Current strategies for monitoring and controlling bacterial biofilm formation on medical surfaces.

Biofilms, intricate microbial communities that attach to surfaces, especially medical devices, form an exopolysaccharide matrix, which enables bacteria to resist environmental pressures and conventional antimicrobial agents, leading to the emergence of multi-drug resistance. Biofilm-related infections associated with medical devices are a significant public health threat, compromising device performance. Therefore, developing effective methods for supervising and managing biofilm growth is imperative. This in-depth review presents a systematic overview of strategies for monitoring and controlling bacterial biofilms. We first outline the biofilm creation process and its regulatory mechanisms. The discussion then progresses to advancements in biosensors for biofilm detection and diverse treatment strategies. Lastly, this review examines the obstacles and new perspectives associated with this domain to facilitate the advancement of innovative monitoring and control solutions. These advancements are vital in combating the spread of multi drug-resistant bacteria and mitigating public health risks associated with infections from biofilm formation on medical instruments.

Alterations and associations between lung microbiota and metabolite profiles in silica-induced lung injury.

Silicosis, caused by silica exposure, is the most widespread and deadliest occupational disease. However, effective treatments are lacking. Therefore, it is crucial to elucidate the mechanisms and targets involved in the development of silicosis. We investigated the basic processes of silicosis development and onset at different exposure durations (2 or 4 weeks) using various techniques such as histopathology, immunohistochemistry, Enzyme linked immunosorbent assay(ELISA),16 S rRNA, and untargeted metabolomics.These results indicate that exposure to silica leads to progressive damage to lung tissue with significant deterioration observed over time. Time-dependent cytokines such as the IL-4, IL-13, and IL-6 are detected in lung lavage fluid, the model group consistently exhibited elevated levels of these cytokines, indicating a persistent and worsening inflammatory response in the lungs. Meanwhile, HE and Masson results show that 4-week exposure to silica causes more obvious lung injury and pulmonary fibrosis. Besides, the model group consistently exhibited a distinct lung bacterial population, known as the Lachnospiraceae_NK4A136_group, regardless of exposure duration. However, with increasing exposure duration, specific temporal changes were observed in lung bacterial populations, including Haliangium, Allobaculum, and Sandaracinus (at 4 weeks; p < 0.05). Furthermore, our study revealed a strong correlation between the mechanism of silica-induced lung injury and three factors: oxidative stress, impaired lipid metabolism, and imbalanced amino acid metabolism. We observed a close correlation between cytokine levels, changes in lung microbiota, and metabolic disturbances during various exposure periods. These findings propose that a possible mechanism of silica-induced lung injury involves the interplay of cytokines, lung microbiota, and metabolites.

A graphene-oxide-based aptasensor for fluorometric determination of chloramphenicol in milk and honey samples utilizing exonuclease III-assisted target recycling and Nb.BbvCI-powered DNA walker cascade amplification.

Herein, a graphene oxide (GO)-based fluorescence aptasensor was developed for the sensitive and selective detection of chloramphenicol (CAP), based on exonuclease III (Exo III)-assisted target recycling and Nb.BbvCI-driven DNA walker cascade amplification. Interactions between CAP, hairpin1(HP1), hairpin2 (HP2), and 3'-amino modified hairpin3 (HP3) labeled with carboxyfluorescein (FAM) and covalently coupled to GO enabled efficient CAP detection. CAP was quantitatively assayed by measuring fluorescence at excitation/emission wavelengths of 480/514 nm, resulting from the accumulation of released FAM. A good linear range of 1 fM to 1 nM and a limit of detection (LOD) of 0.875 fM (signal-to-noise (S/N)= 3) were achieved. This aptasensor can distinguish the CAP from interference antibiotics with good specificity and selectivity, even if the concentration of the interfering substance is ten-fold higher than the target concentration. Moreover, the developed fluorescence aptasensor was successfully applied for the detection of CAP in spiked milk and honey samples. Thus, this method is potentially applicable for assaying CAP in foods and provides a promising strategy for the development of fluorescence aptasensors for environmental sample analysis.

[Maxing Shigan Decoction improves lung and colon tissue damage caused by influenza virus infection through JAK1/2-STAT1 signaling pathway].

