Role of TSP-1 and its receptor ITGB3 in the renal tubulointerstitial injury of focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS), a common cause of primary glomerulonephritis, has a poor prognosis and is pathologically featured by tubulointerstitial injury. Thrombospondin-1 (TSP-1) is an extracellular matrix protein that acts in combination with different receptors in the kidney. Here, we analyzed the tubular expression of TSP-1 and its receptor integrin ß3 (ITGB3) in FSGS. Previously the renal interstitial chip analysis of FSGS patients with tubular interstitial injury showed that the expressions of TSP-1 and ITGB3 were up-regulated. We found that the level of TSP-1 and ITGB3 increased in the tubular cells of FSGS patients. The serum level of TSP-1 increased and was correlated to the degree of tubulointerstitial lesions in FSGS patients. THBS1/ITGB3 signaling induced renal tubular injury in HK-2 cells exposure to BSA and the ADR-induced nephropathy model. THBS1 knockout ameliorated tubular injury and renal fibrosis in ADR-treated mice. THBS1 knockdown decreased the expression of KIM-1 and caspase 3 in the HK-2 cells treated with BSA, while THBS1 overexpression could induce tubular injury. In vivo, we identified cyclo-RGDfK as an agent to block the binding of TSP-1 to ITGB3. Cyclo-RGDfK treatment could alleviate ADR-induced renal tubular injury and interstitial fibrosis in mice. Moreover, TSP-1 and ITGB3 were colocalized in tubular cells of FSGS patients and ADR-treated mice. Taken together, our data showed that TSP-1/ITGB3 signaling contributed to the development of renal tubulointerstitial injury in FSGS, potentially identifying a new therapeutic target for FSGS.

The predictive value of peripheral blood CD4 cells ATP concentration for immune-related adverse events in advanced non-small cell lung cancer patients.

OBJECTIVE: Lung cancer with the highest incidence and mortality in the world. Immune checkpoint inhibitors (ICIs), can bring long-term survival benefits to patients, but also can bring immune-related adverse events (irAEs) in some patients during therapy. Therefore, the aim of this study was to investigate the predictive effect of peripheral blood WBC, NLR, sATPCD4 and nATPCD4 on irAEs in advanced non-small cell lung cancer (NSCLC). METHODS: Clinical data of 112 patients with advanced NSCLC who were treated with PD -1/PD -L1 inhibitor in the Fifth Affiliated Hospital of Guangzhou Medical University from December 15, 2019 to April 30, 2023 were retrospectively analyzed. These patients were divided into the irAEs group (n = 27) and non-irAEs group (n = 85). The clinical data of the two groups were compared. Receiver operating characteristic (ROC) curves were drawn to determine the threshold value of baseline peripheral blood parameters to predict the occurrence of irAEs. Multivariate logistic regression analysis was used to explore the relationship between peripheral blood markers and the incidence of irAEs. RESULTS: The patient characteristics have no significant difference between irAEs and non-irAEs group. But the baseline peripheral blood WBC, sATPCD4 and nATPCD4 of patients in the irAEs group were higher than those in the non-irAEs group (p < 0.05), and the NLR in irAEs group was similar to in the non-irAEs group (p = 0.639).Univariate analysis showed that high WBC, sATPCD4 and nATPCD4 may the risk factors for the occurrence of irAEs (p < 0.05). Multivariate logistic regression analysis showed that high sATPCD4 and nATPCD4 were independent risk factors for the occurrence of irAEs (p < 0.05). The best critical values of WBC, sATPCD4 and nATPCD4 before treatment for predicting the occurrence of irAEs were 8.165 × 109cells/L (AUC = 0.705) ,484.5 ng/mL (AUC = 0.777), and 156 ng/mL (AUC = 0.840), respectively. CONCLUSIONS: sATPCD4 and nATPCD4 were independent risk factors for the occurrence of irAEs in advanced NSCLC patients. This discovery provides a new method to predict the occurrence of irAEs in patients. Based on the prediction results, corresponding treatment measures can be taken to reduce the incidence of adverse events.

Development of a gene-coded biosensor to establish a high-throughput screening platform for salidroside production.

