Streptococcus suis Serotype 2 Type IV Secretion Effector SspA-1 Induces Proinflammatory Cytokine Production via TLR2 Endosomal and Type I Interferon Signaling.

The subtilisin-like protease-1 (SspA-1) plays an important role in the pathogenesis of a highly virulent strain of Streptococcus suis 2. However, the mechanism of SspA-1-triggered excessive inflammatory response is still unknown. In this study, we demonstrated that activation of type I IFN signaling is required for SspA-1-induced excessive proinflammatory cytokine production. Further experiments showed that the TLR2 endosomal pathway mediates SspA-1-induced type I IFN signaling and the inflammatory response. Finally, we mapped the major signaling components of the related pathway and found that the TIR adaptor proteins Mal, TRAM, and MyD88 and the downstream activation of IRF1 and IRF7 were involved in this pathway. These results explain the molecular mechanism by which SspA-1 triggers an excessive inflammatory response and reveal a novel effect of type I IFN in S. suis 2 infection, possibly providing further insights into the pathogenesis of this highly virulent S. suis 2 strain.

Inhibition of Walker-256 Tumor Growth by Combining Microbubble-Enhanced Ultrasound and Endostar.

OBJECTIVES: This research is to investigate the anti-tumor effects by combining anti-vascular effect of microbubble enhanced ultrasound (MEUS) mechanical destruction and anti-angiogenic effect of Endostar. METHODS: Rats bearing Walker-256 tumor were randomly divided into 4 groups treated by Endostar + MEUS combination, Endostar, MEUS or Sham ultrasound (US), respectively. MEUS was induced by Sonazoid microbubble and a focused therapeutic US device. Contrast-enhanced ultrasound (CEUS) was used to assess tumor perfusion before and after treatment. Microvessel density (MVD) was evaluated with immunohistochemical staining of CD31, CD34, and VEGFA. TUNEL assay was used to determine the apoptosis rate of tumor cells. RESULTS: Endostar + MEUS combined group induced the most reduced blood perfusion and most retarded tumor growth compared with other 3 groups. Decreased MVD was shown in Endostar + MEUS, Endostar and MEUS group, but the lowest MVD value was presented in the combined treatment group. Significant increase was observed in the combined therapy group and MEUS group. CONCLUSIONS: This study showed an improved anti-vascular and anti-angiogenic effect achieved by combining Endostar and MEUS, and may provide a new method potential for anti-tumor therapy.

Ultrasensitive and High Reproducible Detection of Urinary Metabolites Using the Tip-Contact Extraction Method Coupled with Negative LDI-MS.

More and more evidence has proved that urinary metabolites can instantly reflect disease state. Therefore, ultra-sensitive and reproducible detection of urinary metabolites in a high-throughput way is urgently desirable for clinical diagnosis. Matrix-free laser desorption/ionization mass spectrometry (LDI-MS) is a high-throughput platform for metabolites detection, but it is encountered by severe interference from numerous salts in urine samples, because the crystallized urine salt on dried samples could result in poor reproducibility in LDI-MS detection. The present work proposed a tip-contact extraction (TCE) technique to eliminate interference from the urine salt. Vertical silicon nanowire arrays decorated with the fluorinated ethylene propylene film (FEP@VSiNWs) could effectively extract metabolites from the urine sample dropping on its surface. High salt tolerance was observed in the subsequent LDI-MS detection of the metabolites extracted on the tip of FEP@VSiNWs even in the presence of 1 M urea. Stable and reproducible mass spectra for non-target metabolic analysis were obtained in real urine samples with different dilution folds. Urinary metabolites collected from bladder cancer (BC) patients were reliably profiled by the TCE method coupled with negative LDI-MS. Based on this platform, potential metabolic biomarkers that can distinguish BC patients and normal controls were uncovered.

Single-Cell Sequencing-Enabled Hexokinase 2 Assay for Noninvasive Bladder Cancer Diagnosis and Screening by Detecting Rare Malignant Cells in Urine.

