HLA-G expression in Merkel cell carcinoma and the correlation with Merkel cell polyomavirus infection.

Merkel cell carcinoma (MCC) is a rare aggressive neuroendocrine cutaneous carcinoma with a high mortality rate. The MCC etiology is not fully understood. Merkel cell-associated polyomavirus (MCPyV) was found in MCC patients, indicating a risk factor for the tumor. Caucasian, elderly, and immunocompromised individuals are more likely to develop this tumor. HLA-G consists of a non-classical class I (Ib) HLA molecule with an immunoregulatory function and was associated with tumor escape in different types of tumors, nonetheless, never been studied in MCC. The purpose of this study was to evaluate the HLA-G expression and also to detect the MCPyV in MCC patients and correlate it with the clinical course of the disease. Forty-five MCC patients were included in a retrospective study. Formalin-fixed paraffin-embedded cutaneous skin biopsies were used by immunohistochemistry and RT-PCR to verify the HLA-G expression and MCPyV infection. HLA-G expression was found in 7 (15.6%), while the presence of MCPyV was detected in 28 (62.2%) of the studied patients. No significant association was found between HLA-G expression and MCPyV infection (p = 0.250). The presence of MCPyV was associated with areas of low sunlight exposure (p = 0.042) and the HLA-G expression with progression to death (p = 0.038). HLA-G expression was detected in MCC patients, as well as the MCPyV presence was confirmed. These markers could represent factors with a possible impact on patient survival; however, further studies with a greater number of patients are needed, to better elucidate the possible role in disease progression.

HLA-G expression in Merkel cell carcinoma and the correlation with Merkel cell polyomavirus infection

Merkel cell carcinoma (MCC) is a rare aggressive neuroendocrine cutaneous carcinoma with a high mortality rate. The MCC etiology is not fully understood. Merkel cell-associated polyomavirus (MCPyV) was found in MCC patients, indicating a risk factor for the tumor. Caucasian, elderly, and immunocompromised individuals are more likely to develop this tumor. HLA-G consists of a non-classical class I (Ib) HLA molecule with an immunoregulatory function and was associated with tumor escape in different types of tumors, nonetheless, never been studied in MCC. The purpose of this study was to evaluate the HLA-G expression and also to detect the MCPyV in MCC patients and correlate it with the clinical course of the disease. Forty-five MCC patients were included in a retrospective study. Formalin-fixed paraffin-embedded cutaneous skin biopsies were used by immunohistochemistry and RT-PCR to verify the HLA-G expression and MCPyV infection. HLA-G expression was found in 7 (15.6%), while the presence of MCPyV was detected in 28 (62.2%) of the studied patients. No significant association was found between HLA-G expression and MCPyV infection (p = 0.250). The presence of MCPyV was associated with areas of low sunlight exposure (p = 0.042) and the HLA-G expression with progression to death (p = 0.038). HLA-G expression was detected in MCC patients, as well as the MCPyV presence was confirmed. These markers could represent factors with a possible impact on patient survival; however, further studies with a greater number of patients are needed, to better elucidate the possible role in disease progression.

Positions and numbers of FKS mutations in Candida albicans selectively influence in vitro and in vivo susceptibilities to echinocandin treatment.

Candidemia is the fourth most common kind of microbial bloodstream infection, with Candida albicans being the most common causative species. Echinocandins are employed as the first-line treatment for invasive candidiasis until the fungal species is determined and confirmed by clinical diagnosis. Echinocandins block the FKS glucan synthases responsible for embedding ß-(1,3)-d-glucan in the cell wall. The increasing use of these drugs has led to the emergence of antifungal resistance, and elevated MICs have been associated with single-residue substitutions in specific hot spot regions of FKS1 and FKS2. Here, we show for the first time the caspofungin-mediated in vivo selection of a double mutation within one allele of the FKS1 hot spot 1 in a clinical isolate. We created a set of isogenic mutants and used a hematogenous murine model to evaluate the in vivo outcomes of echinocandin treatment. Heterozygous and homozygous double mutations significantly enhance the in vivo resistance of C. albicans compared with the resistance seen with heterozygous single mutations. The various FKS1 hot spot mutations differ in the degree of their MIC increase, substance-dependent in vivo response, and impact on virulence. Our results demonstrate that echinocandin EUCAST breakpoint definitions correlate with the in vivo response when a standard dosing regimen is used but cannot predict the in vivo response after a dose escalation. Moreover, patients colonized by a C. albicans strain with multiple mutations in FKS1 have a higher risk for therapeutic failure.

