Engineered acetogenic bacteria as microbial cell factory for diversified biochemicals.

Acetogenic bacteria (acetogens) are a class of microorganisms with conserved Wood-Ljungdahl pathway that can utilize CO and CO2/H2 as carbon source for autotrophic growth and convert these substrates to acetate and ethanol. Acetogens have great potential for the sustainable production of biofuels and bulk biochemicals using C1 gases (CO and CO2) from industrial syngas and waste gases, which play an important role in achieving carbon neutrality. In recent years, with the development and improvement of gene editing methods, the metabolic engineering of acetogens is making rapid progress. With introduction of heterogeneous metabolic pathways, acetogens can improve the production capacity of native products or obtain the ability to synthesize non-native products. This paper reviews the recent application of metabolic engineering in acetogens. In addition, the challenges of metabolic engineering in acetogens are indicated, and strategies to address these challenges are also discussed.

Alginate oligosaccharide assimilation by gut microorganisms and the potential role in gut inflammation alleviation.

Dietary fiber metabolism by gut microorganisms plays important roles in host physiology and health. Alginate, the major dietary fiber of daily diet seaweeds, is drawing more attention because of multiple biological activities. To advance the understanding of alginate assimilation mechanism in the gut, we show the presence of unsaturated alginate oligosaccharides (uAOS)-specific alginate utilization loci (AUL) in human gut microbiome. As a representative example, a working model of the AUL from the gut microorganism Bacteroides clarus was reconstructed from biochemistry and transcriptome data. The fermentation of resulting monosaccharides through Entner-Doudoroff pathway tunes the metabolism of short-chain fatty acids and amino acids. Furthermore, we show that uAOS feeding protects the mice against dextran sulfate sodium-induced acute colitis probably by remodeling gut microbiota and metabolome. IMPORTANCE: Alginate has been included in traditional Chinese medicine and daily diet for centuries. Recently discovered biological activities suggested that alginate-derived alginate oligosaccharides (AOS) might be an active ingredient in traditional Chinese medicine, but how these AOS are metabolized in the gut and how it affects health need more information. The study on the working mechanism of alginate utilization loci (AUL) by the gut microorganism uncovers the role of unsaturated alginate oligosaccharides (uAOS) assimilation in tuning short-chain fatty acids and amino acids metabolism and demonstrates that uAOS metabolism by gut microorganisms results in a variation of cell metabolites, which potentially contributes to the physiology and health of gut.

Recent trends in preparation and biomedical applications of iron oxide nanoparticles.

The iron oxide nanoparticles (IONPs), possessing both magnetic behavior and semiconductor property, have been extensively used in multifunctional biomedical fields due to their biocompatible, biodegradable and low toxicity, such as anticancer, antibacterial, cell labelling activities. Nevertheless, there are few IONPs in clinical use at present. Some IONPs approved for clinical use have been withdrawn due to insufficient understanding of its biomedical applications. Therefore, a systematic summary of IONPs' preparation and biomedical applications is crucial for the next step of entering clinical practice from experimental stage. This review summarized the existing research in the past decade on the biological interaction of IONPs with animal/cells models, and their clinical applications in human. This review aims to provide cutting-edge knowledge involved with IONPs' biological effects in vivo and in vitro, and improve their smarter design and application in biomedical research and clinic trials.

Advanced application of nanotechnology in active constituents of Traditional Chinese Medicines.

Traditional Chinese Medicines (TCMs) have been used for centuries for the treatment and management of various diseases. However, their effective delivery to targeted sites may be a major challenge due to their poor water solubility, low bioavailability, and potential toxicity. Nanocarriers, such as liposomes, polymeric nanoparticles, inorganic nanoparticles and organic/inorganic nanohybrids based on active constituents from TCMs have been extensively studied as a promising strategy to improve the delivery of active constituents from TCMs to achieve a higher therapeutic effect with fewer side effects compared to conventional formulations. This review summarizes the recent advances in nanocarrier-based delivery systems for various types of active constituents of TCMs, including terpenoids, polyphenols, alkaloids, flavonoids, and quinones, from different natural sources. This review covers the design and preparation of nanocarriers, their characterization, and in vitro/vivo evaluations. Additionally, this review highlights the challenges and opportunities in the field and suggests future directions for research. Nanocarrier-based delivery systems have shown great potential in improving the therapeutic efficacy of TCMs, and this review may serve as a comprehensive resource to researchers in this field.

