Conservation Genetics

Impact of habitat loss and fragmentation on reproduction, dispersal and species persistence for an endangered Chilean tree Abstract Survival of rare and endangered plant species following habitat loss and fragmentation is dependent upon in situ reproduction and population persistence, and establishment in new sites, so that stochastic, local extirpation does not lead to species extinction. Here we investigate if and how vegetative and sexual reproduction, propagule dispersal, and seedling establishment promote in situ persistence and establishment in new sites for the endangered, endemic Chilean tree Gomortega keule, in a landscape experiencing extensive habitat loss and fragmentation. Genetic analyses show G. keule reproduces vegetatively, producing large clone groups and providing a mechanism for long-term in situ persistence. Abundant seed set was observed, but seedling establishment was rare. At the single site where seedlings were observed, parentage analysis revealed seed dispersal over greater distances, and in a more aggregated pattern, than would be expected from gravity dispersal alone. We hypothesize that G. keule may represent a 'seed dispersal anachronism', wherein the native seed disperser has been lost and possibly replaced by domesticated cattle. The lack of seedling establishment at most sites raises concerns about the ability of existing populations to expand within existing habitat patches or establish at new locations. Globally, habitat loss and fragmentation, coupled with other environmental changes, pose a particular threat to rare species with small populations, because inbreeding and the loss of mutualists can lead to establishment failure and local extirpation. We propose that analysis of a plant's reproductive strategies as a whole, in its landscape context, is essential for effective species conservation.

Population genomics of the peripheral freshwater fish Polynemus melanochir (Perciformes, Polynemidae) in a changing Mekong Delta Abstract The Mekong River is a vital fisheries resource supporting millions of people in mainland Southeast Asia. However, numerous threats have the potential to negatively impact fish populations in this region including overfishing, pollution, climate change and increased urban, agriculture and upstream hydropower development. Although a few studies have examined the population genetic structure of fishes within the upper Mekong River, no known studies have explored that of fishes within the Mekong Delta (MD). Here, we examine the population structure of an important food fish within the MD, Polynemus melanochir, using a panel of 1735 single nucleotide polymorphisms (SNPs) generated by restriction site-associated DNA (RAD) sequencing across eight locations on the Tien (Mekong) and Hau (Bassac) Rivers in Vietnam. Pairwise FST values, principal component analysis and Structure analysis all indicate high levels of gene flow among the sites sampled across the MD. In contrast to the lack of genetic structure, high levels of relatedness were found, including 26 putatively related pairs, as well as an effective population size (Ne) of less than 500 across the MD. While panmixia indicates that fragmentation of this population is not presently an important threat, a low Ne estimate suggests this species may not be resilient to long-term environmental changes in the MD. The reliance on P. melanochir as a food resource may be contingent on management and mitigation of low effective population sizes.

A morphologically cryptic salamander reveals additional hidden diversity: evidence for ancient genetic divergence in Webster's salamander, Plethodon websteri Abstract We investigated the genetic diversity and phylogenetic structure of Webster's salamander (Plethodon websteri), an understudied species of conservation concern and one of the first morphologically cryptic salamander species described solely on the basis of molecular techniques. Using a combination of mitochondrial and microsatellite data, we discovered significant genetic differentiation across the known distribution (ɸST > 0.868 and FST = 0.261−0.652, p < 0.001), and identified at least two highly divergent clades. These clades, referenced herein as the Semlitsch and Type Locality clades, are of late Miocene origin (approximately 5.3 million years old) and are composed of several geographically and genetically distinct populations. Together, our observations suggest that previously unrecognized species-level diversity may exist in P. websteri, with populations potentially representing distinct but undescribed taxa. We hypothesize that divergence within P. websteri likely resulted from a culmination of radical changes in climate, hydrology, and geology over deep time. Moreover, our results add to mounting evidence suggesting that P. websteri does not belong within the P. welleri group, but instead forms its own discrete species complex separate and genetically distant from P. welleri. This study provides a starting point for future work and reiterates that relationships among, and species-level diversity within, eastern species of Plethodon require additional investigation and potential reevaluation.

Population genetic assessment of two critically endangered freshwater mussel species, Tennessee bean Venustaconcha trabalis and Cumberland bean Venustaconcha troostensis Abstract The Cumberland bean Venustaconcha troostensis (Conrad in Academy of Natural Sciences of Philadelphia. http://biodiversitylibrary.org/page/16346317/, 1834) and Tennessee bean V. trabalis (Lea in Proc Acad Nat Sci Phila 13:38–41, 1861) are both endangered freshwater mussel species endemic to the Cumberland and Tennessee river basins, respectively. These species are closely related and morphologically similar in appearance, and now exist only in small, fragmented populations relative to their historical abundances and distributions throughout the southeastern United States. A critical management need is to understand genetic differences among populations, so that informed decisions can be made to promote demographic and genetic viability of remaining populations. Against this background, we conducted a population genetic analysis by assessing variation at eight microsatellite DNA loci across 127 individuals. Our results showed five population clusters across five rivers, V. trabalis in the Clinch, Obed-Emory, Holston, and Hiwassee rivers; and V. troostensis in the Cumberland river. There was moderate to high differentiation among populations based on FST (range 0.09 − 0.18) and high differentiation among populations based on Jost's D (range 0.33 − 0.67). The highest differentiation was observed between populations in the Cumberland and Tennessee River basins (mean FST = 0.155, mean D = 0.559). Analysis of allele frequencies suggest that all populations likely have lost genetic diversity and are bottlenecked to various degrees. Linkage disequilibrium-based estimates of recent-current effective population sizes (Ne) ranged from 42 in the Cumberland River population to 130 individuals in the Hiwassee River population, suggesting that populations have become small and fragmented by natural historical processes and by man-made impediments to gene flow. Using approximate Bayesian computation to estimate Ne over longer-term coalescent time, median posterior distributions of Ne ranged from 7130 for the Clinch River population to 901 for the Holston River population. The inferences gained in this study may warrant action by conservation managers to artificially cross extant stocks within basins for purposes of genetic and demographic augmentation. However, based on our initial results, we do not recommend interspecific mixing of individuals between the Cumberland and Tennessee River basins.

Conservation implications of widespread polyploidy and apomixis: a case study in the genus Pomaderris (Rhamnaceae) Abstract Polyploidy resulting from whole genome duplication is common in plants and is increasingly being recognised as a critical factor in conservation actions, particularly when within-species variation in ploidy exists. Pomaderris(Rhamnaceae), a genus of 70 species endemic to Australia and New Zealand, has many species listed as threatened and that are subject to conservation management but of unknown ploidy. To provide a better understanding of polyploidy in the genus we sampled 36 of 70 species of Pomaderris and used flow cytometry to establish genome sizes and infer ploidy. Additionally, to examine within-species variation, we screened 104 individuals of eight rare species subject to conservation management. We did not find evidence for infraspecific variation in ploidy, suggesting that from a cytological perspective, conspecific individuals from the screened populations do not need to be kept geographically separated in conservation management. There is, however, considerable variation among species, with genome sizes suggesting the occurrence of diploidy, triploidy, tetraploidy and hexaploidy. Finding several species to be triploid but capable of seed production, we then explored reproductive biology using the flow cytometric seed screen. Results suggested that triploid species produce seeds asexually, as previously reported for two New Zealand congeners. While asexual reproduction through apomixis is generally a means of odd-ploid taxa overcoming sterility, we found that more than half of examined diploids and tetraploids also produced seeds asexually. Asexual reproduction means genetic diversity is potentially low, and these results should therefore be considered in future conservation actions and seed sampling designs.

The role of landscape and history on the genetic structure of peripheral populations of the Near Eastern fire salamander, Salamandra infraimmaculata , in Northern Israel Abstract Genetic studies on core versus peripheral populations have yielded many patterns. This diversity in genetic patterns may reflect diversity in the meaning of "peripheral populations" as defined by geography, gene flow patterns, historical effects, and ecological conditions. Populations at the lower latitude periphery of a species' range are of particular concern because they may be at increased risk for extinction due to global climate change. In this work we aim to understand the impact of landscape and ecological factors on different geographical types of peripheral populations with respect to levels of genetic diversity and patterns of local population differentiation. We examined three geographical types of peripheral populations of the endangered salamander, Salamandra infraimmaculata, in Northern Israel, in the southernmost periphery of the genus Salamandra, by analyzing the variability in 15 microsatellite loci from 32 sites. Our results showed that: (1) genetic diversity decreases towards the geographical periphery of the species' range; (2) genetic diversity in geographically disjunct peripheral areas is low compared to the core or peripheral populations that are contiguous to the core and most likely affected by a founder effect; (3) ecologically marginal conditions enhance population subdivision. The patterns we found lead to the conclusion that genetic diversity is influenced by a combination of geographical, historical, and ecological factors. These complex patterns should be addressed when prioritizing areas for conservation.

