Desmaninae (Talpidae, Mammalia) from the Pliocene of Tollo de Chiclana (Guadix Basin, Southern Spain): Considerations on the phylogeny of the genus Archaeodesmana

The fossil Desmaninae (water-moles) from the Pliocene continental deposits of Tollo de Chiclana (Guadix Basin, Southern Spain) are described. A new species, Archaeodesmana elvirae, is defined from the locality of Tollo de Chiclana-1 (upper Ruscinian). This species is characterized by relatively small canines and premolars (except the P4) and large P4 and molars, besides several morphological features. The presence of Archaeodesmana brailloni is reported from the locality of Tollo de Chiclana-1B (uppermost Ruscinian). A small sample assigned to the genus Archaeodesmana is described from the lower Villafranchian site of Tollo de Chiclana-3, which cannot be determined at the specific level. The phylogenetic relationships between the different species of Archaeodesmana are reconsidered in the light of the recent findings, which support the idea of a more complex phylogeny than previously proposed for this genus. The populations from the Guadix Basin, previously assigned to Dibolia dekkersi (= Archaeodesmana getica), are here considered to belong to a different (unnamed) species, which is the ancestor of A. elvirae. On the other hand, the new species A. elvirae is proposed as the ancestor of A. brailloni.     

Two de novo mutations of MFN2 associated with early-onset Charcot-Marie-Tooth disease type 2A neuropathy

Charcot-Marie-Tooth disease type 2A (CMT2A) is one of the subdivisions of CMT2, an axonal defective form of peripheral neuropathy. Different mutations in the mitochondrial GTPase mitofusin 2 (MFN2) gene produce various degrees of severity of CMT2A phenotype or CMT2A related hereditary motor and sensory neuropathy VI (HMSN VI). The occurrence of de novo mutations in MFN2 is by far the most frequent as compared to other CMT genes. About 26% of the pathogenic MFN2 mutations reported in the Inherited Peripheral Neuropathies Mutations Database are de novo. This study identified two de novo mutations of MFN2, c.1048T>C (S350P) and c.310C>T (R104W), from two Korean CMT2A patients with early onset severe clinical symptoms. The comparative genotype-phenotype correlations of these mutations according to a previously reported case were also viewed. The R104W mutation has been reported recurrently, outspread over different ethnic backgrounds as de novo. The re-occurrence of the same pathogenic de novo variants within and amongst different ethnic groups clearly suggests a susceptible hot spot for mutation in the MFN2 gene. If the deleterious mutations discourage fitness and reproduction, this negative selection factor should ultimately reduce the prevalence of the disease. It appears that spontaneous de novo mutations in turn seem to be maintaining the disease phenotype??s prevalence.     

Identification of genes required for de novo DNA methylation in Arabidopsis

De novo DNA methylation in Arabidopsis thaliana is catalyzed by the methyltransferase DRM2, a homolog of the mammalian de novo methyltransferase DNMT3. DRM2 is targeted to DNA by small interfering RNAs (siRNAs) in a process known as RNA-directed DNA Methylation (RdDM). While several components of the RdDM pathway are known, a functional understanding of the underlying mechanism is far from complete. We employed both forward and reverse genetic approaches to identify factors involved in de novo methylation. We utilized the FWA transgene, which is methylated and silenced when transformed into wild-type plants, but unmethylated and expressed when transformed into de novo methylation mutants. Expression of FWA is marked by a late-flowering phenotype, which is easily scored in mutant versus wild-type plants. By reverse genetics we discovered the requirement for known RdDM effectors AGO6 and NRPE5a for efficient de novo methylation. A forward genetic approach uncovered alleles of several components of the RdDM pathway, including alleles of clsy1, ktf1 and nrpd/e2, which have not been previously shown to be required for the initial establishment of DNA methylation. Mutations were mapped and genes cloned by both traditional and whole genome sequencing approaches. The methodologies and the mutant alleles discovered will be instrumental in further studies of de novo DNA methylation.Key words: DNA methylation, Arabidopsis, de novo, genetic screen, whole-genome sequencing     

