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Abstract Halophilic proteins possess unique structural properties and show high stability under extreme conditions. This distinct characteristic makes them invaluable for application in various aspects such as bioenergy, pharmaceuticals, environmental clean‐up, and energy production. Generally, halophilic proteins are discovered and characterized through labor‐intensive and time‐consuming wet lab experiments. In this study, we introduce the Halophilic Protein Classifier (HPClas), a machine learning‐based classifier developed using the catBoost ensemble learning technique to identify halophilic proteins. Extensive in silico calculations were conducted on a large public dataset of 12,574 samples and HPClas achieved an area under the receiver operating characteristic curve (AUROC) of 0.844 on an independent test set of 200 samples. The source code and curated dataset of HPClas are publicly available at https://github.com/Showmake2/HPClas. In conclusion, HPClas can be explored as a promising tool to aid in the identification of halophilic proteins and accelerate their application in different fields.

Abstract Background To investigate the efficiency of a new method for the prevention of argentinian flag sign during the process of continuous, circular, and centered anterior capsulotomy (CCC) on the anterior capsule in cortically liquefied intumescent cataracts. This study was registered in an appropriate registry and the registration number of registration was xyy11[2022]-XJSFX-087; The date of of registration was 2022-04-29. Methods Preoperative examinations including slit-lamp examination, ocular A-scan ultrasonography, and Ultrasound Biomicroscopy (UBM) UBM were conducted on 61 patients with intumescent cataracts. Cases with cortically liquefied intumescent cataracts were selected and after staining with indocyanine green, the anterior chamber air bubble technique was used to compress the anterior capsule, and liquefied cortex was aspirated using a puncture needle. Corrected Distance Visual Acuity (CDVA) and intraocular pressure were recorded on postoperative days 1, 1 week, 1 month, and 6 months. Intraoperative and postoperative complications were documented and analyzed. Results Fifty eyes were identified as having cortically liquefied intumescent cataracts. No cases of the Argentinian flag sign occurred, and standard capsulorrhexis was achieved, facilitating smooth phacoemulsification. All patients achieved satisfactory outcomes at follow-ups of 1 day, 1 week, 1 month, and 6 months postoperatively. Mild corneal edema was observed in three cases on the first postoperative day, with no other complications noted. Conclusions The anterior chamber air bubble technique combined with cortical fluid release technique can prevent the occurrence of the Argentinian flag sign in cortically liquefied intumescent cataracts, this method is simple, convenient and economic for the clinical promotion.

Abstract Background Slow transit constipation (STC) is highly prevalent and has rising incidence. Shouhui Tongbian capsule (SHTB) is a traditional Chinese Medicine formula with extensive and highly efficacious usage in STC treatment, however, its mechanism of action, especially the regulation of microbiome and lipid metabolites, remains unclear. Methods After quality control of SHTB using LC‒MS to obtain its material basis, we tried to elucidate the cohesive modulatory network of SHTB against STC using hyphenated methods from microbiomics, lipidomics, mass spectrometry imaging (MSI) and molecular methods. Results SHTB could repair intestinal barrier damage, reduce systemic inflammation and increase intestinal motility in a diphenoxylate-induced STC rat model. Based on 16S rDNA sequencing results, SHTB rehabilitated the abnormal changes in Alloprevotella, Coprococcus, Marvinbryantia, etc., which were associated with STC symptoms. Meanwhile, microbial functional prediction showed that lipid metabolism was improved with SHTB administration. The differential lipids, including fatty acids, lysophosphatidylcholine, phosphatidylcholine, sphingomyelin triglyceride and ceramide, that are closely related to STC disease and SHTB efficacy. Furthermore, SHTB significantly reversed the abnormal expression of these key target enzymes in colon samples, including CTP-phosphocholine cytidylyltransferase, CTP-phosphoethanolamine cytidylyltransferase, phosphatidic acid phosphatase, acid sphingomyelinase etc. Conclusions Combined analysis demonstrated that SHTB reducing lipid accumulation and recovery of intestinal microbial homeostasis was the critical mechanism by which SHTB treats STC.

A. argyi is a renowned herb widely used in food and medicine, offering significant economic benefits. H. pylori, a known carcinogen, is associated with numerous diseases. Current treatments primarily rely on antibiotics, but rising resistance has reduced their efficacy. The activity and mechanisms of A. argyi against H. pylori infection remain unclear. A. argyi exhibited strong bacteriostatic and bactericidal effects against multiple H. pylori. It demonstrated no antagonistic effects when combined with antibiotics and disrupted H. pylori morphology and structure, downregulated various virulence genes, and inhibited urease activity. In co-culture systems, A. argyi reduced H. pylori adhesion, urease levels, and NO production, indicating immunomodulatory activity. Among the five components of A. argyi with anti-H. pylori activity, jaceosidin exhibited the strongest effect (MIC: 10 μg/mL), followed by eupatilin. Methylation of jaceosidin reduced its anti-H. pylori activity. A. argyi is an edible herb with anti- H. pylori activity and excellent safety.

BackgroundTriphala, is a composite of three individual botanical drugs: Terminalia chebula, Terminalia bellirica, and Emblica officinalis. It exhibits properties such as heatclearing, anti-inflammatory, anti-fatigue, antioxidant, and antibacterial effects,making it extensively utilized in India and Tibet. It has been found to exhibitinhibitory effects on Helicobacter pylori (H. pylori); however, further comprehensive research is still needed to elucidate its specific antibacterial mechanism. The present study investigates the in vitro antibacterial activity and antibacterial mechanism of Triphala against H. pylori.MethodsOurs research investigates the in vitro inhibitory activity of Triphala on multiple standard and clinical strains using microdilution broth method, time-kill curve, time-bactericidal curve and scanning electron microscopy (SEM). Furthermore, the antibacterial mechanism of Triphala is further explored through experiments on urease activity, biofilm formation, anti-adhesion properties, virulence actor assays using RT-qPCR and Western Blotting techniques.ResultsThe research findings indicate that Triphala exhibits a minimum inhibitory concentration of 80–320 μg/mL against both standard and clinical strains of H. pylori. Triphala exerts its anti-H. pylori effect by perturbing the microstructure of H. pylori, downregulating adhesion-associated genes (alpA, alpB, babA), urease-related genes (ureA, ureB, ureE, ureF), and flagellar genes (flaA, flaB); inhibiting bacterial adhesion, biofilm formation, urease activity as well as CagA protein expression.DiscussionThese findings suggest that Triphala exerts inhibitory effects on H. pylori activity through multiple mechanisms, underscoring its potential as a new drug for the prevention and treatment of H. pylori infection.

BackgroundTerminalia chebula Retz, known as the King of Tibet, is considered a functional food in China, celebrated for its antioxidant, immune-modulating, antibacterial, and anti-inflammatory properties. Chebulinic acid, derived from aqueous extracts of Terminalia chebula Retz, is known for its anti-inflammatory properties. However, its potential as an anti-Helicobacter pylori (HP) agent has not been fully explored.MethodsHerein, we extracted the main compound from Terminalia chebula Retz using a semi-preparative liquid chromatography (LC) system and identified compound 5 as chebulinic acid through Ultra-high performance liquid chromatography-MS/MS (UPLC–MS/MS) and Nuclear Magnetic Resonance (NMR). To evaluate its role, we conducted minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays, scanning electron microscope (SEM) imaging, inhibiting kinetics curves, urea fast test, cell counting kit-8 (CCK-8) assay, western blot analysis, griess reagent system, and molecular docking.ResultsOur results showed that chebulinic acid effectively inhibited the growth of the HP strain ATCC 700392, damaged the HP structure, and exhibited selective antimicrobial activity without affecting normal epithelial cells GES-1. Importantly, it suppressed the expression of Cytotoxin-associated gene A (Cag A) protein, a crucial factor in HP infection. Molecular docking analysis predicted a strong affinity (−9.7 kcal/mol) between chebulinic acid and Cag A protein.ConclusionOverall, our findings suggest that chebulinic acid acts as an anti-adhesive agent, disrupting the adhesion of HP to host cells, which is a critical step in HP infection. It also suppresses the Cag A protein. These results highlight the potential of chebulinic acid against HP infections.