Three-dimensional visualization and evaluation of hilar cholangiocarcinoma resectability and proposal of a new classification.

BACKGROUND: As digital medicine has exerted profound influences upon diagnosis and treatment of hepatobiliary diseases, our study aims to investigate the accuracy of three-dimensional visualization and evaluation (3DVE) system in assessing the resectability of hilar cholangiocarcinoma (hCCA), and explores its potential clinical value. MATERIALS AND METHODS: The discovery cohort, containing 111 patients from April 2013 to December 2019, was retrospectively included to determine resectability according to revised criteria for unresectability of hCCA. 3D visualization models were reconstructed to evaluate resectability parameters including biliary infiltration, vascular involvement, hepatic atrophy and metastasis. Evaluation accuracy were compared between contrast-enhanced CT and 3DVE. Logistic analysis was performed to identify independent risk factors of R0 resection. A new comprehensive 3DVE classification of hCCA based on factors influencing resectability was proposed to investigate its role in predicting R0 resection and prognosis. The main outcomes were also analyzed in cohort validation, including 34 patients from January 2020 to August 2022. RESULTS: 3DVE showed an accuracy rate of 91% (95%CI 83.6-95.4%) in preoperatively evaluating hCCA resectability, significantly higher than 81% (95%CI 72.8-87.7%) of that of CT (p = 0.03). By multivariable analysis, hepatic artery involvement in 3DVE was identified an independent risk factor for R1 or R2 resection (OR = 3.5, 95%CI 1.4,8.8, P < 0.01). New 3DVE hCCA classification was valuable in predicting patients' R0 resection rate (p < 0.001) and prognosis (p < 0.0001). The main outcomes were internally validated. CONCLUSIONS: 3DVE exhibited a better efficacy in evaluating hCCA resectability, compared with contrast-enhanced CT. Preoperative 3DVE demonstrated hepatic artery involvement was an independent risk factor for the absence of R0 margin. 3DVE classification of hCCA was valuable in clinical practice.

Reconstruction of a Large Oncologic Defect Involving Lower Eyelid and Infraorbital Cheek Using MSTAFI Flap.

An oncologic defect that includes both the lower eyelid and the infraorbital cheek often results in complex reconstructive problems because its reconstruction involves 2 distinct tissue types and cosmetic subunits. Herein, we first present a novel combination of modified supratrochlear artery forehead island flap and advancement rotation cheek flap enables reconstructing a large oncologic defect of lower eyelid and infraorbital cheek. Although discoid lupus erythematosus affects the skin, the patient had achieved a satisfying color match and an acceptable aesthetic restoration without tumor recurrence. This novel flap has shown to be feasible, reliable, and advantageous alternative to the repair of such defects.

A formal [4 + 2] annulation of diamines and prop-2-ynyl sulfonium salts for the synthesis of tetrahydroquinoxalines.

A formal [4 + 2] annulation of diamines and prop-2-ynyl sulfonium salts was developed. This strategy enables efficient access to tetrahydroquinoxalines in excellent yields.

Identification of antimalarial targets of chloroquine by a combined deconvolution strategy of ABPP and MS-CETSA.

