Intraspecies Levels Research Articles

Conserved A-to-I RNA editing with non-conserved recoding expands the candidates of functional editing sites

ABSTRACT Adenosine-to-inosine (A-to-I) RNA editing recodes the genome and confers flexibility for the organisms to adapt to the environment. It is believed that RNA recoding sites are well suited for facilitating adaptive evolution by increasing the proteomic diversity in a temporal-spatial manner. The function and essentiality of a few conserved recoding sites are recognized. However, the experimentally discovered functional sites only make up a small corner of the total sites, and there is still the need to expand the repertoire of such functional sites with bioinformatic approaches. In this study, we define a new category of RNA editing sites termed ‘conserved editing with non-conserved recoding’ and systematically identify such sites in Drosophila editomes, figuring out their selection pressure and signals of adaptation at inter-species and intra-species levels. Surprisingly, conserved editing sites with non-conserved recoding are not suppressed and are even slightly overrepresented in Drosophila. DNA mutations leading to such cases are also favoured during evolution, suggesting that the function of those recoding events in different species might be diverged, specialized, and maintained. Finally, structural prediction suggests that such recoding in potassium channel Shab might increase ion permeability and compensate the effect of low temperature. In conclusion, conserved editing with non-conserved recoding might be functional as well. Our study provides novel aspects in considering the adaptive evolution of RNA editing sites and meanwhile expands the candidates of functional recoding sites for future validation.

Agrobacteria deploy two classes of His-Me finger superfamily nuclease effectors exerting different antibacterial capacities against specific bacterial competitors.

The type VI secretion system (T6SS) assembles into a contractile nanomachine to inject effectors across bacterial membranes for secretion. The Agrobacterium tumefaciens species complex is a group of soil inhabitants and phytopathogens that deploys T6SS as an antibacterial weapon against bacterial competitors at both inter-species and intra-species levels. The A. tumefaciens strain 1D1609 genome encodes one main T6SS gene cluster and four vrgG genes (i.e., vgrGa-d), each encoding a spike protein as an effector carrier. A previous study reported that vgrGa-associated gene 2, named v2a, encodes a His-Me finger nuclease toxin (also named HNH/ENDO VII nuclease), contributing to DNase-mediated antibacterial activity. However, the functions and roles of other putative effectors remain unknown. In this study, we identified vgrGc-associated gene 2 (v2c) that encodes another His-Me finger nuclease but with a distinct Serine Histidine Histidine (SHH) motif that differs from the AHH motif of V2a. We demonstrated that the ectopic expression of V2c caused growth inhibition, plasmid DNA degradation, and cell elongation in Escherichia coli using DNAse activity assay and fluorescence microscopy. The cognate immunity protein, V3c, neutralizes the DNase activity and rescues the phenotypes of growth inhibition and cell elongation. Ectopic expression of V2c DNase-inactive variants retains the cell elongation phenotype, while V2a induces cell elongation in a DNase-mediated manner. We also showed that the amino acids of conserved SHH and HNH motifs are responsible for the V2c DNase activity in vivo and in vitro. Notably, V2c also mediated the DNA degradation and cell elongation of the target cell in the context of interbacterial competition. Importantly, V2a and V2c exhibit different capacities against different bacterial species and function synergistically to exert stronger antibacterial activity against the soft rot phytopathogen, Dickeya dadantii.

The evolution of mini-chromosomes in the fungal genus Colletotrichum.

