Reduction of nuclear Y654-p-β-catenin expression through SH3GL2-meditated downregulation of EGFR in chemotolerance TNBC: Clinical and prognostic importance

Triple negative breast cancer (TNBC) originates from a less differentiated ductal cell of breast, which is less sensitive to chemotherapy. The chemotolerance mechanism of TNBC has not yet been studied in detail. For this reason, molecular profiles (expression/genetic/epigenetic) of Y654-p-β-catenin (active) and its kinase epidermal growth factor receptor (EGFR) along with SH3GL2 (regulator of EGFR homeostasis) were compared between neoadjuvant chemotherapy treated (NACT) and pretherapeutic TNBC samples. Reduced nuclear expression of Y654-p-β-catenin protein with low proliferation index and CD44 prevalence showed concordance with reduced expression of EGFR/Y1045-p-EGFR proteins in the NACT samples than the pretherapeutic TNBC samples. Infrequent messenger RNA expression, gene amplification (10–32.5%), and mutation (1%) of EGFR were seen in the TNBC samples irrespective of therapy, suggesting the importance of EGFR protein stabilization in this tumor. The upregulation of SH3GL2 seen in the NACT samples in contrast to the pretherapeutic samples might be due to its promoter hypomethylation, as seen in the quantitative methylation assay. A similar trend of upregulation of SH3GL2 and downregulation of EGFR, Y1045-p-EGFR, Y654-p-β-catenin were seen in the MDA-MB-231 cell line using antharacycline antitumor drugs (doxorubicin/nogalamycin). The NACT patients with reduced expression of Y654-p-β-catenin and/or EGFR and high expression of SH3GL2 showed comparatively better prognosis than the pretherapeutic patients. Thus, our study showed that reduced nuclear expression of Y654-p-β-catenin in NACT samples due to downregulation of EGFR protein through promoter hypomethylation-mediated upregulation of SH3GL2, resulting in low proliferation index/CD44 prevalence with better prognosis of the NACT patients, might have an important role in the chemotolerance of TNBC.     

Influence of Physicochemical Parameters on the Abundance of Coliform Bacteria in an Industrial Site of the Hooghly River, India

Industrial effluents from jute, paper, pulp mills and sewage from households are regularly discharged into the Hooghly River. It generates a potential risk for both humans and animals of the area concerned. In the present study, water quality of the Hooghly River passing by the site of a growing township (Naihati, North 24 Parganas, West Bengal, India) was assessed throughout the year 2010 on the basis of the data collected on the physicochemical and microbiological parameters. The water samples collected on each month revealed the presence of higher amount of coliform bacteria, Streptococcus sp. and Escherichia coli, than the standard limit. Different physicochemical parameters like chemical oxygen demand, biological oxygen demand, dissolved oxygen (DO), total suspended solids, total dissolved solids (TDS), total hardness, alkalinity, chlorinity, nitrate and nitrite of the water at the sampling sites were found to be considerably higher than the levels standardized by WHO (2006). It was found that the relative abundance of Streptococcus and E. coli was influenced by two independent variables (water quality parameters), namely, DO and TDS. The abundance of coliform bacteria in the water sample warrants the adoption of proper measures to reduce the pollution level at the point source on way of scientific disposal of industrial effluents.     

Association of RAGE gene polymorphism with circulating AGEs level and paraoxonase activity in relation to macro-vascular complications in Indian type 2 diabetes mellitus patients

Background and aims

Sustained interaction of advanced glycation end products (AGEs) with their receptor RAGE and subsequent signaling plays an important role in the development of diabetic complications. Genetic variation of RAGE gene may be associated with the development of vascular complications in type 2 diabetes mellitus (T2DM).

Objectives

The present study aimed to explore the possible association of RAGE gene polymorphisms namely − 374T/A, − 429T/C and G82S with serum level of AGEs, paraoxonase (PON1) activity and macro-vascular complications (MVC) in Indian type 2 diabetes mellitus patients (T2DM).

Methods

A total of 265 diabetic patients, including DM without any complications (n = 135), DM-MVC (n = 130) and 171 healthy individuals were enrolled. Genotyping of RAGE variants were assessed by polymerase chain reaction-restriction fragment length polymorphism. Serum AGEs were estimated by ELISA and fluorometrically. and PON1 activity was assessed spectrophotometrically.

