SARS-CoV-2 Research in Dogs (Canis lupus familiaris) and Felines (Felis silvestris catus) Domiciled in an International Border Region (Paraguay and Brazil).

Introduction: COVID-19 is an infectious disease caused by SARS-CoV-2 that has become a serious threat to public health owing to its rapid spread from aerosols from infected people. Despite being considered a strictly human disease, there are reports in the literature about animals with confirmed presence of the virus. Aim: Owing to the scarcity of scientific literature on the potential for infection of animals and their importance for One Health, the objective of this work was to research SARS-CoV-2 RNA in felines (Felis silvestris catus) and dogs (Canis lupus familiaris) domiciled. Materials and Methods: Oropharyngeal swabs were collected from domestic dogs and cats belonging to patients diagnosed with COVID-19 from August to October 2021 and residents of the northwest and west regions of Paraná, Brazil. Results: Of the 34 samples collected, 14 were from dogs and 20 from cats. Three of these samples tested positive in real-time PCR, and two of them were also positive in the immunochromatographic test. After testing positive in real-time PCR, the samples underwent genetic sequencing using the Illumina COVIDSeq test. Of the 34 samples collected, three (9%), all of them female and from the feline species, tested positive in real-time PCR, with two of these (67%) also testing positive in the immunochromatographic test. Regarding sequencing, it was possible to sequence the three samples aligned with the AY.101 lineage, corresponding to the Delta variant. Conclusion: The occurrence of SARS-CoV-2 infection in dogs and cats is seen as an unintended event with significant implications for public health, including its potential transmission to other animal species. Further research is required to enhance our understanding of how this disease spreads among these animals and its broader impact on One Health initiatives.

Missing History of a Modern Domesticate: Historical Demographics and Genetic Diversity in Farm-bred Red Fox Populations.

The first record of captive bred red foxes (Vulpes vulpes) dates to 1896, when a breeding enterprise emerged in the provinces of Atlantic Canada. Because its domestication happened during recent history, the red fox offers a unique opportunity to examine the genetic diversity of an emerging domesticated species in the context of documented historical and economic influences. In particular, the historical record suggests that North American and Eurasian farm-bred populations likely experienced different demographic trajectories. Here, we focus on the likely impacts of founder effects and genetic drift given historical trends in fox farming on North American and Eurasian farms. A total of 15 mitochondrial haplotypes were identified in 369 foxes from 10 farm populations that we genotyped (n=161) or that were previously published. All haplotypes are endemic to North America. Although most haplotypes were consistent with eastern Canadian ancestry, a small number of foxes carried haplotypes typically found in Alaska and other regions of western North America. The presence of these haplotypes supports historical reports of wild foxes outside of Atlantic Canada being introduced into the breeding stock. These putative Alaskan and Western haplotypes were more frequently identified in Eurasian farms compared to North American farms, consistent with historical documentation suggesting that Eurasian economic and breeding practices were likely to maintain low-frequency haplotypes more effectively than in North America. Contextualizing inter- versus intra-farm genetic diversity alongside the historical record is critical to understanding of the origins of this emerging domesticate and the relationships between wild and farm-bred fox populations.

How does hospital organisation influence the use of caesarean sections in low- and middle-income countries? A cross-sectional survey in Argentina, Burkina Faso, Thailand and Vietnam for the QUALI-DEC project.

