Involvement in Fertilization and Expression of Gamete Ubiquitin-Activating Enzymes UBA1 and UBA6 in the Ascidian Halocynthia roretzi.

The extracellular ubiquitin-proteasome system is involved in sperm binding to and/or penetration of the vitelline coat (VC), a proteinaceous egg coat, during fertilization of the ascidian (Urochordata) Halocynthia roretzi. It is also known that the sperm receptor on the VC, HrVC70, is ubiquitinated and degraded by the sperm proteasome during the sperm penetration of the VC and that a 700-kDa ubiquitin-conjugating enzyme complex is released upon sperm activation on the VC, which is designated the "sperm reaction". However, the de novo function of ubiquitin-activating enzyme (UBA/E1) during fertilization is poorly understood. Here, we show that PYR-41, a UBA inhibitor, strongly inhibited the fertilization of H. roretzi. cDNA cloning of UBA1 and UBA6 from H. roretzi gonads was carried out, and their 3D protein structures were predicted to be very similar to those of human UBA1 and UBA6, respectively, based on AlphaFold2. These two genes were transcribed in the ovary and testis and other organs, among which the expression of both was highest in the ovary. Immunocytochemistry showed that these enzymes are localized on the sperm head around a mitochondrial region and the follicle cells surrounding the VC. These results led us to propose that HrUBA1, HrUBA6, or both in the sperm head mitochondrial region and follicle cells may be involved in the ubiquitination of HrVC70, which is responsible for the fertilization of H. roretzi.

Removal of the block to self-fertilization by low-calcium artificial seawater in the ascidian Ciona intestinalis.

Ascidians (Urochordate) are hermaphroditic marine invertebrates that release sperm and eggs to the surrounding seawater. However, several ascidians, including Ciona intestinalis and Halocynthia roretzi, show strict self-sterility due to a self/nonself-recognition mechanism in the interaction between sperm and the vitelline coat (VC) of the eggs. We have previously reported that sperm intracellular Ca2+ level drastically increased immediately after sperm binding to the VC of self eggs but not nonself eggs in C. intestinalis type A, which was potently inhibited by lowering the external Ca2+ concentration, suggesting that sperm Ca2+ influx occurs after sperm self-recognition on the VC. Here, we investigated whether self-sterility was abolished by lowering the external Ca2+ concentration in C. intestinalis. The results showed that the block to self-fertilization was removed by low-Ca2+ (∼1 mM) seawater without decreasing the fertilization rate. Such an effect was not observed with Mg2+ or K+. These results led us to conclude that a low-Ca2+ environment is sufficient to block the self-recognition signal upon fertilization. As low-Ca2+ seawater showed no effect on H. roretzi self-sterility, we propose that the mechanism of self-sterility in Ciona must be distinctive from that in Halocynthia.

The male gamete membrane protein DMP9/DAU2 is required for double fertilization in flowering plants.

All flowering plants exhibit a unique type of sexual reproduction called 'double fertilization' in which each pollen tube-delivered sperm cell fuses with an egg and a central cell. Proteins that localize to the plasma membrane of gametes regulate one-to-one gamete pairing and fusion between male and female gametes for successful double fertilization. Here, we have identified a membrane protein from Lilium longiflorum generative cells using proteomic analysis and have found that the protein is an ortholog of Arabidopsis DUF679 DOMAIN MEMBRANE PROTEIN 9 (DMP9)/DUO1-ACTIVATED UNKNOWN 2 (DAU2). The flowering plant DMP9 proteins analyzed in this study were predicted to have four transmembrane domains and be specifically expressed in both generative and sperm cells. Knockdown of DMP9 resulted in aborted seeds due to single fertilization of the central cell. Detailed imaging of DMP9-knockdown sperm cells during in vivo and semi-in vitro double fertilization revealed that DMP9 is involved in gamete interaction that leads to correct double fertilization.

Vitelline membrane proteins promote left-sided nodal expression after neurula rotation in the ascidian, Halocynthia roretzi.

