A graphene oxide-assisted protein immobilization paper-tip immunosensor with smartphone and naked eye readout for the detection of okadaic acid.

BACKGROUND: Okadaic acid (OA), as a diarrhetic shellfish poisoning, can increase the risk of acute carcinogenic or teratogenic effects for the ingestion of OA contaminated shellfish. At present, much effort has been made to graft immunoassay onto a paper substrate to make paper-based sensors for rapid and simple detection of shellfish toxin. However, the complicated washing steps and low protein fixation efficiency on the paper substrate need to be further addressed. RESULTS: A novel paper-tip immunosensor for detecting OA was developed combined with smartphone and naked eye readout. The trapezoid paper tip was consisted of quantitative and qualitative detection zones. To improve the OA antigen immobilization efficiency on the paper substrate, graphene oxide (GO)-assisted protein immobilization method was introduced. Meanwhile, Au nanoparticles composite probe combined with the lateral flow washing was developed to simplify the washing step. The OA antigen-immobilized zone, as the detection zone Ⅰ, was used for quantitative assay by smartphone imaging. The paper-tip front, as the detection zone Ⅱ, which could qualitatively differentiate OA pollution level within 45 min using the naked eye. The competitive immunoassay on the paper tip exhibited a wide linear range for detecting OA (0.02-50 ng∙mL-1) with low detection limit of 0.02 ng∙mL-1. The recovery of OA in spiked shellfish samples was in the range of 90.3 %-113.%. SIGNIFICANCE: These results demonstrated that the proposed paper-tip immunosensor could provide a simple, low-cost and high-sensitivity test for OA detection without the need for additional large-scale equipment or expertise. We anticipate that this paper-tip immunosensor will be a flexible and versatile tool for on-site detecting the pollution of marine products.

HBB contributes to individualized aconitine-induced cardiotoxicity in mice via interfering with ABHD5/AMPK/HDAC4 axis.

The root of Aconitum carmichaelii Debx. (Fuzi) is an herbal medicine used in China that exerts significant efficacy in rescuing patients from severe diseases. A key toxic compound in Fuzi, aconitine (AC), could trigger unpredictable cardiotoxicities with high-individualization, thus hinders safe application of Fuzi. In this study we investigated the individual differences of AC-induced cardiotoxicities, the biomarkers and underlying mechanisms. Diversity Outbred (DO) mice were used as a genetically heterogeneous model for mimicking individualization clinically. The mice were orally administered AC (0.3, 0.6, 0.9 mg· kg-1 ·d-1) for 7 d. We found that AC-triggered cardiotoxicities in DO mice shared similar characteristics to those observed in clinic patients. Most importantly, significant individual differences were found in DO mice (variation coefficients: 34.08%-53.17%). RNA-sequencing in AC-tolerant and AC-sensitive mice revealed that hemoglobin subunit beta (HBB), a toxic-responsive protein in blood with 89% homology to human, was specifically enriched in AC-sensitive mice. Moreover, we found that HBB overexpression could significantly exacerbate AC-induced cardiotoxicity while HBB knockdown markedly attenuated cell death of cardiomyocytes. We revealed that AC could trigger hemolysis, and specifically bind to HBB in cell-free hemoglobin (cf-Hb), which could excessively promote NO scavenge and decrease cardioprotective S-nitrosylation. Meanwhile, AC bound to HBB enhanced the binding of HBB to ABHD5 and AMPK, which correspondingly decreased HDAC-NT generation and led to cardiomyocytes death. This study not only demonstrates HBB achievement a novel target of AC in blood, but provides the first clue for HBB as a novel biomarker in determining the individual differences of Fuzi-triggered cardiotoxicity.

A capillary-based centrifugal indicator equipped with in situ pathogenic bacteria culture for fast antimicrobial susceptibility testing.

