Conjugal transfer of bacteriocin plasmids from different genera of lactic acid bacteria into Enterococcus faecalis JH2-2

The lactic acid bacteria (LAB) are of great interest because of their food grade quality and industrial importance. In the recent past, the pediocin PA-1 like bacteriocin was found to be synthesized in cross-species and cross-genera. Hence, the present work was carried out in order to determine the transfer of plasmid encoded pediocin PA-1 like bacteriocin among LAB. The objective of this study is to demonstrate the dissemination of bacteriocin-encoded plasmid from Pediococcus acidilactici NCIM 5424, Enterococcus faecium NCIM 5423 and Lactobacillus plantarum Acr2 to Enterococcus faecalis JH2-2 under in vitro (filter mating method) and in situ (soymilk model) conditions. The fermentation of the soymilk was determined by the selected pediocin producers. E. faecium NCIM 5423 was able to transfer the bacteriocin only under in situ conditions, whereas the native pediocin producer P. acidilactici NCIM 5424 transferred the bacteriocin under both the methods used. The in situ method gave more transfer frequency, ranging from 10^?7 to 10^?4 transconjugants per recipient cell. No conjugal transfer by L. plantarum Acr2 was observed. The physiological conditions like pH and temperature were found to influence the production of bacteriocin in the obtained transconjugants. The results suggest the horizontal gene transfer (HGT) and the natural spread of pediocin PA-1-like bacteriocin among LAB present in their close vicinity by means of conjugation. The dissemination of pediocin PA-1-like bacteriocin under in situ conditions is noteworthy, and such bacteriocin producers can be useful in the fermentation of dairy products and construction of novel cultures.     

Cloning, Characterization, and Expression of a New cry1Ab Gene from DOR Bt-1, an Indigenous Isolate of Bacillus thuringiensis

A new cry1Ab gene was cloned from the promising local isolate, DOR Bt-1, a Bacillus thuringiensis strain isolated from castor semilooper (Achaea janata L.) cadavers from castor bean (Ricinus communis L.) field. The nucleotide sequence of the cloned cry1Ab gene indicated that the open reading frame consisted of 3,465 bases encoding a protein of 1,155 amino acid residues with an estimated molecular weight of 130 kDa. Homology comparisons revealed that the deduced amino acid sequence of cry1Ab had a variation of seven amino acid residues compared to those of the known Cry1Ab proteins in the NCBI database and this gene has been designated as cry1Ab26 by the B. thuringiensis δ-endotoxin Nomenclature Committee. cry1Ab26 was cloned into pET 29a(+) vector and expressed in E. coli strain BL21 (DE3) under the control of T7 promoter with IPTG induction. ELISA, SDS-PAGE, and Western blot analysis confirmed the expression of 130-kDa protein. Insect bioassays with neonate larvae of Helicoverpa armigera showed that the partially purified Cry1Ab26 caused 97 % mortality within 5 days of feeding.     

Vitexin prevents Aβ proteotoxicity in transgenic Caenorhabditis elegans model of Alzheimer's disease by modulating unfolded protein response

Alzheimer's disease (AD) accounts for an estimated 60% to 80% of all dementia cases. The present study is aimed at evaluating the neuroprotective efficacy of vitexin, an apigenin flavone glycoside using transgenic Caenorhabditis elegans strain (CL2006) of AD. The neuroprotective effect of vitexin was determined using physiological assays, quantitative polymerase chain reaction, and Western blotting. The results of survival and paralysis assay indicate that vitexin (200 μM) significantly extended the lifespan of the nematodes. Vitexin‐treated nematodes showed a significant reduction in the expression of Aβ, ace‐1, and ace‐2 genes when compared to control. Further, vitexin significantly upregulated the expression of acr‐8 and dnj‐14, and increased the lifespan of the nematodes. Vitexin was also found to modulate the unfolded protein response genes (hsp‐4, pek‐1, ire‐1, and xbp‐1) and suppress the expression of Aβ. Overall, the results show that vitexin acts as a neuroprotective agent and protects transgenic C. elegans strains from Aβ proteotoxicity.     

