NOXA Is Important for Verticillium dahliae’s Penetration Ability and Virulence

NADPH oxidase (Nox) genes are responsible for Reactive Oxygen Species (ROS) production in living organisms such as plants, animals, and fungi, where ROS exert different functions. ROS are critical for sexual development and cellular differentiation in fungi. In previous publications, two genes encoding thioredoxin and NADH-ubiquinone oxidoreductase involved in maintaining ROS balance were shown to be remarkably induced in a highly versus a weakly aggressive Verticillium dahliae isolate. This suggested a role of these genes in the virulence of this pathogen. NoxA (NADPH oxidase A) was identified in the V. dahliae genome. We compared in vitro expression of NoxA in highly and weakly aggressive isolates of V. dahliae after elicitation with extracts from different potato tissues. NoxA expression was induced more in the weakly than highly aggressive isolate in response to leaf and stem extracts. After inoculation of potato detached leaves with these two V. dahliae isolates, NoxA was drastically up-regulated in the highly versus the weakly aggressive isolate. We generated single gene disruption mutants for NoxA genes. noxa mutants had significantly reduced virulence, indicating important roles in V. dahliae pathogenesis on the potato. This is consistent with a significant reduction of cellophane penetration ability of the mutants compared to the wild type. However, the cell wall integrity was not impaired in the noxa mutants when compared with the wild type. The resistance of noxa mutants to oxidative stress were also similar to the wild type. Complementation of noxa mutants with a full length NoxA clones restored penetration and pathogenic ability of the fungus. Our data showed that NoxA is essential for both penetration peg formation and virulence in V. dahliae.

Hypothetical protein gene 1038 contributes to colistin resistance in Aeromonas hydrophila

Inhibition of vital respiratory enzymes, such as NADH: ubiquinone oxidoreductase (complex I), type II NADH-quinone oxidoreductases (NDH-2), and malate: quinone oxidoreductase, in the inner membrane, is a secondary antibacterial mechanism of colistin (1–3). However, colistin resistance mechanisms associated with this secondary mode of action of colistin have rarely been reported. Herein, we confirmed that the hypothetical protein gene 1038 was associated with colistin resistance in Aeromonas hydrophila by reducing antibiotic function in the inner membrane, providing novel knowledge on the generation of colistin resistance.

The Role of Mitochondria in Carcinogenesis

The mitochondria are essential for normal cell functioning. Changes in mitochondrial DNA (mtDNA) may affect the occurrence of some chronic diseases and cancer. This process is complex and not entirely understood. The assignment to a particular mitochondrial haplogroup may be a factor that either contributes to cancer development or reduces its likelihood. Mutations in mtDNA occurring via an increase in reactive oxygen species may favour the occurrence of further changes both in mitochondrial and nuclear DNA. Mitochondrial DNA mutations in postmitotic cells are not inherited, but may play a role both in initiation and progression of cancer. One of the first discovered polymorphisms associated with cancer was in the gene NADH-ubiquinone oxidoreductase chain 3 (mt-ND3) and it was typical of haplogroup N. In prostate cancer, these mutations and polymorphisms involve a gene encoding subunit I of respiratory complex IV cytochrome c oxidase subunit 1 gene (COI). At present, a growing number of studies also address the impact of mtDNA polymorphisms on prognosis in cancer patients. Some of the mitochondrial DNA polymorphisms occur in both chronic disease and cancer, for instance polymorphism G5913A characteristic of prostate cancer and hypertension.

Ubiquinone Binding and Reduction by Complex I—Open Questions and Mechanistic Implications

NADH: ubiquinone oxidoreductase (complex I) is the first enzyme complex of the respiratory chain. Complex I is a redox-driven proton pump that contributes to the proton motive force that drives ATP synthase. The structure of complex I has been analyzed by x-ray crystallography and electron cryo-microscopy and is now well-described. The ubiquinone (Q) reduction site of complex I is buried in the peripheral arm and a tunnel-like structure is thought to provide access for the hydrophobic substrate from the membrane. Several intermediate binding positions for Q in the tunnel were identified in molecular simulations. Structural data showed the binding of native Q molecules and short chain analogs and inhibitors in the access pathway and in the Q reduction site, respectively. We here review the current knowledge on the interaction of complex I with Q and discuss recent hypothetical models for the coupling mechanism.

