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Abstract Torpor encompasses diverse adaptations to extreme environmental stressors such as hibernation, aestivation, brumation, and daily torpor. Here we introduce StrokeofGenus, an analytic pipeline that identifies distinct transcriptomic states and shared gene expression patterns across studies, tissues, and species. We use StrokeofGenus to study multiple and diverse forms of torpor from publicly-available RNA-seq datasets that span eight species and two classes. We identify three transcriptionally distinct states during the cycle of heterothermia: euthermia, torpor, and interbout arousal. We also identify torpor-specific gene expression patterns that are shared both across tissues and between species with over three hundred million years of evolutionary divergence. We further demonstrate the general sharing of gene expression patterns in multiple forms of torpor, implying a common evolutionary origin for this process. Although here we apply StrokeofGenus to analysis of torpor, it can be used to interrogate any other complex physiological processes defined by transient transcriptomic states.

Abstract Health risks due to preventable infections such as human papillomavirus (HPV) are exacerbated by persistent vaccine hesitancy. Due to limited sample sizes and the time needed to roll out, traditional methodologies like surveys and interviews offer restricted insights into quickly evolving vaccine concerns. Social media platforms can serve as fertile ground for monitoring vaccine-related conversations and detecting emerging concerns in a scalable and dynamic manner. Using state-of-the-art large language models, we propose a minimally supervised end-to-end approach to identify concerns against HPV vaccination from social media posts. We detect and characterize the concerns against HPV vaccination pre- and post-2020 to understand the evolution of HPV vaccine discourse. Upon analyzing 653 k HPV-related post-2020 tweets, adverse effects, personal anecdotes, and vaccine mandates emerged as the dominant themes. Compared to pre-2020, there is a shift towards personal anecdotes of vaccine injury with a growing call for parental consent and transparency. The proposed approach provides an end-to-end system, i.e. given a collection of tweets, a list of prevalent concerns is returned, providing critical insights for crafting targeted interventions, debunking messages, and informing public health campaigns.

PurposeTo characterize the effect of sampling window size on maps of foveal cone density derived from adaptive optics scanning light ophthalmoscope (AOSLO) images of the cone mosaic.MethodsForty-four AOSLO-derived montages of the foveal cone mosaic (300 x 300µm) were used for this study (from 44 individuals with normal vision). Cone photoreceptor coordinates were semi-automatically identified by one experienced grader. From these coordinates, cone density matrices across each foveal montage were derived using 10 different sampling window sizes containing 5, 10, 15, 20, 40, 60, 80, 100, 150, or 200 cones. For all 440 density matrices, we extracted the location and value of peak cone density (PCD), the cone density centroid (CDC) location, and cone density at the CDC.ResultsAcross all window sizes, PCD values were larger than those extracted at the CDC location, though the difference between these density values decreased as the sampling window size increased (p

Lymphocytic choriomeningitis virus is an underreported cause of miscarriage and neurologic disease. Surveillance remains challenging because of nonspecific symptomatology, inconsistent case reporting, and difficulties with diagnostic testing. We describe a case of acute lymphocytic choriomeningitis virus disease in a person living with HIV in Connecticut, USA, identified by using quantitative reverse transcription PCR.

Abstract In the development of end-to-end large-scale live virus vaccine (LVV) manufacturing, process analytical technology (PAT) tools enable timely monitoring of critical process parameters (CPP) and significantly guide process development and characterization. In a commercial setting, these very same tools can enable real time monitoring of CPPs on the shop floor and inform harvest decisions, predict peak potency, and serve as surrogates for release potency assays. Here we introduce the development of four advanced PAT tools for upstream and downstream process monitoring in LVV manufacturing. The first tool explores the application of capacitance probes for real time monitoring of viable cell density in bioreactors. The second tool utilizes high content imaging to determine optimum time of infection in a microcarrier process. The third tool uses flow virometry (or nanoscale flow cytometry) to monitor total virus particle counts across upstream and downstream process steps and establishes a robust correlation to virus potency. The fourth and final tool explores the use of nucleic acid dye staining to discriminate between “good” and “damaged” virus particles and uses this strategy to also monitor virus aggregates generated sometimes during downstream processing. Collectively, these tools provide a comprehensive monitoring toolbox and represent a significantly enhanced control strategy for the manufacturing of LVVs.

Abstract Background Despite females comprising 50.8% of the U.S. population, the percentage of females in the physician workforce is only 36.3%. Studies have examined sex trends within select specialties, however there is insufficient literature studying trends across all specialties. In this study, the authors examined trends in the proportion of female residents from 2011 to 2019 across all specialties, including both surgical and non-surgical. Methods Data on the proportion of female residents from 2011 to 2019 in all specialties was extracted from the Accreditation Council for Graduate Medical Education (ACGME) Data Resource Books and analyzed with the chi-square test for trend. Results From 2011 to 2019, there was a statistically significant increase in the percentage of female residents in surgical specialties (p

Abstract The use of geographic information system (GIS) technologies to improve access to health is gaining momentum in Africa. This has become more pertinent with the increasing penetration of mobile-phone technology and internet use, and calls for innovative strategies to support implementation of the World Health Organization Sustainable Development Goals for universal health coverage on the continent. The huge potential benefits of GIS to advance health service delivery in Africa is, however, yet to be fully harnessed due to critical challenges such as proliferation of pilot projects, poor coordination, inadequate preparedness of the health workforce for GIS, lack of interoperability, and inadequate sustainable financing. To discuss these challenges and propose the way forward for rapid, cost-effective, and sustainable deployment of GIS, the African Regional GIS Summit was held in Brazzaville, Republic of the Congo, on 7–10 October 2019 under the umbrella of the AFRO GIS Centre.

Abstract This study examines retinas from a rat glaucoma model for oxidized nucleosides 8OHdG and 8OHG, biomarkers for oxidative damage of DNA and RNA, respectively. Immunohistochemical data indicate a predominant localization of 8OHdG/8OHG in retinal ganglion cells (RGCs). The levels for these oxidized DNA/RNA products were 3.2 and 2.8 fold higher at 1 and 2 weeks after intraocular pressure elevation compared to control retinas, respectively. 8OHdG/8OHG were almost exclusively associated with mitochondrial DNA/RNA: ~ 65% of 8OHdG/8OHG were associated with RNA isolated from mitochondrial fraction and ~ 35% with DNA. Furthermore, we analyzed retinas of the rd10 mouse, a model for retinitis pigmentosa, with severe degeneration of photoreceptors to determine whether high levels of 8OHdG/8OHG staining intensity in RGCs of control animals is related to the high level of mitochondrial oxidative phosphorylation necessary to support light-evoked RGC activity. No significant difference in 8OHdG/8OHG staining intensity between control and rd10 mouse retinas was observed. The results of this study suggest that high levels of 8OHdG/8OHG in RGCs of wild-type animals may lead to cell damage and progressive loss of RGCs observed during normal aging, whereas ocular hypertension-induced increase in the level of oxidatively damaged mitochondrial DNA/RNA could contribute to glaucomatous neurodegeneration.

