Search

Prenatal exposure to maternal diabetes has been recognized as a significant cardiovascular risk factor, increasing the susceptibility to the emergence of conditions such as high blood pressure, atherosclerosis, and heart disease in later stages of life. However, it is unclear if offspring exposed to diabetes in utero have worse vascular outcomes on a high-salt (HS) diet. To test the hypothesis that in utero exposure to maternal diabetes predisposes to HS-induced vascular dysfunction, we treated adult male wild-type offspring (DM_Exp, 6 mo old) of diabetic Ins2 +/C96Y mice ( Akita mice) with HS (8% sodium chloride, 10 days) and analyzed endothelial function via wire myograph and cyclooxygenase (COX)-derived prostanoids pathway by ELISA, quantitative PCR, and immunochemistry. On a regular diet, DM_Exp mice did not manifest any vascular dysfunction, remodeling, or inflammation. However, HS increased aortic contractility to phenylephrine and induced endothelial dysfunction (analyzed by acetylcholine-induced endothelium-dependent relaxation), vascular hydrogen peroxide production, COX2 expression, and prostaglandin E 2 (PGE 2 ) overproduction. Interestingly, ex vivo antioxidant treatment (tempol) or COX1/2 (indomethacin) or COX2 (NS398) inhibitors improved or reverted the endothelial dysfunction in DM_Exp mice fed a HS diet. Finally, DM_Exp mice fed with HS exhibited greater circulating cytokines and chemokines accompanied by vascular inflammation. In summary, our findings indicate that prenatal exposure to maternal diabetes predisposes to HS-induced vascular dysfunction, primarily through the induction of oxidative stress and the generation of COX2-derived PGE 2 . This supports the concept that in utero exposure to maternal diabetes is a cardiovascular risk factor in adulthood. NEW & NOTEWORTHY Using a unique mouse model of prenatal exposure to maternal type 1 diabetes, our study demonstrates the novel observation that prenatal exposure to maternal diabetes results in a predisposition to high-salt (HS) dietary-induced vascular dysfunction and inflammation in adulthood. Mechanistically, we demonstrated that in utero exposure to maternal diabetes and HS intake induces vascular oxidative stress, cyclooxygenase-derived prostaglandin E 2 , and inflammation.

Background: Aldosterone has been described to initiate cardiovascular diseases by triggering exacerbated sterile vascular inflammation. The functions of CCL5 (C-C motif chemokine ligand 5) and its receptor CCR5 (C-C motif chemokine receptor 5) are well known in infectious diseases, their contributions to aldosterone-induced vascular injury and hypertension remain unknown., Methods: We analyzed the vascular profile, blood pressure, and renal damage in wild-type (CCR5 +/+ ) and CCR5 knockout (CCR5 -/- ) mice treated with aldosterone (600 µg/kg per day for 14 days) while receiving 1% saline to drink. Vascular function was analyzed in aorta and mesenteric arteries, blood pressure was measured by telemetry and renal injury and inflammation were analyzed via histology and flow cytometry. Endothelial cells were used to study the molecular signaling whereby CCL5 induces endothelial dysfunction., Results: Aldosterone treatment resulted in exaggerated CCL5 circulating levels and vascular CCR5 expression in CCR5 +/+ mice accompanied by endothelial dysfunction, hypertension, and renal inflammation and damage. CCR5 -/- mice were protected from these aldosterone-induced effects. Mechanistically, we demonstrated that CCL5 increased NOX1 (NADPH oxidase 1) expression, reactive oxygen species formation, NFκB (nuclear factor kappa B) activation, and inflammation and reduced NO production in isolated endothelial cells. These effects were abolished by antagonizing CCR5 with Maraviroc. Finally, aorta incubated with CCL5 displayed severe endothelial dysfunction, which is prevented by blocking NOX1, NFκB, or CCR5., Conclusions: Our data demonstrate that CCL5/CCR5, through activation of NFκB and NOX1, is critically involved in aldosterone-induced vascular and renal damage and hypertension placing CCL5 and CCR5 as potential therapeutic targets for conditions characterized by aldosterone excess., Competing Interests: None.

A key regulator of blood pressure homeostasis is the steroid hormone aldosterone, which is released as the final signaling hormone of the renin-angiotensin-aldosterone-signaling (RAAS) system. Aldosterone increases sodium (Na + ) reabsorption in the kidney distal nephron to regulate blood volume. Unregulated RAAS signaling can lead to hypertension and cardiovascular disease. The serum and glucocorticoid kinase (SGK1) coordinates much of the Na + reabsorption in the cortical collecting duct (CCD) tubular epithelial cells. We previously demonstrated that aldosterone alters the expression of microRNAs (miRs) in CCD principal cells. The aldosterone-regulated miRs can modulate Na + transport and the cellular response to aldosterone signaling. However, the sex-specific regulation of miRs by aldosterone in the kidney distal nephron has not been explored. In this study, we report that miR-19, part of the miR-17-92 cluster, is upregulated in female mouse CCD cells in response to aldosterone activation. Mir-19 binding to the 3'-untranslated region of SGK1 was confirmed using a dual-luciferase reporter assay. Increasing miR-19 expression in CCD cells decreased SGK1 message and protein expression. Removal of this cluster using a nephron-specific, inducible knockout mouse model increased SGK1 expression in female mouse CCD cells. The miR-19-induced decrease in SGK1 protein expression reduced the response to aldosterone stimulation and may account for sex-specific differences in aldosterone signaling. By examining evolution of the miR-17-92 cluster, phylogenetic sequence analysis indicated that this cluster arose at the same time that other Na + -sparing and salt regulatory proteins, specifically SGK1, first emerged, indicating a conserved role for these miRs in kidney function of salt and water homeostasis. NEW & NOTEWORTHY Expression of the microRNA-17-92 cluster is upregulated by aldosterone in mouse cortical collecting duct principal cells, exclusively in female mice. MiR-19 in this cluster targets the serum and glucocorticoid kinase (SGK1) to downregulate both mRNA and protein expression, resulting in a decrease in sodium transport across epithelial cells of the collecting duct. The miR-17-92 cluster is evolutionarily conserved and may act as a novel feedback regulator for aldosterone signaling in females.

Programmed cell death (PCD) is an important mechanism of innate immunity against bacterial pathogens. The innate immune PCD pathway involves the molecules caspase-7 and caspase-8, among others. Brucella abortus is a gram-negative bacterium that causes a zoonotic disease termed brucellosis. The innate immune response against this pathogen involves activation of inflammasome components and induction of pyroptosis. However, no studies so far have revealed the role of caspase-7 or caspase-8 during this bacterial infection. Herein, we demonstrate that caspase-7 is dispensable for caspase-1 processing, IL-1β secretion and cell death in macrophages. Additionally, caspase-7 deficient animals control B. abortus infection as well as the wild type mice. Furthermore, we addressed the role of caspase-8 in inflammasome activation and pyroptosis during this bacterial infection. Macrophages deficient in caspase-8 secreted reduced amounts of IL-1β that parallels with diminished caspase-1 activity when compared to wild type cells. Additionally, caspase-8 KO macrophages showed reduced LDH release when compared to wild type, suggesting that caspase-8 may play an important role in pyroptosis in response to B. abortus . Finally, caspase-8 KO animals were more susceptible to Brucella infection when compared to wild type mice. Overall, this study contributes to a better understanding of the involvement of caspase-7 and caspase-8 in innate immunity against B. abortus infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Santos, Cerqueira, Zamboni and Oliveira.)

