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We investigate the impact of bacterial (Chlamydia trachomatis, Ureaplasma urealyticum/parvum, Mycomplasma hominis/genitalium, Gardnerella vaginalis) and viral (HSV1/2, EBV, CMV, VZV, HHV6/HHV7, HHV8) pathogens, as a potential cause of reproductive failure in women by analysis of menstrual blood. We analyzed DNA extracted from 48 probands selected on the basis of history of infertility. DNA extraction, Real-time qPCR, gel electrophoresis were applied. In 64.6% of all tested menstrual blood samples of infertile women bacterial and/or viral pathogens were detected. In 41.4% of all tested samples we found bacterial, while in 37.5% viral pathogens. Ureaplasma parvum and Gardnerella vaginalis were detected in 58.3% and 54.2%, respectively, of the positive for bacterial pathogens samples. EBV, HHV7 and HHV6 were detected in 38.9%, 55.6%, and, respectively, 11.1% of the positive for viral pathogens samples. Bacterial and viral co-infection was found in 22.6% of all patients. Chlamydia trachomatis, Mycomplasma hominis/genitalium, Ureaplasma urealyticum, HSV1, HSV2, CMV, VZV and HHV8 were not detected in the menstrual blood samples. Our study offers new approach for diagnostics of infections in the upper female genital tract by analysis of menstrual blood. The opportunity to detect asymptomatic bacterial and viral infections in female endometrium contributes to reveal the cause for sterility. Our work contributes to clarify the infectious etiology of reproductive failure which is of a great importance for individualized therapy.

COVID‑19 patients with severe infection have been observed to have elevated auto‑antibodies (AAs) against angiotensin II receptor type 1 (AT1R) and endothelin (ET) 1 receptor type A (ETAR), compared with healthy controls and patients with favorable (mild) infection. AT1R and ETAR are G protein‑coupled receptors, located on vascular smooth muscle cells, fibroblasts, immune and endothelial cells, and are activated by angiotensin II (Ang II) and ET1 respectively. AAs that are specific for these receptors have a functional role similar to the natural ligands, but with a more prolonged vasoconstrictive effect. They also induce the production of fibroblast collagen, the release of reactive oxygen species and the secretion of proinflammatory cytokines (including IL‑6, IL‑8 and TNF‑α) by immune cells. Despite the presence of AAs in severe COVID‑19 infected patients, their contribution and implication in the severity of the disease is still not well understood and further studies are warranted. The present review described the major vascular homeostasis systems [ET and renin‑angiotensin‑aldosterone system (RAAS)], the vital regulative role of nitric oxide, the AAs, and finally the administration of angiotensin II receptor blockers (ARBs), so as to provide more insight into the interplay that exists among these components and their contribution to the severity, prognosis and possible treatment of COVID‑19.

Pathogenic variants in MYH7 cause a wide range of cardiac and skeletal muscle diseases with childhood or adult onset. These include dilated and/or hypertrophic cardiomyopathy, left ventricular non-compaction cardiomyopathy, congenital myopathies with multi-minicores and myofiber type disproportion, myosin storage myopathy, Laing distal myopathy and others (scapulo-peroneal or limb-girdle muscle forms). Here we report the results from molecular genetic analyses (NGS and Sanger sequencing) of 4 patients in two families with variable neuromuscular phenotypes with or without cardiac involvement. Interestingly, variants in MYH7 gene appeared to be the cause in all the cases. A novel nonsense variant c.5746C>T, p.(Gln1916Ter) was found in the patient in Family 1 who deceased at the age of 2 years 4 months with the clinical diagnosis of dilated cardiomyopathy, whose father died before the age of 40 years, due to cardiac failure with clinical diagnosis of suspected limb-girdle muscular dystrophy. A splice acceptor variant c.5560–2A>C in MYH7 was detected in the second proband and her sister, with late onset distal myopathy without cardiac involvement. These different phenotypes (muscular involvement with severe cardiomyopathy and pure late onset neuromuscular phenotype without heart involvement) may result from novel MYH7 variants, which most probably impact the LMM (light meromyosin) domain's function of the mature protein.