Abstract In this study, we designed two deep neural networks to encode 16 features for early seizure detection in intracranial EEG and compared them and their frequency responses to 16 widely used engineered metrics to interpret their properties: epileptogenicity index (EI), phase locked high gamma (PLHG), time and frequency domain Cho Gaines distance (TDCG, FDCG), relative band powers, and log absolute band powers (from alpha, beta, theta, delta, low gamma, and high gamma bands). The deep learning models were pretrained for seizure identification on the time and frequency domains of 1 s, single-channel clips of 127 seizures (from 25 different subjects) using “leave-one-out” (LOO) cross validation. Each neural network extracted unique feature spaces that were interpreted using spectral power modulations before being used to train a Random Forest Classifier (RFC) for seizure identification. The Gini Importance of each feature was calculated from the pretrained RFC, enabling the most significant features (MSFs) for each task to be identified. The MSFs were extracted to train another RFC for UPenn and Mayo Clinic’s Seizure Detection Kaggle Challenge. They obtained an AUC score of 0.93, demonstrating a transferable method to identify and interpret biomarkers for seizure detection.

Abstract Background The recent 2018 Declaration of Astana recognized primary health care (PHC) as a means to achieve universal health coverage (UHC) and the health-related Sustainable Development Goals (SDGs). Following this declaration, country progress on operationalization of the PHC agenda and attainment of UHC has been stalled by the new challenges posed by the COVID-19 pandemic. The pandemic has also disrupted the continuity of essential health service provision and tested the resilience of the region’s health systems. Methods In accordance with this, the WHO Regional Office for Africa convened the Fifth Health Sector Directors’ Planning and Policy Meeting across the 47 Member States of the Region. The two-day forum focused on building health system resilience to facilitate service continuity during health threats, PHC revitalization, and health systems strengthening towards UHC. Results The Regional Forum provided evidence on building resilient health systems in the WHO African Region and engaged participants in meaningful and critical discussion. It is from these discussions that four key themes emerged: (1) working multisectorally/intersectorally, (2) moving from fragmentation to integration, (3) ensuring implementation and knowledge exchange, and (4) rethinking resilience and embracing antifragility. These discussions and associated groupings by thematic areas lend themselves to recommendations for the WHO. Conclusions This paper details the proceedings and key findings on building resilient health systems, the four themes that emerged from participant deliberation, and the recommendations that have emerged from the meeting. Deliberations from the Regional Forum are critical, as they have the potential to directly inform policy and program design, given that the meeting convenes health sector technocrats, who are at the helm of policy design, action, and implementation.

Abstract This article presents the construction of a multimodality platform that can be used for efficient destruction of brain tumor by a combination of photodynamic and sonodynamic therapy. For in vivo studies, U87 patient-derived xenograft tumors were implanted subcutaneously in SCID mice. For the first time, it has been shown that the cell-death mechanism by both treatment modalities follows two different pathways. For example, exposing the U87 cells after 24 h incubation with HPPH [3-(1′-hexyloxy)ethyl-3-devinyl-pyropheophorbide-a) by ultrasound participate in an electron-transfer process with the surrounding biological substrates to form radicals and radical ions (Type I reaction); whereas in photodynamic therapy, the tumor destruction is mainly caused by highly reactive singlet oxygen (Type II reaction). The combination of photodynamic therapy and sonodynamic therapy both in vitro and in vivo have shown an improved cell kill/tumor response, that could be attributed to an additive and/or synergetic effect(s). Our results also indicate that the delivery of the HPPH to tumors can further be enhanced by using cationic polyacrylamide nanoparticles as a delivery vehicle. Exposing the nano-formulation with ultrasound also triggered the release of photosensitizer. The combination of photodynamic therapy and sonodynamic therapy strongly affects tumor vasculature as determined by dynamic contrast enhanced imaging using HSA-Gd(III)DTPA.

Abstract Photobiomodulation (PBM) by far-red (FR) to near-infrared (NIR) light has been demonstrated to restore the function of damaged mitochondria, increase the production of cytoprotective factors and prevent cell death. Our laboratory has shown that FR PBM improves functional and structural outcomes in animal models of retinal injury and retinal degenerative disease. The current study tested the hypothesis that a brief course of NIR (830 nm) PBM would preserve mitochondrial metabolic state and attenuate photoreceptor loss in a model of retinitis pigmentosa, the P23H transgenic rat. P23H rat pups were treated with 830 nm light (180 s; 25 mW/cm2; 4.5 J/cm2) using a light-emitting diode array (Quantum Devices, Barneveld, WI) from postnatal day (p) 10 to p25. Sham-treated rats were restrained, but not treated with 830 nm light. Retinal metabolic state, function and morphology were assessed at p30 by measurement of mitochondrial redox (NADH/FAD) state by 3D optical cryo-imaging, electroretinography (ERG), spectral-domain optical coherence tomography (SD-OCT), and histomorphometry. PBM preserved retinal metabolic state, retinal function, and retinal morphology in PBM-treated animals compared to the sham-treated group. PBM protected against the disruption of the oxidation state of the mitochondrial respiratory chain observed in sham-treated animals. Scotopic ERG responses over a range of flash intensities were significantly greater in PBM-treated rats compared to sham controls. SD-OCT studies and histological assessment showed that PBM preserved the structural integrity of the retina. These findings demonstrate for the first time a direct effect of NIR PBM on retinal mitochondrial redox status in a well-established model of retinal disease. They show that chronic proteotoxic stress disrupts retinal bioenergetics resulting in mitochondrial dysfunction, and retinal degeneration and that therapies normalizing mitochondrial metabolism have considerable potential for the treatment of retinal degenerative disease.

