The Role of Trypan Blue as a Conventional and Fluorescent Dye for the Diagnosis of Superficial Mycoses by Potassium Hydroxide (KOH) Testing.

Direct microscopic examination of samples using potassium hydroxide (KOH) is a fast, simple, and inexpensive method to confirm clinical suspicion of superficial mycosis. However, the lack of color contrast in this test makes it difficult to separate any fungal structures from artifacts. The sensitivity of the KOH mount technique may be enhanced using both fluorochromes and conventional stains that highlight the fungal structures when observed under fluorescence microscopy and bright-field, respectively. Here we study the possibility of using Trypan Blue (TB), an azo dye which is often used as a live/dead marker, in the diagnosis of superficial mycoses by KOH testing. TB at 0.01% displayed a fluorescent staining pattern similar to that of Calcofluor White (CFW), the conventional cell wall fluorophore. Furthermore, by adjusting the TB concentration to 0.1-0.3%, in addition to maintaining the fluorescent staining pattern, the fungal elements were stained in blue under bright-field microscopy. Thus, we demonstrate for the first time that TB has the unique property as a fungal stain that can be used in both bright-field and fluorescence microscopy for diagnosis of superficial mycoses by direct microscopic examination.

Effects of diabetes mellitus on myenteric neuronal density and sodium channel expression in the rat ileum.

Diabetes mellitus (DM) may lead to gastrointestinal motility disorders. Rodent models of DM indicate the presence of morpho-functional abnormalities of the enteric nervous system. Here, we evaluated whether experimental DM can cause changes in the excitatory cholinergic fibers, neuronal density, and voltage-gated sodium channel (Nav) expression in the myenteric plexus of the ileum. After streptozotocin-induced hyperglycemia in female rats progressed for eight weeks, triple immunofluorescence labeling experiments revealed that the neuronal density in DM rats was significantly lower than that in control. On average, the density of total neurons reduced by 52.2% (p = 0.0001), cholinergic neurons by 50.0% (p = 0.0068), and nitrergic neurons by 54.8% (p = 0.0042). The number of neurons per ganglionic area was also significantly reduced (to 28.2% of total neurons, p = 0.0002; 27.7% of cholinergic neurons, p = 0.0002, and 32.1% of nitrergic neurons, p = 0.0016). Furthermore, the density of the cholinergic fibers at the surface of the longitudinal muscle was significantly reduced (DM: 24 ±â€¯3%; p = 0.003, control: 41 ±â€¯2%); however, western-blot analysis did not indicate a reduction in the expression of choline acetyltransferase (ChAT) in the DM group. The Nav1.6 isoform was detected in different myenteric neurons of the ileum. RT-qPCR data did not suggest an alteration of transcripts for ChAT, neuronal nitric oxide synthase, Nav1.3, Nav1.6, or Nav1.7. Our data support the view that chronic DM leads to a reduction of excitatory cholinergic fibers and neuronal density. However, changes in sodium channel expression pattern, which could cause neuronal dysfunction, were not detected.

Selective human inhibitors of ATR and ATM render Leishmania major promastigotes sensitive to oxidative damage.

All cellular processes, including those involved in normal cell metabolism to those responsible for cell proliferation or death, are finely controlled by cell signaling pathways, whose core proteins constitute the family of phosphatidylinositol 3-kinase-related kinases (PIKKs). Ataxia Telangiectasia Mutated (ATM) and Ataxia Telangiectasia and Rad3 related (ATR) are two important PIKK proteins that act in response to DNA damage, phosphorylating a large number of proteins to exert control over genomic integrity. The genus Leishmania belongs to a group of early divergent eukaryotes in evolution and has a highly plastic genome, probably owing to the existence of signaling pathways designed to maintain genomic integrity. The objective of this study was to evaluate the use of specific human inhibitors of ATR and ATM in Leishmania major. Bioinformatic analyses revealed the existence of the putative PIKK genes ATR and ATM, in addition to mTOR and DNA-PKcs in Leishmania spp. Moreover, it was possible to suggest that the inhibitors VE-821 and KU-55933 have binding affinity for the catalytic sites of putative L. major ATR and ATM, respectively. Promastigotes of L. major exposed to these inhibitors show slight growth impairment and minor changes in cell cycle and morphology. It is noteworthy that treatment of promastigotes with inhibitors VE-821 and KU-55933 enhanced the oxidative damage caused by hydrogen peroxide. These inhibitors could significantly reduce the number of surviving L. major cells following H2O2 exposure whilst also decreasing their evaluated IC50 to H2O2 to less than half of that observed for non-treated cells. These results suggest that the use of specific inhibitors of ATR and ATM in Leishmania interferes in the signaling pathways of this parasite, which can impair its tolerance to DNA damage and affect its genome integrity. ATR and ATM could constitute novel targets for drug development and/or repositioning for treatment of leishmaniases.

