The transcriptional repressor Hes1 attenuates inflammation by regulating transcription elongation.

Most of the known regulatory mechanisms that curb inflammatory gene expression target pre-transcription-initiation steps, and evidence for post-initiation regulation of inflammatory gene expression remains scarce. We found that the transcriptional repressor Hes1 suppressed production of CXCL1, a chemokine that is crucial for recruiting neutrophils. Hes1 negatively regulated neutrophil recruitment in vivo in a manner that was dependent on macrophage-produced CXCL1, and it attenuated the severity of inflammatory arthritis. Mechanistically, inhibition of Cxcl1 expression by Hes1 did not involve modification of transcription initiation. Instead, Hes1 inhibited signal-induced recruitment of the positive transcription-elongation complex P-TEFb and thereby prevented phosphorylation of RNA polymerase II at Ser2 and productive elongation. Thus, our results identify Hes1 as a homeostatic suppressor of inflammatory responses that exerts its suppressive function by regulating transcription elongation.

Slc6a8-Mediated Creatine Uptake and Accumulation Reprogram Macrophage Polarization via Regulating Cytokine Responses.

Macrophage polarization is accompanied by drastic changes in L-arginine metabolism. Two L-arginine catalytic enzymes, iNOS and arginase 1, are well-characterized hallmark molecules of classically and alternatively activated macrophages, respectively. The third metabolic fate of L-arginine is the generation of creatine that acts as a key source of cellular energy reserve, yet little is known about the role of creatine in the immune system. Here, genetic, genomic, metabolic, and immunological analyses revealed that creatine reprogrammed macrophage polarization by suppressing M(interferon-γ [IFN-γ]) yet promoting M(interleukin-4 [IL-4]) effector functions. Mechanistically, creatine inhibited the induction of immune effector molecules, including iNOS, by suppressing IFN-γ-JAK-STAT1 transcription-factor signaling while supporting IL-4-STAT6-activated arginase 1 expression by promoting chromatin remodeling. Depletion of intracellular creatine by ablation of the creatine transporter Slc6a8 altered macrophage-mediated immune responses in vivo. These results uncover a previously uncharacterized role for creatine in macrophage polarization by modulating cellular responses to cytokines such as IFN-γ and IL-4.

ILC3s select microbiota-specific regulatory T cells to establish tolerance in the gut.

Microbial colonization of the mammalian intestine elicits inflammatory or tolerogenic T cell responses, but the mechanisms controlling these distinct outcomes remain poorly understood, and accumulating evidence indicates that aberrant immunity to intestinal microbiota is causally associated with infectious, inflammatory and malignant diseases1-8. Here we define a critical pathway controlling the fate of inflammatory versus tolerogenic T cells that respond to the microbiota and express the transcription factor RORγt. We profiled all RORγt+ immune cells at single-cell resolution from the intestine-draining lymph nodes of mice and reveal a dominant presence of T regulatory (Treg) cells and lymphoid tissue inducer-like group 3 innate lymphoid cells (ILC3s), which co-localize at interfollicular regions. These ILC3s are distinct from extrathymic AIRE-expressing cells, abundantly express major histocompatibility complex class II, and are necessary and sufficient to promote microbiota-specific RORγt+ Treg cells and prevent their expansion as inflammatory T helper 17 cells. This occurs through ILC3-mediated antigen presentation, αV integrin and competition for interleukin-2. Finally, single-cell analyses suggest that interactions between ILC3s and RORγt+ Treg cells are impaired in inflammatory bowel disease. Our results define a paradigm whereby ILC3s select for antigen-specific RORγt+ Treg cells, and against T helper 17 cells, to establish immune tolerance to the microbiota and intestinal health.

Atf7ip and Setdb1 interaction orchestrates the hematopoietic stem and progenitor cell state with diverse lineage differentiation.

