Utilization of Microfluidic Droplet-Based Methods in Diagnosis and Treatment Methods of Hepatocellular Carcinoma: A Review.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is associated with high morbidity and mortality. One of the main challenges in the management of HCC is late clinical presentation and thus diagnosis of the disease, which results in poor survival. The pathogenesis of HCC is complex and involves chronic liver injury and genetic alterations. Diagnosis of HCC can be made either by biopsy or imaging; however, conventional tissue-based biopsy methods and serological biomarkers such as AFP have limited clinical applications. While hepatocellular carcinoma is associated with a range of molecular alterations, including the activation of oncogenic signaling pathways, such as Wnt-TGFß, PI3K-AKT-mTOR, RAS-MAPK, MET, IGF, and Wnt-ß-catenin and TP53 and TERT promoter mutations, microfluidic applications have been limited. Early diagnosis is crucial for advancing treatments that would address the heterogeneity of HCC. In this context, microfluidic droplet-based methods are crucial, as they enable comprehensive analysis of the genome and transcriptome of individual cells. Single-cell RNA sequencing (scRNA-seq) allows the examination of individual cell transcriptomes, identifying their heterogeneity and cellular evolutionary relationships. Other microfluidic methods, such as Drop-seq, InDrop, and ATAC-seq, are also employed for single-cell analysis. Here, we examine and compare these microfluidic droplet-based methods, exploring their advantages and limitations in liver cancer research. These technologies provide new opportunities to understand liver cancer biology, diagnosis, treatment, and prognosis, contributing to scientific efforts in combating this challenging disease.

Higher levels of plasma Adrenocorticotropic hormone (ACTH) are associated with lower suicidal ideation in depressed patients compared to controls and suicide attempters, independently from depression severity.

Introduction: Suicidal ideation, an important risk factor for suicide attempts, has an unclear neurobiological basis and is potentially linked to the dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis and immune-inflammatory systems. While inflammatory markers have been associated with suicide attempts and, to a lower extent suicidal ideation, the data on the role of a stress-response system is less robust, with most studies carried out with cortisol showing inconsistent results. The present study extends on the previous studies implicating stress-response and immune-inflammatory systems in suicidal thoughts and behaviours, focusing on the associations of several stress-response (adrenocorticotropic hormone (ACTH), cortisol, and dehydroepiandrosterone (DHEA)) and immune-inflammatory (C-reactive protein (CRP),interle ukin-6 (IL-6), and tumour necrosis factor-alpha (TNF-alpha)) with suicidal ideation severity in recent suicide attempters, patients with major depressive disorder, and non-psychiatric controls. Methods: This observational study included 156 adults from three Vilnius hospitals, recruited into one of the three groups in equal parts: recent suicide attempters, patients with major depressive disorder in current depressive episode, and non-psychiatric controls. Measures included the Hamilton Depression Rating Scale (HDRS-17) and the Beck Scale for Suicide Ideation/Suicide Severity Index (BSS/SSI), alongside sociodemographic data, alcohol, tobacco use, and morning blood samples, measuring plasma ACTH, cortisol, DHEA, CRP, and IL-6. Data were analysed with non-parametric tests, Kendall's tau correlation, and multivariate linear regression adjusted for confounders. Results: We found a negative correlation between the plasma ACTH levels and suicidal ideation severity (tau = -0.130, p = 0.033), which was driven by the patients with major depressive disorder (tau = -0.237, p = 0.031). Suicidal ideation severity was also negatively correlated with TNF-alpha (tau = -0.231; p < 0.001), positively correlated with IL-6 (tau = 0.154, p = 0.015), and CRP levels (tau = 0.153, p = 0.015), but no differences were observed in group-stratified analyses. The association between plasma ACTH levels and suicidal ideation severity in patients with major depressive disorder remained robust to adjustment for major confounders (adjusted for age, sex, education years, body mass index, smoking status, plasma CRP and PEth concentration (measuring chronic alcohol exposure), and antidepressant use) in the linear regression model (t = -2.71, p = 0.011), as well as additionally adjusting for depression severity (t = -2.99, p = 0.006). Discussion: The present study shows an association between plasma ACTH levels and suicidal ideation severity in patients with major depressive disorder, robust to adjustment for antidepressant use and depression severity. This finding highlights the potential role of ACTH, in elucidating the effects of stress and mental health disorders. Our findings underscore the importance of the HPA axis in the diagnosis and treatment of suicidal ideation in major depressive disorder and invite further research on interventions targeting this pathway.

