Current Biotechnology - Current Issue

Thaumatin-like proteins (TLPs) constitute the PR-5 family of plant pathogenesis-related (PR) proteins and are responsible for host defense and various growth and development-related processes. Mints (Mentha sp.) are essential oil-bearing plants, mainly affected by water availability and fungal pathogens.

Identification of candidate TLPs in Mentha longifolia that may help to counter pathogen infection.

Several bioinformatics procedures were used to identify TLP gene sequences from M. longifolia and to characterize them for nucleotide length and composition, their translated proteins, subcellular location, signal peptides, post translational modifications, allergenicity potential, evolutionary relatedness, and miRNA targeting.

19 TLP candidates were identified in M. longifolia (Mlo_TLP001 - Mlo_TLP0019) annotating the available transcriptome data. The molecular mass of Mlo_TLPs ranged between 35.43 kDa - 12.79 kDa. Expression analysis of the Mlo_TLPs revealed that Mlo_TLP003, 006, 008, 010, 012, 013, 014, and 018 were upregulated in M. longifolia stem as compared to the root under control conditions. Further, the analysis of control and V. dahliae infected M. longifolia plants has shown Mlo_TLP006 to be upregulated in both the infected stem and root tissues as compared to the control.

Mlo_TLP006 is a probable candidate that imparts resistance to fungal pathogens in Mentha longifolia.

Cancer is the uncontrolled proliferation of cells leading to metastasis due to genetic alterations resulting in oncogenes activation and tumor suppressor genes deactivation. It is the 2^nd leading cause of death across the world. MMP-9 or gelatinase B, plays an effective role in ECM degradation, normal tissue turnover, and tissue remodelling. Overexpression of MMP-9 has been studied in almost all types of cancers proving the effective role of MMP-9 in accelerating malignant conditions. Thus, targeting MMP-9 to treat cancer seems to be a potential strategy to deal with adverse pathologies of cancer. Chemotherapy and radiotherapy are frequently utilized for the treatment of cancer but are associated with diverse side effects. Flavonoids are natural compounds frequently found in plants and have been analysed for the structural inhibition potential against MMP-9 by several researchers to develop natural treatments against cancer, but none of the flavonoids have landed into clinical use.

In the present study, in-depth in-silico analysis to investigate the synergistic effects of flavonoids for structural inhibition of MMP-9 was done.

The ADMET and bioactive properties analysis revealed effective drug-like properties of the considered flavonoids. Principal component analysis of ADMET and bioactive properties revealed high similarity in the chemical nature of luteolin and quercetin. Molecular docking analysis of MMP-9 with the considered flavonoids individually revealed the highest effective binding energy of luteolin. Combination docking analysis of MMP-9 with different combinations of flavonoids led to the identification of two combinations including Quercetin with Genistein and Luteolin and Genistein revealing high negative binding energies of -15.48kcal/mol and -15.31kcal/mol which were significantly greater than the binding energies identified for respective ligands in individual dockings.

Thus, the present study put forward synergistic natural flavonoid combinations against cancer via the MMP-9 inhibition approach that can be further evaluated to develop high-efficacy treatments.

The waste-to-wealth concept was applied in this study. Our main objective was to identify and evaluate several microorganisms responsible for the production of important industrial enzymes using sludge from palm oil processing. We were able to isolate several bacteria that are tyrosinase producers: Bacillus cereus, Acinobacter seifertii, Klebsiella variicola and Pseudomonas stutzeri. Laccase producers Trametes polyzona, Bacillus subtilis, Bacillus pumilus and Staphylococcus condimenti, as well as lipase producers.

In addition, we focused specifically on B. cereus because we knew that it produces tyrosinase. Lipase is another target enzyme, and S. condimenti was found to be a hyperproducer. Production conditions included a 24-hour incubation period at 40°C and a pH of 6.0, while typical substrates such as starch and coconut oil were used.

Preliminary protein purification studies identified a 43 kDa lipase that is active at pH 7.0 and 40°C. Salts such as NaCl, various detergents such as Triton X-100 and Tween-80 as well as many metal ions enhanced the activity and made the enzyme unique in its biological function.

Even with EDTA (2.5 mM), which does not completely block its function, only 40% inhibition was observed. Only a few organic solvents such as butanol, acetone and DMF are involved in inhibiting its activity.

Enterococci are natural inhabitants of the human gastrointestinal tract and contain plasmids, which are often used to generate plasmid vectors. RGK7, a strain of Enterococcus faecium isolated from neonatal faeces, natively contains a 1935 bp plasmid pRGK7d.

This study focused on the molecular characterization of the Enterococcus faecium plasmid pRGK7d and its exploitation for the generation of shuttle cloning vectors.

The isolate RGK7 was identified as Enterococcus faecium by phenotypic and genotypic methods, including 16S rRNA sequencing. Phylogenetic analysis also confirmed that the strain belongs to the group of E. faecium. BLAST analysis of the pRGK7d plasmid sequence revealed the highest homology with E. faecium genes for replication protein. ORF Finder analysis of the 1.9 kb plasmid pRGK7d showed a total of 17 ORFs in multiple overlapping frames, and the biggest ORF sequence showed 97% homology with the Rep protein of E. faecium. In order to make it an E. coli-Enterococcus shuttle vector, the plasmid was first cloned in pUC18 and then recloned in T vector by incorporating antibiotic selection markers ampicillin resistance (amp^r) and erythromycin resistance (ery^r) for its selectivity in Gram-negative E. coli and Gram-positive E. faecium. The shuttle vector was reduced in size and made more effective by cloning only the Rep protein instead of the whole plasmid along with the ery^r gene.

An E. coli-Enterococcus shuttle vector was generated utilizing the plasmid pRGK7d, which, on transformation, conferred ampicillin and erythromycin resistance to the E. coli and Enterococcus, respectively, that was otherwise sensitive. The shuttle vector was found to be stable in both organisms.

This study investigates the defense responses of tolerant versus susceptible varieties of tomato against the hemi biotroph pathogen Rhizoctonia solani.

The early infection of Rhizoctonia solani was characterized by a tolerant Pusa Ruby and a susceptible tomato variety, Oriental (VT- 946).

The microscopy experiments showed more hyphal growth in a susceptible plant than in a tolerant one. The hyphae formed extensive branching, contacting the host surface to form a greater number of various infection structures for penetration in the susceptible host compared to the tolerant variety. Biochemical assays showed differences in the production of phenolics, proline, hydrogen peroxide, and catalase in the tomato varieties.

The differential expression profiles of the early response allene oxide synthase AOS gene were obtained over time in the two hosts.