Irreversible biosynthesis of D-allulose from D-glucose in Escherichia coli through fine-tuning of carbon flux and cofactor regeneration engineering.

BACKGROUND: As a rare hexose with low calories and various physiological functions, d-allulose has drawn increasing attention. The current industrial production of d-allulose from d-fructose or d-glucose is achieved via epimerization based on the Izumoring strategy; however, the inherent reaction equilibrium during reversible reaction limits its high conversion yield. Although the conversion of d-fructose to d-allulose could be enhanced via phosphorylation-dephosphorylation mediated by metabolic engineering, biomass reduction and byproduct accumulation remain a largely unresolved issue. RESULTS: After modifying the glycolytic pathway of Escherichia coli and optimizing the whole-cell reaction condition, the engineered strain produced 7.57 ± 0.61 g L-1 d-allulose from 30 g L-1 d-glucose after 24 h of catalysis. By developing an ATP regeneration system for enhanced substrate phosphorylation, the cell growth inhibition was alleviated and d-allulose production increased by 55.3% to 11.76 ± 0.58 g L-1 (0.53 g g-1 ). Fine-tuning of carbon flux caused a 48% reduction in d-fructose accumulation to 1.47 ± 0.15 g L-1 . After implementing fed-batch co-substrate strategy, the d-allulose titer reached 15.80 ± 0.31 g L-1 (0.62 g g-1 ) with a d-glucose conversion rate of 84.8%. CONCLUSION: The present study reports a novel strategy for high-yield d-allulose production from low-cost substrate. © 2023 Society of Chemical Industry.

Identification of potential feature genes in non-alcoholic fatty liver disease using bioinformatics analysis and machine learning strategies.

The prevalence of non-alcoholic fatty liver disease (NAFLD) and NAFLD-associated hepatocellular carcinoma (HCC) has continuously increased in recent years. Machine learning is an effective method for screening the feature genes of a disease for prediction, prevention and personalized treatment. Here, we used the "limma" package and weighted gene co-expression network analysis (WGCNA) to screen 219 NAFLD-related genes and found that they were mainly enriched in inflammation-related pathways. Four feature genes (AXUD1, FOSB, GADD45B, and SOCS2) were screened by LASSO regression and support vector machine-recursive feature elimination (SVM-RFE) machine learning algorithms. Therefore, a clinical diagnostic model with an area under the curve (AUC) value of 0.994 was constructed, which was superior to other indicators of NAFLD. Significant correlations existed between feature genes expression and steatohepatitis histology or clinical variables. These findings were also validated in external datasets and a mouse model. Finally, we found that feature genes expression was significantly decreased in NAFLD-associated HCC and that SOCS2 may be a prognostic biomarker. Our findings may provide new insights into the diagnosis, prevention and treatment targets of NAFLD and NAFLD-associated HCC.

A deep learning-based model for detecting depression in senior population.

Objectives: With the attention paid to the early diagnosis of depression, this study tries to use the biological information of speech, combined with deep learning to build a rapid binary-classification model of depression in the elderly who use Mandarin and test its effectiveness. Methods: Demographic information and acoustic data of 56 Mandarin-speaking older adults with major depressive disorder (MDD), diagnosed with the Mini-International Neuropsychiatric Interview (MINI) and the fifth edition of Diagnostic and Statistical Manual of Mental Disorders (DSM-5), and 47 controls was collected. Acoustic data were recorded using different smart phones and analyzed by deep learning model which is developed and tested on independent validation set. The accuracy of the model is shown by the ROC curve. Results: The quality of the collected speech affected the accuracy of the model. The initial sensitivity and specificity of the model were respectively 82.14% [95%CI, (70.16-90.00)] and 80.85% [95%CI, (67.64-89.58)]. Conclusion: This study provides a new method for rapid identification and diagnosis of depression utilizing deep learning technology. Vocal biomarkers extracted from raw speech signals have high potential for the early diagnosis of depression in older adults.

Activation of STAT6 by intranasal allergens correlated with the development of eosinophilic chronic rhinosinusitis in a mouse model.

