Assessment of Genetic Relatedness of Crossbred Chicken Populations Using Microsatellite Markers

To measure genetic relatedness between populations, for breeding purposes, we analyzed 170 birds from six crossbred populations of three pure lines of White Leghorn chickens, using 14 microsatellite markers. All the microsatellites were polymorphic, with 2–6 alleles. The mean number of alleles per locus was 3.21. The effective number of alleles varied from 1.14 to 3.94. The observed heterozygosity varied from 0.133 to 1.00, with a mean of 0.748. The F _IS values were mostly negative, with an average of ?0.345. The mean F _ST value was 0.056. The Nm values ranged from 1.91 to 42.17. The highest genetic identity was observed between IWI × IWK and IWK × IWI. The relation between any two groups of crosses was more than 85%. The results suggest that the crossbred populations were very closely related.     

Human papillomavirus (HPV) oncoprotein E6 facilitates Calcineurin-Nuclear factor for activated T cells 2 (NFAT2) signaling to promote cellular proliferation in cervical cell carcinoma

The calcineurin-NFAT signaling pathway regulates cell proliferation, differentiation, and development in diverse cell types and organ systems. Deregulation of calcineurin-NFAT signaling has been reported in leukaemias and few solid tumors such as breast and colon. In the present study, we found elevated calcineurin protein levels and phosphatase activity in cervical cancer cell lines and depletion of the same attenuated cell proliferation. Additionally, nuclear levels of NFAT2, a downstream target of calcineurin, viz, was found elevated in human papillomavirus (HPV) infected cells, HeLa and SiHa, compared to the HPV negative cells, HaCaT and C33A, indicative of its higher DNA binding activity. The nuclear levels of both NFAT1 and NFAT3 remain unaltered implicating they have little role in cervical carcinogenesis. Similar to the in vitro studies, the HPV infected human squamous cell carcinoma specimens showed higher NFAT2 levels compared to the normal cervical epithelium. Depletion of NFAT2 by RNAi attenuated growth of SiHa cells. Overexpression of HPV16 oncoproteins viz, E6 and E7 increased NFAT2 expression levels and DNA binding activity, while knockdown of E6 by RNAi decreased the same. Briefly, we now report an activation of calcineurin-NFAT2 axis in cervical cancer and a novel role of HPV oncoprotein in facilitating NFAT2 dependent cell proliferation.     

Ligand-dependent structural changes and limited proteolysis of Escherichia coli phosphofructokinase-2

An isoform (rhesus UGT1A01) orthologus to the human UGT1A1 was cloned and sequenced from female rhesus monkey liver cDNA using primers designed from the human nucleotide sequences. Open reading frame analysis of the PCR-generated product encodes a 533-amino acid protein with a proposed 27-residue signal peptide. Nucleotide sequence comparison of rhesus UGT1A01 to other rhesus UGT1A isoforms detected a single-transition mutation at nucleotide 1520 (T-->C), resulting in a neutral F to S substitution at position 507. Rhesus UGT1A01 was greater than 99 and 95% identical to cynomolgus UGT1A01 and human UGT1A1, respectively. The rhesus UGT1A01 was expressed in HK-293 cells for functional analysis. Catalytic activity of UGT1A01 was determined with 7-hydroxy-4-(trifluoromethyl)-coumarin and more specific human UGT1A1 substrates (1-naphthol, beta-estradiol, 17 alpha-ethinylestradiol, and bilirubin). Expression of UGT1A01 protein was also detected by a Western blot utilizing a polyclonal antibody developed against the human UGT1A family.     

In Vitro Modulation of E. coli Community Behavior and Human Innate Immune System by Lantibiotic Nisin

Biofilms are microbial communities with genetically divergent microorganisms. Such communal behavior is known to provide survival benefit to the unicellular organisms in adverse conditions. Pathogenicity of opportunistic bacterial pathogens largely depends on their success in proper quorum establishment and biofilm formation. Thus molecules causing quorum-sensing attenuation, preventing the biofilm formation or instigating preformed biofilm dislodgement could serve as attractive drugs/drug supplements. Here we investigate the effect of nisin??type A lantibiotic naturally produced by Lactococcus lactis??on laboratory developed Escherichia coli biofilms and on isolated human neutrophils. Activity evaluation was done on the biofilms of clinical isolates of E. coli, developed on glass slides in a simple static bioreactor design. Nisin not only inhibited the formation but also effectively dislodged the preformed E. coli biofilms developed on glass surfaces. Presence of nisin also demonstrated a significant decrease in the expression of E. coli virulence factors viz. hemolysin and curli expression. The microorganisms dislodged from the biofilms and set free in the circulation of infected host might later reassociate to form new biofilms after nisin clearance from circulation. Thus complete eradication of infective bacterium will depend on stimulatory effect of nisin (if any) on human immune system cells. Therefore modulation of human neutrophil activity by nisin was also evaluated. Presence of nisin induced neutrophil extracellular trap (NET) formation or NETosis in a manner similar to that demonstrated by LPS (lipopolysaccharide) in vitro. Our results thus present nisin as a plausible molecule to be used in treatment of chronic bacterial infections as it indicated increased fitness for the same.     