Metabolic engineering reconfigures cellular networks to produce value-added compounds from renewable substrates efficiently. However, identifying strains with desired phenotypes from large libraries through rational or random mutagenesis remains challenging. To overcome this bottleneck, an effective high-throughput screening (HTS) method must be developed to detect and analyze target candidates rapidly. Salidroside is an aromatic compound with broad applications in food, healthcare, medicine, and daily chemicals. However, there currently needs to be HTS methods available to monitor salidroside levels or to screen enzyme variants and strains for high-yield salidroside biosynthesis, which severely limits the development of microbial cell factories capable of efficiently producing salidroside on an industrial scale. This study developed a gene-encoded whole-cell biosensor that is specifically responsive to salidroside. The biosensor was created by screening a site-saturated mutagenic library of uric acid response regulatory protein binding bags. This work demonstrates the feasibility of monitoring metabolic flux with whole-cell biosensors for critical metabolites. It provides a promising tool for building salidroside high-yielding strains for high-throughput screening and metabolic regulation to meet industrial needs.

Metabolic engineering of Candida tropicalis for efficient 1,2,4-butanetriol production.

1,2,4-Butanetriol serves as a precursor in the manufacture of diverse pharmaceuticals and the energetic plasticizer 1,2,4-butanetriol trinitrate. The study involved further modifications to an engineered Candida tropicalis strain, aimed at improving the production efficiency of 1,2,4-butanetriol. Faced with the issue of xylonate accumulation due to the low activity of heterologous xylonate dehydratase, we modulated iron metabolism at the transcriptional level to boost intracellular iron ion availability, thus enhancing the enzyme activity by 2.2-fold. Addressing the NADPH shortfall encountered during 1,2,4-butanetriol biosynthesis, we overexpressed pivotal genes in the NADPH regeneration pathway, achieving a 1,2,4-butanetriol yield of 3.2 g/L. The introduction of calcium carbonate to maintain pH balance led to an increased yield of 4 g/L, marking a 111% improvement over the baseline strain. Finally, the use of corncob hydrolysate as a substrate culminated in 1,2,4-butanetriol production of 3.42 g/L, thereby identifying a novel host for the conversion of corncob hydrolysate to 1,2,4-butanetriol.

A retrospective study of chemotherapeutic effect without wide-margin surgery or radiation therapy in dogs with oral malignant melanoma.

Background: Effective treatment for canine oral malignant melanoma (e.g., curative-intent surgery) may not be feasible or radiation therapy may be unavailable. However, chemotherapy is usually an option, and more information is needed regarding its use without adequate local treatments. Objective: Our objective was to investigate the efficacy of chemotherapy in canine oral malignant melanoma without adequate local control, using carboplatin with dose reduction in small-breed dogs and metronomic chemotherapy. Animals and procedure: Client-owned dogs with histopathologically diagnosed oral malignant melanoma were retrospectively enrolled from 2016 to 2022. The chemotherapy protocol in each case was determined by the attending clinician. Results: Thirteen dogs were included. The median progression-free interval of all 13 dogs was 42 d (14 to 953 d). The median overall survival time of dogs with chemotherapy as their only systemic treatment was 181 d (50 to 960 d; n = 11). The median dosage of carboplatin was 250 mg/m2. Response to treatment and clinical stage were significant prognostic factors. Conclusion and clinical relevance: As chemotherapy provided a median survival of 6 mo, it could be considered when adequate local control is infeasible. Earlier clinical stages or achievement of at least stable disease during chemotherapy may indicate better survival in dogs.

Une étude rétrospective de l'effet chimiothérapeutique sur le mélanome malin buccal canin dépourvu de chirurgie et de radiothérapie á large marge : le stade clinique et la réponse au traitement prédisent les résultats du patient. Mise en contexte: Des traitements efficaces pour le mélanome malin oral canin, tels que la chirurgie á visée curative, ne sont parfois pas réalisables ou la radiothérapie n'est pas disponible dans certaines régions. La chimiothérapie reste une option de traitement et davantage d'informations devraient être fournies pour les cas qui n'ont pas eu accés á un traitement local adéquat. Objectif: Cette étude visait á étudier l'efficacité de la chimiothérapie dans le mélanome malin oral canin sans contrôle local adéquat, en utilisant le carboplatine avec réduction de dose chez les chiens de petite race et la chimiothérapie métronomique. Animaux et procédure: Treize chiens appartenant á des clients atteints d'un mélanome malin oral diagnostiqué par histopathologie ont été rétrospectivement inscrits de 2016 á 2022. Le protocole de chimiothérapie a été déterminé par le clinicien traitant. Résultats: L'intervalle médian sans progression des treize chiens était de 42 jours (14­953 jours). La durée médiane de survie globale des chiens ayant reçu une chimiothérapie comme seul traitement systémique était de 181 jours (50­960 jours; n = 11). La dose médiane de carboplatine était de 250 mg/m2. La réponse au traitement et le stade clinique étaient des facteurs pronostiques importants. Conclusion et pertinence clinique: La chimiothérapie pouvait encore être envisagée lorsqu'un contrôle local adéquat était impossible. Des stades cliniques plus précoces ou des patients atteignant au moins une maladie stable pendant la chimiothérapie peuvent indiquer une meilleure survie.(Traduit par les auteurs).