Bladder cancer (BC) is among the most common tumors with a high recurrence rate, necessitating noninvasive and sensitive diagnostic methods. Accurate detection of exfoliated tumor cells (ETCs) in urine is crucial for noninvasive BC diagnosis but suffers from limited sensitivity when ETCs are rare and confounded by reactive, regenerative, or reparative cells. Single-cell sequencing (SCS) enables accurate detection of ETCs by surveying oncogenic driver mutations or genome-wide copy number alternations. To overcome the low-throughput limitation of SCS, we report a SCS-validated cellular marker, hexokinase 2 (HK2), for high-throughput screening cells in urine and detecting ETCs engaging elevated glycolysis. In the SCS-based training set, a total of 385 cells from urine samples of eight urothelial carcinoma (UC) patients were sequenced to establish a HK2 threshold that achieved >90% specificity for ETC detection. This urine-based HK2 assay was tested with a blinded patient group (n = 384) including UC and benign genitourinary disorders as a validation cohort for prospectively evaluating diagnostic accuracy. The sensitivity, specificity, positive predictive value, and negative predictive value of the assay were 90, 88, 83, and 93%, respectively, which were superior to urinary cytology. For investigating the potential to be a screening test, the HK2 assay was tested with a group of healthy individuals (n = 846) and a 6-month follow-up. The specificity was 98.4% in this health group. Three participants were found to have >5 putative ETCs that were sequenced to exhibit recurrent copy number alternations characteristic of malignant cells, demonstrating early BC detection before current clinical methods.

Impact of focused ultrasound on the ethanol ablation of VX2 liver tumours in rabbits.

OBJECTIVES: In this study, a treatment combining ethanol ablation (EA) and focused ultrasound (FUS) was performed to investigate its synergistic ablation effect on normal liver and VX2 liver tumours in rabbits. METHODS: A total of 59 healthy New Zealand white rabbits were included. For normal liver ablation, 39 animals were treated with FUS alone (n = 12), EA alone (n = 12), EA+FUS combination treatment (n = 12), or the control treatment (n = 3). The other 20 rabbits with implanted VX2 liver tumours were treated with EA alone (n = 10) or EA+FUS (n = 10). For FUS, the liver was exposed to 1 MHz FUS with an intensity of 33.0 W/cm2 (ISPTA) for 20 s. The EA group received an injection of absolute ethanol in the liver or liver tumours. For EA+FUS combination therapy, FUS was focused at the EA injection site, and both methods were carried out at the same time. RESULTS: In normal liver tissues, the ablated volume treated by FUS combined with EA (1.46 ± 0.30 cm3) was approximately 3 times larger than that of EA alone (0.51 ± 0.17 cm3); in VX2 liver tumours, the tumour necrosis rate of the combination therapy was 90.27%, which was much higher than that of EA treatment (63.55%). CONCLUSION: The combination of EA and FUS could effectively increase the liver ablation volume and induce more complete tumour necrosis. KEY POINTS: • This study demonstrated a novel method for enhancing ethanol ablation and elucidated its potential to enhance percutaneous ethanol ablation (PEA) in a simple non-invasive way. • Ethanol excited by focused ultrasound (FUS) exposure tended to accumulate at the injection site, which could prevent ethanol from being washed out by the bloodstream. • The combination of EA and FUS could effectively increase the liver ablation volume and induce more complete tumour necrosis.

Laparoscopic nephrectomy outside gerota fascia and En bloc ligation of the renal hilum for management of inflammatory renal diseases.

OBJECTIVES: This study aims to improve laparoscopic nephrectomy techniques for inflammatory renal diseases (IRD) and to reduce complications. MATERIALS AND METHODS: Thirty-three patients underwent laparoscopic nephrectomy for IRD, with a method of outside Gerota fascia dissection and en-bloc ligation and division of the renal pedicle. Operative time, blood loss, complications, analgesia requirement, post-operative recovery of intestinal function and hospital stay were recorded. The degrees of perinephric adhesion were classified based on the observation during operation and post-operative dissection of the specimen, and the association of different types of adhesion with the difficulty of the procedures was examined. RESULTS: Among 33 cases, three were converted to hand-assisted laparoscopy, and one was converted to open surgery. Mean operative time was 99.6±29.2min, and blood loss was 75.2±83.5 mL. Postoperative recovery time of intestinal function was 1.6±0.7 days and average hospital stay was 4.8±1.4 days. By classification and comparison of the perinephric adhesions, whether inflammation extending beyond Gerota fascia or involving renal hilum was found to be not only an important factor influencing the operative time and blood loss, but also the main reason for conversion to hand-assisted laparoscopy or open surgery. CONCLUSIONS: In laparoscopic nephrectomy, outside Gerota fascia dissection of the kidney and en-bloc ligation of the renal pedicle using EndoGIA could reduce the difficulty of procedure and operative time, with satisfactory safety and reliability. Inflammation and adhesion extending beyond Gerota fascia or involving renal hilum is an important predictor of the difficulty related to laparoscopic nephrectomy for IRD.

Development and evaluation of an efficient heterologous gene knock-in reporter system in Lactococcus lactis.

BACKGROUND: Lactococcus lactis is a food grade probiotics and widely used to express heterologous proteins. Generally, target genes are knocked into the L. lactis genome through double-crossover recombination to express heterologous proteins stably. However, creating marker-less heterologous genes knocked-in clones is laborious. In this study, an efficient heterologous gene knock-in reporter system was developed in L. lactis NZ9000. RESULTS: Our knock-in reporter system consists of a temperature-sensitive plasmid pJW and a recombinant L. lactis strain named NZB. The pJW contains homologous arms, and was constructed to knock-in heterologous genes at a fixed locus of NZ9000 genome. lacZ (ß-galactosidase) gene was knocked into the chromosome of NZ9000 as a counter-selective marker through the plasmid pJW to generate NZB. The engineered NZB strain formed blue colonies on X-Gal plate. The desired double-crossover mutants formed white colonies distinctive from the predominantly blue colonies (parental and plasmid-integrated clones) when the embedded lacZ was replaced with the target heterologous genes carried by pJW in NZB. CONCLUSIONS: By using the system, the heterologous gene knocked-in clones are screened by colony phenotype change rather than by checking colonies individually. Our new knock-in reporter system provides an efficient method to create heterologous genes knocked-in clones.

The Dome Wall of Bladder Acts as a Pacemaker Site in Detrusor Instability in Rats.

BACKGROUND The aim of this study was to confirm that the interstitial cells of Cajal (ICCs) in the dome wall of the bladder are pacemaker cells, and that the dome wall of the bladder acts as a pacemaker site in the detrusor instability (DI) rat model. MATERIAL AND METHODS The model of DI in Wistar rats was established and urodynamic studies measuring the bladder volume and pressure were performed. The detrusor excitability was investigated using the amplitude and frequency of phasic contraction of strips. The localization and quantity of ICCs was identified by immunohistochemistry and c-KIT protein expression in the rat bladder. PCR assay and Western blot were used to assess the expression of HCN2 and Cx43. RESULTS The bladder capacity, residual volume, voiding volume, and maximum voiding pressure were significantly increased in the DI group. The contraction frequency and amplitude of the strips from the dome of the bladder in the DI group were higher than the triangle, body, and base parts. Both the concentration of c-KIT positive ICCs cells and expression of the c-KIT protein in the dome wall were higher than in other parts of the bladder. The expression of HCN2 and Cx43 in each part of the DI rat group were obviously higher than each part in the control group. Compared to the body, base, and triangle parts, the expression of HCN2 and Cx43 in the dome wall were obviously higher in the DI group. CONCLUSIONS The quantity of ICCs was higher in the dome wall and the dome wall of bladder acts as a pacemaker site in the DI rat model.

A novel rabbit anti-hepatocyte growth factor monoclonal neutralizing antibody inhibits tumor growth in prostate cancer cells and mouse xenografts.

The hepatocyte growth factor and its receptor c-Met are correlated with castration-resistance in prostate cancer. Although HGF has been considered as an attractive target for therapeutic antibodies, the lack of cross-reactivity of monoclonal antibodies with human/mouse HGFs is a major obstacle in preclinical developments. We generated a panel of anti-HGF RabMAbs either blocking HGF/c-Met interaction or inhibiting c-Met phosphorylation. We selected one RabMAb with mouse cross-reactivity and demonstrated that it blocked HGF-stimulated downstream activation in PC-3 and DU145 cells. Anti-HGF RabMAb inhibited not only the growth of PC-3 cells but also HGF-dependent proliferation in HUVECs. We further demonstrated the efficacy and potency of the anti-HGF RabMAb in tumor xenograft mice models. Through these in vitro and in vivo experiments, we explored a novel therapeutic antibody for advanced prostate cancer.

Efficacy and influential factors of hematoporphyrin monomethyl ether mediated photodynamic therapy in the treatment for port-wine stains.

