Trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite, has been linked to cardiovascular diseases. This study aimed to explore the role of TMAO in cardiac fibrosis by examining its effects on the NLRP3 inflammasome, endoplasmic reticulum stress (ERS), mitochondria-associated membranes (MAMs), and mitochondrial dynamics in cardiac fibroblasts (CFs), alongside clinical data from acute myocardial infarction (AMI) patients and unstable angina (UA) patients and AMI animal model data. Plasma TMAO levels were measured in AMI patients and healthy controls. In vitro, CFs were treated with TMAO to assess cellular activation and fibrosis markers. Western blot, immunofluorescence, and RNA sequencing identified key pathways and proteins related to ERS, NLRP3 inflammasome activation, and mitochondrial dynamics. In vivo, Masson's trichrome staining, Hematoxylin–Eosin (HE) staining and Immunohistochemical were used to evaluate the effects of TMAO on AMI mice. Plasma TMAO levels were significantly higher in the AMI group. TMAO promoted cardiac fibroblast activation and fibrosis by increasing α-SMA and Collagen I expression. It induced ERS, marked by elevated GRP78, p-PERK, and CHOP, and upregulated Sigma-1R, enhancing MAM formation. TMAO also altered mitochondrial dynamics via DRP1 phosphorylation and Mfn2 expression. RNA sequencing identified macrophage migration inhibitory factor (MIF) as a key mediator linking TMAO to NLRP3 inflammasome activation. TMAO exacerbates myocardial injury and fibrotic remodeling in AMI mice. TMAO exacerbates cardiac fibrosis via ERS and NLRP3 activation, with implications for mitochondrial dynamics and MAM formation. Elevated TMAO levels in AMI patients underscore its potential as a therapeutic target for ventricular remodeling fibrosis.

Aging is characterized by systemic inflammation and progressive cognitive decline, yet the molecular pathways linking peripheral aging signals to central nervous system dysfunction remain elusive. Here, we identify plasma extracellular vesicle (EV)-derived long interspersed nuclear element-1 (LINE-1) RNA as a potent systemic aging factor mediating neuroinflammation and cognitive impairment in humans and mice. Plasma EV LINE-1 RNA levels markedly increase with age and strongly correlate with established brain aging biomarkers, including neurofilament light chain (NFL). Utilizing mouse models, we demonstrate that EVs from aged individuals penetrate the blood-brain barrier, deliver LINE-1 RNA to microglia, and initiate cGAS-STING signaling, leading to pronounced neuroinflammation, neuronal damage, and impaired cognition. Pharmacological blockade of LINE-1 reverse transcription by 3TC or inhibition of STING signaling with H151 significantly ameliorates these age-associated deficits. Notably, aged peripheral tissues, especially brain and lung, emerge as primary sources of pro-aging EVs enriched with LINE-1 RNA, revealing a novel mechanism of inter-organ communication in aging. Our findings position EV-derived LINE-1 RNA and its downstream cGAS-STING pathway as critical systemic drivers of brain aging, presenting promising therapeutic targets for mitigating cognitive decline and age-related neurodegenerative diseases.

