Comprehensive detection and identification of bacterial DNA in the blood of patients with sepsis and healthy volunteers using next-generation sequencing method - the observation of DNAemia.

Blood is considered to be a sterile microenvironment, in which bacteria appear only periodically. Previously used methods allowed only for the detection of either viable bacteria with low sensitivity or selected species of bacteria. The Next-Generation Sequencing method (NGS) enables the identification of all bacteria in the sample with their taxonomic classification. We used NGS for the analysis of blood samples from healthy volunteers (n = 23) and patients with sepsis (n = 62) to check whether any bacterial DNA exists in the blood of healthy people and to identify bacterial taxonomic profile in the blood of septic patients. The presence of bacterial DNA was found both in septic and healthy subjects; however, bacterial diversity was significantly different (P = 0.002) between the studied groups. Among healthy volunteers, a significant predominance of anaerobic bacteria (76.2 %), of which most were bacteria of the order Bifidobacteriales (73.0 %), was observed. In sepsis, the majority of detected taxa belonged to aerobic or microaerophilic microorganisms (75.1 %). The most striking difference was seen in the case of Actinobacteria phyla, the abundance of which was decreased in sepsis (P < 0.001) and Proteobacteria phyla which was decreased in the healthy volunteers (P < 0.001). Our research shows that bacterial DNA can be detected in the blood of healthy people and that its taxonomic composition is different from the one seen in septic patients. Detection of bacterial DNA in the blood of healthy people may suggest that bacteria continuously translocate into the blood, but not always cause sepsis; this observation can be called DNAemia.

Are there new biomarkers of the gastroduodenal microbiota useful in the diagnosis of coeliac disease in children? A pilot study.

The changing of microbiome could precede the development of coeliac disease (CeD). We compared the bacterial profile of microbiota of tissues collected simultaneously from the stomach and duodenum in newly diagnosed patients with CeD. Biopsies were collected from 60 children and adolescents aged 2-18 years: (1) 40 patients with CeD; (2) 20 children as control group. The evaluation of the bacterial microbiota was carried out by sequencing the V3-V4 regions of the 16S rRNA subunit, using next-generation sequencing (NGS). The composition of bacterial microbiota was correlated with clinical and blood parameters. The beta diversity analysis revealed a significant dissimilarity in the gastric samples between the CeD and control group (Bray-Curtis index, P = 0.008, and weighted UniFrac distance, P = 0.024). At L2 (phylum level), Campylobacterota was only present in the stomach of the CeD group. A comparison of the abundance of bacteria between the stomach and duodenum showed significant differences in 10 OTUs (operational taxonomic units) in the control and 9 OTUs in the CeD group at L6 (genus) and in 8 OTUs and in 6 OTUs, respectively, at L7 (species). A significant correlation was observed between the genus Novosphingobium in stomach of CeD group and possession of the DQ2.5 and DQ 8 allele, and in the duodenum - between the DQ 8 allele and the species Blautia wexlerae. Significant differences in selected, little-known genera of bacteria suggest their potential role as new biomarkers in the development of CeD. To fully understand the mechanism of CeD development in genetically predisposed individuals, it is necessary to take into account not only the abundance of a given genus or species of bacteria, but also the anatomical location of its occurrence.

Types of cell death and methods of their detection in yeast Saccharomyces cerevisiae.

The occurrence of programmed cell death in unicellular organisms is a subject that arouses great interest of theoreticians and experimental scientists. Already found evolutionarily conserved genes and metabolic pathways confirmed its existence in yeast, protozoa and even bacteria. In the yeast Saccharomyces cerevisiae, at least three main types of death are distinguished: apoptosis, necrosis and autophagy. Their classification suggested by the Nomenclature Committee on Cell Death initially based on the morphological characteristics has now been extended to include the measurable biochemical characteristics. Several laboratory methods previously used to detect the types of cell death of higher eucaryotes and later developed and successfully used for the analysis of yeast cells are here critically reviewed. Their advantages and limitations are described.

Alterations in intestinal Archaea composition in pediatric patients with Crohn's disease based on next-generation sequencing - a pilot study.