Sequence analysis of five new field isolates demonstrates that the chain length of potato spindle tuber viroid (PSTVd) is not strictly conserved but as variable as in other viroids

The sequence analysis of five new field isolates of potato spindle tuber viroid (PSTVd) of different virulence revealed that the lengthof their RNA chain is not strictly conserved to 359 nucleotides (nts), as one could have inferred from the previously sequenced PSTVd strains. It was now found that the chain length is strain-specific like in the case of practically all other viroids, and that it may vary, so far, between 356 and 360 nts. Taking our previously sequenced and least virulent mild strain PSTVd KF6-M as standard, the new mild strains PSTVd WA-M and PSTVd F-M differ from it by one or two nts. The new intermediate-severe strains PSTVd F-IS and PSTV-F 88-IS differ from the standard mild strain by eight and nine nts, respectively, whereas the new severe-lethal strain PSTVd F-SL differes in seven nts. Most of these mutations are located within the virulence-modulating (VM) region and within the variable region (VR), and only in two strains a single mutation is found in the right terminal domain.     

Amber fossils reveal the Early Cenozoic dipterocarp rainforest in central Tibet

The palaeoenvironmental reconstruction of central Tibet is key to understanding the uplift history of the Tibetan Plateau, which had a profound influence on Cenozoic global climate and biotic change. Here we report an amber layer from the lower part of the Dingqing Formation (late Oligocene) in Lunpola of central Tibet, which is the first record of amber from Tibet. Herein we find that Lunpola amber is derived from dipterocarp trees, as determined by gas chromatography-mass spectrometry, which are restricted to and dominant in Asian rainforest nowadays. This amber forest represents the northernmost dipterocarp forest and is consistent with the hypothesis of out-of-India dispersal of Asian dipterocarps. The Lunpola amber most probably was derived from the lower part of the Niubao Formation (early–middle Eocene) and suggests a tropical/subtropical wet forest was present in central Tibet at least before the late Oligocene (probably early–middle Eocene).     

Structural and Functional Properties of Exopolysaccharide Excreted by a Novel <Emphasis Type="Italic">Bacillus anthracis</Emphasis> (Strain PFAB2) of Hot Spring Origin

Exopolysaccharide produced by a unique avirulent Bacillus anthracis strain PFAB2 of hot spring origin has been characterized and its functional properties are investigated which is a first report. Maximum yield of EPS is 7.66 g/l with 2% glucose and 1% peptone as optimum carbon and nitrogen source respectively. The EPS is found to be a homopolymer consisting of only glucose as principle monosaccharide component. Through ¹H NMR study, different dextran-like proton peaks are observed. Molecular weight of the EPS resembles low molecular weight bacterial origin polysaccharides. Melting transition of the EPS has started after 276 °C which indicates good thermal stability. The EPS also shows potent antioxidant activity in terms of DPPH and ABTS mediated free radical scavenging property compared to standard ascorbic acid. Emulsifying property of the EPS is also observed and has shown good emulsification of vegetable oils. The polysaccharide forms a thermo resistant gel during the heating phase, with G′ higher than G″ indicating excellent shear-thinning behaviour and viscoelastic nature of the EPS.     

Role of Cys-295 on subunit interactions and allosteric regulation of phosphofructokinase-2 from Escherichia coli

In a previous work, chemical modification of Cys-238 of Escherichia coli Pfk-2 raised concerns on the importance of the dimeric state of Pfk-2 for enzyme activity, whereas modification of Cys-295 impaired the enzymatic activity and the MgATP-induced tetramerization of the enzyme. The results presented here demonstrate that the dimeric state of Pfk-2 is critical for the stability and the activity of the enzyme. The replacement of Cys-238 by either Ala or Phe shows no effect on the kinetic parameters, allosteric inhibition, dimer stability and oligomeric structure of Pfk-2. However, the mutation of Cys-295 by either Ala or Phe provokes a decrease in the k(cat) value and an increment in the K(m) values for both substrates. We suggest that the Cys-295 residue participates in intersubunit interactions in the tetramer since the Cys-295-Phe mutant exhibits higher tetramer stability, which in turn results in an increase in the fructose-6-P concentration required for the reversal of the MgATP inhibition relative to the wild type enzyme.     

