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Abstract Objective Loeys–Dietz syndrome (LDS) is a heritable disorder of connective tissue closely related to Marfan syndrome (MFS). LDS is caused by loss‐of‐function variants of genes that encode components of transforming growth factor‐β (TGF‐β) signaling; nevertheless, LDS type 1/2 caused by TGFBR1/2 pathogenic variants is frequently found to have paradoxical increases in TGF‐β signaling in the aneurysmal aortic wall. Here, we present a Japanese LDS family having a novel SMAD3 variant. Methods The proband was tested via clinical, genetic, and histological analyses. In vitro analysis was performed for pathogenic evaluation. Results The novel heterozygous missense variant of SMAD3 [c.1262G>A, p.(Cys421Tyr)], located just upstream of the C‐terminal Ser423‐X‐Ser425 phosphorylation motif, was found in this instance of LDS type 3. This variant led to reduced phospho‐SMAD3 (Ser423/Ser425) levels and transcription activity in vitro; however, a paradoxical upregulation of TGF‐β signaling was evident in the aortic wall. Conclusions Our results revealed the presence of TGF‐β paradox in this case with the novel loss‐of‐function SMAD3 variant. The precise mechanism underlying the paradox is unknown, but further research is warranted to clarify the influence of the SMAD3 variant type and location on the LDS3 phenotype as well as the molecular mechanism leading to LDS3 aortopathy.

Abstract Lung transplantation (LT) is the only option for patients with pulmonary arterial hypertension (PAH) refractory to maximal medical therapy. However, some patients referred for LT could survive without LT, and its determinants remain unclear. This study aimed to elucidate prognostic factors of severe PAH at the referral time. We retrospectively analyzed 34 patients referred for LT evaluation. The primary outcome was a composite of death or LT. Over a median follow-up period of 2.56 years, eight patients received LT and eight died. Compared with LT-free survival group, pulmonary arterial systolic pressure (PASP) was higher (p = 0.042), and the ratio of tricuspid annular plane systolic excursion (TAPSE) to PASP (TAPSE/PASP) was lower (p = 0.01) in LT or death group. In receiver operating characteristic analysis, the area under the curve was 0.759 (95% confidence interval 0.589–0.929) for TAPSE/PASP to predict primary outcome, and the optimal cut-off value was 0.30 mm/mmHg (sensitivity 0.875 and specificity 0.667). In a multivariate analysis, TAPSE/PASP was independently associated with death or LT. Kaplan–Meier analysis showed a better LT-free survival in patients with TAPSE/PASP ≧0.30 mm/mmHg than in those with

A network anomaly detection method is proposed for large-scale, wide-range Internet Protocol (IP) networks. Because network behavior is projected onto communication traffic, anomaly detection can be achieved by properly analyzing the communication traffic flows. However, in wide-area IP networks, communication traffic flows are encapsulated by headers assigned by communication carriers and thus are observed as more macroscopic information. Therefore, accurately detecting the occurrence of anomalies in individual communication flows is difficult because the flow observation results obtained by flow measurement protocols such as IP Flow Information Export (IPFIX) are the result of superimposing various communication flows with different characteristics. In this study, we propose an anomaly-detection method based on time-series traffic flows. First, we decompose superimposed traffic flows into individual flows using our implemented system called the Fast xFlow Proxy, which can decompose traffic flows to a fine granularity. Our method detects anomalies in the decomposed flows based on a simple correlation analysis and dynamic threshold configuration. Our extensive simulation shows that, if we observe individual flows using the Fast xFlow Proxy, our method can detect anomalies caused by service failures with almost 100% accuracy. Our method can achieve an accuracy of approximately 80%–90% even in more difficult detection cases, such as small traffic fluctuations or noisy situations.

