A spontaneous missense mutation in the chromodomain helicase DNA-binding protein 8 (CHD8) gene: a novel association with congenital myasthenic syndrome.

AIMS: Congenital myasthenic syndromes (CMS) are characterized by muscle weakness, ptosis and episodic apnoea. Mutations affect integral protein components of the neuromuscular junction (NMJ). Here we searched for the genetic basis of CMS in female monozygotic twins. METHODS: We employed whole-exome sequencing for mutation detection and Sanger sequencing for segregation analysis. Immunohistology was done with antibodies against CHD8, rapsyn, ß-catenin (ßCAT) and golgin on fi-bro-blasts, human and mouse muscle. We recorded superresolution images of the NMJ using 3D-structured illumination microscopy. RESULTS: We discovered a spontaneous missense mutation in CHD8 [chr14:g.21,884,051G>A, GRCh37.p11 | c.1732C>T, NM_00117062 | p.(R578C)], the gene encoding chromodomain helicase DNA-binding protein 8. This is the first missense mutation affecting Duplin, the short 110 kDa isoform of CHD8. It is known that CHD8/Duplin negatively regulates ßCAT signalling in the WNT pathway and plays a role in chromatin remodelling. Inactivating CHD8 mutations are associated with autism spectrum disorder and intellectual disability in combination with facial dysmorphism, overgrowth and macrocephalus. No muscle-specific phenotype has been reported to date. Co-immunostaining with rapsyn on human and mouse muscle revealed a strong presence of CHD8 at the NMJ being located towards the sarcoplasmic side of the rapsyn cluster, where it co-localizes with ßCAT. CONCLUSION: We hypothesize CHD8 to have a role in the maintenance of the structural integrity and function of the NMJ. Both patients benefited from treatment with 3,4-diaminopyridine, a reversible blocker of voltage-gated potassium channels at the nerve terminal that prolongs the action potential and increases acetylcholine release.

Live-imaging of revertant and therapeutically restored dystrophin in the DmdEGFP-mdx mouse model for Duchenne muscular dystrophy.

BACKGROUND: Dmdmdx , harbouring the c.2983C>T nonsense mutation in Dmd exon 23, is a mouse model for Duchenne muscular dystrophy (DMD), frequently used to test therapies aimed at dystrophin restoration. Current translational research is methodologically hampered by the lack of a reporter mouse model, which would allow direct visualization of dystrophin expression as well as longitudinal in vivo studies. METHODS: We generated a DmdEGFP-mdx reporter allele carrying in cis the mdx-23 mutation and a C-terminal EGFP-tag. This mouse model allows direct visualization of spontaneously and therapeutically restored dystrophin-EGFP fusion protein either after natural fibre reversion, or for example, after splice modulation using tricyclo-DNA to skip Dmd exon 23, or after gene editing using AAV-encoded CRISPR/Cas9 for Dmd exon 23 excision. RESULTS: Intravital microscopy in anaesthetized mice allowed live-imaging of sarcolemmal dystrophin-EGFP fusion protein of revertant fibres as well as following therapeutic restoration. Dystrophin-EGFP-fluorescence persisted ex vivo, allowing live-imaging of revertant and therapeutically restored dystrophin in isolated fibres ex vivo. Expression of the shorter dystrophin-EGFP isoforms Dp71 in the brain, Dp260 in the retina, and Dp116 in the peripheral nerve remained unabated by the mdx-23 mutation. CONCLUSION: Intravital imaging of DmdEGFP-mdx muscle permits novel experimental approaches such as the study of revertant and therapeutically restored dystrophin in vivo and ex vivo.

Adgrf5 contributes to patterning of the endothelial deep layer in retina.

Neovascularization of the inner retinal space is a major cause of vision loss. In retinal angiomatous proliferation (RAP) syndrome, newly formed vessels originate from the retinal plexus and invade the inner retinal space. However, the molecular pathways preventing subretinal vascularization remain largely unknown. In most murine models of RAP, pathological neovascularization occurs concomitantly with the development of the retinal vasculature. Here, we demonstrate that disturbing the sequence of morphogenetic events that shape the three-layered retinal vascular network leads to subretinal vascularization. Sprouts emerging from the perivenous region after the first postnatal week extended toward the retinal space where they merged into the deep layer. The small GTPase Rac1 was required for the formation of these vascular extensions and the vascular inner plexus is formed coaxially to the overarching veins. The adhesion receptor Adgrf5 was highly expressed in the endothelium of the central nervous system, where it regulates blood-brain barrier formation. The vascular superficial plexus of Adgrf5 mutant mouse retinae exhibited an increased vascular density in the perivenous areas with increased projections toward the inner plexus where they subsequently created hyper-dense endothelial cells (EC) clusters. Disturbing the perivenous pool of EC thus significantly altered the inner plexus formation. These abnormalities culminated in transient vascular protrusions in the inner retinal space. Taken together, these results reveal a previously unobserved vascular morphogenetic defect in Adgrf5 knockout mice, implicating a role for ADGRF5 in the initiation of subretinal vascularization. Our findings also illustrate how vein-derived EC shape the inner retinal layer formation and could control the appearance of angiomatous malformations.

