Rapid screening of acetylcholinesterase active contaminants in water: A solid phase microextraction-based ligand fishing approach.

Effect-directed analysis (EDA) has been increasingly used for screening toxic contaminants in the environment, but conventional EDA procedures are often time-consuming and labor-extensive. This challenges the use of EDA for toxicant identification in the scenarios when quick answers are demanded. Herein, a solid phase microextraction ligand fishing (SPME-LF) strategy has been proposed as a rapid EDA approach for identifying acetylcholinesterase (AChE) active compounds in water. The feasibility of ligand fishing techniques for screening AChE active chemicals from environmental mixtures was first verified by a membrane separation method. Then, SPME fibers were prepared through self-assembly of boronic acid groups with AChE via co-bonding and applied for SPME-LF. As AChE coated SPME fibers selectively enriched AChE-active compounds from water, comparing sorbing compounds by the SPME fibers with and without AChE coating can quickly distinguish AChE toxicants in mixtures. Compared with conventional EDA, SPME-LF does not require repeating sample separations and bioassays, endowing SPME-LF with the merits of low-cost, labor-saving, and user-friendly. It is believed that cost-efficient and easy-to-use SPME-LF strategy can potentially be a rapid EDA method for screening receptor-specific toxicants in aquatic environment, especially applicable in time-sensitive screening.

A novel tsRNA-5008a promotes ferroptosis in cardiomyocytes that causes atrial structural remodeling predisposed to atrial fibrillation.

Atrial fibrillation (AF) is an extremely common clinical arrhythmia disease, but whether its mechanism is associated with ferroptosis remains unclear. The tRNA-derived small RNAs (tsRNAs) are involved in a variety of cardiovascular diseases, however, their role and mechanism in atrial remodeling in AF have not been studied. We aimed to explore whether tsRNAs mediate ferroptosis in AF progression. The AF models were constructed to detect ferroptosis-related indicators, and Ferrostatin-1 (Fer-1) was introduced to clarify the relationship between ferroptosis and AF. Atrial myocardial tissue was used for small RNA sequencing to screen potential tsRNAs. tsRNA functioned on ferroptosis and AF was explored. Atrial fibrosis and changes in the cellular structures and arrangement were observed in AF mice model, and these alterations were accompanied by ferroptosis occurrence, exhibited by the accumulation of Fe2+ and MDA levels and the decrease of expression of FTH1, GPX4, and SLC7A11. Blocking above ferroptosis activation with Fer-1 resulted in a significant improvement for AF. A total of 7 tsRNAs were upregulated (including tsRNA-5008a) and 2 tsRNAs were downregulated in atrial myocardial tissue in the AF group compared with the sham group. We constructed a tsRNA-mRNA regulated network, which showed tsRNA-5008a targeted 16 ferroptosis-related genes. Knockdown of tsRNA-5008a significantly suppressed ferroptosis through targeting SLC7A11 and diminished myocardial fibrosis both in vitro and in vivo. On the contrary, tsRNA-5008a mimics promoted ferroptosis in cardiomyocytes. Collectively, tsRNA-5008a involved in AF through ferroptosis. Our study provides novel insights into the role of tsRNA-5008a mediated ferroptosis in AF progression.

COMPARISON BETWEEN ACTIVE ABDOMINAL COMPRESSION-DECOMPRESSION CARDIOPULMONARY RESUSCITATION AND STANDARD CARDIOPULMONARY RESUSCITATION IN ASPHYCTIC CARDIAC ARREST RATS WITH MULTIPLE RIB FRACTURES.

ABSTRACT: Background: Active abdominal compression-decompression cardiopulmonary resuscitation (AACD-CPR) is potentially more effective for cardiac arrest (CA) with multiple rib fractures. However, its effect on survival rates and neurological outcomes remains unknown. This study aimed to assess if AACD-CPR improves survival rates and neurological outcomes in a rat model of asphyctic CA with multiple rib fractures. Methods: Adult male Sprague-Dawley rats were randomized into three groups-AACD group (n = 15), standard cardiopulmonary resuscitation (STD-CPR) group (n = 15), and sham group (n = 10)-after bilateral rib fractures were surgically created and endotracheal intubation was performed. AACD-CPR and STD-CPR groups underwent 8 min of asphyxia followed by different CPR techniques. The sham group had venous catheterization only. Physiological variables and arterial blood gases were recorded at baseline and during a 4-h monitoring period. Neurological deficit scores (NDSs) and cumulative survival rates were assessed at 24, 48, and 72 h. NDS, serum biomarkers, and hippocampal neuron analysis were used to evaluate neurological outcomes. Results: No statistical differences were observed in the return of spontaneous circulation (ROSC), 24-, 48-, and 72-h survival rates between the AACD-CPR and STD-CPR groups. AACD-CPR rats had lower serum levels of neuron-specific enolase and S100B at 72 h post-ROSC, and higher NDS at 72 h post-ROSC compared with STD-CPR animals. Cellular morphology analysis, hematoxylin and eosin staining, and TUNEL/DAPI assays showed more viable neurons and fewer apoptotic neurons in the AACD-CPR group than in the STD-CPR group. Conclusions: AACD-CPR can achieve similar survival rates and better neurological outcome after asphyxial CA in rats with multiple rib fractures when compared with STD-CPR.

