Characterization and Cytotoxicity of Pseudomonas Mediated Rhamnolipids Against Breast Cancer MDA-MB-231 Cell Line

A biosurfactant producing bacterium was identified as Pseudomonas aeruginosa DNM50 based on molecular characterization (NCBI accession no. MK351591). Structural characterization using MALDI-TOF revealed the presence of 12 different congeners of rhamnolipid such as Rha-C8-C8:1, Rha-C10-C8:1, Rha-C10-C10, Rha-C10-C12:1, Rha-C16:1, Rha-C16, Rha-C17:1, Rha-Rha-C10:1-C10:1, Rha-Rha-C10-C12, Rha-Rha-C10-C8, Rha-Rha-C10-C8:1, and Rha-Rha-C8-C8. The radical scavenging activity of rhamnolipid (DNM50RL) was determined by 2, 3-diphenyl-1-picrylhydrazyl (DPPH) assay which showed an IC50 value of 101.8 μg/ ml. The cytotoxic activity was investigated against MDA-MB-231 breast cancer cell line by MTT (4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide) assay which showed a very low IC50 of 0.05 μg/ ml at 72 h of treatment. Further, its activity was confirmed by resazurin and trypan blue assay with IC50 values of 0.01 μg/ml and 0.64 μg/ ml at 72 h of treatment, respectively. Thus, the DNM50RL would play a vital role in the treatment of breast cancer targeting inhibition of p38MAPK.

Oribatid mites (Acariformes, Sarcoptiformes: Oribatida) in the gills of Salmo spp. (Actinopterygii: Salmonidae) parr – Short communication

Live oribatid mites of the family Malaconothridae were found on Salmo spp. parr caught in the rivers of Northwest Russia. The mites were localised in the gill filaments and enclosed in connective tissue capsules. The encapsulation was accompanied by hyperplasia and displacement of the respiratory epithelium. One mite specimen was an adult female, while all the other specimens were protonymphs. The adult female and one protonymph specimen were identified as Tyrphonothrus sp. Other protonymphs could be identified only at the family level. The obtained partial 18S rDNA sequence of one protonymph was 100% identical to that of Tyrphonothrus maior (NCBI accession No. KY922215). This is the first report of living malaconothrid mites encapsulated in fish gills, and the phenomenon may assume parasitic behaviour. However, the nature of the relationship between the mites and the fish requires further investigations.

Probiotic Characteristics and Anti-inflammatory Properties of an Indian Indigenous Lactobacillus plantarum against Pathogenic Salmonella Typhimurium

Probiotic should be well characterized for any preventive and therapeutic application. Studies have reported that the population specific indigenous probiotics are more effective. Huge number of probiotics are available globally but indigenous Indian probiotic candidates are limited. Therefore, a study was conducted to evaluate probiotic characteristics and anti-inflammatory properties of an Indian indigenous Lactobacillus plantarum (Lp1) – (NCBI accession no- MN386242) against gastrointestinal (GI) pathogen Salmonella Typhimurium (ST) using Caco-2 cell lines. Anti-inflammatory properties were assessed by evaluating the expression of Immunoglobulins (IgA, IgG) receptors, Toll Like Receptors (TLR2, TLR4) and cytokine (IL10, IL-8, IL17 and IL23) using flowcytometry and Real Time-PCR. Lp1 showed good adhesion and ability to survive in high acidic and bile conditions of the gut. Lp1 demonstrated antagonistic potential against GI pathogens and modulated pro and anti-inflammatory cytokines expression significantly. Observed result indicated that this indigenous Lp1 is a good probiotic candidate with anti-inflammatory potential and can be evaluated further clinically for the treatment or prevention of salmonellosis and other GI inflammatory disorders.

Phylo-geo-network and haplogroup analysis of 611 novel coronavirus (SARS-CoV-2) genomes from India

The novel coronavirus (SARS-CoV-2) from Wuhan China discovered in December 2019 has since developed into a global epidemic. Presently, we constructed and analyzed the phylo-geo-network of SARS-CoV-2 genomes from across India to understand the viral evolution in the country. A total of 611 full-length genomes from different states of India were extracted from the EpiCov repository of GISAID initiative on 6 June, 2020. Their alignment with the reference sequence (Wuhan, NCBI accession number NC_045512.2) uncovered 270 parsimony informative sites. Furthermore, 339 genomes were divided into 51 haplogroups. The network revealed the core haplogroup as that of reference sequence NC_045512.2 (Haplogroup A1) with 157 identical sequences present across 16 states. Remaining haplogroups had <10 identical sequences across a maximum of three states. Some states with fewer samples had more haplogroups. Forty-one haplogroups were localized exclusively to any one state. The two most common lineages are B6 and B1 (Pangolin) whereas clade A2a (Covidex) appears to be the most predominant in India. Because the pandemic is still emerging, the observations need to be monitored.