Gastric cancer is a significant health concern worldwide. Helicobacter pylori (HP) infection is associated with gastric cancer risk, but differences between HP-infected and HP-free gastric cancer have not been studied sufficiently. The objective of this study was to investigate the effects of HP infection on the viability and migration of gastric cancer cells and identify potential underlying genetic mechanisms as well as their clinical relevance. Cell counting kit-8, lactate dehydrogenase, wound healing, and transwell assay were applied in the infection model of multiple clones of HP and multiple gastric cancer cell lines. Genes related to HP infection were identified using bioinformatics analysis and subsequently validated using real-time quantitative PCR. The association of these genes with immunity and drug sensitivity of gastric cancer was analyzed. Results showed that HP has no significant impact on viability but increases the migration of gastric cancer cells. We identified 1405 HP-upregulated genes, with their enriched terms relating to cell migration, drug, and immunity. Among these genes, the 82 genes associated with survival showed a significant impact on gastric cancer in consensus clustering and LASSO prognostic model. The top 10 hub HP-associated genes were further identified, and 7 of them were validated in HP-infected cells using real-time quantitative PCR, including ERBB4, DNER, BRINP2, KCTD16, MAPK4, THPO, and VSTM2L. The overexpression experiment showed that KCTD16 medicated the effect of HP on gastric cancer migration. Our findings suggest that HP infection may enhance the migratory potential of gastric cancer cells and these genes might be associated with immunity and drug sensitivity of gastric cancer. In human subjects with gastric cancer, HP presence in tumors may affect migration, immunity, and drug sensitivity.

BackgroundHelicobacter pylori (H. pylori) is thought to primarily colonize the human stomach and lead to various gastrointestinal disorders, such as gastritis and gastric cancer. Currently, main eradication treatment is triple or quadruple therapy centered on antibiotics. Due to antibiotic resistance, the eradication rate of H. pylori is decreasing gradually. Therefore, searching for anti-H. pylori drugs from herbal sources has become a strategy for the treatment. Our team proposed a Hezi Qingyou Formula (HZQYF), composed of Chebulae Fructus, Ficus hirta Vahl and Cloves, and studied its anti-H. pylori activity and mechanism.MethodsChemical components of HZQYF were studied using UHPLC–MS/MS and HPLC. Broth microdilution method and agar dilution method were used to evaluate HZQYF’s antibacterial activity. The effects of HZQYF on expression of adhesion genes (alpA, alpB, babA), urease genes (ureE, ureF), and flagellar genes (flaA, flaB) were explored using Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) technology. Effects on morphology and permeability of the extracellular membrane were studied using scanning electron microscopy (SEM) and N-phenylnaphthalen-1-amine (NPN) uptake. Effect on urease activity was studied using a urease kinetics analysis in vitro. Immunofluorescence staining method was used to examine the effect on adhesion. Western blot was used to examine the effect on cagA protein.ResultsMinimum inhibitory concentration (MIC) values of the formula against H. pylori clinical strains and standard strains were 80–160 μg/mL, and minimum bactericidal concentration (MBC) values were 160–320 μg/mL. The formula could down-regulate the expression of adhesion genes (alpA, alpB, babA), urease genes (ureE, ureF) and flagellar genes (flaA, flaB), change the morphology of H. pylori, increase its extracellular membrane permeability, and decrease its urease activity.ConclusionPresent studies confirmed that HZQYF had promising in vitro anti-H. pylori activities and demonstrated its possible mechanism of action by down-regulating the bacterial adhesion, urease, and flagellar gene expression, which provided scientific bases for further clinical investigations.

The primary treatment method for eradicating Helicobacter pylori (H. pylori) infection involves the use of antibiotic-based therapies. Due to the growing antibiotic resistance of H. pylori, there has been a surge of interest in exploring alternative therapies. Cetylpyridinium chloride (CPC) is a water-soluble and nonvolatile quaternary ammonium compound with exceptional broad-spectrum antibacterial properties. To date, there is no documented or described specific antibacterial action of CPC against H. pylori. Therefore, this study aimed to explore the in vitro activity of CPC against H. pylori and its potential antibacterial mechanism. CPC exhibited significant in vitro activity against H. pylori, with MICs ranging from 0.16 to 0.62 μg/mL and MBCs ranging from 0.31 to 1.24 μg/mL. CPC could result in morphological and physiological modifications in H. pylori, leading to the suppression of virulence and adherence genes expression, including flaA, flaB, babB, alpA, alpB, ureE, and ureF, and inhibition of urease activity. CPC has demonstrated in vitro activity against H. pylori by inhibiting its growth, inducing damage to the bacterial structure, reducing virulence and adherence factors expression, and inhibiting urease activity.

Rosuvastatin calcium is a widely used 3-hydroxy-3-methylglutaryl coenzyme A-reductase inhibitor developed for the treatment of dyslipidemia. To establish a control strategy for the elemental impurities, a new digestion method combined with an inductively coupled plasma-mass spectrometer (ICP-MS) was developed and validated by our team to determine elements Cd, Pb, As, Hg, Co, V, and Ni in rosuvastatin calcium tablets, which digest the sample perfectly even in the presence of a large number of excipients, especially titanium dioxide. The measurement mode was collision cell mode with kinetic energy discrimination (KED). 209Bi+, 115In+, and 89Y+ were chosen as internal standard elements. The recoveries of the limit of quantitation (LOQ) ranged from 90.5% to 106.4%, concentrations of the abovementioned elements in LOQ were 0.25 µg·L−1, 0.25 µg·L−1, 0.75 µg·L−1, 1.5 µg·L−1, 2.5 µg·L−1, 5 µg·L−1, and 8 µg·L−1 , respectively, linear correlation coefficients were above 0.9997, the recoveries in accuracy item ranged from 91.8% to 103.6%, and relative standard deviations (RSDs) of recovery in precision were not more than 1.8%, reflecting a reliable method of high sensitivity, strong anti-interference capacity, and good precision, and that it was suitable for the determination of elemental impurities in drugs.

Smart manufacturing still remains critical challenges for pharmaceutical manufacturing. Here, an original data-driven engineering framework was proposed to tackle the challenges. Firstly, from sporadic indicators to five kinds of systematic quality characteristics, nearly 2,000,000 real-world data points were successively characterized from Ginkgo Folium tablet manufacturing. Then, from simplex to the multivariate system, the digital process capability diagnosis strategy was proposed by multivariate Cpk integrated Bootstrap-t. The Cpk of Ginkgo Folium extracts, granules, and tablets were discovered, which was 0.59, 0.42, and 0.78, respectively, indicating a relatively weak process capability, especially in granulating. Furthermore, the quality traceability was discovered from unit to end-to-end analysis, which decreased from 2.17 to 1.73. This further proved that attention should be paid to granulating to improve the quality characteristic. In conclusion, this paper provided a data-driven engineering strategy empowering industrial innovation to face the challenge of smart pharmaceutical manufacturing.

1,3,6-Trigalloylglucose is a natural compound that can be extracted from the aqueous extracts of ripe fruit of Terminalia chebula Retz, commonly known as “Haritaki”. The potential anti-Helicobacter pylori (HP) activity of this compound has not been extensively studied or confirmed in scientific research. This compound was isolated using a semi-preparative liquid chromatography (LC) system and identified through Ultra-high-performance liquid chromatography–MS/MS (UPLC-MS/MS) and Nuclear Magnetic Resonance (NMR). Its role was evaluated using Minimum inhibitory concentration (MIC) assay and minimum bactericidal concentration (MBC) assay, scanning electron microscope (SEM), inhibiting kinetics curves, urea fast test, Cell Counting Kit-8 (CCK-8) assay, Western blot, and Griess Reagent System. Results showed that this compound effectively inhibits the growth of HP strain ATCC 700392, damages the HP structure, and suppresses the Cytotoxin-associated gene A (Cag A) protein, a crucial factor in HP infection. Importantly, it exhibits selective antimicrobial activity without impacting normal epithelial cells GES-1. In vitro studies have revealed that 1,3,6-Trigalloylglucose acts as an anti-adhesive agent, disrupting the adhesion of HP to host cells, a critical step in HP infection. These findings underscore the potential of 1,3,6-Trigalloylglucose as a targeted therapeutic agent against HP infections.