A quantitative genetic analysis of life-history traits and lifetime reproductive success in reintroduced Chinook salmon Abstract Reintroductions are widely implemented as a means of reestablishing wild populations and genetic parentage methods can be used in concert with these efforts to monitor and evaluate efficacy. In addition to understanding demographic outcomes, reconstructed pedigrees, when combined with phenotypic data, can provide insight into the adaptive potential of reintroduced individuals. Here, we examined the heritability and evolvability of life-history traits and lifetime reproductive success in two threatened Chinook salmon populations undergoing reintroduction to historical habitats above dams in Oregon, USA, using previously-developed multigenerational genetic pedigrees. All of the examined life-history traits: length-at-maturity, age-at-maturity, and arrival timing to the spawning grounds, and lifetime reproductive success exhibited significant narrow-sense heritabilities and evolvabilities. There was also a detectable influence of parental effects (i.e., paternal or maternal effects) on life-history trait variation, suggesting that in addition to genetic effects, nongenetic inheritance mechanisms are influencing life-history diversity in the populations. Additionally, our analyses revealed evidence of natural selection on the date of reintroduction, although the form and intensity of selection differed between the two populations; the forms of selection also point to the potential for phenotype-environment mismatch under some conditions. Overall, our results suggest that these threatened Chinook salmon populations exhibit significant adaptive potential, a factor that should be important to the longer-term success of recovery efforts.

Genetic mixture analyses in support of restoration of a high value recreational fishery for rainbow trout ( Oncorhynchus mykiss ) from a large lake in interior British Columbia Abstract Genetic mixture analysis is an important tool to apportion catch amongst potential component populations contributing to a fishery. We used variation at 10 microsatellite DNA loci to assess the level of genetic divergence between two ecotypes of rainbow trout (Oncorhynchus mykiss) that naturally co-occur in Kootenay Lake, southeastern British Columbia, and to exploit such divergence in a mixture analysis. One form, "Gerrard" rainbow trout, historically matured at sizes greater than 60 cm and 5 kg, spawns at a lake outlet after upstream migration in a large river tributary to Kootenay Lake, and is highly prized in the recreational fishery. The other form, "non-Gerrard" rainbow trout, is also native to the lake and matures at smaller sizes and spawns in numerous small streams tributary to Kootenay Lake. Recent declines in growth rate of Gerrard rainbow trout, however, has made them difficult to identify by size in fishery samples. Gerrard (N = 130, 6 sites) and non-Gerrard trout N = 312, 15 sites) were highly divergent from one another (FST = 0.14, P < 0.001) and constituted distinct genetic groups in model-based clustering analyses. Genetic mixture analyses of fishery samples indicated a high degree of accuracy in estimating mixture proportions; 100% Gerrard simulated fisheries were estimated to contain 99.9% Gerrards (95% confidence intervals of 99.8–100%) while 100% non-Gerrard rainbow trout simulated fisheries were estimated to contain 100.0% non-Gerrard trout (100–100%). Across eight fishery creel samples obtained between 2015 and 2017 (N = 527 fish), mixture analysis estimated the fishery to contain an average of 73.4% (95% confidence interval = 68.4–74.6%) Gerrard and 26.6% (23.4–31.6%) non-Gerrard trout. Realistic fishery simulations demonstrated strong agreement with empirical results; the average simulated values for Gerrards was 73.4% (65.9–80.0%) and for non-Gerrards was 26.8% (20.0–34.1%). Assignment tests resulted in an average 98.5% (± 0.066%) assignment confidence; 385 fish from the fishery samples were assigned to the Gerrard group (0.73) and 142 (0.27) to the non-Gerrard rainbow trout genetic group. Fitting length-at-age data for genetically assigned fishery samples to a von Bertalanffy growth model found greatest support for a model employing ecotype-specific L∞ (= 59.6 and 52.9 cm for Gerrards and non-Gerrards, respectively, both P < 0.001), and t0 (= − 1.36 and − 2.58, respectively, both P < 0.05), but a common K (= 0.189, P < 0.001). Our mixture analyses are being used to monitor catches and better understand the feeding and migration biology of these sympatric ecotypes of rainbow trout.

Beyond Bonferroni revisited: concerns over inflated false positive research findings in the fields of conservation genetics, biology, and medicine Abstract In 2006, Narum published a paper in Conservation Genetics emphasizing that Bonferroni correction for multiple testing can be highly conservative with poor statistical power (high Type II error). He pointed out that other approaches for multiple testing correction can control the false discovery rate (FDR) with a better balance of Type I and Type II errors and suggested that the approach of Benjamini and Yekutieli (BY) 2001 provides the most biologically relevant correction for evaluating the significance of population differentiation in conservation genetics. However, there are crucial differences between the original Benjamini and Yekutieli procedure and that described by Narum. After carefully reviewing both papers, we found an error due to the incorrect implementation of the BY procedure in Narum (Conserv Genet 7:783–787, 2006) such that the approach does not adequately control FDR. Since the incorrect BY approach has been increasingly used, not only in conservation genetics, but also in medicine and biology, it is important that the error is made known to the scientific community. In addition, we provide an overview of FDR approaches for multiple testing correction and encourage authors first and foremost to provide effect sizes for their results; and second, to be transparent in their descriptions of multiple testing correction. Finally, the impact of this error on conservation genetics and other fields will be study-dependent, as it is related to the number of true to false positives for each study.

Broad-scale patterns of genetic diversity and structure in a foundational salt marsh species black needlerush ( Juncus roemerianus ) Abstract Clonal and genetic diversity in foundational plant species are critical for species resiliency and ecosystem processes, both of which contribute to restoration success; however, genetic data is often lacking for common plant species used in many restoration practices. Only a few plant species dominate salt marshes, ecologically and economically valuable ecosystems targeted for restoration due to global decline. Despite being a foundational species in southeastern United States salt marshes black needlerush (Juncus roemerianus Scheele) is understudied, especially in comparison to the co-occurring smooth cordgrass (Spartina alterniflora Loisel). We used a panel of 18 microsatellite markers on 849 samples of J. roemerianus collected at 17 sites across a majority of the species range from Mississippi to South Carolina to measure clonal and genetic diversity and characterize population structure. Results are consistent with previous genetic studies on J. roemerianus and other clonal plant species in that clonal and genetic diversity were higher than expected based on life history literature, with an average genotypic diversity (GD) of 0.67 and average observed heterozygosity (HO) of 0.56. Differences in diversity between the ecologically and environmentally divergent Gulf (GD = 0.64, HO = 0.52) and Atlantic (GD = 0.80, HO = 0.45) coasts suggest that life history strategy may vary by environment. Hierarchical structure was apparent across the study area, with STRUCTURE analyses identifying three genetic clusters that further subdivided into five clusters and a broad transition zone. The scale of this genetic differentiation should be considered in sourcing plants for salt marsh restoration efforts.

Chromosome Research

MiR-153 regulates cardiomyocyte apoptosis by targeting Nrf2/HO-1 signaling Abstract MicroRNAs (miRNAs) play various roles in the regulation of human disease, including cardiovascular diseases. MiR-153 has been previously shown to be involved in regulating neuron survival during cerebral ischemia/reperfusion (I/R) injury. However, whether miR-153 is involved in I/R-induced cardiomyocyte apoptosis remains to be elucidated. In this study, we aimed to explore the role of miR-153 in the regulation of I/R-induced cardiomyocyte apoptosis and to investigate the miR-153-mediated molecular signaling pathway responsible for its effect on cardiomyocytes using an oxygen-glucose deprivation and reoxygenation (OGD/R) cellular model. We found that OGD/R treatment induced significant upregulation of miR-153 in cardiomyocytes causing reactive oxygen species (ROS) production and cell apoptosis signaling activation and subsequently leading to cardiomyocyte apoptosis. Suppression of miR-153 protected cardiomyocytes against OGD/R treatment. We further identified that nuclear factor-like 2 (Nrf2) is a functional target of miR-153. Nrf2/ heme oxygenase-1 (HO-1) signaling plays a critical role in miR-153 regulated OGD/R-induced cardiomyocyte apoptosis. Our study indicates that the inhibition of miR-153 or restoration of Nrf2 may serve as a potential therapeutic strategy for ischemia/reperfusion injury prevention.

