Previous cell cycle studies have been based on cell-nuclear proliferation only. Eukaryotic cells, however, have double membranes-bound organelles, such as the cell nucleus, mitochondrion, plastids and single-membrane-bound organelles such as ER, the Golgi body, vacuoles (lysosomes) and microbodies. Organelle proliferations, which are very important for cell functions, are poorly understood. To clarify this, we performed a microarray analysis during the cell cycle of Cyanidioschyzon merolae. C. merolae cells contain a minimum set of organelles that divide synchronously. The nuclear, mitochondrial and plastid genomes were completely sequenced. The results showed that, of 158 genes induced during the S or G2-M phase, 93 were known and contained genes related to mitochondrial division, ftsZ1-1, ftsz1-2 and mda1, and plastid division, ftsZ2-1, ftsZ2-2 and cmdnm2. Moreover, three genes, involved in vesicle trafficking between the single-membrane organelles such as vps29 and the Rab family protein, were identified and might be related to partitioning of single-membrane-bound organelles. In other genes, 46 were hypothetical and 19 were hypothetical conserved. The possibility of finding novel organelle division genes from hypothetical and hypothetical conserved genes in the S and G2-M expression groups is discussed.

Most chloroplast and mitochondrial proteins are encoded by nuclear genes, whose functions remain largely unknown because mutant alleles are lacking. A reverse genetics screen for mutations affecting the mitochondrial transcription termination factor (mTERF) family in Arabidopsis thaliana allowed us to identify 75 lines carrying T-DNA insertions. Two of them were homozygous for insertions in the At4g14605 gene, which we dubbed MDA1 (MTERF DEFECTIVE IN Arabidopsis1). The mda1 mutants exhibited altered chloroplast morphology and plant growth, and reduced pigmentation of cotyledons, leaves, stems and sepals. The mda1 mutations enhanced salt and osmotic stress tolerance and altered sugar responses during seedling establishment, possibly as a result of reduced ABA sensitivity. Loss of MDA1 function caused up-regulation of the RpoTp/SCA3 nuclear gene encoding a plastid RNA polymerase and modified the steady-state levels of chloroplast gene transcripts. Double mutant analyses indicated that MDA1 and the previously described mTERF genes SOLDAT10 and RUG2 act in different pathways. Our findings reveal a new role for mTERF proteins in the response to abiotic stress, probably through perturbed ABA retrograde signalling resulting from a disruption in chloroplast homeostasis.

We have characterized a nuclear mutation, mda1-ncc1, that affects mRNA stability for the atpA gene cluster in the chloroplast of Chlamydomonas. Unlike all nuclear mutations altering chloroplast gene expression described to date, mda1-ncc1 is a dominant mutation that still allows accumulation of detectable amounts of atpA mRNAs. At variance with the subset of these mutations that affect mRNA stability through the 5' UTR of a single chloroplast transcript, the mutated version of MDA1 acts on the coding region of the atpA message. We discuss the action of MDA1 in relation to the unusual pattern of expression of atpA that associates particularly short lived-transcripts with a very high translational efficiency.

Mitochondria are not produced de novo but are maintained by division. Mitochondrial division is a coordinated process of positioning and constriction of the division site and fission of double membranes, in which dynamin-related protein is believed to mediate outer membrane fission. Part of the mitochondrial division machinery was purified from M phase-arrested Cyanidioschyzon merolae cells through biochemical fractionation. The dynamin-related protein Dnm1 was one of the two major proteins in the purified fraction and was accompanied by a newly identified protein CMR185C, named Mda1. Mda1 contained a predictable coiled-coil region and WD40 repeats, similarly to Mdv1 and Caf4 in yeasts. Immunofluorescence and immunoelectron microscopy showed that Mda1 localizes as a medial belt or ring on the mitochondrial outer surface throughout the division. The ring formation of Mda1 followed the plane of the ring of FtsZ, a protein that resides in the matrix. Dnm1 consistently colocalized with Mda1 only in the late stages of division. Mda1 protein was expressed through S to M phases and was phosphorylated specifically in M phase when Mda1 transformed from belt into foci and became colocalizing with Dnm1. Dephosphorylation of Mda1 in vitro increased its sedimentation coefficient, suggesting conformational changes of the macromolecule. Disassembly of the purified mitochondrial division machinery was performed by adding GTP to independently release Dnm1, suggesting that Mda1 forms a stable homo-oligomer by itself as a core structure of the mitochondrial division machinery.

