Biomolecule-regulation of fluorescent probe signaling: Homogeneous rapid portable protease sensing in serum.

BACKGROUND: As is well documented, prostate cancer (PCa) being the second most prevalent cancer in men worldwide, emphasizing the importance of early diagnosis for prognosis. However, conventional prostate-specific antigen (PSA) testing lacks sufficient diagnostic efficiency due to its relatively low sensitivity and limited detection range. Mounting evidence suggests that matrix metalloproteinase 9 (MMP-9) expression increases with the aggressive behavior of PCa, highlighting the significance of detecting the serum level of MMP-9 in patients. Developing a non-immune rapid, portable MMP-9 detection strategy and investigating its representativeness of PCa serum markers hold considerable implications. RESULTS: Herein, our study developed a simple, homogeneous dual fluorescence and smartphone-assisted red-green-blue (RGB) visualization peptide sensor of MMP-9, utilizing cadmium telluride quantum dots (CdTe QDs) and calcein as signal reporters. The essence of our approach revolves around the proteolytic ability of MMP-9, exploiting the selective recognition of molecule-Cu2+ complexes with different molecular weights by CdTe QDs and calcein. Under optimized conditions, the limits of detection (LODs) for MMP-9 were 0.5 pg/mL and 6 pg/mL using fluorescence and RGB values readouts, respectively. Indeed, this strategy exhibited robust specificity and anti-interference ability. MMP-9 was quantified in 42 clinical serum samples via dual-fluorescence analysis, with 12 samples being visually identified with a smartphone. According to receiver operating characteristic curve (ROC) analysis, its sensitivity and specificity were 90 % and 100 %, respectively, with an area under curve (AUC) value of 0.903. SIGNIFICANCE AND NOVELTY: Of note, the results of the aforementioned analysis were highly consistent with the serum level of PSA, clinical color Doppler flow imaging (CDFI), and histopathological results. Therefore, this simple, rapid, homogeneous fluorescence and visualization strategy can reliably measure MMP-9 levels and exhibit promising potential in point-of-care testing (POCT) applications for PCa patients.

Evaluation of electrokinetic-assisted phytoremediation efficiency of dibutyl phthalate contaminated soil by maize (Zea mays L.) under different electric field intensities.

The excessive accumulation of dibutyl phthalate (DBP) in soil poses a serious threat to soil ecosystems and crop safety production. Electrokinetic-assisted phytoremediation (EKPR) has been considered as a potential technology for remediating organic contaminated soils. In order to investigate the effect of different electric fields on removal efficiency of DBP, three kinds of electric fields were set up in this study (1 V·cm-1, 2 V·cm-1 and 3 V·cm-1). The results showed that 59 % of DBP in soil was removed by maize (Zea mays L.) within 20 d in low-intensity electric field (1 V·cm-1), and the accumulation of DBP in maize tissues decreased significantly compared to the non-electrified treatment group. Interestingly, it could be observed that the low-intensity electric field could maintain ion homeostasis and improve the photosynthetic efficiency of the plant, thereby relieving the inhibition of DBP on plant growth and increasing the chlorophyll content (94.1 %) of maize. However, the removal efficiency of DBP by maize decreased significantly under the medium-intensity (2 V·cm-1) and high-intensity electric field (3 V·cm-1). Moreover, the important roles of soil enzyme and rhizosphere bacterial community in low-electric field were also investigated and discussed. This study provided a new perspective for exploring the mechanism of removing DBP through EKPR.

Correlations of pathomorphological parameters between lesions at the invasive front and lymph node metastases in colorectal cancer: a retrospective clinical study.