Alzheimer’s disease (AD) is the most common form of dementia. However, there is a lack of effective diagnostic biomarkers to differentiate AD from non-Alzheimer’s (Non-AD) dementias, such as vascular dementia, Lewy body dementia, Parkinson’s disease dementia, and chronic traumatic encephalopathy. This study aims to evaluate the expression variations of LINE-1 retrotransposon in plasma EVs as a potential biomarker to differentiate between AD and Non-AD dementias and explore its diagnostic value in clinical practice. Here, we analyzed two independent clinical cohorts: a training cohort (AD: n = 139; Non-AD: n = 137; Control: n = 79) and a validation cohort (AD: n = 158; Non-AD: n = 133; Control: n = 61). Expression of LINE-1 mRNA in plasma EVs, containing ORF1, ORF2, and 5’UTR regions, was quantified using RT-qPCR, and the differences between control, AD and Non-AD groups were assessed by one-way ANOVA or non-parametric tests. In both training and validation cohorts, LINE-1 mRNA expression was significantly higher in AD patients compared to Non-AD patients and healthy control in the ORF1, 5’UTR, and ORF2 regions. LINE-1 expression was correlated with that of neuroinflammatory and neurodegeneration markers (GFAP and NfL), whereas no significant correlations were revealed between LINE-1 expression and cognitive scores. ROC analysis revealed that while LINE-1 was less effective than traditional biomarkers for general AD diagnosis, it demonstrated superior accuracy in differentiating AD from Non-AD dementias. Specifically, the AUC values for LINE-1_ORF2 and LINE-1_5’UTR were 0.91 and 0.92, respectively, significantly outperforming p-Tau217 (AUC = 0.80) and GFAP (AUC = 0.62) in the training cohort. Multivariate regression confirmed that the LINE-1 composite index was an independent predictor of AD. These findings suggest that the LINE-1 composite score, as an age-independent predictor, holds significant clinical potential for the differential diagnosis of AD.

Brain aging is not an independent process, yet how systemic aging drives neural decline remains unclear. Here, we identified a circulating miR-4433b-3p, packaged within extracellular vesicles (EVs), as a trans-organ effector bridging cardiac aging with central nervous system (CNS) decline. Small RNA sequencing and human cohort validation revealed selective enrichment of miR-4433b-3p in aged plasma EVs (Op-EVs), correlating with blood biomarkers of brain aging. Source tracing in mice identified the aged heart as the major origin of miR-4433b-3p-laden EVs. Functionally, aged cardiac EVs (Oc-EVs) accumulated in the hippocampus, impaired memory and induced neuronal senescence. Mechanistically, miR-4433b-3p suppressed TP53INP2, a facilitator of autophagic flux, leading to disrupted autophagosome maturation. Restoring TP53INP2 or inhibiting miR-4433b-3p rescued neuronal autophagy and improved cognition. Collectively, these findings uncover a heart-brain axis by EV-mediated miRNA signaling, positioning cardiac EV-miR-4433b-3p as a circulating biomarker and potential therapeutic target for age-related cognitive decline.

Sequence type 59 (ST59) is a predominant methicillin-resistant Staphylococcus aureus (MRSA) clone that causes invasive infections in Chinese hospitals. We performed weighted correlation network analysis on clinical MRSA and identified qorA as an ST59-specific redox regulator that sustains intracellular homeostasis by promoting NAD+-to-NADH conversion. Deletion of qorA impaired oxidative stress resistance; reduced the production of SodA, SodM, and staphyloxanthin; triggered metabolic reprogramming; and increased susceptibility to complement C3b deposition and neutrophil-mediated killing; these defects were partially rescued by the antioxidant N-acetylcysteine (NAC). Competitive murine infections demonstrated distinct organ-specific fitness: ΔqorA was outcompeted by the wild type in the liver and spleen but exhibited a fitness advantage in the heart, lungs, and kidneys, with comparable bacterial burdens in subcutaneous abscesses. Our findings indicate that qorA mediates organ-specific adaptation of ST59-MRSA by balancing immune evasion and metabolic trade-offs, highlighting its potential as a therapeutic target against S. aureus.