Efficient production of hydroxytyrosol by directed evolution of HpaB in Escherichia coli.

Hydroxytyrosol (HT) is an olive-derived phenolic phytochemical that has gained increasing commercial interest due to its natural antioxidant properties. It is widely used in the field of food supplement and medicine. It is reported that 4-hydroxyphenylacetate 3-hydroxylase (EcHpaB) and flavin reductase (EcHpaC) from E. coli BL21(DE3) can successfully express and catalyze the production of HT from tyrosol. In this study, the tyrosol production strain YMG5∗R as chassis cells, and a random mutant library of EcHpaB was established using error-prone PCR to improve the ability of EcHpaB to convert tyrosol to HT. Finally, a highly efficient HT synthetic mutant strainYMG5∗R-HpaBTLEHC with high transformation efficiency was screened by directed evolution. The YMG5∗R-HpaBTLEHC strain efficiently converted 50 mM tyrosol, with a yield of hydroxytyrosol reaching 48.2 mM (7.43 g/L) and a space-time yield reached 0.62 g/L·h. Overall, our study demonstrates the successful development of a highly efficient synthetic enzyme mutant for the production of HT, which has the potential to significantly improve the commercial viability of this natural antioxidant.

Efficient production of cembratriene-ol in Escherichia coli via systematic optimization.

BACKGROUND: The tobacco leaf-derived cembratriene-ol exhibits anti-insect effects, but its content in plants is scarce. Cembratriene-ol is difficult and inefficiently chemically synthesised due to its complex structure. Moreover, the titer of reported recombinant hosts producing cembratriene-ol was low and cannot be applied to industrial production. RESULTS: In this study, Pantoea ananatis geranylgeranyl diphosphate synthase (CrtE) and Nicotiana tabacum cembratriene-ol synthase (CBTS) were heterologously expressed to synthsize the cembratriene-ol in Escherichia coli. Overexpression of cbts*, the 1-deoxy-D-xylulose 5-phosphate synthase gene dxs, and isopentenyl diphosphate isomerase gene idi promoted the production of cembratriene-ol. The cembratriene-ol titer was 1.53-folds higher than that of E. coli Z17 due to the systematic regulation of ggpps, cbts*, dxs, and idi expression. The production of cembratriene-ol was boosted via the overexpression of genes ispA, ispD, and ispF. The production level of cembratriene-ol in the optimal medium at 72 h was 8.55-folds higher than that before fermentation optimisation. The cembratriene-ol titer in the 15-L fermenter reached 371.2 mg L- 1, which was the highest titer reported. CONCLUSION: In this study, the production of cembratriene-ol in E. coli was significantly enhanced via systematic optimization. It was suggested that the recombinant E. coli producing cembratriene-ol constructed in this study has potential for industrial production and applications.

Research progress and application of enzymatic synthesis of glycosyl compounds.

Glucoside compounds are widely found in nature and have garnered significant attention in the medical, cosmetics, and food industries due to their diverse pharmaceutical properties, biological activities, and stable application characteristics. Glycosides are mainly obtained by direct extraction from plants, chemical synthesis, and enzymatic synthesis. Given the challenges associated with plant extraction, such as low conversion rates and the potential for environmental pollution with chemical synthesis, our review focuses on enzymatic synthesis. Here, we reviewed the enzymatic synthesis methods of 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G), 2-O-α-D-glucosyl glycerol (α-GG), arbutin and α-glucosyl hesperidin (Hsp-G), and other glucoside compounds. The types of enzymes selected in the synthesis process are comprehensively analyzed and summarized, as well as a series of enzyme transformation strategies adopted to improve the synthetic yield. KEY POINTS: • Glycosyl compounds have applications in the biomedical and food industries. • Enzymatic synthesis converts substrates into products using enzymes as catalysts. • Substrate bias and specificity are key to improving substrate conversion.