Hematopoietic stem and progenitor cells (HSPCs) are a heterogeneous group of cells with expansion, differentiation, and repopulation capacities. How HSPCs orchestrate the stemness state with diverse lineage differentiation at steady condition or acute stress remains largely unknown. Here, we show that zebrafish mutants that are deficient in an epigenetic regulator Atf7ip or Setdb1 methyltransferase undergo excessive myeloid differentiation with impaired HSPC expansion, manifesting a decline in T cells and erythroid lineage. We find that Atf7ip regulates hematopoiesis through Setdb1-mediated H3K9me3 modification and chromatin remodeling. During hematopoiesis, the interaction of Atf7ip and Setdb1 triggers H3K9me3 depositions in hematopoietic regulatory genes including cebpß and cdkn1a, preventing HSPCs from loss of expansion and premature differentiation into myeloid lineage. Concomitantly, loss of Atf7ip or Setdb1 derepresses retrotransposons that instigate the viral sensor Mda5/Rig-I like receptor (RLR) signaling, leading to stress-driven myelopoiesis and inflammation. We find that ATF7IP or SETDB1 depletion represses human leukemic cell growth and induces myeloid differentiation with retrotransposon-triggered inflammation. These findings establish that Atf7ip/Setdb1-mediated H3K9me3 deposition constitutes a genome-wide checkpoint that impedes the myeloid potential and maintains HSPC stemness for diverse blood cell production, providing unique insights into potential intervention in hematological malignancy.

Hsa_circ_0007321 regulates Zika virus replication through miR-492/NFKBID/NF-κB signaling pathway.

IMPORTANCE: Over the past decade, increasing evidence has shown that circular RNAs (circRNAs) play important regulatory roles in viral infection and host antiviral responses. However, reports on the role of circRNAs in Zika virus (ZIKV) infection are limited. In this study, we identified 45 differentially expressed circRNAs in ZIKV-infected A549 cells by RNA sequencing. We clarified that a downregulated circRNA, hsa_circ_0007321, regulates ZIKV replication through targeting of miR-492 and the downstream gene NFKBID. NFKBID is a negative regulator of nuclear factor-κB (NF-κB), and we found that inhibition of the NF-κB pathway promotes ZIKV replication. Therefore, this finding that hsa_circ_0007321 exerts its regulatory role on ZIKV replication through the miR-492/NFKBID/NF-κB signaling pathway has implications for the development of strategies to suppress ZIKV and possibly other viral infections.

Evaluating oleaginous yeasts for enhanced microbial lipid production using sweetwater as a sustainable feedstock.

BACKGROUND: Yeasts exhibit promising potential for the microbial conversion of crude glycerol, owing to their versatility in delivering a wide range of value-added products, particularly lipids. Sweetwater, a methanol-free by-product of the fat splitting process, has emerged as a promising alternative feedstock for the microbial utilization of crude glycerol. To further optimize sweetwater utilization, we compared the growth and lipid production capabilities of 21 oleaginous yeast strains under different conditions with various glycerol concentrations, sweetwater types and pH. RESULTS: We found that nutrient limitation and the unique carbon composition of sweetwater boosted significant lipid accumulation in several strains, in particular Rhodosporidium toruloides NRRL Y-6987. Subsequently, to decipher the underlying mechanism, the transcriptomic changes of R. toruloides NRRL Y-6987 were further analyzed, indicating potential sugars and oligopeptides in sweetwater supporting growth and lipid accumulation as well as exogenous fatty acid uptake leading to the enhanced lipid accumulation. CONCLUSION: Our comparative study successfully demonstrated sweetwater as a cost-effective feedstock while identifying R. toluroides NRRL Y-6987 as a highly promising microbial oil producer. Furthermore, we also suggested potential sweetwater type and strain engineering targets that could potentially enhance microbial lipid production.

Using Bland-Altman plot-based harmonization algorithm to optimize the harmonization for immunoassays.

OBJECTIVES: Harmonization has been recommended by the International Organization for Standard (ISO) to achieve equivalent results across in vitro diagnostic measurement devices (IVD-MDs). We aim to evaluate the effectiveness of Bland-Altman plot-based harmonization algorithm (BA-BHA) created in this study and compare it with weighted Deming regression-based harmonization algorithm (WD-BHA) proposed in ISO 21151:2020. METHODS: Eighty patient sera were used as the harmonization reference material (HRM) to develop IVD-MD-specific harmonization algorithms. Another panel of 40 patient sera was used to validate the effectiveness of harmonization algorithms. We compared regression slopes, intercepts, Bland-Altman plot layouts, percent differences, limits of agreement (LoAs), between-method coefficients of variation (CV) before and after harmonization. RESULTS: After harmonization by WD-BHA, acceptable slopes and intercepts between measured values and HRM targets were observed in weighted Deming regression, but not in Passing-Bablok analysis. Mean differences were -5.5 to 5.0 % and differences at specific levels were -33.9 to 23.9 %. LoAs were -64.6 to 74.6 %. Between-method CV was 22.9 % (±12.9 %). However, after harmonization by BA-BHA, both weighted Deming and Passing-Bablok regressions equations presented harmonized results. Mean differences were -0.3 to 0.2 % and differences at specific levels were -1.1 to 1.6 %. LoAs were -23.3 to 23.2 %. Between-method CV was 8.4 % (±4.0 %). The data points were evenly distributed at both sides of the mean in Bland-Altman plots. CONCLUSIONS: The inequivalence of test results between different methods can be improved but unacceptable analytical differences at specific levels may be hidden in terms of an acceptable slope and intercept on WD-BHA. The new protocol BA-BHA may be a viable alternative to optimize the harmonization for immunoassays.