Anthracnose diseases caused by Colletotrichum species are among the most common fungal diseases. These symptoms typically manifest as dark, sunken lesions on leaves, stems, and fruit. In China, mango anthracnose seriously affects fruit yield and quality. Genome sequencing of several species shows the presence of mini-chromosomes. These are thought to contribute to virulence, but their formation and activity remain to be fully elucidated. Here, we assembled 17 Colletotrichum genomes (16 isolated from mango plus one from persimmon) through PacBio long-read sequencing. Half of the assembled scaffolds had telomeric repeats at both ends indicating full-length chromosomes. Based on comparative genomics analysis at interspecies and intraspecies levels, we identified extensive chromosomal rearrangements events. We analyzed mini-chromosomes of Colletotrichum spp. and found large variation among close relatives. In C. fructicola, homology between core chromosomes and mini-chromosomes suggested that some mini-chromosomes were generated by recombination of core chromosomes. In C. musae GZ23-3, we found 26 horizontally transferred genes arranged in clusters on mini-chromosomes. In C. asianum FJ11-1, several potential pathogenesis-related genes on mini-chromosomes were upregulated, especially in strains with highly pathogenic phenotypes. Mutants of these upregulated genes showed obvious defects in virulence. Our findings provide insights into the evolution and potential relationships to virulence associated with mini-chromosomes. IMPORTANCE Colletotrichum is a cosmopolitan fungal genus that seriously affects fruit yield and quality of many plant species. Mini-chromosomes have been found to be related to virulence in Colletotrichum. Further examination of mini-chromosomes can help us elucidate some pathogenic mechanisms of Colletotrichum. In this study, we generated novel assemblies of several Colletotrichum strains. Comparative genomic analyses within and between Colletotrichum species were conducted. We then identified mini-chromosomes in our sequenced strains systematically. The characteristics and generation of mini-chromosomes were investigated. Transcriptome analysis and gene knockout revealed pathogenesis-related genes located on mini-chromosomes of C. asianum FJ11-1. This study represents the most comprehensive investigation of chromosome evolution and potential pathogenicity of mini-chromosomes in the Colletotrichum genus.

Bacterial communities along parrot digestive and respiratory tracts: the effects of sample type, species and time

Digestive and respiratory tracts are inhabited by rich bacterial communities that can vary between their different segments. In comparison with other bird taxa with developed caeca, parrots that lack caeca have relatively lower variability in intestinal morphology. Here, based on 16S rRNA metabarcoding, we describe variation in microbiota across different parts of parrot digestive and respiratory tracts both at interspecies and intraspecies levels. In domesticated budgerigar (Melopsittacus undulatus), we describe the bacterial variation across eight selected sections of respiratory and digestive tracts, and three non-destructively collected sample types (faeces, and cloacal and oral swabs). Our results show important microbiota divergence between the upper and lower digestive tract, but similarities between respiratory tract and crop, and also between different intestinal segments. Faecal samples appear to provide a better proxy for intestinal microbiota composition than the cloacal swabs. Oral swabs had a similar bacterial composition as the crop and trachea. For a subset of tissues, we confirmed the same pattern also in six different parrot species. Finally, using the faeces and oral swabs in budgerigars, we revealed high oral, but low faecal microbiota stability during a 3-week period mimicking pre-experiment acclimation. Our findings provide a basis essential for microbiota-related experimental planning and result generalisation in non-poultry birds.

Clasnip: a web-based intraspecies classifier and multi-locus sequence typing for pathogenic microorganisms using fragmented sequences.

Bioinformatic approaches for the identification of microorganisms have evolved rapidly, but existing methods are time-consuming, complicated or expensive for massive screening of pathogens and their non-pathogenic relatives. Also, bioinformatic classifiers usually lack automatically generated performance statistics for specific databases. To address this problem, we developed Clasnip (www.clasnip.com), an easy-to-use web-based platform for the classification and similarity evaluation of closely related microorganisms at interspecies and intraspecies levels. Clasnip mainly consists of two modules: database building and sample classification. In database building, labeled nucleotide sequences are mapped to a reference sequence, and then single nucleotide polymorphisms (SNPs) statistics are generated. A probability model of SNPs and classification groups is built using Hidden Markov Models and solved using the maximum likelihood method. Database performance is estimated using three replicates of two-fold cross-validation. Sensitivity (recall), specificity (selectivity), precision, accuracy and other metrics are computed for all samples, training sets, and test sets. In sample classification, Clasnip accepts inputs of genes, short fragments, contigs and even whole genomes. It can report classification probability and a multi-locus sequence typing table for SNPs. The classification performance was tested using short sequences of 16S, 16-23S and 50S rRNA regions for 12 haplotypes of Candidatus Liberibacter solanacearum (CLso), a regulated plant pathogen associated with severe disease in economically important Apiaceous and Solanaceous crops. The program was able to classify CLso samples with even only 1-2 SNPs available, and achieved 97.2%, 98.8% and 100.0% accuracy based on 16S, 16-23S, and 50S rRNA sequences, respectively. In comparison with all existing 12 haplotypes, we proposed that to be classified as a new haplotype, given samples have at least 2 SNPs in the combined region of 16S rRNA (OA2/Lsc2) and 16-23S IGS (Lp Frag 4-1611F/Lp Frag 4-480R) regions, and 2 SNPs in the 50S rplJ/rplL (CL514F/CL514R) regions. Besides, we have included the databases for differentiating Dickeya spp., Pectobacterium spp. and Clavibacter spp. In addition to bacteria, we also tested Clasnip performance on potato virus Y (PVY). 251 PVY genomes were 100% correctly classified into seven groups (PVYC, PVYN, PVYO, PVYNTN, PVYN:O, Poha, and Chile3). In conclusion, Clasnip is a statistically sound and user-friendly bioinformatic application for microorganism classification at the intraspecies level. Clasnip service is freely available at www.clasnip.com.

