ABSTRACT
Recently, biallelic variants in the SORD gene were identified as causal for axonal hereditary neuropathy (HN). We ascertained the spectrum and frequency of SORD variants among a large cohort of Czech patients with unknown cause of HN. Exome sequencing data were analysed for SORD (58 patients). The prevalent c.757del variant was tested with fragment analysis (931 patients). Sanger sequencing in additional 70 patients was done. PCR primers were designed to amplify the SORD gene with the exclusion of the pseudogene SORD2P. Sequence differences between gene and pseudogene were identified and frequencies of SNPs were calculated. Eighteen patients from 16 unrelated families with biallelic variants in the SORD gene were found and the c.757del was present in all patients on at least one allele. Three novel, probably pathogenic, variants were detected, always in a heterozygous state in combination with the c.757del on the second allele. Patients presented with a slowly progressive axonal HN. Almost all patients had moderate pes cavus deformity. SORD neuropathy is frequent in Czech patients and the third most common cause of autosomal recessive HN. The c.757del is highly prevalent. Specific amplification of the SORD gene with the exclusion of the pseudogene is essential for a precise molecular diagnostics.
Subject(s)
Hereditary Sensory and Motor Neuropathy , L-Iditol 2-Dehydrogenase/genetics , Adult , Aged , Cohort Studies , Czech Republic/epidemiology , Female , Hereditary Sensory and Motor Neuropathy/diagnosis , Hereditary Sensory and Motor Neuropathy/epidemiology , Hereditary Sensory and Motor Neuropathy/genetics , Humans , Male , Middle Aged , Mutation , Exome SequencingABSTRACT
Biallelic SBF2 mutations cause Charcot-Marie-Tooth disease type 4B2 (CMT4B2), a sensorimotor neuropathy with autosomal recessive inheritance and association with glaucoma. Since the discovery of the gene mutation, only few additional patients have been reported. We identified seven CMT4B2 families with nine different SBF2 mutations. Revisiting genetic and clinical data from our cohort and the literature, SBF2 variants were private mutations, including exon-deletion and de novo variants. The neuropathy typically started in the first decade after normal early motor development, was predominantly motor and had a rather moderate course. Electrophysiology and nerve biopsies indicated demyelination and excess myelin outfoldings constituted a characteristic feature. While neuropathy was >90% penetrant at age 10 years, glaucoma was absent in ~40% of cases but sometimes developed with age. Consequently, SBF2 mutation analysis should not be restricted to individuals with coincident neuropathy and glaucoma, and CMT4B2 patients without glaucoma should be followed for increased intraocular pressure. The presence of exon-deletion and de novo mutations demands comprehensive mutation scanning and family studies to ensure appropriate diagnostic approaches and genetic counseling.
Subject(s)
Charcot-Marie-Tooth Disease/diagnosis , Charcot-Marie-Tooth Disease/genetics , Genetic Association Studies , Genetic Predisposition to Disease , Mutation , Phenotype , Protein Tyrosine Phosphatases, Non-Receptor/genetics , Adolescent , Adult , Biopsy , Child , Female , Genetic Association Studies/methods , Humans , Male , Young AdultABSTRACT
BACKGROUND: The rabies virus causes a fatal encephalitis and can be transmitted through organ transplantation. In 2013, a man developed rabies 18 months after receiving a kidney from a donor with rabies, who was not known to have been infected when the organs were procured. Three additional persons who received organs from the same donor (liver, kidney, heart), all of whom were not vaccinated for rabies before transplantation, received rabies post-exposure prophylaxis (PEP) with rabies immune globulin and 5 doses of rabies vaccine as soon as the diagnosis of rabies was made in the donor (18 months after their transplant surgeries). We describe their clinical management. METHODS: As the 3 recipients were all on immunosuppressive medications, post-vaccination serologic testing was performed using the rapid fluorescent focus inhibition test to measure rabies virus neutralizing antibodies (RVNAs). An acceptable antibody response to administration of rabies vaccine was defined as detection of RVNAs at a concentration ≥0.1 IU/mL from a serum specimen collected ≥7 days after the fifth vaccine dose. RESULTS: All 3 recipients demonstrated an acceptable antibody response despite their immunosuppressed states. More than 36 months have passed since their transplant surgeries, and all 3 recipients have no evidence of rabies. CONCLUSIONS: The survival of 3 previously unvaccinated recipients of solid organs from a donor with rabies is unexpected. Although the precise factors that led to their survival remain unclear, our data suggest that PEP can possibly enhance transplant safety in settings in which donors are retrospectively diagnosed with rabies.
