Impact of Climate on the Incidence of Acute Coronary Syndrome　- Differences Between Japan and Thailand.

Background: Although there are many reports of temperature being associated with the onset of acute coronary syndrome (ACS), few studies have examined differences in ACS due to climatic differences between Japan and Thailand. The aim of this joint Japan-Thailand study was to compare patients with myocardial infarction in Japanese and Thai hospitals in different climates. Methods and Results: We estimated the climate data in 2021 for the Wakayama Prefecture and Chonburi Province, two medium-sized cities in Japan and Thailand, respectively, and ACS patients who were treated at the Wakayama Medical University (WMU) and Burapha University Hospital (BUH), the two main hospitals in these provinces (ACS patient numbers: WMU, n=177; BUH, n=93), respectively. In the Chonburi Province, although the average temperature was above 25℃, the number of ACS cases in BUH varied up to threefold between months (minimum: July, 4 cases; maximum: October, 14 cases). In Japan and Thailand, there was a mild to moderate negative correlation between temperature-atmospheric pressure at the onset of ACS, but different patterns for temperature-humidity (temperature-atmospheric pressure, temperature-humidity, and atmospheric pressure-humidity: correlation index; r=-0.561, 0.196, and -0.296 in WMU vs. r=-0.356, -0.606, and -0.502 in BUH). Conclusions: The present study suggests that other climatic conditions and factors, not just temperature, might be involved in the mechanism of ACS.

Intracoronary Near-Infrared Spectroscopy to Predict No-Reflow Phenomenon During Percutaneous Coronary Intervention in Acute Coronary Syndrome.

Near-infrared spectroscopy-intravascular ultrasound (NIRS-IVUS) can identify the lipid-rich lesions, described as high lipid-core burden index (LCBI). The aim of this study was to investigate the relation between lipid-core plaque (LCP) in the infarct-related lesion detected using NIRS-IVUS and no-reflow phenomenon during percutaneous coronary intervention (PCI) in patients with acute coronary syndrome (ACS). We investigated 371 patients with ACS who underwent NIRS-IVUS in the infarct-related lesions before PCI. The extent of LCP in the infarct-related lesion was calculated as the maximum LCBI for each of the 4-mm longitudinal segments (maxLCBI4mm) measured by NIRS-IVUS. The patients were divided into 2 groups using a maxLCBI4mm cut-off value of 400. The overall incidence of no-reflow phenomenon was 53 of 371 (14.3%). No-reflow phenomenon more frequently occurred in patients with maxLCBI4mm ≥400 compared with those with maxLCBI4mm<400 (17.5% vs 2.5%, p <0.001). After propensity score matching, multivariable logistic regression analysis demonstrated that maxLCBI4mm (odds ratio: 1.008; 95% confidence interval: 1.005 to 1.012, p <0.001) was independently associated with the no-reflow phenomenon. The maxLCBI4mm of 719 in the infarct-related lesion had the highest combined sensitivity (69.8%) and specificity (72.1%) for the identification of no-reflow phenomenon. In conclusion, in patients with ACS, maxLCBI4mm in the infarct-related lesion assessed by NIRS-IVUS was independently associated with the no-reflow phenomenon during PCI.

Characteristics of Discharged Elderly Patients with Acute Heart Failure Followed by Board-Certified-Cardiologists in a Rural Area of Japan.

The worldwide incidence rates of heart failure (HF) are approaching pandemic status due to aging societies. Board-certified cardiologists (BCCs) of the Japanese Circulation Society (JCS) are cardiologists who have completed the respective fellowship program and passed the examination. However, in rural areas, patients have limited access to medical care for social or geographical reasons. The clinical features of the specialist's follow-up for HF patients in rural areas are unclear.This study consists of 205 consecutive discharged elderly patients who were admitted to our hospital due to acute HF (AHF). All patients were recommended for follow-up with BCCs-JCS by the multidisciplinary HF team at the discharge-care planning meeting. The aim of this study was to investigate the clinical features and impact of BCC follow-up for discharged elderly patients with AHF in rural areas.A total of 156 patients chose follow-up with BCCs-JCS (BCC group), and 49 patients chose follow-up with non-BCCs-JCS (non-BCC group). Patients in the BCC group were younger (83 [76-86] versus 89 [75-93] years old, P < 0.001) and had more frequent use of ß-blockers (67% versus 39%, P < 0.001). The degree of frailty assessed by the clinical frailty scale was more severe in the non-BCC group than in the BCC group (4 [3-5] versus 6 [4-7], P < 0.001). The non-BCC group lived in nursing homes more frequently than the BCC group (16% versus 5%, P = 0.011).The HF patients followed by BCCS-JCS in rural areas were younger and had less frailty.

