Creation of Recombinant Biocontrol Agents by Genetic Programming of Yeast.

Bacterial infections caused by antibiotic-resistant pathogens pose an extremely serious and elusive problem in healthcare. The discovery and targeted creation of new antibiotics are today among the most important public health issues. Antibiotics based on antimicrobial peptides (AMPs) are of particular interest due to their genetically encoded nature. A distinct advantage of most AMPs is their direct mechanism of action that is mediated by their membranolytic properties. The low rate of emergence of antibiotic resistance associated with the killing mechanism of action of AMPs attracts heightened attention to this field. Recombinant technologies enable the creation of genetically programmable AMP producers for large-scale generation of recombinant AMPs (rAMPs) or the creation of rAMP-producing biocontrol agents. The methylotrophic yeast Pichia pastoris was genetically modified for the secreted production of rAMP. Constitutive expression of the sequence encoding the mature AMP protegrin-1 provided the yeast strain that effectively inhibits the growth of target gram-positive and gram-negative bacteria. An antimicrobial effect was also observed in the microculture when a yeast rAMP producer and a reporter bacterium were co-encapsulated in droplets of microfluidic double emulsion. The heterologous production of rAMPs opens up new avenues for creating effective biocontrol agents and screening antimicrobial activity using ultrahigh-throughput technologies.

B Cell Profiling in Patients with Pemphigus Vulgaris.

Pemphigus vulgaris is a severe, socially significant autoimmune disease associated with autoantibodies to the desmoglein 3 antigen. The disease affects all age groups, beginning at 18 years of age; the mortality rate of pemphigus can reach as high as 50%, depending on a patient's age and a number of other factors. There is no highly selective or personalized therapy for pemphigus vulgaris at the moment. One of the well-known therapeutic approaches to the disease is to use rituximab, an anti-CD20 antibody that can help achieve B cell depletion in peripheral blood. To solve the problem of nonspecific elimination of B cells in patients with pemphigus vulgaris, it is reasonable to use specific immunoligands, their choice being based on an assessment of the level of autoantibodies specific to each of the fragments of desmoglein. In this work, the proportion of autoreactive B cells in patients diagnosed with pemphigus vulgaris is found to be 0.09-0.16%; a positive correlation was revealed between the antibody level and the number of autoreactive B cells to various fragments of desmoglein.

The Recombinant Luciferase of the Fungus Neonothopanus nambi: Obtaining and Properties.

A key component of the recently described bioluminescent system of higher fungi is luciferase, a new class of proteins. The properties of fungal luciferase and their relationship with its structure are interesting both for improving autoluminescent systems already created on its basis and for creating new ones. Therefore, it is extremely important to understand the spatial structure of this protein. We have performed heterologous expression and purification of Neonothopanus nambi luciferase, obtained a protein suitable for subsequent crystallization, and also determined some biochemical properties of the recombinant luciferase.

Molecular Pathway Activation Markers Are Associated with Efficacy of Trastuzumab Therapy in Metastatic HER2-Positive Breast Cancer Better than Individual Gene Expression Levels.

Increased expression or amplification of HER2 receptor tyrosine kinase gene ERBB2 is well-known and widely used as a prognostic biomarker of breast cancer (BC) response to the targeted treatment with trastuzumab and its analogs. Considering that part of the BC patients overexpressing HER2 does not respond to trastuzumab, clinical trial NCT03521245 was initiated to identify additional gene expression and molecular pathway activation response biomarkers to trastuzumab treatment in HER2-positive BC. Using RNA sequencing gene expression in 23 formalin-fixed, paraffin embedded HER2 positive BC tissue blocks from patients who either responded or not responded to trastuzumab treatment was profiled. Differentially regulated genes and molecular pathways were identified in the groups of trastuzumab responders and non-responders. These results were next compared with the 42 previously published BC trastuzumab responder and non-responder RNA sequencing profiles from the clinical trials NCT00513292 and NCT00353483. No correlation was observed between the response status and the expression levels of ERBB2 gene in the HER2 positive BC samples. Analysis of the differentially expressed genes and molecular pathways in the combined dataset revealed 15/27 commonly up/down regulated genes and 15/25 pathways, respectively. However, only the intersection of molecular pathways upregulated in trastuzumab responders vs non-responders was statistically significantly enriched compared to the random expectation model. A classifier built using the most significantly upregulated molecular pathway - cAMP Pathway Protein Retention - demonstrated the best performance for prediction of the HER2 positive BC response to trastuzumab for both our experimental and previously reported data. This pathway also predicted time to recurrence in the combined dataset with Log-rank p-value 0.041.

[Isolated Myocardial Infarction of the Right Ventricle Complicated by Myocardial Rupture with Cardiac Tamponade in a Female Patient with Arrhythmogenic Right Ventricular Dysplasia].