Based on Janus kinase 1/2-signal transducer and activator of transcription 1(JAK1/2-STAT1) signaling pathway, this study explored the immune mechanism of Maxing Shigan Decoction in alleviating the lung tissue and colon tissue damage in mice infected with influenza virus. The influenza virus infection was induced in mice by nasal drip of influenza virus. The normal group, model group, oseltamivir group, antiviral granule group, and Maxing Shigan Decoction group were designed. After intragastric administration of corresponding drugs or normal saline for 3 or 7 days, the body mass was measured, and lung index, spleen index, and thymus index were calculated. Based on hematoxylin-eosin(HE) staining, the pathological changes of lung tissue and colon tissue were observed. Enzyme-linked immunosorbent assay(ELISA) was used to detect serum levels of inflammatory factors interleukin-8(IL-8) and interferon-γ(IFN-γ), Western blot and real-time quantitative polymerase chain reaction(RT-qPCR) to determine the protein and mRNA levels of JAK1, JAK2, STAT1, interferon regulatory factor 9(IRF9), and IFN-γ in lung tissue and colon tissue. The results showed that after 3 and 7 days of administration, the body mass, spleen index, and thymus index were lower(P<0.05 or P<0.01), and the lung index was higher(P<0.01) in the model group than in the normal group. Moreover, the model group showed congestion, edema, and infiltration of a large number of lymphocytes and macrophages in the lung tissue, irregular structure of colon mucosa, ulceration and shedding of epithelial cells, and infiltration of a large number of inflammatory cells. The model group had higher levels of serum IFN-γ(P<0.01), higher protein and mRNA expression of JAK1, JAK2, STAT1, IRF9, IFN-γ in lung tissue(P<0.05 or P<0.01), higher level of JAK2 protein in colon tissue(P<0.01), and higher protein and mRNA levels of STAT1 and IRF9(P<0.05 or P<0.01) than the normal group. Compared with the model group, Maxing Shigan Decoction group had high body mass, spleen index, and thymus index(P<0.05 or P<0.01), low lung index(P<0.05 or P<0.01), and significant alleviation of pathological injury in lung and colon. Moreover, lower serum level of IFN-γ(P<0.05 or P<0.01), protein and mRNA levels of JAK1, JAK2, STAT1, IRF9, and IFN-γ in lung tissue(P<0.05 or P<0.01), JAK2 protein level in colon tissue(P<0.01), and protein and mRNA levels of STAT1 and IRF9(P<0.05 or P<0.01) were observed in the Maxing Shigan Decoction group than in the model group. After 3 days of administration, the level of serum IL-8 in the model group was significantly higher than that in the normal group(P<0.01), and the level in the Maxing Shigan Decoction group was significantly reduced(P<0.01). In conclusion, Maxing Shigan Decoction can significantly up-regulate body mass, spleen index, and thymus index, down-regulate lung index, reduce the levels of IL-8 and IFN-γ, and down-regulate protein and mRNA levels of JAK1, JAK2, STAT1, IRF9, and IFN-γ in lung tissue and protein and mRNA levels of JAK2, STAT1, and IRF9 in colon tissue, and alleviate pathological damage of lung tissue and colon tissue. The mechanism is the likelihood that it inhibits the activation of JAK1/2-STAT1 signaling pathway to alleviate the damage to lung and colon tissue damage.

Graphene-based fluorometric determination of agrD gene transcription in methicillin-resistant Staphylococcus aureus using exonuclease III-aided target recycling and DNA walker cascade amplification.

A graphene-based bioassay is described for the fluorometric determination of agrD gene transcription (mRNA) in methicillin-resistant Staphylococcus aureus (MRSA). This method includes exonuclease III (Exo III)-assisted target recycling and DNA walker cascade amplification. Hairpin1 (HP1) consists of a capture probe (CP) and DNA walker sequence. In the absence of the target, 5'-amino modified hairpin2 (HP2) labeled with carboxyfluorescein (FAM) at its 3' terminus is covalently linked to graphene via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) catalysis, resulting in the quenching of the FAM signal. The stem-loop structure of HP1 opens when the target is added to form partially complementary DNA/RNA hybrids. Exo III then initiates the target recycling process by cleaving the CP and DNA walker cascade reaction by automatic walking. This iterative reaction causes the FAM to dissociate from the graphene, and the fluorescence can be measured at excitation/emission wavelengths of 480/514 nm. Therefore, the target can be assayed by fluorescence. This method has a linear relationship with the concentration of target within the range 1 fM to 100 pM with a detection limit of 1 fM. The developed bioassay was used to monitor biofilm formation and investigate the mechanism of drug action with satisfactory results. Schematic representation of the graphene-based fluorescent bioassay for agrD gene transcription in methicillin-resistant Staphylococcus aureus by using exonuclease III-aided target recycling and DNA walker cascade amplification.