Intestinal dysbiosis can lead to the induction of systemic immune-mediated inflammatory diseases, such as Crohn's disease Although archaea are part of the commensal microbiota, they are still one of the least studied microorganisms. The aim of our study was the standardization of the optimal conditions and primers for sequencing of the gut archaeome using Next Generation Sequencing, and evaluation of the differences between the composition of archaea in patients and healthy volunteers, as well as analysis of the changes that occur in the archaeome of patients depending on disease activity. Newly diagnosed patients were characterized by similar archeal profiles at every taxonomic level as in healthy individuals (the dominance of Methanobacteria at the class level, and Methanobrevibacter at the genus level). In turn, in patients previously diagnosed with Crohn's disease (both in active and remission phase), an increased prevalence of Thermoplasmata, Thermoprotei, Halobacteria (at the class level), and Halococcus, Methanospaera or Picrophilus (at the genus level) were observed. Furthermore, we have found a significant correlation between the patient's parameters and the individual class or species of Archaea. Our study confirms changes in archaeal composition in pediatric patients with Crohn's disease, however, only in long-standing disease. At the beginning of the disease, the archeal profile is similar to that of healthy people. However, in the chronic form of the disease, significant differences in the composition of archaeome begin to appear. It seems that some archaea may be a good indicator of the chronicity and activity of Crohn's disease.

Interferon γ is a STAT1-dependent direct inducer of BCL6 expression in imatinib-treated chronic myeloid leukemia cells.

B-cell CLL/lymphoma 6 (BCL6) exerts oncogenic effects in several human hematopoietic malignancies including chronic myeloid leukemia (CML), where BCL6 expression was shown to be essential for CML stem cell survival and self-renewal during imatinib mesylate (IM) treatment. As several lines of evidence suggest that interferon γ (IFNγ) production in CML patients might have a central role in the response to tyrosine kinase inhibitor (TKI) therapy, we analyzed if IFNγ modulates BCL6 expression in CML cells. Although separate IFNγ or IM treatment only slightly upregulated BCL6 expression, combined treatment induced remarkable BCL6 upregulation in CML lines and primary human CD34+ CML stem cells. We proved that during combined treatment, inhibition of constitutive signal transducer and activator of transcription (STAT) 5 activation by IM allowed the specific enhancement of the STAT1 dependent, direct upregulation of BCL6 by IFNγ in CML cells. By using colony-forming assay, we found that IFNγ enhanced the ex vivo colony or cluster-forming capacity of human CML stem cells in the absence or presence of IM, respectively. Furthermore, inhibition of the transcriptional repressor function of BCL6 in the presence of IM and IFNγ almost completely blocked the cluster formation of human CML stem cells. On the other hand, by using small interfering RNA knockdown of BCL6, we demonstrated that in an IM-treated CML line the antiapoptotic effect of IFNγ was independent of BCL6 upregulation. We found that IFNγ also upregulated several antiapoptotic members of the BCL2 and BIRC gene families in CML cells, including the long isoform of MCL1, which proved to be essential for the antiapoptotic effect of IFNγ in an IM-treated CML line. Our results suggest that combination of TKIs with BCL6 and MCL1 inhibitors may potentially lead to the complete eradication of CML stem cells.

Hepatitis C in obstetrics and gynecology.

Because of the risk of perinatal transmission and possible sexual transmission, it is important for obstetrician-gynecologists to keep abreast of the rapidly expanding literature on hepatitis C. Acute hepatitis C represents about 5% of all reported cases of hepatitis. Approximately 50% of acute infections progress to chronic liver disease. Risk factors for infection include intravenous (IV) drug use (21-42% of cases), previous blood transfusion (6-17%), and multiple sexual partners (6%); 40-50% of cases have no identified risk factors. The seroprevalence of anti-hepatitis C antibody is 70.8% in IV drug users, 11.6% in patients with human immunodeficiency virus, 8.8% in prostitutes, 1.2% in hospital personnel, and 0.5-1.4% in volunteer blood donors. The risk of transmission to the neonate depends on the trimester at exposure. No perinatal transmission has been shown after acute maternal infection in the second trimester. Based on the few reported cases, chronic maternal infection or acute infection in the third trimester may result in neonatal infection rates of 45-87.5%. Universal screening is probably not cost-effective because the prevalence is low and over 70% of screening tests can be falsely positive using the currently approved assay. Selective screening of high-risk patients is recommended.

Pregnancy after mustard repair for transposition of the great arteries.

BACKGROUND: Since the introduction of surgical repair procedures, women with complete transposition of the great arteries are surviving into their reproductive years. Only three successful pregnancies in such women have been described previously. CASES: Three women with transposition of the great arteries repaired in childhood became pregnant in 1991. Two pregnancies were complicated by failure of the systemic ventricle and one by preterm labor. Labor was managed with antibiotic prophylaxis against endocarditis, clinical hemodynamic assessment, epidural anesthesia, avoidance of maternal expulsive efforts in the second stage, and low forceps delivery. Three healthy infants were delivered vaginally between 34-39 weeks' gestation. CONCLUSION: With close cooperation between the cardiologist and obstetrician, successful pregnancy is possible after surgical repair of transposition of the great arteries. However, failure of the systemic ventricle is common and should be diagnosed and treated promptly.