BACKGROUNDS: Hematoporphyrin monomethyl ether mediated photodynamic therapy (HMME-PDT) has emerged as an alternative approach for port-wine stain (PWS), which was primarily treated with pulsed dye laser (PDL). This study was aimed to evaluate the efficacy and safety of HMME-PDT for PWS and to explore influential factors on the efficacy. METHODS: A total of 254 patients were enrolled. Patients received an intravenous injection of HMME at 5 mg/kg. Lesion areas were irradiated with 532-nm light for 20-25 min. Efficacy was assessed according to fading of lesions and graded as excellent (≥90 %), good (60 %-89 %), fair (20 %-59 %), or poor (<20 %). Adverse events were recorded. Clinical data were analyzed including gender, age, lesion sub-type, lesion location and number of treatments. RESULTS: Overall, 72.4 % of patients achieved an effective response, with 27.6% showing excellent efficacy, 24.8 % showing good efficacy and 20.1 % showing fair efficacy. Only 27.6 % showed poor efficacy. Patients under the age of 18 obtained a better efficacy than adults. Lesions in face showed a better therapeutic outcome than those in neck or trunk and extremities. A more effective response was seen in pink type compared with nodular thickening type. Multiple HMME-PDT treatments could improve the clinical response. Lesion location, lesion sub-type, number of treatments were independent influential factors on efficacy. Adverse events included edema, blister, crust, hypopigmentation, hyperpigmentation, pain, itch and burning sensation. No severe systemic side events were observed. CONCLUSIONS: HMME-PDT was effective for treating PWS and was safe and well-tolerated by patients. It is worth further investigation in efficacy and safety involving more patients from medical institutions in different regions in China. The optimal treatment parameters and treatment protocols are still being explored in the clinical treatment for PWS.

Development of humanized rabbit monoclonal antibodies against vascular endothelial growth factor receptor 2 with potential antitumor effects.

Vascular endothelial growth factor-A (VEGF-A) plays a critical role in physiologic and pathologic angiogenesis through its receptors especially through VEGFR2. The lack of cross-reactivity of monoclonal antibodies with human VEGFR2/mouse Flk-1 is a major obstacle in preclinical developments. In this study, using a unique hybridoma technique, we generated a panel of 30 neutralization anti-VEGFR2 rabbit monoclonal antibodies (RabMAbs) either blocking VEGF/VEGFR2 interaction or inhibiting VEGF-stimulated VEGFR2 tyrosine kinase phosphorylation. Among 18 RabMAbs with human/mouse VEGFR2 cross-reactivity, we humanized one lead candidate RabMAb by Mutational Lineage Guided (MLG) method and further demonstrated its potent inhibition of tumor growth in xenograft mouse model. Our study suggests that RabMAbs are highly relevant for therapeutic applications.

Comprehensive optimization of urinary exfoliated tumor cells tests in bladder cancer with a promising microfluidic platform.

BACKGROUND: Enrichment of urinary exfoliated tumor cells (UETCs) is a noninvasive way of bladder cancer diagnosis, but the lack of specific capture and identification of tumor cells from the urine remains a limitation that impedes the development of liquid biopsy. METHODS: The CytoBot® 2000, a novel circulating cell isolation and enrichment platform, was used for UETCs isolation after comprehensive optimization. The commercial cell lines of bladder cancer were used in spiking assay for cell recovery test. The flow cytometry and immunofluorescent staining assays were performed for expression validation of capture target and identification markers. The performance of optimized platform was validated by 159 clinical samples and analyzed using receiver operator characteristic curve. RESULTS: The chip that had a pore diameter of 15*20 µm could reduce the background residues while maintaining a higher cell recovery rate. We found that the cell capture ability of chip significantly improved after anti-EpCam antibody encapsulation, but not with T4L6FM1. In identification system optimization, the spiking assay and validation of clinical sample showed that the performance of CK20 and DBC-1 were better that pan-CK in tumor cell identification, in addition, the staining quality is more legible with CK20. CONCLUSION: The optimized capture chip is more specific for UETCs isolation. CK20 and DBC-1 are both sensitive biomarkers of UETCs in bladder cancer diagnosis. The performance of this optimized platform is excellent in clinical test that improves the accuracy of urine cell testing and provides a new alternative for the clinical application of BLCA liquid biopsy assessment.

Progress in overactive bladder: novel avenues from psychology to clinical opinions.