BACKGROUND: Lymph node (LN) metastasis is one of the most important indicators to evaluate stage, choose treatment strategy, and predict outcome of colorectal cancer (CRC). The morphological correlation between primary tumors and LN metastases can help predict the incidence of LN metastasis in CRC more accurately and assist with more individualized risk-stratification management decisions. METHODS: A retrospective study was devised with paired tissue specimens from the invasive front of primary tumors and LN metastases in 426 patients after a radial surgery for CRC. According to the presence (N +) or absence (N-) of regional LN metastasis and the number of LN metastases (pN1a/1b/1c/2a/2b), comparisons were performed regarding tumor budding (TB) and poorly-differentiated clusters (PDC). In addition, their correlation with the incidence of LN metastasis and the extent were explored. RESULTS: The TB and PDC in the invasive front of primary tumors presented significant correlations with the incidence of LN metastasis and the number of LN metastases in CRC (P < 0.001). TB2/3 led to a risk of LN metastasis 6.68-fold higher than TB1, while PDC2/3 resulted in a risk of LN metastasis 8.46-fold higher than PDC1. Additionally, the risk of developing 4 or more LN metastases was 3.08-fold and 2.86-fold higher upon TB2/3 and PDC2/3 than that with TB1 and PDC1, respectively. Moderate positive correlations were found between the invasive front of primary tumors and LN metastases in terms of TB and PDC, respectively. CONCLUSIONS: TB and PDC, at the invasive tumor front are important morphological markers to evaluate LN metastasis in CRC, and they can be employed as reference indicators to assess or predict the potential of LN metastasis in CRC in clinical practice.

Comparative Bleeding Risk of Brand Vs Generic Rivaroxaban in Elderly Inpatients with Atrial Fibrillation.

Objective: Atrial fibrillation (AF) is the most common abnormal heart rhythm in elderly patients. Rivaroxaban has been widely used for stroke prevention. The anticoagulant response to rivaroxaban increases with age, which may make elderly patients susceptible to adverse outcomes resulting from small differences in bioavailability between generic and brand products. Methods: We designed a cohort study of ≥65-year-old inpatients with AF. Sociodemographic and laboratory measures of qualified patients who received brand or generic rivaroxaban for at least 72 hours at the study hospital from January 2021 to June 2023 were collected retrospectively. The primary outcome was the incidence of bleeding. Results: A total of 1008 qualifying patients were included for analysis, with 626 (62.1%) receiving brand rivaroxaban and 382 (37.9%) receiving generic rivaroxaban. After propensity score matching and weighting to account for confounders, the odds ratios comparing brand vs generic rivaroxaban (95% confidence intervals) for the bleeding was 1.15 (0.72-1.82). Results from subgroup analyses of patients with age ≥85, HAS-BLED score ≥ 3, containment of antiplatelet drugs, and female patients were consistent with the primary analysis. Conclusion: It provides evidence regarding the clinical safety outcome of generic rivaroxaban in the elderly AF population that may be particularly susceptible to adverse outcomes resulting from small allowable differences in pharmacokinetics.

In silico study of androgen receptor N-terminal domain and exploration of its modulators.

The androgen receptor (AR, Uniprot: P10275) signaling plays a key role in the progression of prostate cancer, various AR-related ligands have been reported to treat prostate cancer. However, some resistance mechanisms limited the treating effect of these ligands. Since DBD binding or the allosteric binding sites in LBD of AR may allow the circumvention of some drug resistance mechanisms, anti-resistance is expected especially through the NTD (N-terminal domain) targeting. What's more, studies have shown that compounds including EPI-001 and its derivatives which bind to the Tau-5 region on NTD could be promising molecules for AR-based therapeutics. Herein, we employed aMD (accelerated molecular dynamics) simulation to fold Tau-5 unit proteins into native structure correctly. Subsequently, based on the predicted structural features of Tau-5, the virtual screening was conducted to discover new compounds targeting AR-NTD. We picked up 8 compounds (according to their docking scores and partly similar structural consists as known AR ligands) and analyzed their interaction with Tau-5, compared with the positive control EPI-001, four of the pick-up compounds showed better glide scores. Interestingly, although compound 8 had a lower docking score, it consisted of a similar component as the ligand EIQPN and the amide derivatives, this predicts that compound 8 has also the potential to be modified into an excellent AR-NTD binding molecule. These 8 compounds were all commercially available and could be tested to check whether there was a hit compound to bind the AR-NTD and to regulate its bio-activities. Together, this study described an in silico VLS approach to discover AR-NTD ligands and provided more choices for developing AR-targeted therapies.Communicated by Ramaswamy H. Sarma.