Flanker: a tool for comparative genomics of gene flanking regions

Analysing the flanking sequences surrounding genes of interest is often highly relevant to understanding the role of mobile genetic elements (MGEs) in horizontal gene transfer, particular for antimicrobial resistance genes. Here, we present Flanker, a Python package which performs alignment-free clustering of gene flanking sequences in a consistent format, allowing investigation of MGEs without prior knowledge of their structure. Flanker clusters flanking sequences based on Mash distances, allowing for easy comparison of similarity and the extent of this similarity across sequences. Additionally, Flanker can be flexibly parameterised to finetune outputs by characterising upstream and downstream regions separately and investigating variable lengths of flanking sequence. We apply Flanker to two recent datasets describing plasmid-associated carriage of important carbapenemase genes (blaOXA-48 and blaKPC-2/3) and show that it successfully identifies distinct clusters of flanking sequences (flank patterns), including both known and previously uncharacterised structural variants. We demonstrate that flank patterns are linked to geographical regions and carbapenem phenotypes, suggesting they may be useful as epidemiological markers. Flanker is freely available under an MIT license at https://github.com/wtmatlock/flanker.Data SummaryNCBI accession numbers for all sequencing data used in this study is provided in Supplementary Table 1. The analysis performed in this manuscript can be reproduced in a binder environment provided on the Flanker Github page (https://github.com/wtmatlock/flanker).

A NOVEL MULTI-EPITOPE PEPTIDE VACCINE CONSTRUCT AGAINST INFLUENZA A VIRUS AND STREPTOCOCCUS PNEUMONIAE CO-INFECTION: AN IN SILICO APPROACH

Viral and bacterial respiratory tract co-infections in the same host often result in severity and heightened pathology of illness compared to single infections. This has proven to be true for combined infections with Influenza A virus and the bacterium Streptococcus pneumoniae. Separate vaccines do exist for each individual infection but they prove to be ineffective and non-specific when the infection has multiplied in case of co-infection. The study utilised in silico approaches and proposed a structural design for multi-epitope peptide vaccine having the ability to target co-infection caused by A/New York/392/2004 (H3N2) and R6 strains of Influenza A virus (NCBI Accession: PRJNA15622) and Streptococcus pneumoniae (NCBI Accession: PRJNA278), respectively. Epitope prediction followed by protein prioritization was performed using the reference sequence of each strain to short list the epitopes that can later be used for constructing multi-epitope structure. The multi-epitope constructs having Cholera Toxin Subunit B as adjuvant and (Gly4Ser)3 as flexible linker were then analyzed for their ability to induce an effective immune response in human body for which Macrophage receptor with collagenous structure, Toll-like receptor 2, 4 and 5 were taken as Pattern Recognition Receptors. The significant immune response generated through each Pattern Recognition Receptor helped to conclude that multi-epitope peptide structures can be used as probable candidates for the design of vaccine. The combination of the epitopes LWSYNAELL and FTGKQLQVG of Influenza A virus and Streptococcus pneumoniae, respectively, induced highly significant immune response in case of each Pattern Recognition Receptor when tested through in-silico predictive tools.

First report of Fusarium lacertarum causing Fusarium Head Blight on sorghum in North Carolina