Light-driven Proton Pumps as a Potential Regulator for Carbon Fixation in Marine Diatoms.

Diatoms are a major phytoplankton group responsible for approximately 20% of carbon fixation on Earth. They perform photosynthesis using light-harvesting chlo-rophylls located in plastids, an organelle obtained through eukaryote-eukaryote endosymbiosis. Microbial rhodopsin, a photoreceptor distinct from chlo-rophyll-based photosystems, was recently identified in some diatoms. However, the physiological function of diatom rhodopsin remains unclear. Heterologous expression techniques were herein used to investigate the protein function and subcellular localization of diatom rhodopsin. We demonstrated that diatom rhodopsin acts as a light-driven proton pump and localizes primarily to the outermost membrane of four membrane-bound complex plastids. Using model simulations, we also examined the effects of pH changes inside the plastid due to rhodopsin-mediated proton transport on photosynthesis. The results obtained suggested the involvement of rhodopsin-mediated local pH changes in a photosynthetic CO2-concentrating mechanism in rhodopsin-possessing diatoms.

Efficient production of bispecific antibody by FAST-IgTM and its application to NXT007 for the treatment of hemophilia A.

Efficient production of bispecific antibodies (BsAbs) in single mammalian cells is essential for basic research and industrial manufacturing. However, preventing unwanted pairing of heavy chains (HCs) and light chains (LCs) is a challenging task. To address this, we created an engineering technology for preferential cognate HC/LC and HC/HC paring called FAST-Ig (Four-chain Assembly by electrostatic Steering Technology - Immunoglobulin), and applied it to NXT007, a BsAb for the treatment of hemophilia A. We introduced charged amino-acid substitutions at the HC/LC interface to facilitate the proper assembly for manufacturing a standard IgG-type BsAb. We generated CH1/CL interface-engineered antibody variants that achieved > 95% correct HC/LC pairing efficiency with favorable pharmacological properties and developability. Among these, we selected a design (C3) that allowed us to separate the mis-paired species with an unintended pharmacological profile using ion-exchange chromatography. Crystal structure analysis demonstrated that the C3 design did not affect the overall structure of both Fabs. To determine the final design for HCs-heterodimerization, we compared the stability of charge-based and knobs into hole-based Fc formats in acidic conditions and selected the more stable charge-based format. FAST-Ig was also applicable to stable CHO cell lines for industrial production and demonstrated robust chain pairing with different subclasses of parent BsAbs. Thus, it can be applied to a wide variety of BsAbs both preclinically and clinically.

Prognostic value of Toll-like receptor 4 on human monocyte subsets combined with computed tomography-adapted Leaman score assessing coronary artery disease.

BACKGROUND: Upregulation of Toll-like receptor 4 (TLR-4) is associated with coronary plaque vulnerability assessed by coronary computed tomography angiography (CCTA). Computed tomography-adapted Leaman score (CT-LeSc) is an independent long-term predictor of cardiac events. The relationship between the TLR-4 expression of CD14 ++ CD16 + monocytes and future cardiac events is unknown. We investigated this relationship using CT-LeSc in patients with coronary artery disease (CAD). METHODS: We analyzed 61 patients with CAD who underwent CCTA. Three monocyte subsets (CD14 ++ CD16 - , CD14 ++ CD16 + , and CD14 + CD16 + ) and the expression of TLR-4 were measured by flow cytometry. We divided the patients into two groups according to the best cutoff value of the TLR-4 expression on CD14 + CD16 + which could predict future cardiac events. RESULTS: CT-LeSc was significantly greater in the high TLR-4 group than the low TLR-4 group [9.61 (6.70-13.67) vs. 6.34 (4.27-9.09), P  < 0.01]. The expression of TLR-4 on CD14 ++ CD16 + monocytes was significantly correlated with CT-LeSc ( R2  = 0.13, P  < 0.01). The expression of TLR-4 on CD14 ++ CD16 + monocytes was significantly higher in patients who had future cardiac events than in those who did not [6.8 (4.5-9.1) % vs. 4.2 (2.4-7.6) %, P  = 0.04]. High TLR-4 expression on CD14 ++ CD16 + monocytes was an independent predictor for future cardiac events ( P  = 0.01). CONCLUSION: An increase in the TLR-4 expression on CD14 ++ CD16 + monocytes is related to the development of future cardiac events.