[Development of a Catalytic Hairpin Assembly-Based Fluorescent Assay for the Rapid Detection of SARS-CoV-2 Target RNA]. / åŸºäºŽå‚¬åŒ–å‘å¤¹è‡ªç»„è£ æŠ€æœ¯çš„æ–°åž‹å† çŠ¶ç— æ¯’é¶RNAå¿«é€Ÿè§å ‰æ£€æµ‹æ³•çš„å»ºç«‹.

Objective: To develop a catalytic hairpin assembly (CHA)-based fluorescent assay for the detection of the target RNA of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), so as to realize the rapid nucleic acid testing of SARS-CoV-2. Methods: A 24-nt segment of the SARS-CoV-2 nucleocapsid protein gene (N gene, NC_045512.2) was chosen as the target RNA and the hairpin motif 1 (H1) and hairpin motif 2 (H2) were designed based on the principle of CHA reaction. The H1 motif was labelled with a fluorophore group as well as a quencher group. When the target RNA was added to the hairpin motifs, CHA reaction was triggered at room temperature (25 ℃), which led to the amplification of fluorescence signal, thereby enabling the rapid detection of the target RNA. After the optimization of the hairpin motifs and the experimental conditions, the sensitivity and the specificity of the testing method were measured to evaluate its performance. Results: We successfully constructed a CHA-based fluorescent assay specifically for the target RNA of SARS-CoV-2. With this method, testing could be completed at room temperature within 30 min. This testing method exhibited excellent specificity and could be used to accurately distinguish the perfectly-matched target RNA from the target RNA with single-base mutations. In addition, the testing method demonstrated good sensitivity, with a detection limit of 50 pmol/L. Conclusion: The proposed assay enables the simple and rapid detection of the SARS-CoV-2 target RNA with excellent sensitivity and specificity, showing great promise for further optimization and subsequent clinical application for the rapid detection of SARS-CoV-2 nucleic acid.

Platelet-derived drug delivery systems: Pioneering treatment for cancer, cardiovascular diseases, infectious diseases, and beyond.

Platelets play a critical role as circulating cells in the human body and contribute to essential physiological processes such as blood clotting, hemostasis, vascular repair, and thrombus formation. Currently, platelets are extensively employed in the development of innovative biomimetic drug delivery systems, offering significant enhancements in circulation time, biocompatibility, and targeted delivery efficiency compared to conventional drug delivery approaches. Leveraging the unique physiological functions of platelets, these platelet-derived drug delivery systems (DDSs) hold great promise for the treatment of diverse diseases, including cancer, cardiovascular diseases, infectious diseases, wound healing and other diseases. This review primarily focuses on the design and characteristics of existing platelet-derived DDSs, including their preparation and characterization methods. Furthermore, this review comprehensively outlines the applications of these materials across various diseases, offering a holistic understanding of their therapeutic potential. This study aimed to provide a comprehensive overview of the potential value of these materials in clinical treatment, serving as a valuable reference for the advancement of novel platelet-derived DDSs and their broader utilization in the field of disease treatment.

Laccase as a useful assistant for maize to accelerate the phenanthrene degradation in soil.

