Culturing conditions highly affect DNA methylation and gene expression levels in MCF7 breast cancer cell line

The levels of DNA methylation and their role in gene expression are key factors that could affect diagnosis, prognosis, and treatment options of different diseases. In this study, the methylation levels of 22 genes that are mostly correlated to breast cancer were determined using EpiTect methyl II PCR array. This analysis was performed to determine the effect of cells’ passage number and the use of antibiotics in the culturing media on gene methylation levels in MCF7 cell line. DNA methylation levels of PTGS2, ADAM23, HIC1, and PYCARD were found to be significantly different among different passages. While the DNA methylation levels of CCNA1, RASSF1, and THBS1 were found to be affected by the use of 1% of penicillin/streptomycin in the culture media. Gene expression analysis after demethylation using 5-Aza-2′-deoxycytidine showed that the gene expression levels of the hypermethylated genes varied between different passage numbers. This study shows that the presence of antibiotic within cultured media and cell line’s passage number could greatly affect the methylation levels that need to be considered in future studies on cell lines.     

Expression of poliovirus nonstructural proteins in Escherichia coli cells. Modification of membrane permeability induced by 2B and 3A.

The poliovirus nonstructural proteins 2B, 2C, 2C3A, 2C3AB, 3A, and 3AB have been cloned and efficiently expressed in Escherichia coli cells. Each individual protein, or combinations of some of them, were cloned using polymerase chain reaction techniques and correspond to the genuine poliovirus protein plus an additional methionine. The system used to express them uses pET vectors containing the promoter of gene 10 of phage T7. Expression of protein 2C in BL21 (DE3) pLysS cells, which express the T7 lysozyme, is not toxic, and the bacteria synthesize this protein for several hours after induction. In contrast, the expression of proteins 2B, 3A, or 3AB is not tolerated by BL21 (DE3) pLysS cells which could make them only for a limited period of time. Protein 3AB was particularly toxic and induced a rapid lysis of the recombinant clone after its induction with isopropyl-1-thio-beta-D-galactopyranoside alone or with both isopropyl-1-thio-beta-D-galactopyranoside and rifampicin. Further analyses showed that 3AB induced profound modifications in membrane permeability to o-nitrophenyl-beta-D-galactopyranoside, labeled uridine, and nonpermeant translation inhibitors. Cloning and expression of proteins 2B, 3A, and 3AB in BL21 (DE3) cells that do not contain the T7 lysozyme lead to a more sustained expression of these proteins without detectable cell lysis. Changes in permeability to low molecular weight compounds such as radioactive uridine, o-nitrophenyl-beta-D-galactopyranoside, and hygromycin B readily appeared upon induction of 2B, 3A, and 3AB. Our results indicate that the poliovirus nonstructural polypeptides 2B and 3A (or 3AB) are lytic for the bacteria. In fact, both proteins 2B and 3A contain hydrophobic domains in a potential amphipathic helix; this is one characteristic shared with a number of membrane-active peptides.     

Phylogeographical analysis shows the need to protect the wild yaks' last refuge in Nepal

The wild yak Bos mutus was believed to be regionally extinct in Nepal for decades until our team documented two individuals from Upper Humla, north‐western Nepal, in 2014. The International Union for Conservation of Nature (IUCN) seeks further evidence for the conclusive confirmation of that sighting. We conducted line transects and opportunistic sign surveys in the potential wild yak habitats of Humla, Dolpa, and Mustang districts between 2015 and 2017 and collected genetic samples (present and historic) of wild and domestic yaks Bos grunniens. We also sighted another wild yak in Upper Humla in 2015. Phylogenetic and haplotype network analyses based on mitochondrial D‐loop sequences (~450 bp) revealed that wild yaks in Humla share the haplotype with wild yaks from the north‐western region of the Qinghai‐Tibetan Plateau in China. While hybridization with domestic yaks is a major long‐term threat, illegal hunting for meat and trophy put the very small populations of wild yaks in Nepal at risk. Our study indicates that the unprotected habitat of Upper Humla is the last refuge for wild yaks in Nepal. We recommend wild yak conservation efforts in the country to focus on Upper Humla by (i) assigning a formal status of protected area to the region, (ii) raising awareness in the local communities for wild yak conservation, and (iii) providing support for adaptation of herding practice and pastureland use to ensure the viability of the population.     

Interdomain allosteric regulation of Polo kinase by Aurora B and Map205 is required for cytokinesis

Drosophila melanogaster Polo and its human orthologue Polo-like kinase 1 fulfill essential roles during cell division. Members of the Polo-like kinase (Plk) family contain an N-terminal kinase domain (KD) and a C-terminal Polo-Box domain (PBD), which mediates protein interactions. How Plks are regulated in cytokinesis is poorly understood. Here we show that phosphorylation of Polo by Aurora B is required for cytokinesis. This phosphorylation in the activation loop of the KD promotes the dissociation of Polo from the PBD-bound microtubule-associated protein Map205, which acts as an allosteric inhibitor of Polo kinase activity. This mechanism allows the release of active Polo from microtubules of the central spindle and its recruitment to the site of cytokinesis. Failure in Polo phosphorylation results in both early and late cytokinesis defects. Importantly, the antagonistic regulation of Polo by Aurora B and Map205 in cytokinesis reveals that interdomain allosteric mechanisms can play important roles in controlling the cellular functions of Plks.     

