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BACKGROUND: Age-related mechanisms of sarcopenia associated with vascular function have been recently suggested. This study compared and tested associations between muscle mass and strength, microcirculation, inflammatory biomarkers, and oxidative stress in older adults classified as sarcopenic and non-sarcopenic. METHODS: Thirty-three physically inactive individuals (72±7 yrs) were assigned to age-matched sarcopenic (SG) and non-sarcopenic (NSG) groups. Between-group comparisons were performed for appendicular skeletal mass (ASM), handgrip and isokinetic strength, microvascular function and morphology, C-reactive protein, insulin-like growth factor-1, tumor necrosis factor-alpha, interleukin-6 (IL-6), soluble vascular cell adhesion molecule-1, soluble intercellular adhesion molecule-1, endothelin-1, and oxidized low-density lipoprotein. RESULTS: ASM and knee isokinetic strength were lower in SG than NSG (P

The duration of natural immunity in response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a matter of some debate in the literature at present. For example, in a recent publication characterizing SARS-CoV-2 immunity over time, the authors fit pooled longitudinal data, using fitted slopes to infer the duration of SARS-CoV-2 immunity. In fact, such approaches can lead to misleading conclusions as a result of statistical model-fitting artifacts. To exemplify this phenomenon, we reanalyzed one of the markers (pseudovirus neutralizing titer) in the publication, using mixed-effects modeling, a methodology better suited to longitudinal datasets like these. Our findings showed that the half-life was both longer and more variable than reported by the authors. The example selected by us here illustrates the utility of mixed-effects modeling in provide more accurate estimates of the duration and heterogeneity of half-lives of molecular and cellular biomarkers of SARS-CoV-2 immunity.

La investigación denominada Efectos de la aplicación de diferentes niveles de fertilización potásica en el rendimiento y la calidad de frutos en el cultivo de pimiento (Capsicum annum L.) Var: Híbrido Nathalie, en el distrito de Pilar, año agrícola 2017/2018, fue presentada a la Facultad de Ciencias Agropecuarias y Desarrollo Rural, Universidad Nacional de Pilar. Investigación de tipo experimental, enfocado a la evaluación de diversos parámetros en la producción de pimiento de la variedad Híbrido Nathalie, con aplicación de diferentes niveles de fertilización potásica, los resultados obtenidos valorar la influencia de dichos tratamientos en la calidad y el rendimiento del cultivo de pimiento. Las dosis utilizadas fueron las siguientes 50, 75, 100, 125, y 150 kg/ha, incluyendo un testigo el cual fue el tratamiento sin aplicación de fertilizante potásico, contando así con seis tratamientos y cinco repeticiones distribuidos en bloques al azar. Dicho análisis se comprobó con pruebas estadísticas, análisis de la varianza y la comparación de medias a través de la prueba de Duncan al 5%. Respecto a la calidad, las medias de longitud, diámetro y grosor de pared del fruto no presentaron grandes diferencias, destacando que el T4 (100 Kg/ha) siempre presentó un nivel superior entre los tratamientos, y en cuanto al rendimiento el análisis de varianza arrojó que no existe diferencia significativa entre los tratamientos, sin embargo, el T4 tuvo el mayor rendimiento con 55.225 Kg/ha donde la comparación de medias indicó que presentó diferencia significativa sobre el testigo.

Duchenne muscular dystrophy (DMD) affects 1 in 3500 live male births. To date, there is no effective cure for DMD, and the identification of novel molecular targets involved in disease progression is important to design more effective treatments and therapies to alleviate DMD symptoms. Here, we show that protein levels of the Bromodomain and extra-terminal domain (BET) protein BRD4 are significantly increased in the muscle of the mouse model of DMD, the mdx mouse, and that pharmacological inhibition of the BET proteins has a beneficial outcome, tempering oxidative stress and muscle damage. Alterations in reactive oxygen species (ROS) metabolism are an early event in DMD onset and they are tightly linked to inflammation, fibrosis, and necrosis in skeletal muscle. By restoring ROS metabolism, BET inhibition ameliorates these hallmarks of the dystrophic muscle, translating to a beneficial effect on muscle function. BRD4 direct association to chromatin regulatory regions of the NADPH oxidase subunits increases in the mdx muscle and JQ1 administration reduces BRD4 and BRD2 recruitment at these regions. JQ1 treatment reduces NADPH subunit transcript levels in mdx muscles, isolated myofibers and DMD immortalized myoblasts. Our data highlight novel functions of the BET proteins in dystrophic skeletal muscle and suggest that BET inhibitors may ameliorate the pathophysiology of DMD., Duchenne muscular dystrophy (DMD) is characterised by progressive muscle degeneration. Here, the authors show that the BET protein BRD4 is increased in the muscle of DMD mouse models, and that pharmacological inhibition of BRD4 leads to reduced muscle pathology in mice, by modulating NADPH oxidase expression.​

Purpose Does controlled ovarian stimulation (COS) and progesterone (P) luteal supplementation modify the vaginal and endometrial microbiota of women undergoing in vitro fertilization? Methods Fifteen women underwent microbiota analysis at two time points: during a mock transfer performed in the luteal phase of the cycle preceding COS, and at the time of fresh embryo transfer (ET). A vaginal swab and the distal extremity of the ET catheter tip were analyzed using next-generation 16SrRNA gene sequencing. Heterogeneity of the bacterial microbiota was assessed according to both the Bray-Curtis similarity index and the Shannon diversity index. Results Lactobacillus was the most prevalent genus in the vaginal samples, although its relative proportion was reduced by COS plus P supplementation (71.5 ± 40.6% vs. 61.1 ± 44.2%). In the vagina, an increase in pathogenic species was observed, involving Prevotella (3.5 ± 8.9% vs. 12.0 ± 19.4%), and Escherichia coli-Shigella spp. (1.4 ± 5.6% vs. 2.0 ± 7.8%). In the endometrium, the proportion of Lactobacilli slightly decreased (27.4 ± 34.5% vs. 25.0 ± 29.9%); differently, both Prevotella and Atopobium increased (3.4 ± 9.5% vs. 4.7 ± 7.4% and 0.7 ± 1.5% vs. 5.8 ± 12.0%). In both sites, biodiversity was greater after COS (p Conclusions Our findings suggest that COS and P supplementation significantly change the composition of vaginal and endometrial microbiota. The greater instability could affect both endometrial receptivity and placentation. If our findings are confirmed, they may provide a further reason to encourage the freeze-all strategy.

Asparaginase (ASNase) causes pancreatitis in approximately 10% of leukemia patients, and the mechanisms underlying this painful complication are not known. ASNase primarily depletes circulating asparagine, and the endogenously expressed enzyme, asparagine synthetase (ASNS), replenishes asparagine. ASNS was suggested previously to be highly expressed in the pancreas. In this study, we determined the expression pattern of ASNS in the pancreas and the mechanism for increased pancreatic ASNS abundance. Compared with other organs, ASNS was highly expressed in both the human and mouse pancreas, and, within the pancreas, ASNS was present primarily in the acinar cells. The high baseline pancreatic ASNS was associated with higher baseline activation of protein kinase R-like endoplasmic reticulum kinase (PERK) signaling in the pancreas, and inhibition of PERK in acinar cells lessened ASNS expression. ASNase exposure, but not the common pancreatitis triggers, uniquely up-regulated ASNS expression, indicating that the increase is mediated by nutrient stress. The up-regulation of acinar ASNS with ASNase exposure was owing to increased transcriptional rather than delayed degradation. Knockdown of ASNS in the 266-6 acinar cells provoked acinar cell injury and worsened ASNase-induced injury, whereas ASNS overexpression protected against ASNase-induced injury. In summary, ASNS is highly expressed in the pancreatic acinar cells through heightened basal activation of PERK, and ASNS appears to be crucial to maintaining acinar cell integrity. The implications are that ASNS is especially hardwired in the pancreas to protect against both baseline perturbations and nutrient deprivation stressors, such as during ASNase exposure. Keywords: Leukemia, Asparaginase-Associated Pancreatitis, Asparagine Synthetase, PERK Signaling

