CRISPR-AMRtracker: A novel toolkit to monitor the antimicrobial resistance gene transfer in fecal microbiota.

The spread of antibiotic resistance genes (ARGs), particularly those carried on plasmids, poses a major risk to global health. However, the extent and frequency of ARGs transfer in microbial communities among human, animal, and environmental sectors is not well understood due to a lack of effective tracking tools. We have developed a novel fluorescent tracing tool, CRISPR-AMRtracker, to study ARG transfer. It combines CRISPR/Cas9 fluorescence tagging, fluorescence-activated cell sorting, 16S rRNA gene sequencing, and microbial community analysis. CRISPR-AMRtracker integrates a fluorescent tag immediately downstream of ARGs, enabling the tracking of ARG transfer without compromising the host cell's antibiotic susceptibility, fitness, conjugation, and transposition. Notably, our experiments demonstrate that sfGFP-tagged plasmid-borne mcr-1 can transfer across diverse bacterial species within fecal samples. This innovative approach holds the potential to illuminate the dynamics of ARG dissemination and provide valuable insights to shape effective strategies in mitigating the escalating threat of antibiotic resistance.

CRISPR/Cas12a-coupled multiplexed strand displacement amplification for miRNA155 one-tube detection: via a dual-cavity PCR tube.

A CRISPR/Cas12a-coupled multiplexed strand displacement amplification (CMSDA) for the detection of miR155 has been developed. Non-specific amplification was avoided by designing a single-stranded DNA template with a hairpin structure. The detection target miR155 was used as a primer to initiate a multiple-strand displacement reaction to produce abundant ssDNA. ssDNA was recognized by the Cas12a/CrRNA binary complex, activating the trans-cleaving activity of Cas12a. The multiple-strand displacement reaction is more efficiently detected compared with a single-strand displacement reaction. The detection range is from 250 pM to 1 nM, and the limit of the detection is 6.5 pM. The proposed method showed a good applicability in complex serum environments, indicating that the method has a broad prospect for disease detection and clinical application. In addition, we designed a dual-cavity PCR tube, which realized one-tube detection of miRNA155 and avoided open-cap contamination.

CRISPR/Cas12a-Powered EC/FL Dual-Mode Controlled-Release Homogeneous Biosensor for Ultrasensitive and Cross-Validated Detection of Messenger Ribonucleic Acid.

Accurate detection of cancer-associated mRNAs is beneficial to early diagnosis and potential treatment of cancer. Herein, for the first time, we developed a novel CRISPR/Cas12a-powered electrochemical/fluorescent (EC/FL) dual-mode controlled-release homogeneous biosensor for mRNA detection. A functionalized ssDNA P2-capped Fe3O4-NH2 loaded with methylene blue (P2@MB-Fe3O4-NH2) was synthesized as the signal probe, while survivin mRNA was chosen as the target RNA. In the presence of the target mRNA, the nicking endonuclease-mediated rolling circle amplification (NEM-RCA) was triggered to produce significant amounts of ssDNA, activating the collateral activity of Cas12a toward the surrounding single-stranded DNA. Thus, the ssDNA P1 completely complementary to ssDNA P2 was cleaved, resulting in that the ssDNA P2 bio-gate on Fe3O4-NH2 could not be opened due to electrostatic interactions. As a result, there was no or only a little MB in the supernatant after magnetic separation, and the measured EC/FL signal was exceedingly weak. On the contrary, the ssDNA P2 bio-gate was opened, enabling MB to be released into the supernatant, and generating an obvious EC/FL signal. Benefiting from the accuracy of EC/FL dual-mode cross-verification, high amplification efficiency, high specificity of NEM-RCA and CRISPR/Cas12a, and high loading of mesoporous Fe3O4-NH2 on signal molecules, the strategy shows aM-level sensitivity and single-base mismatch specificity. More importantly, the practical applicability of this dual-mode strategy was confirmed by mRNA quantification in complex serum environments and tumor cell lysates, providing a new way for developing a powerful disease diagnosis tool.

Electrochemical detection of the p53 gene using exponential amplification reaction (EXPAR) and CRISPR/Cas12a reactions.

