Origins of asexuality in <Emphasis Type="Italic">Bryobia</Emphasis>mites (Acari: Tetranychidae)

Background  

Obligate asexual reproduction is rare in the animal kingdom. Generally, asexuals are considered evolutionary dead ends that are unable to radiate. The phytophagous mite genus Bryobia contains a large number of asexual species. In this study, we investigate the origin and evolution of asexuality using samples from 111 populations in Europe, South Africa and the United States, belonging to eleven Bryobia species. We also examine intraspecific clonal diversity for one species, B. kissophila, by genotyping individuals from 61 different populations. Knowledge on the origin of asexuality and on clonal diversity can contribute to our understanding of the paradox of sex.     

A mRNA landscape of bovine embryos after standard and MAPK-inhibited culture conditions: a comparative analysis

Background

Genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass (ICM) samples using microarray analysis. As MAPK inhibition can increase the NANOG/GATA6 ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a MAPK inhibitor and changes in gene expression in the inner cell mass were analysed.

Results

Between morula and blastocyst 3,774 genes were differentially expressed and the largest differences were found in blastocyst up-regulated genes. Gene ontology (GO) analysis shows lipid metabolic process as the term most enriched with genes expressed at higher levels in blastocysts. Genes with higher expression levels in morulae were enriched in the RNA processing GO term. Of the 497 differentially expressed genes comparing ICM and TE, the expression of NANOG, SOX2 and POU5F1 was increased in the ICM confirming their evolutionary preserved role in pluripotency. Several genes implicated to be involved in differentiation or fate determination were also expressed at higher levels in the ICM. Genes expressed at higher levels in the ICM were enriched in the RNA splicing and regulation of gene expression GO term. Although NANOG expression was elevated upon MAPK inhibition, SOX2 and POU5F1 expression showed little increase. Expression of other genes in the MAPK pathway including DUSP4 and SPRY4, or influenced by MAPK inhibition such as IFNT, was down-regulated.

Conclusion

The data obtained from the microarray studies provide further insight in gene expression during bovine embryonic development. They show an expression profile in pluripotent cells that indicates a pluripotent, epiblast-like state. The inability to culture ICM cells as stem cells in the presence of an inhibitor of MAPK activity together with the reported data indicates that MAPK inhibition alone is not sufficient to maintain a pluripotent character in bovine cells.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1448-x) contains supplementary material, which is available to authorized users.     

Elevated extracellular matrix production and degradation upon bone morphogenetic protein-2 (BMP-2) stimulation point toward a role for BMP-2 in cartilage repair and remodeling

Bone morphogenetic protein-2 (BMP-2) has been proposed as a tool for cartilage repair and as a stimulant of chondrogenesis. In healthy cartilage, BMP-2 is hardly present, whereas it is highly expressed during osteoarthritis. To assess its function in cartilage, BMP-2 was overexpressed in healthy murine knee joints and the effects on proteoglycan (PG) synthesis and degradation were evaluated. Moreover, the contribution of BMP in repairing damage induced by interleukin-1 (IL-1) was investigated. Ad-BMP-2 was injected intra-articularly into murine knee joints, which were isolated 3, 7, and 21 days after injection for histology, immunohistochemistry, and autoradiography. In addition, patellar and tibial cartilage was isolated for RNA isolation or measurement of PG synthesis by means of ³⁵SO₄ ^2- incorporation. To investigate the role for BMP-2 in cartilage repair, cartilage damage was induced by intra-articular injection of IL-1. After 2 days, Ad-BMP-2, Ad-BMP-2 + Ad-gremlin, Ad-gremlin, or a control virus was injected. Whole knee joints were isolated for histology at day 4 or patellae were isolated to measure ³⁵SO₄ ^2- incorporation. BMP-2 stimulated PG synthesis in patellar cartilage on all days and in tibial cartilage on day 21. Aggrecan mRNA expression had increased on all days in patellar cartilage, with the highest increase on day 7. Collagen type II expression showed a similar expression pattern. In tibial cartilage, collagen type II and aggrecan mRNA expression had increased on days 7 and 21. BMP-2 overexpression also induced increased aggrecan degradation in cartilage. VDIPEN staining (indicating matrix metalloproteinase activity) was elevated on day 3 in tibial cartilage and on days 3 and 7 in patellar cartilage, but no longer was by day 21. Increased NITEGE staining (indicating aggrecanase activity) was found on days 7 and 21. In IL-1-damaged patellar cartilage, BMP-2 boosted PG synthesis. Blocking of BMP activity resulted in a decreased PG synthesis compared with IL-1 alone. This decreased PG synthesis was associated with PG depletion in the cartilage. These data show that BMP-2 boosts matrix turnover in intact and IL-damaged cartilage. Moreover, BMP contributes to the intrinsic repair capacity of damaged cartilage. Increased matrix turnover might be functional in replacing matrix molecules in the repair of a damaged cartilage matrix.     

