Expression and purification of epinecidin-1 variant (Ac-Var-1) by acid cleavage.

The demand for massive quantities of therapeutic active antimicrobial peptides (AMPs) is high due to their potential as alternatives to antibiotics. However, each antimicrobial peptide has unique properties, necessitating distinct synthesis and purification strategies for their large-scale production. In this study, we bio-synthesized and purified a functional enhanced variant of the AMP epinecidin-1, known as Ac-Var-1 (acid-cleavable variant-1). To generate the active peptide, we cloned the gene for Ac-Var-1 with acid-cleavable site (aspartic acid-proline) into the pET-32a expression vector, purified the fusion protein by His tag enrichment chromatography, and performed acid cleavage to release the active Ac-Var-1 peptide. After acid cleavage, the active Ac-Var-1 was purified and characterized by SDS-PAGE and mass spectrometry. The results from both techniques provided confirmation of the intactness of the purified Ac-Var-1. The Ac-Var-1 inhibited the growth of pathogenic Escherichia coli and Staphylococcus aureus. KEY POINTS : • Epinecidin-1 is a well-known antimicrobial peptide having multipotential bioactivities. • Epinecidin-1 variant is developed via the site-directed mutagenesis method to improve its structural stability and bioactivity. • AC-Var-1 development is an economical and easy method to remove peptide from tag protein.

Analysis of the main bioactive compounds from Ocimum basilicum for their antimicrobial and antioxidant activity.

The interest in bioactives especially from botanicals to treat vancomycin-resistant enterococcal (VRE) infections is increased. Many species of Ocimum have a long history in folk medicinal and food industries. Nevertheless, their bioactive compounds remain unexplored. This study is aimed to assess the antimicrobial and antioxidant activity of basil leaf extract prepared using ethanol, methanol, and water. The ethanol and methanol extract have all the phytochemicals (alkaloids, flavonoids, phenolic compounds, tannins, saponins, quinones, carbohydrates, and proteins) except steroids and terpenoids. In addition to steroids and terpenoids, tannin was also absent in the aqueous extract. Total phenolic and flavonoid content was high in ethanol and followed by methanol and aqueous extract. Similarly, ethanol and methanol extract showed strong antimicrobial activity against VRE and MTCC strains at a concentration of 20 mg/mL than aqueous extract. Among the 10 indicators, Staphylococcus aureus is highly susceptible to ethanol extract at a concentration of 8 mg/mL and followed by other MTCC strains. Vancomycin-resistant enterococci pathogens were inhibited at the minimum inhibitory concentration of 14, 16, and 20 mg/mL of ethanol, methanol, and aqueous extract. Further, on the basis of determining the absorbing material (nucleic acid and protein) at 260 nm and scanning electron microscopic, it was confirmed that the loss of cell membrane integrity and cell membrane damage were the effective mechanisms of plant extract antimicrobial activity. All three solvents have shown remarkable antioxidant activity. Gas chromatography-mass spectrometry analysis of basil leaves ethanol extract identified 19 compounds with various therapeutic and food applications.

Antibiotic Susceptibility Patterns and Virulence-Associated Factors of Vancomycin-Resistant Enterococcal Isolates from Tertiary Care Hospitals.

This study explored the prevalence of multi-drug resistance and virulence factors of enterococcal isolates obtained from various clinical specimens (n = 1575) including urine, blood, pus, tissue, catheter, vaginal wash, semen, and endotracheal secretions. Out of 862 enterococcal isolates, 388 (45%), 246 (29%), 120 (14%), and 108 (13%) were identified as Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, and Enterococcus hirae, respectively, using standard morphological and biochemical methods. The antibiotic resistance profile of all these enterococcal isolates was checked using the disc diffusion technique. High-level resistance was observed for benzylpenicillin (70%) and vancomycin (43%) among E. faecalis and E. faecium isolates, respectively. This study also revealed the prevalence of 'multi-drug resistance (resistant to 3 antibiotic groups)' among the vancomycin-resistant enterococcal strains, and this was about 11% (n = 91). The virulence determinants associated with vancomycin resistance (VR) were determined phenotypically and genotypically. About 70 and 39% of E. faecalis and E. faecium isolates showed to be positive for all four virulence factors (gelatinase, protease, hemolysin, and biofilm). Among the several virulence genes, gelE was the most common virulence gene with a prevalence rate of 76 and 69% among E. faecalis and E. faecium isolates, respectively. More than 50% of VRE isolates harbored other virulence genes, such esp, asa, ace, and cylA. Similarly, the majority of the VR enterococcal isolates (n = 88/91) harbored vanA gene and none of them harbored vanB gene. These results disclose the importance of VR E. faecalis and E. faecium and the associated virulence factors involved in the persistence of infections in clinical settings.

