The transcriptional landscape underlying larval development and metamorphosis in the Malabar grouper (Epinephelus malabaricus).

Most teleost fishes exhibit a biphasic life history with a larval oceanic phase that is transformed into morphologically and physiologically different demersal, benthic, or pelagic juveniles. This process of transformation is characterized by a myriad of hormone-induced changes, during the often abrupt transition between larval and juvenile phases called metamorphosis. Thyroid hormones (TH) are known to be instrumental in triggering and coordinating this transformation but other hormonal systems such as corticoids, might be also involved as it is the case in amphibians. In order to investigate the potential involvement of these two hormonal pathways in marine fish post-embryonic development, we used the Malabar grouper (Epinephelus malabaricus) as a model system. We assembled a chromosome-scale genome sequence and conducted a transcriptomic analysis of nine larval developmental stages. We studied the expression patterns of genes involved in TH and corticoid pathways, as well as four biological processes known to be regulated by TH in other teleost species: ossification, pigmentation, visual perception, and metabolism. Surprisingly, we observed an activation of many of the same pathways involved in metamorphosis also at an early stage of the larval development, suggesting an additional implication of these pathways in the formation of early larval features. Overall, our data brings new evidence to the controversial interplay between corticoids and thyroid hormones during metamorphosis as well as, surprisingly, during the early larval development. Further experiments will be needed to investigate the precise role of both pathways during these two distinct periods and whether an early activation of both corticoid and TH pathways occurs in other teleost species.

Prospects and Potential for Chimerism Analysis after Allogeneic Hematopoietic Stem Cell Transplantation.

Chimerism analysis after allogeneic hematopoietic stem cell transplantation serves to confirm engraftment, indicate relapse of hematologic malignancy, and attribute graft failure to either immune rejection or poor graft function. Short tandem repeat PCR (STR-PCR) is the prevailing method, followed by quantitative real-time PCR (qPCR), with detection limits of 1-5% and 0.1%, respectively. Chimerism assays using digital PCR or next-generation sequencing, both of which are more sensitive than STR-PCR, are increasingly used. Stable mixed chimerism is usually not associated with poor outcomes in non-malignant diseases, but recipient chimerism may foretell relapse of hematologic malignancies, so higher detection sensitivity may be beneficial in such cases. Thus, the need for and the type of intervention, e.g., immunosuppression regimen, donor lymphocyte infusion, and/or salvage second transplantation, should be guided by donor chimerism in the context of the feature and/or residual malignant cells of the disease to be treated.

Anemonefish are better taxonomists than humans.

The symbiosis between giant sea anemones, algae of the family Symbiodiniaceae, and anemonefish is an iconic example of a mutualistic trio1,2. Molecular analyses have shown that giant sea anemones hosting anemonefish belong to three clades: Entacmaea, Stichodactyla, and Heteractis3,4,5 (Figure 1A). Associations among 28 species of anemonefish and 10 species of giant sea anemone hosts are complex. Some fish species are highly specialized to only one anemone species (e.g., Amphiprion frenatus with Entacmaea quadricolor), whereas others are more generalist (e.g., Amphiprion clarkii)1,2,6. Reasons for host preferences are obscured, among other things, by the lack of resolution in the giant sea anemone phylogeny. Here, we generated a transcriptomic dataset from 55 sea anemones collected from southern Japan to reconstruct these phylogenetic relationships. We observed that the bubble-tip sea anemone E. quadricolor, currently considered a single species, can be separated into at least four cryptic lineages (A-D). Surprisingly, these lineages can be precisely distinguished by observing their association with anemonefish: A. frenatus only associates with lineage D, whereas A. clarkii lives in the other three lineages.

Brain-specific glycosylation enzyme GnT-IX maintains levels of protein tyrosine phosphatase receptor PTPRZ, thereby mediating glioma growth.

