Cross-species microarray hybridizations: a developing tool for studying species diversity

The use of cross-species hybridization (CSH) to DNA microarrays, in which the target RNA and microarray probe are from different species, has increased in the past few years. CSH is used in comparative, evolutionary and ecological studies of closely related species, and for gene-expression profiling of many species that lack a representative microarray platform. However, unlike species-specific hybridization, CSH is still considered a non-standard use of microarrays. Here, we present the recent developments in the field of CSH for cDNA and oligomer microarray platforms. We discuss issues that influence the quality of CSH results, including platform choice, experiment design and data analysis, and suggest strategies that can lead to improvement of CSH studies to investigate species diversity.     

Impact of enrichment conditions on cross‐species capture of fresh and degraded DNA

By combining high‐throughput sequencing with target enrichment (‘hybridization capture’), researchers are able to obtain molecular data from genomic regions of interest for projects that are otherwise constrained by sample quality (e.g. degraded and contamination‐rich samples) or a lack of a priori sequence information (e.g. studies on nonmodel species). Despite the use of hybridization capture in various fields of research for many years, the impact of enrichment conditions on capture success is not yet thoroughly understood. We evaluated the impact of a key parameter – hybridization temperature – on the capture success of mitochondrial genomes across the carnivoran family Felidae. Capture was carried out for a range of sample types (fresh, archival, ancient) with varying levels of sequence divergence between bait and target (i.e. across a range of species) using pools of individually indexed libraries on Agilent SureSelect^? arrays. Our results suggest that hybridization capture protocols require specific optimization for the sample type that is being investigated. Hybridization temperature affected the proportion of on‐target sequences following capture: for degraded samples, we obtained the best results with a hybridization temperature of 65 °C, while a touchdown approach (65 °C down to 50 °C) yielded the best results for fresh samples. Evaluation of capture performance at a regional scale (sliding window approach) revealed no significant improvement in the recovery of DNA fragments with high sequence divergence from the bait at any of the tested hybridization temperatures, suggesting that hybridization temperature may not be the critical parameter for the enrichment of divergent fragments.     

Oxidation resistance 1 is a novel senolytic target

The selective depletion of senescent cells (SCs) by small molecules, termed senolytic agents, is a promising therapeutic approach for treating age‐related diseases and chemotherapy‐ and radiotherapy‐induced side effects. Piperlongumine (PL) was recently identified as a novel senolytic agent. However, its mechanism of action and molecular targets in SCs was unknown and thus was investigated. Specifically, we used a PL‐based chemical probe to pull‐down PL‐binding proteins from live cells and then mass spectrometry‐based proteomic analysis to identify potential molecular targets of PL in SCs. One prominent target was oxidation resistance 1 (OXR1), an important antioxidant protein that regulates the expression of a variety of antioxidant enzymes. We found that OXR1 was upregulated in senescent human WI38 fibroblasts. PL bound to OXR1 directly and induced its degradation through the ubiquitin‐proteasome system in an SC‐specific manner. The knockdown of OXR1 expression by RNA interference significantly increased the production of reactive oxygen species in SCs in conjunction with the downregulation of antioxidant enzymes such as heme oxygenase 1, glutathione peroxidase 2, and catalase, but these effects were much less significant when OXR1 was knocked down in non‐SCs. More importantly, knocking down OXR1 selectively induced apoptosis in SCs and sensitized the cells to oxidative stress caused by hydrogen peroxide. These findings provide new insights into the mechanism by which SCs are highly resistant to oxidative stress and suggest that OXR1 is a novel senolytic target that can be further exploited for the development of new senolytic agents.     