Made available in DSpace on 2020-12-12T01:00:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Objective: The aim of this study was to investigate the biofilm formation and cell viability of a polymer-infiltrated ceramic (PIC) and an yttria-stabilized polycrystalline zirconium dioxide ceramic (Y-TZP). The null hypothesis was that there would be no difference in biofilm formation and cell viability between the materials. Methods and Materials: Streptococcus mutans biofilm was analyzed with scanning electron microscopy (SEM), confocal laser scanning microscopy, and colony counting (colony-forming units/mL). The cell viability (fibroblasts) of both materials was measured with 3-(4,5-dime-thylthiazol-2yl)-2,5-diphenyl tetrazolium) (MTT) test. Roughness measurements were also performed. Results: The PIC displayed higher roughness but showed similar colony-forming units and biovolume values to those of Y-TZP. SEM showed a higher amount of adhered fibroblasts on the PIC surface on the first day and similar amounts on both materials after seven days. Moreover, the materials were biocompatible with human fibroblasts. Conclusion: PIC and Y-TZP are biocompatible and present the same characteristics for biofilm formation; therefore, they are indicated for indirect restorations and implant abutments. Intitute of Science and Technology Univ. Estadual Paulista UNESP Dental Materials and Prosthodontics University of Taubaté Dentistry Taubaté Institute of Science and Technology Univ. Estadual Paulista UNESP Univ. Estadual Paulista Department of Biosciences and Oral Diagnosis Department of Restorative Dental Sciences Univ. of Florida College of Dentistry Intitute of Science and Technology Univ. Estadual Paulista UNESP Dental Materials and Prosthodontics Institute of Science and Technology Univ. Estadual Paulista UNESP FAPESP: 2014/19357-9

Chronic pancreatitis (CP) is a complex inflammatory disorder with numerous associated genetic and environmental risk factors. The most distressing characteristic of CP is recalcitrant pain, often requiring surgical resection including total pancreatectomy with islet autotransplantation (TPIAT). We studied five consented subjects undergoing pancreatic resection and processed isolated cells for single-cell RNA sequencing (scRNA-Seq). Using high-dimensional transcriptomic cluster analysis, we identified 11 unique cell clusters in the pancreas tissue. These cell clusters include a cluster of undifferentiated/dedifferentiated cells and two unique clusters of acinar cells, one of which appears to be in a transitional stage. To determine the cellular response to protease inhibitor and stimulation, we treated aliquots of cells from one subject with a protease inhibitor cocktail with and without bethanechol (a muscarinic receptor agonist) at 100 and 400 µM and compared gene expression profiles. The protease inhibitors appeared to reduce cell stress. Pancreatic digestive enzymes and islet hormones were upregulated in both doses of bethanechol-treated cells compared with naïve cells. High-dose bethanechol appeared to be toxic and consistent with hyperstimulation. These studies demonstrate the feasibility of investigating human acinar cell physiology at the single-cell level and initial evidence that these cells retain responsiveness to agonist stimulation with predicted second messenger and transcriptomic responses. NEW & NOTEWORTHY We conducted single cell RNA sequencing on pancreas tissue from five individuals. We identified eleven unique cell clusters including a large population of dedifferentiated cells as well as two unique clusters of acinar cells, one of which appears to exist in a transitional state. We also examined the cellular response of pancreas tissue to stimulation and identified affected genes and pathways, including pancreatic digestive enzymes.

Background Type 1 diabetes (T1DM) is a chronic autoimmune disease characterized by T-cell-mediated destruction of insulin-producing beta cells. Evidence shows that patients with T1DM and mice used in specific diabetic models both exhibit changes in their intestinal microbiota and dysregulated microbiota contributes to the pathogenesis of T1DM. Islet transplantation (Tx) is poised to play an important role in the treatment of T1DM. However, whether treatment of T1DM with islet Tx can rescue dysregulated microbiota remains unclear. Methods In this study, we induced diabetic C57BL/6 mice with streptozotocin. Then treatment with either insulin administration, or homogenic or allogenic islet Tx was performed to the diabetic mice. Total DNA was isolated from fecal pellets and high-throughput 16S rRNA sequencing was used to investigate intestinal microbiota composition. Results The overall microbial diversity was comparable between control (nonstreptozotocin treated) and diabetic mice. Our results showed the ratio of the Bacteroidetes: Firmicutes between nondiabetic and diabetic mice was significant different. Treatment with islet Tx or insulin partially corrects the dysregulated bacterial composition. At the genus level, Bacteroides, Odoribacter, and Alistipes were associated with the progression and treatment efficacy of the disease, which may be used as a biomarker to predict curative effect of treatment for patients with T1DM. Conclusions Collectively, our results indicate that diabetic mice show changed microbiota composition and that treatment with insulin and islet Tx can partially correct the dysregulated microbiota.

Aedes aegypti mosquitoes transmit arboviruses of important global health impact, and their intestinal microbiota can influence vector competence by stimulating the innate immune system. Midgut epithelial cells also produce toxic reactive oxygen species (ROS) by dual oxidases (DUOXs) that are essential players in insect immunity. Strigomonas culicis is a monoxenous trypanosomatid that naturally inhabits mosquitoes; it hosts an endosymbiotic bacterium that completes essential biosynthetic pathways of the parasite and influences its oxidative metabolism. Our group previously showed that S. culicis hydrogen peroxide (H2O2)-resistant (WTR) strain is more infectious to A. aegypti mosquitoes than the wild-type (WT) strain. Here, we investigated the influence of both strains on the midgut oxidative environment and the effect of infection on mosquito fitness and immunity. WT stimulated the production of superoxide by mitochondrial metabolism of midgut epithelial cells after 4 days post-infection, while WTR exacerbated H2O2 production mediated by increased DUOX activity and impairment of antioxidant system. The infection with both strains also disrupted the fecundity and fertility of the females, with a greater impact on reproductive fitness of WTR-infected mosquitoes. The presence of these parasites induced specific transcriptional modulation of immune-related genes, such as attacin and defensin A during WTR infection (11.8- and 6.4-fold, respectively) and defensin C in WT infection (7.1-fold). Thus, we propose that A. aegypti oxidative response starts in early infection time and does not affect the survival of the H2O2-resistant strain, which has a more efficient antioxidant system. Our data provide new biological aspects of A. aegypti–S. culicis relationship that can be used later in alternative vector control strategies.

Control of mosquito-borne pathogens using genetically-modified vectors has been proposed as a promising tool to complement conventional control strategies. CRISPR-based homing gene drive systems have made transgenic technologies more accessible within the scientific community. Evaluation of transgenic mosquito performance and comparisons with wild-type counterparts in small laboratory cage trials provide valuable data for the design of subsequent field cage experiments and experimental assessments to refine the strategies for disease prevention. Here, we present three different protocols used in laboratory settings to evaluate transgene spread in anopheline mosquito vectors of malaria. These include inundative releases (no gene-drive system), and gene-drive overlapping and non-overlapping generation trials. The three trials vary in a number of parameters and can be adapted to desired experimental settings. Moreover, insectary studies in small cages are part of the progressive transition of engineered insects from the laboratory to open field releases. Therefore, the protocols described here represent invaluable tools to provide empirical values that will ultimately aid field implementation of new technologies for malaria elimination.

Islet transplantation can restore glycemic control in patients with type 1 diabetes. Using this procedure, the early stages of engraftment are often crucial to long-term islet function, and outcomes are not always successful. Numerous studies have shown that mesenchymal stem cells (MSCs) facilitate islet graft function. However, experimental data can be inconsistent due to variables associated with MSC generation (including donor characteristics and tissue source), thus, demonstrating the need for a well-characterized and uniform cell product before translation to the clinic. Unlike bone marrow- or adipose tissue-derived MSCs, human embryonic stem cell-derived-MSCs (hESC-MSCs) offer an unlimited source of stable and highly-characterized cells that are easily scalable. Here, we studied the effects of human hemangioblast-derived mesenchymal cells (HMCs), (i.e., MSCs differentiated from hESCs using a hemangioblast intermediate), on islet cell transplantation using a minimal islet mass model. The co-transplantation of the HMCs allowed a mass of islets that was insufficient to correct diabetes on its own to restore glycemic control in all recipients. Our in vitro studies help to elucidate the mechanisms including reduction of cytokine stress by which the HMCs support islet graft protection in vivo. Derivation, stability, and scalability of the HMC source may offer unique advantages for clinical applications, including fewer islets needed for successful islet transplantation.