Xinyang tablet ameliorates sepsis-induced myocardial dysfunction by regulating Beclin-1 to mediate macrophage autophagy and M2 polarization through LncSICRNT1 targeting E3 ubiquitin ligase TRAF6.

OBJECTIVE: Xinyang Tablet (XYT) has emerged as a potential intervention to counter sepsis-induced myocardial dysfunction (SMID) by influencing macrophage autophagy and M2 polarization. This study aimed to unravel the underlying mechanism of XYT in sepsis-induced myocardial dysfunction (SIMD). METHODS: A microarray analysis was employed to explore sepsis-related changes, and bioinformatics analysis was used to predict lncRNAs binding to tumor necrosis factor receptor-associated factor 6 (TRAF6). This studio utilized SIMD mouse models induced by lipopolysaccharide (LPS) injection, followed by treatments involving varied doses of XYT, digoxin (positive control), or si-LncSICRNT1. After seven days, evaluations encompassing mouse hair/mental state/diet/weight were measured, and cardiac function via echocardiography were conducted. Myocardial tissue changes were observed using hematoxylin-eosin staining. Additionally, bone marrow-derived macrophages (BMDMs) subjected to LPS for M1 polarization were treated with oe-LncSICRNT1, si-TRAF6 and their negative control, XYT, or autophagy inhibitor 3-Methyladenine (3-MA) (positive control). RT-qPCR and Western blot analyses were employed to assess LncSICRNT1, TRAF6, Beclin-1, LC3II/LC3I, and p62 levels. Immunohistochemistry and flow cytometry were used for M1/M2 polarization markers, while enzyme-linked immunosorbent assay (ELISA) gauged inflammatory factor levels. Interaction between TRAF6 and LncSICRNT1 was probed using RNA pull-down and RNA immunoprecipitation (RIP) assays. RESULTS: Chip analysis obtained 1463 differentially expressed lncRNAs, including LINC01550 (LncSICRNT1). Further prediction indicated that LncSICRNT1 was highly likely to directly bind to TRAF6. XYT treatment in LPS-induced SIMD mice led to notable enhancements in sleep/hair/diet/activity, increased weight/left ventricular end-diastolic diameter (LVEDd)/LV ejection fraction (LVEF)/LV fraction shortening (LVFS). These improvements were associated with elevated LncSICRNT1 expression and decreased TRAF6 protein levels, culminating in reduced myocardial inflammatory responses and improved cardiac function. Notably, XYT was found to suppress macrophage M1 polarization, while enhancing M2 polarization, ultimately benefitting cardiac function via LncSICRNT1 modulation. Furthermore, the study revealed LncSICRNT1 modulated Beclin-1 ubiquitination and restrained macrophage autophagy by targeting TRAF6 expression. CONCLUSION: The study highlights XYT's potential to ameliorate LPS-induced SIMD by elevating LncSICRNT1 expression, influencing TRAF6 expression, and regulating Beclin-1 ubiquitination. These actions collectively inhibit macrophage autophagy and foster M1/M2 polarization, contributing to cardiac function improvement.

Sodium pyruvate exerts protective effects against cigarette smoke extract-induced ferroptosis in alveolar and bronchial epithelial cells through the GPX4/Nrf2 axis.

