Sex-sorting of boar spermatozoa does not influence the localization of glucose transporters

Sex-sorting damages spermatozoa function, shortening their lifespan and fertility. This study used an immunofluorescence technique to investigate the effect of sex-sorting on the localization of glucose transporters (GLUTs) in boar spermatozoa. GLUTs are trans-membrane proteins responsible for glucose transport within cells. Distribution of GLUTs on sperm cells was similar in unsorted and sex-sorted semen, suggesting that the flow cytometric sex-sorting process did not affect the sperm energy apparatus.     

Improved BM212 MmpL3 Inhibitor Analogue Shows Efficacy in Acute Murine Model of Tuberculosis Infection

1,5-Diphenyl pyrroles were previously identified as a class of compounds endowed with high in vitro efficacy against M. tuberculosis. To improve the physical chemical properties and drug-like parameters of this class of compounds, a medicinal chemistry effort was undertaken. By selecting the optimal substitution patterns for the phenyl rings at N1 and C5 and by replacing the thiomorpholine moiety with a morpholine one, a new series of compounds was produced. The replacement of the sulfur with oxygen gave compounds with lower lipophilicity and improved in vitro microsomal stability. Moreover, since the parent compound of this family has been shown to target MmpL3, mycobacterial mutants resistant to two compounds have been isolated and characterized by sequencing the mmpL3 gene; all the mutants showed point mutations in this gene. The best compound identified to date was progressed to dose-response studies in an acute murine TB infection model. The resulting ED₉₉ of 49 mg/Kg is within the range of commonly employed tuberculosis drugs, demonstrating the potential of this chemical series. The in vitro and in vivo target validation evidence presented here adds further weight to MmpL3 as a druggable target of interest for anti-tubercular drug discovery.     

Gene Expression Analysis of PTEN Positive Glioblastoma Stem Cells Identifies DUB3 and Wee1 Modulation in a Cell Differentiation Model

The term astrocytoma defines a quite heterogeneous group of neoplastic diseases that collectively represent the most frequent brain tumors in humans. Among them, glioblastoma multiforme represents the most malignant form and its associated prognosis is one of the poorest among tumors of the central nervous system. It has been demonstrated that a small population of tumor cells, isolated from the brain neoplastic tissue, can reproduce the parental tumor when transplanted in immunodeficient mouse. These tumor initiating cells are supposed to be involved in cancer development and progression and possess stem cell-like features; like their normal counterpart, these cells remain quiescent until they are committed to differentiation. Many studies have shown that the role of the tumor suppressor protein PTEN in cell cycle progression is fundamental for tumor dynamics: in low grade gliomas, PTEN contributes to maintain cells in G1 while the loss of its activity is frequently observed in high grade gliomas. The mechanisms underlying the above described PTEN activity have been studied in many tumors, but those involved in the maintenance of tumor initiating cells quiescence remain to be investigated in more detail. The aim of the present study is to shed light on the role of PTEN pathway on cell cycle regulation in Glioblastoma stem cells, through a cell differentiation model. Our results suggest the existence of a molecular mechanism, that involves DUB3 and WEE1 gene products in the regulation of Cdc25a, as functional effector of the PTEN/Akt pathway.     

Clerocidin-mediated DNA footprinting discriminates among different G-quadruplex conformations and detects tetraplex folding in a duplex environment

Background

G-quadruplexes are polymorphic non-canonical nucleic acid conformations involved both in physiological and pathological processes. Given the high degree of folding heterogeneity and comparable conformational stabilities, different G-quadruplex forms can occur simultaneously, hence rendering the use of basic instrumental methods for structure determination, like X-ray diffraction or NMR, hardly useful. Footprinting techniques represent valuable and relatively rapid alternative to characterize DNA folding. The natural diterpenoid clerocidin is an alkylating agent that specifically reacts at single-stranded DNA regions, with different mechanisms depending on the exposed nucleotide.

Methods

Clerocidin was used to footprint G-quadruplex structures formed by telomeric and oncogene promoter sequences (c-myc, bcl-2, c-kit2), and by the thrombin binding aptamer.

Results

The easy modulability of CL reactivity towards DNA bases permitted to discriminate fully and partially protected sites, highlights stretched portions of the G-quadruplex conformation, and discriminate among topologies adopted by one sequence in different environmental conditions. Importantly, CL displayed the unique property to allow detection of G-quadruplex folding within a duplex context.

Conclusions

CL is a finely performing new tool to unveil G-quadruplex arrangements in DNA sequences under genomically relevant conditions.

General significance

Nucleic acid G-quadruplex structures are an emerging research field because of the recent indication of their involvement in a series of key biological functions, in particular in regulation of proliferation-associated gene expression. The use of clerocidin as footprinting agent to identify G-quadruplex structures under genomically relevant conditions may allow detection of new G-quadruplex-based regulatory regions.     