Ethnopharmacology relevance Pimpinella anisum is used in traditional medicine because of its pharmacological properties which include cardiovascular action. However, no scientific information supports this use. Aim of the study This study investigated the effects of Pimpinella on arterial blood pressure (BP) and its pharmacological mechanism of action. Material and methods Pimpinella seeds were extracted with water, concentrated and freeze-dried yielding the aqueous extract (AE). A non-invasive BP assessment method was used (via the caudal artery) on Wistar, Wistar Kyoto, SHRs and rats that were submitted to high intake of dietary salt. Direct BP and heart rate were evaluated in Wistar rats in the absence or presence of atropine, L-NAME and angiotensin II. Spontaneous diuresis and the effect of AE on depolarized portal vein of Wistar rats was also examined. Results The data revealed that AE reduced BP in all groups evaluated and its effects were not due to diuretic, sympatholytic or parasympathomimetic actions. Additionally, it was shown that AE does not act as an angiotensin receptor blocker and does not induce hypotension by reducing vascular resistance induced by oxide nitric. In the depolarized portal vein, AE inhibited calcium influx, which indicates that AE acts as calcium channel blocker. Conclusion This study validates the cardiovascular actions of Pimpinella and characterizes the hypotensive effects of Pimpinella that are related to the blockade of calcium channels.

Autochthonous particulate organic carbon (POC) is an important precursor of humic substances (HS), and macrophytes represent the major source of POC in tropical aquatic ecosystems. Autochthonous HS influence the carbon supply, light regime, and primary production within freshwater systems. This study addresses the conversion of POC from two macrophyte species into HS and their mineralization under different nutrient conditions (oligotrophic to hypereutrophic). A first-order kinetic model was adopted to describe the conversion routes. The POC conversion rate to HS for detritus derived from Paspalum repens was similar under different nutrient conditions, but eutrophication decreased the kR (global coefficient reaction) for detritus from Pistia stratiotes due to its high detritus quality (C:N:P ratio). Fulvic acids were the main fraction of HS in both plants. The mineralization of humic acids from P. stratiotes was inhibited at higher nutrient availability, while eutrophication increased the mineralization of fulvic acids from P. repens. The main route of POC cycling is humification through fulvic acid formation (up to 40% of POC). The intrinsic characteristics of the source detritus were the main forcing functions that stimulated the cycling of HS. In tropical aquatic ecosystems, the degradation of autochthonous carbon decreased due to eutrophication, thus contributing to the diagenetic process in the long term.

Rapid progress has been made in the last decade related to stem cell-mediated pulp–dentin regeneration, from characterization of dental pulp stem cells (DPSCs) to the first-ever reported clinical case in humans. However, many challenges still need to be addressed before such technology can become a common clinical practice; therefore, further rigorous research is needed. Animal study models are very important to test new ideas, concepts, and technologies. This review summarizes and discusses several key animal models that have been utilized to investigate pulp–dentin regeneration. From a tissue regeneration perspective, we categorize the animal model by the location where the regenerated pulp tissue is formed: ectopic, semiorthotopic, and orthotopic. Several animal species are discussed, including mouse, ferret, dog, and miniswine. Mouse is used for ectopic pulp–dentin regeneration in the dorsum subcutaneous space. A commonly tested approach is hydroxyapatite/tricalcium phosphate (HA-TCP) granules model used to observe ectopic pulp–dentin complex formation. The semiorthotopic model includes tooth slices or fragments with which de novo pulp regeneration in a root canal space can be tested in the mouse subcutaneous space. For orthotopic pulp regeneration, the canine teeth of ferrets are large enough for such purposes. As nonprimate large animal models, dog and miniswine teeth have many aspects quite similar to those of humans, allowing researchers to perform experiments that mimic clinical conditions in humans. The protocols established and the data obtained from large animal studies may directly relate to and apply to future human studies. Complete orthotopic pulp regeneration has been demonstrated in dogs and miniswine. The use of allogeneic and subpopulations of DPSCs for pulp regeneration, and testing of the periapical disease model and aging model have been performed in miniswine or dogs. In sum, all these animal models will help address challenges that still face pulp regeneration in humans. We need to thoroughly utilize these models to test new ideas, technologies, and strategies before reliable and predictable clinical protocols can be established for human clinical trials or treatment. IMPACT STATEMENT: Animal models are essential for tissue regeneration studies. This review summarizes and discusses the small and large animal models, including mouse, ferret, dog, and miniswine that have been utilized to experiment and to demonstrate stem cell-mediated dental pulp tissue regeneration. We describe the models based on the location where the tissue regeneration is tested—either ectopic, semiorthotopic, or orthotopic. Developing and utilizing optimal animal models for both mechanistic and translational studies of pulp regeneration are of critical importance to advance this field.

This study compared macro- and microvascular endothelial function and redox status in active vs inactive HIV-infected patients (HIVP) under antiretroviral therapy. Using a cross-sectional design, macro- and microvascular reactivity, systemic microvascular density, and oxidative stress were compared between 19 HIVP (53.1 ± 6.1 year) enrolled in a multimodal training program (aerobic, strength and flexibility exercises) for at least 12 months (60-minutes sessions performed 3 times/wk with moderate intensity) vs 25 sedentary HIVP (51.2 ± 6.3 year). Forearm blood flow during reactive hyperemia (521.7 ± 241.9 vs 361.4% ± 125.0%; P = 0.04) and systemic microvascular density (120.8 ± 21.1 vs 105.6 ± 25.0 capillaries/mm2 ; P = 0.03) was greater in active than inactive patients. No significant difference between groups was detected for endothelium-dependent and independent skin microvascular vasodilation (P > 0.05). As for redox status, carbonyl groups (P = 0.22), lipid peroxidation (P = 0.86), catalase activity (P = 0.99), and nitric oxide levels (P = 0.72) were similar across groups. However, superoxide dismutase activity was greater in active vs inactive HIVP (0.118 ± 0.013 vs 0.111 ± 0.007 U/mL; P = 0.05). Immune function reflected by total T CD4 and T CD8 counts (cell/mm3 ) did not differ between active and inactive groups (P > 0.82). In conclusion, physically active HIVP exhibited similar immune function, but greater macrovascular reactivity, systemic microvascular density, and superoxide dismutase activity than inactive patients of similar age.

Blood-feeding arthropods are considered an enormous public health threat. They are vectors of a plethora of infectious agents that cause potentially fatal diseases like Malaria, Dengue fever, Leishmaniasis, and Lyme disease. These vectors shine due to their own physiological idiosyncrasies, but one biological aspect brings them all together: the requirement of blood intake for development and reproduction. It is through blood-feeding that they acquire pathogens and during blood digestion that they summon a collection of multisystemic events critical for vector competence. The literature is focused on how classical immune pathways (Toll, IMD, and JAK/Stat) are elicited throughout the course of vector infection. Still, they are not the sole determinants of host permissiveness. The dramatic changes that are the hallmark of the insect physiology after a blood meal intake are the landscape where a successful infection takes place. Dominant processes that occur in response to a blood meal are not canonical immunological traits yet are critical in establishing vector competence. These include hormonal circuitries and reproductive physiology, midgut permeability barriers, midgut homeostasis, energy metabolism, and proteolytic activity. On the other hand, the parasites themselves have a role in the outcome of these blood triggered physiological events, consistently using them in their favor. Here, to enlighten the knowledge on vector–pathogen interaction beyond the immune pathways, we will explore different aspects of the vector physiology, discussing how they give support to these long-dated host–parasite relationships.

Made available in DSpace on 2022-04-29T08:35:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 In several species, mating reduces the estrous length and advances ovulation. The aim of this study was to determine if multiple matings reduces the estrous length and modifies the moment of ovulation, as well as the estradiol and LH patterns in ewes. The estrous cycle of Corriedale ewes was synchronized, and the onset of receptivity was monitored every 3 h with rams, avoiding mating. At the estrous onset, ewes were assigned to two experimental groups (n=10 each): 1) estrous was monitored every 3 h with a ram avoiding mating (group CON), and 2) a ram was allowed to mate and ejaculate once every 3 h (group MAT). The ovaries were scanned with transrectal ultrasonography and blood samples were collected for measuring 17β-estradiol and LH concentrations every 3 h until ovulation. Estrus was shorter in MAT than CON ewes (24.7 ± 1.5 h vs. 30.4 ± 1.5 h, respectively; P=0.02); the proportion of animals that ovulated before the end of estrus was greater in CON ewes: (9/10 vs. 3/10, P=0.009). The area under the LH curve (AUC) was greater in MAT than CON ewes (36.1 ± 3.5 ng.h-1.mL-1 vs 24.9 ± 3.5 ng.h-1.mL-1 P=0.03). However, MAT ewes had a lower 17β-estradiol AUC than CON ewes (41.0 ± 4.9 pg.h'1.mL'1 vs 59.4 ± 4.9 pg.h'1.mL'1 P=0.01). Mating reduced the estrous length, induced a greater secretion of LH but less total 17β-estradiol secreted and, additionally, ovulation occurred more frequently after the end of estrus in mated ewes. Departamento de Biociencias Veterinarias Facultad de Veterinaria Universidad de la República Departamento de Producción Animal y Pasturas Facultad de Agronomía Universidad de la República Departamento de Medicina Veterinária Preventiva e Reprodução Animal Faculdade de Ciências Agrárias e Veterinárias Universidade Estadual Paulista “Júlio de Mesquita Filho” Instituto de Ciencia Animal Facultad de Ciencias Veterinarias Universidad Austral de Chile Departamento de Medicina Veterinária Preventiva e Reprodução Animal Faculdade de Ciências Agrárias e Veterinárias Universidade Estadual Paulista “Júlio de Mesquita Filho”

