Malignant potential of dysplastic endocervical epithelium assessed by ploidy status,S-phase fraction and C-myc expression

The aim of this study was to determine if dysplastic endocervical cells (EC) posses a neoplastic potential as precursor lesions to adenocarcinoma in situ (AIS). The malignant potential was determined by assessing the ploidy status and proliferative activity by flow cytometry and c-myc expression by immunohistochemistry. The studied parameters were assessed separately in morphologically normal, dysplastic and malignant EC. The chi 2 test showed significant association of malignant EC with aneuploidy (p = 0.008) and high proliferative activity (p = 0.042). Since one third of the dysplastic EC are also aneuploid and show high mitotic activity, they probably have malignant potential as well. The dysplastic EC showed a significant association with c-myc oncogene expression (p = 0.028). Our results indicate the existence of pre-malignant glandular lesions, while the immunohistochemical detection of c-myc protooncogene could be helpful in detection of EC with malignant potential, even without any dysplastic morphological changes.     

Paraoxonase activity and concentration of indicators of lipids status in the serum of cardiac patients

In this research, we measured the activity of paraoxonase (basal and activated) enzyme, and components of lipid status components (total cholesterol, LDL cholesterol, HDL cholesterol and Apo A I) in the serum of patients, undergoing bypass surgery. We also tested how the applied EKC affected changes of defined indicators. Measuring of all the given parameters was conducted prior to the operation, 90 minutes, 1.5 hour, 6 hours, 24 hours and 72 hours, on 29 patients (11 of them did undergo myocardium revascularization with the application of EKC, while the rest of them did not). Activity of paraoxonase (both basal and activated) changes significantly during the postoperative period, in relation to pre-operative values, p < 0.05. Total cholesterol concentration is reduced in both examined groups, regardless of the application of EKC. This trend is also accompanied by LDL cholesterol concentration. On the other hand, HDL cholesterol concentration during post-operative period does not indicate any significant statistical change in relation to pre-operative values, while we noticed difference with regard to EKC application, 90 minutes after surgery. This change of lipid status indicator is partly due to heparin, a stimulator of lipoprotein lipase that was applied during the surgery. Our conclusion is that lipid profile changes significantly after the bypass surgery, mostly regardless of the application of EKC.     

Identification of tryptic peptides from large databases using multiplexed tandem mass spectrometry: simulations and experimental results

Multiplexed tandem mass spectrometry (MS/MS) has recently been demonstrated as a means to increase the throughput of peptide identification in liquid chromatography (LC) MS/MS experiments. In this approach, a set of parent species is dissociated simultaneously and measured in a single spectrum (in the same manner that a single parent ion is conventionally studied), providing a gain in sensitivity and throughput proportional to the number of species that can be simultaneously addressed. In the present work, simulations performed using the Caenorhabditis elegans predicted proteins database show that multiplexed MS/MS data allow the identification of tryptic peptides from mixtures of up to ten peptides from a single dataset with only three "y" or "b" fragments per peptide and a mass accuracy of 2.5 to 5 ppm. At this level of database and data complexity, 98% of the 500 peptides considered in the simulation were correctly identified. This compares favorably with the rates obtained for classical MS/MS at more modest mass measurement accuracy. LC multiplexed Fourier transform-ion cyclotron resonance MS/MS data obtained from a 66 kDa protein (bovine serum albumin) tryptic digest sample are presented to illustrate the approach, and confirm that peptides can be effectively identified from the C. elegans database to which the protein sequence had been appended.     

Developmental potential of mouse embryos without extraembryonic membranes in modified organ culture

The long-term stationary culture of postimplanatation embryos without extraembryonic membranes is a method to assess their developmental potential in vitro. The method was almost exclusively used on rat embryos, while mouse embryos were considered unsuitable due to their poor differentiation. In present study the postimplantation mouse embryos were used to verify potential of this method in mice. In addition, the course of in vitro differentiation was compared to embryo development in situ. Embryos were cultivated for maximum of 14 days and morphology and differentiation was analysed on serial semithin sections. Although anatomical relationships were lost from the beginning of the cultivation, the differentiation was only delayed, and the developmental potential after long-term culture was comparable to those observed in rats. Therefore the advantages of long-term cultivation could be utilized to analyse the differentiation of numerous lines of genetically modified mice with impaired postimplantation development.     