Phylogenetic relationships within the Alcidae (Charadriiformes: Aves) inferred from total molecular evidence

The Alcidae is a unique assemblage of Northern Hemisphere seabirds that forage by "flying" underwater. Despite obvious affinities among the species, their evolutionary relationships are unclear. We analyzed nucleotide sequences of 1,045 base pairs of the mitochondrial cytochrome b gene and allelic profiles for 37 allozyme loci in all 22 extant species. Trees were constructed on independent and combined data sets using maximum parsimony and distance methods that correct for superimposed changes. Alternative methods of analysis produced only minor differences in relationships that were supported strongly by bootstrapping or standard error tests. Combining sequence and allozyme data into a single analysis provided the greatest number of relationships receiving strong support. Addition of published morphological and ecological data did not improve support for any additional relationship. All analyses grouped species into six distinct lineages: (1) the dovekie (Alle alle) and auks, (2) guillemots, (3) brachyramphine murrelets, (4) synthliboramphine murrelets, (5) true auklets, and (6) the rhinoceros auklet (Cerorhinca monocerata) and puffins. The two murres (genus Uria) were sister taxa, and the black guillemot (Cepphus grylle) was basal to the other guillemots. The Asian subspecies of the marbled murrelet (Brachyramphus marmoratus perdix) was the most divergent brachyramphine murrelet, and two distinct lineages occurred within the synthliboramphine murrelets. Cassin's auklet (Ptychoramphus aleuticus) and the rhinoceros auklet were basal to the other auklets and puffins, respectively, and the Atlantic (Fratercula arctica) and horned (Fratercula corniculata) puffins were sister taxa. Several relationships among tribes, among the dovekie and auks, and among the auklets could not be resolved but resembled "star" phylogenies indicative of adaptive radiations at different depths within the trees.      

Companion cell-specific inhibition of the potato sucrose transporter SUT1

In many plants, translocation of sucrose from mesnsophyll to phloem for long-distance transport is carrier-mediated. The sucrose H⁺-symporter gene SUT1 from potato is expressed at high levels in the phloem of mature, exporting leaves and at lower levels in other organs. Inhibition of SUT1 by expression of an antisense gene in companion cells under control of the rolC promoter leads to accumulation of high amounts of soluble and insoluble carbohydrates in leaves and inhibition of photosynthesis. The distribution of in situ localized starch does not correspond with areas of reduced photosynthesis as shown by fluorescence imaging. Dissection of antisense effects on sink and source organs by reciprocal grafts shows that inhibition of transporter gene expression in leaves is sufficient to produce chlorosis in leaves and reduced tuber yield. In contrast to the arrest of plasmodesmal development found in plants that express yeast invertase in the apoplast, in mature leaves of sucrose transporter antisense plants plasmodesmata are branched and have median cavities. These data strongly support an apoplastic mode of phloem loading in potato, in which the sucrose transporter located at the plasma membrane of the sieve element/companion cell complex represents the primary route for sugar uptake into the long-distance translocation pathway.     

HUWE1 interacts with PCNA to alleviate replication stress

Defects in DNA replication, DNA damage response, and DNA repair compromise genomic stability and promote cancer development. In particular, unrepaired DNA lesions can arrest the progression of the DNA replication machinery during S‐phase, causing replication stress, mutations, and DNA breaks. HUWE1 is a HECT‐type ubiquitin ligase that targets proteins involved in cell fate, survival, and differentiation. Here, we report that HUWE1 is essential for genomic stability, by promoting replication of damaged DNA. We show that HUWE1‐knockout cells are unable to mitigate replication stress, resulting in replication defects and DNA breakage. Importantly, we find that this novel role of HUWE1 requires its interaction with the replication factor PCNA, a master regulator of replication fork restart, at stalled replication forks. Finally, we provide evidence that HUWE1 mono‐ubiquitinates H2AX to promote signaling at stalled forks. Altogether, our work identifies HUWE1 as a novel regulator of the replication stress response.     

