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Abstract Background In humans, muscle-invasive bladder cancer (MIBC) is highly aggressive and associated with a poor prognosis. With a high mutation load and large number of altered genes, strategies to delineate key driver events are necessary. Dogs and cats develop urothelial carcinoma (UC) with histological and clinical similarities to human MIBC. Cattle that graze on bracken fern also develop UC, associated with exposure to the carcinogen ptaquiloside. These species may represent relevant animal models of spontaneous and carcinogen-induced UC that can provide insight into human MIBC. Results Whole-exome sequencing of domestic canine (n = 87) and feline (n = 23) UC, and comparative analysis with human MIBC reveals a lower mutation rate in animal cases and the absence of APOBEC mutational signatures. A convergence of driver genes (ARID1A, KDM6A, TP53, FAT1, and NRAS) is discovered, along with common focally amplified and deleted genes involved in regulation of the cell cycle and chromatin remodelling. We identify mismatch repair deficiency in a subset of canine and feline UCs with biallelic inactivation of MSH2. Bovine UC (n = 8) is distinctly different; we identify novel mutational signatures which are recapitulated in vitro in human urinary bladder UC cells treated with bracken fern extracts or purified ptaquiloside. Conclusion Canine and feline urinary bladder UC represent relevant models of MIBC in humans, and cross-species analysis can identify evolutionarily conserved driver genes. We characterize mutational signatures in bovine UC associated with bracken fern and ptaquiloside exposure, a human-linked cancer exposure. Our work demonstrates the relevance of cross-species comparative analysis in understanding both human and animal UC.

Abstract Kidney organoids are a promising model to study kidney disease, but their use is constrained by limited knowledge of their functional protein expression profile. Here, we define the organoid proteome and transcriptome trajectories over culture duration and upon exposure to TNFα, a cytokine stressor. Older organoids increase deposition of extracellular matrix but decrease expression of glomerular proteins. Single cell transcriptome integration reveals that most proteome changes localize to podocytes, tubular and stromal cells. TNFα treatment of organoids results in 322 differentially expressed proteins, including cytokines and complement components. Transcript expression of these 322 proteins is significantly higher in individuals with poorer clinical outcomes in proteinuric kidney disease. Key TNFα-associated protein (C3 and VCAM1) expression is increased in both human tubular and organoid kidney cell populations, highlighting the potential for organoids to advance biomarker development. By integrating kidney organoid omic layers, incorporating a disease-relevant cytokine stressor and comparing with human data, we provide crucial evidence for the functional relevance of the kidney organoid model to human kidney disease.

Abstract Background Infants under 6 months of age are often excluded from malaria surveillance and observational studies. The impact of malaria during early infancy on health later in childhood remains unknown. Methods Infants from two birth cohorts in Malawi were monitored at quarterly intervals and whenever they were ill from birth through 24 months for Plasmodium falciparum infections and clinical malaria. Poisson regression and linear mixed effects models measured the effect of exposure to malaria in infancy on subsequent malaria incidence, weight-for-age z-scores (WAZ), and haemoglobin concentrations after 6 months. Results Infants with at least one P. falciparum infection during their first 6 months had increased incidence ratio (IRR) of P. falciparum infection (IRR = 1.27, 95% CI, 1.06–1.52) and clinical malaria (IRR = 2.37, 95% CI, 2.02–2.80) compared to infants without infection. Infants with clinical malaria had increased risk of P. falciparum infection incidence between 6 and 24 months (IRR = 1.64, 95% CI, 1.38–1.94) and clinical malaria (IRR = 1.85, 95% CI, 1.48–2.32). Exposure to malaria was associated with lower WAZ over time (p = 0.02) and lower haemoglobin levels than unexposed infants at every time interval (p = 0.02). Conclusions Infants experiencing malaria infection or clinical malaria are at increased risk of subsequent infection and disease, have poorer growth, and lower haemoglobin concentrations.

