Chemical Composition and Antioxidant,Antiparasitic, Cytotoxicity and Antimicrobial Potential of the Algerian Limonium Oleifolium Mill. Essential Oil and Organic Extracts

This work aimed to investigate, for the first time, the chemical composition, antioxidant, antiparasitic, cytotoxicity, and antimicrobial activities of the aromatic plant Limonium oleifolium Mill. essential oil (EO) and organic extracts. L. oleifolium aerial parts essential oil was analyzed by GC-FID and GC-MS, and 46 constituents representing 98.25±1.12 % of the oil were identified. γ-Muurolene (10.81±0.07 %), cis-caryophyllene (7.71±0.06 %), o-cymene (7.07±0.01 %) and α-copaene (5.02±0.05 %) were the essential oil main compounds. The antioxidant activity of L. oleifolium EO and organic extracts (MeOH, CHCl₃, AcOEt, BuOH) was explored using 2,2-diphenyl-1-picrylhydrazyl (DPPH), ABTS, β-carotene/linoleic acid, cupric reducing antioxidant capacity (CUPRAC), and ferric reducing power assays. The results showed that L. oleifolium EO exhibit antioxidant capacity (IC₅₀=17.40±1.32 μg/mL for DPPH assay, IC₅₀=29.82±1.08 μg/mL for β-carotene assay, IC₅₀=25.23±1.01 μg/mL for ABTS assay, IC₅₀=9.11±0.08 μg/mL for CUPRAC assay and IC₅₀=19.41±2.06 mg/mL for reducing power assay). Additionally, the EO showed significant activity against trophozoite form of Acanthamoeba castellanii (IC₅₀=7.48±0.41 μg/mL) and promastigote form of Leishmania amazonensis (IC₅₀=19.36±1.06 μg/mL) and low cytotoxicity on murine macrophages (LC₅₀ 90.23±1.09 μg/mL), as well as good antimicrobial activity against Staphylococcus aureus, Escherichia coli, Klebsiella oxytoca, and Pseudomonas aeruginosa. These results suggest that L. oleifolium essential oil is a valuable source of bioactive compounds presenting antioxidant, antiparasitic, and antimicrobial activities. Furthermore, it is considered nontoxic.     

A Ruler Protein in a Complex for Antiviral Defense Determines the Length of Small Interfering CRISPR RNAs

Small RNAs undergo maturation events that precisely determine the length and structure required for their function. CRISPRs (clustered regularly interspaced short palindromic repeats) encode small RNAs (crRNAs) that together with CRISPR-associated (cas) genes constitute a sequence-specific prokaryotic immune system for anti-viral and anti-plasmid defense. crRNAs are subject to multiple processing events during their biogenesis, and little is known about the mechanism of the final maturation step. We show that in the Staphylococcus epidermidis type III CRISPR-Cas system, mature crRNAs are measured in a Cas10·Csm ribonucleoprotein complex to yield discrete lengths that differ by 6-nucleotide increments. We looked for mutants that impact this crRNA size pattern and found that an alanine substitution of a conserved aspartate residue of Csm3 eliminates the 6-nucleotide increments in the length of crRNAs. In vitro, recombinant Csm3 binds RNA molecules at multiple sites, producing gel-shift patterns that suggest that each protein binds 6 nucleotides of substrate. In vivo, changes in the levels of Csm3 modulate the crRNA size distribution without disrupting the 6-nucleotide periodicity. Our data support a model in which multiple Csm3 molecules within the Cas10·Csm complex bind the crRNA with a 6-nucleotide periodicity to function as a ruler that measures the extent of crRNA maturation.     

Somatic Variations in Cervical Cancers in Indian Patients

There are very few reports that describe the mutational landscape of cervical cancer, one of the leading cancers in Indian women. The aim of the present study was to investigate the somatic mutations that occur in cervical cancer. Whole exome sequencing of 10 treatment naïve tumour biopsies with matched blood samples, from a cohort of Indian patients with locally advanced disease, was performed. The data revealed missense mutations across 1282 genes, out of 1831 genes harbouring somatic mutations. These missense mutations (nonsynonymous + stop-gained) when compared with pre-existing mutations in the COSMIC database showed that 272 mutations in 250 genes were already reported although from cancers other than cervical cancer. More than 1000 novel somatic variations were obtained in matched tumour samples. Pathways / genes that are frequently mutated in various other cancers were found to be mutated in cervical cancers. A significant enrichment of somatic mutations in the MAPK pathway was observed, some of which could be potentially targetable. This is the first report of whole exome sequencing of well annotated cervical cancer samples from Indian women and helps identify trends in mutation profiles that are found in an Indian cohort of cervical cancer.     

