Studies on the role of SecA, FtsY and Tfh in the insertion of membrane proteins in Escherichia coli

The essential protein SecA plays a key role in post-translational protein secretion across the inner membrane of Escherichia coli. However, the extent of SecA-requirement in co-translational insertion of inner membrane proteins mediated by the signal recognition particle and its membrane-associated receptor FtsY is not clear. To inactivate SecA function a seven amino acid sequence that is specifically recognised by tobacco etch virus protease was introduced into the secA sequence. The resulting secA-constructs were integrated into the chromosome at the 1 attachment site via a specific lambda phage. Chromosomal wild-type secA was then eliminated by using the 1 Red recombinase system. Cleavage of nascent SecA 195 by a trigger factor-TEV protease hybrid protein resulted in effective inhibition of SecA activity. In addition, SecA830 under the control of the lacpromoter could be depleted in cells not supplemented with the inducer IPTG. Biotinylation assays showed that inactivation of SecA via site-specific proteolysis or depletion of SecA affected the proper secretion of the periplasmic protein alkaline phosphatase as well as the proper biogenesis of the inner membrane proteins FtsQ and MalF274. Similar experiments with Ffh- and FtsY-depletion strains confirmed the involvement of these proteins in inner membrane protein assembly. DNA macroarray technique was used to compare gene expression profiles of cells depleted either of Ffh, FtsY or SecA with the profiles obtained from undepleted cells. While depletion of Ffh and FtsY resulted in the induction of heat shock genes, depletion of SecA induced the expression of the phage-shock protein operon. The results strengthen the notion that SecA not only plays the central role in post-translational secretion of preproteins, but is also extensively involved in co-translational protein translocation. In addition, DNA array analysis indicated a possible involvement of cellular chaperones in targeting of inner membrane proteins

Mediating role of C-reactive protein in associations between pre-pregnancy BMI and adverse maternal and neonatal outcomes: the ABCD-study cohort

Objectives: Increased body mass index (BMI) is associated with several adverse pregnancy outcomes, though the underlying mechanism of this association has not been fully elucidated. A mediating role of low-grade systemic inflammation in these associations is suspected but has been understudied. Our objective was to examine the effect of pre-pregnancy BMI (pBMI) on maternal and neonatal pregnancy outcomes and to explore potential mediation of these effects by C-reactive protein (CRP), a first trimester peripheral marker of inflammation. Methods: Data from the prospective community-based ABCD-study cohort (n = 3547) was used to assess associations between self-reported continuous and categorized pBMI and outcome measures gestational hypertension (GH) and preeclampsia (PE), preterm birth (PTB) and small for gestational age (SGA) based on national perinatal registration linkage data. High-sensitivity CRP concentrations determined in serum were used to explore potential mediation of these associations by inflammation. Results: Multivariable logistic regression analyses, adjusted for confounders, showed that pBMI was significantly related to gestational hypertensive disorders (odds ratio (OR) per standard deviation (SD) 1.66, 95% confidence interval (CI) 1.51–1.83) and PTB (OR 1.20, 95% CI 1.05–1.37). Dose–response relationships between categorical pBMI and gestational hypertensive disorders (overweight OR 2.37, 95% CI 1.85–3.03 and obese OR 4.45, 95% CI 2.93–6.72) and PTB (obese OR 2.12, 95% CI 1.16–3.87) were found as well. SGA was only significantly more prevalent in the underweight BMI category (OR 2.06, 95% CI 1.33–3.19). Mediation analyses revealed small but significant indirect effects of pBMI on overall PTB (0.037, bootstrapped 95% CI 0.005–0.065) and spontaneous PTB (0.038, bootstrapped 95% CI 0.002–0.069) through higher CRP. CRP was not a significant mediator of associations between BMI and gestational hypertensive disorders although larger mediation was found for GH than for PE. Conclusion: Our findings provide additional evidence that high(er) pBMI increases the risk of adverse maternal and neonatal outcomes and that systemic inflammation mediates some of these risks. Further research in large cohorts including (morbidly) obese women is warranted to identify pathways that may be incorporated in future interventions to reduce the risk of adverse pregnancy outcomes due to maternal obesity

Studies on the role of SecA, FtsY and Tfh in the insertion of membrane proteins in Escherichia coli