Abstract Hydroxychloroquine (HCQ) is Food and Drug Administration (FDA)‐approved for malaria, systemic and chronic discoid lupus erythematosus, and rheumatoid arthritis. Because HCQ has a proposed multimodal mechanism of action and a well‐established safety profile, it is often investigated as a repurposed therapeutic for a range of indications. There is a large degree of uncertainty in HCQ pharmacokinetic (PK) parameters which complicates dose selection when investigating its use in new disease states. Complications with HCQ dose selection emerged as multiple clinical trials investigated HCQ as a potential therapeutic in the early stages of the COVID‐19 pandemic. In addition to uncertainty in baseline HCQ PK parameters, it was not clear if disease‐related consequences of SARS‐CoV‐2 infection/COVID‐19 would be expected to impact the PK of HCQ and its primary metabolite desethylhydroxychloroquine (DHCQ). To address the question whether SARS‐CoV‐2 infection/COVID‐19 impacted HCQ and DHCQ PK, dried blood spot samples were collected from SARS‐CoV‐2(−)/(+) participants administered HCQ. When a previously published physiologically based pharmacokinetic (PBPK) model was used to fit the data, the variability in exposure of HCQ and DHCQ was not adequately captured and DHCQ concentrations were overestimated. Improvements to the previous PBPK model were made by incorporating the known range of blood to plasma concentration ratios (B/P) for each compound, adjusting HCQ and DHCQ distribution settings, and optimizing DHCQ clearance. The final PBPK model adequately captured the HCQ and DHCQ concentrations observed in SARS‐CoV‐2(−)/(+)participants, and incorporating COVID‐19‐associated changes in cytochrome P450 activity did not further improve model performance for the SARS‐CoV‐2(+) population.

Two-stage hepatectomy (TSH) has been proposed for patients with bilateral liver tumours who have a high risk of posthepatectomy liver failure after one-stage hepatectomy (OSH). This study aimed to determine the outcomes of TSH for extensive bilateral colorectal liver metastases.

Abstract Objective Mucopolysaccharidosis type IIIA (MPSIIIA) caused by recessive SGSH variants results in sulfamidase deficiency, leading to neurocognitive decline and death. No disease‐modifying therapy is available. The AAVance gene therapy trial investigates AAVrh.10 overexpressing human sulfamidase (LYS‐SAF302) delivered by intracerebral injection in children with MPSIIIA. Post‐treatment MRI monitoring revealed lesions around injection sites. Investigations were initiated in one patient to determine the cause. Methods Clinical and MRI details were reviewed. Stereotactic needle biopsies of a lesion were performed; blood and CSF were sampled. All samples were used for viral studies. Immunohistochemistry, electron microscopy, and transcriptome analysis were performed on brain tissue of the patient and various controls. Results MRI revealed focal lesions around injection sites with onset from 3 months after therapy, progression until 7 months post therapy with subsequent stabilization and some regression. The patient had transient slight neurological signs and is following near‐normal development. No evidence of viral or immunological/inflammatory cause was found. Immunohistochemistry showed immature oligodendrocytes and astrocytes, oligodendrocyte apoptosis, strong intracellular and extracellular sulfamidase expression and hardly detectable intracellular or extracellular heparan sulfate. No activation of the unfolded protein response was found. Interpretation Results suggest that intracerebral gene therapy with local sulfamidase overexpression leads to dysfunction of transduced cells close to injection sites, with extracellular spilling of lysosomal enzymes. This alters extracellular matrix composition, depletes heparan sulfate, impairs astrocyte and oligodendrocyte function, and causes cystic white matter degeneration at the site of highest gene expression. The AAVance trial results will reveal the potential benefit–risk ratio of this therapy.