Enhanced Diabetic Rat Wound Healing by Platelet-Rich Plasma Adhesion Zwitterionic Hydrogel.

BACKGROUND: The skin is the largest organ in the human body and serves as a barrier for protective, immune, and sensory functions. Continuous and permanent exposure to the external environment results in different levels of skin and extracellular matrix damage. During skin wound healing, the use of good dressings and addition of growth factors to the wound site can effectively modulate the rate of wound healing. A dressing containing bioactive substances can absorb wound exudates and reduce adhesion between the wound and dressing, whereas growth factors, cytokines, and signaling factors can promote cell motility and proliferation. AIM AND OBJECTIVES: We prepared a functional wound dressing by combining platelet-rich plasma (PRP) and zwitterionic hydrogels. Functional wound dressings are rich in various naturally occurring growth factors that can effectively promote the healing process in various types of tissues and absorb wound exudates to reduce adhesion between wounds and dressings. Furthermore, PRP-incorporated zwitterionic hydrogels have been used to repair full-thickness wounds in Sprague-Dawley rats with diabetes (DM SD). MATERIALS AND METHODS: Fibroblasts and keratinocytes were cultured with PRP, zwitterionic hydrogels, and PRP-incorporated zwitterionic hydrogels to assess cell proliferation and specific gene expression. Furthermore, PRP-incorporated zwitterionic hydrogels were used to repair full-thickness skin defects in DM SD rats. RESULTS: The swelling ratio of hydrogel, hydrogel + PRP1000 (108 platelets/mL), and hydrogel + PRP1000 (109 platelets/mL) groups were similar (~07.71% ± 1.396%, 700.17% ± 1.901%, 687.48% ± 4.661%, respectively) at 144 hours. The tensile strength and Young modulus of the hydrogel and hydrogel + PRP10000 groups were not significantly different. High concentrations of PRP (approximately 108 and 109 platelets/mL) effectively promoted the proliferation of fibroblasts and keratinocytes. The zwitterionic hydrogels were not cytotoxic to any cell type. High PRP concentration-incorporated zwitterionic hydrogels increased the rate of cell proliferation and significantly increased the expression of characteristic genes such as collagen, fibronectin, involucrin, and keratin. Subsequently, zwitterionic hydrogels with high PRP concentrations were used to repair full-thickness skin defects in DM SD rats, and a wound healing rate of more than 90% was recorded on day 12. CONCLUSIONS: PRP contains high concentrations of growth factors that promote cell viability, enhance specific gene expression, and have a high medical value in cell therapy. Zwitterionic hydrogels have a 3-dimensional interconnected microporous structure and can resist cell adhesion without causing cytotoxicity. Platelet-rich plasma-incorporated zwitterionic hydrogels further enhance the cellular properties and provide an effective therapeutic option for wound healing.

Protective Effect and Mechanism of Autophagy in Endothelial Cell Injury Induced by Hyperoxia.

OBJECTIVE: Bronchopulmonary dysplasia is a chronic lung disease in premature infants with alveolar simplification and pulmonary vascular development disorder as the main pathological feature and hyperoxia as the main etiology. Autophagy is a highly conserved cytological behavior of self-degrading cellular components and is accompanied by oxidative stress. Studies have reported that autophagy is regulated by FOXO1 posttranslational modification. However, whether autophagy can be involved in the regulation of endothelial cell injury induced by hyperoxia and its mechanism are still unclear. STUDY DESIGN: We have activated and inhibited autophagy in human umbilical vein endothelial cells under hyperoxia and verified the role of autophagy in endothelial cell-related functions from both positive and negative aspects. RESULTS: Our research showed that the expression level of autophagy-related proteins decreased, accompanied by decreased cell migration ability and tube formation ability and increased cell reactive oxygen species level and cell permeability under hyperoxia conditions. Using an autophagy agonist alleviated hyperoxia-induced changes and played a protective role. However, inhibition of autophagy aggravated the cell damage induced by hyperoxia. Moreover, the decrease in autophagy proteins was accompanied by the upregulation of FOXO1 phosphorylation and acetylation. CONCLUSION: We concluded that autophagy was a protective mechanism against endothelial cell injury caused by hyperoxia. Autophagy might participate in this process by coregulating posttranslational modifications of FOXO1. KEY POINTS: · Hyperoxia induces vascular endothelial cell injury.. · Autophagy may has a protective role under hyperoxia conditions.. · FOXO1 posttranslational modification may be involved in the regulation of autophagy..

