Hemisected molars for additional overdenture support.

A preventive prosthodontic technique has been described which utilizes hemisected teeth for overdenture support. This multidiscipline procedure provides firm denture support and preserves posterior residual alveolar ridges. By retaining posterior roots, proprioceptive mechanisms are maintained, and psychologically, the patient does not seem edentulous. As Brewer and Fenton stated, "The application of this method of treatment is limited only by the imagination of the dentist."     

Identification of a human mutation of DMT1 in a patient with microcytic anemia and iron overload

Divalent metal transporter 1 (DMT1) is a transmembrane protein crucial for duodenal iron absorption and erythroid iron transport. DMT1 function has been elucidated largely in studies of the mk mouse and the Belgrade rat, which have an identical single nucleotide mutation of this gene that affects protein processing, stability, and function. These animals exhibit hypochromic microcytic anemia due to impaired intestinal iron absorption, and defective iron utilization in red cell precursors. We report here the first human mutation of DMT1 identified in a female with severe hypochromic microcytic anemia and iron overload. This homozygous mutation in the ultimate nucleotide of exon 12 codes for a conservative E399D amino acid substitution; however, its pre-dominant effect is preferential skipping of exon 12 during processing of pre-messenger RNA (mRNA). The lack of full-length mRNA would predict deficient iron absorption in the intestine and deficient iron utilization in erythroid precursors; however, unlike the animal models of DMT1 mutation, the patient is iron overloaded. This does not appear to be due to up-regulation of total DMT1 mRNA. DMT1 protein is easily detectable by immunoblotting in the patient's duodenum, but it is unclear whether the protein is properly processed or targeted.     

Rapid Detection and Identification of Mucormycetes from Culture and Tissue Samples by Use of High-Resolution Melt Analysis