Bactericidal and non-cytotoxic activity of bacteriocin produced by Lacticaseibacillus paracasei F9-02 and evaluation of its tolerance to various physico-chemical conditions.

This study aims to explore novel lactic acid bacteria (LAB) from breast-fed infants' faeces towards characterizing their antimicrobial compound, bacteriocin. The LAB, Lacticaseibacillus paracasei F9-02 showed strong antimicrobial activity against clinical pathogens. Their proteinaceous nature was confirmed as the activity was completely abolished when treated with proteinaceous enzymes and retained during neutral pH and catalase treatment. The purified bacteriocin showed antimicrobial activity at the minimum inhibitory concentration (MIC) value of 7.56 µg/ml against vancomycin-resistant Enterococcus sp. [vancomycin-resistant enterococcal (VRE)], and methicillin-resistant Staphylococcus aureus (MRSA), 15.13 µg/ml against Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica serotype typhi and 30.25 µg/ml against Shigella flexneri. Present study also proved the bactericidal, non-cytotoxic and non-hemolytic nature of bacteriocin. Additionally, bacteriocin retained their stability under various physico-chemical conditions, broad range of pH (2-10), temperature (40-121°C), enzymes (amylase, lipase and lysozyme), surfactants [Tween-20, 80, 100 and sodium dodecyl sulfate (SDS)], metal ions (CaCl2 , FeSO4 , ZnSO4 , MgSO4 , MnSO4 , CuCl2 ) and NaCl (2%-8%). The molecular weight of bacteriocin (~28 kDa) was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), functional and active groups were assessed by Fourier Transform-Infrared (FT-IR). To our knowledge, this is the first study reporting L. paracasei from breast-fed infants' faeces and assessing their antimicrobial compound, bacteriocin. The study results furnish the essential features to confirm the therapeutic potential of L. paracasei F9-02 bacteriocin.

Phenotypic Assessment of Probiotic and Bacteriocinogenic Efficacy of Indigenous LAB Strains from Human Breast Milk.

Breast milk is the combination of bioactive compounds and microflora that promote newborn's proper growth, gut flora, and immunity. Thus, it is always considered the perfect food for newborns. Amongst their bioactives, probiotic communities-especially lactic acid bacteria (LAB)-are characterized from breast milk over the first month of parturition. In this study, seven LAB were characterized phenotypically and genotypically as Levilactobacillus brevis BDUMBT08 (MT673657), L. gastricus BDUMBT09 (MT774596), L. paracasei BDUMBT10 (MT775430), L. brevis BDUMBT11 (MW785062), L. casei BDUMBT12 (MW785063), L. casei BDUMBT13 (MW785178), and Brevibacillus brevis M2403 (MK371781) from human breast milk. Their tolerance to lysozyme, acid, bile, gastric juice, pancreatic juice, and NaCl and potential for mucoadhesion, auto-aggregation, and co-aggregation with pathogens are of great prominence in forecasting their gut colonizing ability. They proved their safety aspects as they were negative for virulence determinants such as hemolysis and biofilm production. Antibiogram of LAB showed their sensitivity to more than 90% of the antibiotics tested. Amongst seven LAB, three isolates (L. brevis BDUMBT08 and BDUMBT11, and L. gatricus BDUMBT09) proved their bacteriocin producing propensity. Although the seven LAB isolates differed in their behavior, their substantial probiotic properties with safety could be taken as promising probiotics for further studies to prove their in vivo effects, such as health benefits, in humans.