Clinical effects of Special Pressure Ulcer Intervention Combined with Gel Positioning Pad Intervention on Preventing Acute Stress Injury in Patients Undergoing Long-Term Lateral Position Spinal Surgery.

Objective: Prevention of acute pressure injuries (PS) is critical in patients undergoing certain surgeries. This type of pressure injury can develop during and after surgery, causing unnecessary pain and complications for the patient. However, preventing PS in these high-risk patients may present some challenges and require specific nursing measures. To explore the clinical effects of special pressure ulcer intervention combined with gel positioning pad intervention on the prevention of acute stress injury in patients undergoing long-term lateral position spinal surgery. Methods: The simple randomization method was used in this study; 100 patients with lateral position spinal surgery from March 2022 to March 2023 were selected as research subjects and were divided into an observation group and control group with 50 cases in each group by the random number table method. The control group was given routine intervention, and the observation group carried out special pressure ulcer intervention and gel positioning pad intervention. Special pressure ulcer intervention was performed, using appropriate surface support to relieve pressure, keeping the patient's skin clean and dry, and turning regularly to relieve pressure. In addition, we use a gel positioning pad intervention to disperse pressure, improve local blood circulation, and reduce the risk of pressure injuries. The occurrence of acute stress injury, VAS scores at different time points after surgery, local skin infrared thermography analysis results at 72 hours after surgery, incidence rates of complications and nursing satisfaction were compared between the two groups of patients. Results: The incidence rates of acute stress injury during surgery, within 2 hours after surgery and within 72 hours after surgery in the observation group were lower than those in the control group (P < .0046). The number and area of injury in the observation group were smaller than those in the control group (P < .0037). The National Pressure Ulcer Advisory Panel (NPUAP) grading of acute stress injury in the observation group was lower compared with that in the control group (P < .0021). The pain VAS scores in the observation group at 2 hours, 24 hours and 72 hours after surgery were lower than those in the control group (P < .001). The local skin infrared thermography analysis temperature values of the neck, shoulder, hip, knee and ankle were lower in the observation group than those in the control group at 72 hours after surgery (P < .001). The incidence rates of postoperative lumbago and shoulder-neck pain in the observation group were lower than those in the control group (P < .001). The scores of three aspects of nursing technology and nursing operation satisfaction, service attitude and humanistic care satisfaction, and nursing environment satisfaction were higher in the observation group than compared to the control group (P < .001). The findings of this study highlight the importance of specific pressure ulcer interventions that can be widely used in clinical practice and have the potential to reduce the incidence of pressure injuries and improve patient satisfaction with care. Conclusion: Special pressure ulcer intervention combined with gel positioning pad intervention has a good preventive effect on acute stress injury in patients undergoing long-term lateral position spinal surgery. Limitations of this study include the small sample size and single study institution, which may affect the external validity of the study. In addition, data collection in this study was limited to a specific time period and does not reflect long-term outcomes. Future studies could consider multi-center, broader samples, and longer follow-up to confirm the benefits of these interventions and to investigate in depth the long-term rehabilitation and quality of life of patients.

CRISPR-Cas Technology for Bioengineering Conventional and Non-Conventional Yeasts: Progress and New Challenges.

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (CRISPR-Cas) system has undergone substantial and transformative progress. Simultaneously, a spectrum of derivative technologies has emerged, spanning both conventional and non-conventional yeast strains. Non-conventional yeasts, distinguished by their robust metabolic pathways, formidable resilience against diverse stressors, and distinctive regulatory mechanisms, have emerged as a highly promising alternative for diverse industrial applications. This comprehensive review serves to encapsulate the prevailing gene editing methodologies and their associated applications within the traditional industrial microorganism, Saccharomyces cerevisiae. Additionally, it delineates the current panorama of non-conventional yeast strains, accentuating their latent potential in the realm of industrial and biotechnological utilization. Within this discourse, we also contemplate the potential value these tools offer alongside the attendant challenges they pose.