A De Novo 8q22.2q22.3 Interstitial Microdeletion in a Girl with Developmental Delay and Congenital Defects.

Background and Objectives: Only nine patients with interstitial de novo 8q22.2q22.3 microdeletions have been reported to date. The objective of this report is to present clinical features of a new patient with an 8q22.2q22.3 microdeletion, to compare her phenotype to other previously reported patients, and to further expand the phenotype associated with this microdeletion. Materials and Methods: We describe an 8½-year-old girl with developmental delay, congenital hip dysplasia, a bilateral foot deformity, bilateral congenital radioulnar synostosis, a congenital heart defect, and minor facial anomalies. Results: Chromosomal microarray analysis revealed a 4.9 Mb deletion in the 8q22.2q22.3 region. De novo origin was confirmed by real-time PCR analysis. Conclusions: Microdeletions in the 8q22.2q22.3 region are characterized by moderate to severe intellectual disability, seizures, distinct facial features and skeletal abnormalities. In addition to one already reported individual with an 8q22.2q22.3 microdeletion and unilateral radioulnar synostosis, this report of a child with bilateral radioulnar synostosis provides additional evidence, that radioulnar synostosis is not an incidental finding in individuals with an 8q22.2q22.3 microdeletion. Additional patients with similar microdeletions would be of a great importance for more accurate phenotypic description and further analysis of the genotypic-phenotypic relationship.

The academic impact and value of an international online surgery lecture series.

Due to the worldwide travel restrictions caused by the 2019 coronavirus disease pandemic, many universities and students lost opportunities to engage in international exchange over the past 2 years. Teleconferencing systems have thus been developed to compensate for severe travel restrictions. Kansai Medical University in Japan and Vilnius University in Lithuania have a collaborative research and academic relationship. The two universities have been conducting an online joint international surgery lecture series for the medical students of both universities. Fifteen lectures were given from October 2021 to May 2022. The lectures focused on gastrointestinal surgery, gastroenterology, radiology, pathology, genetics, laboratory medicine, and organ transplantation. A survey of the attendees indicated that they were generally interested in the content and satisfied with attending this lecture series. Our efforts were successful in providing Japanese and Lithuanian medical students with the opportunity to engage in international exchange through lectures held in each other's countries.

Rare disease education in Europe and beyond: time to act.

People living with rare diseases (PLWRD) still face huge unmet needs, in part due to the fact that care systems are not sufficiently aligned with their needs and healthcare workforce (HWF) along their care pathways lacks competencies to efficiently tackle rare disease-specific challenges. Level of rare disease knowledge and awareness among the current and future HWF is insufficient. In recent years, many educational resources on rare diseases have been developed, however, awareness of these resources is still limited and rare disease education is still not sufficiently taken into account by some crucial stakeholders as academia and professional organizations. Therefore, there is a need to fundamentally rethink rare disease education and HWF development across the whole spectrum from students to generalists, specialists and experts, to engage and empower PLWRD, their families and advocates, and to work towards a common coherent and complementary strategy on rare disease education and training in Europe and beyond. Special consideration should be also given to the role of nurse coordinators in care coordination, interprofessional training for integrated multidisciplinary care, patient and family-centered education, opportunities given by digital learning and fostering of social accountability to enforce the focus on socially-vulnerable groups such as PLWRD. The strategy has to be developed and implemented by multiple rare disease education and training providers: universities, medical and nursing schools and their associations, professional organizations, European Reference Networks, patient organizations, other organizations and institutions dedicated to rare diseases and rare cancers, authorities and policy bodies.