Abstract The burden of antibiotic resistance is currently estimated by mathematical modeling, without real count of resistance to key antibiotics. Here we report the real rate of resistance to key antibiotics in bacteria isolated from humans during a 5 years period in a large area in southeast in France. We conducted a retrospective study on antibiotic susceptibility of 539,107 clinical strains isolated from hospital and private laboratories in south of France area from January 2014 to January 2019. The resistance rate to key antibiotics as well as the proportion of bacteria classified as Difficult-to-Treat (DTR) were determined and compared with the Mann–Whitney U test, the χ2 test or the Fisher’s exact test. Among 539,037 isolates, we did not observe any significant increase or decrease in resistance to key antibiotics for 5 years, (oxacillin resistance in Staphylococcus aureus, carbapenem resistance in enterobacteria and Pseudomonas aeruginosa and 3rd generation cephalosporin resistance in Escherichia coli and Klebsiella pneumoniae). However, we observed a significant decrease in imipenem resistance for Acinetobacter baumannii from 2014 to 2018 (24.19–12.27%; p = 0.005) and a significant increase of ceftriaxone resistance in Klebsiella pneumoniae (9.9–24.03%; p = 0.001) and Enterobacter cloacae (24.05–42.05%; p = 0.004). Of these 539,037 isolates, 1604 (0.3%) had a DTR phenotype. Over a 5-year period, we did not observe a burden of AR in our region despite a high rate of antibiotic consumption in our country. These results highlight the need for implementation of real-time AR surveillance systems which use factual data.

Abstract Cardiac stress is a critical determinant of outcomes associated with severe thermal injury. The cardiovascular response to a catecholamine‐mediated surge from severe burns passes through two phases. Initial hypovolemia with myocardial depression leads to a low cardiac output, which then progresses to a hyperdynamic‐hypermetabolic phase with increased cardiac output.

Summary: Background: Radiation therapy (RT) has a suboptimal effect in patients with pancreatic ductal adenocarcinoma (PDAC) due to intrinsic and acquired radioresistance (RR). Comprehensive bioinformatics and microarray analysis revealed that cholesterol biosynthesis (CBS) is involved in the RR of PDAC. We now tested the inhibition of the CBS pathway enzyme, farnesyl diphosphate synthase (FDPS), by zoledronic acid (Zol) to enhance radiation and activate immune cells. Methods: We investigated the role of FDPS in PDAC RR using the following methods: in vitro cell-based assay, immunohistochemistry, immunofluorescence, immunoblot, cell-based cholesterol assay, RNA sequencing, tumouroids (KPC-murine and PDAC patient-derived), orthotopic models, and PDAC patient's clinical study. Findings: FDPS overexpression in PDAC tissues and cells (P < 0.01 and P < 0.05) is associated with poor RT response and survival (P = 0.024). CRISPR/Cas9 and pharmacological inhibition (Zol) of FDPS in human and mouse syngeneic PDAC cells in conjunction with RT conferred higher PDAC radiosensitivity in vitro (P < 0.05, P < 0.01, and P < 0.001) and in vivo (P < 0.05). Interestingly, murine (P = 0.01) and human (P = 0.0159) tumouroids treated with Zol+RT showed a significant growth reduction. Mechanistically, RNA-Seq analysis of the PDAC xenografts and patients-PBMCs revealed that Zol exerts radiosensitization by affecting Rac1 and Rho prenylation, thereby modulating DNA damage and radiation response signalling along with improved systemic immune cells activation. An ongoing phase I/II trial (NCT03073785) showed improved failure-free survival (FFS), enhanced immune cell activation, and decreased microenvironment-related genes upon Zol+RT treatment. Interpretation: Our findings suggest that FDPS is a novel radiosensitization target for PDAC therapy. This study also provides a rationale to utilize Zol as a potential radiosensitizer and as an immunomodulator in PDAC and other cancers. Funding: National Institutes of Health (P50, P01, and R01).

BackgroundThe ligaments in the knee are prone to injury especially during dynamic activities. The resulting instability can have a profound impact on a patient's daily activities and functional capacity. Musculoskeletal knee modelling provides a non-invasive tool for investigating ligament force-strain behaviour in various dynamic scenarios, as well as potentially complementing existing pre-planning tools to optimise surgical reconstructions. However, despite the development and validation of many musculoskeletal knee models, the effect of modelling parameters on ligament mechanics has not yet been systematically reviewed.ObjectivesThis systematic review aimed to investigate the results of the most recent studies using musculoskeletal modelling techniques to create models of the native knee joint, focusing on ligament mechanics and modelling parameters in various simulated movements.Data sourcesPubMed, ScienceDirect, Google Scholar, and IEEE Xplore.Eligibility criteria for selecting studiesDatabases were searched for articles containing any numerical ligament strain or force data on the intact, ACL-deficient, PCL-deficient, or lateral extra-articular reconstructed (LER) knee joints. The studies had to derive these results from musculoskeletal modelling methods. The dates of the publications were between 1 January 1995 and 30 November 2021.MethodA customised data extraction form was created to extract each selected study's critical musculoskeletal model development parameters. Specific parameters of the musculoskeletal knee model development used in each eligible study were independently extracted, including the (1) musculoskeletal model definition (i.e., software used for modelling, knee type, source of geometry, the inclusion of cartilage and menisci, and articulating joints and joint boundary conditions (i.e., number of degrees of freedom (DoF), subjects, type of activity, collected data and type of simulation)), (2) specifically ligaments modelling techniques (i.e., ligament bundles, attachment points, pathway, wrapping surfaces and ligament material properties such as stiffness and reference length), (3) sensitivity analysis, (4) validation approaches, (5) predicted ligament mechanics (i.e., force, length or strain) and (6) clinical applications if available. The eligible papers were then discussed quantitatively and qualitatively with respect to the above parameters.Results and discussionFrom the 1004 articles retrieved by the initial electronic search, only 25 met all inclusion criteria. The results obtained by aggregating data reported in the eligible studies indicate that considerable variability in the predicted ligament mechanics is caused by differences in geometry, boundary conditions and ligament modelling parameters.ConclusionThis systematic review revealed that there is currently a lack of consensus on knee ligament mechanics. Despite this lack of consensus, some papers highlight the potential of developing translational tools using musculoskeletal modelling. Greater consistency in model design, incorporation of sensitivity assessment of the model outcomes and more rigorous validation methods should lead to better agreement in predictions for ligament mechanics between studies. The resulting confidence in the musculoskeletal model outputs may lead to the development of clinical tools that could be used for patient-specific treatments.