BACKGROUND: Improving the understanding of non-clinical factors that lead to the increasing caesarean section (CS) rates in many low- and middle-income countries is currently necessary to meet the challenge of implementing effective interventions in hospitals to reverse the trend. The objective of this study was to study the influence of organizational factors on the CS use in Argentina, Vietnam, Thailand and Burkina Faso. METHODS: A cross-sectional hospital-based postpartum survey was conducted in 32 hospitals (8 per country). We selected women with no potential medical need for CS among a random sample of women who delivered at each of the participating facilities during the data collection period. We used multilevel multivariable logistic regression to analyse the association between CS use and organizational factors, adjusted on women's characteristics. RESULTS: A total of 2,092 low-risk women who had given birth in the participating hospitals were included. The overall CS rate was 24.1%, including 4.9% of pre-labour CS and 19.3% of intra-partum CS. Pre-labour CS was significantly associated with a 24-hour anaesthetist dedicated to the delivery ward (ORa = 3.70 [1.41; 9.72]) and with the possibility to have an individual room during labour and delivery (ORa = 0.28 [0.09; 0.87]). Intra-partum CS was significantly associated with a higher bed occupancy level (ORa = 1.45 [1.09; 1.93]): intrapartum CS rate would increase of 6.3% points if the average number of births per delivery bed per day increased by 10%. CONCLUSION: Our results suggest that organisational norms and convenience associated with inadequate use of favourable resources, as well as the lack of privacy favouring women's preference for CS, and the excessive workload of healthcare providers drive the CS overuse in these hospitals. It is also crucial to enhance human and physical resources in delivery rooms and the organisation of intrapartum care to improve the birth experience and the working environment for those providing care. TRIAL REGISTRATION: The QUALI-DEC trial is registered on the Current Controlled Trials website ( https://www.isrctn.com/ ) under the number ISRCTN67214403.

Enhanced recovery in cranial surgery (ERACraS) - A single-centre quality improvement study.

BACKGROUND: Enhanced Recovery After Surgery (ERAS) is a well-established, protocol-driven, evidence-based approach to peri-operative care. ERAS protocols have been used to improve patient morbidity and mortality outcomes in various surgical specialties. More recently, it has been introduced to neurosurgery. Our aim was to establish an Enhanced Recovery After Cranial Surgery (ERACraS) protocol for patients as part of a quality improvement project (QIP) with the intention of reducing hospital length of stay (HLOS). METHODS: This QIP was carried out in the Department of Neurosciences (DCN), Edinburgh, over two four-month periods. A total of 40 patients over 18 years of age undergoing elective craniotomy surgery under a sole neurosurgeon were invited to take part in this QIP. Subsequently, data was retrospectively collected through our institution's online documentation system. RESULTS: 19 patients received conventional perioperative care (pre-ERACraS group) during December 2021-March 2022, and 21 received care according to the novel ERACraS (ERACraS group) during June-September 2022. Regarding supra-tentorial surgery, there was a reduction of 73% in HLOS in the ERACraS group. No change was observed in infra-tentorial surgery. Overall, the ERACraS protocol reduced HLOS by 50% in cranial surgery. CONCLUSION: The QIP data from ERACraS in our unit has shown that implementing ERAS protocols is feasible. A reduction in HLOS has implications for patient morbidity, mortality, and quality of care. We endeavour to collect long-term data by collaborating with neurosurgical units across the UK and Ireland to validate its feasibility and sustainability as part of a major QIP in neurosurgical practice. This can be potentially adopted by neurosurgical centres across the globe in a safe and sustained manner.

Implementing the QUALI-DEC project in Argentina, Burkina Faso, Thailand and Viet Nam: a process delineation and theory-driven process evaluation protocol.

The project 'Quality Decision-making by women and providers' (QUALI-DEC) combines four non-clinical interventions to promote informed decision-making surrounding mode of birth, improve women's birth experiences, and reduce caesarean sections among low-risk women. QUALI-DEC is currently being implemented in 32 healthcare facilities across Argentina, Burkina Faso, Thailand, and Viet Nam. In this paper, we detail implementation processes and the planned process evaluation, which aims to assess how and for whom QUALI-DEC worked, the mechanisms of change and their interactions with context and setting; adaptations to intervention and implementation strategies, feasibility of scaling-up, and cost-effectiveness of the intervention. We developed a project theory of change illustrating how QUALI-DEC might lead to impact. The theory of change, together with on the ground observations of implementation processes, guided the process evaluation strategy including what research questions and perspectives to prioritise. Main data sources will include: 1) regular monitoring visits in healthcare facilities, 2) quantitative process and output indicators, 3) a before and after cross-sectional survey among post-partum women, 4) qualitative interviews with all opinion leaders, and 5) qualitative interviews with postpartum women and health workers in two healthcare facilities per country, as part of a case study approach. We foresee that the QUALI-DEC process evaluation will generate valuable information that will improve interpretation of the effectiveness evaluation. At the policy level, we anticipate that important lessons and methodological insights will be drawn, with application to other settings and stakeholders looking to implement complex interventions aiming to improve maternal and newborn health and wellbeing.Trial registration: ISRCTN67214403.