An improved electrochemical sensor has been developed for sensitive detection of the p53 gene based on exponential amplification reaction (EXPAR) and CRISPR/Cas12a. Restriction endonuclease BstNI is introduced to specifically identify and cleave the p53 gene, generating primers to trigger the EXPAR cascade amplification. A large number of amplified products are then obtained to enable the lateral cleavage activity of CRISPR/Cas12a. For electrochemical detection, the amplified product activates Cas12a to digest the designed block probe, which allows the signal probe to be captured by the reduced graphene oxide-modified electrode (GCE/RGO), resulting in an enhanced electrochemical signal. Notably, the signal probe is labeled with large amounts of methylene blue (MB). Compared with traditional endpoint decoration, the special signal probe effectively amplifies the electrochemical signals by a factor of about 15. Experimental results show that the electrochemical sensor exhibits wide ranges from 500 aM to 10 pM and 10 pM to 1 nM, as well as a relatively low limit detection of 0.39 fM, which is about an order of magnitude lower than that of fluorescence detection. Moreover, the proposed sensor shows reliable application capability in real human serum, indicating that this work has great prospects for the construction of a CRISPR-based ultra-sensitive detection platform.

Molecular View on the Impact of DHA Molecules on the Physical Properties of a Model Cell Membrane.

Docosahexaenoic acid (DHA) is a ω-3 polyunsaturated fatty acid, which can be uptaken by cells and is essential for proper neuronal and retinal function. However, the detailed physical impact of DHA molecules on the plasma membrane is still unclear. Hence, in this work, we carried out µs-scale coarse-grained molecular dynamics (MD) simulations to reveal the interactions between DHA molecules and a model cell membrane. As is known, the cell membrane can segregate into liquid-ordered (Lo) and liquid-disordered (Ld) membrane domains due to the differential interactions between lipids and proteins. In order to capture this feature, we adopted the three-component phase-separated lipid membranes and considered both anionic and neutral DHA molecules in the current work. Our results showed that DHA molecules can spontaneously self-assemble into nanoclusters, fuse with lipid membranes, and localize preferably in Ld membrane domains. During the membrane fusion process, DHA molecules can change the intrinsic transmembrane potential of the lipid membrane, and the effects of anionic DHA molecules are much more significant. Besides, the presence of DHA molecules mainly in the Ld membrane domains could regulate the differences in the lipid chain order, membrane thickness, cholesterol preference, and cholesterol flip-flop basically in a concentration-dependent manner, which further promote the stability of the intraleaflet dynamics and inhibit the interleaflet dynamics (or promote membrane domain registration) of the membrane domains. In short, the impact of DHA molecules on the physical properties of a model cell membrane on the molecular level revealed in our work will provide useful insights for understanding the biological functions of DHA molecules.

Acute proximal left anterior descending coronary artery occlusion presenting with Normal ECG: A case report.

The middle-aged male was diagnosed with "acute anterior wall myocardial infarction" based on clinical symptoms, laboratory examination, and coronary angiography (CAG), but his ECG showed no significant change in QRS wave or ST-T within 6 h of admission. Thus, a perfect explanation with the existing theory is difficult, and only the case is presented here.

A three-dimensional hydrogel-modified indium tin oxide electrode with enhanced performance for in situ electrochemical detection of extracellular H2O2.

Two different electrochemical sensors (Hemin-G4/Au/GCE and Hemin-G4/Au/ITO) were developed and applied to explore the electrocatalytic capacity of H2O2 reduction. Due to the excellent catalytic activity of Hemin-G4 and high conductivity of gold nanoparticles, both electrodes show excellent electrochemical performances towards H2O2 with a low LOD (0.67 µM for Hemin-G4/Au/GCE and 0.65 µM for Hemin-G4/Au/ITO), rapid response (<4 s), and high selectivity and sensitivity (314.33 µA mM-1 cm-2 for Hemin-G4/Au/GCE and 322.22 µA mM-1 cm-2 for Hemin-G4/Au/ITO). The two electrodes allow sensitive capture of H2O2 produced by A549 cells. Compared with the conventional method of detection in cell suspensions, an ITO electrode with a large specific surface area and good biocompatibility can provide a promising platform for cell adhesion, so as to realize real-time and in situ detection of extracellular H2O2. The experimental results show that A549 cells can adhere to the surface of the Hemin-G4/Au/ITO electrode and grow well. This is benefitted from the three-dimensional structure of the Hemin-G4/Au hydrogel, which provides a suitable microenvironment for cell adhesion and growth. Furthermore, the in situ detection shows a faster response time than that of in-solution detection. This is because the H2O2 generated by the cells can be directly captured by the ITO electrode, which avoids diffusion from the solution to the electrode. These results indicate that the self-supporting hydrogel modified ITO electrode has great application prospects in basic biomedical research and continuous dynamic surveillance of diseases.