Phenotypic assessment of safety and probiotic potential of native isolates from marine fish Moolgarda seheli towards sustainable aquaculture.

Aquaculture is a highly productive and fast-growing agricultural sector. The occurrence of epidemic or sporadic disease outbreak is a major limiting factor in this sector, thus better alternatives are the need of the hour. Use of indigenous probiotics is a promising strategy to control infectious diseases. Thus, the present study was aimed to screen and characterize potent indigenous probiotics from marine fish, Moolgarda seheli, towards enhancing sustainable aquaculture production. Totally 347 bacterial isolates were obtained from M. seheli gastrointestinal tract, out of these, four isolates (KAF121, 124, 135, 136) were confirmed as potent probiotics in terms of biosafety, highly resistant to acidic pH, gastric juice, bile salt, high hydrophobicity to solvents, auto and co-aggregation potential. These four isolates also exhibited virtuous antioxidant activity. Further the isolates, KAF124 and 135 proved their efficiency in growth and survival of fish after challenged againt Aeromonas hydrophila. The isolates were identified based on their 16S rRNA gene sequence and the data were submitted to Genbank as Pseudomonas aeruginosa KAF121 (MH393516), Bacillus cereus KAF124 (MH393226), Bacillus thuringiensis KAF135 (MH393230), and Pseudomonas otitidis KAF136 (MH393230). The results conclude that two isolates, KAF124 and KAF135 are highly safe and potent probiotics which are first time isolated from the marine fish M. seheli. The two Bacillus strains could be used as better alternatives to antibiotics and other chemical-based drugs to prevent/control infectious diseases in aquaculture.

Production of eco-friendly PHB-based bioplastics by Pseudomonas aeruginosa CWS2020 isolate using poultry (chicken feather) waste.

Nowadays, the accumulation of non-degradable plastics and other disposed wastes leads to environmental pollution across the world. The production of eco-friendly and cost-effective poly-ß-hydroxybutyrate (PHB) could be a better alternative to conventional petroleum-based plastics and prevent environmental pollution. Besides, the area in and around Namakkal, Tamil Nadu, India is well known for poultries, currently facing the number of environmental issues due to the accumulation of chicken feather waste. This study focused on the production of eco-friendly PHB by recycling poultry (chicken feather) waste as the substrate. The native PHB producers were screened from the chicken waste disposal site in Namakkal by Sudan black B staining method. Further, the potent bacterial isolate was identified as Pseudomonas aeruginosa (NCBI accession MF18889) by phenotypic and genotypic characteristics. The PHB production media with chicken feather waste was statistically optimized by response surface methodology. The dry weight of PHB produced under optimized condition (15.96 g/L chicken feather waste, 37 °C temperature, 19.8 g/L glucose and 6.85 pH) was found to be 4.8 g/L. Besides, PHB was characterized and confirmed by thin-layer chromatography, Fourier-transform infrared spectroscopy and Gas chromatography-mass spectrometry analysis. Thus, this study concludes that poultry waste could be a complex nitrogen source for improving the growth of PHB producers and substantially increasing the yield of PHB, and it will be an eco-friendly and low-cost production in bioprocess technology.

Bacteriocin-a potential antimicrobial peptide towards disrupting and preventing biofilm formation in the clinical and environmental locales.

Biofilm, a consortium of microbial cells, protected by extracellular polymeric matrix, is considered a global challenge due to the inherent antibiotic resistance conferred by its lifestyle. Besides, it poses environmental threats causing huge damage in food industries, fisheries, refineries, water systems, pharmaceutical industries, medical industries, etc. Living in a community of microbial populations is most critical in the clinical field, making it responsible for about 80% of severe and chronic microbial diseases. The necessity to find an alternative approach is the need of the hour to solve these crises. So far, many approaches have been attempted to disrupt the initial stage of biofilm formation, including adherence and maturation. Bacteriocins are a group of antimicrobial peptides, produced by bacteria having the potential to disrupt biofilm either by itself or in combination with other drugs than antibiotic counterparts. A clear understanding on mechanisms of bacterial biofilm formation, progression, and resistance will surely lead to the development of innovative, effective biofilm control strategies in pharmaceutical, health care industries and environmental locales.