Abstract Background Mutations in genes involved in DNA damage repair (DDR), a hallmark of cancer, are associated with increased cancer cell sensitivity to certain therapies. This study sought to evaluate the association of DDR pathogenic variants with treatment efficacy in patients with advanced non‐small cell lung cancer (NSCLC). Methods A retrospective cohort of consecutive patients with advanced NSCLC attending a tertiary medical center who underwent next‐generation sequencing in 01/2015‐8/2020 were clustered according to DDR gene status and compared for overall response rate (ORR), progression‐free survival (PFS) (patients receiving systemic therapy), local PFS (patients receiving definitive radiotherapy), and overall survival (OS) using log‐rank and Cox regression analyses. Results Of 225 patients with a clear tumor status, 42 had a pathogenic/likely pathogenic DDR variant (pDDR), and 183 had no DDR variant (wtDDR). Overall survival was similar in the two groups (24.2 vs. 23.1 months, p = 0.63). The pDDR group had a higher median local PFS after radiotherapy (median 45 months vs. 9.9 months, respectively; p = 0.044), a higher ORR (88.9% vs. 36.2%, p = 0.04), and a longer median PFS (not reached vs. 6.0 months, p = 0.01) in patients treated with immune checkpoint blockade. There was no difference in ORR, median PFS, and median OS in patients treated with platinum‐based chemotherapy. Conclusion Our retrospective data suggest that in patients with stage 4 NSCLC, pathogenic variants in DDR pathway genes may be associated with higher efficacy of radiotherapy and immune checkpoint inhibitors (ICIs). This should be further explored prospectively.

Antisense oligonucleotide (ASO) therapeutics are being investigated for a broad range of neurological diseases. While ASOs have been effective in the clinic, improving productive ASO internalization into target cells remains a key area of focus in the field. Here, we investigated how the delivery of ASO-loaded lipid nanoparticles (LNPs) affects ASO activity, subcellular trafficking, and distribution in the brain. We show that ASO-LNPs increase ASO activity up to 100-fold in cultured primary brain cells as compared to non-encapsulated ASO. However, in contrast to the widespread ASO uptake and activity observed following free ASO delivery in vivo, LNP-delivered ASOs did not downregulate mRNA levels throughout the brain after intracerebroventricular injection. This lack of activity was likely due to ASO accumulation in cells lining the ventricles and blood vessels. Furthermore, we reveal a formulation-dependent activation of the immune system post dosing, suggesting that LNP encapsulation cannot mask cellular ASO backbone-mediated toxicities. Together, these data provide insights into how LNP encapsulation affects ASO distribution as well as activity in the brain, and a foundation that enables future optimization of brain-targeting ASO-LNPs.

The aberrant activation of HER2 has a pivotal role in bone metastasis implantation and progression in several tumor types, including prostate cancer (PC). Trastuzumab and other anti-HER2 therapies, such as lapatinib, have been used in human breast cancer HER2 positive. Although HER2 overexpression has been reported in PC, anti-HER2 therapy response has revealed conflicting results. We investigated the potential of lapatinib in inhibiting cell migration and inducing apoptosis in two human (LNCaP and PC3) and two canine PC cell lines (PC1 and PC2). Cell migration and apoptosis were evaluated by Annexin V/PI analysis after lapatinib treatment. The transcriptome analysis of all cell lines before and after treatment with lapatinib was also performed. We found increased apoptosis and migration inhibition in LNCaP cells (androgen-sensitive cell line), while PC1, PC2, and PC3 cells showed no alterations after the treatment. The transcriptome analysis of LNCaP and PC3 cell lines showed 158 dysregulated transcripts in common, while PC1 and PC2 cell lines presented 82. At the doses of lapatinib used, we observed transcriptional modifications in all cell lines. PI3K/AKT/mTOR pathway were enriched in human PC cells, while canine PC cells showed enrichment of tyrosine kinase antitumor response and HER2-related pathways. In canine PC cells, the apoptosis failed after lapatinib treatment, possibly due to the downregulation of MAPK genes. Prostate cancer cells insensitive to androgens may be resistant to lapatinib through PI3K gene dysregulation. The association of lapatinib with PI3K inhibitors may provide a more effective antitumor response and clinical benefits to PC patients.