Viral Proteins Originated De Novo by Overprinting Can Be Identified by Codon Usage: Application to the “Gene Nursery” of Deltaretroviruses

A well-known mechanism through which new protein-coding genes originate is by modification of pre-existing genes, e.g. by duplication or horizontal transfer. In contrast, many viruses generate protein-coding genes de novo, via the overprinting of a new reading frame onto an existing (“ancestral”) frame. This mechanism is thought to play an important role in viral pathogenicity, but has been poorly explored, perhaps because identifying the de novo frames is very challenging. Therefore, a new approach to detect them was needed. We assembled a reference set of overlapping genes for which we could reliably determine the ancestral frames, and found that their codon usage was significantly closer to that of the rest of the viral genome than the codon usage of de novo frames. Based on this observation, we designed a method that allowed the identification of de novo frames based on their codon usage with a very good specificity, but intermediate sensitivity. Using our method, we predicted that the Rex gene of deltaretroviruses has originated de novo by overprinting the Tax gene. Intriguingly, several genes in the same genomic region have also originated de novo and encode proteins that regulate the functions of Tax. Such “gene nurseries” may be common in viral genomes. Finally, our results confirm that the genomic GC content is not the only determinant of codon usage in viruses and suggest that a constraint linked to translation must influence codon usage.     

Taxonomic notes on fruit flies of tribes Schistopterini and Eutretini (Diptera: Tephritidae: Tephritinae) in India,with description of a new species of Rhabdochaeta de Meijere

Rhabdochaeta nigroapicalis David, Hancock and Sachin, sp. n., is described from Assam, NE India. A key to all species of Schistopterini and Eutretini recorded from India is provided and taxonomic notes on previously described species are included. DNA barcode sequences of Calloptera asteria (Schiner), Rhabdochaeta pulchella de Meijere, Rhabdochaeta nigroapicalis David, Hancock and Sachin, sp. n. and Rhochmopterum venustum (de Meijere) were obtained and reported. Phylogenetic analysis using 62 mtCOI sequences of Tephritinae revealed Schistopterini to be a monophyletic group and the new species closely related to Rhabdochaeta pulchella, justifying its placement in Rhabdochaeta de Meijere.http://zoobank.org/A97337B3-E2BD-460D-BA62-6D3D4A1A60A2     

Cephalosporium serrae,agente etiologico de micetomas

Cephalosporium serrae is isolated from white granules of a mycetoma of right feet. The histological study of the grains with H.E. P.A.S. and Groccot is reported. The fungus is classified asCephalosporium serrae by its morphological and biological characteristic. Its sensitivity to antifungal drugs and the experimental inoculation to mice is reported. ElCephalosporium serrae fué aislado por primera vez en 1929 por Maffei en Italia de una queratomicosis. En 1932 Focosi comunica dos nuevos aislamientos de lesiones oculares, Coutelen, Cochet & Biguet (10). El trabajo que a continuación se presenta constituye el primer aislamiento delCephalosporium serrae como agente etiológico de un micetoma.     

DNMT3L Modulates Significant and Distinct Flanking Sequence Preference for DNA Methylation by DNMT3A and DNMT3B In Vivo

The DNTM3A and DNMT3B de novo DNA methyltransferases (DNMTs) are responsible for setting genomic DNA methylation patterns, a key layer of epigenetic information. Here, using an in vivo episomal methylation assay and extensive bisulfite methylation sequencing, we show that human DNMT3A and DNMT3B possess significant and distinct flanking sequence preferences for target CpG sites. Selection for high or low efficiency sites is mediated by the base composition at the −2 and +2 positions flanking the CpG site for DNMT3A, and at the −1 and +1 positions for DNMT3B. This intrinsic preference reproducibly leads to the formation of specific de novo methylation patterns characterized by up to 34-fold variations in the efficiency of DNA methylation at individual sites. Furthermore, analysis of the distribution of signature methylation hotspot and coldspot motifs suggests that DNMT flanking sequence preference has contributed to shaping the composition of CpG islands in the human genome. Our results also show that the DNMT3L stimulatory factor modulates the formation of de novo methylation patterns in two ways. First, DNMT3L selectively focuses the DNA methylation machinery on properly chromatinized DNA templates. Second, DNMT3L attenuates the impact of the intrinsic DNMT flanking sequence preference by providing a much greater boost to the methylation of poorly methylated sites, thus promoting the formation of broader and more uniform methylation patterns. This study offers insights into the manner by which DNA methylation patterns are deposited and reveals a new level of interplay between members of the de novo DNMT family.     

