Mortality in a swiftly repurposed hospital in northeast Brazil during the first and second COVID-19 waves: A retrospective cohort study.

Background: The first months of the coronavirus disease 2019 (COVID-19) pandemic demanded rapid re-organization of available local resources. This study evaluated the performance of a private hospital in the Brazilian state of Ceará that was swiftly repurposed into a public tertiary COVID-19 centre during the first wave of the COVID-19 pandemic, and how it improved in the second wave. Methods: This retrospective cohort study included 2492 patients with COVID-19 at Hospital Estadual Leonardo da Vinci (HELV) during the first and second waves. Demographic, clinical and laboratory data were collected using a dedicated web platform (ResCOVID). A Poisson regression model was used to estimate factors associated with in-hospital mortality. Results: Differences in demographics and clinical features were found between the two waves. There was reduced in-hospital mortality during the second wave (36.2%) in comparison with the first wave (48.8%). Invasive mechanical ventilation showed the strongest association with increased risk of death in both waves {first wave: relative risk (RR) 4.28 [95% confidence interval (CI) 2.86-6.41], P<0.001; second wave: RR 12.94 (95% CI 3.4-49.12), P<0.001}. Conclusions: HELV was a pillar in the strategic public health plan to respond to COVID-19 in Ceará, helping to assist a group of moderate-to-severe cases and reduce the pressure on emergency and primary care facilities. Although mortality in intubated individuals remained high, there was an overall decrease in the in-hospital mortality rate in the second wave.

O FeliciLab, a inovação e a pandemia de covid-19 / FeliciLab, innovation and the covid-19 pandemic / El FeliciLab, la innovación y la pandemia de covid-19

Com o fim de propiciar um ambiente que possibilite decisões ágeis, criativas e efetivas no sistema de saúde, o Laboratório de Inovações no SUS do Ceará (FeliciLab) tem adotado o uso de métodos inovadores de gestão de projetos. Este relato de experiência tem como objetivo apresentar como a Escola de Saúde Pública do Ceará (ESP/CE) conseguiu, através do FeliciLab, orquestrar um conjunto de pessoas, métodos e tecnologias capazes de provocar uma quebra em padrões até então apresentados no mercado de tecnologias direcionadas para o sistema de saúde, diante da crise sanitária ocasionada pela pandemia de covid-19. Com adoção de metodologias mais ágeis de condução de trabalho e gerenciamento de crises, ampliou-se a percepção de valor da tecnologia da informação (TI) da ESP/CE, nutrida pela compreensão de que inovar é mais do que produzir e implantar tecnologias: trata-se de transformar os modos de criar e consumir soluções, de forma a impactar os contextos e as experiências das pessoas.

In order to provide an environment with agile, creative, and effective decisions in the health system, the Felicilab laboratory has been adopting the use of innovative project management methods. This experience report aims to present how the Escola de Saúde Pública do Ceará (ESP/CE), Brazil, had managed to orchestrate, through FeliciLab, a set of people, methods, and technologies from a disruptive perspective of standards hitherto presented in the market of technologies directed to the health system, in face of the health crisis caused by the covid-19 pandemic. With the adoption of more agile methodologies for conducting work and managing crisis, the perception of the value of the Information technology (IT) used at ESP/CE was expanded, understanding that innovating is more than producing and implementing technologies: it means transforming the ways of creating and consume solutions in order to impact on personal experiences and their context.

A fin de propiciar un ambiente con decisiones ágiles, creativas y efectivas en el sistema de salud, el Laboratório de Inovações no SUS do Ceará (FeliciLab) tiene adoptado el uso de métodos innovadores de gestión de proyectos. Este relato de experiencia tiene como objetivo presentar cómo la Escola de Saúde Pública do Ceará (ESP/CE), a través del FeliciLab, ha conseguido orquestar un conjunto de personas,nmétodos y tecnologías capaces para quebrar estándares hasta entonces presentados en el mercado de tecnologías dirigidas al sistema de salud, frente a la crisis de salud provocada por la pandemia de Covid-19. Con la adopción de metodologías más ágiles para la realización del trabajo y la gestión de crisis, se amplió la percepción del valor de la tecnología de la información (TI) ESP/CE, alimentada por el entendimiento de que innovar es más que producir e implementar tecnologías: se trata de transformar formas de crear y consumir soluciones para impactar los contextos y las experiencias de las personas.