Cataract is a leading cause of reversible blindness. Secondary cataract is the result of migration, proliferation, and epithelial-mesenchymal transformation (EMT) of lens epithelial cells (LECs), induced by transforming growth factor β (TGFβ). The extracellular signal-regulated kinases 1 and 2 (ERK 1/2) and casein kinase 2 (CK2) are important for fundamental cellular processes. This study aimed to evaluate the role of CK2 in the EMT of LECs. Primary LECs cultures were obtained from age-related and type 2 diabetes-induced cataracts. Diabetic LECs were capable of spontaneous in vitro differentiation. Treatment of LECs with TGFβ2-stimulated ERK1/2 activity, which correlated with the expressed α-smooth muscle actin (α-SMA). siRNA-mediated attenuation of the endogenous catalytic subunit (α) of CK2-inhibited ERK1/2 activity and decreased α-SMA expression. This study shows evidence that the function of ERK1/2 is regulated by the constitutively active heterotetrameric protein kinase CK2 in this cellular phenotype. This study enriches the knowledge on LECs physiology in normal and in pathological conditions.

Cataract is a multifactorial disease with increasing prevalence with age. Adult diabetics develop cataract earlier. Lens epithelial cells (LECs) exposed to oxidative stress (ROS), increased calcium deposit and membrane damage, undergo apoptosis, which results in lens opacification. Remaining LECs post-surgery leads to posterior capsular opacification (PCO). This study’s aim was to investigate the physiological characteristics of LECs from cataractous diabetic and non-diabetic lenses. Leader cells migration from age-related cataracts started on day 5–7 and from type-2 diabetics on day 8–10. Differences were found in the collective migratory activity and colony formation. On day 22, the colonies formed by LECs from age-related cataracts were three times more, than those formed by diabetic LECs. DNA synthesis and FOXM1 expression occurred in 55.76% of age-related cataract LECs, but only in 33.45% of diabetic LECs. The highest level of reactive oxygen species (ROS) was found in diabetic LECs. Extracellular matrix calcification followed the same pattern as ROS. Among the main reasons for the development of age-related and diabetic cataracts is lens damage due to ROS release and elevated calcium levels. Diabetic LECs experience significantly lower in vitro migration and proliferative activities, compared to LECs from age-related cataracts. This is the first study of its kind in Bulgaria, contributing to the elucidation of the mechanisms of primary and secondary cataractogenesis in diabetic and non-diabetic adults.

Purpose: The aim of the present work was to analyse and identify differentially expressed miRNAs in Bulgarian patients with non-functioning pituitary adenomas (NFPA). The relationship between deregulated miRNAs and tumor size, invasiveness, and recurrence was determined.Methods: Twenty patients with NFPAs were selected and fresh pituitary tumor tissues were collected. RNA containing miRNAs were isolated using miRNAeasy mini kit and analysed with LNA miRNA Cancer Focus PCR Panel (Qiagen) by quantitative real-time PCR. Results: Three miRNAs (miR-210-3p, miR-149-3p and miR-29b-3p) were deregulated in invasive compared to non-invasive NFPAs. Differential expression of 4-miRNA signature - miRNA-17, miR-19, miR-106a and miR-20 was informative as a recurrence biomarker. ROC analysis showed that the selected miRNAs could be used as potential biomarkers for the diagnosis, prognosis and therapy monitoring of NFPAs.Conclusion: In this pilot study, we selected a unique miRNA expression profile correlating with NFPA invasiveness and recurrence. Moreover, some of the selected miRNAs are reported for the first time in patients with NFPAs and might elucidate the molecular mechanisms involved in pituitary pathogenesis. The selected miRNAs showed a promising biomarker potential and could be studied for future miRNAs-based anti-NFPAs therapy.

Laryngeal squamous cell carcinoma is a common malignancy in men. Bulgaria is one of the countries in Europe with the highest incidence and mortality rates of the aggressive, severe disease of laryngeal cancer. Proven etiological factors are the abuse of tobacco and alcohol beverages. Despite the progress of technologies of multimodal medical treatment, survival rates have not reached satisfactory levels. Over the last few decades, scientific and clinical research data have led to a growing interest in exploring potential biomarkers. In the last years, non-coding RNAs have become promising biomarkers. They are important key regulators in both normal and tumour specific biological processes as well as in the response to environmental factors and treatment, including chemo- and radiotherapy. Studies have shown ectopic expression of a number of ncRNAs in laryngeal cancer. Published data provide evidence of the lncRNAs and miRNAs that could help us better understand complex carcinogenesis in laryngeal cancer and would provide reliable diagnostic, prognostic and predictive biomarkers.

The hereditary transthyretin amyloidosis (ATTRv amyloidosis) is an autosomal dominant genetic disease characterized by amyloid formation in different tissues due to pathogenic variants in the TTR g...

