Pancreatic ductal adenocarcinomas from Mexican patients present a distinct genomic mutational pattern

Molecular Biology Reports - Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in humans, with less than 5% 5-year survival rate. PDAC is characterized by a small number of...     

Bacillus subtilis YhaM,a member of a new family of 3'-to-5' exonucleases in gram-positive bacteria

A strain of Bacillus subtilis lacking two 3'-to-5' exoribonucleases, polynucleotide phosphorylase (PNPase) and RNase R, was used to purify another 3'-to-5' exoribonuclease, which is encoded by the yhaM gene. YhaM was active in the presence of Mn(2+) (or Co(2+)), was inactive in the presence of Mg(2+), and could also degrade single-stranded DNA. The half-life of bulk mRNA in a mutant lacking PNPase, RNase R, and YhaM was not significantly different from that of the wild type, suggesting the existence of additional activities that can participate in mRNA turnover. Sequence homologues of YhaM were found only in gram-positive organisms. The Staphylococcus aureus homologue, CBF1, which had been characterized as a double-stranded DNA binding protein involved in plasmid replication, was also shown to be an Mn(2+)-dependent exoribonuclease. YhaM protein has a C-terminal "HD domain," found in metal-dependent phosphohydrolases. By structure modeling, it was shown that YhaM also contains an N-terminal "OB-fold," present in many oligosaccharide- and oligonucleotide-binding proteins. The combination of these two domains is unique. Thus, YhaM and 10 related proteins from gram-positive organisms constitute a new exonuclease family.     

Heating and microwave assisted SPPS of C-terminal acid peptides on trityl resin: the truth behind the yield

Despite correct purity of crude peptides prepared on trityl resin by Fmoc/tBu microwave assisted solid phase peptide synthesis, surprisingly, lower yields than those expected were obtained while preparing C-terminal acid peptides. This could be explained by cyclization/cleavage through diketopiperazine formation during the second amino acid deprotection and third amino acid coupling. However, we provide here evidence that this is not the case and that this yield loss was due to high temperature promoted hydrolysis of the 2-chlorotrityl ester, yielding premature cleavage of the C-terminal acid peptides.     

Preservation of RNA and DNA from mammal samples under field conditions

Ecological and conservation genetics require sampling of organisms in the wild. Appropriate preservation of the collected samples, usually by cryostorage, is key to the quality of the genetic data obtained. Nevertheless, cryopreservation in the field to ensure RNA and DNA stability is not always possible. We compared several nucleic acid preservation solutions appropriate for field sampling and tested them on rat (Rattus rattus) blood, ear and tail tip, liver, brain and muscle. We compared the efficacy of a nucleic acid preservation (NAP) buffer for DNA preservation against 95% ethanol and Longmire buffer, and for RNA preservation against RNAlater (Qiagen) and Longmire buffer, under simulated field conditions. For DNA, the NAP buffer was slightly better than cryopreservation or 95% ethanol, but high molecular weight DNA was preserved in all conditions. The NAP buffer preserved RNA as well as RNAlater. Liver yielded the best RNA and DNA quantity and quality; thus, liver should be the tissue preferentially collected from euthanized animals. We also show that DNA persists in nonpreserved muscle tissue for at least 1 week at ambient temperature, although degradation is noticeable in a matter of hours. When cryopreservation is not possible, the NAP buffer is an economical alternative for RNA preservation at ambient temperature for at least 2 months and DNA preservation for at least 10 months.     

Inhibition of NAMPT pathway by FK866 activates the function of p53 in HEK293T cells

Inactivation of p53 protein by endogenous and exogenous carcinogens is involved in the pathogenesis of different human malignancies. In cancer associated with SV-40 DNA tumor virus, p53 is considered to be non-functional mainly due to its interaction with the large T-antigen. Using the 293T cell line (HEK293 cells transformed with large T antigen) as a model, we provide evidence that p53 is one of the critical downstream targets involved in FK866-mediated killing of 293T cells. A reduced rate of apoptosis and an increased number of cells in S-phase was accompanied after knockdown of p53 in these cells. Inhibition of NAMPT by FK866, or inhibition of SIRT by nicotinamide decreased proliferation and triggered death of 293T cells involving the p53 acetylation pathway. Additionally, knockdown of p53 attenuated the effect of FK866 on cell proliferation, apoptosis, and cell cycle arrest. The data presented here shed light on two important facts: (1) that p53 in 293T cells is active in the presence of FK866, an inhibitor of NAMPT pathway; (2) the apoptosis induced by FK866 in 293T cells is associated with increased acetylation of p53 at Lys382, which is required for the functional activity of p53.     

