Biomimetic Activation of N-Nitrosamides with Red Light-Triggered Nitric Oxide Release via Mediated Electron Transfer.

Mediated electron transfer (MET) is fundamental to many biological functions, including cellular respiration, photosynthesis, and enzymatic catalysis. However, leveraging the MET process to enable the release of therapeutic gases has been largely unexplored. Herein, we report the bio-inspired activation of a series of UV-absorbing N-nitrosamide derivatives (NOA) under red light exposure, enabling the quantitative release of nitric oxide (NO) gasotransmitter via an MET process. The cornerstone of our design is the covalent linkage of a 2,4-dinitroaniline moiety, which acts as an electron mediator to the N-nitrosamide groups. This facilitates efficient electron transfer from the excited palladium(II) meso-tetraphenyltetrabenzoporphyrin (PdTPTBP) photocatalyst and the selective activation of NOA. Our approach has been validated with distinct photocatalysts and various N-nitrosamides, including those derived from carbamates, amides, and ureas. Notably, the modulation of the linker length between the electron mediator and N-nitrosamide groups serves as a regulatory mechanism for controlling NO release kinetics. Moreover, this biomimetic NO release platform demonstrates effective operation under both normoxic and hypoxic conditions, and it enables localized delivery of NO under physiological conditions, exhibiting significant anticancer efficacy within the phototherapeutic window and enhanced selectivity towards tumor cells.

Effect of acupuncture on post-stroke dysphagia: a randomized controlled trial.

Introduction: Post-stroke dysphagia (PSD) is associated with various complications that increase morbidity and mortality rates. Acupuncture has been used extensively in China to treat these complications; however, its therapeutic efficacy remains uncertain. We therefore aimed to study the clinical effects of acupuncture on PSD. Methods: Patients (n = 101) were randomly divided into acupuncture (n = 50) and rehabilitation training control (n = 51) groups based on the treatment used. Both groups were treated once daily, 6 days a week, for a total of 4 weeks. Pulse oxygen saturation (SpO2) and standardized swallowing assessment (SSA) were performed before the intervention, 2 weeks into treatment, after the intervention (4 weeks post-intervention), and at a 6-month follow-up (28 weeks). The levels of hemoglobin (Hb) and albumin (ALB), and 5-hydroxytryptamine (5-HT) and dopamine (DA) were measured before the intervention, 2 weeks into treatment, and after the intervention (4 weeks), as nutrition and swallowing function indices, respectively. Results: Following the intervention, significant differences were observed between the acupuncture and control groups. The acupuncture group exhibited considerably superior enhancements in SpO2 and SSA scores at 4 weeks (p < 0.001). Moreover, this group demonstrated significantly greater improvements in Hb, ALB, 5-HT, and DA values 4 weeks post-treatment (p < 0.001). However, sex-based differences were not observed (P > 0.005). Conclusion: Acupuncture treatment can improve the swallowing function and nutritional status of patients with PSD, and increase the levels of 5-HT and DA. These findings strongly support the efficacy of acupuncture as a therapeutic intervention in patients with PSD.Clinical trial registration: identifier, ChiCTR2100052201. (https://www.chictr.org.cn/).

A cell-free fluorescence biosensor based on allosteric transcription factor NalC for detection of pentachlorophenol.

Pentachlorophenol (PCP) was once used as a pesticide, germicide, and preservative due to its stable properties and resistance to degradation. This study aimed to design a biosensor for the quantitative and prompt detection of capable of PCP. A cell-free fluorescence biosensor was developed while employing NalC, an allosteric Transcription Factor responsive to PCP and In Vitro Transcription. By adding a DNA template and PCP and employing Electrophoretic Mobility Shift Assay while monitoring the dynamic fluorescence changes in RNA, this study offers evidence of NalC's potential applicability in sensor systems developed for the specific detection of PCP. The biosensor showed the capability for the quantitative detection of PCP, with a Limit of Detection (LOD) of 0.21 µM. Following the addition of Nucleic Acid Sequence-Based Amplification, the fluorescence intensity of RNA revealed an excellent linear relationship with the concentration of PCP, showing a correlation coefficient (R2) of 0.9595. The final LOD was determined to be 0.002 µM. This study has successfully translated the determination of PCP into a fluorescent RNA output, thereby presenting a novel approach for detecting PCP within environmental settings.