Challenges in the Diagnosis of XY Differences of Sexual Development.

Background: We report the clinical case of female patient with 46,XY difference of sexual development (DSD) and discuss the challenges in the differential diagnosis between complete gonadal dysgenesis (also called Swyer syndrome) and complete androgen insensitivity syndrome. Case Presentation: The patient's with primary amenorrhea gynaecological examination and magnetic resonance imaging (MRI) revealed the absence of the uterus and a very short vagina. Two sclerotic structures, similar to ovaries, were recognised bilaterally in the iliac regions. Hormonal assay tests revealed hypergonadotropic hypogonadism and the testosterone level was above normal. The karyotype was 46,XY and a diagnosis of Swyer syndrome was made. At the age of 41, the patient underwent a gynaecological review and after evaluating her tests and medical history, the previous diagnosis was questioned. Therefore, a molecular analysis of sex-determining region Y (SRY) and androgen receptor (AR) genes was made and the results instead led to a definite diagnosis of complete androgen insensitivity syndrome. Conclusions: The presented case illustrates that differentiating between complete gonadal dysgenesis and complete androgen insensitivity can be challenging. A well-established diagnosis is crucial because the risk of malignancy is different in those two syndromes, as well as the timing and importance of gonadectomy.

Diagnostic Significance of FNAB miRNA Expression in Papillary Thyroid Carcinoma.

The aim of the study was to evaluate the diagnostic utility of specific miRNAs in the preoperative assessment of thyroid nodules. One hundred and sixty thyroid fine needle aspiration biopsy (FNAB) samples with suspected thyroid carcinoma were collected. To detect the levels of miRNA expression in FNAB, next generation small RNA sequencing was performed in 60 samples. Based on the results obtained, three miRNAs (miR125A, miR200B, miR4324) were selected for further analysis. Based on the most frequently reported miRNAs in the literature associated with thyroid papillary carcinoma (PTC), two more miRNA (miR146B, miR221) were selected for further validation, using real-time reverse transcriptase polymerase chain reaction (RT-PCR) in 36 benign and 64 PTC samples. Expression of miR125A, miR146B, miR221, and miR4324 was significantly higher in patients with PTC compared with benign thyroid nodules (p ˂ 0.05). miR125A and miR4324 were also significantly more highly expressed in patients with extrathyroidal tumor extension compared to those without extrathyroidal PTC extension (p < 0.001). We also found a significantly higher expression of miR221 (p = 0.043) in patients with multifocal carcinomas compared to patients with single foci carcinomas. This prospective study showed that the expression analysis of four miRNAs (miR125A, miR146B, miR221, and miR4324) improve accuracy of FNAB, which could allow a better pre-operative diagnostic and prognostic assessment of thyroid malignancies.

E-Cigarettes as a Growing Threat for Children and Adolescents: Position Statement From the European Academy of Paediatrics.

As the tobacco epidemic has waned, it has been followed by the advent of electronic nicotine delivery devices (ENDS) primarily manufactured by the tobacco industry to try to recruit replacements for deceased tobacco addicts. This document sets out the ten recommendations of the European Academy of Paediatrics (EAP) with regard to e-cigarettes and children and young people (CYP). The EAP notes that nicotine is itself a drug of addiction, with toxicity to the foetus, child and adult, and were ENDS only to contain nicotine, their use to create a new generation of addicts would be rigorously opposed. However, e-cigarettes include numerous unregulated chemicals, including known carcinogens, whose acute and long term toxicities are unknown. The EAP asserts that there is incontrovertible evidence that the acute toxicity of e-cigarettes is greater than that of "traditional" tobacco smoking, and a variety of acute pulmonary toxicities, including acute lung injuries, have been recorded due to e-cigarettes usage. The chronic toxicity of e-cigarettes is unknown, but given the greater acute toxicity compared to tobacco, the EAP cannot assume that e-cigarettes are safer in the long term. The high uptake of e-cigarettes by CYP, including under-age children, is partly fuelled by deceitful marketing and internet exposure, which is also unregulated. Although proposed as aids to smoking cessation, there is no evidence that e-cigarettes add anything to standard smoking cessation strategies. In summary, the EAP regards these devices and liquids as very dangerous, and ineluctably opposed to their use, and their direct or indirect marketing.