The anatomy of death.

Medical science does not treat death as an independent physiological phenomenon. It believes that disease is the cause of death and treats both as preventable phenomena. Doctors and relatives nurture a guilt complex when death occurs. The moment of natural death is robbed of its poignancy. There is no cause of death. Death of the physical body is an intrinsic, time governed, built-in ontolytic program. It completes the biologic trajectory of the individual organism which begins at conception and ends with death. In this article, the actual built-in mechanism and operation of the physiological process of death is suggested vis-a-vis biology and medicine. The physical body of an individual is like a biological gadget which is constantly charged with life force received from cosmic energy through the subtle body which is an implement that cannot be objectified in the same way as the gross physical body but is experienced only subjectively by one and all, as mind (antahkaran in Sanskrit and Indian languages, meaning inner instrument, a subtler instrument in contrast to external organs of actions like limbs, tongue, etc. of the physical body). This life force keeps the subtle body and the entire physical body live and functioning. It maintains the body's integrity and internal homeostasis throughout life. The physical body like a gadget has a built-in program which decides how much total capacity, pace and quantum of life force it can use. It is often referred to as breath quota and heartbeat quota, however, they are manifestations and not the mechanism and operation of life and death in the physical body. When the quantum of life force of the physical body is exhausted, the subtle body, a connector for life forces departs, leaving behind the physical body. The vital functions stop and the person is declared dead. It is usually referred to as departure of soul (Pran or Jiva in India) from the physical body in common parlance. This phenomenon of predetermined capacity, pace and fixed quota of life force is operational in all the components of the body. Each cell, each organ, each system and the whole body is a packed unit of bioenergy. They are in structural and functional dynamic homeostasis under constant flux resulting in constant change in the body. The rate of expenditure contributes towards deciding the total life span of the cell, organ, systems and individual. It accounts for the phenomena of cell death, progressive diminution in vitality, change in organ and system functions during life and physical death of an individual at the end. Death is a bio-cosmic phenomenon governed by biological laws. There is variation in life span at the interspecies and intraspecies levels because we measure lifespan of an individual in terms of physical time and not in terms of biological time which is an independent entity, not measurable and predictable as physical time. Everyone lives one full unit of life in terms of biological time which begins at conception and ends with death. The normal distribution of each biological feature, including the biological unit of life, is independent of one another making each individual unique. The biological clock runs at its own dynamic unique pace in every individual. The biological timer starts ticking at conception and stops when the life force quantum is over. When the person dies, the physical body stops functioning and is left behind for others to dispose of. The timing of death is beyond the realm of objective perception and cannot be equated with chronological or physical time. It is a trans-science and trans-technique phenomenon beyond the ken of humankind to alter its mechanism and operation. The article explains the impact of extrinsic and intrinsic diseases, various treatments such as organ transplant, as well as the impact of lifestyle on the biological time scale of an individual. It spells out the difference between natural and unnatural death. With this understanding, death can be appreciated, accepted and respected as a built-in operational intrinsic physiological phenomenon for the end of one's life. It is a must for the survival and healthy continuity of all the species in the biological world including mankind.

