Sequence homolog-based molecular engineering for shifting the enzymatic pH optimum

AbstractCell-free synthetic biology system organizes multiple enzymes (parts) from different sources to implement unnatural catalytic functions. Highly adaption between the catalytic parts is crucial for building up efficient artificial biosynthetic systems. Protein engineering is a powerful technology to tailor various enzymatic properties including catalytic efficiency, substrate specificity, temperature adaptation and even achieve new catalytic functions. However, altering enzymatic pH optimum still remains a challenging task. In this study, we proposed a novel sequence homolog-based protein engineering strategy for shifting the enzymatic pH optimum based on statistical analyses of sequence-function relationship data of enzyme family. By two statistical procedures, artificial neural networks (ANNs) and least absolute shrinkage and selection operator (Lasso), five amino acids in GH11 xylanase family were identified to be related to the evolution of enzymatic pH optimum. Site-directed mutagenesis of a thermophilic xylanase from Caldicellulosiruptor bescii revealed that four out of five mutations could alter the enzymatic pH optima toward acidic condition without compromising the catalytic activity and thermostability. Combination of the positive mutants resulted in the best mutant M31 that decreased its pH optimum for 1.5 units and showed increased catalytic activity at pH < 5.0 compared to the wild-type enzyme. Structure analysis revealed that all the mutations are distant from the active center, which may be difficult to be identified by conventional rational design strategy. Interestingly, the four mutation sites are clustered at a certain region of the enzyme, suggesting a potential “hot zone” for regulating the pH optima of xylanases. This study provides an efficient method of modulating enzymatic pH optima based on statistical sequence analyses, which can facilitate the design and optimization of suitable catalytic parts for the construction of complicated cell-free synthetic biology systems

Study on Damage Mechanism and Repair of Vascular Endothelial Cells Based on Ultrasound Technology

Objective. To detect the endothelial function of external iliac artery in rats with different stages of atherosclerosis by high-resolution ultrasound, so as to provide experimental methodological basis for evaluating the function of vascular endothelial cells by ultrasound. Methods. The animals were randomly divided into the control group (n=6) and the atherosclerosis model group (n=15). The atherosclerosis group was further divided into 4-week group, 8-week group, and 12-week group, with 5 animals in each group. After separating and grinding rat spleen, the obtained cells were cultured by density gradient centrifugation. After the cells adhered, the morphology of the cells was observed under a microscope and identified by DiI-Ac-LDL and FITC-UEA-I double staining. The activities of LDH and SOD, the contents of MDA and GSH, and the contents of NO in plasma were detected by biochemical methods. Results. The protective effect of rosanilin on brain injury in rats with acute hypobaric hypoxia and its regulation on the expression of pAkt protein; ox-LDL inhibited the proliferation activity of EPCs in a concentration-dependent manner. The expression of KLF2 and S1PR1 in HAEC can be knocked down by small interfering RNA, and knocking down KLF2 can not only downregulate the expression of S1PR1 but also downregulate HAVEN. With the development of atherosclerosis, the endothelium-dependent relaxation function and endothelium-independent relaxation function of the control group and the atherosclerosis model at 4, 8, and 12 weeks were damaged in different degrees and gradually aggravated. Conclusion. Atherosclerosis is a disease with both morphological and functional damage, and vascular endothelial function is damaged in the early stage with corresponding pathological changes. Ultrasound is an effective method to evaluate vascular endothelial function

Biomass Accumulation of Chlorella Zofingiensis G1 Cultures Grown Outdoors in Photobioreactors

The complicated and changeable weather conditions and pest invasion increase difficulties in outdoor microalgae cultivation. In this paper, outdoor microalgae cultivation experiments were investigated in Foshan city, South China. During June and August when the temperature is high, the cooling system-water spray combined shade tarpaulin can effectively reduce the temperature to 4–7°C in the 40 L vertical tubular outdoor photobioreactors. Under 300 mg/L urea and 200 mg/L ammonium bicarbonate conditions, Chlorella zofingiensis G1 had the largest biomass accumulation. Addition of acetic acid to adjust the pH of the medium can effectively improve the C. zofingiensis G1 biomass in the photobioreactor, which was four times more than that only through air ventilation conditions. The biomass accumulation of C. zofingiensis G1 was essentially unchanged during night time

Cultivation of Chlorella zofingiensis in bench-scale outdoor ponds by regulation of pH using dairy wastewater in winter, South China

Cultivation of Chlorella zofingiensis and nutrients removal in dairy wastewater were investigated in bench-scale outdoor ponds in winter, South China. The impacts of the two types of pH regulations, 5 similar to 6% CO2 and acetic acid (HAc) on this process were studied. After 6 days cultivation, the removal rates of total nitrogen (TN) and orthophosphate (PO43-) using CO2 regulation were better than those using HAc. The removal rates of PO43- and TN were 97.5% and 51.7%, respectively using CO2 regulation; 79.6% (TN) and 42.0% (PO43-) were obtained using HAc regulation. Higher biomass, protein, sugar content, and stable pH control were found using CO2 regulation. However, significantly higher lipid content (31.8%) was observed using HAc regulation. The dominant differences of fatty acids were the content of C18:1 and C18:3. The growth characteristics and environmental conditions especially during the typical logarithmic phase were also analyzed. (C) 2012 Elsevier Ltd. All rights reserved.</p

Culture of four microalgal strains for bioenergy production and nutrient removal in the meliorative municipal wastewater

