Analysis of heterologous expression of phaCBA promotes the acetoin stress response mechanism in Bacillus subtilis using transcriptomics and metabolomics approaches.

Acetoin, a versatile platform chemical and popular food additive, poses a challenge to the biosafety strain Bacillus subtilis when produced in high concentrations due to its intrinsic toxicity. Incorporating the PHB synthesis pathway into Bacillus subtilis 168 has been shown to significantly enhance the strain's acetoin tolerance. This study aims to elucidate the molecular mechanisms underlying the response of B. subtilis 168-phaCBA to acetoin stress, employing transcriptomic and metabolomic analyses. Acetoin stress induces fatty acid degradation and disrupts amino acid synthesis. In response, B. subtilis 168-phaCBA down-regulates genes associated with flagellum assembly and bacterial chemotaxis, while up-regulating genes related to the ABC transport system encoding amino acid transport proteins. Notably, genes coding for cysteine and D-methionine transport proteins (tcyB, tcyC and metQ) and the biotin transporter protein bioY, are up-regulated, enhancing cellular tolerance. Our findings highlight that the expression of phaCBA significantly increases the ratio of long-chain unsaturated fatty acids and modulates intracellular concentrations of amino acids, including L-tryptophan, L-tyrosine, L-leucine, L-threonine, L-methionine, L-glutamic acid, L-proline, D-phenylalanine, L-arginine, and membrane fatty acids, thereby imparting acetoin tolerance. Furthermore, the supplementation with specific exogenous amino acids (L-alanine, L-proline, L-cysteine, L-arginine, L-glutamic acid, and L-isoleucine) alleviates acetoin's detrimental effects on the bacterium. Simultaneously, the introduction of phaCBA into the acetoin-producing strain BS03 addressed the issue of insufficient intracellular cofactors in the fermentation strain, resulting in the successful production of 70.14 g/L of acetoin through fed-batch fermentation. This study enhances our understanding of Bacillus's cellular response to acetoin-induced stress and provides valuable insights for the development of acetoin-resistant Bacillus strains.

Upregulation of IDO gene expression reduces the immunogenicity of epidermal cells and strengthens the immune protection of epidermal cells during transplantation treatment of wounds.

BACKGROUND: Epidermal cell transplantation is a feasible treatment option for large wounds; however, sources of autologous epidermal cells are often limited. Allogeneic epidermal cells can be cultured conveniently; however, related immune rejection needs to be addressed. Herein, we hypothesized that the immunogenicity of epidermal cells with high indoleamine 2,3-dioxygenase (IDO) expression may be reduced by gene transfection. METHODS/RESULTS: To test this hypothesis, we obtained stable transfectants by transfecting epidermal stem cells with a lentiviral vector encoding the IDO gene and screening them for puromycin resistance (a marker for successful transfection). The phenotype tested using cell counting kit -8 and Transwell assays confirmed that IDO-transfected epidermal cells maintained their characteristics. Co-culture of IDO-transfected epidermal cells with allogeneic CD4+ T cells in vitro showed that the upregulation of IDO expression in epidermal cells inhibited the proliferation of CD4+ T cells (P < 0.001, P < 0.001, and P < 0.001, respectively) and promoted their apoptosis (P = 0.00028, P = 0.0006, and P = 0.00247, respectively) and transformation into functional regulatory T cells (Tregs) (P = 0.0051, P = 0.0132, and P = 0.0248, respectively) compared with Con, NC, and 1-MT groups. The increased proportion of Tregs may be related to the overexpression of IDO, which promoted the expression of transforming growth factor beta (TGF-ß) (P = 0.0001, P = 0.0013, and, P = 0.0009) and interleukin (IL) 10 (IL-10) (P = 0.0062, P = 0.0058, and P = 0.0119) while inhibited the expression of IL-2 (P = 0.0012, P = 0.0126, and P = 0.0066). We further verified these effects in vivo as transplanted IDO-transfected epidermal stem cells were effective in treating wounds in mice. On days 5 and 7, wounds treated with IDO cells healed faster than those in the other groups (day 5: P = 0.012 and P = 0.0136; day 7: P = 0.0242 and P = 0.0187, respectively), whereas this effect was significantly inhibited by 1-methyltryptophan (1-MT) (day 5: P = 0.0303; day 7: P = 0.0105). Immunofluorescence staining detected IDO and CD4+ Foxp3+ Tregs in the transplanted wounds, which may promote Foxp3+ Tregs in the wound tissue (day 5: P < 0.0001, P < 0.0001, and P < 0.0001; day 7: P < 0.0001, P < 0.0001, and P < 0.0001), respectively) and decrease CD4+ T cells (day 5: P < 0.0001, P < 0.0001, and P < 0.0001; day 7: P < 0.0001, P < 0.0001, and P < 0.0001). CONCLUSION: Our results suggest that the upregulation of IDO expression in epidermal stem cells can reduce their immunogenicity by promoting Tregs, thus inducing the immune protection of epidermal stem cells.