Antimicrobial resistance has become a major global health threat due to the misuse and overuse of antibiotics. Rapid, affordable, and high-efficiency antimicrobial susceptibility testing (AST) is among the effective means to solve this problem. Herein, we developed a capillary-based centrifugal indicator (CBCI) equipped with an in situ culture of pathogenic bacteria for fast AST. The bacterial incubation and growth were performed by macro-incubation, which seamlessly integrated the capillary indicator. Through simple centrifugation, all the bacterial cells were confined at the nanoliter-level capillary column. The packed capillary column height could linearly reflect the bacterial count, and the minimum inhibitory concentration (MIC) was determined based on the difference in the column height between the drug-added groups and the control group. The AST results could easily be determined by the naked eye or smartphone imaging. Thus, the CBCI realized the combination of macro-bacterial incubation and early micro assessment, which accelerated the phenotypic AST without complex microscopic counting or fluorescent labelling. The whole operation process was simple and easy to use. AST results could be determined for E. coli ATCC strains within 3.5 h, and the output results for clinical samples were consistent with the hospital reports. We expect this AST platform to become a useful tool in limiting antimicrobial resistance, especially in remote/resource-limited areas or in underdeveloped countries.

Orthostatic Hypotension Promotes the Progression From Mild Cognitive Impairment to Dementia in Type 2 Diabetes Mellitus.

CONTEXT: In type 2 diabetes mellitus (T2DM), orthostatic hypotension (OH) is associated with cognition, but the mechanisms governing the link between OH and cognition are still unclear. OBJECTIVE: We sought to analyze Alzheimer's disease (AD) biomarkers and the part of complement proteins in modulating the association of OH with cognitive impairment and examine whether OH could accelerate the clinical progression of mild cognitive impairment (MCI) to dementia in T2DM. METHODS: We recruited patients with T2DM with MCI and collected general healthy information and blood samples. Complement proteins of astrocyte-derived exosomes were isolated and AD biomarkers of neuronal cell-derived exosomes isolated were quantified by enzyme-linked immunosorbent assay. Cognitive assessments were performed at patient enrollment and follow-up. RESULTS: Mediation analysis showed that the influence of OH on cognition in T2DM was partly mediated by baseline AD biomarkers and complement proteins. Cox proportional-hazards regression proved the OH group had a higher risk of developing dementia compared to the T2DM without OH group. CONCLUSION: In T2DM with MCI patients, AD biomarkers and complement proteins mediate the effects of OH on cognitive impairment and OH may be a risk factor of progression from MCI to dementia in T2DM.

