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1.
Int J Lab Hematol ; 43(6): 1628-1634, 2021 Dec.
Article in English | MEDLINE | ID: mdl-34251753

ABSTRACT

INTRODUCTION: The high-sequence homology of the α-globin-gene cluster is responsible for microhomology-mediated recombination events during meiosis, resulting in a high density of deletion breakpoints within a 10 kb region. Commonly used deletion detection methods, such as multiplex ligation-dependent probe amplification (MLPA) and Southern blot, cannot exactly define the breakpoints. This typically requires long-range PCR, which is not always successful. Targeted locus amplification (TLA) is a targeted enrichment method that can be used to sequence up to 70 kb of neighboring DNA sequences without prior knowledge about the target site. METHODS: Genomic DNA (gDNA) TLA is a technique that folds isolated DNA, ensuring that adjacent loci are in a close spatial proximity. Subsequent digestion and religation form DNA circles that are amplified using fragment-specific inverse primers, creating a library that is suitable for Illumina sequencing. RESULTS: Here, we describe the characterization of a rare 16 771 bp deletion, utilizing gDNA TLA with a single inverse PCR primer set on one end of the breakpoint. Primers for breakpoint PCR were designed to confirm the deletion breakpoints and were consequently used to characterize the same deletion in 10 additional carriers sharing comparable hematologic data and similar MLPA results. CONCLUSIONS: The gDNA TLA technology was successfully used to identify deletion breakpoints within the alpha-globin cluster. The deletion was described only once in an earlier study as the --gb , but as it was not registered correctly in the available databases, it was not initially recognized as such.


Subject(s)
Alleles , Chromosome Breakpoints , Sequence Deletion , alpha-Globins/genetics , alpha-Thalassemia/diagnosis , alpha-Thalassemia/genetics , Genetic Testing , Genomic Library , High-Throughput Nucleotide Sequencing , Humans , Nucleic Acid Amplification Techniques , Polymerase Chain Reaction , alpha-Thalassemia/blood
2.
Methods Mol Biol ; 1492: 185-196, 2017.
Article in English | MEDLINE | ID: mdl-27822865

ABSTRACT

Despite developments in targeted and whole-genome sequencing, the robust detection of all genetic variation, including structural variants, in and around genes of interest and in an allele-specific manner remains a challenge. Targeted locus amplification (TLA) is a cross-linking-based technique that generates complex DNA libraries covering >100 kb of contiguous sequence surrounding one primer pair complementary to a short locus-specific sequence. In combination with next-generation sequencing, TLA enables the complete sequencing and haplotyping of targeted regions of interest. Here we outline the basis of TLA, together with a detailed protocol of the technique.


Subject(s)
High-Throughput Nucleotide Sequencing/methods , Polymerase Chain Reaction/methods , DNA Primers
3.
Tissue Antigens ; 69(2): 181-8, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17257322

ABSTRACT

Genes at the centromeric end of the human leukocyte antigen region influence adaptive autoimmune diseases and cancer. In this study, we characterized protein expression of HKE2, a gene located in the centromeric portion of the class II region of the major histocompatibility complex encoding subunit 6 of prefoldin. Immunohistochemical analysis using an anti-HKE2 antibody indicated that HKE2 protein expression is dramatically upregulated as a consequence of activation. In a tissue microarray and in several tumors, HKE2 was overexpressed in certain cancers compared with normal counterparts. The localization of the HKE2 gene to the class II region, its cytoplasmic expression and putative protein-binding domain suggest that HKE2 may function in adaptive immunity and cancer.


Subject(s)
Genes, MHC Class II/genetics , Molecular Chaperones/metabolism , Neoplasms/metabolism , Amino Acid Sequence , Binding Sites , Chromosome Mapping , Cytoplasm/chemistry , Hematopoietic Stem Cells/chemistry , Hematopoietic Stem Cells/metabolism , Humans , Immunohistochemistry , Molecular Chaperones/analysis , Molecular Chaperones/genetics , Molecular Sequence Data , Neoplasms/chemistry , Protein Conformation , Tumor Cells, Cultured
4.
J Child Neurol ; 16(9): 661-7, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11575607