Background and purpose: Bilateral femoral distraction osteogenesis in patients with achondroplasia is insufficiently reported. We aimed to perform the first study that exclusively analyzed simultaneous bilateral femoral distraction osteogenesis with motorized intramedullary lengthening nails via an antegrade approach in patients with achondroplasia focused on reliability, accuracy, precision, and the evolving complications. Patients and methods: In this retrospective singlecenter study we analyzed patients with achondroplasia who underwent simultaneous bilateral femoral lengthening with antegrade intramedullary lengthening nails between October 2014 and April 2019. 15 patients (30 femoral segments) of median age 14 years (interquartile range [IQR] 12–15) were available for analysis. The median follow-up was 29 months (IQR 27–37) after nail implantation. Results: The median distraction length per segment was 49 mm (IQR 47–51) with a median distraction index of 1.0 mm/day (IQR 0.9–1.0), and a median consolidation index of 20 days/cm (IQR 17–23). Reliability of the lengthening nails was 97% and their calculated accuracy and precision were 96% and 95%, respectively. The most common complication was temporary restriction of knee range of motion during distraction in 10 of 30 of the lengthened segments. 1 patient was treated with 2 unplanned additional surgeries due to premature consolidation. Conclusion: The method is reliable and accurate with few complications.

ObjectivesTo date, there is no evidence regarding the safety of automated titanium fastener compared with hand-tied knots for prosthesis fixation in infective endocarditis.MethodsBetween January 2016 and December 2022, a total of 220 patients requiring surgery for infective endocarditis were included in this retrospective analysis. The primary study endpoint was re-endocarditis during follow-up. The secondary study endpoints included stroke onset, all-cause mortality, and a composite outcome of either re-endocarditis, stroke, or all-cause mortality during follow-up.ResultsSuture-securing with an automated titanium fastener was performed in 114 (51.8%) patients, whereas the conventional technique of hand knot-tying was used in 106 (48.2%) patients. The risk of re-endocarditis was significantly lower in the automated titanium fastener group, as shown in a multivariable proportional competing risk regression model (adjusted sub-hazard ratio 0.33, 95% confidence interval 0.11–0.99, p = 0.048). The multivariable Cox proportional hazards regression analysis showed that the automated titanium fastener group was not associated with an increased risk of stroke-onset or attaining the composite outcome, respectively, (adjusted hazard ratio 0.54, 95% confidence interval 0.27–1.08, p = 0.082), (adjusted hazard ratio 0.65, 95% confidence interval 0.42–1.02, p = 0.061). Also, this group was not associated with an increased risk of all-cause mortality, as demonstrated in the multivariable Poisson regression analysis (adjusted incidence-rate ratio 1.42, 95% confidence interval 0.83–2.42, p = 0.202).ConclusionsThe use of automated titanium fastener device seems to be safe for infective endocarditis. Analyses of larger cohorts are required.

IntroductionAcquired brain injury (ABI) is a prevalent diagnosis in pediatric rehabilitation. Gross motor skills are often affected by ABI and limit the ability to participate in various physical activities. However, as ABI injury location is diverse, children and adolescents (youth) with localized ABI, such as ABI in the posterior fossa (ABI-PF) may present unique and different motor disabilities than youth with ABI on account of traumatic brain injury (TBI).AimsThe aims of the study were: (1) to compare gross motor deficits in youth with TBI vs. ABI-PF; and (2) to compare two methods on scoring BOT2 to determine which is better for identifying motor deficits.MethodsParticipated in this study youth with TBI (N = 50) and ABI-PF (N = 30). Participants were tested on Bruininks-Oseretsky Test of Motor Proficiency-2nd Edition (BOT2) Upper-Limb Coordination, Balance, Strength, Running Speed and Agility, and Bilateral-Coordination subtests. Motor performance deficits were established using two-standard deviations (2SD) and age-equivalent methods. Between-group differences were assessed via independent t-tests and receiver operating characteristic curves (ROC).ResultsAccording to the 2SD method, motor deficits in the ABI-PF group ranged from 20% to 66.66%, whereas in the TBI group 8%–16%. According to the age-equivalent method, in the TBI and ABI-PF groups 40%–66.0% and 46.66%–76.66% of the youth presented motor deficits, respectively. Moreover, ROC analysis showed that motor performance deficits of both groups in all sub-scales except for Bilateral Coordination differed enough to result in medium area under the curve.ConclusionsMotor deficits post-pediatric ABI are prevalent. In comparison to the TBI group, deficits are greater in the ABI-PF group. Moreover, compared to the 2SD method, the extent of motor deficiency is greater in the age-equivalent method. Therefore, using the later might provide a more valid classification of deficits in gross motor proficiency for youth post-ABI.