Formononetin, a bioactive isoflavonoid constituent from Astragalus membranaceus (Fisch.) Bunge, ameliorates type 1 diabetes mellitus via activation of Keap1/Nrf2 signaling pathway: An integrated study supported by network pharmacology and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Type 1 diabetes mellitus (T1DM) results from insulin deficiency due to the destruction of pancreatic ß-cells. Previously, our studies showed that inhibition of Keap1/Nrf2 signaling pathway promoted the onset of T1DM, which suggests that finding drugs that can activate the Keap1/Nrf2 signaling may be a promising therapeutic strategy for the T1DM treatment. Astragalus membranaceus (Fisch.) Bunge is a common traditional Chinese medicine that has been frequently applied in Chinese clinics for the treatment of diabetes and other diseases. Formononetin (FMNT), one of the major isoflavonoid constituents isolated from this herbal medicine, possesses diverse pharmacological benefits and T1DM therapeutic potential. However, the exact molecular mechanisms underlying the action of FMNT in ameliorating T1DM have yet to be fully elucidated. AIMS OF THE STUDY: This study is to investigate the regulation of FMNT on the Keap1/Nrf2 signaling pathway to ameliorate T1DM based on network pharmacology approach combined with experimental validation. MATERIALS AND METHODS: A mouse-derived pancreatic islet ß-cell line (MIN6) was used for the in vitro studies. An alloxan (ALX)-induced T1DM model in wild-type and Nrf2 knockout (Nrf2-/-) C57BL/6J mice were established for the in vivo experiments. The protective effects of FMNT against ALX-stimulated MIN6 cell injury were evaluated using MTT, EdU, apoptosis and comet assays. The levels of blood glucose in mice were measured by using a blood monitor and test strips. The protein expression was detected by Western blot analysis. Furthermore, the binding affinity of FMNT to Keap1 was evaluated using cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) assay, and solvent-induced protein precipitation (SIP) assay. The interaction pattern between FMNT and Keap1 was assessed by molecular docking and molecular dynamics simulation techniques. RESULTS: Network pharmacology analysis revealed that FMNT exerted its therapeutic effect against T1DM by mainly regulating oxidative stress response-associated signaling molecules and pathways, such as Nrf2 regulating anti-oxidant/detoxification enzymes and Keap1-Nrf2 signaling pathway. The in vivo results showed that FMNT significantly deceased the ALX-induced high blood glucose levels and conversely increased the ALX-induced low insulin contents. In vitro, FMNT markedly protected MIN6 cells from ALX-induced cytotoxicity, proliferation inhibition and DNA damage and reduced the ALX-stimulated cell apoptosis. FMNT also inhibited ALX-induced overproduction of intracellular ROS to alleviate oxidative stress. In addition, FMNT could bind to Keap1 to notably activate the Keap1/Nrf2 signaling to upregulate Nrf2 expression and promote the Nrf2 translocation from the cytoplasm to the nucleus, resulting in enhancing the expression of antioxidant proteins HO-1 and NQO1. Inhibition of Keap1/Nrf2 signaling by ALX was also markedly abolished in the cells and mice exposed to FMNT. Moreover, these effects of FMNT in ameliorating T1DM were not observed in Nrf2-/- mice. CONCLUSIONS: This study demonstrates that FMNT could bind to Keap1 to activate the Keap1/Nrf2 signaling to prevent intracellular ROS overproduction, thereby attenuating ALX-induced MIN6 cell injury and ameliorating ALX-stimulated T1DM. Results from this study might provide evidence and new insight into the therapeutic effect of FMNT and indicate that FMNT is a promising candidate agent for the treatment of T1DM in clinics.

Designing of a functional paper-tip substrate for sensitive surface-enhanced Raman spectroscopy (SERS) detection.

A simple and flexible fabrication method of paper SERS substrate was developed by nanoparticles (NPs) droplet self-assembly at the paper tip with a temperature gradient (PTTG). We turned the drawback of the coffee ring effect into an effective way of preparing paper SERS substrate. When the NPs droplets were continuously dripped onto the PTTG, NPs were densely and uniformly distributed at the paper-tip front based on the combination of gravity and the coffee ring effect, which could achieve 91.2-fold improvement of SERS performance compared to a flat filter paper. Meanwhile, the analytes could also be enriched at the paper-tip front, which could achieve 9.3-fold signal enhancement compared to the paper-tip tail. Thus, the PTTG realized an excellent signal amplification for SERS detection. The paper-tip SERS substrate combined with a portable Raman spectrometer yielded an excellent analytical enhancement factor of 1.15 × 105 with the detection limit of 10 nM Rhodamine 6G (R6G). The whole fabrication procedure was completed within 2 h, and the paper-tip substrate showed a satisfactory substrate-to-substrate reproducibility with a relative standard deviation (RSD) of 5.13% (n = 10). It was successfully applied for quantitatively detecting real samples of oxytetracycline and malachite green with recoveries of 83.84-105.25% (n = 3). Meanwhile, we further evaluated the SERS performance of the PTTG using a laboratory-based Raman spectrometer, and it could realize the detection as low as 10 pM R6G. The proposed paper-tip substrate would offer a promising potential application for the on-site SERS analysis of food safety and environmental health.

Salvianolic acid F suppresses KRAS-dependent lung cancer cell growth through the PI3K/AKT signaling pathway.