Single-cell, whole-embryo phenotyping of mammalian developmental disorders.

Mouse models are a critical tool for studying human diseases, particularly developmental disorders1. However, conventional approaches for phenotyping may fail to detect subtle defects throughout the developing mouse2. Here we set out to establish single-cell RNA sequencing of the whole embryo as a scalable platform for the systematic phenotyping of mouse genetic models. We applied combinatorial indexing-based single-cell RNA sequencing3 to profile 101 embryos of 22 mutant and 4 wild-type genotypes at embryonic day 13.5, altogether profiling more than 1.6 million nuclei. The 22 mutants represent a range of anticipated phenotypic severities, from established multisystem disorders to deletions of individual regulatory regions4,5. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibit changes in dozens of trajectories whereas others exhibit changes in only a few cell types. We also identify differences between widely used wild-type strains, compare phenotyping of gain- versus loss-of-function mutants and characterize deletions of topological associating domain boundaries. Notably, some changes are shared among mutants, suggesting that developmental pleiotropy might be 'decomposable' through further scaling of this approach. Overall, our findings show how single-cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution.

Antimyeloperoxidase antibodies modulate inflammatory responses and activate profibrotic pathways in human monocytes.

Antimyeloperoxidase (anti-MPO) and antiproteinase 3 (anti-PR3) antibodies are found in anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV). We investigated the effect of both anti-MPO and anti-PR3 IgG on human monocytes. Peripheral blood monocytes were cultured under a range of conditions that included TLR agonists, anti-MPO IgG and anti-PR3 IgG with appropriate controls. Experiments included whole transcriptome profiling and an assessment of the role of Fc receptors. When monocytes were stimulated with LPS or R848, anti-MPO but not anti-PR3 IgG, caused a reduction in IL-10 secretion and had a profound effect on cell-surface marker expression. Anti-MPO but not anti-PR3 IgG enhanced monocyte survival in the absence of TLR stimulation. These effects depended on the Fc receptor CD32a. With TLR stimulation, the effect of anti-MPO but not anti-PR3 IgG on the transcriptional response at 6 h was variable, but we identified a core set of transcripts likely to be important. Without TLR stimulation, there was a robust effect of anti-MPO but not anti-PR3 IgG on the transcriptional response at 24 h, and there was a highly significant enrichment of genes encoding extracellular matrix and extracellular matrix-associated proteins. Analysis with nCounter confirmed many of the differentially expressed transcripts and supported a role for CD32a. These data show that anti-MPO, but not anti-PR3 IgG, from patients with AAV has wide-ranging effects on monocytes which depend on CD32a. The activation of a profibrotic transcriptional response by anti-MPO but not anti-PR3 IgG may give insights into the differences in disease phenotype.

Introduction of a Single Electronic Health Record for Maternity Units in Ireland: Outline of the Experiences of the Project Management Team.

Electronic health records (EHRs) are being introduced worldwide. The change from paper to electronic records has not always been a seamless or quick process; however, EHRs are viewed as central to updating modern health care, especially organization structures and delivery of sustainable care with the potential for joint decision-making with the patient. The objective of this viewpoint paper is to outline how an EHR is being developed in Ireland. The focus of the Maternal & Newborn Clinical Management System project is the design and implementation of an EHR for all women and babies in the maternity services in the Republic of Ireland. The paper also outlines the lessons learned from the planning to the optimization stage of the project. The paper was developed through discussions with the project management team and their completed reports that outline the lessons they acquired from each project stage. Key lessons learned from each stage of the project are highlighted. This viewpoint paper explains how the national project management team is implementing the EHR and outlines the experiences and lessons learned and the challenges ahead following the phase one introduction. The Maternal & Newborn Clinical Management System is an example of a clinician-led, patient-focused, change management project from its inception to implementation. The introduction of EHRs is essential in modernizing health care and optimizing patient outcomes through the accurate and appropriate use of data.