Hereditary transthyretin amyloidosis is an autosomal dominant genetic disorder caused by missense mutations in the TTR gene resulting in amyloid formation of the transthyretin protein. Depending on the system affection, the manifestations may be different and high heterogeneity in the penetrance is observed. An endemic region in Bulgaria exists where the TTR mutation Glu89Gln is found with high frequency. This is a rare mutation and was probably introduced in the population by a common ancestor. This phenomenon, called "founder effect" was proved in carrier families by haplotype analysis of microsatellite markers showing linkage disequilibrium. Allele frequencies were analyzed and haplotype reconstruction was done with Arlequin v.3.01 software. The common ancestry of the carriers was demonstrated using additional data for their genealogies and microsatellite data from a control group of non-affected individuals. The results show that the mutation Glu89Gln is linked to one haplotype, called "hypothetical founder haplotype" which was compared to published haplotype data from other European patients and no similarity was found. Further population genetics studies of carriers of the Glu89Gln mutation from other endemic regions are required in order to clarify the geographical distribution of the mutation.

Background. Dravet syndrome (DS) is the most severe form of Generalized Epilepsy with Febrile Seizures plus (GEFS+) syndrome with a clear genetic component in 85% of the cases. It is characterized by fever-provoked seizure onset around six months of age and subsequent developmental deterioration later in life. Methods. In the current study, 60 patients with fever-provoked seizures and suspicion either of GEFS+ (50 patients) or of DS (10 patients) were referred for SCN1A gene sequence analysis. Results. SCN1A gene sequencing revealed clinically significant variants in 11 patients (18.3%); seven pathogenic (11.7%) and four likely pathogenic (6.7%). Five of these variants have not been reported previously. Among the preselected group of ten DS patients, five had pathogenic SCN1A variants which confirmed diagnosis of DS. In four patients with preliminary diagnosis GEFS+, the detected SCN1A variant enabled us to specify the diagnosis of DS in these patients. Thus, SCN1A sequencing led to confirmation of the genetic diagnosis in 50% (5/10) of DS patients, as well as clarification of the diagnosis of DS in 8% of GEFS+ patients (4/50). In this study, four patients with truncating mutations had refractory seizures and additional psychomotor abnormalities. Additionally, pathogenic missense mutations were detected in three children with comparable phenotypes, which support the observations that missense mutations in critical channel function regions can cause a devastating epileptic condition. Conclusions. This is the first systematic screening of SCN1A gene in our country, which expands the spectrum of SCN1A variants with five novel variants from Bulgaria and demonstrates the clinical utility of confirmatory SCN1A testing, which helps clinicians make early and precise diagnoses. It is important for a better followup, choice of proper treatment, avoidance of development of refractory seizures and neuropsychological complications. Identification of pathogenic variants in SCN1A in the milder GEFS+ and severe DS cases, will help to offer adequate prenatal diagnosis and improve the genetic counselling provided to affected families.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by the development of hamartomas localized in various tissues which can occur in the skin, brain, kidney and other organs. TSC is caused by mutations in the TSC1 and TSC2 genes. Here we report the results from the first molecular testing of 16 Bulgarian patients and one Romanian patient in whom we found six novel mutations: four in the TSC22 gene, of which one is nonsense, two frame shift and one large deletion of 16 exons; and two in the TSC1 gene, one nonsense and other frame shift. In addition, we detected 10 previously reported mutations; some of which are described only once in the literature. Our data is similar to the previous studies with exception of the larger number of TSC1 mutations than that reported in the literature data. In total, 40% (4/10) of the mutation in the TSC2 gene are located in the GTPase-activating protein domain, while 50% (3/6) are in the TSC1 gene and clustered in exon 15. All the cases represent the typical clinical symptoms and meet the clinical criteria for TSC diagnosis. In 35% of our cases the family history was positive. Our results add novel findings in the genetic heterogeneity and pathogenesis of TSC. The genetic heterogeneity might correlate to the clinical variability among the TSC-affected families, which makes the genetic counselling a real challenge.