Exosomes (EXOs) derived from the plasma of young individuals are believed to have the potential to ameliorate aging-related memory deficits. However, their specific roles and mechanisms in Alzheimer's disease (AD) therapy have not yet been systematically investigated. In this study, the rabies virus glycoprotein-targeting peptide (RVG-29) was conjugated to the surface of young plasma-derived EXOs to construct RVG-engineered EXOs (RVG-EXOs), and their therapeutic potential and underlying mechanisms in AD models were systematically evaluated. In 3×Tg AD model mice, exogenous administration of young plasma-derived EXOs and their engineered product (RVG-EXOs) revealed that RVG-EXOs could more efficiently enter brain tissue and target neurons, significantly reduce Aβ plaque and phosphorylated Tau (P-Tau) pathological deposition, restore synaptic structure, promote neuronal survival, and improve cognitive behavior. Mechanistic studies demonstrated that RVG-EXOs inhibited RPTOR expression, thereby activating the autophagy pathway and promoting the clearance of pathological proteins. Both in vitro and in vivo experiments confirmed that overexpression of RPTOR significantly suppressed the therapeutic effects of RVG-EXOs. single-cell transcriptomic profiling further revealed that RVG-EXOs not only increased neuronal proportion and modulated excitatory/inhibitory neuronal balance but also reshaped the microglial landscape by reducing deleterious disease-associated while increasing homeostatic surveillant microglia. In summary, this study not only reveals for the first time the potential value of young plasma-derived EXOs in AD treatment but also, through RVG engineering strategies and the elucidation of the RPTOR-autophagy mechanism, provides new insights for targeted therapy of neurodegenerative diseases.

Aging significantly impacts brain function, and identifying reliable biomarkers for early detection of age-related neurodegeneration is crucial for improving diagnosis and treatment outcomes. This proof-of-principle study aims to evaluate the abundance of mitochondrial DNA (mtDNA) targets within plasma-derived extracellular vesicles (EVs) and to investigate whether they correlate with established biomarkers of brain aging, independent of chronological age and renal function. mtDNA copy number was quantified using absolute quantitative polymerase chain reaction (qPCR). Brain aging biomarkers were measured by enzyme-linked immunosorbent assay (ELISA). Multivariable regression analysis was performed to examine the associations between EV mitochondrial genes and aging biomarkers. A multi-biomarker model was developed to assess the performance of combined biomarkers in distinguishing between age groups. We observed that EV mitochondrial gene levels were significantly increased with age (p < .001). Levels of neurofilament light chain (NfL), amyloid-beta (Aβ42 and Aβ40), also showed significant age-related increases (p < .001). A multi-biomarker model combining EV mitochondrial genes and brain aging biomarkers showed the optimal performance in distinguishing older adults from younger individuals, with an area under the receiver operating characteristic (ROC) curve (AUC) significantly higher than that of any single biomarker (p < .01). These findings collectively indicate that EV-derived mitochondrial genes, in combination with other biomarkers like NfL, hold great potential as a noninvasive tool for early detection and monitoring of brain aging and neurodegenerative diseases.

This study aimed to assess the therapeutic efficacy of syringaldehyde (SYD) in Alzheimer’s disease (AD) and to investigate its potential underlying molecular mechanisms. The potential of SYD for AD treatment was first explored through a network pharmacology approach. APPswe/PS1dE9 (APP/PS1) transgenic mice were treated with SYD via intraperitoneal injection for 9 weeks, and cognitive and behavioral functions were evaluated using the Y-maze, Morris water maze, and novel object recognition tests. Histopathological analysis was conducted to assess neuronal changes and amyloid plaque deposition in the hippocampus using immunofluorescence, hematoxylin and eosin staining, Nissl staining, and Congo red staining. RNA sequencing and transcription factor prediction analyses were utilized to identify the potential molecular mechanisms underlying SYD’s therapeutic effects. In addition, in vitro experiments were performed on HT22 hippocampal neuronal cells, including ROS assay, TUNEL assay, and quantitative reverse transcription PCR, to validate the mechanisms suggested by the in vivo results. The results demonstrated that SYD treatment significantly reduced amyloid plaque deposition in the hippocampus of APP/PS1 mice, promoted neuronal repair, and improved cognitive performance. Further analysis indicated that these therapeutic effects were mediated by SYD’s ability to enhance resistance to oxidative stress, alleviate neuronal damage, and inhibit the NF-κB/IL-1β/NLRP3 inflammatory pathway, thereby counteracting neuroinflammation induced by neuronal activation. In conclusion, this study provides strong evidence for the potential of SYD to ameliorate cognitive impairment and reduce amyloid plaque deposition in AD, highlighting its promising role as a therapeutic agent in the treatment of Alzheimer’s disease.

