Dependency of ISW1a chromatin remodeling on extranucleosomal DNA

The nucleosome remodeling activity of ISW1a was dependent on whether ISW1a was bound to one or both extranucleosomal DNAs. ISW1a preferentially bound nucleosomes with an optimal length of approximately 33 to 35 bp of extranucleosomal DNA at both the entry and exit sites over nucleosomes with extranucleosomal DNA at only one entry or exit site. Nucleosomes with extranucleosomal DNA at one of the entry/exit sites were readily remodeled by ISW1a and stimulated the ATPase activity of ISW1a, while conversely, nucleosomes with extranucleosomal DNA at both entry/exit sites were unable either to stimulate the ATPase activity of ISW1a or to be mobilized. DNA footprinting revealed that a major conformational difference between the nucleosomes was the lack of ISW1a binding to nucleosomal DNA two helical turns from the dyad axis in nucleosomes with extranucleosomal DNA at both entry/exit sites. The Ioc3 subunit of ISW1a was found to be the predominant subunit associated with extranucleosomal DNA when ISW1a is bound either to one or to both extranucleosomal DNAs. These two conformations of the ISW1a-nucleosome complex are suggested to be the molecular basis for the nucleosome spacing activity of ISW1a on nucleosomal arrays. ISW1b, the other isoform of ISW1, does not have the same dependency for extranucleosomal DNA as ISW1a and, likewise, is not able to space nucleosomes.     

Titration of mitochondrial fusion rescues Mff-deficient cardiomyopathy

Defects in mitochondrial fusion or fission are associated with many pathologies, raising the hope that pharmacological manipulation of mitochondrial dynamics may have therapeutic benefit. This approach assumes that organ physiology can be restored by rebalancing mitochondrial dynamics, but this concept remains to be validated. We addressed this issue by analyzing mice deficient in Mff, a protein important for mitochondrial fission. Mff mutant mice die at 13 wk as a result of severe dilated cardiomyopathy leading to heart failure. Mutant tissue showed reduced mitochondrial density and respiratory chain activity along with increased mitophagy. Remarkably, concomitant deletion of the mitochondrial fusion gene Mfn1 completely rescued heart dysfunction, life span, and respiratory chain function. Our results show for the first time that retuning the balance of mitochondrial fusion and fission can restore tissue integrity and mitochondrial physiology at the whole-organ level. Examination of liver, testis, and cerebellum suggest, however, that the precise balance point of fusion and fission is cell type specific.     

Map-based analysis of genes affecting the brittle rachis character in tetraploid wheat (Triticum turgidum L.)

The mature spike rachis of wild emmer [Triticum turgidum L. ssp. dicoccoides (Körn. ex Asch. and Graebner) Thell.] disarticulates spontaneously between each spikelet leading to the dispersion of wedge-type diaspores. By contrast, the spike rachis of domesticated emmer (Triticum turgidum L. ssp. turgidum) fails to disarticulate and remains intact until it is harvested. This major distinguishing feature between wild and domesticated emmer is controlled by two major genes, brittle rachis 2 (Br-A2) and brittle rachis 3 (Br-A3) on the short arms of chromosomes 3A and 3B, respectively. Because of their biological and agricultural importance, a map-based analysis of these genes was undertaken. Using two recombinant inbred chromosome line (RICL) populations, Br-A2, on chromosome 3A, was localized to a ~11-cM region between Xgwm2 and a cluster of linked loci (Xgwm666.1, Xbarc19, Xcfa2164, Xbarc356, and Xgwm674), whereas Br-A3, on chromosome 3B, was localized to a ~24-cM interval between Xbarc218 and Xwmc777. Comparative mapping analyses suggested that both Br-A2 and Br-A3 were present in homoeologous regions on chromosomes 3A and 3B, respectively. Furthermore, Br-A2 and Br-A3 from wheat and Btr1/Btr2 on chromosome 3H of barley (Hordeum vulgare L.) also were homoeologous suggesting that the location of major determinants of the brittle rachis trait in these species has been conserved. On the other hand, brittle rachis loci of wheat and barley, and a shattering locus on rice chromosome 1 did not appear to be orthologous. Linkage and deletion-based bin mapping comparisons suggested that Br-A2 and Br-A3 may reside in chromosomal areas where the estimated frequency of recombination was ~ 4.3 Mb/cM. These estimates indicated that the cloning of Br-A2 and Br-A3 using map-based methods would be extremely challenging.     