Reports of new-onset diabetes and diabetic ketoacidosis in individuals with COVID-19 have led to the hypothesis that SARS-CoV-2, the virus that causes COVID-19, is directly cytotoxic to pancreatic islet β cells. This would require binding and entry of SARS-CoV-2 into host β cells via cell surface co-expression of ACE2 and TMPRSS2, the putative receptor and effector protease, respectively. To define ACE2 and TMPRSS2 expression in the human pancreas, we examined six transcriptional datasets from primary human islet cells and assessed protein expression by immunofluorescence in pancreata from donors with and without diabetes. ACE2 and TMPRSS2 transcripts were low or undetectable in pancreatic islet endocrine cells as determined by bulk or single cell RNA sequencing, and neither protein was detected in α or β cells from these donors. Instead, ACE2 protein was expressed in the islet and exocrine tissue microvasculature and also found in a subset of pancreatic ducts, whereas TMPRSS2 protein was restricted to ductal cells. The absence of significant ACE2 and TMPRSS2 co-expression in islet endocrine cells reduces the likelihood that SARS-CoV-2 directly infects pancreatic islet β cells through these cell entry proteins.

The increasing detection of infections of Trypanosoma cruzi, the etiological agent of Chagas disease, in non-endemic regions beyond Latin America has risen to be a major public health issue. With an impact in the millions of people, current treatments rely on antiquated drugs that produce severe side effects and are considered nearly ineffective for the chronic phase. The minimal progress in the development of new drugs highlights the need for advances in basic research on crucial biochemical pathways in T. cruzi to identify new targets. Here, we report on the T. cruzi presenilin-like transmembrane aspartyl enzyme, a protease of the aspartic class in a unique phylogenetic subgroup with T. vivax separate from protozoans. Computational analyses suggest it contains nine transmembrane domains and an active site with the characteristic PALP motif of the A22 family. Multiple linear B-cell epitopes were identified by SPOT-synthesis analysis with Chagasic patient sera. Two were chosen to generate rabbit antisera, whose signal was primarily localized to the flagellar pocket, intracellular vesicles, and endoplasmic reticulum in parasites by whole-cell immunofluorescence. The results suggest that the parasitic presenilin-like enzyme could have a role in the secretory pathway and serve as a target for the generation of new therapeutics specific to the T. cruzi.

Isolated reports of new-onset diabetes in individuals with COVID-19 have led to the hypothesis that SARS-CoV-2 is directly cytotoxic to pancreatic islet β cells. This would require binding and entry of SARS-CoV-2 into β cells via co-expression of its canonical cell entry factors, angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2); however, their expression in human pancreas has not been clearly defined. We analyzed six transcriptional datasets of primary human islet cells and found that ACE2 and TMPRSS2 were not co-expressed in single β cells. In pancreatic sections, ACE2 and TMPRSS2 protein was not detected in β cells from donors with and without diabetes. Instead, ACE2 protein was expressed in islet and exocrine tissue microvasculature and in a subset of pancreatic ducts, whereas TMPRSS2 protein was restricted to ductal cells. These findings reduce the likelihood that SARS-CoV-2 directly infects β cells in vivo through ACE2 and TMPRSS2., Graphical Abstract, Highlights • ACE2 and TMPRSS2 mRNA are expressed in the human pancreas • ACE2 protein is expressed in some islet and exocrine capillaries and pericytes • ACE2 and TMPRSS2 proteins are co-expressed in some pancreatic ducts • ACE2 and TMPRSS2 protein is not detected in β cells of the human pancreas, Coate et al. examined expression of canonical SARS-CoV-2 entry proteins ACE2 and TMPRSS2 in the human pancreas and report ACE2 expression in the microvasculature, including islet pericytes, whereas both ACE2 and TMPRSS2 are expressed in some ducts. Conversely, neither protein is detected in β cells, arguing against direct β cell viral infection in vivo.

Oral bacterial infection represents the leading cause of the gradual destruction of tooth and periodontal structures anchoring the teeth. Lately, injectable hydrogels have gained increased attention as a promising minimally invasive platform for localized delivery of personalized therapeutics. Here, an injectable and photocrosslinkable gelatin methacryloyl (GelMA) hydrogel is successfully engineered with ciprofloxacin (CIP)-eluting short nanofibers for oral infection ablation. For this purpose, CIP or its β-cyclodextrin (β-CD)-inclusion complex (CIP/β-CD-IC) has been incorporated into polymeric electrospun fibers, which were subsequently cut into short nanofibers, and then embedded in GelMA to obtain an injectable hybrid antimicrobial hydrogel. Thanks to the solubility enhancement of CIP by β-CD-IC and the tunable degradation profile of GelMA, the hydrogels promote localized, sustained, and yet effective cell-friendly antibiotic doses, as measured by a series of bacterial assays that demonstrated efficacy in attenuating the growth of Gram-positive Enterococcus faecalis. Altogether, we foresee significant potential in translating this innovative hybrid hydrogel as an injectable platform technology that may have broad applications in oral infection ablation, such as periodontal disease and pulpal pathology.

Whipworms are responsible for up to 500 million cases of trichuriasis worldwide, with higher endemicity in tropical and sub-tropical countries. In non-endemic countries, trichuriasis can be accidentally diagnosed upon colonoscopy, often in the presence of negative microscopy. Here, we describe an incidental diagnosis of trichuriasis in an HIV patient residing in a non-endemic area (i.e., Turin, Italy), six months after his return from Antigua. The species-level diagnosis was made thanks to PCR-based molecular identification of Trichuris sp. following optical microscopy detection. Overall, this case highlights the importance of improving parasitic diseases diagnosis through cutting-edge clinical and laboratory diagnostic tools alongside advanced training of specialists in the area of parasitology.

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central nervous system (CNS). The increasing evidences of their involvement in the control of infectious diseases justify the emerging interest in the improvement of methodologies to isolate primary astrocytes and microglia in order to evaluate their responses to infections that affect the CNS. Considering the impact of Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) infection in the CNS, here we provide a method to extract, maintain, dissociate and infect murine astrocytes and microglia cells with protozoa parasites. Extracted cells from newborn cortices are maintained in vitro for 14 days with periodic differential media replacement. Astrocytes and microglia are obtained from the same extraction protocol by mechanical dissociation. After phenotyping by flow cytometry, cells are infected with protozoa parasites. The infection rate is determined by fluorescence microscopy at different time points, thus enabling the evaluation of differential ability of glial cells to control protozoan invasion and replication. These techniques represent simple, cheap and efficient methods to study the responses of astrocytes and microglia to infections, opening the field for further neuroimmunology analysis.

SummaryThe low proliferative rate of adult β-cells presents a challenge for diabetes therapy to increase β-cell numbers while maintaining mature function. Although proliferative quiescence in β-cells is required to maintain functional competence, exemplified by glucose stimulated insulin secretion, the molecular underpinnings of this reciprocal relationship remain enigmatic. Here, we demonstrate that Tead1, the transcription effector of the mammalian-Hippo pathway drives developmental stage-specific β-cell proliferative capacity in conjunction with its functional maturation. Tead1 promotes adult β-cell mature identity by direct transcriptional control of a network of critical β-cell transcription factors, including, Pdx1, Nkx6.1 and MafA, while its regulation of Cdkn2a maintains proliferative quiescence. Consequently, mice with either constitutive or inducible genetic deletion of Tead1 in β-cells developed overt diabetes due to a severe loss of secretory function despite induction of proliferation. Furthermore, we show that Tead1 has a similar regulatory role in human β-cells. We propose that Tead1 is an essential intrinsic molecular switch coordinating adult β-cell proliferative quiescence with mature identity and functional competence to maintain glucose homeostasis.