Phosphatidylinositol 3-Kinase δ Deficiency Protects From Antimyeloperoxidase Vasculitis.

OBJECTIVE: Antineutrophil cytoplasmic antibody-associated vasculitis (AAV) is a systemic autoimmune disease in which glomerulonephritis is an important manifestation. Antibodies against myeloperoxidase (MPO) or proteinase 3 are thought to be important in pathogenesis. Phosphoinositide 3-kinase δ (PI3Kδ) mediates a number of effects in lymphocytes, but its role in myeloid cell responses is less clear. Therefore, this study was undertaken to assess this in a preclinical model of glomerulonephritis induced by the transfer of antibodies to MPO. METHODS: D910A mice with inactive PI3Kδ were compared with wild-type controls. Disease protocols allowed for a comparison of experimental groups in the setting of both mild and more severe disease. Adoptive transfer experiments were performed, with flow cytometric analysis of digested kidneys taken at the end of the experiment. RESULTS: With mild disease, D910A mice had fewer glomerular macrophages, fewer glomerular neutrophils, and reduced albuminuria compared with wild-type controls. With more severe disease, they also had fewer glomerular crescents and lower serum creatinine levels, indicating protection from acute kidney injury. Adoptive transfer experiments showed a defect in the recruitment of D910A monocytes to the diseased kidney. CONCLUSION: Mice with inactive PI3Kδ were protected from anti-MPO vasculitis. This is due to cell intrinsic defect in the recruitment of monocytes to the kidney. These findings suggest that PI3Kδ is a potential therapeutic target in AAV.

Cesarean scar niche: An evolving concern in clinical practice.

Increasing cesarean section rates have led to an increased awareness of associated complications such as the formation of cesarean scar niche, defined as an indentation at the site of the cesarean scar with a depth of at least 2 mm, diagnosed by ultrasound or magnetic resonance imaging. The precise prevalence of cesarean scar niche is unclear. The cause of a cesarean scar niche appears to be multifactorial and likely a combination of technical factors (low incision location), anatomical factors (uterine retroflexion), and patient factors, which might impair healing (body mass index, smoking, maternal age). Most patients with cesarean scar niche are asymptomatic; however, women can present with postmenstrual bleeding, pelvic pain, and subfertility. In pregnancy, cesarean scar niches have been associated with placenta accreta spectrum disorder and uterine rupture. Treatment should be reserved for symptomatic women. Hormonal treatment using either the combined oral contraceptive pill or a progesterone-containing intrauterine device may address irregular vaginal bleeding. Surgical management should be reserved for those in whom hormonal manipulation has failed or is contraindicated. The aim of this review was to summarize current literature pertaining to the cause, prevalence, diagnosis, and symptoms of cesarean scar niche and to make recommendations for managing this relatively new condition.

Disquiet concerning cesarean birth.

Cesarean birth has increased substantially in many parts of the world over recent decades and concerns have been raised about the propriety of this change in obstetric practice. Sometimes, a cesarean is necessary to preserve fetal and maternal health. But in balancing the risks of surgical intervention the implicit assumption has been that cesarean birth is an equivalent alternative to vaginal birth from the standpoint of the immediate and long-term health of the fetus and neonate. Increasingly, we realize this is not necessarily so. Delivery mode per se may influence short-term and abiding problems with homeostasis in offspring, quite independent of the indications for the delivery and other potentially confounding factors. The probability of developing various disorders, including respiratory compromise, obesity, immune dysfunction, and neurobehavioral disorders has been shown in some studies to be higher among individuals born by cesarean. Moreover, many of these adverse effects are not confined to the neonatal period and may develop over many years. Although the associations between delivery mode and long-term health are persuasive, their pathogenesis and causality remain uncertain. Full exploration and a clear understanding of these relationships is of great importance to the health of offspring.

Myeloid expression of the anti-apoptotic protein Mcl1 is required in anti-myeloperoxidase vasculitis but myeloperoxidase inhibition is not protective.

