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Direct-acting antivirals (DAAs) for hepatitis C virus (HCV) infection have delivered high response rates (>95%) and simplified the management of HCV treatment, permitting non-specialists to manage patients without advanced liver disease. We collected and reviewed global data on the registration and reimbursement (government subsidised) of HCV therapies, including restrictions on reimbursement. Primary data collection occurred between Nov 15, 2021, and July 24, 2023, through the assistance of a global network of 166 HCV experts. We retrieved data for 160 (77%) of 209 countries and juristrictions. By mid-2023, 145 (91%) countries had registered at least one of the following DAA therapies: sofosbuvir–velpatasvir, sofosbuvir–velpatasvir–voxilaprevir, glecaprevir–pibrentasvir, sofosbuvir–daclatasvir, or sofosbuvir. 109 (68%) countries reimbursed at least one DAA therapy. Among 102 low-income and middle-income countries (LMICs), 89 (87%) had registered at least one HCV DAA therapy and 53 (52%) reimbursed at least one DAA therapy. Among all countries with DAA therapy reimbursement (n=109), 66 (61%) required specialist prescribing, eight (7%) had retreatment restrictions, seven (6%) had an illicit drug use restriction, five (5%) had an alcohol use restriction, and three (3%) had liver disease restrictions. Global access to DAA reimbursement remains uneven, with LMICs having comparatively low reimbursement compared with high-income countries. To meet WHO goals for HCV elimination, efforts should be made to assist countries, particularly LMICs, to increase access to DAA reimbursement and remove reimbursement restrictions—especially prescriber-type restrictions—to ensure universal access.

Recent advances in molecular genetics and the invention of new technologies led to an advance in the development of gene therapy. Gene therapy is used to correct defective genes in order to cure a disease or help the body better fight a disease. It works by restoring or modifying cellular functions through the introduction of a functional gene into the target cell. The concept of gene therapy is simple, but introducing it to routine clinical practice is not. The main concerns are related to some safety issues as well as to the problem that maintaining a stable and prolonged expression in target cells may not be easily achieved. In spite of the difficulties, gene therapy remains a hope for many hematological disorders that cannot be effectively treated so far. This article reviews the current status of gene therapy with a focus on hematological disorders. In addition, clinically applied approaches are presented through particular examples of approved gene therapy drugs.

Recent advances in molecular genetics and the invention of new technologies led to a development in our knowledge about human microbiota, specifically bacterial one. The microbiota plays a fundamental role in the immunologic, hormonal and metabolic homeostasis of the host. After the initiation of the Human Microbiome Project, it became clear that the human microbiota consists of the 10-100 trillion symbiotic microbial cells harbored by each person, primarily bacteria in the gut, but also in other spots as the skin, mouth, nose, and vagina. Despite of the differences in studying bacterial species, decreased bacterial diversity and persistence has been connected with several diverse human diseases primarily diabetes, IBD (inflammatory bowel disease) and others; attempts were made even to explain psychiatric pathology. Several species emerged as dominant and were clearly linked to certain disorders or accepted as biomarkers of others. The current review aims to discuss key issues of our current knowledge about bacteria in human, the difficulties and methods of its analysis, its contribution to human health and responsibility for human diseases.

Introduction:Azacitidine is one of the hypomethylating agents available for the treatment of elderly patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). It is also used as an appropriate treatment of chronic myelomonocytic leukemia (CMML) in the real life setting. As treatment of AML and CMML is not curative, and allogeneic stem cell transplantation (allo-SCT) remains traditionally the only option, significant clinical benefits by hypomethylating agents have been reported. According to the available data, 16% of subjects with MDS who received azacitidine had a complete or partial normalization of blood cell counts and bone marrow morphology, while two-thirds of patients who required blood transfusions no longer needed them. Nevertheless, it can also be hepatotoxic in patients with severe liver impairment and extensive liver tumors.