Specific and Sensitive Visual Proviral DNA Detection of Major Pathogenic Avian Leukosis Virus Subgroups Using CRISPR-Associated Nuclease Cas13a.

Avian leukosis viruses (ALVs) include a group of avian retroviruses primarily associated with neoplastic diseases in poultry, commonly referred to as avian leukosis. Belonging to different subgroups based on their envelope properties, ALV subgroups A, B, and J (ALV-A, ALV-B, and ALV-J) are the most widespread in poultry populations. Early identification and removal of virus-shedding birds from infected flocks are essential for the ALVs' eradication. Therefore, the development of rapid, accurate, simple-to-use, and cost effective on-site diagnostic methods for the detection of ALV subgroups is very important. Cas13a, an RNA-guided RNA endonuclease that cleaves target single-stranded RNA, also exhibits non-specific endonuclease activity on any bystander RNA in close proximity. The distinct trans-cleavage activity of Cas13 has been exploited in the molecular diagnosis of multiple pathogens including several viruses. Here, we describe the development and application of a highly sensitive Cas13a-based molecular test for the specific detection of proviral DNA of ALV-A, B, and J subgroups. Prokaryotically expressed LwaCas13a, purified through ion exchange and size-exclusion chromatography, was combined with recombinase polymerase amplification (RPA) and T7 transcription to establish the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) molecular detection system for the detection of proviral DNA of ALV-A/B/J subgroups. This novel method that needs less sample input with a short turnaround time is based on isothermal detection at 37 °C with a color-based lateral flow readout. The detection limit of the assay for ALV-A/B/J subgroups was 50 copies with no cross reactivity with ALV-C/D/E subgroups and other avian oncogenic viruses such as reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). The development and evaluation of a highly sensitive and specific visual method of detection of ALV-A/B/J nucleic acids using CRISPR-Cas13a described here will help in ALV detection in eradication programs.

Pheromone cCF10 inhibits the antibiotic persistence of Enterococcus faecalis by modulating energy metabolism.

Introduction: Bacterial resistance presents a major challenge to both the ecological environment and human well-being, with persistence playing a key role. Multiple studies were recently undertaken to examine the factors influencing the formation of persisters and the underlying process, with a primary focus on Gram-negative bacteria and Staphylococcus aureus (Gram-positive bacteria). Enterococcus faecalis (E. faecalis) is capable of causing a variety of infectious diseases, but there have been few studies of E. faecalis persisters. Previous studies have shown that the sex pheromone cCF10 secreted by E. faecalis induces conjugative plasmid transfer. However, whether the pheromone cCF10 regulates the persistence of E. faecalis has not been investigated. Methods: As a result, we investigated the effect and potential molecular mechanism of pheromone cCF10 in regulating the formation of persisters in E. faecalis OG1RF using a persistent bacteria model. Results and discussion: The metabolically active E. faecalis OG1RF reached a persistence state and temporarily tolerated lethal antibiotic concentrations after 8 h of levofloxacin hydrochloride (20 mg/mL) exposure, exhibiting a persistence rate of 0.109 %. During the growth of E. faecalis OG1RF, biofilm formation was a critical factor contributing to antibiotic persistence, whereas 10 ng/mL cCF10 blocked persister cell formation. Notably, cCF10 mediated the antibiotic persistence of E. faecalis OG1RF via regulating metabolic activity rather than suppressing biofilm formation. The addition of cCF10 stimulated the Opp system and entered bacterial cells, inhibiting (p)ppGpp accumulation, thus maintaining the metabolically active state of bacteria and reducing persister cell generation. These findings offer valuable insights into the formation, as well as the control mechanism of E. faecalis persisters.

Regulation of Autophagy Signaling Pathways by Ginseng Saponins: A Review.

Ginseng saponins ( ginsenosides), bioactive compounds derived from ginseng, are widely used natural products with potent therapeutic properties in the management of various ailments, particularly tumors, cardiovascular and cerebrovascular diseases, and immune system disorders. Autophagy, a highly regulated and multistep process involving the breakdown of impaired organelles and macromolecules by autophagolysosomes and autophagy-related genes (ATGs), has gained increasing attention as a potential target for ginsenoside-mediated disease treatment. This review aims to provide a comprehensive overview of recent research advances in the understanding of autophagy-related signaling pathways and the role of ginsenoside-mediated autophagy regulation. By delving into the intricate autophagy signaling pathways underpinning the pharmacological properties of ginsenosides, we highlight their therapeutic potential in addressing various conditions. Our findings serve as a comprehensive reference for further investigation into the medicinal properties of ginseng or ginseng-related products.

