Detection and Exclusion of False-Positive Molecular Formula Assignments via Mass Error Distributions in UHR Mass Spectra of Natural Organic Matter.

Ultrahigh resolution mass spectrometry (UHRMS) routinely detects and identifies thousands of mass peaks in complex mixtures, such as natural organic matter (NOM) and petroleum. The assignment of several chemically plausible molecular formulas (MFs) for a single accurate mass still poses a major problem for the reliable interpretation of NOM composition in a biogeochemical context. Applying sensible chemical rules for MF validation is often insufficient to eliminate multiple assignments (MultiAs)─especially for mass peaks with low abundance or if ample heteroatoms or isotopes are included - and requires manual inspection or expert judgment. Here, we present a new approach based on mass error distributions for the identification of true and false assignments among MultiAs. To this end, we used the mass error in millidalton (mDa), which was superior to the commonly used relative mass error in ppm. We developed an automatic workflow to group MultiAs based on their shared formula units and Kendrick mass defect values and to evaluate the mass error distribution. In this way, the number of valid assignments of chlorinated disinfection byproducts was increased by 8-fold as compared to only applying 37Cl/35Cl isotope ratio filters. Likewise, phosphorus-containing MFs can be differentiated against chlorine-containing MFs with high confidence. Further, false assignments of highly aromatic sulfur-containing MFs ("black sulfur") to sodium adducts in negative ionization mode can be excluded by applying our approach. Overall, MFs for mass peaks that are close to the detection limit or where naturally occurring isotopes are rare (e.g., 15N) or absent (e.g., P and F) can now be validated, substantially increasing the reliability of MF assignments and broadening the applicability of UHRMS analysis to even more complex samples and processes.

Spatiotemporal consistency analysis of cerebral small vessel disease: an rs-fMRI study.

Introduction: Cerebral small vessel disease (SVD) affects older adults, but traditional approaches have limited the understanding of the neural mechanisms of SVD. This study aimed to explore the effects of SVD on brain regions and its association with cognitive decline using the four-dimensional (spatiotemporal) consistency of local neural activity (FOCA) method. Methods: Magnetic resonance imaging data from 42 patients with SVD and 38 healthy controls (HCs) were analyzed using the FOCA values. A two-sample t test was performed to compare the differences in FOCA values in the brain between the HCs and SVD groups. Pearson correlation analysis was conducted to analyze the association of various brain regions with SVD scores. Results: The results revealed that the FOCA values in the right frontal_inf_oper, right temporal_pole_sup, and default mode network decreased, whereas those in the temporal_inf, hippocampus, basal ganglia, and cerebellum increased, in patients with SVD. Most of these varying brain regions were negatively correlated with SVD scores. Discussion: This study suggested that the FOCA approach might have the potential to provide useful insights into the understanding of the neurophysiologic mechanisms of patients with SVD.

Engineered endolysin of Klebsiella pneumoniae phage is a potent and broad-spectrum bactericidal agent against "ESKAPEE" pathogens.

The rise of antimicrobial resistance in ESKAPEE pathogens poses significant clinical challenges, especially in polymicrobial infections. Bacteriophage-derived endolysins offer promise in combating this crisis, but face practical hurdles. Our study focuses on engineering endolysins from a Klebsiella pneumoniae phage, fusing them with ApoE23 and COG133 peptides. We assessed the resulting chimeric proteins' bactericidal activity against ESKAPEE pathogens in vitro. ApoE23-Kp84B (CHU-1) reduced over 3 log units of CFU for A. baumannii, E. faecalis, K. pneumoniae within 1 h, while COG133-Kp84B (CHU-2) showed significant efficacy against S. aureus. COG133-L1-Kp84B, with a GS linker insertion in CHU-2, exhibited outstanding bactericidal activity against E. cloacae and P. aeruginosa. Scanning electron microscopy revealed alterations in bacterial morphology after treatment with engineered endolysins. Notably, CHU-1 demonstrated promising anti-biofilm and anti-persister cell activity against A. baumannii and E. faecalis but had limited efficacy in a bacteremia mouse model of their coinfection. Our findings advance the field of endolysin engineering, facilitating the customization of these proteins to target specific bacterial pathogens. This approach holds promise for the development of personalized therapies tailored to combat ESKAPEE infections effectively.