Efficient Biosynthesis of Acidic/Lactonic Sophorolipids and Their Application in the Remediation of Cyanobacterial Harmful Algal Blooms.

Cyanobacterial harmful algal blooms (CyanoHABs) pose significant threats to human health and natural ecosystems worldwide, primarily caused by water eutrophication, increased surface water temperature, and co-occurring microorganisms. Urgent action is needed to develop an eco-friendly solution to effectively curb the proliferation of CyanoHABs. Sophorolipids (SLs) are fully biodegradable biosurfactants synthesized by Starmerella bombicola. They can be classified into lactone and acid types. The lactone type displays strong antimicrobial activity, while the acid type exhibits good solubility, which make them ideal agents for mitigating CyanoHABs. Nevertheless, the broad utilization of SLs are hindered by their expensive production costs and the absence of effective genetic editing tools in the native host. In this study, we constructed recombinant strains capable of producing either acidic or lactonic SLs using the CRISPR-Cas9 gene editing system. The yields of acidic and lactonic SLs reached 53.64 g/L and 45.32 g/L in a shaking flask, respectively. In a 5 L fermenter, acidic SLs reached 129.7 g/L using low-cost glucose and rapeseed oil as substrates. The addition of 5 mg/L lactonic SLs effectively degraded cyanobacteria within 30 min, and a ratio of 8.25:1.75 of lactonic to acidic SLs showed the highest degradation efficiency. This study offers a safe and promising solution for CyanoHABs treatment.

Krüppel-like Factor 15 Suppresses Ferroptosis by Activating an NRF2/GPX4 Signal to Protect against Folic Acid-Induced Acute Kidney Injury.

Acute kidney injury (AKI) is a common and serious disease with high morbidity and mortality, and its pathophysiological mechanisms are not fully understood. Increasing evidence suggests an important role of ferroptosis in AKI. Krüppel-like factor 15 (KLF15) is a transcription factor involved in several metabolic diseases, but its role in AKI and ferroptosis remains unclear. In this study, we explored the potential role of KLF15 using a folic acid-induced AKI model. Our study showed that KLF15 expression was reduced in kidney tissues of AKI mice, and KLF15 knockout exacerbated folic acid-induced ferroptosis and kidney injury. In vitro studies revealed that the ferroptosis inducer erastin significantly suppressed KLF15 expression in human tubular epithelial cells. Notably, the overexpression of KLF15 attenuated ferroptosis, as evidenced by a decrease in the lipid peroxidation marker of malondialdehyde and the upregulation of glutathione peroxidase 4 (GPX4), while KLF15 knockdown with shRNA exerted the opposite effect. Mechanistically, KLF15 stabilized the protein of nuclear factor erythroid 2-related factor 2 (NRF2) and subsequently increased the GPX4 level. Collectively, KLF15 plays an important role in the modulation of ferroptosis in AKI and may be a potential therapeutic target for treating AKI.

Risk factors of postoperative neurodevelopmental abnormalities in neonates with critical congenital heart disease. / å±é‡å ˆå¤©æ€§å¿ƒè„ç— æ–°ç”Ÿå„¿æœ¯åŽæ–°å‘ç¥žç»å‘è‚²å¼‚å¸¸çš„å±é™©å› ç´ .