BACKGROUND: Malaria is a devastating infectious disease that disproportionally threatens hundreds of millions of people in developing countries. In the history of anti-malaria campaign, chloroquine (CQ) has played an indispensable role, however, its mechanism of action (MoA) is not fully understood. METHODS: We used the principle of photo-affinity labeling and click chemistry-based functionalization in the design of a CQ probe and developed a combined deconvolution strategy of activity-based protein profiling (ABPP) and mass spectrometry-coupled cellular thermal shift assay (MS-CETSA) that identified the protein targets of CQ in an unbiased manner in this study. The interactions between CQ and these identified potential protein hits were confirmed by biophysical and enzymatic assays. RESULTS: We developed a novel clickable, photo-affinity chloroquine analog probe (CQP) which retains the antimalarial activity in the nanomole range, and identified a total of 40 proteins that specifically interacted and photo-crosslinked with CQP which was inhibited in the presence of excess CQ. Using MS-CETSA, we identified 83 candidate interacting proteins out of a total of 3375 measured parasite proteins. At the same time, we identified 8 proteins as the most potential hits which were commonly identified by both methods. CONCLUSIONS: We found that CQ could disrupt glycolysis and energy metabolism of malarial parasites through direct binding with some of the key enzymes, a new mechanism that is different from its well-known inhibitory effect of hemozoin formation. This is the first report of identifying CQ antimalarial targets by a parallel usage of labeled (ABPP) and label-free (MS-CETSA) methods.

Celastrol mitigates inflammation in sepsis by inhibiting the PKM2-dependent Warburg effect.

BACKGROUND: Sepsis involves life-threatening organ dysfunction and is caused by a dysregulated host response to infection. No specific therapies against sepsis have been reported. Celastrol (Cel) is a natural anti-inflammatory compound that shows potential against systemic inflammatory diseases. This study aimed to investigate the pharmacological activity and molecular mechanism of Cel in models of endotoxemia and sepsis. METHODS: We evaluated the anti-inflammatory efficacy of Cel against endotoxemia and sepsis in mice and macrophage cultures treated with lipopolysaccharide (LPS). We screened for potential protein targets of Cel using activity-based protein profiling (ABPP). Potential targets were validated using biophysical methods such as cellular thermal shift assays (CETSA) and surface plasmon resonance (SPR). Residues involved in Cel binding to target proteins were identified through point mutagenesis, and the functional effects of such binding were explored through gene knockdown. RESULTS: Cel protected mice from lethal endotoxemia and improved their survival with sepsis, and it significantly decreased the levels of pro-inflammatory cytokines in mice and macrophages treated with LPS (P < 0.05). Cel bound to Cys424 of pyruvate kinase M2 (PKM2), inhibiting the enzyme and thereby suppressing aerobic glycolysis (Warburg effect). Cel also bound to Cys106 in high mobility group box 1 (HMGB1) protein, reducing the secretion of inflammatory cytokine interleukin (IL)-1ß. Cel bound to the Cys residues in lactate dehydrogenase A (LDHA). CONCLUSION: Cel inhibits inflammation and the Warburg effect in sepsis via targeting PKM2 and HMGB1 protein.

Biofuel and chemical production from carbon one industry flux gas by acetogenic bacteria.

Carbon one industry flux gas generated from fossil fuels, various industrial and domestic waste, as well as lignocellulosic biomass provides an innovative raw material to lead the sustainable development. Through the chemical and biological processing, the gas mixture composed of CO, CO2, and H2, also termed as syngas, is converted to biofuels and high-value chemicals. Here, the syngas fermentation process is elaborated to provide an overview. Sources of syngas are summarized and the influences of impurities on biological fermentation are exhibited. Acetogens and carboxydotrophs are the two main clusters of syngas utilizing microorganisms, their essential characters are presented, especially the energy metabolic scheme with CO, CO2, and H2. Synthetic biology techniques and microcompartment regulation are further discussed and proposed to create a high-efficiency cell factory. Moreover, the influencing factors in fermentation and products in carboxylic acids, alcohols, and others such like polyhydroxyalkanoate and poly-3-hydroxybutyrate are addressed. Biological fermentation from carbon one industry flux gas is a promising alternative, the latest scientific advances are expatiated hoping to inspire more creative transformation.

Prophylactic Closed Suction Drainage Is Irrelevant to Accelerated Rehabilitation after Open Reduction and Internal Fixation for Closed Distal Femur Fractures.

