Membrane restructuring by phospholipase A2 is regulated by the presence of lipid domains

Secretory phospholipase A₂ (sPLA₂) catalyzes the hydrolysis of glycerophospholipids. This enzyme is sensitive to membrane structure, and its activity has been shown to increase in the presence of liquid-crystalline/gel (L_α/L_β) lipid domains. In this work, we explore whether lipid domains can also direct the activity of the enzyme by inducing hydrolysis of certain lipid components due to preferential activity of the enzyme toward lipid domains susceptible to sPLA₂. Specifically, we show that the presence of L_α/L_β and L_α/P_β′ phase coexistence in a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2 distearoyl-sn-glycero-3-phosphocholine (DSPC) system results in the preferential hydrolysis of the shorter-chained lipid component in the mixture, leading to an enrichment in the longer-chained component. The restructuring process is monitored by atomic force microscopy on supported single and double bilayers formed by vesicle fusion. We observe that during preferential hydrolysis of the DMPC-rich L_α regions, the L_β and P_β′ regions grow and reseal, maintaining membrane integrity. This result indicates that a sharp reorganization of the membrane structure can occur during sPLA₂ hydrolysis without necessarily destroying the membrane. We confirm by high-performance liquid chromatography the preferential hydrolysis of DMPC within the phase coexistence region of the DMPC/DSPC phase diagram, showing that this preferential hydrolysis is accentuated close to the solidus phase boundary. Differential scanning calorimetry results show that this preferential hydrolysis in the presence of lipid domains leads to a membrane system with a higher-temperature melting profile due to enrichment in DSPC. Together, these results show that the presence of lipid domains can induce specificity in the hydrolytic activity of the enzyme, resulting in marked differences in the physical properties of the membrane end-product.     

Experimental treatment of Neospora caninum-infected mice with the arylimidamide DB750 and the thiazolide nitazoxanide

The cationic arylimidamide DB750 and the thiazolide nitazoxanide had been shown earlier to be effective against Neospora caninum tachyzoites in vitro with an IC₅₀ of 160 nM and 4.23 μM, respectively. In this study, we have investigated the effects of DB750 and nitazoxanide treatments of experimentally infected Balb/c mice, by applying the drugs either through the oral or the intraperitoneal route. In experiment 1, administration of DB750 (2 mg/kg/day) and nitazoxanide (150 mg/kg/day) started already 3 days prior to experimental infection of mice with 2 × 10⁶ tachyzoites. Following infection, the drugs were further administrated daily for a period of 2 weeks, either orally or intraperitoneally. Intraperitoneal injection of DB750 was well tolerated by the mice, but treatment with nitazoxanide resulted in death of all mice within 3 days. Upon intraperitoneal application of DB750, the cerebral parasite load was significantly reduced compared to all other groups, while oral application of DB750 and nitazoxanide were not as effective, and resulted in significant weight loss. In experiment 2, mice were infected with 2 × 10⁶ tachyzoites and at 2 weeks post-infection, DB750 (2 mg/kg/day) was applied by intraperitoneal injections for 14 days. In the DB750-treated group, only 2 out of 12 mice succumbed to infection, compared to 7 out of 12 mice in the placebo-group. DB750 treatment also resulted in significantly reduced cerebral parasite burden, and reduced numbers of viable tachyzoites. Our data suggest that DB750 exerted its activity also after crossing the blood–brain barrier, and that this class of compounds could be promising for the control of N. caninum-associated disease.     

Deciphering the electron transport pathway for graphene oxide reduction by Shewanella oneidensis MR-1

We determined that graphene oxide reduction by Shewanella oneidensis MR-1 requires the Mtr respiratory pathway by analyzing a range of mutants lacking these proteins. Electron shuttling compounds increased the graphene oxide reduction rate 3- to 5-fold. These results may help facilitate the use of bacteria for large-scale graphene production.     

Using VAAST to identify an X-linked disorder resulting in lethality in male infants due to N-terminal acetyltransferase deficiency

We have identified two families with a previously undescribed lethal X-linked disorder of infancy; the disorder comprises a distinct combination of an aged appearance, craniofacial anomalies, hypotonia, global developmental delays, cryptorchidism, and cardiac arrhythmias. Using X chromosome exon sequencing and a recently developed probabilistic algorithm aimed at discovering disease-causing variants, we identified in one family a c.109T>C (p.Ser37Pro) variant in NAA10, a gene encoding the catalytic subunit of the major human N-terminal acetyltransferase (NAT). A parallel effort on a second unrelated family converged on the same variant. The absence of this variant in controls, the amino acid conservation of this region of the protein, the predicted disruptive change, and the co-occurrence in two unrelated families with the same rare disorder suggest that this is the pathogenic mutation. We confirmed this by demonstrating a significantly impaired biochemical activity of the mutant hNaa10p, and from this we conclude that a reduction in acetylation by hNaa10p causes this disease. Here we provide evidence of a human genetic disorder resulting from direct impairment of N-terminal acetylation, one of the most common protein modifications in humans.     