Exploring causal association between circulating inflammatory cytokines and functional outcomes following ischemic stroke: A bidirectional Mendelian randomization study.

OBJECTIVES: Previous observational studies have indicated correlations between various inflammatory cytokines and functional outcomes following ischemic stroke (IS); however, the causality remains unclear. We aimed to further evaluate the causal association between 41 circulating inflammatory cytokines and functional outcomes following IS. METHODS: Two-sample bidirectional Mendelian randomization (MR) analysis was used in this study. The genetic variation of 41 circulating inflammatory cytokines were derived from genome-wide association study (GWAS) data of European ancestry (n = 8293). The corresponding genetic association of functional outcomes following IS were derived from European ancestry GWAS data (n = 6021). RESULTS: Inverse variance weighted (IVW) analysis showed that genetically predicted increased levels of regulation and activation in normal T-cell expression and secretion factor (RANTES/CCL5) and eosinophilic chemotactic factor (EOTAXIN/CCL11) were positively correlated with the increased adverse functional outcomes (modified Rankin Scale [mRS≥3] following IS (OR: 1.40, 95% CI: 1.002-1.96, p = 0.049; OR: 1.33, 95% CI: 1.15-1.54, p = 0.0001). Interleukin 18 (IL-18) level might be the downstream consequence of adverse functional outcomes following IS (ß: -0.09, p = 0.039). Other inflammatory cytokines and functional outcomes following IS did not appear to be causally related. CONCLUSIONS: This study suggests a causality between inflammation and adverse functional outcomes following IS. RANTES (CCL5) and EOTAXIN (CCL11) may be the upstream factors of adverse functional outcomes following IS, while IL-18 may be the downstream effect of adverse functional outcomes following IS. Whether these cytokines can be used to predict or improve adverse functional outcomes after IS requires further researches.

Repetitive transcranial magnetic stimulation for cerebellar ataxia: a systematic review and meta-analysis.

Background: Repetitive transcranial magnetic stimulation, a non-invasive brain stimulation technique, can manage cerebellar ataxia (CA) by suppressing cerebral cortical excitability. Hence, this study aimed to summarize the efficacy and safety of rTMS for CA patients by meta-analysis. Methods: The PubMed, Embase, Web of Science, and Cochrane Library databases were searched for eligible studies published till 20 May 2023. Weighted mean difference (MD) and 95% confidence intervals (CIs) were used to assess the effect of rTMS treatment. Additionally, the quality of the included studies and the risk of bias were evaluated using the Physiotherapy Evidence Database (PEDro) scale. Results: Overall, eight studies involving 278 CA patients were included in this meta-analysis. rTMS could significantly improve the Scale for the Assessment and Rating of Ataxia (SARA) (MD: -2.00; 95% CI: -3.97 to -0.02, p = 0.05), International Cooperative Ataxia Rating Scale (ICARS) (MD: -3.96; 95% CI: -5.51 to -2.40, p < 0.00001), Timed Up-and-Go test (TUG) (MD: -1.54; 95% CI: -2.24 to -0.84, p < 0.0001), 10-m walk test (10 MWT) (MD10-m steps: -2.44; 95% CI: -4.14 to -0.73, p = 0.005), and Berg Balance Scale (BBS) (MD: 2.59; 95% CI: 1.15-4.03, p = 0.0004) as compared to sham stimulation. Active rTMS was not significantly different from sham rTMS in changing the duration (MD10-m time: -1.29; 95% CI: -7.98 to 5.41, p = 0.71). No severe adverse events were observed in both sham stimulation and active rTMS groups. Conclusion: This meta-analysis provides limited evidence that rTMS may be beneficial in treating CA patients. However, these findings should be treated with caution due to the limitations of the smaller sample size and the inconsistent approach and target of rTMS treatment. Therefore, more large-scale RCTs are required to further validate our analytical findings. Systematic review registration: https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=295726, identifier: CRD42022295726.

A study on the preoperative risk factors for primary healing failure in the reconstruction of deep sternal wound infection with platelet-rich plasma and negative pressure trauma therapy.

Deep sternal wound infection (DSWI) is a relatively complex wound in wound reconstruction surgery. Because plastic surgeons deal with DSWI patients late. The primary healing (healing by first intention) after reconstruction of DSWI is restricted by many preoperative risk factors. The purpose of this study is to explore and analyse the risk factors of primary healing failure in patients with DSWI treated with platelet-rich plasma (PRP) and negative pressure trauma therapy (NPWT). 115 DSWI patients treated with the PRP and NPWT (PRP + NPWT) modality were retrospectively (2013-2021) analysed. They were divided into two groups according to primary healing results after the first PRP + NPWT treatment. Univariate and multivariate analyses were used to compare the data of the two groups to find out the risk factors and their optimal cut-off values were identified by ROC analysis. The primary healing results, debridement history, wound size, sinus, osteomyelitis, renal function, bacterial culture, albumin (ALB), platelet (PLT) between the two groups were significantly different (P < 0.05). Binary logistic regression showed that osteomyelitis, sinus, ALB and PLT were the risk factors affecting primary healing outcomes (P < 0.05). ROC analysis showed that AUC for ALB in the non-primary healing group was 0.743 (95% CI: 0.650-0.836, P < 0.05) and its optimal cutoff value of 31 g/L was associated with primary healing failure with a sensitivity of 96.9% and specificity of 45.1%. AUC for PLT in the non-primary healing group was 0.670 (95% CI: 0.571 ~ 0.770, P < 0.05) its optimal cutoff value of 293 × 109 /L was associated with primary healing failure with a sensitivity of 72.5% and specificity of 56.3%. In the cases included in this study, the success rate of primary healing of DSWI treated with PRP + NPWT was not affected by the most common preoperative risk factors for wound non-union. It is indirectly confirmed that PRP + NPWT is an ideal treatment. However, it should be noted that it will still be adversely affected by sinus osteomyelitis, ALB and PLT. The patients need to be carefully evaluated and corrected before reconstruction.