Polycyclic aromatic hydrocarbon (PAH) pollution has attracted much attention due to their wide distribution in soil environment and serious harm to human health. In order to establish an efficient and eco-friendly technology for remediation of PAH-contaminated soil, phytoremediation utilizing maize assisted with enzyme remediation was explored in this study. The results showed that the participation of laccase could promote the degradation of phenanthrene (PHE) from soil and significantly reduce the accumulation of PHE in maize. The degradation efficiency of PHE in soil could reach 77.19% under laccase-assisted maize remediation treatment, while the accumulation of PHE in maize roots and leaves decreased by 41.23% and 74.63%, respectively, compared to that without laccase treatment, after 24 days of maize cultivation. Moreover, it was found that laccase addition shifted the soil microbial community structure and promoted the relative abundance of some PAH degrading bacteria, such as Pseudomonas and Sphingomonas. In addition, the activities of some enzymes that were involved in PAH degradation process and soil nutrient cycle increased with the treatment of laccase enzyme. Above all, the addition of laccase could not only improve the removal efficiency of PHE in soil, but also alter the soil environment and reduce the accumulation of PHE in maize. This study provided new perspective for exploring the efficiency of the laccase-assisted maize in the remediation of contaminated soil, evaluating the way for reducing the risk of secondary pollution of plants in the phytoremediation process.

The survival of murine hepatitis virus (a surrogate of SARS-CoV-2) on conventional packaging materials under cold chain conditions.

Introduction: The cold chain conditions have been suggested to facilitate long-distance transmission of SARS-CoV-2, but it is unclear how viable the virus is on cold chain packaging materials. Methods: This study used the MHV-JHM strain of murine hepatitis virus as a model organism to investigate the viability of SARS-CoV-2 on foam, plastic, cardboard, and wood sheets at different temperatures (-40°C, -20°C, and 4°C). In addition, the ability of peracetic acid and sodium hypochlorite to eliminate the MHV-JHM on plastic and cardboard sheets were also evaluated. Results: The results indicate that MHV-JHM can survive on foam, plastic, or cardboard sheets for up to 28 days at -40°C and -20°C, and up to 14 days on foam and plastic surfaces at 4°C. Although viral nucleic acids were still detectable after storing at 4°C for 28 days, the corresponding virus titer was below the limit of quantification (LOQ). Discussion: The study highlights that a positive nucleic acid test result may not indicate that the virus is still viable, and confirms that peracetic acid and sodium hypochlorite can effectively eliminate MHV-JHM on packaging materials under cold chain conditions.

The role of microplastics in the process of laccase-assisted phytoremediation of phenanthrene-contaminated soil.

Polycyclic aromatic hydrocarbons (PAHs) are highly toxic organic pollutants widely distributed in terrestrial environments and laccase was considered as an effective enzyme in PAHs bioremediation. However, laccase-assisted phytoremediation of PAHs-contaminated soil has not been reported. Moreover, the overuse of plastic films in agriculture greatly increased the risk of co-existence of PAHs and microplastics in soil. Microplastics can adsorb hydrophobic organics, thus altering the bioavailability of PAHs and ultimately affecting the removal of PAHs from soil. Therefore, this study aimed to evaluate the efficiency of laccase-assisted maize (Zea mays L.) in the remediation of phenanthrene (PHE)-contaminated soil and investigate the effect of microplastics on this remediation process. The results showed that the combined application of laccase and maize achieved a removal efficiency of 83.47 % for soil PHE, and laccase significantly reduced the accumulation of PHE in maize. However, microplastics significantly inhibited the removal of soil PHE (10.88 %) and reduced the translocation factor of PHE in maize (87.72 %), in comparison with PHE + L treatment. Moreover, microplastics reduced the laccase activity and the relative abundance of some PAHs-degrading bacteria in soil. This study provided an idea for evaluating the feasibility of the laccase-assisted plants in the remediation of PAHs-contaminated soil, paving the way for reducing the risk of secondary pollution in the process of phytoremediation.

Genetically Engineered-Cell-Membrane Nanovesicles for Cancer Immunotherapy.

The advent of immunotherapy has marked a new era in cancer treatment, offering significant clinical benefits. Cell membrane as drug delivery materials has played a crucial role in enhancing cancer therapy because of their inherent biocompatibility and negligible immunogenicity. Different cell membranes are prepared into cell membrane nanovesicles (CMNs), but CMNs have limitations such as inefficient targeting ability, low efficacy, and unpredictable side effects. Genetic engineering has deepened the critical role of CMNs in cancer immunotherapy, enabling genetically engineered-CMN (GCMN)-based therapeutics. To date, CMNs that are surface modified by various functional proteins have been developed through genetic engineering. Herein, a brief overview of surface engineering strategies for CMNs and the features of various membrane sources is discussed, followed by a description of GCMN preparation methods. The application of GCMNs in cancer immunotherapy directed at different immune targets is addressed as are the challenges and prospects of GCMNs in clinical translation.