The essential protein SecA plays a key role in post-translational protein secretion across the inner membrane of Escherichia coli. However, the extent of SecA-requirement in co-translational insertion of inner membrane proteins mediated by the signal recognition particle and its membrane-associated receptor FtsY is not clear. To inactivate SecA function a seven amino acid sequence that is specifically recognised by tobacco etch virus protease was introduced into the secA sequence. The resulting secA-constructs were integrated into the chromosome at the 1 attachment site via a specific lambda phage. Chromosomal wild-type secA was then eliminated by using the 1 Red recombinase system. Cleavage of nascent SecA 195 by a trigger factor-TEV protease hybrid protein resulted in effective inhibition of SecA activity. In addition, SecA830 under the control of the lacpromoter could be depleted in cells not supplemented with the inducer IPTG. Biotinylation assays showed that inactivation of SecA via site-specific proteolysis or depletion of SecA affected the proper secretion of the periplasmic protein alkaline phosphatase as well as the proper biogenesis of the inner membrane proteins FtsQ and MalF274. Similar experiments with Ffh- and FtsY-depletion strains confirmed the involvement of these proteins in inner membrane protein assembly. DNA macroarray technique was used to compare gene expression profiles of cells depleted either of Ffh, FtsY or SecA with the profiles obtained from undepleted cells. While depletion of Ffh and FtsY resulted in the induction of heat shock genes, depletion of SecA induced the expression of the phage-shock protein operon. The results strengthen the notion that SecA not only plays the central role in post-translational secretion of preproteins, but is also extensively involved in co-translational protein translocation. In addition, DNA array analysis indicated a possible involvement of cellular chaperones in targeting of inner membrane proteins.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Target‐directed proteolysis In vivo

The experimental problems associated with in vivo studies of essential proteins or integral membrane proteins have triggered geneticists to generate novel approaches that have often led to insights of general relevance (Shuman and Silhavy, 2003). In order to extend the experimental portfolio, we developed target‐directed proteolysis (TDP), an in vivo method allowing structural and functional characterization of target proteins in living cells. TDP is based on the activity of the highly sequence‐specific NIa protease from tobacco etch virus. When its recognition site of seven residues is engineered into target proteins and NIa protease is expressed under tight promoter control, substrates can be conditionally processed while other cellular proteins remain unaffected. Applications include conditional inactivation as well as functional characterization of target proteins

Associations Between Maternal Lipid Blood Levels at the 13th Week of Pregnancy and Offspring's Adiposity at Age 11-12 Years

CONTEXT: There is increasing evidence that intrauterine lipid metabolism influences the adiposity of the newborn and the first years thereafter. It remains unclear if these effects persist when these children grow older. OBJECTIVE: This study examined the associations between maternal lipid blood levels during the 13th week of pregnancy and an offspring's adiposity, measured at age 11-12, and if these associations were moderated by the child's sex. METHODS: Data were obtained from a community-based birth cohort, the Amsterdam Born Children and their Development (ABCD) study. At a median of 13 weeks' gestation, nonfasting blood samples of triglycerides (TGs), total cholesterol (TC), free fatty acids (FFAs), and apolipoprotein B/apolipoprotein A1 ratio (ApoB/ApoA1) were measured. An offspring's body mass index (BMI), subcutaneous fat (SCF), waist-to-height-ratio (WHtR), and fat percentage (fat%) were measured at age 11-12. Mothers with at-term born children were included (n = 1853). Multivariable linear regression analyses were performed to assess the associations between maternal lipids and each offspring's adiposity outcome separately. Sex differences were additionally evaluated. RESULTS: TGs, TC, ApoB/ApoA1, and FFAs were significantly positively associated with BMI, WHtR, and fat% (adjusted for gestational age at blood sampling, child's age, sex, and sexual maturation). After additional adjustments for potential confounders and covariates, only TGs remained significantly associated with WHtR (0.45, 95% CI -0.007; 0.91). There were no associations between maternal lipids and SCF and no clear sex-specific results were found. CONCLUSION: Overall, our results do not strongly support that maternal lipid profile during the 13th week of pregnancy has programming effects on adiposity in preadolescence

Associations Between Maternal Lipid Blood Levels at the 13th Week of Pregnancy and Offspring's Adiposity at Age 11-12 Years

CONTEXT: There is increasing evidence that intrauterine lipid metabolism influences the adiposity of the newborn and the first years thereafter. It remains unclear if these effects persist when these children grow older. OBJECTIVE: This study examined the associations between maternal lipid blood levels during the 13th week of pregnancy and an offspring's adiposity, measured at age 11-12, and if these associations were moderated by the child's sex. METHODS: Data were obtained from a community-based birth cohort, the Amsterdam Born Children and their Development (ABCD) study. At a median of 13 weeks' gestation, nonfasting blood samples of triglycerides (TGs), total cholesterol (TC), free fatty acids (FFAs), and apolipoprotein B/apolipoprotein A1 ratio (ApoB/ApoA1) were measured. An offspring's body mass index (BMI), subcutaneous fat (SCF), waist-to-height-ratio (WHtR), and fat percentage (fat%) were measured at age 11-12. Mothers with at-term born children were included (n = 1853). Multivariable linear regression analyses were performed to assess the associations between maternal lipids and each offspring's adiposity outcome separately. Sex differences were additionally evaluated. RESULTS: TGs, TC, ApoB/ApoA1, and FFAs were significantly positively associated with BMI, WHtR, and fat% (adjusted for gestational age at blood sampling, child's age, sex, and sexual maturation). After additional adjustments for potential confounders and covariates, only TGs remained significantly associated with WHtR (0.45, 95% CI -0.007; 0.91). There were no associations between maternal lipids and SCF and no clear sex-specific results were found. CONCLUSION: Overall, our results do not strongly support that maternal lipid profile during the 13th week of pregnancy has programming effects on adiposity in preadolescence

