THE ORAL SUCKER OF SOME GASTROINTESTINAL TREMATODES: HISTOLOGICAL AND HISTOCHEMICAL RESEARCH

We conducted histological and histochemical studies of the oral sucker of gastrointestinal trematodes of the family Paramphistomatidae, Fischoeder, 1901 living in the rumen of ruminants, namely, Liorchis scotiae, Paramphistomum cervi and Paramphistomum ichikawai. The oral sucker of paramphistomes and the Liorchis scotiae trematode is a complex muscular organ modified into a pharynx-sucker, the muscle complex of which is formed from longitudinal, circular and radial muscle fibers. In the thick pharynx wall, we found secretory cells, single neurosecretory cells and many desmoblastic cells of connective tissue. Histochemical stains showed intense staining with alcian blue, toluidine blue, bromophenol blue and a positive periodic acid Schiff reaction, which indicates the presence of glycosaminoglycans, total proteins and glycoproteins. Due to this structure, the pharynx sucker of gastrointestinal trematodes is involved in capturing food and evacuating its undigested residues from the helminth's body to the outside. In addition, the substances produced by secretory cells of the pharynx carry out a partial chemical treatment of the food consumed by the parasite and protect the parasite from substances that are metabolites of the host tissues and waste products of symbionts and commensals that inhabit the rumen of ruminants. Thus, trophic adaptation of the parasite in its ecological niche, the rumen of polygastric mammals, is ensured.

Histochemical features of the structure of fetal ovaries in different stages of gestation from mothers with physiological pregnancy

In the following article specific hystochemical features of the fetal ovaries’ structure from mothers with the physiological pregnancy are considered. All fetals were divided into subgroups by taking into consideration the term of gestation, as well as main stages of fetal gonads’ formation and laying. Namely: 21-28 weeks, 29-36 weeks, 37-40 weeks. All fetuses had died intranatally, as a result of acute uterine-placental circulation. The course of pregnancy in all cases was psychological, according to appropriate medical documentation. In the aim of reaching the scientific goal the following research methods were used: macroscopic, organometric, histological, histochemichal, morphometric, statistical. The comprehensive study has revealed the following features of the ovaries’ structure, depending on gestational term: weight, length, thickness, width and volume of the ovaries were reaching their minimum levels in case of fetuses with a gestational age of 21-28 weeks, while the maximum one was reached in case of fetuses on 37-42 weeks of gestation. All gonads are represented by cortical and cerebral matter, with the constant ratio despite of different gestational term. According to the growth of pregnancy term the number of germ cells decreases, while the number of apoptotically altered forms oppositely increases. Primarily it is because of the psychological cells’ death during ovarian formation.The follicular component at all stages of gestation is represented by primordial and primary follicles; at the 37-40 weeks though the atretic forms and cystically altered specimens are appearing. Moreover, in accordance with the growth of the pregnancy term, the number of primordial follicles decreases, while oppositely, the number of primary ones increases. The aforementioned changes lead to the decrease in the relative volume of follicular tissue and to the increase of the relative volume of the interstitial component. By the following histochemical methods (Felgen-Rossenbeck reaction, Brache reaction, Schiff-reaction) an increase in the functional activity of the fetal ovaries was revealed, by reaching its maximum point during gestation of the 37-40 weeks. The aforementioned features correspond with gestational terms as well as with stages of the ovarian development of the fetus. That is why they could be used as a control group during leading a research on characteristical features of fetal gonads structure of fetuses from mothers with complicated pregnancies.

Super-Resolution Fluorescence Imaging of Arabidopsis thaliana Transfer Cell Wall Ingrowths using Pseudo-Schiff Labelling Adapted for the Use of Different Dyes