Subject(s)
Antibodies, Viral/blood , Heart Transplantation/adverse effects , Kidney Transplantation/adverse effects , Liver Transplantation/adverse effects , Rabies Vaccines/administration & dosage , Rabies virus/immunology , Rabies/immunology , Adult , Humans , Immunity, Humoral , Male , Middle Aged , Post-Exposure Prophylaxis , Rabies/transmission , Retrospective Studies , Tissue Donors , Treatment OutcomeABSTRACT
Emergency room and hospital discharge data have been used to describe the risk factors and public health impact of dog bites. These data sets are based on financial charges for severe bites and underestimates dog bite burdens within communities. This study expands both the source of information and risk factor data collected to provide demographic analysis of dog bite injury risk factors reported in Bay County, Florida in 2009-2010. Extended data for dog bites reported by various sources from January 1, 2009 to December 31, 2010 were collected by Florida Department of Health in Bay County. Data collected included bite victim's age and gender, primary reported cause of bite, setting, dog's restraint status and relationship between the victim and the dog. A total of 799 bites were reported. Most bites (55%) were reported first by healthcare practitioners, particularly bites involving children<6 years. Bites involving unfamiliar dogs and dogs off the owner's property were more likely to be reported by other sources. Boys aged 6-14 years accounted for 2.24 times more bites than same-aged females (P<0.001) and had the highest incidence with 424 bites per 100,000 persons per year. Persons 6 years or older were 3.6 times more likely to be bitten by an unfamiliar dog. Inappropriate behaviour management was the most common cause of bites (26%), followed by protective behaviour (24%). Bites of unknown cause were 2.5 times more likely in children<6 years. Separating dog fights was the most common cause of bites for persons 15 years or older (24%); females were significantly more likely to be bit than males (P=0.01). Bites by unrestrained dogs off the owner's property (32% of all bites) most commonly involved males. Estimates based solely on healthcare discharge data significantly underestimate dog bite burden within a community. Characterizing these risks by age group or gender provides an opportunity to implement targeted interventions to prevent dog bites.
Subject(s)
Bites and Stings/epidemiology , Dogs , Ownership/statistics & numerical data , Adolescent , Adult , Age Distribution , Animals , Behavior, Animal , Child , Child, Preschool , Female , Florida/epidemiology , Hospital Records , Humans , Male , Middle Aged , Primary Health Care , Risk Factors , Sex Distribution , Young AdultABSTRACT
This article describes and contrasts the public health response to two human rabies cases: one organ recipient diagnosed within days of symptom onset and the transplant donor who was diagnosed 18 months post-symptom onset. In response to an organ-transplant-related rabies case diagnosed in 2013, organ donor and recipient investigations were conducted by multiple public health agencies. Persons with potential exposure to infectious patient materials were assessed for rabies virus exposure. An exposure investigation was conducted to determine the source of the organ donor's infection. Over 100 persons from more than 20 agencies spent over 2700 h conducting contact investigations in healthcare, military and community settings. The 564 persons assessed include 417 healthcare workers [5.8% recommended for post-exposure prophylaxis (PEP)], 96 community contacts (15.6% recommended for PEP), 30 autopsy personnel (50% recommended for PEP), and 21 other persons (4.8% recommended for PEP). Donor contacts represented 188 assessed with 20.2% recommended for PEP, compared with 5.6% of 306 recipient contacts recommended for PEP. Human rabies cases result in substantial use of public health and medical resources, especially when diagnosis is delayed. Although rare, clinicians should consider rabies in cases of encephalitis of unexplained aetiology, particularly for cases that may result in organ donation.