Elevated serum tumor necrosis factor-alpha concentrations and bioactivity in Type 2 diabetics and patients with android type obesity.

The role of tumor necrosis factor-alpha in insulin resistance has been studied in 59 patients with Type 2 diabetes, 28 with android type obesity and 35 healthy lean controls. Immunoreactive concentrations and bioactivity of serum tumor necrosis factor-alpha have repeatedly been determined in 8 weeks intervals for 12 months, five times per patients, by using ELISA and L929 cell cytotoxicity bioassay. Significantly higher immunoreactive tumor necrosis factor-alpha concentrations and bioactivity have been found in both, the Type 2 diabetic and obese groups as compared to the healthy persons. Tumor necrosis factor-alpha concentrations and bioactivity have showed a significant positive linear correlation with the elevated basal serum C-peptide levels and body mass indexes in both groups of patients. According to these data the cytokine might play a role in insulin resistance in obesity as well in Type 2 diabetes.

Calcium metabolism and endocrine functions in a family with familial hypocalciuric hypercalcemia.

OBJECTIVE: We report two Hungarian patients with familial hypocalciuric hypercalcemia (FHH) caused by a mutation of the calcium-sensing receptor (CaSR) at codon 55. The proband and her father were heterozygous for this mutation. DESIGN: We performed detailed clinical and laboratory assessments of this family to characterize the effects of CaSR mutation on several endocrine organs expressing CaSR. RESULTS: Interestingly, we could not detect any failure in the function of any tissues we examined, except in serum calcium levels. CONCLUSIONS: To our knowledge, this has been the first report from Eastern and Central Europe showing P55 L mutation of the CaSR, as well as the first publication discussing the effect of this mutation on several endocrine systems containing CASR.

Mutational activation of H-ras oncogene transformability by alkylnitrosourea-induced DNA damage.

To assess the role of DNA alkylation damage in oncogene activation, plasmid DNA containing H-ras proto-oncogene (p220-EC) and oncogene (p220-EJ) were treated with increasing concentrations of carcinogenic methylnitrosourea (MNU) and ethylnitrosourea (ENU). The modified plasmid DNA were analyzed by transfection-transformation of the NIH/3T3-recipient cells. Treatment with varying doses of MNU (0.1-5 mM) and ENU (1-15 mM) did not result in the inactivation of the plasmid containing target genes. A transformation efficiency of greater than 40% was observed upon treatment of H-ras oncogene with the highest doses of the alkylating agents. The morphologically transformed foci obtained with alkylated p220-EC ranged from 2.8 to 0.3/microgram MNU alkylated and 1.6 to 0.6/microgram ENU alkylated plasmid DNA. A significant proportion of the morphological transformants exhibited growth in soft agar. The HpaII/MspI restriction length polymorphism (RFLP) at codon 12 of H-ras exon-1 was detected with 4 independently isolated clones obtained from MNU-alkylated p220-EC transfections. Allele-specific in situ gel hybridization with a battery of codon 12 and codon 61 oligonucleotide probes confirmed these RFLPs to be due to sequence changes at codon 12. No clone with sequence changes in the H-ras codon 61 could be detected. The data indicate that a high degree of in vitro alkylation damage of the target gene is necessary to elicit mutational activation of H-ras in transfection-transformation assay. Low frequency notwithstanding, the data demonstrate that DNA alkylation damage at critical target sites can initiate neoplastic cellular transformation.

Effects of time compression and time compression plus reverberation on the intelligibility of Northwestern University Auditory Test No. 6.

The Tonal and Speech Materials for Auditory Perceptual Assessment, Disc 1.0 audio compact disc developed in 1992 includes several sets of degraded speech materials, two of which, time compression and reverberation, are described in this paper. The digital techniques used to compress the NU No. 6 materials (female speaker) on an 80386-based computer are described, along with a series of experiments on subjects with normal hearing that document the effects of the time compression on recognition performance. Experiment I examined at 70 dB SPL the effect on word recognition of 45, 55, 65, 70, and 75 percent compressions. Experiment II developed psychometric functions for the 45, 65, and 75 percent time-compression conditions. Experiment III defined the effects that time-compression degradation (45% and 65%) plus reverberation degradation (0.3 sec) had on the recognition performance on the NU No. 6 materials. Based on the experiments, four conditions (45% compression, 65% compression, 45% compression plus 0.3-sec reverberation, and 65% compression plus 0.3-sec reverberation) were selected and recorded on the compact disc. In the compact disc trials, normative data on the four conditions were developed from 120 listeners with normal hearing.