Aborted Cardiac Arrest in LQT2 Related to Novel KCNH2 (hERG) Variant Identified in One Lithuanian Family.

Congenital long QT syndrome (LQTS) is a hereditary ion channelopathy associated with ventricular arrhythmia and sudden cardiac death starting from young age due to prolonged cardiac repolarization, which is represented by QT interval changes in electrocardiogram (ECG). Mutations in human ether-à-go-go related gene (KCNH2 (7q36.1), formerly named hERG) are responsible for Long QT syndrome type 2 (LQT2). LQT2 is the second most common type of LQTS. A resuscitated 31-year-old male with the diagnosis of LQT2 and his family are described. Sequencing analysis of their genomic DNA was performed. Amino acid alteration p.(Ser631Pro) in KCNH2 gene was found. This variant had not been previously described in literature, and it was found in three nuclear family members with different clinical course of the disease. Better understanding of genetic alterations and genotype-phenotype correlations aids in risk stratification and more effective management of these patients, especially when employing a trigger-specific approach to risk-assessment and individually tailored therapy.

Dysregulation of microRNAs as the risk factor of lymph node metastasis in papillary thyroid carcinoma: systematic review.

Papillary thyroid carcinoma (PTC) has an excellent prognosis with a relatively low mortality rate, but a small portion of PTC patients suffer from an aggressive form of the disease. In such cases early detection of lymph node metastasis (LNM) is as paramount as it is problematic. The routine use of central neck lymph node dissection is not recommended. New methods to detect LNM are needed. MicroRNAs are a potential biomarker for diagnosis and prognosis of PTC. In this review we summarise the current knowledge regarding dysregulated miRNAs and their association with LNM in PTS patients. The PubMed and EBSCO databases were searched using terms for "microRNA", "thyroid carcinoma", and "prognosis" by using Boolean operators. Based on eligibility and exclusion criteria, articles were screened and reviewed in full, methodological data of included studies were extracted, and risk of bias analysis performed. In total, 446 unique studies were extracted from the mentioned databases, and based on inclusion and exclusion criteria 27 studies were included in this review. Of them 17 analysed tissue microRNAs, 5 analysed circulating microRNAs, and 5 studies analysed both tissue and circulating samples. MiRNA-146B, miRNA-221, miRNA-222, miRNA-21, miRNA-204, miRNA-451, miRNA-199a-3p, and miRNA-30a-3p were dysregulated in at least 2 separate studies. A sizable portion of studies failed to show statistically significant differences in miRNA expression between LNM-positive and -negative patients. Different methodologies and disparities of patient populations could explain these discrepancies.\ This research supports the statement that specific up- and downregulated miRNAs are associated with LNM in PTC patients. However, the prognostic value of these miRNAs is limited. Additional targeted cohort studies are required to elucidate the role of miRNAs in defining individualised treatment strategies for thyroid cancer patients.

A novel variant in the PDE4D gene is the cause of Acrodysostosis type 2 in a Lithuanian patient: a case report.