We present a method for rapid and simple detection of clinically relevant mucormycetes of the Mucorales order in cultures and clinical samples. This seminested real-time PCR uses mucormycete-specific primers and is followed by species identification using high-resolution melt (HRM) analysis. The method is highly suitable for routine clinical diagnostics.Invasive infections caused by mucormycetes started to occur more frequently in the last decade and are connected with rapid progression and high mortality rates. Early diagnostics and targeted treatment are crucial. Most mucormycosis cases (over 90%) are caused by Rhizopus spp., followed by Mucor spp., Lichtheimia spp., Rhizomucor pusillus, and, rarely, some other species (2, 9, 11, 16).Definitive diagnosis of mucormycosis is usually made after histopathological proof of mucormycete-like hyphae in involved tissue; the causative agent can be determined only by culture (13). So far, no serological test is available and radiological methods are nonspecific.Molecular detection of mucormycetes is complicated by several factors, and we still do not have any standard protocol. Few methods for the detection of mucormycetes have been published, and only some have been evaluated using clinical samples (1, 5, 10, 14, 15, 17) or samples from animal models (6, 7).The aim of this study was to develop a rapid and sensitive technique for the detection and identification of clinically important mucormycetes. We adopted primers from a qualitative method previously published by Bialek et al. (1) that is specific for members of the order Mucorales targeting 18S ribosomal DNA (rDNA). We modified it to seminested real-time PCR with EvaGreen dye, followed by species distinction by high-resolution melt (HRM) analysis. HRM analysis uses amplification of DNA in the presence of intercalation dye. Fluorescence is measured during a controlled melting of PCR product that results in a melt curve that depends mainly on GC content, length, and sequence of the PCR product. This simple method can be used for genotyping or mutation scanning without the need for time-consuming sequencing (4, 12).DNA was isolated from 50 μl of fungal culture (inoculum was prepared by covering sporulating colonies with approximately 2 ml of sterile 0.85% saline) or a piece of fresh tissue (2 by 1 mm) using the ZR fungal/bacterial DNA kit (Zymo Research). Tissue samples were incubated in lysis buffer overnight, and cultures were immediately processed according to the manufacturer''s protocol. Disruption was extended to 15 min (Disruptor Genie; Scientific Industries). DNA from formalin-fixed, paraffin wax-embedded (FFPE) tissue samples was isolated from 2 or 3 scrolls (5 to 10 μm each) of paraffin block using a DNeasy blood and tissue kit (Qiagen). Paraffin was dissolved in 1 ml of xylene, and then the tissue was washed two times using 1 ml of 96% ethanol and incubated in 180 μl of ATL buffer (Qiagen) and 20 μl of proteinase K (600 mAU/ml solution, where one mAU represents the activity of proteinase K that releases folin-positive amino acids and peptides corresponding to 1 μmol of tyrosine per min) at 55°C overnight and then at 90°C for 1 h. The next steps were done in accordance with the manufacturer''s protocol. DNA isolation from clinical samples was done in a biological safety cabinet. An aliquot of sterile water was processed with each set of samples as a control of potential contamination during the isolation process.Five microliters of DNA was amplified in 25 μl of amplification mixture that contained a 0.2 μM concentration each of primers ZM1 and ZM2 (1), 120 μM deoxynucleoside triphosphates (dNTPs; Roche, Germany), 2.5 mM MgCl₂, 1× GeneAmp PCR Gold buffer, and 1.5 U AmpliTaq Gold DNA polymerase (Applied Biosystems). The cycling conditions were 10 min at 95°C, 16 cycles of 30 s at 94°C, 30 s at 50°C, and 60 s at 72°C, and 7 min at 72°C. One microliter of PCR product from the external round was then amplified in duplicate using Rotorgene 6000 (Corbett Research, Australia). Twenty-five microliters of the amplification mixture contained a 0.4 μM concentration each of primers ZM1 and ZM3 (1), 12.5 μl of SensiMix HRM, and 1 μl of EvaGreen (both from a SensiMix HRM kit; Quantace, United Kingdom). The cycling conditions were 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, 20 s at 60°C, and 30 s at 72°C (acquired on the green channel), followed by HRM analysis (ramp from 74°C to 79.5°C, rising by 0.1°C each cycle, acquired on the HRM channel). Rotorgene 6000 series software (version 1.7) was used for analysis of the results. All positive results were confirmed by sequencing of the PCR product. DNA was purified using a QIAquick PCR purification kit (Qiagen, Germany) and sequenced using a BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems) on an ABI Prism 310 genetic analyzer (Applied Biosystems). Sequences were analyzed using the BLAST alignment program of the GenBank database.We used DNA extracted from five mucormycete cultures diluted in Tris-EDTA (TE) buffer as positive controls in every run. A DNA isolation control (sterile water processed with clinical samples) and a negative control of PCR (sterile water) were added to each run as well.In this study, we tested 31 fungal isolates, comprising 10 mucormycete isolates and 21 isolates from other filamentous fungal groups (Department of Clinical Microbiology, University Hospital Brno and Czech Collection of Microorganisms, Czech Republic). All mucormycete isolates were correctly identified. The melting temperatures (T_m) for each species were as follows: for Rhizopus microsporus, 76.46°C; for Rhizopus oryzae, 76.59°C; for Mucor racemosus, 76.78°C; for Mucor circinelloides, 76.98°C; for Rhizomucor pusillus, 77.87°C; and for Lichtheimia corymbifera, 78.56°C. Representative HRM curves for six different mucormycetes are shown in Fig. ​Fig.1.1. All HRM analysis results were confirmed by sequencing. None of the nonmucormycete fungi were positively tested. The results are summarized in Table ​Table11.Open in a separate windowFIG. 1.Representative result of high-resolution melt (HRM) analysis. Shown are HRM curves for six mucormycete isolates (black curves) and one negative and one positive tissue sample (gray curves).

TABLE 1.

List of fungal isolates used in this study and results of HRM analysis^a

Mesalamine modulates intercellular adhesion through inhibition of p-21 activated kinase-1

Mesalamine (5-ASA) is widely used for the treatment of ulcerative colitis, a remitting condition characterized by chronic inflammation of the colon. Knowledge about the molecular and cellular targets of 5-ASA is limited and a clear understanding of its activity in intestinal homeostasis and interference with neoplastic progression is lacking. We sought to identify molecular pathways interfered by 5-ASA, using CRC cell lines with different genetic background. Microarray was performed for gene expression profile of 5-ASA-treated and untreated cells (HCT116 and HT29). Filtering and analysis of data identified three oncogenic pathways interfered by 5-ASA: MAPK/ERK pathway, cell adhesion and β-catenin/Wnt signaling. PAK1 emerged as a consensus target of 5-ASA, orchestrating these pathways. We further investigated the effect of 5-ASA on cell adhesion. 5-ASA increased cell adhesion which was measured by cell adhesion assay and transcellular-resistance measurement. Moreover, 5-ASA treatment restored membranous expression of adhesion molecules E-cadherin and β-catenin. Role of PAK1 as a mediator of mesalamine activity was validated in vitro and in vivo. Inhibition of PAK1 by RNA interference also increased cell adhesion. PAK1 expression was elevated in APC^min polyps and 5-ASA treatment reduced its expression. Our data demonstrates novel pharmacological mechanism of mesalamine in modulation of cell adhesion and role of PAK1 in APC^min polyposis. We propose that inhibition of PAK1 expression by 5-ASA can impede with neoplastic progression in colorectal carcinogenesis. The mechanism of PAK1 inhibition and induction of membranous translocation of adhesion proteins by 5-ASA might be independent of its known anti-inflammatory action.     