Olgotrelvir as a Single-Agent Treatment of Nonhospitalized Patients with Covid-19.

BACKGROUND: Olgotrelvir is an oral antiviral with dual mechanisms of action targeting severe acute respiratory syndrome coronavirus 2 main protease (i.e., Mpro) and human cathepsin L. It has potential to serve as a single-agent treatment of coronavirus disease 2019 (Covid-19). METHODS: We conducted a phase 3, double-blind, randomized, placebo-controlled trial to evaluate the efficacy and safety of olgotrelvir in 1212 nonhospitalized adult participants with mild to moderate Covid-19, irrespective of risk factors, who were randomly assigned to receive orally either 600 mg of olgotrelvir or placebo twice daily for 5 days. The primary and key secondary end points were time to sustained recovery of a panel of 11 Covid-19-related symptoms and the viral ribonucleic acid (RNA) load. The safety end point was incidence of treatment-emergent adverse events. RESULTS: The baseline characteristics of 1212 participants were similar in the two groups. In the modified intention-to-treat population (567 patients in the placebo group and 558 in the olgotrelvir group), the median time to symptom recovery was 205 hours in the olgotrelvir group versus 264 hours in the placebo group (hazard ratio, 1.29; 95% confidence interval [CI], 1.13 to 1.46; P<0.001). The least squares mean (95% CI) changes of viral RNA load from baseline were -2.20 (-2.59 to -1.81) log10 copies/ml in olgotrelvir-treated participants and -1.40 (-1.79 to -1.01) in participants receiving placebo at day 4. Skin rash (3.3%) and nausea (1.5%) were more frequent in the olgotrelvir group than in the placebo group; there were no treatment-related serious adverse events, and no deaths were reported. CONCLUSIONS: Olgotrelvir as a single-agent treatment significantly improved symptom recovery. Adverse effects were not dose limiting. (Funded by Sorrento Therapeutics, a parent company of ACEA Therapeutics; ClinicalTrials.gov number, NCT05716425.).

Phage therapy combats pandrug-resistant Acinetobacter baumannii infection safely and efficiently.

Phage therapy offers a promising approach to combat the growing threat of antimicrobial resistance. Yet, key questions remain regarding dosage, administration routes, combination therapy, and the causes of therapeutic failure. In this study, we focused on a novel lytic phage, ФAb4B, which specifically targeted the A. baumannii strains with KL160 CPS, including the pan-drug resistant A. baumannii YQ4. ФAb4B exhibited the ability to effectively inhibit biofilm formation and eradicate mature biofilms independently of dosage. Additionally, it demonstrated a wide spectrum of antibiotic-phage synergy and did not show any cytotoxic or hemolytic effects. Continuous phage injections, both intraperitoneally and intravenously over 7 days, showed no acute toxicity in vivo. Importantly, phage therapy significantly improved neutrophil counts, outperforming ciprofloxacin (CIP). However, excessive phage injections suppressed neutrophil levels. The combinatorial treatment of phage-CIP rescued 91% of the mice, a superior outcome compared to phage alone (67%). The efficacy of the combinatorial treatment was independent of phage dosage. Notably, prophylactic administration of the combinatorial regimen provided no protection, but even when combined with a delayed therapeutic regimen, it saved all the mice. Bacterial resistance to the phage was not a contributing factor to treatment failure. Our preclinical study systematically describes the lytic phage's effectiveness in both in vitro and in vivo settings, filling in crucial details about phage treatment against bacteriemia caused by A. baumannii, which will provide a robust foundation for the future of phage therapy.

Assessing brain microstructural changes in chronic kidney disease: a diffusion imaging study using multiple models.