OBJECTIVES: To investigate the risk factors of postoperative neuro-developmental abnormalities in neonates with critical congenital heart disease (CCHD). METHODS: Clinical data of 50 neonates with CCHD admitted in the Cardiac Intensive Care Unit, The Children's Hospital, Zhejiang University School of Medicine from November 2020 to December 2021 were retrospectively analyzed. Neurological assessment was performed with cranial ultrasonography, CT/MRI, video electroencephalogram and clinical symptoms before and after surgical treatment for all patients, and neurodevelopmental abnormalities were documented. Binary logistic stepwise regression was used to analyze risk factors of postoperative new-onset neurodysplasia in children with CCHD, and the predictive value of the risk factors on postoperative neurodevelopmental abnormalities were evaluated using the receiver operating characteristic (ROC) curve. RESULTS: Neurodevelopmental abnormalities were detected in 22 cases (44.0%) and not detected in 28 cases (56.0%) before surgery. There were no significant differences in gender, birth weight, age at admission, gestational age, preoperative SpO2 level, prematurity, cyanotic congenital heart disease, and ventilator support between the two groups (all P>0.05). After surgery, there were 22 cases (44.0%) with new-onset neurological abnormalities and 28 cases (56.0%) without new-onset abnormalities. Multivariate logistic regression analysis showed that postoperative 24 h peak lactic acid (OR=1.537, 95%CI: 1.170-2.018, P<0.01) and postoperative length of ICU stay (OR=1.172, 95%CI:1.031-1.333, P<0.05) were independent risk factors for postoperative new-onset neurodevelopmental abnormalities. The area under ROC curve (AUC) of the postoperative 24 h peak lactic acid for predicting the new-onset neurological abnormalities after operation was 0.829, with cut-off value of 4.95 mmol/L. The diagnostic sensitivity and specificity were 90.0% and 64.3%, respectively. The AUC of postoperative length of ICU stay for predicting the new-onset neurological abnormalities after operation was 0.712, with cut-off value of 18.0 d. The diagnostic sensitivity and specificity were 50.0% and 96.4%, respectively. The AUC of the combination of the two indicators was 0.917, the diagnostic sensitivity and specificity were 95.5% and 64.3%, respectively. CONCLUSIONS: The incidence of neurodysplasia in neonatal CCHD is high, and new neurological abnormalities may occur after surgery. The postoperative 24 h peak lactic acid and postoperative length of ICU stay are risk factors for new-onset neurodysplasia after surgery. The combination of the two indicators has good predictive value for neurodevelopmental outcomes after surgery in CCHD infants.

Discovery of the ATPase Activity of a Cobalt-Type Nitrile Hydratase Activator and Its Promoting Effect on Enzyme Maturation.

An activator protein and a metal ion are two factors known to be indispensable for the maturation of nitrile hydratase (NHase). Here, the third key factor, adenosine triphosphate (ATP), was identified to play an important role in the activation of Co-type NHase. Free phosphate measurements revealed that the Co-type activator protein can hydrolyze ATP/GTP with appreciable performance and that such catalytic performance is related to NHase activity. Computational analysis and site-directed mutagenesis identified several potential hot spot residues involved in the binding of ATP to Co-type activator protein, and an E60A/W61A/D62A/I139A/T141A combinatorial variant reduced the ATPase activity to 18% of its original level. Further NHase activation studies using the combinatorial variant demonstrated that although the ATPase activity of the Co-type activator protein correlated with NHase activity, a low ATP concentration of 0.5 mmol/L was optimal for NHase activation, with higher ATP concentrations potentially inhibiting NHase activity.

Application of electrodeposited Cu-metal nanoflake structures as 3D current collector in lithium-metal batteries.

Since uncontrolled lithium (Li) dendrite growth and dendrite-induced dead Li severely limit the development of Li metal batteries, 3D Cu current collectors can effectively alleviate these problems during Li plating/stripping. Herein, one-step galvanostatic electrodeposition method is employed to fabricate a new current collector on Cu foam decorated with large-scale and uniform 3D porous Cu-based nanoflake (NF) structures (abbreviated as 3D Cu NF@Cu foam). This 3D structure with large internal surface areas not only generates lithophilic surface copper oxides and hydroxides as charge centers and nucleation sites for Li insertion/extraction, but also endows abundant space with interlinked NFs for buffering the cell volume expansion and increasing battery performance. As a result, Li-deposited 3D Cu NF@Cu foam current collector can realize stable cycling over 455 cycles with an average Coulombic efficiency of 98.8% at a current density of 1.0 mA cm-2, as well as a prolonged lifespan of >380 cycles in symmetrical cell without short-circuit, which are superior to those of blank Cu foam current collector. This work realizes Li metal anode stabilization by constructing 3D porous Cu NFs current collectors, which can advance the development of Li metal anode for battery industries.

Efficient extracellular production of recombinant proteins in E. coli via enhancing expression of dacA on the genome.