Background: The characterization of hepatic metastases as having neuroendocrine origins is essential and the main markers currently used are chromogranin A (CgA) and synaptophysin (Syn). However, these markers may exhibit certain limitations, and the use of CD56 and CD57 can also be considered, although, due to low specificity, their use is discouraged. Aim: This study sought to compare the immunohistochemical expression of these markers in hepatic metastases of neuroendocrine neoplasms (NEN). Materials and Methods: Eighteen samples, were used for immunohistochemical staining with CgA, Syn, CD56, and CD57 antibodies. The immunostaining reactions were compared according to its intensity (I), the percentage of labeled cells (P), and a final score (I × P). Statistical agreement between the markers was also evaluated. Results: CD57 was expressed in the highest number of cases and also showed the most intense expression. CgA showed the highest number of cases with more than 80% positively stained area (72.2%), followed by CD57 (61.1%). The highest average score (I × P) was obtained for CD57 (9.1 ± 4.1). The best indices of agreement were between CgA and CD57 with respect to positivity (P = 0.021) and score (P = 0.014). According to the primary site, stomach/duodenum, lungs, and undetermined subgroups showed the highest average scores for CD57, followed by CgA. For the small bowel subgroup, the highest average score was obtained for CgA, followed by CD57. Conclusion: Our results highlight the importance of CD57 in the evaluation of hepatic metastases of NEN and indicate that this marker should be included with CgA and Syn in routine diagnostic panels.

The low proliferative rate of adult β-cells presents a challenge for diabetes therapy to increase β-cell numbers while maintaining mature function. Although proliferative quiescence in β-cells is required to maintain functional competence, exemplified by glucose stimulated insulin secretion, the molecular underpinnings of this reciprocal relationship remain enigmatic. Here, we demonstrate that Tead1, the transcription effector of the mammalian-Hippo pathway drives developmental stage-specific β-cell proliferative capacity in conjunction with its functional maturation. Tead1 promotes adult β-cell mature identity by direct transcriptional control of a network of critical β-cell transcription factors, including, Pdx1, Nkx6.1 and MafA, while its regulation of Cdkn2a maintains proliferative quiescence. Consequently, mice with either constitutive or inducible genetic deletion of Tead1 in β-cells developed overt diabetes due to a severe loss of secretory function despite induction of proliferation. Furthermore, we show that Tead1 has a similar regulatory role in human β-cells. We propose that Tead1 is an essential intrinsic molecular switch coordinating adult β-cell proliferative quiescence with mature identity and functional competence to maintain glucose homeostasis.

Most patients affected by Glycogen storage disease type 1a (GSD-1a), an inherited metabolic disorder caused by mutations in the enzyme glucose-6-phosphatase-alpha (G6Pase-α), develop renal and liver complications, including development of hepatocellular adenoma/carcinoma. The purpose of this study was to identify potential biomarkers of the pathophysiology of the GSD1a affected liver. To this end, we utilized the plasma exosomes of a murine model of GSD-1a, LS-G6pc−/− mice, to uncover microRNA expression modulation associated with the disease. Differentially expressed microRNA between LS-G6pc−/− and wild type mice, LS-G6pc−/− mice with hepatocellular adenoma and LS-G6pc−/− mice without adenoma, and LS-G6pc−/− mice with amyloidosis and LS-G6pc−/− mice without amyloidosis were identified. Pathway analysis demonstrated that the target genes of the differentially expressed microRNA were significantly enriched for insulin signaling pathway, glucose and lipid metabolism, Wnt-beta catenin, telomere maintenance and hepatocellular carcinoma, and chemokine and immune regulation signaling pathways. While some microRNA were common to the different pathologic conditions others were unique to cancerous or inflammatory status of the animals. Therefore, the altered expression of several microRNA correlates with various pathologic liver statuses and may help discriminate during the progression of the disease and the development of late GSD1-a associated complications.

The advancement toward a clinical application for porcine islets to cure diabetes in humans must include reproducible long-term successes in non-human primate (NHP) models. Many dedicated researchers around the world are continuing to work toward this goal. In this chapter, we describe procedures for islet isolation of pancreatic islets from adult and neonatal/fetal pigs. We further include procedures for the induction of diabetes in non-human primates and subsequent insulin therapy, islet transplantation, immunosuppression, and also the daily maintenance of xenotransplanted NHPs. The procedures that we outline in this chapter are ones that we have successfully utilized in pig-to-NHP islet transplantation models. However, where appropriate, alternative methods will also be identified.

SMYD3 is a member of the SMYD lysine methylase family and plays an important role in the methylation of various histone and non-histone targets. Aberrant SMYD3 expression contributes to carcinogenesis and SMYD3 upregulation was proposed as a prognostic marker in various solid cancers. Here we summarize SMYD3-mediated regulatory mechanisms, which are implicated in the pathophysiology of cancer, as drivers of distinct oncogenic pathways. We describe SMYD3-dependent mechanisms affecting cancer progression, highlighting SMYD3 interplay with proteins and RNAs involved in the regulation of cancer cell proliferation, migration and invasion. We also address the effectiveness and mechanisms of action for the currently available SMYD3 inhibitors. The findings analyzed herein demonstrate that a complex network of SMYD3-mediated cytoplasmic and nuclear interactions promote oncogenesis across different cancer types. These evidences depict SMYD3 as a modulator of the transcriptional response and of key signaling pathways, orchestrating multiple oncogenic inputs and ultimately, promoting transcriptional reprogramming and tumor transformation. Further insights into the oncogenic role of SMYD3 and its targeting of different synergistic oncogenic signals may be beneficial for effective cancer treatment.

The immunologic barriers to successful xenotransplantation are related to the presence of natural anti-pig antibodies in humans and nonhuman primates that bind to antigens expressed on the transplanted pig organ (the most important of which is galactose-α1,3-galactose [Gal]) and activate the complement cascade, which results in rapid destruction of the graft, a process known as hyperacute rejection. High levels of elicited anti-pig IgG may develop if the adaptive immune response is not prevented by adequate immunosuppressive therapy, resulting in activation and injury of the vascular endothelium. The transplantation of organs and cells from pigs that do not express the important Gal antigen (α1,3-galactosyltransferase gene-knockout [GTKO] pigs) and express one or more human complement-regulatory proteins (hCRP, e.g., CD46, CD55), when combined with an effective costimulation blockade-based immunosuppressive regimen, prevents early antibody-mediated and cellular rejection. However, low levels of anti-nonGal antibody and innate immune cells and/or platelets may initiate the development of a thrombotic microangiopathy in the graft that may be associated with a consumptive coagulopathy in the recipient. This pathogenic process is accentuated by the dysregulation of the coagulation–anticoagulation systems between pigs and primates. The expression on GTKO/hCRP pigs of a human coagulation-regulatory protein, e.g., thrombomodulin, is increasingly being associated with prolonged pig graft survival in nonhuman primates.

Reliance on rodents for understanding pancreatic genetics, development and islet function could limit progress in developing interventions for human diseases like diabetes mellitus. Similarities of pancreas morphology and function suggest that porcine and human pancreas developmental biology may have useful homologies. However, little is known about pig pancreas development. To fill this knowledge gap, we investigated fetal and neonatal pig pancreas at multiple, crucial developmental stages using modern experimental approaches. Purification of islet β-, α- and δ-cells followed by transcriptome analysis (RNA-Seq) and immunohistology identified cell- and stage-specific regulation, and revealed that pig and human islet cells share characteristic features not observed in mice. Morphometric analysis also revealed endocrine cell allocation and architectural similarities between pig and human islets. Our analysis unveiled scores of signaling pathways linked to native islet β-cell functional maturation, including evidence of fetal α-cell GLP-1 production and signaling to β-cells. Thus, the findings and resources detailed here show how pig pancreatic islet studies complement other systems for understanding the developmental programs that generate functional islet cells, and that are relevant to human pancreatic diseases.Summary StatementThis study reveals transcriptional, signaling and cellular programs governing pig pancreatic islet development, including striking similarities to human islet ontogeny, providing a novel resource for advancing human islet replacement strategies.

