[Characteristics of δ-Aminolevulinic Acid Dehydratase of the Cold-Water Sponge Halisarca dujardinii].

δ-Aminolevulinic acid dehydratase (ALAD) is a key enzyme of the cytoplasmic heme biosynthesis pathway. The primary structure of the ALAD gene, the multimeric structure of the ALAD/hemB protein, and ALAD expression during the annual reproductive cycle were studied in the cold-water marine sponge Halisarca dujardinii. The results implicated the GATA-1 transcription factor and DNA methylation in regulating ALAD expression. Re-aggregation of sponge cells was accompanied by a decrease in ALAD expression and a change in the cell content of an active ALAD/hemB form. Further study of heme biosynthesis and the role of ALAD/hemB in morphogenesis of basal animals may provide new opportunities for treating pathologies in higher animals.

Сell clusters isolation in glioblastomas and their functional and molecular characterization using new morphometric approaches.

BACKGROUND: Digital pathology has come a long way in terms of creating tools to improve existing diagnostic approaches. However, several pathology fields, such as neuropathology, are still characterized by low coverage from machine learning tools and neural network analysis, which may be due to the complexity of the internal cellular and molecular structure of the corresponding neoplasms, including glioblastomas. METHOD: In the framework of this study, using advanced proprietary tools for obtaining images of histological slides and their deep morphometric analysis, we studied samples of 198 patients with glioblastoma with the selection of morphometric cell clusters. Also, cells of each cluster were isolated, and their proliferative, migratory, invasive activity, survival ability, aerobic glycolysis activity, and chemo- and radioresistance were studied. RESULTS: Four morphometric clusters were identified, including small-cell cluster, paracirculonuclear cluster, hypochromic cluster, and macronuclear cluster, which significantly differed in morphometric parameters and functional parameters. Hypochromic cluster cells demonstrated the highest proliferation activity; macronuclear cluster was the most active glucose consumer; paracirculonuclear cluster had the most prominent migratory and invasive activity and hypoxia resistance; small-cell cluster demonstrated predominantly average values of all parameters. Moreover, additional analysis revealed the presence of a separate subcluster of stem cell elements that correspond in their molecular properties to glioma stem cells and are present in all four clusters. It also turned out that several key molecular parameters of glioblastoma, such as mutational modifications in the EGFR, PDGFRA, and NF1 genes, along with the molecular GBM subtype, are significantly correlated with the identified cell clusters. CONCLUSIONS: Thus, the results represent an up-and-coming innovation in the practical field of digital pathology and fundamental questions of glioma carcinogenesis.

[Structure of Neuroglobin from Cold-Water Sponge Halisarca dujardinii].

The iron-containing protein neuroglobin (Ngb) involved in the transport of oxygen is generally considered the precursor of all animal globins. In this report, we studied the structure of Ngb of the cold-water sponge Halisarca dujardinii. In sponges, the oldest multicellular organisms, the Ngb gene contains three introns. In contrast to human Ngb, its promoter contains a TATA-box, rather than CG-rich motifs. In sponges, Ngb consists of 169 amino acids showing rather low similarity with its mammalian orthologues. It lacks Glu and Arg residues in positions required for prevention of hypoxia-related apoptosis. Nevertheless, Ngb contains both proximal and distal conserved heme-biding histidines. The primary structure of H. dujardinii neuroglobin predicted by sequencing was confirmed by mass-spectrometry analysis of recombinant Ngb expressed in E. coli. The high level of Ngb expression in sponge tissues suggests its possible involvement in the gas metabolism and presumably in other key metabolic processes in H. dujardinii.

Changes in the Activity of Genes Involved in the Regulation of Hematopoiesis during Tumorigenesis in Irradiated Mice.