MicroRNAs (miRNAs) belong to a class of endogenous small noncoding RNAs that regulate gene expression at the posttranscriptional level, through both translational repression and mRNA destabilization. They are key regulators of kidney morphogenesis, modulating diverse biological processes in different renal cell lineages. Dysregulation of miRNA expression disrupts early kidney development and has been implicated in the pathogenesis of developmental kidney diseases. In this Review, we summarize current knowledge of miRNA biogenesis and function and discuss in detail the role of miRNAs in kidney morphogenesis and developmental kidney diseases, including congenital anomalies of the kidney and urinary tract and Wilms tumor. We conclude by discussing the utility of miRNAs as potentially novel biomarkers and therapeutic agents.

Mammalian nephrons originate from a population of nephron progenitor cells, and changes in these cells' transcriptomes contribute to the cessation of nephrogenesis, an important determinant of nephron number. To characterize microRNA (miRNA) expression and identify putative cis-regulatory regions, we collected nephron progenitor cells from mouse kidneys at embryonic day 14.5 and postnatal day zero and assayed small RNA expression and transposase-accessible chromatin. We detect expression of 1104 miRNA (114 with expression changes), and 46,374 chromatin accessible regions (2103 with changes in accessibility). Genome-wide, our data highlight processes like cellular differentiation, cell migration, extracellular matrix interactions, and developmental signaling pathways. Furthermore, they identify new candidate cis-regulatory elements for Eya1 and Pax8, both genes with a role in nephron progenitor cell differentiation. Finally, we associate expression-changing miRNAs, including let-7-5p, miR-125b-5p, miR-181a-2-3p, and miR-9-3p, with candidate cis-regulatory elements and target genes. These analyses highlight new putative cis-regulatory loci for miRNA in nephron progenitors., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Cryptosporidium infections are one of the most prevalent causes of diarrhea in calves and considered to be one of the major sources of economic loss in livestock production. A global trend is currently underway, in identifying natural and sustainable alternatives to support animal husbandry and production. Isoquinoline alkaloids are known for their anti-inflammatory, antimicrobial, and antioxidant properties in the promotion of gut health. Thus, an experiment was designed to evaluate the effects of natural, herbal-based feed isoquinoline alkaloids to support calves experimentally inoculated with Cryptosporidium parvum. Twenty-six calves were randomly divided into control (CN) (n = 13) and treatment (SG) (n = 13) groups. The SG group received 5 g of feed additive in every milk feeding from 1 to 21 days of age. The CN group received milk without any additives. All calves were orally inoculated on the third day of life with 1 × 10 6 Cryptosporidium parvum oocysts. The animals were evaluated daily, from 3 to 30 days of age, for the occurrence, duration, and intensity of diarrhea. Calves with a base deficit of ≥ 9 mEq/L were hydrated to aid recovery. The SG calves showed a higher average weight gain between 14 and 21 days of age, without mortality and with reduced intensity and duration of diarrhea. In contrast, calves in the CN group showed more serious acid-base disorders, required more hydration support, and had a mortality rate of 15.4 %. These results showed that calves supplemented with isoquinoline alkaloids had decreased intensity and duration of symptoms, reduced requirement for supportive therapy, and prevented mortality among animals., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The kidney is a complex organ composed of more than 30 terminally differentiated cell types that all are required to perform its numerous homeostatic functions. Defects in kidney development are a significant cause of chronic kidney disease in children, which can lead to kidney failure that can only be treated by transplant or dialysis. A better understanding of molecular mechanisms that drive kidney development is important for designing strategies to enhance renal repair and regeneration. In this study, we profiled gene expression in the developing mouse kidney at embryonic day 14.5 at single-cell resolution. Consistent with previous studies, clusters with distinct transcriptional signatures clearly identify major compartments and cell types of the developing kidney. Cell cycle activity distinguishes between the "primed" and "self-renewing" sub-populations of nephron progenitors, with increased expression of the cell cycle-related genes Birc5, Cdca3, Smc2 and Smc4 in "primed" nephron progenitors. In addition, augmented expression of cell cycle related genes Birc5, Cks2, Ccnb1, Ccnd1 and Tuba1a/b was detected in immature distal tubules, suggesting cell cycle regulation may be required for early events of nephron patterning and tubular fusion between the distal nephron and collecting duct epithelia., (© 2021. The Author(s).)

Several direct or indirect methods can be used to assess immunoglobulin G (IgG) concentrations in calves, which evaluates the transfer of passive immunity (TPI). Radial immunodiffusion (RID) is the gold standard method to measure serum IgG in bovines. Previous studies have shown that colostrum provides several molecules in addition to immunoglobulins, which play an important role in the passive immunity of the calf. However, no studies have yet determined the level of interference of these components in the immunity, health and survival of calves. In this sense, the objective of this study is to review the methods of evaluation available for the laboratory and field diagnosis of TPI in calves and discuss the main aspects of each technique. Several methods available for TPI evaluation in calves may provide insights into the various components of colostrum involved in passive immunity.

In this study, we provide evidence that galectin-3 (Gal-3) plays an important role in Brucella abortus infection. Our results showed increased Gal-3 expression and secretion in B. abortus infected macrophages and mice. Additionally, our findings indicate that Gal-3 is dispensable for Brucella-containing vacuoles disruption, inflammasome activation and pyroptosis. On the other hand, we observed that Brucella-induced Gal-3 expression is crucial for induction of molecules associated to type I IFN signalling pathway, such as IFN-β: Interferon beta (IFN-β), C-X-C motif chemokine ligand 10 (CXCL10) and guanylate-binding proteins. Gal-3 KO macrophages showed reduced bacterial numbers compared to wild-type cells, suggesting that Gal-3 facilitates bacterial replication in vitro. Moreover, priming Gal-3 KO cells with IFN-β favoured B. abortus survival in macrophages. Additionally, we also observed that Gal-3 KO mice are more resistant to B. abortus infection and these animals showed elevated production of proinflammatory cytokines when compared to control mice. Finally, we observed an increased recruitment of macrophages, dendritic cells and neutrophils in spleens of Gal-3 KO mice compared to wild-type animals. In conclusion, this study demonstrated that Brucella-induced Gal-3 is detrimental to host and this molecule is implicated in inhibition of recruitment and activation of immune cells, which promotes B. abortus spread and aggravates the infection. TAKE AWAYS: Brucella abortus infection upregulates galectin-3 expression Galectin-3 regulates guanylate-binding proteins expression but is not required for Brucella-containing vacuole disruption Galectin-3 modulates proinflammatory cytokine production during bacterial infection Galectin-3 favours Brucella replication., (© 2021 John Wiley & Sons Ltd.)