BACKGROUND: Acrodysostosis is a rare hereditary disorder described as a primary bone dysplasia with or without hormonal resistance. Pathogenic variants in the PRKAR1A and PDE4D genes are known genetic causes of this condition. The latter gene variants are more frequently identified in patients with midfacial and nasal hypoplasia and neurological involvement. The aim of our study was to analyse and confirm a genetic cause of acrodysostosis in a male patient. CASE PRESENTATION: We report on a 29-year-old Lithuanian man diagnosed with acrodysostosis type 2. The characteristic phenotype includes specific skeletal abnormalities, facial dysostosis, mild intellectual disability and metabolic syndrome. Using patient's DNA extracted from peripheral blood sample, the novel, likely pathogenic, heterozygous de novo variant NM_001104631.2:c.581G > C was identified in the gene PDE4D via Sanger sequencing. This variant causes amino acid change (NP_001098101.1:p.(Arg194Pro)) in the functionally relevant upstream conserved region 1 domain of PDE4D. CONCLUSIONS: This report further expands the knowledge of the consequences of missense variants in PDE4D that affect the upstream conserved region 1 regulatory domain and indicates that pathogenic variants of the gene PDE4D play an important role in the pathogenesis mechanism of acrodysostosis type 2 without significant hormonal resistance.

A Familial 4q12 Deletion Involving KIT Gene Causes Piebaldism.

Piebaldism is a rare, autosomal dominant disorder characterized by the congenital absence of melanocytes in affected areas of the skin and hair. We report on a familial 4q12 deletion that involves the KIT gene and causes piebaldism in affected individuals. Whole-genome genotyping analysis of the proband using HumanCytoSNP-12v2.1 BeadChips (Illumina Inc., San Diego, CA, USA, revealed a 1.34-Mb microduplication of 1q21.1q21.2 and a 2.7-Mb microdeletion of 4q12. The analysis of the parents confirmed the paternal origin of the 4q12 microdeletion. The clinical and molecular findings in the proband and his affected relatives showed that the 2.7-Mb 4q12 microdeletion, the smallest microdeletion reported to date, causes isolated piebaldism due to the loss of the KIT gene.

The First Live Birth in Lithuania After Application of Preimplantation Genetic Testing.

SUMMARY BACKGROUND: Preimplantation genetic testing (PGT) is a genetic testing procedure that is performed before the implantation of embryos for the identification of genetic abnormalities. It is commonly performed when one or both expecting parents have such abnormalities and are at a high risk of passing them to their offspring. The aim of this case report is to describe the first successful IVF/ICSI/PGT procedure in Lithuania. CASE REPORT: A 27-year-old woman and a 31-year-old man, a married couple, were referred to VUHSK Santaros Fertility Center after trying to conceive for 4 years. In a previous relationship, the woman got pregnant spontaneously and decided to terminate the pregnancy. The husband does not have any children. During the medical examination, the transvaginal ultrasound revealed a low antral follicle count and low anti-Müllerian hormone level for the woman. Semen analysis for the male patient showed severe oligoastenospermia, which confirmed the previous abnormal spermogram results. Chromosome analysis revealed normal karyotype for the woman (46,XX) and Robertsonian translocation for the husband (45,XY,der(13;14)(q10;q10)). After the interdisciplinary medical team counselling, an ICSI with PGT-SR was suggested for the couple. The woman underwent controlled ovarian hyperstimulation with GnRH antagonist protocol for 11 days. Only one embryo with no unbalanced rearrangements was identified and transferred to the woman. On the 14th day post oocyte retrieval, the first serum ß-hCG result was received - 39.5 mIU/ml, and the normal gestational sac at 5 weeks and 3 days was confirmed by ultrasound examination. CONCLUSION: the first successful pregnancy was achieved in Lithuania and the first IVF/ICSI/PGT-SR newborn in Lithuania was born in 2019 - a vaginal birth of a healthy girl with gestational age of 38 weeks and 4 days and a weight of 2820 g; the Apgar score was 10/10. The IVF/ICSI/PGT procedure was successfully implemented by the multidisciplinary team in VUHSK.

Autosomal recessive hypercholesterolemia: Case report.

