Harnessing the Phytochemistry Through Pesticidal Potential of Diverse Elsholtzia Species: A Path to Sustainable Agriculture.

This study investigates the phytochemical profiles and pesticidal activities of various Elsholtzia species, including E. ciliata, E. flava, E. fruticosa, and E. eriostachya, to discover their bioactive potential for sustainable pest management. Through comparative phytochemical analysis using GC-MS technique, key compounds in the essential oils were identified. The major components were thymoquinone (44.97%) in E. ciliata, shisofuran (28.66%) in E. flava, perillene (50.88%) in E. fruticosa, and pinocarvone (42.41%) in E. eriostachya. Despite variability in chemical composition, all species primarily contained oxygenated monoterpenes. The bioactivity of the oils was evaluated for their nematicidal and herbicidal bioassays. E. ciliata showed the highest egg hatching inhibition and juvenile mortality of M. incognita, while E. flava exhibited the lowest activity. For herbicidal activity, E. eriostachya achieved 96.70% seed germination inhibition, 100% root growth inhibition, and 95.56% shoot growth inhibition. E. flava showed the lowest inhibition in germination, root length, and shoot length at 66.70%, 81.56%, and 85.28%, respectively. The findings revealed significant variations in phytochemical composition and pesticidal efficacy, emphasizing the importance of species selection for pest management. This research highlights the bioactive potential of Elsholtzia species in sustainable pest management strategies.

Environment dependent expression of mycobacterium hormone sensitive lipases: expression pattern under ex-vivo and individual in-vitro stress conditions in M. tuberculosis H37Ra.

BACKGROUND: Hormone-sensitive lipase (HSL) is a neutral lipase capable of hydrolysing various kinds of lipids. In comparison to single human Hormone Sensitive Lipase (hHSL), that is induced under nutritional stress, twelve serine hydrolases are annotated as HSL in Mycobacterium tuberculosis (mHSL). Mycobacterium is exposed to multiple stresses inside the host. Therefore, the present study was carried out to investigate if mHSL are also expressed under stress condition and if there is any correlation between various stress conditions and expression pattern of mHSL. METHODS AND RESULTS: The expression pattern of mHSL under different environmental conditions (in-vitro and ex-vivo) were studied using qRT-PCR in M. tuberculosis H37Ra strain with 16 S rRNA as internal control. Out of 12, only two genes (lipU and lipY) were expressed at very low level in mid log phase culture under aerobic conditions, while 9 genes were expressed at stationary phase of growth. Ten mHSLs were expressed post-infection under ex-vivo conditions in time dependent manner. LipH and lipQ did not express at any time point under ex-vivo condition. The relative expression of most of the genes under individual stress was much higher than observed in ex-vivo conditions. The expression pattern of genes varied with change in stress condition. CONCLUSIONS: Different sets of mHSL genes were expressed under different individual stress conditions pointing towards the requirement of different mHSL to combat different stress conditions. Overall, most of the mHSLs have demonstrated stress dependent expression pointing towards their role in intracellular survival of mycobacteria.

Characterization of LipN (Rv2970c) of Mycobacterium Tuberculosis H37Rv and its Probable Role in Xenobiotic Degradation.

LipN (Rv2970c) belongs to the Lip family of M. tuberculosis H37Rv and is homologous to the human Hormone Sensitive Lipase. The enzyme demonstrated preference for short carbon chain substrates with optimal activity at 45°C/pH 8.0 and stability between pH 6.0-9.0. The specific activity of the enzyme was 217 U/mg protein with pNP-butyrate as substrate. It hydrolyzed tributyrin to di- and monobutyrin. The active-site residues of the enzyme were confirmed to be Ser216, Asp316, and His346. Tetrahydrolipstatin, RHC-80267 and N-bromosuccinimide inhibited LipN enzyme activity completely. Interestingly, Trp145, a non active-site residue, demonstrated functional role to retain enzyme activity. The enzyme was localized in cytosolic fraction of M. tuberculosis H37Rv. The enzyme was able to synthesize ester of butyric acid, methyl butyrate, in presence of methanol. LipN was able to hydrolyze 4-hydroxyphenylacetate to hydroquinone. The gene was not expressed in in-vitro growth conditions while the expression of rv2970c gene was observed post 6h of macrophage infection by M. tuberculosis H37Ra. Under individual in-vitro stress conditions, the gene was expressed during acidic stress condition only. These findings suggested that LipN is a cytosolic, acid inducible carboxylesterase with no positional specificity in demonstrating activity with short carbon chain substrates. It requires Trp145, a non active site residue, for it's enzyme activity.

Characterization of an acid inducible lipase Rv3203 from Mycobacterium tuberculosis H37Rv.

The Rv3203 (LipV) of Mycobacterium tuberculosis (Mtb) H37Rv, is annotated as a member of Lip family based on the presence of characteristic consensus esterase motif 'GXSXG'. In vitro culture studies of Mtb H37Ra indicated that expression of Rv3203 gene was up-regulated during acidic stress as compared to normal whereas no expression was observed under nutrient and oxidative stress conditions. Therefore, detailed characterization of Rv3203 was done by gene cloning and its further expression and purification as his-tagged protein in microbial expression system. The enzyme was purified to homogeneity by affinity chromatography. It demonstrated broad substrate specificity and preferentially hydrolyzed p-nitrophenyl myristate. The purified enzyme demonstrated an optimum activity at pH 8.0 and temperature 50 °C. The specific activity, K m and V max of enzyme was determined to be 21.29 U mg(-1) protein, 714.28 µM and 62.5 µmol ml(-1) min(-1), respectively. The pH stability assay and circular dichroism spectroscopic analysis revealed that Rv3203 protein is more stable in acidic condition. Tetrahydrolipstatin, a specific lipase inhibitor and RHC80267, a diacylglycerol lipase inhibitor abolished the activity of this enzyme. The catalytic triad residues were determined to be Ser50, Asp180 and His203 residues by site-directed mutagenesis.

