The cadmium tolerance and bioaccumulation mechanism of Tetratostichococcus sp. P1: insight from transcriptomics analysis.

Cadmium (Cd) has become a severe issue in relatively low concentration and attracts expert attention due to its toxicity, accumulation, and biomagnification in living organisms. Cd does not have a biological role and causes serious health issues. Therefore, Cd pollutants should be reduced and removed from the environment. Microalgae have great potential for Cd absorption for waste treatment since they are more environmentally friendly than existing treatment methods and have strong metal sorption selectivity. This study evaluated the tolerance and ability of the microalga Tetratostichococcus sp. P1 to remove Cd ions under acidic conditions and reveal mechanisms based on transcriptomics analysis. The results showed that Tetratostichococcus sp. P1 had a high Cd tolerance that survived under the presence of Cd up to 100 µM, and IC50, the half-maximal inhibitory concentration value, was 57.0 µM, calculated from the change in growth rate based on the chlorophyll content. Long-term Cd exposure affected the algal morphology and photosynthetic pigments of the alga. Tetratostichococcus sp. P1 removed Cd with a maximum uptake of 1.55 mg g-1 dry weight. Transcriptomic analysis revealed the upregulation of the expression of genes related to metal binding, such as metallothionein. Group A, Group B transporters and glutathione, were also found upregulated. While the downregulation of the genes were related to photosynthesis, mitochondria electron transport, ABC-2 transporter, polysaccharide metabolic process, and cell division. This research is the first study on heavy metal bioremediation using Tetratostichococcus sp. P1 and provides a new potential microalga strain for heavy metal removal in wastewater.[Figure: see text]Abbreviations:BP: Biological process; bZIP: Basic Leucine Zipper; CC: Cellular component; ccc1: Ca (II)-sensitive cross complementary 1; Cd: Cadmium; CDF: Cation diffusion facilitator; Chl: Chlorophyll; CTR: Cu TRansporter families; DAGs: Directed acyclic graphs; DEGs: Differentially expressed genes; DVR: Divinyl chlorophyllide, an 8-vinyl-reductase; FPN: FerroportinN; FTIR: Fourier transform infrared; FTR: Fe TRansporter; GO: Gene Ontology; IC50: Growth half maximal inhibitory concentration; ICP: Inductively coupled plasma; MF: molecular function; NRAMPs: Natural resistance-associated aacrophage proteins; OD: Optical density; RPKM: Reads Per Kilobase of Exon Per Million Reads Mapped; VIT1: Vacuolar iron transporter 1 families; ZIPs: Zrt-, Irt-like proteins.

Chromosome-scale genome assembly of an acidophilic microalga Tetratostichococcus sp. P1 isolated from a tropical peatland in Malaysia.

Tetratostichococcus sp. P1 shows an acidophilic phenotype which could allow mass-scale monoculture of this green microalga without severe contamination by environmental microorganisms. In this study, we report a chromosome-scale genome assembly for Tetratostichococcus sp. P1.

Genetic Modification of Aurantiochytrium sp. 18W-13a for Enhancement of Proteolytic Activity by Heterologous Expression of Extracellular Proteases.

A marine thraustochytrid, Aurantiochytrium, is a promising organism to produce docosahexaenoic acid (DHA) and squalene. Utilization of inexpensive substances such as proteins in wastes and by-products from the food industry for cultivation is a considerable option to reduce production cost; however, the proteolytic ability of Aurantiochytrium spp. is low compared to taxonomically close Shizochytrium aggregatum. We previously identified extracellular protease (extracellular protease 1, EP1) in S. aggregatum ATCC 28209 from the supernatant of the culture and found that a similar protease gene (EP2) was located downstream of the EP1 gene. In the present study, we created the transformants expressing SaEP1 and/or SaEP2 to enhance the proteolytic ability of Aurantiochytrium sp. 18W-13a strain and cultivated them in the medium containing casein as a test protein substrate. Through SDS-PAGE analysis, we confirmed that casein in the supernatant was more efficiently degraded by the transformants than the wild type, suggesting that the expressed protease(s) were properly expressed and excreted. After 4-day cultivation in the casein medium, the value of optical density at 660 nm and the cell number in the culture of the transformant that expressed both SaEP1 and SaEP2 (designated as EP12 strain) showed 1.48- and 1.38-fold higher than those of wild type, respectively. The DHA and squalene yield of the EP12 strain were respectively 158.3 and 0.23 mg L-1, and these values were 1.42- and 2.01-fold higher than those of wild type, respectively, suggesting that the EP12 created in the present study is a favorable strain for the cultivation using protein-containing medium.