Genome-wide DNA copy number analysis and targeted transcriptional analysis of canine histiocytic malignancies identifies diagnostic signatures and highlights disruption of spindle assembly complex Abstract Canine histiocytic malignancies (HM) are rare across the general dog population, but overrepresented in certain breeds, such as Bernese mountain dog and flat-coated retriever. Accurate diagnosis relies on immunohistochemical staining to rule out histologically similar cancers with different prognoses and treatment strategies (e.g., lymphoma and hemangiosarcoma). HM are generally treatment refractory with overall survival of less than 6 months. A lack of understanding regarding the mechanisms of disease development and progression hinders development of novel therapeutics. While the study of human tumors can benefit veterinary medicine, the rarity of the suggested orthologous disease (dendritic cell sarcoma) precludes this. This study aims to improve the understanding of underlying disease mechanisms using genome-wide DNA copy number and gene expression analysis of spontaneous HM across several dog breeds. Extensive DNA copy number disruption was evident, with losses of segments of chromosomes 16 and 31 detected in 93% and 72% of tumors, respectively. Droplet digital PCR (ddPCR) evaluation of these regions in numerous cancer specimens effectively discriminated HM from other common round cell tumors, including lymphoma and hemangiosarcoma, resulting in a novel, rapid diagnostic aid for veterinary medicine. Transcriptional analysis demonstrated disruption of the spindle assembly complex, which is linked to genomic instability and reduced therapeutic impact in humans. A key signature detected was up-regulation of Matrix Metalloproteinase 9 (MMP9), supported by an immunohistochemistry-based assessment of MMP9 protein levels. Since MMP9 has been linked with rapid metastasis and tumor aggression in humans, the data in this study offer a possible mechanism of aggression in HM.

Fluorescence in situ hybridization in plants: recent developments and future applications Abstract Fluorescence in situ hybridization (FISH) was developed more than 30 years ago and has been the most paradigm-changing technique in cytogenetic research. FISH has been used to answer questions related to structure, mutation, and evolution of not only individual chromosomes but also entire genomes. FISH has served as an important tool for chromosome identification in many plant species. This review intends to summarize and discuss key technical development and applications of FISH in plants since 2006. The most significant recent advance of FISH is the development and application of probes based on synthetic oligonucleotides (oligos). Oligos specific to a repetitive DNA sequence, to a specific chromosomal region, or to an entire chromosome can be computationally identified, synthesized in parallel, and fluorescently labeled. Oligo probes designed from conserved DNA sequences from one species can be used among genetically related species, allowing comparative cytogenetic mapping of these species. The advances with synthetic oligo probes will significantly expand the applications of FISH especially in non-model plant species. Recent achievements and future applications of FISH and oligo-FISH are discussed.

Ribosomal DNA-connecting ribosome biogenesis and chromosome biology Abstract Ribosomal DNA, the topic of this special issue, has long fascinated biologists. The RNA products of the ribosomal DNA are the ribosomal RNAs that are part of the ribosome. In this special issue, we focus on the sequence, molecular organization, repair, stability, copy number, and peculiar genetics of this region of the genome. The locus can impact not only the translational capability of cells, but also genome organization, stability and integrity, providing a link between translation and chromosome biology.

Dosage effects of human ribosomal genes (rDNA) in health and disease Abstract Human ribosomal RNA genes encoding a pre-transcript of the three major ribosomal RNA (18S, 5.8S, and 28S rRNA) are tandemly repeated in human genome. Their total copy number varies from 250 to 670 per diploid genome with a mean of approximately 420 copies, but only a fraction of them is transcriptionally active. The functional consequences of human ribosomal RNA gene dosage are not widely known and often assumed to be negligible. Here, we review the facts of rRNA gene dosage effects on normal growth and aging, stress resistance of healthy individuals, and survivability of patients with chromosomal abnormalities, as well as on the risk and severity of some multifactorial diseases with proven genetic predisposition. An original hypothesis that rRNA gene dosage can be a modulating factor involved in the pathogenesis of schizophrenia and rheumatoid arthritis is put forward.

Keeping ribosomal DNA intact: a repeating challenge Abstract More than half of the human genome consists of repetitive sequences, with the ribosomal DNA (rDNA) representing two of the largest repeats. Repetitive rDNA sequences may form a threat to genomic integrity and cellular homeostasis due to the challenging aspects of their transcription, replication, and repair. Predisposition to cancer, premature aging, and neurological impairment in ataxia-telangiectasia and Bloom syndrome, for instance, coincide with increased cellular rDNA repeat instability. However, the mechanisms by which rDNA instability contributes to these hereditary syndromes and tumorigenesis remain unknown. Here, we review how cells govern rDNA stability and how rDNA break repair influences expansion and contraction of repeat length, a process likely associated with human disease. Recent advancements in CRISPR-based genome engineering may help to explain how cells keep their rDNA intact in the near future.

Cytogenetic instability of chromosomal nucleolar organizer regions (NORs) in cloned mouse L929 fibroblasts Abstract Ribosomal DNA (rDNA) gene codes for 18S, 5.8S, and 28S rRNA form tandem repetitive clusters, which occupy distinct chromosomal loci called nucleolar organizer regions (NORs). The number and position of NORs on chromosomes are genetic characteristics of the species although within a cell, the NOR sizes can significantly vary due to loss or multiplication of rDNA copies. In the current study, we used mouse L929 fibroblasts, the aneuploid cells which differ in the FISH- and Ag-NOR numbers, to examine whether the parental NOR variability is inherited in clones. By statistical analysis, we showed that the cloned fibroblasts were able to restore the NOR numerical characteristics of the parental cells after long-term culturing. These results support the idea that mammalian cells may have mechanisms which control the number and activity of NORs at the population level. In L929 fibroblasts, we also regularly observed laterally asymmetry of FISH-NORs that evidenced in an unequal distribution of the mother rDNA copies between the daughter cells in mitosis.

Ribosomal DNA and the nucleolus in the context of genome organization Abstract The nucleolus constitutes a prominent nuclear compartment, a membraneless organelle that was first documented in the 1830s. The fact that specific chromosomal regions were present in the nucleolus was recognized by Barbara McClintock in the 1930s, and these regions were termed nucleolar organizing regions, or NORs. The primary function of ribosomal DNA (rDNA) is to produce RNA components of ribosomes. Yet, ribosomal DNA also plays a pivotal role in nuclear organization by assembling the nucleolus. This review is focused on the rDNA and associated proteins in the context of genome organization. Recent advances in understanding chromatin organization suggest that chromosomes are organized into topological domains by a DNA loop extrusion process. We discuss the perspective that rDNA may also be organized in topological domains constrained by structural maintenance of chromosome protein complexes such as cohesin and condensin. Moreover, biophysical studies indicate that the nucleolar compartment may be formed by active processes as well as phase separation, a perspective that lends further insight into nucleolar organization. The application of the latest perspectives and technologies to this organelle help further elucidate its role in nuclear structure and function.

Implications of sequence variation on the evolution of rRNA Abstract The evolution of the multi-copy family of ribosomal RNA (rRNA) genes is unique in regard to its genetics and genome evolution. Paradoxically, rRNA genes are highly homogenized within and between individuals, yet they are globally distinct between species. Here, we discuss the implications for models of rRNA gene evolution in light of our recent discoveries that ribosomes bearing rRNA sequence variants can affect gene expression and physiology and that intra-individual rRNA alleles exhibit both context- and tissue-specific expression.

Nucleoli in embryos: a central structural platform for embryonic chromatin remodeling? Abstract Nucleoli are the site of ribosomal RNA production and subunit assembly. In contrast to active nucleoli in somatic cells, where three basic sub-compartments can be observed, mammalian oocytes and early embryos contain atypical nucleoli termed "nucleolus-like bodies" or "nucleolus precursor bodies", respectively. Unlike their somatic counterparts, these structures are composed of dense homogenous fibrillar material and exhibit no polymerase activity. Irrespective of these unusual properties, they have been shown to be absolutely essential for embryonic development, as their microsurgical removal results in developmental arrest. Historically, nucleolus-like and nucleolus precursor bodies have been perceived as passive storage sites of nucleolar material, which is gradually utilized by embryos to construct fully functional nucleoli once they have activated their genome and have started to produce ribosomes. For decades, researchers have been trying to elucidate the composition of these organelles and provide the evidence for their repository role. However, only recently has it become clear that the function of these atypical nucleoli is altogether different, and rather than being involved in ribosome biogenesis, they participate in parental chromatin remodeling, and strikingly, the artificial introduction of a single NPB component is sufficient to rescue the developmental arrest elicited by the NPB removal. In this review, we will describe and summarize the experiments that led to the change in our understanding of these unique structures.