Aim:This study aimed to identify autophagy-related microRNAs (miRNAs) in plasma exosomes as non-invasive biomarkers for brain aging and explore their potential to improve early detection of age-associated neurodegeneration. With the increasing incidence of neurodegenerative disorders, such as Alzheimer's disease (AD), frontotemporal dementia (FTD), and Parkinson's disease (PD), non-invasive diagnostic tools are urgently needed. Methods:Plasma samples were collected from 200 individuals, divided into three groups, including young (20-40 years), middle-aged (41-60 years), and elderly (> 60 years). Exosomes were isolated, followed by small RNA sequencing (sRNA-seq) to identify differentially expressed miRNAs, and differentially expressed miRNAs related to autophagy were validated using quantitative real-time PCR (qRT-PCR). Spearman correlation analysis was performed to assess the relationship between autophagy-related miRNAs and brain aging biomarkers. Receiver operating characteristic (ROC) curve analysis was conducted to evaluate the diagnostic performance. Results:Nine autophagy-related miRNAs were identified and validated as significantly upregulated in plasma exosomal from elderly, including hsa-miR-2110, hsa-miR-18a-3p, hsa-miR-766-3p, hsa-miR-4446-3p, hsa-miR-4667-5p, hsa-miR-4433b-3p, hsa-miR-146a-5p, hsa-miR-423-5p, and novel_260. These miRNAs were validated by qRT-PCR. Correlation analysis showed that several of these miRNAs, such as hsa-miR-2110 and hsa-miR-766-3p, were strongly correlated with NfL (r = 0.68, p = 0.002), Aβ42 (r = 0.62, p = 0.004), and p-Tau181 (r = 0.55, p = 0.008). ROC curve analysis showed that combining these miRNAs with NfL resulted in an area under the curve (AUC) of 0.92, outperforming NfL alone (AUC = 0.85) and miRNAs alone (AUC = 0.84). Further subgroup analysis revealed that multiple miRNAs, such as miR-2110, miR-4446-3p, and novel_260, achieved high AUCs (>0.83) in distinguishing middle-aged adults (41-60 years) from older adults (>60 years), supporting their potential utility for early detection of age-associated neurodegeneration. Conclusion:This study identifies a set of autophagy-related miRNAs as promising biomarkers for brain aging. The combination of these miRNAs with traditional biomarkers offers a non-invasive and highly sensitive method for early detection of brain aging, providing significant potential to enhance diagnostic accuracy in neurodegenerative diseases.

PURPOSE:This study aimed to systematically explore the growth-inhibitory efficacy of Antimicrobial Blue Light (aBL) against Pseudomonas aeruginosa and comprehensively elucidate its molecular mechanisms of action. METHODS:A network pharmacology framework was employed to evaluate the therapeutic potential of aBL. Experimental assessments included: Bacterial proliferation analysis: Quantification of inhibitory effects via colony-forming unit (CFU) counts and colony area. Kinetic profiling: Analyze the growth curve of bacteria to determine aBL-mediated bactericidal dynamics. Biofilm quantification: Crystal violet staining to assess biofilm formation capacity under aBL exposure. Mechanistic investigation: RNA sequencing and transcription factor prediction to identify molecular pathways modulated by aBL. VALIDATION:Quantitative reverse transcription PCR (qRT-PCR) was performed on both laboratory and clinical strains to confirm transcriptional regulation of key targets. RESULTS:1. aBL significantly inhibited the growth of P. aeruginosa, as evidenced by a reduction in colony count and area. The bacterial growth rate decreased, ultimately leading to a marked decline in bacterial density; 2. RNA-seq results revealed 297 upregulated genes and 247 downregulated genes in the aBL group. GO enrichment analysis indicated that differentially expressed genes were concentrated in biological processes such as oxidoreductase activity and nucleoside phosphate binding. KEGG enrichment analysis demonstrated significant enrichment in pathways including ABC transporters, two-component systems, carbon metabolism, and quorum sensing; 3. qPCR validation results indicate that aBL treatment significantly upregulates the expression of the glyoxylate cycle genes glcE and glcF, as well as the denitrification pathway genes nirM, nosD, and nosZ, suggesting that aBL activated the relevant metabolic pathways. CONCLUSION:1. aBL effectively inhibited the growth of P. aeruginosa and reduced its biofilm formation; 2. aBL activated the glyoxylate cycle and denitrification pathways, thereby influencing bacterial metabolic activity. This study provides experimental evidence and theoretical support for employing aBL as an adjunctive therapeutic strategy for localised clinical infection control.