Gliomas are the most prevalent primary tumor of the central nervous system. Despite advances in imaging technologies, neurosurgical techniques, and radiotherapy, a cure for high-grade glioma remains elusive. Several groups have reported that protein tyrosine phosphatase receptor type Z (PTPRZ) is highly expressed in glioblastoma, and that targeting PTPRZ attenuates tumor growth in mice. PTPRZ is modified with diverse glycan, including the PTPRZ-unique human natural killer-1 capped O-mannosyl core M2 glycans. However, the regulation and function of these unique glycans are unclear. Using CRISPR genome-editing technology, we first demonstrated that disruption of the PTPRZ gene in human glioma LN-229 cells resulted in profoundly reduced tumor growth in xenografted mice, confirming the potential of PTPRZ as a therapeutic target for glioma. Furthermore, multiple glycan analyses revealed that PTPRZ derived from glioma patients and from xenografted glioma expressed abundant levels of human natural killer-1-capped O-Man glycans via extrinsic signals. Finally, since deficiency of O-Man core M2 branching enzyme N-acetylglucosaminyltransferase IX (GnT-IX) was reported to reduce PTPRZ protein levels, we disrupted the GnT-IX gene in LN-229 cells and found a significant reduction of glioma growth both in vitro and in the xenograft model. These results suggest that the PTPR glycosylation enzyme GnT-IX may represent a promising therapeutic target for glioma.

The multi-level regulation of clownfish metamorphosis by thyroid hormones.

Most marine organisms have a biphasic life cycle during which pelagic larvae transform into radically different juveniles. In vertebrates, the role of thyroid hormones (THs) in triggering this transition is well known, but how the morphological and physiological changes are integrated in a coherent way with the ecological transition remains poorly explored. To gain insight into this question, we performed an integrated analysis of metamorphosis of a marine teleost, the false clownfish (Amphiprion ocellaris). We show how THs coordinate a change in color vision as well as a major metabolic shift in energy production, highlighting how it orchestrates this transformation. By manipulating the activity of liver X regulator (LXR), a major regulator of metabolism, we also identify a tight link between metabolic changes and metamorphosis progression. Strikingly, we observed that these regulations are at play in the wild, explaining how hormones coordinate energy needs with available resources during the life cycle.

The chromosome-scale genome assembly of the yellowtail clownfish Amphiprion clarkii provides insights into the melanic pigmentation of anemonefish.

Anemonefish are an emerging group of model organisms for studying genetic, ecological, evolutionary, and developmental traits of coral reef fish. The yellowtail clownfish Amphiprion clarkii possesses species-specific characteristics such as inter-species co-habitation, high intra-species color variation, no anemone specificity, and a broad geographic distribution, that can increase our understanding of anemonefish evolutionary history, behavioral strategies, fish-anemone symbiosis, and color pattern evolution. Despite its position as an emerging model species, the genome of A. clarkii is yet to be published. Using PacBio long-read sequencing and Hi-C chromatin capture technology, we generated a high-quality chromosome-scale genome assembly initially comprised of 1,840 contigs with an N50 of 1,203,211 bp. These contigs were successfully anchored into 24 chromosomes of 843,582,782 bp and annotated with 25,050 protein-coding genes encompassing 97.0% of conserved actinopterygian genes, making the quality and completeness of this genome the highest among all published anemonefish genomes to date. Transcriptomic analysis identified tissue-specific gene expression patterns, with the brain and optic lobe having the largest number of expressed genes. Further analyses revealed higher copy numbers of erbb3b (a gene involved in melanocyte development) in A. clarkii compared with other anemonefish, thus suggesting a possible link between erbb3b and the natural melanism polymorphism observed in A. clarkii. The publication of this high-quality genome, along with A. clarkii's many unique traits, position this species as an ideal model organism for addressing scientific questions across a range of disciplines.

Transcriptomes of Giant Sea Anemones from Okinawa as a Tool for Understanding Their Phylogeny and Symbiotic Relationships with Anemonefish.

The relationship between anemonefish and sea anemones is one of the most emblematic examples of mutualistic symbiosis in coral reefs. Although this is a textbook example, the major aspects of this symbiosis are still not fully understood in mechanistic terms. Moreover, since studies of this relationship have usually been focused on anemonefish, much less is known about giant sea anemones, their similarities, their phylogenetic relationships, and their differences at the molecular level. Since both partners of the symbiotic relationship are important, we decided to explore this well-known phenomenon from the perspective of giant sea anemones. Here, we report reference transcriptomes for all seven species of giant sea anemones that inhabit fringing reefs of Okinawa (Japan) and serve as hosts for six species of local anemonefish. Transcriptomes were used to investigate their phylogenetic relations, genetic differences and repertoires of nematocyte-specific proteins. Our data support the presence of three distinct groups corresponding to three genera: Entacmaea, Heteractis, and Stichodactyla. The basal position among the three groups belongs to Entacmaea, which was the first to diverge from a common ancestor. While the magnitude of genetic difference between the representatives of Entacmaea and Stichodactyla is large, intra-specific variation within Stichodactyla is much smaller and seems to result from recent speciation events. Our data reconfirms that Heteractis magnifica belongs to the genus Stichodactyla, despite an overall morphological similarity with representatives of the genus Heteractis. The availability of reference transcriptomes will facilitate further research into the fascinating relationship between sea anemones and anemonefish.