Mechanism of inhibition of CRISPR-Cas9 by anti-CRISPR protein AcrIIC1.

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems are bacterial and archaeal defense mechanisms against invading phages and viruses. To overcome these defenses, phages and other mobile genetic elements (MGEs) have evolved multiple anti-CRISPR proteins (Acrs) that can inhibit the function of CRISPR-Cas systems. The AcrIIC1 protein has been shown to be able to inhibit the activity of Neisseria meningitidis Cas9 (NmeCas9) in both bacteria and human cells. Here, we solve the structure of AcrIIC1 in complex with the HNH domain of NmeCas9 using X-ray crystallography. The structure shows that AcrIIC1 binds to the catalytic sites of the HNH domain, preventing it from accessing the DNA target. In addition, our biochemical data show that AcrIIC1 is a broad-spectrum inhibitor targeting Cas9 enzymes from different subtypes. Taken together, the structure and biochemical analysis reveal the molecular mechanism of AcrIIC1-mediated Cas9 inhibition and provide new insights into regulatory tools for Cas9-based applications.

Transcriptional and Epigenetic Alterations in the Progression of Non-Alcoholic Fatty Liver Disease and Biomarkers Helping to Diagnose Non-Alcoholic Steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) encompasses a broad spectrum of conditions from simple steatosis (non-alcoholic fatty liver (NAFL)) to non-alcoholic steatohepatitis (NASH), and its global prevalence continues to rise. NASH, the progressive form of NAFLD, has higher risks of liver and non-liver related adverse outcomes compared with those patients with NAFL alone. Therefore, the present study aimed to explore the mechanisms in the progression of NAFLD and to develop a model to diagnose NASH based on the transcriptome and epigenome. Differentially expressed genes (DEGs) and differentially methylated genes (DMGs) among the three groups (normal, NAFL, and NASH) were identified, and the functional analysis revealed that the development of NAFLD was primarily related to the oxidoreductase-related activity, PPAR signaling pathway, tight junction, and pathogenic Escherichia coli infection. The logistic regression (LR) model, consisting of ApoF, THOP1, and BICC1, outperformed the other five models. With the highest AUC (0.8819, 95%CI: 0.8128-0.9511) and a sensitivity of 97.87%, as well as a specificity of 64.71%, the LR model was determined as the diagnostic model, which can differentiate NASH from NAFL. In conclusion, several potential mechanisms were screened out based on the transcriptome and epigenome, and a diagnostic model was built to help patient stratification for NAFLD populations.

Serum neurofilament light chain levels are associated with early neurological deterioration in minor ischemic stroke.