Cu2O-mediated assembly of electrodeposition of Au nanoparticles onto 2D metal-organic framework nanosheets for real-time monitoring of hydrogen peroxide released from living cells.

The development of metal nanoparticles (MNP) combined with a metal-organic framework (MOF) has received more and more attention due to its excellent synergistic catalytic ability, which can effectively broaden the scope of catalytic reactions and enhance the catalytic ability. In this work, we developed a novel ternary nanocomposite named Cu2O-mediated Au nanoparticle (Au NP) grown on MIL-53(Fe) for real-time monitoring of hydrogen peroxide (H2O2) released from living cells. First, Cu2O-MIL-53(Fe) was prepared by redox assembly technology, which provided the growth template, and active sites for AuCl4-. Au@Cu2O-MIL-53(Fe)/GCE biosensor was prepared by further loading nano-Au uniformly on the surface of Cu2O by electrochemical deposition. Compared to individual components, the hybrid nanocomposite showed superior electrochemical properties as electrode materials due to the synergistic effect between AuNPs, Cu2O, and MIL-53(Fe). Electrochemical measurement showed that the Au@Cu2O-MIL-53(Fe)/GCE biosensor presented a satisfactory catalytic activity towards H2O2 with a low detection limit of 1.01 µM and sensitivity of 351.57 µA mM-1 cm-2 in the linear range of 10-1520 µM. Furthermore, this biosensor was successfully used for the real-time monitoring of dynamic H2O2 activated by PMA released from living cells. And the great results of confocal fluorescence microscopy of the co-culture cells with PMA and Au@Cu2O-MIL-53(Fe) verified the reliability of the biosensor, suggesting its potential application to the monitoring of critical pathological processes at the cellular level.

ZIF-67 MOF-derived Co nanoparticles supported on N-doped carbon skeletons for the amperometric determination of hydrogen peroxide.

ZIF-67-derived Co nanoparticles supported on N-doped carbon skeletons have been prepared from melamine foam (Co-NPs/NCs) for non-enzymatic electrochemical H2O2 detection. The synthesis of Co-NPs/NCs was demonstrated via calcination treatment using melamine foam (MF) and ZIF-67 as precursors. The experimental results show that Co-NPs/NCs composites exhibit eminent catalytic activity toward specific determination of H2O2 with high selectivity and sensitivity (252.43 and 203.88 µA mM-1 cm-2), low LOD (0.12 µM), and wide linear ranges (10-2080 and 2080-11,800 µM). The excellent performance might be ascribed to the synergetic effects of MOF and N-doped carbon skeletons. The carbon skeletons serve as a conductive bridge and provide a large specific surface area, which can facilitate electron transfer and well disperse nanoparticles. This non-enzymatic electrochemical sensor based on Co-NPs/NCs can successfully detect H2O2 secreted by living cells, indicating its great potential in the early diagnosis and pathological exploration of disease.

Fabrication of Cu-hemin metal-organic-frameworks nanoflower supported on three-dimensional reduced graphene oxide for the amperometric detection of H2O2.