Toxicokinetics of selenate in earthworm sub-tissues and potential bio-accessibility assessment of earthworm-derived selenium.

Selenium (Se) pollution is mainly caused by anthropogenic activities, and the resulting biosecurity concerns have garnered significant attention in recent years. Using one-compartmental toxicokinetic (TK) modelling, this study explored the kinetic absorption, sub-tissue distribution, and elimination processes of the main Se species (selenate, Se(VI)) in the cultivated aerobic soil of the earthworm Eisenia fetida. The bio-accessibility of earthworm-derived Se was assessed using an in vitro simulated gastrointestinal digestion test to evaluate its potential trophic risk. The results demonstrated that Se accumulated in the pre-clitellum (PC) and total tissues (TT) of earthworms in a time- and dose-dependent manner. The highest Se levels in the PC, post-clitellum (PoC), and TT were 70.54, 57.93, and 64.26 mg/kg during the uptake phase, respectively. The kinetic Se contents in the earthworms PC and TT were consistent with the TK model but not with PoC. The earthworm TT exhibited a faster uptake (Kus = 0.83-1.02 mg/kg/day) and elimination rate of Se (Kee = 0.044-0.049 mg/kg/day), as well as a shorter half-life time (LT1/2 = 15.88-14.22 days) than PC at low soil Se levels (≤5 mg/kg). Conversely, the opposite trend was observed with higher Se concentrations (10 and 20 mg/kg). These results are likely attributable to the tissue specificity and concentration of the toxicant. Earthworms PC and TT exhibited a higher kinetic Se accumulation factor (BAFk) than steady-state BAF (BAFss), with values ranging from 8 to 24 and 3-13, respectively. Furthermore, the bio-accessibility of earthworm-derived Se to poultry ranged from 66.25 % to 84.35 %. As earthworms are at the bottom of the terrestrial food chain, the high bio-accessibility of earthworm-derived Se poses a potential risk to predators. This study offers data support and a theoretical foundation for understanding the biological footprint of soil Se and its toxicological impacts and ecological hazards.

Pharmacokinetic Properties of Baitouweng Decoction in Bama Miniature Pigs: Implications for Clinical Application in Humans.

Traditional Chinese medicine (TCM) serves as a significant adjunct to chemical treatment for chronic diseases. For instance, the administration of Baitouweng decoction (BTWD) has proven effective in the treatment of ulcerative colitis. However, the limited understanding of its pharmacokinetics (PK) has impeded its widespread use. Chinese Bama miniature pigs possess anatomical and physiological similarities to the human body, making them a valuable model for investigating PK properties. Consequently, the identification of PK properties in Bama miniature pigs can provide valuable insights for guiding the clinical application of BTWD in humans. To facilitate this research, a rapid and sensitive UPLC-MS/MS method has been developed for the simultaneous quantification of eleven active ingredients of BTWD in plasma. Chromatographic separation was conducted using an Acquity UPLC HSS T3 C18 column and a gradient mobile phase comprising acetonitrile and water (containing 0.1% acetic acid). The methodology was validated in accordance with the FDA Bioanalytical Method Validation Guidance for Industry. The lower limit of quantitation fell within the range of 0.60-2.01 ng/mL. Pharmacokinetic studies indicated that coptisine chloride, berberine, columbamine, phellodendrine, and obacunone exhibited low Cmax, while fraxetin, esculin, fraxin, and pulchinenoside B4 were rapidly absorbed and eliminated from the plasma. These findings have implications for the development of effective components in BTWD and the adjustment of clinical dosage regimens.

Treatment outcomes amongst older people with HIV infection receiving antiretroviral therapy.