Antioxidant activity and phenolic profile in filamentous cyanobacteria: the impact of nitrogen

In the present study, ethanolic extracts of ten cyanobacterial strains cultivated under different nitrogen conditions were assessed for the phenolic content and antioxidant activity. The amount of detected phenolic compounds ranged from 14.86 to 701.69 μg g^?1 dry weight (dw) and HPLC-MS/MS analysis revealed gallic acid, chlorogenic acid, quinic acid, catechin, epicatechin, kaempferol, rutin and apiin. Only catechin, among the detected phenolics, was present in all the tested strains, while quinic acid was the most dominant compound in all the tested Nostoc strains. The results also indicated the possibility of increasing the phenolic content in cyanobacterial biomass by manipulating nitrogen conditions, such as in the case of quinic acid in Nostoc 2S7B from 70.83 to 594.43 μg g^?1 dw. The highest radical scavenging activity in DPPH assay expressed Nostoc LC1B with IC₅₀ value of 0.04?±?0.01 mg mL^?1, while Nostoc 2S3B with IC₅₀ =?9.47?±?3.61 mg mL^?1 was the least potent. Furthermore, the reducing power determined by FRAP assay ranged from 8.36?±?0.08 to 21.01?±?1.66 mg AAE g^?1, and it was significantly different among the tested genera. The Arthrospira strains exhibited the highest activity, which in the case of Arthrospira S1 was approximately twofold higher in comparison to those in nitrogen-fixing strains. In addition to this, statistical analysis has indicated that detected phenolics were not major contributor to antioxidant capacities of tested cyanobacteria. However, this study highlights cyanobacteria of the genera Nostoc, Anabaena, and Arthrospira as producers of antioxidants and phenolics with pharmacological and health-beneficial effects, i.e., quinic acid and catechin in particular.     

Haloferax volcanii archaeosortase is required for motility,mating, and C‐terminal processing of the S‐layer glycoprotein

Cell surfaces are decorated by a variety of proteins that facilitate interactions with their environments and support cell stability. These secreted proteins are anchored to the cell by mechanisms that are diverse, and, in archaea, poorly understood. Recently published in silico data suggest that in some species a subset of secreted euryarchaeal proteins, which includes the S‐layer glycoprotein, is processed and covalently linked to the cell membrane by enzymes referred to as archaeosortases. In silico work led to the proposal that an independent, sortase‐like system for proteolysis‐coupled, carboxy‐terminal lipid modification exists in bacteria (exosortase) and archaea (archaeosortase). Here, we provide the first in vivo characterization of an archaeosortase in the haloarchaeal model organism Haloferax volcanii. Deletion of the artA gene (HVO_0915) resulted in multiple biological phenotypes: (a) poor growth, especially under low‐salt conditions, (b) alterations in cell shape and the S‐layer, (c) impaired motility, suppressors of which still exhibit poor growth, and (d) impaired conjugation. We studied one of the ArtA substrates, the S‐layer glycoprotein, using detailed proteomic analysis. While the carboxy‐terminal region of S‐layer glycoproteins, consisting of a putative threonine‐rich O‐glycosylated region followed by a hydrophobic transmembrane helix, has been notoriously resistant to any proteomic peptide identification, we were able to identify two overlapping peptides from the transmembrane domain present in the ΔartA strain but not in the wild‐type strain. This clearly shows that ArtA is involved in carboxy‐terminal post‐translational processing of the S‐layer glycoprotein. As it is known from previous studies that a lipid is covalently attached to the carboxy‐terminal region of the S‐layer glycoprotein, our data strongly support the conclusion that archaeosortase functions analogously to sortase, mediating proteolysis‐coupled, covalent cell surface attachment.     

SHBG Is an Important Factor in Stemness Induction of Cells by DHT In Vitro and Associated with Poor Clinical Features of Prostate Carcinomas

Androgen plays a vital role in prostate cancer development. However, it is not clear whether androgens influence stem-like properties of prostate cancer, a feature important for prostate cancer progression. In this study, we show that upon DHT treatment in vitro, prostate cancer cell lines LNCaP and PC-3 were revealed with higher clonogenic potential and higher expression levels of stemness related factors CD44, CD90, Oct3/4 and Nanog. Moreover, sex hormone binding globulin (SHBG) was also simultaneously upregulated in these cells. When the SHBG gene was blocked by SHBG siRNA knock-down, the induction of Oct3/4, Nanog, CD44 and CD90 by DHT was also correspondingly blocked in these cells. Immunohistochemical evaluation of clinical samples disclosed weakly positive, and areas negative for SHBG expression in the benign prostate tissues, while most of the prostate carcinomas were strongly positive for SHBG. In addition, higher levels of SHBG expression were significantly associated with higher Gleason score, more seminal vesicle invasions and lymph node metastases. Collectively, our results show a role of SHBG in upregulating stemness of prostate cancer cells upon DHT exposure in vitro, and SHBG expression in prostate cancer samples is significantly associated with poor clinicopathological features, indicating a role of SHBG in prostate cancer progression.     

