In vitro methods for antifungal susceptibility testing of Trichophyton spp.

In general, methods to test the susceptibility of fungi to antifungal drugs require standardized techniques, but so far there is no methodology that is widely applicable to dermatophytes. Here we introduced modifications to the protocols from documents of the National Committee for Clinical Laboratory Standards (CLSI) M38-A and the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) that are usually applied to moulds and fermentative yeasts, in order to adjust the conditions for the growth of dermatophytes. The modifications included: growth on potato dextrose agar supplemented with 2 % in-house rice flour to encourage sporulation, the addition of 2 % glucose to the culture media (RPMI-1640), and an incubation temperature of 28 °C. In addition, the incubation period was 7 d, the minimum inhibitory concentration (MIC) was defined as 80 % growth inhibition endpoints for azole agents, and the inocula only contained microconidia. Results obtained by both tested methodologies were very similar to the ones reported by other researchers. MIC₉₀ (MIC at which 90% of isolates tested were inhibited) values were identical for four out of five antifungal drugs tested and there was only a difference of one or two dilutions when MIC₅₀ values were compared. Although the modifications introduced did not interfere with the results, more studies are necessary to establish a standard technique to test susceptibility of dermatophytes to antifungal drugs.     

In Silico Insights into Protein-Protein Interactions and Folding Dynamics of the Saposin-Like Domain of Solanum tuberosum Aspartic Protease

The plant-specific insert is an approximately 100-residue domain found exclusively within the C-terminal lobe of some plant aspartic proteases. Structurally, this domain is a member of the saposin-like protein family, and is involved in plant pathogen defense as well as vacuolar targeting of the parent protease molecule. Similar to other members of the saposin-like protein family, most notably saposins A and C, the recently resolved crystal structure of potato (Solanum tuberosum) plant-specific insert has been shown to exist in a substrate-bound open conformation in which the plant-specific insert oligomerizes to form homodimers. In addition to the open structure, a closed conformation also exists having the classic saposin fold of the saposin-like protein family as observed in the crystal structure of barley (Hordeum vulgare L.) plant-specific insert. In the present study, the mechanisms of tertiary and quaternary conformation changes of potato plant-specific insert were investigated in silico as a function of pH. Umbrella sampling and determination of the free energy change of dissociation of the plant-specific insert homodimer revealed that increasing the pH of the system to near physiological levels reduced the free energy barrier to dissociation. Furthermore, principal component analysis was used to characterize conformational changes at both acidic and neutral pH. The results indicated that the plant-specific insert may adopt a tertiary structure similar to the characteristic saposin fold and suggest a potential new structural motif among saposin-like proteins. To our knowledge, this acidified PSI structure presents the first example of an alternative saposin-fold motif for any member of the large and diverse SAPLIP family.     

Mitochondrial Dynamics Protein Drp1 Is Overexpressed in Oncocytic Thyroid Tumors and Regulates Cancer Cell Migration

Oncocytic cell tumors are characterized by the accumulation of morphologically abnormal mitochondria in their cells, suggesting a role for abnormal mitochondrial biogenesis in oncocytic cell transformation. Little is known about the reason for the dysmorphology of accumulated mitochondria. The proteins regulating the morphology of mitochondria, the "mitochondria-shaping" proteins, can modulate their size and number; however, nothing is known hitherto about a possible involvement of mitochondrial dynamics in oncocytic cell transformation in tumors. Our aim was to assess the status of the mitochondria morphology and its role in oncocytic cell transformation. We therefore evaluated the expression pattern of the main mitochondrial fusion and fission proteins in a series of thyroid cell tumor samples, as well as in thyroid tumor cell lines, with and without oncocytic cell features. The expression of mitochondrial fusion (Opa1, Mfn1 and Mfn2) and fission (Drp1 and Fis1) proteins were evaluated by immunohistochemistry (IHC) in a series of 88 human thyroid tumors. In vitro studies, for comparative purposes and to deepen the study, were performed using TPC1 - a papillary thyroid carcinoma derived cell line—and XTC.UC1, an oncocytic follicular thyroid carcinoma-derived cell line. Both IHC and in vitro protein analyses showed an overall increase in the levels of "mitochondrial-shaping" proteins in oncocytic thyroid tumors. Furthermore, overexpression of the pro-fission protein Drp1 was found to be associated with malignant oncocytic thyroid tumors. Interestingly, genetic and pharmacological blockage of Drp1 activity was able to influence thyroid cancer cells’ migration/invasion ability, a feature of tumor malignancy. In this study we show that unbalanced mitochondrial dynamics characterize the malignant features of thyroid oncocytic cell tumors, and participate in the acquisition of the migrating phenotype.     