Transcriptome analysis of hepatic injury caused by delayed resuscitation following severe burns in rats.

BACKGROUND: Delayed resuscitation (DR) can induce hepatic reperfusion injury after severe burns. The underlying molecular mechanisms of DR-induced hepatic injury remain unidentified. This study sought to predict candidate genes and molecular pathways in a DR-induced hepatic injury preclinical model. METHODS: Rats were randomized into three groups: the sham injury (Sham) group; the DR group, which had third-degree burns covering 30% of the body surface area and DR; and the early resuscitation (ER) group, in which ER was administered. The liver tissue was harvested for the purpose of evaluating hepatic injury and performing transcriptome sequencing. Differentially expressed genes (DEGs) for DR versus Sham and ER versus DR were analyzed respectively. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Ingenuity Pathway Analysis were used. The DEGs and critical module genes were intersected to obtain critical genes. Immune infiltration and competing endogenous RNA networks were also analyzed. Validation was conducted using quantitative real-time polymerase chain reaction. RESULTS: Hepatic injury was evident in DR rats. There were 2,430 DEGs between DR and Sham and 261 DEGs between ER and DR. Differentially expressed genes were mostly enriched in metabolic process for DR versus Sham, and immune and inflammatory processes for ER versus DR. Four critical genes (Tff3, C1galt1, Cd48, and MGC105649) were obtained by screening. Five immune cells were significantly different between DR and Sham, and seven immune cells were significantly different between ER and DR in immunoassays. Three critical genes, 75 miRNAs, 7 lncRNAs, and 197 edges constituted the mRNA-miRNA-lncRNA linkages, which included C1galt1-rno-miR-330-5p-Pvt1, among others. CONCLUSION: This is the first attempt to perform a high-throughput analysis of gene expression profiles in DR-induced hepatic injury. It shows that immunity and inflammation-related RNAs and pathways play an important role in the progression of hepatic injury. It also provides insight into some important RNAs and regulatory targets related to disease.

A Potential Resuscitation Route on Battlefield: Immediate Intraperitoneal Fluid Administration Post-burn Shows Satisfactory Fluid Absorption and Anti-shock Effects.

INTRODUCTION: Timely fluid resuscitation remains the key to the early treatment of severe burns. Intraperitoneal (IP) fluid administration is a simple, rapid resuscitation strategy via a puncture in the abdominal wall. This study aimed to evaluate the fluid absorption and anti-shock effects of IP delivery in the early stage after severe burns. MATERIALS AND METHODS: A 30% total body surface area full-thickness burn model was established using male C57BL/6 mice. A total of 126 mice were randomly assigned into six groups (n = 21): the sham injury group (SHAM), the burn group without fluid resuscitation (NR), and the four IP resuscitation groups (IP-A/B/C/D, each being intraperitoneally administered with 60, 80, 100, and 120 mL/kg of sodium lactate Ringer's solution post-injury). Three-hour post-burn, six mice in each group were randomly selected and sacrificed for blood and tissue sampling to detect the IP fluid absorption rate and evaluate organ damage because of low perfusion. The remaining 15 mice in each group were observed for the vital signs within 48-h post-injury, and their survival rate was calculated. RESULTS: The 48-h survival rate increased in the IP-A (40.0%), IP-B (66.7%), IP-C (60.0%), and IP-D (13.3%) groups, compared with the NR group (0%). The mean arterial pressure, body temperature, and heart rate of mice were significantly stabilized in the IP groups. For the first 3-h post-injury, the absorption rates of groups IP-A (74.3% ± 9.5%) and IP-B (73.3% ± 6.9%) were significantly higher than those of groups IP-C (59.7% ± 7.1%) and IP-D (48.7% ± 5.7%). The levels of arterial blood pH, partial pressure of oxygen, partial pressure of carbon dioxide, lactate, and hematocrit were better maintained in the IP groups. Intraperitoneal resuscitation remarkably reduced the injury scores in burn-induced histopathology of the liver, kidneys, lungs, and intestines, accompanied by decreased alanine transaminase, creatinine, interleukin-1, and tumor necrosis factor-α in plasma, and augmented superoxide dismutase 2 and inhibited malondialdehyde in tissues. Group IP-B has the best performance for these indices. CONCLUSIONS: Intraperitoneal administration of isotonic saline post-burn can be adequately and rapidly absorbed, thereby boosting circulation and perfusion, precluding shock, alleviating organ damage caused by ischemia and hypoxia, and significantly increasing the survival rate. This technique, with a potential to be a supplement to existing resuscitation methods on the battlefield, is worth further investigation.