OBJECTIVE: To investigate whether closed suction drainage (CSD) is related to accelerated rehabilitation of patients after open reduction and internal fixation (ORIF) for closed distal femur fractures. METHODS: This study was a prospective, randomized controlled clinical trial. Between October 2018 and June 2020, 160 closed distal femur fracture patients who were prepared for ORIF were prospectively randomized into two groups: a CSD group with the mean age of 57.91 ± 14.38 years (32 [40%] men and 48 [60%] women) and a non-CSD group with the mean age of 59.73 ± 17.55 years (27 [34%] men and 54 [66%] women). Wound visual analogue scale (VAS) pain scores, peri-wound skin temperature, hematocrit (Hct), hemoglobin (Hb) concentrations, hidden blood loss (HBL), dressing change, period of wound oozing, postoperative blood transfusion, and length of postoperative hospital stay were recorded. Postoperative wound complications, namely wound infections, wound haematoma, wound dehiscence, erythema of wound, and lower limb deep vein thrombosis (DVT) were collected. All the patients were administrated by a single surgical team and followed up for 1 month after the ORIF. RESULTS: The patients without CSD were identified with lower peri-wound skin temperature and wound VAS pain scores during the first three postoperative days (36.69 ± 0.33 vs 36.86 ± 0.38 °C, P = 0.002; 1.88 ± 0.82 vs 3.15 ± 1.15, P = 0.000). However, both the peri-wound skin temperature and wound VAS pain scores did not differ significantly between the two groups on the fifth postoperative day. In addition, patients with CSD had a longer length of postoperative hospitalization time (11.45 ± 5.95 vs 9.78 ± 4.64 days, P = 0.049). There was no statistically significant difference between CSD and non-CSD groups within 1 month after the ORIF regarding blood loss, period of wound oozing, and postoperative complications, such as incidence of wound infection, haematoma, erythema, dehiscence, and lower limb DVT. CONCLUSION: Prophylactic CSD after primary ORIF for closed distal femur fractures not only had no significant advantage to minimize blood loss and wound complications, but increased local inflammation and postoperative hospital stay, and thus we suggest that prophylactic CSD after primary ORIF for closed distal femur fractures is not recommended for optimized clinical pathways and accelerated recovery.

Age-related dynamic deformation of the femoral shaft and associated osteoporotic factors: a retrospective study in Chinese adults.

Dynamic skeletal deformation with ageing showed important signs of degenerative and osteoporotic diseases. We found that both femoral bowing and cortical thickness were correlated with ageing in a Chinese population. Further, femoral cortical index, an osteoporotic indicator, was negatively correlated with femoral bowing angle. Hence, more attention should be paid to these femoral morphological changes to avoid fragility fractures and failed internal fixation. PURPOSE: The purpose of this study was to determine whether morphological parameters of the femoral shaft are in age-related deformation and identify correlations between parameters of femoral cortical thickness and femoral shaft bowing. METHODS: One hundred twenty patients (mean 50 years, range 18~104 years) who had received standard long-standing anteroposterior and femoral lateral radiographs from October 2016 to October 2019 were included in this retrospective study. The sagittal femoral bowing angle (sFBA), sagittal femoral cortical index (sFCI), coronal femoral bowing angle (cFBA), and coronal femoral cortical index (cFCI) were measured by two orthopaedists separately. All the participants' demographic data, including age, sex, body laterality, height, and weight, were collected. The Student's t test, Mann-Whitney U test, two-way ANOVA, Pearson correlation, and multiple linear regression were used in the statistical analysis. RESULTS: The mean age of the male and female participants was 46.95 ± 15.25 and 52.22 ± 15.61 years, respectively. Two-way ANOVA revealed that females had a significantly lower sFCI than males at the right side (P < 0.05). There were no significant interactions between sex or body laterality and the sFBA, cFBA, sFCI, and cFCI groups (P > 0.05). Pearson correlation revealed that sFCI was strongly correlated with sFBA (r = - 0.535, P < 0.05) and cFBA (r = - 0.535, P < 0.05). Multiple linear regression analysis demonstrated that both age (ß = 0.304 and 0.308 respectively) and sFCI (ß = - 0.322 and - 0.414 respectively) were two independent predictors associated with sFBA and cFBA respectively. CONCLUSIONS: The femoral shaft bowing of the Chinese population was positively correlated with ageing, whereas the sagittal femoral cortical thickness negatively correlated with ageing. A high FBA occurred in femoral shafts with a low sFCI, which revealed that femoral shaft bowing was associated with femoral cortical thickness. During femur-related surgery in older patients, more attention should be paid to these femoral morphological changes.