Two new species and new records of biting midges of the genus Dasyhelea Kieffer (Diptera: Ceratopogonidae) from India

Pupa, male and female adults of Dasyhelea (Prokempia) flava Carter, Ingram & Macfie, pupa and male adult of D. (Pseudoculicoides) acuta n. sp., and male adult of D. (Pseudoculicoides) comosa n. sp. are described and illustrated.     

Restoration of p53/miR-34a regulatory axis decreases survival advantage and ensures Bax-dependent apoptosis of non-small cell lung carcinoma cells

Tumor-suppressive miR-34a, a direct target of p53, has been shown to target several molecules of cell survival pathways. Here, we show that capsaicin-induced oxidative DNA damage culminates in p53 activation to up-regulate expression of miR-34a in non-small cell lung carcinoma (NSCLC) cells. Functional analyses further indicate that restoration of miR-34a inhibits B cell lymphoma-2 (Bcl-2) protein expression to withdraw the survival advantage of these resistant NSCLC cells. In such a proapoptotic cellular milieu, where drug resistance proteins are also down-regulated, p53-transactivated Bcl-2 associated X protein (Bax) induces apoptosis via the mitochondrial death cascade. Our results suggest that p53/miR-34a regulatory axis might be critical in sensitizing drug-resistant NSCLC cells.     

Comparison of the effects of the superoxide dismutase mimetics EUK-134 and tempol on paraquat-induced nephrotoxicity

Paraquat-induced nephrotoxicity involves severe renal cell damage caused by reactive oxygen species (ROS), specifically via increasing concentrations of superoxide anions in the kidney. Recently, superoxide dismutase (SOD) mimetics (SODm) have been developed that display safe SOD activities but which also possess additional antioxidant enzyme (e.g., catalase) or ROS-scavenging activities. The aim of this study was to compare the effects of two such SODm, specifically, EUK-134, a SODm with catalase activity, and tempol, a SODm with ROS-scavenging properties, on paraquat-induced nephrotoxicity of renal NRK-52E cells. Incubation with paraquat (1 mM) for 24 h reduced cell viability and increased necrosis significantly. Paraquat also generated significant quantities of superoxide anions and hydroxyl radicals. Both EUK-134 (10-300 microM) and tempol (0.3-1.0 mM) were able to improve cell viability and reduced paraquat-induced cell death significantly via dismutation or scavenging of superoxide anions and reduced hydroxyl radical generation. The data presented here suggest that SODm such as EUK-134 and tempol, which possess additional catalase and/or ROS-scavenging activities, can significantly reduce renal cell damage caused by paraquat. These effects were evident at concentrations which avoid the pro-oxidant activities associated with higher concentrations of SOD. Such SODm could therefore prove to be beneficial as therapies for paraquat nephrotoxicity.     

Kinetic Analysis of DNA Strand Joining by Chlorella Virus DNA Ligase and the Role of Nucleotidyltransferase Motif VI in Ligase Adenylylation

Chlorella virus DNA ligase (ChVLig) is an instructive model for mechanistic studies of the ATP-dependent DNA ligase family. ChVLig seals 3'-OH and 5'-PO(4) termini via three chemical steps: 1) ligase attacks the ATP α phosphorus to release PP(i) and form a covalent ligase-adenylate intermediate; 2) AMP is transferred to the nick 5'-phosphate to form DNA-adenylate; 3) the 3'-OH of the nick attacks DNA-adenylate to join the polynucleotides and release AMP. Each chemical step requires Mg(2+). Kinetic analysis of nick sealing by ChVLig-AMP revealed that the rate constant for phosphodiester synthesis (k(step3) = 25 s(-1)) exceeds that for DNA adenylylation (k(step2) = 2.4 s(-1)) and that Mg(2+) binds with similar affinity during step 2 (K(d) = 0.77 mm) and step 3 (K(d) = 0.87 mm). The rates of DNA adenylylation and phosphodiester synthesis respond differently to pH, such that step 3 becomes rate-limiting at pH ≤ 6.5. The pH profiles suggest involvement of one and two protonation-sensitive functional groups in catalysis of steps 2 and 3, respectively. We suggest that the 5'-phosphate of the nick is the relevant protonation-sensitive moiety and that a dianionic 5'-phosphate is necessary for productive step 2 catalysis. Motif VI, located at the C terminus of the OB-fold domain of ChVLig, is a conserved feature of ATP-dependent DNA ligases and GTP-dependent mRNA capping enzymes. Presteady state and burst kinetic analysis of the effects of deletion and missense mutations highlight the catalytic contributions of ChVLig motif VI, especially the Asp-297 carboxylate, exclusively during the ligase adenylylation step.     

