Etiological profile of hearing loss amongst Lithuanian pediatric cochlear implant users

article no. 110043Introduction: Congenital sensorineural hearing loss is a heterogeneous disorder; its etiological profile varies between populations. Pathogenic variants of GJB2 gene are the major cause of non-syndromic hearing loss. Congenital cytomegalovirus infection (cCMV) is the most important prenatal etiological factor causing hearing loss and other disorders. Perinatal events, syndromes, postnatal infections or traumas are less common. Causes of the remaining one third of hearing loss cases are unknown. Objectives: To determine the etiological profile of hearing loss in pediatric cochlear implant users in Lithuanian population. Methods: The data of 122 children (70 male/52 female; aged 7.6 ± 3.3 years) cochlear implant users were analysed. Medical records of all children recruited in Santaros Clinics (Vilnius, Lithuania) were analysed to identify prenatal, perinatal, or postnatal risk factors based on the adapted list proposed by the Joint Committee of Infant Hearing. Genetic counselling and testing according to the scheme were performed to 101 children. DNA of 117 children was extracted from the DBS on Guthrie cards and CMV DNA detected using real time PCR. Results: Non-syndromic hearing loss was diagnosed in 65 cases (53.3%), 58 of which were GJB2 gene-associated; syndromic hearing loss was diagnosed to 8 children (6.6%). Perinatal (prematurity, low birth weight, hypoxia, hyperbilirubinemia, sepsis, ototoxicity, and meningitis) and postnatal (meningitis) risk factors were associated with hearing loss in 16 (13.1%) and 4 (3.3%) study participants respectively. CMV DNA was detected in 12 samples (9.8%). The cause of hearing loss remained unknown only for 17 (13.9%) children. Conclusions: The major cause of HL in the current study was GJB2 gene alterations. [...]Vilniaus universiteta

Une âme de jeune fille [ : Gabrielle***] / Jean Vaudon,...

<p>A. Distribution of CpG-motifs within rDNA (transcribed region of human ribosomal repeat). The digits indicate the nucleotide order number, the vertical bar shows the motif location. Red color is used to mark region A and region B, that were analyzed for the presence of methylated CCGG sites. B. Determination of methylation index of three genes in DNA from cells treated with 20 μM DBP(1–4), 72 h (description is given in Methods).</p

The influence of DBP(1–4) on the MCF-7mitochondria.

<p>A. (1)—control cells (stained with MitoTracker TMRM) in visible light (VIS), in λex = 520 nm (TMRM) and in λex = 380 nm (background). (2)—control cells stained with MitoTracker TMRM and Hoechst 33258 (10μM, 0.5 h). Specially selected field shows single cells with a weak mitochondria staining. Particularly these cells accumulate Hoechst 33258 in the nuclei. B. MCF-7 cells treated with 20μM DBP(2) for 0.5–48 h after incubation with MitoTracker TMRM (15 min). For 0.5 h time point we specially selected a field that shows cells with a weak mitochondria staining. DBP(2) quickly penetrates into these cells, similar to Hoechst 33258. Magnification X 60. The cell in a yellow square is enlarged several times for demonstration.</p

DNA methylation in nuclei of the control MCF-7 cells.

<p>A. Cells were processed for immunofluorescence staining with anti 5-mC antibody. (1)—Magnification X60; (2)—For analysis of the nuclei the photo was enlarged by an order using computer processing. The arrow points to the methylated DNA block close to the nucleolus. B. (1)—DNA methylation in nuclei of the control MCF-7 cells and the cells treated with 10 μM AzaC or 20μM DBP(1–4), 72 h. Background: Control cells were only treated with secondary antibodies conjugated with FITC. (2) The photo was enlarged considerably for the analysis of nuclei size. The nucleus of the cell cultivated with DBP(4) is shown for an example. C. An example of the different DNA methylation level in cells’ nuclei that were cultivated in the presence of 20μM DBP(4), 72 h. A,B(1) and C—Cell nuclei were additionally colored with DAPI.</p

Overall 5-methylcytosine level in DNA of cells treated with 20 μM DBP(1–4) for 72 hours.

<p>A (ELISA). (1, 2)–example of calibration dependence between methylated DNA in samples and integral stain density (I). (3) example of DNA samples analysis from cells treated with DBP(1–4). 10 ng of DNA was applied. pBR322 plasmid was used as a negative control. (4) Relative signal change reflecting difference in DNA methylation level. B (<i>UPLC/MS/MS</i>). Average 5mC content in DNA samples.</p

DBP(n) (20μM, 72h) effect on RNA level in MCF-7.