Connective-Tissue Growth Factor (CTGF/CCN2) Induces Astrogenesis and Fibronectin Expression of Embryonic Neural Cells In Vitro

Connective-tissue growth factor (CTGF) is a modular secreted protein implicated in multiple cellular events such as chondrogenesis, skeletogenesis, angiogenesis and wound healing. CTGF contains four different structural modules. This modular organization is characteristic of members of the CCN family. The acronym was derived from the first three members discovered, cysteine-rich 61 (CYR61), CTGF and nephroblastoma overexpressed (NOV). CTGF is implicated as a mediator of important cell processes such as adhesion, migration, proliferation and differentiation. Extensive data have shown that CTGF interacts particularly with the TGFβ, WNT and MAPK signaling pathways. The capacity of CTGF to interact with different growth factors lends it an important role during early and late development, especially in the anterior region of the embryo. ctgf knockout mice have several cranio-facial defects, and the skeletal system is also greatly affected due to an impairment of the vascular-system development during chondrogenesis. This study, for the first time, indicated that CTGF is a potent inductor of gliogenesis during development. Our results showed that in vitro addition of recombinant CTGF protein to an embryonic mouse neural precursor cell culture increased the number of GFAP- and GFAP/Nestin-positive cells. Surprisingly, CTGF also increased the number of Sox2-positive cells. Moreover, this induction seemed not to involve cell proliferation. In addition, exogenous CTGF activated p44/42 but not p38 or JNK MAPK signaling, and increased the expression and deposition of the fibronectin extracellular matrix protein. Finally, CTGF was also able to induce GFAP as well as Nestin expression in a human malignant glioma stem cell line, suggesting a possible role in the differentiation process of gliomas. These results implicate ctgf as a key gene for astrogenesis during development, and suggest that its mechanism may involve activation of p44/42 MAPK signaling. Additionally, CTGF-induced differentiation of glioblastoma stem cells into a less-tumorigenic state could increase the chances of successful intervention, since differentiated cells are more vulnerable to cancer treatments.     

Leptin and prolactin, but not corticosterone, modulate body weight and thyroid function in protein-malnourished lactating rats.

To understand the role of hormonal changes in the lower food ingestion and body weight in protein-restricted lactating rats as well as the higher serum T (3), higher deiodination, iodide and T (3) milk transfer, we measured maternal serum prolactin, leptin, TSH and corticosterone, which are hormones that could influence those parameters. After birth, dams were separated into: control-fed with a 23 % protein diet (n = 12) and PR (protein-restricted)-fed with an 8 % protein diet (n = 12). At the 4 (th) and 21 (st) day of lactation, half of the animals in each group were sacrificed. PR dams presented hyperleptinemia (day 4: + 20 %; day 21: + 19 %; p < 0.05) and hypoprolactinemia (day 4: - 85 %; day 21: - 92 %; p < 0.05), which could help explain the lower food consumption and body weight in lactating PR rats since leptin is anorexigenic and prolactin is orexigenic. Also, this hyperleptinemia could contribute for the increase in serum T (3) of PR dams, since leptin stimulates T (3) production, especially acting on deiodinases. Serum corticosterone was not different between PR and C groups, and TSH was lower only at the end of lactation. Thus, we suggest that both leptin and prolactin could play an important role in the body weight and thyroid hormone changes observed in protein-malnourished lactating rats.     

Population proteomics: an emerging discipline to study metapopulation ecology

Proteomics research has developed until recently in a relative isolation from other fast-moving disciplines such as ecology and evolution. This is unfortunate since applying proteomics to these disciplines has apparently the potential to open new perspectives. The huge majority of species indeed exhibit over their entire geographic range a metapopulation structure, occupying habitats that are fragmented and heterogeneous in space and/or through time. Traditionally, population genetics is the main tool used to studying metatopulations, as it describes the spatial structure of populations and the level of gene flow between them. In this Viewpoint, we present the reasons why we think that proteomics, because of the level of integration it promotes, has the potential to resolve interesting issues specific to metapopulation biology and adaptive processes.     

