Correction: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3'-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis.

[This corrects the article DOI: 10.1371/journal.pgen.1004749.].

Synthesis of a tetralone derivative of ampicillin to control ampicillin-resistant Staphylococcusaureus.

Due to the rapid emergence of antibiotic resistant new bacterial strains and new infections, there is an urgent need for novel or newly modified and efficient alternatives of treatment. However, conventional antibiotics are still used in therapeutic settings but their efficacy is uncertain due to the rapid evolution of drug resistance. In the present study, we have synthesized a new derivative of conventional antibiotic ampicillin using SN2-type substitution reaction. NMR and mass analysis of the newly synthesized derivative of ampicillin confirmed it as ampicillin-bromo-methoxy-tetralone (ABMT). Importantly, ABMT is revealed to have efficient activity against Staphylococcus aureus (S. aureus) with a MIC value of 32 µg ml-1 while ampicillin was not effective, even at 64 µg ml-1 of concentration. Electron microscopy results confirmed the membrane-specific killing of S. aureus at 1 h of treatment. Additionally, molecular docking analysis revealed a strong binding affinity of ABMT with ß-lactamase via the formation of a closed compact bridge. Our findings, avail a new derivative of ampicillin that could be a potential alternative to fight ampicillin-resistant bacteria possibly by neutralizing the ß-lactamase action.

Nitric oxide mediated hypoxia dynamics in COVID-19.

Several COVID-19 patients frequently experience with happy hypoxia. Sometimes, the level of nitric oxide (NO) in COVID-19 patients was found to be greater than in non-COVID-19 hypoxemics and most of the cases lower. Induced or inhaled NO has a long history of usage as a therapy for hypoxemia. Excessive production of ROS and oxidative stress lower the NO level and stimulates mitochondrial malfunction is the primary cause of hypoxia-mediated mortality in COVID-19. Higher level of NO in mitochondria also the cause of dysfunction, because, excess NO can also diffuse quickly into mitochondria or through mitochondrial nitric oxide synthase (NOS). A precise dose of NO may increase oxygenation while also acting as an effective inhibitor of cytokine storm. NOS inhibitors may be used in conjunction with iNO therapy to compensate for the patient's optimal NO level. NO play a key role in COVID-19 happy hypoxia and a crucial component in the COVID-19 pathogenesis that demands a reliable and easily accessible biomarker to monitor.

Identification of a novel proline-rich antimicrobial protein from the hemolymph of Antheraea mylitta.

Antimicrobial proteins (AMPs) are small, cationic proteins that exhibit activity against bacteria, viruses, parasites, fungi as well as boost host-specific innate immune responses. Insects produce these AMPs in the fat body and hemocytes, and release them into the hemolymph upon microbial infection. Hemolymph was collected from the bacterially immunized fifth instar larvae of tasar silkworm, Antheraea mylitta, and an AMP was purified by organic solvent extraction followed by size exclusion and reverse-phase high-pressure liquid chromatography. The purity of AMP was confirmed by thin-layer chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The molecular mass was determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry as 14 kDa, and hence designated as AmAMP14. Peptide mass fingerprinting of trypsin-digested AmAMP14 followed by de novo sequencing of one peptide fragment by tandem mass spectrometry analysis revealed the amino acid sequences as CTSPKQCLPPCK. No homology was found in the database search and indicates it as a novel AMP. The minimum inhibitory concentration of the purified AmAMP14 was determined against Escherichia coli, Staphylococcus aureus, and Candida albicans as 30, 60, and 30 µg/ml, respectively. Electron microscopic examination of the AmAMP14-treated cells revealed membrane damage and release of cytoplasmic contents. All these results suggest the production of a novel 14 kDa AMP in the hemolymph of A. mylitta to provide defense against microbial infection.

Trends of mutation accumulation across global SARS-CoV-2 genomes: Implications for the evolution of the novel coronavirus.