Subject(s)
Contact Tracing , Organ Transplantation/adverse effects , Public Health , Rabies virus/isolation & purification , Rabies/transmission , Tissue Donors , Cross Infection/virology , Humans , Post-Exposure Prophylaxis , Rabies/virology , Risk AssessmentABSTRACT
Five laboratory-acquired brucellosis (LAB) cases that occurred in the United States between 2008 and 2011 are presented. The Centers for Disease Control and Prevention (CDC) reviewed the recommendations published in 2008 and the published literature to identify strategies to further prevent LAB. The improved prevention strategies are described.
Subject(s)
Brucellosis/diagnosis , Brucellosis/prevention & control , Infection Control/methods , Occupational Exposure , Adult , Child , Female , Health Personnel , Humans , Male , Middle Aged , United StatesABSTRACT
We report a fatal case of Brucella suis endocarditis initially misdiagnosed by automated identification systems as Ochrobactrum anthropi infection in a patient with a history of Marfan syndrome and recreational feral swine hunting. This report emphasizes the need to consider brucellosis as a part of the differential diagnosis of acute febrile illness, particularly in patients with known risk of exposure.
Subject(s)
Brucella suis/isolation & purification , Brucellosis/diagnosis , Diagnostic Errors , Endocarditis, Bacterial/diagnosis , Marfan Syndrome/complications , Automation/methods , Bacteriological Techniques/methods , Brucellosis/microbiology , Brucellosis/pathology , Endocarditis, Bacterial/microbiology , Endocarditis, Bacterial/pathology , Fatal Outcome , Humans , Male , Middle Aged , Ochrobactrum anthropi/isolation & purificationABSTRACT
Earlier studies have established that the average speed of a replication fork is two to three times slower in early S-phase than in late S-phase and that the intracellular 2'-deoxyribonucleoside 5'-triphosphate pools grow during S-phase. In this study, the effect of the exogenous 2'-deoxyribonucleoside 5'-triphosphate (dNTP) supply on the average replication speed in a synchronised population of human HeLa cells was tested. The speed of replication fork movement was measured on extended DNA fibers labelled with 2'-deoxythymidine analogues 5-chloro-2'-deoxyuridine and 5-iodo-2'-deoxyuridine. We show that the introduction of exogenous dNTPs accelerates the replication process at the beginning of DNA synthesis only. In late S-phase, the administration of additional dNTPs has no effect on the speed of replication forks. The availability of 2'-deoxynucleotides seems to be a rate-limiting factor for DNA replication during early S-phase.
Subject(s)
DNA Replication/drug effects , Deoxyribonucleotides/pharmacology , S Phase/drug effects , HeLa Cells , Humans , Kinetics , Microscopy, FluorescenceABSTRACT
The precise location of ribosomal RNA (rRNA) synthesis within the nucleolus is the subject of recent controversy; some investigators have detected nascent RNA in the dense fibrillar components (DFCs) while others have localized transcription to the fibrillar centers (FCs). We endeavored to resolve this controversy by applying a new technique for non-isotopic labeling of RNA and examined the synthesis and movement of non-isotopically labeled rRNA within the nucleolus. We found that rRNA is synthesized only in a restricted area of DFCs, also involving the boundary region with FCs. We traced a movement of RNA from transcription sites through DFCs to granular components. Our results indicate functional compartmentalization of DFCs with respect to the synthesis and processing of precursor rRNA. In situ mapping of the 5' leader sequence of the 5' external transcribed spacer together with transcription labeling indicated that transcription and the first steps in processing of precursor rRNA are spatially separated. Surprisingly, the results also pointed to a partially extended conformation of newly synthesized precursor rRNA transcripts.