Role and Mechanism of Endoplasmic Reticulum Stress in Mice Pancreatic Islet Dysfunction After Severe Burns.

This study attempted to investigate the role and mechanism of endoplasmic reticulum (ER) stress in the islet dysfunction in mice after severe burns. C57BL/6 mice were randomly divided into the sham group, burn group, and burn+4-phenylbutyric acid (4-PBA) group. Mice were burned with full thickness of 30% total surface area (TBSA), and 4-PBA solution was intraperitoneally injected into mice in burn+4-PBA group. Glucose-stimulated insulin secretion (GSIS), Fasting blood glucose (FBG) and glucose tolerance were detected 24 hours post severe burns. The ER stress-related pathway markers immunoglobulin binding protein (BIP), X-box binding protein 1 (XBP1), phosphorylation-PKR-like ER kinase (p-PERK), phosphorylation-eukaryotic translation initiation factor 2α (p-eIF2α), CHOP, activating transcription factor 6 (ATF6), apoptosis-related protein Cleaved-Caspase 3, and islet cell apoptosis were measured. Mice were characterized with elevated FBG, decreased glucose tolerance and GSIS levels post severe burns. The expression of BIP, XBP1, p-PERK, p-eIF2α, CHOP, ATF6, Cleaved-Caspase 3, and islet cell apoptosis were increased significantly after severe burns. 4-PBA treatment contributed to decreased FBG, improved glucose tolerance, increased GSIS, inhibited islet ER stress, and reduced pancreatic islet cell apoptosis in mice post severe burns. ER stress occurs in islets of severely burned mice, which leads to increased apoptosis of islet cells, thus resulting in islet dysfunction.

Fractionated FLASH radiation in xenografted lung tumors induced FLASH effect at a split dose of 2 Gy.

PURPOSE: To explore the minimum split dose of FLASH radiotherapy (FLASH). MATERIAL AND METHODS: Lungs of nude mice were used to verify the capacity of normal tissue sparing of FLASH, while tumor-bearing nude mice were used to evaluate the curative power. Xenografted tumor models were established in Balb/c-nu mice using A549 cells at a concentration of 5×106/100 µL. With the same total dose (20 Gy), the dose rate of FLASH was 200 Gy/s when conventional radiotherapy(CONV) was 0.033 Gy/s. Two schemes of FLASH irradiations were applied: single pulse (FLASH1) and ten pulses (FLASH10). Then, according to the different tissue types and irradiation schemes, mice were divided into eight groups: Control-T, CONV-T, FLASH1-T, FLASH10-T (T for tumor) and Control-L, CONV-L, FLASH1-L, FLASH10-L (L for lung). Evaluation of FLASH effect was based on the changes in tumor volume and pathological analysis of tumor and lung tissues before and after irradiation. RESULTS: Compared to control group, the mean volume of tumors in nude mice increased slowly or decreased after irradiation with both FLASH and CONV (Control-T: 233.6±55.19 mm3, CONV-T: 146.1±50.62 mm3, FLASH1-T: 148±18.83 mm3, FLASH10-T: 119.1±50.62 mm3, p ≤ .05) . Tumor cells of irradiated groups had similar degrees of dissolution damage and inflammation, while the acute radiation pneumonia induced by FLASH was less severe. The pulmonary pathology of FLASH1-L and FLASH10-L were similar, and only a few neutrophils were observed. In addition to inflammatory cells, slight thickening of alveolar septum and obvious interstitial hemorrhage were also observed in the CONV-L group. CONCLUSION: The FLASH effect was successfully reproduced in both single and fractionated irradiation, with 2 Gy being the minimum split dose to achieve the FLASH effect in existing experiments. It is suggested that the transient oxygen depletion might not be the only mechanism behind the FLASH effect.

Production of acetoin and its derivative tetramethylpyrazine from okara hydrolysate with Bacillus subtilis.