We used 183 F1 CBA×C57Bl hybrid mice to study the delayed effects of low-power long-term γ-irradiation at a dose of 12.6 Gy (10 mGy/min) 8 and 10 months after the treatment. Eight months after the treatment we found the increased expression of the transcription factor NFκB and its target genes iNOS and G-SCF in the bone marrow (BM). Ten months after the treatment malignant lymphomas were revealed in 14 of 94 mice in the liver, abdominal cavity, and subcutaneously. In the BM of these mice, the transcription of the PTEN, NFκB, and iNOS genes was inhibited and the contents of long non-coding RNAs (lncRNAs) lnc p21, NEAT1, and microRNA miR-125b were decreased. The expression of the NFκB(p65) gene and miR-125b was inhibited in the BM of irradiated mice without tumors ten months after the treatment. These data show the deregulation of the P53 system supporting the genome stability in the BM of irradiated mice. These indices will be studied as potential markers of risk of development of irradiation-induced tumors.

[Myofascial pain syndrome in the dysfunction of the temporomandibular joint]. / Miofastsial'nyi bolevoi sindrom pri disfunktsii visochno-nizhnecheliustnogo sustava.

AIM: Selection of the clinical characteristics of pain syndrome in the orofacial region in temporomandibular joint pain dysfunction syndrome (TMJ PDS). MATERIAL AND METHODS: One hundred and two patients with TMJ PDS were examined using the Verbal Descriptive Pain Rating Scale of the orofacial area. The Clinical Index of TMJ dysfunction (CID) by Helkimo M. was used for objectification of the level of musculo-tonic disorders in the area of the chewing muscles. The psychopathological status of patients with TMJ PDS was assessed with HRDS and SCL-90-R. RESULTS: A significant proportion of patients with TMJ PDS, in addition to algic symptoms, have hypertonus of the facial muscles, the nature and severity of which require not only a special diagnostic assessment, but also follow-up treatment. The analysis of data allowed us to identify three clinical groups of patients: with isolated (muscular-tonic), neurological symptoms; with mixed (combination of muscular-tonic and somatoform manifestations) neurological and psychopathological symptoms and with somatoform symptoms. CONCLUSION: Patients with TMJ PDS are clinically heterogeneous and require clinical differentiation, distinguishing the common group of patients with muscular dysfunction accompanied by pain and emotional response to it (anxiety and minor depressive symptoms) and the group of patients with a somatoform disorder (and manifestations as general anxiety).

[Early postoperative stabilometric training in patients with spinal diseases who underwent decompressive-stabilizing interventions]. / Stabilometricheskii trening v rannem posleoperatsionnom periode u patsientov s zabolevaniiami pozvonochnika, perenesshikh dekompressivno-stabiliziruiushchie vmeshatel'stva.

AIM: To study the impact of rehabilitation measures on the dynamics of pain syndrome and on the state of postural muscle balance in the early postoperative period in patients with degenerative-dystrophic diseases of the lumbar spine who have undergone decompressive-stabilizing interventions. MATERIAL AND METHODS: This paper comparatively analyzed the level of pain syndrome in the lumbar spine and lower extremities, the stabilometric indicators characterizing the amplitude of center-of-pressure oscillations in patients who had undergone decompressive-stabilizing operations at 7±2 days after surgical treatment for degenerative-dystrophic diseases of the lumbar spine. Two groups of 60 people in each were formed: rehabilitation measures were implemented, starting on day 2 of an early postoperative period (the duration of the latter was 7±2 days after surgery); the basic complex was supplemented with stabilometric training in Group 1 (a study group); only the basic complex was used in Group 2 (a comparison group). RESULTS: In the study group, the frequency of rehabilitation outcomes with an achieved excellent result depending on the level of pain syndrome in the lower extremities and spine on a visual analogue scale was 62.5 and 88.9%, respectively; which significantly statistically exceeds the proportion of patients with the same outcome in the comparison group (37.5 and 11.1%; p<0.01 in both cases). There was a statistically significant more pronounced decrease in the level of pain syndrome in the lumbar spine (p=0.0001) and lower limbs (p=0.003) in the patients of the study group in the early postoperative period. Intergroup comparison revealed a statistically significant decrease in all the indicators monitored in the study, which characterize the amplitude of the center-of-pressure oscillations. Moreover, the value of the oscillation area parameter between the patient groups differed by 1.76 and 1.83 times during Romberg's test with the eyes open or closed, respectively (p=0.0001). CONCLUSION: The findings suggest that the comprehensive treatment supplemented with stabilometric training in the early postoperative period improves the state of postural muscle balance. There was a statistically significant predominance of compliance with normative values in patients of the study group during Romberg's test with the eyes open or closed (p=0.007 and p=0.00002, respectively), which contributes to a more marked decrease in the level of pain syndrome in the lumbar spine (p=0.001) and lower extremities (p=0.003).