Subject(s)
Cell Nucleolus/metabolism , In Situ Hybridization/methods , RNA Processing, Post-Transcriptional , RNA, Ribosomal/biosynthesis , RNA, Ribosomal/metabolism , Animals , Cell Nucleolus/genetics , Cell Nucleolus/ultrastructure , Chromosomal Proteins, Non-Histone/metabolism , Female , Fluorescent Antibody Technique, Direct , Green Fluorescent Proteins , HeLa Cells , Humans , Luminescent Proteins/analysis , Mice , Microscopy, Immunoelectron , RNA Precursors/biosynthesis , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Ribosomal/genetics , Rabbits , Transcription, GeneticABSTRACT
From early April into mid-June 1977, sequential groups of juvenile rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) were each exposed for 10 days to the parasite Myxobolus cerebralis by immersion in a stream inhabited by infected wild trout. Following incubation in a M. cerebralis-free facility, trout were subsequently killed, and heads and gill arches were examined by routine histologic methods. A grading scale to quantify lesion severity was developed and applied. Percentage infected, lesion severity scores, effects of water temperature and flow rates on percentage infected and lesion severity scores, and resulting pathology were determined for each species at each exposure period. The percentage of rainbow trout infected with M. cerebralis was significantly higher than the percentage of brown trout infected for each exposure period. The percentages of rainbow trout infected in exposure periods later in the calendar year were significantly higher than those in earlier periods. The percentages of brown trout infected were not significantly different among exposure periods. Overall average lesion severity scores were significantly higher in rainbow than in brown trout. Lesion severity scores in rainbow trout increased over time (a positive correlation with exposure period). Lesion severity scores were not significantly different for brown trout among exposure periods. A significant correlation existed between water temperature and percentage of rainbow trout infected; a significant correlation also existed between water temperature and lesion severity scores in rainbow trout. Similar correlations did not exist for percentage of brown trout infected or accompanying lesion severity scores. In rainbow trout, ventral calvarium was the most common site of M. cerebralis replication, followed by gill arches. In brown trout, lesions were virtually confined to gill arches. Early lesions consisted of foci of cartilage necrosis with small numbers of M. cerebralis developmental stages. More advanced lesions consisted of multifocal areas of cartilage necrosis with numerous M. cerebralis developmental stages and/or mature myxospores bordered and/or infiltrated by mono- and multinuclear leukocytes. Lesions in brown trout were smaller and had fewer associated leukocytes and M. cerebralis developmental stages and/or mature myxospores. Higher infection rates, lesion severity scores, and differences in lesion location in rainbow versus brown trout explain in part why numbers of rainbow but not brown trout have fallen in western rivers inhabited with M. cerebralis-infected trout.
Subject(s)
Eukaryota/pathogenicity , Fish Diseases/parasitology , Oncorhynchus mykiss/parasitology , Protozoan Infections, Animal/pathology , Salmonidae/parasitology , Animals , Environment , Fish Diseases/epidemiology , Fish Diseases/pathology , Incidence , Larva , Population Dynamics , Protozoan Infections, Animal/epidemiology , TemperatureABSTRACT
The available data concerning the subnucleolar localisation of the individual steps of precursor-ribosomal RNA (pre-rRNA) processing are ambiguous. According to in situ hybridisation studies, the late steps of pre-rRNA processing have been located into the granular component of the nucleolus, but factors engaged in these events were found being enriched in the dense fibrillar component. In this study, by utilisation of permeabilised human cells, we demonstrate that the newly synthesised, bromouridine-labelled pre-rRNAs reside at, or near, the sites of transcription. We provide evidence that processing of pre-rRNA occurs in permeabilised mammalian cells and that the incorporated bromouridine residues do not interfere with pre-rRNA maturation. Our results suggest that the maturation process of ribosomal RNA in permeabilised cells takes place at, or nearby, the site of transcription and that the processing complex is assembled during or early after the rRNA transcription.