Genome‐wide SNP identification in Fraxinus linking genetic characteristics to tolerance of Agrilus planipennis

Ash (Fraxinus spp.) is one of the most widely distributed tree genera in North America. Populations of ash in the United States and Canada have been decimated by the introduced pest Agrilus planipennis (Coleoptera: Buprestidae; emerald ash borer), having negative impacts on both forest ecosystems and economic interests. The majority of trees succumb to attack by A. planipennis, but some trees have been found to be tolerant to infestation despite years of exposure. Restriction site‐associated DNA (RAD) sequencing was used to sequence ash individuals, both tolerant and susceptible to A. planipennis attack, in order to identify single nucleotide polymorphism (SNP) patterns related to tolerance and health declines. de novo SNPs were called using SAMtools and, after filtering criteria were implemented, a set of 17,807 SNPs were generated. Principal component analysis (PCA) of SNPs aligned individual trees into clusters related to geography; however, five tolerant trees clustered together despite geographic location. A subset of 32 outlier SNPs identified within this group, as well as a subset of 17 SNPs identified based on vigor rating, are potential candidates for the selection of host tolerance. Understanding the mechanisms of host tolerance through genome‐wide association has the potential to restore populations with cultivars that are able to withstand A. planipennis infestation. This study was successful in using RAD‐sequencing in order to identify SNPs that could contribute to tolerance of A. planipennis. This was a first step toward uncovering the genetic basis for host tolerance to A. planipennis. Future studies are needed to identify the functionality of the loci where these SNPs occur and how they may be related to tolerance of A. planipennis attack.     

Natural underlying mtDNA heteroplasmy as a potential source of intra‐person hiPSC variability

Functional variability among human clones of induced pluripotent stem cells (hiPSCs) remains a limitation in assembling high‐quality biorepositories. Beyond inter‐person variability, the root cause of intra‐person variability remains unknown. Mitochondria guide the required transition from oxidative to glycolytic metabolism in nuclear reprogramming. Moreover, mitochondria have their own genome (mitochondrial DNA [mtDNA]). Herein, we performed mtDNA next‐generation sequencing (NGS) on 84 hiPSC clones derived from a cohort of 19 individuals, including mitochondrial and non‐mitochondrial patients. The analysis of mtDNA variants showed that low levels of potentially pathogenic mutations in the original fibroblasts are revealed through nuclear reprogramming, generating mutant hiPSCs with a detrimental effect in their differentiated progeny. Specifically, hiPSC‐derived cardiomyocytes with expanded mtDNA mutations non‐related with any described human disease, showed impaired mitochondrial respiration, being a potential cause of intra‐person hiPSC variability. We propose mtDNA NGS as a new selection criterion to ensure hiPSC quality for drug discovery and regenerative medicine.     

A natural fusion of flavodiiron,rubredoxin, and rubredoxin oxidoreductase domains is a self-sufficient water-forming oxidase of Trichomonas vaginalis

Microaerophilic pathogens such as Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis have robust oxygen consumption systems to detoxify oxygen and maintain intracellular redox balance. This oxygen consumption results from H₂O-forming NADH oxidase (NOX) activity of two distinct flavin-containing systems: H₂O-forming NOXes and multicomponent flavodiiron proteins (FDPs). Neither system is membrane bound, and both recycle NADH into oxidized NAD⁺ while simultaneously removing O₂ from the local environment. However, little is known about the specific contributions of these systems in T. vaginalis. In this study, we use bioinformatics and biochemical analyses to show that T. vaginalis lacks a NOX–like enzyme and instead harbors three paralogous genes (FDPF1–3), each encoding a natural fusion product between the N-terminal FDP, central rubredoxin (Rb), and C-terminal NADH:Rb oxidoreductase domains. Unlike a “stand-alone” FDP that lacks Rb and oxidoreductase domains, this natural fusion protein with fully populated flavin redox centers directly accepts reducing equivalents of NADH to catalyze the four-electron reduction of oxygen to water within a single polypeptide with an extremely high turnover. Furthermore, using single-particle cryo-EM, we present structural insights into the spatial organization of the FDP core within this multidomain fusion protein. Together, these results contribute to our understanding of systems that allow protozoan parasites to maintain optimal redox balance and survive transient exposure to oxic conditions.     