Cell Biology and Toxicology

Off-target challenge for base editor-mediated genome editing

Off-target genome editing: a new discipline of gene science and a new of class medicine Abstract With an increasing growth of genome editing, off-target effects such as non-specific genetic modifications resulting from the designed process of genome editing become a new discipline of gene science and new class medicine. The degree of short-term and long-term side effects and toxicity or dynamics of the primary and secondary off-target genome editing varies with the application of different methodologies of gene editing and measuring, readouts of genetic modifications, or comparison reference. Measurements of dynamic off-target effects caused directly or indirectly by genome editing are critical in clinical application of gene editing. The quality of genome editing methods is one of the decisive factors in the occurrence of off-target effects. Mechanisms by which off-target effects of genome editing occurs are more complex and comprehensive than we expected. The heterogeneity of off-target effects of gene-edited cells at single-cell levels should be defined during the development and formation of cell clusters. In addition to off-target effects on gene-edited cells per se, alterations of gene sequence, structure, dimension, and function of related regulators caused by off-target effects may also influence intercellular communications and interactions between gene-edited cells, between gene-edited cells and non-edited cells, or between non-edited cells. Thus, controlling, measuring, defining, categorizing, and predicting off-target genome editing need to be standardized and prioritized before clinical application of gene editing.

Resveratrol inhibits the proliferation of estrogen receptor-positive breast cancer cells by suppressing EZH2 through the modulation of ERK1/2 signaling Abstract Enhancer of zeste homolog 2 (EZH2) is frequently overexpressed in breast cancer and plays an important role in maintaining the cell proliferative capacity. However, the mechanisms underlying the transcriptional regulation of EZH2 in estrogen receptor (ER)-positive breast cancer cells remain unclear. The antitumor effects of resveratrol have been reported. However, whether EZH2 was involved in these effects needs further exploration. Here, we showed that EZH2 is required for estrogen-induced cell proliferation in ER-positive breast cancer. Exposure to 17β-estradiol (E2) upregulated EZH2 via ERα signaling, and this effect was blocked by U0126, a MEK inhibiter. Resveratrol inhibited the proliferation and colony formation in ER-positive breast cancer cells and downregulated EZH2 through inhibition of phospho-ERK1/2. These findings indicated that ERK1/2 and ER signaling–mediated EZH2 upregulation is crucial for the proliferation of ER-positive breast cancer cells. The suppression of EZH2 expression by ERK1/2 dephosphorylation is important for the antiproliferative activities of resveratrol against ER-positive breast cancer cells.

Olive leaf extract counteracts epithelial to mesenchymal transition process induced by peritoneal dialysis, through the inhibition of TGFβ1 signaling Abstract The mesothelial cells (MCs) play an important role in the morpho-functional alterations of the peritoneal membrane (PM) undergoing peritoneal dialysis (PD). MCs, through the epithelial-mesenchymal transition process (EMT), progressively acquire a myofibroblast-like phenotype, promoting peritoneal fibrosis (PF) and failure of peritoneal membrane function. Transforming growth factor β1 (TGFβ1), through canonical and non-canonical pathways, promotes the epithelial-mesenchymal transition (EMT) process leading to PF. To investigate the therapeutic potential of an olive leaf extract (OLE) on preserving peritoneal membrane function, we evaluated the effect of OLE on the TGFβ1-induced EMT in mesothelial cells, Met5A, and elucidated the underlying molecular mechanisms. As assessed by changes in the expression of epithelial, mesenchymal, and fibrotic cell markers (such as E-cadherin, N-cadherin, α-SMA, fibronectin, vimentin), levels of matrix metalloproteinases (MMP2 and MMP9), and cell migration, OLE inhibited the TGFβ1-induced EMT. Importantly, the beneficial effect of OLE was mediated by reduction of the TGFβ1-induced activation of Smad2/3 signaling and the mitigation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Smad/non-Smad signaling pathways, activated by TGFβ1, both reduce expression of epithelial marker E-cadherin which has a crucial role in EMT initiation. Interestingly, we observed that in presence of OLE activity of the E-cadherin, promoter was increased and concomitantly OLE reduced the nuclear content of its co-repressor SNAIL. Our results suggest the potential therapeutic of OLE to counteract fibrotic process in peritoneal dialysis patients.

The mechanistic role of oxidative stress in cigarette smoke-induced cardiac stem cell dysfunction and prevention by ascorbic acid Abstract Cigarette smoking causes a vast array of diseases including cardiovascular diseases. Our laboratory focuses on investigating cigarette smoke (CS)-induced cardiovascular malfunction and the responsible mechanisms utilizing the model, c-kit-positive cardiac stem cells (CSCs). The main objective of our study is to investigate whether CS extracts (CSEs) cause impairment of CSC functions via oxidative damage. We hypothesized that CSE, via oxidative modifications of CSC proteins and antioxidant enzymes, can modulate CSC functions and these modifications can be attenuated by ascorbate treatment. Our specific aims are (1) to investigate CSE-induced oxidative modification of CSC proteins via carbonylation, and prevention by ascorbic acid; (2) to investigate CSE-induced oxidative modification of antioxidant enzymes and ascorbic acid-mediated modulations; and (3) to investigate CSE-induced changes in CSC functions and protection by ascorbic acid. CSCs were cultured, and the aqueous extracts of CSE were prepared. CSE-induced modulations of CSC viability, oxidative modification of proteins, and antioxidant enzyme activities were detected using standard assays including Apostain, bromodeoxyuridine, and Oxiblot. CSE caused oxidative modification of CSC proteins, changed antioxidant enzyme levels, attenuated CSC proliferation, and accelerated CSC apoptosis. Ascorbic acid prevented CSE-induced CSC malfunctions, and ascorbic acid therapy might be useful in smoker CSC recipients and to condition CSCs prior to the transplant in the future. Cardiac stem cell therapy is currently undergoing in clinical trials.

Identification of cancer-type specific expression patterns for active aldehyde dehydrogenase (ALDH) isoforms in ALDEFLUOR assay Abstract Aldehyde dehydrogenases (ALDHs) defend intracellular homeostasis by catalyzing the conversion of toxic aldehydes into non-toxic carboxylic acids, which is of particular importance to the self-renewal of stem cells and cancer stem cells. The widely used ALDEFLUOR assay was initially designed to indicate the activity of ALDH1A1 in leukemia and has been demonstrated to detect the enzyme activity of several other ALDH isoforms in various cancer types in recent years. However, it is still elusive which isoforms, among the 19 ALDH isoforms in human genome, are the potential contributors in catalyzing ALDEFLUOR assay in different cancers. In the current study, we performed a screening via overexpressing each ALDH isoform to assess their ability of catalyzing ALDEFLUOR assay. Our results demonstrate that nine isoforms are active in ALDEFLUOR assay, whose overexpression significantly increases ALDH-positive (ALDH+) population. Further analysis of the expression of these active isoforms in various cancers reveals cancer-type specific expression patterns, suggesting that different cancer types may exhibit ALDEFLUOR activity through expression of specific active ALDH isoforms. This study strongly indicates that a detailed elucidation of the functions for each active ALDH isoform in CSCs is necessary and important for a profound understanding of the underlying mechanisms of ALDH-associated stemness.

Spermine protects alpha-synuclein expressing dopaminergic neurons from manganese-induced degeneration Abstract Manganese exposure is among the many environmental risk factors linked to the progression of neurodegenerative diseases, such as manganese-induced parkinsonism. In animal models, chronic exposure to manganese causes loss of cell viability, neurodegeneration, and functional deficits. Polyamines, such as spermine, have been shown to rescue animals from age-induced neurodegeneration in an autophagy-dependent manner; nonetheless, it is not understood whether polyamines can prevent manganese-induced toxicity. In this study, we used two model systems, the Caenorhabditis elegans UA44 strain and SK-MEL-28 cells, both expressing the protein alpha-synuclein (α-syn) to determine whether spermine could ameliorate manganese-induced toxicity. Manganese caused a substantial reduction in the viability of SK-MEL-28 cells and hastened neurodegeneration in the UA44 strain. Spermine protected both the SK-MEL-28 cells and the UA44 strain from manganese-induced toxicity. Spermine also reduced the age-associated neurodegeneration observed in the UA44 strain compared with a control strain without α-syn expression and led to improved avoidance behavior in a functional assay. Treatment with berenil, an inhibitor of polyamine catabolism, which leads to increased intracellular polyamine levels, also showed similar cellular protection against manganese toxicity. While both translation blocker cycloheximide and autophagy blocker chloroquine caused a reduction in the cytoprotective effect of spermine, transcription blocker actinomycin D had no effect. This study provides new insights on the effect of spermine in preventing manganese-induced toxicity, which is most likely via translational regulation of several candidate genes, including those of autophagy. Thus, our results indicate that polyamines positively influence neuronal health, even when exposed to high levels of manganese and α-syn, and supplementing polyamines through diet might delay the onset of diseases involving degeneration of dopaminergic neurons.