Proteogenomic Analysis of a Thermophilic Bacterial Consortium Adapted to Deconstruct Switchgrass

Thermophilic bacteria are a potential source of enzymes for the deconstruction of lignocellulosic biomass. However, the complement of proteins used to deconstruct biomass and the specific roles of different microbial groups in thermophilic biomass deconstruction are not well-explored. Here we report on the metagenomic and proteogenomic analyses of a compost-derived bacterial consortium adapted to switchgrass at elevated temperature with high levels of glycoside hydrolase activities. Near-complete genomes were reconstructed for the most abundant populations, which included composite genomes for populations closely related to sequenced strains of Thermus thermophilus and Rhodothermus marinus, and for novel populations that are related to thermophilic Paenibacilli and an uncultivated subdivision of the little-studied Gemmatimonadetes phylum. Partial genomes were also reconstructed for a number of lower abundance thermophilic Chloroflexi populations. Identification of genes for lignocellulose processing and metabolic reconstructions suggested Rhodothermus, Paenibacillus and Gemmatimonadetes as key groups for deconstructing biomass, and Thermus as a group that may primarily metabolize low molecular weight compounds. Mass spectrometry-based proteomic analysis of the consortium was used to identify >3000 proteins in fractionated samples from the cultures, and confirmed the importance of Paenibacillus and Gemmatimonadetes to biomass deconstruction. These studies also indicate that there are unexplored proteins with important roles in bacterial lignocellulose deconstruction.     

Microbiological surveillance of the surgical intensive care unit in Zagreb--a pivot for guideline-based therapy of severe sepsis

The aim of this retrospective study was to create guidelines for therapy of severe sepsis in surgical intensive care unit (ICU) for unknown causative agent based on antimicrobial susceptibility of causative bacteria. Seventy-four patients with severe sepsis from surgical ICU in 2003.-2005. were included in study. Their clinical and microbiological data were analyzed from the medical records. Antimicrobial susceptibility of the strains isolated from the blood-culture was tested by disk diffusion method according to CLSI (Clinical Laboratory Standard Institution). APACHE II score was used to predict the severity of illness. Statistical significance difference between results was tested by Mann-Whitney test and chi2 test. Important problem remained type of sepsis: mono-agent sepsis presented less therapeutic problem than sepsis caused with two or more agents (mixed sepsis). Methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and Acinetobacter baumannii were predominant causative agents in both type of sepsis. There was remarkable increase of A. baumannii prevalence in 2005 compared to 2004 and to 2003. There was also decrease of MRSA prevalence in 2004 and 2005 compared to 2003. P. aeruginosa were the predominant causative agents in 2004. MRSA displayed good susceptibility to vancomycin and linezolide, whereas P. aeruginosa showed excellent susceptibility to ceftazidime and carbapenems. A. baumannii, third predominant causative agent, exhibited excellent susceptibility to ampicillin+ sulbactam and carbapenems. The recommended therapy is empirical and should cover all important pathogens.     

Mushroom poisoning

In the research we included a total of 207 subjects with the dismissal diagnosis of "mycetismus", who were treated at the Department of Infectious Diseases, General Hospital Osijek, during the 1983-1992 period. 32 of them were children. There were 44.93% of men, 39.61% of women and 15.45% of children. The latent time > 6 hours was determined in 51 (25%) and < 6 hours in 75% of subjects. In 156 of patients with the latent time > 6 hours, "false" poisoning occured, while 51 patients experienced real mushroom toxins poisoning. At the admission to the hospital, in patients with the latent time > 6 hours, a pathological PT (protrombine time) was established only in women, leukocytosis in both women and children, increased concentration of GGT (gamma-glutamin-transferase) in men, increased AST (aspartate-aminotransferase) and ALT (alanin-aminotransferase) only in women, and increased urea in both women and children. After 24 hours, control measuring established high values of AST and ALT extended PT uremia and exalted amount of ammonia in blood in 11 of patients (2 men, 7 women and 2 children). They had severe liver and kidney damage, the most probably caused by Amanita phalloides toxins. The latent time lasted 9 to 13 hours. Of the 11 above mentioned patients, 2 women, aged 74 and 43, and one girl, aged 6, died. No pathological laboratory parameters were established in 40 of subjects with the latent time of 6 and more hours, and the disease manifested through vomiting and diarrhea that lasted for several days. These subjects most probably suffered from mushroom toxins poisoning. Mushroom toxins irritate the mucuous membrane of the gastrointestinal tract, and there are many such poisonous mushrooms. There were no mortalities in this group of subjects.