Diatom pyrenoids are encased in a protein shell that enables efficient CO2 fixation.

Pyrenoids are subcompartments of algal chloroplasts that increase the efficiency of Rubisco-driven CO2 fixation. Diatoms fix up to 20% of global CO2, but their pyrenoids remain poorly characterized. Here, we used in vivo photo-crosslinking to identify pyrenoid shell (PyShell) proteins, which we localized to the pyrenoid periphery of model pennate and centric diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana. In situ cryo-electron tomography revealed that pyrenoids of both diatom species are encased in a lattice-like protein sheath. Single-particle cryo-EM yielded a 2.4-Å-resolution structure of an in vitro TpPyShell1 lattice, which showed how protein subunits interlock. T. pseudonana TpPyShell1/2 knockout mutants had no PyShell sheath, altered pyrenoid morphology, and a high-CO2 requiring phenotype, with reduced photosynthetic efficiency and impaired growth under standard atmospheric conditions. The structure and function of the diatom PyShell provide a molecular view of how CO2 is assimilated in the ocean, a critical ecosystem undergoing rapid change.

Phenylboronic Acid-Functionalized Micelles Dual-Targeting Boronic Acid Transporter and Polysaccharides for siRNA Delivery into Brown Algae.

Brown algae play essential roles ecologically, practically, and evolutionarily because they maintain coastal areas, capture carbon dioxide, and produce valuable chemicals such as therapeutic drugs. To unlock their full potential, understanding the unique molecular biology of brown algae is imperative. Genetic engineering tools that regulate homeostasis in brown algae are essential for determining their biological mechanisms in detail. However, few methodologies have been developed to control gene expression due to the robust structural barriers of brown algae. To address this issue, we designed peptide-based, small interfering RNA (siRNA)-loaded micelles decorated with phenylboronic acid (PBA) ligands. The PBA ligands facilitated the cellular uptake of the micelles into a model brown alga, Ectocarpus siliculosus (E. Siliculosus), through chemical interaction with polysaccharides in the cell wall and biological recognition by boronic acid transporters on the plasma membrane. The micelles, featuring "kill two birds with one stone" ligands, effectively induced gene silencing related to auxin biosynthesis. As a result, the growth of E. siliculosus was temporarily inhibited without persistent genome editing. This study demonstrated the potential for exploring the characteristics of brown algae through a simple yet effective approach and presented a feasible system for delivering siRNA in brown algae.

The Cell Wall Characterization of Brown Alga Cladosiphon okamuranus during Growth.

The present study provides new insights into the growth of the brown algal cell wall by showing that cell wall polysaccharides play an important role in the process of growth, considering the physicochemical characteristic of young and old Cladosiphon okamuranus. To determine its structural variation in detail, the cell wall was sequentially fractionated into five fractions: hot water (HW), ammonium oxalate, hemicellulose-I (HC-I), HC-II, and cellulose, and analyzed physicochemically. Results showed that almost 80% of the total recovery cell wall from both young and old thalli was HW, and HC-I contained mainly fucoidan composed of Fucose, Glucuronic acid, and sulfate in molar ratios of 1.0:0.3:0.6~0.7 and 1.0:0.3:0.2~0.3, respectively. Fucoidan in HW was a highly sulfated matrix polysaccharide abundance in young thalli, while fucoidan in HC-I was rich in old thalli and functions as hemicellulose in land plants, crosslinking with cellulose and strengthening the cell wall. We found that HW and HC-I were particularly involved in the growth and strength of old thalli appeared to be due to the deposition of HC-I and the reduction in water content during the growth process.

Ruxolitinib altered IFN-ß induced necroptosis of human dental pulp stem cells during osteoblast differentiation.