ObjectiveEstimate associations between the health-related quality of life (HRQoL) and adiposity in a low-income population.MethodsIn a cluster random sample of 3796 Filipinos aged 40-70 years in Nueva Ecija province, we measured body mass index (BMI), waist circumference (WC), waist-to-hip ratio (WHR), and six dimensions of HRQoL using the 20-item Short Form Health Survey. We stratified by sex and used nonparametric regression to graph mean HRQoL in each dimension by BMI, WC, and WHR. We used ordinary least squares regression to estimate differences in each HRQoL dimension by categories of BMI, WC, and WHR adjusted for sociodemographic characteristics and smoking.ResultsMean HRQoL was lowest for health perception (Males: 67.5 (SD = 15.9); Females: 66.7 (15.8)) and highest for role functioning (Males: 97.5 (12.9); Females: 97.4 (13.3)). Mean (SD) values of BMI, WC, and WHR were 22.1 (3.6), 84.8 cm (9.5), and 0.9 (0.1), respectively for males, and 23.7 (4.2), 86.5 cm (10.2), and 0.9 (0.1), respectively, for females. There was no evidence that higher BMI was associated with lower HRQoL. Adjusted mean social functioning was 4.92 (p = 0.076) higher for males with high BMI risk (8.6% prevalence) compared with acceptable BMI risk (50.3%). Mean social functioning was 3.61 (p = 0.012) and 5.48 (p = 0.017) lower for females with high WC (44.7%) and WHR (83.1%), respectively, compared with those with low WC (23.8%) and WHR (3.6%). Mean physical functioning was lower by 2.70 (p = 0.204) and 1.07 (p = 0.198) for males and females, respectively, with high compared with low WC. Mean physical functioning was 3.93 (p = 0.037) lower for males with high (7.6%) compared with low (38.8%) WHR. Mean role functioning was 1.09 (p = 0.124) and 2.46 (p = 0.158) lower for males with borderline and high WHR, respectively.ConclusionsThere is discordance between future adiposity-related health risk and current experience of HRQoL.

ObjectivePolypharmacy, defined as the concurrent use of multiple medications, is a growing concern globally. This study aimed to identify the significant factors that predict the perceived burden of medication and health-related quality of life.MethodsAdults, aged 18 years and above who have used at least two regular medicines, were invited to complete the study questionnaires between June and October 2019. Multiple linear regression analysis was conducted to identify significant predictors for perceived burden of medication and health-related quality of life.ResultsA total of 119 participants completed this study. The average age of the participants was 63 years (SD±16 years). Factors significantly predicting perceived burden of medication were participants' current health condition (p = 0.001), overall burden of treatment (pConclusionsThis study revealed that hypertensive participants in poor health tended to experience higher perceived burden of medication, which in turn was found to be correlated with lower perceived health-related quality of life.

Context: Diverticula are acquired or congenital outpouchings of the gastrointestinal tract, normally occurring at points of weakness in the wall of the alimentary canal. They can be present from esophagus to anus, with colonic diverticulosis accounting for the majority of cases. While often asymptomatic, diverticula of the gastrointestinal (GI) tract are clinically significant due to potential life-threatening complications. Familiarity with key epidemiologic, etiologic, clinical, imaging, and therapeutic features of various diverticula is therefore important. This review is meant to serve as a concise guide highlighting the distribution, epidemiology, presentation, classic imaging findings, and treatment for the spectrum of GI diverticula. Evidence Acquisition: We review fluoroscopic, computed tomography (CT), and magnetic resonance imaging (MRI) features of the most commonly encountered GI diverticula. Diverticula that are anatomically adjacent often share features such as symptomatology, optimal diagnostic imaging modality, and management. Thus below we categorize them by location along the GI tract. Esophageal (Zenker’s, Killian-Jamieson, midthoracic, epiphrenic), false and true gastric, small bowel (duodenal, jejunoileal, and Meckel’s), and large bowel (appendiceal, cecal, ascending/descending/sigmoid colonic) diverticula are discussed. Results and Conclusions: Although commonly incidental, diverticula of the GI tract can be clinically significant due to complications. This pictorial essay describes the epidemiologic, etiologic, clinical, and therapeutic features and imaging findings associated with GI diverticula encountered in clinical practice.

Abstract Background Inadequate access to quality health care services due to weak health systems and recurrent public health emergencies are impediments to the attainment of Universal Health Coverage and health security in Africa. To discuss these challenges and deliberate on plausible solutions, the World Health Organization Regional Office for Africa, in collaboration with the Government of Cabo Verde, convened the second Africa Health Forum in Praia, Cabo Verde on 26–28 March 2019, under the theme Achieving Universal Health Coverage and Health Security: The Africa We Want to See. Methods The Forum was conducted through technical sessions consisting of high-level, moderated panel discussions on specific themes, some of them preceded by keynote addresses. There were booth exhibitions by Member States, World Health Organization and other organizations to facilitate information exchanges. A Communiqué highlighting the recommendations of the Forum was issued during the closing ceremony. More than 750 participants attended. Relevant information from the report of the Forum and notes by the authors were extracted and synthesized into these proceedings. Conclusions The Forum participants agreed that the role of community engagement and participation in the attainment of Universal Health Coverage, health security and ultimately the Sustainable Development Goals cannot be overemphasized. The public sector of Africa alone cannot achieve these three interrelated goals; other partners, such as the private sector, must be engaged. Technological innovations will be a key driver of the attainment of these goals; hence, there is need to harness the comparative advantages that they offer. Attainment of the three goals is also intertwined – achieving one paves the way for achieving the others. Thus, there is need for integrated public health approaches in the planning and implementation of interventions aimed at achieving them. Recommendations To ensure that the recommendations of this Forum are translated into concrete actions in a sustainable manner, we call on African Ministers of Health to ensure their integration into national health sector policies and strategic documents and to provide the necessary leadership required for their implementation. We also call on partners to mainstream these recommendations into their ongoing support to World Health Organization African Member States.