Characterization of Staphylococcus aureus isolates in milk and the milking environment from small-scale dairy farms of São Paulo, Brazil, using pulsed-field gel electrophoresis.

This research aimed to evaluate the occurrence of Staphylococcus aureus isolates in milk and in the milking environment of 10 small-scale farms (<400 L/d) located in the regions of Franca and Ribeirão Preto, state of São Paulo, Brazil. Two-hundred twenty samples of milk were collected from individual cows, along with 120 samples from bulk tank milk, 389 samples from milking equipment and utensils (teat cups, buckets, and sieves), and 120 samples from milkers' hands. Fifty-six Staph. aureus strains were isolated from 849 analyzed samples (6.6%): 12 (5.5%) from milk samples of individual cows, 26 (21.7%) from samples of bulk tank milk, 14 (3.6%) from samples collected from equipment and utensils, and 4 (3.3%) from samples from milkers' hands. Pulsed-field gel electrophoresis typing of the 56 Staph. aureus isolates by SmaI restriction enzyme resulted in 31 profiles (pulsotypes) arranged in 12 major clusters. Results of this study indicate a low incidence, but wide distribution of Staph. aureus strains isolated from raw milk collected from individual cows and surfaces of milkers' hands and milking equipment in the small-scale dairy farms evaluated. However, the high percentage of bulk milk samples found with Staph. aureus is of public health concern because raw, unprocessed milk is regularly consumed by the Brazilian population.

Mesostructured silica aerosol particles: comparison of gas-phase and powder deposit X-ray diffraction data.

We report on the characterization of mesostructured aerosol silica particles in the gas phase using in situ synchrotron small-angle X-ray scattering (SAXS) in order to unveil the influence of the basic production parameters. The investigated system was based on tetraethylorthosilicate (TEOS) as the inorganic precursor and on cetyltrimethyl-ammonium bromide (CTAB) as the surfactant. The heating temperature, surfactant to silicate ratio, and particle flow rate were thoroughly investigated, and for this purpose, an in-house-built aerosol reactor equipped with a special X-ray observation chamber was used. Complementary fine structural analysis was applied on dried deposits of the silica aerosols comprising direct Fourier transforms as well as simple two-phase model fits. This resulted in robust estimates for the silica wall thickness and surfactant core radius of the hexagonally ordered mesostructure. The particle shape and size distribution were examined by scanning electron microscopy (SEM). The quality of the inner nanostructure was revealed from an analysis of the peak width. The comparison of data from the gas phase and powder deposit shows that, in general, slower drying conditions (heating temperature about 80 °C) and a medium surfactant to Si ratio (about 0.14) lead to nanostructures of the best quality in terms of well-defined long-range organization.

Interactions at the bilayer interface and receptor site induced by the novel synthetic pyrrolidinone analog MMK3.

This work presents a thorough investigation of the interaction of the novel synthetic pyrrolidinone analog MMK3 with the model membrane system of dipalmitoylphosphatidylcholine (DPPC) and the receptor active site. MMK3 has been designed to exert antihypertensive activity by functioning as an antagonist of the angiotensin II receptor of subtype 1 (AT(1)). Its low energy conformers were characterized by 2D rotating-frame Overhauser effect spectroscopy (ROESY) in combination with molecular dynamics (MD) simulations. Docking study of MMK3 shows that it fits to the AT(1) receptor as SARTANs, however, its biological activity appears to be lower. Thus, differential scanning calorimetry (DSC), Raman spectroscopy and small angle X-ray scattering (SAXS) experiments on the interaction of MMK3 with DPPC bilayers were carried out and results demonstrate that the drug is well incorporated into the membrane leaflets and furthermore causes partial bilayer interdigitation, although less effective than SARTANs. Thus, it appears that the nature of the bilayer matrix and the stereoelectronic active site requirements of the receptor are responsible for the low bioactivity of MMK3.

Novel in situ setup to study the formation of nanoparticles in the gas phase by small angle x-ray scattering.