BACKGROUND: KRAS mutation is a common driver of NSCLC, and there is a high proportion of lung cancer patients with KRAS G12C and G12D mutation. KRAS was previously considered an "undruggable" target, but the first KRAS G12C mutation-targeted drug AMG510, entered the market in 2021. However, treatments for G12D mutant tumors remain to be discovered. Salvianolic acid F (SalF), a monomer derived from the traditional Chinese medicine Salvia miltiorrhiza (SM), and KRAS had high binding affinity, especially for KRAS G12D. There is an urgent need to investigate effective and safe novel targeted therapies against KRAS G12D-driven NSCLC. METHODS: To evaluate the anticancer effect of SalF, we used KRAS-overexpressing lung cancer cells in vitro, a subcutaneous transplant tumor model, and KRAS G12D mice model in vivo. Then, the binding effect of SalF and KRAS was investigated using molecular docking, proteolytic assays and protein thermal shift assays. More critically, the PI3K/AKT signaling pathway in the lung was investigated utilizing RT-qPCR and Western Blotting. RESULTS: This is the first study to evaluate the anticancer effect of SalF on KRAS-overexpressing lung cancer cells or KRAS G12D lung tumors in vivo. We demonstrated that SalF inhibits OE-KRAS A549 cell migration, proliferation and promotes apoptosis in vitro. In addition, we used a subcutaneous transplant tumor model to show that SalF suppresses the growth of lung cancer cells in vivo. Interestingly, our group found that SalF was strongly bound to G12D and could decrease the stability and promoted the degradation of the KRAS G12D mutant through molecular docking, proteolytic assays and protein thermal shift assays. Further research demonstrated that in the KrasG12D mice model, after SalF treatment, the number and size of mouse lung tumors were significantly reduced. More importantly, SalF can promote apoptosis by inhibiting downstream PI3K/AKT signaling pathway activation. CONCLUSION: SalF activated apoptosis signaling pathways, suppressed anti-apoptotic genes, and inhibited lung cancer cell growth. These datas suggested that SalF could effectively inhibit the growth of lung tumors with KRAS G12D mutation. SalF may be a novel inhibitor against KRAS G12D, providing a strong theoretical basis for the clinical treatment of lung cancer with KRAS mutations.

Evaluation of anti-Xa levels in patients with venous thromboembolism within the first 48 h of anticoagulation with unfractionated heparin.

Background: A 2019 study by Prucnal and colleagues found that the majority of patients treated with unfractionated heparin for pulmonary embolism did not maintain therapeutic activated partial thromboplastin time levels during the first 48 h of therapy. Objective: The purpose of this study was to evaluate the ability of an institution's unfractionated heparin dosing protocol to achieve and maintain therapeutic anti-Xa levels within the first 48 h of therapy in patients with venous thromboembolism. Methods: This retrospective study included 205 patients from May 2016 through September 2020. Patients were divided into two cohorts: bolus plus infusion (N = 89) and infusion only (N = 116). The primary objective was to determine the number of patients who achieved at least one therapeutic level. Results: Overall, 200 patients (97.6%) had at least one therapeutic level with no statistically significant difference between cohorts (p = 0.65). No more than 60% of patients achieved a therapeutic level at any of the 6-h intervals throughout the timeframe. The median time to the first therapeutic level in the overall group was 12.8 h with no statistically significant difference between the bolus plus infusion and infusion-only cohorts (13.3 h versus 12.7 h, respectively, p = 0.48). Conclusions: Most patients were able to achieve at least one therapeutic level within the first 48 h, but fewer were able to maintain therapeutic levels. Further studies are warranted to determine whether alternative dosing strategies would yield consistent achievement of therapeutic levels and affect patient-oriented outcomes.

Acaricidal activity of bioactive compounds isolated from Aspergillus oryzae against poultry red mites, Dermanyssus gallinae (Acari: Dermanyssidae).