Okara, a renewable biomass resource, is a promising fermentative raw material for the bio-production of value-added chemicals due to its abundance and low-costs. we developed a process for the enzymatic hydrolysis of okara, and then engineered Bacillus subtilis to utilize mixed sugars to produce acetoin in okara hydrolysis without the addition of a supplemental nitrogen source. Okara was initially hydrolyzed with cellulase, ß-glucosidase, and pectinase to obtain okara hydrolysate containing mixed sugars (32.78 ± 0.23 g/L glucose, 1.43 ± 0.064 g/L arabinose, 7.74 ± 0.11 g/L galactose) and amino acids. In this study, Bacillus subtilis 168 was used as the acetoin-producing strain, and the key genes bdhA and acoA of the acetoin catabolism pathway were knocked out to improve the fermentation yield of acetoin. In order to utilize the galactose in the hydrolysate, the recombinant strain BS03 (Bacillus subtilis168∆bdhA∆acoA) was used to overexpress the arabinose transporter-encoding gene (araE) drive heterologous expression of the Leloir pathway gene (galKTE). The corn dry powder concentration was optimized to 29 g/L in the reducing sugar okara hydrolysate. The results show that the recombinant bacterium BS03 could still synthesize 11.79 g/L acetoin without using corn dry powder as a nitrogen source. Finally, using okara enzymatic hydrolysate as the carbon and nitrogen source, 11.11 g/L and 29.7 g/L acetoin were obtained by batch fermentation and fed-batch fermentation, respectively, which was further converted to 5.33 g/L and 13.37 g/L tetramethylpyrazine (TTMP) by reaction with an ammonium salt.

Sirt3-dependent regulation of mitochondrial oxidative stress and apoptosis contributes to the dysfunction of pancreatic islets after severe burns.

BACKGROUND: Severe burns are often complicated with hyperglycemia caused by mitochondrial oxidative stress-related pancreatic islet dysfunction. Silent information regulator of transcription 3 (Sirt3) can regulate mitochondrial oxidative stress. However, the role and mechanism of Sirt3 on islet function after severe burns remain unclear. Therefore, this study aimed to investigate whether Sirt3 played a role in both mitochondrial oxidative stress in islets and mediating islet function post severe burns. METHODS: A mouse model of 30% total body surface area full-thickness burn and an in vitro MIN6 cell hypoxia model were established. Sirt3 KO mice were used to demonstrate further the role of Sirt3 in maintaining redox homeostasis and regulating islet function. Fasting blood glucose and glucose-stimulated insulin secretion (GSIS) were detected to assess the islet function. The levels of mitochondrial ROS and deacetylation, and the activities of Mn-SOD and IDH2 were measured to evaluate oxidative stress. The mitochondrial membrane potential (MMP)was detected and the apoptosis rate measured. RESULTS: In vitro MIN6 cells, the hypoxia treatment significantly reduced Sirt3 expression, resulting in increased deacetylation of Mn-SOD and IDH2, which further led to a higher level of mitochondrial ROS. In addition, hypoxia reduced MMP and increased apoptosis rate, which impaired GSIS eventually. Knockdown of Sirt3 caused similar alterations. The hypoxia-induced high level of mitochondrial ROS and apoptosis and impaired GSIS could be reversed by overexpression of Sirt3. Similarly, after severe burns, the expression of Sirt3 in islets decreased significantly with a high level of deacetylation of Mn-SOD, IDH2, mitochondrial ROS and apoptosis, and islet dysfunction. Oxidative stress and apoptosis also occurred in islets of Sirt3 KO mice, accompanied by islet dysfunction. CONCLUSIONS: Sirt3 and downstream signalling are critical in modulating the islet function post severe burns by regulating mitochondrial oxidative stress and apoptosis.

The Epidermal Barrier Structure and Function of Re-Harvested Skin from Non-Scalp Donor Sites.

AIM: The aim of this study was to explore the epidermal barrier structure and function of re-harvested skin from non-scalp donor sites. METHODS: Six patients with large-area deep burns who met the inclusion and exclusion criteria were subjected to split-thickness skin excision three times on the same healthy non-scalp donor sites, with an interval of 14 days. The donor skin thus harvested was labeled as primary skin (S1), secondary skin (S2), and tertiary skin (S3). The transepidermal water loss (TEWL) and stratum corneum water content (SCH) of donor skin were detected before each surgery, and the donor skin was harvested during the surgery. The donor skin was stained with hematoxylin and eosin (HE) and involucrin, loricrin, filaggrin, small molecule proline-rich protein 3 (SPRR3), ZO-3, JAM-A, and JAM-C, or observed by transmission electron microscopy. RESULTS: The epidermal barrier function of the re-harvested skin from the non-scalp donor sites became impaired. The histopathological structure of the re-harvested skin from non-scalp donor sites became abnormal. The barrier of the epidermal stratum corneum of the re-harvested skin from non-scalp donor sites was damaged. The epidermal tight junction barrier in the re-harvested skin from non-scalp donor sites was damaged. CONCLUSIONS: As the number of harvesting increases, the epidermal barrier function of the skin decreased, and the damage to the barrier structure increased. Hence, it is vitally important to restore the epidermal barrier function for re-harvesting in non-scalp donor sites.

Effects and Mechanisms of Peritoneal Resuscitation on Acute Kidney Injury After Severe Burns in Rats.