Background: Trefoil factors (TFF1, TFF2, and TFF3) are small secretory molecules that recently have gained significant attention in multiple studies as an integral component of pancreatic cancer (PC) subtype-specific gene signature. Here, we comprehensively investigated the diagnostic potential of all the member of trefoil family, i.e., TFF1, TFF2, and TFF3 in combination with CA19.9 for detection of PC. Methods: Trefoil factors (TFFs) gene expression was analyzed in publicly available cancer genome datasets, followed by assessment of their expression in genetically engineered spontaneous mouse model (GEM) of PC (KrasG12D; Pdx1-Cre (KC)) and in human tissue microarray consisting of normal pancreas adjacent to tumor (NAT), precursor lesions (PanIN), and various pathological grades of PC by immunohistochemistry (IHC). Serum TFFs and CA19.9 levels were evaluated via ELISA in comprehensive sample set (n = 362) comprised of independent training and validation sets each containing benign controls (BC), chronic pancreatitis (CP), and various stages of PC. Univariate and multivariate logistic regression and receiver operating characteristic curves (ROC) were used to examine their diagnostic potential both alone and in combination with CA19.9. Findings: The publicly available datasets and expression analysis revealed significant increased expression of TFF1, TFF2, and TFF3 in human PanINs and PC tissues. Assessment of KC mouse model also suggested upregulated expression of TFFs in PanIN lesions and early stage of PC. In serum analyses studies, TFF1 and TFF2 were significantly elevated in early stages of PC in comparison to benign and CP control group while significant elevation in TFF3 levels were observed in CP group with no further elevation in its level in early stage PC group. In receiver operating curve (ROC) analyses, combination of TFFs with CA19.9 emerged as promising panel for discriminating early stage of PC (EPC) from BC (AUCTFF1+TFF2+TFF3+CA19.9 = 0.93) as well as CP (AUCTFF1+TFF2+TFF3+CA19.9 = 0.93). Notably, at 90% specificity (desired for blood-based biomarker panel), TFFs combination improved CA19.9 sensitivity by 10% and 25% to differentiate EPC from BC and CP respectively. In an independent blinded validation set, the combination of TFFs and CA19.9 (AUCTFF1+TFF2+TFF3+CA19.9 = 0.82) also improved the overall efficacy of CA19.9 (AUCCA19.9 = 0.66) to differentiate EPC from CP proving unique biomarker capabilities of TFFs to distinguish early stage of this deadly lethal disease. Interpretation: In silico, tissue and serum analyses validated significantly increased level of all TFFs in precursor lesions and early stages of PC. The combination of TFFs enhanced sensitivity and specificity of CA19.9 to discriminate early stage of PC from benign control and chronic pancreatitis groups. Keywords: Biomarker, Trefoil factor, TFF1, TFF2, TFF3, CA19.9, Pancreatic cancer

Introduction Prevalence rates of breastfeeding remain low even though the World Health Organization (WHO) and the American Academy of Pediatrics recommend exclusive breast feeding for the first 6 months of life in combination with appropriate complementary feeding beyond six 6 months of age. There have been several studies that address the implication of drinking animal milk and/or infant formula on children’s health and development when breast feeding is not offered during the first year of life. Vast improvements have been made in infant formula design, which may increase its benefits compared with animal’s milk. The objective of this review is therefore to synthesise the most recent evidence on the effects of the consumption of animal milk compared with infant formula in non-breastfed or mixed breastfed infants aged 6–11 months.Methods and analysis We will conduct a systematic review and meta-analysis of studies that assessed the effect of animal milk compared with formula or mixed-fed (breastmilk and formula) on infants aged 6–11 months. The primary outcomes of interest include anaemia, gastrointestinal blood loss, weight for age, height for age and weight for height. We will include randomised and non-randomised studies with a control group. We will use the Cochrane risk of bias tools to assess the risk of bias. We will use meta-analysis to pool findings if the identified studies are conceptually homogenous and data are available from more than one study. We will assess the overall quality of evidence using the Grading of Recommendations Assessment, Development, and Evaluation approach.Ethics and dissemination This is a systematic review, so no patients will be directly involved in the design or development of this study. The findings from this systematic review will be disseminated to relevant patient populations and caregivers and will guide the WHO’s recommendations on formula consumption versus animal milk in infants aged 6–11 months.Trial registration number CRD42020210925.

Social grooming in the animal kingdom is common and serves several functions, from removing ectoparasites to maintaining social bonds between conspecifics. We examined whether time spent grooming with others in a highly social mammal species was associated with infection status for gastrointestinal parasites. Of six parasites detected, one (Trichuris sp.) was associated with social grooming behaviors, but more specifically with direct physical contact with others. Individuals infected with Trichuris sp. spent significantly less time grooming conspecifics than those not infected, and time in direct contact with others was the major predictor of infection status. One model correctly predicted infection status for Trichuris sp. with a reliability of 95.17% overall when the variables used were time spent in direct contact and time spent grooming others. This decrease in time spent grooming and interacting with others is likely a sickness behavior displayed by individuals with less energy or motivation for non-essential behaviors. This study emphasizes the possible links between host behavior and parasitic infections and highlights the need for an understanding of a study population's parasitic infections when attempting to interpret animal behavior.

As of May 2021, over 286 million coronavirus 2019 (COVID-19) vaccine doses have been administered across the country. This data is promising, however there are still populations that, despite availability, are declining vaccination. We reviewed vaccine likelihood and receptiveness to recommendation from a doctor or nurse survey responses from 101,048 adults (≥18 years old) presenting to 442 primary care clinics in 8 states and the District of Columbia. Occupation was self-reported and demographic information extracted from the medical record, with 58.3% (n = 58,873) responding they were likely to receive the vaccine, 23.6% (n = 23,845) unlikely, and 18.1% (n = 18,330) uncertain. We found that essential workers were 18% less likely to receive the COVID-19 vaccination. Of those who indicated they were not already “very likely” to receive the vaccine, a recommendation from a nurse or doctor resulted in 16% of respondents becoming more likely to receive the vaccine, although certain occupations were less likely than others to be receptive to recommendations. To our knowledge, this is the first study to look at vaccine intent and receptiveness to recommendations from a doctor or nurse across specific essential worker occupations, and may help inform future early phase, vaccine rollouts and public health measure implementations.

Nuclear IGF1R has been linked to poor outcome in cancer. We recently showed that nuclear IGF1R phosphorylates PCNA and increases DNA damage tolerance. In this paper we aimed to describe this mechanism in cancer tissue as well as in cancer cell lines. In situ proximity ligation assay identified frequent IGF1R and PCNA colocalization in many cancer types. IGF1R/PCNA colocalization was more frequently increased in tumor cells than in adjacent normal, and more prominent in areas with dysplasia and invasion. However, the interaction was often lost in tumors with poor response to neoadjuvant treatment and most metastatic lesions. In two independent cohorts of serous ovarian carcinomas and oropharyngeal squamous cell carcinomas, stronger IGF1R/PCNA colocalization was significantly associated with a higher overall survival. Ex vivo irradiation of ovarian cancer tissue acutely induced IGF1R/PCNA colocalization together with γH2AX-foci formations. In vitro, RAD18 mediated mono-ubiquitination of PCNA during replication stress was dependent on IGF1R kinase activity. DNA fiber analysis revealed that IGF1R activation could rescue stalled DNA replication forks, but only in cancer cells with baseline IGF1R/PCNA interaction. We believe that the IGF1R/PCNA interaction is a basic cellular mechanism to increase DNA stress tolerance during proliferation, but that this mechanism is lost with tumor progression in conjunction with accumulated DNA damage and aberrant strategies to tolerate genomic instability. To exploit this mechanism in IGF1R targeted therapy, IGF1R inhibitors should be explored in the context of concomitant induction of DNA replication stress as well as in earlier clinical stages than previously tried.