The control of organelle gene expression in plants is far from fully understood. The characterization of mutants in Arabidopsis thaliana is assigning an increasingly prominent role to the mitochondrial transcription termination factors (mTERFs) in this process. To gain insight into the function of mTERF genes in plants, we took a reverse genetics approach to identify and characterize A. thaliana mTERF-defective mutants. Here we report the characterization of the mterf9 mutant, affected in an mTERF protein functionally conserved in plants and targeted to chloroplasts. Loss of MTERF9 results in defective chloroplast development, which is likely to cause paleness, stunted growth and reduced mesophyll cell numbers. Expression analysis of different plastid genes revealed reduced levels of plastid-encoded polymerase (PEP)-dependent transcripts and increased levels of transcripts dependent of nucleus-encoded polymerase. mterf9 plants exhibited altered responses to sugars, abscisic acid (ABA), salt and osmotic stresses, and the microarray data analysis showed modifications in MTERF9 expression after salt or mannitol treatments. Our genetic interactions results indicate a functional relationship between MTERF9 and the previously characterized MDA1 gene, and between MDA1 and some plastid ribosomal genes. MDA1 and MTERF9 were upregulated in the mterf9 and mda1 mutants, respectively. Moreover, 21 of 50 genes were commonly co-expressed with MDA1 and MTERF9. The analysis of the MDA1 and MTERF9 promoters showed that both were rich in stress-related cis-regulatory elements. Our results highlight the role of the MTERF9 gene in plant biology and deepens the understanding of the functional relationship of plant mTERF genes.

The expression of chloroplast and mitochondrial genes depends on nucleus-encoded proteins, some of which control processing, stability, and/or translation of organellar RNAs. To test the specificity of one such RNA stability factor, we used two known Chlamydomonas reinhardtii nonphotosynthetic mutants carrying mutations in the Mcd1 nuclear gene (mcd1-1 and mcd1-2). We previously reported that these mutants fail to accumulate the chloroplast petD mRNA and its product, subunit IV of the cytochrome b6/f complex, which is essential for photosynthesis. Such mutants are generally presumed to be gene specific but are not tested rigorously. Here, we have used microarray analysis to assess changes in chloroplast, mitochondrial, and nuclear RNAs, and since few other RNAs were significantly altered in these mutants, conclude that Mcd1 is indeed specifically required for petD mRNA accumulation. In addition, a new unlinked nuclear mutation was discovered in mcd1-2, which greatly reduced chloroplast atpA mRNA accumulation. Genetic analyses showed failure to complement mda1-ncc1, where atpA-containing transcripts are similarly affected (D. Drapier, J. Girard-Bascou, D.B. Stern, F.-A. Wollman [2002] Plant J 31: 687-697), and we have named this putative new allele mda1-2. We conclude that DNA microarrays are efficient and useful for characterizing the specificity of organellar RNA accumulation mutants.

Taenia solium taeniasis-cysticercosis and soil-transmitted helminths (STHs) are parasitic Neglected Tropical Diseases endemic throughout Southeast Asia. Within Lao PDR, a remote northern hill tribe village had previously been identified as a hyper endemic focus for T. solium. To reduce this observed prevalence, a One Health intervention covering both pigs and humans was implemented, which included two Mass drug administrations (MDA1 and MDA2) for village residents using a triple dose albendazole 400mg treatment regime. In addition to the effect on T. solium levels, the dual impact of this anthelmintic regime on STHs within the community was also monitored. Faecal samples were collected pre and post MDA1 and MDA2 and analysed for the presence of Taenia species and the STHs Ascaris lumbricoides, Trichuris trichiura and hookworm species. The McMaster technique was used to measure the changes in both prevalence and intensity of infection. Molecular characterisation of Taenia and hookworm species was conducted to detect zoonotic species. The level of taeniasis within the sampled population decreased by 79.4% after MDA1, remained steady during the five month inter-treatment interval and decreased again by 100% after MDA2. The prevalence of STHs decreased by 65.5% and 62.8% after MDA1 and MDA2 respectively; however an increase to 62.1% of pre MDA1 levels was detected during the inter-treatment interval. Individually, hookworm prevalence decreased by 83.4% (MDA1) and 84.5% (MDA2), A. lumbricoides by 95.6% and 93.5% and T. trichiura by 69.2% and 61%. The intensity of infection within the sampled population also decreased, with egg reduction rates of 94.4% and 97.8% for hookworm, 99.4% and 99.3% for A. lumbricoides and 77.2% and 88.5% for T. trichiura. Molecular characterisation identified a T. solium tapeworm carrier from 21.6% (13/60) of households in the village. T. saginata was identified in 5% (3/60) of households. The zoonotic hookworm A. ceylanicum was detected in the resident dog population. These results suggest that the triple dose albendazole 400mg treatment regime achieved a significant reduction in the level of taeniasis whilst simultaneously reducing the STH burden within the village. The increased STH prevalence detected between MDAs reflects the need for behavioural changes and a sustained chemotherapy programme, which may also need to include the resident dog population.