Neurofibromatosis (NF) is a clinically heterogeneous autosomal dominant disorder. Three distinct forms have been identified: neurofibromatosis type 1 (NF1), type 2 (NF2) and schwannomatosis. In the present study, we report clinical and genetic findings in the NF1 and NF2 genes in a cohort of 27 Bulgarian patients, with 18 cases (67%) genetically verified. Both NF1 and NF2 genes were screened by Sanger sequencing on DNA samples. The Sanger negative samples were screened by Multiplex Ligation-dependent Probe Amplification (MLPA) for deletions and duplications. The results from genetic testing revealed three novel mutations and fifteen previously reported ones (13 in the NF1 gene and 2 in the NF2 gene). The novel variants in the NF1 gene are a splice site mutation c.4725-1G>A, a small deletion of five bases c.823delATCTT, p.Leu275ValfsTer14, and a single base duplication c.6547dupC, p.Arg2183ProfsTer11. The novel splice site mutation is manifested by multiple “cafe au lait” macules and neurofibromas. Both novel out of frame mutations were found in patients with multiple “cafe au lait” spots and focal epilepsy. A segmental neurofibromatosis (SNF1) is restricted to one or more body segments. Here we present a case with SNF1 caused by a somatic deletion of exons 1 to 12 of the NF1 gene which is manifested by multiple neurofibromas in the right hand. Two nonsense mutations are found in the NF2 gene. Our study adds three novel mutations to the NF1 mutation spectra and contributes to the clinical-genetic NF1-characterization. Here we report strikingly different phenotypic spectra caused by the same mutation in a single family. Our findings contribute to the genotype- phenotype correlations which are difficult to establish, due to the extremely complex NF phenotype being a combination of clinical features.

Wolman disease is a rare autosomal recessive disorder caused by mutations in the LIPA gene (10q23.31). The LIPA gene encodes lysosomal acid lipase (LAL), which plays a key role in hydrolysis of the cholesteryl esters and triglycerides. Two unrelated families from Bulgaria were referred for genetic testing with clinical diagnosis Wolman disease. Sanger sequencing of all coding exons and exon-intron boundaries of the LIPA gene was performed. The index patients were found to be homozygous for two different mutations in the LIPA gene: a missense mutation, c.260G > T, p.Gly87Val, which affects the enzyme active site and a splice-site change, c.822+1G > A, which most probably destroys the enzyme polypeptide chain. These two completely different types of mutations along the LIPA gene resulted in a very similar phenotype involving liver, kidney, gastrointestinal, muscle and blood disturbances. As consanguinity is not typical for the Bulgarian population, a possible explanation of the homozygosity could be presence of endemic regions for given mutations. To check this hypothesis, selective screening for these mutations was performed in two presumable endemic regions in Bulgaria. Altogether, 100 newborns were screened for p.Gly87Val mutation and the detected carrier frequency was about 1% (1/100), while in the group of 100 newborns screened for the c.822 + 1G > A mutation the detected carrier frequency was 2% (2/100). The results indicate a high recurrence risk of Wolman disease in these particular Bulgarian regions of about 1:10000. These findings are from crucial importance for the inhabitants of the corresponding parts of Bulgaria. They may benefit from early genetic testing and adequate genetic counselling during family planning.

Background Charcot-Marie-Tooth (CMT) disease is the most common inherited neuromuscular disorder characterized by wide clinical, genetic and pathomechanistic heterogeneity. Recently, the gene encoding peripheral myelin protein 2 (PMP2) was identified as a novel cause for CMT neuropathy with three mutations that structurally cluster together (p.Ile43Asn, p.Thr51Pro, p.Ile52Thr) reported in five families. Results Using whole exome sequencing and cohort screening we identified two novel missense substitutions in PMP2 in Bulgarian (p.Met114Thr, c.341C > T) and German (p.Val115Ala, c.344 T > C) families. The mutations affect adjacent and highly conserved amino acid residues outside of the known mutation-rich region in the protein. Crystal structure analysis positions the affected residues within a cluster of highly conserved fatty acid coordinating residues implying their functional significance. The clinical, electrophysiological and imaging features in both families were consistent with a childhood onset polyneuropathy with variable patterns of demyelination, slow to very slow progression, and most severe involvement of the peroneal muscles. Conclusions We expand the genetic and phenotypic spectrum of PMP2-related peripheral neuropathy. Our findings reveal a second mutational cluster in the protein. Electronic supplementary material The online version of this article (10.1186/s13023-019-1162-x) contains supplementary material, which is available to authorized users.

