Endorsing cellular competitiveness in aberrant epithelium of oral submucous fibrosis progression: neighbourhood analysis of immunohistochemical attributes

Epithelial abnormality during the transformation of oral submucous fibrosis (OSF) into oral squamous cell carcinoma has been well studied and documented. However, the differential contribution of atrophy and hyperplasia for malignant potentiality of OSF is yet to be resolved. Existing diagnostic conjectures lack precise diagnostic attributes which may be effectively resolved by substantiation of specific molecular pathology signatures. Present study elucidates existence of cellular competitiveness in OSF conditions using computer-assisted neighbourhood analysis in quantitative immunohistochemistry (IHC) framework. The concept of field cancerization was contributory in finding correspondence among neighbouring cells of epithelial layers with reference to differential expression of cardinal cancer-related genes [c-Myc (oncogene), p53 (tumour suppressor), and HIF-1α (hypoxia regulator)] which are known to be important sensors in recognizing cellular competitive interface. Our analyses indicate that different states of OSF condition may be associated with different forms of competitiveness within epithelial neighbouring cells which might be responsible to shape the present and future of the pre-malignant condition. Analytical findings indicated association of atrophic epithelium with stress-driven competitive environment having low c-Myc, high-p53, and stable HIF-1α (the looser cells) which undergo apoptosis. Whereas, the cells with high c-Myc⁺ (winner cells) give rise to hyperplastic epithelium via possible mutation in p53. The epithelial dysplasia plausibly occurs due to clonal expansion of c-Myc and p53 positive supercompetitor cells. Present study proposes quantitative IHC along with neighbourhood analysis which might help us to dig deeper on to the interaction among epithelial cell population to provide a better understanding of field cancerization and malignant transformation of pre-malignancy.     

Mechanisms of sodium/calcium selectivity in sodium channels probed by cysteine mutagenesis and sulfhydryl modification.

A conserved lysine residue in the "P loop" of domain III renders sodium channels highly selective. Conversion of this residue to glutamate, to mimic the homologous position in calcium channels, enables Ca2+ to permeate sodium channels. Because the lysine-to-glutamate mutation converts a positively charged side chain to a negative one, it has been proposed that a positive charge at this position suffices for Na+ selectivity. We tested this idea by converting the critical lysine to cysteine (K1237C) in mu 1 rat skeletal sodium channels expressed in Xenopus oocytes. Selectivity of the mutant channels was then characterized before and after chemical modification to alter side-chain charge. Wild-type channels are highly selective for Na+ over Ca2+ (PCa/PNa < 0.01). The K1237C mutation significantly increases permeability to Ca2+ (PCa/PNa = 0.6) and Sr2+. Analogous mutations in domains I (D400C), II (E755C), and IV (A1529C) did not alter the selectivity for Na+ over Ca2+, nor did any of the domain IV mutations (G1530C, W1531C, and D1532C) that are known to affect monovalent selectivity. Interestingly, the increase in permeability to Ca2+ in K1237C cannot be reversed by simply restoring the positive charge to the side chain by using the sulfhydryl modifying reagent methanethiosulfonate ethylammonium. Single-channel studies confirmed that modified K1237C channels, which exhibit a reduced unitary conductance, remain permeable to Ca2+, with a PCa/PNa of 0.6. We conclude that the chemical identity of the residue at position 1237 is crucial for channel selectivity. Simply rendering the 1237 side chain positive does not suffice to restore selectivity to the channel.     

Molecular motions within the pore of voltage-dependent sodium channels.

The pores of ion channel proteins are often modeled as static structures. In this view, selectivity reflects rigidly constrained backbone orientations. Such a picture is at variance with the generalization that biological proteins are flexible, capable of major internal motions on biologically relevant time scales. We tested for motions in the sodium channel pore by systematically introducing pairs of cysteine residues throughout the pore-lining segments. Two distinct pairs of residues spontaneously formed disulfide bonds bridging domains I and II. Nine other permutations, involving all four domains, were capable of disulfide bonding in the presence of a redox catalyst. The results are inconsistent with a single fixed backbone structure for the pore; instead, the segments that line the permeation pathway appear capable of sizable motions.     

The co-chaperone CHIP is induced in various stresses and confers protection to cells

The C-terminus of Hsp70 interacting protein (CHIP) is being considered to be a cellular quality control E3 ubiquitin ligase because of its ability to degrade misfolded proteins in association with heat shock chaperones. The neuroprotective role of CHIP also has been implicated in several familial neurodegenerative diseases including polyglutamine diseases. However, the regulation of the expression of CHIP under different stress conditions and its protective role thereon is unknown. Here we have shown that the mRNA level of CHIP is significantly increased in the cells exposed to oxidative, endoplasmic reticulum and proteasomal stress. CHIP also protected from various stress-induced cell death. Finally, we have demonstrated upregulation of CHIP mRNA levels in the expanded polyglutamine protein expressing cells. Our result suggests that the upregulation of CHIP under various stress environments is an adaptive response of the cells to deal with the excess burden of misfolded protein.     

