Molecular Aspects of Pruritus Pathogenesis in Psoriasis

Psoriasis is a chronic, systemic inflammatory disease with a genetic background that involves almost 3% of the general population worldwide. Approximately, 70–90% of patients with psoriasis suffer from pruritus, an unpleasant sensation that provokes a desire to scratch. Despite the enormous progress in understanding the mechanisms that cause psoriasis, the pathogenesis of psoriasis-related pruritus still remains unclear. In order to improve patients’ quality of life, development of more effective and safer antipruritic therapies is necessary. In turn to make it possible, better understanding of complexed and multifactorial pathogenesis of this symptom is needed. In this article we have systematized the current knowledge about pruritus origin in psoriasis.     

Molecular Insights into the Thermal Stability of mAbs with Variable‐Temperature Ion‐Mobility Mass Spectrometry

The aggregation of protein‐based therapeutics such as monoclonal antibodies (mAbs) can affect the efficacy of the treatment and can even induce effects that are adverse to the patient. Protein engineering is used to shift the mAb away from an aggregation‐prone state by increasing the thermodynamic stability of the native fold, which might in turn alter conformational flexibility. We have probed the thermal stability of three types of intact IgG molecules and two Fc‐hinge fragments by using variable‐temperature ion‐mobility mass spectrometry (VT‐IM‐MS). We observed changes in the conformations of isolated proteins as a function of temperature (300–550 K). The observed differences in thermal stability between IgG subclasses can be rationalized in terms of changes to higher‐order structural organization mitigated by the hinge region. VT‐IM‐MS provides insights into mAbs structural thermodynamics and is presented as a promising tool for thermal‐stability studies for proteins of therapeutic interest.     

Autophagy Modulators in Cancer Therapy

Autophagy is a process of self-degradation that plays an important role in removing damaged proteins, organelles or cellular fragments from the cell. Under stressful conditions such as hypoxia, nutrient deficiency or chemotherapy, this process can also become the strategy for cell survival. Autophagy can be nonselective or selective in removing specific organelles, ribosomes, and protein aggregates, although the complete mechanisms that regulate aspects of selective autophagy are not fully understood. This review summarizes the most recent research into understanding the different types and mechanisms of autophagy. The relationship between apoptosis and autophagy on the level of molecular regulation of the expression of selected proteins such as p53, Bcl-2/Beclin 1, p62, Atg proteins, and caspases was discussed. Intensive studies have revealed a whole range of novel compounds with an anticancer activity that inhibit or activate regulatory pathways involved in autophagy. We focused on the presentation of compounds strongly affecting the autophagy process, with particular emphasis on those that are undergoing clinical and preclinical cancer research. Moreover, the target points, adverse effects and therapeutic schemes of autophagy inhibitors and activators are presented.     

Extended depth from focus reconstruction using NIH ImageJ plugins: Quality and resolution of elevation maps

In this work, NIH ImageJ plugins for extended depth‐from‐focus reconstructions (EDFR) based on spatial domain operations were compared and tested for usage optimization. Also, some preprocessing solutions for light microscopy image stacks were evaluated, suggesting a general routine for the ImageJ user to get reliable elevation maps from grayscale image stacks. Two reflected light microscope image stacks were used to test the EDFR plugins: one bright‐field image stack for the fracture of carbon‐epoxy composite and its darkfield corresponding stack at same (x,y,z) spatial coordinates. Image quality analysis consisted of the comparison of signal‐to‐noise ratio and resolution parameters with the consistence of elevation maps, based on roughness and fractal measurements. Darkfield illumination contributed to enhance the homogeneity of images in stack and resulting height maps, reducing the influence of digital image processing choices on the dispersion of topographic measurements. The subtract background filter, as a preprocessing tool, contributed to produce sharper focused images. In general, the increasing of kernel size for EDFR spatial domain‐based solutions will produce smooth height maps. Finally, this work has the main objective to establish suitable guidelines to generate elevation maps by light microscopy. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

The Influence of Bisphenol A (BPA) on the Occurrence of Selected Active Substances in Neuregulin 1 (NRG1)-Positive Enteric Neurons in the Porcine Large Intestine

Bisphenol A (BPA) is a substance used in the manufacture of plastics which shows multidirectional adverse effects on living organisms. Since the main path of intoxication with BPA is via the gastrointestinal (GI) tract, the stomach and intestine are especially vulnerable to the impact of this substance. One of the main factors participating in the regulation of intestinal functions is the enteric nervous system (ENS), which is characterized by high neurochemical diversity. Neuregulin 1 (NRG1) is one of the lesser-known active substances in the ENS. During the present study (performed using the double immunofluorescence method), the co-localization of NRG1 with other neuronal substances in the ENS of the caecum and the ascending and descending colon has been investigated under physiological conditions and after the administration of BPA. The obtained results indicate that NRG1-positive neurons also contain substance P, vasoactive intestinal polypeptide, a neuronal isoform of nitric oxide synthase and galanin and the degree of each co-localization depend on the type of enteric plexus and the particular fragment of the intestine. Moreover, it has been shown that BPA generally increases the degree of co-localization of NRG1 with other substances.     