Objectives: Patients with minor ischemic stroke (MIS) frequently suffer from early neurological deterioration (END) and become disabled. Our study aimed to explore the association between serum neurofilament light chain (sNfL) levels and END in patients with MIS. Methods: We conducted a prospective observational study in patients with MIS [defined as a National Institutes of Health Stroke Scale (NIHSS) score 0-3] admitted within 24 h from the onset of symptoms. sNfL levels were measured at admission. The primary outcome was END, defined as an increase in the NIHSS score by ≥2 points within 5 days after admission. Univariate and multivariate analyses were performed to explore the risk factors associated with END. Stratified analyses and interaction tests were conducted to identify variables that might modify the association between sNfL levels and END. Results: A total of 152 patients with MIS were enrolled, of which 24 (15.8%) developed END. The median sNfL level was 63.1 [interquartile range (IQR), 51.2-83.4] pg/ml on admission, which was significantly higher than that of 40 age- and sex-matched healthy controls (median 47.6, IQR 40.8-56.1 pg/ml; p < 0.001). Patients with MIS with END had a higher level of sNfL (with ND: median 74.1, IQR 59.5-89.8 pg/ml; without END: median 61.2, IQR 50.5-82.2 pg/ml; p = 0.026). After adjusting for age, baseline NIHSS score, and potential confounding factors in multivariate analyses, an elevated sNfL level (per 10 pg/mL) was associated with an increased risk of END [odds ratio (OR) 1.35, 95% confidence interval (CI) 1.04-1.77; p = 0.027). Stratified analyses and interaction tests demonstrated that the association between sNfL and END did not change by age group, sex, baseline NIHSS score, Fazekas' rating scale, hypertension, diabetes mellitus, intravenous thrombolysis, and dual antiplatelet therapy in patients with MIS (all p for interaction > 0.05). END was associated with an increased risk of unfavorable outcomes (modified Rankin scale score ranging from 3 to 6) at 3 months. Conclusion: Early neurological deterioration is common in minor ischemic stroke and is associated with poor prognosis. The elevated sNfL level was associated with an increased risk of early neurological deterioration in patients with minor ischemic stroke. sNfL might be a promising biomarker candidate that can help to identify patients with minor ischemic stroke at high risk of neurological deterioration, for reaching individual therapeutic decisions in clinical practice.

Lnc-AIFM2-1 promotes HBV immune escape by acting as a ceRNA for miR-330-3p to regulate CD244 expression.

Chronic hepatitis B (CHB) virus infection is a major risk factor for cirrhosis and hepatocellular carcinoma (HCC). Hepatitis B virus (HBV) immune escape is regulated by the exhaustion of virus-specific CD8+ T cells, which is associated with abnormal expression of negative regulatory molecule CD244. However, the underlying mechanisms are unclear. To investigate the important roles of non-coding RNAs play in CD244 regulating HBV immune escape, we performed microarray analysis to determine the differential expression profiles of long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and mRNAs in patients with CHB and patients with spontaneous clearance of HBV. Competing endogenous RNA (ceRNA) was analyzed by bioinformatics methods and confirmed by the dual-luciferase reporter assay. Furthermore, gene silencing and overexpression experiments were used to further identify the roles of lncRNA and miRNA in HBV immune escape through CD244 regulation. The results showed that the expression of CD244 on the surface of CD8+ T cells was significantly increased in CHB patients and in the co-culture system of T cells and HBV-infected HepAD38 cells, which was accompanied by the reduction of miR-330-3p and the elevation of lnc-AIFM2-1. The down-regulated miR-330-3p induced the apoptosis of T cells by lifting the inhibition of CD244, which was reversed by miR-330-3p mimic or CD244-siRNA. Lnc-AIFM2-1 promotes the accumulation of CD244, which is mediated by decreased miR-330-3p, and then reduced the clearance ability of CD8+ T cells to HBV through regulated CD244 expression. And the injury in the ability of CD8+ T cells to clear HBV can be reversed by lnc-AIFM2-1-siRNA, miR-330-3p mimic, or CD244-siRNA. Collectively, our findings indicate that lnc-AIFM2-1 on CD244 by acting as a ceRNA of miR-330-3p contributes to HBV immune escape, which may provide novel insights into the roles of interaction networks among lncRNA, miRNA, and mRNA in HBV immune escape, highlighting potential applications of lnc-AIFM2-1 and CD244 for diagnosis and treatment in CHB.

Combined transcriptomic and metabolomic analysis of Salmonella in the presence or absence of PhoP-PhoQ system under low Mg2+ conditions.