WNK1 is an assembly factor for the human ER membrane protein complex

1. Download : Download high-res image (156KB)
2. Download : Download full-size image

     

Size‐spectrum based differential response of phytoplankton to nutrient and iron‐organic matter combinations in microcosm experiments in a Chilean Patagonian Fjord

The Patagonian fjords have been recognized as a major region of relatively high primary productivity systems during spring–summer bloom periods, where iron‐organic matter forms may be essential complexes involved in key growth processes connected to the carbon and nitrogen cycles. We used two dissolved organic matter (DOM) types, marine polysaccharide and siderophore, as a model to understand how they affect the bioavailability of Fe to phytoplankton and bacteria and to assess their ecological role in fjord systems. A 10‐day microcosm study was performed in the Comau Fjord during summer conditions (March 2012). Pico‐, nano‐, and microphytoplankton abundance, total chlorophyll‐a and bacteria abundance, and bacterial secondary production estimates were analyzed in five treatments: (i) control (no additions), (ii) only nutrients (NUT: PO₄, NO₃, Si), (iii) nutrients + Fe(II), (iv) polysaccharide (natural diatoms extracted: 1–3 beta Glucan), and (v) Hexandentate Desferroxiamine B (DFB, siderophore). Our results showed that while DFB reduced Fe bioavailability for almost all phytoplankton assemblages in the fjord, polysaccharide did not have effects on the iron bioavailability. At Nutrients + Fe and Polysaccharide treatments, chlorophyll‐a concentration abruptly increased from 0.9 to 20 mg m^?3 during the first 4–6 days of the experimental period. Remarkably, at the Nutrients + Fe treatment, the development of the bloom was accompanied by markedly high abundances of Synechococcus, picoeukaryotes, and autotrophic nanoflagellates within the first 4 days of the experiment. Our study indicated that small plankton (phytoplankton <20 μm and bacteria) were the first to respond to dissolved Nutrients + Fe compared to large sized micro‐phytoplankton cells (>20 μm). This could be at least partially attributed to biological utilization of Fe (2 to 3 nM) by <20 μm phytoplankton and bacteria through the interaction with organic ligands released by bacteria that eventually could increase solubility of the Fe dissolved fraction thus having a positive effect on the small‐sized phytoplankton community.     

Influenza A viruses limit NLRP3‐NEK7‐complex formation and pyroptosis in human macrophages

Pyroptosis is a fulminant form of macrophage cell death, contributing to release of pro‐inflammatory cytokines. In humans, it depends on caspase 1/4‐activation of gasdermin D and is characterized by the release of cytoplasmic content. Pathogens apply strategies to avoid or antagonize this host response. We demonstrate here that a small accessory protein (PB1‐F2) of contemporary H5N1 and H3N2 influenza A viruses (IAV) curtails fulminant cell death of infected human macrophages. Infection of macrophages with a PB1‐F2‐deficient mutant of a contemporary IAV resulted in higher levels of caspase‐1 activation, cleavage of gasdermin D, and release of LDH and IL‐1β. Mechanistically, PB1‐F2 limits transition of NLRP3 from its auto‐repressed and closed confirmation into its active state. Consequently, interaction of a recently identified licensing kinase NEK7 with NLRP3 is diminished, which is required to initiate inflammasome assembly.     

Anthracyclines: isolation of overproducing strains by the selection and genetic recombination of putative regulatory mutants of Streptomycespeucetius var. caesius

In Streptomyces peucetius var. caesius, the production of anthracyclines was suppressed either by 330 mM d-glucose or 25 mM phosphate. In addition, the anthracycline doxorubicin and the glucose analogue 2-deoxyglucose inhibited the growth of this microorganism at concentrations of 0.025 mM and 10 mM respectively. Spontaneous and induced mutants, resistant to the action of these compounds, were isolated, tested and chosen by their ability to overproduce anthracyclines. Genetic recombination between representative mutants was carried out by the protoplast fusion technique. Some recombinants carrying resistance to doxorubicin, phosphate and 2-deoxyglucose produced more than 40-fold greater levels of anthracyclines than those obtained with the parental strain. This improvement resulted in total antibiotic titres of more than 2 g/l culture medium at 6 days of fermentation. Received: 14 April 1997 / Received revision: 19 June 1997 / Accepted: 4 July 1997     

Biochemical markers of vascular calcification in elderly hemodialysis patients

The increased vascular calcification, cardiovascular morbidity, and mortality in chronic kidney disease (CKD) patients has been associated with disturbances in mineral-bone metabolism. In order to determine markers of the vascular calcification frequently observed in these patients, blood samples of elderly male and female hemodialysis CKD patients were used to measure serum levels of: osteoprotegerin (OPG), total soluble receptor activator of nuclear factor-κB ligand (sRANKL), and fetuin-A by enzyme immunoassay; tartrate-resistant acid phosphatase (TRACP-5b), and bone-specific alkaline phosphatase (BAP) by immunoenzymometric assay; osteocalcin (OC) by ELISA; iPTH by immunoradiometric assay; 25(OH)D₃ and 1,25(OH)₂D₃, by I¹²⁵ radioimmunoassay; and calcium and phosphorus by photometric assay. Serum OPG, BAP, iPTH, phosphorus, and OC levels were higher and serum 25(OH)D₃, 1,25(OH)₂D₃, and fetuin-A levels lower in both male and female CKD patients than in their respective controls. Our results indicate that the bone formation and resorption parameters are altered in elderly male and female hemodialysis CKD patients. These changes may lead to vascular calcifications and cardiovascular complications, given that elevated OPG and OC levels and reduced fetuin-A levels are associated with cardiovascular events.