Stereotyped left-right asymmetry both in external and internal organization is found in various animals. Left-right symmetry is broken by the neurula rotation in the ascidian, Halocynthia roretzi. Neurula embryos rotate along the anterior-posterior axis in a counterclockwise direction, and the rotation stops when the left side of the embryo is oriented downwards, resulting in contact of the left-side epidermis with the vitelline membrane at the bottom of perivitelline space. Then, such contact induces the expression of nodal and its downstream Pitx2 gene in the left-side epidermis. Vitelline membrane is required for the promotion of nodal expression. Here, we showed that a chemical signal from the vitelline membrane promotes nodal gene expression, but mechanical stimulus at the point of contact is unnecessary since the treatment of devitellinated neurulae with an extract of the vitelline membrane promoted nodal expression on both sides. The signal molecules are already present in the vitelline membranes of unfertilized eggs. These signal molecules are proteins but not sugars. Specific fractions in gel filtration chromatography had the nodal promoting activity. By mass spectrometry, we selected 48 candidate proteins. Proteins that contain both a zona pellucida (ZP) domain and epidermal growth factor (EGF) repeats were enriched in the candidates of the nodal inducing molecules. Six of the ZP proteins had multiple EGF repeats that are only found in ascidian ZP proteins. These were considered to be the most viable candidates of the nodal-inducing molecules. Signal molecules are anchored to the entire vitelline membrane, and contact sites of signal-receiving cells are spatially and mechanically controlled by the neurula rotation. In this context, ascidians are unusual with respect to mechanisms for specification of the left-right axis. By suppressing formation of epidermis monocilia, we also showed that epidermal cilia drive the neurula rotation but are dispensable for sensing the signal from the vitelline membrane.

Dual Gene Repertoires for Larval and Adult Shells Reveal Molecules Essential for Molluscan Shell Formation.

Molluscan shells, mainly composed of calcium carbonate, also contain organic components such as proteins and polysaccharides. Shell organic matrices construct frameworks of shell structures and regulate crystallization processes during shell formation. To date, a number of shell matrix proteins (SMPs) have been identified, and their functions in shell formation have been studied. However, previous studies focused only on SMPs extracted from adult shells, secreted after metamorphosis. Using proteomic analyses combined with genomic and transcriptomic analyses, we have identified 31 SMPs from larval shells of the pearl oyster, Pinctada fucata, and 111 from the Pacific oyster, Crassostrea gigas. Larval SMPs are almost entirely different from those of adults in both species. RNA-seq data also confirm that gene expression profiles for larval and adult shell formation are nearly completely different. Therefore, bivalves have two repertoires of SMP genes to construct larval and adult shells. Despite considerable differences in larval and adult SMPs, some functional domains are shared by both SMP repertoires. Conserved domains include von Willebrand factor type A (VWA), chitin-binding (CB), carbonic anhydrase (CA), and acidic domains. These conserved domains are thought to play crucial roles in shell formation. Furthermore, a comprehensive survey of animal genomes revealed that the CA and VWA-CB domain-containing protein families expanded in molluscs after their separation from other Lophotrochozoan linages such as the Brachiopoda. After gene expansion, some family members were co-opted for molluscan SMPs that may have triggered to develop mineralized shells from ancestral, nonmineralized chitinous exoskeletons.

Peanut agglutinin specifically binds to a sperm region between the nucleus and mitochondria in tunicates and sea urchins.

Peanut agglutinin (PNA) is an established marker of the mammalian acrosome. However, we observed that PNA specifically binds to a unique intracellular structure alongside the nucleus in ascidian sperm. Here, we characterize the PNA-binding structure in sperm of marine invertebrates. PNA bound to the region between the mitochondrion and nucleus in spermatozoa of ascidians, sea urchins, and an appendicularian. However, PNA-binding substances were not exposed by the calcium ionophore ionomycin in three ascidian species, indicating that it is a distinct structure from the acrosome. Instead, the ascidian PNA-binding region was shed with the mitochondrion from the sperm head via an ionomycin-induced sperm reaction. The ascidian PNA-binding substance appeared to be solubilized with SDS, but not Triton X-100, describing its detergent resistance. Lectins, PHA-L4 , SSA, and MAL-I were detected at an area similar to the PNA-binding region, suggesting that it contains a variety of glycans. The location and some of the components of the PNA-binding region were similar to known endoplasmic reticulum (ER)-derived structures, although the ER marker concanavalin A accumulated at an area adjacent to but not overlapping the PNA-binding region. Therefore, we conclude that ascidian sperm possess a non-acrosomal, Triton-resistant, glycan-rich intracellular structure that may play a general role in reproduction of tunicates and sea urchins given its presence across a wide taxonomic range.