Results

Of the three examined SNPs, association of − 429T/C polymorphism with MVC in T2DM was observed (OR = 3.001, p = 0.001) in the dominant model. Allele ‘A’ of − 374T/A polymorphism seems to confer better cardiac outcome in T2DM. Patients carrying C allele (− 429T/C) and S allele (G82S) had significantly higher AGEs levels. − 429T/C polymorphism was also found to be associated with low PON1 activity. Interaction analysis revealed that the risk of development of MVC was higher in T2DM patients carrying both a CC genotype of − 429T/C polymorphism and a higher level of AGEs (OR = 1.343, p = 0.040).

Conclusion

RAGE gene polymorphism has a significant effect on AGEs level and PON1 activity in diabetic subjects compared to healthy individuals. Diabetic patients with a CC genotype of − 429T/C are prone to develop MVC, more so if AGEs levels are high and PON1 activity is low.     

YY1 controls Igκ repertoire and B‐cell development,and localizes with condensin on the Igκ locus

Conditional knock‐out (KO) of Polycomb Group (PcG) protein YY1 results in pro‐B cell arrest and reduced immunoglobulin locus contraction needed for distal variable gene rearrangement. The mechanisms that control these crucial functions are unknown. We deleted the 25 amino‐acid YY1 REPO domain necessary for YY1 PcG function, and used this mutant (YY1ΔREPO), to transduce bone marrow from YY1 conditional KO mice. While wild‐type YY1 rescued B‐cell development, YY1ΔREPO failed to rescue the B‐cell lineage yielding reduced numbers of B lineage cells. Although the IgH rearrangement pattern was normal, there was a selective impact at the Igκ locus that showed a dramatic skewing of the expressed Igκ repertoire. We found that the REPO domain interacts with proteins from the condensin and cohesin complexes, and that YY1, EZH2 and condensin proteins co‐localize at numerous sites across the Ig kappa locus. Knock‐down of a condensin subunit protein or YY1 reduced rearrangement of Igκ Vκ genes suggesting a direct role for YY1‐condensin complexes in Igκ locus structure and rearrangement.     

Insights into the Mechanism of Ribosomal Incorporation of Mammalian L13a Protein during Ribosome Biogenesis

In contrast to prokaryotes, the precise mechanism of incorporation of ribosomal proteins into ribosomes in eukaryotes is not well understood. For the majority of eukaryotic ribosomal proteins, residues critical for rRNA binding, a key step in the hierarchical assembly of ribosomes, have not been well defined. In this study, we used the mammalian ribosomal protein L13a as a model to investigate the mechanism(s) underlying eukaryotic ribosomal protein incorporation into ribosomes. This work identified the arginine residue at position 68 of L13a as being essential for L13a binding to rRNA and incorporation into ribosomes. We also demonstrated that incorporation of L13a takes place during maturation of the 90S preribosome in the nucleolus, but that translocation of L13a into the nucleolus is not sufficient for its incorporation into ribosomes. Incorporation of L13a into the 90S preribosome was required for rRNA methylation within the 90S complex. However, mutations abolishing ribosomal incorporation of L13a did not affect its ability to be phosphorylated or its extraribosomal function in GAIT element-mediated translational silencing. These results provide new insights into the mechanism of ribosomal incorporation of L13a and will be useful in guiding future studies aimed at fully deciphering mammalian ribosome biogenesis.     

A comparative study on the interaction of the putative anticancer alkaloids,sanguinarine and chelerythrine,with single- and double-stranded,and heat-denatured DNAs

A detailed investigation on the interaction of two benzophenanthridine alkaloids, sanguinarine (SGR) and chelerythrine (CHL), with the double-stranded (ds), heat-denatured (hd), and single-stranded (ss) DNA was performed by spectroscopy and calorimetry techniques. Binding to the three DNA conformations leads to quenching of fluorescence of SGR and enhancement in the fluorescence of CHL. The binding was cooperative for both of the alkaloids with all the three DNA conformations. The binding constant values of both alkaloids with the ds DNA were in the order of 10⁶ M^?1; binding was weak with hd and much weaker to the ss DNA. The fluorescence emission of the alkaloid molecules bound to the ds and hd DNAs was quenched much less compared to those bound to the ss DNA based on competition with the anionic quencher KI. For both double stranded and heat denatured structures the emission of the bound alkaloid molecules was polarized significantly and strong energy transfer from the DNA bases to the alkaloid molecules occurred. Intercalation of SGR and CHL to ds, hd, and ss DNA was proved from these fluorescence results. Calorimetric studies suggested that the binding to all DNA conformations was both enthalpy and entropy favored. Both the alkaloids preferred double-helical regions for binding, but SGR was a stronger binder than CHL to all the three DNA structures.     