Gaucher disease (GD) is a multi-systemic disease with a low population frequency. It is a lysosomal storage disorder (LSD) that causes accumulation of glucocerebroside in the so called Gaucher cells predominantly in areas like the spleen, liver and bone marrow. Type I GD (GDI) is the most common form and usually does not involve the brain and the spinal cord. The symptoms can range from mild to severe and may appear anytime from childhood to adulthood. Diagnostics can often be challenging and imposes looking at person’s medical history, symptoms, physical exam, and laboratory test results. We present a difficult to diagnose case of a 34-year-old woman admitted to the Clinic of Hematology, “Sv. I. Rilski” hospital with splenomegaly, normal laboratory findings and non-enlarged liver. She didn’t show focal neurological symptoms. A series of tests were assigned including genetic targeted analysis. The case is an example of a rare genetic disease with mild clinical symptoms. Diagnosis of Gaucher disease, type I was confirmed by measurement of a GBA enzyme activity and identification of mutations in the GBA gene inherited in an autosomal recessive manner. Thanks to the efforts of the clinical team, the assignment of adequate clinical and laboratory tests and their correct interpretation, the patient was subjected to enzyme replacement therapy (ERT). Although the diagnosis was settled relatively late (at 34 years of age), the correct therapy slowed down the invalidation and improved the quality of life of the patient.

Myelofibrosis (MF) is characterized by a presence of an extra fibrous tissue in the bone marrow and additional hematopoiesis. The somatic mutation in the Janus kinase 2 (JAK2) gene (V617F) occurs gradually and is detected in about 50.0% of myelofibrosis or essential thrombo-cytopenia (ET) patients. Our aim was to determine the genotype status according to the carriers of the V617F mutation in patients with MF at the Hematology Ward of the University Hospital "Ivan Rilski" in Sofia, Bulgaria. DNA samples were isolated from venous blood of patients with various hematological disorders. DNA was amplified by polymerase chain reaction (PCR) and subsequent restriction analysis was performed using a BsaXI restriction enzyme. The genotype status was determined on 2.0% agarose gel. We analyzed 38 patients initially suspected of carrying MF or osteomyelofibrosis (OMF). After trepanobiopsy, 20 out of 38 patients were confirmed as myelofibrotic (52.6%), 5/38 (13.2%) were diagnosed as ET, 1/38 (2.6%) was diagnosed as myeloproliferative neoplasm (MPN), 6/38 (15.8%) had polycythemia vera (PV). In six patients, the presence of disease was rejected. Patients with MF were divided into three groups according to the JAK2V617F genotype status: homozygous for the mutation (3/20 or 15.0%), heterozygous (9/20 or 45.0%) and homozygous for the wild type allele (8/20 or 40.0%). The triggering factor of MF is still unknown. It was considered that this factor could have a genetic nature. Mutations in three genes were mainly accepted as an actual predisposing events to this disease: point mutations leading to amino acid substitutions in JAK2(V617F) and in MPL(W515L, W515K), as well as insertion or deletion in CALKWe have proven that carriers of the V617F mutation prevailed in the group of patients with MF (altogether 12 patients or 60.0%). Previous studies also showed that JAK2V617F is present in more than half of MF patients within their blood-forming cells. Therefore, the risk of evolution to MF could be associated with V617F-mutant allele burden in patients with MPN.

Mutations in the receptor of the epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) are used as biomarkers for predicting the response of treatment with EGFR tyrosine kinase inhibitors (EGFR TKIs). Non-small cell lung cancer patients usually have activating EGFRmutations that leads to a very good response when they are treated with EGFR TKIs. Our tumor samples were examined for the presence of sensitive mutations in the EGFRgene, resistant mutations or the absence of mutations. To identify the types of the mutation, we used a real-time polymerase chain reaction (RT-PCR) method. Additionally, we evaluated the frequency of EGFRmutations and their association with smoking status, gender and histology. The tumor samples (n= 551) were tested for 29 somatic mutations in the EGFRgene. Sensitive mutations in the EGFRgenes were found in 55 NSCLC samples (10.0%). The prevalence of EGFRmutations was much higher for females than for males (27.1 vs. 3.9%, p<0.001). The prevalence of EGFRmutations was greater in subjects who had never smoked than in smokers (15.0 vs. 6.08%, p<0.003). Additionally, the frequency of EGFRmutations was higher in adenocarcinomas than in other histological types (14.9 vs. 5.1%; p<0.001). Our results show that activating mutations on the EGFRgene are more frequent in females than in males, in adenocarcinoma than other histological types and in non smokers than smokers.

