Developing the host for targeted integration cell line development

Unlike the conventional random integration (RI) cell line development (CLD), the targeted integration (TI) CLD introduces the transgene at a predetermined “hot-spot” in the CHO genome with a defined copy number (1-2 copies). Given the low copy number and the pretested integration site, TI cell lines likely exhibit better stability compared to RI cell lines. In this study, we performed a genome wide screening using transposon based cassette integration and established a TI host (255-3) that has a single landing cassette inserted in its genome. Host 255-3 was able to support the CLD for three test molecules with product titers similar to those of the corresponding RI cell lines. For two regular antibody test cases, the top four TI cell lines achieved ~4-5g/L. For a proven difficult to express antibody, the top four TI lines achieved ~1-1.2g/L. The product titer for this hard to express molecule was increased 3-fold with additional vector improvement. Moreover, the timeline for CLD was shortened by ~2 weeks and resources required per cell line were substantially reduced using the TI method. Together these data indicate that the TI host we developed can be a suitable host to support our clinical / commercial CLD

Angiography-Derived Fractional Flow Reserve in Coronary Assessment: Current Developments and Future Perspectives

Coronary physiology assessment is an important factor in guiding myocardial revascularization. A growing body of research highlights the value of using fractional flow reserve, FFR and other pressure-based indicators for functional assessment of stable coronary stenoses. Invasive functional coronary assessment techniques have evolved from intracoronary wire-based to wire-free approaches as a result of technological advancements. In addition, several software programs on the market have been thoroughly investigated and validated against invasive FFR, and have shown good accuracy and correlation. However, use of FFR remains modest. Hence, this review provides an overview of angiography-based FFR solutions and compares their technologies. Additionally, a systematic scoping review was performed to understand the research landscape in wire-free coronary physiology assessment, to complement the narratives of existing FFR trials on wire-free FFR. Furthermore, future developments and strategies that could expand the use of wire-free computed coronary functional assessment in the Asia Pacific region are discussed

Contributions of the C-Terminal Helix to the Structural Stability of a Hyperthermophilic Fe-Superoxide Dismutase (TcSOD)

Hyperthermophilic superoxide dismutases (SODs) are of particular interest due to their potential industrial importance and scientific merit in studying the molecular mechanisms of protein folding and stability. Compared to the mesophilic SODs, the hyperthermostable Fe-SODs (TcSOD and ApSOD) have an extended C-terminal helix, which forms an additional ion-pairing network. In this research, the role of the extended C-terminus in the structural stability of TcSOD was studied by investigating the properties of two deletion mutants. The results indicated that the ion-pairing network at the C-terminus had limited contributions to the stability of TcSOD against heat- and GdnHCl-induced inactivation. The intactness of the C-terminal helix had dissimilar impact on the two stages of TcSOD unfolding induced by guanidinium chloride. The mutations slightly decreased the Gibbs free energy of the dissociation of the tetrameric enzymes, while greatly affected the stability of the molten globule-like intermediate. These results suggested that the additional ion-pairing network mainly enhanced the structural stability of TcSOD by stabilizing the monomers

Five endometrial cancer risk loci identified through genome-wide association analysis.

We conducted a meta-analysis of three endometrial cancer genome-wide association studies (GWAS) and two follow-up phases totaling 7,737 endometrial cancer cases and 37,144 controls of European ancestry. Genome-wide imputation and meta-analysis identified five new risk loci of genome-wide significance at likely regulatory regions on chromosomes 13q22.1 (rs11841589, near KLF5), 6q22.31 (rs13328298, in LOC643623 and near HEY2 and NCOA7), 8q24.21 (rs4733613, telomeric to MYC), 15q15.1 (rs937213, in EIF2AK4, near BMF) and 14q32.33 (rs2498796, in AKT1, near SIVA1). We also found a second independent 8q24.21 signal (rs17232730). Functional studies of the 13q22.1 locus showed that rs9600103 (pairwise r(2) = 0.98 with rs11841589) is located in a region of active chromatin that interacts with the KLF5 promoter region. The rs9600103[T] allele that is protective in endometrial cancer suppressed gene expression in vitro, suggesting that regulation of the expression of KLF5, a gene linked to uterine development, is implicated in tumorigenesis. These findings provide enhanced insight into the genetic and biological basis of endometrial cancer.I.T. is supported by Cancer Research UK and the Oxford Comprehensive Biomedical Research Centre. T.H.T.C. is supported by the Rhodes Trust and the Nuffield Department of Medicine. Funding for iCOGS infrastructure came from the European Community's Seventh Framework Programme under grant agreement 223175 (HEALTH-F2-2009-223175) (COGS), Cancer Research UK (C1287/A10118, C1287/A10710, C12292/A11174, C1281/A12014, C5047/A8384, C5047/A15007, C5047/A10692 and C8197/A16565), the US National Institutes of Health (R01 CA128978, U19 CA148537, U19 CA148065 and U19 CA148112), the US Department of Defense (W81XWH-10-1-0341), the Canadian Institutes of Health Research (CIHR) for the CIHR Team in Familial Risks of Breast Cancer, the Susan G. Komen Foundation for the Cure, the Breast Cancer Research Foundation and the Ovarian Cancer Research Fund. SEARCH recruitment was funded by a programme grant from Cancer Research UK (C490/A10124). Stage 1 and stage 2 case genotyping was supported by the NHMRC (552402 and 1031333). Control data were generated by the WTCCC, and a full list of the investigators who contributed to the generation of the data is available from the WTCCC website. We acknowledge use of DNA from the British 1958 Birth Cohort collection, funded by UK Medical Research Council grant G0000934 and Wellcome Trust grant 068545/Z/02; funding for this project was provided by the Wellcome Trust under award 085475. NSECG was supported by the European Union's Framework Programme 7 CHIBCHA grant and Wellcome Trust Centre for Human Genetics Core Grant 090532/Z/09Z, and CORGI was funded by Cancer Research UK. BCAC is funded by Cancer Research UK (C1287/A10118 and C1287/A12014). OCAC is supported by a grant from the Ovarian Cancer Research Fund thanks to donations by the family and friends of Kathryn Sladek Smith (PPD/RPCI.07) and the UK National Institute for Health Research Biomedical Research Centres at the University of Cambridge.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/ng.356