Stramenopile-Type Lipid Droplet Protein Functions as a Lipid Droplet Scaffold Protein in the Marine Diatom Phaeodactylum tricornutum.

Oleaginous microalgae are gaining great attention as feedstock for biofuels because of their substantial accumulation capacity for neutral lipids in the cytosolic compartment called the lipid droplet (LD). Understanding the regulatory mechanism of neutral lipid accumulation and degradation, which is mediated by LD-associated proteins, is an important issue in improving lipid productivity. However, LD-associated proteins vary among species and are waiting to be characterized in many microalgae. Stramenopile-type LD protein (StLDP) was previously identified as a primary LD protein in the marine diatom Phaeodactylum tricornutum. We produced a knockout mutant of StLDP by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing. Also, we tried to complement this mutant by expressing recognition site-modified StLDP (RSM-StLDP), which is designed to avoid an attack by Cas9 nuclease expressing in the mutant. The RSM-StLDP:enhanced green fluorescent protein was localized to both LDs and the outer chloroplast-endoplasmic reticulum. The decrease in the LD number per cell, increase in LD size and no alteration of neutral lipid content in the mutant under nitrogen deficiency clearly indicate that StLDP acts as an LD scaffold protein. The number of LDs per cell increased in the complemented strain compared to wild-type (WT) cells. The LD morphology in the mutant is probably over-rescued in the complemented strain by the strong function of the nitrate reductase promoter, which is also supported by high neutral lipid content in the complemented strain. The growth of stldp mutant showed a long lag phase relative to WT cells, suggesting that the low surface-to-volume ratio of fused LD decreased the efficiency of LD hydrolysis during the initial growth phase.

Comparison of the OPTIVUS-Complex PCI Multivessel Cohort With the Historical CREDO-Kyoto Registry Cohort-3.

BACKGROUND: There is a paucity of data on the effect of optimal intravascular ultrasound (IVUS)-guided percutaneous coronary intervention (PCI) compared with standard PCI or coronary artery bypass grafting (CABG) in patients with multivessel disease.Methods and Results: The OPTIVUS-Complex PCI study multivessel cohort was a prospective multicenter single-arm study enrolling 1,021 patients undergoing multivessel PCI including the left anterior descending coronary artery using IVUS aiming to meet the prespecified criteria for optimal stent expansion. We conducted propensity score matching analyses between the OPTIVUS group and historical PCI or CABG control groups from the CREDO-Kyoto registry cohort-3 (1,565 and 899 patients) fulfilling the inclusion criteria for this study. The primary endpoint was a composite of death, myocardial infarction, stroke, or any coronary revascularization. In the propensity score-matched cohort (OPTIVUS vs. historical PCI control: 926 patients in each group; OPTIVUS vs. historical CABG control: 436 patients in each group), the cumulative 1-year incidence of the primary endpoint was significantly lower in the OPTIVUS group than in the historical PCI control group (10.4% vs. 23.3%; log-rank P<0.001) or the historical CABG control group (11.8% vs. 16.5%; log-rank P=0.02). CONCLUSIONS: IVUS-guided PCI targeting the OPTIVUS criteria combined with contemporary clinical practice was associated with superior clinical outcomes at 1 year compared with not only the historical PCI control, but also the historical CABG control.

Immobilization of a Broad Range of Polypeptides on the Frustule of the Diatom Thalassiosira pseudonana.