Long-term risk stratification for hospitalized patients with acute heart failure at a single rural cardiovascular centre.

AIMS: Early risk stratification of patients with acute heart failure (AHF) is critical for appropriate triage and outcome improvement. The serum sodium, blood urea nitrogen, age, serum albumin, systolic blood pressure, and natriuretic peptide level (SOB-ASAP) score can predict in-hospital mortality of AHF. However, the relationship between the SOB-ASAP score and long-term prognosis is unknown. This study investigated the determinants of the long-term prognosis of AHF by evaluating the SOB-ASAP score. METHODS: This retrospective cohort study included all patients with acute decompensated heart failure who were admitted to our hospital between April 2017 and March 2018. And those who discharged were analysed retrospectively. The follow-up period was 3 years. Primary end point was all-cause death. RESULTS: Total of 140 patients were analysed. The median SOB-ASAP score of entire cohort on admission was 3 points (interquartile range; 1-5). The Kaplan-Meier curve demonstrated that patients in the higher SOB-ASAP group (score ≥3) had a higher incidence of all-cause death (log-rank test; P < 0.001) than those in the lower SOB-ASAP (group score <3). CONCLUSIONS: At admission, the SOB-ASAP score may be useful for predicting the long-term prognosis of hospitalized patients with AHF.

A pyrenoid-localized protein SAGA1 is necessary for Ca2+-binding protein CAS-dependent expression of nuclear genes encoding inorganic carbon transporters in Chlamydomonas reinhardtii.

Microalgae induce a CO2-concentrating mechanism (CCM) to maintain photosynthetic affinity for dissolved inorganic carbon (Ci) under CO2-limiting conditions. In the model alga Chlamydomonas reinhardtii, the pyrenoid-localized Ca2+-binding protein CAS is required to express genes encoding the Ci-transporters, high-light activated 3 (HLA3), and low-CO2-inducible protein A (LCIA). To identify new factors related to the regulation or components of the CCM, we isolated CO2-requiring mutants KO-60 and KO-62. These mutants had insertions of a hygromycin-resistant cartridge in the StArch Granules Abnormal 1 (SAGA1) gene, which is necessary to maintain the number of pyrenoids and the structure of pyrenoid tubules in the chloroplast. In both KO-60 and the previously identified saga1 mutant, expression levels of 532 genes were significantly reduced. Among them, 10 CAS-dependent genes, including HLA3 and LCIA, were not expressed in the saga1 mutants. While CAS was expressed normally at the protein levels, the localization of CAS was dispersed through the chloroplast rather than in the pyrenoid, even under CO2-limiting conditions. These results suggest that SAGA1 is necessary not only for maintenance of the pyrenoid structure but also for regulation of the nuclear genes encoding Ci-transporters through CAS-dependent retrograde signaling under CO2-limiting stress.

A YAK1-type protein kinase, triacylglycerol accumulation regulator 1, in the green alga Chlamydomonas reinhardtii is a potential regulator of cell division and differentiation into gametes during photoautotrophic nitrogen deficiency.