Pulmonary hypertension (PH) is a life-threatening disease characterized by a progressive narrowing of pulmonary arterioles. Although VEGF is highly expressed in lung of patients with PH and in animal PH models, the involvement of angiogenesis remains elusive. To clarify the pathophysiological function of angiogenesis in PH, we compared the angiogenic response in hypoxia (Hx) and SU5416 (a VEGFR2 inhibitor) plus Hx (SuHx) mouse PH models using 3D imaging. The 3D imaging analysis revealed an angiogenic response in the lung of the Hx-PH, but not of the severer SuHx-PH model. Selective VEGFR2 inhibition with cabozantinib plus Hx in mice also suppressed angiogenic response and exacerbated Hx-PH to the same extent as SuHx. Expression of endothelial proliferator-activated receptor γ coactivator 1α (PGC-1α) increased along with angiogenesis in lung of Hx-PH but not SuHx mice. In pulmonary endothelial cell–specific Ppargc1a-KO mice, the Hx-induced angiogenesis was suppressed, and PH was exacerbated along with increased oxidative stress, cellular senescence, and DNA damage. By contrast, treatment with baicalin, a flavonoid enhancing PGC-1α activity in endothelial cells, ameliorated Hx-PH with increased Vegfa expression and angiogenesis. Pulmonary endothelial PGC-1α–mediated angiogenesis is essential for adaptive responses to Hx and might represent a potential therapeutic target for PH.

Aquaculture of marine macroalgae (i.e. seaweeds) such as the kelp Undaria pinnatifida is expected to contribute to future food and biomass production. Although macroalgal survival, biomass, and morphology are strongly affected by the density of individual plants in natural environments, little is known about the cultivation density (individuals per 1 m of cultivation rope) of macroalgae required to optimize aquaculture production, commercial profit (sales – labor expenses for processing), and quality as food. The present study examined the effect of increasing the cultivation density of U. pinnatifida from 10 to 200 individuals m-1 on survival rate, biomass production, profit, and morphological features related to quality as food. Survival rate was almost 100% in all treatments, indicating self-thinning did not occur. Biomass production increased with increasing density, suggesting that the maximum density possible is in excess of 200 individuals m-1. However, although profit rose with increasing density from 10 to 120 individuals m-1, it did not rise further if density was further increased. Moreover, some morphological features related to quality increased or decreased with increasing density. On balance, these results suggest that 80-120 individuals m-1 is an appropriate density range to optimize production of this species in terms of both profit and quality as food. However, only 10-30 individuals m-1 was the density best suited to enhance production of the sporophyll form, which is known to be a nutritious food both for humans and sea urchins.

ABSTRACT Leptin replacement therapy (LRT) has drastically improved the prognosis of patients with lipodystrophy, but pro‐inflammatory properties of leptin could become evident in the long term. Here, we report a 30‐year‐old Japanese woman with generalized lipodystrophy‐associated progeroid syndrome due to a heterozygous LMNA variant (c.29C > T; p.T10I), who was diagnosed with severe aortic stenosis (AS) after more than a decade of LRT, which required transcatheter aortic valve implantation. Given her marked hypoadiponectinemia and the LMNA variant, our patient might have been susceptible to progeria‐associated disorders, including aortic stenosis, which could have been exaggerated by the prolonged ‘imbalanced adipokines’ caused by LRT between pro‐inflammatory leptin and anti‐inflammatory adiponectin. Thus, long‐term LRT could be associated with AS in patients with the LMNA variant to cause generalized lipodystrophy‐associated progeroid syndrome and hypoadiponectinemia.

Abstract Background The unicellular red alga Cyanidioschyzon merolae exhibits a very simple cellular and genomic architecture. In addition, procedures for genetic modifications, such as gene targeting by homologous recombination and inducible/repressible gene expression, have been developed. However, only two markers for selecting transformants, uracil synthase (URA) and chloramphenicol acetyltransferase (CAT), are available in this alga. Therefore, manipulation of two or more different chromosomal loci in the same strain in C. merolae is limited. Results This study developed a nuclear targeting and transformant selection system using an antibiotics blasticidin S (BS) and the BS deaminase (BSD) selectable marker by homologous recombination in C. merolae. In addition, this study has succeeded in simultaneously modifying two different chromosomal loci by a single-step cotransformation based on the combination of BSD and CAT selectable markers. A C. merolae strain that expresses mitochondrion-targeted mSCARLET (with the BSD marker) and mVENUS (with the CAT marker) from different chromosomal loci was generated with this procedure. Conclusions The newly developed BSD selectable marker enables an additional genetic modification to the already generated C. merolae transformants based on the URA or CAT system. Furthermore, the cotransformation system facilitates multiple genetic modifications. These methods and the simple nature of the C. merolae cellular and genomic architecture will facilitate studies on several phenomena common to photosynthetic eukaryotes.