BackgroundStreptococcus agalactiae is a recognized pathogen that primarily affects infants and pregnant women. However, its increasingly important role in causing invasive infections among non-pregnant adults has become a significant health concern due to the severity and variety of its clinical impacts.MethodsNonduplicate S. agalactiae clinical strains associated with clinical infections (n=139) were isolated from non-pregnant adults in Shandong, China. Antibiotic susceptibility testing, whole-genome sequencing, and genomic analyses were conducted to characterize the genome and identify resistance features of these strains.ResultsThe strains exhibited universal susceptibility to penicillin, ampicillin, cefotaxime, meropenem, linezolid, and vancomycin. Notably, high resistance rates were observed for erythromycin (91.4%), clindamycin (89.2%), levofloxacin (84.2%), tetracycline (54.0%) and, to a lesser extent, chloramphenicol (12.9%). Serotyping revealed seven serotypes and one non-typeable strain. Serotypes Ia, Ib, III, and V predominated, representing 95.7% of the strains. Nineteen sequence types were categorized into seven clonal complexes, with CC10 being the most prevalent at 48.9%. The resistance genes mreA (100%), ermB (70.5%), and tetM (46.0%) were commonly detected. All the isolates carried at least one pilus backbone determinant and one alpha-like protein gene, with the PI-1+PI-2a and the bca gene being the most frequent at 84.2% and 54.7%, respectively.ConclusionsWhile S. agalactiae strains in non-pregnant adults retain sensitivity to β-lactam antibiotics, the elevated resistance to erythromycin, clindamycin, levofloxacin, and tetracycline is concerning. Given the growing elderly population worldwide, the burden of S. agalactiae infections is significant. Continuous surveillance of serotype distribution and antibiotic resistance patterns is imperative for targeted prevention and therapeutic strategies.

Escherichia coli, a rod-shaped Gram-negative bacterium, is a significant causative agent of severe clinical bacterial infections. This study aimed to analyze the epidemiology of extended-spectrum β-lactamase (ESBL)-producing mcr-1 -positive E. coli in Shandong, China. We collected 668 non-duplicate ESBL-producing E. coli strains from clinical samples at Shandong Provincial Hospital between January and December 2018, and estimated their minimum inhibitory concentrations (MICs) using a VITEK® 2 compact system and broth microdilution. Next-generation sequencing and bioinformatic analyses identified the mcr-1 gene and other resistance genes in the polymyxin B-resistant strains. The conjugation experiment assessed the horizontal transfer capacity of the mcr-1 gene. Of the strains collected, 24 polymyxin B-resistant strains were isolated with a positivity rate of 3.59% and among the 668 strains, 19 clinical strains carried the mobile colistin resistance gene mcr-1, with a positivity rate of approximately 2.8%. All 19 clinical strains were resistant to ampicillin, cefazolin, ceftriaxone, ciprofloxacin, levofloxacin, and polymyxin B. Seventeen strains successfully transferred the mcr-1 gene into E. coli J53. All transconjugants were resistant to polymyxin B, and carried the drug resistance gene mcr-1. The 19 clinical strains had 14 sequence types (STs), with ST155 (n = 4) being the most common. The whole-genome sequencing results of pECO-POL-29_mcr1 revealed that no ISApl1 insertion sequences were found on either side of the mcr-1 gene. Our study uncovered the molecular epidemiology of mcr-1-carrying ESBL-producing E. coli in the region and suggested horizontal transmission mediated by plasmids as the main mode of mcr-1 transmission.

