Exploring optimal Taxol® CYP725A4 activity in Saccharomyces cerevisiae

Background: CYP725A4 catalyses the conversion of the first Taxol® precursor, taxadiene, to taxadiene-5α-ol (T5α-ol) and a range of other mono- and di-hydroxylated side products (oxygenated taxanes). Initially known to undergo a radical rebound mechanism, the recent studies have revealed that an intermediate epoxide mediates the formation of the main characterised products of the enzyme, being T5α-ol, 5(12)-oxa-3(11)-cyclotaxane (OCT) and its isomer, 5(11)-oxa-3(11)-cyclotaxane (iso-OCT) as well as taxadienediols. Besides the high side product: main product ratio and the low main product titre, CYP725A4 is also known for its slow enzymatic activity, massively hindering further progress in heterologous production of Taxol® precursors. Therefore, this study aimed to systematically explore the key parameters for improving the regioselectivity and activity of eukaryotic CYP725A4 enzyme in a whole-cell eukaryotic biocatalyst, Saccharomyces cerevisiae. / Results: Investigating the impact of CYP725A4 and reductase gene dosages along with construction of self-sufficient proteins with strong prokaryotic reductases showed that a potential uncoupling event accelerates the formation of oxygenated taxane products of this enzyme, particularly the side products OCT and iso-OCT. Due to the harmful effect of uncoupling products and the reactive metabolites on the enzyme, the impact of flavins and irons, existing as prosthetic groups in CYP725A4 and reductase, were examined in both their precursor and ready forms, and to investigate the changes in product distribution. We observed that the flavin adenine dinucleotide improved the diterpenoids titres and biomass accumulation. Hemin was found to decrease the titre of iso-OCT and T5α-ol, without impacting the side product OCT, suggesting the latter being the major product of CYP725A4. The interaction between this iron and the iron precursor, δ-Aminolevulinic acid, seemed to improve the production of these diterpenoids, further denoting that iso-OCT and T5α-ol were the later products. While no direct correlation between cellular-level oxidative stress and oxygenated taxanes was observed, investigating the impact of salt and antioxidant on CYP725A4 further showed the significant drop in OCT titre, highlighting the possibility of enzymatic-level uncoupling event and reactivity as the major mechanism behind the enzyme activity. To characterise the product spectrum and production capacity of CYP725A4 in the absence of cell growth, resting cell assays with optimal neutral pH revealed an array of novel diterpenoids along with higher quantities of characterised diterpenoids and independence of the oxygenated product spectra from the acidity effect. Besides reporting on the full product ranges of CYP725A4 in yeast for the first time, the highest total taxanes of around 361.4 ± 52.4 mg/L including 38.1 ± 8.4 mg/L of T5α-ol was produced herein at a small, 10-mL scale by resting cell assay, where the formation of some novel diterpenoids relied on the prior existence of other diterpenes/diterpenoids as shown by statistical analyses. / Conclusions: This study shows how rational strain engineering combined with an efficient design of experiment approach systematically uncovered the promoting effect of uncoupling for optimising the formation of the early oxygenated taxane precursors of Taxol®. The provided strategies can effectively accelerate the design of more efficient Taxol®-producing yeast strains

Heterogeneity in Blood Pressure Response to 4 Antihypertensive Drugs: A Randomized Clinical Trial

Importance: Hypertension is the leading risk factor for premature death worldwide. Multiple blood pressure-lowering therapies are available but the potential for maximizing benefit by personalized targeting of drug classes is unknown. Objective: To investigate and quantify the potential for targeting specific drugs to specific individuals to maximize blood pressure effects. Design, Setting, and Participants: A randomized, double-blind, repeated crossover trial in men and women with grade 1 hypertension at low risk for cardiovascular events at an outpatient research clinic in Sweden. Mixed-effects models were used to assess the extent to which individuals responded better to one treatment than another and to estimate the additional blood pressure lowering achievable by personalized treatment. Interventions: Each participant was scheduled for treatment in random order with 4 different classes of blood pressure-lowering drugs (lisinopril [angiotensin-converting enzyme inhibitor], candesartan [angiotensin-receptor blocker], hydrochlorothiazide [thiazide], and amlodipine [calcium channel blocker]), with repeated treatments for 2 classes. Main Outcomes and Measures: Ambulatory daytime systolic blood pressure, measured at the end of each treatment period. Results: There were 1468 completed treatment periods (median length, 56 days) recorded in 270 of the 280 randomized participants (54% men; mean age, 64 years). The blood pressure response to different treatments varied considerably between individuals (P <.001), specifically for the choices of lisinopril vs hydrochlorothiazide, lisinopril vs amlodipine, candesartan vs hydrochlorothiazide, and candesartan vs amlodipine. Large differences were excluded for the choices of lisinopril vs candesartan and hydrochlorothiazide vs amlodipine. On average, personalized treatment had the potential to provide an additional 4.4 mm Hg-lower systolic blood pressure. Conclusions and Relevance: These data reveal substantial heterogeneity in blood pressure response to drug therapy for hypertension, findings that may have implications for personalized therapy. Trial Registration: ClinicalTrials.gov Identifier: NCT02774460