Biological properties of cardiac mesenchymal stem cells in rats with diabetic cardiomyopathy.

Cardiomyopathy is a major outcome in patients with diabetes mellitus (DM) and contributes to the high morbidity/mortality observed in this disease. AIMS: To evaluate several biological properties of cardiac mesenchymal stem cells (cMSCs) in a rat model of streptozotocin-induced DM with concomitant diabetic cardiomyopathy. MAIN METHODS: After 10weeks of DM induction, diabetic and control rats were assessed using ECG and ventricular hemodynamics monitoring. Then, the hearts were excised and processed for histology and for extracting non-cardiomyocytic cells. A pool of these cells was plated for a colony forming units-fibroblasts (CFU-F) assay in order to estimate the number of cMSCs. The remaining cells were expanded to assess their proliferation rate as well as their osteogenic and adipogenic differentiation ability. KEY FINDINGS: DM rats presented intense hyperglycemia and changes in ECG, LV hemodynamic, cardiac mass index and fibrosis, indicating presence of DCM. The CFU-F assay revealed a higher number of cardiac CFU-Fs in DM rats (10.4±1.1CFU-F/105 total cells versus 7.6±0.7CFU-F/105 total cells in control rats, p<0.05), which was associated with a significantly higher proliferative rate of cMSCs in DM rats. In contrast, cMSCs from DM rats presented a lower capacity to differentiate into both osteogenic (20.8±4.2% versus 10.1±1.0% in control rats, p<0.05) and adipogenic lineages (4.6±1.0% versus 1.3±0.5% in control rats, p<0.05). SIGNIFICANCE: The findings suggest, for the first time, that in chronic DM rats with overt DCM, cMSCs increase in number and exhibit changes in several functional properties, which could be implicated in the pathogenesis of diabetic cardiomyopathy.

Influence of the Expression of Inflammatory Markers on Kidney after Fetal Programming in an Experimental Model of Renal Failure.

Objective. To evaluate the expression of inflammatory markers in experimental renal failure after fetal programming. Methods. The offspring aged two and five months were divided into four groups: CC (control dams, control offspring); DC (diabetic dams, control offspring); CFA (control dams, folic acid offspring, 250 mg/Kg); and DFA (diabetic dams, folic acid offspring). Gene expression of inflammatory markers MCP-1, IL-1, NOS3, TGF-ß, TNF-α, and VEGF was evaluated by RT-PCR. Results. MCP-1 was increased in the CFA and DFA groups at two and five months of age, as well as in DC5 when compared to CC5. There was a higher expression of IL-1 in the CFA2, DFA2, and DC2 groups. There was a decrease in NOS3 and an increase in TNF-α in DFA5 in relation to CFA5. The gene expression of TGF-ß increased in cases that had received folic acid at two and five months, and VEGF decreased in the CFA5 and DFA5 groups. DC5 showed increased VEGF expression in comparison with CC5. Conclusions. Gestational diabetes mellitus and folic acid both change the expression of inflammatory markers, thus demonstrating that the exposure to harmful agents in adulthood has a more severe impact in cases which underwent fetal reprogramming.

Morphological and functional aspects of acute kidney injury after fetal programing in the offspring of diabetic rats.

OBJECTIVE: To evaluate the effects of folic acid (FA)-induced renal failure in young offspring of diabetic mothers. METHODS: The offspring of streptozotocin-induced diabetic dams were divided into four groups: CC (controls receiving vehicle); DC (diabetics receiving vehicle); CA (controls receiving FA solution, 250 mg/kg) and DA (diabetics receiving FA solution, 250 mg/kg). Renal function tests and morphometry results were analyzed. RESULTS: An increase in creatinine and urea levels was observed in CA and DA groups at two and five months. FA administration caused a significant reduction in the number of glomeruli in the offspring of diabetic dams. The diabetes group treated with FA had fewer glomeruli compared to controls at two and five months. FA caused an increase in the area of the urinary space both in controls and offspring of diabetic dams at two and five months. The number of glomeruli and area of the urinary space at two months were negatively correlated. CONCLUSIONS: Fetal programing promotes remarkable changes in kidney morphology and function in offspring. We suggest that the morphological changes in the kidneys are more pronounced when fetal programing is associated with newly acquired diseases, e.g. renal failure induced by FA.