Objectives: The mechanism of olfactory disorder (OD) in patients with COVID-19 is unclear. Our study aimed to elucidate the relationships between inflammatory factors and OD in a sample of patients infected with the Omicron variant, with a high vaccination rate in China. Methods: The Sniffin’ Sticks 12-item test was performed in a cross-sectional study of 148 recovered patients who were infected with the Omicron variant to evaluate OD severity. We compared demographic, laboratory, and clinical data. Results: A total of 148 patients infected with the Omicron variant were enrolled. A total of 129 cases of OD were detected. Increased inflammation contributed to OD severity, especially in the adult group. OD was shown to be aggravated by an increase in interleukin-6 levels. The adjusted odds ratio (OR) was 2.22 (95% confidence interval 0.98-5.05, P = 0.056) after adjustment for age, sex, and vaccine characteristics. Conclusion: These findings indicated that the prevalence of OD remains high in vaccinated patients infected with the Omicron variant and that the Sniffin’ Sticks 12-item test might be a feasible method to screen for OD. Interleukin-6 may play a role in the biochemical and pathological processes underlying OD.

Summary: Mitochondria play important roles in angiogenesis. However, the mechanisms remain elusive. In this study, we found that mitochondrial ubiquinol-cytochrome c reductase complex assembly factor 3 (UQCC3) is a key regulator of angiogenesis. TALEN-mediated knockout of Uqcc3 in mice caused embryonic lethality at 9.5–10.5 days postcoitum, and vessel density was dramatically reduced. Similarly, knockout of uqcc3 in zebrafish induced lethality post-fertilization and impaired vascular development. Knockout of UQCC3 resulted in slower tumor growth and angiogenesis. Mechanistically, UQCC3 was upregulated under hypoxia, promoted reactive oxygen species (ROS) generation, enhanced HIF-1α stability and increased VEGF expression. Finally, higher expression of UQCC3 was associated with poor prognosis in multiple types tumors, implying a role for UQCC3 in tumor progression. In conclusion, our findings highlight the important contribution of UQCC3 to angiogenesis under both physiological and pathological conditions, indicating the potential of UQCC3 as a therapeutic target for cancer.

Previous studies suggested that Syzygium aromaticum dried buds (S. aromaticum), which is an edible and medicinal herb, possess potential effect on innate immunity and Helicobacter pylori (H. pylori) infection-related inflammation, but the specific action and especially the molecular mechanism have not been well elucidated. The objective of this work was to further explore the effects of Syzygium aromaticum aqueous extract (AE) on innate immunity and H. pylori related-inflammation, especially the potential molecular mechanisms involved. In order to accomplish this, analyses of the phagocytic rate, NO content, ROS level, as well as ELISA, RT-qPCR, and Western blotting et al. assays were carried out. Results indicated that AE increased phagocytic rate, ROS, and cytokine expression levels, meanwhile activated TLR4/MyD88 mediated NF-κB and MAPK pathways and decreased Nrf2/HO-1 signaling. AE also prevented the release of pro-inflammatory substances made by H. pylori-infected RAW 264.7 and GES-1 cells. All things considered, it was shown that AE's ability to boost innate immunity involves inducing macrophage M1 polarization by increasing TLR4/MyD88-mediated NF-κB and MAPK pathways and blocking Nrf2/HO-1 signaling. Additionally, by lowering the production of pro-inflammatory molecules, AE can lessen the inflammation brought on by H. pylori. Considering that S. aromaticum is an edible herbal medicine with immune-enhancing activity and good safety, it deserves further study and utilization for treating diseases including H. pylori infection-related disorders.

Irritable bowel syndrome (IBS) is characterized by visceral pain, impaired intestinal barrier and a disorder of the microbiota. DXL-A-24 has analgesic and anti-inflammatory effects by inhibiting neuropeptides and inflammatory factors. In this study, we used chronic unpredictable mild stress (CUMS) induced IBS model, to assess the action of DXL-A-24 on visceral hypersensitivity, barrier function and microbiota. Visceral sensation was assessed by colorectal distension in a model of IBS. The expressions of substance P (SP) and calcitonin gene-related peptide (CGRP) were detected by immunohistochemistry and western blot, the contents of diamine oxidase (DAO) and D-lactic acid were detected by ELISA, and 16S rRNA to detect the diversity of gut microbiota. CUMS reduced visceral pain threshold and increased colonic permeability of rats. DXL-A-24 for 28 days inhibited these changes. DXL-A-24 also decreased the expression of SP, CGRP in colon and D-LA, DAO in serum. Besides, DXL-A-24 increased the richness and diversity of intestinal microbiota. In conclusions, DXL-A-24 reduced visceral sensitivity, improved intestinal barrier and regulated gut microbiota in rats with IBS.

Sanguisorba officinalis L., a traditional Chinese medicine (TCM) called DiYu (DY) in China, has a strong tradition of utilization as a scorching, blood-cooling, and hemostatic medication, and was used for cancer prevention and treatment due to its potential immune-enhancing and hematological toxicity-reducing effects. Previous studies have reported significant effects of DY on cancers including colorectal cancer (CRC), which is one of the most common malignancies worldwide. The first-line cure 5-fluorouracil (5-FU) plays decisive commerce in the sedative of CRC as a clinically available chemotherapeutic agent. One of the primary causes of cancer treatment failure is the acquisition of chemotherapy drug resistance. In order to successfully combat the emergence of chemoresistance, it is essential to identify herbs or traditional Chinese medicine that have adjuvant therapeutic effects on CRC. Therefore, this study aimed to determine whether DY could improve the sensitivity, conquer the chemoresistance of 5-FU-resistant CRC cells, and investigate its intrinsic mechanism. Materials and methods: MTT, Hoechst 33258 staining, and flow cytometry assays were used to determine the anticancer activity of DY alone or in combination with 5-FU against 5-FU-resistant CRC cells (RKO-R and HCT15-R) and wound healing assays were conducted to detect cell migration. Transcriptomic techniques were carried out to explore the effect and mechanism of DY on drug-resistant CRC cells. Western Blot and RT q-PCR assays were performed to validate the mechanism by which DY overcomes drug-resistant CRC cells. Results: These results indicated that DY alone or in combination with 5-FU significantly inhibited the proliferation and the migration of resistant CRC cells, and potentiated the susceptibility of 5-FU to drug-resistant CRC cells. GO and KEGG enrichment analysis showed that the mechanisms of drug resistance in CRC cells and DY against drug-resistant CRC cells highly overlapped, involved in the modulation of biological processes such as cell migration, positive regulation of protein binding and cytoskeleton, and MAPK (Ras-ERK-MEK), PI3K/Akt, and other signaling pathways. Moreover, DY can mediate the expression of p-R-Ras, p-ERK1/2, p-MEK1/2, p-PI3K, p-AKT, HIF-1A and VEGFA proteins. In addition, DY significantly suppressed the expression of AKT3, NEDD9, BMI-1, and CXCL1 genes in resistant CRC cells. Conclusion: In conclusion, DY could inhibit the proliferation and migration of 5-FU-resistant cells and strengthen the sensitivity of 5-FU to CRC-resistant cells. Furthermore, DY may prevail over chemoresistance through the Ras/MEK/ERK and PI3K/Akt pathways. These findings imply that DY may be a potential drug for clinical treatment or adjuvant treatment of drug-resistant CRC.

Abstract The ‘angiogenic switch’ is critical for tumor progression. However, the pathological details and molecular mechanisms remain incompletely characterized. In this study, we established mammal xenografts in zebrafish to visually investigate the first vessel growth (angiogenic switch) in real-time, by inoculating tumor cells into the perivitelline space of live optically transparent Transgenic (flk1:EGFP) zebrafish larvae. Using this model, we found that hypoxia and hypoxia-inducible factor (HIF) signaling were unnecessary for the angiogenic switch, whereas vascular endothelial growth factor A gene (Vegfa) played a crucial role. Mechanistically, transcriptome analysis showed that the angiogenic switch was characterized by inhibition of translation, but not hypoxia. Phosphorylation of eukaryotic translation initiation factor 2 alpha (Eif2α) and the expression of Vegfa were increased in the angiogenic switch microtumors, and 3D tumor spheroids, and puromycin-treated tumor cells. Vegfa overexpression promoted early onset of the angiogenic switch, whereas Vegfa knockout prevented the first tumor vessel from sprouting. Pretreatment of tumor cells with puromycin promoted the angiogenic switch in vivo similarly to Vegfa overexpression, whereas Vegfa knockdown suppressed the increase. This study provides direc and dynamic in vivo evidences that inhibition of translation, but not hypoxia or HIF signaling promotes the angiogenic switch in tumor by increasing Vegfa transcription.