Fluorometric determination of agrA gene transcription in methicillin-resistant Staphylococcus aureus with a graphene oxide-based assay using strand-displacement polymerization recycling and hybridization chain reaction.

A graphene oxide (GO)-based fluorescent bioassay was developed to quantify agrA gene transcription (its mRNA) in methicillin-resistant Staphylococcus aureus (MRSA). This method is based on the use of Klenow fragment (KF)-assisted target recycling amplification and hybridization chain reaction (HCR). A triple complex was designed that contained a capture probe (CP), a trigger probe (TP), and a help probe (HP), which were partially complementary to one another. In the absence of the target, all the oligonucleotides labeled with carboxyfluorescein (FAM) are adsorbed onto the surface of GO by π-stacking interactions. This adsorption quenches the FAM signal. On the contrary, the target RNA causes the triple complex to disintegrate and initiates strand-displacement polymerization reaction (SDPR) and HCR in the presence of the appropriate raw materials, including the primer, KF, dNTPs, hairpin 1 (H1), and hairpin 2 (H2), generating double-stranded DNA (dsDNA) products. These dsDNA products are repelled by GO and produce strong fluorescence, measured at excitation/emission wavelengths of 480/514 nm. The fluorescent signal is greatly amplified by SYBR Green I (SGI) due to the synergistic effect of dsDNA-SGI. The target was assayed with this method at concentrations in the range 10 fM to 100 pM, and the detection limit (LOD) was 10 fM. This method also displayed good applicability in the analysis of real samples. It provides a new way of monitoring biofilm formation and studying the mechanisms of drug actions. Graphical abstract Schematic representation of the graphene oxide-based fluorescent bioassay for agrA gene transcription in methicillin-resistant Staphylococcus aureus by using strand-displacement polymerization recycling and hybridization chain reaction.

Fluorometric determination of mercury(II) via a graphene oxide-based assay using exonuclease III-assisted signal amplification and thymidine-Hg(II)-thymidine interaction.

A highly sensitive and selective fluorometric method is described for determination of mercury(II). It is based on (a) the use of graphene oxide (GO) acting as a quencher of the fluoresence of the carboxy-fluorescein (FAM), and (b) of Hg(II)-triggered cleavage of the newly formed nucleic acid sequences harbored blunt 3'-hydroxyl termini by exonuclease III (Exo III) that leads to signal amplification. Two DNA probes are used, viz. a capture probe (CP) and a help probe; HP) that is partially complementary. In the absence of Hg(II), the FAM-labeled hairpin (signal probe, SP) is adsorbed onto the surface of GO via π-stacking interactions. CP blocks the release of the HP for binding to SP. This results in quenching of the green fluorescence of the label. Upon addition of Hg(II), the linear structure of CP is converted to a hairpin structure due to the formation of thymidine-Hg(II)-thymidine duplexes. HP is released from the CP/HP hybrids, and this causes SP to be released from from GO and fluorescence to be recovered. The signal is strongly amplified by using Exo III-assisted targeting and recycling of HP. Hence, Hg(II) can be detected via the strong increase in fluorescence. The method has a linear response in the 0.1 to 30 nM Hg(II) concentration range and a 10 pM detection limit. It was applied to the determination of Hg(II) in three (spiked) Chinese medicines. Graphical abstract Schematic representation of fluorescence sensing strategy for Hg2+ by using graphene oxide as a quencher and exonuclease III-assisted signal amplification.

Curcumin Nicotinate Selectively Induces Cancer Cell Apoptosis and Cycle Arrest through a P53-Mediated Mechanism.