Green synthesis of silver nanoparticles from aqueous extract of Scutellaria barbata and coating on the cotton fabric for antimicrobial applications and wound healing activity in fibroblast cells (L929)

Scutellaria barbata is a perennial herb which was vastly prescribed in Chinese medicine to treat inflammations, infections and it is also used a detoxifying agent. We synthesized silver nanoparticles with Scutellaria barbata extract and characterized the nanoparticles with UV–Vis spectroscopic analysis, TEM, AFM, FTIR and XRD. The biofilm inhibiting property of synthesized silver nanoparticles were examined with XTT reduction assay and the antimicrobial property was examined with well diffusion method. The silver nanoparticles were also coated with cotton fabrics and their efficacy against antimicrobials was analyzed to prove its application. The cytotoxic property of synthesized silver nanoparticles was examined with L929 fibroblast cells using MTT assay. Finally we analyzed the wound healing property of synthesized silver nanoparticles with wound scratch assay. The result of our UV–Vis spectroscopic analysis confirms Scutellaria barbata aqueous extract reduced silver ions and synthesized silver nanoparticles. The characterization studies TEM, AFM, FTIR and XRD confirms the synthesized silver nanoparticles are in ideal shape and size to be utilized as a drug. The XTT reduction assay proves silver nanoparticles effectively inhibits the biofilm formation in both resistant and sensitive strains. Antimicrobial sensitivity tests confirms synthesized silver nanoparticles and cotton coated synthesized silver nanoparticles both are effective against gram positive, gram negative and fungal species. Further the results of MTT assay confirms the synthesized silver nanoparticles are non toxic and finally the wound healing potency of the nanoparticles was confirmed with wound scratch assay. Over all our results authentically confirms the silver nanoparticles synthesized with Scutellaria barbata aqueous extract is potent wound healing drug.     

A Leap Forward Towards Unraveling Newer Anti-infective Agents from an Unconventional Source: a Draft Genome Sequence Illuminating the Future Promise of Marine Heterotrophic Bacillus sp. Against Drug-Resistant Pathogens

Marine Biotechnology - During the previous decade, genome-built researches on marine heterotrophic microorganisms displayed the chemical heterogeneity of natural product resources coupled with the...     

Purification and characterization of recombinant FAD synthetase from Neurospora crassa

FAD Synthetase (FADS) [EC 2.7.7.2], the second enzyme in flavin cofactor biosynthetic pathway converts FMN to FAD, plays an important role in many redox reactions. Neurospora crassa FADS (NcFADS) was cloned and overexpressed in E. coli cells. Recombinant NcFADS was purified in high yields of ～8 mg per liter of bacterial culture using a single step glutathione sepharose affinity chromatography. SDS-PAGE and MALDI-MS revealed that NcFADS has a molecular mass of ～31 kDa. Enzyme kinetic analysis monitored by reverse phase HPLC demonstrate a specific activity and k_cat of 1356 nmol/min/mg and 0.69sec^?1 respectively. Steady state kinetic analysis of NcFADS exhibited a K_m of NcFADS for FMN is 2.7 μM and for MgATP^?2 is 88.7 μM. Isothermal titration calorimetry experiments showed that the recombinant protein binds to the substrates with apparent K_d of 20.8 μM for FMN and 16.6 μM for MgATP^?2. Biophysical characterization using intrinsic fluorescence suggests that the enzyme is in folded conformation. Far-UV CD data suggest that the backbone of the enzyme is predominantly in a helical conformation. Differential scanning calorimetry data shows that the T_m is 53 °C ± 1. This is the first report on cloning, purification and characterization of FADS from N. crassa. The specific activity of NcFADS is the highest than any of the reported FADS from any other source. The results obtained in this study is expected to pave way for intensive research aimed to understand the molecular basis for the extraordinarily high turnover rate of NcFADS.     