Arrhythmogenic dysplasia of the right ventricle is a rare pathology of the myocardium, the diagnosis of which is difficult. Isolated myocardial infarction of the right ventricle occurs and is diagnosed extremely rarely. In this article we describe a case of arrhythmogenic right ventricular dysplasia, complicated by transmural infarction of the anterolateral wall of the right ventricle, myocardial rupture, and cardiac tamponade.

[Mycological and entomological methods for estimation of the time since death in forensic medicine].

The objective of the present paper was to summarize the results of experimental studies and expert estimation carried out by the authors to survey the process of cadaver biodecomposition under different conditions with special reference to the characteristics of soil biota at different burial sites. Criteria have been developed for the estimation of prescription of death coming and the identification of biotic communities of the corpse buried in the soil, saline and fresh water.

[Parallels between clinical and morphological characteristics in fetal and newborn asphyxia from the standpoint of forensic medicine].

This paper deals with morphological methods for diagnostics of pre-, intra-, and postnatal asphyxia in newborn infants. Parallels between clinical and morphological characteristics in fatal cases resulting from this condition are described. The analysis of the voluminous literature has demonstrated the imperative necessity to broaden the scope of diagnostic morphological and pathomorphological methods for the study of newborn baby corpses in order to elucidate the causes of death or clarify whether a baby was born alive or stillborn.

[The modern concepts of placental pathology and its role in perinatal mortality in the context of forensic medical practice].

The modern concepts of placental pathology are considered and analysed in the context of forensic medical practice. The most promising approaches to the application of fundamental knowledge of placental pathology (including morphometric methods) for the purpose of forensic medical expertise are discussed.

[Pancreatitis as a fatal iatrogenic complication of opisthorchiasis].

A case of pancreonecrosis is reported that developed in a patient with symptoms of chronic cholangitis and pancreatitis associated with opisthorchiasis in response to endoscopic probing of hepatic and pancreatic ducts with the administration of a contrast agent.

[New criteria for diagnostics of sudden cardiac death in forensic medicine].

New data on characteristics of sudden cardiac death are provided. It is characterized by hypohydration of the brain, elevated cerebral impedance, a sharp fall of the index of coronary heart arteries passability, mostly areactive affection of the stem nuclei of the brain.

Analysis of altered gene expression profiles in retinoic acid or CpG-treated Sprague-Dawley rats with MNU-induced mammary adenocarcinoma by cDNA macro array.

In the present work the effects of 13-cis retinoic acid (RA) and CpG-containing oligodeoxynucleotides (CpG-ODN) on the gene expression profile of spleen and tumor tissue in a MNU-induced mammary gland carcinoma ratmodel were investigated by the use of a commercial cDNA macro array (Atlas rat toxicology array 1.2, Clontech). Treatment with these components, either alone or in combination, induced differences of the expression profiles between the distinct treatment groups in both tissues. The large number of genes with altered expression (> 200) points to a highly complex process in vivo.

Reduction of 1-methyl-1-nitrosourea-induced tumor burden with DNA vaccines encoding mutated ras epitopes and the costimulatory molecule B7.1.

1-methyl-1-nitrosourea (MNU), a well characterized carcinogen, was used to induce adenocarcinomas in rat mammary gland. 150 days after the first injection of MNU, the animals were treated with DNA minigene vaccines encoding ras T cell epitopes together with the co-stimulatory molecule B7.1 (CD 80). Five injections with a biolistic device (gene gun) in monthly intervals significantly reduced the tumor burden. A therapeutic effect could be measured with both, DNA vaccines encoding ras epitopes and B7.1, as well as with a DNA vaccine expressing solely the B7.1 molecule thus indicating the potential of genetic vaccination for turnor treatment.

Effect of cadmium and mercury on the nuclear retinoic acid receptors.

The actions of retinoic acids (RA) are mediated by their cognate nuclear receptors--ligand inducible transcription factors (retinoic acid receptors (RAR)). Possible interactions of toxic heavy metals on the RAR system are of interest due to involvement of the RAR system in multiple systemic processes. We assayed cadmium chloride and mercury chloride for their influence on the RAR system in rat and in cell culture. Mercury chloride was observed to decrease the maximal binding capacity in vitro of RARs for all-trans RA in liver nuclear fraction containing sets of nuclear receptors by seventy percent at a concentration of 0.1 mmol/l, though not cadmium chloride. Neither mercury chloride nor cadmium chloride induced any changes with respect to mRNA levels of RAR and binding properties of nuclear receptor fraction for RA or retinoic acid responsive elements (RARE) in male Wistar rats receiving tap water with cadmium chloride (9.7 mg/l) or mercury chloride (11.5 mg/l) for six weeks. In rat pituitary GH4C1 cells, neither mRNA levels nor binding properties for RARE in cell culture were affected by non-toxic concentrations of these heavy metals. From the data obtained it is suggested that, in vivo, cadmium or mercury have no significant impact on RA nuclear receptor system.

[Characteristics of exogenous dormancy of Aspergillus niger conidia]. / Osobennosti ékzogennogo pokoia konidii Aspergillus niger.