Curcumin is an anticancer agent, but adverse effects and low bioavailability are its main drawbacks, which drives efforts in chemical modifications of curcumin. This study evaluated antiproliferative activity and cancer cell selectivity of a curcumin derivative, curcumin nicotinate (CN), in which two niacin molecules were introduced. Our data showed that CN effectively inhibited proliferation and clonogenic growth of colon (HCT116), breast (MCF-7) and nasopharyngeal (CNE2, 5-8F and 6-10B) cancer cells with IC50 at 27.7 µM, 73.4 µM, 64.7 µM, 46.3 µM, and 31.2 µM, respectively. In cancer cells, CN induced apoptosis and cell cycle arrest at G2/M phase through a p53-mediated mechanism, where p53 was activated, p21 and pro-apoptotic proteins Bid and Bak were upregulated, and PARP was cleaved. In non-transformed human mammary epithelial cells MCF10A, CN at 50 µM had no cytotoxicity and p53 was not activated, but curcumin at 12.5 µM activated p53 and p21 and inhibited MCF10A cell growth. These data suggest that CN inhibits cell growth and proliferation through p53-mediated apoptosis and cell cycle arrest with cancer cell selectivity.

Graphene oxide-based fluorometric determination of methicillin-resistant Staphylococcus aureus by using target-triggered chain reaction and deoxyribonuclease-assisted recycling.

The authors describe a method for the fluorometric determination of methicillin-resistant Staphylococcus aureus (MRSA) by exploiting target-triggered chain reactions and deoxyribonuclease I (DNase I)-aided target recycling. It is making use of a carboxy-fluorescein (FAM)-labeled single-stranded probe containing two sections. One is complementary to the 5' terminus of the target, while the 3' terminus of the other target is adsorbed on the surface of graphene oxide (GO) via π-stacking interactions without the target (16S rRNA). This adsorption results in quenching of the fluorescence of the label and protects it from being cleaved by DNase I. However, upon addition of the target, DNA/RNA hybrids are repelled by GO. This leads to fluorescence recovery as measured at excitation/emission wavelengths of 480/514 nm due to a chain reaction that is triggered by the target. The signal is strongly amplified by using DNase I-mediated target recycling. The 16S rRNA of MRSA can be detected by this method in the 1 to 30 nM concentration range, and the detection limit is 0.02 nM. The method was applied to analyze bacterial samples, and the detection limit is as low as 30 CFU . mL-1. The assay is highly sensitive and selective and in our percpetion has a large potential in diagnosis of drug-resistant bacteria. Graphical abstract Schematic of the graphene oxide-based fluorescent bioassay for Methicillin-resistant Staphylococcus aureus detection by using target-triggered chain reaction and deoxyribonuclease I-aided signal amplification.

[Effects of Maxing Shigan Decoction Decocted by Different Methods and Its Drug Containing Serum on Neuraminidase Activity of Influenza A Virus].

Objective To observe the effects of Maxing Shigan Decoction (MSD) decocted by different methods and its drug containing serum on neuraminidase ( NA ) activity of influenza A virus (IAV). Methods The effects of MSD decocted by different methods, its corresponding drug containing serums , and drug containing serum in inhibiting the proliferation of virus on NA activity of IAV were detected using 2-(4-methyl umbelliferyl )-α-D-N-acetyl neuraminic acid (MUNANA) as substrate. Results (1) Effect of MSD on NA activity of IAV: OD value was less in groups with Ephedra decocted 25-min earlier and 30-min earlier than the group with four drugs decocted at the same time (P <0. 05, P <0. 01) ; (2) Effect of MSD on NA activity of IAV: OD value was less in groups with Ephedra decocted 30-min earlier and 40-min earlier than the group with four drugs decocted at the same time (P <0. 05, P <0. 01) ; (3) In the process of inhibiting viral multiplication, effect of MSD containing serum on NA activity of IAV: OD value was less in groups with Ephedra decocted 5 -20 min earlier, 30 min earlier, 35 min earlier than the group with four drugs decocted at the same time (P <0. 05, P <0. 01). In terms of drug concentration, OA value decreased more in 6. 25% and 12. 50% MSD containing serums than in 25. 00% MSD containing serum (P <0. 01). Conclusion MSD decocted by different methods might lead to different anti-IAV effects.

