Thachillath Pramathan

Centromeric inactivation in a dicentric human Y;21 translocation chromosome

Abstract. A de novo dicentric Y;21 (q11.23;p11) translocation chromosome with one of its two centromeres inactive has provided the opportunity to study the relationship between centromeric inactivation, the organization of alphoid satellite DNA and the distribution of CENP-C. The proband, a male with minor features of Down’s syndrome, had a major cell line with 45 chromosomes including a single copy of the translocation chromosome, and a minor one with 46 chromosomes including two copies of the translocation chromosome and hence effectively trisomic for the long arm of chromosome 21. Centromeric activity as defined by the primary constriction was variable: in most cells with a single copy of the Y;21 chromosome, the Y centromere was inactive. In the cells with two copies, one copy had an active Y centromere (chromosome 21 centromere inactive) and the other had an inactive Y centromere (chromosome 21 centromere active). Three different partial deletions of the Y alphoid array were found in skin fibroblasts and one of these was also present in blood. Clones of single cell origin from fibroblast cultures were analysed both for their primary constriction and to characterise their alphoid array. The results indicate that (1) each clone showed a fixed pattern of centromeric activity; (2) the alphoid array size was stable within a clone; and (3) inactivation of the Y centromere was associated with both full-sized and deleted alphoid arrays. Selected clones were analysed with antibodies to CENP-C, and staining was undetectable at both intact and deleted arrays of the inactive Y centromeres. Thus centromeric inactivation appears to be largely an epigenetic event.

In vitro biocompatibility of calcined mesoporous silica particles and fetal blood cells

Background: The biocompatibility of two forms of calcined mesoporous silica particles, labeled as MCM41-cal and SBA15-cal, with fetal blood mononuclear cells was assessed in vitro. Methods and results: Fetal mononuclear cells were isolated from umbilical cord blood and exposed to 0.5 mg/mL of MCM41-cal or SBA15-cal for several hours. Transmission electron micrographs confirmed the presence of particles in the cytosol of macrophages, neutrophils, and lymphocytes without noticeable damage to the cellular organelles. The particles (especially MCM41-cal) were in close proximity to plasma, and nuclear and mitochondrial membranes. Biocompatibility was assessed by a functional assay that measured cellular respiration, ie, mitochondrial O2 consumption. The rate of respiration (kc, in μM O2 per minute per 107 cells) for untreated cells was 0.42 ± 0.16 (n = 10), for cells treated with MCM41-cal was 0.39 ± 0.22 (n = 5, P > 0.966) and for cells treated with SBA15-cal was 0.44 ± 0.13 (n = 5, P > 0.981). Conclusion: The results show reasonable biocompatibility of MCM41-cal and SBA15-cal in fetal blood mononuclear cells. Future studies are needed to determine the potential of collecting fetal cells from a fetus or neonate, loading the cells in vitro with therapeutic MCM41-cal or SBA15-cal, and reinfusing them into the fetus or neonate.

Lung tissue bioenergetics and caspase activity in rodents

BackgroundThis study aimed to establish a suitable in vitro system for investigating effects of respiratory pathogens and toxins on lung tissue bioenergetics (cellular respiration and ATP content) and caspase activity. Wistar rats and C57Bl/6 mice were anesthetized by sevoflurane inhalation. Lung fragments were then collected and incubated at 37°C in a continuously gassed (with 95% O2:5% CO2) Minimal Essential Medium (MEM) or Krebs-Henseleit buffer. Phosphorescence O2 analyzer that measured dissolved O2 concentration as a function of time was used to monitor the rate of cellular mitochondrial O2 consumption. Cellular ATP content was measured using the luciferin/luciferase system. The caspase-3 substrate N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) was used to monitor intracellular caspase activity; cleaved AMC moieties (reflecting caspase activity) were separated on HPLC and detected by fluorescence. Lung histology and immunostaining with anti-cleaved caspase-3 antibody were also performed.ResultsFor Wistar rats, the values of kc and ATP for 0 < t ≤ 7 h (mean ± SD) were 0.15 ± 0.02 μM O2 min-1 mg-1 (n = 18, coefficient of variation, Cv = 13%) and 131 ± 69 pmol mg-1 (n = 16, Cv = 53%), respectively. The AMC peak areas remained relatively small despite a ~5-fold rise over 6 h. Good tissue preservation was evident despite time-dependent increases in apoptotic cells. Lung tissue bioenergetics, caspase activity and structure were deleterious in unoxygenated or intermittently oxygenated solutions. Incubating lung tissue in O2 depleted MEM for 30 min or anesthesia by urethane had no effect on lung bioenergetics, but produced higher caspase activity.ConclusionsLung tissue bioenergetics and structure could be maintained in vitro in oxygenated buffer for several hours and, thus, used as biomarkers for investigating respiratory pathogens or toxins.