OBJECTIVE: This study aimed to evaluate the role of ruxolitinib in the interferon beta (IFN-ß) mediated osteoblast differentiation using human dental pulp stem cells (hDPSCs). DESIGN: hDPSCs from five deciduous teeth of healthy patients were stimulated by adding human recombinant IFN-ß protein (1 or 2 ng/ml) to the osteogenic differentiation induction medium. Substrate formation was determined using Alizarin Red staining, calcium concentration, and osteoblast marker expression levels. Ruxolitinib was used to inhibit the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway. Apoptosis was detected using terminal deoxynucleotidyl nick-end labeling (TUNEL) staining, and necroptosis was detected using propidium iodide staining and phosphorylated mixed lineage kinase domain-like protein (pMLKL) expression. RESULTS: In the IFN-ß-treated group, substrate formation was inhibited by a reduction in alkaline phosphatase (ALP) expression in a concentration-dependent manner. Although the proliferation potency was unchanged between the IFN-ß-treated and control groups, the cell number was significantly reduced in the experimental group. TUNEL-positive cell number was not significantly different; however, the protein level of necroptosis markers, interleukin-6 (IL-6) and pMLKL were significantly increased in the substrate formation. Cell number and ALP expression level were improved in the group administered ruxolitinib, a JAK-STAT inhibitor. Additionally, ruxolitinib significantly suppressed IL-6 and pMLKL levels. CONCLUSION: Ruxolitinib interfered with the IFN-ß-mediated necroptosis and osteogenic differentiation via the JAK-STAT pathway.

Localization and characterization θ carbonic anhydrases in Thalassiosira pseudonana.

Carbonic anhydrase (CA) is a crucial component for the operation of CO2-concentrating mechanisms (CCMs) in the majority of aquatic photoautotrophs that maintain the global primary production. In the genome of the centric marine diatom, Thalassiosira pseudonana, there are four putative gene sequences that encode θ-type CA, which was a type of CA recently identified in marine diatoms and green algae. In the present study, specific subcellular locations of four θCAs, TpθCA1, TpθCA2, TpθCA3, and TpθCA4 were determined by expressing GFP-fused proteins of these TpθCAs in T. pseudonana. As a result, C-terminal GFP fusion proteins of TpθCA1, TpθCA2, and TpθCA3 were all localized in the chloroplast; TpθCA2 was at the central chloroplast area, and the other two TpθCAs were throughout the chloroplast. Immunogold-labeling transmission electron microscopy was further performed for the transformants expressing TpθCA1:GFP and TpθCA2:GFP with anti-GFP-monoclonal antibody. TpθCA1:GFP was localized in the free stroma area, including the peripheral pyrenoid area. TpθCA2:GFP was clearly located as a lined distribution at the central part of the pyrenoid structure, which was most likely the pyrenoid-penetrating thylakoid. Considering the presence of the sequence encoding the N-terminal thylakoid-targeting domain in the TpθCA2 gene, this localization was likely the lumen of the pyrenoid-penetrating thylakoid. On the other hand, TpθCA4:GFP was localized in the cytoplasm. Transcript analysis of these TpθCAs revealed that TpθCA2 and TpθCA3 were upregulated in atmospheric CO2 (0.04% CO2, LC) levels, while TpθCA1 and TpθCA4 were highly induced under 1% CO2 (HC) condition. The genome-editing knockout (KO) of TpθCA1, by CRISPR/Cas9 nickase, gave a silent phenotype in T. pseudonana under LC-HC conditions, which was in sharp agreement with the case of the previously reported TpθCA3 KO. In sharp contrast, TpθCA2 KO is so far unsuccessful, suggesting a housekeeping role of TpθCA2. The silent phenotype of KO strains of stromal CAs suggests that TpαCA1, TpθCA1, and TpθCA3 may have functional redundancy, but different transcript regulations in response to CO2 of these stromal CAs suggest in part their independent roles.

Transmission of COVID-19 in Nightlife, Household, and Health Care Settings in Tokyo, Japan, in 2020.

