Engineering of a wheat germ expression system to provide compatibility with a high throughput pET-based cloning platform

Wheat germ cell-free methods provide an important approach for the production of eukaryotic proteins. We have developed a protein expression vector for the TNT® SP6 High-Yield Wheat Germ Cell-Free (TNT WGCF) expression system (Promega) that is also compatible with our T7-based Escherichia coli intracellular expression vector pET15_NESG. This allows cloning of the same PCR product into either one of several pET_NESG vectors and this modified WGCF vector (pWGHisAmp) by In-Fusion LIC cloning (Zhu et al. in Biotechniques 43:354–359, 2007). Integration of these two vector systems allowed us to explore the efficacy of the TNT WGCF system by comparing the expression and solubility characteristics of 59 human protein constructs in both WGCF and pET15_NESG E. coli intracellular expression. While only 30% of these human proteins could be produced in soluble form using the pET15_NESG based system, some 70% could be produced in soluble form using the TNT WGCF system. This high success rate underscores the importance of eukaryotic expression host systems like the TNT WGCF system for eukaryotic protein production in a structural genomics sample production pipeline. To further demonstrate the value of this WGCF system in producing protein suitable for structural studies, we scaled up, purified, and analyzed by 2D NMR two 15N-, 13C-enriched human proteins. The results of this study indicate that the TNT WGCF system is a successful salvage pathway for producing samples of difficult-to-express small human proteins for NMR studies, providing an important complementary pathway for eukaryotic sample production in the NESG NMR structure production pipeline

Multinucleation followed by an acytokinetic cell division in myxofibrosarcoma with giant cell proliferation

<p>Abstract</p> <p>Background</p> <p>Multinucleated cells are frequently seen in association with a malignant neoplasm. Some of these multinucleated cells are considered to be neoplastic. The mechanism of neoplastic multinucleation remains unknown, but is considered to be induced by either cell-cell fusion or acytokinetic cell division. Myxofibrosarcoma consists of spindled and pleomorphic tumor cells and bizarre multinucleated giant cells. Some of these multinucleated cells are considered to be neoplastic.</p> <p>Methods</p> <p>We studied the mitotic activity of the multinucleated cells by Ki-67 immunohistochemistry, and the dynamics and differentiation by live-cell video microscopy in the two myxofibrosarcoma cell lines to determine whether the mechanism of multinucleation is cell-cell fusion or acytokinetic cell division</p> <p>Results</p> <p>A Ki-67 immunohistochemical analysis revealed a high positive rate of multinucleated cells, as well as mononuclear cells, and mitotic ability was shown in the multinucleated cells. In live-cell video microscopy, most of the multinucleated cells were induced via the process of acytokinetic cell division.</p> <p>Conclusion</p> <p>The current study indicates that a vulnerability of the cytoskeleton components, such as the contractile ring, causes multinucleation to occur from the telophase to the cytokinesis of the cell cycle.</p

New insights into the distribution and conservation status of the Golden-White Tassel-Ear Marmoset Mico chrysoleucos (Primates, Callitrichidae)

Among the 13 Mico species recognized by the IUCN Red List of Threatened Species, six are listed as "Data Deficient". The geographic range of most of the Mico species has been estimated from only a few records. We report new localities and the geographic extension of Mico chrysoleucos. In addition, we confirmed the presence of the species in two distinct protected areas. We modeled the habitat suitability of M. chrysoleucos using the maximum entropy method and including new records obtained by the authors in the state of Amazonas, Brazil. From the total area of occurrence calculated for the species, 22.8% is covered by protected areas and indigenous lands. The annual mean deforestation rate estimated between 2000 and 2015 was 2.95%, and the total area deforested by 2015 was 3354 km2 or 8.6% of the total distribution limits of the species. The habitat lost between 2000 and 2015 was 3.2% (1131 km2 ) of the total potential distribution, while the habitat loss area legally protected was 31 km2, and the habitat loss in settlements was equal to 691 km2. Our results extend the geographic distribution of the species about 100 km farther south, with the Maracanã River being a possible geographic barrier for the species. The significantly low rate of habitat loss inside protected areas and indigenous land, when compared to unprotected areas, points out the importance of these areas to M. chrysoleucos conservation. The species is relatively wide-ranging, legally protected, and resilient to regional anthropic threats. However, the hydroelectric schemes and the improvement of the road system in southern Amazonia pose an imminent threat to the species