Background: Damage to the renal microvasculature is a hallmark of renal ischemia-reperfusion injury (IRI)-mediated AKI. The miR-17∼92 miRNA cluster (encoding miR-17 , -18a , -19a , -20a , -19b-1 , and -92a-1 ) regulates angiogenesis in multiple settings, but no definitive role in renal endothelium during AKI pathogenesis has been established., Methods: Antibodies bound to magnetic beads were utilized to selectively enrich for renal endothelial cells from mice. Endothelial-specific miR-17∼92 knockout ( miR-17∼92 endo-/- ) mice were generated and given renal IRI. Mice were monitored for the development of AKI using serum chemistries and histology and for renal blood flow using magnetic resonance imaging (MRI) and laser Doppler imaging. Mice were treated with miRNA mimics during renal IRI, and therapeutic efficacies were evaluated., Results: miR-17 , -18a , -20a , -19b , and pri-miR-17∼92 are dynamically regulated in renal endothelial cells after renal IRI. miR-17∼92 endo-/- exacerbates renal IRI in male and female mice. Specifically, miR-17∼92 endo-/- promotes renal tubular injury, reduces renal blood flow, promotes microvascular rarefaction, increases renal oxidative stress, and promotes macrophage infiltration to injured kidneys. The potent antiangiogenic factor thrombospondin 1 (TSP1) is highly expressed in renal endothelium in miR-17∼92 endo-/- after renal IRI and is a target of miR-18a and miR-19a/b . miR-17∼92 is critical in the angiogenic response after renal IRI, which treatment with miR-18a and miR-19b mimics can mitigate., Conclusions: These data suggest that endothelial-derived miR-17∼92 stimulates a reparative response in damaged renal vasculature during renal IRI by regulating angiogenic pathways., (Copyright © 2021 by the American Society of Nephrology.)

Interleukin-6 (IL-6) is a pleiotropic cytokine promptly produced in response to infections, which contributes to host defense through the stimulation of acute phase immune responses. Brucella abortus is an intracellular bacterium that causes chronic disease in humans and domestic animals and triggers a robust immune response, characterized by the production of inflammatory cytokines. However, the mechanisms of IL-6-related immune responses in the context of Brucella infections are not completely understood. In this report, we describe an increased susceptibility of IL-6 knockout (KO) mice in the early phase of Brucella infection. Furthermore, we demonstrate that IL-6 is required for interferon (IFN)-γ and tumor necrosis factor (TNF)-α induction by infected splenocytes, indicating a protective role for IL-6 against B. abortus that parallels with Th1 type of immune response. Additionally, IL-6 KO mice exhibited reduced splenomegaly during the early phase of the infection. Corroborating this result, IL-6 KO mice displayed reduced numbers of macrophages, dendritic cells, and neutrophils in the spleen and reduced myeloperoxidase activity in the liver compared to wild-type infected mice. However, we demonstrate that IL-6 is not involved in B. abortus intracellular restriction in mouse macrophages. Taken together, our findings demonstrate that IL-6 contributes to host resistance during the early phase of B. abortus infection in vivo, and suggest that its protective role maybe partially mediated by proinflammatory immune responses and immune cell recruitment.

Background: The Brazilian health regulatory agency (Agência Nacional de Vigilância Sanitária, ANVISA) has embarked on transformational initiatives to fulfill its mandate to provide timely access to safe, effective, and quality therapeutics. A new Brazilian law was enacted to provide the agency with greater flexibility. Optimizing Efficiencies in Regulatory Agencies (OpERA) is a regulatory-strengthening program that seeks to provide benchmarking data that can be used to define performance targets and focus performance improvement. The objective of this study was to use OpERA methodology to undertake a retrospective analysis of the timelines associated with important components of the ANVISA regulatory review process to establish a baseline against which the influence of the new law could be measured., Methods: The OpERA tool was used to collect specific milestone data that identify time periods, review stages, and data points for products approved by ANVISA 2013-2016., Results: For the 138 products approved in this cohort, the overall median approval time was 795 days. ANVISA and submitting companies will need to reduce their review and response times by approximately half in order to meet the total time goal of 365 days., Conclusions: The observations from this baseline study have identified opportunities for ANVISA and sponsor companies to collaborate to reduce regulatory assessment times while assuring the timely approval of safe and effective, quality medicines. These analyses will be repeated to determine how the provisions of the new Law will impact the activities of ANVISA and the extent of sponsors' contributions to this effort.

Background: Vesicoureteral reflux (VUR), backflow of urine into the kidney, is associated with urinary tract infections and chronic kidney disease. Integrity of the vesicoureteral junction (VUJ), where reflux occurs, is determined largely by proper induction of the ureteric bud from the Wolffian duct. Induction is modulated by signals from the surrounding peri-Wolffian duct stroma. We evaluated whether miRNAs in the peri-Wolffian duct stroma are necessary for proper ureteric induction, VUJ formation, and suppression of VUR., Methods: We generated a mouse with loss of miRNAs in the peri-Wolffian duct stroma. We evaluated embryos for ureteric bud induction defects and expression of genes that regulate induction. We performed cystograms to assess for reflux and assessed VUJs in postnatal mice., Results: Mutant embryos had cranially displaced ureteric bud induction sites vs. controls. We observed no changes in expression of genes known to regulate induction. While mutants were early postnatal lethal, they had high rates of VUR vs. controls. Mutant VUJs that refluxed had low inserting ureters and shortened intravesicular tunnels vs. non-refluxing mice., Conclusions: We found that miRNAs in the peri-Wolffian duct stroma are required for normal ureteric bud induction, VUJ formation, and prevention of VUR.

Low nephron number results in an increased risk of developing hypertension and chronic kidney disease. Intrauterine growth restriction is associated with a nephron deficit in humans, and is commonly caused by placental insufficiency, which results in fetal hypoxia. The underlying mechanisms by which hypoxia impacts kidney development are poorly understood. microRNA-210 is the most consistently induced microRNA in hypoxia and is known to promote cell survival in a hypoxic environment. In this study, the role of microRNA-210 in kidney development was evaluated using a global microRNA-210 knockout mouse. A male-specific 35% nephron deficit in microRNA-210 knockout mice was observed. Wnt/β-catenin signaling, a pathway crucial for nephron differentiation, was misregulated in male kidneys with increased expression of the canonical Wnt target lymphoid enhancer binding factor 1. This coincided with increased expression of caspase-8-associated protein 2, a known microRNA-210 target and apoptosis signal transducer. Together, these data are consistent with a sex-specific requirement for microRNA-210 in kidney development., (© 2020 Federation of American Societies for Experimental Biology.)

Proteinuric chronic kidney disease (CKD) remains a major health problem worldwide. While it is well established that the progression of primary glomerular disease induces tubulointerstitial lesions, how tubular injury triggers glomerular damage is poorly understood. We hypothesized that injured tubules secrete mediators that adversely affect glomerular health. To test this, we used conditional knockout mice with tubule-specific ablation of β-catenin (Ksp-β-cat-/-) and subjected them to chronic angiotensin II (Ang II) infusion or Adriamycin. Compared with control mice, Ksp-β-cat-/- mice were dramatically protected from proteinuria and glomerular damage. MMP-7, a downstream target of β-catenin, was upregulated in treated control mice, but this induction was blunted in the Ksp-β-cat-/- littermates. Incubation of isolated glomeruli with MMP-7 ex vivo led to nephrin depletion and impaired glomerular permeability. Furthermore, MMP-7 specifically and directly degraded nephrin in cultured glomeruli or cell-free systems, and this effect was dependent on its proteolytic activity. In vivo, expression or infusion of exogenous MMP-7 caused proteinuria, and genetic ablation of MMP-7 protected mice from Ang II-induced proteinuria and glomerular injury. Collectively, these results demonstrate that β-catenin-driven MMP-7 release from renal tubules promotes glomerular injury via direct degradation of the key slit diaphragm protein nephrin.