ANISEED 2015: a digital framework for the comparative developmental biology of ascidians.

Ascidians belong to the tunicates, the sister group of vertebrates and are recognized model organisms in the field of embryonic development, regeneration and stem cells. ANISEED is the main information system in the field of ascidian developmental biology. This article reports the development of the system since its initial publication in 2010. Over the past five years, we refactored the system from an initial custom schema to an extended version of the Chado schema and redesigned all user and back end interfaces. This new architecture was used to improve and enrich the description of Ciona intestinalis embryonic development, based on an improved genome assembly and gene model set, refined functional gene annotation, and anatomical ontologies, and a new collection of full ORF cDNAs. The genomes of nine ascidian species have been sequenced since the release of the C. intestinalis genome. In ANISEED 2015, all nine new ascidian species can be explored via dedicated genome browsers, and searched by Blast. In addition, ANISEED provides full functional gene annotation, anatomical ontologies and some gene expression data for the six species with highest quality genomes. ANISEED is publicly available at: http://www.aniseed.cnrs.fr.

Two HAP2-GCS1 homologs responsible for gamete interactions in the cellular slime mold with multiple mating types: Implication for common mechanisms of sexual reproduction shared by plants and protozoa and for male-female differentiation.

Fertilization is a central event in sexual reproduction, and understanding its molecular mechanisms has both basic and applicative biological importance. Recent studies have uncovered the molecules that mediate this process in a variety of organisms, making it intriguing to consider conservation and evolution of the mechanisms of sexual reproduction across phyla. The social amoeba Dictyostelium discoideum undergoes sexual maturation and forms gametes under dark and humid conditions. It exhibits three mating types, type-I, -II, and -III, for the heterothallic mating system. Based on proteome analyses of the gamete membranes, we detected expression of two homologs of the plant fertilization protein HAP2-GCS1. When their coding genes were disrupted in type-I and type-II strains, sexual potency was completely lost, whereas disruption in the type-III strain did not affect mating behavior, suggesting that the latter acts as female in complex organisms. Our results demonstrate the highly conserved function of HAP2-GCS1 in gamete interactions and suggest the presence of additional allo-recognition mechanisms in D. discoideum gametes.

Impact of BNP level and peak VO2 on future heart failure events: comparison between sinus rhythm and atrial fibrillation.

This study aimed to examine the discrete impacts of peak oxygen consumption (VO2) and brain natriuretic peptide (BNP) levels on future heart failure (HF) events in sinus rhythm (SR) and atrial fibrillation (AF). A total of 1447 patients who underwent symptom-limited cardiopulmonary exercise testing and whose BNP values were determined simultaneously were analysed (SR, N = 1151 and AF, N = 296). HF events were defined as HF hospitalization or HF death. Over a mean follow-up period of 1472 days, 140 HF events were observed. A high BNP value (dichotomized by median value) was independently associated with HF events in SR (HR 8.08; 95 % CI 4.02-16.26; p < 0.0001), but not in AF patients (HR 1.97; 95 % CI 0.91-4.28; p = 0.087) with a significant interaction between the rhythms. By contrast, low-peak VO2 was independently associated with HF events in both rhythms (AF; HR 5.81; 95 % CI 1.75-19.30; p = 0.004, SR; HR 2.04; 95 % CI 1.19-3.49; p = 0.009), with a marginal interaction between them. In bivariate Cox models, low-peak VO2 had much stronger predictive power for HF events than high-BNP in AF, whereas high-BNP was more powerful than low-peak VO2 in SR. The prognostic value of BNP and peak VO2 for future HF events seemed to be different between SR and AF.

Effects of Smoking on Ischemic Stroke, Intracranial Hemorrhage, and Coronary Artery Events in Japanese Patients With Non-Valvular Atrial Fibrillation.

