Amphibians and reptiles from a protected area in western Brazilian Amazonia (Reserva Extrativista do Baixo Juruá)

Sampling gaps across the logistically challenging and extremely biodiverse Amazonia largely hamper our understanding of broad-scale amphibian and reptile diversity patterns in this ecosystem. The Juruá River basin, a southwestern tributary of the Amazon River, is one of these undersampled areas, with only punctual information documented for these vertebrates that are spatially or temporally biased. This is especially the case for the lower-middle courses of the Juruá River, which also has comparatively less protected areas than its upper course. In order to fill some biodiversity knowledge gaps associated with amphibians and reptiles in this river basin, we combined results of our field expeditions carried out in 1992, 2005-2007, and 2018 to the Reserva Extrativista (Extractive Reserve) do Baixo Juruá, a Brazilian protected area in the right bank of the lower Juruá River. Amphibians and reptiles were sampled using four complementary methods: active surveys, pitfall traps, funnel traps, and trammel nets. We identified species or updated their taxonomic status with a reanalysis of the external morphology of the preserved material in the light of novel taxonomic literature (more than 1,500 specimens) and employment of DNA barcoding analyses for some newly collected specimens with contentious taxonomic status. Our combined sampling evidenced 149 species of amphibians and reptiles occurring in this protected area (72 amphibians, 68 squamates, six chelonians, and three crocodilians). Recorded species highlight the value of the lower Juruá River region as harboring quite diverse assemblages for these vertebrates, with species typical of the western and southwestern Amazonia sub-regions. Remarkable species records are presented, as well as accounts on species with lower taxonomic resolution. Furthermore, we discuss the biogeographic affinities of recorded assemblages based on the species geographic range and preferred habitats, and the value of this protected area to preserve the regional biological diversity.(AU)

Corneal structure and endothelial morphological changes after uneventful phacoemulsification in type 2 diabetic and nondiabetic patients / Alterações da estrutura e endotélio da cornea após facoemulsificação sem complicações em pacientes diabéticos tipo 2 e não diabéticos

ABSTRACT Purpose: The aim of this study was to compare corneal structure and endothelial morphological changes after uneventful phacoemulsification cataract surgery between type 2 diabetic and nondiabetic patients and to determine the preoperative and intraoperative factors that may predict greater endothelial cell density loss. Methods: Forty-five diabetic pa­tients (45 eyes) and 43 controls (43 eyes) with age-related cataract were enrolled in this prospective observational study. Corneal (thickness and volume) and anterior segment parameters were measured by Scheimpflug tomography; endothelial cell density and morphology (coefficient of variation of cell size, hexagonal cells) were recorded using noncontact specular microscopy. Patients were evaluated preoperatively and at one and six months after surgery. Univariate and multivariate linear regression analyses were performed to evaluate the relationship between demographic, clinical, ocular, and intraoperative parameters and postoperative endothelial cell density changes at six months. Results: Significant postoperative endothelial cell loss occurred one month after surgery in both groups (p<0.001), which remained stable until month 6; there were no differences between patients with and without diabetes mellitus at any time point. The mean postoperative central corneal thickness at one and six months did not change significantly from the mean preoperative value in either group (p>0.05). Multivariate linear regression analysis showed that older age (p=0.042) and higher cataract grades (p=0.001) were significantly associated with greater endothelial cell density reduction at six-month follow-up. Conclusion: This study showed that older age and denser cataracts might be associated with greater endothelial cell density reduction after cataract surgery. Other factors, such as diabetes mellitus and preoperative anterior segment parameters, did not influence postoperative changes in endothelial cell density.

