Engineering allosteric inhibition of homoserine dehydrogenase by semi-rational saturation mutagenesis screening

Allosteric regulation by pathway products plays a vital role in amino acid metabolism. Homoserine dehydrogenase (HSD), the key enzyme for the biosynthesis of various aspartate family amino acids, is subject to feedback inhibition by l-threonine and l-isoleucine. The desensitized mutants with the potential for amino acid production remain limited. Herein, a semi-rational approach was proposed to relieve the feedback inhibition. HSD from Corynebacterium glutamicum (CgHSD) was first characterized as a homotetramer, and nine conservative sites at the tetramer interface were selected for saturation mutagenesis by structural simulations and sequence analysis. Then, we established a high-throughput screening (HTS) method based on resistance to l-threonine analog and successfully acquired two dominant mutants (I397V and A384D). Compared with the best-ever reported desensitized mutant G378E, both new mutants qualified the engineered strains with higher production of CgHSD-dependent amino acids. The mutant and wild-type enzymes were purified and assessed in the presence or absence of inhibitors. Both purified mutants maintained >90% activity with 10 mM l-threonine or 25 mM l-isoleucine. Moreover, they showed >50% higher specific activities than G378E without inhibitors. This work provides two competitive alternatives for constructing cell factories of CgHSD-related amino acids and derivatives. Moreover, the proposed approach can be applied to engineering other allosteric enzymes in the amino acid synthesis pathway

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Effects of Accumulated Damage on the Dynamic Properties of Coal Measures Sandstone

The coupling effect of accumulated damage and impact load substantially affects the integrity of the surrounding rock structure in deep coal mining engineering, which has inhibited safe and effective coal mining. Therefore, dynamic compression tests were performed on coal measures sandstone specimens with accumulated damage using the SHPB device. The effects of a high strain rate and accumulated damage on the sandstone’s mechanical behavior and damage evolution were investigated. The results reveal that accumulated damage has a considerable impact on specimen stress–strain curves and lowers dynamic compressive strength and deformation modulus substantially. The sandstone failure mode looks to be shear failure from a macroscopic perspective, while it appears to be intergranular fracture between the mineral particles from a microscopic perspective. The macroscopic and microscopic failure mechanisms of the sandstone specimens likewise conformed to the energy absorption law. The accumulated damage factor and the accumulated damage correction coefficient were presented in order to construct a statistical damage constitutive model of rocks based on the Weibull distribution. This model provides a decent description of the effects of accumulated damage and the strain rate on sandstone’s mechanical behavior, with parameters that are all of evident physical significance

Detecting DNA synthesis of neointimal formation after catheter balloon injury in GK and in Wistar rats: using 5-ethynyl-2'-deoxyuridine

<p>Abstract</p> <p>Background</p> <p>Neointimal formation plays an important role in the pathogenesis of coronary restenosis after percutaneous coronary intervention (PCI), especially in patients with diabetes mellitus. Recently, some studies have shown that 5-ethynyl-2'-deoxyuridine (EdU) incorporation can serve as a novel alternative to the 5-bromo-2'-deoxyuridine (BrdU) antibody detection method for detection of DNA synthesis in regenerating avian cochlea, chick embryo and the adult nervous system. However, few studies have been performed to assess the suitability of EdU for detecting DNA synthesis in vascular neointima.</p> <p>Methods</p> <p>The carotid artery balloon injury model was established in Goto-Kakizaki (GK) and Wistar rats. A Cell-Light^TM EdU Kit was used to detect EdU-labeled cell nuclei of common carotid arteries at day 7 after catheter balloon injury. Different methods of injecting EdU were tested. The protein levels of proliferating cell nuclear antigen (PCNA) and p-Akt (Ser473), as well as the mRNA levels of PCNA were evaluated by Western blotting and quantitative real-time PCR (qRT-PCR), respectively. Immunohistochemical staining was also employed to visualize PCNA-positive cells.</p> <p>Results</p> <p>At day 7 after catheter balloon injury, far more EdU-positive and PCNA-positive cells were observed in GK rats. When comparing groups that received different EdU doses, it was found that the percentage of EdU-positive cells at a dose of 100 mg/kg body weight was than at doses of 25 mg/kg and 50 mg/kg. The number of positive cells was significantly higher in the repeated injection group compared to the single injection group. Further, after balloon injury DNA synthesis in GK rats was more notable than in Wistar rats. Neointimal formation in GK rats was more obvious than in Wistar rats. The protein levels of PCNA and p-Akt (Ser473) and the mRNA levels of PCNA were increased in injured rats as compared to uninjured rats, and were significantly higher in GK rats than in Wistar rats.</p> <p>Conclusion</p> <p>By intraperitoneal injections of EdU at a dose of 100 mg/kg three times, EdU incorporation can detect carotid arterial DNA synthesis caused by neointimal formation in GK rats and Wistar rats at day 7 after balloon injury by the EdU click reaction quickly and effectively. Moreover, more obvious DNA synthesis in the vascular neointima could be observed in GK rats than in Wistar rats.</p

Dynamic Compressive Mechanical Property Characteristics and Fractal Dimension Applications of Coal-Bearing Mudstone at Real-Time Temperatures

Coal-bearing rocks are inevitably exposed to high temperatures and impacts (rapid dynamic load action) during deep-earth resource extraction, necessitating the study of their mechanical properties under such conditions. This paper reports on dynamic compression tests conducted on coal-bearing mudstone specimens at real-time temperatures (the temperature of the rock remains constant throughout the impact process) ranging from 25 °C to 400 °C using a temperature Hopkinson (T-SHPB) test apparatus developed in-house. The objective is to analyze the relationship between mechanical properties and the fractal dimension of fractured fragments and to explore the mechanical response of coal-bearing mudstone specimens to the combined effects of temperature and impact using macroscopic fracture characteristics. The study found that the peak stress and dynamic elastic modulus initially increased and then decreased with increasing temperature, increasing in the 25–150 °C range and monotonically decreasing in the 150–400 °C range. Based on the distribution coefficients and fractal dimensions of the fractured fragments, it was found that the degree of damage of coal-bearing mudstone shows a trend of an initial decrease and then an increase with increasing temperature. In the temperature range of 25–150 °C, the expansion of clay minerals within the mudstone filled the voids between the skeletal particles, resulting in densification and decreased damage. In the temperature range of 150–400 °C, thermal stresses increased the internal fractures and reduced the overall strength of the mudstone, resulting in increased damage. Negative correlations between fractal dimensions, the modulus of elasticity, and peak stress could be used to predict rock properties in engineering