Objectives: To explore the effectiveness of diffusion quantitative parameters derived from advanced diffusion models in detecting brain microstructural changes in patients with chronic kidney disease (CKD). Methods: The study comprised 44 CKD patients (eGFR<59 mL/min/1.73 m2) and 35 age-and sex-matched healthy controls. All patients underwent diffusion spectrum imaging (DSI) and conventional magnetic resonance imaging. Reconstructed to obtain diffusion MRI models, including diffusion tensor imaging (DTI), neurite orientation dispersion and density imaging (NODDI) and Mean Apparent Propagator (MAP)-MRI, were processed to obtain multi-parameter maps. The Tract-Based Spatial Statistics (TBSS) analysis was utilized for detecting microstructural differences and Pearson correlation analysis assessed the relationship between renal metabolism markers and diffusion parameters in the brain regions of CKD patients. Receiver operating characteristic (ROC) curve analysis assessed the diagnostic performance of diffusion models, with AUC comparisons made using DeLong's method. Results: Significant differences were noted in DTI, NODDI, and MAP-MRI parameters between CKD patients and controls (p < 0.05). DTI indicated a decrease in Fractional Anisotropy(FA) and an increase in Mean and Radial Diffusivity (MD and RD) in CKD patients. NODDI indicated decreased Intracellular and increased Extracellular Volume Fractions (ICVF and ECVF). MAP-MRI identified extensive microstructural changes, with elevated Mean Squared Displacement (MSD) and Q-space Inverse Variance (QIV) values, and reduced Non-Gaussianity (NG), Axial Non-Gaussianity (NGAx), Radial Non-Gaussianity (NGRad), Return-to-Origin Probability (RTOP), Return-to-Axis Probability (RTAP), and Return-to-Plane Probability (RTPP). There was a moderate correlation between serum uric acid (SUA) and diffusion parameters in six brain regions (p < 0.05). ROC analysis showed the AUC values of DTI_FA ranged from 0.70 to 0.793. MAP_NGAx in the Retrolenticular part of the internal capsule R reported a high AUC value of 0.843 (p < 0.05), which was not significantly different from other diffusion parameters (p > 0.05). Conclusion: The advanced diffusion models (DTI, NODDI, and MAP-MRI) are promising for detecting brain microstructural changes in CKD patients, offering significant insights into CKD-affected brain areas.

METTL3-deficiency m6A-dependently degrades MALAT1 to suppress NLRP3-mediated pyroptotic cell death and inflammation in Mycobacterium tuberculosis (H37Ra strain)-infected mouse macrophages.

Mycobacterium tuberculosis (Mtb)-infected macrophages aggravated the development of pulmonary tuberculosis, but its detailed molecular mechanisms are still largely unknown. Here, the mouse primary peritoneal macrophages were infected with the attenuated strain of Mtb H37Ra, and we firstly verified that targeting a novel METTL3/N6-Methyladenosine (m6A)/LncRNA MALAT1/miR-125b/TLR4 axis was effective to suppress pyroptotic cell death in the Mtb-infected macrophages. Specifically, through performing Real-Time qPCR and Western Blot analysis, we validated that METTL3, LncRNA MALAT1 and TLR4 were elevated, whereas miR-125b and the anti-oxidant agents (Nrf2 and HO-1) were downregulated in Mtb-infected mouse macrophages. In addition, functional experiments confirmed that both ROS scavenger NAC and METTL3-ablation downregulated NLRP3, GSDMD-C, cleaved Caspase-1 and ASC to restrain pyroptotic cell death and decreased the expression levels of IL-1ß, IL-18, IL-6 and TNF-α to restrain inflammatory cytokines expression in Mtb-infected macrophages. Next, METTL3-ablation induced m6A-demethylation and instability in LncRNA MALAT1, and low-expressed LncRNA MALAT1 caused TLR4 downregulation through sponging miR-125b, resulting in the inactivation of NLRP3 inflammasome. Finally, silencing of METTL3-induced protective effects in Mtb-infected macrophages were all abrogated by overexpressing LncRNA MALAT1 and downregulating miR-125b. Thus, we concluded that targeting METTL3-mediated m6A modifications suppressed Mtb-induced pyroptotic cell death in mouse macrophages, and the downstream LncRNA MALAT1/miR-125b/TLR4 axis played critical role in this process.

