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Urinary tract infections (UTIs) by extended-spectrum β-lactamase producing Enterobacteriaceae (ESBL-E) have become an important public health problem with a substantial economic burden, as they lead to more complicated infections, longer duration of treatment, and increases in patient mortality. A total of 1267 urine samples were collected from patients during the period of 2018 to 2020. The isolates were identified based on colony morphology and biochemical tests. Antimicrobial susceptibility testing was done by modified Kirby Bauer disc diffusion method. Resistant isolates to third generation cephalosporins were tested for ESBL production by double-disk synergy test (DDST), DDST methods on MH agar plates supplemented with cloxacillin and by confirmatory double disk test (DDT). 400 strains causing UTIs were identified and classified as members of Enterobacteriaceae. The results of antimicrobial susceptibility tests showed that 68.5% (274/400) of the Enterobacteriaceae isolates were multi-drug resistant (MDR) strains. The overall prevalence of ESBL-E was 59.5% (238/400). The highest frequencies of ESBL-E were observed in internal medicine devices (68/400) and the highest ESBL production was observed in E. coli (85/238). The ESBL producing isolates were significantly resistant to penicillins and cephalosporins compared to non-ESBL producers. However, those ESBL-E strains were sensitive to imipenem (80.2%) and nitrofurantoin (87.8%). High levels of MDR strains and ESBL-E in our study highlights the need for applying specific infection control measures, and accordingly we urge physicians to opt for specific strategies for regular surveillance of uropathogenic bacteria.

We study the nonsteady Stokes flow in a thin tube structure composed by two thin rectangles with lateral elastic boundaries which are connected by a domain with rigid boundaries. After a variational approach of the problem which gives us existence, uniqueness, regularity results, and some a priori estimates, we construct an asymptotic solution. The existence of a junction region between the two rectangles imposes to consider, as part of the asymptotic solution, some boundary layer correctors that correspond to this region. We present and solve the problems for all the terms of the asymptotic expansion. For two different cases, we describe the order of steps of the algorithm of solving the problem and we construct the main term of the asymptotic expansion. By means of the a priori estimates, we justify our asymptotic construction, by obtaining a small error between the exact and the asymptotic solutions.

This study aims at preparing and optimizing lacidipine (LCDP) polymeric micelles using thin film hydration technique in order to overcome LCDP solubility-limited oral bioavailability. A two-factor three-level central composite face-centered design (CCFD) was employed to optimize the formulation variables to obtain LCDP polymeric micelles of high entrapment efficiency and small and uniform particle size (PS). Formulation variables were: Pluronic to drug ratio (A) and Pluronic P123 percentage (B). LCDP polymeric micelles were assessed for entrapment efficiency (EE%), PS and polydispersity index (PDI). The formula with the highest desirability (0.959) was chosen as the optimized formula. The values of the formulation variables (A and B) in the optimized polymeric micelles formula were 45% and 80%, respectively. Optimum LCDP polymeric micelles had entrapment efficiency of 99.23%, PS of 21.08 nm and PDI of 0.11. Optimum LCDP polymeric micelles formula was physically characterized using transmission electron microscopy. LCDP polymeric micelles showed saturation solubility approximately 450 times that of raw LCDP in addition to significantly enhanced dissolution rate. Bioavailability study of optimum LCDP polymeric micelles formula in rabbits revealed a 6.85-fold increase in LCDP bioavailability compared to LCDP oral suspension.

Abstract Background Hepatic cancer endures a major health scourge as the consequence of a high incidence of > 1 million cases by 2025. Plant-based products are typically effective in ameliorating health conditions. Pomegranate peel extract (PE) with its high polyphenolic content has anticancer effects against different types of cancer. Herein, we aimed to maximize the PE chemotherapeutic efficacy by loading it in a suitable delivery system to overcome the limitations of PE, to control its release and to achieve liver targeting. Method A nanoprecipitation procedure was adopted to incorporate PE into biodegradable and biocompatible natural polymeric zein (ZN)-based nanoparticles (NPs) (PE-ZN NPs). A full factorial design (22 × 31) was developed to study the effects of the formulation variables, namely pH of dispersion, PE-to-ZN ratio and surfactant concentration. Results The optimization revealed a surfactant-free stable PE-ZN NPs formula with a small particle size of 99.5 ± 6.43 nm, high PE encapsulation efficiency % of 99.31% ± 3.64 (w/w) and controlled release of PE over 24 h. Conclusion Moreover, the cytotoxicity of the optimum formula against hepatic cancer HepG2 cell lines was assessed and attained about a 2.5-fold reduction in the inhibitory concentration (IC50) values compared to the free PE affording a promising green platform to combat hepatic cancer. Graphical abstract

The current study aimed to fabricate curcumin-loaded bilosomal hydrogel for topical wound healing purposes, hence alleviating the poor aqueous solubility and low oral bioavailability of curcumin. Bilosomes were fabricated via the thin film hydration technique using cholesterol, Span® 60, and two different types of bile salts (sodium deoxycholate or sodium cholate). Bilosomes were verified for their particle size (PS), polydispersity index (PDI), zeta potential (ZP), entrapment efficiency (EE%), and in vitro drug release besides their morphological features. The optimum formulation was composed of cholesterol/Span® 60 (molar ratio 1:10 w/w) and 5 mg of sodium deoxycholate. This optimum formulation was composed of a PS of 246.25 ± 11.85 nm, PDI of 0.339 ± 0.030, ZP of −36.75 ± 0.14 mv, EE% of 93.32% ± 0.40, and the highest percent of drug released over three days (96.23% ± 0.02). The optimum bilosomal formulation was loaded into alginate dialdehyde/chitosan hydrogel cross-linked with calcium chloride. The loaded hydrogel was tested for its water uptake capacity, in vitro drug release, and in vivo studies on male Albino rats. The results showed that the loaded hydrogel possessed a high-water uptake percent at the four-week time point (729.50% ± 43.13) before it started to disintegrate gradually; in addition, it showed sustained drug release for five days (≈100%). In vivo animal testing and histopathological studies supported the superiority of the curcumin-loaded bilosomal hydrogel in wound healing compared to the curcumin dispersion and plain hydrogel, where there was a complete wound closure attained after the three-week period with a proper healing mechanism. Finally, it was concluded that curcumin-loaded bilosomal hydrogel offered a robust, efficient, and user-friendly dosage form for wound healing.