BACKGROUND: Ferroptosis in alveolar and bronchial epithelial cells is one of the main mechanisms underlying the development of chronic obstructive pulmonary disease (COPD). Sodium pyruvate (NaPyr) is a natural antioxidant in the body, exhibiting anti-inflammatory and antioxidant activities. NaPyr has been used in a Phase II clinical trial as a novel therapy for COPD; however, the mechanism underlying NaPyr-mediated therapeutic benefits in COPD is not well understood. OBJECTIVE: We aimed to assess the protective effects of NaPyr and elucidate its potential mechanism in cigarette smoke extract (CSE)-induced ferroptosis.To minic the inflammatory response and ferroptosis triggered by cigarette smoke in COPD in an invitro cell based system, we expose a human bronchial epithelial cells to CSE. METHODS: To minic the inflammatory response and ferroptosis triggered by cigarette smoke in COPD in an invitro cell based system, the A549 (human lung carcinoma epithelial cells) and BEAS-2B (bronchial epithelial cells) cell lines were cultured, followed by treatment with CSE. To measure cellular viability and iron levels, we determined the levels of malondialdehyde (MDA), glutathione (GSH), reactive oxygen species (ROS), mitochondrial superoxide (MitoSOX), membrane potential (MMP), and inflammatory factors (tumor necrosis factor [TNF] and interleukin [IL]-8), and examined CSE-induced pulmonary inflammation and ferroptosis. To clarify the molecular mechanisms of NaPyr in COPD therapy, we performed western blotting and real-time PCR (qPCR) to determine the expression of glutathione peroxidase 4 (GPX4), nuclear factor E2-related factor 2 (Nrf2), and cyclooxygenase 2 (COX2). RESULTS: We found that NaPyr effectively mitigated CSE-induced apoptosis and improved apoptosis induced by erastin, a ferroptosis inducer. NaPyr significantly decreased iron and MDA levels and increased GSH levels in CSE-induced cells. Furthermore, NaPyr suppressed ferroptosis characteristics, such as decreased levels of ROS, MitoSOX, and MMP. NaPyr significantly increases the expression levels of GPX4 and Nrf2, indicating that activation of the GPX4/Nrf2 axis could inhibit ferroptosis in alveolar and bronchial epithelial cells. More importantly, NaPyr inhibited the secretion of downstream inflammatory factors, including TNF and IL-8, by decreasing COX2 expression levels to suppress CSE-induced inflammation. CONCLUSION: Accordingly, NaPyr could mitigate CSE-induced ferroptosis in alveolar and bronchial epithelial cells by activating the GPX4/Nrf2 axis and decreasing COX2 expression levels. In addition, NaPyr reduced the secretion of inflammatory factors (TNF and IL-8), affording a novel therapeutic candidate for COPD.

Environmental DNA metabarcoding revealed the impacts of anthropogenic activities on phytoplankton diversity in Dianchi Lake and its three inflow rivers.

Phytoplankton diversity is closely related to environmental variables and has been widely used in ecological health assessment of rivers and lakes. Combining advantages of DNA-based identification and high-throughput sequencing technology, environmental DNA (eDNA) metabarcoding permits a new measurement for biodiversity monitoring in aquatic ecosystems. However, it had rarely been used to explore the variability and similarity of phytoplankton diversity between lake and its inflow rivers and the effects of environmental variables on phytoplankton. This study utilized eDNA metabarcoding to investigate the spatial distribution of phytoplankton and the impacts of environmental variables on the phytoplankton diversity in Dianchi Lake (one of the most polluted urban lakes in China) and its main inflow rivers (Panlong River, Baoxiang River, and Chai River). A total of 243 distinct phytoplankton taxa were detected, covering 9 phyla, 30 classes, 84 orders, and 132 families, and the taxonomic richness of rivers was higher than that of Dianchi Lake. Distinct biodiversity patterns (e.g., community structure, dominant taxon, ɑ-diversity) were exhibited among Dianchi Lake and its three inflow rivers, but similar biodiversity patterns were also observed in Dianchi Lake and the estuarine sites. The patterns of phytoplankton diversity were closely related to environmental variables, which were associated with pollution sources from different anthropogenic activities (e.g., urbanization, water diversion, industrial and agricultural activities). The primary environmental variables correlated with phytoplankton diversity varied in different habitats. The total phosphorus (TP) and chemical oxygen demand (COD) positively correlated with the phytoplankton community structures in Dianchi Lake, whereas negatively correlated in Panlong River and Baoxiang River. The total nitrogen (TN) positively correlated with the phytoplankton community structures in Baoxiang River and Chai River but negatively correlated in Dianchi Lake. Overall, this study provides insights on the phytoplankton diversity monitoring and the conservation of its diversity and healthy management of Dianchi Lake.

Effects of commercial beverages on the neurobehavioral motility of Caenorhabditis elegans.