NMR redox studies of Desulfovibrio vulgaris Cytochrome c3. Electron transfer mechanisms

The 300-MHz proton NMR spectra of the tetrahaem cytochrome c3 from Desulfovibrio vulgaris were examined while varying the pH and the redox potential. The analysis of the complete NMR reoxidation pattern was done taking into account all the 16 redox states that can be present in the redox titration of a tetra-redox-center molecule. A network of saturation transfer experiments performed at different oxidation stages, between the fully reduced and the fully oxidized states, allowed the observation of different resonances for some of the haem methyl groups. In the present experimental conditions, some of the haems show a fast intramolecular electron exchange rate, but the intermolecular electron exchange is always slow. In intermediate reoxidation stages, large shifts of the resonances of some haem methyl groups were observed upon changing the pH. These shifts are discussed in terms of a pH dependence of the haem midpoint redox potentials. The physiological relevance of this pH dependence is discussed.     

Impact of unilateral spatial neglect on chronic patient's post-stroke quality of life

Abstract

Background: Unilateral spatial neglect (USN) is the prevalent feature in patients with right-sided stroke. It is diagnosed through the behavior inattention test (BIT) and has a negative impact on patients affecting both their functional capacity and quality of life.

Objective: Here, we aimed to evaluate the impact of USN on the quality of life of patients in the chronic phase of stroke.

Methods: This is a cross-sectional study with stroke patients with USN. After confirming the presence of stroke through neuroimaging examinations and of USN through the BIT, patients’ quality of life was evaluated by using the EUROQOL scale. Spearman’s correlation was used to validate the correlation between patients’ USN and quality of life, with a p?<?.05 representing significant results.

Results: Eighteen individuals were included. When correlating the value of each domain of the EUROQOL scale with the results of the BIT, we observed a negative correlation between mobility (r?=?–0.97; p?=?.000), self-care (r?=?–0.82; p?=?.013), usual activities (r?=?–0.87; p?=?.005); pain or discomfort (r?=?–0.88; p?=?.004), anxiety or depression (r?=?–0.97; p?=?.000), and EUROQOL total score (r?=?–0.97, p?=?.000).

Conclusion: After a correlation between the overall EUROQOL and BIT scores, we suggest that the higher the USN degree is in stroke patients, the worse their perceived quality of life tends to be.     

Early prepubertal testis criteria, seminiferous epithelium and hormone concentrations as related to testicular development in beef bulls

The present study was conducted to evaluate testis size, spermatogenesis and hormone concentrations before and when peripheral testosterone reached 1 ng/ml as related to further gonad development of beef bulls (n=28). Blood samples were taken weekly starting at 10 weeks (wk) and when testosterone reached 1 ng/ml (AGE1), the left testis was surgically excised. From AGE1 until 54 wk, blood samples were collected to follow basal and GnRH-stimulated hormone profiles. At 54 wk, the second testis was removed. Testosterone reached 1 ng/ml at 20±0.6 wk and, at this developmental state, the seminiferous tubules occupied 57±1.1% of the testis parenchyma. At this phase, 79.3±1.4% of tubule sections had no germ cells and only 2.4±0.3% of the remaining tubules had spermatocytes as the most advanced germ cell type. Also at AGE1, testis size was correlated with the number of Sertoli cells per testis (r=0.67; P<0.05), but not (P>0.05) with the percentage of tubules with germ cells. There was a consistent increase in body weight and testis size throughout the study showing that hemicastration did not impair the development of the bulls. At 54 wk, seminiferous tubules represented 76±0.7% of the testis parenchyma and 72.3±1.7% of tubule sections were found with either round or elongated spermatids. Quantitative criteria of spermatogenesis in the second testis (excised at 54 wk) were not correlated (P>0.05) with the percentage of seminiferous tubules with germ cells in the first testis (excised at AGE1). As determined by regression analysis, testis diameter measured between 30 and 44 wk (AVTD) was associated with AGE1 and testis diameter averaged at 12 wk and AGE1 (R(2)=0.77; P<0.01). Also, AVTD was related to AGE1, testis diameter at 12 wk and concentrations of 17β-estradiol (estradiol; basal+GnRH-stimulated) averaged between 10 wk and AGE1 (R(2)=0.79; P<0.01). Yearling testis weight, in turn, was linked to AGE1 and testis weight at AGE1 (R(2)=0.49, P<0.01). In conclusion, early detection of 1 ng of testosterone/ml, larger testis size and greater estradiol before and at that developmental period positively relate to future testis attributes. When testosterone reached 1 ng/ml, the seminiferous tubules had Sertoli cells, spermatogonia and a few spermatocytes and events occurring before and at that phase are potential markers of testis growth and sperm-producing capacity of sires.