We isolated peripheral blood mononuclear cells (PBMCs) from a healthy female donor, and successfully converted into induced pluripotent stem cells (iPSCs) by using non-integrating episomal vectors. These iPSCs displayed a normal karyotype, expressed markers of pluripotency, and showed the capacity to differentiate into three germ layers in vitro, which could be utilized for future research endeavors.

Successfully transforming isolated peripheral blood mononuclear cells (PBMCs) from a healthy male into induced pluripotent stem cells (iPSCs) was achieved using non-integrating episomal vectors containing OCT4, SOX2, c-MYC, KLF4, and BCL-XL. The iPSCs exhibited a normal karyotype, expressed pluripotency markers, and demonstrated the ability to differentiate into three germ layers in vitro. This iPSC line may be employed for subsequent research purposes.

Background:Enterobacter cloacae complex (ECC), which includes major nosocomial pathogens, causes urinary, respiratory, and bloodstream infections in humans, for which colistin is one of the last-line drugs. Objective:This study aimed to analyse the epidemiology and resistance mechanisms of colistin-resistant Enterobacter cloacae complex (ECC) strains isolated from Shandong, China. Methods:Two hundred non-repetitive ECC strains were collected from a tertiary hospital in Shandong Province, China, from June 2020 to June 2022. Whole-genome sequencing and bioinformatics analyses were performed to understand the molecular epidemiology of the colistin-resistant ECC strains. The nucleotide sequences of heat shock protein (hsp60) were analyzed by using BLAST search to classify ECC. The gene expression levels of ramA, soxS, acrA, acrB, phoP, and phoQ were assessed using RT-qPCR. MALDI-TOF MS was used to analyse the modification of lipid A. Results:Twenty-three colistin-resistant strains were detected among the 200 ECC clinical strains (11.5%). The hsp60 cluster analysis revealed that 20 of the 23 ECC strains belonged to heterogeneous resistance clusters. Variants of mgrB, phoPQ, and pmrAB, particularly phoQ and pmrB, were detected in the 23 ECC strains. The soxS and acrA genes were significantly overexpressed in all 23 colistin-resistant ECC strains (P < 0.05). Additionally, all 23 ECC strains contained modified lipid A related to colistin resistance, which showed five ion peaks at m/z 1876, 1920, 1955, 2114, and 2158. Among the 23 ECC strains, 6 strains possessed a phosphoethanolamine (pETN) moiety, 16 strains possessed a 4-amino-4-deoxy-L-arabinose (-L-Ara4N) moiety, and one strain had both pETN and -L-Ara4N moieties. Conclusion:This study suggests that diverse colistin resistance existed in ECC, including unknown resistance mechanisms, exist in ECC. Mechanistic investigations of colistin resistance are warranted to optimise colistin use in clinical settings and minimise the emergence of resistance.

Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) co-producing blaKPC and blaNDM poses a serious threat to public health. This study aimed to investigate the mechanisms underlying the resistance and virulence of CR-hvKP isolates collected from a Chinese hospital, with a focus on blaKPC and blaNDM dual-positive hvKP strains. Five CR-hvKP strains were isolated from a teaching hospital in China. Antimicrobial susceptibility and plasmid stability testing, plasmid conjugation, pulsed-field gel electrophoresis, and whole-genome sequencing (WGS) were performed to examine the mechanisms of resistance and virulence. The virulence of CR-hvKP was evaluated through serum-killing assay and Galleria mellonella lethality experiments. Phylogenetic analysis based on 16 highly homologous carbapenem-resistant K. pneumoniae (CRKP) producing KPC-2 isolates from the same hospital was conducted to elucidate the potential evolutionary pathway of CRKP co-producing NDM and KPC. WGS revealed that five isolates individually carried three unique plasmids: an IncFIB/IncHI1B-type virulence plasmid, IncFII/IncR-type plasmid harboring KPC-2 and IncC-type plasmid harboring NDM-1. The conjugation test results indicated that the transference of KPC-2 harboring IncFII/IncR-type plasmid was unsuccessful on their own, but could be transferred by forming a hybrid plasmid with the IncC plasmid harboring NDM. Further genetic analysis confirmed that the pJNKPN26-KPC plasmid was entirely integrated into the IncC-type plasmid via the copy-in route, which was mediated by TnAs1 and IS26. KPC-NDM-CR-hvKP likely evolved from a KPC-2-CRKP ancestor and later acquired a highly transferable blaNDM-1 plasmid. ST11-KL64 CRKP exhibited enhanced plasticity. The identification of KPC-2-NDM-1-CR-hvKP highlights the urgent need for effective preventive strategies against aggravated accumulation of resistance genes.

