Molecular evolution of gas cavity in [NiFeSe] hydrogenases resurrected in silico

Oxygen tolerance of selenium-containing [NiFeSe] hydrogenases (Hases) is attributable to the high reducing power of the selenocysteine residue, which sustains the bimetallic Ni–Fe catalytic center in the large subunit. Genes encoding [NiFeSe] Hases are inherited by few sulphate-reducing δ-proteobacteria globally distributed under various anoxic conditions. Ancestral sequences of [NiFeSe] Hases were elucidated and their three-dimensional structures were recreated in silico using homology modelling and molecular dynamic simulation, which suggested that deep gas channels gradually developed in [NiFeSe] Hases under absolute anaerobic conditions, whereas the enzyme remained as a sealed edifice under environmental conditions of a higher oxygen exposure risk. The development of a gas cavity appears to be driven by non-synonymous mutations, which cause subtle conformational changes locally and distantly, even including highly conserved sequence regions

Transcriptional Repression of Cdc25B by IER5 Inhibits the Proliferation of Leukemic Progenitor Cells through NF-YB and p300 in Acute Myeloid Leukemia

The immediately-early response gene 5 (IER5) has been reported to be induced by γ-ray irradiation and to play a role in the induction of cell death caused by radiation. We previously identified IER5 as one of the 2,3,4-tribromo-3-methyl-1-phenylphospholane 1-oxide (TMPP)-induced transcriptional responses in AML cells, using microarrays that encompassed the entire human genome. However, the biochemical pathway and mechanisms of IER5 function in regulation of the cell cycle remain unclear. In this study, we investigated the involvement of IER5 in the cell cycle and in cell proliferation of acute myeloid leukemia (AML) cells. We found that the over-expression of IER5 in AML cell lines and in AML-derived ALDHhi (High Aldehyde Dehydrogenase activity)/CD34+ cells inhibited their proliferation compared to control cells, through induction of G2/M cell cycle arrest and a decrease in Cdc25B expression. Moreover, the over-expression of IER5 reduced colony formation of AML-derived ALDHhi/CD34+ cells due to a decrease in Cdc25B expression. In addition, over-expression of Cdc25B restored TMPP inhibitory effects on colony formation in IER5-suppressed AML-derived ALDHhi/CD34+ cells. Furthermore, the IER5 reduced Cdc25B mRNA expression through direct binding to Cdc25B promoter and mediated its transcriptional attenuation through NF-YB and p300 transcriptinal factors. In summary, we found that transcriptional repression mediated by IER5 regulates Cdc25B expression levels via the release of NF-YB and p300 in AML-derived ALDHhi/CD34+ cells, resulting in inhibition of AML progenitor cell proliferation through modulation of cell cycle. Thus, the induction of IER5 expression represents an attractive target for AML therapy

Weighted Fair RIO (WF-RIO) for Fair AF Bandwidth Allocation in a DiffServ-Capable MPLS Network

Abstract. RIO is the primary active queue management technology for handling AF(assured forwarding) class traffic for services that have minimum bandwidth guarantees. However, RIO unfairly allocates the excess AF bandwidth among LSPs(label-switched paths) which have TCP flows aggregated as AF class in a DiffServ-Capable MPLS Network. This issue obstructs the business model in which ISPs promote LSP users to expand the LSP-required bandwidth for enriching the quality of AF traffic. In this paper, we propose a way, called weighted fair RIO (WF-RIO), to resolve this issue. WF-RIO can allocate the excess AF bandwidth among LSPs in proportion to their AF minimum guaranteed bandwidth by multiplying the dropping probability of RIO by an LSP-specific weight which is simply calculated from the traffic rates for the individual LSPs. We evaluate the proposed method by computer simulation and demonstrate its effectiveness

Molecular evolution of gas cavity in [NiFeSe] hydrogenases resurrected in silico

Oxygen tolerance of selenium-containing [NiFeSe] hydrogenases (Hases) is attributable to the high reducing power of the selenocysteine residue, which sustains the bimetallic Ni–Fe catalytic center in the large subunit. Genes encoding [NiFeSe] Hases are inherited by few sulphate-reducing δ-proteobacteria globally distributed under various anoxic conditions. Ancestral sequences of [NiFeSe] Hases were elucidated and their three-dimensional structures were recreated in silico using homology modelling and molecular dynamic simulation, which suggested that deep gas channels gradually developed in [NiFeSe] Hases under absolute anaerobic conditions, whereas the enzyme remained as a sealed edifice under environmental conditions of a higher oxygen exposure risk. The development of a gas cavity appears to be driven by non-synonymous mutations, which cause subtle conformational changes locally and distantly, even including highly conserved sequence regions

Pituitary dysfunction induced by immune checkpoint inhibitors is associated with better overall survival in both malignant melanoma and non-small cell lung carcinoma: a prospective study