The incidence of diabetes mellitus has significantly increased among women of childbearing age, and it has been shown that prenatal exposure to maternal diabetes increases the risk of associated congenital anomalies of the kidney. Congenital anomalies of the kidney are among the leading causes of chronic kidney disease in children. To better understand the effect of maternal diabetes on kidney development, we analyzed wild-type offspring (DM_Exp) of diabetic Ins2 +/C96Y mice (Akita mice). DM_Exp mice at postnatal day 34 have a reduction of ~20% in the total nephron number compared with controls, using the gold standard physical dissector/fractionator method. At the molecular level, the expression of the nephron progenitor markers sine oculis homeobox homolog 2 and Cited1 was increased in DM_Exp kidneys at postnatal day 2 . Conversely, the number of early developing nephrons was diminished in DM_Exp kidneys. This was associated with decreased expression of the intracellular domain of Notch1 and the canonical Wnt target lymphoid enhancer binding factor 1. Together, these data suggest that the diabetic intrauterine environment impairs the differentiation of nephron progenitors into nephrons, possibly by perturbing the Notch and Wnt/β-catenin signaling pathways.

The immune system is armed with a broad range of receptors to detect and initiate the elimination of bacterial pathogens. Inflammasomes are molecular platforms that sense a diverse range of microbial insults to develop appropriate host response. In that context, noncanonical inflammasome arose as a sensor for Gram-negative bacteria-derived LPS leading to the control of infections. This review describes the role of caspase-11/gasdermin-D-dependent immune response against Gram-negative bacteria and presents an overview of guanylate-binding proteins (GBPs) at the interface of noncanonical inflammasome activation. Indeed, caspase-11 acts as a receptor for LPS and this interaction elicits caspase-11 autoproteolysis that is required for its optimal catalytic activity. Gasdermin-D is cleaved by activated caspase-11 generating an N-terminal domain that is inserted into the plasmatic membrane to form pores that induce pyroptosis, a cell death program involved in intracellular bacteria elimination. This mechanism also promotes IL-1β release and potassium efflux that connects caspase-11 to NLRP3 activation. Furthermore, GBPs display many features to allow LPS recognition by caspase-11, initiating the noncanonical inflammasome response prompting the immune system to control bacterial infections. In this review, we discuss the recent findings and nuances related to this mechanism and its biological functions., (©2019 Society for Leukocyte Biology.)

Innate immune response against Brucella abortus involves activation of Toll-like receptors (TLRs) and NOD-like receptors (NLRs). Among the NLRs involved in the recognition of B. abortus are NLRP3 and AIM2. Here, we demonstrate that B. abortus triggers non-canonical inflammasome activation dependent on caspase-11 and gasdermin-D (GSDMD). Additionally, we identify that Brucella-LPS is the ligand for caspase-11 activation. Interestingly, we determine that B. abortus is able to trigger pyroptosis leading to pore formation and cell death, and this process is dependent on caspase-11 and GSDMD but independently of caspase-1 protease activity and NLRP3. Mice lacking either caspase-11 or GSDMD were significantly more susceptible to infection with B. abortus than caspase-1 knockout or wild-type animals. Additionally, guanylate-binding proteins (GBPs) present in mouse chromosome 3 participate in the recognition of LPS by caspase-11 contributing to non-canonical inflammasome activation as observed by the response of Gbpchr3-/- BMDMs to bacterial stimulation. We further determined by siRNA knockdown that among the GBPs contained in mouse chromosome 3, GBP5 is the most important for Brucella LPS to be recognized by caspase-11 triggering IL-1β secretion and LDH release. Additionally, we observed a reduction in neutrophil, dendritic cell and macrophage influx in spleens of Casp11-/- and Gsdmd-/- compared to wild-type mice, indicating that caspase-11 and GSDMD are implicated in the recruitment and activation of immune cells during Brucella infection. Finally, depletion of neutrophils renders wild-type mice more susceptible to Brucella infection. Taken together, these data suggest that caspase-11/GSDMD-dependent pyroptosis triggered by B. abortus is important to infection restriction in vivo and contributes to immune cell recruitment and activation., Competing Interests: The authors have declared that no competing interests exist.

Immunity against microbes depends on recognition of pathogen-associated molecular patterns by innate receptors. Signaling pathways triggered by Brucella abortus DNA involves TLR9, AIM2, and stimulator of IFN genes (STING). In this study, we observed by microarray analysis that several type I IFN-associated genes, such as IFN-β and guanylate-binding proteins (GBPs), are downregulated in STING knockout (KO) macrophages infected with Brucella or transfected with DNA. Additionally, we determined that STING and cyclic GMP-AMP synthase (cGAS) are important to engage the type I IFN pathway, but only STING is required to induce IL-1β secretion, caspase-1 activation, and GBP2 and GBP3 expression. Furthermore, we determined that STING but not cGAS is critical for host protection against Brucella infection in macrophages and in vivo. This study provides evidence of a cGAS-independent mechanism of STING-mediated protection against an intracellular bacterial infection. Additionally, infected IFN regulatory factor-1 and IFNAR KO macrophages had reduced GBP2 and GBP3 expression and these cells were more permissive to Brucella replication compared with wild-type control macrophages. Because GBPs are critical to target vacuolar bacteria, we determined whether GBP2 and GBP chr3 affect Brucella control in vivo. GBP chr3 but not GBP2 KO mice were more susceptible to bacterial infection, and small interfering RNA treated-macrophages showed reduction in IL-1β secretion and caspase-1 activation. Finally, we also demonstrated that Brucella DNA colocalizes with AIM2, and AIM2 KO mice are less resistant to B. abortus infection. In conclusion, these findings suggest that the STING-dependent type I IFN pathway is critical for the GBP-mediated release of Brucella DNA into the cytosol and subsequent activation of AIM2., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Legionella pneumophila is a Gram-negative, flagellated bacterium that survives in phagocytes and causes Legionnaires' disease. Upon infection of mammalian macrophages, cytosolic flagellin triggers the activation of Naip/NLRC4 inflammasome, which culminates in pyroptosis and restriction of bacterial replication. Although NLRC4 and caspase-1 participate in the same inflammasome, Nlrc4-/- mice and their macrophages are more permissive to L. pneumophila replication compared with Casp1/11-/-. This feature supports the existence of a pathway that is NLRC4-dependent and caspase-1/11-independent. Here, we demonstrate that caspase-8 is recruited to the Naip5/NLRC4/ASC inflammasome in response to flagellin-positive bacteria. Accordingly, caspase-8 is activated in Casp1/11-/- macrophages in a process dependent on flagellin, Naip5, NLRC4 and ASC. Silencing caspase-8 in Casp1/11-/- cells culminated in macrophages that were as susceptible as Nlrc4-/- for the restriction of L. pneumophila replication. Accordingly, macrophages and mice deficient in Asc/Casp1/11-/- were more susceptible than Casp1/11-/- and as susceptible as Nlrc4-/- for the restriction of infection. Mechanistically, we found that caspase-8 activation triggers gasdermin-D-independent pore formation and cell death. Interestingly, caspase-8 is recruited to the Naip5/NLRC4/ASC inflammasome in wild-type macrophages, but it is only activated when caspase-1 or gasdermin-D is inhibited. Our data suggest that caspase-8 activation in the Naip5/NLRC4/ASC inflammasome enable induction of cell death when caspase-1 or gasdermin-D is suppressed.