Quantitative Proteomics Reveals a Role for Epigenetic Reprogramming During Human Monocyte Differentiation

The differentiation of monocytes into macrophages and dendritic cells involves mechanisms for activation of the innate immune system in response to inflammatory stimuli, such as pathogen infection and environmental cues. Epigenetic reprogramming is thought to play an important role during monocyte differentiation. Complementary to cell surface markers, the characterization of monocytic cell lineages by mass spectrometry based protein/histone expression profiling opens a new avenue for studying immune cell differentiation. Here, we report the application of mass spectrometry and bioinformatics to identify changes in human monocytes during their differentiation into macrophages and dendritic cells. Our data show that linker histone H1 proteins are significantly down-regulated during monocyte differentiation. Although highly enriched H3K9-methyl/S10-phos/K14-acetyl tri-modification forms of histone H3 were identified in monocytes and macrophages, they were dramatically reduced in dendritic cells. In contrast, histone H4 K16 acetylation was found to be markedly higher in dendritic cells than in monocytes and macrophages. We also found that global hyperacetylation generated by the nonspecific histone deacetylase HDAC inhibitor Apicidin induces monocyte differentiation. Together, our data suggest that specific regulation of inter- and intra-histone modifications including H3 K9 methylation, H3 S10 phosphorylation, H3 K14 acetylation, and H4 K16 acetylation must occur in concert with chromatin remodeling by linker histones for cell cycle progression and differentiation of human myeloid cells into macrophages and dendritic cells.The linker histone H1s “beads-on-a-string” structure aids chromatin folding into highly compacted 30 nm chromatin fibers (1). Previous studies demonstrated that histone H1s are differentially expressed and incorporated into chromatin during embryonic stem cell differentiation and reprogramming to pluripotency (2). More than being accumulated after differentiation, the three histone H1 isoforms, H1.3, H1.4, and H1.5, are required for embryonic stem cell differentiation as demonstrated by in vivo H1.3/H1.4/H1.5 triple null experiments (3). Histone H1 null cells exhibit altered nucleosome architecture (4) which may cause epigenetic reprogramming (2), specific changes in gene regulation including repression of pluripotency gene Oct4 expression (3, 5), and cell growth (6, 7). In human blood or bone marrow, hematopoietic stem cells give rise to two major pluripotent progenitor cell lineages, myeloid and lymphoid progenitors, from which are derived mature blood cells including erythrocytes, megakaryocytes, and cells of the myeloid and lymphoid lineages. However, epigenetic regulation or reprogramming in this complex differentiation system has not yet been fully understood. As a follow up to our proteomics studies on epigenetic networks in U937 cell differentiation (8), we have performed proteomics studies on primary human monocyte differentiation. In this report, using proteomics and bioinformatics tools in lieu of microarray analysis of gene expression, we describe the presence of unique protein expression profiles, specifically the linker histones, in monocyte differentiation into macrophages and dendritic cells.Differentiation of monocytes from primary leukemia cell lines or from human peripheral blood mononuclear cells into macrophages or macrophage-like cells using different differentiating reagents has been frequently used as a mimic model for understanding the process of innate and adaptive immune responses to inflammatory stimuli, viral infection, and environmental cues. Either phorbol myristate acetate (PMA)¹ or granulocyte-macrophage colony-stimulating factor (GMCSF) has normally been used for differentiation of monocytes, though the former is generally for differentiation of primary monocytic cell lines, while the latter for differentiation of human blood monocytes (9–11). In our experiments, CD14+ monocytes were treated with PMA, PMA + ionomycin, GMCSF, or GMCSF + IL4. After treatment, monocyte differentiation into macrophages or dendritic cells was monitored by mass spectrometry and bioinformatics analyses. We report here that monocytic cell lineages can be distinguished based on protein expression profiles, specifically, histone H1.4 and H1.5 expression patterns. We identified H3K9-methyl/S10-phos/K14-acetyl tri-modification forms in the monocyte and macrophages but not in dendritic cells. In addition, histone H4 K16 acetylation was low in monocytes and macrophages but significantly higher in dendritic cells. Our findings suggest a switch from H3 tri-modification and linker histone expression to histone H4 K16 acetylation occurs during the monocyte-to-dendritic cell transition.