Background Several immune-related adverse events (irAEs) are reported to be associated with therapeutic efficacy of immune checkpoint inhibitors, yet whether pituitary dysfunction, a life-threatening irAE, affects overall survival (OS) in patients with malignancies is unclear. This prospective study examined the association of pituitary dysfunction (pituitary-irAE) with OS of patients with non-small cell lung carcinoma (NSCLC) or malignant melanoma (MM).Methods A total of 174 patients (NSCLC, 108; MM, 66) treated with ipilimumab, nivolumab, pembrolizumab, or atezolizumab at Nagoya University Hospital were evaluated for OS and the development of pituitary-irAE. Kaplan-Meier curves of OS as a function of the development of pituitary-irAE were produced with the log-rank test as a primary endpoint.Results Pituitary-irAE was observed in 16 patients (4 (3.7%) with NSCLC, 12 (18.2%) with MM) having two different disease types: hypophysitis with deficiency of multiple anterior pituitary hormones accompanied by pituitary enlargement, and isolated adrenocorticotropic hormone (ACTH) deficiency without pituitary enlargement. Among these patients, 6 developed pituitary-irAE while being treated with ipilimumab (6/25 patients (24.0%) treated with ipilimumab) and 10 developed pituitary-irAE during treatment with nivolumab or pembrolizumab (10/167 (6.0%)). All 16 patients had ACTH deficiency and were treated with physiological doses of hydrocortisone. The development of pituitary-irAE was associated with better OS in patients with NSCLC (not reached vs 441 (95% CI not calculated) days, p<0.05) and MM (885 (95% CI 434 to 1336) vs 298 (95% CI 84 to 512) days, p<0.05).Conclusions In our study cohort, the incidence of pituitary-irAE was higher than previously reported and the development of pituitary-irAE predicted better prognosis for both NSCLC and MM when patients were treated with physiological doses of hydrocortisone.Clinical trials registration UMIN000019024

Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology. Nature Methods

We developed a rapid single nucleotide polymorphism (SNP) detection system named smart amplification process version 2 (SMAP 2). Because DNA amplification only occurred with a perfect primer match, amplification alone was sufficient to identify the target allele. To achieve the requisite fidelity to support this claim, we used two new and complementary approaches to suppress exponential background DNA amplification that resulted from mispriming events. SMAP 2 is isothermal and achieved SNP detection from whole human blood in 30 min when performed with a new DNA polymerase that was cloned and isolated from Alicyclobacillus acidocaldarius (Aac pol). Furthermore, to assist the scientific community in configuring SMAP 2 assays, we developed software specific for SMAP 2 primer design. With these new tools, a high-precision and rapid DNA amplification technology becomes available to aid in pharmacogenomic research and molecular-diagnostics applications. The availability of the human genome sequence 1,2 and genome diversity databases 3-5 at the beginning of the 21 st century are causing a paradigm shift away from the standard protocol of medical care toward genotyped medicine. This new type of medicine is based on the accumulating knowledge of gene polymorphisms (SNPs) and their relationship to specific phenotypes, such as disease predisposition, drug metabolism and disease development. A key step for the development of individualized medicine is the ability to rapidly test patients for these SNPs and/or other mutations correlated to diseases and disease predisposition. Supporting this point, the US Food and Drug Administration has required the drug industry to publicly provide SNP data examined in the process of procuring a drug license. Today SNP genotyping technologies 6-9 are still a bottleneck in drug discovery research and clinical applications. But high-throughput gene analysis and SNP detection technologies will inevitably become both cheaper and faster in the future. Besides SNP genotyping, these improved sequence-detection technologies would also allow and advance studies in other disciplines such as population genetics, the global surveillance of infectious disease and the study of somatic mutations in human cancer. Almost all previously developed SNP-detection systems consist of two steps: amplification (usually by PCR) and detection of SNP (using DNA fragments amplified in the first step). This approach is reasonably fast, but to shorten the time required and simplify the detection, it is ideal to develop a one-step method, in which the amplification itself can be the SNP detection signal. The difficulty in developing such a technology is in the suppression of the background amplification. For example, primers for allele-specific primer PCR are designed with the nucleotide mismatch at the 3¢ end of the PCR primers, but the misamplified PCR products primed from mismatched primers are still exponentially amplified, producing background signals that must be addressed. Here we report SMAP 2, the first rapid one-step SNP detection technology in which the amplification of the targeted DNA is the signal of the target SNP itself

IER5 expression inhibited the colony formation of AML ALDH^hi/CD34⁺ cells through the regulation of Cdc25B expression.

<p>A. ALDH^hi/CD34⁺ cells were purified from an AML patient (M1) and were cultured in semisolid methylcellulose media. The ALDH^hi/CD34⁺ cells were either untransfected or were transfected with <i>IER5</i> cDNA or with shRNA-#1, or -#2. After 14 days culture with or without TMPP (5 µM), the cells were then analyzed for their colony forming ability. Colony formation of these ALDH^hi/CD34⁺ cells (3×10² to 5×10² cells/plate) was assessed after plating in semisolid methylcellulose media. B. Analysis of the mRNA expression of <i>IER5</i> and <i>Cdc25B</i> in each colony using QRT-PCR and RT-PCR. C. The cells were viewed using phase-contrast microscopy after 14 days culture with or without TMPP (5 µM). Original magnification ×4. D. ALDH^hi/CD34⁺ cells were purified from an AML patient (M1) and were cultured in semisolid methylcellulose media. The ALDH^hi/CD34⁺ cells were either untransfected or were transfected with <i>IER5</i> cDNA or <i>Cdc25B</i> cDNA. After 14 days culture with or without TMPP (5 µM), the cells were then analyzed for their colony forming ability. Colony formation of these ALDH^hi/CD34⁺ cells (3×10² to 5×10² cells/plate) was assessed after plating in semisolid methylcellulose media. Colony formation was evaluated as a percentage of the corresponding control. E. Analysis of the mRNA expression of <i>IER5</i> and <i>Cdc25B</i> in each colony using QRT-PCR and RT-PCR. The levels of the QRT-PCR products were normalized t<i>o GAPDH</i> expression in the same sample and were then expressed relative to the mRNA level of a normal control which was assigned a value of 1. RT-PCR results representative of three independent experiments are shown. <i>GAPDH</i> mRNA expression is shown as an internal control. The results are the means ± SD of three independent experiments. *<i>P</i><0.01 compared with untreated control cells. F. The cells transfected with <i>Cdc25B</i> or <i>IER5</i> cDNA were viewed using phase-contrast microscopy after 14 days culture with or without TMPP (5 µM). Original magnification ×4.</p