ObjectivesTo examine interocular asymmetry of foveal avascular zone (FAZ) and parafoveal capillary density metrics in sickle cell retinopathy (SCR) using optical coherence tomography angiography (OCT-A).MethodsThis cross-sectional, retrospective study evaluated SCR patients and unaffected controls who underwent 3x3mm macular OCT-A imaging using a spectral domain-OCT system. FAZ (area, perimeter, and acircularity index) and parafoveal capillary density metrics were computed for both eyes of each participant. In unaffected controls, interocular difference in FAZ and parafoveal capillary density metrics were evaluated using Bland-Altman plots. SCR patients with interocular difference outside the upper 97.5% and lower 2.5% limits of agreement from controls were defined as having interocular asymmetry. Area under receiver operating characteristic curve (AROC) was also performed to determine the ability of the absolute interocular difference to differentiate between subjects with SCR-including non-proliferative SCR (NP-SCR) and proliferative SCR (P-SCR)-and unaffected controls.ResultsThirty-one patients with SCR (21 NP-SCR and 10 P-SCR) and 14 race-matched and age-matched controls were included for analysis. Interocular asymmetry was seen for all FAZ and parafoveal capillary density metrics in NP-SCR and P-SCR subjects. SCR subjects showed greater disease severity in the left-eye for FAZ and parafoveal capillary density metrics.ConclusionsNP-SCR and P-SCR patients demonstrated quantifiable interocular asymmetry in FAZ and parafoveal capillary density metrics compared to unaffected subjects, with left-eye predominance in disease severity.

Abstract Imaging with an adaptive optics scanning light ophthalmoscope (AOSLO) enables direct visualization of the cone photoreceptor mosaic in the living human retina. Quantitative analysis of AOSLO images typically requires manual grading, which is time consuming, and subjective; thus, automated algorithms are highly desirable. Previously developed automated methods are often reliant on ad hoc rules that may not be transferable between different imaging modalities or retinal locations. In this work, we present a convolutional neural network (CNN) based method for cone detection that learns features of interest directly from training data. This cone-identifying algorithm was trained and validated on separate data sets of confocal and split detector AOSLO images with results showing performance that closely mimics the gold standard manual process. Further, without any need for algorithmic modifications for a specific AOSLO imaging system, our fully-automated multi-modality CNN-based cone detection method resulted in comparable results to previous automatic cone segmentation methods which utilized ad hoc rules for different applications. We have made free open-source software for the proposed method and the corresponding training and testing datasets available online.

We observed that heat shock of Caenorhabditis elegans leads to the formation of nuclear double-stranded RNA (dsRNA) foci, detectable with a dsRNA-specific monoclonal antibody. These foci significantly overlap with nuclear HSF-1 granules. To investigate the molecular mechanism(s) underlying dsRNA foci formation, we used RNA-seq to globally characterize total RNA and immunoprecipitated dsRNA from control and heat shocked worms. We find a subset of both sense and antisense transcripts enriched in the dsRNA pool by heat shock overlap with dsRNA transcripts enriched by deletion of tdp-1, which encodes the C. elegans ortholog of TDP-43. Interestingly, transcripts involved in translation are over-represented in the dsRNAs induced by either heat shock or deletion of tdp-1. Also enriched in the dsRNA transcripts are sequences downstream of annotated genes (DoGs), which we globally quantified with a new algorithm. To validate these observations, we used fluorescence in situ hybridization (FISH) to confirm both antisense and downstream of gene transcription for eif-3.B, one of the affected loci we identified.

BACKGROUND:The 13-lined ground squirrel (13-LGS) possesses a cone-dominant retina that is highly amenable to non-invasive high-resolution retinal imaging. The ability for longitudinal assessment of a cone-dominant photoreceptor mosaic with an adaptive optics scanning light ophthalmoscope (AOSLO) has positioned the 13-LGS to become an accessible model for vision research. Here, we examine the interocular symmetry, repeatability, and reliability of cone density measurements in the 13-LGS. METHODS:Thirteen 13-LGS (18 eyes) were imaged along the vertical meridian with a custom AOSLO. Regions of interest were selected superior and inferior to the optic nerve head, including the cone-rich visual streak. Non-confocal split-detection was used to capture images of the cone mosaic. Five masked observers each manually identified photoreceptors for 26 images three times and corrected an algorithm's cell identification outputs for all 214 images three times. Intraobserver repeatability and interobserver reliability of cone density were characterized using data collected from all five observers, while interocular symmetry was assessed in five animals using the average values of all observers. The distribution of image quality for all images in this study was assessed with open-sourced software. RESULTS:Manual identification was less repeatable than semi-automated correction for four of the five observers. Excellent repeatability was seen from all observers (ICC = 0.997-0.999), and there was good agreement between repeat cell identification corrections in all five observers (range: 9.43-25.71 cells/degree2). Reliability of cell identification was significantly different in two of the five observers, and worst in images taken from hibernating 13-LGS. Interocular symmetry of cone density was seen in the five 13-LGS assessed. Image quality was variable between blur- and pixel intensity-based metrics. CONCLUSIONS:Interocular symmetry with repeatable cone density measurements suggest that the 13-LGS is well-suited for longitudinal examination of the cone mosaic using split-detection AOSLO. Differences in reliability highlight the importance of observer training and automation of AOSLO cell detection. Cone density measurements from hibernating 13-LGS are not repeatable. Additional studies are warranted to assess other metrics of cone health to detect deviations from normal 13-LGS in future models of cone disorder in this species.

OBJECTIVES/GOALS: Provider and hospital factors influence quality, but granular data is lacking to assess their impact on renal cancer surgery. The Maryland Health Service Cost Review Commission (HSCRC) is an independent state agency that promotes cost containment, access to care and accountability. Within HSCRC, we aimed to assess the impact of surgeon and hospital volume on 30-day outcomes after renal cancer surgery. METHODS/STUDY POPULATION: Data on renal surgery were abstracted from the Maryland HSCRC from 2000-2018. We excluded patients younger than 18, patients without a diagnosis of renal cancer, and patients concurrently receiving another major surgery. Volume categories were derived from the distribution of mean cases performed per year. We used adjusted multivariable logistic and linear regression models to identify associations of surgeon and hospital volume with the length of stay, days in intensive care, cost, 30-day mortality, readmission, and complications. RESULTS/ANTICIPATED RESULTS: A total of 10,590 surgeries, completed by 669 surgeons at 48 hospitals, met criteria. The 25th percentile for cases per year was 1, the 50th percentile was 1.2, and the 75th percentile was 2.6. After adjusting for patient factors and cumulative surgeon experience, high volume surgeons had the greatest decrease in length of stay (β: −1.65, P