Curcumin or turmeric is the active constituent of Curcuma longa L. It has marvelous medicinal applications in many diseases. When the skin integrity is compromised due to either acute or chronic wounds, the body initiates several steps leading to tissue healing and skin barrier function restoration. Curcumin has very strong antibacterial and antifungal activities with powerful wound healing ability owing to its antioxidant activity. Nevertheless, its poor oral bioavailability, low water solubility and rapid metabolism limit its medical use. Tailoring suitable drug delivery systems for carrying curcumin improves its pharmaceutical and pharmacological effects. This review summarizes the most recent reported curcumin-loaded delivery systems for wound healing purposes, chiefly hydrogels, films, wafers, and sponges. In addition, curcumin nanoformulations such as nanohydrogels, nanoparticles and nanofibers are also presented, which offer better solubility, bioavailability, and sustained release to augment curcumin wound healing effects through stimulating the different healing phases by the aid of the small carrier.

In this work, sucrose acetate isobutyrate (SAIB) and polylactic co-glycolic acid (PLGA) were used alone or in combination as a matrix-former (MF) to prepare long-acting injectable rivastigmine (RV) in situ-forming microparticles (ISM). RV-ISM were prepared by the emulsification of an internal phase, containing the drug and the matrix former(s), into an external oily phase containing a stabilizer. The statistical design, Central Composite Design (CCD), was adopted as a quality by design (QbD) approach to optimize the formulation of RV-ISM systems. The fabricated RV-ISM systems was designed to minimize the initial burst drug release and maximize the sustainment of RV release from the ISM and ease of injection. The influence of critical formulation variables such as the matrix-former to drug (MF/D) ratio and SAIB to PLGA (S/P) ratio in the internal phase with respect to critical quality attributes (CQAs), such as the percentage drug release within the first day (Q1), the time required for 50% drug release (T50%) and the rate of injection, were studied using the CCD. The optimal RV-ISM system with the highest desirability value (0.74) was predicted to have an MF/D ratio of 11.7:1 (w/w) and an S/P ratio of 1.64:1 (w/w). The optimal RV-ISM system was assessed for its release profile, injectability, rheological properties, morphology, effect on cell viability, tolerance to γ-sterilization and in vivo performance in male albino rabbits. In vitro release studies revealed that the optimal RV-ISM system released 100% of its drug content throughout a release period of 30 days with only 15.5% drug release within the first day (Q1) and T50% of 13.09 days. Moreover, the optimal system showed a high injection rate of 1.012 mL/min, pseudoplastic flow, uniform spherical globules with homogenous particle size, minimal cytotoxicity and high tolerability to γ-sterilization. In vivo pharmacokinetic (PK) studies revealed that the rate of absorption of RV from the optimal RV-ISM system was controlled compared to a drug solution following either intramuscular (IM) or subcutaneous (SC) injection. Furthermore, the optimal RV-ISM was found to follow flip-flop PK with poor correlation between in vitro release and in vivo findings. These findings suggest that the optimal RV-ISM is a promising tool to achieve a sustained release therapy for RV; however, further investigation is still required to optimize the in vivo performance of RV-ISM.

Background: Mycosis fungoides (MF) is a type of cutaneous T-cell lymphoma where red rash exists on the skin. Understanding the role of miRNAs and ncRNAs in p53-response has become an open discussion, as they can regulate p53 or its downstream targets, and ncRNAs themselves., Objectives: To evaluate the serum levels of NEAT-1, miR-34a, and p53 in MF patients and its relation to healthy controls to indicate whether it has a potential role in the pathogenesis of the disease. Subjects and Methods . This prospective case-control study was carried out on 75 subjects subdivided into two groups, 35 MF patients (stages 1 and II) and 40 matched healthy controls. Their clinical investigations and serum biomarkers (NEAT-1, miR-34a, and p53) were measured., Results: There were significant elevations in the expression levels of both NEAT-1 (5.10 ± 1.16) and p53 (277.28 ± 62.02) in the serum of MF patients in comparison with controls (1.01 ± 0.031) and (194.29 ± 16.039), respectively, while the level of miR-34a tends to decrease in MF patients (0.24 ± 0.15). There are no significant difference between MF stages and the level of miR-34a, while in NEAT-1 and p53, there are significant differences with p value <0.05 between the stages and the biomarkers. There is a positive correlation between the %BSA and miR-34a and a slightly positive correlation between NEAT-1 and P53 with ( r  = 0.353, p =0.037) and ( r  = 0112, p =0.05), respectively. There were also negative correlations between disease duration and NEAT-1 with ( r  = -0.341, p =0.045) and between B2 microglobulin level and p53 ( r  = -0.373, p =0.027)., Conclusion: The combination of miR-34a, NEAT-1, and p53 may be considered as potential biomarkers that play an active role in the disease process of MF for helping in its early diagnosis and stage identification as well., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2024 Reham Fares et al.)