Abstract Most seven transmembrane receptors (7TMRs) are G protein-coupled receptors; however, some 7TMRs evoke intracellular signals through β-arrestin as a biased receptor. As several β-arrestin-biased agonists have been reported to be cardioprotective, we examined the role of the chemokine receptor CXCR7 as a β-arrestin-biased receptor in the heart. Among 510 7TMR genes examined, Cxcr7 was the most abundantly expressed in the murine heart. Single-cell RNA-sequencing analysis revealed that Cxcr7 was abundantly expressed in cardiomyocytes and fibroblasts. Cardiomyocyte-specific Cxcr7 null mice showed more prominent cardiac dilatation and dysfunction than control mice 4 weeks after myocardial infarction. In contrast, there was no difference in cardiac phenotypes between fibroblast-specific Cxcr7-knockout mice and control mice even after myocardial infarction. TC14012, a specific agonist of CXCR7, significantly recruited β-arrestin to CXCR7 in CXCR7-expressing cells and activated extracellular signal-regulated kinase (ERK) in neonatal rat cardiomyocytes. Cxcr7 expression was significantly increased and ERK was activated in the border zone of the heart in control, but not Cxcr7 null mice. These results indicate that the abundantly expressed CXCR7 in cardiomyocytes may play a protective role in the heart as a β-arrestin-biased receptor and that CXCR7 may be a novel therapeutic target for myocardial infarction.

Abstract Percutaneous coronary intervention (PCI) is sometimes considered as an alternative therapeutic strategy to surgical revascularization in patients with coronary artery disease (CAD) and reduced left ventricular ejection fraction (LVEF). However, the types or conditions of patients that receive the clinical benefit of left ventricular reverse remodelling (LVRR) remain unknown. The purpose of this study was to investigate the determinants of LVRR following PCI in CAD patients with reduced LVEF. From 4394 consecutive patients who underwent PCI, a total of 286 patients with reduced LV systolic function (LVEF

Abstract Some clinical trials showed that omega-3 fatty acid (FA) reduced cardiovascular events, but it remains unknown whether omega-3 FA supplementation changes the composition of FAs and their metabolites in the heart and how the changes, if any, exert beneficial effects on cardiac structure and function. To clarify these issues, we supplied omega-3 FA to mice exposed to pressure overload, and examined cardiac structure and function by echocardiography and a proportion of FAs and their metabolites by gas chromatography and liquid chromatography-tandem mass spectrometry, respectively. Pressure overload induced cardiac hypertrophy and dysfunction, and reduced concentration of all FAs’ components and increased free form arachidonic acid and its metabolites, precursors of pro-inflammatory mediators in the heart. Omega-3 FA supplementation increased both total and free form of eicosapentaenoic acid, a precursor of pro-resolution mediators and reduced free form arachidonic acid in the heart. Omega-3 FA supplementation suppressed expressions of pro-inflammatory cytokines and the infiltration of inflammatory cells into the heart and ameliorated cardiac dysfunction and fibrosis. These results suggest that omega-3 FA-induced changes of FAs composition in the heart have beneficial effects on cardiac function via regulating inflammation.

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Photosynthesis generates reactive oxygen species that can damage cells. Here, the authors show that unicellular predators of photosynthetic prey have shared responses to photosynthetic oxidative stress and these may also have been important for the evolution of endosymbiosis.