The effects of smoking on the prognosis of non-valvular atrial fibrillation (NVAF) patients are unclear.The Shinken Database 2004-11 (n = 17,517) includes all new patients visiting the Cardiovascular Institute between June 2004 and March 2012. Among these cases, 2,102 NVAF patients were identified. The effects of smoking on ischemic stroke (IS), intracranial hemorrhage (ICH), and coronary artery events including percutaneous coronary intervention (PCI) and acute coronary syndrome (ACS) were analyzed. Smokers were younger and had lower risk profiles compared with non-smokers. A similar tendency was observed between current and former smokers. In contrast, patients with high tobacco consumption were older and had higher risk profiles, including uncontrolled hypertension, compared with those with low tobacco consumption. In 8,159 patient-years, IS, ICH, PCI, and ACS occurred at rates of 7.7, 2.7, 12.4, and 3.0 per 1000 patient-years. In multivariate Cox regression analysis, smoking was not significantly associated with any adverse event. However, different effects of smoking were observed when stratified by age. In patients ≥ 65 years old, current smokers were independently associated with PCI. Moreover, current smokers and smokers with a total tobacco amount ≥ 800 were marginally and independently associated with IS. In patients < 65 years, current smokers were independently associated with ICH.Age appears to be one of the contributors to differentiation of the effects of smoking on cardiovascular events in our NVAF patients. In elderly patients who still smoke, smoking was associated with the promotion of atherosclerosis or thromboembolism, whereas in young patients it was associated with bleeding.

Transcriptional Framework of Male Gametogenesis in the Liverwort Marchantia polymorpha L.

In land plants, there are two types of male gametes: one is a non-motile sperm cell which is delivered to the egg cell by a pollen tube, and the other is a motile sperm cell with flagella. The molecular mechanism underlying the sexual reproduction with the egg and pollen-delivered sperm cell is well understood from studies using model plants such as Arabidopsis and rice. On the other hand, the sexual reproduction with motile sperm has remained poorly characterized, due to the lack of suitable models. Marchantia polymorpha L. is a model basal land plant with sexual reproduction involving an egg cell and bi-flagellated motile sperm. To understand the differentiation process of plant motile sperm, we analyzed the gene expression profile of developing antheridia of M. polymorpha. We performed RNA-sequencing experiments and compared transcript profiles of the male sexual organ (antheridiophore and antheridium contained therein), female sexual organ (archegoniophore) and a vegetative organ (thallus). Transcriptome analysis showed that the antheridium expresses nearly half of the protein-coding genes predicted in the genome, but it also has unique features. The antheridium transcriptome shares some common features with male gamete transcriptomes of angiosperms and animals, and homologs of genes involved in male gamete formation and function in angiosperms and animals were identified. In addition, we showed that some of them had distinct expression patterns in the spermatogenous tissue of developing antheridia. This study provides a transcriptional framework on which to study the molecular mechanism of plant motile sperm development in M. polymorpha as a model.

Follicle cell trypsin-like protease HrOvochymase: Its cDNA cloning, localization, and involvement in the late stage of oogenesis in the ascidian Halocynthia roretzi.

We previously reported that the sperm trypsin-like protease HrAcrosin and its precursor HrProacrosin participate in fertilization of the ascidian Halocynthia roretzi. The HrProacrosin gene is annotated in the H. roretzi genome database as Harore.CG.MTP2014.S89.g15383; our previously reported sequence of HrProacrosin gene appeared to include four nucleotides inserted near the 3'-end of HrProacrosin, resulting in a frame-shift mutation and a premature termination codon. The gene architecture of HrProacrosin and Harore.CG.MTP2014.S89.g15383 resembles that of Xenopus laevis ovochymase-1/OVCH1 and ovochymase-2/OVCH2, which encode egg extracellular polyproteases. Considering these new observations, we evaluated the cDNA cloning, expression, localization, and function of Harore.CG.MTP2014.S89.g15383, herein designated as HrOvochymase/HrOVCH. We found that HrOVCH cDNA consists of a single open reading frame of 1,575 amino acids, containing a signal peptide, three trypsin-like protease domains, and six CUB domains. HrOVCH was transcribed by the testis and ovary, but the majority of protein exists in ovarian follicle cells surrounding eggs. An anti-HrOVCH antibody inhibited elevation of the vitelline coat at a late stage of oogenesis, during the period when self-sterility is acquired. As trypsin inhibitors are reported to block the acquisition of self-sterility during oogenesis, whereas trypsin induces the acquisition of self-sterility and elevation of the vitelline coat in defolliculated ovarian eggs, we propose that HrOVCH may play a role in the acquisition of self-sterility by late-stage H. roretzi oocytes.