Epigenetic changes, in particular DNA methylation processes, play a role in the pathogenesis and progression of type 2 diabetes mellitus (T2DM) linking genetic and environmental factors. To clarify this role, we have analyzed in patients with different duration of T2DM: (i) expression levels of methyl-CpG-binding domain protein 2 (MBD2) as marker of DNA methylation, and ii) methylation changes in 22 genes connected to cellular stress and toxicity. We have analyzed MBD2mRNA expression levels in16 patients and 12 controls and the methylation status of stress and toxicity genes in four DNA pools: (i) controls; (ii) newly-diagnosed T2DM patients; (iii) patients with T2DM duration of <5 years and (iv) of >5 years. The MBD2expression levels were 10.4-times increased on average in T2DM patients compared to controls. Consistent increase in DNA methylation fraction with the increase in T2DM duration was observed in Prdx2and SCARA3genes, connected to oxidative stress protection and in BRCA1and Tp53tumor-suppressor genes. In conclusion, increased MBD2expression in patients indicated general dysregulation of DNA methylation in T2DM. The elevated methylation of Prdx2and SCARA3genes suggests disturbance in oxidative stress protection in T2DM. The increased methylation of BRCA1and Tp53genes unraveled an epigenetic cause for T2DM related increase in cancer risk.

* CLINICAL QUESTION Do antioxidants prevent gastrointestinal cancers? * BOTTOM LINE Antioxidants do not prevent gastrointestinal cancers. In fact, in pooled results of high-quality studies, antioxidants increased overall mortality. (Level [...]

ABSTRACTFilamentous fungi are widely used for the production of homologous and heterologous proteins. Recently, there has been increasing interest in Aspergillus oryzae PP and Aspergillus awamori K-1 because of its ability to produce heterologous proteins in submerged (liquid) cultures. The goal of this investigation was to determine the α-amylase and endoxylanase enzyme production ability and molecular characteristics of fungal strains Aspergillus oryzae PP and Aspergillus awamori K-1 and its mutant strains R5 and A45. The strains were cultivated in liquid culture and maximum enzyme production of parent and mutant strains was determined after 72–96 h of cultivation. Extracellulars α-amylase and endoxylanase were partially purified from the culture filtrates, using molecular sieve chromatography with Gel permeation chromatography. The molecular weight of the partial purified enzymes has been estimated to be 57 kDa for α-amylase and 31 kDa for endoxylanase on SDS-polyacrylamide gel electrophoresis. The temperature optimum of the enzyme α-amylase was 30°C, respectively for endoxylanase was 40°C and the pH optimum was 4.7 and 4.0.

Apoptosis Gene Expression Profile in Early-Stage non Small Cell Lung CancerNon small cell lung cancer (NSCLC) is a highly aggressive malignancy with survival rates limited to some patients in early stages (I and II). Apoptosis resistance is a hallmark of solid tumors that is tightly concerned with their biology. We analyzed the expression of 84 apoptosis-related genes in a group of Bulgarian patients with early-stage NSCLC.RNA samples extracted from 12 early-stage NSCLC patients [five squamous cell carcinomas (SCC) and seven adenocarcinomas (AC)] and eight adjacent non neoplastic pulmonary tissues were used for gene expression analysis. We applied pathway-focused expression profiling of 84 apoptosis-related genes using real-time PCR.Apoptosis-related genes down regulated in NSCLC compared to non tumor lung tissue (p<0.05) included representatives of the tumor necrosis factor (TNF) ligand family [TNF superfamily 8 (TNFSF8)], caspase cascade (CASP8 and CASP10) and caspase recruitment domain (CARD) family (BCL10), the positive apoptosis regulator DAPK1 and BCL2 family member MCL1. The potential of apoptosis-related genes as prognostic and predictive markers should be validated in future studies.