To understand SARS-CoV-2 microevolution, this study explored the genome-wide frequency, gene-wise distribution, and molecular nature of all point-mutations detected across its 71,703 RNA-genomes deposited in GISAID till 21 August 2020. Globally, nsp1/nsp2 and orf7a/orf3a were the most mutation-ridden non-structural and structural genes respectively. Phylogeny of 4618 spatiotemporally-representative genomes revealed that entities belonging to the early lineages are mostly spread over Asian countries, including India, whereas the recently-derived lineages are more globally distributed. Of the total 20,163 instances of polymorphism detected across global genomes, 12,594 and 7569 involved transitions and transversions, predominated by cytidine-to-uridine and guanosine-to-uridine conversions, respectively. Positive selection of nonsynonymous mutations (dN/dS >1) in most of the structural, but not the non-structural, genes indicated that SARS-CoV-2 has already harmonized its replication/transcription machineries with the host metabolism, while it is still redefining virulence/transmissibility strategies at the molecular level. Mechanistic bases and evolutionary/pathogenicity-related implications are discussed for the predominant mutation-types.

Purification and characterization of a novel antimicrobial peptide (QAK) from the hemolymph of Antheraea mylitta.

Antheraea mylitta, a tropical non-mulberry silkworm, is cultivated for tasar silk production in India. Several defense molecules including few antimicrobial peptides (AMPs) and proteins have been identified from this insect. Here, we have isolated and purified an antimicrobial tri-peptide by sequential chromatographic separation procedures. The amino acid sequence of the peptide was determined as NH2-Gln-Ala-Lys-COOH (QAK) using MALDI MS/MS fragmentation analysis. Further, the peptide was synthesized in vitro following solid phase chemistry of peptide synthesis and acetylated by acetic anhydride reaction. Antimicrobial activities of non-acetylated and acetylated QAK were tested against both Escherichia coli and Staphylococcus aureus bacteria. Acetylated peptide inhibited bacterial growth more effectively and its minimum inhibitory concentration (MICs) was found lower than non-acetylated peptide. SEM studies revealed more membrane damage and release of intracellular materials like ß-galactosidase enzyme from acetylated peptide treated bacteria in comparison to non-acetylated QAK. At MIC, acetylated peptide did not show any significant hemolytic activity against rabbit erythrocytes. The results suggest that acetylated-QAK is a promising new antimicrobial peptide and can be used for therapeutic purpose.

Electrochemical communication in biofilm of bacterial community.

Electrochemical communication during biofilm formation has recently been identified. Bacteria within biofilm-adopt different strategies for electrochemical communication such as direct contact via membrane-bound molecules, diffusive electron transfer via soluble redox-active molecules, and ion channel-mediated long-range electrochemical signaling. Long-range electrical signals are important to communicate with distant members within the biofilm, which function through spatially propagating waves of potassium ion (K+ ) that depolarizes neighboring cells. During propagation, these waves coordinate between the metabolic states of interior and peripheral cells of the biofilm. The understanding of electrochemical communication within the biofilm may provide new strategies to control biofilm-mediated drug resistance. Here, we summarized the different mechanisms of electrochemical communication among bacterial populations and suggested its possible role in the development of high level of antibiotic resistance. Thus, electrochemical signaling opens a new avenue concerning the electrophysiology of bacterial biofilm and may help to control the biofilm-mediated infection by developing future antimicrobials.

Identification of a novel humoral antifungal defense molecule in the hemolymph of tasar silkworm Antheraea mylitta.

Humoral defenses are the major components of insect innate immune system that include the production of several soluble effector molecules from fat body and hemocytes, and released in to the hemolymph upon microbial infection. Hemolymph was collected from the fungal immunized fifth instar larvae of tasar silkworm, Antheraea mylitta, extracted with a mixture of solvent (methanol/glacial acetic acid/water) and fractionated through RP-HPLC. Several fractions were collected, lyophilized and their antifungal activity was tested against Candida albicans. Only the fraction showing strong antifungal activity was further purified via gel filtration chromatography and the purity of active compound was confirmed by thin layer chromatography which showed only single spot after staining with ninhydrin. The molecular mass of this purified compound was determined by high resolution mass spectrometry as 531 Da and analysis of 1H and 13C NMR spectral data along with mass fragmentation pattern indicated the probable structure of the isolated compound as symmetric bis-decanoate derivative. Scanning electron microscopic study revealed that the compound degraded fungal cell wall leading to its lysis and may be the major target for its antifungal activity. These results indicate that presence of this compound in the hemolymph of A. mylitta provides defense against fungal infection.