ABSTRACT

We retrospectively compared electroencephalographic (EEG) dipoles of interictal spikes from prolonged video-EEG monitoring with magnetoencephalographic dipoles from short-term recording in four children with extratemporal lobe epilepsy. We analyzed both sets of dipoles using individual interictal spikes and single moving dipole modeling and evaluated the profiles of spike appearance, dipole position, and orientation in EEG and magnetoencephalography. We obtained more than 100 magnetoencephalographic spikes in two patients who manifested frequent interictal EEG spikes throughout both day and night but fewer than 40 magnetoencephalographic spikes in two patients who had interictal EEG spikes mainly during sleep. The dipole positions of EEG and magnetoencephalography were in close proximity and included in the surgical resection area. Most of the dipoles between EEG and magnetoencephalography were oriented perpendicularly. A combination of EEG dipole analysis from prolonged video-EEG monitoring and magnetoencephalographic dipole analysis provides complementary information for presurgical evaluation in children with intractable extratemporal lobe epilepsy.


Subject(s)
Electroencephalography , Epilepsies, Partial/diagnosis , Epilepsy, Frontal Lobe/diagnosis , Epilepsy, Temporal Lobe/diagnosis , Magnetoencephalography , Parietal Lobe , Video Recording , Adolescent , Brain Mapping , Child , Diagnosis, Differential , Epilepsies, Partial/physiopathology , Epilepsies, Partial/surgery , Epilepsy, Frontal Lobe/physiopathology , Epilepsy, Frontal Lobe/surgery , Epilepsy, Temporal Lobe/physiopathology , Epilepsy, Temporal Lobe/surgery , Evoked Potentials/physiology , Female , Humans , Length of Stay , Male , Monitoring, Physiologic , Parietal Lobe/physiopathology , Parietal Lobe/surgery , Predictive Value of Tests , Retrospective Studies , Signal Processing, Computer-Assisted
5.
J Trauma ; 49(4): 654-8; discussion 658-9, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11038082

ABSTRACT

BACKGROUND: Adult brain injury studies recommend maintaining cerebral perfusion pressure (CPP) above 70 mm Hg. We evaluated CPP and outcome in brain-injured children. METHODS: We retrospectively reviewed the hospital courses of children at two Level I trauma centers who required insertion of intracranial pressure (ICP) monitors for management of traumatic brain injury. ICP, CPP, and mean arterial pressure were evaluated hourly, and means were calculated for the first 48 hours after injury. RESULTS: Of 188 brain-injured children, 118 had ICP monitors placed within 24 hours of injury. They suffered severe brain injury, with average admitting Glasgow Coma Scale scores of 6 +/- 3. Overall mortality rate was 28%. No patient with mean CPP less than 40 mm Hg survived. Among patients with mean CPP in deciles of 40 to 49, 50 to 59, 60 to 69, or 70 mm Hg, no significant difference in Glasgow Outcome Scale distribution existed. CONCLUSION: Low mean CPP was lethal. In children with survivable brain injury (mean CPP > 40 mm Hg), CPP did not stratify patients for risk of adverse outcome.


Subject(s)
Blood Pressure , Brain Injuries/diagnosis , Cerebrovascular Circulation , Intracranial Pressure , Trauma Severity Indices , Adolescent , Analysis of Variance , Brain Injuries/mortality , Child , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Male , Odds Ratio , Oregon/epidemiology , Retrospective Studies , Risk , Survival Rate
6.
Lancet ; 356(9234): 961-7, 2000 Sep 16.
Article in English | MEDLINE | ID: mdl-11041396