Resveratrol Attenuates Hyperoxia Lung Injury in Neonatal Rats by Activating SIRT1/PGC-1α Signaling Pathway.

OBJECTIVES: Our previous study showed that resveratrol (Res) attenuates apoptosis and mitochondrial dysfunction in alveolar epithelial cell injury induced by hyperoxia by activating the SIRT1/PGC-1α signaling pathway. In the present study, we investigated whether Res protects against hyperoxia-induced lung injury in neonatal rats by activating SIRT1/PGC-1α signaling pathway. METHODS: Naturally delivered neonatal rats were randomly divided into six groups: normoxia + normal saline, normoxia + dimethyl sulfoxide (DMSO), normoxia + Res, hyperoxia + normal saline, hyperoxia + DMSO, and hyperoxia + Res. Lung tissue samples were collected on postnatal days 1, 7, and 14. Hematoxylin and eosin staining was used to evaluate lung development. Dual-immunofluorescence staining, real-time polymerase chain reaction, and western blotting were used to evaluate the levels of silencing information regulator 2-related enzyme 1 (SIRT1), peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), nuclear respiratory factor 1 (Nrf1), Nrf2, transcription factor A (TFAM) and citrate synthase, the number of mitochondrial DNA (mtDNA) and mitochondria, the integrity of mtDNA, and the expression of TFAM in mitochondria. RESULTS: We found that hyperoxia insulted lung development, whereas Res attenuated the hyperoxia lung injury. Res significantly upregulated the levels of SIRT1, PGC-1α, Nrf1, Nrf2, TFAM, and citrate synthase; promoted TFAM expression in the mitochondria; and increased the copy number of ND1 and the ratio of ND4/ND1. CONCLUSIONS: Our data suggest that Res attenuates hyperoxia-induced lung injury in neonatal rats, and this was achieved, in part, by activating the SIRT1/PGC-1α signaling pathway to promote mitochondrial biogenesis. KEY POINTS: · Hyperoxia insulted lung development in neonatal rats.. · Resveratrol promoted mitochondrial biogenesis to attenuate hyperoxia lung injury in neonatal rats.. · Resveratrol, at least in part, promoted mitochondrial biogenesis by activating the SIRT1/PGC-1α signaling pathway..

An In Vitro Study of the Effects of Mechanical and Enzymatic Isolation of Stromal Vascular Fraction on Wound Healing.

ABSTRACT: The adipose-derived stromal vascular fraction (SVF) is considered to be an attractive source of stem cells in cell therapy. Besides stem cells, it also contains functional cells, such as macrophages, precursor cells, somatic stem cells, and pericytes. Collagenase digestion is the most frequently used method to isolate SVF, but it is time-consuming and costly and has some problems, such as infectious agents and immune reactions. In this research, we compared the yield, cell population ratios, and cell viability when isolating SVF by the ultrasonic physics (U-SVF) method and traditional enzymatic method (E-SVF). Then, we isolated exosomes from U-SVF and E-SVF, respectively, and cocultured them with fibroblasts to investigate the potential of applying this cell secretion in wound repair. The results showed that there was no significant difference between the ultrasonic method and enzymatic method in terms of cell viability, cell numbers, or the expression of CD markers of stem cells. However, exosome analysis identified a greater number and smaller size of exosome particles obtained by U-SVF. In terms of cell proliferation efficiency, although the proliferation efficiency of U-SVF was lower than that of E-SVF. Trilineage differentiation experiments revealed that both E-SVF and U-SVF had good differentiation ability, owing to high stem cell content. Finally, E-SVF and U-SVF exosomes were cocultured with fibroblasts. The efficiency of fibroblast migration increased in the SVF exosome treated groups, and the expression of related genes (integrin α5ß1) was slightly upregulated; however, the expression of FAK, AKT, ERK, and RhoA was significantly upregulated at 24 hours. From the abovementioned experiments, we found that there was no significant difference in stem cell-related characteristics between SVF isolated by ultrasonic cavitation and SVF isolated by the enzymatic method. In addition, exosomes secreted by SVF may have excellent therapeutic effect on skin injuries, which provides a new viewpoint and therapeutic strategy for soft tissue repair.