To study the effects of different types of commercially available drinks/beverages on neurobehavior using the model organism C. elegans, and critically review their potential health hazards. Eighteen kinds of beverages from the supermarket were randomly selected and grouped into seven categories namely functional beverage, tea beverage, plant protein beverage, fruit juice beverage, dairy beverage, carbonated beverage and coffee beverage. The pH value, specific gravity and osmotic pressure were also examined. The L4 stage N2 worms were exposed to different concentration of tested beverages (0, 62.5, 125, 250 and 500 µL/mL) for 24 h to measure the survival rate and locomotory behavior such as head thrashing, body bending as well as pharyngeal pumping. All the 18 beverages tested did not induce any visible lethal effects in the nematodes. However, exposure to different types of tested beverages exhibited different effects on the behavioral ability of C. elegans: (1) sports functional beverage and herbal tea drink accelerated the head thrashing and body bending of nematodes when compared to the control group (P < 0.05). (2) The vibration frequency of the pharyngeal pump of nematodes was significantly accelerated after treated with three plant protein beverages (almond milk, coconut milk and milk tea) and dairy products A and B (P < 0.05), and decelerated after treatment with other tested beverages. (3) Carbonated beverage significantly inhibits the head thrashing, body bending and pharyngeal pumping vibration (P < 0.05). Our results indicate that 18 kinds of popular beverages in the market have different influence on the neurobehavior in C. elegans, which may be related to their different components or properties. Further research would be required to conduct a systematic analysis of the effect of beverages by appropriate kinds, taking into consideration other endpoints such as reproduction, lifespan and molecular stress response, etc., and to elucidate the mechanism for its potential health hazards.

Aggregation and Size Attributes Analysis of Unadsorbed and Adjuvant-adsorbed Antigens using a Multispectral Imaging Flow Cytometer Platform.

Various vaccine quality attributes should be monitored to ensure consistency, potency, purity, and safety of vaccine products prior to lot release. Vaccine particle size and protein antigen aggregation are two important considerations for particle-adsorbed vaccines. In this study, we evaluated the use of imaging flow cytometry as a potential all-in-one platform to measure adjuvant particle size and to detect protein aggregates through a combination of brightfield microscopy, side scatter detection, and fluorescence microscopy. An aluminum phosphate adjuvant was analyzed for size using the brightfield function, and the size measurement was compared against laser diffraction. Heat-induced protein aggregates of either unadsorbed antigens or aluminum phosphate adjuvant-adsorbed antigens were stained with the fluorescent ProteoStat aggregation dye, followed by detection and analysis using a combination of the brightfield and fluorescence microscopy functions. The change in aggregation of unadsorbed antigens was confirmed using dynamic light scattering. These results demonstrate the versatility of the imaging flow cytometry platform for the evaluation of multiple vaccine quality characteristics.

The indole alkaloids from the roots of Isatidis Radix.

The root of Isatis indigotica is used as a traditional Chinese medicine (termed Isatidis Radix) due to its antiviral effects. We examined compounds isolated from Isatidis Radix and elucidated the structures of three new natural alkaloids, and we examined the possible mechanisms or active targets of indole alkaloids occurring in blood of rats treated by gavage. Three new natural products were isolated from Radix Isatidis for the first time, including 1-methoxy-2-indoleacetonitrile, 1-hydroxy-3-indoleacetonitrile, 8-Methoxy-1, 2-dihydroquinoline, and 4 compounds isolated from this medicinal material for the first time. Their structures were elucidated using nuclear magnetic resonance. The components of Isatidis Radix were analysed using liquid chromatography tandem mass spectrometry, and 33 compounds were detected in plasma of treated rats; 24 of these compounds were indole alkaloids, and they included the newly identified compounds. Molecular docking and in vitro antithrombin activity tests showed HA inhibition activity of indoleacetonitriles.

Indole-3-carboxaldehyde regulates RSV-induced inflammatory response in RAW264.7 cells by moderate inhibition of the TLR7 signaling pathway.