Pleiotropic roles of autophagy in stem cell–based therapies

Stem cells (SCs) have been proven to possess regenerative and immunomodulatory properties and can be used to treat diseases that involve loss of cells due to tissue damage or inflammation. For this approach to succeed, SCs or their derivatives should be able to engraft in the target tissue at least for a short period of time. Unfortunately, once injected, therapeutic SCs will encounter a hostile environment, including hypoxia, lack of nutrients and stromal support, and cells may also be targeted and rejected by the immune system. Therefore, SC's stress-response mechanisms likely play a significant role in survival of injected cells and possibly contribute to their therapeutic efficacy. Autphagy, a stress-response pathway, is involved in many different cellular processes, such as survival during hypoxia and nutrient deprivation, cellular differentiation and de-differentiation, and it can also contribute to their immunovisibility by regulating antigen presentation and cytokine secretion. Autophagy machinery interacts with many proteins and signaling pathways that regulate SC properties, including PI3K/Akt, mammalian target of rapamycin (mTOR), Wnt, Hedgehog and Notch, and it is also involved in regulating intracellular reactive oxygen species (ROS) levels. In this review, we contend that autophagy is an important therapeutic target that can be used to improve the outcome of SC-based tissue repair and regeneration. Further research should reveal whether inhibition or stimulation of autophagy increases the therapeutic utility of SCs and it should also identify appropriate therapeutic regimens that can be applied in the clinic.     

Targeting cancer stem cells with phytochemicals

Cancer, second only to heart disease, is the leading cause of death in the US. Although progress has been made in the early detection of cancer and in improvements of cancer therapies, the ability to provide long-term survival has been limited. Increasing evidence suggests that a minute, biologically unique population of cancer stem cells (SCs) exists in most neoplasms and may be responsible for tumor initiation, progression, metastasis, and relapse. Characterization of cancer SCs has led to the identification of key cellular activities that may make cancer SCs vulnerable to therapeutic interventions that target drug-effluxing capabilities, stem cell pathways, anti-apoptotic mechanisms, and induction of differentiation. Phytochemicals, compounds made from fruits, vegetables, and grains, possess anti-cancer properties and represent a promising therapeutic approach for the prevention and treatment of many cancers. This review summarizes the evidence for the cancer SC hypothesis and discusses the potential mechanisms by which phytochemicals might target cancer SCs.     

Standing out from the crowd: Spotting your targets in a mixed plankton sample

The diversity of marine organisms is staggering, and this fact is readily appreciated by microscopic examination of the contents of a plankton net after a short tow across the ocean surface. Although this diversity is beautiful, it can present a significant problem for those seeking to extract information about a single species of interest. Enumeration of the eggs and larvae of a specific target species can provide a quantitative window into reproductive dynamics that are of great use for fisheries stock assessment and management. But how do you efficiently sort through the mass of plankton and identify target species’ eggs and larvae that may be morphologically indistinguishable from those of a number of other local species? In this issue of Molecular Ecology Resources, Oxley et al. ( 2017 ) describe an innovative in situ hybridization (ISH) approach that successfully solves this important problem and opens an exciting new avenue to ichthyoplankton analysis that may be widely adopted by both fish ecologists and fisheries managers.     

The relationship between genome size and synaptonemal complex length in higher plants

There appears to be only a weak correlation between genome size and the corresponding total length of a complete set of synaptonemal complexes (SCs) based on published evidence for several fungal, plant, and animal species. This result is unexpected, considering the strong positive correlations between genome size (DNA amount) and total chromosome length and volume and between relative lengths of chromosomes and SCs. Because the observed weak correlation was based on limited data, we systematically investigated the relationship between genome size and SC length, using ten higher plant species. Two-dimensional spreads of SCs from primary microsporocytes at pachytene were prepared using a hypotonic bursting technique. The SC spreads were examined either by light or electron microscopy, and the lengths of at least ten complete sets of SCs were measured for each of the ten species. Additionally, the genome size of each species was determined from pollen tetrad protoplasts using flow cytometry. A strong correlation (r = 0.97) between total SC length and genome size was observed for higher plants, indicating a constant amount of DNA is associated with a given length of SC, at least when averaged over the whole genome.     