Eosinophilic chronic rhinosinusitis (ECRS) is a chronic inflammatory disease characterized by prominent eosinophilic infiltration along with a T-helper-2 (Th2) response. It has been well documented that signal transducer and activator of transcription 6 (STAT6) is a nuclear transcription factor that mediates Th2-type immunity and is implicatory of STAT1 and STAT3 in the pathogenesis of allergic airway diseases. However, little is known about the association between STATs and ECRS. Here, we explored the relationship between STAT1, STAT3, and/or STAT6 and eosinophilic inflammation accompanied by Th2-type immunity in a mouse model of ECRS. An ovalbumin (OVA)-staphylococcal enterotoxin B (SEB)-induced ECRS murine model was first established. The mucosal histological alterations were determined using hematoxylin and eosin staining. The number of eosinophils in peripheral blood was measured using a blood cell analyzer. The cytokine (IL-4, IL-5, IL17 A and IFN-γ) expression levels in the sinonasal mucosa and total and OVA-specific IgE from serum were measured using ELISA. Then, the protein levels of STAT1, STAT3, STAT6, phosphorylated STAT1 (p-STAT1), p-STAT3, p-STAT6, T-box expressed in T-cells (T-bet), GATA binding protein 3 (GATA-3), and retinoic acid receptor-related orphan receptor γ (RORγt) in the sinonasal mucosa were examined by immunohistochemical staining or Western blotting. Local administration of OVA combined with SEB (OVA + SEB) induced multiple polyp-like lesions, accompanied by prominent eosinophilic infiltration in the sinonasal mucosa. The OVA- and OVA+SEB-treated groups showed significantly higher eosinophil counts from peripheral blood and total and OVA-specific IgE levels from serum than those in the PBS- and SEB-treated groups. The levels of p-STAT6 were markedly increased by OVA + SEB exposure, as well as GATA-3, IL-4, and IL-5, but did not affect STAT6, p-STAT1, p-STAT3, T-bet, RORγt, IFN-γ, or IL-17A. Furthermore, an eosinophil count in the sinonasal mucosa showed a positive correlation with the level of p-STAT6 in the ECRS mouse model. Signal transducer and activator of transcription 6 signaling could be activated in the OVA+SEB-induced ECRS model and might be a crucial signal transducer in the development of Th2-skewed ECRS.

Association of Antibiotic Alterations in Gut Microbiota With Decreased Osseointegration of an Intramedullary Nail in Mice With and Without Osteomyelitis.

Treatment of osteomyelitis requires prolonged antibiotic therapy which significantly alters the gut microbiota. While the influences on bone mass and microstructure have been extensively studied, it is poorly understood what impact the changes in gut microbiota may have on the host response to osseointegration around an intramedullary nail implanted. Here, we explored the influence of gut microbiota on the bone osseointegration process around an implant under two conditions: implantation of an intramedullary nail in the bone marrow cavity and chronic osteomyelitis (CO) induced by Staphylococcus aureus infection. Body weight, hepatorenal functions, serum levels of proinflammatory cytokines were monitored. The composition of gut microbiota was assessed via 16S rRNA sequencing, and the bone condition was analyzed via micro-computed tomography, hematoxylin and eosin staining, Safranin O-fast green and Goldner's trichrome staining. Osteoblastogenesis and osteoclastogenesis were assessed by detecting tartrate-resistant acid phosphatase and osterix expression. We found that perturbation of gut microbiota (increase in Proteobacteria and decrease in Bacteroidetes) associated with delayed osseointegration and increased levels of proinflammatory cytokines in the serum (p<0.05), lower bone mass (p<0.05), deficient endochondral ossification and bone formation, reduced osteoblastogenesis (p<0.05) and enhanced osteoclastogenesis (p<0.001). Survival rates (p=0.002) and bacterial loads (p=0.0363) in bone differed significantly between the CO and antibiotic-treated CO mice, but cytokines levels, bone mineral density, and bone formation did not differ, likely because of the severely damaged bone structure. In summary, antibiotic treatment perturbed the gut microbiota and significantly interfered with the bone osseointegration around the nail by increasing proinflammatory cytokine levels in circulation, inhibiting osteoblastogenesis, enhancing osteoclastogenesis, and thus leading to higher pathogen colonization as well as higher mortality postinfection. This report of ours is the first to demonstrate antibiotic-induced alterations in the gut microbiota affect bone osseointegration, helping us understand the role of gut microbiota disorders in osteoblastogenesis and osteoclastogenesis following implant insertion with or without infection.

Phosphoproteomics and Proteomics Reveal Metabolism as a Key Node in LPS-Induced Acute Inflammation in RAW264.7.