Human respiratory syncytial virus (RSV) is highly contagious and the leading cause of severe respiratory tract illness in infants, elderly, and immunocompromised individuals. Toll-like receptor 7 (TLR7), a pattern recognition receptor recognising the ssRNA of RSV, activates proinflammatory pathways and triggers secretion of interferons (IFNs). On the one hand, the inflammatory responses help clear out virus. On the other hand, they lead to severe lung damage. Banlangen is a traditional Chinese herbal medicine commonly prescribed for respiratory virus infection treatment, but the mechanisms of action and active components remain largely unknown. In the present study, we investigated the effects of the main active components of total alkaloids from banlangen (epigoitrin, indole-3-carboxaldehyde, indole-3-acetonitrile and 4-methoxyindole-3-acetonitrile) on the RSV-induced inflammatory responses in mouse macrophage cells (RAW264.7). Our results demonstrated that RSV-induced IFN-α excessive secretion was moderately inhibited by indole-3-carboxaldehyde through downregulation of mRNA expression in a dose-dependent manner, in comparison, the inhibitory effects of ribavirin were too strong. Furthermore, we revealed that indole-3-carboxaldehyde suppressed transcription of IFN-α by inhibiting RSV-induced TLR7 expression in RAW264.7 cells. Additionally, indole-3-carboxaldehyde inhibited RSV-induced NF-κB signalling activation in a TLR7-MyD88-dependent manner. Together, our findings suggest that indole-3-carboxaldehyde inhibited RSV-induced inflammatory injury by moderate regulation of TLR7 signaling pathway and did not significantly affect the viral clearance competence of the innate immune system.

Comparative effects of intensive ganglionated plexus ablation in treating paroxysmal atrial fibrillation and vasovagal syncope.

BACKGROUND: Ganglionated plexus (GP) ablation is used to treat atrial fibrillation (AF) and vasovagal syncope (VVS). However, the comparative effects of GP ablation in treating paroxysmal atrial fibrillation (PAF) and VVS have not been well studied. OBJECTIVE: The purpose of this study was to investigate the effects of intensive GP ablation on PAF and VVS. METHODS: PAF and VVS patients were enrolled in this study. Pulmonary vein isolation (PVI) was performed in the PAF group, and additional ablation was performed at GP sites. Anatomic ablation of left atrial GPs was performed in the VVS group. The primary endpoint was freedom from AF or other sustained atrial tachycardia and syncope recurrence. RESULTS: A total of 195 patients were enrolled: 146 patients with PAF, including eight patients with combined VVS (PAF group), and 49 patients with VVS (VVS group). Vasovagal response (VR) was achieved in 78 (53.4%) patients in the PAF group and 48 patients (98.0%) in the VVS group (P < .05). During the 17.8 ± 10.5 (range, 3-42) month follow-up, 126 (86.3%) patients were free of AF in the PAF group, and 45 (91.8%) patients in the VVS group had no syncope recurrence and significantly improved symptoms. CONCLUSIONS: Anatomically guided intensive GP ablation showed efficient clinical outcomes for both groups of patients. Compared with PAF patients, VVS patients had more VR during ablation in the left atrium. Furthermore, VR during ablation indicated a better prognosis in PAF patients.

Personalized Fluoroscopic Angles in Watchman™ Left Atrial Appendage Closure Landing Zone Assessment: A Three-Dimensional Printed Simulation Study.

Background Atrial fibrillation causes ischemic stroke when thrombi dislodge from a cardiac outpouching, the left atrial appendage (LAA), and embolize to the brain. LAA occlusion with the Watchman™ device (Boston Scientific Corporation, MA, USA), which prevents stroke, requires accurate LAA measurements for device sizing. We explore whether standard fluoroscopic LAA measurements improve when obtained at CT-derived viewing angles personalized to LAA anatomy while concurrently referring to three-dimensional (3D) CT. Methods Left atrial 3D reconstructions created from contrast CT (n=28) were analysed to identify personalized viewing angles wherein LAA dimensions (LAA maximum landing zone diameter and LAA length) were best observed. The 3D-CT reconstructions were then 3D printed with stands. Fluoroscopy of anatomically oriented models in the catheter lab simulated LAA angiography. Fluoroscopic images were acquired at standard (caudal 20˚/right anterior oblique 30˚) and personalized viewing angles. Repeated measurements of LAA dimensions were taken from CT (Control), fluoroscopy at standard angles (Standard), personalized angles (Blinded), and personalized angles while concurrently referring to 3D CT (Referred). Results Control measurements correlated and agreed better with Referred and Blinded measurements than with Standard measurements (diameter correlation and agreement: Control/Standard r=.554, limits of agreement [LOAs]=6.83/-5.91; Control/Blinded r=.641, LOA =5.67/-5.54; Control/Referred r=.741, LOA=4.69/-4.14; length correlation and agreement: Control/Standard rs=.829, LOA=9.61/-3.02; Control/Blinded rs=0.789, LOA=7.13/-4.94; Control/Referred rs=.907, LOA=4.84/-4.13). Personalized angles resulted in hypothetical device size predictions more consistent with Control (device size correlation: Control/Standard rs=.698, Control/Blinded rs=.731, Control/Referred rs=.893, P<0.001). False ineligibility rates were Standard=6/28, Blinded=6/28, and Referred=2/28. Conclusion This simulation suggests that personalized fluoroscopic viewing angles with in-procedural reference to 3D CT may improve the accuracy of LAA maximum landing zone diameter and length measurements at the Watchman landing zone. This improvement may result in more consistent device size selection and procedural eligibility assessment. Further clinical research on these interventions is merited.