Replication of the circular bacterial chromosome is initiated at a unique origin (oriC) in a DnaA-dependent manner in which replication proceeds bidirectionally from oriC to ter. The nucleotide compositions of most bacteria differ between the leading and lagging DNA strands. Thus, the chromosomal DNA sequence typically exhibits an asymmetric GC skew profile. Further, free-living bacteria without genomes encoding dnaA were unknown. Thus, a DnaA-oriC-dependent replication initiation mechanism may be essential for most bacteria. However, most cyanobacterial genomes exhibit irregular GC skew profiles. We previously found that the Synechococcus elongatus chromosome, which exhibits a regular GC skew profile, is replicated in a DnaA-oriC-dependent manner, whereas chromosomes of Synechocystis sp. PCC 6803 and Nostoc sp. PCC 7120, which exhibit an irregular GC skew profile, are replicated from multiple origins in a DnaA-independent manner. Here we investigate the variation in the mechanisms of cyanobacterial chromosome replication. We found that the genomes of certain free-living species do not encode dnaA and such species, including Cyanobacterium aponinum PCC 10605 and Geminocystis sp. NIES-3708, replicate their chromosomes from multiple origins. Synechococcus sp. PCC 7002, which is phylogenetically closely related to dnaA-lacking free-living species as well as to dnaA-encoding but DnaA-oriC-independent Synechocystis sp. PCC 6803, possesses dnaA. In Synechococcus sp. PCC 7002, dnaA was not essential and its chromosomes were replicated from a unique origin in a DnaA-oriC independent manner. Our results also suggest that loss of DnaA-oriC-dependency independently occurred multiple times during cyanobacterial evolution and raises a possibility that the loss of dnaA or loss of DnaA-oriC dependency correlated with an increase in ploidy level.

In many eukaryotes, cytokinesis proceeds in two successive steps: first, ingression of the cleavage furrow and second, abscission of the intercellular bridge. In animal cells, the actomyosin contractile ring is involved in the first step, while the endosomal sorting complex required for transport (ESCRT), which participates in various membrane fusion/fission events, mediates the second step. Intriguingly, in archaea, ESCRT is involved in cytokinesis, raising the hypothesis that the function of ESCRT in eukaryotic cytokinesis descended from the archaeal ancestor. In eukaryotes other than in animals, the roles of ESCRT in cytokinesis are poorly understood. To explore the primordial core mechanisms for eukaryotic cytokinesis, we investigated ESCRT functions in the unicellular red alga Cyanidioschyzon merolae that diverged early in eukaryotic evolution. C. merolae provides an excellent experimental system. The cell has a simple organelle composition. The genome (16.5 Mb, 5335 genes) has been completely sequenced, transformation methods are established, and the cell cycle is synchronized by a light and dark cycle. Similar to animal and fungal cells, C. merolae cells divide by furrowing at the division site followed by abscission of the intercellular bridge. However, they lack an actomyosin contractile ring. The proteins that comprise ESCRT-I–IV, the four subcomplexes of ESCRT, are partially conserved in C. merolae. Immunofluorescence of native or tagged proteins localized the homologs of the five ESCRT-III components [charged multivesicular body protein (CHMP) 1, 2, and 4–6], apoptosis-linked gene-2-interacting protein X (ALIX), the ESCRT-III adapter, and the main ESCRT-IV player vacuolar protein sorting (VPS) 4, to the intercellular bridge. In addition, ALIX was enriched around the cleavage furrow early in cytokinesis. When the ESCRT function was perturbed by expressing dominant-negative VPS4, cells with an elongated intercellular bridge accumulated—a phenotype resulting from abscission failure. Our results show that ESCRT mediates cytokinetic abscission in C. merolae. The fact that ESCRT plays a role in cytokinesis in archaea, animals, and early diverged alga C. merolae supports the hypothesis that the function of ESCRT in cytokinesis descended from archaea to a common ancestor of eukaryotes.