Importance: There have been few studies on the heterogeneous interconnection of COVID-19 outbreaks occurring in different social settings using robust, surveillance epidemiological data. Objectives: To describe the characteristics of COVID-19 transmission within different social settings and to evaluate settings associated with onward transmission to other settings. Design, Setting, and Participants: This is a case series study of laboratory-confirmed COVID-19 cases in Tokyo between January 23 and December 5, 2020, when vaccination was not yet implemented. Using epidemiological investigation data collected by public health centers, epidemiological links were identified and classified into 7 transmission settings: imported, nightlife, dining, workplace, household, health care, and other. Main Outcomes and Measures: The number of cases per setting and the likelihood of generating onward transmissions were compared between different transmission settings. Results: Of the 44 054 confirmed COVID-19 cases in this study, 25 241 (57.3%) were among male patients, and the median (IQR) age of patients was 36 (26-52) years. Transmission settings were identified in 13 122 cases, including 6768 household, 2733 health care, and 1174 nightlife cases. More than 6600 transmission settings were detected, and nightlife (72 of 380 [18.9%]; P < .001) and health care (119 [36.2%]; P < .001) settings were more likely to involve 5 or more cases than dining, workplace, household, and other settings. Nightlife cases appeared in the earlier phase of the epidemic, while household and health care cases appeared later. After adjustment for transmission setting, sex, age group, presence of symptoms, and wave, household and health care cases were less likely to generate onward transmission compared with nightlife cases (household: adjusted odds ratio, 0.03; 95% CI, 0.02-0.05; health care: adjusted odds ratio, 0.57; 95% CI, 0.41-0.79). Household settings were associated with intergenerational transmission, while nonhousehold settings mainly comprised transmission between the same age group. Among 30 932 cases without identified transmission settings, cases with a history of visiting nightlife establishments were more likely to generate onward transmission to nonhousehold settings (adjusted odds ratio, 5.30 [95% CI, 4.64-6.05]; P < .001) than those without such history. Conclusions and Relevance: In this case series study, COVID-19 cases identified in nightlife settings were associated with a higher likelihood of spreading COVID-19 than household and health care cases. Surveillance and interventions targeting nightlife settings should be prioritized to disrupt COVID-19 transmission, especially in the early stage of an epidemic.

Pleiotropic Functions of Nitric Oxide Produced by Ascorbate for the Prevention and Mitigation of COVID-19: A Revaluation of Pauling's Vitamin C Therapy.

Linus Pauling, who was awarded the Nobel Prize in Chemistry, suggested that a high dose of vitamin C (l-ascorbic acid) might work as a prevention or treatment for the common cold. Vitamin C therapy was tested in clinical trials, but clear evidence was not found at that time. Although Pauling's proposal has been strongly criticized for a long time, vitamin C therapy has continued to be tested as a treatment for a variety of diseases, including coronavirus infectious disease 2019 (COVID-19). The pathogen of COVID-19, SARS-CoV-2, belongs to the ß-coronavirus lineage, which includes human coronavirus, severe acute respiratory syndrome (SARS), and Middle East respiratory syndrome (MERS). This review intends to shed new light on vitamin C antiviral activity that may prevent SARS-CoV-2 infection through the chemical production of nitric oxide (NO). NO is a gaseous free radical that is largely produced by the enzyme NO synthase (NOS) in cells. NO produced by upper epidermal cells contributes to the inactivation of viruses and bacteria contained in air or aerosols. In addition to enzymatic production, NO can be generated by the chemical reduction of inorganic nitrite (NO2-), an alternative mechanism for NO production in living organisms. Dietary vitamin C, largely contained in fruits and vegetables, can reduce the nitrite in saliva to produce NO in the oral cavity when chewing foods. In the stomach, salivary nitrite can also be reduced to NO by vitamin C secreted from the epidermal cells of the stomach. The strong acidic pH of gastric juice facilitates the chemical reduction of salivary nitrite to produce NO. Vitamin C contributes in multiple ways to the host innate immune system as a first-line defense mechanism against pathogens. Highlighting chemical NO production by vitamin C, we suggest that controversies on the therapeutic effects of vitamin C in previous clinical trials may partly be due to less appreciation of the pleiotropic functions of vitamin C as a universal bioreductant.

Fractionation and characterization of cell wall polysaccharides from the brown alga Cladosiphon okamuranus.

