Project description:Plasmid-based gene expression is a fundamental tool in the field of biotechnology. However, overexpression of genes of interest with multi-copy plasmids often causes detrimental effects on host cells. To overcome this problem, chromosomal integration of target genes has been used for decades; however, insufficient protein expression occurred with this method. In this study, we developed a novel cloning and expression system named the chromosomal vector (ChroV) system, that has features of stable and high expression of target genes on the F' plasmid in the Escherichia coli JM109(DE3) strain. We used an RMT cluster (KCTC 11994BP) containing a silent cat gene from a previous study to clone a gene into the F' plasmid. The ChroV system was applied to clone two model targets, GFPuv and carotenoids gene clusters (4 kb), and successfully used to prove the inducible tightly regulated protein expression in the F' plasmid. In addition, we verified that the expression of heterologous genes in ChroV system maintained stably in the absence of antibiotics for 1 week, indicating ChroV system is applicable to antibiotics-free production of valuable proteins. This protocol can be widely applied to recombinant protein expression for antibiotics-free, stable, and genome-based expression, providing a new platform for recombinant protein synthesis in E. coli. Overall, our approach can be widely used for the economical and industrial production of proteins in E. coli.
Project description:Although leading causes of death are regularly reported, there is disagreement on which long-term conditions (LTCs) reduce disability-free life expectancy (DFLE) the most. We aimed to estimate increases in DFLE associated with elimination of a range of LTCs. This is a comprehensive systematic review and meta-analysis of studies assessing the effects of LTCs on health expectancy (HE). MEDLINE, Embase, HMIC, Science Citation Index, and Social Science Citation Index were systematically searched for studies published in English from July 2007 to July 2020 with updated searches from inception to April 8, 2021. LTCs considered included: arthritis, diabetes, cardiovascular disease including stroke and peripheral vascular disease, respiratory disease, visual and hearing impairment, dementia, cognitive impairment, depression, cancer, and comorbidity. Studies were included if they estimated HE outcomes (disability-free, active or healthy life expectancy) at age 50 or older for individuals with and without the LTC. Study selection and quality assessment were undertaken by teams of independent reviewers. Meta-analysis was feasible if three or more studies assessed the impact of the same LTC on the same HE at the same age using comparable methods, with narrative syntheses for the remaining studies. Studies reporting Years of Life Lost (YLL), Years of Life with Disability (YLD) and Disability Adjusted Life Years (DALYs = YLL+YLD) were included but reported separately as incomparable with other HE outcomes (PROSPERO registration: CRD42020196049). Searches returned 6072 unique records, yielding 404 eligible for full text retrieval from which 30 DFLE-related and 7 DALY-related were eligible for inclusion. Thirteen studies reported a single condition, and 17 studies reported on more than one condition (two to nine LTCs). Only seven studies examined the impact of comorbidities. Random effects meta-analyses were feasible for a subgroup of studies examining diabetes (four studies) or respiratory diseases (three studies) on DFLE. From pooled results, individuals at age 65 without diabetes gain on average 2.28 years disability-free compared to those with diabetes (95% CI: 0.57–3.99, p<0.01, I2 = 96.7%), whilst individuals without respiratory diseases gain on average 1.47 years compared to those with respiratory diseases (95% CI: 0.77–2.17, p<0.01, I2 = 79.8%). Eliminating diabetes, stroke, hypertension or arthritis would result in compression of disability. Of the seven longitudinal studies assessing the impact of multiple LTCs, three found that stroke had the greatest effect on DFLE for both genders. This study is the first to systematically quantify the impact of LTCs on both HE and LE at a global level, to assess potential compression of disability. Diabetes, stroke, hypertension and arthritis had a greater effect on DFLE than LE and so elimination would result in compression of disability. Guidelines for reporting HE outcomes would assist data synthesis in the future, which would in turn aid public health policy.
Project description:Slippery lubricant-infused surfaces allow easy removal of liquid droplets on surfaces. They consist of textured or porous substrates infiltrated with a chemically compatible lubricant. Capillary forces help to keep the lubricant in place. Slippery surfaces hold promising prospects in applications including drag reduction in pipes or food packages, anticorrosion, anti-biofouling, or anti-icing. However, a critical drawback is that shear forces induced by flow lead to depletion of the lubricant. In this work, a way to overcome the shear-induced lubricant depletion by replenishing the lubricant from the flow of emulsions is presented. The addition of small amounts of positively charged surfactant reduces the charge repulsion between the negatively charged oil droplets contained in the emulsion. Attachment and coalescence of oil droplets from the oil-in-water emulsion at the substrate surface fills the structure with the lubricant. Flow-induced lubrication of textured surfaces can be generalized to a broad range of lubricant-solid combinations using minimal amounts of oil.