Altering O-Linked β-N-Acetylglucosamine Cycling Disrupts Mitochondrial Function

Mitochondrial impairment is commonly found in many diseases such as diabetes, cancer, and Alzheimer disease. We demonstrate that the enzymes responsible for the addition or removal of the O-GlcNAc modification, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively, are critical regulators of mitochondrial function. Using a SILAC (stable isotope labeling of amino acids in cell culture)-based proteomics screen, we quantified the changes in mitochondrial protein expression in OGT- and OGA-overexpressing cells. Strikingly, overexpression of OGT or OGA showed significant decreases in mitochondria-localized proteins involved in the respiratory chain and the tricarboxylic acid cycle. Furthermore, mitochondrial morphology was altered in these cells. Both cellular respiration and glycolysis were reduced in OGT/OGA-overexpressing cells. These data demonstrate that alterations in O-GlcNAc cycling profoundly affect energy and metabolite production.     

Cardiomyocyte-specific Loss of Diacylglycerol Acyltransferase 1 (DGAT1) Reproduces the Abnormalities in Lipids Found in Severe Heart Failure

Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final step in triglyceride synthesis, the conversion of diacylglycerol (DAG) to triglyceride. Dgat1^−/− mice exhibit a number of beneficial metabolic effects including reduced obesity and improved insulin sensitivity and no known cardiac dysfunction. In contrast, failing human hearts have severely reduced DGAT1 expression associated with accumulation of DAGs and ceramides. To test whether DGAT1 loss alone affects heart function, we created cardiomyocyte-specific DGAT1 knock-out (hDgat1^−/−) mice. hDgat1^−/− mouse hearts had 95% increased DAG and 85% increased ceramides compared with floxed controls. 50% of these mice died by 9 months of age. The heart failure marker brain natriuretic peptide increased 5-fold in hDgat1^−/− hearts, and fractional shortening (FS) was reduced. This was associated with increased expression of peroxisome proliferator-activated receptor α and cluster of differentiation 36. We crossed hDgat1^−/− mice with previously described enterocyte-specific Dgat1 knock-out mice (hiDgat1^−/−). This corrected the early mortality, improved FS, and reduced cardiac ceramide and DAG content. Treatment of hDgat1^−/− mice with the glucagon-like peptide 1 receptor agonist exenatide also improved FS and reduced heart DAG and ceramide content. Increased fatty acid uptake into hDgat1^−/− hearts was normalized by exenatide. Reduced activation of protein kinase Cα (PKCα), which is increased by DAG and ceramides, paralleled the reductions in these lipids. Our mouse studies show that loss of DGAT1 reproduces the lipid abnormalities seen in severe human heart failure.     

Dosage Changes of a Segment at 17p13.1 Lead to Intellectual Disability and Microcephaly as a Result of Complex Genetic Interaction of Multiple Genes

The 17p13.1 microdeletion syndrome is a recently described genomic disorder with a core clinical phenotype of intellectual disability, poor to absent speech, dysmorphic features, and a constellation of more variable clinical features, most prominently microcephaly. We identified five subjects with copy-number variants (CNVs) on 17p13.1 for whom we performed detailed clinical and molecular studies. Breakpoint mapping and retrospective analysis of published cases refined the smallest region of overlap (SRO) for microcephaly to a genomic interval containing nine genes. Dissection of this phenotype in zebrafish embryos revealed a complex genetic architecture: dosage perturbation of four genes (ASGR1, ACADVL, DVL2, and GABARAP) impeded neurodevelopment and decreased dosage of the same loci caused a reduced mitotic index in vitro. Moreover, epistatic analyses in vivo showed that dosage perturbations of discrete gene pairings induce microcephaly. Taken together, these studies support a model in which concomitant dosage perturbation of multiple genes within the CNV drive the microcephaly and possibly other neurodevelopmental phenotypes associated with rearrangements in the 17p13.1 SRO.     

Parent of Origin,Mosaicism, and Recurrence Risk: Probabilistic Modeling Explains the Broken Symmetry of Transmission Genetics

Most new mutations are observed to arise in fathers, and increasing paternal age positively correlates with the risk of new variants. Interestingly, new mutations in X-linked recessive disease show elevated familial recurrence rates. In male offspring, these mutations must be inherited from mothers. We previously developed a simulation model to consider parental mosaicism as a source of transmitted mutations. In this paper, we extend and formalize the model to provide analytical results and flexible formulas. The results implicate parent of origin and parental mosaicism as central variables in recurrence risk. Consistent with empirical data, our model predicts that more transmitted mutations arise in fathers and that this tendency increases as fathers age. Notably, the lack of expansion later in the male germline determines relatively lower variance in the proportion of mutants, which decreases with paternal age. Subsequently, observation of a transmitted mutation has less impact on the expected risk for future offspring. Conversely, for the female germline, which arrests after clonal expansion in early development, variance in the mutant proportion is higher, and observation of a transmitted mutation dramatically increases the expected risk of recurrence in another pregnancy. Parental somatic mosaicism considerably elevates risk for both parents. These findings have important implications for genetic counseling and for understanding patterns of recurrence in transmission genetics. We provide a convenient online tool and source code implementing our analytical results. These tools permit varying the underlying parameters that influence recurrence risk and could be useful for analyzing risk in diverse family structures.