The mTERF gene family encodes for nucleic acid binding proteins that are predicted to regulate organellar gene expression in eukaryotes. Despite the implication of this gene family in plant development and response to abiotic stresses, a precise molecular function was assigned to only a handful number of its ~30 members in plants. Using a reverse genetics approach in Arabidopsis thaliana and combining molecular and biochemical techniques, we revealed new functions for the chloroplast mTERF protein, MDA1. We demonstrated that MDA1 associates in vivo with components of the plastid-encoded RNA polymerase and transcriptional active chromosome complexes. MDA1 protein binds in vivo and in vitro with specificity to 27-bp DNA sequences near the 5'-end of psbE and ndhA chloroplast genes to stimulate their transcription and additionally promote the stabilization of the 5'-ends of processed psbE and ndhA mRNAs. Finally, we provided evidence that MDA1 function in gene transcription likely coordinates RNA folding and the action of chloroplast RNA binding proteins on mRNA stabilization. Our results provide examples for the unexpected implication of DNA binding proteins and gene transcription in the regulation of mRNA stability in chloroplasts blurring the boundaries between DNA and RNA metabolism in this organelle.

Present-day chloroplast and mitochondrial genomes contain only a few dozen genes involved in ATP synthesis, photosynthesis, and gene expression. The proteins encoded by these genes are only a small fraction of the many hundreds of proteins that act in chloroplasts and mitochondria. Hence, the vast majority, including components of organellar gene expression (OGE) machineries, are encoded by nuclear genes, translated into the cytosol and imported to these organelles. Consequently, the expression of nuclear and organellar genomes has to be very precisely coordinated. Furthermore, OGE regulation is crucial to chloroplast and mitochondria biogenesis, and hence, to plant growth and development. Notwithstanding, the molecular mechanisms governing OGE are still poorly understood. Recent results have revealed the increasing importance of nuclear-encoded modular proteins capable of binding nucleic acids and regulating OGE. Mitochondrial transcription termination factor (mTERF) proteins are a good example of this category of OGE regulators. Plant mTERFs are located in chloroplasts and/or mitochondria, and have been characterized mainly from the isolation and analyses of Arabidopsis and maize mutants. These studies have revealed their fundamental roles in different plant development aspects and responses to abiotic stress. Fourteen mTERFs have been hitherto characterized in land plants, albeit to a different extent. These numbers are limited if we consider that 31 and 35 mTERFs have been, respectively, identified in maize and Arabidopsis. Notwithstanding, remarkable progress has been made in recent years to elucidate the molecular mechanisms by which mTERFs regulate OGE. Consequently, it has been experimentally demonstrated that plant mTERFs are required for the transcription termination of chloroplast genes (mTERF6 and mTERF8), transcriptional pausing and the stabilization of chloroplast transcripts (MDA1/mTERF5), intron splicing in chloroplasts (BSM/RUG2/mTERF4 and Zm-mTERF4) and mitochondria (mTERF15 and ZmSMK3) and very recently, also in the assembly of chloroplast ribosomes and translation (mTERF9). This review aims to provide a detailed update of current knowledge about the molecular functions of plant mTERF proteins. It principally focuses on new research that has made an outstanding contribution to unravel the molecular mechanisms by which plant mTERFs regulate the expression of chloroplast and mitochondrial genomes.

Keywords: Arabidopsis; chloroplast; maize; mitochondria; mitochondrial transcription termination factor; organellar gene expression.

Malondialdehyde (MDA), a product of lipid peroxidation and biomarker of oxidative stress, is measured over the long term in spruce Picea abies needles under real conditions in three Czech mountain border areas. The trends presented collate the MDA content in spruce needles with ambient ozone, temperature and precipitation as casual, and defoliation as a subsequent factor for the period 1994-2006. We have found the overall decreasing trends in MDA and defoliation. The highest MDA and defoliation are recorded in the Jizerske, the lowest in the Krusne hory Mts. Out of the examined variables the MDA is predicted best by mean temperature in vegetation season, median of O(3) concentrations and AOT40; these three variables account for 34% of MDA1 and 36% of MDA2 variability. Our hypothesis that higher ambient O(3) exposure results in higher MDA contents in P. abies needles under real conditions has not been approved.