Desmoid tumors (DTs) are non-metastasizing and locally aggressive soft-tissue mesenchymal neoplasms. Those that become enlarged often become locally invasive and cause significant morbidity. DTs have a varied pattern of clinical presentation, with up to 50-60% not growing after diagnosis and 20-30% shrinking or even disappearing after initial progression. Enlarging tumors are considered unstable and progressive. The management of symptomatic and enlarging DTs is challenging, and primarily consists of chemotherapy. Despite wide surgical resection, DTs carry a rate of local recurrence as high as 50%. There is a consensus that contrast-enhanced magnetic resonance imaging (MRI) or, alternatively, computerized tomography (CT) is the preferred modality for monitoring DTs. Each uses Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1), which measures the largest diameter on axial, sagittal, or coronal series. This approach, however, reportedly lacks accuracy in detecting response to therapy and fails to detect tumor progression, thus calling for more sophisticated methods. The objective of this study was to detect unique features identified by deep learning that correlate with the future clinical course of the disease. Between 2006 and 2019, 51 patients (mean age 41.22 ± 15.5 years) who had a tissue diagnosis of DT were included in this retrospective single-center study. Each had undergone at least three MRI examinations (including a pretreatment baseline study), and each was followed by orthopedic oncology specialists for a median of 38.83 months (IQR 44.38). Tumor segmentations were performed on a T2 fat-suppressed treatment-naive MRI sequence, after which the segmented lesion was extracted to a three-dimensional file together with its DICOM file and run through deep learning software. The results of the algorithm were then compared to clinical data collected from the patients' medical files. There were 28 males (13 stable) and 23 females (15 stable) whose ages ranged from 19.07 to 83.33 years. The model was able to independently predict clinical progression as measured from the baseline MRI with an overall accuracy of 93% (93 ± 0.04) and ROC of 0.89 ± 0.08. Artificial intelligence may contribute to risk stratification and clinical decision-making in patients with DT by predicting which patients are likely to progress.

The principle of proportionality of the systolic area of the central aortic pressure to stroke volume (SV) has been long known. The aim of the present work was to evaluate an in silico solution derived from this principle for modelling SV (iSV model) in cardiovascular safety pharmacology studies by telemetry. Blood pressure was measured in the abdominal aorta in accordance with standard practice. Central aortic pressure was modelled from the abdominal aortic pressure waveform using the N-point moving average (NPMA) method for beat-to-beat estimation of SV. First, the iSV was compared to the SV measured by ultrasonic flowmetry in the ascending aorta (uSV) after various pharmacological challenges in beagle dogs anaesthetised with etomidate/fentanyl. The iSV showed minimal bias (0.2 mL i.e. 2%) and excellent agreement with uSV. Then, previous telemetry studies including reference vasoactive and inotropic compounds were retrospectively reanalysed to model drug effects on stroke volume (iSV), cardiac output (iCO) and systemic vascular resistance (iSVR). Among them, the examples of nicardipine and isoprenaline highlight risks of erroneous or biased estimation of drug effects from the abdominal aortic pressure due to pulse pressure amplification. Furthermore, the examples of verapamil, quinidine and moxifloxacin show that iSV, iCO and iSVR are earlier biomarkers than blood pressure itself for predicting drug effect on blood pressure. This in silico modelling approach included in vivo telemetry safety pharmacology studies can be considered as a New Approach Methodology (NAM) that provides valuable additional information and contribute to improving non-clinical translational research to the clinic., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Hepatitis E virus (HEV) causes acute hepatitis in humans, which can progress to chronicity in immunosuppressed individuals. Almost all reported HEV infections are caused by Paslahepevirus balayani genotypes 1-4. The structural ORF2 protein is the major antigen detected in the blood of HEV-infected individuals. ELISA assays to detect IgM antibodies to HEV are the first-line diagnostic tests; however, they showed variable performance with frequently discordant results. A qualitative HEV antigen (ORF2) ELISA is currently available for research use. Here, we report a novel quantitative sandwich ELISA to measure HEV ORF2 protein in 3 matrix types. An optimal pair of capture and detection antibodies was selected among 12 unique combinations tested. A sandwich ELISA protocol was developed using these mAbs and biotin-streptavidin technology. The protocol was further optimized to quantify ORF2 antigen in different matrices by interpolating from a standard curve with a linear range of 3.17 to 50.8 femtomoles/mL. Using this method, ORF2 protein was detected in the cell culture medium of Huh7 cells as early as 2-3 days after transfection with HEV genome RNA and in a medium of human hepatocytes infected with HEV. ORF2 antigen was readily detected in the first 2 weeks post-HEV infection in gerbil sera. In immunosuppressed gerbils, ORF2 was detected up to 6 weeks, and the levels were significantly higher between 3 and 6 weeks post-infection. HEV ORF2 antigen levels showed a strong positive correlation with HEV RNA levels in both cell culture medium and gerbil sera. Our novel sandwich ELISA detected at least 7.3 femtomoles/mL ORF2 protein in human plasma spiked with cell culture propagated HEV and detected ORF2 protein in human plasma samples that tested positive for HEV RNA but negative for anti-HEV antibodies. Further, the assay was nonreactive, with negative human plasma, and HBV or HCV-positive human plasma demonstrating specificity. Overall, our ORF2 antigen ELISA will be useful for quantifying ORF2 antigen in cell culture medium, gerbil serum, and human plasma. Further studies are warranted to evaluate its utility in HEV clinical diagnosis.

Introduction: Evidence has shown that neurological soft signs are strongly associated with neurocognitive dysfunction. Therefore, the primary objective of this study was to assess the association between NSS and cognitive impairments in a sample of inpatients with schizophrenia. The secondary objective was to explore the association between NSS total scores and functioning. Methods: The study enrolled 95 inpatients diagnosed with schizophrenia disorders and 45 healthy controls. The neurological evaluation scale (NES) was used to assess neurological soft sign while the Brief Assessment of Cognition in Schizophrenia (BACS) was used to evaluate cognitive functioning in patients with schizophrenia. Results: Patients with schizophrenia had significantly higher mean scores on the NES total test and subtests than the control group. Higher cognition was significantly associated with lower NES total and subtest scores. Higher functional independence was significantly associated with a lower NES total score (Beta = -.25), lower motor coordination subtest score (Beta = -.04), and lower others subtest (Beta = -.12). When taking the functional independence scale as the dependent variable, a higher NES total score was significantly associated with lower functioning (Beta = -0.03). Conclusion: NSS were associated to neurocognitive impairments in almost every domain among patients with schizophrenia. Further prospective research is still needed to confirm this role.