Putative Virulence Traits and Pathogenicity of Vibrio cholerae Non-O1, Non-O139 Isolates from Surface Waters in Kolkata, India

Vibrio cholerae non-O1, non-O139 was isolated from natural surface waters from different sites sampled in diarrhea endemic zones in Kolkata, India. Twenty-one of these isolates were randomly selected and included in the characterization. The multiserogroup isolates were compared by their virulence traits with a group of clinical non-O1, non-O139 isolates from the same geographic area. Of the 21 environmental isolates, 6 and 14 strains belonged to Heiberg groups I and II, respectively. Three of the environmental isolates showed resistance to 2,2-diamine-6,7-diisopropylpteridine phosphate. All of the non-O1, non-O139 strains were positive for toxR, and except for one environmental isolate, none of them were positive for tcpA in the PCR assay. None of the isolates were positive for genes encoding cholera toxin (ctxA), heat-stable toxin (est), heat-labile toxin (elt), and Shiga toxin variants (stx) of Escherichia coli. Additionally, except for one environmental isolate (PC32), all were positive for the gene encoding El Tor hemolysin (hly). The culture supernatants of 86% (18 of 21) of the environmental isolates showed a distinct cytotoxic effect on HeLa cells, and some of these strains also produced cell-rounding factor. The lipase, protease, and cell-associated hemagglutination activities and serum resistance properties of the environmental and clinical isolates did not differ much. However, seven environmental isolates exhibited very high hemolytic activities (80 to 100%), while none of the clinical strains belonged to this group. The environmental isolates manifested three adherence patterns, namely, carpet-like, diffuse, and aggregative adherence, and the clinical isolates showed diffuse adherence on HeLa cells. Of the 11 environmental isolates tested for enteropathogenic potential, 8 (73%) induced positive fluid accumulation (≥100) in a mouse model, and the reactivities of these isolates were comparable to those of clinical strains of non-O1, non-O139 and toxigenic O139 V. cholerae. Comparison of the counts of the colonized environmental and clinical strains in the mouse intestine showed that the organisms of both groups had similar colonizing efficiencies. These findings indicate the presence of potentially pathogenic V. cholerae non-O1, non-O139 strains in surface waters of the studied sites in Kolkata.     

Structure-function analysis of the OB and latch domains of chlorella virus DNA ligase

Chlorella virus DNA ligase (ChVLig) is a minimized eukaryal ATP-dependent DNA sealing enzyme with an intrinsic nick-sensing function. ChVLig consists of three structural domains, nucleotidyltransferase (NTase), OB-fold, and latch, that envelop the nicked DNA as a C-shaped protein clamp. The OB domain engages the DNA minor groove on the face of the duplex behind the nick, and it makes contacts to amino acids in the NTase domain surrounding the ligase active site. The latch module occupies the DNA major groove flanking the nick. Residues at the tip of the latch contact the NTase domain to close the ligase clamp. Here we performed a structure-guided mutational analysis of the OB and latch domains. Alanine scanning defined seven individual amino acids as essential in vivo (Lys-274, Arg-285, Phe-286, and Val-288 in the OB domain; Asn-214, Phe-215, and Tyr-217 in the latch), after which structure-activity relations were clarified by conservative substitutions. Biochemical tests of the composite nick sealing reaction and of each of the three chemical steps of the ligation pathway highlighted the importance of Arg-285 and Phe-286 in the catalysis of the DNA adenylylation and phosphodiester synthesis reactions. Phe-286 interacts with the nick 5'-phosphate nucleotide and the 3'-OH base pair and distorts the DNA helical conformation at the nick. Arg-285 is a key component of the OB-NTase interface, where it forms a salt bridge to the essential Asp-29 side chain, which is imputed to coordinate divalent metal catalysts during the nick sealing steps.