Mutations in KCNQ2 are associated with a range of electroclinical syndromes with dominant inheritance that are differentiated by the age at onset of the seizures and are associated with good prognosis. These are benign familial neonatal seizures (BFNS), benign familial neonatal-infantile seizures (BFNIS) and benign familial infantile seizures. Herein, we report the results of a systematic screening of KCNQ2 in 27 unrelated Bulgarian patients with compatible clinical diagnoses. Two pathogenic point mutations were identified: a novel splice-site c. 1526-2A > G variation causing BFNS and a missense c. 998G > A alteration in a patient with BFNIS, who subsequently developed benign epilepsy with centro-temporal spikes. Additionally, multiplex ligation-dependent probe amplification analysis and array comparative genomic hybridization assay detected a de novo deletion on 20q13.3 encompassing 0.41 Mb genomic region and covering 11 genes, including KCNQ2 and CHRNA4. This large-scale rearrangement was found in a patient with typical BFNS and no additional developmental abnormalities. Overall, KCNQ2 genetic defects were found in 11% of the patients in our cohort. These findings enrich the genetic epidemiology and mutation spectrum of KCNQ2 and allow adequate genetic counselling in the affected families.

To describe and provide a comprehensive overview on the development, use and efficacy of autologous platelet concentrates in different in vitro and in vivo studies focusing on oral and maxillofacial pathologies.Present work employs an extensive critical overview of the literature on the development and application of platelet concentrates.Platelet concentrates are innovative endogenous therapeutic agents which gained a lot of interest in different medical and dental disciplines due to their potential ability to stimulate and increase regeneration of soft and hard tissues. The effect of platelet-derived products is considered to be a result of the high number of platelets which contain a wide range of growth factors. They are not just therapeutic products but autologous blood concentrates containing active molecules. The quality of platelet concentrates may vary according to the individual physical state of donors making it difficult to to compare the outcomes of their application. Although, there are many studies analyzing the properties of these biomaterials both in vivo and in vitro, a consensus regarding their efficacy still has to be reached.Evidences described in the literature on the efficacy of platelet concentrates in procedures in oral and maxillofacial region are controversial and limited. In order to clarify the real advantages and priorities for the patients, when the blood-derived products are applied, further in vitro and in vivo research about the activity of PRP and PRF on the dental cells biology should be conducted.

Purpose: Homozygosity mapping is an effective approach for detecting molecular defects in consanguineous families by delineating stretches of genomic DNA that are identical by descent. Constant developments in next-generation sequencing created possibilities to combine whole-exome sequencing (WES) and homozygosity Mapping in a single step. Methods: Basic optimization of homozygosity mapping parameters was performed in a group of families with autosomal-recessive (AR) mutations for which both single-nucleotide polymorphism (SNP) array and WES data were available. We varied the criteria for SNP extraction and PLINK thresholds to estimate their effect on the accuracy of homozygosity mapping based on WES. Results: Our protocol showed high specificity and sensitivity for homozygosity detection and facilitated the identification of novel mutations in GAN, GBA2, and ZFYVE26 in four families affected by hereditary spastic paraplegia or Charcot-Marie-Tooth disease. Filtering and mapping with optimized parameters was integrated into the HOMWES (homozygosity mapping based on WES analysis) tool in the GenomeComb package for genomic data analysis. Conclusion: We present recommendations for detection of homozygous regions based on WES data and a bioinformatics tool for their identification, which can be widely applied for studying AR disorders.

Ethno-pharmacological relevance Historically, aphrodisiacs have had a reputation for making sex more achievable and satisfying. It has been long believed that Tribulus terrestris L. (TT), an annual plant of the family Zygophyllaceae , possesses aphrodisiac properties purportedly attributed to its ability to influence levels or mimic function of sex hormones. Due to this appealing beliefs, the popularity of medicinal products from TT is expanding at a remarkable pace among consumers who are attempting to enhance their sexual health. However, reliable scientific evidence supporting these purported bioactivities are scant and far from conclusive. Aim of the review To critically analyze and updated the evidence supporting a role for TT as an aphrodisiac and to reappraise the widely believed view of TT as an androgen enhancing botanical supplement. Material and method An extensive review of the literature was carried out based on systematic search of major scientific databases (PubMed, Elsevier, Springer Link, Google Scholar, Medline Plus, and Web of Science) for studies of phytochemical, pharmacological and traditional uses of TT published between 1968 and 2015. In addition, the reference lists of the available articles were reviewed and relevant studies including material in journals which are not indexed internationally were reviewed. Results Analysis of phytochemical and pharmacological studies in humans and animals revealed an important role for TT in treating erectile dysfunction and sexual desire problems; however, empirical evidence to support the hypothesis that this desirable effects are due to androgen enhancing properties of TT is, at best, inconclusive, and analysis of empirical evidence from a comprehensive review of available literature proved this hypothesis wrong. While the mechanisms underlying TT aphrodisiac activity remain largely unknown, there is emerging compelling evidence from experimental studies in animals for possible endothelium and nitric oxide-dependent mechanisms underlying TT aphrodisiac and pro-erectile activities. Conclusion It is becoming increasingly clear that the deep-seated traditional view of TT bioactivity focused exclusively on its androgen enhancing properties is outdated and incapable for accommodating the emerging evidence from recent clinical and experimental studies pointing toward new and, perhaps, more plausible modes of action. Novel paradigms guiding the development of new testable hypotheses for TT aphrodisiac properties are needed to stimulate further investigations into potential biological mechanisms in which many apparently conflicting observations can be reconciled.