Microalgae have been considered as the most promising sources of alternative bioenergy. For the purpose of saving costs, the present work focused on the potential use of microalgae in the meliorative municipal wastewater, which contains 90% municipal wastewater and 10% dairy wash wastewater. Four microalgal species, Palmellococcus miniatus, Neochloris oleoabundans, Scenedesmus quadricanda #507, and Chlorella zofingiensis, were cultured in pure municipal wastewater and meliorative municipal wastewater, respectively, for 5 days. Their biomass accumulation and removal rates of nitrogen and phosphate were measured. Results showed that the growth rates of Neochloris oleoabundans, Palmellococcus miniatus, and Chlorella zofingiensis in meliorative municipal wastewater (> 0.8 gL(-1)d(-1)) were significantly higher than that in municipal wastewater (2.6 gL(-1)d(-1)), while there was no significant difference between the growth rates of Scenedesmus quadricanda #507 in meliorative municipal wastewater and in municipal wastewater. Neochloris oleoabundans exhibited the highest growth rate (0.86 gL(-1)d(-1)) and relatively high nutrient removal capacity. Scenedesmus quadricanda #507 had the highest P removal rate of over 94%. The four species have a similar N removal rate at about 90%. The results showed that the highest average removal rate of N and P were about 23.1 mgL(-1)d(-1) and 7.1 mgL(-1)d(-1). Furthermore, the content of lipid or carbohydrates increased and a different profile of fatty acids were found compared to those in municipal wastewater. Cellular components changes of microalgae in meliorative municipal wastewater were favorable as raw materials for bioethanol and biodiesel production. Cultivation with meliorative municipal wastewater is a win-win culture mode that facilitates the biomass production, lipid and carbohydrate accumulation, and wastewater purification

Iterative Convolutional Encoder-Decoder Network with Multi-Scale Context Learning for Liver Segmentation

Rapid and accurate extraction of liver tissue from abdominal computed tomography (CT) and magnetic resonance (MR) images has critical importance for diagnosis and treatment of hepatic diseases. Due to adjacent organs with similar intensities and anatomical variations between different subjects, the performance of segmentation approaches based on deep learning still has room for improvement. In this study, a novel convolutional encoder-decoder network incorporating multi-scale context information is proposed. The probabilistic map from previous classifier is iteratively fed into the encoder layers, which fuses high-level shape context with low-level appearance features in a multi-scale manner. The dense connectivity is adopted to aggregate feature maps of varying scales from the encoder and decoder. We evaluated the proposed method with 2D and 3D application on abdominal CT and MR images of three public datasets. The proposed method generated liver segmentation with significantly higher accuracy (p <0.05), in comparison to several competing methods. These promising results suggest that the novel model could offer high potential for clinical workflow

Improved Electrochemical Performance of Li-Rich Cathode Materials via Spinel Li₂MoO₄ Coating

Li-rich manganese-based cathode materials (LRMs) are considered one of the most promising cathode materials for the next generation of lithium-ion batteries (LIBs) because of their high energy density. However, there are problems such as a capacity decay, poor rate performance, and continuous voltage drop, which seriously limit their large-scale commercial applications. In this work, Li1.2Mn0.54Co0.13Ni0.13O2 coated with Li2MoO4 with a unique spinel structure was prepared with the wet chemistry method and the subsequent calcination process. The Li2MoO4 coating layer with a spinel structure could provide a 3D Li+ transport channel, which is beneficial for improving rate performance, while protecting LRMs from electrolyte corrosion, suppressing interface side reactions, and improving cycling stability. The capacity retention rate of LRMs coated with 3 wt% Li2MoO4 increased from 69.25% to 81.85% after 100 cycles at 1 C, and the voltage attenuation decreased from 7.06 to 4.98 mV per cycle. The lower Rct also exhibited an improved rate performance. The results indicate that the Li2MoO4 coating effectively improves the cyclic stability and electrochemical performance of LRMs

Status and treatment of patients with uterine fibroids in hospitals in central China: a retrospective study from 2018 to 2021

Objective To evaluate the hospitalised patients with uterine fibroids (UFs) and describe treatment patterns in hospital-treated patients in central China from 2018 to 2021.Design A retrospective analysis.Setting The gynaecology departments of class A and class B secondary and tertiary hospitals in Hubei Province, China.Participants 101 008 patients diagnosed with UFs from 1 January 2018 to 31 December 2021.Results The hospitalised patients with UFs increased with age, reaching a peak at ages 45–49 years and then gradually decreasing. Among these patients, 19.05% had anaemia symptoms. Women aged 25–29 years were more likely to be treated with laparoscopic myomectomy (62.22%), while women aged 20–24 years tend to choose open myomectomy (34.58%). Women over age 45 years who had entered perimenopause tended to be treated with laparoscopic hysterectomy (64.85% for those aged 65–69 years). Patients with fibroid with moderate-to-severe anaemia mostly chose hysterectomy. As a whole, the proportion of patients who chose laparoscopic hysterectomy was similar to that of patients who chose laparoscopic myomectomy (31.38% vs 31.14%). Only 2.08% of UFs were treated with high-frequency MRI-guided focused ultrasound surgery (MRgFUS). The number of patients who choose laparoscopic surgery or MRgFUS treatment was increasing year by year. After stratifying by hospital grade, we found that women treated at class A tertiary hospitals were more likely to have laparoscopic than open surgery (66.12% vs 31.26%). At class B secondary hospitals, 61.9% of the patients underwent myomectomy. By contrast, hysterectomy was used to treat the majority of patients at class A secondary hospitals and class B tertiary hospitals (57.79% and 57.57%, respectively). Use of MRgFUS was mainly concentrated within class A tertiary hospitals.Conclusion UFs affect mainly women in childbearing period. Most patients chose to receive treatment at class A tertiary hospitals, among which laparoscopic myomectomy was the mainstream surgical method for patients in Hubei Province.Trial registration number NCT05840042