OBJECTIVES: There is conflicting data regarding the response of older people with HIV (PWH) to antiretroviral therapy (ART). The objective of this study was to evaluate the long-term immunological and virological responses, changes in regimen, and adverse drug reactions (ADRs) in older participants (50+ years) compared with younger (18-34 years) and middle-aged (35-49 years) PWH. METHODS: A retrospective review of medical records was conducted on 1622 participants who received ART in Yunnan Province, China, from 2010 to 2019. The study compared CD4+ T-cell counts, CD4+/CD8+ ratio, and relative numbers between different groups using the Kruskal-Wallis test. Cox proportional hazards regression models were used to identify variables associated with the occurrence of immune reconstitution insufficiency. The rates of immune reconstitution, incidence of ADRs, and rates of treatment change were analyzed using the chi-squared test or Fisher's exact test. RESULTS: Over 95% achieved viral load 200 copies/ml or less, with no age-related difference. However, older participants exhibited significantly lower CD4+ T-cell counts and CD4+/CD8+ recovery post-ART (P < 0.001), with only 32.21% achieving immune reconstitution (compared with young: 52.16%, middle-aged: 39.29%, P < 0.001) at the end of follow-up. Middle-aged and elderly participants changed ART regimens more because of ADRs, especially bone marrow suppression and renal dysfunction. CONCLUSION: Although the virological response was consistent across age groups, older individuals showed poorer immune responses and higher susceptibility to side effects. This underscores the need for tailored interventions and comprehensive management for older patients with HIV.

Cylindrospermopsin enhances the conjugative transfer of plasmid-mediated multi-antibiotic resistance genes through glutathione biosynthesis inhibition.

Cylindrospermopsin (CYN), a cyanobacterial toxin, has been detected in the global water environment. However, information concerning the potential environmental risk of CYN is limited, since the majority of previous studies have mainly focused on the adverse health effects of CYN through contaminated drinking water. The present study reported that CYN at environmentally relevant levels (0.1-100 µg/L) can significantly enhance the conjugative transfer of RP4 plasmid in Escherichia coli genera, wherein application of 10 µg/L of CYN led to maximum fold change of ∼6.5- fold at 16 h of exposure. Meanwhile, evaluation of underlying mechanisms revealed that environmental concentration of CYN exposure could increase oxidative stress in the bacterial cells, resulting in ROS overproduction. In turn, this led to an upregulation of antioxidant enzyme-related genes to avoid ROS attack. Further, inhibition of the synthesis of glutathione (GSH) was also detected, which led to the rapid depletion of GSH in cells and thus triggered the SOS response and promoted the conjugative transfer process. Increase in cell membrane permeability, upregulation of expression of genes related to pilus generation, ATP synthesis, and RP4 gene expression were also observed. These results highlight the potential impact on the spread of antimicrobial resistance in water environments.

Mitochondrial dysfunction of peripheral blood mononuclear cells is associated with lung carcinogenesis.

The composition, quantity, and function of peripheral blood mononuclear cells (PBMCs) are closely correlated with tumorigenesis. However, the mechanisms of PBMCs in lung cancer are not clear. Mitochondria are energy factories of cells, and almost all cellular functions rely on their energy metabolism level. The present study aimed to test whether the mitochondrial function of PBMCs directly determines their tumor immune monitoring function. We recruited 211 subjects, including 105 healthy controls and 106 patients with recently diagnosed with lung cancer. The model of lung carcinogenesis induced by BaP was used in animal experiment, and the Bap carcinogenic metabolite, Benzo(a)pyren-7,8-dihydrodiol-9,10-epoxide (BPDE), was used in cell experiment. We found that mitochondrial function of PBMCs decreased significantly in patients with new lung cancer, regardless of age. In vivo, BaP caused PBMC mitochondrial dysfunction in mice before the appearance of visible malignant tissue. Moreover, mitochondrial function decreased significantly in mice with lung cancers induced by BaP compared to those without lung cancer after BaP intervention. In vitro, BPDE also induced mitochondrial dysfunction and reduced the aggressiveness of PBMCs toward cancer cells. Furthermore, the changes in mitochondrial energy metabolism gene expression caused by BPDE are involved in this process. Thus, the mitochondrial function of PBMCs is a potential prognostic biomarker or therapeutic target to improve clinical outcomes in patients with lung cancer.

Chlorite and bromate alter the conjugative transfer of antibiotic resistance genes: Co-regulation of oxidative stress and energy supply.