In this work, the modified attachment energy model was used to predict the crystal morphology of isosorbide mononitrate (ISMN) in the dichloromethane (CH2Cl2) solvent system and dichloromethane-n-hexane (CH2Cl2-C6H14) mixed solvent system. The solvent effect can significantly affect the crystal morphology, which can profoundly impact both the drug’s physicochemical properties and the subsequent technological treatment process. In addition, the interactions between solvent molecules and crystal faces were investigated using molecular dynamics simulation, and radial distribution function (RDF) analysis was performed to determine the types of interactions. The structural parameter S was introduced to characterize the roughness of each crystal surface; the change in the CH2Cl2 diffusion coefficient before and after the addition of C6H14 was analyzed using mean square displacement (MSD). The calculation results of the modified attachment energy from the two solvent systems revealed that C6H14 could accelerate crystal growth, while the crystal morphology was not greatly affected, which is of some significance as a guide for the industrial crystallization process.

As a potential drug candidate for the treatment of hypertension and complications, it is speculated that the component-based Chinese medicine of Ginkgo biloba leaves (GBCCM) which mainly composed of flavonoid aglycones (FAs) and terpene lactones (TLs) may have different pharmacological effects at different doses or ratios. Taking the normal mice as the study object, metabonomics was conducted by giving different doses of GBCCM. Based on the components of GBCCM absorbed into the blood, the network pharmacological prediction was carried out. By integrating the results of metabonomics and network pharmacology, predict the possible pharmacological effects of GBCCM and conduct experimental verification. It was found that eight of the 19 compounds in GBCCM could be absorbed into the blood. GBCCM mainly affected the signal pathways of unsaturated fatty acid, pyruvate, bile acid, melanin and stem cells. It was speculated that GBCCM might have activities such as lowering blood pressure, regulating stem cell proliferation and melanogenesis. By establishing the models of mushroom tyrosinase, rat bone marrow mesenchymal stem cells (BMSCs) and spontaneously hypertensive rats (SHRs), we found that FAs and TLs showed synergistic effect in hypertension and tyrosinase models, and the optimal ratio was 3:2 (4.4 mg/kg) and 1:1 (0.4 mg/ml), respectively. As effective substances, FAs significantly promoted the proliferation of rat BMSCs on the third and fifth days at the concentration of 0.2 μg/ml (p < 0.05). GBCCM showed a variety of pharmacological effects at different doses and ratios, which provided an important reference for the druggability of GBCCM.

Background Icaritin (ICT) has been previously demonstrated to display protective effects against cerebral ischemic reperfusion (I/R) by inhibiting oxidative stress, but the mechanism remains unclear. This study aimed to explore the mechanism from the perspective of metabolomics. Methods A mice cerebral artery occlusion/reperfusion (MCAO/R) model was explored to mimic cerebral ischemic reperfusion and protective effect of ICT was assessed by neurologic deficit scoring, infarct volume and brain water content. Ultra-high-performance liquid chromatography electrospray ionization orbitrap tandem mass spectrometry (UHPLC-ESI-QE-Orbitrap-MS) based metabolomic was performed to explore potential biomarkers. Brain tissue metabolic profiles were analyzed and metabolic biomarkers were identified through multivariate data analysis. The protein levels of Nrf2, HO-1 and HQO1 were assayed by western blot. The release of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) were detected using corresponding assay kits. Results The results showed that after ICT treatment, the neurological deficit, cerebral infarction area, brain edema and the level of MDA in brain tissue of MCAO/R mice were significantly reduced. Meanwhile, ICT enhanced the activity of SOD, CAT and GSH-Px. Western blot results confirmed that ICT up-regulated the protein levels of antioxidant-related protein including Nrf2, HO-1 and NQO1. According to the metabolomic profiling of brain tissues, clear separations were observed among the Sham, Model and ICT groups. A total of 44 biomarkers were identified, and the identified biomarkers were mainly related to linoleic acid metabolism, arachidonic acid metabolism, alanine, aspartate and glutamate metabolism, arginine biosynthesis, arginine and proline metabolism, D-glutamine and D-glutamate metabolism, taurine and hypotaurine metabolism and purine metabolism, respectively. At the same time, the inhibitory effect of ICT on arachidonic acid and linoleic acid in brain tissue, as well as the promoting effect on taurine, GABA, NAAG, may be the key factors for the anti-neurooxidative function of mice after MCAO/R injury. Conclusion Our results demonstrate that ICT has benefits for MCAO/R injury, which are partially related to the suppression of oxidative stress via stimulating the Nrf2 signaling and regulating the production of arachidonic acid, linoleic acid, taurine, GABA, NAAG in brain tissue.

Scedosporium apiospermum is a ubiquitous organism present in the environment and is rarely identified in rhinosinusitis. We report a case of invasive rhinosinusitis with Scedosporium apiospermum which made a definite diagnosis by metagenomic next-generation sequencing (mNGS) from a biopsy sample. The resection of the Scedosporium apiospermum pathological mass was performed with low-temperature plasma radiofrequency ablation. Six months of continuous oral voriconazole treatment was followed. The patient was asymptomatic with no signs of recurrence during the next 1-year follow-up.

The rapid and precise acquisition of the agricultural plastic greenhouse (PG) spatial distribution is essential in understanding PG usage and degradation, ensuring agricultural production, and protecting the ecological environment and human health. It is of great practical significance to realize the effective utilization of remote sensing images in the agricultural field and improve the extraction accuracy of PG remote sensing data. In this study, Landsat operational land imager (OLI) remote sensing images were used as data sources, and Shandong Province, which has the largest PG distribution in China, was selected as the study area. PGs in the study area were identified by means of contour recognition, feature set construction of the spatial structure, and machine learning. The results were as follows. (1) Through an optimal segmentation parameter approach, it was determined that the optimal segmentation scale for size, shape, and compactness should be set at 20, 0.8, and 0.5, respectively, which significantly improved PG contour recognition. (2) Among the 72 feature variables for PG spatial recognition, the number of features and classification accuracy showed a trend of first gradually increasing and then decreasing. Among them, fifteen feature variables, including the mean of bands 2 and 5; six index features (NDWI, GNDVI, SWIR1_NIR, NDVI, and PMLI); two shape features, the density and shape index; and two texture features, the contrast and standard deviation, played an important role. (3) According to the recall rate, accuracy rate, and F-value of three machine learning methods, random forest (RDF), CART decision tree (CART), and support vector machine (SVM), SVM had the best classification effect. The classification method described in this paper can accurately extract continuous plastic greenhouses through remote sensing images and provide a reference for the application of facility agriculture and non-point-source pollution control.

Abstract Synovial hyperplasia is the main cause of chronic rheumatoid arthritis (RA), but the mechanism of synovial hyperplasia is still unclear. Etodolac (ETD) is a selective COX-2 inhibitor for relieving pain and stiffness in RA, but the disease modifying effect is still lack of evidence. Proteomics method was used to study the differential proteome of synovial tissue in collagen induced arthritis (CIA) in rats. With the help of STRING analysis, the upregulated proteins enriched in the cluster of complement and coagulation cascades and platelet degranulation were highlighted, these proteins with fibrogenic factors Lum, CIV, CXI and Tgfbi participated in the synovial inflammation, fibrosis and hyperplasia in CIA. Based on KOG function class analysis, the proteins involved in the events of the central dogma was explored. They might be hyperplasia related proteins for most of them are related to the proliferation of cancer. ETD significantly attenuated synovial inflammation, fibrosis and hyperplasia in CIA rats by downregulating these proteins. Several proteins have not been observed in RA so far, such as Tmsb4x, Pura, Nfic, Ruvbl1, Snrpd3, U2af2, Srrm2, Srsf7, Elavl1, Hnrnph1, Wars, Yars, Bzw2, Mcts1, Eif4b, Ctsh, Lamp1, Dpp7, Ptges3, Cdc37 and Septin9, they might be potentials targets for RA. Blood biochemistry tests showed the safety of 7 months use of ETD on rats. In conclusion, present study displayed a comprehensive mechanism of synovial hyperplasia in CIA rats, on this basis, the clinical value of ETD in the treatment of RA was well confirmed.