Hepatitis E virus (HEV) causes self-limited acute hepatitis in immunocompetent individuals and can establish chronic infection in solid organ transplant recipients taking immunosuppressive drugs. A well characterized small animal model is needed to understand HEV pathogenesis. In this study, we established a robust model to study acute and persistent HEV infection using Mongolian gerbils (Meriones unguiculatus) with or without immunosuppression. Gerbils were implanted subcutaneously with continuous release tacrolimus pellet to induce immunosuppression. Gerbils with or without tacrolimus treatment were inoculated with HEV intraperitoneally. Viremia, fecal virus shedding, serum antibody and ALT levels, liver histopathological lesions, hepatocyte apoptosis, and liver macrophage distribution were assessed. Mild to moderate self-limited hepatitis and IgM and IgG antibody responses against HEV ORF2 were observed in immunocompetent gerbils. Levels of HEV-specific IgM responses were higher and lasted longer in immunocompetent gerbils with higher peak viremia. Persistent viremia and fecal virus shedding with either weak, or absent HEV antibody levels were seen in immunosuppressed gerbils. Following HEV infection, serum ALT levels were increased, with lower and delayed peaks observed in immunosuppressed compared to immunocompetent gerbils. In immunocompetent gerbils, foci of apoptotic hepatocytes were detected that were distributed with inflammatory infiltrates containing CD68+ macrophages. However, these foci were absent in immunosuppressed gerbils. The immunosuppressed gerbils showed no inflammation with no increase in CD68+ macrophages despite high virus replication in liver. Our findings suggest adaptive immune responses are necessary for inducing hepatocyte apoptosis, CD68+ macrophage recruitment, and inflammatory cell infiltration in response to HEV infection. Our studies show that Mongolian gerbils provide a promising model to study pathogenesis during acute and persistent HEV infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Psoriasis is a persistent inflammatory skin disorder driven by T cells. The disease is characterized by aberrant keratinocytes (KCs) differentiation, epidermal proliferation, and excessive hyperplasia of veins and arteries. The purpose of the study was to identify the levels of circulating lnc-HULC , miR-122 , and Sirtuin 1 ( SIRT-1 ) in psoriatic patients, evaluate their possible roles as diagnostic biomarkers, and link their levels with the development of metabolic syndrome during psoriasis progression. This study included 176 participants. The subjects were divided into four groups, with 44 participants in each group. All patients have undergone a complete history taking and clinical examination. Laboratory investigations included Low-density lipoprotein (LDL), High-density lipoprotein (HDL), Triglycerides (TG), Fasting blood sugar (FBS), and cholesterol plasma levels. Serum levels of miR-122 and lnc-HULC were examined by qRT-PCR. Serum levels of SIRT-1 were examined by ELISA. The serum concentrations of lnc-HULC and miR-122 were significantly higher in psoriatic participants compared to controls. Psoriatic patients' serum concentrations of SIRT-1 were much lower than those of healthy individuals. There was a negative association between SIRT-1 concentration and BMI, disease duration, PASI score, LDL, and cholesterol levels. The blood levels of lnc-HULC , miR-122 , and SIRT-1 in psoriasis patients provide a promising role as diagnostic biomarkers in patients with and without metabolic syndrome., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors.)

Central nervous (CNS) and respiratory systems are routinely investigated in safety pharmacology core battery studies. For small molecules, the assessment of both vital organ systems is frequently done in rats in two distinct studies. With the advent of a miniaturized technology of jacketed external telemetry for rats (DECRO system), the simultaneous assessment of modified Irwin's or functional observational battery (FOB) test and respiratory (Resp) studies has become possible within a single study. Therefore, the objectives of this study were to perform the FOB and the Resp studies simultaneously in pair-housed rats fitted with jacketed telemetry, and to assess the feasibility and the outcome of this combination in control, baclofen, caffeine, and clonidine treated groups, i.e., with three agents having both respiratory and CNS effects. Our results provided evidence that performing both Resp and FOB assessment simultaneously in the same rat was feasible and the outcome was successful. The expected CNS and respiratory effects of the 3 reference compounds were accurately captured in each assay confirming the results' relevance. In addition, heart rate and activity level were recorded as additional parameters making this design as an enhanced approach for nonclinical safety assessment in rats. This work provides clear evidence that the "3Rs" principles can be effectively applied in core battery safety pharmacology studies while remaining in compliance with worldwide regulatory guidelines. Both reduction in animal use and refinements in procedures are demonstrated with this model., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Parental alienation is a relatively newly described disorder, with a growing prevalence, as divorce and custody battles are becoming more and more complex with increased difficulty of joint custody. In parental alienation, one parent, the alienating parent, forms an alliance with the child involved in the custody dispute and manages to effectively alienate the targeted parent completely. The child and the alienating parent manifest a form of folie à deux and, hence, are in complete synchrony in the hatred and denigration of the targeted parent. Issues, such as potentially false allegations of sexual, physical, and emotional abuse of the child by the targeted parent, arise. The child and the alienating parent become mutually convinced of the targeted parent's transgressions. Consequently, it becomes difficult for the courts and psychiatric professionals to differentiate true abuse from parental alienation., Case Presentation: In this case study, we aimed to conduct an in-depth psychological and psychiatric evaluation of a Lebanese family (white race) where a father was wrongly accused by the mother and his 11-year-old white boy of both physical and sexual abuse. The data for this study were collected through unstructured and semi-structured interviews, observations, and psychological tests (Rorschach test for the parents and Blacky test for the child), and through the analysis of documented evidence presented in the trial., Conclusion: This case manifested most criteria set forth for the diagnosis of parental alienation and created serious doubt regarding the validity of the allegations set forth by the mother and the child. Uncovered parental alienation often misleads mental health professionals at the expense of the child's mental health ., (© 2023. The Author(s).)