ABSTRACTXylanolytic enzymes of microbial origin have received great attention due to their biotechnological utility and potential applications in a range of industrial processes but the observed effects vary depending on xylanase specificities.The goal of this investigation was to determine the endoxylanase enzyme production ability and molecular characteristics of fungal strains Aspergillus awamori K-1 and its mutant strains A59, A50, A45 and A60. The strains were cultivated in liquid culture with 1% wheat bran and 2% ground corn-cobs as inducer. Maximum enzyme production of parent strain Aspergillus awamori K-1 of 42,35 IU/ml and of mutant strains A59 - 96,91 IU/ml, A50 - 71,67 IU/ml, A45 - 74,04 IU/ml and A60 - 62,59 IU/ml was determined after 72–96 h of cultivation.Extracellular endoxylanase was partially purified from the culture filtrates of five strains, using molecular sieve chromatography with two type of Sephadex—G 50 and G100. The purification conditions were optimized and the main endoxylanase activity was determined in second fraction in case of Sephadex G100. The partial purified enzyme fractions showed the same band for all strains on SDS polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular weight of 32 kDa.

ABSTRACTMus spicilegus is an outdoor species from southern-eastern Europe, adapted to agroecosystems. In the beginning of autumn, to spend the winter, mound-building mice build complex mounds. These mounds are built using accumulation of seeds covered with earth. This behavior is unique among mice species. However, there is evidence that at higher temperatures and a larger availability of food in winter, mice can optimize this particular behavior in direction to simpler constructions or even not build them at all. To clarify if mice display building activity under laboratory conditions we studied behavior of M. spicilegus in 5 intraspecific cage groups during autumn-winter period. The observations were carried out in 100 × 100 cm glass cages provided with earth and sawdust as bedding and straw and hay as nesting material. The groups were formed by 3–4 mice captured from a wild population in northern Bulgaria in early September of 2008, where mounds were already built. Mice were grouped according to the mound they belong to. The results showed that mound-building mice display high level of building activity, especially in the first month of their time in the laboratory. In majority of groups mice built domed nests made from straw and hay, where they lived together. Seeds in the cages were covered with earth, as far as that was possible. However, mice in some groups made burrows of simple design. Based on these findings it could be assumed that building behavior of M. spicilegus is genetically determined, but as microtine rodents M. spicilegus can be quite flexible in employing behavior to suit the local environment.

ABSTRACTSelection of fusant cultures, producers of bioactive compounds is of significant importance for the biotechnology industry. Protoplast fusion between prototrophic strains of Aspergillus oryzae PP, producer of alfa-amylase and Apergillus awamori K-1, producer of xylanase was obtained. The interspecific nature of the fusants was determined by morphology and screening for combined enzymatic activity. The aim of this investigation was to quantitate the dominant parental genome in the fusants by applying whole genome typing strategy based on the Amplified Fragment Length Polymorphisms (AFLP) technique. In addition we investigated the genome decomposition of the fusant cultures from first to fourth generation. Our results demonstrate that the appearances of the fusants varied but they basically resembled Aspergillus awamori. On the bases of more than 80 polymorphic markers, the genomic analysis confirmed that Aspergillus awamori is the dominant genome (>85%) in the fusants. The stability of the fusants was examined by successive subcultures. The genomic decomposition of the fusants between first and forth generation is as little as 3%. From our studies we could conclude that AFLP is an efficient and discriminatory method for evaluation of the dominant genome in fusant cultures and the extent of genomic decomposition in the generations.

ABSTRACTDifferent aspects, including safety, functional and technological characteristics, have to be taken into consideration in the selection of each probiotic microorganism. The aim of the present work was to determine the kinetic growth parameters of L. plantarum strain after cultivation in media with different carbon sources. The strain was isolated from traditional Bulgarian white cheese and previously characterized as putative probiotic, based on commonly accepted in vitro criteria. For further biotechnological implementation was necessary to select a suitable and economically relevant growth media. Thus, reconstituted permeate (6% w/v) and the following modification of de Man, Rogose Sharpe media (MRS): (i) MRS-glucose; (ii) MRS-lactose; (iii) MRS-galactooligosaccharide; (iv) MRS-fructooligosaccharide were used. The strain growth, lactic acid production and carbon source utilization were monitored by pH and cell number determination, and HPLC analysis at different time points of the cultivation process. The highest cell growth and carbohydrate conversation were detected in the presence of glucose and lactose. The main product of the fermentation was lactate with detectable level of acetate. The permeate and MRS-galactooligosaccharide also support good growth and lactic acid production, which indicate a great potential for industrial applications of studied L. plantarum strain into the food system.