INTRODUCTION: Autosomal recessive hypercholesterolemia (ARH; OMIM #603813) is a very rare monogenic disorder affecting less than 1 in 1000,000 people and is characterized by very high levels of low-density lipoprotein cholesterol (LDL-C), leading to aggressive and premature atherosclerotic cardiovascular disease if left untreated. Lowering of LDL-C is the main target of the treatment. We report on a 29-year-old male patient born in nonconsanguineous Lithuanian family homo(hemi-)zygous for LDLRAP1 gene variant causing ARH. This variant is not present in population databases and, to our knowledge, has not been reported in scientific literature before. METHODS AND RESULTS: The earliest clinical sign, noticed at the age of 5 years, was painful and enlarging nodules on Achilles tendons. At the age of 10 years, xanthomas of the metacarpal joint area on both hands emerged. The first lipid panel was performed at the age of 12 years. In accordance with Dutch Lipid Clinic Network diagnostic criteria for familial hypercholesterolemia (FH), definite FH (type IIA hyperlipoproteinemia) was diagnosed and the treatment with cholestyramine 4 grams per day was initiated. As the patient was 15 years old, direct adsorption of low-density lipoprotein apheresis was started and repeated monthly. At the age of 20 years, along with lipoprotein apheresis, 10 mg of rosuvastatin daily intake was prescribed. At the age of 28 years, the dose of rosuvastatin was increased to 40 mg per day, and 10 mg of ezetimibe daily intake was added. At the age of 28 years, homozygous LDLRAP1 gene variant NM_015627.2:c.488A>C, NP_056442.2:p.(Gln163Pro) causing autosomal recessive hypercholesterolemia was determined by genetic testing. CONCLUSIONS: This case report implies that ARH, being an extremely rare disorder, is a severe disease. As there is limited routine testing, including genetic testing, patients suffering from both this disease and FH may remain undiagnosed. Cascade screening and genetic counseling differ for ARH as compared with FH, as the carrier of a pathogenic variant in the LDLRAP1 gene does not have marked total cholesterol and LDL-C elevations. However, genetic testing of the proband and their relatives is essential to evaluate the risk of development of FH and to provide prognosis as well as adequate, timely treatment. To improve the quality of life of patients with FH and prolong their life expectancy, national registries of FH and wider laboratory and genetic testing are undoubtedly necessary. A national FH screening program was set up in Lithuania, which helps to identify, monitor, and treat subjects with FH.

Novel GLI3 variant causes Greig cephalopolysyndactyly syndrome in three generations of a Lithuanian family.

BACKGROUND: Preaxial polydactyly type IV, also referred as polysyndactyly, has been described in a few syndromes. We present three generations of a family with preaxial polydactyly type IV and other clinical features of Greig cephalopolysyndactyly syndrome (GCPS). METHODS AND RESULTS: Sequencing analysis of the GLI3 coding region identified a novel donor splice site variant NC_000007.14(NM_000168.6):c.473+3A>T in the proband and the same pathogenic variant was subsequently identified in other affected family members. Functional analysis based on Sanger sequencing of the proband's complementary DNA (cDNA) sample revealed that the splice site variant c.473+3A>T disrupts the original donor splice site, thus leading to exon 4 skipping. Based on further in silico analysis, this pathogenic splice site variant consequently results in a truncated protein NP_000159.3:p.(His123Argfs*57), which lacks almost all functionally important domains. Therefore, functional cDNA analysis confirmed that the haploinsufficiency of the GLI3 is the cause of GCPS in the affected family members. CONCLUSION: Despite the evidence provided, pathogenic variants in the GLI3 do not always definitely correlate with syndromic or nonsyndromic clinical phenotypes associated with this gene. For this reason, further transcriptomic and proteomic evaluation could be suggested.

DNA methyltransferases inhibitors effectively induce gene expression changes suggestive of cardiomyogenic differentiation of human amniotic fluid-derived mesenchymal stem cells via chromatin remodeling.