ABSTRACT

BACKGROUND: Endotoxin is a primary trigger of the inflammatory processes that lead to shock, multiorgan failure, and purpura fulminans in meningococcal sepsis. Bactericidal/permeability-increasing protein (BPI) is a natural protein, stored within the neutrophil granules, that binds to and neutralises the effects of endotoxin in vitro, in laboratory animals, and in humans. To establish whether a recombinant 21-kDa modified fragment of human BPI (rBPI21), containing the active antimicrobial and endotoxin-neutralising moiety, would decrease death and long-term disability from meningococcal sepsis, we did a randomised, double-blind, placebo-controlled trial of rBPI21 in children with severe meningococcal sepsis. METHODS: We enrolled children (2 weeks to 18 years of age) presenting to 22 centres in the UK and the USA with a clinical picture suggestive of meningococcal sepsis, and with evidence of severe disease. Children were randomly assigned rBPI21 (2 mg/kg over 30 min followed by 2 mg/kg over 24 h) or placebo (0.2 mg/mL human albumin solution) in addition to conventional medical therapy. Primary outcome variables were mortality, amputations, and change in paediatric overall performance category (POPC) from before illness to day 60. Analysis was by intention to treat. FINDINGS: Of 1287 patients screened, 892 were excluded, including 57 patients who died or who met criteria for imminent death before receiving the study drug. 190 patients received rBPI21, and 203 placebo. 34 (8.7%) of 393 patients died during the study: 14 (7.4%) in the rBPI21 group and 20 (9.9%) in the placebo group (odds ratio 1.31 [95% CI 0.62-2.74], p=0.48). Compared with patients randomised to placebo, fewer patients treated with rBPI21 had multiple severe amputations (six of 190 [3.2%] vs 15 of 203 [7.4%], odds ratio 2.47 [0.94-6.51], p=0.067), and more had a functional outcome similar to that before illness (as measured by the POPC scale) at day 60 (136 of 176 [77.3%] vs 126 of 190 [66.3%], p=0.019). INTERPRETATION: Because most deaths occurred in the interval between identification of patients and study drug administration, the mortality rate in the placebo group was substantially lower than predicted. The trial was therefore underpowered to detect significant differences in mortality. However, patients receiving rBPI21 had a trend towards improved outcome in all primary outcome variables. Given the excellent severity match between placebo and rBPI21 groups at study entry, the results overall indicate that rBPI21 is beneficial in decreasing complications of meningococcal disease.


Subject(s)
Bacteremia/drug therapy , Membrane Proteins/therapeutic use , Meningococcal Infections/drug therapy , Adolescent , Amputation, Surgical/statistics & numerical data , Bacteremia/classification , Bacteremia/mortality , Chemotherapy, Adjuvant , Child , Child, Preschool , Double-Blind Method , Endotoxins , Female , Glasgow Coma Scale , Humans , Infant , Male , Meningococcal Infections/classification , Meningococcal Infections/mortality , Treatment Outcome , United Kingdom , United States
7.
J Mol Med (Berl) ; 77(1): 165-8, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9930955

ABSTRACT

In a specific animal model the induction of heterologous enchondral ossification was examined in long term follow-up. Physiologically normal and devitalized frozen porcine epiphyseal cartilage as a control group were transplanted as s.c. xenograft into athymic nude mice (nu/nu). 15 weeks after transplantation the porcine growth plate cartilage showed the development of viable cartilage tissue in the recipients, further differentiating to juvenile long bone with epiphysis, diaphysis and growth plate. Porcine growth plate cartilage increased s.c. in the recipient nude mice, showing enchondral ossification without any physiological load, and finally an unexpected development towards complete juvenile long bone.


Subject(s)
Growth Plate/physiology , Growth Plate/transplantation , Osteogenesis , Animals , Bone Development , Disease Models, Animal , Graft Survival , Mice , Mice, Nude , Swine , Transplantation, Heterologous
8.
Lancet ; 350(9089): 1439-43, 1997 Nov 15.
Article in English | MEDLINE | ID: mdl-9371168