Subject(s)
RNA Processing, Post-Transcriptional , RNA, Ribosomal/metabolism , Bromouracil/analogs & derivatives , Cell Nucleolus/metabolism , Fluorescent Antibody Technique, Indirect , HeLa Cells , Humans , In Situ Hybridization , Microinjections , Transcription, Genetic , Uridine/analogs & derivatives , Uridine/metabolismABSTRACT
Bromouridine-triphosphate is commonly used for in situ immunocytochemical labeling of newly synthesized RNA in living cells. While extranucleolar transcripts do not require special conditions for visualization, special treatment prior to fixation (e.g. incubation with alpha-amanitine) is necessary for immunofluorescence detection of bromouridine-labeled nucleolar RNA in previous studies. We show in the present investigation that bromouridine-triphosphate is efficiently used by both extranucleolar and nucleolar RNA polymerases in living cultured cells. The failure to detect incorporated bromouridine within nucleoli is entirely due to improper treatment of cells after bromouridine incorporation. When methanol/acetone fixation is used, fluorescence signals within nucleoli can be routinely found.
Subject(s)
Nucleolus Organizer Region/metabolism , RNA, Ribosomal/biosynthesis , Transcription, Genetic , Uridine/analogs & derivatives , Animals , Bromouracil/analogs & derivatives , Cell Nucleolus/metabolism , Cells, Cultured , Dogs , Fibroblasts/cytology , Fibroblasts/metabolism , Fluorescent Antibody Technique, Indirect , HeLa Cells/cytology , HeLa Cells/metabolism , Humans , In Situ Hybridization, Fluorescence , Kidney/cytology , Kidney/metabolism , RNA Polymerase I/metabolism , Uridine/metabolismABSTRACT
A new procedure for introduction of hydrophilic molecules into living cells based on efficient uptake of these molecules into the cells during hypotonic treatment is presented and its use is demonstrated by a variety of applications. Experiments with cultured vertebrate and Drosophila cells and various animal tissues demonstrated that the increase in cell membrane permeability under hypotonic conditions is a general phenomenon in all animal cells tested. The efficiency of the method depends on the composition and temperature of the hypotonic buffer, the duration of the hypotonic treatment and the molecular weight of the molecules introduced into living cells. The versatility of this approach is demonstrated with various types of molecules such as modified nucleotides, nucleotides with conjugated fluorochrome, peptides, phosphatase substrates and fluorescent dyes. The method opens new possibilities for the direct investigation of a variety of biological problems as documented here with data on the functional organization of the cell nucleus.
Subject(s)
Cell Membrane Permeability/drug effects , Cell Nucleus/metabolism , Hypotonic Solutions/pharmacology , Pharmaceutical Preparations/metabolism , Amanitins/metabolism , Animals , Cell Survival , Cells, Cultured , Chemical Phenomena , Chemistry, Physical , Coloring Agents/metabolism , Diffusion , Dogs , Drosophila melanogaster/metabolism , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression Profiling , HeLa Cells/drug effects , HeLa Cells/metabolism , Humans , Immunoglobulin G/metabolism , Kidney , Liver/metabolism , Male , Microinjections , Microscopy, Immunoelectron , Molecular Weight , Nucleotides/metabolism , Organ Culture Techniques , Rats , Uridine Triphosphate/analogs & derivatives , Uridine Triphosphate/metabolism , Xenopus laevisABSTRACT
Nucleolar transcription was analysed in permeabilized pre-implantation mouse embryos at the four-cell, eight-cell, morula and early blastocyst stages using confocal microscopy to detect incorporated 5-bromouridine. The results demonstrated that the patterns of nucleolar transcription sites were common for all embryonic stages studied. They consisted most frequently of tightly associated groups of transcription foci similar to those encountered in somatic interphase cells. In addition, the nucleologenesis accompanying each cell cycle apparently gave rise to a different fluorescent pattern, that is to spatially separated fluorescent foci in the cells just after the resumption of rRNA synthesis. An immunoelectron microscopic analysis of the nucleolar transcription was also performed in the eight-cell embryos. A signal, usually consisting of clustered gold particles, was found specifically within nucleolar dense fibrillar components. This result was in agreement with established findings, which identify dense fibrillar component as the major site of nucleolar transcription in somatic cells.