[The risk of suicidal behavior in patients with epilepsy]. / Risk razvitiia suitsidal'nogo povedenia u bol'nykh épilepsiei.

AIM: To study a role of psychopathological and social factors in the development of suicidal behavior in patients with different forms of epilepsy. MATERIAL AND METHODS: One hundred and nineteen patients with epilepsy, 57 men and 62 women, mean age 40.7±0.54 years, were studied using neurological and psychiatric examinations, along with a large battery of scales for assessment patient's state and suicidal behavior. RESULTS AND CONCLUSION: Clinical and psychological risk factors for suicidal behavior as well as for motivation of suicide prevention are identified. The formation of psychopathology determines the model of personality changes associated with epilepsy and the patients' response to social exclusion and stigma that lead to suicide.

[A comparative study of the efficacy of personality-oriented (reconstructive) and cognitive-behavioral psychotherapy in neurotic anxiety disorders with insomnia]. / Sravnitel'noe issledovanie éffektivnosti lichnostno-orientirovannoi (rekonstruktivnoi) i kognitivno-povedencheskoi psikhoterapii pri trevozhnykh rasstroistvakh nevroticheskogo urovnia s insomniei.

AIM: To study personality-oriented (reconstructive) and cognitive-behavioral psychotherapy in patients with neurotic anxiety disorders with insomnia. MATERIAL AND METHODS: Ninety-eight patients with disorders coded in ICD-10 as F 40.0; F40.1; F41.0; F41.1; F48.0 were studied. Clinical, psychometric, experimental-psychological methods and polysomnography were used. RESULTS AND CONCLUSION: Phenomenological insomnia types characteristic of neurotic anxiety disorders and their relationship with types of intrapersonal conflicts are described. A quantitative analysis of the dynamics of symptomatic complains, anxiety level, and insomnia is presented. The efficacy of the both psychotherapeutic treatments is shown. However the personality oriented (reconstructive) psychotherapy has demonstrated better and more stable results. This can be explained first of all by its pathogenesis-oriented nature and involvement of the cognitive emotional and behavioral spheres as well as the resolution of the intrapersonal conflicts, which are the cornerstone of neurotic disorders.

The Role of Epibionts of Bacteria of the Genus Pseudoalteromonas and Cellular Proteasomes in the Adaptive Plasticity of Marine Cold-Water Sponges.

It was found that cells of different color morphs of the cold-water marine sponges Halichondria panicea (Pallas, 1766) of the class Demospongiae differ in the content of epibionts of bacteria of the genus Pseudoalteromonas. The sponge cells with elevated levels of epibionts of bacteria of the genus Pseudoalteromonas showed an increased expression of Hsp70 proteins but had a reduced level of the proteasomal catalytic beta 5 subunit, which was accompanied by a change in their activity. Probably, epibionts of bacteria of the genus Pseudoalteromonas may affect the ubiquitin-proteasome system in the cells of cold-water marine sponges and, thereby, ensure their adaptive plasticity.

Time Dependence of the Electron and Positron Components of the Cosmic Radiation Measured by the PAMELA Experiment between July 2006 and December 2015.

Cosmic-ray electrons and positrons are a unique probe of the propagation of cosmic rays as well as of the nature and distribution of particle sources in our Galaxy. Recent measurements of these particles are challenging our basic understanding of the mechanisms of production, acceleration, and propagation of cosmic rays. Particularly striking are the differences between the low energy results collected by the space-borne PAMELA and AMS-02 experiments and older measurements pointing to sign-charge dependence of the solar modulation of cosmic-ray spectra. The PAMELA experiment has been measuring the time variation of the positron and electron intensity at Earth from July 2006 to December 2015 covering the period for the minimum of solar cycle 23 (2006-2009) until the middle of the maximum of solar cycle 24, through the polarity reversal of the heliospheric magnetic field which took place between 2013 and 2014. The positron to electron ratio measured in this time period clearly shows a sign-charge dependence of the solar modulation introduced by particle drifts. These results provide the first clear and continuous observation of how drift effects on solar modulation have unfolded with time from solar minimum to solar maximum and their dependence on the particle rigidity and the cyclic polarity of the solar magnetic field.