Purpose: to evaluate the relationship between religious hallucinations and religious coping among Lebanese patients with schizophrenia., Methods: We have studied the prevalence of religious hallucinations (RH) among 148 hospitalized Lebanese patients suffering from schizophrenia or schizoaffective disorder in November 2021 exhibiting religious delusions (RD), and their relationship with religious coping using the brief Religious Coping Scale (RCOPE). The PANSS scale was used to evaluate psychotic symptoms., Results: After adjustment over all variables, more psychotic symptoms (higher total PANSS scores) (aOR = 1.02) and more religious negative coping (aOR = 1.11) were significantly associated with higher odds of having religious hallucinations, whereas watching religious programs (aOR = 0.34) was significantly associated with lower odds of having religious hallucinations., Conclusion: This paper highlights the important role of religiosity that has to play in the formation of religious hallucinations in schizophrenia. Significant association was found between negative religious coping and the emergence of religious hallucinations., (© 2023. The Author(s).)

Background: To harmonize assays for detection of HEV RNA, a World Health Organization International Standard (WHO IS) was established. The WHO IS represents the highest order standard for HEV RNA but is limited in quantity. Secondary standards are needed to limit the use of WHO IS and minimize the need to replace it., Objective: Establish secondary standards for HEV NAAT assays and to calibrate these against the WHO IS., Methods: Stocks of genotype 3 HEV were prepared using both cell lysates and cell culture supernatants to produce non-enveloped and quasi-enveloped virus stocks, respectively. Both stocks were heat-inactivated, diluted in negative human plasma, and lyophilized to produce two candidate secondary standards: HEV-RR (non-enveloped virus) and HEV-RR.1 (quasi-enveloped virus). Both candidate standards were characterized and calibrated against the WHO IS for HEV RNA in an international collaborative study., Results: The collaborative study returned a total of 15 data sets, with different RNA extraction and amplification methods. The estimated mean values relative to the WHO IS (250,000 IU/ml) are 229,000 IU/ml and 355,000 IU/ml for HEV-RR and HEV-RR.1, respectively., Conclusion: We have established two secondary standards for HEV RNA calibrated against the WHO IS. These standards are non-infectious and stable under different storage temperatures., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Dengue virus (DENV) is a disease-causing agent normally transmitted from person to person through the bite of an infected mosquito. In addition to mosquito-borne cases of dengue, there are instances of transmission of dengue after receipt of blood products or donor organs or tissue. To improve blood safety, we developed a quantitative risk assessment model to estimate the dengue risk of transmission to blood transfusion recipients from preclinical and subclinical blood donors. We derived predictive coefficients from model simulations for predicting the risk outcomes such as monthly infectious blood units and transfusion-transmitted DENV cases based on the rate of reported clinical cases. The model was validated with a previous study where donor blood samples from the 2012 dengue transmission season in Rio de Janeiro, Brazil were tested for DENV RNA by a transcription-mediated amplification (TMA) assay. In that study, about 69·4% of donations were tested by the TMA assay and 78 samples were found positive, indicating that 112 DENV RNA-positive donations would have been detected if testing screening had been performed on all donations. Our model estimated a mean of 93 (2.5-97.5th%ile: 47-186) infected units among the donations, which was consistent with the reported numbers., (Published 2022. This article is a U.S. Government work and is in the public domain in the USA.)

Respiratory and cardiovascular systems are among the vital organ systems that should be studied in safety pharmacology core battery test. Non-invasive jacketed external telemetry technology that enables concomitant monitoring of both systems has been available and used widely for non-rodent species. Recently, the DECRO system, a miniaturized technology system in line with the "3Rs" principles, has been developed to provide a similar approach in rats. However, data to evaluate this system in socially-housed rats is lacking. Therefore, the objectives of this study were to determine the tolerability and the material integrity of this novel solution in pair-housed rats in two conditions: i) in a single session of 22 h simulating a stand-alone safety pharmacology study design, and ii) in three repeated sessions of 22 h each, simulating the inclusion of safety pharmacology endpoints in a 1-month toxicology study. In both conditions, the GABA B receptor agonist baclofen was used as a reference compound inducing cardiorespiratory changes. Our results provided evidence that this novel solution was well tolerated, the material was resistant to deterioration and that it allowed the accurate recording, in a non-invasive manner, of cardiorespiratory parameters and activity level in freely moving, pair-housed rats in the above two conditions. In addition, the expected respiratory depressant effects of baclofen were recorded. These results pave the way for considering this novel solution as an enhanced approach for nonclinical safety assessment in rats., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: TF is cofounder at ETISENSE SAS (www.etisense.com) and RF is a former employee of ETISENSE. ETISENSE supported this study by lending the DECRO system and providing the connected jackets., (Copyright © 2022. Published by Elsevier Inc.)