IntroductionAttachment styles are crucial in human relationships and have been explored through neurophysiological responses and EEG data analysis. This study investigates the potential of EEG data in predicting and differentiating secure and insecure attachment styles, contributing to the understanding of the neural basis of interpersonal dynamics.MethodsWe engaged 27 participants in our study, employing an XGBoost classifier to analyze EEG data across various feature domains, including time-domain, complexity-based, and frequency-based attributes.ResultsThe study found significant differences in the precision of attachment style prediction: a high precision rate of 96.18% for predicting insecure attachment, and a lower precision of 55.34% for secure attachment. Balanced accuracy metrics indicated an overall model accuracy of approximately 84.14%, taking into account dataset imbalances.DiscussionThese results highlight the challenges in using EEG patterns for attachment style prediction due to the complex nature of attachment insecurities. Individuals with heightened perceived insecurity predominantly aligned with the insecure attachment category, suggesting a link to their increased emotional reactivity and sensitivity to social cues. The study underscores the importance of time-domain features in prediction accuracy, followed by complexity-based features, while noting the lesser impact of frequency-based features. Our findings advance the understanding of the neural correlates of attachment and pave the way for future research, including expanding demographic diversity and integrating multimodal data to refine predictive models.

Obstructed hemivagina and ipsilateral renal anomaly (OHVIRA) is a rare congenital developmental syndrome manifested by uterine duplication, lower genital tract obstruction, and unilateral renal anomaly. Literature on reproductive outcomes in this patient population is limited. The aim of this study is to describe obstetric outcomes after surgical correction of obstructed hemivagina in a longitudinal cohort of patients with a diagnosis of OHVIRA. All cases of OHVIRA presenting to a single tertiary care children's hospital from 1990 to 2021 were retrospectively reviewed. Three cases demonstrating a variety of clinically important reproductive outcomes are described in detail including risks such as retained products, endometritis, preterm labor, and malpresentation. Understanding the reproductive outcomes associated with this diagnosis is important for practitioners seeking to counsel and care for patients with this diagnosis. This case series demonstrates a wide array of potential gynecologic and obstetric risks, though ultimately with successful term and near-term pregnancies.

Self-compacting concrete has constructive advantages over conventional concrete, such as reducing labor and construction time, mainly because of its fluidity in the fresh state. However, in the hardened state, it maintains low performance when tensioned, and the fibers can be added to the mixture, maintaining a portion of the resistance after cracking. Steel fibers are usually added to concrete, but recently synthetic fibers have been used, due to their lower cost and non-corrosive nature, but with lower tensile strength. Thus, by combining the two types of fibers, the benefits of each material can be used. This work presents the results of an experimental program to evaluate the effect of the hybridization of metallic and synthetic fibers on the shear strength of self-compacting concrete beams without stirrups. The results demonstrate that both steel and hybrid fibers result in greater shear strength compared with the reference concrete without fibers before shear crack formation; however, the greatest advantages are attributed to post-cracking residual strength. The experimental results were compared with estimates calculated using equations published in the literature, demonstrating the feasibility of using some existing equations for concretes with the addition of hybrid fibers.

Summary: The lymph node (LN) is a critical biological site for immune maturation after vaccination as it includes several cell populations critical for priming the antibody response. Here, we present a protocol for sampling the LN and isolating cell populations to evaluate immunogens targeting germline cells. We describe steps for media and tube preparation and sample collection using an ultrasound-guided LN fine-needle aspiration procedure. This protocol is safe, quick, low-cost, and less invasive than excisional biopsy.For complete details on the use and execution of this protocol, please refer to Leggat et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