Project description:High failure rates of drug candidates in the clinics, restricted-use warnings as well as withdrawals of drugs in postmarketing stages are of substantial concern for the pharmaceutical industry and drug-induced liver injury (DILI) constitutes one of the most frequent reasons for such safety failures. Importantly, as DILI cannot be accurately predicted using animal models, animal safety tests are commonly complemented with assessments in human in vitro systems. 3D spheroid cultures of primary human hepatocytes in chemically defined conditions, hereafter termed CD-spheroids, have recently emerged as a microphysiological model system in which hepatocytes retain their molecular phenotypes and hepatic functions for multiple weeks in culture. However, their predictive power for the detection of hepatotoxic liabilities has not been systematically assessed. Therefore, we here evaluated the hepatotoxicity of 123 drugs with or without direct implication in clinical DILI events. Importantly, using ATP quantifications as the single endpoint, the model accurately distinguished between hepatotoxic and nontoxic structural analogues and exceeded both sensitivity and specificity of all previously published in vitro assays at substantially lower exposure levels, successfully detecting 69% of all hepatotoxic compounds without producing any false positive results (100% specificity). Furthermore, the platform supports the culture of spheroids of primary hepatocytes from preclinical animal models, thereby allowing the identification of animal-specific toxicity events. We anticipate that CD-spheroids represent a powerful and versatile tool in drug discovery and preclinical drug development that can reliably flag hepatotoxic drug candidates and provide guidance for the selection of the most suitable animal models.
Project description:The Kramers escape problem is a paradigmatic model for the kinetics of rare events, which are usually characterized by Arrhenius law. So far, analytical approaches have failed to capture the kinetics of rare events in the important case of non-Markovian processes with long-term memory, as occurs in the context of reactions involving proteins, long polymers, or strongly viscoelastic fluids. Here, based on a minimal model of non-Markovian Gaussian process with long-term memory, we determine quantitatively the mean FPT to a rare configuration and provide its asymptotics in the limit of a large energy barrier E. Our analysis unveils a correction to Arrhenius law, induced by long-term memory, which we determine analytically. This correction, which we show can be quantitatively significant, takes the form of a second effective energy barrier E'<E and captures the dependence of rare event kinetics on initial conditions, which is a hallmark of long-term memory. Altogether, our results quantify the impact of long-term memory on rare event kinetics, beyond Arrhenius law.
Project description:The epicardium contributes both multi-lineage descendants and paracrine factors to the heart during cardiogenesis and cardiac repair, underscoring its potential for cardiac regenerative medicine. Yet little is known about the cellular and molecular mechanisms that regulate human epicardial development and regeneration. Here, we show that the temporal modulation of canonical Wnt signaling is sufficient for epicardial induction from 6 different human pluripotent stem cell (hPSC) lines, including a WT1-2A-eGFP knock-in reporter line, under chemically-defined, xeno-free conditions. We also show that treatment with transforming growth factor beta (TGF-β)-signalling inhibitors permitted long-term expansion of the hPSC-derived epicardial cells, resulting in a more than 25 population doublings of WT1+ cells in homogenous monolayers. The hPSC-derived epicardial cells were similar to primary epicardial cells both in vitro and in vivo, as determined by morphological and functional assays, including RNA-seq. Our findings have implications for the understanding of self-renewal mechanisms of the epicardium and for epicardial regeneration using cellular or small-molecule therapies.
Project description:There is tremendous interest in the production of recombinant proteins, particularly bispecific antibodies and antibody-drug conjugates for research and therapeutic use. Here, we demonstrate a highly versatile plasmid system that allows the rapid generation of stable Expi293 cell pools by episomal retention of transfected DNA. By linking protein expression to puromycin resistance through an attenuated internal ribosome entry site, we achieve stable cell pools producing proteins of interest. In addition, split intein-split puromycin-mediated selection of two separate protein expression cassettes allows the stable production of bispecific antibody-like molecules or antibodies with distinct C-terminal heavy chain modifications, such as an antigen on one chain and a sortase tag on the other chain. We also use this novel expression system to generate stable Expi293 cell pools that secrete sortase A Δ59 variant Srt4M. Using these reagents, we prepared a site-specific drug-to-antibody ratio of 1 antibody-siRNA conjugate. We anticipate the simple, robust, and rapid stable protein expression systems described here being useful for a wide variety of applications.