[Effect of Chinese Herbal Extract HNA-1 on the Thymic Output Function in Simian Immunodeficiency Virus Chronically Infected Chinese Rhesus Macaques].

OBJECTIVE: To observe the effect of Chinese herbal extract HuNan A-1 (HNA-1) on the thymic output function in Simian immunodeficiency virus (SIV) chronically infected rhesus macaques. METHODS: Eight Chinese rhesus macaques had been infected by SIVmac239 for 16 to 21 months, and then they were randomly divided into the treatment group and the control group, 4 in each group. Monkeys in the treatment group were administered with HNA-1 by gastrogavage, once daily for 2 successive months, while those in the control group were administered with equal volume of normal saline by gastrogavage, once daily for 2 successive months. The general condition and body weight of monkeys were observed. Plasma viral loads were detected using real-time fluorescent quantitative PCR assay. CD4 percentages and counts, as well as naive CD subsets were detected using flow cytometry. T-cell receptor excision circles (TREC) were detected using real-time fluorescent quantitative PCR assay. The thymus tissue was pathologically observed using routine HE staining. The correlation between lesions of the thymus tissue, CD4 counts, naive CD counts, and TREC were analyzed. RESULTS: There was no statistical difference in body weight, viral loads, absolute CD ratios between the two groups after treatment (P > 0.05). The altered TREC multiple showed an obvious decreasing tendency in the control group, while it showed an increasing tendency in the treatment group (P < 0.05). In both groups, destroyed structures of the thymus tissue could be seen, filled with pink unstructured material. Increased connective tissues, lowered connective cell density, and confused arrangement could also be seen in the two groups, with no obvious difference. TREC contents were positively correlated with naive CD4 counts after removing extremum (r = 0.926, P = 0.001). Naive CD4 counts were positively correlated with CD4 counts (r = 0.961, P = 0.005). CONCLUSIONS: TREC content determination, as a marker of newly thymic emigrants, could be taken as a testing method for evaluating the thymic output function. Besides, HNA-1 treatment increased the thymic output significantly in SIV chronically infected monkeys. Correlation existed among TREC contents, naive CD4 counts, and pathologies of thymus tissues, especially in late infection stage.

Fluorometric determination of mecA gene in MRSA with a graphene-oxide based bioassay using flap endonuclease 1-assisted target recycling and Klenow fragment-triggered signal amplification.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant bacterium that causes a wide range of illnesses, necessitating the development of new technologies for its detection. Herein, we propose a graphene oxide (GO)-based sensing platform for the detection of mecA gene in MRSA using flap endonuclease 1 (FEN1)-assisted target recycling and Klenow fragment (KF)-triggered signal amplification. Without the target, all the DNA probes were adsorbed onto GO, resulting in fluorescence quenching of the dye. Upon the addition of the target, a triple complex was formed that triggered FEN1-assisted target recycling and initiated two polymerization reactions with the assistance of KF polymerase, generating numerous dsDNA that were repelled by GO. These dsDNAs triggered fluorescence enhancement when SYBR Green I was added. Therefore, the target DNA was quantified by measuring the fluorescence at excitation and emission wavelengths of 480/526 nm. This mecA gene assay showed a good linear range from 1 to 50 nM with a lower limit of detection of 0.26 nM, and displayed good applicability to the analysis of real samples. Thus, a new method for monitoring MRSA has been developed that has great potential for early clinical diagnosis and treatment.

Exploring the immune-inflammatory mechanism of Maxing Shigan Decoction in treating influenza virus A-induced pneumonia based on an integrated strategy of single-cell transcriptomics and systems biology.