INTRODUCTION: Acute kidney injury (AKI) is a common complication in severe burn patients with poor prognosis and high mortality. Reduced kidney perfusion induced by the decreased effective circulating blood volume after severe burn is a common cause of AKI. Routine intravenous resuscitation (IR) is difficult or delayed in extreme conditions such as war and disaster sites. Peritoneal resuscitation (PR) is a simple, rapid resuscitation strategy via a puncture in the abdominal wall. This study investigated whether PR is a validated resuscitation strategy for AKI after severe burns in rats and explored its mechanisms. MATERIALS AND METHODS: Eighty Sprague-Dawley rats were randomized into four groups: (1) sham group; (2) IR group, which was characterized by the full thickness burn of 50% of the total body surface area received IR immediately post-injury; (3) early PR group, in which rats with the same burn model received PR immediately post-injury; and (4) delayed resuscitation (DR) group, in which rats with the same burn model received no resuscitation within 3-hour post-injury. PR and DR groups animals received IR after 3-hour post-injury. The survival rate, mean arterial pressure, renal histopathology, renal function, indicators of renal injury, and renal hypoxia-inducible factor-1α and NADPH oxidase 4 (NOX4) proteins of rats were measured at 3 h, 12 h, and 24 h post-injury. RESULTS: Compared with rats in the DR group, rats in the PR group had a significantly improved survival rate (100% vs. 58.3% at 24 h, P = 0.0087), an increased mean arterial pressure (92.6 ± 6.6 vs. 65.3 ± 10.7, 85.1 ± 5.7 vs. 61.1 ± 6.9, 90.1 ± 8.7 vs. 74.9 ± 7.4 mmHg, at 3 h, 12 h, and 24 h, P < 0.01), a reduced renal water content rate (51.6% ± 5.0% vs. 70.1% ± 6.8%, 57.6% ± 7.7% vs. 69.5% ± 8.7%, at 12 h and 24 h, P < 0.01), attenuated histopathological damage, reduced serum creatinine expression (36.36 ± 4.27 vs. 49.98 ± 2.42, 52.29 ± 4.31 vs. 71.32 ± 5.2, 45.25 ± 2.55 vs. 81.15 ± 6.44 µmol/L, at 3 h, 12 h, and 24 h, P < 0.01) and BUN expression (7.62 ± 0.30 vs. 10.80 ± 0.58, 8.61 ± 0.32 vs. 28.58 ± 1.99, 8.09 ± 0.99 vs. 20.95 ± 1.02 mmol/L, at 3 h, 12 h, and 24 h, P < 0.01), increased kidney injury markers neutrophil gelatinase-associated lipocalin expression (95.09 ± 7.02 vs. 101.75 ± 6.23, 146.77 ± 11.54 vs. 190.03 ± 9.87, 112.79 ± 15.8 vs. 194.43 ± 11.47 ng/mL, at 3 h, 12 h, and 24 h, P < 0.01) and cystatin C expression (0.185 ± 0.006 vs. 0.197 ± 0.006, 0.345 ± 0.036 vs. 0.382 ± 0.013, 0.297 ± 0.012 vs. 0.371 ± 0.028 ng/mL, at 3 h, 12 h, and 24 h, P < 0.01), and reduced renal hypoxia-inducible factor-1α and NADPH oxidase 4 protein expression (P < 0.01). There was no significant difference between rats in the PR group and the IR group in the above indicators. CONCLUSIONS: Early PR could protect severe burn injury rats from AKI. It may be an alternative resuscitation strategy in severe burn injury when IR cannot be achieved.

Transcriptome reveals the dysfunction of pancreatic islets after wound healing in severely burned mice.

BACKGROUND: Severely burned patients have a higher risk of diabetes mellitus after healing, but its mechanism remains unclear. Therefore, the purpose of the study was to explore the influence of burns on pancreatic islets of mice after wound healing. METHODS: Forty-two male C57BL/6 mice were randomized into a sham group and a burn group and subjected to sham treatment or a third-degree burn model of 30% total body surface area. Fasting blood glucose was detected weekly for 8 weeks after severe burns. Glucose-stimulated insulin secretion was measured 8 weeks post severe burns. Islets of the two groups were isolated and mRNA libraries were sequenced by the Illumina sequencing platform. The expressions of differentially expressed genes (DEGs) related to the cell cycle and the amounts of mitochondrial DNA were detected by quantitative real-time polymerase chain reaction after gene ontology, gene set enrichment analysis, and protein-protein network analysis. Hematoxylin-eosin staining of pancreatic tail tissue and adenosine triphosphate (ATP) assay of islets were performed. RESULTS: The levels of fasting blood glucose were significantly higher within 8 weeks post severe burns. Glucose-stimulated insulin secretion was impaired at the eighth week post severe burns. Totally 128 DEGs were selected. Gene ontology and gene set enrichment analysis indicated that the pathways related to the cell cycle, protein processing, and oxidative phosphorylation were downregulated. The expressions of DEGs related to the cell cycle showed a consistent trend with mRNA sequencing data, and most of them were downregulated post severe burns. The cell mass of the burn group was less than that of the sham group. Also, the concentration of ATP and the amount of mitochondrial DNA were lower in the burn group. CONCLUSION: In the model of severe-burned mice, disorders in glucose metabolism persist for 8 weeks after burns, which may be related to low islet cell proliferation, downregulation of protein processing, and less ATP production.