Background and Purpose: HERG blocking drugs known for their propensity to trigger Torsades de Pointes (TdP) were reported to induce a sympatho-vagal coactivation and to enhance High Frequency heart rate (HFHR) and QT oscillations (HFQT) in telemetric data. The present work aimed to characterize the underlying mechanism(s) leading to these autonomic changes., Experimental Approach: Effects of 15 torsadogenic hERG blocking drugs (astemizole, chlorpromazine, cisapride, droperidol, ibutilide, dofetilide, haloperidol, moxifloxacin, pimozide, quinidine, risperidone, sotalol, sertindole, terfenadine, and thioridazine) were assessed by telemetry in beagle dogs. Haemodynamic effects on diastolic and systolic arterial pressure were analysed from the first doses causing QTc prolongation and/or HFQT oscillations enhancement. Autonomic control changes were analysed using the high frequency autonomic modulation (HFAM) model., Key Results: Except for moxifloxacin and quinidine, all torsadogenic hERG blockers induced parasympathetic activation or sympatho-vagal coactivation combined with enhancement of HFQT oscillations. These autonomic effects result from reflex compensatory mechanisms in response to mild haemodynamic side effects. These haemodynamic mechanisms were characterized by transient HR acceleration during HF oscillations. A phenomenon of concealed QT prolongation was unmasked for several torsadogenic hERG blockers under β-adrenoceptor blockade with atenolol. Resulting enhancement of HFQT oscillations was shown to contribute directly to triggering dofetilide-induced ventricular arrhythmias., Conclusion and Implications: This work supports for the first time a contribution of haemodynamic side properties to ventricular arrhythmias triggered by torsadogenic hERG blocking drugs. These haemodynamic side effects may constitute a second component of their arrhythmic profile, acting as a trigger alongside their intrinsic arrhythmogenic electrophysiological properties., (© 2022 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background: Active recovery is believed to offer positive benefits related to exercise by improving recovery and potentially managing several symptoms following strenuous exercise. The current study aimed to verify the effects of a session of low-volume and uphill high-intensity interval exercise on muscle soreness and exercise performance within the recovery period after an exercise-induced muscle damage protocol., Methods: Thirty-one young physically active subjects completed two identical test sessions following an exercise-induced muscle damage protocol, separated by a three-week period, in which they performed uphill high-intensity interval exercise or a passive recovery. The uphill high-intensity interval exercise consisted of four bouts of 30 seconds at maximum velocity, interspersed by 4 minutes of passive rest on an uphill treadmill. Rating of perceived exertion, muscle soreness, serum concentration of Creatine Kinase, muscle circumference, countermovement jump, sprint time, and 1 repetition maximum strength of quadriceps femoris were measured. The assessments were made for four consecutive days, before the exercise-induced muscle damage protocol and 24, 48, and 72 hours afterwards., Results: A significant effect of time was found for all the outcome measures, but there were no significant differences between groups either in pain perception, muscle damage variables, nor in performance outcome measures at any point of time (P>0.05)., Conclusions: Uphill high-intensity interval exercise performed after an exercise-induced muscle damage protocol does not exacerbate muscle soreness or worsens exercise performance in comparison with passive recovery.

Introduction: The release of troponin in patients with acute pulmonary embolism (PE) is assumed to be secondary to elevated intracardiac chamber pressure. Since right ventricular (RV) hypertrophy does not develop in the acute setting, pressure overload correlates with chamber dilatation, causing myocardial injury. The aim of the present study was to investigate correlations between cardiac chamber volume, troponin and subsequent early mortality in patients with acute PE and refine risk stratification., Materials and Methods: Patients who underwent a computerized tomographic pulmonary angiogram (CTPA) and a troponin test within 24 h of the CTPA were included. Automated software calculated the volumes of the four cardiac chambers indexed to body surface area (BSA) and correlated them to troponin and early all-cause mortality., Results: The final cohort consisted of 370 patients (56% females) with acute PE. RV volume and right to left ventricular volume ratio (VVR) were the most significant indicators for elevated troponin (receiving operating characteristic [ROC] 0.796, confidence interval [CI]: 0.749-0.843, p < 0.001, and ROC 0.802, CI: 0.753-0.851, p < 0.001, respectively). VVR cutoff values, which are predictive of elevated troponins, correlated with higher 30-day mortality (odds ratio = 3.1, CI 1.5-6.7, p = 0.003) for a VVR >3 compared to a VVR <2., Conclusion: Cardiac chamber volume correlates to elevated troponin in patients with acute PE. A higher VVR reflects an increased likelihood for myocardial ischemia, as well as an increased short-term mortality risk. These data are available seconds after CTPA performance and may contribute to refining patients' risk stratification., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

While high risk of failure is an inherent part of developing innovative therapies, it can be reduced by adherence to evidence-based rigorous research practices. Supported through the European Union's Innovative Medicines Initiative, the EQIPD consortium has developed a novel preclinical research quality system that can be applied in both public and private sectors and is free for anyone to use. The EQIPD Quality System was designed to be suited to boost innovation by ensuring the generation of robust and reliable preclinical data while being lean, effective and not becoming a burden that could negatively impact the freedom to explore scientific questions. EQIPD defines research quality as the extent to which research data are fit for their intended use. Fitness, in this context, is defined by the stakeholders, who are the scientists directly involved in the research, but also their funders, sponsors, publishers, research tool manufacturers, and collaboration partners such as peers in a multi-site research project. The essence of the EQIPD Quality System is the set of 18 core requirements that can be addressed flexibly, according to user-specific needs and following a user-defined trajectory. The EQIPD Quality System proposes guidance on expectations for quality-related measures, defines criteria for adequate processes (i.e. performance standards) and provides examples of how such measures can be developed and implemented. However, it does not prescribe any pre-determined solutions. EQIPD has also developed tools (for optional use) to support users in implementing the system and assessment services for those research units that successfully implement the quality system and seek formal accreditation. Building upon the feedback from users and continuous improvement, a sustainable EQIPD Quality System will ultimately serve the entire community of scientists conducting non-regulated preclinical research, by helping them generate reliable data that are fit for their intended use., Competing Interests: AB AB is an employee and/or shareholder at PAASP GmbH, PAASP US LLC, Exciva GmbH, Synventa LLC, Ritec Pharma, RB, MA, LB, Nd, EC, AD, RF, VG, SH, MH, MK, MP, PP, EP, LR, GR, MR, JS, MS, CS, VV, JV, KW No competing interests declared, AG AG are employees of Janssen / Johnson & Johnson and shareholders at Johnson & Johnson, BG, CE BG and CE are employees and shareholders at PAASP GmbH. JG JG is an employee of AAALAC International that is an EQIPD Associated Collaborator. VC, CF VC and CFC are employees of Porsolt. IL, FD IAL and FD are employees of Sanofi. LM LM is an employee and shareholder of UCB. SB SB is an employee of Novartis Pharma. BA BA is an employee and shareholder of Pfizer. The views and opinions expressed in this article are those of the individual author and should not be attributed to Pfizer, its directors, officers, employees, affiliates, or any organization with which the author is employed or affiliated. CF AB, BA, NdB, UD, CFB, PK, MK, MM, PM, PP, GR, JS, and TS are members of the Preclinical Data Forum (co-chairs - AB and TS), a network financially and organizationally supported by ECNP and Cohen Veterans Bioscience. PK PK is an employee and shareholder at PAASP US LLC. CK UD and CK receive funding from Volkswagen Foundation, PM PM is owner of Cerbascience Consulting, HP HP has received during the last three years consulting and speaking fees and/or funding for collaborative projects from Bayer, Roche, Zogenix, and Eisai. KW KW is a consultant of Avertim, Brussels, Belgium, support for this contribution was funded by Janssen Pharmaceutica NV. MM MM, UD and TS are members of the Advisory Board at PAASP. MM, UD and TS are members of the ARRIVE guidelines working group. UD UD and CK receive funding from Volkswagen Foundation. MM, UD and TS are members of the Advisory Board at PAASP. MM, UD and TS are members of the ARRIVE guidelines working group. TS MM, UD and TS are members of the Advisory Board at PAASP. MM, UD and TS are members of the ARRIVE guidelines working group. TS is an AAALAC ad-hoc specialist. TS and AG are employees of Janssen / Johnson & Johnson and shareholders at Johnson & Johnson., (© 2021, Bespalov et al.)

