The interest in microalgae production deals with its role as the third generation of feedstock to recover renewable energy. Today, there is a need to analyze the ultimate research and advances in recovering the microalgae biomass from the culture medium. Therefore, this review brings the current research developments (over the last three years) in the field of harvesting microalgae using membrane-based technologies . Initially, the principles of membrane technologies are given to outline the main parameters influencing their operation. The main strategies adopted by the research community for the harvesting of microalgae using membranes are subsequently addressed, paying particular attention to the novel achievements made for improving filtration performance and alleviating fouling. Moreover, this contribution also gives an overview of the advantages of applying membrane technologies for the efficient extraction of the high added-value compounds in microalgae cells, such as lipids, proteins and carbohydrates, which together with the production of renewable biofuels could boost the development of more sustainable and cost-effective microalgae biorefineries.  Today, there is a big interest in implementing renewable energies as a way of replacing the conventional fossil fuels derived from coal, natural gas and crude oil, which in fact are highly polluting to the environment . The gloThe production of microalgae becomes more attractive since they can basically grow in harsh conditions  with minimal competition of fresh water; moreover, when using closed culture systems, they do not depend directly on the weather conditions, allowing their cultivation during the whole year ,6. To daImportantly, considering the small size of algae cells in the order of micrometers to tens of micrometers, the harvesting and dewatering of the biomass from cultivation media becomes a great challenge. Flocculation, flotation, sedimentation and electroflocculation are among the primary harvesting operations, which display operational issues due to the microalgae in suspension owning a similar density to water , while sAs in all membrane technologies, the membrane is the main physical element used for the separation of molecules in liquid and gas states. The membrane is defined as a semi-permeable barrier that displays preferential selectivity. According to the principle of membrane technology, the molecules can be successfully separated using the membrane depending on the driving force . In the case of pressure-driven membrane technologies, the membrane is able to differentiate between molecules due to their difference in size and molecular weight. The pore size is the main criteria used to categorize MF and UF membranes, as specified in When dealing with microalgae harvesting, MF and UF membranes are likely to be the most investigated by the research community . By applAsymmetry properties of a membrane: In the case of polymer membranes, phase inversion technique is commonly used for the preparation of porous membranes [embranes . Such a Intrinsic properties of a membrane: As it is well known, the membranes, depending on the polymer or inorganic material, may either present hydrophilic or hydrophobic properties. In MF and UF processes, hydrophobic materials are preferred  since they repel water molecules, along with all those water-soluble compounds. In addition to this, the surface morphology influences the separation performance, but more importantly, contributes to some specific issues on the operation; for example, the membrane roughness, especially a rougher membrane surface, contributes to fouling. To some extent, the protuberances on a surface allow the capture of organic matter. Here, if there is an accumulation of organic material that may represent a source of microorganism proliferation, the membranes will be susceptible to biofouling formation as well [ as well .Membrane–molecule interactions: In general, electrostatic interactions may occur between membrane surfaces and specific solutes that present any charge. Of course, the membrane should also reveal any type of charge, which is often associated with the availability of functional groups on the membrane surface. Eventually, specific solute–membrane interactions, such as the hydrophobic interaction, Coulombic intermolecular attraction and repulsion, are among the most identified forces in membrane processes [rocesses .Membrane fouling: This factor acts as the main bottleneck of membrane processes since it can lower the flux by pore blocking. The membrane fouling depends crucially on the physicochemical composition of the feed solution to be treated. Here, the possible interactions among the solutes and the membrane can introduce the degree and type of membrane fouling [ fouling . HoweverOperating parameters: The permeate flux is usually increased as a function of the driving force; this is possible until the limiting transmembrane pressure is reached [ reached , in whic reached . On the As reported in As it can be seen, various factors and parameters play a crucial role in membrane performance once