Production of trimeric SARS-CoV-2 spike protein by CHO cells for serological COVID-19 testing.

We describe scalable and cost-efficient production of full length, His-tagged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein trimer by Chinese hamster ovary (CHO) cells that can be used to detect SARS-CoV-2 antibodies in patient sera at high specificity and sensitivity. Transient production of spike in both human embryonic kidney (HEK) and CHO cells mediated by polyethyleneimine was increased significantly (up to 10.9-fold) by a reduction in culture temperature to 32°C to permit extended duration cultures. Based on these data GS-CHO pools stably producing spike trimer under the control of a strong synthetic promoter were cultured in hypothermic conditions with combinations of bioactive small molecules to increase yield of purified spike product 4.9-fold to 53 mg/L. Purification of recombinant spike by Ni-chelate affinity chromatography initially yielded a variety of co-eluting protein impurities identified as host cell derived by mass spectrometry, which were separated from spike trimer using a modified imidazole gradient elution. Purified CHO spike trimer antigen was used in enzyme-linked immunosorbent assay format to detect immunoglobulin G antibodies against SARS-CoV-2 in sera from patient cohorts previously tested for viral infection by polymerase chain reaction, including those who had displayed coronavirus disease 2019 (COVID-19) symptoms. The antibody assay, validated to ISO 15189 Medical Laboratories standards, exhibited a specificity of 100% and sensitivity of 92.3%. Our data show that CHO cells are a suitable host for the production of larger quantities of recombinant SARS-CoV-2 trimer which can be used as antigen for mass serological testing.

Resveratrol Relieves Hyperoxia-Induced Brain Injury in Neonatal Rats by Activating Sirt1.

OBJECTIVE: Neonatal rats with hyperoxia-induced brain injury were treated with resveratrol to investigate its protective effects through analyzing changes in reactive oxygen species (ROS), Sirt1, p53, and acetylated p53 levels. STUDY DESIGN: Neonatal rats were randomly divided into hyperoxia and resveratrol intervened groups. Rats in both groups were placed in a hyperoxia chamber for 7 days to induce hyperoxia-induced brain injury. The rats in the resveratrol intervened group were administered resveratrol 60 µg/g body weight daily, whereas those in the hyperoxia group were administered a dimethyl sulfoxide-based solvent. Brain tissues were collected, and hematoxylin and eosin (H&E) and TUNEL staining, ROS measurements, real time-polymerase chain reaction, and western blotting were performed. RESULTS: H&E and TUNEL staining revealed increased cell damage and apoptosis in brain tissue from hyperoxia-exposed animals compared with the findings in animals in the resveratrol intervened group. Real time-polymerase chain reaction and western blotting identified increases in Sirt1 expression and decreases in p53 expression in the resveratrol intervened group. In addition, acetylated p53 protein expression was lower in the intervened group than in the hyperoxia group. CONCLUSION: Resveratrol alleviated brain apoptosis induced by hyperoxia in neonatal rats by upregulating Sirt1-mediated pathways, suggesting its potentially beneficial role in the treatment of brain injury induced by hyperoxia.

Beneficial Effects of Astragaloside IV-Treated and 3-Dimensional-Cultured Endothelial Progenitor Cells on Angiogenesis and Wound Healing.