BACKGROUND: Influenza is an acute respiratory infection caused by influenza virus. Maxing Shigan Decoction (MXSGD) is a commonly used traditional Chinese medicine prescription for the prevention and treatment of influenza. However, its mechanism remains unclear. METHOD: The mice model of influenza A virus pneumonia was established by nasal inoculation. After 3 days of intervention, the lung index was calculated, and the pathological changes of lung tissue were detected by HE staining. Firstly, transcriptomics technology was used to analyze the differential genes and important pathways in mouse lung tissue regulated by MXSGD. Then, real-time fluorescent quantitative PCR (RT-PCR) was used to verify the changes in mRNA expression in lung tissues. Finally, intestinal microbiome and intestinal metabolomics were performed to explore the effect of MXSGD on gut microbiota. RESULTS: The lung inflammatory cell infiltration in the MXSGD group was significantly reduced (p < 0.05). The results of bioinformatics analysis for transcriptomics results show that these genes are mainly involved in inflammatory factors and inflammation-related signal pathways mediated inflammation biological modules, etc. Intestinal microbiome showed that the intestinal flora Actinobacteriota level and Desulfobacterota level increased in MXSGD group, while Planctomycetota in MXSGD group decreased. Metabolites were mainly involved in primary bile acid biosynthesis, thiamine metabolism, etc. This suggests that MXSGD has a microbial-gut-lung axis regulation effect on mice with influenza A virus pneumonia. CONCLUSION: MXSGD may play an anti-inflammatory and immunoregulatory role by regulating intestinal microbiome and intestinal metabolic small molecules, and ultimately play a role in the treatment of influenza A virus pneumonia.

Non-coding RNAs in breast cancer: with a focus on glucose metabolism reprogramming.

Breast cancer is the tumor with the highest incidence in women worldwide. According to research, the poor prognosis of breast cancer is closely related to abnormal glucose metabolism in tumor cells. Changes in glucose metabolism in tumor cells are an important feature. When sufficient oxygen is available, cancer cells tend to undergo glycolysis rather than oxidative phosphorylation, which promotes rapid proliferation and invasion of tumor cells. As research deepens, targeting the glucose metabolism pathway of tumor cells is seen as a promising treatment. Non-coding RNAs (ncRNAs), a recent focus of research, are involved in the regulation of enzymes of glucose metabolism and related cancer signaling pathways in breast cancer cells. This article reviews the regulatory effect and mechanism of ncRNAs on glucose metabolism in breast cancer cells and provides new ideas for the treatment of breast cancer.

Immune infiltration patterns and identification of new diagnostic biomarkers GDF10, NCKAP5, and RTKN2 in non-small cell lung cancer.

This study aimed to identify potential biomarkers for non-small cell lung cancer (NSCLC) and analyze the role of immune cell infiltration in NSCLC. R software was used to screen differentially expressed genes (DEGs) from NSCLC datasets obtained from the Gene Expression Omnibus (GEO) database, and functional correlation analysis was performed. The machine learning algorithms were used to screen the potential biomarkers of NSCLC. The diagnostic values were assessed through receiver operating characteristic (ROC) curves. The protein and mRNA expression levels of potential biomarkers were verified based on the Human Protein Atlas (HPA) database and qRT-PCR. CIBERSORT was used to evaluate the infiltration of immune cells in NSCLC tissues, and the correlation between potential biomarkers and infiltrated immune cell was analyzed. Finally, specific siRNAs were utilized to reduce the GDF10, NCKAP5, and RTKN2 expression in A549 and H1975 cells. The proliferation ability of A549 and H1975 cells was detected by MTT assay. A total of 848 upregulated DEGs and 1308 downregulated DEGs were identified. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the DEGs were mainly related to cell division. Disease ontology (DO) enrichment analysis showed that the diseases with these DEGs were mainly lung diseases, including NSCLC. In addition,three potential biomarkers were identified: GDF10, NCKAP5, and RTKN2. Immune cell infiltration analysis showed that resting NK cells, activated dendritic cells, and Tregs may be involved in the pathogenesis of NSCLC. Meanwhile, GDF10, NCKAP5, and RTKN2 were negatively correlated with Tregs and naïve B cells but were positively correlated with activated dendritic cells and resting NK cells. Immunohistochemical staining showed that the expression of GDF10, NCKAP5, and RTKN2 in the lung tissue of patients with NSCLC was lower than that of normal lung tissue. qRT-PCR also confirmed that the mRNA expression of three biomarkers in NSCLC cell lines A549 and H1975 were significantly lower than those in human normal lung epithelial cells BEAS-2B. An MTT assay showed that GDF10, NCKAP5, and RTKN2 knockdown significantly promoted the proliferation of A549 and H1975 cells. The in vitro experiments showed that GDF10, NCKAP5, and RTKN2 played the inhibitory effects on NSCLC cell lines proliferation. Hence, GDF10, NCKAP5, and RTKN2 can be used as diagnostic biomarkers for NSCLC.

