IntroductionNew levels of gene regulation with microRNA (miR) and gene copy number alterations (CNAs) have been identified as playing a role in various cancers. We have previously reported that sporadic breast cancer tissues exhibit significant alteration in H2AX gene copy number. However, how CNA affects gene expression and what is the role of miR, miR-24-2, known to regulate H2AX expression, in the background of the change in copy number, are not known. Further, many miRs, including miR-24-2, are implicated as playing a role in cell proliferation and apoptosis, but their specific target genes and the pathways contributing to them remain unexplored.MethodsChanges in gene copy number and mRNA/miR expression were estimated using real-time polymerase chain reaction assays in two mammalian cell lines, MCF-7 and HeLa, and in a set of sporadic breast cancer tissues. In silico analysis was performed to find the putative target for miR-24-2. MCF-7 cells were transfected with precursor miR-24-2 oligonucleotides, and the gene expression levels of BRCA1, BRCA2, ATM, MDM2, TP53, CHEK2, CYT-C, BCL-2, H2AFX and P21 were examined using TaqMan gene expression assays. Apoptosis was measured by flow cytometric detection using annexin V dye. A luciferase assay was performed to confirm BCL-2 as a valid cellular target of miR-24-2.ResultsIt was observed that H2AX gene expression was negatively correlated with miR-24-2 expression and not in accordance with the gene copy number status, both in cell lines and in sporadic breast tumor tissues. Further, the cells overexpressing miR-24-2 were observed to be hypersensitive to DNA damaging drugs, undergoing apoptotic cell death, suggesting the potentiating effect of mir-24-2-mediated apoptotic induction in human cancer cell lines treated with anticancer drugs. BCL-2 was identified as a novel cellular target of miR-24-2.Conclusionsmir-24-2 is capable of inducing apoptosis by modulating different apoptotic pathways and targeting BCL-2, an antiapoptotic gene. The study suggests that miR-24-2 is more effective in controlling H2AX gene expression, regardless of the change in gene copy number. Further, the study indicates that combination therapy with miR-24-2 along with an anticancer drug such as cisplatin could provide a new avenue in cancer therapy for patients with tumors otherwise resistant to drugs.

This study was designed to understand the mechanism and functional implication of the two heterozygous mutations (H391Y and K422R) of human pyruvate kinase M2 isozyme (PKM 2 ) observed earlier in a Bloom syndrome background. The co-expression of homotetrameric wild type and mutant PKM 2 in the cellular milieu resulting in the interaction between the two at the monomer level was substantiated further by in vitro experiments. The cross-monomer interaction significantly altered the oligomeric state of PKM 2 by favoring dimerization and heterotetramerization. In silico study provided an added support in showing that hetero-oligomerization was energetically favorable. The hetero-oligomeric populations of PKM 2 showed altered activity and affinity, and their expression resulted in an increased growth rate of Escherichia coli as well as mammalian cells, along with an increased rate of polyploidy. These features are known to be essential to tumor progression. This study provides insight in understanding the modulated role of large oligomeric multifunctional proteins such as PKM 2 by affecting cellular behavior, which is an essential observation to understand tumor sustenance and progression and to design therapeutic intervention in future.A variety of genetic diseases and experimental situations within the heterozygous state depict an allelic relationship where the mutant recessive allele overrides the function of its normal (wild type) dominant allele. This condition, referred to as dominant negative, is usually observed in the case of oligomeric or multidomain proteins with a possibility of cross-monomer interaction. Such mutant proteins by acting as competitive inhibitors of the normal protein function could generate polymorphic forms in a single cell. A phenomenon observed in collagen, where dominant negative mutations cause the production of abnormal oligomers (1, 2), and in transcription factors like helix-loop-helix and leucine zippers, where mutant monomers sequestering the function of wild type in a dimer bound to DNA, leads to an altered gene expression (3-5). Dominant negative mutations are also reported to affect some multifunctional molecules like p53 with differential impact on cell physiology (6 -10).Pyruvate kinase (EC 2.7.1.40) catalyzes irreversibly the transphosphorylation from P-enolpyruvate to ADP-generating pyruvate and ATP in glycolysis (11,12). Depending upon the differential metabolic requirements of the tissues, the enzyme is expressed in four different isoforms, L, R, M 1 , and M 2 in vertebrates (13). PKM 2 2 is a ubiquitous, prototype enzyme, present in all tissues during embryonic stage, and is gradually replaced by other isozymic forms in specific tissues, during development. It is necessary for cellular division irrespective of the type of tissue and reappears during cellular division and tumor formation (14 -17). PKM 2 is known to regulate its activity by switching between an active tetramer and inactive dimer form in a fructose 1,6-bisphosphate-dependent manner to shift the cellular met...

Aim:  To examine the zinc (Zn) solubilization potential and nematicidal properties of Gluconacetobacter diazotrophicus. Methods and Results:  Atomic Absorption Spectrophotometer, Differential Pulse Polarography and Gas Chromatography Coupled Mass Spectrometry were used to estimate the total Zn and Zn2+ ions and identify the organic acids present in the culture supernatants. The effect of culture filtrate of Zn‐amended G. diazotrophicus PAl5 on Meloidogyne incognita in tomato was examined under gnotobiotic conditions. Gluconacetobacter diazotrophicus PAl5 effectively solubilized the Zn compounds tested and 5‐ketogluconic acid was identified as the major organic acid aiding the solubilization of zinc oxide. The presence of Zn compounds in the culture filtrates of G. diazotrophicus enhanced the mortality and reduced the root penetration of M. incognita under in vitro conditions. Conclusions:  5‐ketogluconic acid produced by G. diazotrophicus mediated the solubilization process and the available Zn2+ ions enhanced the nematicidal activity of G. diazotrophicus against M. incognita. Significance and Impact of the Study:  Zn solubilization and enhanced nematicidal activity of Zn‐amended G. diazotrophicus provides the possibility of exploiting it as a plant growth promoting bacteria.

Bacterial resistance is a serious threat to human health. The production of β-lactamase, which inactivates β-lactams is most common cause of resistance to the β-lactam antibiotics. The Class A enzymes are most frequently encountered among the four β-lactamases in the clinic isolates. Mutations in class A β-lactamases play a crucial role in substrate and inhibitor specificity. SHV and TEM type are known to be most common class A β-lactamases. In the present study, we have analyzed the effect of inhibitor resistant S130G point mutation of SHV type Class-A β-lactamase using molecular dynamics and other in silico approaches. Our study involved the use of different in silico methods to investigate the affect of S130G point mutation on the major physico-chemical properties of SHV type class A β-lactamase. We have used molecular dynamics approach to compare the dynamic behaviour of native and S130G mutant form of SHV β-lactamase by analyzing different properties like root mean square deviation (RMSD), H-bond, Radius of gyration (Rg) and RMS fluctuation of mutation. The results clearly suggest notable loss in the stability of S130G mutant that may further lead to decrease in substrate specificity of SHV. Molecular docking further indicates that S130G mutation decreases the binding affinity of all the three inhibitors in clinical practice.