Biocides and health-care agents are more than just antibiotics: Inducing cross to co-resistance in microbes.

Health-care chemicals are used worldwide as important components of different industries as consumer products, food industry, animal husbandry and agribusiness. There are innumerable reports on the effect of these chemicals (biocides) impacting the development of cross to co-resistance in pathogenic bacteria. However, reports are limited on the concurrent use of agricides (pesticides, herbicides, fungicides and insecticides) which influence the microbial activities in soils and contribute to the increase in incidences of co-resistance. Undoubtedly, indiscriminate use of biocides and agricides has contaminated both water and soil environments. This review describes the onset of cross and co-resistance to biocides and antibiotics which is increasingly being exhibited by specific bacteria under a persistent selective pressure. It also re-examines the significance of mobile genetic platforms and horizontal gene transfer from one to another bacterial species, for understanding the kinetics and efficiency of genetic exchange in stressed environments leading to natural selection of tolerant strains over susceptible ones. The investigation is much warranted, particularly with respect to agricides that commonly occur in recalcitrant states in soil and water ecosystem, livestock, etc and is transmitted either directly or via the food-chain to human beings, facilitating the switch from cross to co-resistance.

Transcriptional regulation of human defense peptides: a new direction in infection control.

While antibiotics remain as a major therapy against life threatening pathogenic infections, they often lead to side effects like rashes, gastrointestinal and central nervous system reactions to serious allergies or organ damage. These adverse effects alongside the emergence of multi-antibiotic resistant bacteria and the decline in the development of new antibiotics, have posed a serious impediment for effective antibiotic therapy. A paradigm shift in attitudes has led us to think about the possibility of controlling infections with the indigenous antimicrobial peptides synthesized by human beings. It has been observed that few transcription factors can stimulate more than three dozen defense peptides in the human system. Hence, during the infection stage, if we can induce these common factors, most of the infections could be healed from inside without the administration of any antibiotics. The efficiency of such peptides is being proven in clinical tests leading to the development of drugs.

Self-Assembled Tea Tannin Graft Copolymer as Nanocarriers for Antimicrobial Drug Delivery and Wound Healing Activity.

Green chemistry polymers from renewable resources have recently received much more attention from pharmaceutical researchers. However, the appropriate application of a polymer depends on its chemical nature, biocompatibility and microstructure. Here, tannin polyphenols from the common beverage, tea, are used to develop a novel self-assembled porous capsule as a microstructure of hydrogel for versatile biological applications, such as drug delivery, antioxidant and wound healing activity. Hydrogel has been successfully used for the delivery of both anticancer and antimicrobial drugs. The developed material shows excellent biocompatibility and antioxidant activity in vitro. The scratch assay for in vitro wound healing activity reveals their higher potential to repair the damaged cells in comparison to control.

The role of the mammalian DNA end-processing enzyme polynucleotide kinase 3'-phosphatase in spinocerebellar ataxia type 3 pathogenesis.

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3'-P and 5'-OH, are processed by mammalian polynucleotide kinase 3'-phosphatase (PNKP), a bifunctional enzyme with 3'-phosphatase and 5'-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14-41 to 55-82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP's 3' phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3'-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients' brain. Finally, long amplicon quantitative PCR analysis of human MJD patients' brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.

PdII/AgI-Catalyzed Room-Temperature Reaction of γ-Hydroxy Lactams: Mechanism, Scope, and Antistaphylococcal Activity.

The present work reports a PdII/AgI-promoted amidoalkylation reaction involving various γ-hydroxy lactams and C/O/S nucleophiles at room temperature. The dual mode of activation of both the electrophile and nucleophile by in situ generated catalytically active cationic PdII species facilitates the reaction at room temperature. Among the synthesized isoindoline derivatives, three compounds are found to be active against vancomycin and methicillin-resistant S. aureus strain with appreciable MIC values.