Can a Byte Improve Our Bite? An Analysis of Digital Twins in the Food Industry

The food industry faces many challenges, including the need to feed a growing population, food loss and waste, and inefficient production systems. To cope with those challenges, digital twins that create a digital representation of physical entities by integrating real-time and real-world data seem to be a promising approach. This paper aims to provide an overview of digital twin applications in the food industry and analyze their challenges and potentials. Therefore, a literature review is executed to examine digital twin applications in the food supply chain. The applications found are classified according to a taxonomy and key elements to implement digital twins are identified. Further, the challenges and potentials of digital twin applications in the food industry are discussed. The survey revealed that the application of digital twins mainly targets the production (agriculture) or the food processing stage. Nearly all applications are used for monitoring and many for prediction. However, only a small amount focuses on the integration in systems for autonomous control or providing recommendations to humans. The main challenges of implementing digital twins are combining multidisciplinary knowledge and providing enough data. Nevertheless, digital twins provide huge potentials, e.g., in determining food quality, traceability, or designing personalized foods

Mediating role of C-reactive protein in associations between pre-pregnancy BMI and adverse maternal and neonatal outcomes: the ABCD-study cohort

Objectives: Increased body mass index (BMI) is associated with several adverse pregnancy outcomes, though the underlying mechanism of this association has not been fully elucidated. A mediating role of low-grade systemic inflammation in these associations is suspected but has been understudied. Our objective was to examine the effect of pre-pregnancy BMI (pBMI) on maternal and neonatal pregnancy outcomes and to explore potential mediation of these effects by C-reactive protein (CRP), a first trimester peripheral marker of inflammation. Methods: Data from the prospective community-based ABCD-study cohort (n = 3547) was used to assess associations between self-reported continuous and categorized pBMI and outcome measures gestational hypertension (GH) and preeclampsia (PE), preterm birth (PTB) and small for gestational age (SGA) based on national perinatal registration linkage data. High-sensitivity CRP concentrations determined in serum were used to explore potential mediation of these associations by inflammation. Results: Multivariable logistic regression analyses, adjusted for confounders, showed that pBMI was significantly related to gestational hypertensive disorders (odds ratio (OR) per standard deviation (SD) 1.66, 95% confidence interval (CI) 1.51–1.83) and PTB (OR 1.20, 95% CI 1.05–1.37). Dose–response relationships between categorical pBMI and gestational hypertensive disorders (overweight OR 2.37, 95% CI 1.85–3.03 and obese OR 4.45, 95% CI 2.93–6.72) and PTB (obese OR 2.12, 95% CI 1.16–3.87) were found as well. SGA was only significantly more prevalent in the underweight BMI category (OR 2.06, 95% CI 1.33–3.19). Mediation analyses revealed small but significant indirect effects of pBMI on overall PTB (0.037, bootstrapped 95% CI 0.005–0.065) and spontaneous PTB (0.038, bootstrapped 95% CI 0.002–0.069) through higher CRP. CRP was not a significant mediator of associations between BMI and gestational hypertensive disorders although larger mediation was found for GH than for PE. Conclusion: Our findings provide additional evidence that high(er) pBMI increases the risk of adverse maternal and neonatal outcomes and that systemic inflammation mediates some of these risks. Further research in large cohorts including (morbidly) obese women is warranted to identify pathways that may be incorporated in future interventions to reduce the risk of adverse pregnancy outcomes due to maternal obesity

Target-directed proteolysis at the ribosome

Target directed proteolysis allows specific processing of proteins in vivo. This method uses tobacco etch virus (TEV) NIa protease that recognizes a seven-residue consensus sequence. Because of its specificity, proteins engineered to contain a cleavage site are proteolysed, whereas other proteins remain unaffected. Therefore, this approach can be used to study the structure and function of target proteins in their natural environment within living cells. One application is the conditional inactivation of essential proteins, which is based on the concept that a target containing a recognition site can be inactivated by coexpressed TEV protease. We have previously identified one site in the secretion factor SecA that tolerated a TEV protease site insert. Coexpression of TEV protease in the cytoplasm led to incomplete cleavage and a mild secretion defect. To improve the efficiency of proteolysis, TEV protease was attached to the ribosome. We show here that cleaving SecA under these conditions is one way of increasing the efficiency of target directed proteolysis. The implications of recruiting novel biological activities to ribosomes are discussed