Antibodies to neutrophil and monocyte myeloperoxidase and proteinase 3 are a feature of anti-neutrophil cytoplasmic antibody vasculitis, a disease with significant morbidity for which new treatments are needed. Mice with a myeloid-specific deletion of the anti-apoptotic protein Mcl1 have reduced numbers of circulating neutrophils. Here, we assessed if myeloid-specific Mcl1 was required in murine anti-myeloperoxidase vasculitis and whether inhibition of myeloperoxidase was protective. In a murine model of anti-neutrophil cytoplasmic antibody vasculitis, induced by anti-myeloperoxidase antibody, mice with a myeloid-specific deletion of Mcl1 were protected from disease. They had fewer crescents, neutrophils, and macrophages in the glomeruli, lower serum creatinine levels and reduced albuminuria compared with controls. At baseline and day six after disease induction they had fewer circulating neutrophils than controls. At day six there were also fewer circulating monocytes. Myeloperoxidase inhibition with AZD5904 had no effect on histological or biochemical parameters of disease, and there was also no reduction in albuminuria at day one, two, five or seven after disease induction. These findings persisted when disease was induced without granulocyte-colony stimulating factor, which increases disease severity. A second myeloperoxidase inhibitor, AZM198, also showed no evidence of an effect, although both AZD5904 and AZM198 inhibited human neutrophil extracellular trap formation in vitro. Thus, our results show that while myeloid-specific Mcl1 is required in this model of anti-myeloperoxidase vasculitis, myeloperoxidase inhibition is not protective.

Entomo-virological surveillance followed by serological active survey of symptomatic individuals is helpful to identify hotspots of early arbovirus transmission.

Arboviruses transmitted by Aedes aegypti in urban environments have spread rapidly worldwide, causing great impacts on public health. The development of reliable and timely alert signals is among the most important steps in designing accurate surveillance systems for vector-borne diseases. In July and September 2017, we conducted a pilot study to improve an existing integrated surveillance system by using entomo-virological surveillance to prioritize areas to conduct active searches for individuals with arbovirus infection symptoms. Foz do Iguaçu City has a permanent entomo-virological surveillance system with approximately 3,500 traps to capture Aedes sp. in the adult stage. The Aedes aegypti females are captured alive and human samples are submitted to RT-qPCR (real-time qPCR) screening for DENV, ZIKV, and CHIKV diagnosis. Of the 55 Ae. aegypti mosquitoes tested in July 2017, seven (12.7%) were considered positive for DENV-2 and three (5.4%) for CHIKV. In September, we tested a sample of 54 mosquitoes, and 15 (27.7%) were considered infected by DENV-2. We created 25 circumferences with 150-m radius each to perform an active survey to identify symptomatic householders. In July, we selected one circumference, and five (35.7%) patients were positive for DENV, whereas two (14.3%) for CHIKV. In September, we selected four circumferences, and, from the 21 individuals sampled, nine (42.8%) were positive for DENV-2. A statistical model with a binomial response was used to estimate the number of cases in areas without active surveys, i.e., 20 circumferences. We estimated an additional 83 symptomatic patients (95% CI: 45-145) to be found in active searches, with 38 (95% CI: 18-72) of them confirming arbovirus infection. Arbovirus detection and serotyping in mosquitoes, but also in symptomatic individuals during active surveys, can provide an alert signal of early arbovirus transmission.

Adaptation of a Human Diagnostic Kit to Detect Dengue, Zika, and Chikungunya Viruses in Mosquito Samples (Aedes aegypti and Aedes albopictus): A Contribution to Public Health in the International Triple Border (Brazil, Paraguay, and Argentina).

