A histone deacetylase 3 and mitochondrial complex I axis regulates toxic formaldehyde production.

Cells produce considerable genotoxic formaldehyde from an unknown source. We carry out a genome-wide CRISPR-Cas9 genetic screen in metabolically engineered HAP1 cells that are auxotrophic for formaldehyde to find this cellular source. We identify histone deacetylase 3 (HDAC3) as a regulator of cellular formaldehyde production. HDAC3 regulation requires deacetylase activity, and a secondary genetic screen identifies several components of mitochondrial complex I as mediators of this regulation. Metabolic profiling indicates that this unexpected mitochondrial requirement for formaldehyde detoxification is separate from energy generation. HDAC3 and complex I therefore control the abundance of a ubiquitous genotoxic metabolite.

The HIF complex recruits the histone methyltransferase SET1B to activate specific hypoxia-inducible genes.

Hypoxia-inducible transcription factors (HIFs) are fundamental to cellular adaptation to low oxygen levels, but it is unclear how they interact with chromatin and activate their target genes. Here, we use genome-wide mutagenesis to identify genes involved in HIF transcriptional activity, and define a requirement for the histone H3 lysine 4 (H3K4) methyltransferase SET1B. SET1B loss leads to a selective reduction in transcriptional activation of HIF target genes, resulting in impaired cell growth, angiogenesis and tumor establishment in SET1B-deficient xenografts. Mechanistically, we show that SET1B accumulates on chromatin in hypoxia, and is recruited to HIF target genes by the HIF complex. The selective induction of H3K4 trimethylation at HIF target loci is both HIF- and SET1B-dependent and, when impaired, correlates with decreased promoter acetylation and gene expression. Together, these findings show SET1B as a determinant of site-specific histone methylation and provide insight into how HIF target genes are differentially regulated.

IKCa agonist (NS309)-elicited all-or-none dehydration response of human red blood cells is cell-age dependent.

Elevated [Ca(2+)](i) in human red blood cells (RBCs) activates IK1 K(+) channels leading to cell dehydration. NS309, a powerful IK1 agonist, has been shown to activate IK1 channels even at sub-physiological [Ca(2+)](i) levels. An intriguing feature of this response is its all-or-none nature, with responsive cells dehydrating fully and refractory cells retaining normal volume. We investigated the mechanism of this response suspecting cell-age involvement. We expected the younger cells, with the more vigorous plasma membrane Ca(2+) pumps (PMCA), to be the refractory cells because of their lower [Ca(2+)](i). Osmotic fragility measurements and density separation through phthalate oil were used to monitor red cell dehydration. The fraction of glycosilated haemoglobin (Hb A1c) was used to estimate the mean age of density fractionated cells. The results showed that inhibition of the PMCA by vanadate abolished the all-or-none response, that the mean age of the responsive cells was young, contrary to expectations, and that pump inhibition subsequent to an all-or-none response caused the refractory cells to dehydrate fully, suggesting that the all-or-none response resulted from a reduced efficiency of NS309 to increase the Ca(2+) sensitivity of IK1 channels in aged RBCs.

Associations between paternally transmitted fetal IGF2 variants and maternal circulating glucose concentrations in pregnancy.

OBJECTIVE: To test the hypothesis that polymorphic variation in the paternally transmitted fetal IGF2 gene is associated with maternal glucose concentrations in the third trimester of pregnancy. RESEARCH DESIGN AND METHODS: A total of 17 haplotype tag single nucleotide polymorphisms in the IGF2 gene region were genotyped in 1,160 mother/partner/offspring trios from the prospective Cambridge Baby Growth Study (n = 845 trios) and the retrospective Cambridge Wellbeing Study (n = 315 trios) (3,480 samples in total). Associations were tested between inferred parent-of-origin fetal alleles, z scores of maternal glucose concentrations 60 min. after an oral glucose load performed at week 28 of pregnancy, and offspring birth weights. RESULTS: Using the minimum P value test, paternally transmitted fetal IGF2 polymorphisms were associated with maternal glucose concentrations; specifically, paternally transmitted fetal rs6578987 (P = 0.006), rs680 (P = 0.01), rs10770125 (P = 0.0002), and rs7924316 (P = 0.01) alleles were associated with increased maternal glucose concentrations in the third trimester of pregnancy and placental IGF-II contents at birth (P = 0.03). In contrast, there were no associations between maternal glucose concentrations and maternal or maternally transmitted fetal IGF2 genotypes. CONCLUSIONS: Polymorphic variation in paternally transmitted fetal IGF2 is associated with increased maternal glucose concentrations in pregnancy and could potentially alter the risk of gestational diabetes in the mother. The association may be at least partially mediated by changes in placental IGF2 expression.