Gene Expression of Putative Pathogenicity-Related Genes in Verticillium dahliae in Response to Elicitation with Potato Extracts and during Infection Using Quantitative Real-Time PCR

Quantitative real-time PCR was used to monitor the expression of 15 Verticillium dahliae’s genes, putatively involved in pathogenicity, highly (HAV) and weakly aggressive (WAV) V. dahliae isolates after either (i) elicitation with potato leaf, stem, or root extracts, or (ii) inoculation of potato detached petioles. These genes, i.e., coding for Ras-GAP-like protein, serine/threonine protein kinase, Ubiquitin-conjugating enzyme variant-MMS2, NADH-ubiquinone oxidoreductase, Thioredoxin, Pyruvate dehydrogenase E1 VdPDHB, myo-inositol 2-dehydrogenase, and HAD-superfamily hydrolase, showed differential upregulation in the HAV versus WAV isolate in response to plant extracts or after inoculation of potato leaf petioles. This suggests their potential involvement in the observed differential aggressiveness between isolates. However, other genes like glucan endo-1,3-alpha-glucosidase and nuc-1 negative regulatory protein VdPREG showed higher activity in the WAV than in the HAV in response to potato extracts and/or during infection. This, in contrast, may suggest a role in their lower aggressiveness. These findings, along with future functional analysis of selected genes, will contribute to improving our understanding of V. dahliae’s pathogenesis. For example, expression of VdPREG negatively regulates phosphorus-acquisition enzymes, which may indicate a lower phosphorus acquisition activity in the WAV. Therefore, integrating the knowledge about the activity of both genes enhancing pathogenicity and those restraining it will provide a guild line for further functional characterization of the most critical genes, thus driving new ideas towards better Verticillium wilt management.

Translational Proteomics for Transfusion Medicine: Resolution of the IVIG Proteomes of Different Geographically Sourced and Prepared IVIG Immunotherapies

Intravenous Immunoglobulin’s (IVIG’s) are prepared from thousands of donor plasmas and as a result comprise an extreme broad mix and depth of Antibody (Ab) specificities. IVIG formulations available in Australia are from both local and overseas donor sources and extracted using a variety of purification methods and immunoglobulin purities, with the Australia-derived and prepared IVIG listed at >98% and the overseas-derived preparations at ≥ 95%. Because of these differences it was predicted that the formulations might individually vary in composition and that together with obvious genetic and geographic antigenic (Ag) environment differences may result in notable variability between formulations. Hence a focussed comparative proteomic profiling of IVIG formulations was undertaken to identify notable similarities and differences across products. Comparisons between formulations did reveal marked qualitative differences in 2D-gel Antibody profiling that included parameters of isoelectric charge (pI), as well as immunoglobulin (Ig) monomer to dimer ratio variability between products, including high molecular weight (MW) immunoglobulin multimers for some. These notable differences were in part quite likely a product of the respective purification methods used, and capacity to select (or de-select) for antibodies of such different properties. Furthermore, for identification of non-Ig proteins carried over from plasma through purifications Mass spectrometry was performed. This identified a few such ancillary proteins, and their identities, in general, differed between formulations. Proteins detected included the most abundant protein of plasma, albumin, as well as other mostly large and abundant proteins; RAG1 - V(D)J recombination activating protein1, gelsolin, complement Factor-B, serotransferrin, tetranectin, NADH ubiquinone oxidoreductase, caspase 3 and VEGFR1. An alternate strategy used commercial Multiplex xMAP assay to detect cytokines, which are small and present in plasma at trace but highly active quantities. This revealed various different cytokine profiles across the formulations studied. The identification of additional proteins, and especially cytokines in IVIGs, is particularly notable, and the positive, negative or null biological relevance for clinical use, needs resolution. Collectively these findings reveal marked differences between Australian and overseas-derived (non-Australian) IVIGs in immunoglobulin composition and biochemical characteristics, and presence of additional carry-over proteins from plasma. These findings prompt the need for further evaluation of the micro-compositions of individual formulations. Perhaps detailed mining and improved comparative understanding of each IVIG formulation, may enable highly tailored and strategic clinical use of certain formulations that are personalised best-fit treatments for particular conditions. Such as in treatment of a neuropathy, as compared to another formulation, more suited for treating a particular infectious disease. The most salient and overarching study conclusion is need for caution in attributing equivalence across IVIGs.