Objective: The objective of this work was to adapt a diagnostic kit developed for humans to identify Dengue (DENV1, DENV2, DENV3, DENV4), Zika (ZIKV) and Chikungunya virus (CHIKV) in females of Aedes aegypti and Aedes albopictus and to verify if the occurrence of mosquitoes infected with these three arboviruses are being found in regions with high occurrence of these diseases in humans. Materials and Methods: For this purpose, live mosquitoes were captured between January and June 2020 using 3,476 traps permanently installed in the field were used. After capture, the species were identified, then the females were placed in a pool of 2 to 10 specimens and sent to the laboratory for detection of DENV1, DENV2, DENV3, DENV4, ZIKV and CHIKV by RT-PCR using a commercial human kit for arboviruses. Results: Of the 76 mosquito pools collected, six (7.9%) pools tested positive for the DENV2 virus. The DENV-positive mosquitoes were collected in regions with a high incidence of reported cases of Dengue or in adjacent areas. Conclusion: The absence of kits for the detection of these arboviruses in Aedes is a limiting factor and the adequacy of commercial kits, already used for the diagnosis of arboviruses in humans, the results presented demonstrate that it is possible to identify the presence of DENV2 in mosquitoes with the respective kit, reinforcing the use of RT-qPCR as a robust diagnostic tool for epidemiological surveillance allowing managers to receive timely results for decision-making regarding prevention and control actions.

Manipulation of tissue factor-mediated basal PAR-2 signalling on macrophages determines sensitivity for IFNγ responsiveness and significantly modifies the phenotype of murine DTH.

Background: Tissue factor (TF) generates proteases that can signal through PAR-1 and PAR-2. We have previously demonstrated PAR-1 signalling primes innate myeloid cells to be exquisitely sensitive to interferon-gamma (IFNγ). In this work we explored how TF mediated PAR-2 signalling modulated responsiveness to IFNγ and investigated the interplay between PAR-1/-2 signalling on macrophages. Methodology: We characterised how TF through PAR-2 influenced IFNγ sensitivity in vitro using PCR and flow cytometry. and how it influenced oxazolone-induced delayed type hypersensitivity (DTH) responses in vivo. We investigated how basal signalling through PAR-2 influenced PAR-1 signalling using a combination of TF-inhibitors and PAR-1 &-2 agonists and antagonists. Finally, we investigated whether this system could be targeted therapeutically using 3-mercaptopropionyl-F-Cha-Cha-RKPNDK (3-MP), which has actions on both PAR-1 and -2. Results: TF delivered a basal signal through PAR-2 that upregulated SOCS3 expression and blunted M1 polarisation after IFNγ stimulation, opposing the priming achieved by signalling through PAR-1. PAR-1 and -2 agonists or antagonists could be used in combination to modify this basal signal in vitro and in vivo. 3-MP, by virtue of its PAR-2 agonist properties was superior to agents with only PAR-1 antagonist properties at reducing M1 polarisation induced by IFNγ and suppressing DTH. Tethering a myristoyl electrostatic switch almost completely abolished the DTH response. Conclusions: TF-mediated signalling through PARs-1 and -2 act in a homeostatic way to determine how myeloid cells respond to IFNγ. 3-MP, an agent that simultaneously inhibits PAR-1 whilst delivering a PAR-2 signal, can almost completely abolish immune responses dependent on M1 polarisation, particularly if potency is enhanced by targeting to cell membranes; this has potential therapeutic potential in multiple diseases.

Repression and 3D-restructuring resolves regulatory conflicts in evolutionarily rearranged genomes.

Regulatory landscapes drive complex developmental gene expression, but it remains unclear how their integrity is maintained when incorporating novel genes and functions during evolution. Here, we investigated how a placental mammal-specific gene, Zfp42, emerged in an ancient vertebrate topologically associated domain (TAD) without adopting or disrupting the conserved expression of its gene, Fat1. In ESCs, physical TAD partitioning separates Zfp42 and Fat1 with distinct local enhancers that drive their independent expression. This separation is driven by chromatin activity and not CTCF/cohesin. In contrast, in embryonic limbs, inactive Zfp42 shares Fat1's intact TAD without responding to active Fat1 enhancers. However, neither Fat1 enhancer-incompatibility nor nuclear envelope-attachment account for Zfp42's unresponsiveness. Rather, Zfp42's promoter is rendered inert to enhancers by context-dependent DNA methylation. Thus, diverse mechanisms enabled the integration of independent Zfp42 regulation in the Fat1 locus. Critically, such regulatory complexity appears common in evolution as, genome wide, most TADs contain multiple independently expressed genes.