Rapid KRAS, EGFR, BRAF and PIK3CA Mutation Analysis of Fine Needle Aspirates from Non-Small-Cell Lung Cancer Using Allele-Specific qPCR

Endobronchial Ultrasound Guided Transbronchial Needle Aspiration (EBUS-TBNA) and Trans-esophageal Ultrasound Scanning with Fine Needle Aspiration (EUS-FNA) are important, novel techniques for the diagnosis and staging of non-small cell lung cancer (NSCLC) that have been incorporated into lung cancer staging guidelines. To guide and optimize treatment decisions, especially for NSCLC patients in stage III and IV, EGFR and KRAS mutation status is often required. The concordance rate of the mutation analysis between these cytological aspirates and histological samples obtained by surgical staging is unknown. Therefore, we studied the extent to which allele-specific quantitative real-time PCR with hydrolysis probes could be reliably performed on EBUS and EUS fine needle aspirates by comparing the results with histological material from the same patient. We analyzed a series of 43 NSCLC patients for whom cytological and histological material was available. We demonstrated that these standard molecular techniques can be accurately applied on fine needle cytological aspirates from NSCLC patients. Importantly, we show that all mutations detected in the histological material of primary tumor were also identified in the cytological samples. We conclude that molecular profiling can be reliably performed on fine needle cytology aspirates from NSCLC patients

Frequently increased epidermal growth factor receptor (EGFR) copy numbers and decreased BRCA1 mRNA expression in Japanese triple-negative breast cancers

<p>Abstract</p> <p>Background</p> <p>Triple-negative breast cancer (estrogen receptor-, progesterone receptor-, and HER2-negative) (TNBC) is a high risk breast cancer that lacks specific therapy targeting these proteins.</p> <p>Methods</p> <p>We studied 969 consecutive Japanese patients diagnosed with invasive breast cancer from January 1981 to December 2003, and selected TNBCs based on the immunohistochemical data. Analyses of epidermal growth factor receptor (<it>EGFR</it>) gene mutations and amplification, and <it>BRCA</it>1 mRNA expression were performed on these samples using TaqMan PCR assays. The prognostic significance of TNBCs was also explored. Median follow-up was 8.3 years.</p> <p>Results</p> <p>A total of 110 (11.3%) patients had TNBCs in our series. Genotyping of the <it>EGFR </it>gene was performed to detect 14 known <it>EGFR </it>mutations, but none was identified. However, <it>EGFR </it>gene copy number was increased in 21% of TNBCs, while only 2% of ER- and PgR-positive, HER2-negative tumors showed slightly increased <it>EGFR </it>gene copy numbers. Thirty-one percent of TNBCs stained positive for EGFR protein by immunohistochemistry. <it>BRCA1 </it>mRNA expression was also decreased in TNBCs compared with controls. Triple negativity was significantly associated with grade 3 tumors, TP53 protein accumulation, and high Ki67 expression. TNBC patients had shorter disease-free survival than non-TNBC in node-negative breast cancers.</p> <p>Conclusion</p> <p>TNBCs have an aggressive clinical course, and <it>EGFR </it>and <it>BRCA1 </it>might be candidate therapeutic targets in this disease.</p

Potent antitumor effects of combined therapy with a telomerase-specific, replication-competent adenovirus (OBP-301) and IL-2 in a mouse model of renal cell carcinoma

OBP-301 (a telomerase-specific, replication-competent adenovirus with hTERT promoter) was constructed in a previous study and it showed a strong anticancer effect by inducing cell lysis in human lung and prostate cancer cells. This study investigated the effectiveness of a combination therapy of OBP-301 and interleukin-2 (IL-2) in a mouse model of renal cell carcinoma (RCC). The cell-killing effect of OBP-301 was confirmed in vitro in the RENCA cancer cells. In in vivo experiment, luciferase-expressing RENCA cells were implanted in the left kidney and lung of BALB/c mice to prepare the RCC metastatic model. The animals were randomly divided into four treatment groups: PBS, IL-2 alone, OBP-301 alone and the combination. The analyses of orthotopic tumor weight, lung metastasis and luciferin-stained tumor images 14 days after each treatment showed significant tumor growth inhibition in the combination group in comparison with that in the OBP-301- or IL-2-treated groups. In addition, the percentage of regulatory T-cells (Tregs) in the combination group was significantly suppressed in comparison with that in the PBS and single-agent treatment groups. The outcomes of this study suggest that tumor-specific oncolytic immunovirotherapy may become an attractive strategy for the treatment of human RCC