Iodine supplementation activates folliculogenesis in rabbit ovary.

The aim of this study was to assess the biological effect of the product Jodis Concentrate (JC) on the rabbit ovaries by evaluating the folliculogenesis and expression of oocyte-specific growth differentiation factor 9 (GDF9).The experiment was conducted with 30 female two month old New Zealand rabbits that were the F1 offspring born to mothers differently treated with Jodis concentrate. The control group (n=10), consisted of F1 offspring born to mothers without iodine treatment, and was not supple- mented with JC. The first experimental group (n=10), consisted of F1 offspring born to mothers treated with JC during pregnancy and the suckling period, and was supplemented with JC daily at a dose of 2 ml/L drinking. The second experimental group (n=10), consisted of F1 offspring born to mothers without iodine treatment, and was also supplemented daily with the same dose of JC - 2 ml/L drinking. All groups were fed with total mixed ration for growing rabbits. The trial lasted 48 days. The ovaries were weighed and prepared for histological examination. The GDF9 protein expression in the ovary was determined by immunohistochemical analysis. The addition of JC to the drinking water of female rabbits led to more active development of the ovarian follicles from primordial to tertiary stage in both experimental groups. More intensive GDF9 protein expression in the oocytes and cumulus cells of rabbits, supplemented with JC was observed.

Epidemiological evidence for the non-random clustering of the components of the metabolic syndrome: multicentre study of the Mediterranean Group for the Study of Diabetes.

OBJECTIVE: To determine: (a) whether the components of metabolic syndrome (MetS) cluster more frequently than predicted by chance alone and (b) whether increased risk for MetS is associated also with values of each component below, but close to the cutoff points defining MetS. RESEARCH DESIGN AND METHODS: Anthropometrical and biochemical measurements were performed and a dietary questionnaire was filled-in in 1833 randomly selected non-diabetic subjects, 916 men and 917 women, 20-74 years old, in nine centres in five Mediterranean countries. The prevalence of MetS and of possible combinations of its individual components was measured. The expected frequencies of the above combinations were calculated according to the mathematical formula of probabilities. RESULTS: The overall prevalence of MetS was 27.2%, but varied greatly among countries, from 5.8% in Algeria to 37.3% in Greece. The observed prevalence of each combination diagnostic of MetS was higher than the expected by chance. Thus, the observed overall prevalence of MetS was also higher than the expected, 27.2 vs 24.0%, P=0.03. Furthermore, for each individual component (except high-density lipoprotein), as values in the normal range, approached the cutoff point, the risk of having MetS (i.e. clustering of the other components) increased significantly (odds ratio 2.2-4.6, P<0.001). CONCLUSIONS: The MetS is not related to the Mediterranean type of diet and its prevalence varies greatly among five Mediterranean countries. The clustering of the components defining the MetS is not due to chance and moreover even 'high normal' levels of each component confer increased risk for the syndrome.

Nutritional habits in the Mediterranean Basin. The macronutrient composition of diet and its relation with the traditional Mediterranean diet. Multi-centre study of the Mediterranean Group for the Study of Diabetes (MGSD).

OBJECTIVE: To compare the nutritional habits among six Mediterranean countries and also with the various official recommendations and the 'Mediterranean diet' as originally described. DESIGN: Cross-sectional study. SETTINGS: Three centres in Greece, two in Italy and one in Algeria, Bulgaria, Egypt and Yugoslavia. SUBJECTS: Randomly selected non-diabetic subjects from the general population, of age 35-60, not on diet for at least 3 months before the study. INTERVENTIONS: A dietary questionnaire validated against the 3-Day Diet Diary was used. Demographic data were collected and anthropometrical measurements done. RESULTS: All results were age adjusted. Energy intake varied in men, from 1825 kcal/day in Italy-Rome to 3322 kcal/day in Bulgaria and in women, from 1561 kcal/day in Italy-Rome to 2550 kcal/day in Algeria. Protein contribution (%) to the energy intake varied little, ranging from 13.4% in Greece to 18.5% in Italy-Rome, while fat ranged from 25.3% in Egypt to 40.2% in Bulgaria and carbohydrates from 41.5% in Bulgaria to 58.6% in Egypt. Fibre intake, g/1000 kcal, ranged from 6.8 in Bulgaria to 13.3 in Egypt and the ratio of plant to animal fat from 1.2 in Bulgaria to 2.8 in Greece. The proportion of subjects following the WHO and the Diabetes and Nutrition Study Group (DNSG) of the EASD recommendations for carbohydrates, fat and protein ranged from 4.2% in Bulgaria to 75.7% in Egypt. Comparison with the Mediterranean diet, as defined in the seven Country Study, showed significant differences especially for fruit, 123-377 vs 464 g/day of the Mediterranean diet, meat, 72-193 vs 35 g/day, cheese, 15-79 vs 13 g/day, bread, 126-367 vs 380 g/day. CONCLUSIONS: (a) Dietary habits of the 'normal' population vary greatly among the Mediterranean countries studied. (b) Egypt is closest to the DNSG recommendations. (c) Significant differences from the originally described Mediterranean diet are documented in most Mediterranean countries, showing a Westernization of the dietary habits.