High‐affinity cooperative Ca2+ binding by MICU1–MICU2 serves as an on–off switch for the uniporter

The mitochondrial calcium uniporter is a Ca²⁺‐activated Ca²⁺ channel that is essential for dynamic modulation of mitochondrial function in response to cellular Ca²⁺ signals. It is regulated by two paralogous EF‐hand proteins—MICU1 and MICU2, but the mechanism is unknown. Here, we demonstrate that both MICU1 and MICU2 are stabilized by Ca²⁺. We reconstitute the MICU1–MICU2 heterodimer and demonstrate that it binds Ca²⁺ cooperatively with high affinity. We discover that both MICU1 and MICU2 exhibit affinity for the mitochondria‐specific lipid cardiolipin. We determine the minimum Ca²⁺ concentration required for disinhibition of the uniporter in permeabilized cells and report a close match with the Ca²⁺‐binding affinity of MICU1–MICU2. We conclude that cooperative, high‐affinity interaction of the MICU1–MICU2 complex with Ca²⁺ serves as an on–off switch, leading to a tightly controlled channel, capable of responding directly to cytosolic Ca²⁺ signals.     

Discovery and evaluation of novel FAAH inhibitors in neuropathic pain model

Conceptual design and modification of urea moiety in chemotype PF-3845/04457845, the bench marking irreversible inhibitor of fatty acid amide hydrolase (FAAH), led to discovery of a novel nicotinamide-based lead 12a having reversible mechanism of action. Focused SAR around the pyridine heterocycle (Ar) in 12a (Tables 1 and 2) resulted into four shortlisted compounds, (?)-12a, (?)-12i, (?)-12l–m. The required (?)-enantiomers were obtained via diastereomeric resolution of a novel chiral dissymmetric intermediate 15. Based on comparative profile of FAAH potency, metabolic stability in liver microsome, liability of inhibiting major hCYP450 isoforms, rat PK, and brain penetration ability, two SAR optimized compounds, (?)-12l and (?)-12m, were selected for efficacy study in rat model of chemotherapy-induced peripheral neuropathy (CIPN). Both the compounds exhibited dose related antihyperalgesic effects, when treated with 3–30?mg/kg po for 7?days. The effects at 30?mg/kg are comparable to that of PF-04457845 (10?mg/kg) and Tramadol (40?mg/kg).     

Homologous protein subunits from Escherichia coli NADH:quinone oxidoreductase can functionally replace MrpA and MrpD in Bacillus subtilis

The complex I subunits NuoL, NuoM and NuoN are homologous to two proteins, MrpA and MrpD, from one particular class of Na+/H+ antiporters. In many bacteria MrpA and MrpD are encoded by an operon comprising 6-7 conserved genes. In complex I these protein subunits are prime candidates for harboring important parts of the proton pumping machinery. Deletion of either mrpA or mrpD from the Bacillus subtilis chromosome resulted in a Na+ and pH sensitive growth phenotype. The deletion strains could be complemented in trans by their respective Mrp protein, but expression of MrpA in the B. subtilis ΔmrpD strain and vice versa did not improve growth at pH 7.4. This corroborates that the two proteins have unique specific functions. Under the same conditions NuoL could rescue B. subtilis ΔmrpA, but improved the growth of B. subtilis ΔmrpD only slightly. NuoN could restore the wild type properties of B. subtilis ΔmrpD, but had no effect on the ΔmrpA strain. Expression of NuoM did not result in any growth improvement under these conditions. This reveals that the complex I subunits NuoL, NuoM and NuoN also demonstrate functional specializations. The simplest explanation that accounts for all previous and current observations is that the five homologous proteins are single ion transporters. Presumably, MrpA transports Na+ whereas MrpD transports H+ in opposite directions, resulting in antiporter activity. This hypothesis has implications for the complex I functional mechanism, suggesting that one Na+ channel, NuoL, and two H+ channels, NuoM and NuoN, are present.