With the rapid aging of the population, the control of age-related disease susceptibility and prognosis faces greater challenges. There is an urgent need for a strategy to maintain the vitality of elderly people. In this study, the effect of Renshen Guben (RSGB) oral liquid was investigated on an accelerated aging mice model of thyrotoxicosis by conventional detection methods combined with multiomics technology. The results showed that RSGB increased the number of neutrophils and lymphocytes, enhanced the function of lymphocytes, and increased the levels of complement and antimicrobial peptides, which indicated that RSGB improved the immunity of thyrotoxicosis mice at the cellular and molecular levels. RSGB corrected malnutrition in thyrotoxicosis mice by improving anemia, hypoalbuminemia, ion transporters, and vitamin-binding proteins. RSGB significantly reduced the lipotoxicity by reducing the level of fatty acids, triglyceride, sphingolipids, and glucocorticoids, thus increasing the level of docosapentaenoic acid (DPA) and bile acids, which contributed to improve immunosenescence. The intestinal defense ability of thyrotoxicosis mice was enhanced with the increase of bile acids and lactic acid bacteria by the RSGB treatment. The plant metabolomics analysis showed that there were various active components in RSGB oral liquid and medicated serum, including terpenoids, phenolic acids, flavonoids, tannin, alkaloids, organic acids, phenolamines, amino acids, and others. They have antioxidant, immune regulation, and anti-aging effects, which was the material basis of RSGB. Totally, RSGB protected the thyrotoxicosis mice against aging by improving immunosenescence, hypoproteinemia, lipotoxicity, and the intestinal flora. It will be beneficial for improving the disease susceptibility and prognosis of the elderly.

Wang et al. developed a fluorescence-assisted single-cell methodology (split protease-E. coli ClpXP (SPEC)) to characterise protein-protein interactions for both eukaryotic and prokaryotic species in E. coli cells. Their method can quantify these interactions with high sensitivity, easy manipulation, and broad temperature adaptability

The problem of aging is mainly the increase of age-related diseases, and elderly patients have longer hospitalization and worse prognosis. Poorer nutritional status and immunosenescence may be predisposing and severe factors. The mechanism of the high incidence of diseases and poor prognosis behind aging is complex. Finding suitable aging models is of great significance to find strategies to prevent aging related events. In this study, the relationship between thyrotoxicosis and aging was investigated in mice. The results of routine blood tests and flow cytometry showed that immunosenescence occurred in thyrotoxicosis mice, which was characterized by a significant decrease in neutrophils, lymphocytes, CD4+/CD8+ and CD4+IFN-γ+ lymphocytes. Biochemical examination results showed that there were hypocholesterolemia, hypolipoproteinemia, and hyperlipidemia in thyrotoxicosis mice. Serum proteomics analysis showed that the downregulation of complement and coagulation proteins was another manifestation of declined immunity. Moreover, proteomics analysis showed that many downregulated proteins were related to homeostasis, mainly transport proteins. Their downregulation led to the disturbance of osmotic pressure, ion homeostasis, vitamin utilization, lipid transport, hyaluronic acid processing, and pH maintenance. Serum metabolomics analysis provided more detailed evidence of homeostasis disturbance, especially lipid metabolism disorder, including the downregulation of cholesterol, vitamin D, bile acids, docosanoids, and the upregulation of glucocorticoids, triglycerides, sphingolipids, and free fatty acids. The upregulated lipid metabolites were related to lipotoxicity, which might be one cause of immunosenescence and many aging related syndromes. This study provides evidence for the aging model of thyrotoxicosis mice, which can be used for exploring anti-aging drugs and strategies.

Abstract Chitinase and chitin-oligosaccaride can be used in multiple field, so it is important to develop a high-yield chitinase producing strain. Here, a recombinant Pichia pastoris with 4 copies of ChiA gene from Bacillus licheniformis and co-expression of molecular chaperon HAC1 was constructed. The amount of recombinant ChiA in the supernatant of high-cell-density fermentation reaches a maximum of 12.7 mg/mL, which is 24-fold higher than that reported in the previous study. The recombinant ChiA can hydrolyze 30% collodidal chitin with 74% conversion ratio, and GlcNAc is the most abundant hydrolysis product, followed by N, N′-diacetylchitobiose. Combined with BsNagZ, the hydrolysate of ChiA can be further transformed into GlcNAc with 88% conversion ratio. Additionally, the hydrolysate of ChiA can obviously accelerate the germination growth of rice and wheat, increasing the seedling height and root length by at least 1.6 folds within 10 days.

Mastitis is the most common malignant disease in women, which seriously threatens women's physical and mental health. Jingfang Granules (JF) is a Chinese medicine compound, derived from the classic famous prescription-''Jingfang Baidu Powder''. JF has been widely used to relieve symptoms such as fever, headache, and cough caused by a cold. Our previous studies have shown that JF contains various active ingredients (β-sitosterol, isorhamnetin, hesperetin), which has the effect on antibacterial, anti-inflammatory and anti-apoptotic. Previous studies conveyed that JF had the potential to treat metabolic abnormalities and inflammation, which promoted us to investigate its therapeutic effects on LPS-induced mastitis and the underlying mechanisms. The pathologic changes suggested that the mammary gland edema and inflammatory factor infiltration were alleviated by JF in LPS-induced mastitis model. Furthermore, The ELISA assays suggested that the activity of Myeloperoxidase (MPO) and the expression of pro-inflammatory factors such as IL-1β, IL-6 and TNF-α were reduced by JF. The protein levels of TLR4, P-NF-κB, NLRP3, ASC, Caspase-1, IL-1β, P-P38, P-ERK1/2, and P-AKT were detected by western blotting. And then we found that JF alleviated mastitis inflammation by regulating NF-κB, NLRP3, PI3K/AKT, MAPK signaling pathways. In addition, we found that JF improved the integrity of the blood-milk barrier by increasing the expression of ZO-1, Claudin-3 and Occludin. Interestingly, JF could also significantly inhibit LPS-induced apoptosis. In summary, we successfully established a model of LPS-induced mastitis in mice and confirmed that JF could inhibit inflammation, improve the integrity of the blood-milk barrier and inhibit apoptosis, which also indicates the potential applicability of JF for the prevention and treatment of mastitis.

Parecoxib sodium is a widely used parenteral cyclooxygenase 2 selective inhibitor to relieve acute postoperative pain following gynecologic laparotomy surgery. To ensure the quality of the drug, a detailed quality specification is indispensable. Nevertheless, it is unavoidable to introduce inorganic impurities during the drug preparation process and how to assess and control them matters. This study proposed an analytical procedure for the determination of elemental impurities (Cd, Pb, As, Hg, Co, V, Ni, Li, Sb, and Cu) in parecoxib sodium, where an easier and safer digestion protocol, graphite digestion, combined with an inductively coupled plasma-mass spectrometer (ICP-MS) was adopted when compared with microwave digestion. Moreover, the study also discussed whether should they be listed in specification to comply with ICH Q3D guidelines after test of process validation batches. Limit of quantitation (LOQ) of the above elemental impurities reached to 0.05, 0.125, 0.375, 0.075, 0.125, 0.25, 0.5, 6.25, 2.25 and 7.5 ppm, respectively, and recovery in accuracy item ranged from 90.2% to 129.9%, reflecting a sensitive and accurate method.