Investigation of the efficacy of paclitaxel on some miRNAs profiles in breast cancer stem cells

Understanding of the functions of microRNAs in breast cancer and breast cancer stem cells have been a hope for the development of new molecular targeted therapies. Here, it is aimed to investigate the differences in the expression levels of let-7a, miR-10b, miR-21, miR-125b, miR-145, miR-155, miR-200c, miR-221, miR-222 and miR-335, which associated with gene and proteins in MCF-7 (parental) and MCF-7s (Mammosphere/stem cell-enriched population/CD44+/CD24-cells) cells treated with paclitaxel. MCF-7s were obtained from parental MCF-7 cells. Cytotoxic activity of paclitaxel was determined by ATP assay. Total RNA isolation and cDNA conversion were performed from the samples. Changes in expression levels of miRNAs were examined by RT-qPCR. Identified target genes and proteins of miRNAs were analyzed with RT-qPCR and western blot analysis, respectively. miR-125b was significantly expressed (2.0946-fold; p = 0.021) in MCF-7s cells compared to control after treatment with paclitaxel. Downregulation of SMO, STAT3, NANOG, OCT4, SOX2, ERBB2 and ERBB3 and upregulation of TP53 genes were significant after 48 h treatment in MCF-7s cells. Protein expressions of SOX2, OCT4, SMAD4, SOX2 and OCT4 also decreased. Paclitaxel induces miR-125b expression in MCF-7s cells. Upregulation of miR-125b may be used as a biomarker for the prediction of response to paclitaxel treatment in breast cancer.     

Hydrophilic Acylated Surface Protein A (HASPA) of <Emphasis Type="Italic">Leishmania donovani</Emphasis>: Expression,Purification and Biophysico-Chemical Characterization

Hydrophilic acylated surface proteins (HASPs) are acidic surface proteins which get localized on the surface of Leishmania parasite during infective stages through a “non-classical” pathway. In this study, we report the heterologous expression and purification of Leishmania donovani HASPA (r-LdHASPA) in E. coli system and its partial characterization. The structural aspects of the purified protein were analyzed using CD spectroscopy and modeling studies which indicate that r-LdHASPA consists of random coils. Studies in mouse macrophage RAW264.7 cell lines indicate that r-LdHASPA enhances reactive oxygen species (ROS) production. Co-immunoprecipitation (IP) studies indicate that r-LdHASPA interacts with certain macrophage proteins which however could not be identified unambiguously. The present study provides key insights into the structural and functional aspects of an important Leishmania protein, HASPA, which we believe could be useful for further research on vaccine/drug development.     

Continuous monitoring of phytoplankton dynamics in Lake Balaton (Hungary) using on-line delayed fluorescence excitation spectroscopy

1. This study introduces delayed fluorescence (DF) excitation spectroscopy as an on‐line tool for in situ monitoring of the composition and biomass of various colour classes of phytoplankton when they are photosynthetically active (cyanobacteria, chlorophytes, chromophytes and cryptophytes). The DF data are validated by comparison with those from conventional methods (weekly microscopic counts and the measurement of chlorophyll concentration). 2. The composition of phytoplankton as assessed by DF agreed reasonably well with the results from microscopic counts, particularly when differences in chlorophyll‐specific DF integrals of the various colour classes were taken into account. 3. Integrals of DF spectra were converted into concentration of chlorophyll a using empirical factors derived from field data. The value of the conversion factor was nearly twice as high when the relative abundance of cyanobacteria was low (<15%) than when it was high. The converted DF‐chl time series agreed well with chlorophyll measurements particularly when blooms were developing. As the DF method is inherently free of the interference caused by pigment degradation products, the discrepancy between the two data sets increased during the collapse of blooms and when sediment resuspension was intense. 4. Fourier spectrum analysis of the time series of DF‐chl indicated that samples must be taken, at a minimum, every 2–3 days to capture the dynamics of phytoplankton. As a consequence, the dynamics of various algal blooms, including their timing, duration and net growth rate, could be estimated with greater confidence than by using conventional methods alone. 5. On‐line DF spectroscopy is an advanced technique for monitoring daily the biomass and composition of the photosynthetically active phytoplankton in aquatic environments, including turbid shallow lakes. At present, the detection limit is around 1 mg DF‐chl a m^?3 in terms of total biomass but confidence in estimates of phytoplankton composition declines sharply below about 5 mg chl a m^?3. 6. On‐line DF spectroscopy represents a promising approach for monitoring phytoplankton. It will be useful in water management where it can act as an early‐warning system of declines in water quality. In basic ecological research it can supplement manual methods. While default calibration spectra may be acceptable for routine monitoring, we suggest a careful individual calibration of the DF spectrometer for basic research. The statistical methods developed here help to assess the adequacy of various calibration sets.