Dermanyssus gallinae, the poultry red mite (PRM), is an obligate ectoparasite feeding on poultry blood, seriously affecting the health of layers and egg production. The control of PRMs mainly relies on chemical drugs, which is facing several challenges such as the environment pollution and drug resistance. Using fungal metabolites is an environmentally friendly alternative for the control of pests. However, few studies have been conducted on the efficacy of fungal metabolites against D. gallinae. In this study, five strains of fungi were isolated from D. gallinae under laboratory conditions, and their extracts with ethyl acetate were tested for acaricidal activity on D. gallinae. The crude extract of Aspergillus oryzae caused 75.55 ± 6.94% mortality of mites at a concentration of 12.5 mg/mL, showing the highest acaricidal effect in all extracts. Subsequently, the extract of A. oryzae was isolated by bio-guided fractionation, and ten major compounds were identified by LC-MS/MS analysis. The results of bioassays indicated that five compounds exhibited acaricidal activity against D. gallinae. N, N-dimethyldecylamine N-oxide was the optimal acaricidal compound with LC50 of 0.568 mg/mL. Additionally, palmitic acid, triethanolamine, cuminaldehyde, and 2,4-dimethylbenzaldehyde also showed acaricidal activity. These compounds have great application potential in the mite control, and the analysis of these fungal acaricidal substances provides a new idea and basis for the subsequent development of PRM control technology.

Structure-activity relationship between gluten and dough quality of sprouted wheat flour based on air classification-induced component recombination.

BACKGROUND: Air classification can separate sprouted wheat flour (SWF) into three types: coarse wheat flour (F1), medium wheat flour (F2) and fine wheat flour (F3). The gluten quality of SWF can be indirectly improved by removing inferior parts (F3). In order to reveal the underlying mechanism of this phenomenon, the composition and structural changes of gluten, as well as the rheological properties and fermentation characteristics of gluten in recombinant dough in the process of air classification of all three SWF types, were analyzed in this study. RESULTS: Overall, sprouting significantly reduced the content of high-molecular-weight subunits, such as glutenin subunit and ω-gliadin. It also destroyed the structural content, such as disulfide bonds, α-helix and ß-turn contents, which maintained the stability of gluten gel. Air classification made the above changes in F3 more severe but reversed them in F1. Moreover, rheological properties were more affected by gluten composition, whereas fermentation characteristics were more affected by gluten structure. CONCLUSION: After air classification, particles rich in high molecular weight subunits from SWF are enriched in F1, and the gluten of F1 has more secondary structure that maintain gel stability, which ultimately lead to improved rheology properties and fermentation characteristics. F3 relatively exhibits the opppsite phenomenon. These results further reveal the potential mechanism of improvement of SWF gluten by air classification. Moreover, Thus, this study provides new perspectives for the utilization of SWF. © 2023 Society of Chemical Industry.

Complement proteins in serum astrocyte-derived exosomes are associated with mild cognitive impairment in type 1 diabetes mellitus patients.

BACKGROUND: The complement system plays a crucial role in cognitive impairment. The aim of this study is to investigate the correlation between the complement proteins levels in serum astrocyte-derived exosomes (ADEs) and mild cognitive impairment (MCI) in type 1 diabetes mellitus (T1DM) patients. METHODS: In this cross-sectional study, the patients with immune-mediated T1DM were enrolled. Healthy subjects matched for age and sex with T1DM patients were selected as controls. The cognitive function was evaluated by a Beijing version of the Montreal Cognitive Assessment (MoCA) questionnaire. The complement proteins including C5b-9, C3b and Factor B in serum ADEs were measured by ELISA kits. RESULTS: This study recruited 55 subjects immune-mediated T1DM patients without dementia, including 31 T1DM patients with MCI, 24 T1DM patients without MCI. 33 healthy subjects were enrolled as controls. The results showed higher complement proteins including C5b-9, C3b and Factor B levels in ADEs from T1DM patients with MCI than those in the controls (P < 0.001, P < 0.001, P = 0.006) and T1DM patients without MCI (P = 0.02, P = 0.02, P = 0.03). The C5b-9 levels in ADEs were independently associated with MCI in T1DM patients(OR: 1.20, 95% CI: 1.00-1.44, P = 0.04). The C5b-9 levels in ADEs were significantly correlated with global cognitive scores (ß = -0.360, P＜0.001) and visuo-executive (ß = -0.132, P＜0.001), language(ß = -0.036, P = 0.026) and delayed recall score (ß = -0.090,P = 0.007). There was no correlation between the C5b-9 levels in ADEs and the fasting glucose, HbA1c, fasting c-peptide and GAD65 antibody in T1DM patients. Furthermore, the C5b-9, C3b and Factor B levels in ADEs exhibited a fair combined diagnostic value for MCI, with an area under the curve of 0.76 (95% CI: 0.63-0.88, P = 0.001). CONCLUSION: The elevated C5b-9 levels in ADEswere significantly associated with theMCI in T1DM patients. The C5b-9 in ADEs may be used as a marker of MCI in T1DM patients.