Regulation of Glutamine Synthetase by Light and during Nitrogen Deficiency in Synchronous Chlorella sorokiniana

A method is described to achieve density labeling of proteins in unicellular algae by using ¹³CO₂. This is a satisfactory procedure especially for work on nitrogen metabolism. The increase in activity of glutamine synthetase (EC 6.3.1.2.) and glutamate synthase (EC 1.4.7.1.) in Chlorella sorokiniana mediated by a dark/light shift and by nitrogen starvation were investigated. Using the method of density labeling and isopycnic centrifugation, we demonstrated that the increase in enzyme activity after a dark/light shift is based on activation rather than de novo synthesis. The increase in enzyme activity after transfer to nitrogen-deficient medium is based both on activation and de novo synthesis.     

Flanking sequence preference modulates de novo DNA methylation in the mouse genome

Mammalian de novo DNA methyltransferases (DNMT) are responsible for the establishment of cell-type-specific DNA methylation in healthy and diseased tissues. Through genome-wide analysis of de novo methylation activity in murine stem cells we uncover that DNMT3A prefers to methylate CpGs followed by cytosines or thymines, while DNMT3B predominantly methylates CpGs followed by guanines or adenines. These signatures are further observed at non-CpG sites, resembling methylation context observed in specialised cell types, including neurons and oocytes. We further show that these preferences result from structural differences in the catalytic domains of the two de novo DNMTs and are not a consequence of differential recruitment to the genome. Molecular dynamics simulations suggest that, in case of human DNMT3A, the preference is due to favourable polar interactions between the flexible Arg836 side chain and the guanine that base-pairs with the cytosine following the CpG. By exchanging arginine to a lysine, the corresponding side chain in DNMT3B, the sequence preference is reversed, confirming the requirement for arginine at this position. This context-dependent enzymatic activity provides additional insights into the complex regulation of DNA methylation patterns.     

Glucose-dependent de Novo Lipogenesis in B Lymphocytes: A REQUIREMENT FOR ATP-CITRATE LYASE IN LIPOPOLYSACCHARIDE-INDUCED DIFFERENTIATION*

Bacterially derived lipopolysaccharide (LPS) stimulates naive B lymphocytes to differentiate into immunoglobulin (Ig)-secreting plasma cells. Differentiation of B lymphocytes is characterized by a proliferative phase followed by expansion of the intracellular membrane secretory network to support Ig production. A key question in lymphocyte biology is how naive B cells reprogram metabolism to support de novo lipogenesis necessary for proliferation and expansion of the endomembrane network in response to LPS. We report that extracellularly acquired glucose is metabolized, in part, to support de novo lipogenesis in response to LPS stimulation of splenic B lymphocytes. LPS stimulation leads to increased levels of endogenous ATP-citrate lyase (ACLY), and this is accompanied by increased ACLY enzymatic activity. ACLY produces cytosolic acetyl-CoA from mitochondrially derived citrate. Inhibition of ACLY activity in LPS-stimulated B cells with the selective inhibitor 2-hydroxy-N-arylbenzenesulfonamide (compound-9; C-9) blocks glucose incorporation into de novo lipid biosynthesis, including cholesterol, free fatty acids, and neutral and acidic phospholipids. Moreover, inhibition of ACLY activity in splenic B cells results in inhibition of proliferation and defective endomembrane expansion and reduced expression of CD138 and Blimp-1, markers for plasma-like B cell differentiation. ACLY activity is also required for LPS-induced IgM production in CH12 B lymphoma cells. These data demonstrate that ACLY mediates glucose-dependent de novo lipogenesis in response to LPS signaling and identify a role for ACLY in several phenotypic changes that define plasma cell differentiation.     