Yet another kinase (YAK) 1 is a conserved eukaryotic protein kinase coordinating growth and development. We previously isolated a mutant of Chlamydomonas reinhardtii defective in the YAK1 ortholog triacylglycerol (TAG) accumulation regulator 1 (TAR1). The mutant tar1-1 displayed higher levels of chlorophyll, starch, TAG, and biomass than the parental strain C9 (renamed as C9-3) in photoautotrophic nitrogen (N)-deficient conditions. However, we found that the parental C9-3 showed faster chlorosis upon N-deficiency than the original C9 (C9-1) freshly recovered from cryopreservation, suggesting that C9-3 had acquired particular characteristics during long-term subculturing. To exclude phenotypes dependent on a particular parental strain, we newly created tar1 mutants from two wild-types, C9-1 and CC 125. Like tar1-1, the new tar1 mutants showed higher levels of chlorophyll and TAG/starch than the parental strain. Upon removal of N, Chlamydomonas cells divide once before ceasing further division. Previously, the single division after N-removal was arrested in tar1-1 in photomixotrophic conditions, but this phenotype was not observed in photoautotrophic conditions because of the particular characteristics of the parental C9-3. However, using C9- 1 and CC-125 as parental strains, we showed that cell division after N-removal was impaired in new tar1 mutants in photoautotrophic conditions. Consistent with the view that the division under N-deficiency is necessary for gametic differentiation, new tar1 mutants showed lower mating efficiency than the parental strains. Taken together, TAR1 was suggested to promote differentiation into gametes through the regulation of cell division in response to N-deficiency.

A potential EARLY FLOWERING 3 homolog in Chlamydomonas is involved in the red/violet and blue light signaling pathways for the degradation of RHYTHM OF CHLOROPLAST 15.

Light plays a major role in resetting the circadian clock, allowing the organism to synchronize with the environmental day and night cycle. In Chlamydomonas the light-induced degradation of the circadian clock protein, RHYTHM OF CHLOROPLAST 15 (ROC15), is considered one of the key events in resetting the circadian clock. Red/violet and blue light signals have been shown to reach the clock via different molecular pathways; however, many of the participating components of these pathways are yet to be elucidated. Here, we used a forward genetics approach using a reporter strain that expresses a ROC15-luciferase fusion protein. We isolated a mutant that showed impaired ROC15 degradation in response to a wide range of visible wavelengths and impaired light-induced phosphorylation of ROC15. These results suggest that the effects of different wavelengths converge before acting on ROC15 or at ROC15 phosphorylation. Furthermore, the mutant showed a weakened phase resetting in response to light, but its circadian rhythmicity remained largely unaffected under constant light and constant dark conditions. Surprisingly, the gene disrupted in this mutant was found to encode a protein that possessed a very weak similarity to the Arabidopsis thaliana EARLY FLOWERING 3 (ELF3). Our results suggest that this protein is involved in the many different light signaling pathways to the Chlamydomonas circadian clock. However, it may not influence the transcriptional oscillator of Chlamydomonas to a great extent. This study provides an opportunity to further understand the mechanisms underlying light-induced clock resetting and explore the evolution of the circadian clock architecture in Viridiplantae.

Serial changes of coronary flow reserve over one year after transcatheter aortic valve implantation in patients with severe aortic stenosis.

Background: Impaired coronary flow reserve (CFR) portends a poor prognosis in patients with aortic stenosis. The present study aims to investigate how CFR changes over one year after transcatheter aortic valve implantation (TAVI) in patients with severe aortic stenosis, and to explore factors related to the changes. Methods: Consecutive patients undergoing TAVI were registered. CFR in the left anterior descending artery was measured by transthoracic echocardiography on three occasions pre-TAVI, one-day post-TAVI, and one-year post-TAVI. Results: A total of 59 patients were enrolled, 46 of whom completed one-year follow-up. CFR was impaired in 35 (59.3%) patients pre-TAVI, but the impairment was only seen in 2 patients (4%) one-year post-TAVI. CFR value improved from 1.75 (1.50-2.10) cm/s pre-TAVI, to 2.00 (1.70-2.30) one-day post-TAVI, and further to 2.60 (2.30-3.10) one-year post-TAVI (P < 0.001). The median difference in CFR between pre-TAVI and one-year post-TAVI was 0.90 (0.53-1.20). Patients with significant improvement of CFR (more than the median value of 0.9) had larger aortic valve area (1.55 [1.38-1.92] vs. 1.36 cm2 [1.26-1.69], P = 0.042) and greater improvement in left ventricular ejection fraction (3.10 [-1.67-4.24] vs. -1.46 [-3.42-1.48] percentage points, P = 0.019) than those without. Conclusions: CFR is impaired in a considerable proportion of patients with severe aortic stenosis, but improvement is seen immediately after TAVI, and one year later. Patients with significant improvement of CFR had larger aortic valve area and greater increase in left ventricular ejection fraction after TAVI.