Brown algae contain a polysaccharide-rich cell wall, mainly composed of alginate and fucoidan which have been extensively studied for their individual structure and bioactivities. Particularly, the cell wall of Cladosiphon okamuranus is rich in fucoidan rather than alginate. However, little is known about its arrangement or interlinking with other polysaccharides such as cellulose in the cell wall. To determine its structure in detail, the cell wall was sequentially fractionated into five fractions: hot water (HW), ammonium oxalate, hemicellulose-I (HC-I), HC-II, and cellulose. Almost 80% of the total cell wall recovered from alcohol insoluble residue in C. okamuranus consisted of HW and HC-I, which mainly contained fucoidan composed of fucose, glucuronic acid, and sulfate in molar ratios of 1.0:0.3:0.9 and 1.0:0.2:0.3, respectively. Methylation analysis revealed that fucoidan in HW and HC-I structurally differed in terms of content of sulfate, and sugar residue which was 1,4-linked xylose and 1,4-linked fucose. Small angle X-ray scattering measurements also showed distinct conformational differences between HW and HC-I. These structural heterogeneities of fucoidan may be related to their localization, and fucoidan in HC-I may be involved in reinforcing cell wall structure by cross-linking to cellulose.

Automatic detection of primary and metastatic lesions on cervicothoracic region and whole-body bone using a uniform machine-learnable approach for [18F]-FDG-PET/CT image analysis.

We propose a method to detect primary and metastatic lesions with Fluorine-18 fluorodeoxyglucose (FDG) accumulation in the lung field, neck, mediastinum, and bony regions on the FDG-PET/CT images. To search for systemic lesions, various anatomical structures must be considered. The proposed method is addressed by using an extraction process for anatomical regions and a uniform lesion detection approach. The uniform approach does not utilize processes that reflect any region-specific anatomical aspects but has a machine-learnable framework. Therefore, it can work as a lesion detection process for a specific anatomical region if it machine-learns the specific region data. In this study, three lesion detection processes for the whole-body bone region, lung field, or neck-mediastinum region are obtained. These detection processes include lesion candidate detection and false positive (FP) candidate elimination. The lesion candidate detection is based on a voxel anomaly detection with a one-class support vector machine. The FP candidate elimination is performed using an AdaBoost classifier ensemble. The image features used by the ensemble are selected sequentially during training and are optimal for candidate classification. Three-fold cross-validation was used to detect performance with the 54 diseased FDG-PET/CT images. The mean sensitivity for detecting primary and metastatic lesions at 3 FPs per case was 0.89 with a 0.10 standard deviation (SD) in the bone region, 0.80 with a 0.10 SD in the lung field, and 0.87 with a 0.10 SD in the neck region. The average areas under the ROC curve were 0.887 with a 0.125 SD for detecting bone metastases, 0.900 with a 0.063 SD for detecting pulmonary lesions, and 0.927 with a 0.035 SD for detecting the neck-mediastinum lesions. These detection performances indicate that the proposed method could be applied clinically. These results also show that the uniform approach has high versatility for providing various lesion detection processes.

Massive hemoptysis managed by prolonged anticoagulation-free veno-venous extracorporeal membrane oxygenation with which thoracic surgeries were accompanied: a case report.

BACKGROUND: Massive hemoptysis causing inadequate ventilation results in life-threatening consequences. We present a patient who developed respiratory insufficiency produced by bronchiectatic massive hemoptysis and underwent prolonged anticoagulation-free veno-venous extracorporeal membrane oxygenation (VV-ECMO) during which thoracic surgeries were performed. CASE PRESENTATION: A 79-year-old woman suffered massive hemoptysis resulting in respiratory failure during fiberoptic bronchoscopy. Bronchial intubation followed by one lung ventilation failed to ensure adequate oxygenation. Anticoagulation-free VV-ECMO, therefore, was installed immediately. Since conservative hemostatic measures including bronchial arterial embolization were not effective, resection of the culprit lung was performed while on VV-ECMO. Next day an exploratory thoracotomy and intercostal artery embolization were needed for recurrent bleeding. The VV-ECMO was withdrawn after five days of operation. CONCLUSIONS: Massive hemoptysis can be fatal and needs instantaneous and intensive treatments. In our case, long-term anticoagulation-free VV-ECMO during which thoracic surgeries and endovascular interventions were performed provided a favorable outcome.