Abstract Background The volvocine lineage, containing unicellular Chlamydomonas reinhardtii and differentiated multicellular Volvox carteri, is a powerful model for comparative studies aiming at understanding emergence of multicellularity. Tetrabaena socialis is the simplest multicellular volvocine alga and belongs to the family Tetrabaenaceae that is sister to more complex multicellular volvocine families, Goniaceae and Volvocaceae. Thus, T. socialis is a key species to elucidate the initial steps in the evolution of multicellularity. In the asexual life cycle of C. reinhardtii and multicellular volvocine species, reproductive cells form daughter cells/colonies by multiple fission. In embryogenesis of the multicellular species, daughter protoplasts are connected to one another by cytoplasmic bridges formed by incomplete cytokinesis during multiple fission. These bridges are important for arranging the daughter protoplasts in appropriate positions such that species-specific integrated multicellular individuals are shaped. Detailed comparative studies of cytokinesis between unicellular and simple multicellular volvocine species will help to elucidate the emergence of multicellularity from the unicellular ancestor. However, the cytokinesis-related genes between closely related unicellular and multicellular species have not been subjected to a comparative analysis. Results Here we focused on dynamin-related protein 1 (DRP1), which is known for its role in cytokinesis in land plants. Immunofluorescence microscopy using an antibody against T. socialis DRP1 revealed that volvocine DRP1 was localized to division planes during cytokinesis in unicellular C. reinhardtii and two simple multicellular volvocine species T. socialis and Gonium pectorale. DRP1 signals were mainly observed in the newly formed division planes of unicellular C. reinhardtii during multiple fission, whereas in multicellular T. socialis and G. pectorale, DRP1 signals were observed in all division planes during embryogenesis. Conclusions These results indicate that the molecular mechanisms of cytokinesis may be different in unicellular and multicellular volvocine algae. The localization of DRP1 during multiple fission might have been modified in the common ancestor of multicellular volvocine algae. This modification may have been essential for the re-orientation of cells and shaping colonies during the emergence of multicellularity in this lineage.

Abstract Background Acute kidney injury (AKI) requiring continuous renal replacement therapy (CRRT) is associated with high mortality in critically ill patients. However, the optimal timing to initiate CRRT in patients with AKI is unknown. The purpose of this study is to investigate whether the timing of initiation of CRRT according to severity of AKI is associated with in-hospital mortality. Methods We retrospectively reviewed 189 patients treated with CRRT for AKI in the intensive care unit between January 2009 and February 2013. Patients aged

Abstract Background Sarcopenia is generally complicated with patients with chronic heart failure (CHF) and its presence negatively affects the course of heart failure, however effective nutritional intervention had not been elucidated yet. The primary objective of this study is to explore whether the addition of a branched-chain amino acid (BCAA) preparation for cardiac rehabilitation (CR) of patients with CHF further improves cardiopulmonary functions, skeletal muscle functions, and metabolism in comparison with conventional CR. Methods This is a randomized, parallel-group comparative study. The elderly patients that were participated in CR and complicated with left ventricular systolic or diastolic dysfunction are randomized into two groups, CR + BCAA and CR. 20 weeks later, the second randomization is performed, which divide subjects into two groups with and without BCAA intervention without CR. Primary outcome measure is the rate of change of the anaerobic threshold workload from baseline to post-intervention. Secondary outcome include parameters of exercise capacity, cardiac function and psychological status. Discussion In the current study the effect of a promising new intervention, BCAA, will be assessed to determine whether its addition to CR improve exercise capacity in patients with heart failure, who are generally complicated with sarcopenia. Trial registration This clinical trial was registered with the University Hospital Medical Information Network—Clinical Trials Registry (UMIN–CTR; JPRN–UMIN R000022440 ).