Molecular characterization of oxidative stress-inducible LipD of Mycobacterium tuberculosis H37Rv.

The Mycobacterium tuberculosis has developed intricate strategies to evade the killing of microorganism and support its survival in phagocytes. The genome sequence of bacterium revealed the presence of several genes for lypolytic enzymes. Rv1923 gene, a member of Lip family in M. tuberculosis demonstrated the least sequence similarity with its counterpart in non-pathogenic strain M. smegmatis. The expression of Rv1923 gene (LipD) was not observed in in vitro growing cultures of M. tuberculosis H37Ra while an upregulation of transcription of Rv1923 gene was noticed in oxidative conditions. For detailed characterization of LipD enzyme the Rv1923 gene was cloned in pQE30-UA vector and expressed in E. coli M15 cells. LipD was purified from inclusion bodies and refolded with nearly 40 % protein yield. The specific activity of enzyme was calculated to be 16 U/mg with pNP-palmitate as a preferred substrate. Kinetic analysis showed K(m) 0.645 mM and V(max) 24.75 U/ml with pNP-palmitate. Ser-102, Asp-342, and His-369, predicted as the members of the catalytic triad, were confirmed by mutagenesis. Mutagenesis studies revealed that catalytic serine residues located in ß-lactamase motifs (S-X-X-K) were responsible for lipolytic activity. Secondary structure analysis by CD spectroscopy demonstrated the presence of α helices and ß sheets in the canonical structure of LipD. The enzyme was stable up to 50 °C and was active even at pH 6.0. The expression of enzyme under stress conditions and its activity and stability at high temperature and low pH suggested the possible role of LipD in the survival of mycobacterium in macrophage compartment.

Recent changes to adult national immunization programs for pneumococcal vaccination in Europe and how they impact coverage: A systematic review of published and grey literature.

Despite widespread use of pneumococcal vaccines throughout Europe, the burden of pneumococcal disease (PD) in adults is considerable. To mitigate this burden, National Immunization Technical Advisory Groups (NITAGs) and Health Technology Assessment (HTA) agencies assess the value of different vaccine schedules for protecting against PD. The aim of this review was to assess the evidence and rationales used by NITAGs/HTA agencies, when considering recent changes to National Immunization Programs (NIPs) for adults, and how identified changes affected vaccine coverage rates (VCRs). A systematic review was conducted of published literature from PubMed® and Embase®, and gray literature from HTA/NITAG websites from the last 5 y, covering 31 European countries. Evidence related to NIP recommendations, epidemiology (invasive PD, pneumonia), health economic assessments and VCRs were collected and synthesized. Eighty-four records providing data for 26 countries were identified. Of these, eight described explicit changes to NIPs for adults in seven countries. Despite data gaps, some trends were observed; first, there appears to be a convergence of NIP recommendations in many countries toward sequential vaccination, with a pneumococcal conjugate vaccine (PCV), followed by pneumococcal polysaccharide vaccine 23. Second, reducing economic or healthcare burden were common rationales for implementing changes. Third, most health economic analyses assessing higher-valency PCVs for adults found its inclusion in NIPs cost-effective. Finally, higher coverage rates were seen in most cases where countries had expanded their NIPs to cover at-risk populations. The findings can encourage agencies to improve surveillance systems and work to reach the NIP's target populations more effectively.

Characterization of a novel esterase Rv1497 of Mycobacterium tuberculosisH37Rv demonstrating ß-lactamase activity.

The Rv1497 (LipL) of the Mycobacterium tuberculosis H37Rv was predicted to be similar to hypothetical esterases and penicillin binding proteins ofM. tuberculosis as well as to be involved in lipid metabolism. Sequence alignment revealed that Rv1497 protein contains characteristic consensus ß-lactamase motif 'SXXK' in addition to a conserve pentapeptide -GXSXG-, characteristic of lipolytic enzymes, at the C-terminus of protein in contrast to its usual N-terminus location. For detailed characterization of protein, the rv1497 gene was cloned, expressed with N-terminal His-tag and purified to homogeneity on Ni-NTA column. Rv1497 demonstrated both esterase and ß-lactamase activities. A serine located within consensus ß-lactamase motif 'SXXK' was identified as catalytic residue in both esterase and ß-lactamase enzymatic activities whereas serine residue located within conserved pentapeptide did not show any effect on both enzyme activities. The catalytic residues of Rv1497 for ß-lactamase activity were determined to be Ser88, Tyr-175 and His355 residues by site-directed mutagenesis. The enzyme demonstrated preference for short chain esters (pNP-butyrate). The expression of lipL gene was significantly up-regulated during acidic stress as compared to normal conditions in in vitro culture of M. tuberculosis H37Ra. This is perhaps the first report demonstrating an esterase of mycobacterium showing ß-lactamase activity.

Differential expression of two members of Rv1922-LipD operon in Mycobacterium tuberculosis: Does rv1923 qualify for membership?

rv1922 and rv1923 (lipD) are members of Rv1922-LipD operon in the genome of Mycobacterium tuberculosis. rv1922 was expressed under aerobic and stress conditions, whereas rv1923 was not expressed in aerobically grown bacteria but expressed moderately under oxidative stress conditions. Different expression of both the operonic genes under normal and stress conditions posed apprehensions for the inclusion of rv1922 and rv1923 in the same operon. The results from this study indicated that although the genes were expressed in an operonic manner, there existed the possibility of differential regulation for rv1923 which has been supported by in silico analysis for the presence of putative internal regulatory sites in the operon.