Series resistance compensation for whole-cell patch-clamp studies using a membrane state estimator

Whole-cell patch-clamp techniques are widely used to measure membrane currents from isolated cells. While suitable for a broad range of ionic currents, the series resistance (R(s)) of the recording pipette limits the bandwidth of the whole-cell configuration, making it difficult to measure rapid ionic currents. To increase bandwidth, it is necessary to compensate for R(s). Most methods of R(s) compensation become unstable at high bandwidth, making them hard to use. We describe a novel method of R(s) compensation that overcomes the stability limitations of standard designs. This method uses a state estimator, implemented with analog computation, to compute the membrane potential, V(m), which is then used in a feedback loop to implement a voltage clamp; we refer to this as state estimator R(s) compensation. To demonstrate the utility of this approach, we built an amplifier incorporating state estimator R(s) compensation. In benchtop tests, our amplifier showed significantly higher bandwidths and improved stability when compared with a commercially available amplifier. We demonstrated that state estimator R(s) compensation works well in practice by recording voltage-gated Na(+) currents under voltage-clamp conditions from dissociated neonatal rat sympathetic neurons. We conclude that state estimator R(s) compensation should make it easier to measure large rapid ionic currents with whole-cell patch-clamp techniques.     

The role of photorespiration during drought stress: an analysis utilizing barley mutants with reduced activities of photorespiratory enzymes

C _i, intercellular CO₂ concentration
F_v/F_m, quantum efficiency of excitation capture by open photosystem II centres
FBPase, fructose-1,6-bisphosphatase
GAPDH, glyceraldehyde-3-phosphate dehydrogenase
GDC, glycine decarboxylase
GS-2, chloroplastic glutamine synthetase
HPR, hydroxypyruvate reductase
PFD, photon flux density
ΦCO₂, quantum efficiency of CO₂ assimilation
ΦPSII, quantum efficiency of photosystem II electron transport
ψ, water potential
q_N, non-photochemical chlorophyll a fluorescence quenching
q_P, photochemical chlorophyll a fluorescence quenching
RuBP, ribulose-1,5-bisphosphate
Rubisco, ribulose-1,5-bisphosphate carboxylase-oxygenase
SBPase, sedoheptulose-1,7-bisphosphatase
SGAT, serine : glyoxylate aminotransferase

The significance of photorespiration in drought-stressed plants was studied by withholding water from wild-type barley (Hordeum vulgare L.) and from heterozygous mutants with reduced activities of chloroplastic glutamine synthetase (GS-2), glycine decarboxylase (GDC) or serine : glyoxylate aminotransferase (SGAT). Well-watered plants of all four genotypes had identical rates of photosynthesis. Under moderate drought stress (leaf water potentials between –1 and –2 MPa), photosynthesis was lower in the mutants than in the wild type, indicating that photorespiration was increased under these conditions. Analysis of chlorophyll a fluorescence revealed that, in the GDC and SGAT mutants, the lower rates of photosynthesis coincided with a decreased quantum efficiency of photosystem II and increased non-photochemical dissipation of excitation energy. Correspondingly, the de-epoxidation state of xanthophyll-cycle carotenoids was increased several-fold in the drought-stressed GDC and SGAT mutants compared with the wild type. Accumulation of glycine in the GDC mutant was further evidence for increased photorespiration in drought-stressed barley. The effect of drought on the photorespiratory enzymes was determined by immunological detection of protein abundance. While the contents of GS-2 and P- and H-protein of the GDC complex remained unchanged as drought stress developed, the content of NADH-dependent hydroxypyruvate reductase increased. Enzymes of the Benson–Calvin cycle, on the other hand, were either not affected (ribulose-1,5-bisphosphate carboxylase-oxygenase and plastidic fructose-1,6-bisphosphatase) or declined (sedoheptulose- 1,7-bisphosphatase and NADP-dependent glyceraldehyde-3-phosphate dehydrogenase). These data demonstrate that photorespiration was enhanced during drought stress in barley and that the control exerted by photorespiratory enzymes on the rate of photosynthetic electron transport and CO₂ fixation was increased.     

Gene-based microsatellites for cassava (Manihot esculenta Crantz): prevalence, polymorphisms, and cross-taxa utility

Background  

Cassava (Manihot esculenta Crantz), a starchy root crop grown in tropical and subtropical climates, is the sixth most important crop in the world after wheat, rice, maize, potato and barley. The repertoire of simple sequence repeat (SSR) markers for cassava is limited and warrants a need for a larger number of polymorphic SSRs for germplasm characterization and breeding applications.