Influence of incubation conditions on cell surface hydrophobicity of Candida species fungi

Cell surface hydrophobicity (CSH) is considered to be one of several virulence factors of Candida yeast-like fungi. The aim of the study was a measurment of hydrophobic properties of Candida sp. depending on growth conditions. A total of 139 strains of Candida (80 - C. albicans and 59 - C. non-albicans) were examined. The method of salt aggregation test (SAT) was used. The strains were cultured on three different media, in two variants of incubation temperature and time. The incubation temperature and microbiological medium affected CSH of just C. albicans strains. The influence of incubation time on CSH of examined species of Candida was not occurred. There was a strong correlation between CSH and species of Candida demonstrated in the study Hydrophobic properties were more frequent and stronger among strains of C. non-albicans than C. albicans species. The results of the study indicates that CSH of Candida spp. is a dynamic feature. The ability to change surface properties may play a role in pathogenesis of candidosis.     

The differential proliferative ability of satellite cells in Lantang and Landrace pigs

Here, for the first time, we evaluate the hypothesis that the proliferative abilities of satellite cells (SCs) isolated from Lantang (indigenous Chinese pigs) and Landrace pigs, which differ in muscle characteristics, are different. SCs were isolated from the longissimus dorsi muscle of neonatal Lantang and Landrace pigs. Proliferative ability was estimated by the count and proliferative activity of viable cells using a hemocytometer and MTT assay at different time points after seeding, respectively. Cell cycle information was detected by flow cytometry. Results showed that there was a greater (P<0.05) number of SCs in Lantang pigs compared with Landrace pigs after 72 h of culture. The percentage of cell population in S phase and G(2)/M phases in Lantang pigs were higher (P<0.05), while in G(0)/G(1) phase was lower (P<0.05) in comparison with the Landrace pigs. The mRNA abundances of MyoD, Myf5, myogenin and Pax7 in SCs from Lantang pigs were higher (P<0.05), while those of myostatin, Smad3 and genes in the mammalian target of rapamycin (mTOR) pathway (with the exception of 4EBP1) were lower (P<0.05) than the Landrace pigs. Protein levels of MyoD, myogenin, myostatin, S6K, phosphorylated mTOR and phosphorylated eIF4E were consistent with the corresponding mRNA abundance. Collectively, these findings suggested that SCs in the two breeds present different proliferative abilities, and the proliferative potential of SCs in Lantang pigs is higher than in Landrace pigs.     

Reversed type of color-pattern modifications of butterfly wings: a physiological mechanism of wing-wide color-pattern determination

Application of cold shock or tungstate to butterfly pupae produces a unique color-pattern modification type on the adult wings, in which the color-pattern elements are dislocated toward the reduced focal elements. This modification-inducing activity has been primarily attributed to the putative cold-shock hormone (CSH) that is secreted into the hemolymph upon cold shock. Here, using a species of nymphalid butterfly Junonia almana, a new "reversed" type of the color-pattern modifications of butterfly wings was obtained by the application of heat shock or thapsigargin, a calcium-ATPase inhibitor, in which most elements were dislocated away from the enlarged focal elements. This result suggests that the endocrine secretion of CSH is sensitive to a wide range of temperature shocks, which then affects the cellular interpretation of the wing-wide positional information that is emitted from the focal locations. Ecdysteroid contributes to the wing-wide patterning primarily independently from CSH, but these two systems negatively interact with each other, probably in the intracellular signaling pathways.     

Searching for telomeric sequences in two Allium species.

Some Alliaceae species have no tandemly repeated TTTAGGG sequences. Instead, at the very end of their chromosomes, there are highly repetitive satellite and (or) rDNA sequences. These sequences apparently replace the canonical plant telomeric sequences in these species. A method of preparing two-dimensional surface spreads of plant synaptonemal complexes (SCs), combined with fluorescent in situ hybridization, has revealed that telomeric chromatin is tightly condensed at the ends of SCs in plants and animals. Using this method, we have tested the organization and location of those sequences postulated to cap the chromosomes in two species of the genus Allium: A. cepa and A. altaicum. We have also extended this study to other putative telomere candidates, such as LTR (long terminal repeat) and non-LTR retrotransposons. None of the DNA sequences analyzed showed the characteristic telomeric organization at pachytene.     