Endothelial expression of human amyloid precursor protein leads to amyloid ß in the blood and induces cerebral amyloid angiopathy in knock-in mice.

The deposition of amyloid ß (Aß) in blood vessels of the brain, known as cerebral amyloid angiopathy (CAA), is observed in most patients with Alzheimer's disease (AD). Compared with the pathology of CAA in humans, the pathology in most mouse models of AD is not as evident, making it difficult to examine the contribution of CAA to the pathogenesis of AD. On the basis of biochemical analyses that showed blood levels of soluble amyloid precursor protein (APP) in rats and mice were markedly lower than those measured in human samples, we hypothesized that endothelial APP expression would be markedly lower in rodents and subsequently generated mice that specifically express human WT APP (APP770) in endothelial cells (ECs). The resulting EC-APP770+ mice exhibited increased levels of serum Aß and soluble APP, indicating that endothelial APP makes a critical contribution to blood Aß levels. Even though aged EC-APP770+ mice did not exhibit Aß deposition in the cortical blood vessels, crossing these animals with APP knock-in mice (AppNL-F/NL-F) led to an expanded CAA pathology, as evidenced by increased amounts of amyloid accumulated in the cortical blood vessels. These results highlight an overlooked interplay between neuronal and endothelial APP in brain vascular Aß deposition. We propose that these EC-APP770+:AppNL-F/NL-F mice may be useful to study the basic molecular mechanisms behind the possible breakdown of the blood-brain barrier upon administration of anti-Aß antibodies.

Myeloproliferative neoplasm-driving Calr frameshift promotes the development of pulmonary hypertension in mice.

Frameshifts in the Calreticulin (CALR) exon 9 provide a recurrent driver mutation of essential thrombocythemia (ET) and primary myelofibrosis among myeloproliferative neoplasms (MPNs). Here, we generated knock-in mice with murine Calr exon 9 mimicking the human CALR mutations, using the CRISPR-Cas9 method. Knock-in mice with del10 [Calrdel10/WT (wild-type) mice] exhibited an ET phenotype with increases of peripheral blood (PB) platelets and leukocytes, and accumulation of megakaryocytes in bone marrow (BM), while those with ins2 (Calrins2/WT mice) showed a slight splenic enlargement. Phosphorylated STAT3 (pSTAT3) was upregulated in BM cells of both knock-in mice. In BM transplantation (BMT) recipients from Calrdel10/WT mice, although PB cell counts were not different from those in BMT recipients from CalrWT/WT mice, Calrdel10/WT BM-derived macrophages exhibited elevations of pSTAT3 and Endothelin-1 levels. Strikingly, BMT recipients from Calrdel10/WT mice developed more severe pulmonary hypertension (PH)-which often arises as a comorbidity in patients with MPNs-than BMT recipients from CalrWT/WT mice, with pulmonary arterial remodeling accompanied by an accumulation of donor-derived macrophages in response to chronic hypoxia. In conclusion, our murine model with the frameshifted murine Calr presented an ET phenotype analogous to human MPNs in molecular mechanisms and cardiovascular complications such as PH.

Amyloid precursor protein 770 is specifically expressed and released from platelets.