D, D-carboxypeptidase DacA plays an important role in the synthesis and stabilization of Escherichia coli cell wall peptidoglycan. The production level of extracellular recombinant proteins in E. coli can be enhanced by high D, D-carboxypeptidase activity. Construction of expression systems under optimal promoters is one of the main strategies to realize high protein production in E. coli. In this study, the promoter PdacA-3 from DacA on the genome of E. coli BL21 (DE3) was verified to be efficient for recombinant green fluorescent protein using the plasmid mutant pET28a-PdacA with PdacA-3. Meanwhile, the promoter PdacA-3 was engineered to increase the production level of proteins via inserting one or two Shine-Dalgarno (SD) sequences between the promoter PdacA-3 and the target genes. The expression level of dacA on the genome was increased by the improved transcription of the engineered promoters (especially after inserting one additional SD sequence). The engineered promoters increased cell membrane permeabilities to significantly enhance the secretion production of extracellular recombinant proteins in E. coli. Among them, the extracellular recombinant amylase activities in E. coli BL21::1SD-pET28a-amyK and E. coli BL21::2SD-pET28a-amyK were increased by 2.0- and 1.6-fold that of the control (E. coli BL21-pET28a-amyK), respectively. Promoter engineering also affected the morphology and growth of the E. coli mutants. It was indicated that the engineered promoters enhanced the expression of dacA on the genome to disturb the synthesis and structural stability of cell wall peptidoglycans.

Focal Segmental Glomerulosclerosis Superimposed on Transplant Glomerulopathy: Implications for Graft Survival.

INTRODUCTION: Transplant glomerulopathy (TG) is a morphological lesion resulting from chronic glomerular endothelium injury, and it is strongly associated with poor graft survival. TG coexisting with focal segmental glomerulosclerosis (FSGS) can be found in renal allograft biopsies, but few related studies are available. METHODS: Consecutive kidney transplant recipients with biopsy-proven TG were studied retrospectively. Patients concomitant with FSGS were identified and compared with those without FSGS. The influence of FSGS on allograft outcomes was assessed using univariate and multivariate Cox regression models. RESULTS: Of the 66 patients with TG, 40 (60.6%) had concomitant FSGS. TG patients with FSGS had higher proteinuria (median, 2.6 vs. 0.8 g/24 h, p < 0.001) and serum creatinine levels (median, 2.5 vs. 2.1 mg/dL, p = 0.04), lower serum albumin levels, higher chronic glomerulopathy (cg) score, larger glomerular tuft area, lower number of podocytes, and higher incidences of podocyte hyperplasia, pseudotubule formation, and diffuse foot process effacement than those without FSGS (all p < 0.05). The kidney allograft loss rate of patients with FSGS was higher than that of patients without FSGS (65.7% vs. 37.5%, p = 0.03). The presence of FSGS was independently associated with allograft loss in TG (hazard ratio (HR) = 3.42, 95% confidence interval (CI): 1.30-8.98, p = 0.01). Other independent predictors were proteinuria (HR = 1.18, 95% CI: 1.02-1.37, p = 0.02), estimated glomerular filtration rate (HR = 0.94, 95% CI: 0.91-0.97, p < 0.001), and panel reactive antibody (HR = 3.99, 95% CI: 1.14-13.99, p = 0.03). Moreover, FSGS (odds ratio (OR) = 4.39, 95% CI: 1.29-14.92, p = 0.02) and cg (OR = 5.36, 95% CI: 1.56-18.40, p = 0.01) were independent risk factors for proteinuria. CONCLUSION: In this cohort of patients with TG, the presence of FSGS was strongly associated with more severe clinicopathological features and worse allograft survival.

Development of thermostable sucrose phosphorylase by semi-rational design for efficient biosynthesis of alpha-D-glucosylglycerol.

Sucrose phosphorylase (SPase) can specifically catalyze transglycosylation reactions and can be used to enzymatically synthesize α-D-glycosides. However, the low thermostability of SPase has been a bottleneck for its industrial application. In this study, a SPase gene from Leuconostoc mesenteroides ATCC 12,291 (LmSPase) was synthesized with optimized codons and overexpressed successfully in Escherichia coli. A semi-rational design strategy that combined the FireProt (a web server designing thermostable proteins), structure-function analysis, and molecular dynamic simulations was used to improve the thermostability of LmSPase. Finally, one single-point mutation T219L and a combination mutation I31F/T219L/T263L/S360A (Mut4) with improved thermostability were obtained. The half-lives at 50 °C of T219L and Mut4 both increased approximately two-fold compared to that of wild-type LmSPase (WT). Furthermore, the two variants T219L and Mut4 were used to produce α-D-glucosylglycerol (αGG) from sucrose and glycerol by incubating with 40 U/mL crude extracts at 37 °C for 60 h and achieved the product concentration of 193.2 ± 12.9 g/L and 195.8 ± 13.1 g/L, respectively, which were approximately 1.3-fold higher than that of WT (150.4 ± 10.0 g/L). This study provides an effective strategy for improving the thermostability of an industrial enzyme. KEY POINTS: • Predicted potential hotspot residues directing the thermostability of LmSPase by semi-rational design • Screened two positive variants with higher thermostability and higher activity • Synthesized α-D-glucosylglycerol to a high level by two screened positive variants.