ApoE Mimetic Peptide COG1410 Kills Mycobacterium smegmatis via Directly Interfering ClpC's ATPase Activity.

Antimicrobial peptides (AMPs) hold promise as alternatives to combat bacterial infections, addressing the urgent global threat of antibiotic resistance. COG1410, a synthetic peptide derived from apolipoprotein E, has exhibited potent antimicrobial properties against various bacterial strains, including Mycobacterium smegmatis. However, our study reveals a previously unknown resistance mechanism developed by M. smegmatis against COG1410 involving ClpC. Upon subjecting M. smegmatis to serial passages in the presence of sub-MIC COG1410, resistance emerged. The comparative genomic analysis identified a point mutation in ClpC (S437P), situated within its middle domain, which led to high resistance to COG1410 without compromising bacterial fitness. Complementation of ClpC in mutant restored bacterial sensitivity. In-depth analyses, including transcriptomic profiling and in vitro assays, uncovered that COG1410 interferes with ClpC at both transcriptional and functional levels. COG1410 not only stimulated the ATPase activity of ClpC but also enhanced the proteolytic activity of Clp protease. SPR analysis confirmed that COG1410 directly binds with ClpC. Surprisingly, the identified S437P mutation did not impact their binding affinity. This study sheds light on a unique resistance mechanism against AMPs in mycobacteria, highlighting the pivotal role of ClpC in this process. Unraveling the interplay between COG1410 and ClpC enriches our understanding of AMP-bacterial interactions, offering potential insights for developing innovative strategies to combat antibiotic resistance.

Novel lncRNA 803 related to Marek's disease inhibits apoptosis of DF-1 cells.

Marek's disease (MD) is a neoplastic disease that significantly affects the poultry industry. Long non-coding RNAs (lncRNAs) are crucial regulatory factors in various biological processes, including tumourigenesis. However, the involvement of novel lncRNAs in the course of MD virus (MDV) infection is still underexplored. Here, we present the first comprehensive characterization of differentially expressed lncRNAs in chicken spleen at different stages of MDV infection. A series of differentially expressed lncRNAs was identified at each stage of MDV infection through screening. Notably, our investigation revealed a novel lncRNA, lncRNA 803, which exhibited significant differential expression at different stages of MDV infection and was likely to be associated with the p53 pathway. Further analyses demonstrated that the overexpression of lncRNA 803 positively regulated the expression of p53 and TP53BP1 in DF-1 cells, leading to the inhibition of apoptosis. This is the first study to focus on the lncRNA expression profiles in chicken spleens during MDV pathogenesis. Our findings highlight the potential role of the p53-related novel lncRNA 803 in MD pathogenesis and provide valuable insights for decoding the molecular mechanism of MD pathogenesis involving non-coding RNA.RESEARCH HIGHLIGHTS Differentially expressed lncRNAs in spleens of chickens infected with Marek's disease virus at different stages were identified for the first time.The effects of novel lncRNA 803 on p53 pathway and apoptosis of DF-1 cells were reported for the first time.

Evaluating the effects of tannic acid on rabbit growth performance, digestibility, antioxidant status, intestinal morphology and caecal fermentation and microbiota.