Effect of Atherectomy on Lesion Preparation in Heavily Calcified Coronary Artery Disease.

Background: Percutaneous coronary intervention (PCI) of heavily calcified lesions remains challenging. This study examined whether calcified lesion preparation is better with an ablation-based than balloon-based technique. Methods and Results: Results of lesion preparations with and without atherectomy devices were compared in 121 patients undergoing optical coherence tomography (OCT)-guided PCI of heavily calcified lesions. Lesion preparation was performed with the ablation-based technique in 59 patients (atherectomy group) and with the balloon-based technique in 62 patients (balloon group). Lower grades of angiographic coronary dissections (National Heart, Lung, and Blood Institute [NHLBI] classification) occurred in the atherectomy than balloon group (atherectomy group: none, 33%; NHLBI A, 59%; B, 8%; C, 0%; D, 0%; balloon group: none, 1%; NHLBI A, 24%; B, 58%; C, 15%; D, 2%). On OCT, a large dissection was less common (49% vs. 90%; P<0.001) and calcium fractures were more frequent (75% vs. 18%; P<0.001) in the atherectomy than balloon group. In multivariable analyses, the ablation-based technique was associated with a lower grade of angiographic coronary dissection (adjusted odds ratio [aOR] 0.04; 95% confidence interval [CI] 0.01-0.12; P<0.001), a lower incidence of OCT-detected large dissection (aOR 0.09; 95% CI 0.03-0.30; P<0.001), and a higher incidence of OCT-detected calcium fracture (aOR 18.19; 95% CI 6.45-58.96; P<0.001). Conclusions: The ablation-based technique outperformed the balloon-based technique in the lesion preparation of heavily calcified lesions.

Telecardiology in Rural Practice: Global Trends.

The management of cardiovascular diseases in rural areas is plagued by the limited access of rural residents to medical facilities and specialists. The development of telecardiology using information and communication technology may overcome such limitation. To shed light on the global trend of telecardiology, we summarized the available literature on rural telecardiology. Using PubMed databases, we conducted a literature review of articles published from January 2010 to December 2020. The contents and focus of each paper were then classified. Our search yielded nineteen original papers from various countries: nine in Asia, seven in Europe, two in North America, and one in Africa. The papers were divided into classified fields as follows: seven in tele-consultation, four in the telemedical system, four in the monitoring system, two in prehospital triage, and two in tele-training. Six of the seven tele-consultation papers reported the consultation from rural doctors to urban specialists. More reports of tele-consultations might be a characteristic of telecardiology specific to rural practice. Further work is necessary to clarify the improvement of cardiovascular outcomes for rural residents.

Hyperosmotic stress-induced microtubule disassembly in Chlamydomonas reinhardtii.

BACKGROUND: Land plants respond to drought and salinity by employing multitude of sophisticated mechanisms with physiological and developmental consequences. Abscisic acid-mediated signaling pathways have evolved as land plant ancestors explored their habitats toward terrestrial dry area, and now play major roles in hyperosmotic stress responses in flowering plants. Green algae living in fresh water habitat do not possess abscisic acid signaling pathways but need to cope with increasing salt concentrations or high osmolarity when challenged with adverse aquatic environment. Hyperosmotic stress responses in green algae are largely unexplored. RESULTS: In this study, we characterized hyperosmotic stress-induced cytoskeletal responses in Chlamydomonas reinhardtii, a fresh water green algae. The Chlamydomonas PROPYZAMIDE-HYPERSENSITEVE 1 (PHS1) tubulin kinase quickly and transiently phosphorylated a large proportion of cellular α-tubulin at Thr349 in G1 phase and during mitosis, which resulted in transient disassembly of microtubules, when challenged with > 0.2 M sorbitol or > 0.1 M NaCl. By using phs1 loss-of-function algal mutant cells, we demonstrated that transient microtubule destabilization by sorbitol did not affect cell growth in G1 phase but delayed mitotic cell cycle progression. Genome sequence analyses indicate that PHS1 genes evolved in ancestors of the Chlorophyta. Interestingly, PHS1 genes are present in all sequenced genomes of freshwater Chlorophyta green algae (including Chlamydomonas) but are absent in some marine algae of this phylum. CONCLUSION: PHS1-mediated tubulin phosphorylation was found to be partly responsible for the efficient stress-responsive mitotic delay in Chlamydomonas cells. Ancient hyperosmotic stress-triggered cytoskeletal remodeling responses thus emerged when the PHS1 tubulin kinase gene evolved in freshwater green algae.