To understand plant growth and development, it is often necessary to investigate the organization of plant cells and plant cell walls. Plant cell walls are often fluorescently labeled for confocal imaging with the dye propidium iodide using a pseudo-Schiff reaction. This reaction binds free amine groups on dye molecules to aldehyde groups on cellulose that result from oxidation with periodic acid. We tested a range of fluorescent dyes carrying free amine groups for their ability to act as pseudo-Schiff reagents. Using the low-pH solution historically used for the Schiff reaction, these alternative dyes failed to label cell walls of Arabidopsis cotyledon vascular tissue as strongly as propidium iodide but replacing the acidic solution with water greatly improved fluorescence labeling. Under these conditions, rhodamine-123 provided improved staining of plant cell walls compared to propidium iodide. We also developed protocols for pseudo-Schiff labeling with ATTO 647N-amine, a dye compatible for super-resolution Stimulated Emission Depletion (STED) imaging. ATTO 647N-amine was used for super-resolution imaging of cell wall ingrowths that occur in phloem parenchyma transfer cells of Arabidopsis, structures whose small size is only slightly larger than the resolution limit of conventional confocal microscopy. Application of surface-rendering software demonstrated the increase in plasma membrane surface area as a consequence of wall ingrowth deposition and suggests that STED-based approaches will be useful for more detailed morphological analysis of wall ingrowth formation. These improvements in pseudo-Schiff labeling for conventional confocal microscopy and STED imaging will be broadly applicable for high-resolution imaging of plant cell walls.

STRUCTURE OF SOME STRIGEATAS' ORGAN OF BRANDES

The purpose of the research: the analysis of histology and histochemistry of the Brandes organ of Strigea strigis and Alaria alata. The trematode marites of Strigea striges and Alaria alata were the material. Fixation of the material was carried out in 10% neutral formalin. The treatment of the specimens was carried out using the conventional histological and histochemical methods: with hematoxylin-eosin according to the method of Van Gizon, Mallory, with Sudan black B, sulema-Bromphenol blue according to Bonhage, with periodic acid Schiff reaction by Mac-Manus, alcian blue according to Stedman and Mowry and with toluidine blue. Histochemical reactions were performed with appropriate controls. The studies have shown that the structure of the Brandes organ of Strigea strigis and Alaria alata differs by their constituents and morphology of glandular cells. The histochemical reactions are similar. The cells of the glandular complex show bromophenolophilia, toluudinophilia and fuchsinophilia in periodic acid Schiff reaction speaks about the glycoprotein nature of the secreted substances. Bromphenophilia and sudanophilia of glandular cells cytoplasm indicate the presence of lipoprotein substances in them. The Brandes of S. strigis and A. alata is a morphofunctional unit, to which the principle of multi-functionality is inherent. It performs the main function – digestion of food components by means of developed glandular structures and specialized secretory activity. Its ability to fix the helminth tightly in the endostatin can be considered a secondary function of the organ.

A Transplantable Syngeneic Allograft Mouse Model for Nongestational Choriocarcinoma of the Ovary

Nongestational choriocarcinoma is a rare malignancy in humans with poor prognosis. Naturally occurring choriocarcinoma is also rare in laboratory mice, and no genetic mouse model accurately recapitulates the features of this cancer. Here we report development of a genetically engineered mouse (GEM) model with alterations in Brca2, Trp53, and RB that develops ovarian tumors. Most of the ovarian tumors displayed histological characteristics of nongestational choriocarcinoma of the ovary (NGCO) (47%) with abundant syncytiotrophoblasts and cytotrophoblasts, positive immunolabeling for human chorionic gonadotropin, and positive periodic acid–Schiff reaction. The rest of the ovarian tumors were serous epithelial ovarian carcinoma (SEOC) (26%) or mixed tumors consisting of NGCO and SEOC (26%). We further established syngeneic orthotopic mouse models for NGCO by in vivo passaging of GEM tumors. These metastatic models provide a platform for evaluating new treatment strategies in preclinical studies aimed at improving outcomes in choriocarcinoma patients.