Genetic Diversity and Phylogenetic Position of Traditional Rice (Oryza sativa L.) Landraces: A Case Study of South Kalimantan in Indonesia

Traditional rice (Oryza sativa L.) landraces provide many essential genes for improving yield, disease resistance, abiotic stress tolerance, and other parameters for future rice breeding. This study aimed to analyze the genetic diversity and determine the phylogenetic position of the traditional rice landraces from the tidal swamp areas of South Kalimantan, Indonesia, compared to other rice germplasm, including wild relatives, obtained from the GenBank database, using a cpDNA-rbcL marker. In this case, six traditional rice landraces from this region were collected and analyzed molecularly using the rbcL marker and compared with 16 similar others and 25 wild relatives from the GenBank database. The genetic diversity of this germplasm was determined using the nucleotide diversity index (π), whereas the phylogenetic analysis by maximum likelihood with bootstrap for 1 000 replicates. The principal component analysis (PCA) was employed to confirm this grouping. Based on this marker, the traditional rice landraces have a genetic diversity of 0.38, lower than intra-species and inter-species levels, i.e., 0.44 and 0.83, respectively. The phylogenetic analysis shows that this germplasm has separated from most O. sativa rice cultivars and their wild relatives, except for the ‘GBVN’ and ‘NARC’ (comparison cultivars obtained from GenBank). This information has substantial implications for future rice breeding and conservation efforts, locally and globally.

Systemic Loxoscelism, Less Frequent but More Deadly: The Involvement of Phospholipases D in the Pathophysiology of Envenomation.

Bites of Loxosceles spiders can lead to a set of clinical manifestations called loxoscelism, and are considered a public health problem in many regions. The signs and symptoms of loxoscelism are divided into cutaneous and systemic forms. The former is more frequent and includes signs of envenoming at the bite site or neighboring regions. Systemic loxoscelism, although much less frequent, is associated with complications, and can even lead to death. It may include intravascular hemolysis, acute renal failure, and thrombocytopenia. Loxosceles venoms are enriched with phospholipases D (PLDs), which are a family of isoforms found at intra-species and inter-species levels. Under experimental conditions, these enzymes reproduce the main clinical signs of loxoscelism, including an exacerbated inflammatory response at the bite site and dermonecrosis, as well as thrombocytopenia, intravascular hemolysis, and acute renal failure. The role of PLDs in cutaneous loxoscelism was described over forty years ago, when studies identified and purified toxins featured as sphingomyelinase D. More recently, the production of recombinant PLDs and discoveries about their structure and mechanism has enabled a deeper characterization of these enzymes. In this review, we describe these biochemical and functional features of Loxosceles PLDs that determine their involvement in systemic loxoscelism.

Start Codon Targeted (SCoT) markers for the assessment of genetic diversity in yeast isolated from Turkish sourdough

Molecular markers are valuable tools for assessing the genetic variation in yeast. Here, we investigated the utility of SCoT markers for the genetic characterization of yeast strains at inter and intraspecies levels. A total of 345 endogenous yeast strains were isolated from 65 Type I sourdough samples collected from six different regions of Turkey. The seven SCoT primers produced 221 bands, of which 95.47% were polymorphic. Each primer could successfully differentiate species, supported by PIC and RP values. The ITS sequencing of isolates selected from the UPGMA dendrogram revealed that Saccharomyces cerevisiae predominated the microflora, followed by Kazachstania servazzii, K. humilis, Wickerhamomyces anomalus, Torulaspora delbrueckii, and Pichia kudriavzevii, respectively. The AMOVA revealed a high genetic variation between (49%) and within populations (51%) for S. cerevisiae. The high gene flow observed among S. cerevisiae populations suggests that it may have contributed to the geographical evolution of S. cerevisiae via the transportation of the sourdough samples. The different geographical origins were most likely to group separately on the UPGMA and PCoA. Saccharomyces cerevisiae strains from more distant populations generally displayed more significant genetic variation. SCoT markers can successfully be used alone or with the other existing DNA markers for DNA fingerprinting and analyzing the genetic variation between and within species.