Xinbao Pill attenuated chronic heart failure by suppressing the ubiquitination of ß-adrenergic receptors.

BACKGROUD: Xinbao Pill (XBP) is extensively used in the adjuvant treatment of chronic heart failure in China. However, the pharmacological effect and underlying mechanism on CHF remains unclear. PURPOSE: Our research was performed to investigate the cardioprotective effect of XBP against CHF and uncover the potential mechanism. METHODS: Male Sprague-Dawley (SD) rats were subjected to the left anterior descending (LAD) artery ligation for 8 weeks and were treated with different doses of XBP (from the 4th week to the end). Cardiac function and morphology assessment were performed by using M-mode echocardiography, H&E and Masson staining. Western blotting analysis, co-immunoprecipitation (IP) assays, siRNA transfection were used to evaluate the mechanism of XBP. RESULTS: XBP improved cardiac function and alleviated cardiac fibrosis in LAD-induced chronic heart failure rats. Meanwhile, XBP protected cardiomyocytes against oxygen-glucose deprivation (OGD) injury in AC16 cells and H9c2 cells. Additionally, XBP could increase the expression of ß1-AR and ß2-AR and inhibit their ubiquitanation. Further mechanism study showed that XBP upregulated USP18 expression, while silence of USP18 attenuated the cardioprotective effect of XBP and the increase of ß1-AR by XBP. Moreover, XBP increased MDM2 and ß-arrestin2, and disrupted the interaction between Nedd4 and ß2-AR. After using the inhibitor of MDM2, SP141, the cardioprotective effect of XBP and the inhibitory effect on the ubiquitanation of ß2-AR were also blocked. CONCLUSION: Our study firstly revealed that XBP improved cardiac function against CHF through suppressing USP18 and MDM2/ß-arrestin2/Nedd4-mediated the ubiquitination of ß1-AR and ß2-AR.

Function of glycogen synthase kinase3 in embryogenesis of Dermanyssus gallinae.

In the life cycle of Dermanyssus gallinae, the embryo is a developmental stage that does not require blood meals, but needs glucose to produce adenosine triphosphate (ATP) through glycolysis or oxidative phosphorylation, providing energy for embryonic development. Glycogen synthase kinase 3 (GSK3), belonging to the serine/threonine kinase family, is a key enzyme involved in glycogen metabolism in many eukaryotes, but not be described in D. gallinae. The present study was conducted to explore the role of Dg-GSK3 in the embryogenesis of D. gallinae. The results of qPCR showed that Dg-GSK3 mRNA was expressed in different development stages of D. gallinae embryos. RNA interference (RNAi) was performed on the female mites and eggs by immersion, and it was found that lowering GSK3 expression level could significantly decrease the female egg laying rate and egg hatching rate (P < 0.05). Some eggs became shrunken and shriveled in appearance. The fecundity of female D. gallinae obtained from the rDg-GSK3-immunized group of chickens (2.56 ± 0.35 eggs per mite, P < 0.0001) decreased significantly from that of the control group (3.49 ± 0.35). The oviposition rate of rDg-GSK3-immunized group (75.94 ± 7.28 %, P = 0.0003）was significantly lower that of the control group (89.69 ± 2.63 %). In conclusion, Dg-GSK3 is a crucial gene during the embryogenesis of D. gallinae, which can affect both the female fecundity and the egg hatching, which help us understand the function of GSK3 gene in the embryogenesis of mites.

Costunolide Protects Myocardium From Ischemia Reperfusion Injury by Inhibiting Oxidative Stress Through Nrf2/Keap1 Pathway Activation.