The widespread use of disinfectants during the global response to the 2019 coronavirus pandemic has increased the co-occurrence of disinfection byproducts (DBPs) and antibiotic resistance genes (ARGs). Although DBPs pose major threats to public health globally, there is limited knowledge regarding their biological effects on ARGs. This study aimed to investigate the effects of two inorganic DBPs (chlorite and bromate) on the conjugative transfer of RP4 plasmid among Escherichia coli strains at environmentally relevant concentrations. Interestingly, the frequency of conjugative transfer was initially inhibited when the exposure time to chlorite or bromate was less than 24 h. However, this inhibition transformed into promotion when the exposure time was extended to 36 h. Short exposures to chlorite or bromate were shown to impede the electron transport chain, resulting in an ATP shortage and subsequently inhibiting conjugative transfer. Consequently, this stimulates the overproduction of reactive oxygen species (ROS) and activation of the SOS response. Upon prolonged exposure, the resurgent energy supply promoted conjugative transfer. These findings offer novel and valuable insights into the effects of environmentally relevant concentrations of inorganic DBPs on the conjugative transfer of ARGs, thereby providing a theoretical basis for the management of DBPs.

Tuning the Mechanical and Dynamic Properties of Elastic Vitrimers by Tailoring the Substituents of Boronic Ester.

Elastic vitrimers, i.e., elastic polymers with associative dynamic covalent bonds, can afford elastomers with recyclability while maintaining their thermal and chemical stability. Herein, we report a series of boronic ester-based vitrimers with tunable mechanical properties and recyclability by varying the substitute groups of boronic acid in polymer networks. The dynamic polymer networks are formed by reacting diol-containing tetra-arm poly(amidoamine) with boronic acid-terminated tetra-arm poly(ethylene glycol), which possesses different substituents adjacent to boronic acid moieties. Varying the substituent adjacent to the boronic ester unit will significantly affect the binding strength of the boronic ester, therefore affecting their dynamics and mechanical performance. The electron-withdrawing substituents noticeably suppress the dynamics of boronic ester exchange and increase the activation energy and relaxation time while enhancing the mechanical strength of the resulting elastic vitrimers. On the other hand, the presence of electron-rich substituent affords relatively reduced glass transition temperature (Tg), faster relaxation, and prominent recyclability and malleability at lower temperatures. The developed pathway will guide the rational design of elastomers with well-tunable dynamics and processabilities.

20(R)-ginsenoside Rg3 attenuates cerebral ischemia-reperfusion injury by mitigating mitochondrial oxidative stress via the Nrf2/HO-1 signaling pathway.

Reducing mitochondrial oxidative stress has become an important strategy to prevent neuronal death in ischemic stroke. Previous studies have shown that 20(R)-ginsenoside Rg3 can significantly improve behavioral abnormalities, reduce infarct size, and decrease the number of apoptotic neurons in cerebral ischemia/reperfusion injury rats. However, it remains unclear whether 20(R)-ginsenoside Rg3 can inhibit mitochondrial oxidative stress in ischemic stroke and the potential molecular mechanism. In this study, we found that 20(R)-ginsenoside Rg3 notably inhibited mitochondrial oxidative stress in middle cerebral artery occlusion/reperfusion (MCAO/R) rats and maintained the stability of mitochondrial structure and function. Treatment with 20(R)-ginsenoside Rg3 also decreased the levels of mitochondrial fission proteins (Drp1 and Fis1) and increased the levels of fusion proteins (Opa1, Mfn1, and Mfn2) in MCAO/R rats. Furthermore, we found that 20(R)-ginsenoside Rg3 promoted nuclear aggregation of nuclear factor erythroid2-related factor 2 (Nrf2) but did not affect Kelch-like ECH-associated protein-1 (Keap1), resulting in the downstream expression of antioxidants. In in vitro oxygen-glucose deprivation/reperfusion stroke models, the results of PC12 cells treated with 20(R)-ginsenoside Rg3 were consistent with animal experiments. After transfection with Nrf2 short interfering RNA (siRNA), the protective effect of 20(R)-ginsenoside Rg3 on PC12 cells was reversed. In conclusion, the inhibition of mitochondrial oxidative stress plays a vital position in the anti-cerebral ischemia-reperfusion injury of 20(R)-ginsenoside Rg3, and its neuroprotective mechanism is related to the activation of the nuclear factor erythroid2-related factor 2/heme oxygenase 1 signaling pathway.