Raphidiopsis raciborskii is the dominant species in several subtropical aquatic ecosystems. This study addressed the influence of nutrient concentration on cyanobacteria growth and saxitoxin synthesis. Therefore, we performed bioassays of interaction between the Chlorophyceae Monoraphidium contortum and R. raciborskii simulating oligotrophic and supereutrophic environments. Experiments were carried out in a climatized room for 15 days in pure cultures of each species (control) and mixed (interaction). The biomass growth (biovolume) and specific growth rates were measured. Saxitoxin was analyzed using the ELISA biochemical method. In the oligotrophic environment, the cell volume of R. raciborskii decreased. This species also showed senescence in interaction with M. contortum. However, there were no statistical differences in the saxitoxin synthesis in both conditions. In the supereutrophic environment, the growth of R. raciborskii and saxitoxin production was similar in both the control and interaction conditions. However, M. contortum growth decreased in interaction with the cyanobacteria. The increase in trophic status from oligo- to supereutrophic contributes to the growth of M. contortum in subtropical aquatic ecosystems, but the decrease in the area/volume ratio of cyanobacteria and the saxitoxin synthesis is an evident survival strategy in oligotrophic environments.

Four killer cell Ig-like receptor (KIR) genes, collectively referred to as framework genes, characterize almost all KIR haplotypes. In particular, KIR3DL3 and KIR3DL2 mark the ends of the locus, whereas KIR3DP1 and KIR2DL4 are located in the central part. A recombination hot spot, mapped between KIR3DP1 and KIR2DL4, splits the haplotypes into two regions: a centromeric (Cen) region (spanning from KIR3DL3 to KIR3DP1) and a telomeric region (from KIR2DL4 to KIR3DL2), both varying in KIR gene content. In this study, we analyzed KIR3DP1 polymorphism in a cohort of 316 healthy, unrelated individuals. To this aim, we divided KIR3DP1 alleles into two groups by the use of a sequence-specific primer– PCR approach. Our data clearly indicated that KIR3DP1 alleles present on haplotypes carrying Cen-A or Cen-B1 regions differ from those having Cen-B2 motifs. Few donors (∼3%) made exceptions, and they were all, except one, characterized by uncommon haplotypes, including either KIR deletions or KIR duplications. Consequently, as KIR2DL1 is present in Cen-A and Cen-B1 regions but absent in Cen-B2 regions, we demonstrated that KIR3DP1 polymorphism might represent a suitable marker for KIR2DL1 gene copy number analysis. Moreover, because Cen-B1 and Cen-B2 regions are characterized by different KIR3DP1 alleles, we showed that KIR3DP1 polymorphism analysis also provides information to dissect between Cen-B1/Cen-B1 and Cen-B1/Cen-B2 donors. Taken together, our data suggest that the analysis of KIR3DP1 polymorphism should be included in KIR repertoire evaluation.

In neoplastic patients, an effective immune response ideally should be achieved by the coordinated action of different immune cells with tumor-suppressive functions. These include the more cytolytic members of the Innate Lymphoid Cells (ILCs) family represented by the Natural Killer (NK) cells, whose activities in cancer patients, however, can be hampered by several inhibitory signals. These are generated by membrane-bound and soluble molecules that, interacting with specific inhibitory receptors, create inhibitory axes impacting the NK cell differentiation and effector functions. These breaks, which now represent major immunotherapeutic targets, may be sensitive to interferon (IFN)-γ, whose source, in vivo, is represented by different cell types including the NK and ILC1. Since also ILCs can express receptors of the inhibitory axes like PD-1 and TIGIT, their therapeutic blockade might further amplify the IFN-γ release that, as an unwanted side effect, would promote the onset of NK cell-resistant tumor variants (NKRTV) expressing ligands involved in inhibitory axes. These variants might also arise from the activity of other cytokines such as IL-27, which can increase the expression of HLA class I and PD-Ls in different cell types, including tumor cells. Besides the amplification of membrane-bound inhibitory axes, tumors can reduce the number of infiltrating cytolytic ILCs, promote the recruitment of poorly cytolytic NK cell subsets, and manipulate to their advantage the infiltrating immune cells, which acquire tumor-promoting activities. This occurs thanks to the production of soluble factors including TGF-β1 and IL-18 that, alone or in combination, modify the activating and chemokine receptor repertoire of NK cells, and induce the ILCs differentiation towards cells ineffective in fighting cancer or, even worse, with tumor-promoting functions. The present review aims to present and discuss major inhibitory axes impacting on ILCs functions, migration, and differentiation with a major focus on tumor context.

Novel therapies that enhance ADCC have the potential to overcome Rituximab (RTX) resistance in lymphoma, which can occur due to e.g. CD20 loss, immunogenicity-induced reduction in RTX exposure, or SNPs resulting in lower CD16:Fc affinity. Nonlinear interactions between effector cells (ECs), drug, and target cell (TC) factors in ADCC, however, complicate the prediction of the net effect of potential therapeutic strategies that modulate one or more factors such as CD16 expression ([CD16]) on ECs, CD16:Fc binding affinity, or intrinsic killing propensity (g).We have developed a QSP model of the molecular-level binding reactions and cell-cell interactions governing ADCC using the anti-CD20 IgG1 antibody RTX in lymphoma as a case study. This model takes as inputs [CD16] on ECs, g, Ab Fc:CD16 binding affinity, [RTX], RTX:CD20 affinity, and [CD20] on TCs and predicts the degree of ADCC killing of tumor cells after RTX exposure. Simulations can then be used to estimate how much increase in an input parameter a potential RTX combination partner must provide to compensate for a given mechanism of RTX resistance (MoRR). The model has been successfully calibrated to ex vivo [RTX]-ADCC assays in SUDHL4 and Z138 cell lines (Herter, Mol Cancer Ther, 2013). The model and estimated parameter set well described the observed [RTX]-dependent %ADCC in both cell lines with ECs (PBMCs) from two donors with V158 and F158 CD16 SNP variants. The table below provides the model predictions of the fold increase in model parameters required to offset various MoRRs. For example, a 10-fold loss of [CD20] on TCs is predicted to cause a 100*(1 - 0.16) = 84% drop in ADCC activity, which can be overcome by a 7.8-fold increase in [CD16] on ECs. We have developed a mechanism-based simulation tool which can be used to predict the ability of novel ADCC-enhancing agents to overcome different mechanisms of IgG1 Ab resistance. Mechanism of RTX resistance (MoRR)ADCC fold change due to MoR(90% CI)Fold change in parameter required to restore ADCC (90% CI)CD16 expression on effector cells [CD16]CD16:Fc binding affinity kon16NK killing propensity g10x loss of CD20 on tumor0.16 (0.13,0.20)7.8 (6.3,9.2)10.6 (10.4,10.9)7.4 (6.1,8.7)10x loss of RTX exposure0.17(0.14,0.21)7.0(5.7,8.1)9.36(9.35,9.37)6.9(5.7,8.1)10x decrease in CD16:Fc affinity0.16(0.13,0.20)7.4(6.1,8.7)10(10,10)7.4(6.1,8.6) Citation Format: Kaitlyn E. Johnson, Maria Veronica Ciocanel, Josua Aponte, Nicolas Bajeux, Fanwang Meng, Dean Bottino. Evaluation of pharmacologic mechanisms to overcome IgG1 antibody (Ab) resistance via quantitative systems pharmacology (QSP) modeling of antibody-dependent cell mediated cytotoxicity (ADCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 231.

Aquaporin-8 (AQP8) allows the bidirectional transport of water and hydrogen peroxide across biological membranes. Depending on its concentration, H2O2 exerts opposite roles, amplifying growth factor signaling in physiological conditions, but causing severe cell damage when in excess. Thus, H2O2 permeability is likely to be tightly controlled in living cells. Aims: In this study, we investigated whether and how the transport of H2O2 through plasma membrane AQP8 is regulated, particularly during cell stress. Results: We show that diverse cellular stress conditions, including heat, hypoxia, and ER stress, reversibly inhibit the permeability of AQP8 to H2O2 and water. Preventing the accumulation of intracellular reactive oxygen species (ROS) during stress counteracts AQP8 blockade. Once inhibition is established, AQP8-dependent transport can be rescued by reducing agents. Neither H2O2 nor water transport is impaired in stressed cells expressing a mutant AQP8, in which cysteine 53 had been replaced by serine. Cells expressing this mutant are more resistant to stress-, drug-, and radiation-induced growth arrest and death. Innovation and Conclusion: The control of AQP8-mediated H2O2 transport provides a novel mechanism to regulate cell signaling and survival during stress. Antioxid. Redox Signal. 24, 1031–1044.