ОЦІНКА ЕФЕКТИВНОСТІ ПОКАЗНИКІВ ІНФОРМАТИВНОСТІ ISSR-МАРКЕРІВ ДЛЯ АНАЛІЗУ ГЕНЕТИЧНОГО ПОЛІМОРФІЗМУ РОСЛИН

Introduction. A number of generations of different types of molecular genetic markers have changed to the present day.The DNA nucleotide sequence marker system allows testing genetic diversity at the gene level. The creation of molecular markers and their use in biological researches made it possible to study genetic polymorphism in more detail, as well as to investigate the level of kinship at the inter- and intra-species levels. The use of various DNA markers that are associated with different sequences of the genome is effective at the time of studying genetic polymorphism. At the same time, it is also important to reduce the cost of polymerase chain reaction analysis, which is associated with the use of a small number of primers, and to choose the most informative primers.Purpose. The purpose of the research is to select the most informative ISSR primers for assessing the genetic polymorphism of plant populations using the example of representatives of the Gentiana L. genus, as well as to determine the most effective indicators of the informativeness of DNA markers.Methods. Molecular genetic markers, statistical methods, calculation of indicators of informativeness of primers.Results. It was established that all the used indicators of the informativeness of the primers to a certain extent characterize the informativeness of the ISSR-markers, which are used for learning of the genetic polymorphism and genetic structure of G. lutea.Three ISSR primers (UBC#807, UBC#811 and UBC#840) with the highest indicators of discriminating(D) and resolving power (Rp) were selected by us. It was established that in general the value of the percentageof polymorphic amplicons was similar, and the values of the indicators of expected heterozygosity and the Shannon index were higher.This was established as a result of comparing the genetic polymorphism indices of the G. luteapopulations obtained using three ISSR primers (UBC#807, UBC#811 and UBC#840) with the genetic polymorphism indices estimated by nine ISSR primers (UBC #807, UBC#809, UBC#810, UBC#811, UBC#827, UBC#835, UBC#840, UBC#857, UBC#889). The increase in genetic diversity is probably caused by an increase in the polymorphic primers due to the removal from the sample of those that form monomorphic amplicons.Originality. For the first time, an effective indicator of the informativeness of primers was selected and the most effective ISSR markers were selected for the assessment of the genetic diversity of plants of the genus Gentiana L. on the example of seven populations of G. lutea from the Ukrainian Carpathians.Conclusions. The most informative primers were selected and their effectiveness was demonstrated for assessing the genetic diversity of a sample of plants from seven populations located in the mountain ranges of the Ukrainian Carpathians. In general, UBC#811 and UBC#807 were identified as the most informative, and UBC#827 and UBC#889 were the least effective in evaluating the genetic polymorphism of G. lutea. This conclusion was made by evaluating indicators(total number of amplicons, number of polymorphic amplicons, PIC (polymorphism information content), MI (marker index), Rp, D). The relationship between all the parameters of the evaluation of the effectiveness of the primers was characterized by a high level of reliability (р<0.001, р=0.05), only in some cases it was higher than 5%. The level of genetic polymorphism of G. lutea for UBC#807, UBC#811 and UBC#840 was evaluated.Key words: Gentianalutea L.; genetic diversity; resolving power; discriminating power, number of polymorphic amplicons

AHL-Lactonase Producing Psychrobacter sp. From Palk Bay Sediment Mitigates Quorum Sensing-Mediated Virulence Production in Gram Negative Bacterial Pathogens.

Quorum sensing (QS) is a signaling mechanism governed by bacteria used to converse at inter- and intra-species levels through small self-produced chemicals called N-acylhomoserine lactones (AHLs). Through QS, bacteria regulate and organize the virulence factors’ production, including biofilm formation. AHLs can be degraded by an action called quorum quenching (QQ) and hence QQ strategy can effectively be employed to combat biofilm-associated bacterial pathogenesis. The present study aimed to identify novel bacterial species with QQ potential. Screening of Palk Bay marine sediment bacteria for QQ activity ended up with the identification of marine bacterial isolate 28 (MSB-28), which exhibited a profound QQ activity against QS biomarker strain Chromobacterium violaceum ATCC 12472. The isolate MSB-28 was identified as Psychrobacter sp. through 16S-rRNA sequencing. Psychrobacter sp. also demonstrated a pronounced activity in controlling the biofilm formation in different bacteria and biofilm-associated virulence factors’ production in P. aeruginosa PAO1. Solvent extraction, heat inactivation, and proteinase K treatment assays clearly evidence the enzymatic nature of the bioactive lead. Furthermore, AHL’s lactone ring cleavage was confirmed with experiments including ring closure assay and chromatographic analysis, and thus the AHL-lactonase enzyme production in Psychrobacter sp. To conclude, this is the first report stating the AHL-lactonase mediated QQ activity from marine sediment bacteria Psychrobacter sp. Future work deals with the characterization, purification, and mass cultivation of the purified protein and should pave the way to assessing the feasibility of the identified protein in controlling QS and biofilm-mediated multidrug resistant bacterial infections in mono or multi-species conditions.