Acute exposure to physiological doses of triiodothyronine does not induce gonadal caspase 3 activity in goldfish in vitro.

Seasonally spawning fish rely on a dynamic and complex hormonal interplay to regulate cycles of gonadal development and the regression. Thyroid hormones have been shown to be a key player during gonadal development, and can regulate the activity of a number of essential reproductive hormones. Apoptosis is a vital cellular process that contributes to the hormonal control of gonadal development and regression, but the roles of thyroid hormones on gonadal apoptosis in goldfish have not been explored. The present study examines the role of acute T3 exposure on caspase 3-dependent apoptosis in dispersed goldfish gonadal tissue in vitro. We examined the levels of caspase 3 activity in early, mid, and late recrudescent gonadal tissue after exposure to physiological doses of T3 for up to 24 h. Acute treatment with T3 did not alter basal caspase 3 activity in goldfish gonads in vitro in these reproductive stages. This initial study suggests that transient increases in T3 levels are unlikely to directly contribute to basal caspase 3-dependent apoptosis in the gonadal tissue of goldfish, although we cannot rule out an interaction of T3 with other hormones involved in the control of apoptosis in the testis and ovary.

Clearance of schistosome parasites by resistant genotypes at a single genomic region in Biomphalaria glabrata snails involves cellular components of the hemolymph.

Schistosomiasis is one of the most detrimental neglected tropical diseases. Controlling the spread of this parasitic illness requires effective sanitation, access to chemotherapeutic drugs, and control over populations of the freshwater snails, such as Biomphalaria glabrata, that are essential intermediate hosts for schistosomes. Effectively controlling this disease, while minimising ecological implications of such control, will require an extensive understanding of the immunological interactions between schistosomes and their molluscan intermediate hosts. Here we histologically characterise the clearance of schistosome larvae by snails that exhibit allelic variation at a single genomic region, the Guadeloupe resistance complex. We show that snails with a resistant Guadeloupe resistance complex genotype clear schistosomes within the first 24-48 h, and that this resistance can be transferred to susceptible snails via whole hemolymph but not cell-free plasma. These findings imply that Guadeloupe resistance complex-coded proteins help to coordinate hemocyte-mediated immune responses to schistosome infections in Guadeloupean snails.

Primary cilia on porcine testicular somatic cells and their role in hedgehog signaling and tubular morphogenesis in vitro.

The primary cilium is a microtubule-based sensory organelle found on nearly all eukaryotic cells but little is understood about its function in the testis. We investigate the role of primary cilia on testis cells in vitro by inhibiting formation of the primary cilium with Ciliobrevin D, a cell-permeable, reversible chemical inhibitor of ATPase motor cytoplasmic dynein. We analyzed cultured cells for the presence of primary cilia and their involvement in hedgehog signaling. Primary cilia were present on 89.3 ± 2.3 % of untreated testicular somatic cells compared to 3.1 ± 2.5 % cells with primary cilia for Ciliobrevin D-treated cells. Protein levels of Gli-2 and Smoothened were lower on Western blots after suppression of cilia with Ciliobrevin D. The inhibitor did not affect centrosome localization or cell proliferation, indicating that changes were due to ablation of the primary cilium. Testicular somatic cells have the ability to form three-dimensional tubules in vitro. In vitro-formed tubules were significantly longer and wider in the control group than in the Ciliobrevin D-treated group (9.91 ± 0.35 vs. 5.540 ± 1.08 mm and 339.8 ± 55.78 vs. 127.2 ± 11.9 µm, respectively) indicating that primary cilia play a role in tubule formation. Our results establish that the inhibition of ATPase motor cytoplasmic dynein perturbs formation of primary cilia in testicular somatic cells, affects the hedgehog signaling pathway and impairs tubule formation in vitro. These findings provide evidence for a role of cilia in the testis in cell signaling and tubular morphogenesis in vitro.