We previously showed that Arabidopsis mda1 and mterf9 mutants, defective in the chloroplast-targeted mitochondrial transcription termination factors mTERF5 and mTERF9, respectively, display altered responses to abiotic stresses and the abscisic acid (ABA) hormone, as well as perturbed development, likely due to abnormal chloroplast biogenesis. To advance in the functional analysis of mTERF5 and mTERF9, we obtained and characterized overexpression (OE) lines. Additionally, we studied genetic interactions between sca3-2, affected in the plastid-RNA polymerase RpoTp, and the mda1-1 and mterf9 mutations. We also investigated mTERF5 and mTERF9 role in plastid translation and plastid-to-nucleus signalling. We found that mTERF9 OE reduces salt and ABA tolerance, while mTERF5 or mTERF9 OE alters the expression of nuclear and plastid genes. We determined that mda1-1 and mterf9 mutations genetically interact with sca3-2. Further, plastid 16S rRNA levels were reduced in mda1-1 and mterf9 mutants and mterf9 was more sensitive to chemical inhibitors of chloroplast translation. The expression of the photosynthesis gene LHCB1, a retrograde signalling marker, was differentially affected in mda1-1 and/or mterf9 compared to Col-0, after treatments with inhibitors of carotenoid biosynthesis (norflurazon) or chloroplast translation (lincomycin). Moreover, mterf9, but not mda1-1, synergistically interacts with gun1-1, defective in GUN1 a central integrator of plastid retrograde signals. Our results show that mTERF9, and to a lesser extent mTERF5, are negative regulators of salt tolerance and both genes are functionally related with RpoTp, and mTERF9 is likely required for plastid ribosomal stability and/or assembly. Furthermore, our findings support a role for mTERF9 in retrograde signalling.

In Chlamydomonas reinhardtii, chloroplast gene expression is tightly regulated post-transcriptionally by gene-specific trans-acting protein factors. Here, we report the molecular identification of an OctotricoPeptide Repeat (OPR) protein, MDA1, which governs the maturation and accumulation of the atpA transcript, encoding subunit α of the chloroplast ATP synthase. As does TDA1, another OPR protein required for the translation of the atpA mRNA, MDA1 targets the atpA 5'-untranslated region (UTR). Unexpectedly, it binds within a region of approximately 100 nt in the middle of the atpA 5'-UTR, at variance with the stabilization factors characterized so far, which bind to the 5'-end of their target mRNA to protect it from 5' → 3' exonucleases. It binds the same region as TDA1, with which it forms a high-molecular-weight complex that also comprises the atpA mRNA. This complex dissociates upon translation, promoting degradation of the atpA mRNA. We suggest that atpA transcripts, once translated, enter the degradation pathway because they cannot reassemble with MDA1 and TDA1, which preferentially bind to de novo transcribed mRNAs.

Oxidative stress is associated with cardiometabolic traits in observational studies, yet the underlying causal relationship remains unclear. Apolipoprotein E-deficient (Apoe^-/-) mice develop significant hyperlipidemia and hyperglycemia on a Western diet. Here we conducted linkage analysis to investigate genetic connections between cardiometabolic traits and oxidative stress.

-/- mice and fed 12 weeks of Western diet. Plasma levels of HDL, LDL cholesterol, triglycerides, glucose and malondialdehyde (MDA) and atherosclerosis in aortic root and left carotid artery were measured. 127 microsatellite markers across the genome were genotyped.

One significant locus at 78.3 cM on chromosome (Chr) 1 (LOD score: 3.85), named Mda1, and two suggestive loci near 60.3 cM on Chr1 (LOD score: 2.32, named Mda2 due to replication in a separate cross) and 19.6 cM on Chr4 (LOD score: 2.34) were identified for MDA levels. Mda1 coincided precisely with loci for LDL, triglyceride, glucose, and body weight and overlapped with a locus for atherosclerosis in the aortic root. Plasma LDL, triglyceride, and glucose explained 25.5, 19.2, and 24.2% of the variation in MDA levels of F2 mice, respectively. After correction for triglyceride or LDL, QTLs for MDA on Chr1 and Chr4 disappeared. QTLs on Chr1 disappeared, remained on Chr4, and additional QTLs on Chr12 and Chr13 were detected after correction for glucose. The QTL on Chr12, named Mda3, had a significant LOD score of 8.034 and peaked 62.22 at cM.We demonstrated a causative role for cardiometabolic traits in oxidative stress and identified hyperlipidemia and hyperglycemia as a major driver of oxidative stress.