KIF14 Binds Tightly to Microtubules and Adopts a Rigor-Like Conformation

The mitotic kinesin motor protein KIF14 is essential for cytokinesis during cell division and has been implicated in cerebral development and a variety of human cancers. Here we show that the mouse KIF14 motor domain binds tightly to microtubules and does not display typical nucleotide-dependent changes in this affinity. It also has robust ATPase activity but very slow motility. A crystal structure of the ADP-bound form of the KIF14 motor domain reveals a dramatically opened ATP-binding pocket, as if ready to exchange its bound ADP for Mg·ATP. In this state, the central β-sheet is twisted ~ 10° beyond the maximal amount observed in other kinesins. This configuration has only been seen in the nucleotide-free states of myosins—known as the “rigor-like” state. Fitting of this atomic model to electron density maps from cryo-electron microscopy indicates a distinct binding configuration of the motor domain to microtubules. We postulate that these properties of KIF14 are well suited for stabilizing midbody microtubules during cytokinesis.     

Purification and biochemical characterization of a native invertase from the hydrogen-producing Thermotoga neapolitana (DSM 4359)

This is the first report describing the purification and enzymatic properties of a native invertase (β-D-fructosidase) in Thermotogales. The invertase of the hydrogen-producing thermophilic bacterium Thermotoga neapolitana DSM 4359 (hereby named Tni) was a monomer of about 47 kDa having an amino acid sequence quite different from other invertases studied up to now. Its properties and substrates specificity let us classify this protein as a solute-binding protein with invertase activity. Tni was specific for the fructose moiety and the enzyme released fructose from sucrose and raffinose and the fructose polymer inulin was hydrolyzed in an endo-type fashion. Tni had an optimum temperature of 85°C at pH 6.0. At temperatures of 80–85°C, the enzyme retained at least 50% of its initial activity during a 6 h preincubation period. Tni had a K _m and k _cat /K _m values (at 85°C and pH 6.0) of about 14 mM and 5.2 × 10⁸ M⁻¹ s⁻¹, respectively. Dedicated to the memory of Prof. R. A. Nicolaus, founder of the Institute (1968).     

B cell depletion in diffuse progressive systemic sclerosis: safety,skin score modification and IL-6 modulation in an up to thirty-six months follow-up open-label trial

Introduction  

An over-expression of CD19 has been shown in B cells of systemic sclerosis (SSc) and B cells are thought to contribute to the induction of skin fibrosis in the tight skin mouse model. The aim was to define the outcome on safety and the change in skin score after rituximab therapy in SSc patients and to correlate the clinical characteristics with the levels of interleukin (IL)-6 and with the immune cell infiltrate detected by immunohistochemistry.     

Gene Variation, Population Differentiation, and Sociogenetic Structure of Nests of Partamona seridoensis (Hymenoptera: Apidae, Meliponini)

Gene variation and the differentiation of two populations of Partamona seridoensis (Hymenoptera: Apidae: Meliponini) from the Caatinga biome, a semiarid ecosystem unique to Brazil, were estimated through allozymic and microsatellite analyses. These populations exhibited similar low degrees of enzyme gene variation. Observed genotype frequencies at the allozyme and microsatellite loci were in accordance with Hardy-Weinberg equilibrium in the two populations. Both markers demonstrated that the two populations are not genetically homogeneous and must be considered distinct populations. The occurrence of private alleles at the allozyme and microsatellite loci corroborates this differentiation, sustaining the hypothesis of a low level of interpopulation gene flow. The phenotypic segregations clearly demonstrated that the progeny inside each nest were the result of mating between the queen of the colony and only one male.     

Tissue-specific organic acid metabolism in reproductive and non-reproductive parts of the fig fruit is partially induced by pollination

Organic acids are important components of overall fruit quality through flavor, taste, nutritional and medicinal values. Pollinated fig (Ficus carica L.) fruit quality is enhanced by increased acidity. We quantified the major organic acids and characterized the expression pattern of organic acid metabolic pathway-related genes in the reproductive part – inflorescence and non-reproductive part – receptacle of parthenocarpic and pollinated fig fruit during ripening. Essentially, pollinated fruit contains seeds in the inflorescence, as opposed to no seeds in the parthenocarpic inflorescence. The major organic acids – citrate and malate – were found in relatively high quantities in the inflorescence compared to the receptacle of both parthenocarpic and pollinated fig fruit. Notably, pollination increased citric acid content significantly in both inflorescence and receptacle. Genes related to the phosphoenolpyruvate carboxylase (PEPC) cycle, tricarboxylic acid cycle, citrate catabolism and glyoxylate cycle were identified in fig fruit. Expression levels of most of these genes were higher in inflorescences than in receptacles. In particular, FcPEPC and FcFUM (encoding fumarase) had significantly higher expression in the inflorescence of pollinated fruit. Most importantly, expression of the glyoxylate cycle genes FcMLS and FcICL (encoding malate synthase and isocitrate lyase, respectively) was induced to strikingly high levels in the inflorescence by pollination, and their expression level was highly positively correlated with the contents of all organic acids. Therefore, the glyoxylate cycle may be responsible for altering the accumulation of organic acids to upgrade the fruit taste during ripening, especially in the pollinated, seeded inflorescence.