Purpose This study aimed to characterise the whole-genome structure of two clinical Klebsiella pneumoniae strains co-harbouring mcr-8.1 and tmexCD1-toprJ1 , both resistant to colistin and tigecycline. Methods K. pneumoniae strains TGC-02 (ST656) and TGC-05 (ST273) were isolated from urine samples of different patients hospitalised at separate times in 2021. Characterisation involved antimicrobial susceptibility testing (AST), conjugation assays, whole-genome sequencing (WGS), and bioinformatics analysis. Comparative genomic analysis was conducted on mcr-8.1-carrying and tmexCD1-toprJ1 -carrying plasmids. Results Both K. pneumoniae isolates displayed a multidrug-resistant phenotype, exhibiting resistance or reduced susceptibility to ampicillin, ampicillin/sulbactam, cefazolin, aztreonam, amikacin, gentamicin, tobramycin, ciprofloxacin, levofloxacin, nitrofurantoin, trimethoprim/sulfamethoxazole, apramycin, tigecycline and colistin. WGS analysis revealed that clinical strain TGC-02 carried the TmexCD1-toprJ1 gene on a 200-Kb IncFII/IncFIB-type plasmid, while mcr-8 was situated on a 146-Kb IncFII-type plasmid. In clinical strain TGC-05, TmexCD1-toprJ1 was found on a 300-Kb IncFIB/IncHI1B/IncR-type plasmid, and mcr-8 was identified on a 137-Kb IncFII/IncFIA-type plasmid. Conjugation experiments assessed the transferability of these plasmids. While transconjugants were not obtained for TGC-05 despite multiple screening with tigecycline or colistin, pTGC-02-tmex and pTGC-02-mcr8 from clinical K. pneumoniae TGC-02 demonstrated self-transferability through conjugation. Notably, the rearrangement of pTGC-02-tmex and pTGC-02-mcr8 via IS26 -based homologous recombination was observed. Moreover, the conjugative and fusion plasmids of the transconjugant co-harboured the tmexCD1-toprJ1 gene cluster and mcr-8.1 , potentially resulting from IS26 -based homologous recombination. Conclusion The emergence of colistin- and tigecycline-resistant K. pneumoniae strains is concerning, and effective surveillance measures should be implemented to prevent further dissemination.

The global health threat posed by carbapenem-resistant Klebsiella pneumoniae (CRKP) is exacerbated by the limited availability of effective treatments. Bacteriophages are promising alternatives to conventional antimicrobial agents. However, current phage databases are limited. Thus, identifying and characterizing new phages could provide biological options for the treatment of multi-drug resistant bacterial infections. Here, we report the characterization of a novel lytic phage, vB_KpnP_23, isolated from hospital sewage. This phage exhibited potent activity against carbapenemase-producing CRKP strains and was characterised by an icosahedral head, a retractable tail, and a genome comprising 40,987 base pairs, with a G + C content of 51 %. Capable of targeting and lysing nine different capsule types (K-types) of CRKP, including the clinically relevant ST11-K64, it demonstrated both high bacteriolytic efficiency and stability in various environmental contexts. Crucially, vB_KpnP_23 lacks virulence factors, antimicrobial resistance genes, or tRNA, aligning with the key criteria for therapeutic application. In vitro evaluation of phage-antibiotic combinations revealed a significant synergistic effect between vB_KpnP_23 and meropenem, levofloxacin, or amikacin. This synergy could lead to an 8-fold reduction in the minimum inhibitory concentration (MIC), suggesting that integrated treatments combining this phage with the aforementioned antibiotics may substantially enhance drug effectiveness. This approach not only extends the clinical utility of these antibiotics but also presents a strategic advance in combating antibiotic resistance. Specifically, it underscores the potential of phage-antibiotic combinations as a powerful tool in the treatment of infections caused by CRKP, offering a promising avenue to mitigate the public health challenges of antibiotic-resistant pathogens.