RESUMO Objetivo: Comparar a estrutura da córnea e as alterações mor­fológicas endoteliais após cirurgia de catarata por facoemulsificação sem intercorrências entre pacientes com diabetes mellitus tipo 2 e não diabéticos; e determinar quais fatores pré e intra-operatórios relacionados com a maior redução da densidade celular endotelial. Métodos: Quarenta e cinco diabéticos (45 olhos) e 43 (43 olhos) controlos com catarata relacionada à idade foram incluídos neste estudo observacional prospectivo. Os parâmetros da córnea (espessura e volume) e do segmento anterior foram medidos pela tomografia Scheimpflug; a densidade e morfologia celular endotelial (coeficiente de variação do tamanho das células, células hexagonais) foram registrados usando microscopia especular não contato. Os pacientes foram avaliados no pré-operatório, 1 e 6 meses após a cirurgia. Foi realizada uma análise de regressão linear uni e multivariada para avaliar a relação entre os parâmetros demográficos, clínicos, oculares e intra-operatórios com a redução da densidade celular endotelial aos 6 meses. Resultados: Nos dois grupos houve uma perda significativa de células endoteliais ao 1º mês pós-ope­ratório (p<0,001), que permaneceu estável até ao 6º mês; sem diferenças estatisticas entre os grupos diabetes mellitus e não diabetes mellitus em qualquer avaliação. A espessura média da córnea no pós-operatório central aos 1 e 6 meses não mudou significativamente em relação ao valor médio pré-operatório nos dois grupos (p>0.05). A análise de regressão multivariada linear mostrou que a idade avançada (p=0.042) e os graus mais elevados de catarata (p=0.001) foram significativamente associados à maior redução densidade celular endotelial aos 6 meses de seguimento. Conclusão: Este estudo mostrou que a idade avançada e as cataratas mais densas podem predispor a uma maior redução densidade celular endotelial após a cirurgia de catarata. Outros fatores, como diabetes mellitus e parâmetros pré-operatórios do segmento anterior, não influenciaram significativamente as alterações pós-operatórias da densidade celular endotelial.

Clotrimazole presents anticancer properties against a mouse melanoma model acting as a PI3K inhibitor and inducing repolarization of tumor-associated macrophages.

The immune system is a key component of tumorigenesis, with the latter promoting the development of cancer, its progression and metastasis. In fact, abundant infiltration of tumor-associated macrophages (TAM), which are M2-like macrophages, has been associated with a poor outcome in most types of cancers. Here, we show that lactate produced by murine melanoma B16F10 cells induces an M2-like profile in cultured macrophages. Further, we demonstrate that clotrimazole (CTZ), an off-target anti-tumor drug, abolishes lactate effects on the activation of macrophages and induces the expression of M1-like markers. We show that clotrimazole has cytotoxic effects on tumor cells by negatively modulating PI3K, which inhibits glycolytic metabolism and leads to a diminishing lactate production by these cells. These effects are more pronounced in cancer cells exposed to conditioned media of M2-polarized macrophages. Moreover, clotrimazole inhibits tumor growth in a murine model of implanted melanoma, reduces lactate content in a tumor microenvironment and decreases vascular endothelial growth factor expression. Finally, clotrimazole drastically diminishes TAM infiltration in the tumors, thereby inducing M1 polarization. Collectively, these findings identify a new antitumor mechanism of clotrimazole by modulating the tumor microenvironment (TME), particularly the activation and viability of TAM.

Corneal structure and endothelial morphological changes after uneventful phacoemulsification in type 2 diabetic and nondiabetic patients.

