Production of recombinant human G protein-coupled estrogen receptor (GPER) and establishment of a ligand binding assay using graphene quantum dots (GQDs).

G protein-coupled estrogen receptor (GPER) is a member of the GPCR family and a key mediator of the rapid, nongenomic actions of estrogens as membrane estrogen receptors. In this study, we established a procedure for the expression and purification of recombinant human membrane estrogen receptor (hGPER) protein via the expression system using the methylotrophic yeast Pichia pastoris. By optimizing codon usage, we successfully expressed hGPER at a level that can be purified by column chromatography. The recombinant protein was purified via three chromatography steps. Purified hGPER showed specific estrogen-binding activity (Kd = 9.9 nM and Bmax = 1.76 nM) in a radiolabeled steroid-binding assay. We subsequently established a homogeneous assay for hGPER ligands by conjugating semiconductor nanoparticles known as graphene quantum dots (GQDs) to hGPER. GQDs coupled with hGPER (GQD-hGPER) caused a decrease in fluorescence at 520 nm from E2-BSA-FITC, which was activated by 370 nm light upon the addition of free estradiol to the reaction mixture. Fluorescence was decreased by the administration of hGPER ligands but not by steroids that do not interact with hGPER. Thus, we successfully established a ligand-binding assay for hGPER that is suitable for screening potential compounds. hGPER is a promising candidate for drug discovery for nongenomic estrogen-stimulating effects. The homogeneous assay established in this study will be usable for that purpose

Biochemical characterization of zebrafish Paqr5b

Previously, we established a gene knock-out strain of paqr5b in zebrafish and showed that the gene is essential for the formation of neurons in the zebrafish olfactory rosette. The results suggested that Paqr5b might play a role as a receptor for neurosteroids and contribute to the differentiation of olfactory neurons.In this study, we attempted to express the recombinant zebrafish Paqr5b protein and analyze its affinity for steroids. Full-length zebrafish Paqr5b (zPaqr5b) was expressed in Pichia pastris according to the method established for goldfish and human Paqr7. Solubilized zPaqr5b was purified by two column chromatography steps, nickel-nitrilotriacetic acid (Ni-NTA) column and gel column (Sephacryl S-300). The protein fraction showed a binding affinity of Kd = 4.6 nM and Bmax = 0.72 nM for progesterone. The result showed that zPaqr5b was successfully fractionated as the active form. The specificity of zPaqr5b against steroids was then analyzed by steroid binding assay. The zPaqr5b showed specific binding to progesterone as well as to the neurosteroid, allopregnanolone (ALLO). In addition, zPaqr5b showed high affinity for 17α,20β-dehydroxyprogesterone (DHP), a pheromone used to induce sexual behavior. In contrast, it was observed that other steroids, estradiol, testosterone and cortisol, showed no affinity, even when present at high doses. These results suggest that zPaqr5b is responsible for a receptor of progestogenic neurosteroids in the differentiation of neurons in the olfactory rosette (OR) and for a receptor for pheromones in developed neuronal cells in the OR

Convalescent plasma transfusion therapy in severe COVID-19 patients- a safety, efficacy and dose response study: A structured summary of a study protocol of a phase II randomized controlled trial