Rationale: Overactive bladder (OAB) is a common, distressing condition that worsens with age and impacts quality of life significantly. As a results of its clinical symptoms, patients suffer from serious physical and mental health issues, have a poor quality of life, and participate in a serious economic burden. The key social-psychological factors include living habits, eating habits, and personality characteristics on this disease, even though the pathogenesis of OAB is complex. However, there is few cognitions and research on OAB in the field of psychology. Methods/Search Strategy: Between 2000 and 2022, two electronic databases were systematically searched in accordance with Cochrane library guidelines (PubMed/Medline, Web of Science). An analysis of the remaining articles with relevant information was conducted using a data extraction sheet. An itemized flow diagram was adopted and used to report systematic reviews and meta-analysis. A systematic review of studies published from 2000 to 2022 in English language were conducted and included in the review. The intended audience: Urological surgeon and psychologists majoring in urinary diseases. Implication: As a result of this information, we are able to develop a better understanding of the role of psychological factors in the development of OAB and suggest potential therapeutic directions for OAB patients. This may benefit the recovery of OAB patients.

Multifunctional Evaluation Technology for Diagnosing Malfunctions of Regional Pelvic Floor Muscles Based on Stretchable Electrode Array Probe.

The pelvic floor dysfunction (PFD) has become a serious public health problem. Accurate diagnosis of regional pelvic floor muscle (PFM) malfunctions is vitally important for the prevention and treatment of PFD. However, there is a lack of reliable diagnostic devices to evaluate and diagnose regional PFM abnormality. In this work, we developed a multifunctional evaluation technology (MET) based on a novel airbag-type stretchable electrode array probe (ASEA) for the diagnosis of malfunctions of regional PFM. The inflatable ASEA has specifically distributed 32 electrodes along the muscles, and is able to adapt to different human bodies for tight contact with the muscles. These allow synchronous collection of high-quality multi-channel surface electromyography (MC-sEMG) signals, and then are used to diagnose regional PFM malfunctions and evaluate inter-regional correlation. Clinical trial was conducted on 15 postpartum stress urinary incontinence (PSUI) patients and 15 matched asymptomatic women. Results showed that SUI patients responded slowly to the command and have symptoms of muscle strength degeneration. The results were consistent with the relevant clinical manifestations, and proved the reliability of MET for multifunctional PFM evaluation. Furthermore, the MET can diagnose malfunctions of regional PFM, which is inaccessible with existing technology. The results also showed that the dysfunction of PSUI patients is mainly located in iliococcygeus, pubococcygeus, and urethral sphincter regions, and there is a weak correlation between these specific regions and nearby regions. In conclusion, MET provides a point-of-care diagnostic method for abnormal function of regional PFM, which has a potential for the targeted point-to-point electrical stimulation treatment and PFD pathology research.

A photoactivatable and phenylboronic acid-functionalized nanoassembly for combating multidrug-resistant gram-negative bacteria and their biofilms.

Background: Multidrug-resistant (MDR) gram-negative bacteria-related infectious diseases have caused an increase in the public health burden and mortality. Moreover, the formation of biofilms makes these bacteria difficult to control. Therefore, developing novel interventions to combat MDR gram-negative bacteria and their biofilms-related infections are urgently needed. The purpose of this study was to develop a multifunctional nanoassembly (IRNB) based on IR-780 and N, N'-di-sec-butyl-N, N'- dinitroso-1,4-phenylenediamine (BNN6) for synergistic effect on the infected wounds and subcutaneous abscesses caused by gram-negative bacteria. Methods: The characterization and bacteria-targeting ability of IRNB were investigated. The bactericidal efficacy of IRNB against gram-negative bacteria and their biofilms was demonstrated by crystal violet staining assay, plate counting method and live/dead staining in vitro. The antibacterial efficiency of IRNB was examined on a subcutaneous abscess and cutaneous infected wound model in vivo. A cell counting kit-8 assay, Calcein/PI cytotoxicity assay, hemolysis assay and intravenous injection assay were performed to detect the biocompatibility of IRNB in vitro and in vivo. Results: Herein, we successfully developed a multifunctional nanoassembly IRNB based on IR-780 and BNN6 for synergistic photothermal therapy (PTT), photodynamic therapy (PDT) and nitric oxide (NO) effect triggered by an 808 nm laser. This nanoassembly could accumulate specifically at the infected sites of MDR gram-negative bacteria and their biofilms via the covalent coupling effect. Upon irradiation with an 808 nm laser, IRNB was activated and produced both reactive oxygen species (ROS) and hyperthermia. The local hyperthermia could induce NO generation, which further reacted with ROS to generate ONOO-, leading to the enhancement of bactericidal efficacy. Furthermore, NO and ONOO- could disrupt the cell membrane, which converts bacteria to an extremely susceptible state and further enhances the photothermal effect. In this study, IRNB showed a superior photothermal-photodynamic-chemo (NO) synergistic therapeutic effect on the infected wounds and subcutaneous abscesses caused by gram-negative bacteria. This resulted in effective control of associated infections, relief of inflammation, promotion of re-epithelization and collagen deposition, and regulation of angiogenesis during wound healing. Moreover, IRNB exhibited excellent biocompatibility, both in vitro and in vivo. Conclusions: The present research suggests that IRNB can be considered a promising alternative for treating infections caused by MDR gram-negative bacteria and their biofilms.