<p>DBP(n) (20μM, 72h) effect on RNA level in MCF-7.</p

Incorporation of DBP(1–4) in MCF-7 cells.

<p>Excitation of fluorescence at 380 nm. Magnification X 20. MCF-7 were incubated with DBP(1–4) (7, 10 or 20 μM) for 24 h and the fluorescence was analyzed in unfixed cells. Staining of the cells was heterogeneous and practically did not depend on the concentrations of DBP(1–4). The 20 μM concentrations of DBP(1–4) were used for an example. The photos of cells were taken with identical exposure. For example the most common nuclei staining types are shown with arrows and numbers: 1 –nucleus is stained only, 2 –nucleus and the cytoplasm are stained. 3- cells show strong blue fluorescence signal while nuclei do not contrast. Background: the photo of control cells in visible light (VIS) and at λex = 380 nm (FL), the exposure is increased two times.</p

A (FL-reader).

<p>(1)–Example of reaction rate constant determination for DCF formation when DCFH reacts with ROS. Cells were cultivated for 24 hours in the presence of 40 μM DBP(1). The cultivation environment was replaced by 5 μm H2DCFH-DA in PBS solution and a relative fluorescence intensity increase was detected. I_t,I0 –sample’s signal at time t and immediately after H2DCFH-DA addition respectively. The slope of the line– <a href="http://universal_ru_en.academic.ru/378785/reaction_rate_constant" target="_blank">reaction rate constant</a> for DCF formation (k). ROS index Δk/k₀ = (k_DBP−k₀)×100/k₀ (%). (2)—Relationship between ROS index Δk/k₀ and DBP(1–4) concentration. Time of cultivation is shown on the figure. B (FCA). Proliferation of MCF-7 cells exposed to DBP(1–4) at final concentration 20 μM for 24 hours. (1)—Distribution of fluorescence of fixed cells stained with anti-Ki-67 antibodies (control—dark green color, DBP(2) as example—blue color). Background fluorescence was quantified using FITC-conjugated secondary antibodies (green color). (2)—The median signal intensity of FL1 (Ki-67+) (gate R). Each experiment was repeated at least three times. Results represent the average of the medians of the FL parameter in independent experiments. C (FCA). (1)—Distribution of fluorescence of fixed cells stained with propidium iodide. The fractions of (1–4) cells with different DNA amounts are shown. (2)–Proportion MCF-7 culture cells with DNA amount corresponding to the G1-, S—and G2/M phases of the cell cycle. The data shown is an average value from three independent experiments. (3) The total number of cells in culture. (4) The number hypodiploid cells (fraction SubG0/G1).D. The proportion of cells in culture with signs of apoptotic nuclei (condensed chromatin, the irregular nucleus shape, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189826#pone.0189826.g004" target="_blank">Fig 4</a>). B, C, D–cells were cultivated in the presence of 20 μM DBP(1–4) for 24 hours.</p

An international survey of current management practices for polymyalgia rheumatica by general practitioners and rheumatologists

Objectives: To explore current management practices for PMR by general practitioners (GPs) and rheumatologists including implications for clinical trial recruitment.Methods: An English language questionnaire was constructed by a working group of rheumatologists and GPs from six countries. The questionnaire focused on: 1: Respondent characteristics; 2: Referral practices; 3: Treatment with glucocorticoids; 4: Diagnostics; 5: Comorbidities; and 6: Barriers to research. The questionnaire was distributed to rheumatologists and GPs worldwide via members of the International PMR/Giant Cell Arteritis Study Group.Results: In total, 394 GPs and 937 rheumatologists responded to the survey. GPs referred a median of 25% of their suspected PMR patients for diagnosis and 50% of these were returned to their GP for management. In general, 39% of rheumatologists evaluated patients with suspected PMR &gt;2 weeks after referral, and a median of 50% of patients had started prednisolone before rheumatologist evaluation. Direct comparison of initial treatment showed that the percentage prescribing &gt;25 mg prednisolone daily for patients was 30% for GPs and 12% for rheumatologists. Diagnostic imaging was rarely used. More than half (56%) of rheumatologists experienced difficulties recruiting people with PMR to clinical trials.Conclusion: This large international survey indicates that a large proportion of people with PMR are not referred for diagnosis, and that the proportion of treatment-naive patients declined with increasing time from referral to assessment. Strategies are needed to change referral and management of people with PMR, to improve clinical practice and facilitate recruitment to clinical trials.</p