Use of Stirred Suspension Bioreactors for Male Germ Cell Enrichment.

Spermatogenesis is a stem cell based system. Both therapeutic and biomedical research applications of spermatogonial stem cells require a large number of cells. However, there are only few germ line stem cells in the testis, contained in the fraction of undifferentiated spermatogonia. The lack of specific markers makes it difficult to isolate these cells. The long term maintenance and proliferation of nonrodent germ cells in culture has so far been met with limited success, partially due to the lack of highly enriched starting populations. Differential plating, which depends on the differential adhesion properties of testicular somatic and germ cells to tissue culture dishes, has been the method of choice for germ cell enrichment, especially for nonrodent germ cells. However, for large animals, this process becomes labor intensive and increases variability due to the need for extensive handling. Here, we describe the use of stirred suspension bioreactors, as a novel system for enriching undifferentiated germ cells from 1-week-old pigs. This method capitalizes on the adherent properties of somatic cells within a controlled environment, thus promoting the enrichment of progenitor cells with minimal handling and variability.

Primary cilia in the developing pig testis.

In vertebrates, a variety of cell types generate a primary cilium. Cilia are implicated in determination and differentiation of a wide variety of organs and during embryonic development. However, there is little information on the presence or function of primary cilia in the mammalian testis. Therefore, the objective of this study was to characterize expression of primary cilia in the developing pig testis. Testicular tissue from pigs at 2-10 weeks of age was analyzed for primary cilia by immunocytochemistry. Expression of primary cilia was also analyzed in testicular tissue formed de novo from a single cell suspension ectopically grafted into a mouse host. Functionality of primary cilia was monitored based on cilia elongation after exposure to lithium. Analysis showed that the primary cilium is present in testis cords as well as in the interstitium of the developing pig testis. Germ cells did not express primary cilia. However, we identified Sertoli cells as one of the somatic cell types that produce a primary cilium within the developing testis. Primary cilium expression was reduced from the second to the third week of pig testis development in situ and during de novo morphogenesis of testis tissue from a single cell suspension after xenotransplantation. In vitro, primary cilia were elongated in response to lithium treatment. These results indicate that primary cilia on Sertoli cells may function during testicular development. De novo morphogenesis of testis tissue from single cell suspensions may provide an accessible platform to study and manipulate expression and function of primary cilia.

De novo morphogenesis of testis tissue: an improved bioassay to investigate the role of VEGF165 during testis formation.

De novo formation of testis tissue from single-cell suspensions allows manipulation of different testicular compartments before grafting to study testicular development and the spermatogonial stem cell niche. However, the low percentages of newly formed seminiferous tubules supporting complete spermatogenesis and lack of a defined protocol have limited the use of this bioassay. Low spermatogenic efficiency in de novo formed tissue could result from the scarcity of germ cells in the donor cell suspension, cell damage caused by handling or from hypoxia during tissue formation in the host environment. In this study, we compared different proportions of spermatogonia in the donor cell suspension and the use of Matrigel as a scaffold to support de novo tissue formation and spermatogenesis. Then, we used the system to investigate the role of vascular endothelial growth factor 165 (VEGF165) during testicular morphogenesis on blood vessel and seminiferous tubule formation, and on presence of germ cells in the de novo developed tubules. Our results show that donor cell pellets with 10×10(6) porcine neonatal testicular cells in Matrigel efficiently formed testis tissue de novo. Contrary to what was expected, the enrichment of the cell suspension with germ cells did not result in higher numbers of tubules supporting spermatogenesis. The addition of VEGF165 did not improve blood vessel or tubule formation, but it enhanced the number of tubules containing spermatogonia. These results indicate that spermatogenic efficiency was improved by the addition of Matrigel, and that VEGF165 may have a protective role supporting germ cell establishment in their niche.