Platelets not only play an essential role in hemostasis after vascular injury but are also involved in the development of coronary artery disease (CAD) and cerebrovascular lesions. Patients with CAD and cerebral ischemia are recommended to undergo antiplatelet therapy, but they have an increased incidence of major bleeding complications. Both assessment of the platelet activation status and response to antiplatelet therapy in each patient are highly desired. ß-Amyloid precursor protein (APP) 770 is expressed in vascular endothelial cells, and its extracellular region, a soluble form of APP770 (sAPP770, also called nexin-2), is proteolytically cleaved for shedding. Abundant sAPP770 is also released from activated platelets. In this study, we used peripheral blood samples from patients with CAD and control subjects and evaluated sAPP770 as a specific biomarker for platelet activation. First, the plasma levels of sAPP770 correlated well with those of the soluble form CD40 ligand (CD40L), an established biomarker for platelet activation. Additionally, flow cytometry analysis using peripheral blood cells showed that CD40L expression is up-regulated in activated T cells, whereas APP770 expression is negligible in all blood cell types except platelets. Following stimulation with collagen or ADP, aggregating platelets immediately released sAPP770. Finally, patients with dual antiplatelet therapy showed significantly lower levels of plasma sAPP770 than those with no therapy. Taken together, our data show that plasma sAPP770 could be a promising biomarker for platelet activation.

Epigenetic regulation of spurious transcription initiation in Arabidopsis.

In plants, epigenetic regulation is critical for silencing transposons and maintaining proper gene expression. However, its impact on the genome-wide transcription initiation landscape remains elusive. By conducting a genome-wide analysis of transcription start sites (TSSs) using cap analysis of gene expression (CAGE) sequencing, we show that thousands of TSSs are exclusively activated in various epigenetic mutants of Arabidopsis thaliana and referred to as cryptic TSSs. Many have not been identified in previous studies, of which up to 65% are contributed by transposons. They possess similar genetic features to regular TSSs and their activation is strongly associated with the ectopic recruitment of RNAPII machinery. The activation of cryptic TSSs significantly alters transcription of nearby TSSs, including those of genes important for development and stress responses. Our study, therefore, sheds light on the role of epigenetic regulation in maintaining proper gene functions in plants by suppressing transcription from cryptic TSSs.

Transcriptional regulation of genes bearing intronic heterochromatin in the rice genome.

Intronic regions of eukaryotic genomes accumulate many Transposable Elements (TEs). Intronic TEs often trigger the formation of transcriptionally repressive heterochromatin, even within transcription-permissive chromatin environments. Although TE-bearing introns are widely observed in eukaryotic genomes, their epigenetic states, impacts on gene regulation and function, and their contributions to genetic diversity and evolution, remain poorly understood. In this study, we investigated the genome-wide distribution of intronic TEs and their epigenetic states in the Oryza sativa genome, where TEs comprise 35% of the genome. We found that over 10% of rice genes contain intronic heterochromatin, most of which are associated with TEs and repetitive sequences. These heterochromatic introns are longer and highly enriched in promoter-proximal positions. On the other hand, introns also accumulate hypomethylated short TEs. Genes with heterochromatic introns are implicated in various biological functions. Transcription of genes bearing intronic heterochromatin is regulated by an epigenetic mechanism involving the conserved factor OsIBM2, mutation of which results in severe developmental and reproductive defects. Furthermore, we found that heterochromatic introns evolve rapidly compared to non-heterochromatic introns. Our study demonstrates that heterochromatin is a common epigenetic feature associated with actively transcribed genes in the rice genome.

Epigenetic Regulation of Intronic Transgenes in Arabidopsis.

Defense mechanisms of plant genomes can epigenetically inactivate repetitive sequences and exogenous transgenes. Loss of mutant phenotypes in intronic T-DNA insertion lines by interaction with another T-DNA locus, termed T-DNA suppression, has been observed in Arabidopsis thaliana, although the molecular basis of establishment and maintenance of T-DNA suppression is poorly understood. Here we show that maintenance of T-DNA suppression requires heterochromatinisation of T-DNA sequences and the nuclear proteins, INCREASED IN BONSAI METHYLATION 2 (IBM2) and ENHANCED DOWNY MILDEW 2 (EDM2), which prevent ectopic 3' end processing of mRNA in atypically long introns containing T-DNA sequences. Initiation of T-DNA suppression is mediated by the canonical RdDM pathway after hybridisation of two T-DNA strains, accompanied by DNA hypermethylation of T-DNA sequences in the F1 generation. Our results reveal the presence of a genome surveillance mechanism through genome hybridisation that masks repetitive DNAs intruding into transcription units.

Opposite-directional sex change in functional female protandrous anemonefish, Amphiprion clarkii: effect of aromatase inhibitor on the ovarian tissue.