Sézary syndrome is a primary cutaneous T-cell lymphoma characterized by pruritic erythroderma, peripheral lymphadenopathy and the presence of malignant T cells in the blood. Unequivocal detection of malignant cells in patients with Sézary syndrome is of important diagnostic, prognostic and therapeutic value. However, no single Sézary syndrome specific cell surface marker has been identified. In a cohort of patients with Sézary syndrome, CD164 expression on total CD4+ lymphocytes was significantly upregulated compared with healthy controls. CD164 expression was in most cases limited to CD4+CD26- malignant T lymphocytes, unequivocally identified using flow-cytometry by the expression of a specific Vβ clone for each patient. Increased expression of CD164 may be a promising diagnostic parameter and a potential target for a CD164-linked therapeutic approach in Sézary syndrome.

TTR-related amyloidosis (ATTR) is manifested in two allelic forms: familial amyloid polyneuropathy (TTR-FAP) and cardiomyopathy (TTR-FAC), both caused by mutations in the TTR gene. The most prevalent mutation in Bulgaria is p.Glu89Gln. Markedly different age at onset and disease penetrance is noticed in Bulgarian p.Glu89Gln cases even in a single family or between genetically identical twins. The present study aimed to evaluate the transcription profile of the TTR gene in order to better understand the difference in disease onset and penetrance. Six p.Glu89Gln positive families were selected from our registry, based on intrafamilial differences in disease onset and penetrance. Plasma and urine specimens were collected from 13 patients and subjected to transcription analysis. Both mutant and wild type transcripts were visualized in a mixed transcription profile, which is the traditional model of autosomal gene expression. The results from a relative quantification of the mutant versus wild type transcript showed presence of the mutant transcript between 0.14 and 1.14 times against the wild type. In addition, monoallelic expression signature was also detected. Based on our results we propose a model of natural selection, which includes age-related allele exclusion or suppression: predominant expression of a wild type (at an early age) and mutant (at the process of ageing) alleles. The intrafamilial differences in disease onset and penetrance need to be considered in genetic counselling and in follow-up of mutation carriers.

FGF8 specifies early tooth development by directing the migration of the early tooth founder cells to the site of tooth emergence. To date the effect of the FGF8 in adult dental pulp has not been studied. We have assessed the regenerative potential of FGF8 by evaluating changes in the proteome landscape of dental pulp following short- and long-term exposure to recombinant FGF8 protein. In addition, we carried out qRT PCR analysis to determine extracellular/adhesion gene marker expression and assessed cell proliferation and mineralization in response to FGF8 treatment. 2D and mass spectrometry data showed differential expression of proteins implicated in cytoskeleton/ECM remodeling and migration, cell proliferation and odontogenic differentiation as evidenced by the upregulation of gelsolin, moesin, LMNA, WDR1, PLOD2, COPS5 and downregulation of P4HB. qRT PCR showed downregulation of proteins involved in cell-matrix adhesion such as ADAMTS8, LAMB3 and ANOS1 and increased expression of the angiogenesis marker PECAM1. We have observed that, FGF8 treatment was able to boost dental pulp cell proliferation and to enhance dental pulp mineralization. Collectively, our data suggest that, FGF8 treatment could promote endogenous healing of the dental pulp via recruitment of dental pulp progenitors as well as by promoting their angiogenic and odontogenic differentiation. Significance Dental pulp cells (DP) have been studied extensively for the purposes of mineralized tissue repair, particularly for the reconstruction of hard and soft tissue maxillofacial defects. Canonical FGF signaling has been implicated throughout multiple stages of tooth development by regulating cell proliferation, differentiation, survival as well as cellular migration. FGF8 expression is indispensible for normal tooth development and particularly for the migration of early tooth progenitors to the sites of tooth emergence. The present study provides proteome and qRT PCR data with regard to the future application and biological relevance of FGF8 in dental regenerative medicine. Authors with ORCID Rozaliya Tsikandelova - 0000-0003-0178-3767 Zornitsa Mihaylova - 0000-0003-1748-4489 Sebastien Planchon - 0000-0002-0455-0574 Nikolay Ishkitiev - 0000-0002-4351-5579