Cell–cell communication: old mystery and new opportunity

Biochemical effects of some CeO 2 , SiO 2 , and TiO 2 nanomaterials in HepG2 cells Abstract The potential mammalian hepatotoxicity of nanomaterials was explored in dose-response and structure-activity studies in human hepatic HepG2 cells exposed to between 10 and 1000 μg/ml of five different CeO2, three SiO2, and one TiO2-based particles for 3 days. Various biochemical parameters were then evaluated to study cytotoxicity, cell growth, hepatic function, and oxidative stress. Few indications of cytotoxicity were observed between 10 and 30 μg/ml. In the 100 to 300 μg/ml exposure range, a moderate degree of cytotoxicity was often observed. At 1000 μg/ml exposures, all but TiO2 showed a high degree of cytotoxicity. Cytotoxicity per se did not seem to fully explain the observed patterns of biochemical parameters. Four nanomaterials (all three SiO2) decreased glucose 6-phosphate dehydrogenase activity with some significant decreases observed at 30 μg/ml. In the range of 100 to 1000 μg/ml, the activities of glutathione reductase (by all three SiO2) and glutathione peroxidase were decreased by some nanomaterials. Decreased glutathione concentration was also found after exposure to four nanomaterials (all three nano SiO2 particles). In this study, the more responsive and informative assays were glucose 6-phosphate dehydrogenase, glutathione reductase, superoxide dismutase, lactate dehydrogenase, and aspartate transaminase. In this study, there were six factors that contribute to oxidative stress observed in nanomaterials exposed to hepatocytes (decreased glutathione content, reduced glucose 6-phosphate dehydrogenase, glutathione reductase, glutathione peroxidase, superoxide dismutase, and increased catalase activities). With respect to structure-activity, nanomaterials of SiO2 were more effective than CeO2in reducing glutathione content, glucose 6-phosphate dehydrogenase, glutathione reductase, and superoxide dismutase activities.

Definition of clinical gene tests Abstract Clinical tests of gene sequence, structure, and function are to predict, diagnose, monitor, and prognose human disease–specific phenomes, characters severities, durations, stages, and responses to therapy. The concept and content of gene tests for clinical application mainly include chromosome/chromatins, DNA, and RNA. Structures and functions of chromosomes and chromatins vary among various durations, phases, and conditions, with the priority consideration in clinical gene tests. Sequences and functions of DNA and associated regulators are an important partial of clinical gene test. Another large group of RNA and RNA-associated factors also contribute to gene expression, regulation, and function. DNA/RNA sequencing is used to measure tumor mutation and heterogeneity, recategorize molecular phenomes and types of cancer, or guide and predict target-based therapies. The structure and function of genome dimensions and regulations as well as various factor involvement and contributions should be seriously considered in clinical gene tests, although there are a number of challenges to be overcome, e.g., method sensitivity, specificity, stability, analysis, and clinical significance. It is also critical to have the national and international standardization, guideline, and consortium of sample handling, experimental operation, quality control, data analysis, and clinical interpretation, when clinical gene tests are developed and applied for clinical application. Thus, there is an urgent need to discover and validate those gene tests according to disease phenomes, subtypes, severity, duration, phase, progression, prognosis, and response to therapy.

Cell Biology and Toxicology

Expression of OPA1 and Mic60 genes and their association with mitochondrial cristae morphology in Tibetan sheep Abstract In order to investigate the relationship between the expression of OPA1 and Mic60 genes and the shape of mitochondrial cristae and to explore the mechanism of Tibetan sheep adapting to a high altitude hypoxia environment, we investigate respiratory rate, mitochondrial cristae and the expression of OPA1 and Mic60 in four different tissues (myocardial, skeletal muscle, spleen and kidney) in Tibetan sheep and Small Tail Han sheep. Tibetan sheep had a higher respiratory rate than Small Tail Han sheep (p < 0.01). In the same tissue, the expression of OPA1 and Mic60 was higher (p < 0.05) in Tibetan sheep than Small Tail Han sheep. Between tissues, the expression of OPA1 and Mic60 was found to be lower (p < 0.05) in spleen than the other three tissues in both breeds. Mitochondrial cristae was dense and clear in myocardial and skeletal muscle but was relatively sparse and slightly swollen in kidney. In spleen, cristae was least and swollen and the gap between the cristae was large. The width of the mitochondrial cristae in the spleen was significantly larger than the width between the inner and outer membranes; however, it had little difference in the other three tissues. The width of mitochondrial cristae was significantly larger in the spleen than that in other tissues (p < 0.05). The numbers of mitochondrial cristae in the four tissues of Tibetan sheep were larger than those in Small Tail Han sheep (p < 0.05). The unique characters of the mitochondrial cristae in Tibetan sheep may be related to its adaption to a high altitude hypoxia environment.

Identification of calretinin-expressing retinal ganglion cells projecting to the mouse superior colliculus Abstract In mice, retinal ganglion cells (RGCs), which consist of around 30 subtypes, exclusively transmit retinal information to the relevant brain systems through parallel visual pathways. The superior colliculus (SC) receives the vast majority of this information from several RGC subtypes. The objective of the current study is to identify the types of calretinin (CR)-expressing RGCs that project to the SC in mice. To label RGCs, we performed CR immunoreactivity in the mouse retina after injections of fluorescent dye, dextran into mouse SC. Subsequently, the neurons double-labeled for dextran and CR were iontophoretically injected with the lipophilic dye, DiI, to characterize the detailed morphological properties of these cells. The analysis of various morphological parameters, including dendritic arborization, dendritic field size and stratification, indicated that, of the ten different types of CR-expressing RGCs in the retina, the double-labeled cells consisted of at least eight types of RGCs that projected to the SC. These cells tended to have small-medium field sizes. However, except for dendritic field size, the cells did not exhibit consistent characteristics for the other morphometric parameters examined. The combination of a tracer and single-cell injections after immunohistochemistry for a particular molecule provided valuable data that confirmed the presence of distinct subtypes of RGCs within multiple-labeled RGCs that projected to specific brain regions.

Oxidative stress modulates the expression of apoptosis-associated microRNAs in bovine granulosa cells in vitro Abstract Despite its essential role in ovulation, oxidative stress (OS) has been found to be cytotoxic to cells, while microRNAs (miRNAs) are known as a major regulator of genes involved in cellular defense against cytotoxicity. However, a functional link between OS and miRNA expression changes in granulosa cells (GCs) remains to be investigated. Here, we investigate the OS modulation of apoptosis-associated miRNAs and their biological relevance in bovine GCs. Following the evaluation of cell viability, accumulation of reactive oxygen species (ROS), cytotoxicity and mitochondrial activity, we used a ready-to-use miRNA PCR array to identify differentially regulated miRNAs. The results showed that exposure to 150 μM H2O2 for 4 h creates remarkable signs of OS in GCs characterized by more than 50% loss of cell viability, higher nuclear factor erythroid 2–related factor 2 (NRF2) nuclear translocation, significantly (p < 0.05) higher abundance of antioxidant genes, significantly (p < 0.001) higher accumulation of ROS, lower mitochondrial activity and a higher (p < 0.001) number of apoptotic nuclei compared to that of the control group. miRNA expression analysis revealed that a total of 69 miRNAs were differentially regulated in which 47 and 22 miRNAs were up- and downregulated, respectively, in stressed GCs. By applying the 2-fold and p < 0.05 criteria, we found 16 miRNAs were upregulated and 10 miRNAs were downregulated. Target prediction revealed that up- and downregulated miRNAs potentially targeted a total of 6210 and 3575 genes, respectively. Pathway analysis showed that upregulated miRNAs are targeting the genes involved mostly in cell survival, intracellular communication and homeostasis, cellular migration and growth control and disease pathways. Our results showed that OS modulates the expression of apoptosis-associated miRNAs that might have effects on cellular or molecular damages.

Dental pulp stem cell transplantation ameliorates motor function and prevents cerebellar atrophy in rat model of cerebellar ataxia Abstract Cerebellar ataxias (CA) include a range of neurodegenerative disorders hallmarked by deterioration of the cerebellum. Cell replacement therapy (CRT) offers a potential remedy for the diseases associated with the central nervous system (CNS). This study was designed to assess the neurorestorative/protective effects of dental pulp stem cell (DPSC) implantation on a rat model of CA induced by 3-acetylpyridine (3-AP) as a neurotoxin. To begin, human DPSCs were extracted, cultured and phenotypically characterized. Then, experimental ataxia was induced in 20 male adult rats by a single injection of 3-AP and bilateral DPSC transplantation was performed 3 days after 3-AP administration, followed by stereological analysis of cerebellar layers along with assessment of motor skills and inflammatory response. The findings showed that transplantation of DPSCs in a 3-AP model of ataxia ameliorated motor coordination and muscle activity, increased cerebellar volumes of molecular and granular layers plus white matter, reduced the levels of inflammatory cytokines and thwarted the degeneration of Purkinje cells against 3-AP toxicity. Taken together, human DPSCs could be considered as a suitable candidate for CRT-based therapies with a specific focus on CA.