Video laryngoscope-guided urethral catheterization in female minipigs.

OBJECTIVE: The objective of this work was to anatomically locate the urethral orifice in female minipigs and describe the use of video laryngoscopes in urethral catheterization. METHODS: Urethral catheterization guided by a video laryngoscope was attempted in 16 adult female Bama minipigs. The anatomical location of urethral orifices, operating time and complications (mucosal edema and bleeding in the vaginal vestibule, and the numbers of red blood cells (RBCs) and white blood cells (WBCs) in mid-stream urine samples) were recorded. RESULTS: The anatomical location of the urethral orifice: the depth of the urethral orifice in female Bama minipigs was 4.2 ± 1.2 cm; all the urethral orifices were covered by mucosal folds of the vaginal vestibule. In the supine position, the orifice of the urethra at 9-12 and 1-3 o'clock accounted for 6.25%, 6.25%, 18.75%, 50%, 12.5%, 6.25% and 6.25%, respectively. All animals were successfully catheterized and the operating time was 9.0 (6.0-12.8) min. Complications: no bleeding in the vaginal vestibule was observed; the incidence of mucosal edema was 12.5%, all of which were mild; of urine samples collected 1 h after catheterization, 12.5% were found to contain RBCs and no RBCs were detected 6 h after catheterization; no WBCs were detected 1 h or 6 h after catheterization. CONCLUSIONS: The urethral orifice of female minipigs was located deep in the vagina at variable clock directions and was unexceptionally covered by mucosal folds. Applying a video laryngoscope in urethral catheterization allowed quick and accurate exposure of the urethral orifice and minimal operational injury in female minipigs.

Elevated Serum Procalcitonin to Predict Severity and Prognosis of Extensive Burns.

PURPOSE: To analyze the factors affecting the elevation of serum procalcitonin (PCT) in patients with extensive burns, and explore its potential value in evaluating the severity and prognosis. METHODS: Clinical data of 139 patients with extensive burns admitted to our burn center from January 2014 to December 2019 were retrospectively analyzed. Spearman's Rank correlation coefficient was used to analyze the factors influencing the elevated PCT levels. The predictive power of PCT for death was evaluated by receiver operating characteristic (ROC) and multiple logistic regression analysis. RESULTS: 72 cases exhibited elevated serum PCT concentrations during the shock phase, but none of them had obvious signs of infection. PCT level in the shock phase was positively correlated with burn area, depth, degree of inhalation injury, delay in fluid resuscitation, APACHE II, and SOFA scores. The peak values of PCT during shock and infection phases were significantly higher in the non-survivors than in the survivors. The areas under the ROC curve for predicting death were 0.788 and 0.926, respectively, and 5.4 ng/mL (OR = 5.33) and 8.5 ng/mL (OR = 14.49) were the high-risk thresholds for death prediction. CONCLUSIONS: Serum PCT level in the shock phase is a potential indicator for evaluating the severity of burns, while the PCT level during the infection period can be used as an early warning indicator for severe systemic infection. High levels of PCT peaks during the shock and infection periods indicate an increased risk of poor prognosis, and targeted treatment is required accordingly.

Nicotinamide mononucleotide promotes pancreatic islet function through the SIRT1 pathway in mice after severe burns.

OBJECTIVE: The high levels of oxidative stress and apoptosis of pancreatic islet cells after severe burns lead to the dysfunction of islets and glucose metabolism disorders. Silent information regulator of transcription 1 (SIRT1) can decrease oxidative stress and apoptosis of islets in diabetes mellitus. This study aimed to investigate the role of SIRT1 on pancreatic islets and whether nicotinamide mononucleotide (NMN) can impact the function of pancreatic islets after severe burns. METHODS: A 30% total body surface area full-thickness burn model was established using male C57BL/6 mice, and mice were randomized into sham group, burn group, burn + NMN group and burn + NMN + EX-527 group. The concentration of nicotinamide adenine dinucleotide (NAD), the expression of SIRT1, apoptosis induction, mitochondrial function and related signalling pathways of pancreatic islets at 24 h after severe burns were tested. RESULTS: Severe burns led to decreased NAD level and SIRT1 expression of pancreatic islets, increased apoptosis rate, and mitochondrial dysfunction of pancreatic islets. NAD repletion by NMN and upregulation of SIRT1 expression reduced the phosphorylation and acetylation levels of NF-κB p65 and burn-induced apoptosis. Meanwhile, the mitochondrial function of islets was rescued by NMN treatment through the SIRT1/UCP2 axis and SIRT1/PGC1-α axis. In addition, the fasting blood glucose decreased and glucose-stimulated insulin secretion was improved with NMN treatment after severe burns. This protective effect of NMN could be abolished by EX-527, the inhibitor of SIRT1. CONCLUSION: NMN can increase the concentration of NAD+ of pancreatic islets and regulate SIRT1 and its downstream targets, thereby reducing apoptosis, maintaining mitochondrial function and improving pancreatic islet function after severe burn injury.