Novel boronic acid derivatives of bis(indolyl) methane as anti-MRSA agents.

Towards the search for a new generation of antibiotics to control methicillin-resistant Staphylococcus aureus (MRSA), the design and synthesis of various bis indolyl methane (BIM) derivatives based on their different electron donor and acceptor properties of the substituents have been made, in which boronic acid derivatives of BIM are found to be active against MRSA. The observed evidence with the lead compound reveals their strong anti-MRSA activity, which paves the way of design and further development of a new generation antibiotics.

A self-assembled clavanin A-coated amniotic membrane scaffold for the prevention of biofilm formation by ocular surface fungal pathogens.

Amniotic membrane (AM) is frequently used in ophthalmologic surgery for rapid ocular surface reconstruction. Sometimes it may create a major problem with associated infections after biofilm formation over the membrane. To overcome this problem, AM was coated with the antimicrobial peptide clavanin A. The antifungal activity of clavanin A in the native and self-assembled form was determined against the common ocular surface pathogens Candida albicans, Aspergillus fumigatus, Alternaria sp. and Fusarium sp. Biofilm formation over the coated surface was significantly reduced in comparison with the uncoated membrane. The coated membrane revealed effectiveness in terms of biocompatibility, cell attachment colonization when tested in non-cancerous 3T3 and human embryonic kidney (HEK)-293 cell lines. Clavanin A-coated AM also exhibited excellent physical, morphological and antifungal characteristics, indicating potential applicability for ocular surface infection control.

Cm-p5: an antifungal hydrophilic peptide derived from the coastal mollusk Cenchritis muricatus (Gastropoda: Littorinidae).

Antimicrobial peptides form part of the first line of defense against pathogens for many organisms. Current treatments for fungal infections are limited by drug toxicity and pathogen resistance. Cm-p5 (SRSELIVHQRLF), a peptide derived from the marine mollusk Cenchritis muricatus peptide Cm-p1, has a significantly increased fungistatic activity against pathogenic Candida albicans (minimal inhibitory concentration, 10 µg/ml; EC50, 1.146 µg/ml) while exhibiting low toxic effects against a cultured mammalian cell line. Cm-p5 as characterized by circular dichroism and nuclear magnetic resonance revealed an α-helical structure in membrane-mimetic conditions and a tendency to random coil folding in aqueous solutions. Additional studies modeling Cm-p5 binding to a phosphatidylserine bilayer in silico and isothermal titration calorimetry using lipid monophases demonstrated that Cm-p5 has a high affinity for the phospholipids of fungal membranes (phosphatidylserine and phosphatidylethanolamine), only moderate interactions with a mammalian membrane phospholipid, low interaction with ergosterol, and no interaction with chitin. Adhesion of Cm-p5 to living C. albicans cells was confirmed by fluorescence microscopy with FITC-labeled peptide. In a systemic candidiasis model in mice, intraperitoneal administration of Cm-p5 was unable to control the fungal kidney burden, although its low amphiphaticity could be modified to generate new derivatives with improved fungicidal activity and stability.

Induction of nodD Gene in a Betarhizobium Isolate, Cupriavidus sp. of Mimosa pudica, by Root Nodule Phenolic Acids.

A range of phenolic acids, viz., p-coumaric acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, protocatechuic acid, caffeic acid, ferulic acid, and cinnamic acid have been isolated and identified by LC-MS analysis in the roots and root nodules of Mimosa pudica. The effects of identified phenolic acids on the regulation of nodulation (nod) genes have been evaluated in a betarhizobium isolate of M. pudica root nodule. Protocatechuic acid and p-hydroxybenzoic acid were most effective in inducing nod gene, whereas caffeic acid had no significant effect. Phenylalanine ammonia lyase, peroxidase, and polyphenol oxidase activities were estimated, indicating regulation and metabolism of phenolic acids in root nodules. These results showed that nodD gene expression of betarhizobium is regulated by simple phenolic acids such as protocatechuic acid and p-hydroxybenzoic acid present in host root nodule and sustains nodule organogenesis.

Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins.

Base oxidation by endogenous and environmentally induced reactive oxygen species preferentially occurs in replicating single-stranded templates in mammalian genomes, warranting prereplicative repair of the mutagenic base lesions. It is not clear how such lesions (which, unlike bulky adducts, do not block replication) are recognized for repair. Furthermore, strand breaks caused by base excision from ssDNA by DNA glycosylases, including Nei-like (NEIL) 1, would generate double-strand breaks during replication, which are not experimentally observed. NEIL1, whose deficiency causes a mutator phenotype and is activated during the S phase, is present in the DNA replication complex isolated from human cells, with enhanced association with DNA in S-phase cells and colocalization with replication foci containing DNA replication proteins. Furthermore, NEIL1 binds to 5-hydroxyuracil, the oxidative deamination product of C, in replication protein A-coated ssDNA template and inhibits DNA synthesis by DNA polymerase δ. We postulate that, upon encountering an oxidized base during replication, NEIL1 initiates prereplicative repair by acting as a "cowcatcher" and preventing nascent chain growth. Regression of the stalled replication fork, possibly mediated by annealing helicases, then allows lesion repair in the reannealed duplex. This model is supported by our observations that NEIL1, whose deficiency slows nascent chain growth in oxidatively stressed cells, is stimulated by replication proteins in vitro. Furthermore, deficiency of the closely related NEIL2 alone does not affect chain elongation, but combined NEIL1/2 deficiency further inhibits DNA replication. These results support a mechanism of NEIL1-mediated prereplicative repair of oxidized bases in the replicating strand, with NEIL2 providing a backup function.

Glucose directly promotes antifungal resistance in the fungal pathogen, Candida spp.

Effects of glucose on the susceptibility of antifungal agents were investigated against Candida spp. Increasing the concentration of glucose decreased the activity of antifungal agents; voriconazole was the most affected drugs followed by amphotericin B. No significant change has been observed for anidulafungin. Biophysical interactions between antifungal agents with glucose molecules were investigated using isothermal titration calorimetry, Fourier transform infrared, and (1)H NMR. Glucose has a higher affinity to bind with voriconazole by hydrogen bonding and decrease the susceptibility of antifungal agents during chemotherapy. In addition to confirming the results observed in vitro, theoretical docking studies demonstrated that voriconazole presented three important hydrogen bonds and amphotericin B presented two hydrogen bonds that stabilized the glucose. In vivo results also suggest that the physiologically relevant higher glucose level in the bloodstream of diabetes mellitus mice might interact with the available selective agents during antifungal therapy, thus decreasing glucose activity by complex formation. Thus, proper selection of drugs for diabetes mellitus patients is important to control infectious diseases.

Screening of serine protease inhibitors with antimicrobial activity using iron oxide nanoparticles functionalized with dextran conjugated trypsin and in silico analyses of bacterial serine protease inhibition.

Plants produce a variety of proteins and peptides which are involved in their defense against pathogens. Serine protease inhibitors are a well-established class of inhibitors correlated with plant defense. Increased levels of protease inhibitors delay cell damage and expand the cell's life-span. Recently, the rapid emergence of antibiotic-resistant microbial pathogens has prompted immense interest in purifying novel antimicrobial proteins or peptides from plant sources. Usually, the purification of protease inhibitors is accomplished by salt-extraction, ultrafiltration and affinity chromatography. Here, we developed a novel approach based on iron oxide nanoparticles conjugated to dextran functionalized with trypsin beads that accelerate the quick screening and purification of antimicrobial peptides with serine protease inhibitor activity. The method described here also works for screening other inhibitors using particular protein kinases, and it is therefore a novel tool for use as the leading method in the development of novel antimicrobial agents with protease inhibitory activity. Finally, and no less important, molecular modelling and dynamics studies of a homologous inhibitor studied here with Escherichia coli trypsin and chymotrypsin are provided in order to shed some light on inhibitor-enzyme interactions.