In August 2018, sorghum plants (Sorghum bicolor (L.) Moench) from research field plots in Wake County, North Carolina were observed with head blight symptoms including panicles with red lesions, visible mycelium, and necrosis. At the time of collection, all plants in research plots displayed symptoms of Fusarium head blight and panicles averaged 33% area affected by symptoms and signs. From these affected plants, samples (n = 5) were collected for further identification. Symptomatic grains were surface sterilized for one minute in 0.825% sodium hypochlorite solution and rinsed for one minute in sterile, deionized water. After drying on sterile paper towels, grains were plated onto water agar. Resulting fungal hyphal tips were then transferred to antibiotic-amended potato dextrose agar (PDA) and incubated at 25oC. Cultures were incubated for 3 to 5 days. Isolates had abundant white hyphae accompanied with peach-colored pigment production. Macroconidia with 5-6 septations were 23.47 ± 7.74 µm long and 3.47 ± 0.66 µm wide with foot-shaped basal cells, tapering to hooked apical cells. Chlamydospores were present in chains but microconidia were not present. Morphological species recognition (MSR) criteria tentatively identified the isolate as Fusarium lacertarum Subrahm., in the Fusarium incarnatum-equiseti species complex (FIESC) using characteristics described by Leslie and Summerell 2006. Molecular characterization using translation elongation factor 1α (TEF-1 α, primers EF1 and EF2 from O’Donnell et al. 1998), β tubulin (TUB2, primers T1 and T22 from O’Donnell and Cigelnik 1997), and ribosomal protein subunit II (RPB2, primers 5F2 and 11AR from Cerón-Bustamante et al. 2018) was conducted to confirm morphological identification. DNA from the hyphae of pure cultures was extracted using the DNeasy PowerSoil DNA extraction kit according to manufacturer’s guidelines. DNA amplification conditions followed the protocols for each primer set (O’Donnell et al. 1998; O’Donnell and Cigelnik 1997; Cerón-Bustamante et al. 2018). BLASTn analysis of TEF-1α (Isolate Accession MT149915, 573bp) alignment had 99.8% identity to F. lacertarum (NCBI accession: JF740828), TUB2 (Isolate Accession MT149914, 1,183bp) alignment had 99.3% identity to F. equiseti (NCBI accession: KJ396338), and RPB2 (Isolate Accession MT184173, 1,538bp) concatenated sequences had 95.3% identity to F. lacertarum (NCBI accession: MH582185). The TUB2 region most closely aligns to F. equiseti, which is likely due to an absence of TUB2 sequences labeled for F. lacertarum in the NCBI database. Pathogenicity was confirmed by spray-inoculating Southern Harvest 80G4 sorghum panicles (n = 9) at anthesis with four ml of conidial suspension (3.3×104 conidia/ml). Control plants (n = 9) were sprayed with sterile water. Plastic bags were placed around panicles for 24 hours to ensure moist conditions during the infection period. Plants were maintained in a greenhouse under a 12-hour light cycle and fertilized bi-weekly with 20-20-20 fertilizer. Symptoms were observed on inoculated panicles after 14 days, and the F. lacertarum isolate was recovered from inoculated plants and confirmed using methods described above. Fusarium spp. were not re-isolated from non-inoculated control plants. Members of FIESC are known to contribute to the Fusarium Head Blight disease complex and may be capable of producing mycotoxins associated with infections (Lincy et al. 2011; Marin et al. 2012; Moretti 2017); however, mycotoxin characterization in F. lacertarum has not been characterized. To our knowledge, this is the first report of F. lacertarum causing disease to sorghum in North Carolina and the United States. Fusarium lacertarum may cause impactful losses to sorghum producers due to direct yield and quality losses by the pathogen as well as the potential for mycotoxins to impact trade.

Whole-Genome Sequence of an Indian Group A Streptococcus emm Type 1-2 Strain Isolated from a Blood Sample in North India

Group A Streptococcus emm type 1-2 is more prevalent than emm type 1 in India. Only partial information is available about the genetic characteristics of this type. Here, genome sequencing of emm type 1-2 strain 1085 (from blood) was conducted. A contig 2,010,300 bp long, with a total of 1,877 annotated proteins, was obtained (NCBI accession number CP047120, assembly accession number ASM983284v1).

Development of Staphylococcus Enzybiotics: The Ph28 Gene of Staphylococcus epidermidis Phage PH15 Is a Two-Domain Endolysin

Given the worldwide increase in antibiotic resistant bacteria, bacteriophage derived endolysins represent a very promising new alternative class of antibacterials in the fight against infectious diseases. Endolysins are able to degrade the prokaryotic cell wall, and therefore have potential to be exploited for biotechnological and medical purposes. Staphylococcus epidermidis is a Gram-positive multidrug-resistant (MDR) bacterium of human skin. It is a health concern as it is involved in nosocomial infections. Genome-based screening approach of the complete genome of Staphylococcus virus PH15 allowed the identification of an endolysin gene (Ph28; NCBI accession number: YP_950690). Bioinformatics analysis of the Ph28 protein predicted that it is a two-domain enzyme composed by a CHAP (22-112) and MurNAc-LAA (171-349) domain. Phylogenetic analysis and molecular modelling studies revealed the structural and evolutionary features of both domains. The MurNAc-LAA domain was cloned, and expressed in E. coli BL21 (DE3). In turbidity reduction assays, the recombinant enzyme can lyse more efficiently untreated S. epidermidis cells, compared to other Staphylococcus strains, suggesting enhanced specificity for S. epidermidis. These results suggest that the MurNAc-LAA domain from Ph28 endolysin may represent a promising new enzybiotic.

Identification and Functional Characterization of Xanthomonas oryzae Pv. oryzae Isolates

Rice is a model crop for studying host - pathogen interaction with one of the most devastating pathogens viz. Xanthomonas oryzae pv. oryzae (Xoo). In the present investigation, an attempt was made to isolate a virulent strain of Xathomonas oryzae from infected rice leaves and production of antioxidant enzymes, which are widely used in studying host - pathogen interactions. Among five isolates of X. oryzae pv. oryzae, SboBLB3 showed greater virulence as it showed susceptibility symptoms in infected rice leaves. The NCBI accession number of SboBLB3 was MH986180, which was obtained by sequencing 16s rDNA. The increased activity of antioxidant enzymes after SboBLB3 further confirms its virulence. Induction of antioxidant enzymes showed that SboBLB3 is a virulent strain of X. oryzae and can be used in host - pathogen interaction at molecular level.