To better understand the acute inflammatory mechanisms, the modulation, and to investigate the key node in predicting inflammatory diseases, high-sensitivity LC-MS/MS-based proteomics and phosphoproteomics approaches were used to identify differential proteins in RAW264.7 macrophages with lipopolysaccharide (LPS). Furthermore, differential proteins and their main biological process, as well as signaling pathways, were analyzed through bioinformatics techniques. The biological process comparison revealed 219 differential proteins and 405 differential phosphorylation proteins, including major regulatory factors of metabolism (PFKL, PGK1, GYS1, ACC, HSL, LDHA, RAB14, PRKAA1), inflammatory signaling transduction (IKKs, NF-κB, IRAK, IKBkb, PI3K, AKT), and apoptosis (MCL-1, BID, NOXA, SQSTM1). Label-free proteome demonstrated canonical inflammation signaling pathways such as the TNF signaling pathway, NF-κB signaling pathway, and NOD-like receptor signaling pathway. Meanwhile, phosphoproteome revealed new areas of acute inflammation. Phosphoproteomics profiled that glycolysis was enhanced and lipid synthesis was increased. Overall, the AMPK signaling pathway is the key regulatory part in macrophages. These revealed that the early initiation phase of acute inflammation primarily regulated the phosphoproteins of glucose metabolic pathway and lipid synthesis to generate energy and molecules, along with the enhancement of pro-inflammatory factors, and further induced apoptosis. Phosphoproteomics provides new evidence for a complex network of specific but synergistically acting mechanisms confirming that metabolism has a key role in acute inflammation.

Amelioration of dry eye syndrome in db/db mice with diabetes mellitus by treatment with Tibetan Medicine Formula Jikan Mingmu Drops.

ETHNOPHARMACOLOGICAL RELEVANCE: Jikan Mingmu Drops (JMD), a traditional Tibetan medicine containing six herbs, has been used to treat dry eye syndrome (DES) in individuals with diabetes mellitus. AIM OF STUDY: However, the activity of JMD ameliorates DES with diabetes mellitus has not been previously examined. The aim of the study is to investigate the molecular mechanism of JMD on db/db mice. MATERIALS AND METHODS: The main chemical constituents of JMD were analyzed by high-performance liquid chromatography and gas chromatography-mass spectrometry. DES was then induced in db/db mice by applying 0.2% benzalkonium chloride to the ocular surface for 7 days. Eye drops containing JMD (0.25, 0.5, or 1 g/mL) or vehicle subsequently were administered three times daily for another 7 days, and the therapeutic effects were evaluated by phenol red thread tear and sodium fluorescein tests. Conjunctival specimens were subjected to hematoxylin and eosin staining and periodic acid-Schiff staining to examine pathological changes and number of goblet cells. ELISA was performed to assess the levels of various inflammatory cytokines. RESULTS: JMD contains hydroxysafflor yellow A, magnoflorine, jatrorrhizine hydrochloride, palmatine hydrochloride, berberine hydrochloride, gallic acid, ellagic acid, tauroursodeoxycholic acid, camphor, isoborneol, borneol, trans-cinnamic acid, and muscone. JMD treatment significantly increased the tear volume, decreased the corneal fluorescein staining score, restored the morphology and structure of conjunctival epithelial cells, and markedly downregulated the levels of interleukin (IL)-6, IL-17α, IL-1ß, tumor necrosis factor-α, and vascular endothelial growth factor in the conjunctiva. Further data showed that these protective effects were accompanied by inhibition of inflammation in a dose-dependent manner. CONCLUSIONS: Amelioration of DES in db/db mice with diabetes mellitus by treatment with Tibetan medicine formula JMD maybe related to its anti-inflammatory effects.

[Effects of staphylococcal enterotoxin of different concentrations on the expression of GATA-3 and Th1/Th2 cytokines in the maxillary sinus mucosa of rabbits].