ABSTRACTThis study aimed to isolate and characterize viable lactic acid bacteria (LAB) from the most popular in Bulgaria fermented milk products. Different samples from home-made cheeses, yoghurt and katak, from ecological regions of Stara Planina, Rila and Rodopi mountains were collected. A total of 25 LAB cultures (coci and rods) were isolated and polyphasic taxonomic characterization was performed. Eight of the strains from yoghurt were phenotypically similar to Lactobacillus delbrueckii. Other seven isolates from cheese and yoghurt were identified as Lactobacillus plantarum, according to classical tests and API LAB profiles. Their species affiliation was confirmed by species-specific and multiplex PCR. The molecular- based methods allowed reclassifying two cheese isolates determined as L. paracasei. L. plantarum was the predominant species in cheese samples and referred as member of non-starter lactic acid bacteria. Two of the new isolated L. plantarum strains showed anti-E. coli activity.

ABSTRACTIn recent years interest in the probiotic lactobacilli has been stimulated by the use of these bacteria in products that are claimed to confer health benefits on the consumer. The probiotic effects are usually strain-specific, meaning that a correct identification is important to link the strain to the specific health effect. Taxonomical characterization of probiotic strains only in phenotypic and phisyological characteristics is often with low level of discrimination, probably due to their co-evolution in the same ecological niches. Thus, the nucleotide base techniques provide an accurate basis for phylogenetic analysis and identification. With this aim a probiotic strain Lactobacillus spp. 50P1 was studied.Trough the physiological characterization with API 50 CH the strain 50P1, was determinate as Lactobacillus helveticus, with low probability- 77,5%. Three PCR-based methods, species-specific PCR, of 16S rRNA gene sequencing, and restriction enzyme of 16S rDNA ARDRA analysis were used for reliable taxonomic characterization of probiotic strain Lactobacillus sp. 50P1. The sequence obtained from the strain was compared to those of Lactobacillus species held in GenBank and the belonging of the strain 50P1 to the species Lactobacillus helveticus was confirmed.

Whole Genome Analysis by Array-Based Comparative Genomic Hybridization in Patients with Congenital MalformationsCongenital malformations present at delivery of an infant are due to genetic or non genetic factors and occur in 15-20% of stillborn children. Most can be diagnosed prenatally by ultrasound examination, but some can only be diagnosed after birth. Seven to 10% of infants with abnormal phenotype have numerical or structural chromosomal abnormalities that require identification for accurate diagnosis and genetic counseling. Molecular-cytogenetic and array-based techniques have enabled screening at higher resolution for congenital anomalies that result from genomic imbalances. We have examined four children with congenital anomalies, with or without mental retardation, of unclear etiology. In one child, we detected a deletion (about 28 Mb) of the region 18q21.1-18q23, in mosaic form. This abnormality was missed in a routine cytogenetic examination. We detected different polymorphic copy number variations (CNVs) in the other children. We conclude that array-based comparative genomic hybridization (CGH) is a powerful diagnostic tool for the detection of low level mosaicism.

SUMMARYThe changes in morphology, structure and chemical composition of the surface of stainless steel 316 L roughened by an electrochemical treatment have been studied by means of profilometry, SEM and XPS methods. The steel was subjected to a two-stage electrochemical anodic treatment based on the use of electrolytes containing an organic acid and a mixture of inorganic acids, respectively. The anodic treatment caused an essential change in surface morphology and structure, this resulting in an increase of the averaged surface depth Rzfrom 0.28 to 2.58 μm. XPS depth profiling has shown that the iron and nickel concentrations in the surface layer of the roughened steel are slightly reduced.

The percentage of thermal dehydration and decomposition of ordinary and deuterated hydrates of barium acetate (tri- and monohydrates) were studied with a derivatograph and by DSC method. The observed phase transitions were identified and their corresponding enthalpy changes determined. The latter were compared with those estimated on thermodynamic data. The conclusions made on the dehydration process in correlation with IR spectra were used as information about the structure of the dehydrated water in the compounds investigated. The end product was also identified as barium carbonate.