[WEAK COMBINED MAGNETIC FIELDS ADJUSTED TO THE PARAMETRIC RESONANCE FOR Ca2+ INTENSIFY DYSTROPHIN SYNTHESIS IN MDX MICE SKELETAL MUSCLES AFTER CELL THERAPY].

The mdx mice are an X-linked myopathic mutants, an animal model for human Duchenne muscular dystrophy (DMD). Mdx mice muscles are characterized by high level of striated muscle fibers (SMF) death followed by regeneration. As a result most SMFs of mdx mice have centrally located nuclei. The possibility of using stem cells therapy for the correction of DMD is actively being studied. One of the approaches to the usage of bone marrow stem cells for cellular therapy of DMD is the replacement of bone marrow after irradiation by X-rays. This method however does not give significant increase of dystrophin synthesis in mdx mice muscles fibers. We have tried to affect the mice after bone marrow transplantation by weak combined magnetic fields adjusted to the parametric resonance for Ca2+(Ca(2+)-MF) based on the data that the weak combined magnetic fields influence on tissues regeneration. We observed a significant increase in the proportion of dystrophin-positive SMFs in group of mdx mice radiation chimera 5 Gy and 3 Gy which was additionally exposed in Ca(2+)-MF in comparison with the control mdx mice and the group of mdx mice radiation chimera 5 Gy and 3 Gy which was kept in terrestrial magnetic field 2 months after chimera preparation--up to 15.8 and 18.3%, respectively. Also, there was an accumulation of SMFs without central nuclei. These data indicate a significanly increased efficacy of cell therapy in the case of additional exposition in Ca(2+)-MF. Thus, the efficiency of bone marrow transplantation mdx mice after both in doses 3 and 5 Gy was considerably enhanced by additional exposition to Ca(2+)-MF. Apparently, such magnetic field can intensify functioning of donor's nuclei which had been incorporated into muscle fibers.

Role of proteasomes in non-specific immune response of marine annelids.

We investigated functioning of proteasomes and chaperones in Arenicola marina coelomocytes in conditions of lipopolysaccharide-induced inflammation. We observed the increase of chymotrypsin-like proteasome activity in coelomocytes 1 h after induction. Amount of proteasome subunits alpha- and beta-5 types increased as well. We also detected appearance of a new form of Hsp70 chaperone in infected coelomocytes. Our results allow us to consider the changes in proteasome structure and induction of chaperones as principle mechanisms in stress adaptation and defensive reactions development in annelids.

CHARACTERISTICS OF AMYLOID DEPOSITS DETECTED IN THE INTERNAL ORGANS OF mdx MICE.

The dystrophin-deficient mdx mouse is the most commonly used experimental model of Duchenne muscular dystrophy (DMD). Although the amyloid has been shown in the muscle biopsies of patients with different types of muscular dystrophies, there are no data on the amyloid accumulations in the biopsy of DMD patients or mdx mouse. Therefore, the aim of the present study was to testify the hypothesis of probable accumulation of amyloid in the visceral organs of mdx mouse. Specimens of myocardium, kidneys, and liver of male and female mdx mice aged from 2 months to 1.5 years (n = 9) were used in the study. The histochemical staining with Congo red demonstrated amyloid accumulations in the studied organs of the mdx mice. Morphology and localization of the found accumulations were described in details and analyzed. The mass-spectrometric study determined the vitronectin and apolipoprotein A-II as the most probable components of the amyloid accumulations in the mdx mouse.

Investigation of Thionins from Black Cumin (Nigella sativa L.) Seeds Showing Cytotoxic, Regulatory and Antifungal Activity.