ABSTRACT: Costunolide (Cos) is a naturally occurring sesquiterpene lactone that exhibits antioxidative properties. In this study, we demonstrate the protective mechanism of Cos against ischemia/reperfusion (I/R)-induced myocardial injury. Cos significantly decreased levels of reactive oxygen species and ameliorated apoptosis of I/R cardiomyocytes both in vitro and in vivo. Further investigation revealed that Cos increased expression of the antioxidant proteins HO-1 and NQO-1 and decreased the Bax/Bcl-2 ratio, thus protecting cardiac cells. NF-E2-related factor 2 (Nrf2) silencing significantly attenuated the protective effects of Cos in tert-butyl hydroperoxide (TBHP)-treated H9C2 cells. Additionally, Cos significantly intensified the I/R- or TBHP-induced dissociation of the Kelch-like ECH-associated protein 1 (Keap1)/Nrf2 complex both in vitro and in vivo. These results suggest that activation of Nrf2/Keap1 using Cos may be a therapeutic strategy for myocardial I/R injury.

Metabolic Engineering of Bacillus subtilis for Riboflavin Production: A Review.

Riboflavin (vitamin B2) is one of the essential vitamins that the human body needs to maintain normal metabolism. Its biosynthesis has become one of the successful models for gradual replacement of traditional chemical production routes. B. subtilis is characterized by its short fermentation time and high yield, which shows a huge competitive advantage in microbial fermentation for production of riboflavin. This review summarized the advancements of regulation on riboflavin production as well as the synthesis of two precursors of ribulose-5-phosphate riboflavin (Ru5P) and guanosine 5'-triphosphate (GTP) in B. subtilis. The different strategies to improve production of riboflavin by metabolic engineering were also reviewed.

Bent-Capillary-Centrifugal-Driven Monodisperse Droplet Generator with Its Application for Digital LAMP Assay.

We developed a bent-capillary-centrifugal-driven (BCCD) monodisperse droplet generator, which could achieve a perfect combination of driving and segmentation for the dispersed phase only using a rotating bent capillary immersed in the continuous phase (mineral oil). The sample could flow continuously to the bent-capillary outlet to form the droplet precursors, which were segmented into homogeneous droplets in the continuous phase. Through the investigation of influence factors on droplet size and stability, we found that the droplet size could be conveniently controlled by the rotational speed of the bent capillary. The droplet volumes could be adjusted with the range from 34 pL to 1 µL, and the coefficient variations (CVs) were less than 3%. Meanwhile, the BCCD droplet generator could realize the controllable droplet output with a high-efficiency sample utilization of 99.75 ± 1.15%, which offered a significant advantage in reducing the waste of precious samples in the droplet generation process. We validated this system with a digital loop-mediated isothermal amplification (dLAMP) assay for the absolute quantification of Mycobacterium tuberculosis complex nucleic acids. The results demonstrated that the BCCD droplet generator was easy to build, was of low cost, and was convenient to operate, as well as avoided sample loss and cross-contamination by coupling with a 96-well plate. Overall, the present platform, as a simple chip-free droplet generator, will provide an especially valuable droplet generation solution for biochemical applications based on droplets.

Identification of guanine and hematin as arrestment pheromones of poultry red mites, Dermanyssus gallinae (Acari: Dermanyssidae) and their application in mite control.

Dermanyssus gallinae, also known as the poultry red mite (PRM), is one of widespread ectoparasite in the poultry industry worldwide, causing direct and indirect detriments to poultry as well as substantial financial losses. Novel control methods are urgently needed to improve the current acaricide-based control of D. gallinae. The control approach based on arrestment pheromone is environment-friendly but the related research is limited in PRMs. In the present study we found two compounds from the mite feces acting as arrestment pheromones of D. gallinae, which could lead to mite arrestment upon contact. One is guanine, which was also found in unfed female mites' acid-saline extract. The other is hematin. Moreover, it was found that the ferric ion of hematin played a pivotal role in stimulating the arrestment of mites. Finally, it was found the combination of guanine or/and hematin plus cypermethrin led to significantly improved mite-killing performance compared with cypermethrin, showing a promising potential of novel control method based on the arrestment pheromone.