Microarray profile analysis identifies ETS1 as potential biomarker regulated by miR-23b and modulates TCF4 in gastric cancer.

BACKGROUND: Gastric cancer (GC), a common malignancy of the human digestive system, represents the second leading cause of cancer-related deaths worldwide. Early detection of GC has a significant impact on clinical outcomes. The aim of this study was to identify potential GC biomarkers. METHODS: In this study, we conducted a multi-step analysis of expression profiles in GC clinical samples downloaded from TCGA database to identify differentially expressed miRNAs (DEMs) and differentially expressed mRNAs (DEGs). Potential prognostic biomarkers from the available DEMs were then established using the Cox regression method. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to investigate the biological role of the predicted target genes of the miRNA biomarkers. Then, the prognostic DEM-mediated regulatory network was constructed based on transcription factor (TF)-miRNA-target interaction. Subsequently, the consensus genes were further determined based on the overlap between DEGs and these target genes of DEMs. Besides, expression profile, co-expression analysis, immunity, and prognostic values of these prognostic genes were also investigated to further explore the roles in the mechanism of GC tumorigenesis. RESULTS: We got five miRNAs, including miR-23b, miR-100, miR-143, miR-145, and miR-409, which are associated with the overall survival of GC patients. Subsequently, enrichment analysis of the target genes of the miRNA biomarkers shown that the GO biological process terms were mainly enriched in mRNA catabolic process, nuclear chromatin, and RNA binding. In addition, the KEGG pathways were significantly enriched in fatty acid metabolism, extracellular matrix (ECM) receptor interaction, and proteoglycans in cancer pathways. The transcriptional regulatory network consisting of 68 TFs, 4 DEMs, and 58 targets was constructed based on the interaction of TFs, miRNAs, and targets. The downstream gene ETS1 of miR-23b and TCF4 regulated by ETS1 were obtained by the regulatory network construction and co-expression analysis. High expression of ETS1 and TCF4 indicated poor prognosis in GC patients, particularly in the advanced stages. The expression of ETS1 and TCF4 was correlated with CD4+ T cells, CD8+ T cells, and B cells. CONCLUSIONS: miR-23b, ETS1, and TCF4 were identified as the prognostic biomarkers. ETS1 and TCF4 had potential immune function in GC, which provided a theoretical basis for molecular-targeted combined immunotherapy in the future.

In vitro metabolism and in vivo pharmacokinetics of bentysrepinine (Y101), an investigational new drug for anti-HBV-infected hepatitis: focus on interspecies comparison.

The objective of this study was to clarify the species differences of pharmacokinetics of Y101 (N-[N-benzoyl-O-(2-dimethylaminoethyl)-l-tyrosyl]-l-phenylalaninol hydrochloride), a derivative of herbal ingredient with anti-HBV hepatitis activity, in rats, dogs, monkeys and humans.The metabolic stability and metabolite identification studies using liver microsomes in vitro, plasma protein binding using a rapid equilibrium dialysis in vitro, pharmacokinetic studies in vivo were carried out to evaluate the interspecies differences. The toxicokinetic study in monkeys was also investigated.The metabolic profiles were similar in monkeys and humans, which were significant different from rats and dogs in vitro. In vitro plasma protein binding showed no major differences between species with medium to high protein binding rates. After single oral dose to rats, dogs, and monkeys, the absolute oral bioavailability of Y101 was 44.9%, 43.1%, and 19.2%, respectively. There was no accumulation for Y101 toxicokinetics in monkeys after oral administration for 90 d.The metabolic profiles indicated monkey was the very animal model for preclinical safety evaluation of Y101. Our results have demonstrated the favorable pharmacokinetics profile of Y101, which supports the clinical trials in humans.