A graphene-oxide-based fluorometric assay for norA gene transcription in MRSA using Nb.BbvCI-assisted target recycling and T7 exonuclease-triggered cascade dual recycling signal amplification.

We describe a graphene oxide (GO)-based bioassay for the fluorometric determination of norA gene transcription (mRNA) in methicillin-resistant Staphylococcus aureus (MRSA). This approach is based on Nb.BbvCI-assisted target recycling (NATR) and T7 exonuclease (T7 Exo)-triggered cascade dual-recycling signal amplification (TTCDRSA). The system included GO, a capture probe (CP), an assistant probe (AP), two carboxyfluorescein (FAM)-labeled hairpins (HP1 and HP2), endonuclease Nb.BbvcI, and exonuclease T7. In the presence of a target, AP, together with the target RNA, can hybridise with CP via partial complementarity to one another and open its hairpin structure to form a triple complex that is recognised by Nb.BbvCI. Once the CP is cleaved, the released AP and target RNA can walk on the carboxylated graphene oxide (CGO) surface to bind with another CP which induces the next round of cleavage, accumulating many trigger probes (TPs). The TPs then activate TTCDRSA with the assistance of T7 Exo, HP1, and HP2 to produce large amounts of free FAMs. These free FAMs are repelled by GO and exhibit enhanced fluorescence signals at excitation/emission wavelengths of 480/514 nm. The limit of detection (LOD) of the bioassay was calculated to be 0.37 fM, and the linear range of the method ranged from 1 fM to 1 nM. More importantly, the bioassay also exhibited high sensitivity and selectivity for target RNA detection in real samples, which may open a new promising avenue for monitoring drug efflux and studying the mechanisms of drug actions.

Research on Pachymaran to Ameliorate CsA-Induced Immunosuppressive Lung Injury by Regulating Microflora Metabolism.

Pachymaran (PCP), the major medicinal constituent of Poria cocos, has a regulatory effect on immunosuppressive lung injury, but its mechanism of action with respect to gut microorganisms and their metabolites is not clear. The aim of this study was to investigate the protective effect of PCP against immunosuppressive lung injury caused by cyclosporine A (CsA), and to reveal its possible mechanism of action via the comprehensive analysis of 16S rRNA and LC-MS. We demonstrated that PCP was effective at alleviating CsA-induced immunosuppressive lung injury by restoring the organ indices and lung tissue morphology and structure. PCP significantly altered the composition of the gut and lung microbiota in mice with CsA-induced immunosuppressive lung injury by increasing the number of beneficial bacteria from the Eubacterium nodatum group, Eubacterium ventriosum group, Akkermansia, and Ruminococcus, and reducing the pathogenic Rikenellaceae RC9 gut group to fulfill its immunomodulatory role. In lung tissue microecology, PCP intervention significantly reduced the abundance of Chryseobacterium, Lawsonella, Paracoccus, and Sediminibacterium and increased the abundance of Alloprevotella. The LC-MS results showed that PCP alleviated the CsA-induced immunosuppression of lung tissue injury. The model serum metabolite Americine decreased the expression of PC(O-18:1(4Z)/0:0). Our results suggest that PCP may be involved in regulating the composition, function, and metabolism of the gut and lung microbiota to reverse CsA-induced immunosuppressive lung injury.

Network pharmacology and experimental validation of Maxing Shigan decoction in the treatment of influenza virus-induced ferroptosis.