Maternally transmitted foetal H19 variants and associations with birth weight.

This study was designed to test the hypothesis that polymorphic variation in maternally transmitted foetal H19 alleles is associated with offspring size at birth and alterations in maternal glucose concentrations in pregnancy. Inferred parent of origins of transmitted alleles from 13 haplotype tag SNPs in the H19 gene region from 845 family (mother, partner, offspring) trios from the prospective Cambridge Baby Growth Study and 315 trios from the retrospective Cambridge Wellbeing Study cohorts were tested for association with offspring size at birth measures, as well as maternal glucose concentrations 1 h after a glucose load at week 28 of pregnancy. The foetal rs2071094 allele inherited from the mother was associated with increased birth weight (p = 0.0015) adjusted for gestational age, parity and sex. In the Cambridge Baby Growth Study it was also associated with increased head circumference (p = 0.004), length (p = 0.017) and sum of skinfold thicknesses (p = 0.017) at birth. In contrast to these results there was no association between offspring birth weight and either the maternal rs2071094 genotype or the foetal allele from the father. None of the foetal alleles or maternal genotypes were associated with maternal glucose concentrations, neither were there any other associations with offspring birth weight. In conclusion, consistent with imprinting, common polymorphic variation in foetal H19 alleles transmitted only from the mother are associated with birth weight and other markers of size at birth. Polymorphic variation in H19 is not associated with significant changes in maternal glucose tolerance in the third trimester of pregnancy.

X-ray microanalysis investigation of the changes in Na, K, and hemoglobin concentration in plasmodium falciparum-infected red blood cells.

Plasmodium falciparum is responsible for severe malaria. During the ∼48 h duration of its asexual reproduction cycle in human red blood cells, the parasite causes profound alterations in the homeostasis of the host red cell, with reversal of the normal Na and K gradients across the host cell membrane, and a drastic fall in hemoglobin content. A question critical to our understanding of how the host cell retains its integrity for the duration of the cycle had been previously addressed by modeling the homeostasis of infected cells. The model predicted a critical contribution of excess hemoglobin consumption to cell integrity (the colloidosmotic hypothesis). Here we tested this prediction with the use of electron-probe x-ray microanalysis to measure the stage-related changes in Na, K, and Fe contents in single infected red cells and in uninfected controls. The results document a decrease in Fe signal with increased Na/K ratio. Interpreted in terms of concentrations, the results point to a sustained fall in host cell hemoglobin concentration with parasite maturation, supporting a colloidosmotic role of excess hemoglobin digestion. The results also provide, for the first time to our knowledge, comprehensive maps of the elemental distributions of Na, K, and Fe in falciparum-infected red blood cells.

Hydrogen ion dynamics in human red blood cells.