Otolith Shape Analysis and its Utilily for Identification of Two Smelt Species, Hypomesus japonicus and H. nipponensis (Osteichthyes, Osmeridae) from the Northwestern Sea of Japan with Inferences in Stock Discrimination of H. japonicus

In this study, otolith shape analysis has been used to perform identification of two smelt species specimens (genus Hypomesus), H. japonicus and H. nipponensis, as well as differentiation of local gatherings in the first species, in the north-western Sea of Japan (SJ). Based on the otolith shape and using several multidimension statistical approaches obvious differences were defined for both inter- and intraspecies levels of otolith variation. Species discrimination by otolith shape indices and other variables extends well to within the species H. japonicus gatherings, the individuals sampled from the northern sample in Olga Bay (OB) are most similar morphologically to those from the coastal waters off the village of Terney to the north of OB. In contrast, the individuals of this species from the coastal waters off Russky Island (RI), including another southern sample and those from northern samples, manifest the greatest differences, thus providing evidence on the existence of at least two local stocks of the species in the waters of the north-western Sea of Japan.

Evolution and Developmental System Drift in the Endoderm Gene Regulatory Network of Caenorhabditis and Other Nematodes.

Developmental gene regulatory networks (GRNs) underpin metazoan embryogenesis and have undergone substantial modification to generate the tremendous variety of animal forms present on Earth today. The nematode Caenorhabditis elegans has been a central model for advancing many important discoveries in fundamental mechanistic biology and, more recently, has provided a strong base from which to explore the evolutionary diversification of GRN architecture and developmental processes in other species. In this short review, we will focus on evolutionary diversification of the GRN for the most ancient of the embryonic germ layers, the endoderm. Early embryogenesis diverges considerably across the phylum Nematoda. Notably, while some species deploy regulative development, more derived species, such as C. elegans, exhibit largely mosaic modes of embryogenesis. Despite the relatively similar morphology of the nematode gut across species, widespread variation has been observed in the signaling inputs that initiate the endoderm GRN, an exemplar of developmental system drift (DSD). We will explore how genetic variation in the endoderm GRN helps to drive DSD at both inter- and intraspecies levels, thereby resulting in a robust developmental system. Comparative studies using divergent nematodes promise to unveil the genetic mechanisms controlling developmental plasticity and provide a paradigm for the principles governing evolutionary modification of an embryonic GRN.

Contribution of Functional Divergence Through Copy Number Variations to the Inter-Species and Intra-Species Diversity in Specialized Metabolites.

There is considerable diversity in the specialized metabolites within a single plant species (intra-species) and among different plant species (inter-species). The functional divergence associated with gene duplications largely contributes to the inter-species diversity in the specialized metabolites, whereas the intra-species diversity is due to gene dosage changes via gene duplications [i.e., copy number variants (CNVs)] at the intra-species level of evolution. This is because CNVs are thought to undergo associated with less functional divergence at the intra-species level of evolution. However, functional divergence caused by CNVs may induce specialized metabolite diversity at the intra-species and inter-species levels of evolution. We herein discuss the functional divergence of CNVs in metabolic quantitative trait genes (mQTGs). We focused on 5,654 previously identified mQTGs in 270 Arabidopsis thaliana accessions. The ratio of nonsynonymous to synonymous variations tends to be higher for mQTGs with CNVs than for mQTGs without CNVs within A. thaliana accessions, suggesting that CNVs are responsible for the functional divergence among mQTGs at the intra-species level of evolution. To evaluate the contribution of CNVs to inter-species diversity, we calculated the ratio of nonsynonymous to synonymous substitutions in the Arabidopsis lineage. The ratio tends to be higher for the mQTGs with CNVs than for the mQTGs without CNVs. Additionally, we determined that mQTGs with CNVs are subject to positive selection in the Arabidopsis lineage. Our data suggest that CNVs are closely related to functional divergence contributing to adaptations via the production of diverse specialized metabolites at the intra-species and inter-species levels of evolution.