Human amniotic fluid-derived mesenchymal stem cells (AF-MSCs) are a new potential stem cell source for cell therapy and regenerative medicine. These are fetal mesenchymal stem cells with multilineage differentiation potential found in amniotic fluid. The aim of the present study was to evaluate in vitro differentiation initiation of AF-MSCs into cardiac progenitors upon application of inhibitors of DNA methyltransferases (DNMT), such as Decitabine (DEC; 5-aza-2'-deoxycytidine) and Zebularine (ZEB). We assessed epigenetic changes and explored patterns of genes, enriched in association with hyperacetylated H4 after induced differentiation. Upregulation of cardiomyogenesis-related genes (TNNT2, MYH6, ACTN2, and DES) and cardiac ion channels genes, downregulation of pluripotency genes markers as well as increase in Connexin43 expression indicated cardiomyogenic commitment. Evaluation of global epigenetic changes showed that levels of chromatin modifying enzymes, such as Polycomb repressive complex 2 proteins (EZH2, SUZ12), DNMT1, histone deacetylases 1 and 2 were reduced to the similar extent by both differentiation agents. Levels of specific histone marks keeping active state of chromatin (H3K4me3, H3K9Ac, and H4hyperAc) increased and marks of repressed chromatin state (H3K27me3 and H3K9me3) decreased after DEC or ZEB treatment. Chip-Seq analysis after chromatin immunoprecipitation with H4hyperAc demonstrated enrichment of around 100 functionally annotated genes, related to chromatin reorganization and cardiomyogenesis and confirmed relation between H4 hyperacetylation and gene expression. Our results demonstrate that both DEC and ZEB can be potentially used as cardiomyogenic differentiation inducers in AF-MSCs, and they cause various genetic and epigenetic changes resulting in global chromatin remodeling.

Epigenetic alterations in amniotic fluid mesenchymal stem cells derived from normal and fetus-affected gestations: A focus on myogenic and neural differentiations.

Amniotic fluid-derived mesenchymal stem cells (AF-MSCs) are autologous to the fetus and represent a potential alternative source for the regenerative medicine and treatment of perinatal disorders. To date, AF-MSCs differentiation capacity to non-mesodermal lineages and epigenetic regulation are still poorly characterized. The present study investigated the differentiation potential of AF-MSCs toward neural-like cells in comparison to the mesodermal myogenic lineage and assessed epigenetic factors involved in tissue-specific differentiation. Myogenic and neural differentiation assays were performed by the incubation with specific induction media. Typical MSCs markers were determined by flow cytometry, the expression of lineage-specific genes, microRNAs and chromatin modifying proteins were examined by RT-qPCR and Western blot, respectively. AF-MSCs of normal and fetus-affected gestations had similar stem cells characteristics and two-lineage potential, as characterized by cell morphology and the expression of myogenic and neural markers. Two-lineage differentiation process was associated with the down-regulation of miR-17 and miR-21, the up-regulation of miR-34a, miR-146a and DNMT3a/DNMT3b along with the gradual decrease in the levels of DNMT1, HDAC1, active marks of chromatin (H4hyperAc, H3K9ac, H3K4me3) and the repressive H3K9me3 mark. Differentiation was accompanied by the down-regulation of PRC1/2 proteins (BMI1/SUZ12, EZH2) and the retention of the repressive H3K27me3 mark. We report that both AF-MSCs of normal and fetus-affected gestations possess differentiation capacity toward myogenic and neural lineages through rather similar epigenetic mechanisms that may provide potential applications for further investigation of the molecular basis of prenatal diseases and for the future autologous therapy.

X-linked juvenile retinoschisis: phenotypic and genetic characterization.