ABSTRACT

BACKGROUND: Meningococcal sepsis remains an important cause of morbidity and mortality. We hypothesised that children with severe meningococcaemia might benefit from inhibition of the inflammatory processes thought responsible for fulminant disease. rBPI21 is a recombinant, N-terminal fragment of human bactericidal/permeability-increasing protein, which kills meningococci and binds to and clears bacterial endotoxin, these being the primary inducers of the systemic inflammation. The aim of this study was to determine the safety and kinetics of rBPI21 in children with severe meningococcaemia and to make a preliminary assessment of clinical outcome. METHODS: In this open-label, dose-escalation, phase I/II trial in severe meningococcaemia (Glasgow meningococcal prognostic septicaemia score [GMSPS] > or = 8), 26 patients aged 1-18 years, who had received their first dose of antibiotics no more than 8 hours earlier were given rBPI21 by infusion at total doses of 1.0, 2.0, and 4.0 mg/kg. FINDINGS: The patients had significantly raised plasma concentrations of bacterial endotoxin and cytokines. Peak and steady state BPI concentrations were comparable with pharmacokinetic data in healthy adults. All complications were compatible with the expected pattern for severe meningococcal sepsis. Only one patient died. This outcome was found to compare favourably with a predicted mortality of > or = 30% by GMSPS, > or = 15% by plasma endotoxin values, > or = 28% by plasma interleukin-6 concentrations, 29-49% by severity of coagulopathy, and 20% (11/54) by comparison with recent historical patients consecutively treated in participating centres before this study. INTERPRETATION: This, the first clinical trial or rBPI21, shows that rBPI21 can be safely administered to children with severe meningococcaemia and that the pharmacokinetics are consistent with patterns seen in healthy adults. Predicted mortality, on the basis of GMSPS, laboratory indices of inflammation and coagulopathy, and historical controls, was for between four and eight deaths. These findings have prompted a phase III randomised trial.


Subject(s)
Anti-Infective Agents/therapeutic use , Bacteremia/drug therapy , Blood Proteins/therapeutic use , Membrane Proteins , Meningococcal Infections/drug therapy , Adult , Anti-Infective Agents/adverse effects , Anti-Infective Agents/pharmacokinetics , Antimicrobial Cationic Peptides , Bacteremia/microbiology , Bacteremia/mortality , Blood Bactericidal Activity , Blood Proteins/adverse effects , Blood Proteins/pharmacokinetics , Child , Dose-Response Relationship, Drug , Female , Humans , Male , Meningococcal Infections/mortality , Recombinant Proteins/adverse effects , Recombinant Proteins/pharmacokinetics , Recombinant Proteins/therapeutic use , Treatment Outcome
9.
Am J Emerg Med ; 15(4): 383-5, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9217533

ABSTRACT

A 4-year-old boy with a history of seizures triggered by fever presented at an emergency department (ED) with tachycardia, skin vasoconstriction, and a rectal temperature of 42.2 degrees C. However, his ear temperature (as repeatedly measured in two ears, by two experienced nurses, and with two infrared thermometers) was between 36.4 degrees C and 37.6 degrees C. Antipyretic therapy resulted in skin vasodilation, a rapid decrease of rectal temperature, restoration of heart rate, and disappearance of the difference between the two temperatures. Seizures did not occur. This case shows that infrared ear thermometry cannot be recommended in EDs as the procedure of choice for detecting fever in small children, especially when they are vasoconstricted.


Subject(s)
Body Temperature , Fever/physiopathology , Child, Preschool , Ear , Fever/complications , Hemodynamics , Humans , Male , Rectum , Seizures/complications , Skin/blood supply , Tachycardia/physiopathology , Thermometers , Vasoconstriction
10.
Psychopharmacology (Berl) ; 118(1): 1-9, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7597114

ABSTRACT

This study was designed to confirm the hypothetical dose-dependent effect of alprazolam on memory and to compare its effects on tests measuring different aspects of cognitive and psychomotor functioning. A secondary purpose was to compare the sensitivity of newly developed telephone tests with a standard laboratory test of memory. Twenty healthy male volunteers (aged 18-35 years) participated in a five-way double-blind crossover design. Treatments were single oral doses of alprazolam (0.25, 0.5 and 1.0 mg), placebo and lorazepam 2.0 mg. The latter was included as an internal control for demonstrating sensitivity of the method. Lorazepam significantly impaired all performance measures except two: verbal fluency and accuracy of music recognition were insensitive to drug effects. Alprazolam had clear dose-dependent effects on different aspects of memory: 0.25 mg did not affect any parameter; 0.5 mg impaired performance in a Word Learning Test and a Spatial Memory Test and also psychomotor performance; 1.0 mg additionally impaired Syntactic Reasoning, Semantic Verification, and Critical Flicker Fusion performance. Alprazolam's effects were not selective for any of the cognitive functions as measured in this study. The telephone tests were clearly less sensitive than the standard test, though sufficient for showing significant effects of alprazolam 1 mg and lorazepam 2 mg.