OBJECTIVE: The aim of this study was to determine the expression of GATA-3 and the level of Th1 and Th2 cytokines upon repeated exposure to staphylococcal enterotoxin B(SEB) of different concentrations in the maxillary sinus mucosa of rabbits. METHOD: The rabbits were randomly divided into 2 groups (24 rabbits per group): low-dose SEB group and high-dose SEB group. The low-dose SEB group and high-dose SEB group received daily injections of 0.6 ng of SEB (2 ml) and 60 ng of SEB (2 ml) into the left maxillary sinus of rabbits for 28 days, respectively. Concurrent treatment of the right maxillary sinus with normal saline was used as a control. Six rabbits chosen randomly in two groups were killed on days 3, 7, 14, and 28, and to obtain the sinus mucosa from the two-side maxillary sinuses for measurement. Mucosal levels of IL-2, IL-4, IL-5, and IFN-γ were measured using ABC-ELISA. Tissue expression of GATA-3 were examined using Real-time PCR and immunohistochemistry. RESULT: IFN-γ and IL-2 levels were significantly elevated in the high-dose SEB group compared with the low-dose SEB and control groups on days 7, 14, and 28 (P < 0.05). However, IL-4 and IL-5 levels were markedly enhanced in the low-dose SEB group compared with the high-dose SEB and control groups on days 14 and 28 (P < 0.05). Real-time PCR showed that the expression of GATA-3 mRNA in the low-dose SEB group was markedly enhanced, and immunohistochemical staining illustrated that the number of GATA-3 positive cells was markedly increased in the low-dose SEB group as compared with the high-dose SEB group (P < 0.05). No significant differences were observed in GATA-3 expression between the high-dose SEB and the control groups (P > 0.05). CONCLUSION: SEB promoted Th1 cytokines production at high concentrations, and enhanced Th2 cytokines expression and Th2 immune response at low concentrations.

Staphylococcal enterotoxin burden determines the type of T helper cell response and pathology of the maxillary sinus mucosa in rabbits.

Staphylococcal enterotoxin (SE) induces T lymphocyte activation along with nasal allergic inflammation during rhinosinusitis, but it is under debate on which types of T helper (Th) cells respond exclusively or whether they respond synergically. We hypothesize that their responses may vary based on dose of SE. To test this hypothesis, we initiated to determine the nature of the T cell response and pathological feature upon repeated exposure to staphylococcal enterotoxin A (SEA) at different doses in the maxillary sinus of rabbits. SEA (0.6 or 60 ng) was instilled daily into the left maxillary sinus of rabbits for 28 days. The right maxillary sinus receiving normal saline was used as control. Mucosal histological changes were examined by hematoxylin-eosin and toluidine blue staining. Tissue expression of myeloperoxidase (MPO), eosinophil cationic protein (ECP), T-box expressed in T cells (T-bet), and GATA binding protein 3 (GATA-3) were examined using immunohistochemistry. Mucosal levels of representative pro-inflammatory cytokines were measured using ELISA. SEA at 60 ng/day induced acute rhinosinusitis, as confirmed by CT scan. Histopathologic examination revealed epithelial disruption, subepithelial edema, and inflammatory cell infiltration. MPO and T-bet expression, as well as interleukin (IL)-2 and interferon (IFN)-γ levels, were up-regulated. However, 0.6 ng/day SEA did not cause discharge. Histological examination revealed prominent eosinophilic infiltration. ECP and GATA-3 expression, as well as IL-4 and IL-5 levels, were increased at this lower dose. In conclusion, SEA induces acute rhinosinusitis associated with a Th1-type immune response at high dose, and a predominantly Th2-biased allergic inflammation at low dose.

[The staphylococcal enterotoxin burden determines the ultrastructure of ciliated epithelia and inflammatory changes in maxillary sinus mucosa of rabbits].