Thionins (NsW1 and NsW2), earlier isolated from the seeds of endemic Middle-Asian black cumin (Aligella sativa L.), showing signilicant inhibitory action on some bacterial and yeast pathogens were investigated for cytotoxic properties against several tumor cell lines (AsPC-1, Colo357, RD and Jukart) in vitro within nano- and micromolar ranges of the active concentrations and as modulators of expression of the genes controlling conversion of normal cells to malignant ones. Suppression of the expression of the genes from MMP, RhoA, miR21 families in human rhabdomyosarcoma (RD) cells was observed, whereas the influence of the molecules on the genes in normal blood cells was not identified. It was shown that the thionins from black cumin induced almost 90% of the cell death in RD and Jukart lines. Moreover, the polypeptides inhibited clinical isolates of Aspergillus ochraceus and A.fimigatus at the level comparable with that of amphotericin B. The data demonstrated that the peptides could be considered as perspective antitumor and antimycotic agents.

[Searching Radiation Countermeasures using the Model of Prolonged Irradiation of Mice with Low Dose Rate and Evaluation of Their Influence on Heat Shock Protein Genes Expression].

Different radiomodificators (cytokine betaleukine, antioxidant phenoxan, antigipoksant limontar and nucleoside riboxin) were investigated on mice for evaluating their radiation protective capacity against prolonged (21 h) exposure at a dose of 12.6 Gy at a low dose rate of 10 mGy/min. Bone marrow cellularity and endogenic CFUs were used as evaluation criteria 9 days after exposure. Simultaneously, expression of the heat shock proteins of 25, 70 and 90 kDa in unexposed mice bone marrow was studied 2, 24 and 48 h after injections. Betaleukine only had a positive significant effect in both tests in the variants of 50 mcg/kg and 3 mcg/kg when administered 2 h and 22 h before exposure, correspondingly. Effects of betaleukine HSPs on expression were both stimulating and inhibiting, that was in contradiction with a constant positive effect in 5 experiments on exposed mice for each betaleukine variant. It argues against the vital role of HSPs in the betaleukine antiradiation effect. In 2 experiments with high temperatures betaleukine administered at a dose of 50 mcg/kg evoked a very high HSP-70 gene expression after 24 h, and mice exposed to irradiation at that time in a parallel experiment showed an increased radiation effect. It corresponds to the idea that HSPs serve a stress indicator.

New upper limit on strange quark matter abundance in cosmic rays with the PAMELA space experiment.

In this work we present results of a direct search for strange quark matter (SQM) in cosmic rays with the PAMELA space spectrometer. If this state of matter exists it may be present in cosmic rays as particles, called strangelets, having a high density and an anomalously high mass-to-charge (A/Z) ratio. A direct search in space is complementary to those from ground-based spectrometers. Furthermore, it has the advantage of being potentially capable of directly identifying these particles, without any assumption on their interaction model with Earth's atmosphere and the long-term stability in terrestrial and lunar rocks. In the rigidity range from 1.0 to ∼1.0×10^{3} GV, no such particles were found in the data collected by PAMELA between 2006 and 2009. An upper limit on the strangelet flux in cosmic rays was therefore set for particles with charge 1≤Z≤8 and mass 4≤A≤1.2×10^{5}. This limit as a function of mass and as a function of magnetic rigidity allows us to constrain models of SQM production and propagation in the Galaxy.

[Comparative analysis of natural and synthetic antimutagens as regulators of gene expression in human cells under exposure to ionizing radiation].

This paper studies the effect of plant peptides of thionine Ns-W2 extracted from seeds of fennel flower (Nigella sativa) and ß-purothionine from wheat germs (Triticum kiharae), as well as a synthetic antimutagen (crown-compound), on the expression of several genes involved in the.control of cellular homeostasis, processes of carcinogenesis, and radiation response in human rhabdomyosarcoma cells (RD cells), T-lymphoblastoid cell line Jurkat, and blood cells. All of these agents acted as antimutagens-anticarcinogens, reducing the expression of genes involved in carcinogenesis (genes of families MMP, TIMP, and IAP and G-protein genes) in a tumor cell. A pronounced reduction in the mRNA level of these genes was caused by thionine Ns-W2, and the least effect was demonstrated by ß-purothionine. Antimutagens had very little effect on the mRNA levels of the several studied genes in normal blood cells.