Low nephron endowment at birth has been associated with an increased risk for developing hypertension and chronic kidney disease. We demonstrated in an earlier study that conditional deletion of the microRNA (miRNA)-processing enzyme Dicer from nephron progenitors results in premature depletion of the progenitors and increased expression of the proapoptotic protein Bim (also known as Bcl-2L11). In this study, we generated a compound mouse model with conditional deletion of both Dicer and Bim , to determine the biologic significance of increased Bim expression in Dicer -deficient nephron progenitors. The loss of Bim partially restored the number of nephron progenitors and improved nephron formation. The number of progenitors undergoing apoptosis was significantly reduced in kidneys with loss of a single allele, or both alleles, of Bim compared to mutant kidneys. Furthermore, 2 miRNAs expressed in nephron progenitors ( miR-17 and miR-106b) regulated Bim levels in vitro and in vivo Together, these data suggest that miRNA-mediated regulation of Bim controls nephron progenitor survival during nephrogenesis, as one potential means of regulating nephron endowment.-Cerqueira, D. M., Bodnar, A. J., Phua, Y. L., Freer, R., Hemker, S. L., Walensky, L. D., Hukriede, N. A., Ho, J. Bim gene dosage is critical in modulating nephron progenitor survival in the absence of microRNAs during kidney development., (© FASEB.)

Gram-negative bacteria from the Legionella genus are intracellular pathogens that cause a severe form of pneumonia called Legionnaires' disease. The bacteria replicate intracellularly in macrophages, and the restriction of bacterial replication by these cells is critical for host resistance. The activation of the NAIP5/NLRC4 inflammasome, which is readily triggered in response to bacterial flagellin, is essential for the restriction of bacterial replication in murine macrophages. Once activated, this inflammasome induces pore formation and pyroptosis and facilitates the restriction of bacterial replication in macrophages. Because investigations related to the NLRC4-mediated restriction of Legionella replication were performed using mice double deficient for caspase-1 and caspase-11, we assessed the participation of caspase-1 and caspase-11 in the functions of the NLRC4 inflammasome and the restriction of Legionella replication in macrophages and in vivo. By using several species of Legionella and mice singly deficient for caspase-1 or caspase-11, we demonstrated that caspase-1 but not caspase-11 was required for pore formation, pyroptosis, and restriction of Legionella replication in macrophages and in vivo. By generating F1 mice in a mixed 129 × C57BL/6 background deficient (129 × Casp-11(-/-) ) or sufficient (129 × C57BL/6) for caspase-11 expression, we found that caspase-11 was dispensable for the restriction of Legionella pneumophila replication in macrophages and in vivo. Thus, although caspase-11 participates in flagellin-independent noncanonical activation of the NLRP3 inflammasome, it is dispensable for the activities of the NLRC4 inflammasome. In contrast, functional caspase-1 is necessary and sufficient to trigger flagellin/NLRC4-mediated restriction of Legionella spp. infection in macrophages and in vivo., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Overactivation of the Wnt/β-catenin pathway in adult tissues has been implicated in many diseases, such as colorectal cancer. Finding chemical substances that can prevent this phenomenon is an emerging problem. Recently, several natural compounds have been described as Wnt/β-catenin inhibitors and might be promising agents for the control of carcinogenesis. Here, we describe two natural substances, derricin and derricidin, belonging to the chalcone subclass, that show potent transcriptional inhibition of the Wnt/β-catenin pathway. Both chalcones are able to affect the cell distribution of β-catenin, and inhibit Wnt-specific reporter activity in HCT116 cells and in Xenopus embryos. Derricin and derricidin also strongly inhibited canonical Wnt activity in vitro, and rescued the Wnt-induced double axis phenotype in Xenopus embryos. As a consequence of Wnt/β-catenin inhibition, derricin and derricidin treatments reduce cell viability and lead to cell cycle arrest in colorectal cancer cell lines. Taken together, our results strongly support these chalcones as novel negative modulators of the Wnt/β-catenin pathway and colon cancer cell growth in vitro.

Flavonoids are plant-derived polyphenolic molecules that have potential biological effects including anti-oxidative, anti-inflammatory, anti-viral, and anti-tumoral effects. These effects are related to the ability of flavonoids to modulate signaling pathways, such as the canonical Wnt signaling pathway. This pathway controls many aspects of embryonic development and tissue maintenance and has been found to be deregulated in a range of human cancers. We performed several in vivo assays in Xenopus embryos, a functional model of canonical Wnt signaling studies, and also used in vitro models, to investigate whether isoquercitrin affects Wnt/β-catenin signaling. Our data provide strong support for an inhibitory effect of isoquercitrin on Wnt/β-catenin, where the flavonoid acts downstream of β-catenin translocation to the nuclei. Isoquercitrin affects Xenopus axis establishment, reverses double axes and the LiCl hyperdorsalization phenotype, and reduces Xnr3 expression. In addition, this flavonoid shows anti-tumoral effects on colon cancer cells (SW480, DLD-1, and HCT116), whereas exerting no significant effect on non-tumor colon cell (IEC-18), suggesting a specific effect in tumor cells in vitro. Taken together, our data indicate that isoquercitrin is an inhibitor of Wnt/β-catenin and should be further investigated as a potential novel anti-tumoral agent., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The kidney is a homeostatic organ required for waste excretion and reabsorption of water, salts and other macromolecules. To this end, a complex series of developmental steps ensures the formation of a correctly patterned and properly proportioned organ. While previous studies have mainly focused on the individual signaling pathways, the formation of higher order receptor complexes in lipid rafts is an equally important aspect. These membrane platforms are characterized by differences in local lipid and protein compositions. Indeed, the cells in the Xenopus pronephric kidney were positive for the lipid raft markers ganglioside GM1 and Caveolin-1. To specifically interfere with lipid raft function in vivo, we focused on the Sterol Carrier Protein 2 (scp2), a multifunctional protein that is an important player in remodeling lipid raft composition. In Xenopus, scp2 mRNA was strongly expressed in differentiated epithelial structures of the pronephric kidney. Knockdown of scp2 did not interfere with the patterning of the kidney along its proximo-distal axis, but dramatically decreased the size of the kidney, in particular the proximal tubules. This phenotype was accompanied by a reduction of lipid rafts, but was independent of the peroxisomal or transcriptional activities of scp2. Finally, disrupting lipid micro-domains by inhibiting cholesterol synthesis using Mevinolin phenocopied the defects seen in scp2 morphants. Together these data underscore the importance for localized signaling platforms in the proper formation of the Xenopus kidney., (Copyright © 2014 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) are major posttranscriptional regulators of a wide variety of biological processes. However, redundancy among most miRNAs has made it difficult to identify their in vivo functions. We previously demonstrated that global inhibition of miRNA biogenesis in Xenopus resulted in a dramatically smaller pronephric kidney. This suggested that microRNAs play a pivotal role in organ size control. Here we now provide a detailed mechanistic explanation for this phenotype. We identified that the activation of the mechanistic target of rapamycin complex 1 (mTORC1) by Insulin and insulin-like growth factor (Igf) 2 is an important regulator in kidney growth, which in turn is modulated by microRNAs. Molecular analyses demonstrate that microRNAs set a threshold for mTORC1 signaling by down-regulating one of its core negative regulators, tuberous sclerosis 1 (Tsc1). Most importantly, this rheostat can be reprogrammed experimentally. Whereas knockdown of miRNAs causes growth arrest, concomitant knockdown of Tsc1 restores mTORC1 activity and proximal tubular size. Together, these data establish a previously unidentified in vivo paradigm for the importance of posttranscriptional regulation in organ size control.