Integrated network pharmacology, molecular docking and pharmacodynamic study reveals protective effects and mechanisms of corilagin against cerebral ischemia-induced injury.

BACKGROUND: Stroke is one of the leading causes of death and long-term disability worldwide. Previous studies have found that corilagin has antioxidant, anti-inflammatory, anti-atherosclerotic and other pharmacological activities and has a protective effect against cardiac and cerebrovascular injury. OBJECTIVES: The aim of this study was to investigate the protective effects of corilagin against ischemic stroke and to elucidate the underlying molecular mechanisms using network pharmacology, molecular docking, and animal and cell experiments. METHODS: We investigated the potential of corilagin to ameliorate cerebral ischemia-reperfusion injury using in vivo rat middle cerebral artery occlusion/reperfusion (MCAO/R) and in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) models. RESULTS: Our results suggest that corilagin may exert its anti-ischemic stroke effect by interacting with 92 key targets, including apoptosis-associated proteins (Bcl-2, Bax, caspase-3) and PI3K/Akt signaling pathway-related proteins. In vivo and in vitro experiments showed that corilagin treatment improved neurological deficits, attenuated cerebral infarct volume, and mitigated neuronal damage in MCAO/R rats. Corilagin treatment also enhanced the survival of PC12 cells exposed to OGD/R, reduced the rate of LDH leakage, inhibited cell apoptosis, and activated the PI3K/Akt signaling pathway. Importantly, the effects of corilagin on the PI3K/Akt signaling pathway and apoptosis-associated proteins were reversed by the PI3K-specific inhibitor LY294002. CONCLUSIONS: These results indicate that the molecular mechanism of the anti-ischemic effect of corilagin involves inhibiting neuronal apoptosis and activating the PI3K/Akt signaling pathway. These findings provide a theoretical and experimental basis for the further development and application of corilagin as a potential anti-ischemic stroke agent.

Red light-triggered release of ROS and carbon monoxide for combinational antibacterial application.

The abuse of antibiotics has led to the emergence of a wide range of drug-resistant bacteria. To address the challenge of drug-resistant bacterial infections and related infectious diseases, several effective antibacterial strategies have been developed. To achieve enhanced therapeutic effects, combinational treatment approaches should be employed. With this in mind, a metal-organic framework (MOF) based nanoreactor with integrated photodynamic therapy (PDT) and gas therapy which can release reactive oxygen species (ROS) and carbon monoxide (CO) under red light irradiation has been developed. The release of ROS and CO under red light irradiation exerts a preferential antibacterial effect on Gram-positive/Gram-negative bacteria. The bactericidal effects of ROS and CO on Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA) are better than ROS only, showing a combinational antibacterial effect. Furthermore, the fluorescence emission properties of porphyrin moieties can be leveraged for real-time tracking and imaging of the nanoreactors. The simple preparation procedures of this material further enhance its potential as a versatile and effective antibacterial candidate, thereby presenting a new strategy for PDT and gas combinational treatment.

Multidrug-resistant plasmid RP4 inhibits the nitrogen removal capacity of ammonia-oxidizing archaea, ammonia-oxidizing bacteria, and comammox in activated sludge.

In wastewater treatment plants (WWTPs), ammonia oxidation is primarily carried out by three types of ammonia oxidation microorganisms (AOMs): ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and comammox (CMX). Antibiotic resistance genes (ARGs), which pose an important public health concern, have been identified at every stage of wastewater treatment. However, few studies have focused on the impact of ARGs on ammonia removal performance. Therefore, our study sought to investigate the effect of the representative multidrug-resistant plasmid RP4 on the functional microorganisms involved in ammonia oxidation. Using an inhibitor-based method, we first evaluated the contributions of AOA, AOB, and CMX to ammonia oxidation in activated sludge, which were determined to be 13.7%, 41.1%, and 39.1%, respectively. The inhibitory effects of C2H2, C8H14, and 3,4-dimethylpyrazole phosphate (DMPP) were then validated by qPCR. After adding donor strains to the sludge, fluorescence in situ hybridization (FISH) imaging analysis demonstrated the co-localization of RP4 plasmids and all three AOMs, thus confirming the horizontal gene transfer (HGT) of the RP4 plasmid among these microorganisms. Significant inhibitory effects of the RP4 plasmid on the ammonia nitrogen consumption of AOA, AOB, and CMX were also observed, with inhibition rates of 39.7%, 36.2%, and 49.7%, respectively. Moreover, amoA expression in AOB and CMX was variably inhibited by the RP4 plasmid, whereas AOA amoA expression was not inhibited. These results demonstrate the adverse environmental effects of the RP4 plasmid and provide indirect evidence supporting plasmid-mediated conjugation transfer from bacteria to archaea.