Proteins immobilized on biosilica which have superior reactivity and specificity and are innocuous to natural environments could be useful biological materials in industrial processes. One recently developed technique, living diatom silica immobilization (LiDSI), has made it possible to immobilize proteins, including multimeric and redox enzymes, via a cellular excretion system onto the silica frustule of the marine diatom Thalassiosira pseudonana. However, the number of application examples so far is limited, and the type of proteins appropriate for the technique is still enigmatic. Here, we applied LiDSI to six industrially relevant polypeptides, including protamine, metallothionein, phosphotriesterase, choline oxidase, laccase, and polyamine synthase. Protamine and metallothionein were successfully immobilized on the frustule as protein fusions with green fluorescent protein (GFP) at the N terminus, indicating that LiDSI can be used for polypeptides which are rich in arginine and cysteine. In contrast, we obtained mutants for the latter four enzymes in forms without green fluorescent protein. Immobilized phosphotriesterase, choline oxidase, and laccase showed enzyme activities even after the purification of frustule in the presence of 1% (wt/vol) octylphenoxy poly(ethyleneoxy)ethanol. An immobilized branched-chain polyamine synthase changed the intracellular polyamine composition and silica nanomorphology. These results illustrate the possibility of LiDSI for industrial applications. IMPORTANCE Proteins immobilized on biosilica which have superior reactivity and specificity and are innocuous to natural environments could be useful biological materials in industrial processes. Living diatom silica immobilization (LiDSI) is a recently developed technique for in vivo protein immobilization on the diatom frustule. We aimed to explore the possibility of using LiDSI for industrial applications by successfully immobilizing six polypeptides: (i) protamine (Oncorhynchus keta), a stable antibacterial agent; (ii) metallothionein (Saccharomyces cerevisiae), a metal adsorption molecule useful for bioremediation; (iii) phosphotriesterase (Sulfolobus solfataricus), a scavenger for toxic organic phosphates; (iv) choline oxidase (Arthrobacter globiformis), an enhancer for photosynthetic activity and yield of plants; (v) laccase (Bacillus subtilis), a phenol oxidase utilized for delignification of lignocellulosic materials; and (vi) branched-chain polyamine synthase (Thermococcus kodakarensis), which produces branched-chain polyamines important for DNA and RNA stabilization at high temperatures. This study provides new insights into the field of applied biological materials.

Evolution of a chimeric mitochondrial carbonic anhydrase through gene fusion in a haptophyte alga.

Carbonic anhydrases (CAs) are a universal enzyme family that catalyses the interconversion of carbon dioxide and bicarbonate, and they are localized in most compartments including mitochondria and plastids. Thus far, eight classes of CAs (α-, ß-, γ-, δ-, ζ-, η-, θ- and ι-CA) have been characterized. This study reports an interesting gene encoding a fusion protein of ß-CA and ι-CA found in the haptophyte Isochrysis galbana. Recombinant protein assays demonstrated that the C-terminal ι-CA region catalyses CO2 hydration, whereas the N-terminal ß-CA region no longer exhibits enzymatic activity. Considering that haptophytes generally have mitochondrion-localized ß-CAs and plastid-localized ι-CAs, the fusion CA would show an intermediate stage in which mitochondrial ß-CA is replaced by ι-CA in a haptophyte species.

Enhancement of Squalene Production by Constitutive Expression of the 3-Hydroxy-3-Methylglutaryl-CoA Reductase in Aurantiochytrium sp. 18W-13a.

Squalene has a wide range of applications in the industry sectors of dietary supplements, cosmetics, immunization, and pharmaceuticals. Yet, suitable organisms as the source of squalene are limited. It is reported that the thraustochytrid Aurantiochytrium sp. strain 18W-13a can accumulate high content of squalene. However, squalene production in this organism is fluctuated under various conditions and is not yet optimized for commercialization. In this organism, the mevalonate pathway supplies isopentenyl pyrophosphate, the initial substrate for squalene production. In this pathway, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is the rate-limiting enzyme. We found that the HMGR activity had a strong positive correlation with the squalene contents in the strain. We constitutively expressed the HMGR in this organism and found that the transformant showed increased and stable production of squalene as well as carotenoids and biomass. These results clearly indicated that the HMGR expression is the bottleneck of squalene synthesis in Aurantiochytrium sp.

Enhancing the use of computed tomography and cardiac catheterization angiography in Zambia: A project report on a global extension of medical technology in Japan.

Cardiovascular disease (CVD) is one of the leading causes of death in adults in Zambia among the non-communicable diseases. The Government of the Republic of Zambia through the Ministry of Health procured Japanese radiological systems, computed tomography, and angiography for the University Teaching Hospitals (UTHs) - Adult in 2015. However, the operation of these diagnostic systems has not been optimal due to lack of a proper maintenance service plan, lack of competent health professionals, and erratic supply of medical consumables. In this study, we report our experiences of providing intensive training to multidisciplinary healthcare teams of the radiology department at UTHs - Adult from 2017 to 2019 to strengthen the quality management system of the radiological equipment so as to provide effective healthcare services. However, the COVID-19 pandemic has had enormous negative impact on essential healthcare. Long-term support through continuous hands-on training must be provided to establish sustainable healthcare services.

Over Ten Years' Follow-Up of Chronic Total Coronary Occlusion Angioplasty.