We thank Drs Kow and Hasan [1] for their comments about our review on the role of angiotensin receptor blockers (ARBs) in cardiovascular disease (CVD) risk management [2]. Drs Kow and Hasan [1] pre...

Vertebrate blood composition is heavily biased towards proteins, and hemoglobin, which is a hemeprotein, is by far the most abundant protein. Typically, hematophagous insects ingest blood volumes several times their weight before the blood meal. This barbarian feast offers an abundance of nutrients, but the degradation of blood proteins generates toxic concentrations of amino acids and heme, along with unparalleled microbiota growth. Despite this challenge, hematophagous arthropods have successfully developed mechanisms that bypass the toxicity of these molecules. While these adaptations allow hematophagous arthropods to tolerate their diet, they also constitute a unique mode of operation for cell signaling, immunity, and metabolism, the study of which may offer insights into the biology of disease vectors and may lead to novel vector-specific control methods.

The objective was to assess the influence of polyunsaturated fatty acid supplementation on the quality of fresh, cooled, and frozen-thawed stallion semen. Ten stallions received their normal diet (control group) or normal diet plus 150 mL of polyunsaturated fatty acid (PUFA) linseed-based oil (PUFA group). Semen was collected every 15 days during 60 days. Stallions were reversed across the treatments after a sixty-day interval. Semen was evaluated at 2, 6, 12, and 24 hours after cooling and 24 hours after freezing. Motility (MOT), vigor, membrane viability, morphology, acrosome integrity, and osmotic tolerance test (OTT) were evaluated. In the frozen-thawed semen, sperm dynamic characteristics were analyzed by computer-assisted sperm analysis and thiobarbituric acid reactive substances (TBARs) determined. The effects of treatment, time, semen type, and their interactions were submitted to PROCMIX (SAS) and means compared by the Tukey test. There was no treatment effect on the quality of fresh and cooled semen. However, frozen-thawed semen MOT, vigor, and OTT were superior ( P P = .002) in the PUFA group after 15, 30, 45, and 60 days from the beginning of supplementation. Thus, sperm may become more susceptible to the reactive oxygen species, probably due to the incorporation of polyunsaturated fat in the cell membrane. The addition of PUFA-enriched oil may be an alternative for improving frozen-thawed semen quality by increasing its MOT and resistance to osmotic changes to which sperm cells are submitted during the freezing process.

The study tested the hypothesis that measurement, using multivariate Principal Components Analy-sis (PCA), of the niche-breadth of river macrophyte species in southern tropical Africa, may predicttheir larger-scale biogeographical range. Two measures of niche-breadth were calculated for 44 riverinemacrophyte species, from 20 families commonly occurring in Zambia, using an approach based on PCAordination with 16 bio-physico-chemical input variables. These included altitude, stream order, streamflow, pH, conductivity and soluble reactive phosphate concentration (SRP). In the absence of additionalchemical water quality data for Zambian rivers, invertebrate-based measures of general water qualitywere also used. These were benthic macroinvertebrate Average Score per Taxon (ASPT), and individualabundance of nine macroinvertebrate families with differing water quality tolerance, indicated by theirSensitivity Weightings within the Zambian Invertebrate Scoring System (ZISS). Macrophyte large-scalelatitudinal range was derived from world geopositional records held by online databases, and additionalrecords held by the authors. The two niche-breadth metrics divided the species into narrow-niche andintermediate/broad-niche categories, showing significant variation (from one or both of correlation andANOVA test outcomes) in altitude, stream flow, conductivity, SRP, pH and ASPT, but not stream order.Macrophyte alpha-diversity (as a measure of number of individual niches co-existing per habitat) showedno significant relationship with individual species niche-breadth. Narrow-niche species included a higherproportion of Afrotropical endemics than did species with broader niche size. There were significant pre-dictive relationships between macrophyte niche-breadth and latitudinal range of the target species atglobal and Afrotropical scales, but not for the Neotropics. Fil: Kennedy, Michael. University Of Aberdeen; Reino Unido Fil: Lang, Pauline. University of Glasgow; Reino Unido Fil: Tapia Grimaldo, Julissa. University of Glasgow; Reino Unido Fil: Varandas Martins, Sara. University of Glasgow; Reino Unido Fil: Bruce, Alannah. University of Glasgow; Reino Unido Fil: Moore, Isabel. University of Glasgow; Reino Unido Fil: Taubert, Rebeca. University of Glasgow; Reino Unido Fil: Macleod-Nolan, Chantal. University of Glasgow; Reino Unido Fil: McWaters, Stephanie. University of Glasgow; Reino Unido Fil: Briggs, John. University of Glasgow; Reino Unido Fil: Lowe, Steve. University of Glasgow; Reino Unido Fil: Saili, Kochelani. University Of Zambia; Fil: SICHINGABULA, Henry. University Of Zambia; Fil: Dallas, Helen. Nelson Mandela Metropolitan University, Sudafrica; Sudáfrica Fil: Morrison, Sean. University of Glasgow; Reino Unido Fil: Franceschini, Maria Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Centro de Ecología Aplicada del Litoral. Universidad Nacional del Nordeste. Centro de Ecología Aplicada del Litoral; Argentina Fil: Willems, Frank. The Kasanka Trust; Zambia Fil: Bottino, Flavia. Universidad Federal de San Carlos; Brasil Fil: MURPHY Kevin. University of Glasgow; Reino Unido

The performance of 3 blood culture bottles (BACTEC Plus Aerobic/F, Plus Anaerobic/F, and Anaerobic Lytic/F) were analyzed with clinical specimens collected from 688 Emergency Department patients. A total of 270 strains belonging to 33 species were identified, with E. coli and S. aureus as the most frequently detected. Overall recovery rate (RR) of bacteria and yeast was equivalent in the Plus Aerobic/F vials (208 of 270 isolates; 77.0%) and Anaerobic Lytic/F vials (206 isolates; 76.3%) and significantly better than in the Plus Anaerobic/F vials (189 isolates; 70.0%). Median time to detection (TTD) was earliest with the Anaerobic Lytic/F vials (12.0 h) compared with the Plus Aerobic/F (14.6 h) and Plus Anaerobic/F vials (15.4 h). Positivity rate (PR) was similar for Anaerobic Lytic/F vials (76.9%) and Plus Aerobic/F vials (76.5%), but better if compared with Plus Anaerobic/F vials (69.4%). The PR and TTD for the combination of Plus Aerobic/F with Anaerobic Lytic/F (94.5% and 12.3 h, respectively) was significantly better than with Plus Aerobic/F with Plus Anaerobic/F (87.8% and 14.1 h).

A major therapeutic goal for type 1 diabetes (T1D) is to induce autoantigen-specific tolerance of T cells. This could suppress autoimmunity in those at risk for the development of T1D, as well as in those with established disease who receive islet replacement or regeneration therapy. Because functional studies of human autoreactive T cell responses have been limited largely to peripheral blood-derived T cells, it is unclear how representative the peripheral T cell repertoire is of T cells infiltrating the islets. Our knowledge of the insulitic T cell repertoire is derived from histological and immunohistochemical analyses of insulitis, the identification of autoreactive CD8+ T cells in situ, in islets of human leukocyte antigen (HLA)-A2+ donors and isolation and identification of DQ8 and DQ2-DQ8 heterodimer-restricted, proinsulin-reactive CD4+ T cells grown from islets of a single donor with T1D. Here we present an analysis of 50 of a total of 236 CD4+ and CD8+ T cell lines grown from individual handpicked islets or clones directly sorted from handpicked, dispersed islets from nine donors with T1D. Seventeen of these T cell lines and clones reacted to a broad range of studied native islet antigens and to post-translationally modified peptides. These studies demonstrate the existence of a variety of islet-infiltrating, islet-autoantigen reactive T cells in individuals with T1D, and these data have implications for the design of successful immunotherapies.