A novel electrochemical sensor based on Cu-hemin metal-organic-frameworks nanoflower/three-dimensional reduced graphene oxide (Cu-hemin MOFs/3D-RGO) was constructed to detect H2O2 released from living cells. The nanocomposite was synthesized via a facile co-precipitation method using hemin as the ligand, then decorated with 3D-RGO. The prepared Cu-hemin MOFs showed a 3D hollow spherical flower-like structure with a large specific surface area and mesoporous properties, which could load more biomolecules and greatly enhance the stability by protecting the activity of hemin. In addition, the introduction of 3D-RGO effectively enhanced the conductivity of Cu-hemin MOFs. Thus, the proposed sensor (Cu-hemin MOFs/3D-RGO/GCE) showed excellent electrochemical performances towards H2O2 with a wide linear range (10-24,400 µM), high sensitivity (207.34 µA mM-1 cm-2), low LOD (0.14 µM), and rapid response time (less than 3 s). Most importantly, we prepared a Cu-hemin MOFs/3D-RGO/ITO electrode with cells growing on it. Compared with detecting H2O2 in cell suspension by GCE-based electrode, adhesion of cells on ITO could shorten the diffusion distance of H2O2 from solution to the surface of the electrode and achieve in situ and a real-time monitor of H2O2 released by living cells. This self-supported sensing electrode showed great potential applications in monitoring the pathological and physiological dynamics of cancer cells.

Synthesis of dopamine-derived N-doped carbon nanotubes/Fe3O4 composites as enhanced electrochemical sensing platforms for hydrogen peroxide detection.

A novel preparation of dopamine-derived N-doped carbon nanotubes/Fe3O4 composites (N-CNTs/Fe3O4 Cs) is demonstrated via facile hydrothermal route and calcination treatment. In this approach, dopamine was selected as N-containing precursor, which can promote Fe3O4 nanocrystal deposition uniformly on the surface of CNTs and effectively modulated the graphitic structure with doped pyridinic N and graphitic N to improve the electrochemical performance of carbon composites. More interestingly, the inhibited growth of the Fe3O4 crystal during calcination can be effectively avoided by soaking PDA-CNT/Fe3O4 Cs in a phosphate solution before calcination. The N-CNTs/Fe3O4 Cs have an enhanced electrocatalytic activity toward hydrogen peroxide with high sensitivity (316.27 mA M-1 cm-2) and wide linear range (0.006-2.057 mM). The N-CNTs/Fe3O4 Cs modified sensor was successfully applied to real-time detection of H2O2 released from living cancer cells, displaying a potential application in the study of oxidative stress-related diseases. This work demonstrates a rational way for high-performance electrocatalytic material synthesis and bioanalysis. Graphical abstract An enzyme-free biosensor of H2O2 was constructed on the basis of dopamine-derived N-doped carbon nanotubes/Fe3O4 composites (N-CNTs/Fe3O4 Cs), and it exhibited the enhanced electrocatalytic activity for H2O2 detection with high sensitivity, wide linear range, and outstanding reproducibility and stability.

Matrilin-2 regulates proliferation, apoptosis and cell cycle during radiation-induced injury in HPAEpiC cell.

Radiation pulmonary injury is related to the accumulation of extracellular matrix proteins in the alveolar interstitial space. Matrilin-2 as a component of extracellular filamentous networks, present higher level in the lung tissue from irradiated mice and irradiated pulmonary epithelial cell line, HPAEpiC cells. Knockdown of endogenous matrilin-2 prevents the apoptosis of HPAEpiC cell induced by the irradiation injury. Consistently, over-expression of matrilin-2 reduced the proliferation and induced apoptosis of HPAEpiC cells. Matrilin-2 promotes the expression of p21 via increasing the transcriptional activity of p53, by which induces the G1 phase arresting in HPAEpiC cells. In summary, matrilin-2, increased by irradiation, reduced the proliferation and induces apoptosis of pulmonary epithelial cells via p53/p21 pathway.

High glucose-induced Matrilin-2 expression in mouse mesangial cells was mediated by transforming growth factor beta 1 (TGF-ß1).