Gastrointestinal dysfunction plays an important role in the occurrence and development of Parkinson’s disease (PD). This study investigates the composition of the gut microbiome using shotgun metagenomic sequencing in PD patients in central China. Fecal samples from 39 PD patients (PD group) and the corresponding 39 healthy spouses of the patients (SP) were collected for shotgun metagenomics sequencing. Results showed a significantly altered microbial composition in the PD patients. Bilophila wadsworthia enrichment was found in the gut microbiome of PD patients, which has not been reported in previous studies. The random forest (RF) model, which identifies differences in microbiomes, reliably discriminated patients with PD from controls; the area under the receiver operating characteristic curve was 0.803. Further analysis of the microbiome and clinical symptoms showed that Klebsiella and Parasutterella were positively correlated with the duration and severity of PD, whereas hydrogen-generating Prevotella was negatively correlated with disease severity. The Cluster of Orthologous Groups of protein database, the KEGG Orthology database, and the carbohydrate-active enzymes of gene-category analysis showed that branched-chain amino acid–related proteins were significantly increased, and GH43 was significantly reduced in the PD group. Functional analysis of the metagenome confirmed differences in microbiome metabolism in the PD group related to short-chain fatty acid precursor metabolism.

Internet of Things (IoT) terminals have firmware with heterogeneous, closed-source, and heavy business but light security characteristics, whereas on the edge, there are limited resources and a high code reuse rate. Once there are security risks at the firmware level, these risks are difficult to detect and discover, and the resulting impact quickly spreads over a wide range. Therefore, a similarity and homology analysis of firmware codes in an IoT terminal will be helpful for further research on firmware malicious code detection, vulnerability mining, backdoor discovery and copyright protection. Inspired by biological genes, this paper attempts to break away from the traditional feature-centered approach and focuses on code classification and the qualitative description of code features to discuss the idea of code similarity and homology analysis. Additionally, the proposed approach is information-centric, focusing on the informativeness (essentiality, stability, antivariability, and heritability) of the firmware code genes and the quantitative analysis of firmware code similarity and homology by discussing common methods and mechanisms. This paper presents security detection technology for IoT terminal firmware by measuring the gene distance between the codes. A prototype firmware security detection system (FSDS) for IoT terminals based on firmware code genes is designed and implemented. The experimental results show that this method has a good search matching effect and has certain advantages over traditional firmware security detection methods based on similarity theory.

Face recognition is one of the hottest issues in the field of computer vision and pattern recognition. Deep learning-based recognition models already have more exceptional recognition ability than the human being on open datasets, but still cannot fully undertake the identity recognition task in real scenarios without human assistance. In this paper, we mainly analyze two obstacles, i.e., domain gap and training data shortage. We propose the unpaired Domain Transfer Generative Adversarial Network (DT-GAN) to relieve these two obstacles. We improve the GAN baseline to bridge the domain gap among datasets by generating images conforming to the style of a target domain by learning the mapping between the source domain and target domain. The generator could synthesize face with an arbitrary viewpoint at the same time. The model is trained with a combination of style transfer loss, identity loss, and pose loss, which ensures the successive domain transfer and data augment. We conduct experiments to testify the effectiveness and reasonability of DT-GAN. Experimental results demonstrate the recognition performance is dramatically boosted after domain transfer and data augment.

Abstract Background For enzymes with buried active sites, transporting substrates/products ligands between active sites and bulk solvent via access tunnels is a key step in the catalytic cycle of these enzymes. Thus, tunnel engineering is becoming a powerful strategy to refine the catalytic properties of these enzymes. The tunnel-like structures have been described in enzymes catalyzing bulky substrates like glycosyl hydrolases, while it is still uncertain whether these structures involved in ligands exchange. Till so far, no studies have been reported on the application of tunnel engineering strategy for optimizing properties of enzymes catalyzing biopolymers. Results In this study, xylanase S7-xyl (PDB: 2UWF) with a deep active cleft was chosen as a study model to evaluate the functionalities of tunnel-like structures on the properties of biopolymer-degrading enzymes. Three tunnel-like structures in S7-xyl were identified and simultaneously reshaped through multi-sites saturated mutagenesis; the most advantageous mutant 254RL1 (V207N/Q238S/W241R) exhibited 340% increase in specific activity compared to S7-xyl. Deconvolution analysis revealed that all three mutations contributed synergistically to the improved activity of 254RL1. Enzymatic characterization showed that larger end products were released in 254RL1, while substrate binding and structural stability were not changed. Dissection of the structural alterations revealed that both the tun_1 and tun_2 in 254RL1 have larger bottleneck radius and shorter length than those of S7-xyl, suggesting that these tunnel-like structures may function as products transportation pathways. Attributed to the improved catalytic efficiency, 254RL1 represents a superior accessory enzyme to enhance the hydrolysis efficiency of cellulase towards different pretreated lignocellulose materials. In addition, tunnel engineering strategy was also successfully applied to improve the catalytic activities of three other xylanases including xylanase NG27-xyl from Bacillus sp. strain NG-27, TSAA1-xyl from Geobacillus sp. TSAA1 and N165-xyl from Bacillus sp. N16-5, with 80%, 20% and 170% increase in specific activity, respectively. Conclusions This study represents a pilot study of engineering and functional verification of tunnel-like structures in enzymes catalyzing biopolymer. The specific activities of four xylanases with buried active sites were successfully improved by tunnel engineering. It is highly likely that tunnel reshaping can be used to engineer better biomass-degrading abilities in other lignocellulolytic enzymes with buried active sites.

Abstract Background Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. Results Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR–Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR–Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR–Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. Conclusions This study applied CRISPR–Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR–Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.

Abstract Background Xylanases have been successfully used in food, paper, and pulp industries and are considered to be a key player in the biodegradation of xylan to valuable end products. However, most of the natural xylanases present poor activity in high-temperature and high-alkali environment. Therefore, it is necessary to modify the enzymes to meet the increasing demands of industries. Results Directed evolution was used to improve the specific activity and pH stability of the xylanase (XynHBN188A) that originated from Bacillus pumilus HBP8. The xylanase XynHBN188A was mutated by error-prone PCR. The mutant, XynHBN188A217, was screened from the mutant library by functional screening. The specific activity of XynHBN188A217 was 3986.7 U/mg, which was 2.8-fold higher than that of wild type. The optimum temperature of XynHBN188A and XynHBN188A217 was 50 °C and 55 °C, respectively. The optimum pH of XynHBN188A and XynHBN188A217 was pH 8.0 and pH 7.5, respectively. The half-life at 60 °C of XynHBN188A217 was 20 min. Moreover, the pH stability of XynHBN188A217 was significantly better than that of XynHBN188A. Finally, homology models and molecular docking were used to identify the location of mutation sites and to explore the mechanism of the improved properties. Conclusion The xylanase XynHBN188A has been improved in the specific activity and pH stability by directed evolution. Also, the enlarged catalytic channel of mutant is beneficial for the substrates access and products release. It may contribute to the improved activity. The mutant XynHBN188A217 will be a potential candidate to be used for industrial application.

Abstract Background Several anaerobic bacteria produce butyric acid, a commodity chemical with use in chemical, pharmaceutical, food and feed industries, using complex media with acetate as a co-product. Butyrate titer of various recombinant Escherichia coli did not exceed 10 g l−1 in batch fermentations in any of the media tested. Results A recombinant E. coli (strain LW393) that produced butyrate as the major fermentation product was constructed with genes from E. coli, Clostridium acetobutylicum and Treponema denticola. Strain LW393 produced 323 ± 6 mM (28.4 ± 0.4 g l−1) butyric acid in batch fermentations in mineral salt medium with glucose as C source at a yield of 0.37 ± 0.01 g (g glucose consumed)−1. Butyrate accounted for 90% of the total products produced by the culture. Supplementing this medium with yeast extract further increased butyric acid titer to 375 ± 4 mM. Average volumetric productivity of butyrate with xylose as C source was 0.89 ± 0.07 g l−1 h−1. Conclusions The butyrate titer reported in this study is about 2.5–3-times higher than the values reported for other recombinant E. coli and this is achieved in mineral salt medium with an expectation of lower purification and production cost of butyrate.

Summary: Hypoxia is known to stimulate mitochondrial reactive oxygen species (mROS) in cells. Here, we present a detailed protocol to detect mROS using MitoSOX staining in live cells under normoxia and hypoxia. Flow cytometry allows sensitive and reliable quantification of mROS by FlowJo software. We optimized several aspects of the procedure including hypoxic treatment, working concentrations of the staining buffer, and quantitative analyses. Here, we use HepG2 cells, but the protocol can be applied to other cell lines.For complete details on the use and execution of this protocol, please refer to Yang et al. (2020).