The Chlamydomonas bZIP transcription factor BLZ8 confers oxidative stress tolerance by inducing the carbon-concentrating mechanism.

Photosynthetic organisms are exposed to various environmental sources of oxidative stress. Land plants have diverse mechanisms to withstand oxidative stress, but how microalgae do so remains unclear. Here, we characterized the Chlamydomonas reinhardtii basic leucine zipper (bZIP) transcription factor BLZ8, which is highly induced by oxidative stress. Oxidative stress tolerance increased with increasing BLZ8 expression levels. BLZ8 regulated the expression of genes likely involved in the carbon-concentrating mechanism (CCM): HIGH-LIGHT ACTIVATED 3 (HLA3), CARBONIC ANHYDRASE 7 (CAH7), and CARBONIC ANHYDRASE 8 (CAH8). BLZ8 expression increased the photosynthetic affinity for inorganic carbon under alkaline stress conditions, suggesting that BLZ8 induces the CCM. BLZ8 expression also increased the photosynthetic linear electron transfer rate, reducing the excitation pressure of the photosynthetic electron transport chain and in turn suppressing reactive oxygen species (ROS) production under oxidative stress conditions. A carbonic anhydrase inhibitor, ethoxzolamide, abolished the enhanced tolerance to alkaline stress conferred by BLZ8 overexpression. BLZ8 directly regulated the expression of the three target genes and required bZIP2 as a dimerization partner in activating CAH8 and HLA3. Our results suggest that a CCM-mediated increase in the CO2 supply for photosynthesis is critical to minimize oxidative damage in microalgae, since slow gas diffusion in aqueous environments limits CO2 availability for photosynthesis, which can trigger ROS formation.

CO2-dependent migration and relocation of LCIB, a pyrenoid-peripheral protein in Chlamydomonas reinhardtii.

Most microalgae overcome the difficulty of acquiring inorganic carbon (Ci) in aquatic environments by inducing a CO2-concentrating mechanism (CCM). In the green alga Chlamydomonas reinhardtii, two distinct photosynthetic acclimation states have been described under CO2-limiting conditions (low-CO2 [LC] and very low-CO2 [VLC]). LC-inducible protein B (LCIB), structurally characterized as carbonic anhydrase, localizes in the chloroplast stroma under CO2-supplied and LC conditions. In VLC conditions, it migrates to aggregate around the pyrenoid, where the CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase is enriched. Although the physiological importance of LCIB localization changes in the chloroplast has been shown, factors necessary for the localization changes remain uncertain. Here, we examined the effect of pH, light availability, photosynthetic electron flow, and protein synthesis on the localization changes, along with measuring Ci concentrations. LCIB dispersed or localized in the basal region of the chloroplast stroma at 8.3-15 µM CO2, whereas LCIB migrated toward the pyrenoid at 6.5 µM CO2. Furthermore, LCIB relocated toward the pyrenoid at 2.6-3.4 µM CO2, even in cells in the dark or treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and cycloheximide in light. In contrast, in the mutant lacking CCM1, a master regulator of CCM, LCIB remained dispersed even at 4.3 µM CO2. Meanwhile, a simultaneous expression of LCIC, an interacting protein of LCIB, induced the localization of several speckled structures at the pyrenoid periphery. These results suggest that the localization changes of LCIB require LCIC and are controlled by CO2 concentration with ∼7 µM as the boundary.

Characterization of a CO2-Concentrating Mechanism with Low Sodium Dependency in the Centric Diatom Chaetoceros gracilis.