[Preparation of colloidal gold test strips for the detection of antibodies to peste des petits ruminants based on monoclonal antibodies to N protein].

A simple, fast, and visual method for detecting antibodies against peste des petits ruminants virus (PPRV) using colloidal gold strips was developed. In this study, the pET-32a-N was transformed into Escherichia coli Rosetta (DE3) for expression. Hybridoma cell lines were generated by fusing SP2/0 myeloma cells with splenocytes from immunized mice with the expressed and purified N protein of PPRV. The PPRV N protein was labeled with colloidal gold particles as the gold-labeled antigen. The N protein served as the gold standard antigen and as the test (T) line-coated antigen, while the monoclonal antibody served as the quality control (C) line-coated antibody to assemble the colloidal gold immunochromatographic test strips for detecting antibodies against the N protein of PPRV. Hybridoma cell line designated as 1F1 was able to stably secrete the monoclonal antibody against the N protein of PPRV. The titer of 1F1 monoclonal antibody in ascites was 1:128 000 determined by indirect enzyme-linked immunosorbent assays (ELISA), and the immunoglobulin subtype of the monoclonal antibody was IgG1, with kappa chain. The obtained monoclonal antibody was able to specifically recognize the N protein of PPRV, as shown by Western blotting and indirect immunofluorescent assay (IFA). The developed colloidal gold test strip method was able to detect PPRV antibodies specifically, and there was no difference between different batches of the test strips. Testing of a total of 122 clinical sera showed that the compliance rate of the test strip with ELISA test was 97.6%.The test strip assay developed in this study has good specificity, reproducibility, and sensitivity, and it can be used for the rapid detection of PPRV antibodies.

A Single-Component Dual Donor Enables Ultrasound-Triggered Co-release of Carbon Monoxide and Hydrogen Sulfide.

The development of dual gasotransmitter donors can not only provide robust tools to investigate their subtle interplay under pathophysiological conditions but also optimize therapeutic efficacy. While conventional strategies are heavily dependent on multicomponent donors, we herein report an ultrasound-responsive water-soluble copolymer (PSHF) capable of releasing carbon monoxide (CO) and hydrogen sulfide (H2 S) based on single-component sulfur-substituted 3-hydroxyflavone (SHF) derivatives. Interestingly, sulfur substitution can not only greatly improve the ultrasound sensitivity but also enable the co-release of CO/H2 S under mild ultrasound irradiation. The co-release of CO/H2 S gasotransmitters exerts a bactericidal effect against Staphylococcus aureus and demonstrates anti-inflammatory activity in lipopolysaccharide-challenged macrophages. Moreover, the excellent tissue penetration of ultrasound irradiation enables the local release of CO/H2 S in the joints of septic arthritis rats, exhibiting superior therapeutic efficacy without the need for any antibiotics.

Development of a cell-free toehold switch for hepatitis A virus type I on-site detection.

Picornavirus hepatitis A virus (HAV) is a common cause of hepatitis worldwide. It is spread primarily through contaminated food and water or person-to-person contact. HAV I has been identified as the most common type of human HAV infection. Here, we have developed a cell-free toehold switch sensor for HAV I detection. We screened 10 suitable toehold switch sequences using NUPACK software, and the VP1 gene was used as the target gene. The optimal toehold switch sequence was selected by in vivo expression. The best toehold switch concentration was further found to be 20 nM in a cell-free system. 5 nM trigger RNA activated the toehold switch to generate visible green fluorescence. The minimum detection concentration decreased to 1 pM once combined with NASBA. HAV I trigger RNA could be detected accurately with excellent specificity. In addition, the cell-free toehold switch sensor was verified in HAV I entities. The successful construction of the cell-free toehold switch sensor provided a convenient, rapid, and accurate method for HAV I on-site detection, especially in developing countries, without the involvement of expensive facilities and additional professional operators.