Human umbilical cord blood mesenchymal stem cells expansion via human fibroblast-derived matrix and their potentials toward regenerative application Abstract Large expansion of human mesenchymal stem cells (MSCs) is of great interest for clinical applications. In this study, we examine the feasibility of human fibroblast-derived extracellular matrix (hFDM) as an alternative cell expansion setting. hFDM is obtained from decellularized extracellular matrix (ECM) derived from in vitro cultured human lung fibroblasts. Our study directly compares conventional platforms (tissue culture plastic (TCP), fibronectin (FN)-coated TCP) with hFDM using umbilical cord blood-derived MSCs (UCB-MSCs). Early cell morphology shows a rather rounded shape on TCP but highly elongated morphology on hFDM. Cell proliferation demonstrates that MSCs on hFDM were significantly better compared to the others in both 10 and 2% serum condition. Cell migration assay suggests that cell motility was improved and a cell migration marker CXCR4 was notably up-regulated on hFDM. MSCs differentiation into osteogenic lineage on hFDM was also very effective as examined via gene expression, von Kossa staining and alkaline phosphatase activity. In addition, as the MSCs were expanded on each substrate, transferred to 3D polymer mesh scaffolds and then cultivated for a while, the data found better cell proliferation and more CXCR4 expression with MSCs pre-conditioned on hFDM. Moreover, higher gene expression of stemness and engraftment-related markers was noticed with the hFDM group. Furthermore when UCB-MSCs expanded on TCP or hFDM were injected into emphysema (a lung disease) animal model, the results indicate that MSCs pre-conditioned on hFDM (with 2% serum) retain more advanced therapeutic efficacy on the improvement of emphysema than those on TCP. Current works demonstrate that compared to the conventional platforms, hFDM can be a promising source of cell expansion with a naturally derived biomimetic ECM microenvironment and may find some practical applications in regenerative medicine.

A novel serotonin-containing tuft cell subpopulation in mouse intestine Abstract In this study, a novel subset of doublecortin-like kinase 1 (DCLK1)-immunoreactive (IR) tuft cells that also contain serotonin (5-hydroxytryptamine, 5HT) is described, in terms of their number, regional distribution, possible synthesis or reuptake of 5HT and proximity to 5-HT-containing enterochromaffin (EC) cells. The small intestine from C57BL/6J mice was divided into five segments while the large intestine was kept undivided. Double immunostaining was used to estimate numbers and topographic distribution of 5HT-IR (DCLK1/5HT) tuft cells and their possible expression of tryptophan hydroxylase (TPH) and serotonin transporter (SERT). Also, possible contacts between tuft cells and 5HT-IR EC cells were studied. In the small intestine, up to 80% of all tuft cells were identified as DCLK1/5HT-IR; in the large intestine, such cells were rare. The highest number of DCLK1/5HT-IR cells was found in the upper small intestine. The numbers of DCLK1/5HT-IR cells gradually decreased distally. DCLK1-IR tuft cells were not found to contain TPH, the rate-limiting enzyme in 5HT synthesis. SERT, the selective transporter for 5HT reuptake, could not convincingly be demonstrated in tuft cells. In villi and crypts, 3% and 10%, respectively, of all DCLK1-IR cells were in close proximity to EC cells. EC cells in close proximity to DCLK1-IR cells were, in villi and crypts, 3 and 8%, respectively. We conclude that DCLK1/5HT-IR cells constitute a novel subset of tuft cells that may have unique roles in the GI tract.

Vascularized composite allograft rejection is delayed by infusion of IFN-γ-conditioned BMSCs through upregulating PD-L1 Abstract Mesenchymal stromal cells (MSCs) have been applied in prevention from allograft rejection based on their immunomodulatory effects. However, conflicting results have been presented among recent studies, for which one possibility being acknowledged is that the exact effect is determined by the microenvironment when MSCs are applied in vivo. Using a hind limb composite tissue allograft model, we investigate the influence of IFN-γ-preconditioning on the immunomodulatory effects of MSCs and the subsequent allograft survival. Firstly, different doses of IFN-γ were respectively used to incubate with bone marrow–derived MSCs (BMSCs). We found that IFN-γ altered the expression of PD-L1, a major suppressor gene in the immune system during allograft rejection, in a strictly dose-dependent manner in BMSCs. Ten nanograms per milliliter IFN-γ-incubated BMSCs significantly stimulated PD-L1 expression and suppressed T cell proliferation and differentiation, while 50 ng/mL IFN-γ-incubated BMSCs sharply reduced PD-L1 expression. Moreover, we observed that, in contrast to the naive BMSC transplantation group, BMSCs pre-conditioned with 10 ng/mL IFN-γ (BMSCs-IFN-γ) significantly delayed the allograft rejection in vivo. In vitro mixed lymphocyte reaction (MLR) indicated that BMSCs-IFN-γ inhibited T lymphocyte proliferation and activation via PD-L1. Moreover, BMSCs-IFN-γ did not influence the proliferation and activation of T lymphocytes when PD-L1 protein was neutralized by the PD-L1 antibody. These data collectively reveal a role of recipient ongoing immune microenviroment in BMSC-based immunesuppressive therapy. Graphical abstract ᅟ

Sox11-modified mesenchymal stem cells accelerate cartilage defect repair in SD rats Abstract Cartilage has a limited capacity to heal. Previously, we have shown that overexpression of Sox11 in rMSCs (Rat Mesenchymal Stem Cells) by lentivirus-mediated gene transfer leads to enhanced tri-lineage differentiation and accelerated bone formation in fracture model of rats. We observed that the fracture repair in the rats that received Sox11-modified rMSCs injection proceeded through an endochondral ossification process much faster than those in the control groups. However, the detailed role of Sox11 in rMSCs chondrogenic differentiation, as well as cartilage defect, is still not clearly clarified. Therefore, this study tests the hypothesis that Sox11 promotes chondrogenesis and cartilage defect repair by regulating β-catenin. Sox11 was transduced into rMSCs using lentiviruses. The expression levels of β-catenin and its downstream genes were evaluated by quantitative RT-PCR. The transcriptional activation of β-catenin was proved by dual-luciferase reporter assay and co-immunoprecipitation was performed to evaluate Sox11-β-catenin interaction. In addition, a cartilage defect model in SD rats was used to evaluate the cartilage regeneration ability of Sox11-modified rMSCs in vivo. We found that Sox11 transcriptionally activated β-catenin expression and discovered the core promoter region (from − 242 to − 1414) of β-catenin gene for Sox11 binding. In addition, Sox11 might regulate β-catenin at the post-transcriptional level by protein-protein interaction. Finally, using a cartilage defect model in rats, we found Sox11-modified rMSCs could improve cartilage regeneration. Taken together, our study shows that Sox11 is an important regulator of chondrogenesis and Sox11-modified rMSCs may have clinical implication for accelerating cartilage defect healing.

Salutary effect of fenofibrate on type 1 diabetic retinopathy via inhibiting oxidative stress–mediated Wnt/β-catenin pathway activation Abstract Fenofibrate has been shown to have therapeutic effects on diabetic retinopathy (DR). Our previous studies demonstrated that the oxidative stress–activated Wnt/β-catenin pathway plays a pathogenic role in diabetic complications. In the present study, we evaluate the effect and mechanism of fenofibrate on regulating the oxidative stress–activated Wnt/β-catenin pathway by using the genetic type 1 diabetes model of C57BL/6J-Ins2Akitamice and high glucose (HG)–treated ARPE-19. Our results demonstrated that retinal phosphorylation of LRP6 and nuclear β-catenin were increased in C57BL/6J-Ins2Akita mice suggesting activation of Wnt/β-catenin signaling. Meanwhile, C57BL/6J-Ins2Akita showed upregulation of oxidant enzyme Nox4 and Nox2 and downregulation of antioxidant enzyme SOD1 and SOD2. All these alterations were reversed in C57BL/6J-Ins2Akitamice with fenofibrate treatment. Moreover, fenofibrate significantly ameliorated diabetes-induced retinal vascular leakage in C57BL/6J-Ins2Akita mice. In cultured ARPE-19, fenofibrate decreased HG-induced Nox2 and Nox4 upregulation, attenuated SOD1 and SOD2 downregulation and inhibited LRP6 phosphorylation. Moreover, activation of Wnt/β-catenin by Wnt3a conditional medium (WCM) reduced SOD1 and SOD2 and did not affect Nox2 and Nox4. Fenofibrate suppressed WCM-induced LRP6 phosphorylation and reversed SOD downregulation. Importantly, Nox4 overexpression directly phosphorylated LPR6 in ARPE19; conversely, Nox4 knockdown suppressed HG-induced LPR6 phosphorylation. Taken together, Nox-mediated oxidative stress contributes to Wnt/β-catenin activation in DR. Fenofibrate ameliorated DR through coordinate attenuation of oxidative stress and blockade of Wnt/β-catenin signaling.