INTRODUCTION: The anemonefish, Amphiprion clarkii, is a protandrous hermaphrodite. Under appropriate social conditions, male fish can become female. Previous studies indicated that estrogens are important regulators of sex change in this fish. However, the mechanism of sexual plasticity in the gonad of this fish is still unknown. To elucidate the mechanisms underlying the sexual plasticity in the ovary of female anemonefish, an aromatase inhibitor (AI, 500 µg/g diet) was administered to the functional female fish for 80 days. RESULTS: The levels of estradiol-17ß (E2) in the fish treated with AI were significantly lower than those in the control group. Three out of five fish had ambisexual gonads with active spermatogenic germ cells in the ovarian tissue. However, female fish in the AI-treated group prior to treatment and those in the control group displayed no testicular characteristics in their developed ovaries. This result strongly suggests that germ cells with bipotentiality or spermatogonial cells remain in the functional ovaries of anemonefish following sex change from functional males to functional females. There is a possibility that estrogen depletion due to AI treatment might have caused the opposite-directional sex change from functional female to male in the anemonefish. CONCLUSIONS: The anemonefish keeps their high sexual bipotential in the ovary after sex change.

Detection of monoclonal integration of bovine leukemia virus proviral DNA as a malignant marker in two enzootic bovine leukosis cases with difficult clinical diagnosis.

Monoclonal integration of bovine leukemia virus (BLV) proviral DNA into bovine genomes was detected in peripheral blood from two clinical cases of enzootic bovine leukosis (EBL) without enlargement of superficial lymph nodes. A BLV-specific probe hybridized with 1 to 3 EcoRI and HindIII fragments in these 2 atypical EBL cattle by Southern blotting and hybridization, as well as in 3 typical EBL cattle. The probe also hybridized to a large number of EcoRI and HindIII fragments in 5 cattle with persistent leukosis. These results suggest that the detection of monoclonal integration of BLV provirus into the host genome may serve as a marker of monoclonal proliferation and malignancy in difficult to diagnose EBL cattle.

Efficacy of 1.0% chlorhexidine-gluconate ethanol compared with 10% povidone-iodine for long-term central venous catheter care in hematology departments: a prospective study.

The efficacy of 1% chlorhexidine-gluconate ethanol and 10% povidone-iodine for skin antisepsis of central venous catheter (CVC) sites were compared among hematology patients. The CVC site colonization rates of those groups were 11.9% and 29.2%, respectively, and the catheter-associated blood stream infections were 0.75 and 3.62 per 1,000 catheter-days, respectively. One percent chlorhexidine-gluconate ethanol was superior to povidone-iodine to reduce skin colonizers at CVC sites even when catheters were used for long duration.

Estrogen favors the differentiation of ovarian tissues in the ambisexual gonads of anemonefish Amphiprion clarkii.

All undifferentiated gonads of anemonefish first differentiate into ovaries, and then testicular tissue appear among ovarian tissue, and finally form ambisexual gonads with both ovarian and testicular tissues. The role of estradiol-17beta (E2) in differentiation of ovarian cells is well conserved across phyla; however, its role in development of ambisexual gonads is poorly understood. Here we demonstrate that the E2 produced during the differentiation of ovarian cells does not allow testicular cells to differentiate in the prospective ambisexual gonad. We examined the immunolocalization of the steroidogenic enzyme cytochrome aromatase (P450(arom)), which is involved in E2 production. In the gonads, numbers of the P450(arom) -positive cells increased during ovarian differentiation. However, immunopositive cells with weak signal intensity were seen in the interstitial areas among oocytes and between oocytes and testicular tissue undergoing testicular differentiation. In contrast, P450(arom) -positive cells were not found in any testicular tissues of the ambisexual gonads. We also examined changes in E2 production in vitro in the gonads during testicular differentiation. E2 was high in the ovaries before the appearance all of testicular tissue, and decreased accompanying the differentiation of testicular tissue. These results suggest a balance of estrogen/androgen seems to be important during sex differentiation, and then a shift from estrogen to androgen production may induce testicular differentiation in the ovary. Further, exogenous E2 treatment suppressed naturally occurring differentiation of testicular cells forming exclusively ovarian tissues in the gonad in vivo, suggesting the increase of estrogen blocks the differentiation of testicular tissue and the formation of ambisexual gonad.