Axonal iron transport in the brain modulates anxiety-related behaviors.

Iron is essential for a broad range of biochemical processes in the brain, but the mechanisms of iron metabolism in the brain remain elusive. Here we show that iron functionally translocates among brain regions along specific axonal projections. We identified two pathways for iron transport in the brain: a pathway from ventral hippocampus (vHip) to medial prefrontal cortex (mPFC) to substantia nigra; and a pathway from thalamus (Tha) to amygdala (AMG) to mPFC. While vHip-mPFC transport modulates anxiety-related behaviors, impairment of Tha-AMG-mPFC transport did not. Moreover, vHip-mPFC iron transport is necessary for the behavioral effects of diazepam, a well-known anxiolytic drug. By contrast, genetic or pharmacological promotion of vHip-mPFC transport produced anxiolytic-like effects and restored anxiety-like behaviors induced by repeated restraint stress. Taken together, these findings provide key insights into iron metabolism in the brain and identify the mechanisms underlying iron transport in the brain as a potential target for development of novel anxiety treatments.

[Cloning, expression, purification and identification of multi-potent transcription factor CTCF recombinant and its segments].

OBJECTIVE: To clone ctc f cDNA, N, Zn and C fragments separately into expresstion vector, purify and identify the expressed proteins. METHODS: Using the recombinant plasmid pGEM7Zf (-)-ctc f as template for PCR, pGEX-4T-2-ctc f, pGEX-4T-2-ctc f-N, pGEX-4T-2- ctc f-Zn and pGEX-4T-2- ctc f-C recombinants were constructed successfully. After transformed into E. coli BL21 cells, the recombinants were confirmed by enzyme digestion and sequence analysis. After optimizing the IPTG inducing condition, the purified GST fusion proteins with affinity chromatography were conformed by Far-Western blotting. RESULTS: The recombinant plasmids pGEX-4T-2-ctc f, pGEX-4T-2- ctc f-N, pGEX-4T-2-ctc f-Zn and pGEX-4T-2-ctc f-C were confirmed by restriction enzyme assay and sequencing. All GST fusion proteins, CTCF, CTCF-N, CTCF-Zn and CTCF-C were successfully expressed at the optimal parameters and purified with affinity chromatography, and specifically recognized by anti-GST antibody. CONCLUSION: Ctc f, ctc f-n, ctc f-Zn and ctc f-c gene recombinants were constructed successfully and their corresponding fusion proteins were successfully purified with affinity chromatography and identified.

[Reactivation of hypermethylated GSTP1 promoter activity in MCF-7 cells by treatment with a component of natural drug].

OBJECTIVE: To investigate the reactivation of hypermethylated GSTP1 (glutathione S-transferase P1) promoter activity by a component of natural drug, CDP. METHODS: The recombinant plasmid of pGL3-GSTP1 pro(m) containing hypermethylated GSTP1 gene promoter was constructed and then used to transiently transfect the human breast cancer cell line MCF-7 cell. After treatment with CDP and 5-aza-C, The luciferase activity in cell lysates were assayed. RESULTS: Low promoter activities were found in hypermethylated GSTP1 promoter. The promoter activities were reactivated and in a CDP dose-dependent mode. CONCLUSION: CDP has the ability to reactivate the hypermethylated GSTP1 gene promoter activity.