This study aimed at evaluating the effect of high glucose on the expression of extracellular matrix (ECM) protein Matrilin-2 and the mechanism underlying this effect by using a mouse mesangial cell line. Mouse mesangial cells (MMCs) were cultured in media containing normal (5 mM d-glucose) or high concentrations of glucose (30 mM d-glucose). The expression of Matrilin-2 was assessed by either RT-PCR or western blot. Additionally, transforming growth factor beta 1 (TGF-ß1) inhibitors and TGF-ß1 were used to determine whether glucose-regulated Matrilin-2 expression was mediated by the TGF-ß1/Smad3 signaling pathway. Our data demonstrated that Matrilin-2 expression was markedly induced by high glucose and TGF-ß1. High glucose-induced Matrilin-2 expression was inhibited by TGF-ß1/Smad3 inhibitors, indicating that Matrilin-2 was markedly induced by high glucose and this induction was mediated by the TGF-ß1/Smad3 pathway. Taken together, our results showed that high-glucose-induced Matrilin-2 expression that was mediated by the TGF-ß1/Smad3 signaling pathway might play a role in Diabetic nephropathy (DN) pathogenesis and our finding provided a potential diagnostic and/or therapeutic target for DN.

En bloc transurethral resection with 2-micron continuous-wave laser for primary non-muscle-invasive bladder cancer: a randomized controlled trial.

PURPOSE: To evaluate the practicability of en bloc transurethral resection with 2-micron continuous-wave laser as treatment for primary non-muscle-invasive bladder cancer (NMIBC). METHODS: This was a single-center, randomized, controlled trial involving 142 patients with newly diagnosed NMIBC. All patients were randomly assigned in a 1:1 ratio to receive either laser treatment or conventional transurethral resection of bladder tumor (TURBT). All patients received intravesical chemotherapy. Follow-up was performed in 18 months. Primary outcome measure was difference of tumor recurrence rate at the end of study. RESULTS: Baseline characteristics did not differ between patients in two groups. Operation time was longer in laser group than in TURBT group (56.5 ± 37.4 vs. 41.0 ± 29.4 min, P = 0.017). Obturator nerve reflection was noted during TURBT in 18 patients, whereas none was noted during laser resection. Number of T1 tumors was higher in the laser group (25 vs. 15, P = 0.047). According to Kaplan-Meier survival curves, there was no statistical difference in the rate of recurrence in 18 months (P = 0.383). All recurrences were out of the site of first resection, and there was no progression in tumor grade. CONCLUSION: Two-micron continuous-wave laser did not diminish tumor recurrence rate in primary NMIBC for 18-months observation. However, T1 tumors were significantly higher among laser group. Clear and complete tumor bases were easily conserved by laser resection, which may enable pathologists to distinguish the T stages of bladder cancer more easily. Further studies need to be done in future.

Rapid, portable, and sensitive detection of CaMV35S by RPA-CRISPR/Cas12a-G4 colorimetric assays with high accuracy deep learning object recognition and classification.

Fast, sensitive, and portable detection of genetic modification contributes to agricultural security and food safety. Here, we developed RPA-CRISPR/Cas12a-G-quadruplex colorimetric assays that can combine with intelligent recognition by deep learning algorithms to achieve sensitive, rapid, and portable detection of the CaMV35S promoter. When the crRNA-Cas12a complex recognizes the RPA amplification product, Cas12 cleaves the G-quadruplex, causing the G4-Hemin complex to lose its peroxide mimetic enzyme function and be unable to catalyze the conversion of ABTS2- to ABTS, allowing CaMV35S concentration to be determined based on ABTS absorbance. By utilizing the RPA-CRISPR/Cas12a-G4 assay, we achieved a CaMV35S limit of detection down to 10 aM and a 0.01 % genetic modification sample in 45 min. Deep learning algorithms are designed for highly accurate classification of color results. Yolov5 objective finding and Resnet classification algorithms have been trained to identify trace (0.01 %) CaMV35S more accurately than naked eye colorimetry. We also coupled deep learning algorithms with a smartphone app to achieve portable and rapid photo identification. Overall, our findings enable low cost ($0.43), high accuracy, and intelligent detection of the CaMV35S promoter.

Hairpin probe-based one-pot multiplex isothermal amplification combined with bifunctional G-quadruplex (IHP-GT) for the detection of alkaline phosphatase.