implemented in the membrane process. This becomes more challenging when complex solutions such as microalgae culture are filtrated. Therefore, the following section collects the main strategies adopted by the research community for the harvesting of microalgae. For this, the most relevant outcomes in the field have been discussed.Scenedesmus acuminatus [Chlorella zofingiensis [C. pyrenoidosa [Dictyosphaerium sp. [Nannochloropsis sp. [Microcystis sp. [Dunaliella salina [Pavlova lutheri [Considering various criteria , filtration is considered among the most applicable methods for separating microalgae from their culture medium, even for different applications, including biofuel, human and animal food, high-marketable products and water quality restoration ,27. As suminatus , Chlorelngiensis , C. pyreenoidosa , Dictyosrium sp. , Nannochpsis sp. , Microcystis sp. , as wella salina , Pavlova lutheri  and many lutheri . However lutheri ,37. Memb lutheri ,36,38. T lutheri ; algal s lutheri ,40; unli lutheri ,42; an i lutheri . Thus, e lutheri ,44, dyna lutheri  and vibr lutheri ,46,47, f lutheri ,49, the  lutheri ,50,51,52 lutheri  jet-assi lutheri , tilted  lutheri ,56.3/m2 h) resulted in 2.7 times higher permeation rates than those of the vertical panel, while the use of a two-sided membrane did not further improve the cleaning efficiency. Similar filtration behaviors were reported by Lau et al. [2 bar h, when employed a one-sided panel at an aeration rate of 1.8 L/min and a tilting angle of 20°. In other study, Ismail et al. [Chlorella vulgaris using an improved nylon 6,6 nanofiber membrane, achieving a permanence and a flux as high as 40.2 L/m2 h and 402.3 L/m2 h bar, respectively [2 h bar at 1.5 L/m aeration for the harvesting of Chlorella vulgaris [In submerged filtration systems, where recirculation provides the cross-flow velocity, membranes are set above micro-porous pipes that generate air bubbles, scouring the foulants along the membrane surface ; thus, ru et al. , who repl et al.  found anl et al. . The effectively . Other hectively . A novelvulgaris . Moreove3, and NaOH; however, the unsuitable application of chemicals could deteriorate the membrane or cause cell lysis. Tailor-made back washable membranes have shown a better performance than commercially available membranes, which have also been proven to be efficient for polishing purposes, allowing the recycling of water and nutrients while removing bacteria and algal debris [Membrane cleaning frequency also impacts on the operation flux and fouling. It has been reported that operating in a filtration/backwashing regime may outperform the regular filtration/relaxation operation . The effl debris . The ope2 h bar, which are 1.5–5 times higher compared with those accommodated by conventional tangential cross-flow filtration [3) [3, equivalent to 1.39–1.46 kWh/kg of harvested microalgae [2 h) during the harvesting of Chlorella vulgaris in comparison with that exhibited by a shear-enhanced microfiltration system equipped with unperforated disks as the control [2 h [In dynamic membrane filtration systems, the hydrodynamic conditions on the membrane surface are altered, by vibrating the membrane instead of the surrounding fluid or by moving a mass at the liquid–membrane interface, in order to achieve a high membrane shear rate and alleviate fouling . Turbuleltration ,62. Regation [3) . Using acroalgae . Moreovecroalgae , dynamiccroalgae ,64,65,66 control ; indeed,rol [2 h .Dictyosphaerium sp. as the membrane material and model microalgae strain, respectively. This strategy exhibited enhanced antifouling properties (which might improve membrane lifetime) due to the increase in the filtration active area per m2 of the membrane and the cross-flow behavior, particularly, the enhancement of local turbulences near to the membrane surface; therefore, ensuring a very high membrane permeances as high as 110 L/m2 h bar using a low cross-flow velocity of 0.0025 m/s. The energy inputs and total harvesting costs were estimated as 0.28 kWh and EUR 0.16 per kg of harvested microalgae. In flocculation-assisted membrane systems, the flocculation step needs to be optimized as it can affect membrane permeance and harvesting efficiency; optimized flocculant type and dossing will result in an improved filterability by giving bigger flocs and less extracellular organic matter content [Desmodesmus sp. with membrane permeances of up to 1000 L/m2 h bar [Membrane surface patterning has been also employed as an alternative to tackle membrane fouling , enhanci content ,71. Prev content ,48,49. C content . Electro content . Introdum2 h bar .Nannochloropsis sp., showing good anti-fouling properties with enhanced hydrophilicity [Regarding the materials used for the fabrication of membranes, the ideal membrane should pose a robust mechanical strength, high permeability, excellent chemical properties  and low investment costs . Polyvinhilicity .Besides membrane material, membrane pore size greatly affects the harvesting performance; thus, its adequate selection is of utmost importance to reduce fouling risk . The sizTetraselmis