INTRODUCTION: Astragaloside IV (AS-IV) is a natural herb extract and a popular compound used in traditional Chinese medicine because of its effect on multiple biological processes, such as promotion of cell proliferation, improvement in cardiopulmonary and vascular function, and promotion of angiogenesis around wounds, leading to accelerated wound healing. Vascular regeneration primarily results from the differentiation of endothelial progenitor cells (EPCs). Biomedical acceleration of angiogenesis and differentiation of EPCs around the wound remain challenging. MATERIALS AND METHODS: In this study, we treated human umbilical cord blood-derived EPCs with AS-IV and cultured them on 2-dimensional (tissue culture polystyrene) and 3-dimensional culture plates (3DPs). These cultured cells were then combined with human blood plasma gel and applied on the skin of nude mice in an attempt to repair full-thickness skin defects. RESULTS: The results show that using 3DP culture could increase vascular-related gene expression in EPCs. Furthermore, 12.5 µg/mL AS-IV-treaded EPCs were combined with plasma gels (P-3DP-EPC12.5) and showed enhanced vascular-related protein expression levels after 3 days of culture. Finally, P-3DP-EPC12.5s were used to repair full-thickness skin defects in nude mice, and we could register a wound healing rate greater than 90% by day 14. CONCLUSIONS: Based on these results, we concluded that we have developed a potential therapeutic approach for wound healing using plasma gel containing 3-dimensional surface-cultured AS-IV-treated EPCs.

MicroRNA-29b-3p suppresses oral squamous cell carcinoma cell migration and invasion via IL32/AKT signalling pathway.

Oral squamous cell carcinoma (OSCC) is aggressive accompanied with poor prognosis. We previously isolated the most invasive cells resembling the invasive tumour front by microfluidic technology and explored their differentially expressed microRNAs (miRNAs) in our previous work. Here, we verified the miR-29b-3p as a guarder that suppressed migration and invasion of OSCC cells and was down-regulated in the most invasive cells. Besides that, the invasion suppression role of miR-29b-3p was achieved through the IL32/AKT pathway. Thus, miR-29b-3p and IL32 might serve as therapeutic targets for blocking the progression and improving the outcome of OSCC.

LncRNA KCNQ1OT1 contributes to cardiomyocyte apoptosis by targeting FUS in heart failure.

Long noncoding RNAs (lncRNAs) have recently been recognized as the important regulators in cardiac diseases. This study was aimed to investigate the role and molecular mechanism of lncRNA KCNQ1OT1 in regulating cardiomyocyte apoptosis in heart failure (HF). The mouse model of HF was induced by doxorubicin (ADR). Cell apoptosis was detected by Hoechst and TUNEL staining. Molecule expressions were determined by qRT-PCR and western blot. The interaction between KCNQ1OT1 and Fused in sarcoma (FUS) was assessed by RNA immunoprecipitation (RIP) and RNA pull-down assays. KCNQ1OT1 was up-regulated in the myocardial tissues of HF mice and the ADR-stimulated mouse myocardial cell line (HL-1). KCNQ1OT1 overexpression promoted apoptosis of ADR-stimulated HL-1 cells, while KCNQ1OT1 knockdown caused the opposite effect. The RIP and RNA pull-down results showed that KCNQ1OT1 - bound to FUS and negatively regulated its protein level. Knockdown of FUS inhibited apoptosis of ADR-stimulated HL-1 cells and reversed the effect of KCNQ1OT1 overexpression on cardiomyocyte apoptosis. In vivo experiment showed that KCNQ1OT1 ovexpression improved myocardial histopathological changes, reduced myocardial fibrosis areas, down-regulated FUS expression, and inhibited cell apoptosis of HF mice. In conclusion, KCNQ1OT1 facilitates cardiomyocyte apoptosis by - targeting FUS in ADR-induced HF.

Evaluation of Polycaprolactone/Gelatin/Chitosan Electrospun Membrane for Peritoneal Adhesion Reduction.

In this study, a novel antiadhesion membrane made of polycaprolactone, gelatin, and chitosan was fabricated using the electrospinning technique. A series of polycaprolactone/gelatin/chitosan (PGC) electrospun membranes with different amounts of chitosan (0%, 0.5%, 1%, and 2% in weight percentage) was synthesized. The physicochemical properties and biocompatibility of the fabricated membranes were examined and compared with the aim to select an effective antiadhesion membrane. Scanning electron microscopy showed that these 4 electrospun membranes had similar fiber diameter and pore area, with no statistical differences between them. Furthermore, the contact angle decreased with increased chitosan content, indicating that chitosan may contribute to increased hydrophilic properties. The in vitro degradation test revealed that the higher chitosan content corresponded to a lower degradation rate in PGC membranes within 7 days. All PGC membranes exhibited similar cell proliferation; however, cell proliferation was lower than tissue culture polystyrene (P < 0.05). To compare antiadhesion ability, the adhesion between the cecum and abdominal wall was created in a rat model. Assessment after implantation of electrospun membranes revealed that PGCs with higher chitosan content (PGC2) had better antiadhesion effects, as evaluated by an adhesion score at day 14 postsurgery. Thus, PGC2 was effective in reducing the formation of tissue adhesion. Therefore, PGC electrospun membrane may provide a potential peritoneal antiadhesion barrier for clinical use.