Acoustic communication signals are inevitably challenged by ambient noise. In response to noise, many animals adjust their calls to maintain signal detectability. However, the mechanisms by which the auditory system adapts to the adjusted pulses are unclear. Our previous study revealed that the echolocating bat, Hipposideros pratti, increased its pulse intensity in the presence of background white noise. In vivo single-neuron recording demonstrated that the auditory midbrain neurons tuned to the second harmonic (H2 neurons) increased their minimal threshold (MT) to a similar degree as the increment of pulse intensity in the presence of the background noise. Furthermore, the H2 neurons exhibited consistent spike rates at their best amplitudes and sharper intensity tuning with background white noise compared with silent conditions. The previous data indicated that sound intensity analysis by auditory midbrain neurons was adapted to the increased pulse intensity in the same noise condition. This study further examined the echolocation pulse frequency and frequency analysis of auditory midbrain neurons with noise conditions. The data revealed that H. pratti did not shift the resting frequency in the presence of background noise. The auditory midbrain neuronal frequency analysis highly linked to processing the resting frequency with the presence of noise by presenting the constant best frequency (BF), frequency sensitivity, and frequency selectivity. Thus, our results suggested that auditory midbrain neuronal responses in background white noise are adapted to process echolocation pulses in the noise conditions.

Patient’s poor compliance and the high risk of toxic effects limit the clinical use of galantamine hydrobromide. To overcome these drawbacks, the sustained-release galantamine pamoate microspheres (GLT-PM-MS) were successfully developed using an oil/water emulsion solvent evaporation method in this study. Physicochemical properties of GLT-PM-MS were carefully characterized, and the in vitro and in vivo drug release behaviors were well studied. Results showed that the morphology of optimized microspheres were spherical with smooth surfaces and core-shell interior structure. Mean particle size, drug loading and entrapment efficiency were 75.23 ± 1.79 μm, 28.01 ± 0.81% and 87.12 ± 2.71%, respectively. The developed GLT-PM-MS were found to have a sustained release for about 24 days in vitro and the plasma drug concentration remained stable for 17 days in rats. These results indicated that GLT-PM-MS could achieve the sustained drug release purpose and be used in clinical trial.

N-acetyl-β-D glucosamine (GlcNAc) is wildly used in cosmetics, nutraceuticals and pharmaceuticals. The traditional chemical process for GlcNAc production from chitin causes serious acidic pollution. Therefore, the enzymatic hydrolysis becomes a great promising and alternative strategy to produce GlcNAc. β-N-acetylglucosaminidase (NAGase) can hydrolyze chitin to produce GlcNAc. Here, a GH3 family NAGase encoding gene BlNagZ from Bacillus licheniformis was expressed extracellularly in Escherichia coli guided by signal peptide PelB. The recombinant BlNagZ presented the best activity at 60°C and pH 5.5 with a high specific activity of 13.05 U/mg. The BlNagZ activity in the fermentation supernatant can reach 13.62 U/mL after optimizing the culture conditions, which is 4.25 times higher than optimization before. Finally, combining BlNagZ with chitinase ChiA we identified before, chitin conversion efficiency to GlcNAc can reach 89.2% within 3.5 h. In all, this study provided not only a high active NAGase, and a secreted expression strategy to reduce the cost of production, which is conducive to the industrial application.

In this study, we performed a retrospective and prospective study of preoperative predictors of the length of stay (LOS) in three groups of surgical patients and conducted a clinical retrospective study of the current research status of preoperative predictors of LOS prolongation in three groups of patients under ERAS (enhanced recovery after surgery) mode, such as patient characteristics and comorbidities. Information such as patients’ exercise preferences, exercise time, frequency and duration, footwear, location of knee osteoarthritis, whether there is a past history of knee injury, and smoking and drinking history was collected, and the research data of 312 patients undergoing the three operations were analyzed by SPSS. Meniscal injury-knee arthroscopy sample included a total of 104 people. Surgical sample for anterior cruciate ligament reconstruction included a total of 100 subjects. Knee osteoarthritis-knee replacement surgery sample included 148 people who were divided into two groups in a ratio of 1 : 1: one group used Mailuo Shutong pills during hospitalization (intervention group) and the other group did not (control group). The research conclusions are as follows. Meniscal Knee Arthroscopy. (1) Samples from different causes of injury showed significant differences for all injured sites. (2) Samples with different smoking and drinking histories all showed significant differences for the causes of injury. (3) Exercise hobby, exercise frequency, duration of each exercise and duration of exercise, and warm-up time before exercise all showed positive correlation. Anterior Cruciate Ligament Reconstruction Surgery. (4) Samples from different causes of injury showed significant differences for all the injured sites. (5) Age has a significant negative influence on the wearing of shoes at ordinary times. (6) Exercise hobby: the warm-up time before exercise had a significant negative influence on the injured area. (7) Two groups of analysis items of exercise frequency, exercise duration and exercise duration, preexercise warm-up time, and exercise hobby were typically positively correlated. Total Knee Arthroplasty. (8) There was a significant difference of 0.01 between the hospitalization days of the intervention group and the control group (p

Summary: Bioenergetic reprogramming during hypoxia adaption is critical to promote hepatocellular carcinoma (HCC) growth and progression. However, the mechanism underlying the orchestration of mitochondrial OXPHOS (oxidative phosphorylation) and glycolysis in hypoxia is not fully understood. Here, we report that mitochondrial UQCC3 (C11orf83) expression increases in hypoxia and correlates with the poor prognosis of HCC patients. Loss of UQCC3 impairs HCC cell proliferation in hypoxia in vitro and in vivo. Mechanistically, UQCC3 forms a positive feedback loop with mitochondrial reactive oxygen species (ROS) to sustain UQCC3 expression and ROS generation in hypoxic HCC cells and subsequently maintains mitochondrial structure and function and stabilizes HIF-1α expression to enhance glycolysis under hypoxia. Thus, UQCC3 plays an indispensable role for bioenergetic reprogramming of HCC cells during hypoxia adaption by simultaneously regulating OXPHOS and glycolysis. The positive feedback between UQCC3 and ROS indicates a self-modulating model within mitochondria that initiates the adaptation of HCC to hypoxic stress.

The aim of this study was to investigate the precise clinical use of Sinitang decoction (SNT) in ulcerative colitis (UC). Network pharmacology-based analysis of the drug components–targets–diseases–pathways was used to predict the possible clinical applications of SNT. Next, 2,4,6-trinitrobenzenesulfonic acid (TNBS) was used to establish a rat model of UC, and the efficacy of SNT against UC was tested, followed by a proteomic analysis of the specific signatures regulated by SNT against UC. SNT was predicted to be effective in inflammatory bowel disease, UC, and several other diseases. In the rats with UC, SNT decreased the disease activity index and colon mucosal damage index compared to the untreated UC model rats. Additionally, SNT reversed the upregulated levels of serum tumor necrosis factor (TNF)-α, prostaglandin E2 (PGE2), interleukin (IL)-6, and nitric oxide (NO) in UC model rats. The proteomic analysis identified 78 proteins that were differentially regulated by SNT in the rats with UC, which were associated with the Gene Ontology terms sulfur compound binding, calcium ion binding, and Toll-like receptor (TLR)-4 binding. Among these differentially regulated proteins, C-reactive protein (CRP) and collagen alpha-1(XII) chain (COL12A1) were found to be signature proteins associated with the efficacy of SNT against UC. This study represents the first precise investigation of the efficacy and mechanisms of SNT against UC, and shows that SNT is a promising candidate for personalized management of UC.

With the development of research on noncontrol data attacks and defense, the threat of data-oriented programming (DOP) attacks has attracted increasing attention from the security research community. DOP attacks can manipulate security-critical noncontrol data to alter program behavior without violating control-flow integrity (CFI) and can circumvent the most effective defenses against control-data attacks. Among DOP attacks, the misuse of user input data is a major contributor. Moreover, existing defense methods, e.g., DOPdefender, currently lack security protection for user input data. To effectively defend against DOP attacks, we propose a novel technique, DOPdefenderPlus, which draws on the idea of divide-and-conquer and uses the modular authentication technique to make DOPdefender scalable for complex software that is designed modularly, as well as introduce the Inputguard technique to protect the program input data. The DOPdefenderPlus is an enhanced version of DOPdefender, which overcomes some limitations of DOPdefender. We implement DOPdefenderPlus on a Linux operating system and use it to defend against multiple realistic DOP attacks. We also evaluate the performance of our method, and all the results show that DOPdefenderPlus can overcome the two limitations of DOPdefender while introducing a moderate runtime overhead.