Juvenile X-linked retinoschisis (XLRS, MIM#312700) belongs to a group of the vitreoretinal dystrophies. We aimed to describe the phenotype-genotype correlation of three XLRS cases in juveniles with different novel mutations from the Lithuanian population. The patients demonstrated macular retinoschisis and typical cyst-like cavities on spectral-domain optical coherence tomography (SD-OCT) images. The mean central foveal thickness was 569.7 µm. Two patients presented with peripheral retinoschisis. Flash electroretinogram demonstrated a reduced b/a ratio (<1.0) in all patients. RS1 (NM_000330.3) gene coding exons Sanger sequencing was performed. RS1 c.599G>T (p.R200L) mutation was detected in one case, showing to be pathogenic in silico analysis. c. (92_97) insC (p.W33fs) mutation was identified for another patient, indicating the variant is possibly damaging in silico analysis. The third case was identified with a pathogenic mutation c.422C>G (p.R141H), HGMD CM981753. These are the first cases of XLRS in the Lithuanian population confirmed by molecular genotyping. Presented patients had a different genotype but similar phenotypic traits.

Histone Modifications Pattern Associated With a State of Mesenchymal Stem Cell Cultures Derived From Amniotic Fluid of Normal and Fetus-Affected Gestations.

Human amniotic fluid (AF)-derived mesenchymal stem cells (MSCs) sharing embryonic and adult stem cells characteristics are interesting by their multipotency and the usage for regenerative medicine. However, the usefulness of these cells for revealing the fetal diseases still needs to be assessed. Here, we have analyzed the epigenetic environment in terms of histone modifications in cultures of MSCs derived from AF of normal pregnancies and those with fetal abnormalities. The comparison of MSCs samples from AF of normal pregnancies (N) and fetus-affected (P) revealed two distinct cultures by their proliferation potential (P I and P II). Cell populations from N and P I samples had similar growth characteristics and exhibited quite similar cell surface (CD44, CD90, CD105) and stemness markers (Oct4, Nanog, Sox2, Rex1) profile that was distinct in slower growing and faster senescent P II cultures. Those differences were associated with changes in 5-Cyt DNA methylation and alterations in the expression levels of chromatin modifiers (DNMT1, HDAC1/2), activating (H4ac, H3K4me3), and repressive (H3K9me2/me3, H3K27me3) histone marks. MSCs isolated from AF with the genetic or multifactorial fetal diseases (P II samples) were enriched with repressive histone marks and H4K16ac, H3K9ac, H3K14ac modifications. This study indicates that differential epigenetic environment reflects a state of AF-MSCs dependently on their growth, phenotype, and stemness characteristics suggesting a way for better understanding of epigenetic regulatory mechanisms in AF-MSCs cultures in normal and diseased gestation conditions. J. Cell. Biochem. 118: 3744-3755, 2017. © 2017 Wiley Periodicals, Inc.

Perinatal manifestation of mevalonate kinase deficiency and efficacy of anakinra.

BACKGROUND: Mevalonate kinase deficiency is a metabolic autoinflammatory syndrome caused by mutations in the MVK gene, mevalonate kinase, the key enzyme in the non-sterol isoprenoid biosynthesis pathway. Two phenotypes of mevalonate kinase deficiency are known based on the level of enzymatic deficiency, mevalonic aciduria and hyperimmunoglobulinemia D syndrome, but a wide spectrum of intermediate phenotypes has been reported. Currently one of the most effective treatments is biological therapy (with interleukin-1 antagonist anakinra or tumour necrosis factor-α inhibitor etanercept). CASE PRESENTATION: The patient in this case has a phenotype contributing to a severe disease that caused the symptoms to manifest very early, in the prenatal period. Mevalonate kinase deficiency was suspected on the basis of clinical (hydrops fetalis, hepatosplenomegaly, hypotonia) and laboratory signs (anaemia, intense acute phase reaction, increased urinary excretion of mevalonic acid). Mutation analysis of the MVK gene confirmed the biochemical diagnosis. Treatment with the interleukin-1 antagonist anakinra was started (minimal dose of 1 mg/kg/day) and revealed its efficacy after three days. CONCLUSIONS: Our case highlights the need for a very detailed clinical and laboratory assessment in new-borns with any suggestion of autoinflammatory disorders. It is important that patients are diagnosed as early as possible to provide better multidisciplinary follow-up and therapy when needed.