Viral transduction of male germline stem cells results in transgene transmission after germ cell transplantation in pigs.

Genetic modification of germline stem cells (GSCs) is an alternative approach to generate large transgenic animals where transgenic GSCs are transplanted into a recipient testis to generate donor-derived transgenic sperm. The objective of the present study was to explore the application of viral vectors in delivering an enhanced green fluorescent protein (EGFP) transgene into GSCs for production of transgenic gametes through germ cell transplantation. Both adeno-associated virus (AAV)- and lentivirus (LV)-based vectors were effective in transducing pig GSCs, resulting in the production of transgenic sperm in recipient boars. Twenty-one boars treated with busulfan to deplete endogenous GSCs and nine nontreated boars received germ cell transplantation at 12 wk of age. Semen was collected from recipient boars from 5 to 7 mo posttransplantation when boars became sexually mature, and semen collection continued for as long as 5 yr for some boars. The percentage of ejaculates that were positive for the EGFP transgene ranged from 0% to 54.8% for recipients of AAV vector-transduced germ cells (n = 17) and from 0% to 25% for recipients of LV vector-transduced germ cells (n = 5). When semen from two AAV recipients was used for in vitro fertilization (IVF), 9.09% and 64.3% of embryos were transgenic. Semen collected from two LV-vector recipients produced 7.7% and 26.3% transgenic IVF embryos. Here, we not only demonstrated AAV-mediated GSC transduction in another large animal model (pigs) but also showed, to our knowledge for the first time, that LV-mediated GSC transduction resulted in transgene transmission in pigs.

From in vitro culture to in vivo models to study testis development and spermatogenesis.

The testis is a complex organ playing host to one of the most intricate mass cell divisions occurring in postnatal life. Since the beginning of the 20th century, great efforts have been made to recapitulate spermatogenesis and elucidate spermatogonial stem cell function. These efforts have resulted in the development of a variety of model systems that provide invaluable knowledge regarding testis organogenesis, key cell types and their interactions, and signaling pathways controlling testis function. The goal of this review is to elaborate on the evolution of the techniques available from in vitro culture systems to in vivo bioassays by providing up to date information and weighing their particular strengths and weaknesses. Each technique offers a different approach to the elucidation of male reproduction, the enhancement of germ-lineage genetic manipulation, the preservation of gametes, the restoration of fertility, and the improvement in our understanding of stem cell biology.

Lymphoid-specific helicase (HELLS) is essential for meiotic progression in mouse spermatocytes.

Lymphoid-specific helicase (HELLS; also known as LSH) is a member of the SNF2 family of chromatin remodeling proteins. Because Hells-null mice die at birth, a phenotype in male meiosis cannot be studied in these animals. Allografting of testis tissue from Hells(-/-) to wild-type mice was employed to study postnatal germ cell differentiation. Testes harvested at Day 18.5 of gestation from Hells(-/-), Hells(+/-), and Hells(+/+) mice were grafted ectopically to immunodeficient mice. Bromodeoxyuridine incorporation at 1 wk postgrafting revealed fewer dividing germ cells in grafts from Hells(-/-) than from Hells(+/+) mice. Whereas spermatogenesis proceeded through meiosis with round spermatids in grafts from Hells heterozygote and wild-type donor testes, spermatogenesis arrested at stage IV, and midpachytene spermatocytes were the most advanced germ cell type in grafts from Hells(-/-) mice at 4, 6, and 8 wk after grafting. Analysis of meiotic configurations at 22 days posttransplantation revealed an increase in Hells(-/-) spermatocytes with abnormal chromosome synapsis. These results indicate that in the absence of HELLS, proliferation of spermatogonia is reduced and germ cell differentiation arrested at the midpachytene stage, implicating an essential role for HELLS during male meiosis. This study highlights the utility of testis tissue grafting to study spermatogenesis in animal models that cannot reach sexual maturity.