Quantitative assessment of walking time and postural change in patients with COPD using a new triaxial accelerometer system.

BACKGROUND: The purpose of this study was to quantify the walking time and frequency of postural changes in daily life in patients with chronic obstructive pulmonary disease (COPD) using a new triaxial accelerometer system. METHODS: Twenty-six elderly patients with stable COPD (age 76.8 ± 6.2 years; percent forced expiratory volume in one second [%FEV1] 52.9% ± 26.3%) and 20 age-matched elderly subjects (age 73.0 ± 4.2 years; %FEV1 124.0% ± 22.3%) participated in the study. The subjects' time spent walking (slow, fast), standing, sitting, and lying down and the frequency of their postural changes (getting up, standing up) were assessed for 7 consecutive days using an Activity Monitoring And Evaluation System (A-MES™). We analyzed the relationships among walking times, frequency of postural changes, and physiologic factors in both COPD patients and controls. RESULTS: The COPD patients' total walking time, including slow (<2 km/hour) and fast (≥2 km/hour) walking, and their frequency of standing up were significantly lower than those of the age-matched controls (P < 0.01). The fast walking time in daily life was significantly correlated with the 6-minute walking distance, quadriceps femoris muscle force, and dyspnea (P < 0.01). CONCLUSION: These results suggest that both slow (<2 km/hour) and fast (≥2 km/hour) walking time and frequency of postural changes is significantly decreased in COPD patients compared with healthy elderly subjects. The data also suggest that the COPD patients' different walking times in daily life are significantly correlated with exercise capacity and dyspnea. The 6-minute walking distance had the strongest correlation with fast walking time.

Mechanism for full-length RNA processing of Arabidopsis genes containing intragenic heterochromatin.

Genomes of higher eukaryotes contain many transposable elements, which often localize within the transcribed regions of active genes. Although intragenic transposable elements can be silenced to form heterochromatin, the impact of intragenic heterochromatin on transcription and RNA processing remains largely unexplored. Here we show using a flowering plant, Arabidopsis, that full-length transcript formation over intragenic heterochromatin depends on a protein named IBM2 (Increase in Bonsai Methylation 2), which has a Bromo-Adjacent Homology domain and an RNA recognition motif. Mutation of ibm2 triggers premature termination of transcripts with 3' RNA processing around intragenic heterochromatin at loci including the H3K9 demethylase gene IBM1. The need for IBM2 is circumvented in variant alleles that lack the heterochromatic domain. Our results reveal a mechanism that masks deleterious effects of intragenic heterochromatin, providing evolutionary sources for genetic and epigenetic variations.

Utility of serum thymidine kinase activity measurements for cases of bovine leukosis with difficult clinical diagnoses.

This study evaluated the clinical usefulness of serum thymidine kinase (TK) activity for diagnosing bovine leukosis cases for which clinical diagnosis was difficult ('BL with difficult diagnosis'). Median TK activity values in 24 'BL with difficult diagnosis' and 36 cattle for which BL was clinically confirmed by cytology findings of enlarged superficial lymph nodes ('clinically confirmed BL') were 36.8 and 39.4 U/l, respectively (no significant difference). The percentage with positive TK activity (> 5.4 U/l) was also similar in both groups (83.3% for 'BL with difficult diagnosis' and 97.2% for 'clinically confirmed BL'). TK activity was significantly higher in cows with 'BL with difficult diagnosis' compared to those with other tumors (N = 13) and those with inflammatory diseases (N = 14). Maximum TK activity in cows with other tumors and inflammatory diseases was not high (< 10 U/l). Median TK activities in cows with other tumors and those with inflammatory diseases were 1.8 and 1.4 IU/l, respectively. Positive TK activity was found in a significantly higher percentage of cows with 'BL with difficult diagnosis' (83.3%) relative to the percentages of cows with other tumors (15.3%) and inflammatory diseases (21.4%). Thus, TK activity is an appropriate marker for detecting BL onset in cows with 'BL with difficult diagnosis' as well as 'clinically confirmed BL' group. While the specificity of TK activity required for BL diagnosis is not clear, simultaneous evaluation of serum lactate dehydrogenase activity may assist in the differential diagnoses of other tumors and inflammatory diseases from BL.