Retraction notice to "Peptide-induced formation of protein aggregates and amyloid fibrils in human and Guinea pig αA-crystallins under physiological conditions of temperature and pH" [Exp. Eye Res. 179 (2019) 193-205].

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors. The senior author contacted the journal in a forthright manner, in an effort to preserve the scientific integrity of the literature, after discovering a significant error in the results reported in the article. The authors were recently made aware of a paper by Kim et al. (Nature Commun. 2019) which shows a spirosome structure (the enzyme aldehyde-alcohol dehydrogenase) present in E. coli (Fig. 5a) that is very similar to the structure the authors thought formed when synthetic alpha A crystallin (66-80) peptide was incubated for 24 h with recombinant guinea pig alpha A insert crystallin (see Kumarasamy et al. Figs. 7C and F, and Fig. 9). Subsequent to publication of their report, the authors later found a number of images that showed what appeared to be the same structure present in samples of their presumably purified recombinant guinea pig alpha A insert crystallin which had been incubated without peptide for 24 h. Hence, the authors now conclude that the structures shown in Figs. 7C and F, and Fig. 9 of their article published in this journal are actually due to E. coli contaminant aldehyde-alcohol dehydrogenase. The authors deeply regret this error and any inconvenience it may have caused.

Antiquorum sensing and antibiofilm potential of biosynthesized silver nanoparticles of Myristica fragrans seed extract against MDR Salmonella enterica serovar Typhi isolates from asymptomatic typhoid carriers and typhoid patients.

Globally, Salmonella infection poses a major public health problem. Here, we report antibiofilm activity and quorum sensing inhibition of aqueous seeds extract of Myristica fragrans (nutmeg) and biosynthesized silver nanoparticles (AgNPs) against multidrug resistant (MDR) Salmonella enterica serovar Typhi (S. Typhi) isolated from typhoid patients and asymptomatic carriers. S. Typhi isolates revealed higher percentage (46%) of biofilm production identified by tissue culture plate (TCP) than Congo red agar (CRA) and tube adherence (TA) methods. The inhibition of biofilm-producing MDR S. Typhi isolates and pigment production of Chromobacterium violaceum (indicator bacteria) demonstrated the quorum sensing potential of nutmeg. The aqueous seed extract of nutmeg exhibited 87% of antibiofilm activity, while the biosynthesized AgNPs showed 99.1% of antibiofilm activity. Molecular docking studies of bioactive compounds of nutmeg against transcriptional regulatory protein RcsB and sensor kinase protein RcsC revealed interaction with the target proteins. It is proposed that biosynthesized AgNPs could be used as one of the effective candidates in treating asymptomatic typhoid carriers or typhoid patients and to control the subsistence of biofilm-producing S. Typhi strains or other pathogenic bacteria in the environment or industrial settings.

Syntheses, physicochemical characterization, antibacterial studies on potassium morpholine dithiocarbamate nickel (II), copper (II) metal complexes and their ligands.

Organic molecule dithiocarbamate transition metal complexes are novel and very attractive pharmaceutical targets for the management and control of antibiotic resistant bacteria. The direct reaction has synthesized new transition metal nickel (II), copper (II) complexes of potassium morpholine dithiocarbamate (K+C5H8NOS2 -) ligands and characterized by UV-visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), as well as NMR physicochemical techniques. Antibacterial bioefficacy of the ligand and its metal complexes has been investigated in vitro on the growth of Gram-positive (Staphylococcus aureus MTCC 737, Bacillus cereus MTCC 1272) and the Gram-negative (Listeria monocytogenes MTCC 657, Shigella flexeneri MTCC 1457) bacteria. The obtained electronic spectral bands are characteristic and consistent with the proposed composition of the ligand as well as its metal complexes. It also provides a further example of the bidentate coordination of dithiocarbamate ligands. Absorption peak values of FTIR are characteristic of the ligand as well as dithiocarbamate group molecules and exhibit their metal coordination. NMR 1H signal variations also correlate with the coordination mediated chemical shifts. Both the metal complexes showed significant antibacterial activity. However, enhanced antimicrobial activity of the ligands than metal complexes against Gram positive and Gram negative bacteria were observed. Thus, further study on this approach could pave a way for the development of dithiocarbamate-metal complex based antibacterial agent.