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

The Role and Responsibility of Clinical Pharmacists in Rheumatology Clinic: An exploratory study in Hong Kong

In Hong Kong, arthritis is the third leading chronic disorder among the older people. It could create a huge amount of burden on the health care system if patients cannot achieve good diseases control and have recurrent flare up of diseases. To maintain stable control and minimize acute flare up, medication adherence is essential. Studies revealed that drug education has the highest evidence in improving medication adherence. Pharmacists who are expert in drugs can provide counseling to arthritis patients, improving their drug adherence and disease activities. As a result, research evaluating the effectiveness of pharmacist counseling service on improving arthritis patients’ medication adherence and disease activities in Hong Kong has been initiated in a specialist out-patient clinic of a local acute hospital. This research aims to improve patient medication adherence and enhance medication safety. A validated Compliance Questionnaire on Rheumatology (CQR-19) is used to measure the medication adherence. From the preliminary data, over 90% of the recruited subjects are non-adherence at baseline and thus, detailed drug counseling is necessary. During the first visit, pharmacists will provide a 20 minutes drug counseling service to the referred patients. This includes discussion on drug administration schedule, importance of drug adherence, side effects management and pain management. Besides, pharmacists also have different roles in other aspects such as drug information and procurement. Pharmacists would assist in providing evidence-based information and recommendations to physicians and nurses for drug-related enquiries. These attempts to improve therapeutic outcomes and minimize medication errors, enhance medication safety and reduce hospitalization. Design of the multidisciplinary care model and results of this study would provide a reference for the future development of clinical pharmacist service in rheumatology.&nbsp

Molecular Cloning and Expression of a Novel Cytochrome P450 from Turkey Liver with Aflatoxin B1 Oxidizing Activity

Cytochromes P450 are members of a superfamily of oxidative hemoprotein enzymes that metabolize a variety of endogenous and exogenous compounds. Previous studies in our laboratory have shown that efficient P450-mediated activation underlies the extreme sensitivity of poultry, specifically turkeys, to the toxic effects of the mycotoxin aflatoxin B1 (AFB1). Using 3‘- and 5‘-rapid amplification of cDNA ends (RACE), we amplified from turkey liver RNA a full-length 1.73 kb cDNA predicted to be 528 amino acids with 94.7% sequence identity to a CYP1A5 from chicken liver. A truncated construct of the turkey CYP1A5 gene with 29 amino acids deleted from the hydrophobic NH2-terminal region was cloned and heterologously expressed in Escherichia coli. The expressed protein from E. coli membranes had a CO-binding spectrum typical of P450s, and it catalyzed the O-dealkylation of the CYP1A prototype substrates ethoxyresorufin and methoxyresorufin. CYP1A5-mediated O-dealkylation of methoxyresorufin was completely inhibited by α-naphthoflavone, a specific CYP1A inhibitor. Inhibitors to other mammalian P450s (3A4, 2D, 2E, and 3A1) either slightly inhibited this activity or not at all. CYP1A5 oxidized AFB1 to form two metabolites: the reactive intermediate, AFB1-8,9-epoxide (AFBO), and aflatoxin M1 (AFM1). Because of the importance of AFBO and AFM1 in the toxicity of AFB1, we conclude that this P450 probably plays some role in the well-known hypersensitivity of turkeys to AFB1. To our knowledge, this is the first P450 cloned and sequenced from turkeys, the species in which the toxicity of AFB1 was first discovered