Rheinheimera japonica sp. nov., a novel bacterium with antimicrobial activity from seashore sediments of the Sea of Japan.

Two gram-negative, aerobic, brown-pigmented, motile rod-shaped bacteria KMM 9512 and KMM 9513(T) were isolated from a sediment sample collected from the Sea of Japan seashore, Russia. On the basis of the 16S rRNA gene sequence analysis, novel strains KMM 9512 and KMM 9513(T) positioned within the genus Rheinheimera (class Gammaproteobacteria) as a separate subline adjacent to Rheinheimera baltica DSM 14885(T) sharing highest gene sequence similarities of 98.6-97.6 % to their closest phylogenetic relatives, Rheinheimera muenzenbergensis LMG 27269(T), R. baltica DSM 14885(T), Rheinheimera aquimaris JCM 14331(T), Rheinheimera nanhaiensis KACC 14030(T), and Rheinheimera pacifica KMM 1406(T). Strains KMM 9512 and KMM 9513(T) belong to the same separate genospecies on the basis of the 16S rRNA gene sequence similarity (99.8 %) and their DNA relatedness to each other (89 %) and to closely related Rheinheimera species (25-53 %). The major isoprenoid quinone was Q-8, polar lipids consisted of phosphatidylserine, phosphatidylglycerol, phosphatidylethanolamine, unknown aminolipids, unknown aminophospholipids, unknown phospholipids, and unknown lipids, and major fatty acid were C16:0, C16:1 ω7c, C17:1 ω8c, C12:0 3-OH followed by C17:0 and C18:1 ω7c in both strains. Strains KMM 9512 and KMM 9513(T) revealed a remarkable antagonistic activity toward a number of gram-positive and gram-negative microorganisms. On the basis of phylogenetic analysis, DNA-DNA hybridization results, and phenotypic differences, strains KMM 9512 and KMM 9513(T) are proposed to be classified as a novel species of the genus Rheinheimera, Rheinheimera japonica sp. nov. The type strain of this species is KMM 9513(T) = NRIC 0918(T).

[A Simple and Efficient Method of Inducing Targeted Deletions in the Drosophila Genome].

Deletion mutagenesis is one of the most efficient approaches to studying gene function. However, conventional methods of inducing targeted mutations in the drosophila genome are time- and labor-consuming. This work proposes a new, simple, and effective method of producing drosophila mutants with gene deletions. The method involves the insertion of I-Scel and I-CreI recognition sites and a fragment homologous to the target sequence into the chromosome region of interest by means of an attB-containing construct, the induction of double-strand DNA breaks by the appropriate meganuclease, and their repair by homologous recombination. The procedure results in a deletion extending from the attP-site to the target locus. A cassette was designed to enable single-step construct production for the deletion of any given genomic region. A set of markers facilitates the selection of recombination events. The efficacy of the proposed technique was confirmed by the induction of a 47-kb deletion containing the qtc gene.

[Effect of bone marrow cells transplantation on the decidua formation in pseudopregnant rats].

One of the most common causes of the current pregnancy loss is the failure of the decidual reaction of endometrial cells. It is assumed that a partial source of decidual cells in endometrial tissue is bone marrow cells (BMCs). In the present work, we have studied possible effect of BMCs transplantation on the process of decidualization using the model of pseudopregnancy in rats. BMCs were flushed from the rat femurs and tibias. The obtained suspension of single BMCs was injected into one of rat uterine horns on the 5th day of pseudopregnancy. PBS without cells was injected into the contralateral horn served as the control. Rats were sacrificed on the 11th day of pseudopregnancy. Decidua formed in the experimental uterine horn showed an increase in the meso-antimezometral direction of their diameter of about 1.5-2 times as compared with a control horn. The weight of decidual tissue in the experimental horn exceeded 3 times the weight of the control one. The presence of transplanted BMCs in decidual tissue was documented by preliminary double staining of BMCs with membrane dye PKH 26 Red and nuclear dye Hoechst 33342. Histological analysis of decidua sections after transplantation revealed any alterations neither in cell differentiation nor in tissue structure. We conclude that BMCs transplantation stimulates decidualization in animals.