Nine-Year Trend of Anticoagulation Use, Thromboembolic Events, and Major Bleeding in Patients With Non-Valvular Atrial Fibrillation　- Shinken Database Analysis.

BACKGROUND: Trends of oral anticoagulant (OAC) prescription and incidence of thromboembolism (TE) and/or major bleeding (MB) in patients with non-valvular atrial fibrillation (NVAF) in Japan are still unclear. METHODS AND RESULTS: We used data from Shinken Database 2004-2012, which included all new patients attending the Cardiovascular Institute between June 2004 and March 2013. Of them, 2,434 patients were diagnosed with NVAF. Patients were divided into 3 time periods according to the year of initial visit: 2004-2006 (n=681), 2007-2009 (n=833), and 2010-2012 (n=920). OAC prescription rate steadily increased from 2004-2006 to 2010-2012. Between 2004-2006 and 2007-2009, irrespective of increased warfarin usage, MB tended to decrease, presumably due to low-intensity therapy and avoidance of concomitant use of dual antiplatelets, but TE did not improve. In 2010-2012, direct OACs (DOAC), preferred in low-risk patients, may have contributed to not only decrease TE, but also increase MB, especially extracranial bleeds. In high-risk patients in that time period, mostly treated with warfarin, incidence of TE and MB did not improve. CONCLUSIONS: The 9-year trend of stroke prevention indicated a steady increase of OAC prescription and a partial improvement of TE and MB. Even in the era of DOAC, TE prevention was insufficient in high-risk patients, and DOAC were associated with increased extracranial bleeding. (Circ J 2016; 80: 639-649).

Left Atrial Remodeling Assessed by Transthoracic Echocardiography Predicts Left Atrial Appendage Flow Velocity in Patients With Paroxysmal Atrial Fibrillation.

Atrial fibrillation (AF) is associated with an increased risk of stroke and other thromboembolic events. Left atrial (LA) thrombus formation is closely related to LA dysfunction, particularly to decreased LA appendage flow velocity (LAA-FV) in patients with AF. We estimated LAA-FV using parameters noninvasively obtained by transthoracic echocardiography (TTE) in patients with paroxysmal AF.Echocardiographic and clinical parameters were assessed in 190 patients with nonvalvular paroxysmal AF showing sinus heart rhythm during transesophageal echocardiography (TEE) and TTE.LAA-FV (60 ± 22 cm/s) significantly correlated with the time interval between the initiation of the P-wave on ECG and that of the A-wave of transmitral flow on TTE (PA-TMF, correlation coefficient, -0.32; P < 0.001), LA dimension (LAD, -0.31; P < 0.001), septal a' velocity of tissue Doppler imaging (TDI, 0.35; P < 0.001), E/e' ratio (-0.28, P < 0.001), E velocity of transmitral flow (-0.20, P = 0.008), E/A ratio of transmitral flow (-0.18, P = 0.02), CHA2DS2-VASc score (-0.15, P = 0.04), and BNP plasma level (-0.32, P = 0.002). Multivariate analysis revealed that PA-TMF (standardized partial regression coefficient, -0.17; P = 0.03), a' velocity (0.24, P = 0.004), and LAD (-0.20, P = 0.01) were independent predictors of LAA-FV (multiple correlation coefficient R, 0.44; P < 0.001).Parameters of atrial remodeling, ie, decreased a' velocity, increased LAD, and PA-TMF during sinus rhythm may be useful predictors of LA blood stasis in patients with nonvalvular PAF. LAA-FV can be estimated using these TTE parameters instead of TEE.