Influenza is an acute viral respiratory infection that has caused high morbidity and mortality worldwide. Influenza A virus (IAV) has been found to activate multiple programmed cell death pathways, including ferroptosis. Ferroptosis is a novel form of programmed cell death in which the accumulation of intracellular iron promotes lipid peroxidation, leading to cell death. However, little is known about how influenza viruses induce ferroptosis in the host cells. In this study, based on network pharmacology, we predicted the mechanism of action of Maxing Shigan decoction (MXSGD) in IAV-induced ferroptosis, and found that this process was related to biological processes, cellular components, molecular function and multiple signaling pathways, where the hypoxia inducible factor-1(HIF-1) signaling pathway plays a significant role. Subsequently, we constructed the mouse lung epithelial (MLE-12) cell model by IAV-infected in vitro cell experiments, and revealed that IAV infection induced cellular ferroptosis that was characterized by mitochondrial damage, increased reactive oxygen species (ROS) release, increased total iron and iron ion contents, decreased expression of ferroptosis marker gene recombinant glutathione peroxidase 4 (GPX4), increased expression of acyl-CoA synthetase long chain family member 4 (ACSL4), and enhanced activation of hypoxia inducible factor-1α (HIF-1α), induced nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF) in the HIF-1 signaling pathway. Treatment with MXSGD effectively reduced intracellular viral load, while reducing ROS, total iron and ferrous ion contents, repairing mitochondrial results and inhibiting the expression of cellular ferroptosis and the HIF-1 signaling pathway. Finally, based on animal experiments, it was found that MXSGD effectively alleviated pulmonary congestion, edema and inflammation in IAV-infected mice, and inhibited the expression of ferroptosis-related protein and the HIF-1 signaling pathway in lung tissues.

[Effects of zhibal dihuang decoction on the pathological changes and the ultrastructure of the testicular tissue in the ureaplasma urealyticum-infected rats].

OBJECTIVE: To study the effects of Zhibai Dihuang Decocion (ZDD) on the pathological changes and the ultrastructure of the testicular tissue in the ureaplasma urealyticum (UU)-infected rats. METHODS: The UU infected animal models were established by the bladder inoculation. The 45 UU infected SD rats were randomly divided into four groups, i.e., the ZDD treatment group (at the daily dose of 2 g/100 g), the Minocycline group (at the daily dose of 10 mg/100 g), the model group, 15 in each group. Besides, another 15 rats were recruited as the sham-operation group. The medication was started 10 days after vaccination. Equal volume of normal saline was given to rats in the model group and the sham-operation group by gastrogavage for 22 successive days. Rats were sacrificed on the 2nd day of medication discontinuation. The testicle mass index was detected. The ultra-structure and the pathological changes of the testicular tissue were observed by optical microscope and transmission electron microscope. RESULTS: There was no significant difference in the rat testicular mass index (P>0.05). UU infection can lead to the pathological changes such as atrophy of seminiferous tubules, germ cell loss, and reduction of sperm cells in lumen, and to the ultrastructural changes such as spermatogenic cell nuclear membrane shrinkage, nuclear breakdown, and obvious edema of mitochondria. The pathological changes and the ultrastructures were improved in the medication groups. Rm and Rs the were not overlapping, and the difference was statistically significant (P<0.05). Rm, Rzh, and Rx were not overlapping, and the difference was statistically significant (P<0.05). Rzh and Rx were overlapping in 95% Cl with no statistical difference (P>0.05). CONCLUSIONS: UU infection can cause the pathological changes and the ultrastructural changes of the testicular tissue at the organic level and the cellular level. ZDD played therapeutic effects through ameliorating its pathological changes and the ultrastructural changes of spermatogenic cells.

Targeted inhibition of methicillin-resistant Staphylococcus aureus biofilm formation by a graphene oxide-loaded aptamer/berberine bifunctional complex.

Biofilm formation is known to promote drug resistance in methicillin-resistant Staphylococcus aureus (MRSA), which is closely related to persistent infections in hospital settings. In this study, a DNA aptamer specific to penicillin-binding protein 2a (PBP2a) with a dissociation constant (Kd) of 82.97 ± 8.86 nM was obtained after 14 cycles of systematic evolution of ligands by exponential enrichment (SELEX). Next, a bifunctional complex containing the aptamer intercalated by berberine into the double-strand region was prepared and adsorbed onto the surface of graphene oxide (GO) by π-stacking interactions. The GO-loaded aptamer/berberine bifunctional complex showed significantly higher inhibition of MRSA biofilm formation than the control. Furthermore, this study shows that the complex possesses anti-biofilm activity, which can be attributed to the ability of the aptamer to reduce cell-surface attachment by blocking the function of PBP2a and berberine to attenuate the level of the accessory gene regulator (agr) system, which plays an important role in mediating MRSA biofilm formation. Therefore, the simultaneous delivery of berberine and PBP2a-targted aptamer using GO may have potential for the treatment of chronic infections caused by MRSA biofilms. It also provides a new avenue for multitarget treatment of bacterial biofilms.