BackgroundDown syndrome (DS) is characterized by a genome-wide profile of differential DNA methylation that is skewed towards hypermethylation in most tissues, including brain, and includes pan-tissue differential methylation. The molecular mechanisms involve the overexpression of genes related to DNA methylation on chromosome 21. Here, we stably overexpressed the chromosome 21 gene DNA methyltransferase 3L (DNMT3L) in the human SH-SY5Y neuroblastoma cell line and assayed DNA methylation at over 26 million CpGs by whole genome bisulfite sequencing (WGBS) at three different developmental phases (undifferentiated, differentiating, and differentiated).ResultsDNMT3L overexpression resulted in global CpG and CpG island hypermethylation as well as thousands of differentially methylated regions (DMRs). The DNMT3L DMRs were skewed towards hypermethylation and mapped to genes involved in neurodevelopment, cellular signaling, and gene regulation. Consensus DNMT3L DMRs showed that cell lines clustered by genotype and then differentiation phase, demonstrating sets of common genes affected across neuronal differentiation. The hypermethylated DNMT3L DMRs from all pairwise comparisons were enriched for regions of bivalent chromatin marked by H3K4me3 as well as differentially methylated sites from previous DS studies of diverse tissues. In contrast, the hypomethylated DNMT3L DMRs from all pairwise comparisons displayed a tissue-specific profile enriched for regions of heterochromatin marked by H3K9me3 during embryonic development.ConclusionsTaken together, these results support a mechanism whereby regions of bivalent chromatin that lose H3K4me3 during neuronal differentiation are targeted by excess DNMT3L and become hypermethylated. Overall, these findings demonstrate that DNMT3L overexpression during neurodevelopment recreates a facet of the genome-wide DS DNA methylation signature by targeting known genes and gene clusters that display pan-tissue differential methylation in DS.

BackgroundEffective prevention against coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently limited to nonpharmaceutical strategies. Laboratory and observational data suggested that hydroxychloroquine had biological activity against SARS-CoV-2, potentially permitting its use for prevention.ObjectiveTo test hydroxychloroquine as postexposure prophylaxis for SARS-CoV-2 infection.DesignHousehold-randomized, double-blind, controlled trial of hydroxychloroquine postexposure prophylaxis. (ClinicalTrials.gov: NCT04328961).SettingNational U.S. multicenter study.ParticipantsClose contacts recently exposed ( 0.20). The frequency of participants experiencing adverse events was higher in the hydroxychloroquine group than the control group (66 [16.2%] versus 46 [10.9%], respectively; P = 0.026).LimitationThe delay between exposure, and then baseline testing and the first dose of hydroxychloroquine or ascorbic acid, was a median of 2 days.ConclusionThis rigorous randomized controlled trial among persons with recent exposure excluded a clinically meaningful effect of hydroxychloroquine as postexposure prophylaxis to prevent SARS-CoV-2 infection.Primary funding sourceBill & Melinda Gates Foundation.

ObjectiveInvestigate the impact of increased access to new and refilled prescriptions by means of an automated pickup kiosk (Asteres ScriptCenter) on prescription abandonment rates, patient experience, and pharmacist consultations.DesignNonrandomized, observational study using retrospective, deidentified data from the filling pharmacy, the kiosk, and a pharmacist-completed counseling documentation log over a 35-month study period.Setting and participantsHospital employees opting to use a kiosk located in the lobby with 24 hours a day, 7 days a week access for pickups and a telephone pharmacist consultation service compared with employees using the regular counter at the filling pharmacy.Outcome measuresReturn to stock (RTS) rate to assess prescription abandonment, time to prescription pickup, consultation duration, kiosk user assessment, and pharmacist assessment of counseling ability.ResultsApproximately 9% of employees (440) enrolled to use the kiosk, with 5062 kiosk pickups recorded for new prescriptions (29%), refill prescriptions (33%), and over-the-counters (38%). The mean kiosk RTS (4.3% ± 3.2) was lower than that at the regular counter (5.6% ± 0.8), P = 0.04, whereas the mean time to pickup was approximately 1 day greater at the kiosk than the regular counter (2.8 ± 0.4 vs. 1.8 ± 0.2, P < 0.001). The average kiosk consultation was approximately 1 minute shorter (2.0 ± 1.4) than that of the regular counter (3.4 ± 1.9, P < 0.001), and fewer patients using the kiosk (15.7%) had additional questions at the end of a consultation session than patients at the regular counter (38.8%, P < 0.001). Most of the kiosk users agreed that their prescription questions were answered and that kiosk convenience was an important reason for using the filling pharmacy. Almost all (>90%) pharmacists indicated that they were able to effectively counsel patients at the kiosk and the regular counter.ConclusionThe kiosk, used by self-selected health care workers located in a hospital workplace setting with 24 hours a day, 7 days a week access, was a convenient, contactless pickup extension of the filling pharmacy with a lower prescription abandonment rate and similar pickup and consultation characteristics as at the regular pharmacy counter.