Attachment and Biofilm Forming Capabilities of Staphylococcus epidermidis Strains Isolated from Preterm Infants

Staphylococcus epidermidis, a human commensal, is an important opportunistic, biofilm-forming pathogen and the main cause of late onset sepsis in preterm infants, worldwide. In this study we describe the characteristics of S. epidermidis strains causing late onset (>72 h) bloodstream infection in preterm infants and skin isolates from healthy newborns. Attachment and biofilm formation capability were analyzed in microtiter plates and with transmission electron microscopy (TEM). Clonal relationship among strains was studied with pulsed-field gel electrophoresis. Antimicrobial susceptibility testing was performed, as well as the detection of biofilm-associated genes and of the invasiveness marker IS256 with polymerase chain reaction. Blood and skin isolates had similar attachment and biofilm-forming capabilities and biofilm formation was not related to the presence of specific genes. Filament-like membrane structures were seen by TEM early in the attachment close to the device surface, both in blood and skin strains. Nine of the ten blood isolates contained the IS256 and were also resistant to methicillin and gentamicin in contrast to skin strains. S. epidermidis strains causing bloodstream infection in preterm infants exhibit higher antibiotic resistance and are provided with an invasive genetic equipment compared to skin commensal strains. Adhesion capability to a device surface seems to involve bacterial membrane filaments.     

Direct amplification of new cellulase genes from woodland soil purified DNA

Eight genes encoding cellulolytic enzymes were obtained by direct PCR amplification of genomic DNA recovered from woodland soil samples. The direct amplifications were carried out by using primers designed from available online cellulase nucleotide sequences. The isolated genes were all different from each other and homologous to endo-β-1,4-glucanases of Bacillus subtilis. The cellulases were functionally expressed in Escherichia coli and tested on soluble substrate at 37 and 60 °C, showing different cellulolytic activities. Among these, the enzyme renamed CelWS6 exhibited good activity at higher temperatures. Further analysis of CelWS6 showed a high performance in acid environments (between pH 4.0 and 6.0) and at elevated temperatures with its maximum activity at pH 5.0 and 50 °C. At the optimum pH, it was very stable since more than 80 % of its original activity was maintained after an incubation of 120 min at 60 °C. Because the cellulases had different cellulolytic activities, but similar amino acid sequences, it was possible to assess the relationship between sequence and protein function.     

11 Findings and hurdles on the path leading from formamide to the spontaneous generation of RNA

Our laboratories analyze the synthetic reactions leading from formamide, NH₂COH to prebiotically relevant compounds in the presence of catalysts. We have described the formation of all the biological nucleic bases of carboxylic acids of two aminoacids, and of condensing agents in the presence of catalysts of terrestrial origin (Saladino et al., 2012) and of one meteorite. Heat-dependent synthetic reactions from NH₂COH lead to the synthesis of acyclonucleosides, not (yet?) to that of nucleosides [hurdle # 1]. Nucleosides are phosphorylated in the presence of NH₂COH and a phosphate source yielding cyclic nucleotides as well. (Costanzo et al., 2007). 3′,5′-cyclic GMP nonenzymatically polymerizes up to at least 25mers, as shown by PAGE, MALDI ToF, ³¹P-NMR, specific RNAse and inhibitors analyses (Costanzo et al., 2012).The reaction is stimulated by 1,8-diazabicycloundec-7-ene and dimethylformamide. 3′,5′-cUMP does not polymerize spontaneously [hurdle # 2], 3′,5′-cAMP polymerizes very poorly [hurdle # 3]. We will discuss data on the polymerization of 3′,5′-cCMP and on a ribozyme activity exerted by oligomers neosynthesized from cyclic nucleotides. This approach finds its larger perspective in the evolutionary scenario depicted by Trifonov (2009).     

Ectopic F0F1 ATP synthase contains both nuclear and mitochondrially-encoded subunits

Over the past few years, several reports have described the presence of F₀F₁ ATP synthase subunits at the surface of hepatocytes, where the hydrolytic activity of F₁ sector faces outside and triggers HDL endocytosis. An intriguing question is whether the ectopic enzyme has same subunit composition and molecular mass as that of the mitochondrial ATP synthase. Also due to the polar nature of hepatocytes, the enzyme may be localized to a particular cell boundary. Using different methods to prepare rat liver plasma membranes, which have been subjected to digitonin extraction, hr CN PAGE, immunoblotting, and mass spectrometry analysis, we demonstrate the presence of ecto-F₀F₁ complexes which have a similar molecular weight to the monomeric form of the mitochondrial complexes, containing both nuclear and mitochondrially-encoded subunits. This finding makes it unlikely that the enzyme assembles on the plasma membranes, but suggest it to be transported whole after being assembled in mitochondria by still unknown pathways. Moreover, the plasma membrane preparation enriched in basolateral proteins contains much higher amounts of complete and active F₀F₁ complexes, consistent with their specific function to modulate the HDL uptake on hepatocyte surface.     

Cyclic N-Terminal Loop of Amylin Forms Non Amyloid Fibers

We report for the first time, to our knowledge, that the N-terminal loop (N_loop) of amylin (islet amyloid polypeptide (IAPP) residues 1–8) forms extremely long and stable non-β-sheet fibers in solution under the same conditions in which human amylin (hIAPP) forms amyloid fibers. This observation applies to the cyclic, oxidized form of the N_loop but not to the linear, reduced form, which does not form fibers. Our findings indicate a potential role of direct N_loop-N_loop interactions in hIAPP aggregation, which has not been previously explored, with important implications for the mechanism of hIAPP amyloid fiber formation, the inhibitory action of IAPP variants, and the competition between ordered and disordered aggregation in peptides of the calcitonin peptide family.