Disease-only alleles at the extreme ends of the human ZMYM3 exceptionally long 5′ UTR short tandem repeat in bipolar disorder: A pilot study

ObjectiveThe X-linked ZMYM3 gene (also known as ZNF261) contains the longest STR, (GA)32, identified in a human protein-coding gene 5′UTR (ENST00000373998.5: ZMYM3-207). This STR reaches maximum length in human, and is located in a complex string of four consecutive GA-STRs with a human-specific formula across the complex. A previous study in Iranian male schizophrenia (SCZ) patients revealed co-occurrence of the extreme short and long alleles of the STR with SCZ. Here we studied the allelic distribution of this STR in bipolar disorder (BD) type I. The interval encompassing the human ZMYM3 STR complex was PCR-amplified and sequenced in 546 male subjects, consisting of 157 BD patients and 389 controls. ResultsWe found three alleles at the extreme short (17-repeat) and long (38- and 43-repeat) ends of the allele distribution curve in the BD cases (4.4% of the BD alleles) that were not detected in the controls (Mid p < 0.0001). These alleles overlapped with the extreme disease-only alleles detected previously in the SCZ patients. Domain reconstruction of the GA-STR complex revealed significant structural alteration as a result of various sequence repeats and nucleotide compositions at the inter and intraspecies levels. ConclusionThe ZMYM3 “exceptionally long” 5′ UTR STR findings may alter our perspective of disease pathogenesis in psychiatric disorders, and set an example in which the low frequency alleles at the extreme short and long ends of the human STRs are, at least in part, a result of natural selection against these alleles and their unambiguous link to major human disorders.

The occurrence of nitrate and nitrite in Mediterranean fresh salad vegetables and its modulation by preharvest practices and postharvest conditions

Winter and summer nitrate/nitrite concentrations in 11 salad vegetables were surveyed using a validated HPLC-DAD method. Nitrate was highest in rocket, both in winter (x̅ = 3974 mg kg−1 fw) and summer (x̅ = 3819 mg kg−1 fw). High nitrate accumulators included spinach, purslane, chards, dill, coriander and parsley. Wide intra-species variability and levels in excess of permitted maxima highlighted the importance of monitoring vegetable production methods to protect consumer health. Occurrence of detectible nitrite (14–352 mg kg−1 fw) was most frequent in winter head cabbage. Three additional experiments examined the seasonal effects of nitrogen (N) fertilization rate, application method, formulation and postharvest storage on nitrate and nitrite levels in lettuce, rocket and spinach. Violation of current nitrate limits is likely when total N exceeds 200 kg ha−1, particularly in rocket and spinach. Postharvest nitrate reduction requires exogenous microbial nitrate reductase activity, which is unlikely to be achieved without visible loss of quality.

A workflow of massive identification and application of intron markers using snakes as a model.

Relative to the commonly used mitochondrial and nuclear protein‐coding genes, the noncoding intron sequences are a promising source of informative markers that have the potential to resolve difficult phylogenetic nodes such as rapid radiations and recent divergences. Yet many issues exist in the use of intron markers, which prevent their extensive application as conventional markers. We used the diverse group of snakes as an example to try paving the way for massive identification and application of intron markers. We performed a series of bioinformatics screenings which identified appropriate introns between single‐copy and conserved exons from two snake genomes, adding particular constraints on sequence length variability and sequence variability. A total of 1,273 candidate intron loci were retrieved. Primers for nested polymerase chain reaction (PCR) were designed for over a hundred candidates and tested in 16 snake representatives. 96 intron markers were developed that could be amplified across a broad range of snake taxa with high PCR successful rates. The markers were then applied to 49 snake samples. The large number of amplicons was subjected to next‐generation sequencing (NGS). An analytic strategy was developed to accurately recover the amplicon sequences, and approximately, 76% of the marker sequences were recovered. The average p‐distances of the intron markers at interfamily, intergenus, interspecies, and intraspecies levels were .168, .052, .015, and .004, respectively, suggesting that they were useful to study snake relationships of different evolutionary depths. A snake phylogeny was constructed with the intron markers, which produced concordant results with robust support at both interfamily and intragenus levels. The intron markers provide a convenient way to explore the signals in the noncoding regions to address the controversies on the snake tree. Our improved strategy of genome screening is effective and can be applied to other animal groups. NGS coupled with appropriate sequence processing can greatly facilitate the extensive application of molecular markers.