Subject(s)
Alprazolam/administration & dosage , Lorazepam/administration & dosage , Memory/drug effects , Adolescent , Adult , Alprazolam/adverse effects , Cross-Over Studies , Dose-Response Relationship, Drug , Double-Blind Method , Flicker Fusion/drug effects , Humans , Lorazepam/adverse effects , Male , Mental Processes/drug effects , Psychomotor Performance/drug effects , Reaction Time/drug effects , Space Perception/drug effects , Verbal Learning/drug effects
11.
Eur J Surg Oncol ; 15(4): 383-5, 1989 Aug.
Article in English | MEDLINE | ID: mdl-2759258

ABSTRACT

A 50-year-old patient who underwent locoregional radiotherapy and surgery for a tonsillar carcinoma, developed osteomyelitis with Actinomyces israelii with fistulization in the treated area, during chemotherapy treatment. This rare complication is discussed.


Subject(s)
Actinomycosis, Cervicofacial/etiology , Carcinoma, Squamous Cell/complications , Tonsillar Neoplasms/complications , Actinomycosis, Cervicofacial/pathology , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/therapy , Cheek , Combined Modality Therapy , Fistula/etiology , Fistula/pathology , Humans , Male , Middle Aged , Osteomyelitis/etiology , Osteomyelitis/pathology , Tonsillar Neoplasms/pathology , Tonsillar Neoplasms/therapy
13.
Arch Belg ; 47(1-4): 113-7, 1989.
Article in Dutch | MEDLINE | ID: mdl-2610549

ABSTRACT

Although cancer in children is uncommon, it represents the second cause of death before the age of 15. One of the determinants in the development of malignancies in children may be formed by the parental occupational exposure to mutagenic or carcinogenic agents. In November 1988, the Department of Public Health of the Free University Brussels, started a case-control study to look for an association between parental occupational exposure and the development of cancer in their offspring. The cases are formed by all patients with leukemia, a brain tumors or a neuroblastoma treated in three pediatric hospitals in Brussels. For each case, two controls matched for age and sex, are recruited from the same hospitals. One control group is formed by all other cancer patients and a second group includes children hospitalized in the departments of surgery. The parents of the cases and the controls are interviewed about their occupations and occupational exposure in the past and the present. The occupational physicians are asked to supply a questionnaire about the occupational history of the employee. At the end of March 1990 the study will be closed. 20 parents of cases and 8 parents of control persons are already interviewed but we have no results yet.


Subject(s)
Carcinogens , Environmental Exposure , Neoplasms/etiology , Parents , Belgium , Case-Control Studies , Child , Child, Preschool , Humans , Infant , Neoplasms/chemically induced
14.
Int J Epidemiol ; 17(4): 724-31, 1988 Dec.
Article in English | MEDLINE | ID: mdl-3225078

ABSTRACT

Different multivariate methods have been applied to obtain an overview of the Belgian female cancer mortality distribution. The resulting maps and figures show the patterns of female cancer mortality to be strongly geographically determined. Two major trends can be derived namely along the north-south axis and along the east-west axis of the country. The cancer sites responsible for these patterns are identified.


Subject(s)
Neoplasms/mortality , Belgium , Data Display , Female , Humans , Pattern Recognition, Automated , Space-Time Clustering , Statistics as Topic
16.
Pathol Res Pract ; 180(4): 392-9, 1985 Oct.
Article in English | MEDLINE | ID: mdl-3906606

ABSTRACT

Human osteosarcoma specimens were sliced in a cryomicrotome under strict morphological guidance. Serial sections of ten 10 micron slices each were collected in two groups according to morphologic criteria, one containing mostly undifferentiated tumor tissue, the other predominantly well-differentiated tumor tissue. The two series were analysed chemically for alkaline phosphatase (APase) acid phosphatase (acPase), beta-glucuronidase and proteolytic activities; protein, phosphorus, hydroxyproline, hexosamine, water and collagen contents were also determined. Four different types of osteosarcoma were studied: case 1 was a highly malignant osteoblastic osteosarcoma, case 2 a small cell sclerosing osteosarcoma case 3 a well-differentiated osteosarcoma, and case 4 a highly malignant anaplastic osteosarcoma. The types of cases 1, 2 and 3 are known as osteoid-forming tumors. In their less well differentiated areas APase activity was about twice as high as in better differentiated osteosarcoma. In contrast, no APase was found in the wholly undifferentiated areas of case 4, while the enzyme showed a marked increase in the areas of incipient differentiation of this tumor. The matrix of tumors differs with regard to collagen and hexosamine contents, in accordance with the general state of differentiation. In general, increasing hexosamine contents together with decreasing hydroxyproline contents will reflect the anaplastic, dedifferentiated osteosarcoma. Calcification evident in the better differentiated areas of osteosarcoma is indicated by the phosphorus content, highest in case 2, with cases 3, 1, and 4 following in sequential order.