Gonadal mosaicism in ARID1B gene causes intellectual disability and dysmorphic features in three siblings

The gene encoding the AT‐rich interaction domain‐containing protein 1B (ARID1B) has recently been shown to be one of the most frequently mutated genes in patients with intellectual disability (ID). The phenotypic spectrums associated with variants in this gene vary widely ranging for mild to severe non‐specific ID to Coffin–Siris syndrome. In this study, we evaluated three children from a consanguineous Emirati family affected with ID and dysmorphic features. Genomic DNA from all affected siblings was analyzed using CGH array and whole‐exome sequencing (WES). Based on a recessive mode of inheritance, homozygous or compound heterozygous variants shared among all three affected children could not be identified. However, further analysis revealed a heterozygous variant (c.4318C>T; p.Q1440*) in the three affected children in an autosomal dominant ID causing gene, ARID1B. This variant was absent in peripheral blood samples obtained from both parents and unaffected siblings. Therefore, we propose that the most likely explanation for this situation is that one of the parents is a gonadal mosaic for the variant. To the best of our knowledge, this is the first report of a gonadal mosaicism inheritance of an ARID1B variant leading to familial ID recurrence.

In vitro study on the pulmonary cytotoxicity of amiodarone

Abstract Context: Amiodarone (an iodinated benzofuran) is a Class III antiarrhythmic drug that produces significant pulmonary disease. Proposed mechanisms of this cytotoxicity include necrosis, apoptosis, mitochondrial dysfunction and glutathione depletion. Objective: This study was designed primarily to explore whether amiodarone impairs lung tissue cellular bioenergetics in BALB/c and Taylor Outbred mice. Materials and methods: Cellular respiration (mitochondrial O2 consumption), ATP, caspase activity and glutathione were measured in lung fragments incubated in vitro with 22 µM amiodarone for several hours. Results: Without amiodarone, lung tissue cellular mitochondrial O2 consumption decayed exponentially with time, showing two distinct phases sharply separated at t ≥ 150 min. The rate of cellular respiration was 6–10-fold higher in the late phase compared to the early phase (p < 0.0001). Lung tissue ATP also decayed exponentially with time, suggesting “uncoupling oxidative phosphorylation” was the responsible mechanism (low cellular ATP with high mitochondrial O2 consumption, resulting in rapid depletion of cellular metabolic fuels). Although intracellular caspase activity increased exponentially with time, the uncoupling was not prevented by the pancaspase inhibitor zVAD-fmk (N-benzyloxycarbonyl-val-ala-asp (O-methyl)-fluoromethylketone). The same profiles were noted in the presence of amiodarone; but cellular ATP decayed 50% faster. Cellular glutathione for untreated tissue was 560 ± 287 pmol mg−1 (n = 12) and for treated tissue was 490 ± 226 pmol mg−1 (n = 12, p = 0.5106). Conclusion: Uncoupling oxidative phosphorylation was demonstrated in untreated mouse lung tissues. Amiodarone lowered cellular ATP. Further studies are needed to explore the susceptibility of the lung to these deleterious insults and their relevance to human diseases.

Impaired Forebrain Cellular Bioenergetics Following Acute Exposure to Ammonia

Introduction: The main purpose of this study was to report on the acute neurotoxicity of ammonia, using cellular respiration and ATP as surrogate metabolic biomarkers. Cellular respiration (mitochondrial O2 consumption), ATP and glutathione (GSH) were measured in forebrain specimens after intraperitoneal administration of 3.8 to 28.8 μmol/g ammonia to Taylor Outbred mice; the lethal dose was ≥ 30 μmol/g. Methods: Cortical fragments were collected in phosphate-buffered saline plus 5 mM glucose and immediately processed for measuring O2 consumption, using a phosphorescence O2 analyzer. Cellular ATP was determined with the luciferase/luciferin system. Cellular GSH was labeled with monobromobimane and the bimane derivatives were separated on HPLC and detected by fluorescence. In the presence of the tissue specimen in a vial sealed from air, O2 concentration declined linearly with time, confirming zero-order kinetics of O2 consumption. This process was inhibited by cyanide, confirming the oxidation occurred in the respiratory chain. Results: For untreated mice, cellular respiration was 0.32 ± 0.12 μM O2 min-1 mg-1 (n=36 mice), ATP was 238 ± 45 pmol.mg-1 (n=9 mice) and GSH was 300 ± 50 pmol.mg-1 (n=6 mice). For treated mice, cellular respiration was 0.26 ± 0.09 μM O2 min-1 mg-1 (n=22 mice; 19% lower, p=0.034), ATP was 183 ± 34 pmol mg-1 (n=9 mice; 23% lower, p=0.008) and GSH was 320 ± 160 pmol mg-1 (n=6 mice, p=0.700). Conclusion: Acute ammonia intoxication significantly lowered forebrain cellular bioenergetics. These changes would be are difficult to monitor at lethal doses, but the results are expected to be more pronounced.

Mitochondrial Oxygen Consumption by the Foreskin and its Fibroblast-rich Culture.