Early injection of human adipose tissue-derived mesenchymal stem cell after inflammation ameliorates dextran sulfate sodium-induced colitis in mice through the induction of M2 macrophages and regulatory T cells Abstract Inflammatory bowel diseases (IBDs) are sometimes refractory to current therapy or associated with severe adverse events during immunosuppressive therapy; thus, new therapies are urgently needed. Recently, mesenchymal stem cells (MSCs) have attracted attention based on their multitude of functions including anti-inflammatory effects. However, proper timing of MSC therapy and the mechanisms underlying the therapeutic effects of MSCs on colitis are not fully elucidated. Human adipose tissue-derived mesenchymal stem cells (hAdMSCs; 1 × 106) were administrated via the tail vein on day 3 (early) or 11 (delayed) using a 7-day dextran sulfate sodium (DSS)-induced mouse model of colitis. The effects were evaluated based on colon length, disease activity index (DAI) and histological score. Cytokine-encoding mRNA levels T cells and macrophages were evaluated by real-time PCR and flow cytometry. Regarding the timing of administration, early (day 3) injection significantly ameliorated DSS-induced colitis in terms of both DAI and histological score, compared to those parameters with delayed (day 11) injection. With early cell injection, the tissue mRNA levels of anti-inflammatory cytokine genes (Il10, Tgfb) increased, whereas those of inflammatory cytokine genes (Il6, Tnfa and Il17a) decreased significantly. Regarding the associated mechanism, hAdMSCs suppressed T cell proliferation and activation in vitro, increased the number of regulatory T cells in vivo and changed the polarity of macrophages (into the anti-inflammatory M2 phenotype) in vitro. Timing of injection is critical for the effective therapeutic effects of hAdMSCs. Furthermore, part of the associated mechanism includes T cell activation and expansion and altered macrophage polarization.

Aminoacids

Clinical significance of LOXL4 expression and features of LOXL4-associated protein–protein interaction network in esophageal squamous cell carcinoma Abstract Lysyl oxidase-like 4 (LOXL4), a member of the LOX family proteins, catalyzes oxidative deamination of lysine residues in collagen and elastin, which are responsible for maintaining extracellular matrix homeostasis. In this study, the mRNA expression of LOXL4 in seven esophageal squamous cell carcinoma (ESCC) cell lines and 15 ESCC pairs of clinical samples were examined. Furthermore, LOXL4 protein levels in the ESCC cell lines were determined using western blotting. With the use of immunofluorescence, LOXL4 was observed to be localized primarily in the cytoplasm, but was also present in the nucleus. In addition, the results indicated that the upregulated expression of LOXL4 was associated with poor survival in patients with ESCC even following curative resection (P = 0.010). Similar Kaplan–Meier estimator curves for proteins that interact with LOXL4, SUV39H1 (P = 0.014) and COL2A1 (P = 0.011), were plotted. The analyses based on the protein–protein interaction network depicted the expression of LOXL4 and its associated proteins as well as their functions, suggesting that LOXL4 and its associated proteins may serve a significant role in the development and progression of ESCC. In conclusion, the results of the present study suggest that LOXL4 is a potential biomarker for patients with ESCC, as well as SUV39H1 and COL2A1, and high expression levels of these genes are associated with poor prognosis in patients with ESCC.

Silver cluster–amino acid interactions: a quantum-chemical study Abstract Binding of silver ion (Ag+) and two atomic neutral silver cluster (Ag2) with a set of amino acids has been studied using Density Functional Theory (DFT) and ab initio MP2 method. We show that binding energy with Ag2 is higher for deprotonated anionic amino acids. Cysteine, aspartic acid, and tyrosine with deprotonated side chain exhibit the highest binding energy (Gbind) values among all the amino acids: − 30.1 kcal mol−1, − 30.7 kcal mol−1, and − 30.9 kcal mol−1, respectively. Binding energies of deprotonated cysteine, glutamic acid, tyrosine, and aspartic acid with silver ion Ag+ are reported here for the first time. Natural bond orbital (NBO) analysis has also been performed to calculate charge transfer, natural populations, occupancies, and Wiberg bond indices of the amino acid–Ag2 complexes. Atoms-in-molecules (AIM) theory was used to establish the nature of interactions. It was shown that, in most cases, the bonds between amino acid and Ag2 cluster are partially electrostatic and partially covalent.

Elevated circulating asymmetric dimethylarginine levels in rheumatoid arthritis: a systematic review and meta-analysis Abstract Rheumatoid arthritis (RA) patients have increased risk of cardiovascular disease (CVD) death. Elevated asymmetric dimethylarginine (ADMA) levels have been reported to be an independent predictor of CVD morbidity and mortality, however, the role of ADMA in RA remains undetermined. To derive a more accurate estimation on circulating ADMA levels in RA patients, a meta-analysis was performed. Embase, PubMed, and The Cochrane Library database (up to October 7 2018) were used to acquire published literatures. Heterogeneity test was performed by the Q statistic and quantified using I2. Publication bias was evaluated using a funnel plot and Egger's linear regression test. A total of 174 articles were identified, 16 studies with 1365 subjects (666 RA patients and 699 healthy individuals) were ultimately included. Plasma/serum ADMA levels appeared to be higher in RA patients than healthy controls (SMD = 0.84, 95% CI 0.32, 1.35). By assessing the BMI, age, disease duration and disease activity as subgroups, BMI ≥ 24 and BMI < 24 groups both showed elevated ADMA levels than controls, disease duration ≥ 8, age < 50 and disease activity ≥ 3.2 and < 5.1 group had a higher ADMA level than control groups. However, disease duration < 8, disease activity ≥ 5.1 and age ≥ 50 groups showed no difference between two groups. Circulating ADMA levels are higher in RA patients compared with healthy controls. In addition, ADMA levels are influenced by age, disease duration and disease activity.

Maternal l -proline supplementation during gestation alters amino acid and polyamine metabolism in the first generation female offspring of C57BL/6J mice Abstract We recently reported that dietary supplementation with l-proline (proline) during gestation improved embryonic survival in C57BL/6J mice. The objective of the present study was to test the hypothesis that the effect of maternal proline supplementation on embryonic survival can be carried forward to the first generation female offspring. In the F0 generation, pregnant dams were fed a purified diet supplemented with 0 (control) or 5 g proline/kg diet. The F1 female adult offsprings were bred to fertile males. Fetal survival at embryonic day (E)12.5 and reproductive outcomes at term birth were recorded. The concentrations of amino acids, ammonia, and urea in plasma and amniotic fluid, as well as concentrations of polyamines in placental tissues and amniotic fluid at E12.5 were determined. Results showed that the F1 generation female offspring from proline-supplemented dams had higher (P < 0.05) concentrations of glutamate and taurine in plasma; of putrescine and spermidine in placental tissues; and of glycine, taurine, and spermidine in amniotic fluid at E12.5, as compared with F1 generation female offsprings from dams without proline supplementation. Concentration of proline in the plasma of offspring mice from proline-supplemented dams were lower (P < 0.05), as compared with the control group. No differences in fetal survival, reproductive outcomes, or concentrations of ammonia and urea in plasma and amniotic fluid were observed between the two groups of F1 female offspring. Collectively, our results indicate that the benefits of maternal proline supplementation during gestation on improving embryonic survival and fetal growth in F0 females are not transmitted to their F1 generation females.

Comparative stability of ficin and papain in acidic conditions and the presence of ethanol Abstract Proteolytic enzymes are used for proteolysis and peptide synthesis which can be run in various conditions including low pH value and the presence of ethanol. The most common cysteine protease applied in acidic–alcoholic conditions is well-characterized papain. Ficin, which is closely related to papain in terms of proteolytic activity and substrate specificity, could potentially be applied in the alcoholic beverage industry and peptide synthesis. The aim of this study was to compare papain and ficin stability in process conditions. Comparative stability study showed that ficin as a mixture of different isoforms has a broader range of stability in respect of pH and cold storage stability, in comparison to papain. It retains about 70% of initial activity after 3-week cold storage at low pH and in the presence of ethanol. Unlike ficin, papain loses about 70% of initial activity in the same incubation period as it is more prone to non-native aggregation that was confirmed by FTIR analysis. The presence of multiple isoforms of ficin stabilizes the protease against cold denaturation and aggregation, making it more suitable for biotechnological and laboratory usage than single papain isoform. It is more cold-stable in alcoholic–acidic and acidic conditions suggesting possible replacement of papain with even lower enzyme concentration.