The fetal mouse is a sensitive genotoxicity model that exposes lentiviral-associated mutagenesis resulting in liver oncogenesis

This article is available open access through the publisher’s website at the link below. Copyright @ 2013 The American Society of Gene & Cell Therapy.Genotoxicity models are extremely important to assess retroviral vector biosafety before gene therapy. We have developed an in utero model that demonstrates that hepatocellular carcinoma (HCC) development is restricted to mice receiving nonprimate (np) lentiviral vectors (LV) and does not occur when a primate (p) LV is used regardless of woodchuck post-translation regulatory element (WPRE) mutations to prevent truncated X gene expression. Analysis of 839 npLV and 244 pLV integrations in the liver genomes of vector-treated mice revealed clear differences between vector insertions in gene dense regions and highly expressed genes, suggestive of vector preference for insertion or clonal outgrowth. In npLV-associated clonal tumors, 56% of insertions occurred in oncogenes or genes associated with oncogenesis or tumor suppression and surprisingly, most genes examined (11/12) had reduced expression as compared with control livers and tumors. Two examples of vector-inserted genes were the Park 7 oncogene and Uvrag tumor suppressor gene. Both these genes and their known interactive partners had differential expression profiles. Interactive partners were assigned to networks specific to liver disease and HCC via ingenuity pathway analysis. The fetal mouse model not only exposes the genotoxic potential of vectors intended for gene therapy but can also reveal genes associated with liver oncogenesis.Imperial College London, the Wellcome Trust, and Brunel University

Cellular Barcoding Identifies Clonal Substitution as a Hallmark of Local Recurrence in a Surgical Model of Head and Neck Squamous Cell Carcinoma

Local recurrence after surgery for head and neck squamous cell carcinoma (HNSCC) remains a common event associated with a dismal prognosis. Improving this outcome requires a better understanding of cancer cell populations that expand from postsurgical minimal residual disease (MRD). Therefore, we assessed clonal dynamics in a surgical model of barcoded HNSCC growing in the submental region of immunodeficient mice. Clonal substitution and massive reduction of clonal heterogeneity emerged as hallmarks of local recurrence, as the clones dominating in less heterogeneous recurrences were scarce in their matched primary tumors. These lineages were selected by their ability to persist after surgery and competitively expand from MRD. Clones enriched in recurrences exhibited both private and shared genetic features and likely originated from ancestors shared with clones dominating in primary tumors. They demonstrated high invasiveness and epithelial-to-mesenchymal transition, eventually providing an attractive target for obtaining better local control for these tumors

Laboratory features of severe vs. non-severe COVID-19 patients in Asian populations: a systematic review and meta-analysis

BACKGROUND: More severe cases of COVID- 19 are more likely to be hospitalized and around one-fifth, needing ICU admission. Understanding the common laboratory features of COVID-19 in more severe cases versus non-severe patients could be quite useful for clinicians and might help to predict the model of disease progression. This systematic review and meta-analysis aimed to compare the laboratory test findings in severe vs. non-severe confirmed infected cases of COVID-19. METHODS: Electronic databases were systematically searched in PubMed, EMBASE, Scopus, Web of Science, and Google Scholar from the beginning of 2019 to 3rd of March 2020. Heterogeneity across included studies was determined using Cochrane's Q test and the I2 statistic. We used the fixed or random-effect models to pool the weighted mean differences (WMDs) or standardized mean differences and 95% confidence intervals (CIs). FINDINGS: Out of a total of 3009 citations, 17 articles (22 studies, 21 from China and one study from Singapore) with 3396 ranging from 12 to1099 patients were included. Our meta-analyses showed a significant decrease in lymphocyte, monocyte, and eosinophil, hemoglobin, platelet, albumin, serum sodium, lymphocyte to C-reactive protein ratio (LCR), leukocyte to C-reactive protein ratio (LeCR), leukocyte to IL-6 ratio (LeIR), and an increase in the neutrophil, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, blood urea nitrogen (BUN), creatinine (Cr), erythrocyte Sedimentation Rate (ESR), C-reactive protein (CRP), Procalcitonin (PCT), lactate dehydrogenase (LDH), fibrinogen, prothr

Analyzing the Number of Common Integration Sites of Viral Vectors – New Methods and Computer Programs

Vectors based on γ-retroviruses or lentiviruses have been shown to stably express therapeutical transgenes and effectively cure different hematological diseases. Molecular follow up of the insertional repertoire of gene corrected cells in patients and preclinical animal models revealed different integration preferences in the host genome including clusters of integrations in small genomic areas (CIS; common integrations sites). In the majority, these CIS were found in or near genes, with the potential to influence the clonal fate of the affected cell. To determine whether the observed degree of clustering is statistically compatible with an assumed standard model of spatial distribution of integrants, we have developed various methods and computer programs for γ-retroviral and lentiviral integration site distribution. In particular, we have devised and implemented mathematical and statistical approaches for comparing two experimental samples with different numbers of integration sites with respect to the propensity to form CIS as well as for the analysis of coincidences of integration sites obtained from different blood compartments. The programs and statistical tools described here are available as workspaces in R code and allow the fast detection of excessive clustering of integration sites from any retrovirally transduced sample and thus contribute to the assessment of potential treatment-related risks in preclinical and clinical retroviral gene therapy studies