Microalgae induce a CO2-concentrating mechanism (CCM) to overcome CO2-limiting stress in aquatic environments by coordinating inorganic carbon (Ci) transporters and carbonic anhydrases (CAs). Two mechanisms have been suggested to facilitate Ci uptake from aqueous media: Na+-dependent HCO3- uptake by solute carrier (SLC) family transporters and accelerated dehydration of HCO3- to CO2 by external CA in model diatoms. However, studies on ecologically and industrially important diatoms including Chaetoceros gracilis, a common food source in aquacultures, are still limited. Here, we characterized the CCM of C. gracilis using inhibitors and growth dependency on Na+ and CO2. Addition of a membrane-impermeable SLC inhibitor, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), or the transient removal of Na+ from the culture medium did not impair photosynthetic affinity for Ci in CO2-limiting stress conditions, but addition of a membrane-impermeable CA inhibitor, acetazolamide, decreased Ci affinity to one-third of control cultures. In culture medium containing 0.23 mM Na+ C. gracilis grew photoautotrophically by aeration with air containing 5% CO2, but not with the air containing 0.04% CO2. These results suggested that C. gracilis utilizes external CAs in its CCM to elevate photosynthetic affinity for Ci rather than plasma-membrane SLC family transporters. In addition, it is possible that low level of Na+ may support the CCM in processes other than Ci-uptake at the plasma membrane specifically in CO2-limiting conditions. Our findings provide insights into the diversity of CCMs among diatoms as well as basic information to optimize culture conditions for industrial applications.

Seasonal Variation in Epidemiology of Kawasaki Disease-Related Coronary Artery Abnormalities in Japan, 1999-2017.

BACKGROUND: Epidemiological studies show a U-shaped tendency in Kawasaki disease (KD)-related coronary artery abnormalities (CAAs) across age categories. Since studies suggest seasonal variations in KD onset, this study aimed to clarify the epidemiologic features of CAAs, considering the seasons of KD-occurrence. METHODS: We analyzed 2,106 (males = 1,215, females = 891) consecutive KD cases from October 1999 through September 2017 using our electronic database of annual surveys, targeting all hospitals with pediatric departments across Wakayama, Japan. The primary outcome was the presence/absence of CAAs measured by echocardiography 1 month after KD onset. Odds ratios (ORs) and 95% confidence intervals (CIs) of combined patient age and sex for CAAs were calculated using logistic regression models adjusted for four seasons. RESULTS: The median age was 25 (range, 1-212) months. The proportion of males decreased with increasing age. The youngest age group (<6 months) showed an inverse summer/autumn to winter/spring ratio (>1.0) in KD-occurrence. CAAs were observed in 2.8% of cases (males = 3.4%, females = 2.1%), which significantly lessened in summer than in other seasons. Moreover, 50% (n = 4/8) of cases with giant aneurysms experienced KD in autumn. Adjusted ORs for CAAs among males aged ≥60 months (3.0; 95%, CI 1.2-7.5) and females aged <6 months (3.6; 95%, CI 1.1-11.8) were significantly higher than those among males aged 12-35 months. CONCLUSIONS: Cumulative 18-year data of consecutive KD cases from one area suggest the influence of interactions between patient age and sex on the development of KD-related CAAs. The season of KD-occurrence may reflect the diversity of agents.

Chlamydomonas reinhardtii tubulin-gene disruptants for efficient isolation of strains bearing tubulin mutations.

The single-cell green alga Chlamydomonas reinhardtii possesses two α-tubulin genes (tua1 and tua2) and two ß-tubulin genes (tub1 and tub2), with the two genes in each pair encoding identical amino acid sequences. Here, we screened an insertional library to establish eight disruptants with defective tua2, tub1, or tub2 expression. Most of the disruptants did not exhibit major defects in cell growth, flagellar length, or flagellar regeneration after amputation. Because few tubulin mutants of C. reinhardtii have been reported to date, we then used our disruptants, together with a tua1 disruptant obtained from the Chlamydomonas Library Project (CLiP), to isolate tubulin-mutants resistant to the anti-tubulin agents propyzamide (pronamide) or oryzalin. As a result of several trials, we obtained 8 strains bearing 7 different α-tubulin mutations and 12 strains bearing 7 different ß-tubulin mutations. One of the mutations is at a residue similar to that of a mutation site known to confer drug resistance in human cancer cells. Some strains had the same amino acid substitutions as those reported previously in C. reinhardtii; however, the mutants with single tubulin genes showed slightly stronger drug-resistance than the previous mutants that express the mutated tubulin in addition to the wild-type tubulin. Such increased drug-resistance may have facilitated sensitive detection of tubulin mutation. Single-tubulin-gene disruptants are thus an efficient background of generating tubulin mutants for the study of the structure-function relationship of tubulin.