The main functions of the kidney are to excrete metabolic waste products and actively reabsorb essential molecules such as amino acids, ions, glucose and water. In humans, a wide range of genetic disorders exist characterized by wasting of metabolically important compounds. At the cellular level, more than 20 highly specialized renal epithelial cell types located in different segments of the nephron contribute to the reabsorption process. In particular, proximal tubular cells play a crucial role and are uniquely adapted to maximize reabsorption efficiency. They accommodate high numbers of transporters and channels by increasing the apical surface area in contact with the primary filtrate by forming a brush border as well as undergoing hypertrophy and hyperplasia. This adaptation is evolutionarily conserved and is detected in the primitive pronephric kidney of fish and amphibians as well as the metanephric kidney of higher vertebrates. Surprisingly, signaling pathways regulating these three processes have remained largely unknown. Here we summarize recent studies that highlight the early phases of kidney development as a critical juncture in establishing proximal tubule size.

Bradykinin is not only important for inflammation and blood pressure regulation, but also involved in neuromodulation and neuroprotection. Here we describe novel functions for bradykinin and the kinin-B2 receptor (B2BkR) in differentiation of neural stem cells. In the presence of the B2BkR antagonist HOE-140 during rat neurosphere differentiation, neuron-specific β3-tubulin and enolase expression was reduced together with an increase in glial protein expression, indicating that bradykinin-induced receptor activity contributes to neurogenesis. In agreement, HOE-140 affected in the same way expression levels of neural markers during neural differentiation of murine P19 and human iPS cells. Kinin-B1 receptor agonists and antagonists did not affect expression levels of neural markers, suggesting that bradykinin-mediated effects are exclusively mediated via B2BkR. Neurogenesis was augmented by bradykinin in the middle and late stages of the differentiation process. Chronic treatment with HOE-140 diminished eNOS and nNOS as well as M1-M4 muscarinic receptor expression and also affected purinergic receptor expression and activity. Neurogenesis, gliogenesis, and neural migration were altered during differentiation of neurospheres isolated from B2BkR knock-out mice. Whole mount in situ hybridization revealed the presence of B2BkR mRNA throughout the nervous system in mouse embryos, and less β3-tubulin and more glial proteins were expressed in developing and adult B2BkR knock-out mice brains. As a underlying transcriptional mechanism for neural fate determination, HOE-140 induced up-regulation of Notch1 and Stat3 gene expression. Because pharmacological treatments did not affect cell viability and proliferation, we conclude that bradykinin-induced signaling provides a switch for neural fate determination and specification of neurotransmitter receptor expression.

Cholesterol-rich membrane microdomains (CRMMs) are specialized structures that have recently gained much attention in cell biology because of their involvement in cell signaling and trafficking. However, few investigations, particularly those addressing embryonic development, have succeeded in manipulating and observing CRMMs in living cells. In this study, we performed a detailed characterization of the CRMMs lipid composition during early frog development. Our data showed that disruption of CRMMs through methyl-β-cyclodextrin (MβCD) cholesterol depletion at the blastula stage did not affect Spemann's organizer gene expression and inductive properties, but impaired correct head development in frog and chick embryos by affecting the prechordal plate gene expression and cellular morphology. The MβCD anterior defect phenotype was recapitulated in head anlagen (HA) explant cultures. Culture of animal cap expressing Dkk1 combined with MβCD-HA generated a head containing eyes and cement gland. Together, these data show that during Xenopus blastula and gastrula stages, CRMMs have a very dynamic lipid composition and provide evidence that the secreted Wnt antagonist Dkk1 can partially rescue anterior structures in cholesterol-depleted head anlagen., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Maternal Wnt/β-Catenin signaling is essential to establish dorsal-specific gene expression required for axial patterning in Xenopus. Deregulation of this pathway causes axis phenotypes in frog embryos. In adult life, mutations in the Wnt pathway components are associated with many diseases, such as polyposis coli; osteoporosis-pseudoglioma syndrome (OPPG); skeletal dysplasia; neural tube defects, cancer and many others. Thus, a better understanding of Wnt/β-catenin signaling will have great and significant impact on Biology and Medicine. In this aspect, natural compounds are potential targets as novel molecules that could modulate the Wnt pathway. For instance, flavonoids are a large group of natural compounds found in plants that modulate important cellular and molecular mechanisms related to diseases, but the specific in vivo mechanism of action of most flavonoids remain unknown. In this way, Xenopus embryos may provide an efficient model, since it is frequently used to test and identify the role of molecules that affect Wnt/β-catenin signaling. Here, we describe a combination of approaches to outline and characterize the role of two flavonoids, quercetin and rutin, on Wnt/β-catenin signaling, using Xenopus embryos as an experimental model. Our data support that quercetin is potential in vivo modulator of canonical Wnt signaling and that this effect might depend on the structure of this molecule, as we did not observe any effect with rutin treatment, a flavonol structurally-related to quercetin. This model is useful to analyze effects of quercetin and other flavonoids in vivo and to provide further understanding of how natural compounds can modulate signaling pathways, using Xenopus embryos as a fast and efficient reading of in vivo effects of those compounds.

Flavonoids are polyphenolic compounds found throughout the plant kingdom. They occur in every organ but are usually concentrated in leaves and flowers. During the last two decades, in vitro and in vivo studies demonstrated that flavonoids have inhibitory effects on human diseases through targeting of multiple cellular signaling components. Wnt/β-catenin signaling regulates proliferation, differentiation and fate specification in developmental stages and controls tissue homeostasis in adult life. For these reasons, this pathway has received great attention in the last years as potential pathway involved in distinct Human pathologies. In this review we discuss the emerging potential mechanisms for flavonoids on Wnt/β-catenin signaling in cancer and possible investigation strategies to understand flavonoids mode of action on this signaling pathway., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Introduction: Several studies related that different human papillomavirus (HPV) types and intratype variants can present different oncogenic potential. In opposite to HPVs 16 and 18 variants, information about variants of other carcinogenic HPV types is still scarce. The aim of this study was to investigate the genetic variability of HPVs 53, 56, and 66 from Central Brazil isolates., Methods: The long control region (LCR), E6, and L1 genomic regions were amplified and sequenced. We evaluate for nucleotide variations in relation to the reference sequence of each HPV type and also the conservation of physicochemical properties of the deduced amino acid substitutions. In silico analysis was performed to locate binding sites for transcriptional factors within the LCR. Moreover, we performed a phylogenetic analysis with the Central Brazilian and worldwide sequences available at genomic databases., Results: Gathering LCR, E6, and L1 genomic regions, the highest genetic variability was found among HPV-53 isolates with 52 nucleotide variations, followed by HPVs 56 and 66 with 24 and 16 nucleotide substitutions, respectively. The genetic analysis revealed 11 new molecular variants of all HPV types analyzed, totalizing 31 new nucleotide and 3 new amino acid variations. Eight nonconservative amino acid substitutions were detected, which may indicate a biological and pathogenic diversity among HPV types. Furthermore, 8 nucleotide substitutions were localized at putative binding sites for transcription factors in the LCR with a potential implication on viral oncogene expression. The HPVs 53, 56, and 66 phylogenetic analysis confirmed a dichotomic division only described to HPV subtypes and different from the patterns described for HPVs 16 and 18 variants., Conclusions: The high genetic variability observed emphasizes the importance of investigating polymorphisms in types other than HPVs 16 or 18 to better understand the molecular genomic profile of viral infection by different HPV types.