Ribosomal protein S19 gene mutations in patients with diamond-blackfan anemia and identification of ribosomal protein S19 pseudogenes

Diamond-Blackfan anemia (DBA) is a rare congenital pure red cell hypoplasia characterized by a selective defect of erythropoiesis with a normochromic macrocytic anemia and reticulocytopenia often accompanied by various congenital anomalies. The critical region responsible for the pathogenesis of DBA has been mapped in some patients to chromosome 19q13.2 (P Gustavsson, E Garelli, N Draptchinskaia, et al. Am. J. Hum. Genet. 63:1388-1395, 1998) and the gene encoding ribosomal protein S19 (RPS19) is believed to be the candidate gene. Here we present molecular analysis of the RPS19 gene in DBA patients from the Czech National DBA Registry. We found that the RPS19 gene was mutated in 25% (5/20) of DBA patients (insertion, deletion, and point mutations, but no nonsense or splice site mutations). Point mutations were localized to hot spots defined by Willig (TN Willig, N Draptchinskaia, I Dianzani, et al. Blood 94:4294-4306, 1999). Moreover, we describe two processed RPS19 pseudogenes, which were not expressed. Possible models of the DBA pathogenesis in the view of RPS19 mutations are discussed.     

Long-term results of allogeneic hematopoietic stem cell transplantation after reduced-intensity conditioning with busulfan, fludarabine, and antithymocyte globulin

Reduced-intensity conditioning (RIC) is widely used for allogeneic stem cell transplantation (SCT). Here we present our long-term experience with RIC regimen consisting of fludarabine (30 mg/m2/day on days -10 to -5), busulfan (4mg/kg/day on days -6 and -5) and antithymocyte globulin (ATG Fresenius, 10 mg/kg/day on days -4 to -1) (Flu-Bu-ATG) in a cohort of 71 patients with various hematological malignancies including chronic myeloid leukemia (24 patients), acute myeloid leukemia (19 patients), lymphoma (20 patients), multiple myeloma (3 patients), myelodysplastic syndrome (3 patients), and myelofibrosis (2 patients). The median age was 50 years. The overall response rate was 87%, including 83% CR and 4% PR. The incidence of acute and chronic GVHD was 35% and 52% and the cumulative incidence of non-relapse mortality at 1 year and 4 years was 8% and 14%. With the median follow-up of 55.0 months, the 2- and 4-year event-free survival (EFS) was 49.0% and 40.3%, and the overall survival (OS) was 73.2% and 62.6%, respectively. Gender, age at SCT, type of donor, disease status at SCT, previous autologous transplantation, and complete chimerism by day +100 did not significantly influence EFS and OS. In a multivariate analysis, no presence of chronic GVHD (p=0.029, HR: 2.5),and diagnosis other than CML (p=0.018, HR: 4.6), and CD34+ dose < 5x106/kg (p=0.010, HR: 2.8) were significant predictors of poor OS. Flu-Bu-ATG protocol is a RIC regimen that combines effective disease control with low non-relapse mortality and acceptable toxicity profile. Keywords: reduced-intensity conditioning, fludarabine, busulfan, antithymocyte globulin.     

Prediabetes/early diabetes-associated neuropathy predominantly involves sensory small fibres

The aim of this study was to investigate the characteristics of prediabetes (preDM) and early (<3 years) diabetes mellitus type 2 (eDM2)-associated neuropathy and the value of recently proposed diagnostic criteria for diabetic sensorimotor polyneuropathy (DSPN). A prospective case-control study in a group of 48 consecutive patients with eDM2, 16 preDM patients and 40 age- and sex-matched normoglycaemic controls was performed. Clinical and laboratory diagnostic tests were used to detect neuropathic abnormalities; these were further classified in terms of recent diagnostic criteria. Criteria for confirmed DSPN based on abnormal nerve conduction (NC) studies were met in 7 (14.6%) eDM2 patients compared to no control (p < 0.05), and the proportion significantly increased to 37.5% compared to 2.5% controls (p < 0.001), if intraepidermal nerve fibre density (IENFD) was used as an alternative criterion in addition to NC. The subclinical DSPN criteria based on NC abnormalities were met in 4.2% eDM2 patients, while the proportion of preDM and eDM2 cases with subclinical sensory small-fibre involvement documented by IENFD reached 12.5% and 22.9% compared with 2.5% controls (p = 0.005 for eDM2). The absolute IENFD values from distal leg were significantly lower in both eDM2 (p < 0.0001) and preDM patients (p = 0.005) compared to controls. Neuropathy associated with preDM/eDM2 predominantly involves sensory small fibres.