Genetic diversity and differentiation of 12 eastern Adriatic and western Dinaric native sheep breeds using microsatellites.

Nuclear genetic diversity and differentiation of 341 sheep belonging to 12 sheep breeds from Croatia and Bosnia and Herzegovina were examined. The aim of the study was to provide the understanding of the genetic structure and variability of the analysed pramenka sheep populations, and to give indications for conservation strategies based on the population diversity and structure information. The genetic variation of the sheep populations, examined at the nuclear level using 27 microsatellite loci, revealed considerable levels of genetic diversity, similar to the diversity found in other European indigenous low-production sheep breeds. Population-specific alleles were detected at most loci and in breeds analysed. The observed heterozygosity ranged from 0.643 (in Lika pramenka) to 0.743 (in Vlasic pramenka), and the expected heterozygosity ranged from 0.646 (in Lika pramenka) to 0.756 (in Dalmatian pramenka). Significant inbreeding coefficients were found for half of the populations studied and ranged from 0.040 (Pag island sheep) to 0.091 (Kupres pramenka). Moderate genetic differentiation was found between the studied sheep populations. The total genetic variability observed between different populations was 5.29%, whereas 94.71% of the variation was found within populations. Cres island sheep, Lika pramenka and Istrian sheep were identified as the most distinct populations, which was confirmed by the factorial analysis of correspondence and supported through a bootstrapping adjustment to correct for the difference in the sample sizes. The population structure analysis distinguished 12 clusters for the 12 sheep breeds analysed. However, the cluster differentiation was low for Dalmatian, Vlasic, Stolac and Krk pramenka. This systematic study identified Lika pramenka and Rab island sheep as those with the lowest diversity, whereas Istrian sheep and Pag island sheep had the highest. Conservation actions are proposed for Istrian, Rab and Cres island sheep, Lika and Kupres pramenka because of high estimated coefficients of inbreeding.

Frequency of PLA1 in blacks.

Racial frequencies of platelet-specific antigens in racial groups other than whites have not been studied. The prevalence of PLA1 was determined in white and black blood donors. Two hundred forty-two of 243 blacks were PLA1-positive (99.6%), compared with 242 of 250 whites (96.8%) (p = 0.021). This black population has the highest frequency of PLA1 yet reported.

De novo DNA methylation at nonrandom founder sites 5' from an unmethylated minimal origin of DNA replication in latent Epstein-Barr virus genomes.

Latent episomal genomes of Epstein-Barr virus, a human gammaherpesvirus, represent a suitable model system for studying replication and methylation of chromosomal DNA in mammals. We analyzed the methylation patterns of CpG dinucleotides in the latent origin of DNA replication of Epstein-Barr virus using automated fluorescent genomic sequencing of bisulfite-modified DNA samples. We observed that the minimal origin of DNA replication was unmethylated in 8 well-characterized human cell lines or clones carrying latent Epstein-Barr virus genomes as well as in a prototype virus producer marmoset cell line. This observation suggests that unmethylated DNA domains can function as initiation sites or zones of DNA replication in human cells. Furthermore, 5' from this unmethylated region we observed focal points of de novo DNA methylation in nonrandom positions in the majority of Burkitt's lymphoma cell lines and clones studied while the corresponding CpG dinucleotides in viral genomes carried by lymphoblastoid cell lines and marmoset cells were completely unmethylated. Clustering of highly methylated CpG dinucleotides suggests that de novo methylation of unmethylated double-stranded episomal viral genomes starts at discrete founder sites in vivo. This is the first comparative high-resolution methylation analysis of a latent viral origin of DNA replication in human cells.

Protein-DNA interaction and CpG methylation at rep*/vIL-10p of latent Epstein-Barr virus genomes in lymphoid cell lines.

The viral interleukin-10 promoter (vIL-10p), overlapping the rep* element in the Epstein-Barr virus (EBV) genome, is a promoter element active mostly in the late phase of the lytic cycle and immediately upon infection of B cells. rep* was, through transfection experiments with small plasmids, characterised as a cis element supporting oriP replicative function. In this study, in vivo protein binding and CpG methylation at rep*/vIL-10p were analysed in five cell lines that harbour strictly latent EBV genomes. Contrary to the invariably unmethylated dyad symmetry element (DS) of oriP, rep*/vIL-10p was highly methylated and showed only traces of protein binding in all examined cell lines. This result is in agreement with vIL-10p being an inactive promoter of EBV genomes, and makes it less likely that rep* functions as a replicative element of latent EBV genomes.