The objective of this study was to assess the effects of dietary supplementation with tannic acid (TA) on the growth performance, digestibility, antioxidant status, intestinal morphology and the caecal fermentation and microbiota in rabbits. A total number of 120 Ira rabbits (30 days of age) were randomly allotted to four dietary treatment groups: TA 0 (control), TA 0.75, TA 1.5 and TA 3, administered basal diets with 0, 0.75, 1.5 and 3 g TA/kg of feed for 28 days. Compared to the control group, dietary 3 g TA/kg inclusion decreased the average daily feed intake (p < 0.05). No significant differences were found in the digestibility among the groups (p > 0.05). Serum total antioxidant capacity was significantly higher in the 3 g/kg TA group than in the other groups (p < 0.05). There was a significant increase in the concentration of propionic acid and butyric acid in the 3 g/kg TA group. The addition of TA had no effect on villus height and crypt depth of small intestine (p > 0.05). The 16S rRNA high-throughput sequencing results showed that at the phylum level, dietary 3 g/kg TA increased the abundance of Bacteroidetes in the caecum of rabbits (p < 0.05). Based on the results, dietary TA is effective in antioxidant capacity of rabbits, improving caecal fermentation and optimizing the caecal microflora. However, the appropriate dosage supplementation of TA in rabbits needs further research.

The novel lncRNA-9802/miR-1646 axis affects cell proliferation of DF-1 by regulating Bax/Bcl-2 signaling pathway.

Marek's disease (MD) is a severe infectious and immunosuppressive neoplastic condition that significantly impacts the global poultry industry. Investigating the role of non-coding RNA in pathogenic mechanisms of MD virus (MDV) offers valuable insights for the effective prevention and management of MD. A higher expression of the novel lncRNA-9802 can be found in spleen tissues of MDV-infected chickens from our prior research, and there is a potential association between lncRNA-9802 and cell proliferation. In this study, we further demonstrated that over-expression of lncRNA-9802 could promote the proliferation of DF-1 cells. It has been established that lncRNA-9802 mediated its effects by binding to miR-1646, and further modulated the expression of the Bax and Bcl-2 genes. Deciphering the role of the recently discovered MD-associated lncRNA-9802/miR-1646 axis provides valuable theoretical basis for decoding the molecular mechanisms underlying MDV pathogenesis.

Adsorption Performance of Heavy Metal Ions under Multifactorial Conditions by Synthesized Organic-Inorganic Hybrid Membranes.

A series of hybridized charged membrane materials containing carboxyl and silyl groups were prepared via the epoxy ring-opening reaction and sol-gel methods using 3-glycidoxypropyltrimethoxysilane (WD-60) and polyethylene glycol 6000 (PEG-6000) as raw materials and DMF as a solvent. Scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FTIR), and thermal gravimetric analyzer/differential scanning calorimetry (TGA/DSC) analysis showed that the heat resistance of the polymerized materials could reach over 300 °C after hybridization. A comparison of the results of heavy metal lead and copper ions' adsorption tests on the materials at different times, temperatures, pHs, and concentrations showed that the hybridized membrane materials have good adsorption effects on heavy metals and better adsorption effects on lead ions. The maximum capacity obtained from optimized conditions for Cu2+ and Pb2+ ions were 0.331 and 5.012 mmol/g. The experiments proved that this material is indeed a new environmentally friendly, energy-saving, high-efficiency material. Moreover, their adsorptions for Cu2+ and Pb2+ ions will be evaluated as a model for the separation and recovery of heavy metal ions from wastewater.

ApoE Mimetic Peptide COG1410 Exhibits Strong Additive Interaction with Antibiotics Against Mycobacterium smegmatis.

