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.     

Emerging role of roots in plant responses to aboveground insect herbivory

Plants have evolved complex biochemical mechanisms to counter threats from insect herbivory. Recent research has revealed an important role of roots in plant responses to above ground herbivory (AGH). The involvement of roots is integral to plant resistance and tolerance mechanisms. Roots not only play an active role in plant defenses by acting as sites for biosynthesis of various toxins and but also contribute to tolerance by storing photoassimilates to enable future regrowth. The interaction of roots with beneficial soil‐borne microorganisms also influences the outcome of the interaction between plant and insect herbivores. Shoot‐to‐root communication signals are critical for plant response to AGH. A better understanding of the role of roots in plant response to AGH is essential in order to develop a comprehensive picture of plant‐insect interactions. Here, we summarize the current status of research on the role of roots in plant response to AGH and also discuss possible signals involved in shoot‐to‐root communication.     

GeneOrder3.0: Software for comparing the order of genes in pairs of small bacterial genomes

Background  

An increasing number of whole viral and bacterial genomes are being sequenced and deposited in public databases. In parallel to the mounting interest in whole genomes, the number of whole genome analyses software tools is also increasing. GeneOrder was originally developed to provide an analysis of genes between two genomes, allowing visualization of gene order and synteny comparisons of any small genomes. It was originally developed for comparing virus, mitochondrion and chloroplast genomes. This is now extended to small bacterial genomes of sizes less than 2 Mb.     

Mastering seeds for genomic size nucleotide BLAST searches

One of the most common activities in bioinformatics is the search for similar sequences. These searches are usually carried out with the help of programs from the NCBI BLAST family. As the majority of searches are routinely performed with default parameters, a question that should be addressed is how reliable the results obtained using the default parameter values are, i.e. what fraction of potential matches have been retrieved by these searches. Our primary focus is on the initial hit parameter, also known as the seed or word, used by the NCBI BLASTn, MegaBLAST and other similar programs in searches for similar nucleotide sequences. We show that the use of default values for the initial hit parameter can have a big negative impact on the proportion of potentially similar sequences that are retrieved. We also show how the hit probability of different seeds varies with the minimum length and similarity of sequences desired to be retrieved and describe methods that help in determining appropriate seeds. The experimental results described in this paper illustrate situations in which these methods are most applicable and also show the relationship between the various BLAST parameters.     

MRD: a microsatellite repeats database for prokaryotic and eukaryotic genomes

MRD is a database system to access the microsatellite repeats information of genomes such as archea, eubacteria, and other eukaryotic genomes whose sequence information is available in public domains. MRD stores information about simple tandemly repeated k-mer sequences where k= 1 to 6, i.e. monomer to hexamer. The web interface allows the users to search for the repeat of their interest and to know about the association of the repeat with genes and genomic regions in the specific organism. The data contains the abundance and distribution of microsatellites in the coding and non-coding regions of the genome. The exact location of repeats with respect to genomic regions of interest (such as UTR, exon, intron or intergenic regions) whichever is applicable to organism is highlighted. MRD is available on the World Wide Web at and/or . The database is designed as an open-ended system to accommodate the microsatellite repeats information of other genomes whose complete sequences will be available in future through public domain.     

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).     

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.     

Meclizine Inhibits Mitochondrial Respiration through Direct Targeting of Cytosolic Phosphoethanolamine Metabolism

We recently identified meclizine, an over-the-counter drug, as an inhibitor of mitochondrial respiration. Curiously, meclizine blunted respiration in intact cells but not in isolated mitochondria, suggesting an unorthodox mechanism. Using a metabolic profiling approach, we now show that treatment with meclizine leads to a sharp elevation of cellular phosphoethanolamine, an intermediate in the ethanolamine branch of the Kennedy pathway of phosphatidylethanolamine biosynthesis. Metabolic labeling and in vitro enzyme assays confirmed direct inhibition of the cytosolic enzyme CTP:phosphoethanolamine cytidylyltransferase (PCYT2). Inhibition of PCYT2 by meclizine led to rapid accumulation of its substrate, phosphoethanolamine, which is itself an inhibitor of mitochondrial respiration. Our work identifies the first pharmacologic inhibitor of the Kennedy pathway, demonstrates that its biosynthetic intermediate is an endogenous inhibitor of respiration, and provides key mechanistic insights that may facilitate repurposing meclizine for disorders of energy metabolism.     

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.