Deuterium labeling for investigating de novo synthesis of terpene volatiles in Achyranthes bidentata

Purpose of work

Plants synthesize and accumulate secondary metabolites as defensive volatiles against diverse stresses. We aim to unravel the jasmonate-inducible volatile de novo synthetic metabolites in plants using a deuterium-labeling technique. Jasmonic acid and its methyl ester (MeJA) are well-documented for inducing defensive volatiles. Here, we have developed an efficient deuterium oxide (D₂O)-based labeling approach to determine the extent of de novo synthetic metabolites in a model plant A. bidentata bidentata. The labeling approach was demonstrated on quantitative profiling of terpene volatile organic compounds (VOCs) elicited by airborne MeJA in Achyranthes plants. We show, for the first time that airborne MeJA-elicited terpene VOCs are predominantly and differentially de novo synthesized except for a homoterpene, (3E)-4,8-dimethyl-1,3,7-nonatriene, which is weakly and least labelled with deuterium. D₂O is therefore an efficient labeling source for investigating de novo synthetic metabolites of terpene VOCs in planta.     

alpha-linolenic acid biosynthesis in Cyanidium caldarium.

Although α-linolenic acid is nearly absent from Cyanidium caldarium cultured at 53 °C, it is the most abundant unsaturated fatty acid in 20 °C-grown cells. A sudden growth temperature shift of 55 to 25 °C does not stimulate the immediate biosynthesis of α-linolenic acid. However, after an induction period of 48 h, synthesis of α-linolenic acid from acetate can be detected, and the fatty acid accumulates in phosphatidyl choline and sulfolipid. The newly synthesized α-linolenic acid appears to be formed primarily by de novo synthesis and to a much lesser extent from the elongation of a previously formed hexadecatrienoic acid precursor. On the other hand, when a cell-free algal preparation was presented with a hexadecatrienoic acid precursor in the presence of [¹⁴C] malonyl-CoA, the α-linolenic acid formed demonstrated a synthesis by elongation of the precursor. While the cell appears enzymatically capable of α-linolenic acid biosynthesis by both the de novo and elongation processes, de novo synthesis of α-linolenic acid appears to be the more significant mode of synthesis.     

An ensemble strategy that significantly improves de novo assembly of microbial genomes from metagenomic next-generation sequencing data

Next-generation sequencing (NGS) approaches rapidly produce millions to billions of short reads, which allow pathogen detection and discovery in human clinical, animal and environmental samples. A major limitation of sequence homology-based identification for highly divergent microorganisms is the short length of reads generated by most highly parallel sequencing technologies. Short reads require a high level of sequence similarities to annotated genes to confidently predict gene function or homology. Such recognition of highly divergent homologues can be improved by reference-free (de novo) assembly of short overlapping sequence reads into larger contigs. We describe an ensemble strategy that integrates the sequential use of various de Bruijn graph and overlap-layout-consensus assemblers with a novel partitioned sub-assembly approach. We also proposed new quality metrics that are suitable for evaluating metagenome de novo assembly. We demonstrate that this new ensemble strategy tested using in silico spike-in, clinical and environmental NGS datasets achieved significantly better contigs than current approaches.     

Évaluation de l'action prédatrice deStethorus madecassus [ColéoptèreCoccinellidae] surTetranychus neocaledonicus [AcarienTetranychidae]

Afin de préciser l'action prédatrice deStethorus madecassus surTetranychus neocaledonicus, la consommation des différents stades du prédateur a été étudiée dans une enceinte à température constante. A 25°, la consommation totale est de 494 ?ufs de tétranyque pour l'ensemble des 4 stades larvaires. A cette température, elle est de 46,8 femelles de tétranyque par jour pour une femelle fécondée deStethorus, 21,3 pour un mâle, et 13,1 pour une femelle vierge. Des essais complémentaires en conditions ambiantes naturelles ont permis de s'assurer de la validité de ces résultats. Les tests à 20° et 30° montrent que, dans cet intervalle, la consommation des adultes double sensiblement pour une élévation de température de 5°, et qu'une forte corrélation positive existe entre la voracité et la fécondité individuelles des femelles.     