Proteomics of ionomycin-induced ascidian sperm reaction: Released and exposed sperm proteins in the ascidian Ciona intestinalis.

Sperm proteins mediating sperm-egg interaction should be exhibited on the sperm surface, or exposed or released when sperm approach an egg. In ascidians (protochordates), sperm undergo a sperm reaction, characterized by enhanced sperm motility and mitochondrial swelling and shedding on contact with the vitelline coat (VC) or by treatment with Ca(2+) ionophore. Here, proteomic analysis was conducted on sperm exudates and sperm surface proteins using ionomycin-induced sperm reaction and cell-impermeable labeling in Ciona intestinalis type A (C. robusta). In the exudate from sperm treated with ionomycin, membrane proteins including a possible VC receptor CiUrabin were abundant, indicating the release of membranous compartments during sperm reaction. Among the surface proteins XP_009859314.1 (uncharacterized protein exhibiting homology to HrTTSP-1) was most abundant before the sperm reaction, but XP_004227079.1 (unknown Ig superfamily protein) appears to be most abundantly exposed by the sperm reaction. Moreover, proteins containing a notable set of domains, astacin-like metalloprotease domain and thrombospondin type 1 repeat(s), were found in this fraction. Possible roles in fertilization as well as localizations and behaviors of these proteins are discussed.

ABO Blood Type and Response of Activated Partial Thromboplastin Time to Dabigatran in Nonvalvular Atrial Fibrillation Patients.

BACKGROUND: The association between ABO blood type and the activated partial thromboplastin time (aPTT) under dabigatran therapy in nonvalvular atrial fibrillation (NVAF) patients is unclear. METHODS AND RESULTS: Between 2011 March and 2015 May, data on ABO blood type and aPTT under dabigatran were obtained for 396 NVAF patients (baseline aPTT, 166). The prevalence of blood type O tended to increase or significantly increase according to baseline aPTT, aPTT under dabigatran, and their difference (∆aPTT) (P=0.054, 0.001, and 0.012, respectively). CONCLUSIONS: In these NVAF patients, a high aPTT value under dabigatran therapy was associated with blood type O.

Self-incompatibility response induced by calcium increase in sperm of the ascidian Ciona intestinalis.

Many hermaphroditic organisms possess a self-incompatibility system to avoid self-fertilization. Recently, we identified the genes responsible for self-sterility in a hermaphroditic primitive chordate (ascidian), Ciona intestinalis: sperm-side polycystin 1-like receptors s-Themis-A/B and egg-side fibrinogen-like ligands on the vitelline coat (VC) v-Themis-A/B. Here, we investigated the sperm behavior and intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to self/nonself-recognition. We found that sperm motility markedly decreased within 5 min after attachment to the VC of self-eggs but not after attachment to the VC of nonself-eggs and that the apparent decrease in sperm motility was suppressed in low Ca(2+) seawater. High-speed video analysis revealed that sperm detached from the self-VC or stopped motility within 5 min after binding to the self-VC. Because s-Themis-B contains a cation channel domain in its C terminus, we monitored sperm [Ca(2+)](i) by real-time [Ca(2+)](i) imaging using Fluo-8H-AM (AAT Bioquest, Inc.). Interestingly, we found that sperm [Ca(2+)](i) rapidly and dramatically increased and was maintained at a high level in the head and flagellar regions when sperm interacted with the self-VC but not when the sperm interacted with the nonself-VC. The increase in [Ca(2+)](i) was also suppressed by low-Ca(2+) seawater. These results indicate that the sperm self-recognition signal triggers [Ca(2+)](i) increase and/or Ca(2+) influx, which elicits a self-incompatibility response to reject self-fertilization in C. intestinalis.

Incidence and predictors of rehospitalization of acute heart failure patients.