Abnormal alkaline phosphatase (ALP) levels have been linked to breast cancer, prostate cancer, bone damage, gingivitis and abnormal liver function. Monitoring ALP levels is important for better diagnosis and treatment of these diseases. Detection of ALP by colorimetric methods is very portable in terms of signal reading, but still suffers from low sensitivity. SERS can achieve high sensitivity detection, but cannot be separated from large precision instruments. Therefore, researchers have worked to optimize various aspects of the sensor, such as sensitivity, detection time, and operating procedures, to enable portable and rapid ALP detection. Isothermal amplification using simple system components meets the current demand for rapid, portable assays. We have developed a novel one-pot high-efficiency ALP assay strategy called IHP-GT. IHP-GT performs a one-step cascade amplification using only one probe (IGHP) as a template. The phosphorylated primer P binds to IGHP, forming a P/IGHP structure. At this point, the G-quadruplex closes and no signal is generated. In the presence of ALP, primer P is dephosphorylated to remove the restriction and then amplified in a cascade using IGHP as a template to release the full G-quadruplex structure. The single-stranded G-quadruplex will bend to form a secondary structure, facilitating secondary amplification starting with primer AT to produce PrG and P'. The PrG structure will trigger triple amplification, enabling cascade amplification. The G-quadruplex structure produced by cascade amplification has the dual role of promoting amplification of primer AT and binding to ThT to produce a fluorescent signal. The IHP-GT method provides a highly sensitive detection of ALP in less than 90 min and has been successfully used to analyze ALP in human serum samples. In addition, IHP-GT can be used to screen for ALP inhibitors. Importantly, we lyophilized the IHP-GT reaction components into powder form for user-friendly poc testing.

Mycobacterium tuberculosis FadD18 Promotes Proinflammatory Cytokine Secretion to Inhibit the Intracellular Survival of Bacillus Calmette-Guérin.

Mycobacterium tuberculosis causes 6.4 million cases of tuberculosis and claims 1.6 million lives annually. Mycobacterial adhesion, invasion of host cells, and subsequent intracellular survival are crucial for the infection and dissemination process, yet the cellular mechanisms underlying these phenomena remain poorly understood. This study created a Bacillus Calmette-Guérin (BCG) transposon library using a MycomarT7 phage carrying a Himar1 Mariner transposon to identify genes related to mycobacteria adhesion and invasion. Using adhesion and invasion model screening, we found that the mutant strain B2909 lacked adhesion and invasion abilities because of an inactive fadD18 gene, which encodes a fatty-acyl CoA ligase, although the specific function of this gene remains unclear. To investigate the role of FadD18, we constructed a complementary strain and observed that fadD18 expression enhanced the colony size and promoted the formation of a stronger cord-like structure; FadD18 expression also inhibited BCG growth and reduced BCG intracellular survival in macrophages. Furthermore, FadD18 expression elevated levels of the proinflammatory cytokines IL-6, IL-1ß, and TNF-α in infected macrophages by stimulating the NF-κB and MAPK signaling pathways. Overall, the FadD18 plays a key role in the adhesion and invasion abilities of mycobacteria while modulating the intracellular survival of BCG by influencing the production of proinflammatory cytokines.

Targeted inhibition of gut bacterial ß-glucuronidases by octyl gallate alleviates mycophenolate mofetil-induced gastrointestinal toxicity.

Mycophenolate mofetil (MMF) is a viable therapeutic option against various immune disorders as a chemotherapeutic agent. Nevertheless, its application has been undermined by the gastrotoxic metabolites (mycophenolic acid glucuronide, MPAG) produced by microbiome-associated ß-glucuronidase (ßGUS). Therefore, controlling microbiota-produced ßGUS underlines the potential strategy to improve MMF efficacy by overcoming the dosage limitation. In this study, the octyl gallate (OG) was identified with promising inhibitory activity on hydrolysis of PNPG in our high throughput screening based on a chemical collection of approximately 2000 natural products. Furthermore, OG was also found to inhibit a broad spectrum of BGUSs, including mini-Loop1, Loop 2, mini-Loop 2, and mini-Loop1,2. The further in vivo experiments demonstrated that administration of 20 mg/kg OG resulted in predominant reduction in the activity of BGUSs while displayed no impact on the overall fecal microbiome in mice. Furthermore, in the MMF-induced colitis model, the administration of OG at a dosage of 20 mg/kg effectively mitigated the gastrointestinal toxicity, and systematically reverted the colitis phenotypes. These findings indicate that the OG holds promising clinical potential for the prevention of MMF-induced gastrointestinal toxicity by inhibition of BGUSs and could be developed as a combinatorial therapy with MFF for better clinical outcomes.