Gene therapy for monogenic liver diseases: clinical successes, current challenges and future prospects

Over the last decade, pioneering liver-directed gene therapy trials for haemophilia B have achieved sustained clinical improvement after a single systemic injection of adeno-associated virus (AAV) derived vectors encoding the human factor IX cDNA. These trials demonstrate the potential of AAV technology to provide long-lasting clinical benefit in the treatment of monogenic liver disorders. Indeed, with more than ten ongoing or planned clinical trials for haemophilia A and B and dozens of trials planned for other inherited genetic/metabolic liver diseases, clinical translation is expanding rapidly. Gene therapy is likely to become an option for routine care of a subset of severe inherited genetic/metabolic liver diseases in the relatively near term. In this review, we aim to summarise the milestones in the development of gene therapy, present the different vector tools and their clinical applications for liver-directed gene therapy. AAV-derived vectors are emerging as the leading candidates for clinical translation of gene delivery to the liver. Therefore, we focus on clinical applications of AAV vectors in providing the most recent update on clinical outcomes of completed and ongoing gene therapy trials and comment on the current challenges that the field is facing for large-scale clinical translation. There is clearly an urgent need for more efficient therapies in many severe monogenic liver disorders, which will require careful risk-benefit analysis for each indication, especially in paediatrics

Risk of stroke in hospitalized SARS-CoV-2 infected patients: A multinational study

Background: There is an increased attention to stroke following SARS-CoV-2. The goal of this study was to better depict the short-term risk of stroke and its associated factors among SARS-CoV-2 hospitalized patients. Methods: This multicentre, multinational observational study includes hospitalized SARS-CoV-2 patients from North and South America (United States, Canada, and Brazil), Europe (Greece, Italy, Finland, and Turkey), Asia (Lebanon, Iran, and India), and Oceania (New Zealand). The outcome was the risk of subsequent stroke. Centres were included by non-probability sampling. The counts and clinical characteristics including laboratory findings and imaging of the patients with and without a subsequent stroke were recorded according to a predefined protocol. Quality, risk of bias, and heterogeneity assessments were conducted according to ROBINS-E and Cochrane Q-test. The risk of subsequent stroke was estimated through meta-analyses with random effect models. Bivariate logistic regression was used to determine the parameters with predictive outcome value. The study was reported according to the STROBE, MOOSE, and EQUATOR guidelines. Findings: We received data from 26,175 hospitalized SARS-CoV-2 patients from 99 tertiary centres in 65 regions of 11 countries until May 1st, 2020. A total of 17,799 patients were included in meta-analyses. Among them, 156(0.9) patients had a stroke�123(79) ischaemic stroke, 27(17) intracerebral/subarachnoid hemorrhage, and 6(4) cerebral sinus thrombosis. Subsequent stroke risks calculated with meta-analyses, under low to moderate heterogeneity, were 0.5 among all centres in all countries, and 0.7 among countries with higher health expenditures. The need for mechanical ventilation (OR: 1.9, 95 CI:1.1�3.5, p = 0.03) and the presence of ischaemic heart disease (OR: 2.5, 95 CI:1.4�4.7, p = 0.006) were predictive of stroke. Interpretation: The results of this multi-national study on hospitalized patients with SARS-CoV-2 infection indicated an overall stroke risk of 0.5(pooled risk: 0.9). The need for mechanical ventilation and the history of ischaemic heart disease are the independent predictors of stroke among SARS-CoV-2 patients. Funding: None. © 2020 The Author

Critical methodological considerations in recruiting and engaging non-native English speaking workers with a head injury: a Canadian perspective

Abstract Objective Non-native English speaking workers with a mild work-related traumatic brain and/or head injury are a vulnerable and underrepresented population in research studies. The researchers present their experiences with recruiting and performing qualitative interviews with non-native English speaking individuals with a work-related mild traumatic brain injury, and provide recommendations on how to better include this vulnerable population in future research studies. This paper presents considerations regarding ethics, recruitment challenges, interview preparation and debriefing, sex & gender and language and cultural issues must be made when working with this vulnerable population. Results The researchers discuss critical issues and provide recommendations in recruiting and engaging with non-native English language workers including ethics, recruitment challenges, interview preparation and debriefing, sex & gender and language, and cultural considerations that must be made when working with this population. The study recommendations advise investigators to spend more time to learn about the non-native English participants in the mild wrTBI context, to be familiar with the vulnerabilities and specific circumstances that these workers experience. By increasing their awareness of the challenging facing this vulnerable population, the intention is to provide better care and treatment options through evidence-based research and practice