An investigation of the "Ancyloplanorbidae" (Gastropoda, Pulmonata, Hygrophila): preliminary evidence from DNA sequence data

The Planorbidae is the largest family of freshwater pulmonate snails, yet an understanding of their intrafamily phylogenetic relationships is lacking and existing inferences are tentative. Moreover, it has been suggested that the Ancylidae, limpet-like freshwater pulmonates, should be merged with Planorbidae according to analysis of internal organ morphology. The present study explicitly tests this hypothesis by phylogenetic inference from partial DNA sequences of three molecular markers, nuclear ribosomal small subunit 18S and the mitochondrial cytochrome oxidase, and large subunit 16S. A molecular phylogeny was inferred based upon 22 taxa representing 12 ancylid and planorbid genera; additional taxa were included from the authors' database and from available sequences from GenBank, to further explore this basic data set. Taxa from Acroloxidae, Lymnaeidae, and Physidae were used as outgroups. Ancylidae and Planorbidae were found to be paraphyletic, with Planorbidae including some members of Ancylidae. "Ancyloplanorbidae" was also found to be paraphyletic because Acroloxus (Acroloxidae) surprisingly was included. Burnupia was found to be ancestral to "Ancyloplanorbidae" (including Acroloxus). The following clades of Planorbidae were supported: Bulininae and Planorbinae, Biomphalarini (including Helisoma and Planorbarius), and Planorbini and Segmentini.     

Activation of the de novo pathway for pyridine nucleotide biosynthesis prior to ricinine biosynthesis in castor beans

The ricinine content of etiolated seedlings of Ricinus communis increased nearly 12-fold over a 4-day period. In plants quinolinic acid is an intermediate in the de novo pathway for the synthesis of pyridine nucleotides. The only known enzyme in the de novo pathway for pyridine nucleotide biosynthesis, quinolinic acid phosphoribosyltransferase, increased 6-fold in activity over a 4-day period which preceded the onset of ricinine biosynthesis by 1 day. The activity of the remainder of the pyridine nucleotide cycle enzymes in the seedlings, as monitored by the specific activity of nicotinic acid phosphoribosyltransferase and nicotinamide deamidase, was similar to that found in the mature green plant. In the roots of Nicotiana rustica, where the pyridine alkaloid nicotine is synthesized, the level of quinolinic acid phosphoribosyltransferase was 38-fold higher than the level of nicotinic acid phosphoribosyltransferase, whereas in most other plants examined, the specific activity of quinolinic acid phosphoribosyltransferase was similar to the level of activity of enzymes in the pyridine nucleotide cycle itself. A positive correlation therefore exists between the specific activity of a de novo pathway enzyme catalyzing pyridine nucleotide biosynthesis in Ricinus communis and Nicotiana rustica and the biosynthesis of ricinine and nicotine, respectively.     

Extension of a de novo TIM barrel with a rationally designed secondary structure element

The ability to construct novel enzymes is a major aim in de novo protein design. A popular enzyme fold for design attempts is the TIM barrel. This fold is a common topology for enzymes and can harbor many diverse reactions. The recent de novo design of a four‐fold symmetric TIM barrel provides a well understood minimal scaffold for potential enzyme designs. Here we explore opportunities to extend and diversify this scaffold by adding a short de novo helix on top of the barrel. Due to the size of the protein, we developed a design pipeline based on computational ab initio folding that solves a less complex sub‐problem focused around the helix and its vicinity and adapt it to the entire protein. We provide biochemical characterization and a high‐resolution X‐ray structure for one variant and compare it to our design model. The successful extension of this robust TIM‐barrel scaffold opens opportunities to diversify it towards more pocket like arrangements and as such can be considered a building block for future design of binding or catalytic sites.