Background There is strong evidence for partial overlap of genetic influence on Schizophrenia (SCZ), Schizoaffective Disorder (SAD) and Bipolar Affective Disorder (BAD). Part of this is due to common genetic variants, detected by GWAS, with a small individual effect on risk. Recent studies using next generation sequencing reveal growing evidence of the role of rare genetic variants in both diseases. We performed targeted NGS on 187 candidate genes to further investigate the genetic architecture of Bulgarian patients with bipolar disorder and schizophrenia. Methods A total of 300 individuals with BAD, 151 with SCZ, 15 SAD according to DSMIV and ICD-10, as well as 85 healthy population controls and 40 healthy relatives were recruited. The samples were sequenced on the Ion PROTON platform. The sequencing panel comprised of 187 preselected strong candidate genes, based on the results from GWAS and previous NGS studies. Only samples with coverage of at least 95% of the target region at 20x were included in the analyses. Case-control association testing was done using PLINK. The rare variants have been evaluated and divided into groups based on their functional relevance: LOF variants (frameshift and nonsense); with potentially damaging effect on protein function (splice site, missense variants, 3’-UTR/5’-UTR variants); and with no known effect on function (synonymous and intron variants). Genes were prioritized by mutation burden analysis based on the average number of functional occurrences per 1000 bp of the gene CDS. The RVIS scoring system for assessing the intolerance of individual genes to protein-altering variants (Petrovski et al., 2013) was used in conjunction with the burden score for the final ordering. Results 5373 variants were detected, of which 2826 were found in affecteds (1831 singletons and 995 recurrent). No significant associations between cases and controls could be detected, that survived the correction for multiple testing. In total we found in patients 14 rare LOF variants; 889 non-synonymous variants (243 of which were potentially damaging); 109 splice site variants and 32 in regulatory regions. Discussion We could find no significant association of common or rare variants in the analyzed samples, most likely due to small sample size. However, we could identify both recurrent and singleton rare variants with potential functional relevance in genes associated with ion channels, postsynaptic plasticity, neurogenesis and signalling cascade pathways.

High resolution chromosomal microarray analysis (CMA) has facilitated the identification of small chromosomal rearrangements throughout the genome, associated with various neurodevelopmental phenotypes, including ID/DD. Recently, it became evident that intellectual disability (ID)/developmental delay (DD) can occur with associated co-morbidities like epileptic seizures, autism and additional congenital anomalies. These observations require whole genome approach in order to detect the genetic causes of these complex disorders. In this study, we examined 92 patients of Bulgarian origin at age between 1 and 22 years with ID, generalized epilepsy, autistic signs and congenital anomalies. CMA was carried out using SurePrint G3 Human CGH Microarray Kit, 4 × 180 K and SurePrint G3 Unrestricted CGH ISCA v2, 4 × 180 K oligo platforms. Referral indications for selection of the patients were the presence of generalized refractory seizures disorders and co-morbid ID. Clearly pathogenic copy number variations (CNVs) were detected in eight patients (8.7%) from our cohort. Additionally, possibly pathogenic rearrangements of unclear clinical significance were detected in six individuals (6.5%), which make for an overall diagnostic yield of 15.2% among our cohort of patients. We report here the patients with clearly pathogenic CNVs, discuss the potential causality of the possibly pathogenic CNVs and make genotype - phenotype correlations. One novel possibly pathogenic heterozygous deletion in 15q22.31 region was detected in a case with ID/DD. Additionally, whole APBA2 gene duplication in 15q13.1 was found in three generations of a family with epilepsy, ID and psychiatric abnormalities. The results from this study allow us to define the genetic diagnosis in a subset of Bulgarian patients and improve the genetic counseling of the affected families. To our knowledge, this is the first aCGH evaluation of a Bulgarian cohort of children with epilepsy and ID so far.