Innovative Delivery System Combining CRISPR-Cas12f for Combatting Antimicrobial Resistance in Gram-Negative Bacteria.

Antimicrobial resistance poses a significant global challenge, demanding innovative approaches, such as the CRISPR-Cas-mediated resistance plasmid or gene-curing system, to effectively combat this urgent crisis. To enable successful curing of antimicrobial genes or plasmids through CRISPR-Cas technology, the development of an efficient broad-host-range delivery system is paramount. In this study, we have successfully designed and constructed a novel functional gene delivery plasmid, pQ-mini, utilizing the backbone of a broad-host-range Inc.Q plasmid. Moreover, we have integrated the CRISPR-Cas12f system into the pQ-mini plasmid to enable gene-curing in broad-host of bacteria. Our findings demonstrate that pQ-mini facilitates the highly efficient transfer of genetic elements to diverse bacteria, particularly in various species in the order of Enterobacterales, exhibiting a broader host range and superior conjugation efficiency compared to the commonly used pMB1-like plasmid. Notably, pQ-mini effectively delivers the CRISPR-Cas12f system to antimicrobial-resistant strains, resulting in remarkable curing efficiencies for plasmid-borne mcr-1 or blaKPC genes that are comparable to those achieved by the previously reported pCasCure system. In conclusion, our study successfully establishes and optimizes pQ-mini as a broad-host-range functional gene delivery vector. Furthermore, in combination with the CRISPR-Cas system, pQ-mini demonstrates its potential for broad-host delivery, highlighting its promising role as a novel antimicrobial tool against the growing threat of antimicrobial resistance.

Mechanisms of gastrointestinal toxicity in neuromyelitis optica spectrum disorder patients treated with mycophenolate mofetil: insights from a mouse model and human study.

Mycophenolate mofetil (MMF) is commonly utilized for the treatment of neuromyelitis optica spectrum disorders (NMOSD). However, a subset of patients experience significant gastrointestinal (GI) adverse effects following MMF administration. The present study aims to elucidate the underlying mechanisms of MMF-induced GI toxicity in NMOSD. Utilizing a vancomycin-treated mouse model, we compiled a comprehensive data set to investigate the microbiome and metabolome in the GI tract to elucidate the mechanisms of MMF GI toxicity. Furthermore, we enrolled 17 female NMOSD patients receiving MMF, who were stratified into non-diarrhea NMOSD and diarrhea NMOSD (DNM) groups, in addition to 12 healthy controls. The gut microbiota of stool samples was analyzed using 16S rRNA gene sequencing. Vancomycin administration prevented weight loss and tissue injury caused by MMF, affecting colon metabolomes and microbiomes. Bacterial ß-glucuronidase from Bacteroidetes and Firmicutes was linked to intestinal tissue damage. The DNM group showed higher alpha diversity and increased levels of Firmicutes and Proteobacteria. The ß-glucuronidase produced by Firmicutes may be important in causing gastrointestinal side effects from MMF in NMOSD treatment, providing useful information for future research on MMF. IMPORTANCE: Neuromyelitis optica spectrum disorder (NMOSD) patients frequently endure severe consequences like paralysis and blindness. Mycophenolate mofetil (MMF) effectively addresses these issues, but its usage is hindered by gastrointestinal (GI) complications. Through uncovering the intricate interplay among MMF, gut microbiota, and metabolic pathways, this study identifies specific gut bacteria responsible for metabolizing MMF into a potentially harmful form, thus contributing to GI side effects. These findings not only deepen our comprehension of MMF toxicity but also propose potential strategies, such as inhibiting these bacteria, to mitigate these adverse effects. This insight holds broader implications for minimizing complications in NMOSD patients undergoing MMF therapy.