Nuciferine attenuates atherosclerosis by regulating the proliferation and migration of VSMCs through the Calm4/MMP12/AKT pathway in ApoE(-/-) mice fed with High-Fat-Diet.

BACKGROUND: Atherosclerosis (AS) is the pathological basis of multiple cardiovascular diseases. The pathogenesis of AS is closely related to the abnormal proliferation and migration of vascular smooth muscle cells (VSMCs). Nuciferine, an aporphine alkaloid from lotus leaf, has various pharmacological activities. However, the effect and mechanism of nuciferine on regulating proliferation and migration of VSMCs against AS is still unclear. PURPOSE: To elucidate the pharmacological effect and molecular mechanism of nuciferine on AS in ApoE(-/-) mice fed with High-Fat-Diet (HFD). STUDY DESIGN: HFD-fed ApoE(-/-) mice and 3% fetal bovine serum (FBS) induced mouse aortic vascular smooth muscle cells (MOVAS) were used to investigate the protective effect and mechanism of nuciferine on AS. METHODS: Oil red O staining was used to detect the atherosclerotic lesions. Western blotting and immunofluorescence were used to determine calmodulin 4 (Calm4) expression and localization. CCK-8 assay, transwell and wound-healing assays were used to measure the migration and proliferation of MOVAS cells. RESULTS: Nuciferine at 40 mg/kg significantly ameliorated the aortic lesion and vascular plaque in AS model, which was equal to the effect of the positive control drug (atorvastatin). In addition, nuciferine attenuated the migration and proliferation of VSMCs in vivo and in vitro. Importantly, nuciferine down-regulated the increase of Calm4 induced by HFD-fed in ApoE(-/-) mice or 3% FBS induced MOVAS cells. However, the inhibitory effect of nuciferine on the migration and proliferation of MOVAS cells was blocked when Calm4 was overexpressed. Furthermore, we found that nuciferine suppressed MMP12 and PI3K/Akt signaling pathway via Calm4. CONCLUSION: Our results illustrated that Calm4 promoted the proliferation and motility of MOVAS by activating MMP12/Akt signaling pathway in AS. Nuciferine has a significant anti-atherogenic effect by regulating the proliferation and migration of VSMCs through the Calm4/MMP12/AKT signaling pathway. Thus, Calm4 could potentially be a new target for AS therapy, and nuciferine could be a potential drug against AS.

Single-dose rAAV5-based vaccine provides long-term protective immunity against SARS-CoV-2 and its variants.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus and its variants, has posed unprecedented challenges worldwide. Existing vaccines have limited effectiveness against SARS-CoV-2 variants. Therefore, novel vaccines to match mutated viral lineages by providing long-term protective immunity are urgently needed. We designed a recombinant adeno-associated virus 5 (rAAV5)-based vaccine (rAAV-COVID-19) by using the SARS-CoV-2 spike protein receptor binding domain (RBD-plus) sequence with both single-stranded (ssAAV5) and self-complementary (scAAV5) delivery vectors and found that it provides excellent protection from SARS-CoV-2 infection. A single-dose vaccination in mice induced a robust immune response; induced neutralizing antibody (NA) titers were maintained at a peak level of over 1:1024 more than a year post-injection and were accompanied by functional T-cell responses. Importantly, both ssAAV- and scAAV-based RBD-plus vaccines produced high levels of serum NAs against the circulating SARS-CoV-2 variants, including Alpha, Beta, Gamma and Delta. A SARS-CoV-2 virus challenge showed that the ssAAV5-RBD-plus vaccine protected both young and old mice from SARS-CoV-2 infection in the upper and lower respiratory tracts. Whole genome sequencing demonstrated that AAV vector DNA sequences were not found in the genomes of vaccinated mice one year after vaccination, demonstrating vaccine safety. These results suggest that the rAAV5-based vaccine is safe and effective against SARS-CoV-2 and several variants as it provides long-term protective immunity. This novel vaccine has a significant potential for development into a human prophylactic vaccination to help end the global pandemic.