Subject(s)
Blastocyst/metabolism , Cell Nucleolus/metabolism , Embryonic Development , Transcription, Genetic , Animals , Blastocyst/ultrastructure , Bromouracil/analogs & derivatives , Female , Mice , Mice, Inbred C57BL , Microscopy, Confocal , Microscopy, Immunoelectron , Morula/metabolism , Morula/ultrastructure , Pregnancy , Uridine/analogs & derivatives , Uridine/metabolismABSTRACT
OBJECTIVE: To determine a target recognized by anti-Bh autoantibody, found in the serum of a patient with the unusual coexistence of systemic sclerosis (SSc) and psoriatic arthritis (PsA). METHODS: Antigens recognized by the anti-Bh serum were characterized by indirect immunofluorescence on HeLa cells, by conventional immunoblotting using nuclear extract or partially purified preparation of heterogenous nuclear RNP (hnRNP) proteins, and by 2-dimensional immunoblotting. For the analysis of cross-reactivity and immunofluorescence patterns, autoantibodies were affinity-purified by blot elution and then retested. RESULTS: Comparison of the reactivity of the anti-Bh antibody with the monoclonal antibody 4F4 against both the hnRNP C proteins, together with the determination of biochemical properties of the autoantigens, led to the identification of C1 and C2 core proteins as the targets for the anti-Bh autoantibody. CONCLUSION: Several essential components of the spliceosome are targeted by autoantibodies that are present in the sera of patients with systemic rheumatic diseases. We also found that the hnRNP core proteins C1 and C2 are recognized by the autoantibody present in the serum of a patient with SSc and PsA. C1 and C2 hnRNP proteins should be added to the several intracellular autoantigens recently shown to be cleaved by interleukin-1beta-converting enzyme-like enzymes during apoptosis.
Subject(s)
Arthritis, Psoriatic/immunology , Autoantibodies/immunology , RNA, Heterogeneous Nuclear/immunology , Ribonucleoproteins/immunology , Scleroderma, Systemic/immunology , Aged , Antibodies, Monoclonal , Arthritis, Psoriatic/complications , Autoantigens/analysis , Chromatography, Affinity , Cross Reactions/immunology , Fluorescent Antibody Technique, Indirect , HeLa Cells/immunology , HeLa Cells/metabolism , Heterogeneous-Nuclear Ribonucleoprotein Group C , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Male , Scleroderma, Systemic/complicationsABSTRACT
There is substantial evidence that the intra- and intercellular messenger nitric oxide, generated enzymatically from L-arginine by nitric oxide synthase in different isoforms, is involved in the development of nervous tissue. In this study we investigated the nitric oxide expression in the pre- and postnatally developing rat brain. With regard to messenger RNA, all of the basic nitric oxide synthase isoforms (neuronal, endothelial and macrophage nitric oxide synthase) were already expressed at embryonic day 10 and showed a temporary decrease at embryonic day 17. Western blot analysis of the three isoform proteins revealed a time pattern that was different from those of messenger RNAs. Although the endothelial nitric oxide synthase isoform was also expressed at embryonic day 10, no quantitative changes were observed over the whole time period studied. Protein amounts of brain and inducible nitric oxide synthase were first detectable at embryonic day 15, with a tendency to rise. A parallel time pattern was found for the NADPH-diaphorase activity in our light microscopic studies, whereas ultrastructurally the reaction product was seen in the brain pallium even of 13-day-old embryos. The data indicate a permanent presence of the transcripts for all nitric oxide synthase isoforms in the rat central nervous system from embryonic day 10 onwards, although the expression of respective proteins and staining patterns may differ.