Adapting CRISPR/Cas9 System for Targeting Mitochondrial Genome

Gene editing of the mitochondrial genome using the CRISPR-Cas9 system is highly challenging mainly due to sub-efficient delivery of guide RNA and Cas9 enzyme complexes into the mitochondria. In this study, we were able to perform gene editing in the mitochondrial DNA by appending an NADH-ubiquinone oxidoreductase chain 4 (ND4) targeting guide RNA to an RNA transport-derived stem loop element (RP-loop) and expressing the Cas9 enzyme with a preceding mitochondrial localization sequence. We observe mitochondrial colocalization of RP-loop gRNA and a marked reduction of ND4 expression in the cells carrying a 11205G variant in their ND4 sequence coincidently decreasing the mtDNA levels. This proof-of-concept study suggests that a stem-loop element added sgRNA can be transported to the mitochondria and functionally interact with Cas9 to mediate sequence-specific mtDNA cleavage. Using this novel approach to target the mtDNA, our results provide further evidence that CRISPR-Cas9-mediated gene editing might potentially be used to treat mitochondrial-related diseases.

Shared evolutionary footprints suggest mitochondrial oxidative damage underlies multiple complex I losses in fungi

Oxidative phosphorylation is among the most conserved mitochondrial pathways. However, one of the cornerstones of this pathway, the multi-protein complex NADH : ubiquinone oxidoreductase (complex I) has been lost multiple independent times in diverse eukaryotic lineages. The causes and consequences of these convergent losses remain poorly understood. Here, we used a comparative genomics approach to reconstruct evolutionary paths leading to complex I loss and infer possible evolutionary scenarios. By mining available mitochondrial and nuclear genomes, we identified eight independent events of mitochondrial complex I loss across eukaryotes, of which six occurred in fungal lineages. We focused on three recent loss events that affect closely related fungal species, and inferred genomic changes convergently associated with complex I loss. Based on these results, we predict novel complex I functional partners and relate the loss of complex I with the presence of increased mitochondrial antioxidants, higher fermentative capabilities, duplications of alternative dehydrogenases, loss of alternative oxidases and adaptation to antifungal compounds. To explain these findings, we hypothesize that a combination of previously acquired compensatory mechanisms and exposure to environmental triggers of oxidative stress (such as hypoxia and/or toxic chemicals) induced complex I loss in fungi.

A new species of geckos of the genus Cyrtodactylus Gray, 1827 from Arunachal Pradesh, India

We here describe a new species of bent-toed geckos from the northeastern Indian state of Arunachal Pradesh, which is widespread across the Dafla and Mishmi hills, occurring at elevations ranging from 179 m to 1400 m. The new species is recovered as sister to the Cyrtodactylus khasiensis clade based on a molecular phylogeny inferred from mitochondrial NADH-ubiquinone oxidoreductase, subunit 2 gene. Intraspecific uncorrected pairwise sequence divergence (p-distance) for the new species was found to be between 0 and 5%, whereas the interspecific divergence from the closely-related congeners was between 19 and 30%. The new species can be differentiated from members of the C. khasiensis clade using a suite of morphological characters: moderate body size (SVL 64.9–81.7); 8–11 supralabials; 8–10 infralabials; 24–26 bluntly conical, feebly keeled dorsal tubercles; 50–60 paravertebral tubercles; ~38 ventral scales between ventrolateral folds; no precloacal groves; 6–10 precloacofemoral pores in a continuous series; 10–16 distal subdigital lamellae on IV of pes; subcaudal scalation of original tail without enlarged plates. This is the fourth reptile species described from Arunachal Pradesh from the expedition led by the team, and this further highlights the need for further herpetological investigations into the region.