RETRACTED: Peptide-induced formation of protein aggregates and amyloid fibrils in human and guinea pig αA-crystallins under physiological conditions of temperature and pH.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors. The senior author contacted the journal in a forthright manner, in an effort to preserve the scientific integrity of the literature, after discovering a significant error in the results reported in the article. The authors were recently made aware of a paper by Kim et al. (Nature Commun. 2019) which shows a spirosome structure (the enzyme aldehyde-alcohol dehydrogenase) present in E. coli (Fig. 5a) that is very similar to the structure the authors thought formed when synthetic alpha A crystallin (66-80) peptide was incubated for 24 h with recombinant guinea pig alpha A insert crystallin (see Kumarasamy et al., Figs. 7C and F, and Fig. 9). Subsequent to publication of their report, the authors later found a number of images that showed what appeared to be the same structure present in samples of their presumably purified recombinant guinea pig alpha A insert crystallin which had been incubated without peptide for 24 h. Hence, the authors now conclude that the structures shown in Figs. 7C and F, and Fig. 9 of their article published in this journal are actually due to E. coli contaminant aldehyde-alcohol dehydrogenase. The authors deeply regret this error and any inconvenience it may have caused.

Effect of Bacillus cereus peptide conjugated with nanoporous silica on inactivation of Listeria monocytogenes in apple juice, as an ecofriendly preservative.

Bacteriocins are ribosomally synthesized antimicrobial proteins/peptides. They are of great interest in the food processing industries as potential natural preservative agent to control food-borne pathogens. Bacillus spp. are one among the potential probiotics receiving more attention since they produce a broad spectrum of antimicrobial bioactive peptides. In this study, a small-scale medium composition and bioprocessing parameters were statistically optimized to increase the yield of bacteriocin namely cerein from Bacillus cereus NS02 showing antagonism against a wide range of food-borne pathogens. The cerein was partially purified, characterized, and evaluated for their optimal reaction condition. It was subjected to surface adsorption onto food-grade silica to evaluate its maximal adsorption, reached at 4 h, 40 °C, pH 6-7, and at the initial concentration of 200 AU mL-1. The effectiveness of silica-adsorbed and silica-free cerein was checked in Listeria monocytogenes inoculated fresh apple juice and demonstrated biopreservative activity. In juice treated with silica-cerein, the colony forming unit (CFU) was found to be less in count on the 15th day of storage at 4 °C whereas, free-cerein was found to contain 3.8 log CFU mL-1. While, on the same day of storage, the control juice contained the strength of 14.6 log CFU mL-1. Based on the above, this study concludes that the identified heat stable low molecular weight peptide cerein from B. cereus NS02 could serve as a potential biopreservative with effective antilisterial activity in the food system. However, a more detailed study is required to determine if their quality change especially the effect of cerein in organoleptic and nutritional properties of food beyond their addition is necessary, before it is to be exploited as an ecofriendly biopreservative.

Multiple aggregates and aggresomes of C-terminal truncated human αA-crystallins in mammalian cells and protection by αB-crystallin.

BACKGROUND: Cleavage of 11 (αA162), 5 (αA168) and 1 (αA172) residues from the C-terminus of αA-crystallin creates structurally and functionally different proteins. The formation of these post-translationally modified αA-crystallins is enhanced in diabetes. In the present study, the fate of the truncated αA-crystallins expressed in living mammalian cells in the presence and absence of native αA- or αB-crystallin has been studied by laser scanning confocal microscopy (LSM). METHODOLOGY/PRINCIPAL FINDINGS: YFP tagged αAwt, αA162, αA168 and αA172, were individually transfected or co-transfected with CFP tagged αAwt or αBwt, expressed in HeLa cells and studied by LSM. Difference in protein aggregation was not caused by different level of α-crystallin expression because Western blotting results showed nearly same level of expression of the various α-crystallins. The FRET-acceptor photo-bleaching protocol was followed to study in situ protein-protein interaction. αA172 interacted with αAwt and αBwt better than αA168 and αA162, interaction of αBwt being two-fold stronger than that of αAwt. Furthermore, aggresomes were detected in cells individually expressing αA162 and αA168 constructs and co-expression with αBwt significantly sequestered the aggresomes. There was no sequestration of aggresomes with αAwt co-expression with the truncated constructs, αA162 and αA168. Double immunocytochemistry technique was used for co-localization of γ-tubulin with αA-crystallin to demonstrate the perinuclear aggregates were aggresomes. CONCLUSIONS/SIGNIFICANCE: αA172 showed the strongest interaction with both αAwt and αBwt. Native αB-crystallin provided protection to partially unfolded truncated αA-crystallins whereas native αA-crystallin did not. Aggresomes were detected in cells expressing αA162 and αA168 and αBwt co-expression with these constructs diminished the aggresome formation. Co-localization of γ-tubulin in perinuclear aggregates validates for aggresomes.