Manifestations of blood coagulation and its relation to clinical outcomes in severe COVID-19 patients: Retrospective analysis.

INTRODUCTION: Characteristics of blood coagulation and its relation to clinical outcomes in COVID-19 patients are still rarely reported. We aimed to investigate the blood coagulation function and its influences on clinical outcomes of patients with syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: A total of 71 severe patients with confirmed SARS-CoV-2 infection who were treated in Wuhan First Hospital from February 12 to March 20, 2020, were enrolled. The blood coagulation data in these patients and in 61 healthy controls were collected. The patients with COVID-19 were divided into two groups: the aggravated group and the nonaggravated group, respectively, basing on whether the patients' conditions turned to critically ill or not after admission. RESULTS: Compared with healthy controls, patients with COVID-19 had significant performances with coagulation dysfunction, including dramatically elevated values of FIB, PT, APTT, INR, FDP, and D-Dimers but markedly reduced AT value (P < .05). Importantly, more noteworthy coagulation disorders similar to the differences between patients and controls were found in the aggravated patients with conditions deterioration after admission than those in the nonaggravated patients without conditions deterioration (P < .05). Moreover, the aggravated patients possessed a longer hospital stay and a higher mortality compared with the nonaggravated patients (P < .001). The coagulation parameters of COVID-19 patients were widely and closely related to the indexes of liver function and inflammation (P < .05), indicating the coagulation dysfunction of these patients may be caused by liver injury and inflammatory storm. CONCLUSION: Severe patients with SARS-CoV-2 infection often possess coagulation dysfunction on admission. A certain correlation exists in coagulation disorder and adverse clinical outcome among severe COVID-19 patients.

Development of a Pertactin-Coated Beads Approach for Screening of Functional Monoclonal Antibodies.

Vaccine manufacturers have recently focused on the development of in vitro potency assays to promote 3R's strategy to replace animal testing. To be able to develop an in vitro potency assay, the immunological characteristics of the monoclonal antibodies used in the assay should be well understood as these antibodies likely reflect the biological activity of a vaccine product. The PRN antigen is one of the immunogenic antigens included in many commercialized acellular pertussis vaccines. Development of an in vitro potency assay for PRN is challenging as the biological properties of PRN are not well understood. In addition, binding of Bordetella pertussis to human cells occurs through multiple bacterial molecules, which makes it very challenging to assess if antibodies contribute to prevention of bacterial adhesion. To overcome these challenges, the functionality of several in-house anti-PRN mAbs has been investigated through a novel approach using PRN-coated beads. We were able to consistently quantify the inhibition of PRN-mediated adhesion for each anti-PRN mAb. Application of the protein-coated beads model has not only enabled screening of functional anti-PRN mAbs but can also be expanded for screening of antibodies against other bacterial or viral antigens.

Anti-FIM and Anti-FHA Antibodies Inhibit Bordetella pertussis Growth and Reduce Epithelial Cell Inflammation Through Bacterial Aggregation.

The pertussis vaccination is highly recommended for infants, children, and pregnant women. Despite a high coverage of vaccination, pertussis continues to be of public health concern as a re-emerging infectious disease. The mechanism by which vaccine-elicited anti-pertussis antibodies mediate direct bactericidal effects is poorly understood. In this study, we showed that the interaction of B. pertussis with A549 epithelial cells induce release of biological factors which enhance bacteria growth. Complement-depleted antisera from vaccine-immunized guinea pigs or monoclonal antibodies targeting FHA and FIM mediate bacteria aggregation and elicit bactericidal effects. Our in vitro results indicated that aggregation of bacteria through anti-FIM and anti-FHA specific antibodies is one of the major biological mechanisms to clear bacterial infections and restore epithelial cell survival in vitro. Our data also indicates that the anti-pertussis antibodies reduce secretion of proinflammatory chemokines and cytokines by preventing interaction of B. pertussis with host cells. The results of this study not only demonstrate mechanism of action of anti-FIM and anti-FHA antibodies, but also opens translational applications for potential therapeutic approaches or development of analytical assays such as in vitro potency assays.