An in-house built aerosol generator setup for in situ gas phase studies of aerosol and nanoparticles is described. The aerosol generator with an ultrasonic ceramic disk mist maker provides high enough particle concentrations for structural gas phase analysis by synchrotron small angle x-ray scattering (for water approximately 4 x 10(8) droplets/s with a droplet size of approximately 2.5 microm). The working principle was proved by scattering of gold nanoparticles. For evaporation induced self-assembly studies of nanostructured particles, an additional thermal treatment chamber was included in the setup. The first on-line gas phase data with our setup for mesostructured silica particles are presented for different thermal treatments. Scanning electron microscope imaging revealed the average particle size to be approximately 1 microm. Furthermore, to quantify their internal nanostructure, diffraction experiments of deposited silica aerosols were carried out and the corresponding electron density map indicates a silica wall thickness of about 1 nm.

Effect of sodium dodecyl sulfate at different hydration conditions on dioleoyl phosphatidylcholine bilayers studied by grazing incidence X-ray diffraction.

The effect of the surfactant content and hydration conditions in the phases of dioleoyl phosphatidylcoline (DOPC)/sodium dodecyl sulfate (SDS) mixtures was studied. To this end, surface X-ray diffraction experiments have been performed on bilayers of the mixtures deposited on hydrophobic silicon wafers by dip coating. To investigate the effect of relative humidity (RH) on bilayer organization, a humidity chamber with dry-wet air control was used, and RH values were fixed between 1 and 65%. Our results showed, in addition to the lamellar phase, a rhombohedral phase in mixtures at low hydration conditions (RH < 30%). The d spacing between lamellae increased with the RH and SDS content. This fact could be associated with a swelling effect that is probably due to the localization of water molecules between the polar headgroups of the DOPC and SDS forming the bilayers. The electron-density profiles calculated by Fourier reconstruction of the lamellar stacking for the different samples also confirmed this fact. In addition, the increase in d spacing could be related to the increase in the hydrophilic character of the mixture when the SDS content increases. The rhombohedral phase was more clearly observed in mixtures with high SDS content. Thus, the stalk structure of the rhombohedral phase could be facilitated because of the SDS contribution to inverse structures.

Mycobacterium tuberculosis isolates belonging to katG gyrA group 2 are associated with clustered cases of tuberculosis in Italian patients.

Fifty-one consecutive isolates of Mycobacterium tuberculosis, collected during a 2-year period in the north-east of Italy, were subjected to IS6110-RFLP analysis to detect the presence of clusters and assigned to one of the three genotypic groups delineated by single nucleotide polymorphisms in the genes katG and gyrA. All the isolates collected from the local population belonged to group 2 or 3, while group 1 isolates were found only in specimens collected from African immigrants. Clustered cases of tuberculosis, which are likely to be related to recently transmitted infection, were found to be significantly associated with katG gyrA group 2. In the local situation, strains belonging to this group may therefore present a higher risk of transmission.

Enzymatic and molecular properties of the Clostridium tertium sialidase.

Clostridium tertium metabolizes sialoglycoconjugates via a secreted sialidase [EC 3.2.1.18] and an intracellular acylneuraminate pyruvate lyase [EC 4.1.3.3]. The sialidase was enriched 1,900-fold from the culture medium with a specific activity of 0.7 U per mg protein. It exhibits a temperature optimum of 50 degreesC and tolerates mercury ions at relatively high concentrations (50% inhibition at 5.2 mM Hg2+). The sialidase gene was detected on two restriction fragments (HincII, HindIII) of chromosomal DNA and their correct recombination resulted in an active enzyme expressed by Escherichia coli. The structural gene is represented by 2,319 bp encoding a protein of 773 amino acids with a molecular mass of 85.4 kDa. The first 28 amino acids possess the character of a signal peptide. The protein reveals a FRIP-region and four Asp-boxes common in all bacterial sialidases. It has 42.6% identical amino acids when compared with the sialidase of Clostridium septicum and 64.8% with a sialidase gene amplified from Macrobdella decora. A further open reading frame was detected 30 bp downstream from the sialidase gene, which exhibits significant homology with acylneuraminate pyruvate lyases. For the first time, both genes were found in close proximity on a bacterial chromosome, probably being part of one operon.