OBJECTIVE: To investigate the ultrastructure of ciliated epithelia and inflammatory changes upon repeated exposure to staphylococcal enterotoxin A (SEA) of different concentrations in the maxillary sinus mucosa of rabbits. METHODS: The rabbits were randomly divided into 2 groups (24 rabbits per group): low-dose SEA group and high-dose SEA group. The low-dose SEA group and high-dose SEA group received daily injections of 0.6 ng of SEA (2 ml) and 60 ng of SEA (2 ml) into the left maxillary sinus of rabbits for 28 days, respectively. Concurrent treatment of the right maxillary sinus with normal saline was used as control. Six rabbits chosen randomly in two groups were examined by computed tomography (CT) scans and then sacrificed to obtain the sinus mucosa from the two-side of maxillary sinuses for histological assessment on days 3, 7, 14 and 28. To characterize the inflammatory changes of the sinus mucosa examined using light microscope, hematoxylin and eosin (HE) and toluidine blue staining was performed. Scanning and transmission electron microscopy were performed to observe ultrastructure of ciliated epithelia in the maxillary sinus mucosa. SPSS 13.0 software was used to analyze the data. RESULTS: On days 14 and 28, CT images showed opacification of the left maxillary sinus in the high-dose SEA group. The percentage of epithelial disruption was (22.73 ± 5.72) % and (30.79 ± 4.30)% in the high-dose SEA group respectively, and were significantly greater than those in the low-dose SEA group (5.12% ± 1.98% and 5.38% ± 1.64%, q value was 10.079 and 19.132) and control group (4.08% ± 1.29% and 4.81% ± 1.62%, q value was 11.016 and 19.592, respectively, all P < 0.01). The subepithelial thickness in the high-dose SEA group was (113.34 ± 14.81)µm and (120.86 ± 12.35) µm respectively, and were significantly different from those of the low-dose SEA group [(71.08 ± 10.39)µm and (81.63 ± 9.32)µm, q value was 8.090 and 8.782] and control group [(37.45 ± 7.67)µm and (38.79 ± 7.68)µm, q value was 15.759 and 19.541, all P < 0.01]. Viewed under the electron microscope, loss of cilia was observed, a few compound cilia and cytoplasmic protrusion were found, an obvious stretching of the endoplasmic reticulum and an obvious turgescence of the mitochondria was also observed. However, in the low-dose SEA group on days 14 and 28, CT scan of the left maxillary sinus showed transparency; light microscopy observations of the maxillary sinus mucosa showed the number of eosinophils was markedly increased as compared with the high-dose SEA and control groups, the differences were significant (q value was 5.871 and 6.766 on day 14, and q value was 7.572 and 8.970 on day 28, respectively, all P < 0.05). But no significant differences were observed in epithelial disruption between the low-dose SEA and the control groups on days 14 and 28 (q value was 1.512 and 0.859 respectively, all P > 0.05); inordinate array and adhesion of cilia was observed, but cilia loss, compound cilia, cytoplasmic protrusions, mitochondrial swelling and endoplasmic reticulum stretching were not found. CONCLUSIONS: SEA may induce allergic inflammation of the sinus mucosa without damaging the structure of ciliated epithelia at low concentration. Whereas SEA impairs the structure of mitochondria and endoplasmic reticulum in ciliated epithelial cells at high concentration, and results in cilia loss and epithelial disruption, which may be one of the main reasons to induce acute sinusitis.

[Observation on ultrastructure of maxillary sinus mucosa in experimental acute sinusitis].

OBJECTIVE: To observe ultrastructure of maxillary sinus mucosa of experimental acute sinusitis in rabbits. METHOD: Twenty-five rabbits were randomly divided into experimental group (20 rabbits) and blank control group (5 rabbits). We established a rhinogenic model of experimental acute sinusitis in experimental group. Five rabbits chosen randomly in experimental group were sacrificed and dissected after 1, 2, 3, and 4 weeks, and the tissue (0.3 cm x 0.3 cm) of sinus mucosa were prepared for visualization by transmission electron microscope (TEM). Animals in blank control group were sacrificed after 1 week. RESULT: Under the transmission electron microscope, in the blank control group, cilia of maxillary sinus mucosa lined up in order without ciliary loss, no stretched endoplasmic reticulum or turgescent mitochondria was observed. However, in the experimental group, inordinate array and loss of cilia was observed, a few compound cilia and cytoplasmic protrusion were also found. Both endoplasmic reticulum and mitochondria were swelling, and the lymphocytes were infiltrating with fibroblast proliferation in the submucosa. There was statistically significant difference between the experimental group and the blank control group (P < 0.05). In the experimental group, the number of compound cilia increased from 1 to 4 weeks, and the amount of compound cilia of the mucosa at 3 and 4 weeks was significantly higher than that at 1 week (P < 0.05). Swelling of mitochondria and endoplasmic reticulum was severe at 2 weeks and abated gradually with time, the results at 2 weeks were different from those of experimental group at 4 weeks (P < 0.05). CONCLUSION: The obstruction of nasal sinuses and the bacterial infection might lead to ultrastructural changes of maxillary sinus mucosa, and these ultrastructural changes were believed to the important processes of pathological changes in acute sinusitis.