Following publication of the original article [1], the authors reported the following author name error is the article:

Rbfox1 is a splicing regulator that has been associated with various neurological conditions such as autism spectrum disorder, mental retardation, epilepsy, attention-deficit/hyperactivity disorder and schizophrenia. We show that in adult rodent retinas, Rbfox1 is expressed in all types of retinal ganglion cells (RGCs) and in certain subsets of amacrine cells (ACs), within the inner nuclear (INL) and ganglion cell (GCL) layers. In the INL, all Rbfox1-positive cells were colocalized with GABAergic ACs, however not all GABAergic ACs were immunostained for Rbfox1. In the GCL, a vast majority of GABAergic dACs were Rbfox1-immunopositive. Furthermore, all cholinergic starburst ACs (SACs) in the INL (type a) and in the GCL (type b) were Rbfox1 positive. The expression of Rbfox1 in the retina significantly overlapped with expression of Rbfox2, another member of Rbfox family of proteins. Rbfox2, in addition to RGCs and ACs, was also expressed in horizontal cells. In developing retinas at E12 and E15, Rbfox1 is localized to the cytoplasm of differentiating RGCs and ACs. Between P0 and P5, Rbfox1 subcellular localization switched from cytoplasmic to predominantly nuclear. Downregulation of Rbfox1 in adult Rbfox1loxP/loxP mice had no detectable effect on retinal gross morphology. However, the visual cliff test revealed marked abnormalities of depth perception of these animals. RNA sequencing of retinal transcriptomes of control and Rbfox1 knockout animals identified a number of Rbfox1-regulated genes that are involved in establishing neuronal circuits and synaptic transmission, including Vamp1, Vamp2, Snap25, Trak2, and Slc1A7, suggesting the role of Rbfox1 in facilitating synaptic communications between ACs and RGCs.

The purpose of this study was to elaborate on the anastomoses between the paraumbilical and systemic veins, particularly the ensiform veins. The connections with the ensiform veins have received little attention in the anatomical and radiological literature, and remain incompletely described. Too small to be reliably traced in normal CT scans, the paraumbilical veins can dilate in response to increased blood flow from systemic veins in superior vena cava obstruction (SVCO), allowing a study of their arrangement and connections. Collateral paraumbilical veins were therefore analyzed retrospectively in 28 patients with SVCO using CT. We observed inferior and superior groups of collateral vessels in 23/28 (82%) and 17/28 (61%) patients, respectively. Inferior veins ascended towards the liver and drained into portal veins (19/28, 68%) or the umbilical vein (8/28, 29%); superior veins descended and drained into portal veins. The inferior veins (N = 27) could be traced to ensiform veins in almost all of the cases (26/27, 96%), and a little over half (14/27, 52%) were also traceable to subcutaneous and deep epigastric veins. They were opacified by ensiform (25/27, 93%), deep epigastric (4/27, 15%) and subcutaneous (4/27, 15%) veins. The superior veins (N = 17) were supplied by diaphragmatic (13/17, 76%) and ensiform veins (4/17, 24%); the diaphragmatic veins were branches of collateral internal thoracic, left pericardiacophrenic and anterior mediastinal veins. Collateral ensiform veins were observed in 22 patients and anastomosed with internal thoracic (19/22, 86%), superior epigastric (9/22, 41%), diaphragmatic (4/22, 18%), subcutaneous (3/22, 14%) and anterior mediastinal veins (1/22, 5%). These observations show that the paraumbilical veins communicate with ensiform, deep epigastric, subcutaneous and diaphragmatic veins, joining the liver to the properitoneal fat pad, anterior trunk, diaphragm and mediastinum. In SVCO, the most common sources of collateral flow to the paraumbilical veins are the ensiform and diaphragmatic branches of the internal thoracic veins.

PURPOSE:To present a method for age-matched deviation mapping in the assessment of disease-related changes to the radial peripapillary capillaries (RPCs). METHODS:We reviewed 4.5x4.5mm en face peripapillary OCT-A scans of 133 healthy control eyes (133 subjects, mean 41.5 yrs, range 11-82 yrs) and 4 eyes with distinct retinal pathologies, obtained using spectral-domain optical coherence tomography angiography. Statistical analysis was performed to evaluate the impact of age on RPC perfusion densities. RPC density group mean and standard deviation maps were generated for each decade of life. Deviation maps were created for the diseased eyes based on these maps. Large peripapillary vessel (LPV; noncapillary vessel) perfusion density was also studied for impact of age. RESULTS:Average healthy RPC density was 42.5±1.47%. ANOVA and pairwise Tukey-Kramer tests showed that RPC density in the ≥60yr group was significantly lower compared to RPC density in all younger decades of life (p

We evaluated performance characteristics of a laboratory-developed, non-invasive prenatal screening (NIPS) assay for fetal aneuploidies. This assay employs massively parallel shotgun sequencing with full automation. GC sequencing bias correction and statistical smoothing were performed to enhance discrimination of affected and unaffected pregnancies. Maternal plasma samples from pregnancies with known aneuploidy status were used for assay development, verification, and validation. Assay verification studies using 2,085 known samples (1873 unaffected, 69 trisomy 21, 20 trisomy 18, 17 trisomy 13) demonstrated complete discrimination between autosomal trisomy (Z scores >8) and unaffected (Z scores

To assess the effect of image registration and averaging on the visualization and quantification of the radial peripapillary capillary (RPC) network on optical coherence tomography angiography (OCTA).Twenty-two healthy controls were imaged with a commercial OCTA system (AngioVue, Optovue, Inc.). Ten 10x10° scans of the optic disc were obtained, and the most superficial layer (50-μm slab extending from the inner limiting membrane) was extracted for analysis. Rigid registration was achieved using ImageJ, and averaging of each 2 to 10 frames was performed in five ~2x2° regions of interest (ROI) located 1° from the optic disc margin. The ROI were automatically skeletonized. Signal-to-noise ratio (SNR), number of endpoints and mean capillary length from the skeleton, capillary density, and mean intercapillary distance (ICD) were measured for the reference and each averaged ROI. Repeated measures analysis of variance was used to assess statistical significance. Three patients with primary open angle glaucoma were also imaged to compare RPC density to controls.Qualitatively, vessels appeared smoother and closer to histologic descriptions with increasing number of averaged frames. Quantitatively, number of endpoints decreased by 51%, and SNR, mean capillary length, capillary density, and ICD increased by 44%, 91%, 11%, and 4.5% from single frame to 10-frame averaged, respectively. The 10-frame averaged images from the glaucomatous eyes revealed decreased density correlating to visual field defects and retinal nerve fiber layer thinning.OCTA image registration and averaging is a viable and accessible method to enhance the visualization of RPCs, with significant improvements in image quality and RPC quantitative parameters. With this technique, we will be able to non-invasively and reliably study RPC involvement in diseases such as glaucoma.