T-cell receptor diversity prevents T-cell lymphoma development.

Mature T-cell lymphomas (MTCLs) have an extremely poor prognosis and are much less frequent than immature T-cell leukemias. This suggests that malignant outgrowth of mature T lymphocytes is well controlled. Indeed, in a previous study we found that mature T cells are resistant to transformation with known T-cell oncogenes. Here, however, we observed that T-cell receptor (TCR) mono-/oligoclonal mature T cells from TCR transgenic (tg) mice (OT-I, P14) expressing the oncogenes NPM/ALK or ΔTrkA readily developed MTCLs in T-cell-deficient recipients. Analysis of cell surface markers largely ruled out that TCR tg lymphomas were derived from T-cell precursors. Furthermore, cotransplanted non-modified TCR polyclonal T cells suppressed malignant outgrowth of oncogene expressing TCR tg T lymphocytes. A dominant role of an anti-leukemic immune response or Tregs in the control of MTCLs seems unlikely as naïve T cells derived from oncogene expressing stem cells, which should be tolerant to leukemic antigens, as well as purified CD4 and CD8 were resistant to transformation. However, our results are in line with a model in which homeostatic mechanisms that stabilize the diversity of the normal T-cell repertoire, for example, clonal competition, also control the outgrowth of potentially malignant T-cell clones. This study introduces a new innate mechanism of lymphoma control.

Mammary gland papilloma--diagnostic and therapeutic behavior.

Papillary lesions of the mammary gland represent a serious disease and incorporate a broad spectrum of lesions. The goal of the current article is to reveal the diagnostic methods and therapeutic behavior in patients with pathological discharge from the nipple of the mammary gland. In this article we present 15 cases of histologically proven mammary gland papilloma. Mammographic, ultrasonographic and cytologic methods are applied for diagnostic specification. The chosen therapeutic behavior in all of the women is surgical. We take into consideration the possibilities of the different diagnostic methods as well as the therapeutic behavior-conservative or surgical approach. We reckon that when there is suspicion for mammary gland papilloma a surgical intervention is necessary to be carried out--due to the functional discomfort and due to the possibility intraductal carcinoma in situ to be missed out in some cases.

[Scientometric characteristics of the published scientific works of Prof. Konstantin Chilov]. / Naukometrichna kharakteristika na publikuvanoto nauchno tvorchestvo na professor Konstantin Chilov.

A comprehensive scientificometric study was carried out on the publications of Corresponding member Prof Dr. K. Cilov, covering 127 monographs, textbooks and papers, published within the period 1929-1957. The results obtained were mathematically--statistically processed according to the methods of statistical grouping and alternative analysis and illustrated via table method and graphic analysis. The criteria of observation concern the quantitative, type and thematic characteristic of the co-authors of Prof K. Cilov. The analysis established an evenness in the scientific and publication creative work of Prof K. Cilov during the periods of his scientific maturing, his scientific interests directed mainly to the clinical-laboratory and cardiological problems, infectious and endocrine diseases. The percentage of his publications, with he as a sole author is 69,29%, and of team publications--30,71%. The personified co-authors are 32, and non-personified ones--65, with a predomination of the inconstant collaborators, so 70,87% of the co-authors are with a duration of the collaboration to 1 year.

[Serial publications of the World Health Organization. Scientifico-metric study]. / Seriini izdaniia na svetovnata zdravna organizatsiia naukometrichno prouchvane.