Abstract Objectives General: To assess the safety, efficacy and dose response of convalescent plasma (CP) transfusion in severe COVID-19 patients Specific: a. To identify the appropriate effective dose of CP therapy in severe patients b. To identify the efficacy of the therapy with their end point based on clinical improvement within seven days of treatment or until discharge whichever is later and in-hospital mortality c. To assess the clinical improvement after CP transfusion in severe COVID-19 patients d. To assess the laboratory improvement after CP transfusion in severe COVID-19 patients Trial Design This is a multicentre, multi-arm phase II Randomised Controlled Trial. Participants Age and sex matched COVID-19 positive (by RT-PCR) severe cases will be enrolled in this trial. Severe case is defined by the World Health Organization (W.H.O) clinical case definition. The inclusion criteria are 1. Respiratory rate &gt; 30 breaths/min; PLUS 2. Severe respiratory distress; or SpO2 ≤ 88% on room air or PaO2/FiO2≤ 300 mm of Hg, PLUS 3. Radiological (X-ray or CT scan) evidence of bilateral lung infiltrate, AND OR 4. Systolic BP &lt; 90 mm of Hg or diastolic BP &lt;60 mm of Hg. AND/OR 5. Criteria 1 to 4 AND or patient in ventilator support Patients’ below18 years, pregnant and lactating women, previous history of allergic reaction to plasma, patients who have already received plasma from a different source will be excluded. Patients will be enrolled at Bangabandhu Sheikh Mujib Medical University (BSMMU) hospital, Dhaka medical college hospital (DMCH) and Mugda medical college hospital (MuMCH). Apheretic plasma will be collected at the transfusion medicine department of SHNIBPS hospital, ELISA antibody titre will be done at BSMMU and CMBT and neutralizing antibody titre will be checked in collaboration with the University of Oxford. Patients who have recovered from COVID-19 will be recruited as donors of CP. The recovery criteria are normality of body temperature for more than 3 days, resolution of respiratory symptoms, two consecutively negative results of sputum SARS-CoV-2 by RT-PCR assay (at least 24 hours apart) 22 to 35 days of post onset period, and neutralizing antibody titre ≥ 1:160. Intervention and comparator This RCT consists of three arms, a. standard care, b. standard care and 200 ml CP and c. standard care and 400 ml CP. Patients will receive plasma as a single transfusion. Intervention arms will be compared to the standard care arm. Main outcomes The primary outcome will be time to clinical improvement within seven days of treatment or until discharge whichever is later and in-hospital mortality. The secondary outcome would be improvement of laboratory parameters after therapy (neutrophil, lymphocyte ratio, CRP, serum ferritin, SGPT, SGOT, serum creatinine and radiology), length of hospital stay, length of ICU stay, reduction in proportion of deaths, requirement of ventilator and duration of oxygen and ventilator support. Randomisation Randomization will be done by someone not associated with the care or assessment of the patients by means of a computer generated random number table using an allocation ratio of 1:1:1. Blinding (masking) This is an open level study; neither the physician nor the patients will be blinded. However, the primary and secondary outcome (oxygen saturations, PaO2/FiO2, BP, day specific laboratory tests) will be recorded using an objective automated method; the study staff will not be able to influence the recording of these data. Number to be randomised (sample size) No similar study has been performed previously. Therefore no data are available that could be used to generate a sample size calculation. This phase II study is required to provide some initial data on efficacy and safety that will allow design of a larger study. The trial will recruit 60 participants (20 in each arm). Trial Status Protocol version 1.4 dated May 5, 2020 and amended version 1.5, dated June 16, 2020. First case was recruited on May 27, 2020. By August 10, 2020, the trial had recruited one-third (21 out of 60) of the participants. The recruitment is expected to finish by October 31, 2020. Trial registration Clinicaltrials.gov ID: NCT04403477. Registered 26 May, 2020 Full Protocol The full protocol is attached as an additional file, accessible from the Trial’s website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this letter serves as a summary of the key elements of the full protocol. </jats:sec

Physiological and Yield Contributing Characters Depicting A High Yield Potential Triticale Line (x Triticosecale Wittm.)

&lt;p&gt;Triticale is used as a dual-purposes. In Bangladesh, dairy and poultry farms are being extended faster day by day. Therefore, the demand of fodder and feed increases swiftly. Moreover, triticale tolerates biotic and abiotic stressors better than wheat and durum. The trials were conducted in two consecutive years to search out a high yielding triticale line whereas two varieties were used as check viz. BARI Triticale 1 (E1) and BARI Triticale 2 (E2) and four lines (expressed as E3, E4, E5, and E6). In the research areas, temperature, rainfall and relative humidity were higher in 2018-19 than in 2019-20, but rainfall was lower. As a result, days to heading (DH), days to maturity (DM), fertile spikes per meter square (FSPMS), and grain yield (GY) were more in 2019-20 than in 2018-19. In 2018-19, thousand grain weight (TGW) correlated negatively with DH, DM, and FSPMS. In 2019-20, a positive association was established between FSPMS and GY. Furthermore, E5 and E6 lines had lower plant height (PH), but higher FSPMS and TGW than the control in both seasons. In addition, E5 and E6 yielded more GY than check and other lines. These findings suggested that E5 and E6 lines may be released as high yielding triticale variety (ies) to boost triticale production in Bangladesh.&lt;/p&gt