PURPOSE: The aim of this study was to compare corneal structure and endothelial morphological changes after uneventful phacoemulsification cataract surgery between type 2 diabetic and nondiabetic patients and to determine the preoperative and intraoperative factors that may predict greater endothelial cell density loss. METHODS: Forty-five diabetic pa-tients (45 eyes) and 43 controls (43 eyes) with age-related cataract were enrolled in this prospective observational study. Corneal (thickness and volume) and anterior segment parameters were measured by Scheimpflug tomography; endothelial cell density and morphology (coefficient of variation of cell size, hexagonal cells) were recorded using noncontact specular microscopy. Patients were evaluated preoperatively and at one and six months after surgery. Univariate and multivariate linear regression analyses were performed to evaluate the relationship between demographic, clinical, ocular, and intraoperative parameters and postoperative endothelial cell density changes at six months. RESULTS: Significant postoperative endothelial cell loss occurred one month after surgery in both groups (p<0.001), which remained stable until month 6; there were no differences between patients with and without diabetes mellitus at any time point. The mean postoperative central corneal thickness at one and six months did not change significantly from the mean preoperative value in either group (p>0.05). Multivariate linear regression analysis showed that older age (p=0.042) and higher cataract grades (p=0.001) were significantly associated with greater endothelial cell density reduction at six-month follow-up. CONCLUSION: This study showed that older age and denser cataracts might be associated with greater endothelial cell density reduction after cataract surgery. Other factors, such as diabetes mellitus and preoperative anterior segment parameters, did not influence postoperative changes in endothelial cell density.

Dietary citrate acutely induces insulin resistance and markers of liver inflammation in mice.

Citrate is widely used as a food additive being part of virtually all processed foods. Although considered inert by most of the regulatory agencies in the world, plasma citrate has been proposed to play immunometabolic functions in multiple tissues through altering a plethora of cellular pathways. Here, we used a short-term alimentary intervention (24 hours) with standard chow supplemented with citrate in amount corresponding to that found in processed foods to evaluate its effects on glucose homeostasis and liver physiology in C57BL/6J mice. Animals supplemented with dietary citrate showed glucose intolerance and insulin resistance as revealed by glucose and insulin tolerance tests. Moreover, animals supplemented with citrate in their food displayed fed and fasted hyperinsulinemia and enhanced insulin secretion during an oral glucose tolerance test. Citrate treatment also amplified glucose-induced insulin secretion in vitro in INS1-E cells. Citrate supplemented animals had increased liver PKCα activity and altered phosphorylation at serine or threonine residues of components of insulin signaling including IRS-1, Akt, GSK-3 and FoxO1. Furthermore, citrate supplementation enhanced the hepatic expression of lipogenic genes suggesting increased de novo lipogenesis, a finding that was reproduced after citrate treatment of hepatic FAO cells. Finally, liver inflammation markers were higher in citrate supplemented animals. Overall, the results demonstrate that dietary citrate supplementation in mice causes hyperinsulinemia and insulin resistance both in vivo and in vitro, and therefore call for a note of caution on the use of citrate as a food additive given its potential role in metabolic dysregulation.

Western diet leads to aging-related tumorigenesis via activation of the inflammatory, UPR, and EMT pathways.

Among the principal causative factors for the development of complications related to aging is a diet rich in fats and sugars, also known as the Western diet. This diet advocates numerous changes that might increase the susceptibility to initiate cancer and/or to create a tissue microenvironment more conducive to the growth of malignant cells, thus favoring the progression of cancer and metastasis. Hypercaloric diets in general lead to oxidative stress generating reactive oxygen species and induce endoplasmic reticulum stress. Our results demonstrate that mice bearing tumors fed with a Western diet presented bigger tumor mass with increased insulin sensitivity in these tissues. Several markers of insulin signaling, such as AKT phosphorylation and mTOR pathway, are promoted in tumors of Western diet-fed animals. This process is associated with increased macrophage infiltration, activation of unfolded protein response pathway, and initiation of epithelial-mesenchymal transition (EMT) process in these tumor tissues. Summing up, we propose that the Western diet accelerates the aging-related processes favoring tumor development.

Acetylsalicylic acid and salicylic acid present anticancer properties against melanoma by promoting nitric oxide-dependent endoplasmic reticulum stress and apoptosis.