Raman Research on Bleomycin-Induced DNA Strand Breaks and Repair Processes in Living Cells

Even several thousands of DNA lesions are induced in one cell within one day. DNA damage may lead to mutations, formation of chromosomal aberrations, or cellular death. A particularly cytotoxic type of DNA damage is single- and double-strand breaks (SSBs and DSBs, respectively). In this work, we followed DNA conformational transitions induced by the disruption of DNA backbone. Conformational changes of chromatin in living cells were induced by a bleomycin (BLM), an anticancer drug, which generates SSBs and DSBs. Raman micro-spectroscopy enabled to observe chemical changes at the level of single cell and to collect hyperspectral images of molecular structure and composition with sub-micrometer resolution. We applied multivariate data analysis methods to extract key information from registered data, particularly to probe DNA conformational changes. Applied methodology enabled to track conformational transition from B-DNA to A-DNA upon cellular response to BLM treatment. Additionally, increased expression of proteins within the cell nucleus resulting from the activation of repair processes was demonstrated. The ongoing DNA repair process under the BLM action was also confirmed with confocal laser scanning fluorescent microscopy.     

Changes in Polychlorinated Biphenyl Residues in Milk during Lactation: Levels of Contamination,Influencing Factors,and Infant Risk Assessment

Given the importance of breastfeeding infants, the contamination of human milk is a significant public concern. The aim of this study was to assess the contamination of human milk with dioxin-like PCBs (dl-PCBs) and non-dioxin-like PCBs (ndl-PCBs) in relation to the duration of lactation and other influencing factors, especially the frequency of the consumption of selected foods during pregnancy. Based on this, the health risk to infants was assessed and compared to the tolerable daily intake (TDI). PCB determinations were performed using gas chromatography/mass spectrometry. The ∑ndl-PCB content ranged from 0.008 to 0.897 ng/g w.w., at an average of 0.552 ng/g wet weight, which was 55% of the maximum level according to the EU guidelines for foods for infants and young children. The toxic equivalent (TEQ) was in the range of 0.033–5.67 pg-TEQ/g w.w. The content of non-ortho, mono-ortho, and ndl-PCBs in human milk decreased the longer lactation continued. Moreover, when pregnant women smoked tobacco, this correlated significantly with increases in the concentrations of PCB congeners 156, 118, and 189 in human milk. The human milk contents of PCB congeners 77, 81, 186, 118, and 189 were strongly positively correlated with the amount of fish consumed. The content of stable congeners PCB 135 and PCB 153 increased with age.     

Comparison of Enzyme Activity in Order to Describe the Metabolic Profile of Dairy Cows during Early Lactation

Enzymatic diagnostics have practical applications in diseases of the liver, heart, pancreas, muscles, blood, and neoplastic diseases. This study aimed to compare enzyme activity to describe dairy cows’ metabolism during early lactation. Based on their general health symptoms, the cows were assigned to one of three groups: acidotic, healthy and ketotic. Samples of milk, blood and rumen fluid were collected at 12 ± 5 days postpartum. Ketotic cows were characterized by the highest malondialdehyde (MDA, 76.098 nM/mL), glutathione reductase (GluRed, 109.852 U/L), superoxide dismutase (SOD, 294.22 U/L) and gamma-glutamyltranspeptidase (GGTP, 71.175 U/L) activity. In comparing ketotic and acidotic cows, MDA, GluRed, SOD and GGTP activity were higher by a factor of almost: 1.85, 1.89, 0.79 and 2.50, respectively. Acidotic cows were characterized by the highest aspartate aminotransferase activity (AspAT, 125.914 U/L). In comparing acidotic and ketotic cows, AspAT activity was higher by a factor of almost 1.90. The use of enzymatic markers could limit the frequency of sampling for laboratory analyses and may result in a faster diagnosis of metabolic disorders. AspAT activity in blood serum seems to be a good indicator of acidosis; GGTP may participate in the pathogenesis of ketosis.     

Antifungal Effect of Penicillamine Due to the Selective Targeting of L-Homoserine O-Acetyltransferase

Due to the apparent similarity of fungal and mammalian metabolic pathways, the number of established antifungal targets is low, and the identification of novel ones is highly desirable. The results of our studies, presented in this work, indicate that the fungal biosynthetic pathway of L-methionine, an amino acid essential for humans, seems to be an attractive perspective. The MET2 gene from Candida albicans encoding L-homoserine O-acetyltransferase (CaMet2p), an enzyme catalyzing the first step in that pathway, was cloned and expressed as the native or the oligo-His-tagged fusion protein in Escherichia coli. The recombinant enzymes were purified and characterized for their basic molecular properties and substrate specificities. The purified MET2 gene product revealed the appropriate activity, catalyzed the conversion of L-homoserine (L-Hom) to O-acetyl-L-homoserine (OALH), and exhibited differential sensitivity to several L-Hom or OALH analogues, including penicillamine. Surprisingly, both penicillamine enantiomers (L- and D-Pen) displayed comparable inhibitory effects. The results of the docking of L- and D-Pen to the model of CaMet2p confirmed that both enantiomeric forms of the inhibitor are able to bind to the catalytic site of the enzyme with similar affinities and a similar binding mode. The sensitivity of some fungal cells to L-Pen, depending on the presence or absence of L-Met in the medium, clearly indicate Met2p targeting. Moreover, C. glabrata clinical strains that are resistant to fluconazole displayed a similar susceptibility to L-Pen as the wild-type strains. Our results prove the potential usefulness of Met2p as a molecular target for antifungal chemotherapy.