A total of 36 consecutive clinical and two fecal-screening carbapenem-resistant Klebsiella pneumoniae isolates from two Bulgarian university hospitals (Varna and Pleven) were investigated. Susceptibility testing, conjugation experiments, and plasmid replicon typing were carried out. Beta-lactamases were characterized by isoelectric focusing, PCR, and sequencing. Clonal relatedness was investigated by RAPD and multilocus sequence typing (MLST). Most of the isolates demonstrated multidrug resistance profile. Amikacin and tigecycline retained good activity with susceptibility rates of 95 and 87%, respectively. The resistance rate to colistin was 63%. Six RAPD- and MLST-types were identified: the dominating MLST-type was ST15 (27 isolates), followed by ST76 (six isolates), and ST1350 (two isolates). ST101, ST258, and ST151 were detected once. All except one of the K. pneumoniae produced KPC-2, mostly in combination with CTX-M-15, while for one isolate (ST101) the enzymes OXA-48 and CTX-M-14 were found. All KPC-2-producing transconjugants revealed the presence of IncFII plasmid. The OXA-48- and CTX-M-14-producing isolate showed the presence of L/M replicon type. The dissemination of KPC-2-producing K.pneumoniae in Bulgaria is mainly due to the sustained spread of successful ST15 clone and to a lesser extent of ST76 clone. This is the first report of OXA-48 producing ST101 K. pneumoniae in Bulgaria.

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive disorder characterized by intractable seizures in neonates and infants. The seizures cannot be controlled with antiepileptic medications but respond both clinically and electrographically to large daily supplements of pyridoxine (vitamin B6). PDE is caused by mutations in the ALDH7A1 gene. Molecular genetic analysis of the ALDH7A1 gene was performed in seven patients, referred with clinical diagnosis of PDE. Mutations were detected in a dizygotic twin pair and a non-related boy with classical form of PDE. Direct sequencing of the ALDH7A1 gene revealed one novel (c.297delG, p.Trp99*) and two already reported (c.328C>T, p.Arg110*; c.584A>G, p.Asn195Ser) mutations. Here, we report the first genetically proven cases of PDE in Bulgaria.

The heavy chain 1 of cytoplasmic dynein (DYNC1H1) is responsible for movement of the motor complex along microtubules and recruitment of dynein components. Mutations in DYNC1H1 are associated with spinal muscular atrophy (SMA), hereditary motor and sensory neuropathy (HMSN), cortical malformations, or a combination of these. Combining linkage analysis and whole-exome sequencing, we identified a novel dominant defect in the DYNC1H1 tail domain (c.1792C>T, p.Arg598Cys) causing axonal HMSN. Mutation analysis of the tail region in 355 patients identified a de novo mutation (c.791G>T, p.Arg264Leu) in an isolated SMA patient. Her phenotype was more severe than previously described, characterized by multiple congenital contractures and delayed motor milestones, without brain malformations. The mutations in DYNC1H1 increase the interaction with its adaptor BICD2. This relates to previous studies on BICD2 mutations causing a highly similar phenotype. Our findings broaden the genetic heterogeneity and refine the clinical spectrum of DYNC1H1, and have implications for molecular diagnostics of motor neuron diseases.

The current definitive treatment for acute or chronic liver condition, that is, cirrhosis, is liver transplantation from a limited number of donors, which might cause complications after donation. Hence, bone marrow stem cell transplantation has been developed, but the risk of carcinogenesis remains. We have recently developed a protocol for hepatic differentiation of CD117(+) stem cells from human exfoliated deciduous teeth (SHED). In the present study, we examine whether SHED hepatically differentiated (hd) in vitro could be used to treat acute liver injury (ALI) and secondary biliary cirrhosis. The CD117(+) cell fraction was magnetically separated from SHED and then differentiated into hepatocyte-like cells in vitro. The cells were transplanted into rats with either ALI or induced secondary biliary cirrhosis. Engraftment of human liver cells was determined immunohistochemically and by in situ hybridization. Recovery of liver function was examined by means of histochemical and serological tests. Livers of transplanted animals were strongly positive for human immunohistochemical factors, and in situ hybridization confirmed engraftment of human hepatocytes. The tests for recovery of liver function confirmed the presence of human hepatic markers in the animals' blood serum and lack of fibrosis and functional integration of transplanted human cells into livers. No evidence of malignancy was found. We show that in vitro hdSHED engraft morphologically and functionally into the livers of rats having acute injury or secondary biliary cirrhosis. SHED are readily accessible adult stem cells, capable of proliferating in large numbers before differentiating in vitro. This makes SHED an appropriate and safe stem cell source for regenerative medicine.