The Molecular Mechanism of Sirt1 Signaling Pathway in Brain Injury of Newborn Rats Exposed to Hyperoxia.

The aim of the study was to investigate the changes in the reactive oxygen species (ROS), Sirt1, p53 and acetylated p53 in brain tissue of newborn rats exposed to hyperoxia to clarify the role of Sirt1 signaling pathway in brain injury. Neonate rats were randomly divided into normoxic group and hyperoxic group. Rats in the normoxic group were exposed to room air while the rats in the hyperoxic group were put in a hyperoxic chamber (80 ± 5% oxygen) for 1 to 14 d. Data, including weight growth, the water content of brain tissue, hematoxyline and eosin (H&E) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (Tunel) stain, ROS expression, the relative expression of Sirt1 mRNA and p53 mRNA, and the protein relative expression of Sirt1, p53 and acetylated p53 were analyzed at 1, 7 and 14 d after exposure. A reduced body weight and increased water content were observed in the brain tissue of hyperoxic group compared to normoxic group. HE staining and Tunel staining of brain tissue suggested that cell damaged after hyperoxic exposure. RT-PCR and Western blot results showed that the expression of Sirt1 in the hyperoxic group was lower than that in the normoxic group while the expression of p53 was higher than that in the normoxic group. In addition, Western blot data indicated acetylated p53 expression was higher in the hyperoxic group. Hyperoxic exposure can lead to brain injury in newborn Sprague-Dawley (SD) rats. These events might be regulated by the Sirt1 pathway, which downregulated the deacetylation of p53.

PTO-QuickStep: A Fast and Efficient Method for Cloning Random Mutagenesis Libraries.

QuickStep is a cloning method that allows seamless point integration of a DNA sequence at any position within a target plasmid using only Q5 High-Fidelity DNA Polymerase and DpnI endonuclease. This efficient and cost-effective method consists of two steps: two parallel asymmetric PCRs, followed by a megaprimer-based whole-plasmid amplification. To further simplify the workflow, enhance the efficiency, and increase the uptake of QuickStep, we replaced the asymmetric PCRs with a conventional PCR that uses phosphorothioate (PTO) oligos to generate megaprimers with 3' overhangs. The ease and speed of PTO-QuickStep were demonstrated through (1) right-first-time cloning of a 1.8 kb gene fragment into a pET vector and (2) creating a random mutagenesis library for directed evolution. Unlike most ligation-free random mutagenesis library creation methods (e.g., megaprimer PCR of whole plasmid [MEGAWHOP]), PTO-QuickStep does not require the gene of interest to be precloned into an expression vector to prepare a random mutagenesis library. Therefore, PTO-QuickStep is a simple, reliable, and robust technique, adding to the ever-expanding molecular toolbox of synthetic biology and expediting protein engineering via directed evolution.

Adaptive Laboratory Evolution of Cupriavidus necator H16 for Carbon Co-Utilization with Glycerol.

Cupriavidus necator H16 is a non-pathogenic Gram-negative betaproteobacterium that can utilize a broad range of renewable heterotrophic resources to produce chemicals ranging from polyhydroxybutyrate (biopolymer) to alcohols, alkanes, and alkenes. However, C. necator H16 utilizes carbon sources to different efficiency, for example its growth in glycerol is 11.4 times slower than a favorable substrate like gluconate. This work used adaptive laboratory evolution to enhance the glycerol assimilation in C. necator H16 and identified a variant (v6C6) that can co-utilize gluconate and glycerol. The v6C6 variant has a specific growth rate in glycerol 9.5 times faster than the wild-type strain and grows faster in mixed gluconate-glycerol carbon sources compared to gluconate alone. It also accumulated more PHB when cultivated in glycerol medium compared to gluconate medium while the inverse is true for the wild-type strain. Through genome sequencing and expression studies, glycerol kinase was identified as the key enzyme for its improved glycerol utilization. The superior performance of v6C6 in assimilating pure glycerol was extended to crude glycerol (sweetwater) from an industrial fat splitting process. These results highlight the robustness of adaptive laboratory evolution for strain engineering and the versatility and potential of C. necator H16 for industrial waste glycerol valorization.