The protective effects of resveratrol on Schwann cells with toxicity induced by ethanol in vitro

Schwann cells (SCs) are the myelin forming cells in the peripheral nervous system, they play a key role in the pathology of various polyneuropathies and provide trophic support to axons via expression of various neurotrophic factors, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). Ethanol (EtOH) adversely affected both SCs proliferation and myelin formation in culture. Resveratrol (Res) has been shown to regulate many cellular processes and to display multiple protective and therapeutic effects. Whether Res has protective effects on SCs with EtOH-induced toxicity is still unclear. The protective efficacy of Res on EtOH-treated SCs in vitro was investigated in the present study. Res improved cell viability of the EtOH-treated SCs. Hoechst 33342 staining and terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick-end labeling analysis showed that the EtOH-induced apoptosis was inhibited by Res. The effects of Res were blocked by the 5′-adenosine monophosphate-activated protein kinase inhibitor Compound C and the silencing information regulator T1 inhibitor nicotinamide. Res could increase the mRNA and protein levels of BDNF and GDNF in the EtOH-treated SCs. However, the EtOH-induced increase of NGF in the SCs is inhibited by Res. The data from the present study indicate that Res protects SCs from EtOH-induced cell death and regulates the expression of neurotrophic factors. Res and its derivative may be effective for the treatment of neuropathic diseases induced by EtOH.     

Tests of rRNA hybridization to microarrays suggest that hybridization characteristics of oligonucleotide probes for species discrimination cannot be predicted

Hybridization of rRNAs to microarrays is a promising approach for prokaryotic and eukaryotic species identification. Typically, the amount of bound target is measured by fluorescent intensity and it is assumed that the signal intensity is directly related to the target concentration. Using thirteen different eukaryotic LSU rRNA target sequences and 7693 short perfect match oligonucleotide probes, we have assessed current approaches for predicting signal intensities by comparing Gibbs free energy (ΔG°) calculations to experimental results. Our evaluation revealed a poor statistical relationship between predicted and actual intensities. Although signal intensities for a given target varied up to 70-fold, none of the predictors were able to fully explain this variation. Also, no combination of different free energy terms, as assessed by principal component and neural network analyses, provided a reliable predictor of hybridization efficiency. We also examined the effects of single-base pair mismatch (MM) (all possible types and positions) on signal intensities of duplexes. We found that the MM effects differ from those that were predicted from solution-based hybridizations. These results recommend against the application of probe design software tools that use thermodynamic parameters to assess probe quality for species identification. Our results imply that the thermodynamic properties of oligonucleotide hybridization are by far not yet understood.     

Bony labyrinth morphometry indicates locomotor adaptations in the squirrel-related clade (Rodentia,Mammalia)

The semicircular canals (SCs) of the inner ear detect angular acceleration and are located in the bony labyrinth of the petrosal bone. Based on high-resolution computed tomography, we created a size-independent database of the bony labyrinth of 50 mammalian species especially rodents of the squirrel-related clade comprising taxa with fossorial, arboreal and gliding adaptations. Our sampling also includes gliding marsupials, actively flying bats, the arboreal tree shrew and subterranean species. The morphometric anatomy of the SCs was correlated to the locomotion mode. Even if the phylogenetic signal cannot entirely be excluded, the main significance for functional morphological studies has been found in the diameter of the SCs, whereas the radius of curvature is of minor interest. Additionally, we found clear differences in the bias angle of the canals between subterranean and gliding taxa, but also between sciurids and glirids. The sensitivity of the inner ear correlates with the locomotion mode, with a higher sensitivity of the SCs in fossorial species than in flying taxa. We conclude that the inner ear of flying and gliding mammals is less sensitive due to the large information flow into this sense organ during locomotion.     

Organization of highly repeated sequences in surface-spread pachytene chromosomes of rye.