OBJECTIVES: It has been reported that successful percutaneous coronary intervention for chronic total occlusion (CTO-PCI) might be associated with symptom relief, a lower rate of subsequent myocardial infarction and coronary artery bypass graft surgery, and improved long-term survival, compared with unsuccessful PCI for CTO. However, the long-term benefit of percutaneous recanalization of CTO remains unclear. Therefore, we aimed to evaluate the long-term benefit of percutaneous recanalization of CTO. METHODS: We analyzed consecutive cases of CTO-PCI performed between January 2000 and December 2006. The health status of all patients on September 2017 was obtained via letter or from medical records. We collected relevant patient information as well as angiographic and procedural characteristics. RESULTS: A total of 477 patients (82.8% men, mean age, 65.7 years) underwent CTO-PCI. The procedural was successful in 382 cases (80.3%). Reference vessel diameter, occlusion length and angiographic stump of CTO site were associated with the success of CTO intervention. During the mean follow-up period of 139.8 months, successful CTO-PCI was associated with a higher survival rate when compared with failed CTO-PCI (Log-rank test: P = 0.0147). When categorized by target vessel, successful revascularization of left anterior descending (LAD) -CTO improved long-term survival (Log-rank test: P = 0.0041). On the other hand, successful revascularization of right coronary artery or left circumflex -CTO was not associated with improved long-term survival [Log-rank test: P = 0.5631 (RCA), P = 0.2774 (LCX)]. CONCLUSIONS: Successful CTO-PCI, especially the successful revascularization of LAD-CTO, improved long-term survival of patients.

Functional assessment of tandem coronary artery stenosis by intracoronary optical coherence tomography-derived virtual fractional flow reserve: a case series.

BACKGROUND: Optical coherence tomography (OCT)-derived fractional flow reserve (FFR)-which may be calculated using fluid dynamics-demonstrated an excellent correlation with the wire-based FFR. However, the applicability of the OCT-derived FFR in the assessment of tandem lesions is currently unclear. CASE SUMMARY: We present two cases of tandem lesions in the mid segment of the left anterior descending (LAD) artery which could have assessed accurately by OCT-derived FFR. The first patient underwent wire-based FFR at the far distal site of LAD, showed a value of 0.66. The OCT-derived FFR was calculated, yielding a value of 0.64. In the absence of stenosis at the proximal lesion, the OCT-derived FFR was calculated as 0.79, which was as same as the wire-based FFR obtained after stenting to the proximal lesion. Thus, additional stenting was performed at the distal lesion. The second patient underwent wire-based FFR at the far distal site of LAD, showed a value of 0.76 which was as same vale as OCT-derived FFR. Considering the absence of stenosis in the proximal lesion, the OCT-derived FFR was estimated as 0.88. After coronary stenting in the proximal lesion, the wire-based FFR yielded a value of 0.90. Therefore, additional intervention to the distal lesion was deferred. DISCUSSION: The described reports are the first two cases which performed physiological assessment using OCT in tandem lesions. The OCT-derived FFR might be able to estimate the wire-based FFR and the severity of each individual lesion in patients with tandem lesions.

Human-rat chimeric anti-occludin monoclonal antibodies inhibit hepatitis C virus infection.

Occludin (OCLN), an integral tetra-spanning plasma membrane protein, is a host entry factor essential for hepatitis C virus (HCV) infection, making it a promising host-targeting molecule for HCV therapeutic intervention. We previously generated rat anti-OCLN monoclonal antibodies (mAbs) that strongly prevented HCV infection in vitro and in vivo. In the present study, we attempted to improve the druggability of the extracellular loop domain-recognizing anti-OCLN mAbs, namely clones 1-3 and 37-5, using genetic engineering. To avoid adverse reactions induced by antibody-dependent cellular cytotoxicity and enhance the antibody stability, we developed human-rat chimeric immunoglobulin G4 S228P mutant (IgG4m) forms of clones 1-3 and 37-5 (named Xi 1-3 and Xi 37-5, respectively) by grafting the variable regions of the light and heavy chains of each rat anti-OCLN mAb into those of human IgG4m. The constructed Xi 1-3 and Xi 37-5 chimeras demonstrated levels of affinity and specificity similar to each parental rat anti-OCLN mAb, and the Fcγ receptor Ⅲa was not activated by the antigen-bound chimeric mAbs, as expected. Both chimeric mAbs inhibited in vitro infection with various HCV genotypes. These results indicate that the IgG4m forms of human-rat chimeric anti-OCLN mAbs may be potential candidate molecules of host-targeting antivirals with pan-genotypic anti-HCV activity.