Different types of effective fractions from Radix Isatidis revealed a multiple-target synergy effect against respiratory syncytial virus through RIG-I and MDA5 signaling pathways, a pilot study to testify the theory of superposition of traditional Chinese Medicine efficacy.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Isatidis, a commonly used traditional Chinese medicine, is also documented in "Dictionary of Chinese Ethnic Medicine" being as an ethnic herb clinically utilized by different nations in China such as Mongol, Uygur, and Dong et al. It has been reported to have a very strong efficacy on respiratory viruses, but to date the mechanism remains unknown. Similarly, it is unclear how different types of effective fractions of Radix Isatidis interact to exert antiviral effects. AIM OF STUDY: To reveal the underlying mechanisms for the inhibitory effects of three active fractions from Radix Isatidis, i.e. total alkaloids, lignans and organic acids, on respiratory syncytial virus when used alone or in combination. In addition, we investigated whether these three parts worked synergistically in vivo and in vitro. MATERIALS AND METHODS: A mouse model of RSV infection was constructed by intranasal infection, and the pathological changes of lung tissues in different parts were observed. The level changes of IFNß and inflammatory cytokines in the mouse alveolar lavage fluid were detected by enzyme-linked immunosorbent assay (ELISA). The anti-RSV effects of different effective fractions were evaluated by the plaque reduction test. The mRNA and protein expressions of RIG-I, MDA-5, MAVS and IRF3 in RAW264.7 cells were detected by RT-PCR and Western blot respectively. RESULTS: HE staining showed that Radix Isatidis extracts alone or in combination relieved virus-induced mouse lung lesions. Compared with individual drugs, the lung lesions were alleviated more significantly after treatment with the three fractions in combination. ELISA demonstrated that the expression levels of IFNß and inflammatory cytokines were maintained balanced between antiviral and proinflammatory effects. The plaque reduction test indicated that the antiviral effect of combination treatment was much stronger than those of individual drugs. RT-qPCR and Western blot suggested that the mRNA and protein expression levels of key signaling molecules in the RIG-I and MDA5 pathways in mouse macrophages were down-regulated by different effective parts alone or in combination. CONCLUSIONS: The three effective fractions of Radix Isatidis have remarkable synergistic anti-RSV effects in vitro and in vivo, and total alkaloids and lignans show multi-target synergistic effects via the RIG-I and MDA5 signaling pathways.

In vitro assessment of biological activity and stability of the ALVAC-HIV vaccine.

The first evidence in humans that a safe and effective preventive vaccine for HIV is possible came from the phase III HIV clinical trial RV144 in Thailand. This trial was based on a prime/boost combination of a recombinant canarypox vaccine and two glycoprotein 120 proteins (ALVAC-HIV and AIDSVAX B/E). A pivotal phase IIb/III trial has recently commenced in the Republic of South Africa, for which the infectious titer assay was applied as the quantitative release test for the ALVAC-HIV vaccine. The infectious titer assay measures the ability of the vaccine vector to infect target permissive cells, but does not indicate if the vaccine transgenes are expressed. We have developed a high-throughput biological activity assay that provides results in agreement with the infectious titer assay. This assay uses flow cytometry to quantify expression of ALVAC-HIV encoded proteins gp120 and p24 in human cells. This transgene expression is detected by two cross-clade-reactive, biologically functional human anti-gp120 monoclonal antibodies isolated from clinical trial participants and a commercial mouse anti-p24 monoclonal antibody. The relative biological activity of the vaccine test sample is calculated by comparison of the test sample dose-response curve against that of a reference standard. We show that the novel biological activity assay is specific, accurate, precise, stability-indicating, and robust. The assay is being used for characterization of ALVAC-HIV (vCP2438) product, the efficacy of which is being evaluated in the pivotal phase IIb/III clinical trial HVTN702. The biological activity assay has the potential to indicate vaccine consistency and quality as a complement to the infectious titer assay.

Resveratrol inhibits VEGF-induced angiogenesis in human endothelial cells associated with suppression of aerobic glycolysis via modulation of PKM2 nuclear translocation.