OBJECTIVES This study investigated the feasibility of using a phosphorescence oxygen analyser to measure cellular respiration (mitochondrial O2 consumption) in foreskin samples and their fibroblast-rich cultures. METHODS Foreskin specimens from normal infants were collected immediately after circumcision and processed for measuring cellular respiration and for culture. Cellular mitochondrial O2 consumption was determined as a function of time from the phosphorescence decay of the Pd (II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. RESULTS In sealed vials containing a foreskin specimen and glucose, O2 concentration decreased linearly with time, confirming the zero-order kinetics of O2 consumption by cytochrome oxidase. Cyanide inhibited O2 consumption, confirming that the oxidation occurred mainly in the mitochondrial respiratory chain. The rate of foreskin respiration (mean ± SD) was 0.074 ± 0.02 μM O2 min(-1) mg(-1) (n = 23). The corresponding rate for fibroblast-rich cultures was 9.84 ± 2.43 μM O2 min(-1) per 10(7) cells (n = 15). Fibroblast respiration was significantly lower in a male infant with dihydrolipoamide dehydrogenase gene mutations, but normalised with the addition of thiamine or carnitine. CONCLUSION The foreskin and its fibroblast-rich culture are suitable for assessment of cellular respiration. However, the clinical utility of foreskin specimens to detect disorders of impaired cellular bioenergetics requires further investigation.

In Vitro Study on the Response of Fibroblast Cellular Respiration to Lipoic acid, Thiamine and Carnitine in Patients with Dihydrolipoyl Dehydrogenase Deficiency

Objectives: This study examined in vitro responses of fibroblast cellular respiration to lipoic acid, thiamine and carnitine in patients with dihydrolipoyl dehydrogenase (DLD) deficiency. This disorder impairs cellular bioenergetics and these compounds are used to improve clinical manifestations of the disease. The study aimed to utilize mitochondrial O2 consumption as a surrogate biomarker for examining cellular responses to metabolic therapies. Methods: Cultured fibroblasts from three patients were treated with therapeutic concentrations of the compounds for 24 hours. Cells were then harvested and processed for measuring respiration using phosphorescence oxygen analyzer. Patients 1 and 2 were severely symptomatic infants with homozygous c.1436A>T mutation in the DLD gene. Patient-3 was a mildly symptomatic adolescent with homozygous c.685G>T mutation. Results: The rate of respiration (mean ± SD, n=6, μM O2 min-1/107 cells) in fibroblasts from a normal infant was 9.3 ± 1.6, in fibroblasts from Patient-1 was 5.1 ± 0.9 (p=0.001), in fibroblasts from Patient-2 was 7.4 ± 1.4 (p=0.051), and in fibroblasts from Patient-3 was 10.3 ± 3.3 (p=0.836). In normal fibroblasts, respiration decreased by the thiamine (p=0.012) and carnitine (p=0.023) treatments. In Patient-1, respiration increased by the lipoic acid (p<0.002), thiamine (p<0.001), and carnitine (p=0.018) treatments; this patient clinically responded to thiamine. In Patient-2, respiration decreased by the thiamine (p=0.026) and carnitine (p=0.008) treatments; this patient did not respond to these drugs. In Patient-3, respiration increased by the carnitine (p=0.012) treatment; the patient clinically responded to carnitine. Conclusions: The results show cellular respiration is a suitable biomarker for the disease. The significance of using this tool to assess responses to therapies requires further studies.

725 Lymphocyte Respiration in Children with Trisomy 21

Aims This study aimed to measure lymphocyte mitochondrial O2 consumption (cellular respiration) in children with trisomy 21. Methods Peripheral blood mononuclear cells were isolated from whole blood of trisomy 21 and control children and immediately used to measure the respiratory rate. [O2] was determined as function of time from the phosphorescence decay rates (1/t) of Pd (II)-meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. In sealed vials containing cells and glucose as a respiratory substrate, [O2] declined linearly with time, confirming the zero-order kinetics of O2 consumption (conversion to H2O) by cytochrome oxidase. Results The rate of respiration (k, in mM O2 per min), thus, was the negative of the slope of [O2] vs. time. NaCN inhibited O2 consumption, confirming the oxidation occurred in the mitochondrial respiratory chain. For control children (age = 8.8±5.6 yr, n=26), the mean (± SD) value of k c (in mM O2 per min per 107 cells) was 1.36±0.79 (coefficient of variation = 58%; median = 1.17; range = 0.60 to 3.12; –2SD = 0.61). For children with trisomy 21 (age = 7.2±4.6 yr, n=26), the value of k c was 0.82±0.62 (coefficient of variation = 76%; median = 0.60; range = 0.20 to 2.80), p<0.001. Fourteen of 26 (54%) children with trisomy 21 had k c values of 0.20 to 0.60 (i.e., < –2SD). Conclusion Thus, it appears that some children with trisomy 21 have relatively reduced lymphocyte bioenergetics. The biological implication of this finding (variation) requires further studies.