Older age at the time of infection with hepatitis viruses is associated with an increased risk of liver fibrosis progression. We hypothesized that the pace of fibrosis progression may reflect changes in gene expression within the aging liver. We compared gene expression in liver specimens from 54 adult donors without evidence of fibrosis, including 36 over 40 y old and 18 between 18 and 40 y old. Chitinase 3-like 1 ( CHI3L1 ), which encodes chitinase-like protein YKL-40/CHI3L1, was identified as the gene with the greatest age-dependent increase in expression in liver tissue. We investigated the cellular source of CHI3L1 in the liver and its function using liver tissue specimens and in vitro models. CHI3L1 expression was significantly higher in livers of patients with cirrhosis of diverse etiologies compared with controls represented by patients who underwent liver resection for hemangioma. The highest intrahepatic CHI3L1 expression was observed in cirrhosis due to hepatitis D virus, followed by hepatitis C virus, hepatitis B virus, and alcohol-induced cirrhosis. In situ hybridization of CHI3L1 messenger RNA (mRNA) identified hepatocytes as the major producers of CHI3L1 in normal liver and in cirrhotic tissue, wherein hepatocytes adjacent to fibrous septa showed higher CHI3L1 expression than did those in more distal areas. In vitro studies showed that recombinant CHI3L1 promotes proliferation and activation of primary human hepatic stellate cells (HSCs), the major drivers of liver fibrosis. These findings collectively demonstrate that CHI3L1 promotes liver fibrogenesis through a direct effect on HSCs and support a role for CHI3L1 in the increased susceptibility of aging livers to fibrosis progression., Competing Interests: The authors declare no competing interest.

Zika virus (ZIKV) caused a public health threat in the United States in 2016, leading to rapid development and implementation of blood screening assays for ZIKV RNA. Several ZIKV sequences from clinical cases have been reported, but none from asymptomatic/pre-symptomatic infections. We isolated and sequenced ZIKV from asymptomatic/pre-symptomatic blood donor (ABD-ZIKV) samples and compared with reported clinical sequences. Twelve ABD-ZIKV isolates were produced from 67 cultivated samples, and isolates were genetically similar among themselves. Most isolates shared mutations with the clinical isolate PRVABC59 2015, whereas two ABD-ZIKV isolates shared specific mutations with U.S. clinical isolates from 2016. The ABD-ZIKV strains clustered into two distinct subclades: one comprised mostly ABD-ZIKV from Puerto Rico, and another one comprised ABD-ZIKV from Florida and QTX-02 isolate (Puerto Rico). In this study, we showed the circulation of two slightly distinct virus strains among Puerto Rico blood donors, one of which was also reported in Florida.

Infections with dengue virus (DENV), West Nile virus (WNV) and Zika virus (ZIKV) usually present similar mild symptoms at early stages, and most infections (~80%) are asymptomatic. However, these infections may progress to severe disease with different clinical manifestations. In this study we attempted to identify unique characteristics for each infection at the presymptomatic/asymptomatic stage of infection and compared levels of soluble immune markers that have been shown to be altered during clinical course of these viral infections. Levels of soluble markers were determined by Luminex-based assays or by ELISA in plasma samples from asymptomatic blood donors who were reactive for RNA from DENV (n = 71), WNV (n = 52) or ZIKV (n = 44), and a control or non-infected (NI) group (n = 22). Results showed that even in the absence of symptoms, increased interleukin (IL) levels of IL-12, IL-17, IL-10, IL-5, CXCL9, E-Selectin and ST2/IL-1R4; and decreased levels of IL-13 and CD40 were found in all flavivirus group samples, compared to those from NI donors. DENV-infected donors demonstrated variation in expression of IL-1ra and IL-2; WNV-infected donors demonstrated variation in expression of IL-1ra, P-Selectin, IL-4 and IL-5; ZIKV-infected donors demonstrated variation in expression of IL-1ra, P-Selectin, IL-4, RANK-L, CD40L and C3a. The findings suggest that, even in the presymptomatic/asymptomatic phase of the infection, different immunomodulation profiles were associated with DENV, WNV and ZIKV infections.