Computational characterization of structural and functional roles of DREB1A , DREB1B and DREB1C in enhancing cold tolerance in rice plant Abstract Rice serves as the major food for almost half of the world population. Because of its origin in the tropical and subtropical area, rice is more sensitive towards cold stress. Three homologs of DREB1, namely DREB1A, DREB1B and DREB1C are induced Queryduring cold stress and after binding with GCC-box in the promoter region of the target gene, they enhance cold tolerance in rice plants. Though the majority of DREBs bind GCC-box, the degree of activation varies among DREBs. The protein encoded via these three transcription factors contains a common domain, namely AP2/ERF. In silico method was utilised to predict 3D structure of each AP2/ERF domain. The molecular dynamic analysis suggests, under the normal environmental condition, in each AP2/ERF domain, a positive correlation exists between β-strands and the movement of C-α is constrained. However, during cold stress, when AP2/ERF domain binds with GCC-box present in the promoter region of the target gene, mean pressure of each three AP2/ERF domain gets lowered and final potential energy increases. A positive correlation between β-strands gets disrupted and C-α experiences random movement suggesting enhanced activity of DREB1A, DREB1B and DREB1C during cold stress and enhancement of cold tolerance in plants. Further, MM/PBSA calculations for protein–DNA affinities reveal that, due to lack of α2 in DREB1C, the binding affinity of GCC-box with AP2/ERF domain of DREB1A > DREB1B > DREB1C. Thus, due to a better binding affinity with GCC-box, DREB1A and DREB1B can be utilised in near future for increasing cold tolerance of rice plant and increasing yield.

The additive effect of type 2 diabetes on fibrinogen, von Willebrand factor, tryptophan and threonine in people living with HIV Abstract Chronic immune activation and ensuing inflammation that accompany HIV infection lead to adverse metabolic consequences and an increased risk of type 2 diabetes (T2D). We examined the additive effects of T2D on circulating biomarkers involved in inflammation, coagulation, and vascular function along with plasma amino acids in people living with HIV (PLWH). This cross-sectional study included PLWH with and without T2D (n = 32 total). Analyses involved a multiplex platform for circulating biomarkers and gas chromatography–vacuum ultraviolet spectroscopy for plasma amino acids. In PLWH and T2D, both fibrinogen (2.0 ± 0.6 vs 1.6 ± 0.4 µg/mL, p = 0.02) and von Willebrand factor (vWF) (40.8 ± 17.2 vs 26.7 ± 13.8 µg/mL, p = 0.02) were increased and tryptophan (47 ± 6 vs 53 ± 8 nmol/mL, p = 0.03) and threonine (102 ± 25 vs 125 ± 33 nmol/mL, p = 0.03) were decreased. Fibrinogen, as a biomarker of inflammation, and vWF, as a biomarker of endothelial dysfunction, are augmented by the combined effects of HIV and T2D and may contribute to the pathogenesis of T2D in PLWH. Chronic immune activation and inflammation compromise the integrity of the intestinal mucosa, which increases mucus production. Tryptophan metabolism is altered by a loss of intestinal membrane integrity and threonine is consumed in the production of mucus. Metabolic competition arising from increased protein synthesis in the setting of chronic inflammation along with the associated loss in intestinal membrane integrity may be a primary mechanism in the pathogenesis of T2D in PLWH and requires further investigation.

A novel comprehensive paradigm for the etiopathogenesis of multiple sclerosis: therapeutic approaches and future perspectives on its treatment Abstract It is well recognized that variation in the geographical distribution of prevalence of multiple sclerosis (MS) exists: increasing the latitude its prevalence increases as well, but the underlying causes of such dissimilarity still remained elusive as of today. Currently, the most accredited hypothesis is that the closer to the equator the more pronounced is the amount of sunlight which, in turn, increases the production of vitamin D. Cholecalciferol is indeed deficient in MS patients, but this factor does not explain by itself the etiopathogenesis of the disease. In the present study, to search for a pattern and provide a model of the disease's etiology consistent with this regional factor, as well with its changing ethnic, sex-ratio, lifestyle variations and the other unexplained aspects of MS, an extensive analysis of peer-reviewed literature and data was conducted. The arisen hypothesis was that, increasing the latitude, the factor that varies and can have the stronger effect on the human organism, is the continuous and ever-increasing diversity of the natural light–dark cycle. The consequent effort of the suprachiasmatic nucleus to entrain the organism's circadian rhythm affects the hypothalamic–pituitary–adrenal axis resulting in desynchronizing the central and peripheral circadian clocks and pathologizing the immunitary system. To verify such hypothesis, a theoretical framework of the etiopathogenesis, coherent with the gathered literature, was conceived and a demonstration to corroborate it was eventually devised and performed. The results underscored that people living in countries subjected to a further circadian disruptive factor, as daylight saving time, have a 6.35 times higher prevalence of MS than States placed on their same latitude that do not observe it, thus strongly supporting the hypothesis. As further reinforcement of the conclusions, it is worth mentioning that the levels of polyamines rise abruptly in autoimmune diseases. Moreover, among their numerous roles, these polycations participate to the regulation of the circadian clock so their sudden variation might disrupt it. Following these interesting findings, new perspectives in therapies are, therefore, proposed.

Carnosine's inhibitory effect on glioblastoma cell growth is independent of its cleavage Abstract The naturally occurring dipeptide carnosine (β-alanyl-l-histidine) inhibits the growth of tumor cells. As its component l-histidine mimics the effect, we investigated whether cleavage of carnosine is required for its antineoplastic effect. Using ten glioblastoma cell lines and cell cultures derived from 21 patients suffering from this malignant brain tumor, we determined cell viability under the influence of carnosine and l-histidine. Moreover, we determined expression of carnosinases, the intracellular release of l-histidine from carnosine, and whether inhibition of carnosine cleavage attenuates carnosine's antineoplastic effect. We observed a significantly higher response of the cells to l-histidine than to carnosine with regard to cell viability in all cultures. In addition, we detected protein and mRNA expression of carnosinases and a low but significant release of l-histidine in cells incubated in the presence of 50 mM carnosine (p < 0.05), which did not correlate with carnosine's effect on viability. Furthermore, the carnosinase 2 inhibitor bestatin did not attenuate carnosine's effect on viability. Interestingly, we measured a ~ 40-fold higher intracellular abundance of l-histidine in the presence of 25 mM extracellular l-histidine compared to the amount of l-histidine in the presence of 50 mM carnosine, both resulting in a comparable decrease in viability. In addition, we also examined the expression of pyruvate dehydrogenase kinase 4 mRNA, which was comparably influenced by l-histidine and carnosine, but did not correlate with effects on viability. In conclusion, we demonstrate that the antineoplastic effect of carnosine is independent of its cleavage.

Comparison between the effects of selenomethionine and S -adenosylmethionine in preventing cholestasis-induced rat liver damage Abstract We aimed to evaluate whether two methionine-related compounds, S-adenosylmethionine (SAM), and selenomethionine (SM), could lessen liver damage induced by regurgitated bile in a model of rat bile duct ligation (BDL). Hepatoprotective potentials of S-adenosylmethionine and selenomethionine were estimated based on the changes of serum liver damage parameters (aminotransferases, alkaline phosphatase, gamma-glutamyltranspeptidase and lactate dehydrogenase activity, and bilirubin concentration), tissue oxidative [xanthine oxidase (XO) and catalase activity, thiobarbituric acid reactive substances (TBARS) levels] and inflammatory [tumor necrosis factor-alfa (TNF-α) concentration] parameters, and morphological liver tissue alterations that follow cholestasis. The treatment regimens proved themselves able to prevent significant liver damage induced by cholestasis. Both SAM and SM decreased XO activity and TBARS levels and increased catalase activity, while only SM significantly reduced TNF-α concentration. Morphological changes related to bile-induced liver damage were also found to be partially diminished by SAM and SM. In view of the mechanisms of action of the two tested methionine-derived compounds, one might say that SM predominantly acted as an antioxidant, while SAM exerted its activity by potentially modulating different gene expression and protein structures. It is also worth mentioning that this is the first study (to the best of our knowledge) that dealt with the effects of SM on BDL-induced liver injury in rats and of the findings that speak favorably of this powerful antioxidant.