The thermal decompositions of Ba(OH)2 · 8H2O, Ba(OH)2 · 8D2O, Ba(OH)2 · H2O and Ba(OH)2 · D2O were studied and the phase transitions were identified by DSC and DTA methods. The corresponding enthalpy changes were determined and compared with those calculated from the thermodynamic data. A decrease of the thermal stability was demonstrated for the deuterated crystal hydrates in comparison with the normal ones.

Abstract: DTA and DSC were used to study the thermal behaviour of Ca(NO3)2·4H2O, Cd(NO3)2·4H2O, Mg(NO3)2·6H2O and their deuterated analogues. Evidence was found concerning the process of melting of the initial hydrates and deuterates, followed by a one-stage dehydration of the melt to vield the respective anhydrous salt. T m, ΔH mo , ΔS mo and ΔH deho were determined and the ΔH fo values for the investigated hydrates were calculated from the ΔH deho data.

In the present work the isotope effect has been studied by DTA and DSC technique not only in the thermal dehydration process but also in the thermal decomposition of water-free compounds formed from normal and deuterated hydrates. The differences in the thermal stability of the hydrates were characterized byTDeh,TPh, δHDeh and kinetic parameters (E* andA). The degree of crystallinity and particle size of the oxides formed as products of thermal decomposition of normal and deuterated hydrates of nitrates have been determined by electron microscopy.

IntroductionBuschke-Lowenstein tumour (BLT) is a very rare sexually-transmitted disease associated with human papillomavirus (HPV) type 6 and 11, but rare cases of oncogenic HPV types including HPV 16 and HPV 18 were also reported. BLT is located in the genital, anorectal and perianal regions. It is regarded as a type of verrucous carcinoma occurring on anogenital mucosal surfaces where it is locally invasive but displays a benign cytology. Buschke-Lowenstein tumour can be associated with a high rate of recurrence and a risk of malignant transformation to invasive SCC, especially in patients with oncogenic types of HPV.MethodsWe report the case of a 59-year-old female patient who addressed our clinic for a large, exophytic, cauliflower-like tumour involving the vulva, perineum and perianal regions with 20 years duration. The first lesions had been appeared on vulva and after 3 years period they grown slowly and covered perineum and perianal area. They had cauliflower like surface, with different sizes. In some points erosions and yellowish secretion with odour are observed.ResultsHistologic examination of a biopsy specimen of large tumour presented nets of well-differentiated squamous cell carcinom, as well as a marked mononuclear cell infiltrate and conspicuous koilocytosis. HPV DNA for 6, 11 types was detected with PCR.ConclusionThe patient was sent to the gynaecology surgery department for excision and remains under the supervision of the dermatology and oncology department for rapid treatment of relapses and early detection of malignant transformation.

The article studies the dielectric properties, dc conductivity and ac conductivity of Be(IO3)2⋅4H2O single crystals. The dielectric constant ɛ has been defined for the three directions of the vectors a, b and c in the crystals in the temperature interval 280–340 K and frequency range 100 Hz–106 Hz. The crystals show strongly expressed anisotropy, at 20 ∘C and frequency 100 Hz ɛa = 235, ɛb = 30 and ɛc = 85. The frequency dependence of ɛ is evidence of the presence of low-frequency relaxation polarization in the crystals. The activation energies of the three directions in the crystals have been derived from the temperature dependence of dc conductivity, and they are 1.03 eV, 0.836 eV and 1.2 eV respectively.

The authors of the Short Communication “[PbAsSiiPr3]6— the First Structurally Characterized Compound with Chemical Bonds between Lead and Arsenic” (C. von Hänisch, D. Nikolova, Z. Anorg. Allg. Chem.2004, 630, 345—346) overlooked the quaternary phase K8NbPbAs5, published by Gascoinund Sevov[1]. Its crystal structure reveals the presence of [NbPbAs5]8—anions. Therefore this is the first structurally characterized compound with bonds between lead and arsenic. Nevertheless, [PbAsSiiPr3]6remains the sole example of a molecular compound, characterized by single‐crystal X‐ray crystallography, containing such Pb—As bonds and organic substituents. The authors apologize for their oversight.

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