Implementation of massively parallel sequencing platforms can bring a great contribution to Forensic Genetics field, with a great saving of time and costs, as well as allowing reliable results to be obtained from small or extremely degraded samples. The aim of this work was to analyze 124 SNP loci (90 autosomal and 34 Y-SNP) included in HID-Ion Ampliseq Identity Panel in a small sample from Rio de Janeiro state, Brazil. Samples from 12 non-related individuals were amplified with HID-Ion Ampliseq Identity Panel and sequenced on the Ion Torrent PGM platform (Thermo Fisher Scientific); genotypes were generated with HID SNP Genotyper plugin and forensic parameters were calculated with PowerStats v.12. All samples were successfully genotyped and were used to calculate allele frequencies, homozygosity, heterozygosity, random match probability (RMP) and exclusion power for all 90 autosomal SNP loci. Using the formula proposed by Budowle et al. (1996), only 4 of the 90 loci genotyped (4,4%) showed allele frequencies below the minimum required. It means that although a small set of individuals was used on this study, it may have shown a good perspective of Rio de Janeiro state allele frequencies. Among the 11 male samples analyzed, a prevalence of the haplogroup R1b of Y chromosome was observed, followed by the haplogroups E, Q and J. Such distribution reflects the results demonstrated in other studies for the population of Rio de Janeiro. All results together demonstrate the usefulness and applicability of SNP analysis on Ion Torrent PGM.

Objective: To assess the prevalence of hepatitis E virus (HEV) RNA and antibodies among kidney transplant recipients (KTR) in Central Brazil. The presence of chronic HEV infection was also investigated. Methods: A cohort study was conducted among 316 KTR treated at a referral center for kidney transplantation in Goiânia, Brazil. All serum samples were tested for the presence of HEV RNA (real-time PCR) and anti-HEV IgG/IgM (ELISA). Anti-HEV-positive samples were confirmed using an immunoblot test. HEV chronicity was investigated in a subgroup of patients with elevated alanine aminotransferase (ALT >40 IU/l) through HEV RNA detection in additional serum samples collected 3 and 6 months apart. Results: A seroprevalence of 2.5% (95% confidence interval 1.2–5.1%) was found for anti-HEV IgG. There was no difference in characteristics between the anti-HEV IgG seropositive and seronegative KTR groups. Anti-HEV IgM was detected in only one patient (0.3%). All KTR were negative for HEV RNA. Conclusions: These results show that HEV infection is infrequent in KTR in Central Brazil, with low seroprevalence rates of past and recent infection, and also an absence of active and chronic HEV infections. Keywords: Hepatitis E virus, Kidney transplant recipients, Prevalence, Brazil

C-reactive protein (CRP) rise might be different in patients with obesity due to chronic inflammation.The aim was to analyze postoperative CRP rise and its role as an early prognostic marker of infectious complications.Center of maximum care in Germany.Patients who underwent laparoscopic sleeve gastrectomy, laparoscopic Roux-en-Y gastric bypass, or laparoscopic one-anastomosis gastric bypass as primary treatment for severe obesity were included. Serum CRP and leukocyte count were measured preoperatively, on postoperative days (POD) 1 and 4 and were analyzed regarding sex, body mass index, waist circumference, obesity-associated diseases, laboratory measurements (glycosylated hemoglobin, triglycerides, cholesterol), surgical procedure, infectious complications, and infectious with anastomotic leakage.Four hundred seventy-one patients underwent surgery. Postoperative CRP rise was similar across sexes but lower in the super-super obese group (P.05) and higher in the gastric bypass groups (P.05). Linear regression model showed, that the higher preoperative value of waist circumference, the higher the preoperative CRP (beta value: .159, P = .006) and the lower the postoperative CRP rise on POD1 (beta value: -.171, P = .004) and 4 (beta value: -.170, P = .003). Only in the laparoscopic one-anastomosis gastric bypass group did a higher glycosylated hemoglobin predict a higher postoperative CRP rise (POD1: beta value: .434, P = .012; POD4: beta value: .513, P = .006). Fourteen patients (3%) developed infections, 7 of whom (1.5%) had anastomotic leakage. Leukocyte count was no predictor of infectious complications. The cut-off for CRP was 80.5 mg/L (POD1) and 164 mg/L (POD4), with 57.1% and 85.7% sensitivity and 97.9% and 99.6% specificity for anastomotic leakage.Standard postoperative CRP rises less in patients with higher waist circumference and super-super obesity, but more after gastric bypass procedures. CRP but not leukocyte count predicts early anastomotic healing after obesity surgery. These findings should be considered when interpreting CRP values in the routine clinical setting.

Impaired counterregulatory response of glucagon to hypoglycemia is a common complication of type 1 diabetes (T1D). One proposed contributor to abnormal glucagon secretion is decreased sympathetic innervation in T1D islets. To systematically assess the innervation of human pancreatic tissues, we examined samples from donors with recent-onset T1D (10 years, age 27-63 years, n = 3), and nondiabetic controls (age 10-52 years, n = 4). Islet and exocrine tissue innervation was visualized by pan-neuronal marker tubulin β-3 and analyzed by a 2-D morphometry and 3-D rendering. By quantifying the nerve fiber length (1.9±0.4 nm/μm2) and density (646±94 fibers/mm2), we found that innervation in control human islets was nearly 20-fold less than previously reported in mouse islets. In contrast to mouse, human exocrine tissue was far more innervated than islets with fiber length of 6.8±0.4 nm/μm2(p0.05), and so was the 3-D arrangement of nerve fibers in exocrine tissue and islets. Regardless of T1D duration, the islet fiber length (recent-onset: 1.5±0.2 nm/μm2; long-standing: 1.0±0.2 nm/μm2) and density (recent-onset: 623±61 fibers/mm2; longstanding: 486±49 fibers/mm2), as well as exocrine fiber length (recent-onset: 5.2±.5 nm/μm2; longstanding: 5.8±0.4 nm/μm2) and density (recent-onset: 1119±77 fibers/mm2; longstanding: 1537±72 fibers/mm2) did not differ from controls (p>0.05). These data indicate that neuronal patterning of human pancreas is significantly different from that of mouse and islet innervation does not appear to be altered in T1D. Disclosure T.M. Richardson: None. R. Haliyur: None. R. Aramandla: None. R. Bottino: Research Support; Self; Imagine Pharma. A.C. Powers: None. M. Brissova: None.

International audience; The legume-rhizobium symbiosis is a major supplier of fixed nitrogen in the biosphere and constitutes a key step of the nitrogen biogeochemical cycle. In some legume species belonging to the Inverted Repeat Lacking Clade (IRLC) and the Dalbergioids, the differentiation of rhizobia into intracellular nitrogen-fixing bacteroids is terminal and involves pronounced cell enlargement and genome endoreduplication, in addition to a strong loss of viability. In the Medicago truncatula-Sinorhizobium spp. system, the extent of bacteroid differentiation correlates with the level of symbiotic efficiency. Here, we used different physiological measurements to compare the symbiotic efficiency of photosynthetic bradyrhizobia in different Aeschynomene spp. (Dalbergioids) hosts inducing different bacteroid morphotypes associated with increasing ploidy levels. The strongly differentiated spherical bacteroids were more efficient than the less strongly differentiated elongated ones, providing a higher mass gain to their hosts. However, symbiotic efficiency is not solely correlated with the extent of bacteroid differentiation especially in spherical bacteroid-inducing plants, suggesting the existence of other factors controlling symbiotic efficiency.

Introduction Curcumin incorporation into polymeric fibers was tested for its antimicrobial properties and potential use in root canal disinfection. Methods Curcumin-modified fibers were processed via electrospinning and tested against a 7-day old established Actinomyces naeslundii biofilm. The medicaments tested were as follows: curcumin-modified fibers at 2.5 and 5.0 mg/mL, curcumin-based irrigant at 2.5 and 5.0 mg/mL, saline solution (negative control), and the following positive controls: 2% chlorhexidine, 1% sodium hypochlorite, and triple antibiotic paste (TAP, 1 mg/mL). All medicaments, except for the positive controls, were allocated according to the light exposure protocol (ie, photoactivation with a light-emitting diode every 30 seconds for 4 minutes or without photoactivation). After treatment, the medicaments were removed, and 1 mL saline solution was added; the biofilm was scraped from the well and used to prepare a 1:2000 dilution. Spiral plating was performed using anaerobic blood agar plates. After 24 hours, colony-forming units (colony-forming units/mL, n = 11/group) were counted to determine the antimicrobial effects. Results Data exhibited significant antimicrobial effects on the positive control groups followed by the curcumin irrigants and, lastly, the photoactivated curcumin-modified fibers. There was a significant reduction of viable bacteria in curcumin-based irrigants, which was greater than the TAP-treated group. Curcumin-free fibers, saline, and the nonphotoactivated curcumin-modified fibers did not display antimicrobial activity. Conclusions Curcumin seems to be a potential alternative to TAP when controlling infection, but it requires a minimal concentration (2.5 mg/mL) to be effective. Photoactivation of curcumin-based medicaments seems to be essential to obtain greater antibiofilm activity.