Nobiletin, a citrus polymethoxy flavonoid with antiapoptotic and antioxidative properties, could safeguard against cisplatin-induced nephrotoxicity and neurotoxicity. Cisplatin, as the pioneer of anti-cancer drug, the severe ototoxicity limits its clinical applications, while the effect of nobiletin on cisplatin-induced ototoxicity has not been identified. The current study investigated the alleviating effect of nobiletin on cisplatin-induced ototoxicity and the underlying mechanisms. Apoptosis and ROS formation were evaluated using the CCK-8 assay, Western blotting, and immunofluorescence, indicating that nobiletin attenuated cisplatin-induced apoptosis and oxidative stress. LC3B and SQSTM1/p62 were determined by Western blotting, qPCR, and immunofluorescence, indicating that nobiletin significantly activated autophagy. Nobiletin promoted the nuclear translocation of NRF2 and the transcription of its target genes, including Hmox1, Nqo1, and ferroptosis markers (Gpx4, Slc7a11, Fth, and Ftl), thereby inhibiting ferroptosis. Furthermore, RNA sequencing analysis verified that autophagy, ferroptosis, and the NRF2 signaling pathway served as crucial points for the protection of nobiletin against ototoxicity caused by cisplatin. Collectively, these results indicated, for the first time, that nobiletin alleviated cisplatin-elicited ototoxicity through suppressing apoptosis and oxidative stress, which were attributed to the activation of autophagy and the inhibition of NRF2/GPX4-mediated ferroptosis. Our study suggested that nobiletin could be a prospective agent for preventing cisplatin-induced hearing loss.

Abstract Background: Drug addiction can seriously damage human physical and mental health, while detoxification is a long and difficult process. Although studies have reported changes in the oral microbiome of methamphetamine (METH) addicts, the role of the microbiome plays in this process is still unknown. This study aims to explore the function of the microbiome based on analysis of the variations in the oral microbiome and metabolome of METH addicts. Results: We performed the 16S rRNA sequencing analysis based on the oral saliva samples collected from 278 METH addicts and 105 healthy controls (CTL) undergoing detoxification at the detoxification center in Shandong, China. In addition, the untargeted metabolomic profiling was conducted based on 220 samples (170 METH addicts and 50 CTL) to identify the biomarkers and build classifiers for both oral microbiota and metabolites. Compared to the CTL group, alpha diversity was reduced in the group of METH addicts, with significant differences in the microbiota and changes in oral metabolic pathways, including enhanced tryptophan metabolism, lysine biosynthesis, purine metabolism, and steroid biosynthesis. Conversely, the metabolic pathways of porphyrin metabolism, glutathione metabolism, and pentose phosphate were significantly reduced. It was speculated that four key microbial taxa, i.e., Peptostreptococcus, Gemella, Campylobacter, and Aggregatibacter, could be involved in the toxicity and addiction mechanisms of METH by affecting the above metabolic pathways. In addition, microbial prediction models were more effective than metabolite-based prediction models in identifying METH addiction. Conclusions: Our study identified the potential functional connections between the oral microbiome and metabolic profile of METH addicts, providing novel insights into exploring the toxic damage and addiction mechanisms underlying the METH addiction.