5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) for acneform rash induced by erlotinib: A case report.

Erlotinib is an anticancer drug approved for the treatment of non-small cell lung cancer. It inhibits growth and proliferation of tumor cells by targeting epidermal growth factor receptor (EGFR). Dermatological toxicities are common side effects associated with EGFR inhibition. Here we describe a patient with acneform rash following oral medication of erlotinib, presented as facial erythema, papules and pustules. Two sessions of 5-aminolevulinic acid induced photodynamic therapy (ALA-PDT) with a 2-week interval were performed. No significant side effects or scarring were observed. The patient showed no recurrence within 6 months. Thus, we conclude that ALA-PDT is an effective treatment for skin lesions induced by erlotinib, especially for patients with need to sustain medication.

Observation on the Efficacy of Moxibustion Combined with Ear Acupoint Pressing Beans in Treating Patients with Phlegm Stasis Syndrome Vertigo.

Objective: The aim of this study is to investigate the efficacy of moxibustion combined with an ear acupoint pressing bean in the treatment of patients with phlegm stasis syndrome vertigo. Methods: 60 patients with vertigo identified as phlegm stasis syndrome who were hospitalized in our department from May 2020 to May 2021 were selected and divided into a control group and a treatment group of 30 cases each according to the random number method. The control group was treated with conventional treatment and care, and the treatment group was treated with moxibustion combined with ear acupressing beans on top of the conventional group. The treatment effects, the dizziness disorder inventory (DHI), Pittsburgh sleep quality index (PSQI), Hamilton anxiety score (HAMA), TCM symptom score, and blood flow parameters (left vertebral artery flow velocity (LVA), right vertebral artery flow velocity (RVA), and basilar artery flow velocity (BA)) of the two groups were compared with each other during and after the treatment. Results: After implementation, the treatment efficiency of the treatment group was higher than that of the control group, and the treatment group had lower PSQI, HAMA, and DHI scores as well as TCM symptom scores such as vertigo, head heavy as a wrap, chest tightness, and nausea and vomiting than the control group (P < 0.05). In addition, LVA, RVA, and BA were all higher in the treatment group than in the control group after treatment (P < 0.05). Conclusion: Moxibustion combined with ear acupoint pressing bean treatment can clearly improve patients' sleep quality, psychological state, relieve patients' various symptoms caused by vertigo, improve blood flow parameters, and have better efficacy in the treatment of phlegm stasis syndrome vertigo.

Inhibition of efflux pump encoding genes and biofilm formation by sub-lethal photodynamic therapy in methicillin susceptible and resistant Staphylococcus aureus.

BACKGROUND: Photodynamic therapy (PDT) is an effective method to inactivate microorganisms which is based on reactive oxygen species (ROS) generated by photosensitizer and light at certain wavelength. Exposure to sub-lethal dose of PDT (sPDT) could activate the regulatory systems in the surviving bacteria in response to oxidative stress. This study aimed to evaluate the effect of sPDT on efflux pump and biofilm formation in Staphylococcus aureus (S. aureus), which are two important virulence related factors. METHODS: Different light irradiation time and toluidine blue O (TBO) concentrations were tested to select a sPDT in methicillin-susceptible and methicillin-resistant S. aureus (MSSA and MRSA). Efflux function was evaluated with EtBr efflux experiment. Biofilm formation was evaluated by crystal violet staining. Gene expressions of norA, norB, sepA, mepA and mdeA following sPDT were analyzed with real-time PCR. RESULTS: Sub-lethal PDT was set at 40 J/cm2 associated with 0.5 µM TBO. Efflux function was significantly inhibited in both strains. The average expression levels of mdeA and mepA in MSSA and MRSA were increased by (3.09, 1.77, 1.57) and (3,44, 1.59, 6.29) fold change respectively, norB and sepA were decreased by (3.77, 6.14) and (3.02, 3.47) fold change respectively. Expression level of norA was decreased by 5.44-fold change in MSSA but increased by 2.80-fold change in MRSA. Biofilm formation in both strains was impeded. CONCLUSIONS: TBO-mediated sPDT could inhibit efflux pump function, alter efflux pump encoding gene expression levels and retard biofilm formation in MSSA and MRSA. Therefore, sPDT is proposed as a potential adjuvant therapy for infections.