Aspergillus niger conidia are characterized by exogenous dormancy: the first stage of their germination is accomplished in twice distilled water. However, germ tube formation requires the availability of carbon and nitrogen sources. Exogenous dormancy in A. niger conidia exhibits the following peculiar features: (i) nitrogen-containing substances are active stimulators of germination; (ii) temperature-dependent changes in the lipid bilayer and in the neutral lipid composition of conidia are virtually identical to those occurring in growing mycelium under temperature stress; and (iii) the spore viability threshold does not exceed 45 degrees C; i.e., the spores are more heat-resistant than the mycelium, but they are less heat-resistant than the spores that are in the state of endogenous dormancy. According to the current classification of the types of cell metabolism arrest, the exogenous dormancy of A. niger conidia resembles the pattern of metabolism characteristic of vegetative cells during the idiophase.

[Detection of sarcocystosis in slaughterhouse animals during a veterinary inspection]. / Záchytnost sarkocystózy u jatocných zvierat pri veterinárnej prehliadke.

The objective of our study was to acquire objective data on the finds of sarcocystiosis incidence in particular species of farm animals during veterinary inspection and to compare the efficiency of different direct diagnostic methods: compression and digestion methods. The examined and observed animals involved sheep, cattle, pigs and goats slaughtered in the packing plants at Ruzomberok, Rimavská Sobota, Trstená, Sabinov and Kosice. The animals were subjected to regular veterinary inspection including aspection focused on predilection spots of sarcocyst incidence. Sarcocysts were investigated in detail in 353 head of sheep, 27 goats, 350 head of cattle and 1,409 pigs. The examination consisted in inspection of the whole body, of cross-sections of muscles and organs. The rate of sarcocyst invasion in meat as well as the size of cysts were largely variable. The highest incidence in sheep was observed on the inner surface of ventral muscles in the region of diaphragmatic ribs (21.4%) and in the region of intercostal muscles (39.6%). The rate of sarcocyst invasion in the organs was highest in the gullet with cysts of various forms and size (in 47% of sheep). Veterinary inspection of the total number of 27 goats revealed at aspection the presence of macrocysts in eight goats, which makes 29.62%, and in six head of cattle (1.7%) and in 13 pigs (0.9%). Regular finds of sarcocysts on a slaughter line were confronted with direct diagnostic methods: compression and digestion methods (Fig. 1, Tabs. I, II and III). Direct diagnostic method confirmed different results, while only the digestion method--trypsin digestion of muscle--can be considered as diagnostically reliable.

Cumulation NOx in foodstuffs and raw materials of animal origin.

In recent years quality of all raw materials processed by agricultural-food industry complex has been influenced by penetration of contaminating hazardous substances into the food chain as a result of high chemization, as well as by the exhalation fall-outs of industrial enterprises. In our study we followed the cumulation of nitrates and nitrites in food and raw materials of animal origin in the exposed industrial area of East Slovak Ironworks in Kosice. Determined were the residual levels of nitrates and nitrites in meat and organs of slaughterhouse cattle, milk and water from individual agricultural enterprises. Consistent control of the content of these hazardous substances in foodstuffs of vegetable and animal origin, water, soil and air represents a considerable contribution from viewpoint of consumer protection.

[Transport of nitrates and nitrites into the milk of dairy cows through the digestive system]. / Prestup dusicnanov a dusitanov do mlieka dojníc cestou tráviaceho traktu.

Increased nitrate concentrations in milk are not only dangerous to human health as the milk is the material for production of baby and infant food, but they cause also many problems in technological milk processing. The study was aimed at the transfer of nitrates and nitrites into milk of dairy cows following nitrate loading. An experiment included 6 dairy cows of the Slovakian Spotted breed at the Experimental Veterinary Centre at Zemplínska Teplica. Prior to start of the experiment samples of feedstuffs and feed water, milk were taken and examined for the presence of nitrates and nitrites. KNO3 in water solution was applied to selected dairy cows in two-week intervals in single peroral doses of 150; 75; 37.5; 18.75 and 9.5 g two hours before evening milking. Nitrate and nitrite residue contents were studied in individual milk samples obtained from manual milking 2, 14, 26, 38 and 50 hours after application of appropriate KNO3 amount. Following the peroral application of KNO3 to dairy cows, a marked increase in nitrate content in milk appeared in dependence on applied KNO3 (Tab. I). Average value of residual nitrate in milk two hours after administration of 150 g of KNO3 was 34.60 mg of NO3-/l. Increased levels of residual nitrate in milk were found also 38 hours after KNO3 application. Nitrate content in milk after 50 hours was almost identical with that that was determined in milk of experimental cows from morning milking on the day of administration of 150 g of KNO3, considered as the control samples. The values of residual nitrate exceeded 0.05 mg neither in single sample of NaNO2/l.(ABSTRACT TRUNCATED AT 250 WORDS)