Synthetic, structural and biochemical approach of the macro cyclic complexes of manganese (II) and tin (II)

This study presents a brief account of the synthesis, spectroscopic and biochemical aspects of tetraazamacrocyclic complexes of managenese (II) and tin (II). The complexes of manganese (II) and tin (II) were prepared by the template condensation of metal salts with phthalic acid and diamines (1,3-diaminobutane and 1,4- diaminobutane) in 1:2:2 molar ratio. All the complexes are soluble in polar solvent. The structures were investigated using elemental analyses, molecular weight determinations, conductivity measurements, electronic, infrared and X-Ray diffraction spectral studies. The elemental analyses are consistent with the formation of complexes of the type [M(Macn)Cl2] (where, n=1-2, M=Mn(II), Sn (II)). An octahedral geometry around the metal ion is suggested for these complexes. All the complexes were screened against several fungi and bacteria to assess its biological properties and results are discussed.The importance of this method includes shorter reaction time and high yield.