Increased transforming growth factor-β (TGF-β) signaling contributes to the pathophysiology of aortic aneurysm in Marfan syndrome (MFS). Recent reports indicate that a small but significant number of inflammatory cells are infiltrated into the aortic media and adventitia in MFS. However, little is known about the contribution of myeloid cells to aortic aneurysmal formation. In this study, we ablated the TGF-β type II receptor gene Tgfbr2 in myeloid cells of Fbn1C1039G/+ MFS mice (Fbn1C1039G/+;LysM-Cre/+;Tgfbr2fl/fl mice, hereinafter called Fbn1C1039G/+;Tgfbr2MyeKO) and evaluated macrophage infiltration and TGF-β signaling in the aorta. Aneurysmal formation with fragmentation and disarray of medial elastic fibers observed in MFS mice was significantly ameliorated in Fbn1C1039G/+;Tgfbr2MyeKO mice. In the aorta of Fbn1C1039G/+;Tgfbr2MyeKO mice, both canonical and noncanonical TGF-β signals were attenuated and the number of infiltrated F4/80-positive macrophages was significantly reduced. In vitro, TGF-β enhanced the migration capacity of RAW264.7 macrophages. These findings suggest that TGF-β signaling in myeloid cells promotes aortic aneurysmal formation and its inhibition might be a novel therapeutic target in MFS.

ABSTRACT The transition from G1 to S phase and subsequent nuclear DNA replication in the cells of many species of eukaryotic algae occur predominantly during the evening and night in the absence of photosynthesis; however, little is known about how day/night changes in energy metabolism and cell cycle progression are coordinated and about the advantage conferred by the restriction of S phase to the night. Using a synchronous culture of the unicellular red alga Cyanidioschyzon merolae, we found that the levels of photosynthetic and respiratory activities peak during the morning and then decrease toward the evening and night, whereas the pathways for anaerobic consumption of pyruvate, produced by glycolysis, are upregulated during the evening and night as reported recently in the green alga Chlamydomonas reinhardtii. Inhibition of photosynthesis by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) largely reduced respiratory activity and the amplitude of the day/night rhythm of respiration, suggesting that the respiratory rhythm depends largely on photosynthetic activity. Even when the timing of G1/S-phase transition was uncoupled from the day/night rhythm by depletion of retinoblastoma-related (RBR) protein, the same patterns of photosynthesis and respiration were observed, suggesting that cell cycle progression and energy metabolism are regulated independently. Progression of the S phase under conditions of photosynthesis elevated the frequency of nuclear DNA double-strand breaks (DSB). These results suggest that the temporal separation of oxygenic energy metabolism, which causes oxidative stress, from nuclear DNA replication reduces the risk of DSB during cell proliferation in C. merolae. IMPORTANCE Eukaryotes acquired chloroplasts through an endosymbiotic event in which a cyanobacterium or a unicellular eukaryotic alga was integrated into a previously nonphotosynthetic eukaryotic cell. Photosynthesis by chloroplasts enabled algae to expand their habitats and led to further evolution of land plants. However, photosynthesis causes greater oxidative stress than mitochondrion-based respiration. In seed plants, cell division is restricted to nonphotosynthetic meristematic tissues and populations of photosynthetic cells expand without cell division. Thus, seemingly, photosynthesis is spatially sequestrated from cell proliferation. In contrast, eukaryotic algae possess photosynthetic chloroplasts throughout their life cycle. Here we show that oxygenic energy conversion (daytime) and nuclear DNA replication (night time) are temporally sequestrated in C. merolae. This sequestration enables “safe” proliferation of cells and allows coexistence of chloroplasts and the eukaryotic host cell, as shown in yeast, where mitochondrial respiration and nuclear DNA replication are temporally sequestrated to reduce the mutation rate.