Identification of a Major Lipid Droplet Protein in a Marine Diatom Phaeodactylum tricornutum.

Various kinds of organisms, including microalgae, accumulate neutral lipids in distinct intracellular compartments called lipid droplets. Generally, lipid droplets are generated from the endoplasmic reticulum, and particular proteins localize on their surface. Some of these proteins function as structural proteins to prevent fusion between the lipid droplets, and the others could have an enzymatic role or might be involved in intracellular membrane trafficking. However, information about lipid droplet proteins in microalgae is scarce as compared with that in animals and land plants. We focused on the oil-producing, marine, pennate diatom Phaeodactylum tricornutum that forms lipid droplets during nitrogen deprivation and we investigated the proteins located on the lipid droplets. After 6 d of cultivation in a nitrate-deficient medium, the mature lipid droplets were isolated by sucrose density gradient centrifugation. Proteomic analyses revealed five proteins, with Stramenopile-type lipid droplet protein (StLDP) being the most abundant protein in the lipid droplet fraction. Although the primary sequence of StLDP did not have homology to any known lipid droplet proteins, StLDP had a central hydrophobic domain. This structural feature is also detected in oleosin of land plants and in lipid droplet surface protein (LDSP) of Nannochloropsis species. As a proline knot motif of oleosin, conservative proline residues existed in the hydrophobic domain. StLDP was up-regulated during nitrate deprivation, and fluctuations of StLDP expression levels corresponded to the size of the lipid droplets.

Sirolimus-eluting stent vs. everolimus-eluting stent for coronary intervention in patients on chronic hemodialysis.

BACKGROUND: Even in the drug-eluting stent era, adverse cardiac events, including restenosis after percutaneous coronary intervention (PCI), have been more frequently seen in patients on hemodialysis (HD) than in non-HD patients. The objective of this study was to compare the sirolimus-eluting stent (SES) and everolimus-eluting stent (EES) for prevention of adverse cardiac events, including restenosis, in HD patients. METHODS AND RESULTS: A total of 100 consecutive patients on HD who underwent PCI were enrolled and randomly assigned to receive SES or EES. Although there was no difference between the 2 groups in baseline patient and lesion characteristics, the angiographic restenosis rate at 8-month follow-up was 21.2% in the SES group and 8.7% in the EES group (P = 0.041). Significant differences were also seen in % diameter stenosis (%DS), minimal lumen diameter, and late lumen loss at 8-month follow-up (P = 0.0024, P = 0.0040, and P = 0.033, respectively). During the 1-year follow-up, major adverse cardiac events occurred in 11 (22.0%) patients in the SES group and in 5 (10.0%) patients in the EES group (P = 0.10). CONCLUSIONS: The use of EES was as safe as that of SES. Moreover, EES significantly prevented restenosis in patients on maintenance HD compared with SES.

The potential benefits and risks of the use of dual antiplatelet therapy beyond 6 months following sirolimus-eluting stent implantation for low-risk patients.

BACKGROUND: The optimal duration of dual antiplatelet therapy (DAT) in patients undergoing intracoronary sirolimus-eluting stent implantation remains controversial. OBJECTIVE: To evaluate the clinical effects of long duration DAT in patients undergoing intracoronary sirolimus-eluting stent implantation in daily practice. In addition, to attempt to identify the optimal duration of DAT after implantation of a sirolimus-eluting stent. METHODS: We retrospectively report on 1293 consecutive patients who underwent successful intracoronary sirolimus-eluting stent implantation. We analyzed the cumulative incidence of stent thrombosis, non-fatal myocardial infarction (MI), death from cardiac causes, and the cumulative incidence of bleeding complications. RESULTS: We compared the study end point in patients who received DAT for <6 months (n=1136) with that for patients who received DAT for >6 months (n=157). The median follow-up period was 1260 ± 462 days. Major bleeding occurred in 35 patients and intracranial hemorrhage in 8. In patients on DAT for >6 months, the incidence of any bleedings, major bleedings, and intracranial hemorrhage was significantly increased. On the other hand, there was no significant difference between the two groups in the risk of the primary end points (stent thrombosis, non-fatal MI, death from cardiac causes, death or MI). CONCLUSIONS: Prolonged DAT for more than 6 months was not significantly more beneficial than aspirin monotherapy in reducing the risk of the occurrence of acute MI, stent thrombosis, and death, although it was associated with an increase in bleeding complications for low-risk patients.