In 2015, Zika virus (ZIKV) appeared as an emerging pathogen, generating a global and urgent need for accurate diagnostic devices. During this public health crisis, several nucleic acid testing (NAT)-based Zika assays were submitted to the US Food and Drug Administration (FDA) for Emergency Use Authorization. The FDA's Center for Devices and Radiological Health, in collaboration with the FDA's Center for Biologics Evaluation and Research, responded to this Zika emergency by developing and producing a reference panel (RP) for Zika RNA (Zika FDA-RP) suitable for performance assessment of ZIKV NAT-based in vitro diagnostic devices. Reference panels are a fundamental tool for performance assessment of molecular tests. The panel is composed of five vials: two different heat-inactivated ZIKV strains (PRVABC59 and FSS13025) in concentrated stocks and three blinded concentrations prepared from those strains. The Zika FDA-RP was shared with developers who had devices in the final stages of validation. In vitro diagnostic developers tested the Zika FDA-RP using the FDA-provided protocol. Depending on sample type, 85% (12/14) of the NAT assays had analytical sensitivities between 500 and 5000 RNA NAT-detectable units/mL (NDUs/mL). One device showed better performance (100 NDUs/mL), and another one showed lower performance (10,000 to 30,000 NDUs/mL). Vials of the Zika FDA-RP are available on request to developers who have interacted with the FDA through the review process., (Published by Elsevier Inc.)

Background: The emergence of Zika virus (ZIKV) in 2015 to 2016 created a global public health crisis and an urgent need for accurate detection assays. Nucleic acid testing (NAT) is the most specific and sensitive technology for early detection of ZIKV. Various NAT protocols have been created, but until recently, assessment of assay performance and comparative studies were hampered by the lack of available standards and reference reagents., Study Design and Methods: The Center for Biologics Evaluation and Research/Food and Drug Administration responded to this crisis with the generation of two ZIKV-RNA reference reagents (ZIKV-RRs) for use in the development, validation, and assessment of performance of ZIKV-NAT assays. These reagents were produced from heat-inactivated (HI) ZIKV culture supernatant stock from two strains (PRVABC59 and FSS13025) diluted in dialyzed, defibrinated human plasma and lyophilized for evaluation in collaborative studies. The liquid, HI stock had been shared with the Paul-Ehrlich-Institute (Germany) and were included in the collaborative validation studies for the World Health Organization International Standard for ZIKV (WHO ZIKV IS)., Results: NAT-detectable units (NDUs)/mL were determined in a collaborative study that led to the assignment of 5.77 log NDUs/mL for PRVABC59 and 5.54 log NDUs/mL for FSS13025 as the final concentrations of the FDA ZIKV-RRs., Conclusion: We have established well-characterized reference reagents for ZIKV to facilitate evaluation of existing NAT assays and development of novel ZIKV assays which are correlated to that of the First WHO ZIKV IS. Vials of the ZIKV-RRs are available to qualified organizations upon request., (© 2018 AABB.)

We report here the complete genome sequences of two Zika virus strains (FSS13025 and PRVABC59) used for formulation of CBER/FDA RNA reference reagents and lot release panels for use with nucleic acid technology (NAT) testing.

Background: Lymph node status is one of the most important prognostic factors in endometrial cancer and crucial for deciding adjuvant therapy., Objective: The aim of the study was to assess the different models used to predict lymphatic nodal disease., Search Strategy: A literature search was conducted to detect the relevant studies., Inclusion Criteria: Relevant papers comparing the preoperative modality with the final histopathological results including randomized clinical trials, case-control studies, and any publications with a minimum of 50 patients in the report., Results: Molecular-based predictors are still far from a practical application. Preoperative radiological scans (positron emission tomography, computed tomography, magnetic resonance imaging, and ultrasound) have shown the best predictor of lymphatic dissemination. However, there is currently no ideal model available, which can be used within standard clinical care., Conclusions: Surgical staging still remains the criterion standard in the determination of lymph node status in endometrial cancer.

In this study the selection of in vivo predictive in vitro dissolution experimental set-ups using a multivariate analysis approach, in line with the Quality by Design (QbD) principles, is explored. The dissolution variables selected using a design of experiments (DoE) were the dissolution apparatus [USP1 apparatus (basket) and USP2 apparatus (paddle)], the rotational speed of the basket/or paddle, the operator conditions (dissolution apparatus brand and operator), the volume, the pH, and the ethanol content of the dissolution medium. The dissolution profiles of two nifedipine capsules (poorly soluble compound), under conditions mimicking the intake of the capsules with i. water, ii. orange juice and iii. an alcoholic drink (orange juice and ethanol) were analysed using multiple linear regression (MLR). Optimised dissolution set-ups, generated based on the mathematical model obtained via MLR, were used to build predicted in vitro-in vivo correlations (IVIVC). IVIVC could be achieved using physiologically relevant in vitro conditions mimicking the intake of the capsules with an alcoholic drink (orange juice and ethanol). The multivariate analysis revealed that the concentration of ethanol used in the in vitro dissolution experiments (47% v/v) can be lowered to less than 20% v/v, reflecting recently found physiological conditions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The impact of hydrodynamics and media composition on nifedipine dissolution profile from IR (immediate release) soft capsules was investigated using dissolution apparatus USP1, USP2, USP3 and USP4 (United State Pharmacopoeia). Media composition was varied in terms of pH and content, to mimic the dosage form intake with water or non-alcoholic beverages (orange juice) and alcoholic beverages (orange juice/ethanol mixture (47% v/v)). Through construction of in vitro-in vivo correlations (IVIVC) with corresponding in vivo data from the literature, it was possible to evaluate the in vitro conditions that are likely to simulate the in vivo formulation behaviour. Both linear and nonlinear correlations were obtained depending on experimental set-ups. Testing of 20mg nifedipine capsules in FaSSGFst (Fasted State Simulated Gastric Fluid pH 1.6; water administration) produced IVIVC with the USP3 (after time scaling) and USP4 apparatus. IVIVC were obtained for USP2, USP3 and USP4 in FaSSGFoj (Fasted State Simulated Gastric Fluid pH 3.4; orange juice administration). Linear and nonlinear correlations were obtained with the USP1, USP2 and USP3 apparatus when testing the capsules in FaSSGFoj/EtOH (orange juice/ethanol administration). This study highlighted that selection of physiologically relevant dissolution set-ups is critical for predicting the in vivo impact of formulations co-administration with water, non-alcoholic and alcoholic beverages., (Copyright © 2015 Elsevier B.V. All rights reserved.)