Podocytes are highly specialized cells in the vertebrate kidney. They participate in the formation of the size-exclusion barrier of the glomerulus/glomus and recruit mesangial and endothelial cells to form a mature glomerulus. At least six transcription factors (wt1, foxc2, hey1, tcf21, lmx1b and mafb) are known to be involved in podocyte specification, but how they interact to drive the differentiation program is unknown. The Xenopus pronephros was used as a paradigm to address this question. All six podocyte transcription factors were systematically eliminated by antisense morpholino oligomers. Changes in the expression of the podocyte transcription factors and of four selected markers of terminal differentiation (nphs1, kirrel, ptpru and nphs2) were analyzed by in situ hybridization. The data were assembled into a transcriptional regulatory network for podocyte development. Although eliminating the six transcription factors individually interfered with aspects of podocyte development, no single gene regulated the entire differentiation program. Only the combined knockdown of wt1 and foxc2 resulted in a loss of all podocyte marker gene expression. Gain-of-function studies showed that wt1 and foxc2 were sufficient to increase podocyte gene expression within the glomus proper. However, the combination of wt1, foxc2 and Notch signaling was required for ectopic expression in ventral marginal zone explants. Together, this approach demonstrates how complex interactions are required for the correct spatiotemporal execution of the podocyte gene expression program.

Isoquercitrin isolated from the aerial parts of Hyptis fasciculata was evaluated according to its capacity to interfere with glioblastoma (Gbm) cell growth. Gbm cells were incubated with isoquercitrin, quercetin, or rutin at concentrations of 25, 50, and 100 mumol/l for 24, 48, and 72 h. Quercetin and rutin affected Gbm cell proliferation after treatment times of longer than 24 h. However, increasing concentrations of isoquercitrin inhibited 50% of Gbm cell proliferation at 24 h and further reached nearly 90% inhibition at 72 h. This effect did not affect cell morphology, cell viability, or cleaved capase-3 levels, indicating that isoquercitrin did not induce Gbm cell death. A marked reduction in cyclin D1 levels and an increase in p27 levels were observed when 100 micromol/l of isoquercitrin was added to Gbm cells. Interestingly, nuclear beta-catenin staining observed in a subpopulation of untreated Gbm cells was found in the cytoplasm after 100-micromol/l isoquercitrin treatment. Collectively, these data show that isoquercitrin reduces Gbm cell growth without inducing apoptosis, possibly by modulating the control of the cell cycle. Our data also suggest that beta-catenin-mediated signaling may be involved on the antiproliferative activity of isoquercitrin.

Reactive oxygen species (ROS) are thought to underline the process of ageing and the pathogenicity of various diseases, such as neurodegenerative disorders and cancer. The use of traditional medicine is widespread and plants still present a large source of natural antioxidants that might serve as leads for the development of novel drugs. In this paper, the alcoholic extract from leaves of Hyptis fasciculata, a Brazilian medicinal plant, and isoquercitrin, a flavonoid identified in this species, showed to be active as 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavengers. The extract of Hyptis fasciculata and isoquercitrin were also able to increase tolerance of the eukaryotic microorganism Saccharomyces cerevisiae to both hydrogen peroxide and menadione, a source of superoxide. Cellular protection was correlated with a decrease in oxidative stress markers, such as levels of ROS, protein carbonylation and lipid peroxidation, confirming the antioxidant potential of Hyptis fasciculata and isoquercitrin.

More than 100 HPV types have been described, 13 of which are classified as high-risk due to their association with the development of cervical cancer. The intratype genomic diversity of HPV-16 and -18 has been studied extensively, while little data have been generated for other less common high-risk types. The present study explores the nucleotide variability and phylogeny of the high-risk HPV-31, -33, -35, -52, and -58, in samples from Central Brazil. For this purpose, the LCR and the E6 and L1 genes were sequenced. Several variants of these HPV types were detected, some of which have been detected in other parts of the world. Furthermore, new variants of all types examined were characterized in a total of 13 new variants. Based on the E6 and L1 sequences, variants were described comprising conservative and non-conservative amino acid changes. For phylogenetic tree construction, samples characterized in this study were compared to others described and submitted to GenBank previously. The phylogenetic analysis of HPV-31, -33, -35, and -58 isolates did not reveal ethnic or geographical clustering as observed previously for HPV-16 and -18. HPV-35 analysis showed a dichotomic branching characteristic of viral subtypes. Interestingly, four clusters relative to the analysis of HPV-52 isolates were identified, two of which could be classified as Asian and European branches. The genomic characterization of HPV variants is crucial for understanding the intrinsic geographical relatedness and biological differences of these viruses and contributes further to studies on their infectivity and pathogenicity., ((c) 2009 Wiley-Liss, Inc.)

HPV-18 is the second most prevalent human papillomavirus genotype found in cervical cancer. Nucleotide variations in HPV-18 sequence can interfere with the viral oncogenic potential. However, the knowledge about HPV-18 variants in Brazil is still limited. The present study aims to determine the LCR, E6, and L1 genetic variability of HPV-18 variants found in women co-infected with HIV-1 in Central Brazil. Four HPV-18 samples were identified and had the LCR, E6, and L1 genomic regions sequenced. It was possible to characterize three European variants and one African variant of HPV-18. All of them are new variants, showing nucleotide substitutions not previously reported. Nucleotide variations in binding sites for transcriptional factors were observed. Phylogenetic analysis was also performed, evidencing the three clusters related to the Asian-American, African, and European variants. The characterization of HPV genetic variability is of pivotal importance to the understanding of the viral pathogenicity.

HPV-16 is the most prevalent human papillomavirus genotype found in cervical intraepithelial neoplasias. The regulatory region of the HPV genome, LCR, has several binding sites for cellular and viral transcription factors, and nucleotide substitutions in this genomic region can interfere with the viral oncogenic expression. The present study aims to determine the LCR variability of European and non-European HPV-16 variants found in Brazil. Through automated sequencing, it was possible to characterize the LCR of ten non-European (eight Asian-American, one African 1, one African 2) and twelve European isolates. Among the 22 isolates analyzed, nine may be new variants of HPV-16, with different combinations of previously reported nucleotide substitutions, and three showed new substitutions not previously reported. Two new nucleotide substitutions, the insertion of T at position 7621 and the substitution of A to G at position 7836, were found in a single isolate, Bsb-14, a putative new African 1 variant. The characterization of the LCR of human papillomaviruses can be of pivotal importance to the understanding of the viral replication and pathogenicity.

The present study on genetic diversity of human papillomaviruses in women infected by HIV in Brazil describes the frequency, the genotypes, and five new variants of HPV. One hundred fifty cervical smears of HIV-positive women were subjected to cytological examination, and the DNA samples obtained were assayed by MY09/MY11 amplification, followed by RFLP typing. The overall HPV-DNA-positive rate was 42.7%. One hundred twenty-two samples (81.3%) had benign cellular alterations or normal cytological results, and HPV DNA frequency among them was 30.3%. Otherwise, 96.4% of samples with altered cytology were positive for HPV DNA. A high diversity of genotypes was observed. HPVs-16 and 81 were the most prevalent (14.1%) and were followed by HPVs 52, 35, 62, 33, 53, 56, 66, 70, 18, 58, 6b, 11, 31, 39, 40, 61, 71, 32, 54, 59, 67, 68, 85, and 102. Five new variants of the high-risk HPVs 18, 33, 53, 59, and 66 were detected. Possible associations between the detection of HPV genotypes and the cytological classification, HIV viral load, CD4 count, and antiretroviral treatment were also examined. We observed that a high proportion of HIV-infected women are infected with HPV and may carry oncogenic genotypes, even when cytological evaluation shows normal results.