OBJECTIVE:According to recent estimates, at least 11% of the total global burden of disease is attributable to surgically-treatable diseases. In children, the burden is even more striking with up to 85% of children in low-income and middle-income countries (LMIC) having a surgically-treatable condition by age 15. Using population data from four countries, we estimated pediatric surgical needs amongst children residing in LMICs. METHODS:A cluster randomized cross-sectional countrywide household survey (Surgeons OverSeas Assessment of Surgical Need) was done in four countries (Rwanda, Sierra Leone, Nepal and Uganda) and included demographics, a verbal head to toe examination, and questions on access to care. Global estimates regarding surgical need among children were derived from combined data, accounting for country-level clustering. RESULTS:A total of 13,806 participants were surveyed and 6,361 (46.1%) were children (0-18 years of age) with median age of 8 (Interquartile range [IQR]: 4-13) years. Overall, 19% (1,181/6,361) of children had a surgical need and 62% (738/1,181) of these children had at least one unmet need. Based on these estimates, the number of children living with a surgical need in these four LMICs is estimated at 3.7 million (95% CI: 3.4, 4.0 million). The highest percentage of unmet surgical conditions included head, face, and neck conditions, followed by conditions in the extremities. Over a third of the untreated conditions were masses while the overwhelming majority of treated conditions in all countries were wounds or burns. CONCLUSION:Surgery has been elevated as an "indivisible, indispensable part of health care" in LMICs and the newly formed 2015 Sustainable Development Goals are noted as unachievable without the provision of surgical care. Given the large burden of pediatric surgical conditions in LMICs, scale-up of services for children is an essential component to improve pediatric health in LMICs.

PURPOSE:Choroideremia is a progressive X-linked recessive dystrophy, characterized by degeneration of the retinal pigment epithelium (RPE), choroid, choriocapillaris, and photoreceptors. We examined photoreceptor structure in a series of subjects with choroideremia with particular attention to areas bordering atrophic lesions. METHODS:Twelve males with clinically-diagnosed choroideremia and confirmed hemizygous mutations in the CHM gene were examined. High-resolution images of the retina were obtained using spectral domain optical coherence tomography (SD-OCT) and both confocal and non-confocal split-detector adaptive optics scanning light ophthalmoscope (AOSLO) techniques. RESULTS:Eleven CHM gene mutations (3 novel) were identified; three subjects had the same mutation and one subject had two mutations. SD-OCT findings included interdigitation zone (IZ) attenuation or loss in 10/12 subjects, often in areas with intact ellipsoid zones; RPE thinning in all subjects; interlaminar bridges in the imaged areas of 10/12 subjects; and outer retinal tubulations (ORTs) in 10/12 subjects. Only split-detector AOSLO could reliably resolve cones near lesion borders, and such cones were abnormally heterogeneous in morphology, diameter and density. On split-detector imaging, the cone mosaic terminated sharply at lesion borders in 5/5 cases examined. Split-detector imaging detected remnant cone inner segments within ORTs, which were generally contiguous with a central patch of preserved retina. CONCLUSIONS:Early IZ dropout and RPE thinning on SD-OCT are consistent with previously published results. Evidence of remnant cone inner segments within ORTs and the continuity of the ORTs with preserved retina suggests that these may represent an intermediate state of retinal degeneration prior to complete atrophy. Taken together, these results supports a model of choroideremia in which the RPE degenerates before photoreceptors.

Purpose: The readiness assurance process (RAP) of team-based learning (TBL) is an important element that ensures that students come prepared to learn. However, the RAP can use a significant amount of class time which could otherwise be used for application exercises. The authors administered the TBL-associated RAP in class or individual readiness assurance tests (iRATs) at home to compare medical student performance and learning preference for physiology content. Methods: Using cross-over study design, the first year medical student TBL teams were divided into two groups. One group was administered iRATs and group readiness assurance tests (gRATs) consisting of physiology questions during scheduled class time. The other group was administered the same iRAT questions at home, and did not complete a gRAT. To compare effectiveness of the two administration methods, both groups completed the same 12-question physiology assessment during dedicated class time. Four weeks later, the entire process was repeated, with each group administered the RAP using the opposite method. Results: The performance on the physiology assessment after at-home administration of the iRAT was equivalent to performance after traditional in-class administration of the RAP. In addition, a majority of students preferred the at-home method of administration and reported that the at-home method was more effective in helping them learn course content. Conclusion: The at-home administration of the iRAT proved effective. The at-home administration method is a promising alternative to conventional iRATs and gRATs with the goal of preserving valuable in-class time for TBL application exercises.

Blood tests are necessary, easy-to-perform and low-cost alternatives for monitoring of oncolytic virotherapy and other biological therapies in translational research. Here we assessed three candidate proteins with the potential to be used as biomarkers in biological fluids: two glucuronidases from E. coli (GusA) and Staphylococcus sp. RLH1 (GusPlus), and the luciferase from Gaussia princeps (GLuc). The three genes encoding these proteins were inserted individually into vaccinia virus GLV-1h68 genome under the control of an identical promoter. The three resulting recombinant viruses were used to infect tumor cells in cultures and human tumor xenografts in nude mice. In contrast to the actively secreted GLuc, the cytoplasmic glucuronidases GusA and GusPlus were released into the supernatants only as a result of virus-mediated oncolysis. GusPlus resulted in the most sensitive detection of enzyme activity under controlled assay conditions in samples containing as little as 1 pg/ml of GusPlus, followed by GusA (25 pg/ml) and GLuc (≥375 pg/ml). Unexpectedly, even though GusA had a lower specific activity compared to GusPlus, the substrate conversion in the serum of tumor-bearing mice injected with the GusA-encoding virus strains was substantially higher than that of GusPlus. This was attributed to a 3.2 fold and 16.2 fold longer half-life of GusA in the blood stream compared to GusPlus and GLuc respectively, thus a more sensitive monitor of virus replication than the other two enzymes. Due to the good correlation between enzymatic activity of expressed marker gene and virus titer, we conclude that the amount of the biomarker protein in the body fluid semiquantitatively represents the amount of virus in the infected tumors which was confirmed by low light imaging. We found GusA to be the most reliable biomarker for monitoring oncolytic virotherapy among the three tested markers.