Endothelial cells (ECs) mainly depend on aerobic glycolysis to generate angiogenesis. Deregulation of glycolysis is often observed in human endothelial cells during angiogenesis. In the present study, we first report that resveratrol (RST), which has been intensively studied in glucose metabolism of various cancer cells, has a profound inhibitory effect on tube formation and migration via suppression of glycolysis in human umbilical vein endothelial cells (HUVECs) induced by vascular endothelial growth factor (VEGF). Moreover, we further reveal that RST reduced the mRNA and protein level of glucose transporter-1(GLUT1), hexokinase II (HK2), phosphofructokinase-1(PFK1) and pyruvate kinase M2 (PKM2) through modulation of ERK-mediated PKM2 nuclear translocation. Our results provide a novel mechanism to account for the inhibition of RST on VEGF-mediated angiogenesis and suggest that targeting aerobic glycolysis or nuclear PKM2 may be a new approach for pathological angiogenesis prevention or treatment.

Catheter radiofrequency ablation for arrhythmias under the guidance of the Carto 3 three-dimensional mapping system in an operating room without digital subtraction angiography.

Several studies have reported the efficacy of a zero-fluoroscopy approach for catheter radiofrequency ablation of arrhythmias in a digital subtraction angiography (DSA) room. However, no reports are available on the ablation of arrhythmias in the absence of DSA in the operating room. To investigate the efficacy and safety of catheter radiofrequency ablation for arrhythmias under the guidance of a Carto 3 three-dimensional (3D) mapping system in an operating room without DSA. Patients were enrolled according to the type of arrhythmia. The Carto 3 mapping system was used to reconstruct heart models and guide the electrophysiologic examination, mapping, and ablation. The total procedure, reconstruction, electrophysiologic examination, and mapping times were recorded. Furthermore, immediate success rates and complications were also recorded. A total of 20 patients were enrolled, including 12 males. The average age was 51.3 ±â€Š17.2 (19-76) years. Nine cases of atrioventricular nodal re-entrant tachycardia, 7 cases of frequent ventricular premature contractions, 3 cases of Wolff-Parkinson-White syndrome, and 1 case of typical atrial flutter were included. All arrhythmias were successfully ablated. The procedure time was 127.0 ±â€Š21.0 (99-177) minutes, the reconstruction time was 6.5 ±â€Š2.9 (3-14) minutes, the electrophysiologic study time was 10.4 ±â€Š3.4 (6-20) minutes, and the mapping time was 11.7 ±â€Š8.3 (3-36) minutes. No complications occurred. Radiofrequency ablation of arrhythmias without DSA is effective and feasible under the guidance of the Carto 3 mapping system. However, the electrophysiology physician must have sufficient experience, and related emergency measures must be present to ensure safety.

Identification of differentially expressed circular RNAs during TGF-ß1-induced endothelial-to-mesenchymal transition in rat coronary artery endothelial cells.

OBJECTIVE: Although differentially expressed circRNAs have been proposed to be closely associated with epithelial-mesenchymal transition (EMT), the roles of circRNAs remain unclear in endothelial-to-mesenchymal transition (EndMT), which is a subcategory of EMT. Herein, we characterized the expression and potential function of circRNAs during TGF-ß1-induced EndMT in rat coronary artery endothelial cells (CAEC). METHODS: High-throughput RNA sequencing was performed for unbiasedly profiling the expression of circRNAs. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway analysis were performed using online forecasting databases. Real-time quantitative polymerase chain reaction (RT-qPCR) was used for confirming the circRNA expression obtained from the sequencing data. RESULTS: Among the candidated circRNAs, 102 circRNAs were differentially expressed, among which 66 circRNAs and 36 circRNAs were up-regulated and down-regulated, respectively, in TGF-ß1-treated rat CAEC. GO analysis findings revealed that numerous differentially expressed circRNAs were closely associated with the biological process. KEGG signaling pathway analysis suggested that the abnormal expression of circRNAs had been implicated in regulating the dynamics endothelial cell junctions. Furthermore, we also found that three EndMT-related circRNAs, chr5:90817794|90827570, chr8:71336875|71337745, and chr6:22033342|22038870, were significantly up-regulated in TGF-ß1-treated rat CAEC. CONCLUSION: The findings of this study reveal a comprehensive expression and potential functions of differentially expressed circRNAs during TGF-ß1-induced EndMT. These findings provide mechanistic insights into the role of circRNAs in EndMT-related cardiovascular diseases (CVDs).