In this work, 22 alcoholic extracts, obtained from 14 species of plants belonging to four families, used for different food and medicinal purposes in Brazil, were evaluated for their capacity to inhibit the reduction of the free radical, 1,1-diphenyl-2-picrylhydrazyl (DPPH), and to protect Saccharomyces cerevisiae cells, an eukaryotic cell model, against the lethal oxidative stress caused by tert-butylhydroperoxide (TBH). Five extracts, two from Lamiaceae family (ethanol and butanol extracts from aerial parts of Hyptis fasciculata) and three from Palmae family (Copernicia cerifera leaves and mesocarp of fruits and the endocarp/mesocarp of fruits from Orbignya speciosa) were able to increase the tolerance of S. cerevisiae to TBH and showed to be active as DPPH radical scavengers, thus indicating that these plant extracts could be considered as potential sources of antioxidants. With the exception of ethanol extract of H. fasciculata, the remainder four extracts exhibited a DPPH radical scavenging activity higher than that obtained from Ginkgo biloba, a reference plant with well documented antioxidant activity. Interestingly, the ethanol extract of G. biloba were not effective for yeast cell protection, reinforcing the antioxidant potential of these extracts.

The present study on molecular characterization of a human papillomavirus (HPV) isolated in Central Brazil describes the L1 gene sequence from a new variant of HPV-58, the isolate Bsb-02. The sample was from a smear obtained from a woman with cervical intraepithelial neoplasia grade II. The whole L1 gene from isolate Bsb-02 was sequenced automatically, showing 99.1% nucleotide identity with the gene from the HPV-58 reference. The clustering between Bsb-02 and HPV-58 reference sequence was also supported by phylogenetic analysis. Fourteen nucleotide substitutions were observed: eight were synonymous and six were associated with amino acid substitutions. A10V and V144I have not been previously described. At GenBank, the only complete L1 sequence from HPV-58 in addition to the HPV-58 reference one is that of Bsb-02. These data provide information that may be relevant to HPV diagnosis and to rational vaccine strategies. HPV variants may also be associated with host immune responses and with the risk of cervical neoplasia.

The objective of this study was to characterize hepatitis C virus (HCV) genotypes in blood donors from the Federal District, Central Brazil, and to compare HCV screening by serological assays and reverse transcriptase polymerase chain reaction (RT-PCR). Plasma samples from 57 individuals with reactive or indeterminate results in serological anti-HCV screening assays (ELISA or EIA) were tested for HCV RNA by RT-PCR. The results from a confirmatory LIA serological assay were also evaluated. The 5' non-coding region of the HCV genome was amplified from 41 PCR positive samples (71.9%), which were further characterized by nucleotide sequencing analysis. Of these, 60.9% were of HCV genotype 1 and 39.1% of genotype 3.

In the context of universal access to antiretroviral therapy, the surveillance of human immunodeficiency virus type 1 (HIV-1) genetic diversity and resistance becomes pivotal. In this work our purpose was to describe the genetic variability; prevalence of drug-resistance mutations; and genotypic resistance profiles in HIV-1 infected individuals under antiretroviral treatment, from the Federal District, Brasilia, Central Brazil. The entire viral protease and codons 19 to 234 of the reverse transcriptase gene from 45 HIV-1 isolates were amplified and sequenced for subtyping and genotyping. By phylogenetic analysis, 96% of the samples clustered with subtype B and the remaining 4% with HIV-1 subtype F sequences. One major protease inhibitor resistance-associated mutation, I50V, was detected in 38% of the samples. Minor mutations were also found at the protease gene: L10I/V (7%), K20M (2%), M36I (11%), L63P (20%), A71T (2%), and V77I (7%). Many mutations associated with reduced susceptibility to nucleoside or non-nucleoside reverse transcriptase inhibitors were detected: M41L (11%), E44D (4%), D67N (11%), T69D (2%), K70R (11%), L74V (2%), L100I (4%), K103N (18%), V118I (9%), Y181C (11%), M184V (18%), G190A (4%), T215Y (4%), and K219E (4%). This study has shown that 84% of the studied population from the Federal District, showing evidences of therapy failure, presented viral genomic mutations associated with drug resistance. The main antiretrovirals to which this population showed resistance were the PI amprenavir (38%), the NNRTIs delavirdine, nevirapine (31%), and efavirenz (24%), and the NRTIs lamivudine (18%), abacavir, and zidovudine (13%).

We report the prevalence of human papillomavirus type 16 (HPV-16) variants in women with cervical lesions from the Federal District, Central Brazil. We analyzed 34 HPV-16 samples, identifying the sequence variations of E6 and L1 genes and correlating variant frequency with disease status. The most prevalent HPV-16 variant was the European (50%), followed by Asian-American (41.2%), African-1 (5.9%), and African-2 (2.9%). European and non-European variants appeared in equal frequencies among the cytological types of lesions - atypical squamous or glandular cells of undetermined significance, cytological alterations suggesting HPV infection, cervical intraepithelial neoplasias, squamous cell carcinoma, and adenocarcinoma.

As a contribution to the public health authorities in planning prophylactic and therapeutic vaccine strategies, we describe the prevalence of human papillomavirus (HPV) types in women presenting abnormal cytological results in Pap smear screening tests in the Federal District, Central Brazil. We studied 129 cervical scraping samples from women whose cytological tests showed either pre-neoplastic or neoplastic lesions. Amplification of HPV DNA was performed by polymerase chain reaction using consensus primers MY09 and MY11 followed by identification of isolates by restriction fragment length polymorphism. We detected HPV DNA in 62% of the samples, including HPV-16 in 43.8%, HPV-58 in 12.5%, HPV-31 in 10%, HPV-53 in 6.3%, each of HPV-18 and HPV-33 in 3.8% of the isolates. Other types (HPV-35, -52, -66, -CP8304, -6, -11, and -CP8061) were less frequent (= or < 2.5% each). The prevalence of HPV-58 was relatively higher in this population than in data in South America, but similar to results obtained in other studies in Latin America, Europe, and Eastern Asia. Case-control studies need to be carried out to establish the association between the prevalence of HPV types specially the less frequent high-risk types and cervical cancer.

The present study on molecular characterization of human papillomaviruses occurring in Central Brazil, describes two variants each of HPV-53 and HPV-58 and one variant of HPV-66 detected in samples from smears of women showing cervical intraepithelial neoplasia grade II (CIN II). Samples were assayed by PCR using MY09/ MY11 consensus primers, followed by restriction fragment length polymorphism typing. The five isolates showed atypical restriction fragment length profile and MY09/MY11 L1 PCR products were subsequently sequenced. Isolate Bsb-02 and Bsb-08 showed, respectively, 99% similarity to HPV-58 IS404 and 100% to HPV-58 IS417 previously described in the African Continent. Isolates Bsb-61 and Bsb-63 showed 98% similarity to HPV-53, and isolate Bsb-68, 97% similarity to HPV-66. Amino acid substitutions were found in two samples: one in Bsb-02 (T to N) at position 375 and the other in Bsb-61 (S to C) at position 343. Although all the substitutions in Bsb-68 proved to be silent, this sample showed the highest value of pairwise evolutionary distance (2.05%). In countries such as Brazil, where the virus prevalence is high and ethnicity, as well as socio-demographic characteristics, vary according to different regions, HPV variability must be wider and not yet clearly defined.