Background: Drug-resistant tuberculosis (TB) is an emerging threat to public health worldwide. Antimicrobial peptide (AMP) is a promising solution to solve the antimicrobial resistance crisis. The apolipoprotein E mimetic peptide COG1410 has been confirmed to simultaneously have neuroprotective, anti-inflammatory, and antibacterial activity. However, whether it is effective to inhibit growth of mycobacteria has not been investigated yet. Methods: The peptide COG1410 was synthesized with conventional solid-phase peptide synthesis and qualified by HPLC and mass spectrometry. Micro-dilution method was used to determine the minimal inhibitory concentration. A time-kill assay was used to determine the bactericidal dynamics of antimicrobial peptide and relative antibiotics. Static biofilm formation was conducted in 24-well plate and the biofilm was separated from planktonic cells and collected. The mechanism of action of COG1410 was explored by TEM observation and ATP leak assay. The localization of COG1410 was observed by confocal laser scan microscopy. The drug-drug interaction was determined by a checkerboard assay. Results: COG1410 was a potent bactericidal agent against M. smegmatis in vitro and within the macrophages with MIC 16 µg/mL, but invalid against M. abscess and M. tuberculosis. A time-kill assay showed that COG1410 killed M. smegmatis as potent as clarithromycin, but faster than LL-37, another short synthetic cationic peptide. 1× MIC COG1410 almost reduced 90% biofilm formation of M. smegmatis. Additionally, COG1410 was able to penetrate the cell membrane of macrophage and inhibit intracellular M. smegmatis growth. TEM observation and ATP leak assay found that COG1410 disrupted cell membrane and caused release of cell contents. Confocal fluorescence microscopy showed that FITC-COG1410 aggregated around cell membrane instead of entering the cytoplasm. Although COG1410 had relative high cytotoxicity, it exhibited strong additive interaction with regular anti-TB antibiotics, which reduced the working concentration of COG1410 and expanding safety window. After 30 passages, there was no induced drug resistance for COG1410. Conclusion: COG1410 was a novel and potent AMP against M. smegmatis by disrupting the integrity of cell membrane.

Association of mental health status between self-poisoning suicide patients and their family members: a matched-pair analysis.

BACKGROUND: The objective of this study was to examine the relationship of mental health status between self-poisoning suicide patients and their family members, and it also sought to identify potential patient's risk and parental factors for the prediction of suicide attempt, anxiety, and depression. METHODS: In this study, 151 poisoned patients were prospectively included, and they were matched 1:1 with 151 family members. We gathered information on patient's and their matched family member's demographics, lifestyle choices, mental health status, level of intimacy, and history of psychiatry disease. The relationship of patient's and their family member's mental health state was investigated using a correlation matrix. Multivariable analyses (multiple logistic regression) were conducted among patients and their matched family members, to identify potential risk factors for self-poisoning suicide, anxiety, and depression. RESULTS: Of the total patients, 67.55% (102/151) attempted self-poisoning suicide. Poisoned patients had more severe anxiety and depression symptoms than their matched family members, and this difference was even more pronounced among patients with self-poisoning suicide. Generalized anxiety disorder-7 (GAD-7) score for family members was significantly and favorably correlated with patient's GAD-7 score after eliminating non-suicide patients and their matched family members. The patient health questionnaire-9 (PHQ-9) score showed a similar pattern, and the family member's PHQ-9 score was strongly and favorably associated with patient's PHQ-9 and Beck hopelessness scale-20 (BHS-20) score. Multivariable analysis showed that married marital status (P = 0.038), quitting smoking (P = 0.003), sedentary time of 1 to 6 h (P = 0.013), and participation in a sports more than five times per week (P = 0.046) were all significantly associated with a lower risk of suicide by self-poisoning, while a more serious anxiety state (P = 0.001) was significantly associated with a higher risk of self-poisoning suicide. Multivariable analysis demonstrated that, specifically among self-poisoning suicide patients, married marital status (P = 0.011) and no history of psychiatry disease (P < 0.001) were protective factors for anxiety, while divorced or widowed marital status (P = 0.004), a sedentary time of 1 to 3 h (P = 0.022), and a higher monthly income (P = 0.027) were significant contributors to anxiety. The propensity of additional family-matched characteristics to predict patient's suicidality, anxiety, and depression was also examined. CONCLUSIONS: Self-poisoning suicide patients have severe mental health issues. Patients who self-poison have a close connection to their family member's mental health, particularly their levels of anxiety and depression. According to the findings, being married and adopting healthy lifestyle habits, such as quitting smoking and drinking, increasing their physical activity levels, and managing their idle time, are able to help patients with mental health concerns and even suicidal thoughts.