Abstract The demand for decellularized xenogeneic tissues used in reconstructive heart surgery has increased over the last decades. Complete decellularization of longer and tubular aortic sections suitable for clinical application has not been achieved so far. The present study aims at analyzing the effect of pressure application on decellularization efficacy of porcine aortas using a device specifically designed for this purpose. Fresh porcine descending aortas of 8 cm length were decellularized using detergents. To increase decellularization efficacy, detergent treatment was combined with pressure application and different treatment schemes. Quantification of penetration depth as well as histological staining, scanning electron microscopy, and tensile strength tests were used to evaluate tissue structure. In general, application of pressure to aortic tissue does neither increase the decellularization success nor the penetration depth of detergents. However, it is of importance from which side of the aorta the pressure is applied. Application of intermittent pressure from the adventitial side does significantly increase the decellularization degree at the intimal side (compared to the reference group), but had no influence on the penetration depth of SDC/SDS at both sides. Although the present setup does not significantly improve the decellularization success of aortas, it is interesting that the application of pressure from the adventitial side leads to improved decellularization of the intimal side. As no adverse effects on tissue structure nor on mechanical properties were observed, optimization of the present protocol may potentially lead to complete decellularization of larger aortic segments. Graphical Abstract

Abstract Background Control of malaria parasite transmission can be enhanced by understanding which human demographic groups serve as the infectious reservoirs. Because vector biting can be heterogeneous, some infected individuals may contribute more to human-to-mosquito transmission than others. Infection prevalence peaks in school-age children, but it is not known how often they are fed upon. Genotypic profiling of human blood permits identification of individual humans who were bitten. The present investigation used this method to estimate which human demographic groups were most responsible for transmitting malaria parasites to Anopheles mosquitoes. It was hypothesized that school-age children contribute more than other demographic groups to human-to-mosquito malaria transmission. Methods In a region of moderate-to-high malaria incidence in southeastern Malawi, randomly selected households were surveyed to collect human demographic information and blood samples. Blood-fed, female Anopheles mosquitoes were sampled indoors from the same houses. Genomic DNA from human blood samples and mosquito blood meals of human origin was genotyped using 24 microsatellite loci. The resultant genotypes were matched to identify which individual humans were sources of blood meals. In addition, Plasmodium falciparum DNA in mosquito abdomens was detected with polymerase chain reaction. The combined results were used to identify which humans were most frequently bitten, and the P. falciparum infection prevalence in mosquitoes that resulted from these blood meals. Results Anopheles females selected human hosts non-randomly and fed on more than one human in 9% of the blood meals. Few humans contributed most of the blood meals to the Anopheles vector population. Children ≤ 5 years old were under-represented in mosquito blood meals while older males (31–75 years old) were over-represented. However, the largest number of malaria-infected blood meals was from school age children (6–15 years old). Conclusions The results support the hypothesis that humans aged 6–15 years are the most important demographic group contributing to the transmission of P. falciparum to the Anopheles mosquito vectors. This conclusion suggests that malaria control and prevention programmes should enhance efforts targeting school-age children and males.

Objectives: Although long COVID-19 is widely recognized in adults, less information is available about this condition in children, especially in developing countries. Here, we studied the long-term symptoms of SARS-CoV-2 infection beyond 3 months and the associated risk factors in a pediatric population. Methods: This observational study included 639 Argentinian children and adolescents with previously confirmed COVID-19 from June 2020-June 2021 and 577 children without previous COVID-19. Parents completed a survey about symptoms that their child had for >3 months after the diagnosis of SARS-CoV-2 infection. Results: At least one persistent symptom was observed more frequently in children with previous COVID-19 than in the non-COVID-19 group (34% vs 13%, P