Chandipura Virus Induces Neuronal Death through Fas-Mediated Extrinsic Apoptotic Pathway

Chandipura virus (CHPV; genus Vesiculovirus, family Rhabdoviridae) is an emerging tropical pathogen with a case fatality rate of 55 to 75% that predominantly affects children in the age group of 2 to 16 years. Although it has been established as a neurotropic virus causing encephalitis, the molecular pathology leading to neuronal death is unknown. The present study elucidates for the first time the mechanism of cell death in neurons after CHPV infection that answers the basic cause of CHPV-mediated neurodegeneration. Through various cell death assays in vitro and in vivo, a relationship between viral replication within neuron and neuronal apoptosis has been established. We report that expression of CHPV phosphoprotein increases up to 6 h postinfection and diminishes thereafter in neuronal cell lines, signifying the replicative phase of CHPV. Various analyses conducted during the investigation established that CHPV-infected neurons are undergoing apoptosis through an extrinsic pathway mediated through the Fas-associated death domain (FADD) following activation of caspase-8 and -3 and prominent cleavage of poly(ADP-ribose) polymerase (PARP). Knocking down the expression of caspase-3, the final executioner of apoptosis, in a neuronal cell line by endoribonuclease-prepared small interfering RNA (siRNA) validated its pivotal role in CHPV-mediated neurodegeneration by showing reduction in apoptosis after CHPV infection.     

Magnetopriming circumvents the effect of salinity stress on germination in chickpea seeds

Chickpea seeds of Pusa 1053 (Mediterranean) and Pusa 256 (native) were magnetoprimed with 100 mT static magnetic field for 1 h to evaluate the effect of magnetopriming on germination of seeds under saline conditions. Enhanced rate of germination and seedling growth parameters (root and shoot length, and vigour indices) under different salinity levels indicated that magnetopriming was more effective in alleviating salinity stress at early seedling stage in Pusa 1053 as compared to Pusa 256. Dynamics of seed water absorption in magnetoprimed seeds showed increased water uptake in Pusa 1053 under non-saline as compared to saline conditions. This could have resulted in faster hydration of enzymes in primed seeds leading to higher rate of germination. Total amylase, protease and dehydrogenase activities were higher in primed seeds as compared to unprimed seeds under both non-saline and saline conditions. Production of superoxide radicals was enhanced in germinating seeds of both the genotypes under salinity irrespective of priming. Increased levels of hydrogen peroxide in germinating magnetoprimed seeds, under both the growing conditions, suggested its role in promotion of germination. Our results showed that magnetopriming of dry seeds of chickpea can be effectively used as a pre-sowing treatment for mitigating adverse effects of salinity at seed germination and early seedling growth.     

Structural Mechanism of Replication Stalling on a Bulky Amino-Polycyclic Aromatic Hydrocarbon DNA Adduct by a Y Family DNA Polymerase

Polycyclic aromatic hydrocarbons and their nitro derivatives are culprits of the detrimental health effects of environmental pollution. These hydrophobic compounds metabolize to reactive species and attach to DNA producing bulky lesions, such as N-[deoxyguanosine-8-yl]-1-aminopyrene (APG), in genomic DNA. The bulky adducts block DNA replication by high-fidelity polymerases and compromise replication fidelities and efficiencies by specialized lesion bypass polymerases. Here we present three crystal structures of the DNA polymerase Dpo4, a model translesion DNA polymerase of the Y family, in complex with APG-lesion-containing DNA in pre-insertion and extension stages. APG is captured in two conformations in the pre-insertion complex; one is highly exposed to the solvent, whereas the other is harbored in a shallow cleft between the finger and unique Y family little finger domain. In contrast, APG is in a single conformation at the extension stage, in which the pyrene ring is sandwiched between the little finger domain and a base from the turning back single-stranded template strand. Strikingly, a nucleotide intercalates the DNA helix to form a quaternary complex with Dpo4, DNA, and an incoming nucleotide, which stabilizes the distorted DNA structure at the extension stage. The unique APG DNA conformations in Dpo4 inhibit DNA translocation through the polymerase active site for APG bypass. We also modeled an insertion complex that illustrates a solvent-exposed pyrene ring contributing to an unstable insertion state. The structural work combined with our lesion replication assays provides a novel structural mechanism on bypass of DNA adducts containing polycyclic aromatic hydrocarbon moieties.