Project description:ObjectiveRecent evidence suggests that antibiotic use, which alters the gut microbiome, is associated with an increased risk of colorectal cancer. However, the association between antibiotic use and risk of colorectal adenoma, the precursor for the majority of colorectal cancers, has not been investigated.DesignWe prospectively evaluated the association between antibiotic use at age 20-39 and 40-59 (assessed in 2004) and recent antibiotic use (assessed in 2008) with risk of subsequent colorectal adenoma among 16 642 women aged ≥60 enrolled in the Nurses' Health Study who underwent at least one colonoscopy through 2010. We used multivariate logistic regression to calculate ORs and 95% CIs.ResultsWe documented 1195 cases of adenoma. Increasing duration of antibiotic use at age 20-39 (ptrend=0.002) and 40-59 (ptrend=0.001) was significantly associated with an increased risk of colorectal adenoma. Compared with non-users, women who used antibiotics for ≥2 months between age 20 and 39 had a multivariable OR of 1.36 (95% CI 1.03 to 1.79). Women who used ≥2 months of antibiotics between age 40 and 59 had a multivariable OR of 1.69 (95% CI 1.24 to 2.31). The associations were similar for low-risk versus high-risk adenomas (size ≥1 cm, or with tubulovillous/villous histology, or ≥3 detected lesions), but appeared modestly stronger for proximal compared with distal adenomas. In contrast, recent antibiotic use within the past four years was not associated with risk of adenoma (ptrend=0.44).ConclusionsLong-term antibiotic use in early-to-middle adulthood was associated with increased risk of colorectal adenoma.
Project description:Chronic granulomatous disease (CGD) is associated with significant morbidity and mortality from infection. The first CGD gene therapy trial resulted in only short-term marking of 0.01% to 0.1% of neutrophils. A recent study, using busulfan conditioning and an SFFV retrovirus vector, achieved more than 20% marking in 2 patients with X-linked CGD. However, oxidase correction per marked neutrophil was less than normal and not sustained. Despite this, patients clearly benefited in that severe infections resolved. As such, we initiated a gene therapy trial for X-CGD to treat severe infections unresponsive to conventional therapy. We treated 3 adult patients using busulfan conditioning and an MFGS retroviral vector encoding gp91(phox), achieving early marking of 26%, 5%, and 4% of neutrophils, respectively, with sustained long-term marking of 1.1% and 0.03% of neutrophils in 2 of the patients. Gene-marked neutrophils have sustained full correction of oxidase activity for 34 and 11 months, respectively, with full or partial resolution of infection in those 2 patients. Gene marking is polyclonal with no clonal dominance. We conclude that busulfan conditioning together with an MFGS vector is capable of achieving long-term correction of neutrophil oxidase function sufficient to provide benefit in management of severe infection. This study was registered at www.clinicaltrials.gov as #NCT00394316.
Project description:Methylmalonic acidemia is a severe metabolic disorder caused by a deficiency of the ubiquitously expressed mitochondrial enzyme, methylmalonyl-CoA mutase (MUT). Liver transplantation has been used to treat a small number of patients with variable success, and whether liver-directed gene therapy might be employed in such a pleiotropic metabolic disorder is uncertain. In this study, we examined the therapeutic effects of hepatocyte-directed delivery of the Mut gene to mice with a severe form of methylmalonic acidemia. We show that a single intrahepatic injection of recombinant adeno-associated virus serotype 8 expressing the Mut gene under the control of the liver-specific thyroxine-binding globulin (TBG) promoter is sufficient to rescue Mut(-/-) mice from neonatal lethality and provide long-term phenotypic correction. Treated Mut(-/-) mice lived beyond 1 year of age, had improved growth, lower plasma methylmalonic acid levels, and an increased capacity to oxidize [1-(13)C]propionate in vivo. The older treated mice showed increased Mut transcription, presumably mediated by upregulation of the TBG promoter during senescence. The results indicate that the stable transduction of a small number of hepatocytes with the Mut gene can be efficacious in the phenotypic correction of an inborn error of organic acid metabolism and support the rapid translation of liver-directed gene therapy vectors already optimized for human subjects to patients with methylmalonic acidemia.