INTRODUCTION: Previous reports revealed the role played by Salmonella PhoP-PhoQ system in virulence activation, antimicrobial tolerance and intracellular survival, the impact of PhoP-PhoQ on cell metabolism has been less extensively described. OBJECTIVES: The aim of this study is to address whether and how the PhoP-PhoQ system affects the cell metabolism of Salmonella. METHODS: We constructed a Salmonella phoP deletion mutant strain TT-81 (PhoP-OFF), a Salmonella PhoP constitutively expressed strain TT-82 (PhoP-ON) and a wild-type Salmonella PhoP strain TT-80 (PhoP-N), using P22-mediated generalized transduction or λ Red-mediated targeted mutagenesis. We then measured the in vitro growth kinetics of all test strains and determined their metabolomic and transcriptomic profiles using gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) and RNA-seq technique, respectively. RESULTS: Low-Mg2+ conditions impaired the growth of the phoP deletion mutant strain TT-81 (PhoP-OFF) dramatically. 42 metabolites in the wild-type PhoP strain TT-80 (PhoP-N) and 28 metabolites in the PhoP constitutively expressed strain TT-82 (PhoP-ON) changed by the absence of phoP. In contrast, the level of 19 compounds in TT-80 (PhoP-N) changed comparing to the PhoP constitutively expressed strain TT-82 (PhoP-N). The mRNA level of 95 genes in TT-80 (PhoP-N) changed when phoP was disrupted, wherein 78 genes downregulated and 17 genes upregulated. 106 genes were determined to be differentially expressed between TT-81 (PhoP-OFF) and TT-82 (PhoP-ON). While only 16 genes were found to differentially expressed between TT-82 (PhoP-ON) and TT-80 (PhoP-N). CONCLUSION: Our findings confirmed the impact of PhoP-PhoQ system on lipopolysaccharide (LPS) modification, energy metabolism, and the biosynthesis or transport of amino acids. Most importantly, we demonstrated that the turnover of a given metabolite could respond differentially to the level of phoP. Taken together, the present study provided new insights into the adaptation of Salmonella to the host environment and helped to characterize the impact of the PhoP-PhoQ system on the cell metabolism.

Recognition of cyclic dinucleotides and folates by human SLC19A1.

Cyclic dinucleotides (CDNs) are ubiquitous signalling molecules in all domains of life1,2. Mammalian cells produce one CDN, 2'3'-cGAMP, through cyclic GMP-AMP synthase after detecting cytosolic DNA signals3-7. 2'3'-cGAMP, as well as bacterial and synthetic CDN analogues, can act as second messengers to activate stimulator of interferon genes (STING) and elicit broad downstream responses8-21. Extracellular CDNs must traverse the cell membrane to activate STING, a process that is dependent on the solute carrier SLC19A122,23. Moreover, SLC19A1 represents the major transporter for folate nutrients and antifolate therapeutics24,25, thereby placing SLC19A1 as a key factor in multiple physiological and pathological processes. How SLC19A1 recognizes and transports CDNs, folate and antifolate is unclear. Here we report cryo-electron microscopy structures of human SLC19A1 (hSLC19A1) in a substrate-free state and in complexes with multiple CDNs from different sources, a predominant natural folate and a new-generation antifolate drug. The structural and mutagenesis results demonstrate that hSLC19A1 uses unique yet divergent mechanisms to recognize CDN- and folate-type substrates. Two CDN molecules bind within the hSLC19A1 cavity as a compact dual-molecule unit, whereas folate and antifolate bind as a monomer and occupy a distinct pocket of the cavity. Moreover, the structures enable accurate mapping and potential mechanistic interpretation of hSLC19A1 with loss-of-activity and disease-related mutations. Our research provides a framework for understanding the mechanism of SLC19-family transporters and is a foundation for the development of potential therapeutics.

Functional characterization of a gamma-glutamyl phosphate reductase ProA in proline biosynthesis and promoting expression of type three secretion system in Ralstonia solanacearum.