Synthesis of N,N-Diethyl, N-Methyl Chitosan Chloride with Certain Quaternization Degree and Molecular Spectroscopic and Thermo-Morphological Study of the Alkylation

The quartenizeid chloride derivative of natural polyaminosaccharide chitosan was synthesized in two stages with acetate aldehyde and methyl iodide chemical reaction and ion replacement, which could be soluble in the water and wide pH ranges. The synthesis of the homopolymer was initially carried out with acetate aldehyde in Schiff reaction, and reduction was held on with the presence of NaBH4. The quaternization was accomplished in the acetonitrile medium with methyl iodine by continuous exposure of N2.7-8% quartenized N,N-diethyl, N-methyl chitosan iodine were synthesized with 89-91% yield, obtained by deprotonation of amine groups, with reaction of CH3J and N,N-diethyl chitosan. The ion exchange was carried out at 10% NaCl solution during 24 hours and N,N-diethyl, N-methyl chitosan chloride was obtained. Synthesis was performed with simpler and chemically effective methods compared to previous studies. The structure of product was characterized by FT-IR, UV-Vis, NMR, SEM, TGA, DTA and elemental analysis was determined. Functional changes in the structure of macromolecules were monitored with NMR and UV-Vis, and it was proved that, the main intermediate product was composed to be N,N-diethyl carbocation carrying >C=N-chromophore group. The increasing percent of carbon in content while alkylation is depeering and the presence of halogenated ions (Cl-or J-) after quaternization were observed. It has been determined that, the solubility of N,N-diethyl,N-methyl chitosan chloride or iodide in water and in pH =1-10 increased frequently. Key words. Chitosan; alkylation; diethylmethyl chitosan iodine; quartenization; UV-Vis; NMR

Activation and activity of glycosylated KLKs 3, 4 and 11

Human kallikrein-related peptidases 3, 4, 11, and KLK2, the activator of KLK3/PSA, belong to the prostatic group of the KLKs, whose major physiological function is semen liquefaction during the fertilization process. Notably, these KLKs are upregulated in prostate cancer and are used as clinical biomarkers or have been proposed as therapeutic targets. However, this potential awaits a detailed characterization of these proteases. In order to study glycosylated prostatic KLKs resembling the natural proteases, we used Leishmania (LEXSY) and HEK293 cells for secretory expression. Both systems allowed the subsequent purification of soluble pro-KLK zymogens with correct propeptides and of the mature forms. Periodic acid-Schiff reaction, enzymatic deglycosylation assays, and mass spectrometry confirmed the glycosylation of these KLKs. Activation of glycosylated pro-KLKs 4 and 11 turned out to be most efficient by glycosylated KLK2 and KLK4, respectively. By comparing the glycosylated prostatic KLKs with their non-glycosylated counterparts from Escherichia coli, it was observed that the N-glycans stabilize the KLK proteases and change their activation profiles and their enzymatic activity to some extent. The functional role of glycosylation in prostate-specific KLKs could pave the way to a deeper understanding of their biology and to medical applications.

Histochemical study of Encephalitozoon cuniculi spores in the kidneys of naturally infected New Zealand rabbits

Encephalitozoon cuniculi is an important microsporidian pathogen that is considered an emergent, zoonotic, and opportunistic. It infects both domestic and laboratory rabbits, generating severe chronic interstitial and granulomatous nephritis with fibrosis and granulomatous encephalitis. Encephalitozoonosis is diagnosed in paraffin-embedded sections by examining the spores in the host tissues. The spores are difficult to observe when the samples are stained with hematoxylin and eosin (H&E), particularly when there is an inflammatory reaction and tissue damage. The spores are easily mistaken for other microorganisms, such as fungi (yeasts), protozoa, and bacteria. In our study, we used kidney samples from E. cuniculi–positive rabbits and employed 14 recommended histologic stains for detecting microsporidia spores: alcian blue, calcofluor white, Giemsa, Gram, Grocott, H&E, Luna, Luxol fast blue, Masson trichrome, modified trichrome stain (MTS), periodic acid–Schiff reaction (PAS), Van Gieson, Warthin–Starry (WS), and Ziehl–Neelsen (ZN).We concluded that MTS and Gram stain, detected by light microscopy, and calcofluor white stain, detected by ultraviolet light microscopy, are the best stains for detecting spores of E. cuniculi in paraffin-embedded tissues from infected rabbits. These stains were superior to WS, ZN, Giemsa, and PAS for identifying spores without background “noise” or monochromatic interference. Also, they allow individual spores to be discerned in paraffin-embedded tissues. MTS allows observation of the polar tube, polaroplast, and posterior vacuole, the most distinctive parts of the spore.