Mitochondria play a key role in oxidative stress-induced pancreatic islet dysfunction after severe burns.

BACKGROUND: Severe burns are often complicated with hyperglycemia in part caused by pancreatic islet dysfunction. Previous studies have revealed that in diabetes mellitus, the pancreatic islet dysfunction is partly attributed to oxidative stress. However, the role and mechanism of oxidative stress in hyperglycemia after severe burns remain unclear. Therefore, the purpose of this study was to explore the level and mechanism of oxidative stress in pancreatic islets after severe burns and the antioxidant effect of sodium pyruvate. METHODS: A 30% total body surface area full-thickness burn model was established using male C57BL/6 mice. Fasting blood glucose and glucose-stimulated insulin secretion (GSIS) 24 hours post severe burns were detected. The levels of reactive oxygen species (ROS) and mitochondrial ROS of islets were detected. The activities of complexes in the mitochondrial respiratory chain of islets were measured. The main antioxidant defense system, glutaredoxin system, and thioredoxin system-related indexes were detected, and the expression of manganese superoxide dismutase (Mn-SOD) was measured. In addition, the antioxidant activity of sodium pyruvate was evaluated post severe burns. RESULTS: After severe burns, fasting blood glucose levels increased, while GSIS levels decreased, with significantly elevated ROS levels of pancreatic islets. The activity of complex III decreased and the level of mitochondrial ROS increased significantly post severe burns. For the detoxification of ROS, the expressions of thioredoxin 2, thioredoxin reductase 2, and Mn-SOD located in mitochondria decreased. Sodium pyruvate reduced the level of mitochondrial ROS in islet cells and improved the GSIS of islets after severe burns. CONCLUSION: The high level of mitochondrial ROS of islets is caused by reducing the activity of complex III in mitochondrial respiratory chain, inhibiting mitochondrial thioredoxin system, and downregulating Mn-SOD post severe burns. Sodium pyruvate plays an antioxidant role post severe burns in mice islets and improves the islet function.

Tofu processing wastewater as a low-cost substrate for high activity nattokinase production using Bacillus subtilis.

BACKGROUND: Even though tofu is a traditional Chinese food loved by Asian people the wastewater generated during the production of tofu can pollute the environment, and the treatment of this generated wastewater can increase the operating cost of the plant. In this study, the production of nattokinase could be achieved by using the nitrogen source in tofu processing wastewater (TPW) instead of using the traditional nattokinase medium. This meets the need for the low-cost fermentation of nattokinase and at the same time addresses the environmental pollution concerns caused by the wastewater. Bacillus subtilis 13,932 is, a high yielding strain of nattokinase, which is stored in our laboratory. To increase the activity of nattokinase in the tofu process wastewater fermentation medium, the medium components and culture parameters were optimized. Nattokinase with high enzymatic activity was obtained in 7 L and 100 L bioreactors when TPW was used as the sole nitrogen source catalyzed by Bacillus subtilis. Such a result demonstrates that the production of nattokinase from TPW fermentation using B. subtilis can be implemented at an industrial level. RESULTS: The peptide component in TPW is a crucial factor in the production of nattokinase. Box-Behnken design (BBD) experiments were designed to optimize various critical components, i.e., Glucose, TPW, MgSO4·7H2O, CaCl2, in nattokinase fermentation media. A maximum nattokinase activity was recorded at 37 °C, pH 7.0, 70 mL liquid medium, and 200 rpm. The highest nattokinase activities obtained from 7 to 100 L bioreactors were 8628.35 ± 113.87 IU/mL and 10,661.97 ± 72.47 IU/mL, respectively. CONCLUSIONS: By replacing the nitrogen source in the original medium with TPW, there was an increase in the enzyme activity by 19.25% after optimizing the medium and culture parameters. According to the scale-up experiment from conical flasks to 100 L bioreactors, there was an increase in the activity of nattokinase by 47.89%.

Expression, Characterization and Its Deinking Potential of a Thermostable Xylanase From Planomicrobium glaciei CHR43.

Genome mining is more and more widely used in identifying new enzymes from database. In the present study, we reported a putative xylanase, Pg-Xyn (WP_053166147.1), which originated from a psychrotolerant strain Planomicrobium glaciei CHR 43, and was identified from Genbank by genome mining. Sequence analysis and homology modeling showed that Pg-Xyn belongs to glycosyl hydrolase family 10. On the basis of heterologous expression in E. coli and biochemical characterization, we found Pg-Xyn was most active at pH 9.0 and 80°C and exhibited good stability from pH 5.0 to 12.0 and below 90°C. Pg-Xyn was slightly activated in the presence of Ca2+ and Mg2+, while it was strongly inhibited by Mn2+. The analysis of hydrolysis products showed that Pg-Xyn was an endo-ß-1,4-xylanase. In addition, Pg-Xyn performed good deinking ability in a paper deinking test. In consideration of its unique properties, Pg-Xyn might be a promising candidate for application in the paper and pulp industries.