Abstract The unicellular thermoacidophilic red alga Cyanidioschyzon merolae is an emerging model organism of photosynthetic eukaryotes. Its relatively simple genome (16.5 Mbp) with very low‐genetic redundancy and its cellular structure possessing one chloroplast, mitochondrion, peroxisome, and other organelles have facilitated studies. In addition, this alga is genetically tractable, and the nuclear and chloroplast genomes can be modified by integration of transgenes via homologous recombination. Recent studies have attempted to clarify the structure and function of the photosystems of this alga. However, it is difficult to obtain photosynthesis‐defective mutants for molecular genetic studies because this organism is an obligate autotroph. To overcome this issue in C. merolae, we expressed a plasma membrane sugar transporter, GsSPT1, from Galdieria sulphuraria, which is an evolutionary relative of C. merolae and capable of heterotrophic growth. The heterologously expressed GsSPT1 localized at the plasma membrane. GsSPT1 enabled C. merolae to grow mixotrophically and heterotrophically, in which cells grew in the dark with glucose or in the light with a photosynthetic inhibitor 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU) and glucose. When the GsSPT1 transgene multiplied on the C. merolae chromosome via the URACm‐Gs selection marker, which can multiply itself and its flanking transgene, GsSPT1 protein level increased and the heterotrophic and mixotrophic growth of the transformant accelerated. We also found that GsSPT1 overexpressing C. merolae efficiently formed colonies on solidified medium under light with glucose and DCMU. Thus, GsSPT1 overexpresser will facilitate single colony isolation and analyses of photosynthesis‐deficient mutants produced either by random or site‐directed mutagenesis. In addition, our results yielded evidence supporting that the presence or absence of plasma membrane sugar transporters is a major cause of difference in trophic properties between C. merolae and G. sulphuraria.

Symbiodinolide is a polyol marine natural product with a molecular weight of 2860. Herein, a streamlined synthesis of the C79–C97 fragment of symbiodinolide is described. In the synthetic route, a spiroacetalization, a Julia–Kocienski olefination, and a Sharpless asymmetric dihydroxylation were utilized as the key transformations.

The cell of the unicellular red alga Cyanidioschyzon merolae contains a single chloroplast and mitochondrion, the division of which is tightly synchronized by a light/dark cycle. The genome content is extremely simple, with a low level of genetic redundancy, in photosynthetic eukaryotes. In addition, transient transformation and stable transformation by homologous recombination have been reported. However, for molecular genetic analyses of phenomena that are essential for cellular growth and survival, inducible gene expression/suppression systems are needed. Here, we report the development of a heat-shock inducible gene expression system in C. merolae. CMJ101C, encoding a small heat shock protein, is transcribed only when cells are exposed to an elevated temperature. Using a superfolder GFP as a reporter protein, the 200-bp upstream region of CMJ101C orf was determined to be the optimal promoter for heat-shock induction. The optimal temperature to induce expression is 50°C, at which C. merolae cells are able to proliferate. At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression. After the heat shock, the mRNA level decreases rapidly. As an example of the system, the expression of a dominant negative form of chloroplast division DRP5B protein, which has a mutation in the GTPase domain, was induced. Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure. This result suggests that the dominant negative DRP5B inhibited the final scission of the chloroplast division site, but not the earlier stages of division site constriction. It is also suggested that cell cycle progression is not arrested by the impairment of chloroplast division at the final stage.

The unicellular red alga Cyanidioschyzon merolae is an emerging model organism for studying organelle division and inheritance: the cell is composed of an extremely simple set of organelles (one nucleus, one mitochondrion and one chloroplast), and their genomes are completely sequenced. Although a fruitful set of cytological and biochemical methods have now been developed, gene targeting techniques remain to be fully established in this organism. Thus far, only a single selection marker, URA Cm-Gs , has been available that complements the uracil-auxotrophic mutant M4. URA Cm-Gs , a chimeric URA5.3 gene of C. merolae and the related alga Galdieria sulphuraria, was originally designed to avoid gene conversion of the mutated URA5.3 allele in the parental strain M4. Although an early example of targeted gene disruption by homologous recombination was reported using this marker, the genome structure of the resultant transformants had never been fully characterized. In the current study, we showed that the use of the chimeric URA Cm-Gs selection marker caused multicopy insertion at high frequencies, accompanied by undesired recombination events at the targeted loci. The copy number of the inserted fragments was variable among the transformants, resulting in high yet uneven levels of transgene expression. In striking contrast, when the authentic URA5.3 gene (URA Cm-Cm ) was used as a selection marker, efficient single-copy insertion was observed at the targeted locus. Thus, we have successfully established a highly reliable and reproducible method for gene targeting in C. merolae. Our method will be applicable to a number of genetic manipulations in this organism, including targeted gene disruption, replacement and tagging.