Analytical approaches to photobiological hydrogen production in unicellular green algae

Several species of unicellular green algae, such as the model green microalga Chlamydomonas reinhardtii, can operate under either aerobic photosynthesis or anaerobic metabolism conditions. A particularly interesting metabolic condition is that of “anaerobic oxygenic photosynthesis”, whereby photosynthetically generated oxygen is consumed by the cell’s own respiration, causing anaerobiosis in the culture in the light, and induction of the cellular “hydrogen metabolism” process. The latter entails an alternative photosynthetic electron transport pathway, through the oxygen-sensitive FeFe-hydrogenase, leading to the light-dependent generation of molecular hydrogen in the chloroplast. The FeFe-hydrogenase is coupled to the reducing site of photosystem-I via ferredoxin and is employed as an electron-pressure valve, through which electrons are dissipated, thus permitting a sustained electron transport in the thylakoid membrane of photosynthesis. This hydrogen gas generating process in the cells offers testimony to the unique photosynthetic metabolism that can be found in many species of green microalgae. Moreover, it has attracted interest by the biotechnology and bioenergy sectors, as it promises utilization of green microalgae and the process of photosynthesis in renewable energy production. This article provides an overview of the principles of photobiological hydrogen production in microalgae and addresses in detail the process of induction and analysis of the hydrogen metabolism in the cells. Furthermore, methods are discussed by which the interaction of photosynthesis, respiration, cellular metabolism, and H(2) production in Chlamydomonas can be monitored and regulated

International Consensus Statement on Rhinology and Allergy: Rhinosinusitis

Background: The 5 years since the publication of the first International Consensus Statement on Allergy and Rhinology: Rhinosinusitis (ICAR‐RS) has witnessed foundational progress in our understanding and treatment of rhinologic disease. These advances are reflected within the more than 40 new topics covered within the ICAR‐RS‐2021 as well as updates to the original 140 topics. This executive summary consolidates the evidence‐based findings of the document. Methods: ICAR‐RS presents over 180 topics in the forms of evidence‐based reviews with recommendations (EBRRs), evidence‐based reviews, and literature reviews. The highest grade structured recommendations of the EBRR sections are summarized in this executive summary. Results: ICAR‐RS‐2021 covers 22 topics regarding the medical management of RS, which are grade A/B and are presented in the executive summary. Additionally, 4 topics regarding the surgical management of RS are grade A/B and are presented in the executive summary. Finally, a comprehensive evidence‐based management algorithm is provided. Conclusion: This ICAR‐RS‐2021 executive summary provides a compilation of the evidence‐based recommendations for medical and surgical treatment of the most common forms of RS

Preliminary studies on the antibacterial activity of crude extracts and alkaloids from species of Aspidosperma

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Screening tests of hydroethanolic crude extracts of six species of Aspidosperma (Apocynaceae) against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa were performed. Aspidosperma ramiflorum Muell. Arg. showed good activity against Bacillus subtilis with MIC and MBC of 15.7 and 125 mu g/mL, moderate activity against Staphylococcus aureus with MIC and MBC of 250 and 500 mu g/mL, and weak activity against Escherichia coli with MIC and MBC of 1000 mu g/mL. Aspidosperma pyricolum Muell. Arg. (MIC/MBC 125/250 mu g/mL) and Aspidosperma olivaceum Muell. Arg. (MIC/MBC 250/> 1000 mu g/mL) displayed moderate antibacterial activity against Bacillus subtilis. Separation of the crude extract of Aspidosperma ramiflorum was performed according to the usual acid-base process, which produces alkaloid mixtures and closely related metabolites. The basic fraction was active against Bacillus subtilis, Staphylococcus aureus, and Escherichia coli, with MICs of 31.2, 62.5, and 250 mu g/mL, respectively. The basic fractions were more active than the acid fractions, probably because they contained some active alkaloids and/or closely related metabolites absent from the other fractions, or they contained a higher concentration of these active compounds.471110851089Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundacao AraucariaPrograma de Pos-graduacao em Ciencias Farmaceuticas da Universidade Estadual de MaringaConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES