Genetic differentiation among distinct karyomorphs of the wolf fish Hoplias malabaricus species complex (Characiformes,Erythrinidae) and report of unusual hybridization with natural triploidy

In this study, genetic differentiation between karyomorphs A (2n = 42) and D (2n = 39/40) of the wolf fish Hoplias malabaricus, which is comprised of several cryptic species that present a wide variety of diploid chromosome numbers and sex chromosome systems, resulting in the identification of seven distinct karyomorphs (A–G), was investigated using a combination of molecular and cytogenetic tools. Deep sequence divergences for both karyomorphs were observed and indicate a long period of reproductive isolation between karyomorphs A and D. Additionally, one individual with 61 chromosomes was identified, which, as far as is known, is the first case of natural triploidy resulting from the hybridization between these highly differentiated karyomorphs of H. malabaricus. Molecular and cytogenetic analyses revealed that this allotriploid specimen carries two sets of maternal chromosomes from karyomorph D (2n = 40) and one set of chromosomes from karyomorph A (n = 21). Moreover, ribosomal sites and active nucleolus organizer regions from both parental contributors were found in the triploid hybrid. Considering the significant genetic distances between karyomorphs A and D, one of the primary reasons for the lack of recurrent reports of hybridization in the H. malabaricus species complex may be due to post‐zygotic barriers, such as hybrid sterility or unviability.     

Adjusting for selection bias in retrospective, case-control studies

Retrospective case–control studies are more susceptibleto selection bias than other epidemiologic studies as by designthey require that both cases and controls are representativeof the same population. However, as cases and control recruitmentprocesses are often different, it is not always obvious thatthe necessary exchangeability conditions hold. Selection biastypically arises when the selection criteria are associatedwith the risk factor under investigation. We develop a methodwhich produces bias-adjusted estimates for the odds ratio. Ourmethod hinges on 2 conditions. The first is that a variablethat separates the risk factor from the selection criteria canbe identified. This is termed the "bias breaking" variable.The second condition is that data can be found such that a bias-correctedestimate of the distribution of the bias breaking variable canbe obtained. We show by means of a set of examples that suchbias breaking variables are not uncommon in epidemiologic settings.We demonstrate using simulations that the estimates of the oddsratios produced by our method are consistently closer to thetrue odds ratio than standard odds ratio estimates using logisticregression. Further, by applying it to a case–controlstudy, we show that our method can help to determine whetherselection bias is present and thus confirm the validity of studyconclusions when no evidence of selection bias can be found.     

Development of pyrF-Based Genetic System for Targeted Gene Deletion in Clostridium thermocellum and Creation of a pta Mutant

We report development of a genetic system for making targeted gene knockouts in Clostridium thermocellum, a thermophilic anaerobic bacterium that rapidly solubilizes cellulose. A toxic uracil analog, 5-fluoroorotic acid (5-FOA), was used to select for deletion of the pyrF gene. The ΔpyrF strain is a uracil auxotroph that could be restored to a prototroph via ectopic expression of pyrF from a plasmid, providing a positive genetic selection. Furthermore, 5-FOA was used to select against plasmid-expressed pyrF, creating a negative selection for plasmid loss. This technology was used to delete a gene involved in organic acid production, namely pta, which encodes the enzyme phosphotransacetylase. The C. thermocellum Δpta strain did not produce acetate. These results are the first examples of targeted homologous recombination and metabolic engineering in C. thermocellum, a microbe that holds an exciting and promising future in the biofuel industry and development of sustainable energy resources.Conversion of cellulosic biomass using saccharolytic fermentative microorganisms without the addition of purified cellulase and hemicellulase enzymes is a promising approach for low-cost production of renewable fuels and chemicals (22, 23). Thermophilic, cellulolytic bacteria are one departure point for development of microorganisms with the requisite capabilities for such consolidated bioprocessing (CBP), with Clostridium thermocellum being exemplary in this regard. As reviewed elsewhere (6, 22), C. thermocellum is a Gram-positive organism able to ferment cellulose and products of cellulose solubilization to ethanol, acetic acid, lactic acid, formic acid, hydrogen, and CO₂. C. thermocellum appears to be a cellulose-utilizing specialist (6, 8) and produces a multienzyme cellulose-solubilizing complex termed a cellulosome (2, 3, 9).Metabolic engineering is required in order to increase the yield of ethanol or other desired products from mixed-product fermentation, such as that carried out by Clostridium thermocellum. Comprehensive work directed to this end has been carried out with genetically tractable organisms, such as Escherichia coli, resulting in high or near-theoretical yields achieved for ethanol (35, 36), other native products (21, 25), and nonnative products (7, 12). In these organisms, genetic systems involving both positive and negative selection markers have been employed in order to facilitate reuse of the same marker and to develop marker-free strains. One prominent system in the category involves use of the gene encoding the enzyme orotidine 5-phosphate decarboxylase (PyrF) (4, 11, 20, 27-29, 39). PyrF participates in de novo pyrimidine biosynthesis but is also a target for the antimetabolite 5-fluoroorotic acid (5-FOA) (4). Thus, cells lacking pyrF are uracil auxotrophs and resistant to 5-FOA, creating an opportunity whereby ectopic expression of pyrF can be selected or counterselected (4).Reliable genetic tractability has been elusive for Clostridium species. Prior to this report, the only Clostridia species in which gene deletion via homologous recombination has been demonstrated are Clostridium acetobutylicum, Clostridium perfringens, and Clostridium septicum. In the first organism, the use of a replicating plasmid for transformation followed by selection and screening for plasmid segregation resulted in a single clone that when analyzed contained a disruption in the gene of interest but not by the expected recombination events (13). The last two organisms have either an unusually high transformation frequency or feasibility for acquiring DNA from E. coli via conjugation, allowing the use of suicide plasmids (1, 16, 19). By comparison, the recently reported method of C. thermocellum transformation consists of a complex and cumbersome electroporation protocol using a custom pulse delivery system (37, 38). In our hands, efficiency of the C. thermocellum electrotransformation system does not compare with that of typical model organisms and does not enable the use of nonreplicating plasmids as a means of gene manipulation. Alternatively, group II intron technology has been used to inactivate gene targets in clostridia that were previously characterized as genetically intractable, but systems described to date have a temperature restriction that make such approaches incompatible with thermophilic clostridia (14, 15, 34).The only C. thermocellum mutant characterized genetically was isolated following a random mutagenesis and enrichment for cells that did not adhere to cellulose (43). The random mutagenesis approach is limited, in the sense that it does not lend itself well to reverse genetics, as many desired mutations lack selectable or screenable phenotypes. For instance, attempts have been made, with little success, to isolate saccharolytic thermophiles containing lesions in the pta-ack operon responsible for acetate production by selective enrichment using antimetabolites (26). In contrast, the creation of a Thermoanaerobacterium saccharolyticum Δpta-ack strain has been achieved using selectable markers that serve as a proxy for the events leading to targeted gene deletion (32). Motivated by the potential of microbial cellulose processing and the attributes of C. thermocellum, we undertook to develop a gene deletion system based on the pyrF gene.     

Saccharomyces cerevisiae-Based Molecular Tool Kit for Manipulation of Genes from Gram-Negative Bacteria

A tool kit of vectors was designed to manipulate and express genes from a wide range of gram-negative species by using in vivo recombination. Saccharomyces cerevisiae can use its native recombination proteins to combine several amplicons in a single transformation step with high efficiency. We show that this technology is particularly useful for vector design. Shuttle, suicide, and expression vectors useful in a diverse group of bacteria are described and utilized. This report describes the use of these vectors to mutate clpX and clpP of the opportunistic pathogen Pseudomonas aeruginosa and to explore their roles in biofilm formation and surface motility. Complementation of the rhamnolipid biosynthetic gene rhlB is also described. Expression vectors are used for controlled expression of genes in two pseudomonad species. To demonstrate the facility of building complicated constructs with this technique, the recombination of four PCR-generated amplicons in a single step at >80% efficiency into one of these vectors is shown. These tools can be used for genetic studies of pseudomonads and many other gram-negative bacteria.     

Chromosome polymorphism in Astyanax fasciatus (Teleostei, Characidae). 1. Karyotype analysis, Ag-NORs and mapping of the 18S and 5S ribosomal genes in sympatric karyotypes and their possible hybrid forms

Astyanax fasciatus may be characterized as a chromosomally diversified 'species' presenting distinct cytotypes, each with its specific variants. The sympatric and syntopic occurrence of different cytotypes reinforces the hypothesis in which A. fasciatus may represent a group of species currently placed under a single common designation. Specimens from three collection points spread along the Mogi-Gua?u River in southeast Brazil were examined in the present work: (1) near its headwaters (Ouro Fino--MG), (2) in the middle region of the river (Cachoeira de Emas, Pirassunun ga--SP) and (3) close to its confluence with the Pardo River (Barrinha--SP). The 2n = 48 chromosomes cytotype was found in all sampling points, while cytotype 2n = 46 was only encountered in Barrinha and Cachoeira de Emas. In the latter locality, cytotype 2n = 46 predominated; nevertheless, other karyotype forms with 2n = 45 and 47 chromosomes also occurred, besides a structural variant of cytotype 2n = 46. One specimen with 2n = 47 chromosomes was also found in Ouro Fino. The Ag-NOR analysis, as well as the location of the 18S and 5S ribosomal genes, were conserved in all cytotypes. The data indicate that the variant karyotypes are a consequence of interbreeding between the standard cytotypes (2n = 46 and 48) and/or its descendants. This suggests a karyotype plasticity for this species, where at least a few variant karyotypes would not have deleterious effects on their bearers.     

ZZ/ZW sex chromosome system in an undescribed species of the genus Apareiodon (Characiformes, Parodontidae)

The chromosomes of an undescribed species of the genus Apareiodon (Characiformes, Parodontidae) from the Verde River, a headwater affluent of the Tibagi River (Paraná State, Brazil), were investigated using conventional Giemsa and Ag stainings, C-banding, CMA(3) fluorescence and fluorescent in situ hybridization (FISH) using 18S and 5S rDNA probes. The diploid chromosome number was 2n = 54, with the karyotype composed of 48 meta/submetacentric and six subtelocentric chromosomes in males, and 47 meta/submetacentric + seven subtelocentric chromosomes in females. The difference is hypothesized to be due to a ZZ/ZW heteromorphic sex chromosome system, a cytotaxonomic characteristic previously observed only in some species of the genus Parodon (family Parodontidae). The presence of similar and/or identical heteromorphic sex chromosome systems might suggest that species of the genera Parodon and Apareiodon bearing ZZ/ZW heteromorphic sex chromosomes likely constitute a monophyletic group, a hypothesis to be tested by a robust phylogeny of the family.     

Activation of the selenoprotein SEPS1 gene expression by pro-inflammatory cytokines in HepG2 cells

SEPS1 (also called selenoprotein S, SelS) plays an important role in the production of inflammatory cytokines and its expression is activated by endoplasmic reticulum (ER) stress. In this report, we have identified two binding sites for the nuclear factor kappa B in the human SEPS1 promoter. SEPS1 gene expression, protein levels and promoter activity were all increased 2-3-fold by TNF-alpha and IL-1beta in HepG2 cells. We have also confirmed that the previously proposed ER stress response element GGATTTCTCCCCCGCCACG in the SEPS1 proximate promoter is fully functional and responsive to ER stress. However, concurrent treatment of HepG2 cells with IL-1beta and ER stress produced no additive effect on SEPS1 gene expression. We conclude that SEPS1 is a new target gene of NF-kappaB. Together with our previous findings that SEPS1 may regulate cytokine production in macrophage cells, we propose a regulatory loop between cytokines and SEPS1 that plays a key role in control of the inflammatory response.     

Genome wide gene-expression analysis of facultative reproductive diapause in the two-spotted spider mite Tetranychus urticae

Background

Diapause or developmental arrest, is one of the major adaptations that allows mites and insects to survive unfavorable conditions. Diapause evokes a number of physiological, morphological and molecular modifications. In general, diapause is characterized by a suppression of the metabolism, change in behavior, increased stress tolerance and often by the synthesis of cryoprotectants. At the molecular level, diapause is less studied but characterized by a complex and regulated change in gene-expression. The spider mite Tetranychus urticae is a serious polyphagous pest that exhibits a reproductive facultative diapause, which allows it to survive winter conditions. Diapausing mites turn deeply orange in color, stop feeding and do not lay eggs.

Results

We investigated essential physiological processes in diapausing mites by studying genome-wide expression changes, using a custom built microarray. Analysis of this dataset showed that a remarkable number, 11% of the total number of predicted T. urticae genes, were differentially expressed. Gene Ontology analysis revealed that many metabolic pathways were affected in diapausing females. Genes related to digestion and detoxification, cryoprotection, carotenoid synthesis and the organization of the cytoskeleton were profoundly influenced by the state of diapause. Furthermore, we identified and analyzed an unique class of putative antifreeze proteins that were highly upregulated in diapausing females. We also further confirmed the involvement of horizontally transferred carotenoid synthesis genes in diapause and different color morphs of T. urticae.

Conclusions

This study offers the first in-depth analysis of genome-wide gene-expression patterns related to diapause in a member of the Chelicerata, and further adds to our understanding of the overall strategies of diapause in arthropods.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-14-815) contains supplementary material, which is available to authorized users.     

PKCα-Specific Phosphorylation of the Troponin Complex in Human Myocardium: A Functional and Proteomics Analysis

Aims

Protein kinase Cα (PKCα) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKCα is implicated in heart failure and serves as a potential therapeutic target, however, the exact consequences for contractile function in human myocardium are unclear. This study aimed to investigate the effects of PKCα phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved.

Methods and Results

Endogenous cTn from permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (∼69%) with PKCα-treated recombinant human cTn (cTn (DD+PKCα)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out in vitro cross-phosphorylation of the PKA sites by PKCα. Isometric force was measured at various [Ca²⁺] after exchange. The maximal force (F_max) in the cTn (DD+PKCα) group (17.1±1.9 kN/m²) was significantly reduced compared to the cTn (DD) group (26.1±1.9 kN/m²). Exchange of endogenous cTn with cTn (DD+PKCα) increased Ca²⁺-sensitivity of force (pCa₅₀ = 5.59±0.02) compared to cTn (DD) (pCa₅₀ = 5.51±0.02). In contrast, subsequent PKCα treatment of the cells exchanged with cTn (DD+PKCα) reduced pCa₅₀ to 5.45±0.02. Two PKCα-phosphorylated residues were identified with mass spectrometry: Ser198 on cTnI and Ser179 on cTnT, although phosphorylation of Ser198 is very low. Using mass spectrometry based-multiple reaction monitoring, the extent of phosphorylation of the cTnI sites was quantified before and after treatment with PKCα and showed the highest phosphorylation increase on Thr143.

Conclusion

PKCα-mediated phosphorylation of the cTn complex decreases F_max and increases myofilament Ca²⁺-sensitivity, while subsequent treatment with PKCα in situ decreased myofilament Ca²⁺-sensitivity. The known PKC sites as well as two sites which have not been previously linked to PKCα are phosphorylated in human cTn complex treated with PKCα with a high degree of specificity for Thr143.     

Whole chromosome painting reveals independent origin of sex chromosomes in closely related forms of a fish species

The wolf fish Hoplias malabaricus includes well differentiated sex systems (XY and X₁X₂Y in karyomorphs B and D, respectively), a nascent XY pair (karyomorph C) and not recognized sex chromosomes (karyomorph A). We performed the evolutionary analysis of these sex chromosomes, using two X chromosome-specific probes derived by microdissection from the XY and X₁X₂Y sex systems. A putative-sex pair in karyomorph A was identified, from which the differentiated XY system was evolved, as well as the clearly evolutionary relationship between the nascent XY system and the origin of the multiple X₁X₂Y chromosomes. The lack of recognizable signals on the sex chromosomes after the reciprocal cross-FISH experiments highlighted that they evolved independently from non-homologous autosomal pairs. It is noteworthy that these distinct pathways occur inside the same nominal species, thus exposing the high plasticity of sex chromosome evolution in lower vertebrates. Possible mechanisms underlying this sex determination liability are also discussed.