Autoinducer-2 Quorum Sensing Influences Viability of Escherichia coli O157:H7 under Osmotic and In Vitro Gastrointestinal Stress Conditions.

Bacteria use autoinducer molecules to communicate both at intra-species and inter-species levels by quorum sensing. One such cell density-dependent signaling system is the luxS-mediated universal quorum sensing using autoinducer-2 (AI-2). Virulence of several pathogens is determined by an AI-2 system and is related to colonization and infection of the host. From this concept, numerous papers have suggested that AI-2 inhibition is an important strategy toward designing of new antimicrobial agents. However, recent studies indicate that the AI-2 system is also involved in adaptation and survival under environmental stress conditions. Therefore, we hypothesized that interaction between quorum sensing and environmental conditions may be critical in influencing predicted results in a control and when combating of target pathogens. We investigated the growth of enterohemorrhagic Escherichia coli O157:H7 (EHEC) and its luxS-deficient (non AI-2 producing) mutant strain under various stress conditions, and found significant differences in the growth rate under osmotic stress. Moreover, we could also show the impact of the AI-2 molecule on viability in the gastrointestinal tract model representing a complex environmental condition. Differences in vital responses of the strains suggest that AI-2 quorum sensing has a significant influence on the viability of EHEC under environmental stress conditions.

Leaf epidermal features of Quercus Group Ilex (Fagaceae) and their application to species identification

Leaf epidermal features are useful for elucidating the taxonomy and systematics of oak species. Quercus Group Ilex has a wide distribution mainly in subtropical areas of Eurasia with rich fossil records since the Oligocene, but no comprehensive study has ever been conducted on its diversity of leaf epidermal features. We compared the leaf epidermal features of 37 species of Quercus Group Ilex and Q. suber from Group Cerris using light microscopy (LM) and scanning electron microscopy (SEM). A total of five trichome types (including three glandular and two non-glandular types) and two trichome base types were found. Leaf epidermal features of Group Ilex show typical xeromorphic features. The stomatal complexes are stephanocytic. The leaf trichome types and trichome base types are stable at inter- and intra-species levels. The trichomes, stomatal sizes and density and the ray arm lengths show a wide variation during developing stages within species. The leaf epidermal features show a highly convergent pattern in the genus Quercus based on tree-mapping analysis, except for the multiple epidermal layers on the adaxial surface supports the monophyletic origin of section Heterobalanus. Cluster analysis of leaf epidermal characters infers two morphological subgroups within Group Ilex: (1) the capitate and (2) the branched uniseriate. The trichomes and trichome base types, and their density are useful for species identification, which can improve the accuracy of assigning fossil leaves to their nearest living relatives in Group Ilex.

Role of invasions in formation of phylogeographic structure of house mouse (Mus musculus) in some areas of Russia and the near abroad

The role of invasions in formation of the phylogeographic structure of a synanthropic species of house mouse (Mus musculus) is investigated. The probable ways of its expansion in some areas of Russia and the near abroad are analyzed. The genetic variability of subspecies and populations of M. musculus is evaluated by the data on polymorphism of the mitochondrial DNA control region (D loop). The data show that there are three phylogenetic varieties of house mice living in Russia and the near abroad: (i) house mice from the hybrid zone in Transcaucasia and М. m. musculus from Moscow; (ii) house mice of southern European Russia and southern Western Siberia; (iii) populations of M. m. musculus from Eastern Europe. The invasions caused nonuniformity of the phylogeographic structure of M. musculus. None of the studied subspecies (M. m. musculus, M. m. wagneri, and M. m. gansuensis) formed a uniform phylogroup. This is probably related to hybridization of different parapatric taxa of house mice at the species and intraspecies levels.