Melanoma is the most aggressive and fatal type of skin cancer due to being highly proliferative. Acetylsalicylic acid (ASA; Aspirin) and salicylic acid (SA) are ancient drugs with multiple applications in medicine. Here, we showed that ASA and SA present anticancer effects against a murine model of implanted melanoma. These effects were also validated in 3D- and 2D-cultured melanoma B16F10 cells, where the drugs promoted pro-apoptotic effects. In both in vivo and in vitro models, SA and ASA triggered endoplasmic reticulum (ER) stress, which culminates with the upregulation of the pro-apoptotic transcription factor C/EBP homologous protein (CHOP). These effects are initiated by ASA/SA-triggered Akt/mTOR/AMPK-dependent activation of nitric oxide synthase 3 (eNOS), which increases nitric oxide and reactive oxygen species production inducing ER stress response. In the end, we propose that ASA and SA instigate anticancer effects by a novel mechanism, the activation of ER stress.

A new nurse frog of Allobates masniger-nidicola complex (Anura, Aromobatidae) from the east bank of Tapajós River, eastern Amazonia.

The small nurse frogs of the genus Allobates (Anura, Aromobatidae) represent one of the most challenging taxonomic issues of the Neotropics. During several amphibian surveys in the Middle Tapajós River region, state of Pará, Brazil, we collected phenotypic, ecological, and molecular data on species of this genus, leading to the identification of a new species included in the Allobates masniger-nidicola complex. The new species is characterized by a large body size (snout-vent length 19.2-21.7 mm in males and 19.3-22.0 mm in females), finger III not swollen in adult males; cryptic external coloration, with dorsum uniformly ochre; a dark brown lateral stripe and a pale cream ventrolateral stripe; limbs ranging from ochre to orange; throat and chest violaceous in males and yellowish in females. The advertisement call is usually arranged in bouts of four closely spaced notes, which we term 4-pulsed units of repetition (UR), 0.317 s long on average, followed by silent intervals, and an average dominant frequency of 4.163 kHz. The new species also has exotrophic tadpoles with a unique fin morphology, which begins after the body-tail insertion and is deeper posteriorly to half of the caudal length. Sequencing of the 16S and COI regions of the mitochondrial DNA show a genetic p-distance of approximately 6-10% compared to closely related congeners. We discuss the biogeography of the new species based on phylogenetic relationships of the species within the Allobates masniger-nidicola complex and the allopatric geographic distribution in relation to sister taxa. Functional characteristics and geographic restrictions make this species particularly sensitive to the increasing human impact in eastern Amazonia.

A Novel Triazole Derivative Drug Presenting In Vitro and In Vivo Anticancer Properties.

BACKGROUND: Cancer is a major cause of death worldwide, despite many different drugs available to treat the disease. This high mortality rate is largely due to the complexity of the disease, which results from several genetic and epigenetic changes. Therefore, researchers are constantly searching for novel drugs that can target different and multiple aspects of cancer. EXPERIMENTAL: After a screening, we selected one novel molecule, out of ninety-four triazole derivatives, that strongly affects the viability and proliferation of the human breast cancer cell line MCF-7, with minimal effects on non-cancer cells. The drug, named DAN94, induced a dose-dependent decrease in MCF-7 cells viability, with an IC50 of 3.2 ± 0.2 µM. Additionally, DAN94 interfered with mitochondria metabolism promoting reactive oxygen species production, triggering apoptosis and arresting the cancer cells on G1/G0 phase of cell cycle, inhibiting cell proliferation. These effects are not observed when the drug was tested in the non-cancer cell line MCF10A. Using a mouse model with xenograft tumor implants, the drug preventing tumor growth presented no toxicity for the animal and without altering biochemical markers of hepatic function. RESULTS AND CONCLUSION: The novel drug DAN94 is selective for cancer cells, targeting the mitochondrial metabolism, which culminates in the cancer cell death. In the end, DAN94 has been shown to be a promising drug for controlling breast cancer with minimal undesirable effects.

Effects of Food Additives on Immune Cells As Contributors to Body Weight Gain and Immune-Mediated Metabolic Dysregulation.