Repeated hospitalization due to acute decompensated heart failure (HF) is a pandemic health problem in Japan. However, it is difficult to predict rehospitalization after discharge for acute decompensated HF. We used a single hospital-based cohort from the Shinken Database 2004-2012, comprising all new patients (n = 19,994) who visited the Cardiovascular Institute Hospital. A total of 282 patients discharged after their first acute HF admission were included in the analysis. The median follow-up period was 908 ± 865 days. Of these patients, rehospitalization due to worsening HF occurred in 55 patients. The cumulative rate of rehospitalization was 17.5% at 1 year, 21.4% at 2 years, and 25.5% at 3 years. Patients with rehospitalization were older than those without rehospitalization. Prevalence of diabetes mellitus (DM) was more common in patients with rehospitalization. Average heart rate (HR) tended to be higher in patients with rehospitalization. Loop diuretics were more commonly used at hospital discharge in patients with rehospitalization. Multivariate Cox regression analysis revealed that age ≥ 75 years, DM, HR ≥ 75 bpm at discharge, and use of loop diuretics at discharge were independent predictors for rehospitalization. The number of these independent risk factors could be used to clearly discriminate between the HF rehospitalization low-, middle- and high-risk patients. HF rehospitalization commonly occurred in patients who were discharged after their first acute HF admission. Older age, DM, increased HR, and loop diuretics use at discharge were independently associated with HF rehospitalization. By simply counting these risk factors, we might be able to predict the risk of HF rehospitalization after discharge.

Self/non-self recognition mechanisms in sexual reproduction: new insight into the self-incompatibility system shared by flowering plants and hermaphroditic animals.

Sexual reproduction is an essential process for generating a genetic variety in the next generation. However, most flowering plants and hermaphroditic animals potentially allow self-fertilization. Approximately 60% of angiosperms possess a self-incompatibility (SI) system to avoid inbreeding. The SI system functions at a process of interaction between pollen (or pollen tube) and the pistil. These SI-responsible factors (S-determinants) in pollen and the pistil are encoded by highly polymorphic multiallelic genes in the S-locus, which are tightly linked making a single haplotype. Different taxonomic families utilize different types of S-determinant proteins. In contrast to the plant system, the mechanisms of SI in simultaneously hermaphroditic animals are largely unknown. Among them, promising candidates for SI in ascidians (primitive chordates) were recently identified. The SI system in the ascidian Cionaintestinalis was found to be very similar to those in flowering plants: The products of sperm- and egg-side multiallelic SI genes, which are tight linked and highly polymorphic, appear to be responsible for the SI system as revealed by genetic analysis. These findings led us to speculate that the SI systems in plants and animals evolved in a manner of convergent evolution. Here, we review the current understanding of the molecular mechanisms of the SI system in flowering plants, particularly Brassicacea, and in ascidians from the viewpoint of common mechanisms shared by plants and animals.

Integrative omics analysis reveals differentially distributed proteins in dimorphic euspermatozoa of the squid, Loligo bleekeri.

In the coastal squid Loligo bleekeri, each male produces one of two types of fertilization-competent spermatozoa (eusperm) that exhibit morphological and behavioral differences. Large "consort" males produce short-tailed spermatozoa that display free-swimming behavior when ejaculated into seawater. Small "sneaker" males, on the other hand, produce long-tailed spermatozoa that exhibit a self-swarming trait after ejaculation. To understand the molecular basis for adaptive traits employed by alternative male mating tactics, we performed the transcriptome deep sequencing (RNA-seq) and proteome analyses to search for differences in testicular mRNAs and sperm proteins, respectively. From mature male testes we identified a total of 236,455 contigs (FPKM ≧1) where 3789 and 2789 were preferentially (≧10-fold) expressed in consort and sneaker testes, respectively. A proteomic analysis detected 4302 proteins in the mature sperm as post-translational products. A strongly biased (≧10-fold) distribution occurred in 55 consort proteins and 61 sneaker proteins. There was no clear mRNA-protein correlation, making a ballpark estimate impossible for not only overall protein abundance but also the degree of biased sperm type expressed in the spermatozoa. A family encoding dynein heavy chain gene, however, was found to be biased towards sneakers, whereas many enzymes involving energy metabolism were heavily biased towards consort spermatozoa. The difference in flagellar length matched exactly the different amount of tubulins. From these results we hypothesize that discrete differential traits in dimorphic eusperm arose from a series of innovative alterations in the intracellular components of spermatozoa.