Interaction of C-terminal truncated human alphaA-crystallins with target proteins.

BACKGROUND: Significant portion of alphaA-crystallin in human lenses exists as C-terminal residues cleaved at residues 172, 168, and 162. Chaperone activity, determined with alcohol dehydrogenase (ADH) and betaL-crystallin as target proteins, was increased in alphaA(1-172) and decreased in alphaA(1-168) and alphaA(1-162). The purpose of this study was to show whether the absence of the C-terminal residues influences protein-protein interactions with target proteins. METHODOLOGY/PRINCIPAL FINDINGS: Our hypothesis is that the chaperone-target protein binding kinetics, otherwise termed subunit exchange rates, are expected to reflect the changes in chaperone activity. To study this, we have relied on fluorescence resonance energy transfer (FRET) utilizing amine specific and cysteine specific fluorescent probes. The subunit exchange rate (k) for ADH and alphaA(1-172) was nearly the same as that of ADH and alphaA-wt, alphaA(1-168) had lower and alphaA(1-162) had the lowest k values. When betaL-crystallin was used as the target protein, alphaA(1-172) had slightly higher k value than alphaA-wt and alphaA(1-168) and alphaA(1-162) had lower k values. As expected from earlier studies, the chaperone activity of alphaA(1-172) was slightly better than that of alphaA-wt, the chaperone activity of alphaA(1-168) was similar to that of alphaA-wt and alphaA(1-162) had substantially decreased chaperone activity. CONCLUSIONS/SIGNIFICANCE: Cleavage of eleven C-terminal residues including Arg-163 and the C-terminal flexible arm significantly affects the interaction with target proteins. The predominantly hydrophilic flexible arm appears to be needed to keep the chaperone-target protein complex soluble.

Role of the specifically targeted lysine residues in the glycation dependent loss of chaperone activity of alpha A- and alpha B-crystallins.

Earlier studies have shown significant loss of chaperone activity in alpha-crystallin from diabetic lenses. In vitro glycation studies have suggested that glycation of alpha-crystallin could be the major cause of chaperone activity loss. The following lysine (K) residues in alpha-crystallin have been identified as the major glycation sites: K11, K78, and K166 in alpha A-crystallin and K90, K92, and K166 in alpha B-crystallin. The present study was aimed to assess the contribution of each of the above glycation site in the overall glycation and loss of chaperone activity by mutating them to threonine followed by in vitro glycation with fructose. Level of glycated protein (GP) was determined by phenylboronate affinity chromatography, advanced glycation end products (AGEs) by direct ELISA using anti-AGE polyclonal antibody, and chaperone activity by using alcohol dehydrogenase as the target protein. K11T, K78, and K166T mutants of alpha A showed 33, 17, and 27% decrease in GP and 32, 18, and 21% decrease in AGEs, respectively, as compared to alpha A-wt. Likewise, K90T, K92T, K90T/K92T, and K166T mutants of alpha B showed 18, 21, 29, and 12% decrease in GP and 22, 24, 32, and 16% decrease in AGEs, respectively. Chaperone activity also showed concomitant increase with decreasing glycation and AGEs formation. alpha A-K11T and alpha B-K90T/K92T mutants showed the largest decrease in glycation and increase in chaperone activity.