A comprehensive study was carried out on the serial editions of the World Health Organization, European Regional Bureau and of the International Agency of cancer studies for the period 1950-1981. A total of 1060 publications were analyzed concerning the type, quantitative characteristic and their thematic trends. The science-metric analysis revealed that the series "Technical reports" had the longest duration--32 years. A firm increase both of the number of the publications and the series is outlined. Thematically, the publications concerning the organization and management of public health have the greatest relative share. The publications about infectious diseases and the campaign against them are also widely covered as well as the oncologic diseases, nutrition, environmental preservation, psychic diseases, etc. The study revealed that the system for scientific communications of WHO in medicine and public health is an example of collective team scientific work and international character of medical service of modern type--international both as creation and consumption.

[Differential provision of scientific medical information to the management and specialists in a hospital]. / Diferentsirano osiguriavane s nauchna meditsinska informatsiia na rukovodstvoto i spetsialistite v bolnichno zavedenie.

A research was carried out, of the order of an experiment, on the activity of DIPM for active provision with scientific medical drug and technical information of the management personnel and specialties from the hospital for governmental officials. The main functions of the system are indicated, affording information on 10 problems with 196 information materials. DIPM system is a form of active addressed, information attendance, orientated according to problems affording documentary and factographic information and as a modification of information attendance in the regime IRI, it is more flexible, more effective and cheaper.

[Need for scientific medical information by internists working in the therapeutic and prophylactic system]. / Potrebnost ot nauchna meditsinska informatsiia na internistite, raboteshti v lechebno-profilaktichnata mrezha.

A study was carried out on the necessity of scientific medical information (SMI) among the internists, engaged in the practical public health care according to the methods: inquiry, collective expert evaluation and entropic. The results revealed that the scientific medical periodicals, books and drug catalogues are preferred as a source of SMI, whereas the secondary sources indexes, surveys, congress materials are less known and used. Of occupational and scientific interest is the scientific information on the fresh news of the methods about: a) diagnostics; b) treatment and c) new drugs. The inquired physicians gave the priority to addressed and current information. Sixty one per cent of them spend 60 min. weekly in retrieving the necessary SMI, and 40 per cent--over 240 min weekly, processing the relevant literature found.

[Case of primary hypogonadism in congenital dyskeratosis: Zinsser-Cole-Engmann syndrome]. / Sluchai na purvichen khipogonadisum pri kongenitalna diskeratoza--sindrom na Zinsser-Cole-Engmann.

A rare case is described with congenital dermatosis, leading to systemic lesions of epithelium of skin and mucose, combined with primary hypogonadism. A very low level of testosterone was established with high gonadotropic hormones and prolactin. Hypogonadism was established to be the result from systemic changes of skin, its accessory and epithelium of seminiferous tubules in testes.

[Insulin needs of insulin-dependent diabetics during biostator control and subcutaneous use of insulin]. / Insulinovi nuzhdi pri insulinozavisimi diabetitsi po vreme na biostatoren kontrol i podkozhno prilagane na insulin.

20 insulin-dependent diabetics, aged 17 to 52 years, duration of the diabetes from 1 to 22 years and mean value of the glycated hemoglobin 13.5 +/- 3.6% were studied. The 24 h insulin needs were determined with the help of an artificial pancreas (Biostator) at the following constants of the algorhithm: KR = 70, KF = 67, BI = 80, RI = 12, QI = 40, FI = 400, BD = 55, RD = 25, QD = 20, FD = 200 The insulin needs determined by the biostator were compared with those given subcutaneously which had, led to compensation of the diabetes before discharge of the patients. The mean insulin dose by the biostator was 1.0% U/kg and that of the subcutaneous insulin treatment was 0.86 U kg the difference is statistically insignificant). The above mentioned constants are recommended as being more physiological for the determination of 24-h insulin needs for a subcutaneous insulin treatment of diabetes.

[Study of the "dawn phenomenon" using an artificial endocrine pancreas]. / Prouchvaniia vurkhu "utrinniia fenomen" s pomoshtta na izkustven endokrin pankreas.

30 insulin-dependent, C-peptide negative diabetic patients were examined with the help of biostator. The mean age of the patients was 29 years, the mean duration of the diabetes was 7 years and the mean index of body mas was 22. For the period from 0 to 8 o'clock in the morning the blood sugar level and the speed of the insulin infusion in order to maintain euglycemic level were examined at 1/2 hour intervals. The mean blood sugar level for the period 5-8 o'clock (5.71 +/- 0.31 mmol/l) was reliably higher (p less than 0.05) than that for the period 0-5 o'clock (5.4 +/- 0.18 mmol/l). The speed of the insulin infusion for the period 5-8 o'clock (0.52 +/- 0.09 IU/kg/min) was also reliably higher (p less than 0.02) than that for the period 0-5 o'clock (0.4 +/- 0.06 IU/kg/min). These two indices were correlated with the somatotropic hormone and hydrocortisone levels at 2 and 8 o'clock. The possible pathogenetic mechanisms of this "morning phenomenon" in diabetes is discussed.