NMR spectroscopy based metabolic profiling of urine and serum for investigation of physiological perturbations during radiation sickness

Radiation accidents are rare events that induce radiation syndrome, a complex pathology which is difficult to treat. In medical management of radiation victims, life threatening damage to different physiological systems should be taken into consideration. The present study was proposed to identify metabolic and physiological perturbations in biofluids of mice during different phases of radiation sickness using ¹H nuclear magnetic resonance (¹H NMR) spectroscopy and pattern recognition (PR) technique. The ¹H NMR spectra of the biofluids collected from mice irradiated with 5 Gray (Gy) at different time points during radiation sickness were analysed visually and by principal components analysis. Urine and serum spectral profile clearly showed altered metabolic profiles during different phases of radiation sickness. Increased concentration of urine metabolites viz. citrate, α ketoglutarate, succinate, hippurate, and trimethylamine during prodromal and clinical manifestation phase of radiation sickness shows altered gut microflora and energy metabolism. On the other hand, serum nuclear magnetic resonance (NMR) spectra reflected changes associated with lipid, energy and membrane metabolism during radiation sickness. The metabonomic time trajectory based on PR analysis of ¹H NMR spectra of urine illustrates clear separation of irradiated mice group at different time points from pre dose. The difference in NMR spectral profiles depicts the pathophysiological changes and metabolic disturbances observed during different phases of radiation sickness, that in turn, demonstrate involvement of multiple organ dysfunction. This could further be useful in development of multiparametric approach for better evaluation of radiation damage as well as for medical management during radiation sickness.     

Dengue virus activates polyreactive, natural IgG B cells after primary and secondary infection

Background

Dengue virus is transmitted by mosquitoes and has four serotypes. Cross-protection to other serotypes lasting for a few months is observed following infection with one serotype. There is evidence that low-affinity T and/or B cells from primary infections contribute to the severe syndromes often associated with secondary dengue infections. such pronounced immune-mediated enhancement suggests a dengue-specific pattern of immune cell activation. This study investigates the acute and early convalescent B cell response leading to the generation of cross-reactive and neutralizing antibodies following dengue infection.

Methodology/Principal Findings

We assayed blood samples taken from dengue patients with primary or secondary infection during acute disease and convalescence and compared them to samples from patients presenting with non-dengue related fever. Dengue induced massive early plasmablast formation, which correlated with the appearance of polyclonal, cross-reactive IgG for both primary and secondary infection. Surprisingly, the contribution of IgG to the neutralizing titer 4–7 days after fever onset was more than 50% even after primary infection.

Conclusions/Significance

Poly-reactive and virus serotype cross-reactive IgG are an important component of the innate response in humans during both primary and secondary dengue infection, and “innate specificities” seem to constitute part of the adaptive response in dengue. While of potential importance for protection during secondary infection, cross-reactive B cells will also compete with highly neutralizing B cells and possibly interfere with their development.     

Virulence characteristics and genetic affinities of multiple drug resistant uropathogenic Escherichia coli from a semi urban locality in India

Extraintestinal pathogenic Escherichia coli (ExPEC) are of significant health concern. The emergence of drug resistant E. coli with high virulence potential is alarming. Lack of sufficient data on transmission dynamics, virulence spectrum and antimicrobial resistance of certain pathogens such as the uropathogenic E. coli (UPEC) from countries with high infection burden, such as India, hinders the infection control and management efforts. In this study, we extensively genotyped and phenotyped a collection of 150 UPEC obtained from patients belonging to a semi-urban, industrialized setting near Pune, India. The isolates representing different clinical categories were analyzed in comparison with 50 commensal E. coli isolates from India as well as 50 ExPEC strains from Germany. Virulent strains were identified based on hemolysis, haemagglutination, cell surface hydrophobicity, serum bactericidal activity as well as with the help of O serotyping. We generated antimicrobial resistance profiles for all the clinical isolates and carried out phylogenetic analysis based on repetitive extragenic palindromic (rep)-PCR. E. coli from urinary tract infection cases expressed higher percentages of type I (45%) and P fimbriae (40%) when compared to fecal isolates (25% and 8% respectively). Hemolytic group comprised of 60% of UPEC and only 2% of E. coli from feces. Additionally, we found that serum resistance and cell surface hydrophobicity were not significantly (p = 0.16/p = 0.51) associated with UPEC from clinical cases. Moreover, clinical isolates exhibited highest resistance against amoxicillin (67.3%) and least against nitrofurantoin (57.3%). We also observed that 31.3% of UPEC were extended-spectrum beta-lactamase (ESBL) producers belonging to serotype O25, of which four were also positive for O25b subgroup that is linked to B2-O25b-ST131-CTX-M-15 virulent/multiresistant type. Furthermore, isolates from India and Germany (as well as global sources) were found to be genetically distinct with no evidence to espouse expansion of E. coli from India to the west or vice-versa.