Toluidine blue O-induced photoinactivation inhibit the biofilm formation of methicillin-resistant Staphylococcus aureus.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to conventional antimicrobial therapies, allowing for high morbidity and mortality. Photodynamic antimicrobial chemotherapy (PACT) is one method that combines visible harmless light with the optimum wavelength with photosensitizers or dyes, producing singlet oxygen (1O2) and reactive oxygen strains (ROS), making permanent damages to the target cells. The purpose of this research is to evaluate the suppression efficacy of toluidine blue O (TBO)-mediated PACT on mature MRSA biofilm in vitro. METHODS: In this study, the 48 h mature biofilm of the multidrug-resistant Staphylococcus aureus strain MRSA252 was used. The photodynamic therapy (PDT) group was treated with different concentrations of TBO (0.5, 0.75, 1.0 or 1.25 µM) and different doses of red light (635 ± 5 nm wavelength; 30 or 50 J/cm2). The biofilms viability after PDT were evaluated by crystal violet (CV) staining assay and {2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetra-zolium hydroxide} (XTT) assay; meanwhile, the morphological changes were detected by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), separately. Moreover, the biofilms virulence was evaluated by red blood cell (RBC) hemolysis assay and staphylococcal virulence factor enterotoxins A (SEA) detected by enzyme linked immunosorbent assay (ELISA). After PDT, the biofilm was re-cultured for extra 48 h. Its formation viability and virulence were detected again. All data were analyzed by ANOVAs followed by the Games Howell post hoc test (α = 0.05). RESULTS: The biofilm was inactivated about 2.3 log10 at 1.25 µM with 30 J/cm2 illumination, and 3.5 log10 with 50 J/cm2 after PDT (P<0.05). XTT assays demonstrated the viability of mature MRSA biofilms was reduced after PACT. PDT group shows a distinct reduction in RBC hemolysis rate and the concentration of SEA compared to the control groups. The morphological features of the biofilms showed great changes, such as shrinkage, fissure, fragmentation, and rarefaction after being treated by TBO-PDT and observed by SEM. The recovery of the structure and virulence of biofilm were suppressed after PDT. CONCLUSION: TBO-mediated PDT could destroy the biofilm structure, reduce its virulence and depress its self-recovery.

A Multi-Center, Randomized, Blind, Controlled Clinical Trial of the Safety and Efficacy of Micro Radio Frequency Therapy System for the Treatment of Overactive Bladder.

Purpose: The purpose of this study was to evaluate the efficacy and safety of low power micro radiofrequency (RF) therapy (µRFthera®) through urethra in the treatment of overactive bladders (OAB) through a prospective, single-blind, placebo-controlled, multi-center clinical protocol. Materials and Methods: One hundred and fourteen patients with refractory OAB were randomized at 2:1 ratio, treatment to control undergoing same procedures except only the micro-RF treatment group at turned "on" setting in energy. Bladder diaries recorded during the screening period (3 days before enrollment) and during follow-up period on week 1, 3, and 7, respectively. The patients in control could choose receiving an energized treatment during extension stage. Results: The treatment efficacy was 76.1%. There was 49.80% rate improvement compared to control (95%CL 32.48%, 67.13%). The crude rate ration (RR) was 2.89, 95% CI (1.67-5.01) with p < 0.001 in uni-variate analysis, while the RR became 2.94, 95% CI (1.67-5.16) with p < 0.001 after adjusted potential confounding factors in multi-variate analysis. Statistically significant improvements have been demonstrated in the frequency of urination, urgency, nocturia, and quality of life (QoL) scores. Conclusions: Micro RF therapy is safe and effective for the treatment of OAB. The main treatment-related complications were catheterization related complications. Clinical Trial Registration: Zhejiang Device Registration Certificate No. 202090909, www.chictr.org.cn, Clinical Trial Accession Number: ChiCTR2100050096.