Subject(s)
Bone Neoplasms/pathology , Osteosarcoma/pathology , Acid Phosphatase/analysis , Alkaline Phosphatase/analysis , Bone Neoplasms/enzymology , Collagen/analysis , Glucuronidase/analysis , Hexosamines/analysis , Humans , Hydroxyproline/analysis , Neoplasm Proteins/analysis , Osteosarcoma/enzymology , Peptide Hydrolases/analysis
17.
Pathol Res Pract ; 180(4): 383-91, 1985 Oct.
Article in English | MEDLINE | ID: mdl-3865164

ABSTRACT

A new technique was applied to the study of human osteosarcoma. Ten slices of 10 micron were cut serially from 2 X 2 X 6 mm shock frozen blocks of human osteosarcoma for chemical analysis. Before and after each series of 10 slices, one slice of 10 micron was separated for morphological analysis. Four different types of osteosarcoma were investigated: Case 1 was an atypical osteoblastic osteosarcoma, case 2 a small cell sclerosing osteosarcoma, case 3 a well-differentiated parosteal osteosarcoma grade I, and case 4 a highly malignant anaplastic osteosarcoma. Alkaline phosphatase, acid phosphatase, beta-glucuronidase and proteolytic activities were analysed as well as matrix collagen and hexosamine, phosphorus (Pi and Po), protein, DNA, and water content. In accordance with the morphology, the obtained data illustrate the great heterogeneity of osteosarcomas. Although case 1, 2 and 3 all represent calcifying types of the tumor, characteristic differences exist with regard to the matrix and the degree of calcification. In contrast to these three, case 4 presents a noncalcified type of osteosarcoma whose matrix contains relatively high amounts of hexosamine and low amounts of collagen, whereas DNA and water contents are high. The data from the analysis of osteosarcoma were compared with previous results from the calf epiphyseal growth plate in order to define differences and similarities between the formation of tumor bone and the physiological formation of hard tissue.


Subject(s)
Bone Neoplasms/pathology , Osteosarcoma/pathology , Acid Phosphatase/analysis , Alkaline Phosphatase/analysis , Bone Neoplasms/classification , Bone Neoplasms/enzymology , Glucuronidase/analysis , Humans , Osteosarcoma/classification , Osteosarcoma/enzymology , Registries
18.
Cell Tissue Res ; 242(1): 217-9, 1985.
Article in English | MEDLINE | ID: mdl-4042136

ABSTRACT

Electron-dense particles with a diameter of 50-200 nm have been observed at the cell membrane of chondrocytes in the zone of the initiation and advance of mineralization, using the dark field STEM mode. Electron-probe x-ray microanalysis and laser microprobe mass analysis indicate that these particles contain predominantly K and Na. They appear only in dry thin sections of shock-frozen, freeze-dried embedded tissue and not in sections of water-treated samples; hence they contain water-extractable potassium and sodium. The function of the two elements at these special sites is not yet clear. On the one hand, they might reflect exocytotic processes connected with a Na-K-ATPase; on the other hand, they might exist as a transitory state before being replaced by Ca and phosphate in the mineralizing matrix and later transported elsewhere by the blood vessels.


Subject(s)
Growth Plate/analysis , Potassium/metabolism , Animals , Cell Membrane/analysis , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Electron Probe Microanalysis , Female , Freeze Drying , Growth Plate/metabolism , Growth Plate/ultrastructure , Guinea Pigs , Microscopy, Electron, Scanning , Sodium/metabolism , Tibia
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