A method of preparing two-dimensional surface spreads of plant synaptonemal complexes (SCs) associated with fluorescence in situ hybridization (FISH) has been applied to analyze the location and organization of five different highly repeated DNA sequences in rye. Our observations indicate that, depending on the type of sequence, the chromatin displays different types of organization. Telomeric sequences were seen tightly associated with the SC while other repetitive DNA sequences were found to form loops that are associated with SCs only at their bases. On the contrary, the FISH signal of a centromeric satellite had a granular appearance, reflecting that the hybridization occurs only with parts of the chromatin loops.     

Differences in Characteristics of Reserve Network Selection Using Population Data Versus Habitat Surrogates

The use of species data versus environmental surrogates used in lieu of species data in systematic reserve site selection is still highly debated. We analyse in a case study whether and how the results of reserve network selection are affected by the use of species data versus habitat surrogates (habitat models) for qualitative (presence/absence) and quantitative (population size/habitat quality) information. In a model region, the post-mining landscape south of Leipzig/Germany, we used iterative algorithms to select a network for 29 animal target species from a basic set of 127 sites. The network results differ markedly for the two information types: depending on the representation goal, 18–45% of the selected sites chosen in response to one information type do not appear in the results for the other type. Given the availability of quantitative and hence deeper information, evaluation rules can be used to filter out the best habitats and the largest populations. In our model study, 0–40% less suitable areas were selected when instead of quantitative details only qualitative data were used. In view of various advantages and limitations of the two information types, we propose improving the methodological approach to the selection of networks for animal species by combining different information types.     

Genome‐wide sequence information reveals recurrent hybridization among diploid wheat wild relatives

Many conflicting hypotheses regarding the relationships among crops and wild species closely related to wheat (the genera Aegilops, Amblyopyrum, and Triticum) have been postulated. The contribution of hybridization to the evolution of these taxa is intensely discussed. To determine possible causes for this, and provide a phylogeny of the diploid taxa based on genome‐wide sequence information, independent data were obtained from genotyping‐by‐sequencing and a target‐enrichment experiment that returned 244 low‐copy nuclear loci. The data were analyzed using Bayesian, likelihood and coalescent‐based methods. D statistics were used to test if incomplete lineage sorting alone or together with hybridization is the source for incongruent gene trees. Here we present the phylogeny of all diploid species of the wheat wild relatives. We hypothesize that most of the wheat‐group species were shaped by a primordial homoploid hybrid speciation event involving the ancestral Triticum and Am. muticum lineages to form all other species except Ae. speltoides. This hybridization event was followed by multiple introgressions affecting all taxa except Triticum. Mostly progenitors of the extant species were involved in these processes, while recent interspecific gene flow seems insignificant. The composite nature of many genomes of wheat‐group taxa results in complicated patterns of diploid contributions when these lineages are involved in polyploid formation, which is, for example, the case for tetraploid and hexaploid wheats. Our analysis provides phylogenetic relationships and a testable hypothesis for the genome compositions in the basic evolutionary units within the wheat group of Triticeae.     

A Disintegrin and Metalloprotease 10 (ADAM10) Is Indispensable for Maintenance of the Muscle Satellite Cell Pool

Satellite cells (SCs) are muscle-specific stem cells that are essential for the regeneration of damaged muscles. Although SCs have a robust capacity to regenerate myofibers, the number of SCs decreases with aging, leading to insufficient recovery after muscle injury. We herein show that ADAM10 (a disintegrin and metalloprotease 10), a membrane-bound proteolytic enzyme with a critical role in Notch processing (S2 cleavage), is essential for the maintenance of SC quiescence. We generated mutant mice in which ADAM10 in SCs can be conditionally abrogated by tamoxifen injection. Tamoxifen-treated mutant mice did not show any apparent defects and grew normally under unchallenged conditions. However, these mice showed a nearly complete loss of muscle regeneration after chemically induced muscle injury. In situ hybridization and flow cytometric analyses revealed that the mutant mice had significantly less SCs compared with wild type controls. Of note, we found that inactivation of ADAM10 in SCs severely compromised Notch signaling and led to dysregulated myogenic differentiation, ultimately resulting in deprivation of the SC pool in vivo. Taken together, the present findings underscore the role of ADAM10 as an indispensable component of Notch signaling in SCs and for maintaining the SC pool.