Ralstonia solanacearum RSc2741 has been predicted as a gamma-glutamyl phosphate reductase ProA catalyzing the second reaction of proline formation from glutamate. Here, we experimentally demonstrated that proA mutants were proline auxotrophs that failed to grow in a minimal medium, and supplementary proline, but not glutamate, fully restored the diminished growth, confirming that ProA is responsible for the biosynthesis of proline from glutamate in R. solanacearum. ProA was previously identified as one of the candidates regulating the expression of genes for type three secretion system (T3SS), one of the essential pathogenicity determinants of R. solanacearum. Supplementary proline significantly enhanced the T3SS expression both in vitro and in planta, indicating that proline is a novel inducer of the T3SS expression. Deletion of proA substantially impaired the T3SS expression both in vitro and in planta even under proline-supplemented conditions, indicating that ProA plays additional roles apart from proline biosynthesis in promoting the expression of the T3SS genes. It was further revealed that the involvement of ProA in the T3SS expression was mediated through the pathway of PrhG-HrpB. Both the proA mutants and the wild-type strain grew in the intercellular spaces of tobacco leaves, while their ability to invade and colonize tobacco xylem vessels was substantially impaired, which was about a 1-day delay for proA mutants to successfully invade xylem vessels and was about one order of magnitude less than the wild-type strain to proliferate to the maximum densities in xylem vessels. It thus resulted in substantially impaired virulence of proA mutants toward host tobacco plants. The impaired abilities of proA mutants to invade and colonize xylem vessels were not due to possible proline insufficiency in the rhizosphere soil or inside the plants. All taken together, these results extend novel insights into the understanding of the biological function of ProA and sophisticated regulation of the T3SS and pathogenicity in R. solanacearum.

Expert consensus statement on therapeutic drug monitoring and individualization of linezolid.

Linezolid is an oxazolidinone antibacterial drug, and its therapeutic drug monitoring and individualized treatment have been challenged since its approval. With the in-depth clinical research of linezolid, we have changed our attitude toward its therapeutic drug monitoring and our view of individualized treatment. On the basis of summarizing the existing clinical studies, and based on the practical experience of each expert in their respective professional fields, we have formed this expert consensus. Our team of specialists is a multidisciplinary team that includes pharmacotherapists, clinical pharmacology specialists, critical care medicine specialists, respiratory specialists, infectious disease specialists, emergency medicine specialists and more. We are committed to the safe and effective use of linezolid in patients in need, and the promotion of its therapeutic drug monitoring.

Medical expenditure of women during pregnancy, childbirth and puerperium at the beginning of China's universal two-child policy enactment: a population-based retrospective study.

OBJECTIVES: To describe and explore women's medical expenditures during pregnancy, childbirth and puerperium at the beginning of the universal two-child policy enactment in China. DESIGN: Population-based retrospective study. SETTING: Dalian, China. PARTICIPANTS: Under the System of Health Accounts 2011 framework, the macroscopic dataset was obtained from the annual report at the provincial and municipal levels in China. The research sample incorporated 65 535 inpatient and outpatient records matching International Classification of Diseases, 10th Revision codes O00-O99 in Dalian city from 2015 through 2017. PRIMARY AND SECONDARY OUTCOME MEASURES: The study delineates women's current curative expenditure (CCE) during pregnancy, childbirth and puerperium at the beginning of the universal two-child policy in China. The temporal changes of medical expenditure of women during pregnancy, childbirth and puerperium at the beginning of China's universal two-child policy enactment were assessed. The generalised linear model and structural equation model were used to test the association between medical expenditure and study variables. RESULTS: Unlike the inverted V-shaped trend in the number of live newborns in Dalian over the 3 studied years, CCE on pregnancy, childbirth and puerperium dipped slightly in 2016 (¥260.29 million) from 2015 (¥263.28 million) and saw a surge in 2017 (¥288.65 million). The ratio of out-of-pocket payment/CCE reduced year by year. There was a rapid increase in CCE in women older than 35 years since 2016. Length of stay mediated the relationship between hospital level, year, age, reimbursement ratio and medical expenditure. CONCLUSIONS: The rise in CCE on pregnancy, delivery and puerperium lagged 1 year behind the surge of newborns at the beginning of China's universal two-child policy. Length of stay acted as a crucial mediator driving up maternal medical expenditure. Reducing medical expenditure by shortening the length of stay could be a feasible way to effectively address the issue of cost in women during pregnancy, childbirth and puerperium.