Pectate lyases (Pels) can be used in the textile industrial process for cotton scouring and ramie degumming, and its hydrolyzed products oligo galacturonic acid, are high-value added agricultural and health products. In our previous studies, an alkaline pectate lyase PEL168 mutant, PEL3, was obtained with improved specific activity and thermostability. Here, a facile and rapid method for preparing an immobilized PEL3-inorganic hybrid nanoflower was developed, as it could improve its biocatalytic performance. With 0.02 mg/mL (112.2 U/mL) PEL3 in PBS buffer, five different divalent ions, including Mn2+, Ca2+, Co2+, Zn2+, and Cu2+, were used as inorganic component. The results showed that PEL3/Cu3(PO4)2 hybrid nanoflowers presented the highest relative activity with 2.5-fold increase, compared to the free PEL3. X-ray diffraction analysis confirmed that the composition of PEL3/Cu3(PO4)2 hybrid nanoflowers were pectate lyase PEL3 and Cu3(PO4)2⋅5H2O. The optimum temperature and pH of PEL3/Cu3(PO4)2 hybrid nanoflowers were ascertained to be 55°C and pH 9.0, respectively, exhibiting subtle difference from the free PEL3. However, the PEL3/Cu3(PO4)2 hybrid nanoflowers maintained 33% residual activity after 24 h incubation at 55°C, while the free PEL3 completely lost its activity after 18 h incubation at 55°C. Furthermore, over 50% residual activity of the PEL3/Cu3(PO4)2 hybrid nanoflowers was remained, even after four times of repetitive utilization, demonstrating its promising stability for practical application.

Human lysozyme (hLYZ), known for its bacteriolytic activity, is widely applied in the food and pharmaceutical industries as an antimicrobial agent. However, its extensive application was limited by its low large-scale production efficiency. In this study, a combinational method of integrating codon optimization, multiple gene copies, and ER molecular chaperone co-expression was developed to improve the heterologous production of hLYZ in Pichia pastoris GS115. Our results showed that increasing the copy number of the optimized hLYZ gene in P. pastoris could enhance its secretory production level up to 1.57-fold. The recombinant opt-hLYZ-6C strain that contains six copies of opt-hLYZ gene exhibited the highest mRNA transcription levels, giving the highest production of 0.22 ± 0.02 mg/mL of hLYZ in the medium supernatant with a bacteriolytic activity of 14,680 ± 300 U/mL against Micrococcus lysodeikticus in the shaking flask experiment. Moreover, co-overexpression of ER retention molecular chaperones, such as Pdi1 or Ero1, in the recombinant opt-hLYZ-6C strain both presented positive effects on the secretory production of hLYZ. Our further characterization indicated that tandem co-expression of Ero1 and Pdi1 together presented an added-up effect. The secretory production of hLYZ in the medium supernatant reached 0.34 ± 0.02 mg/mL of the recombinant opt-hLYZ-6C-EP strain in the shaking flask experiment, with a bacteriolytic activity of 21,200 ± 400 U/mL. Compared to the recombinant opt-hLYZ-1C strain, these final improvements were calculated as 2.43-fold and 2.30-fold on secretory protein levels and antibacterial activity, respectively. Finally, the recombinant opt-hLYZ-6C-EP strain was applied for high-density cultivation in 5 L of fermenter, in which the secretory yield of hLYZ reached 2.34 ± 0.02 mg/mL in the medium supernatant, with a bacteriolytic activity of 1.76 ± 0.02 × 105 U/mL against M. lysodeikticus. All these numbers presented the highest heterologous production levels of hLYZ in microbial systems.

Background: The present study aimed to explore the correlation between calcium-activated potassium channels, left atrial flow field mechanics, valvular atrial fibrillation (VAF), and thrombosis. The process of transforming mechanical signals into biological signals has been revealed, which offers new insights into the study of VAF. Methods: Computational fluid dynamics simulations use numeric analysis and algorithms to compute flow parameters, including turbulent shear stress (TSS) and wall pressure in the left atrium (LA). Real-time PCR and western blotting were used to detect the mRNA and protein expression of IKCa2.3/3.1, ATK1, and P300 in the left atrial tissue of 90 patients. Results: In the valvular disease group, the TSS and wall ressure in the LA increased, the wall pressure increased in turn in all disease groups, mainly near the mitral valve and the posterior portion of the LA, the increase in TSS was the most significant in each group near the mitral valve, and the middle and lower part of the back of the LA and the mRNA expression and protein expression levels of IKCa2.3/3.1, AKT1, and P300 increased (p < 0.05) (n = 15). The present study was preliminarily conducted to elucidate whether there might be a certain correlation between IKCa2.3 and LA hemodynamic changes. Conclusions: The TSS and wall pressure changes in the LA are correlated with the upregulation of mRNA and protein expression of IKCa2.3/3.1, AKT1, and P300.

Abstract Background Valvular heart disease is a leading cause of cardiovascular mortality, especially in China. More than a half of valvular heart diseases are caused by acute rheumatic fever. microRNA is involved in many physiological and pathological processes. However, the miRNA profile of the rheumatic valvular heart disease is unknown. This research is to discuss microRNAs and their target gene pathways involved in rheumatic heart valve disease. Methods Serum miRNA from one healthy individual and four rheumatic heart disease patients were sequenced. Specific differentially expressed miRNAs were quantified by Q-PCR in 40 patients, with 20 low-to-moderate rheumatic mitral valve stenosis patients and 20 severe mitral valve stenosis patients. The target relationship between certain miRNA and predicted target genes were analysis by Luciferase reporter assay. The IL-1β and IL1R1 expression levels were analyzed by immunohistochemistry and western blot in the mitral valve from surgery of mitral valve replacement. Results The results showed that 13 and 91 miRNAs were commonly upregulated or downregulated in all four patients. Nine miRNAs, 1 upregulated and 8 downregulated, that had a similar fold change in all 4 patients were selected for quantitative PCR verification. The results showed similar results from miRNA sequencing. Within these 9 tested miRNAs, hsa-miR-205-3p and hsa-miR-3909 showed a low degree of dispersion between the members of each group. Hsa miR-205-3p and hsa-miR-3909 were predicted to target the 3’UTR of IL-1β and IL1R1 respectively. This was verified by luciferase reporter assays. Immunohistochemistry and Western blot results showed that the mitral valve from rheumatic valve heart disease showed higher levels of IL- 1β and IL1R1 expression compared with congenital heart valve disease. This suggested a difference between rheumatic heart valve disease and other types of heart valve diseases, with more inflammatory responses in the former. Conclusion In the present study, by next generation sequencing of miRNAs, it was revealed that interleukin 1β and interleukin 1 receptor 1 was involved in rheumatic heart diseases. And this is useful for diagnosis and understanding of mechanism of rheumatic heart disease.

The bolt support quality of coal roadways is one of the important factors for the efficiency and security of coal production. By means of a self-developed technique and equipment of random non-destructive testing, non-destructive detection and pre-warning analysis on the quality of bolt support in deep roadways of mining districts were performed in a number of mining areas. The measured data were obtained in the detection instances of abnormal in-situ stress and support invalidation etc. The corresponding relation between axial bolt load variation and roadway surrounding rock deformation and stability was summarized in different mining service stages. Pre-warning technology of roadway surrounding rock stability is proposed based on the detection of axial bolt load. Meanwhile, pre-warning indicators of axial bolt load in different mining service stages are offered and some successful pre-warning cases are also illustrated. The research results show that the change rules of axial bolt load in different mining service stages are quite similar in different mining areas. The change of axial bolt load is in accord with the adjustment of surrounding rock stress, which can consequently reflect the deformation and stability state of roadway surrounding rock. Through the detection of axial bolt load in different sections of roadways, the status of real-time bolt support quality can be reflected; meanwhile, the rationality of bolt support design can be evaluated which provides reference for bolting parameters optimization. Keywords: Deep roadways, Bolt support quality, Random nondestructive testing, Surrounding rock stability, Prediction and pre-warning