Numerous studies have indicated that the oxidative modification of low-density lipoprotein (LDL) plays a critical role in the pathogenesis of atherosclerosis. Malondialdehyde-modified LDL (MDA-LDL) is one of the candidate oxidative products. Therefore, to allow the assessment of oxidized LDL in human serum, we developed monoclonal antibodies for MDA-LDL. Two of these-MDA1 and MDA2-bound to oxidized LDL but not to native LDL by Western blot analysis. The murine monoclonal antibodies to oxidized LDL have potential clinical implications, as imaging agents, for defining the compositions of atherosclerotic vessels in vivo.

To enhance our understanding of the roles of mitochondrial transcription termination factors (mTERFs) in plants, we have taken a reverse genetic approach in Arabidopsis thaliana. One of the mutants isolated carried a novel allele of the mTERF6 gene, which we named mterf6-5. mTERF6 is a chloroplast and mitochondrial localised protein required for the maturation of chloroplast isoleucine tRNA. The mterf6-5 plants are pale and exhibit markedly reduced growth, and altered leaf and chloroplast development. Our qRT-PCR analyses revealed mis-expression of several plastid, mitochondrial and nuclear genes in mterf6-5 plants. Synergistic phenotypes were observed in double mutant combinations of mterf6-5 with alleles of other mTERF genes as well as with scabra3-2, affected in the plastid RpoTp RNA polymerase; these observations suggest a functional relationship between mTERF6, other mTERFs and SCA3. The mterf6-5 mutation also enhanced the leaf dorsoventral polarity defects of the asymmetric leaves1-1 (as1-1) mutant, which resulted in radial leaves. This interaction seemed specific of the impaired mTERF6 function because mutations in the mTERF genes MDA1 or TWR-1/mTERF9 did not result in radialised leaves. Furthermore, the mterf6-5 mutation dramatically increased the leaf phenotype of as2-1 and caused lethality early in vegetative development. Our results uncover a new role for mTERF6 in leaf patterning and highlight the importance of mTERFs in plant development.

As the most widely-used single cell whole genome amplification (WGA) approach, multiple displacement amplification (MDA) has a superior performance, due to the high-fidelity and processivity of phi29 DNA polymerase. However, chimeric reads, generated in MDA, cause severe disruption in many single-cell studies. Herein, we constructed ChimeraMiner, an improved chimeric read detection pipeline for analyzing the sequencing data of MDA and classified the chimeric sequences. Two datasets (MDA1 and MDA2) were used for evaluating and comparing the efficiency of ChimeraMiner and previous pipeline. Under the same hardware condition, ChimeraMiner spent only 43.4% (43.8% for MDA1 and 43.0% for MDA2) processing time. Respectively, 24.4 million (6.31%) read pairs out of 773 million reads, and 17.5 million (6.62%) read pairs out of 528 million reads were accurately classified as chimeras by ChimeraMiner. In addition to finding 83.60% (17,639,371) chimeras, which were detected by previous pipelines, ChimeraMiner screened 6,736,168 novel chimeras, most of which were missed by the previous pipeline. Applying in single-cell datasets, all three types of chimera were discovered in each dataset, which introduced plenty of false positives in structural variation (SV) detection. The identification and filtration of chimeras by ChimeraMiner removed most of the false positive SVs (83.8%). ChimeraMiner revealed improved efficiency in discovering chimeric reads, and is promising to be widely used in single-cell sequencing.

Lipid peroxidation is a free radical-mediated process. Malonaldehyde (MDA) is one of the products of reduction in this process. Selenium (Se) is a physiological free radical scavenger. In this paper the research on serum selenium (Se), serum MDA (MDAp) and MDA in low density lipoprotein (MDA1) in 52 men (age 38-52 years) will be presented. Plasma total cholesterol (T-chol), free cholesterol (F-chol), esterified cholesterol (E-chol) and triglyceride (Tg) concentrations were estimated also. It was found a significant negative correlation between MDA1 and T-chol (r = -0.4140, p = 0.0023), E-chol (r = -0.3947, p = 0.0038), F-chol (r = 0.4104, p = 0025). The patients were divided according to T-chol level (group I: T-chol greater than 5.2 mmol/l, group II: T-chol less than 5.2 mmol/l). Statistically significant negative correlation were found between: Se and MDAp in both groups (I: r = -0.7734, p = 0.0002, II: r = -0.6695, p = 0.0012), Se and MDA1 only in II group (r = -0.4693, p = 00368). There was no apparent relation between MDAp and T-chol, E-chol, F-chol and Tg.