Our understanding of pH regulation within red blood cells (RBCs) has been inferred mainly from indirect experiments rather than from in situ measurements of intracellular pH (pH(i)). The present work shows that carboxy-SNARF-1, a pH fluorophore, when used with confocal imaging or flow cytometry, reliably reports pH(i) in individual, human RBCs, provided intracellular fluorescence is calibrated using a 'null-point' procedure. Mean pH(i) was 7.25 in CO(2)/HCO(3)(-)-buffered medium and 7.15 in Hepes-buffered medium, and varied linearly with extracellular pH (slope of 0.77). Intrinsic (non-CO(2)/HCO(3)(-)-dependent) buffering power, estimated in the intact cell (85 mmol (l cell)(-1) (pH unit)(-1) at resting pH(i)), was somewhat higher than previous estimates from cell lysates (50-70 mmol (l cell)(-1) (pH unit)(-1)). Acute displacement of pH(i) (superfusion of weak acids/bases) triggered rapid pH(i) recovery. This was mediated via membrane Cl(-)/HCO(3)(-) exchange (the AE1 gene product), irrespective of whether recovery was from an intracellular acid or base load, and with no evident contribution from other transporters such as Na(+)/H(+) exchange. H(+)-equivalent flux through AE1 was a linear function of [H(+)](i) and reversed at resting pH(i), indicating that its activity is not allosterically regulated by pH(i), in contrast to other AE isoforms. By simultaneously monitoring pH(i) and markers of cell volume, a functional link between membrane ion transport, volume and pH(i) was demonstrated. RBC pH(i) is therefore tightly regulated via AE1 activity, but modulated during changes of cell volume. A comparable volume-pH(i) link may also be important in other cell types expressing anion exchangers. Direct measurement of pH(i) should be useful in future investigations of RBC physiology and pathology.

Detection of Plasmodium falciparum-infected red blood cells by optical stretching.

We present the application of a microfluidic optical cell stretcher to measure the elasticity of malaria-infected red blood cells. The measurements confirm an increase in host cell rigidity during the maturation of the parasite Plasmodium falciparum. The device combines the selectivity and sensitivity of single-cell elasticity measurements with a throughput that is higher than conventional single-cell techniques. The method has potential to detect early stages of infection with excellent sensitivity and high speed.

Effects of elevated intracellular calcium on the osmotic fragility of human red blood cells.

High throughput methodologies that measure the distribution of osmotic fragilities in red blood cell populations have enabled the investigation of dynamic changes in red cell homeostasis and membrane permeability in health and disease. The common assumption in the interpretation of dynamic changes in osmotic fragility curves is that left or right shifts reflect a decreased or increased hydration state of the cells, respectively, allowing direct inferences on membrane transport from osmotic fragility measurements. However, the assumed correlation between shifts in osmotic fragility and hydration state has never been directly explored, and may prove invalid in certain conditions. We investigated here whether this correlation holds for red cells exposed to elevated intracellular calcium. The results showed that elevated cell calcium causes a progressive increase in osmotic fragility with minimal contribution from cell hydration (<8%). Loss of membrane area by the release of 160+/-40nm diameter (mean+/-SD) vesicles is shown to be a major contributor, but may not account for the full non-hydration component. The rest must reflect a specific calcium-induced lytic vulnerability of the membrane causing rupture before the cells attain their maximal spherical volumes. The implications of these findings are discussed.

Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders.

The analysis of rare chromosomal translocations in myeloproliferative disorders has highlighted the importance of aberrant tyrosine kinase signaling in the pathogenesis of these diseases. Here we have investigated samples from 679 patients and controls for the nonreceptor tyrosine kinase JAK2 V617F mutation. Of the 480 myeloproliferative disorder (MPD) samples, the proportion of positive cases per disease subtype was 30 (20%) of 152 for atypical or unclassified MPD, 2 of 134 (2%) for idiopathic hypereosinophilic syndrome, 58 of 72 (81%) for polycythemia vera, 24 of 59 (41%) essential thrombocythemia (ET), and 15 of 35 (43%) for idiopathic myelofibrosis. V617F was not identified in patients with systemic mastocytosis (n = 28), chronic or acute myeloid leukemia (n = 35), secondary erythrocytosis (n = 4), or healthy controls (n = 160). Homozygosity for V617F was seen in 43% of mutant samples and was closely correlated with chromosome 9p uniparental disomy. Homozygosity was significantly less common in ET compared with other MPD subtypes. In 53 cases analyzed, the median level of PRV1 expression was significantly higher in V617F-positive cases compared with cases without the mutation. We conclude that V617F is widespread in MPDs. Detection of this acquired mutation is likely to have a major impact on the way patients with MPD are diagnosed, as well as serving as an obvious target for signal transduction therapy.