Food additives are compounds used in order to improve food palatability, texture, and shelf life. Despite a significant effort to assure safety of use, toxicological analysis of these substances, generally, rely on their direct toxicity to target organs (liver and kidney) or their genotoxic effects. Much less attention is paid to the effects of these compounds on cells of the immune system. This is of relevance given that metabolic dysregulation and obesity have a strong immune-mediated component. Obese individuals present a state of chronic low-grade inflammation that contributes to the establishment of insulin resistance and other metabolic abnormalities known as the metabolic syndrome. Obesity and metabolic syndrome are currently recognized as worldwide epidemics that pose a profound socioeconomic impact and represent a concern to public health. Cells of the immune system contribute to both the maintenance of "lean homeostasis" and the metabolic dysregulation observed in obese individuals. Although much attention has been drawn in the past decades to obesity and metabolic syndrome as a result of ingesting highly processed food containing large amounts of fat and simple sugars, mounting evidence suggest that food additives may also be important contributors to metabolic derangement. Herein, we review pieces of evidence from the literature showing that food additives have relevant effects on cells of the immune system that could contribute to immune-mediated metabolic dysregulation. Considering their potential to predispose individuals to develop obesity and metabolic syndrome, their use should be taken with caution or maybe revisited.

Reference genes for quantitative PCR in the adipose tissue of mice with metabolic disease.

Obesity and diabetes are metabolic diseases and they are increasing in prevalence. The dynamics of gene expression associated with these diseases is fundamental to identifying genes involved in related biological processes. qPCR is a sensitive technique for mRNA quantification and the most commonly used method in gene-expression studies. However, the reliability of these results is directly influenced by data normalization. As reference genes are the major normalization method used, this work aims to identify reference genes for qPCR in adipose tissues of mice with type-I diabetes or obesity. We selected 12 genes that are commonly used as reference genes. The expression of these genes in the adipose tissues of mice was analyzed in the context of three different experimental protocols: 1) untreated animals; 2) high-fat-diet animals; and 3) streptozotocin-treated animals. Gene-expression stability was analyzed using four different algorithms. Our data indicate that TATA-binding protein is stably expressed across adipose tissues in control animals. This gene was also a useful reference when the brown adipose tissues of control and obese mice were analyzed. The mitochondrial ATP synthase F1 complex gene exhibits stable expression in subcutaneous and perigonadal adipose tissue from control and obese mice. Moreover, this gene is the best reference for qPCR normalization in adipose tissue from streptozotocin-treated animals. These results show that there is no perfect stable gene suited for use under all experimental conditions. In conclusion, the selection of appropriate genes is a prerequisite to ensure qPCR reliability and must be performed separately for different experimental protocols.

Exogenous citrate impairs glucose tolerance and promotes visceral adipose tissue inflammation in mice.

Overweight and obesity have become epidemic worldwide and are linked to sedentary lifestyle and the consumption of processed foods and drinks. Citrate is a metabolite that plays central roles in carbohydrate and lipid metabolism. In addition, citrate is the additive most commonly used by the food industry, and therefore is highly consumed. Extracellular citrate can freely enter the cells via the constitutively expressed plasma membrane citrate transporter. Within the cytosol, citrate is readily metabolised by ATP-citrate lyase into acetyl-CoA - the metabolic precursor of endogenously produced lipids and cholesterol. We therefore hypothesised that the citrate ingested from processed foods and drinks could contribute to increased postprandial fat production and weight gain. To test our hypothesis, we administered citrate to mice through their drinking water with or without sucrose and monitored their weight gain and other metabolic parameters. Our results showed that mice receiving citrate or citrate+sucrose did not show increased weight gain or an increase in the weight of the liver, skeletal muscles or adipose tissues (AT). Moreover, the plasma lipid profiles (TAG, total cholesterol, LDL and HDL) were similar across all groups. However, the group receiving citrate+sucrose showed augmented fasting glycaemia, glucose intolerance and the expression of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6 and IL-10) in their AT. Therefore, our results suggest that citrate consumption contributes to increased AT inflammation and altered glucose metabolism, which is indicative of initial insulin resistance. Thus, citrate consumption could be a previously unknown causative agent for the complications associated with obesity.