sp. The results showed that, although both evaluated systems mediated similar total concentrations factors of 37.3, the use of forward osmosis as a post-harvesting process resulted in 24% less energy consumption compared to the dual stage ultrafiltration process [2 h [4Cl [2 h with associated volumetric concentration factors of up to 6× [2+ to harvest microalgae  due to the in situ hydroxyl radical (•OH) generation which can generate electrostatic repulsion excluding away negatively charged microalgal cells and organic matter from the membrane surface and degrade selectively extracellular organic matter, including proteins, polysaccharides and hydrophobic humic-like substances, on the surface of the membrane and inside the pores. Moreover, a flow cytometry analysis revealed a 3.8% reduction in live cells after a 3 h filtration, mainly due to the fact that hydroxyl radical is highly selective to organic matter and diffuses slowly through the algal membrane, suggesting that the electro-Fenton system may slightly affect the microbial activity. On the other hand, Kim et al. [Chlorella, less cake formation was observed and pore-clogging without deteriorating the integrity of the cells.Forward osmosis has been also applied for the harvesting of microalgae, mainly as an initial dewatering step ,85,86,87 process . Compareup to 6× . A conceup to 6× . Other sup to 6× , electriup to 6× , and turup to 6×  have beeup to 6×  evaluatecroalgae ; the memm et al.  evaluate3 [Experiences gained from pilot-scale membrane microalgae harvesting are needed to provide a solid proof of its technical viability for commercialization and datasets for future economic–environmental assessments . Unlike 3 .Microalgae biorefinery aims at coupling the cultivation of microalgal biomass with the production of biofuels and high-value co-products such as pigments, proteins, lipids, carbohydrates, vitamins, anti-oxidants, among others, which have application in several sectors, including food, cosmetics, as well as pharma industries ,93. HowePorphyridium using a Cogent M1 pilot-scale tangential-flow filtration unit configured to operate in an ultrafiltration and diafiltration mode using PES flat membranes [Nannochloropsis gaditana, which had an initial biomass concentration of 100 g/L. The lowest membrane cut-off led to the highest protein yield of 25%. By contrast, the filtration process was not improved by further increasing the cut-off of the membrane due to adsorptive fouling of polysaccharides.Membrane filtration is seen as a promising method for the recovery of proteins from microalgae not only because of its green nature , but also because it may preserve both their functional and nutritional properties, which become particularly relevant in the production of health beneficial products ,95. The embranes . By applembranes  also appChlorella sorokiniana) was recently reported using a three-step membrane filtration process encompassing a prefiltration step  to remove cell fragments, followed by diafiltration  to recover the proteins previously retained and, finally, a concentration step  in which the proteins are concentrated while washing out salts and small sugars [A proof-of-concept study for the recovery, purification and concentration of proteins derived from microalgal biomass  [Gelidium pusillum (68% yield and a purity index of 0.49), which has a market value of 180–250 USD/mg and commercial applications in immunology, diagnostics, cosmetics and foods [Grateloupia turuturu, obtaining a purity index of 1.07 despite the use of a single ultrafiltration step [In another study, Böcker et al. isolated proteins with emulsification potential from factants . Food grltration . More rere size) . The PVDre size) ) via a tnd foods . Finallyion step .Porphyridium is one of the leading microalgae genera used as a source of EPS having nutraceutical and pharmaceutical activities [P. cruentum has been exploited for the production of EPS, and other valuable compounds such as pigments, at large scale [P. cruentum [2 h. It is important to note that EPS solutions have a strong fouling capacity, even at diluted concentrations, because of their ability to form highly viscous gels and compacts deposits on the membrane’s surface. At a low EPS concentration of 0.1 g glucose equiv./L, irreversible and reversible fouling was ascertained as 88 and 12%, respectively.In the case of algal exopolysaccharides (EPS), tivities . Indeed,ge scale . Lab-scacruentum . The volP. cruentum has been also extracted using cross-flow filtration in the diafiltration mode with a 300 kDa molecular weight cut-off, but with an EPS loss in the permeate of 34% [2 h at four bar depending on the cross-flow velocity, recovering more than 80% of the EPS in a concentrated fraction  [It has been reported that carbohydrates lead to a higher fouling risk than proteins when hydrophilic polyvinylchloride (PVC) membranes in ultrafiltration configuration were tested . Howevere of 34% . Diafilt.26 g/L) .Nostoc sphaeroides and Haematococcus pluvialis, achieving average permeate fluxes close to 37 L/m2 h at a transmembrane pressure of 0.5–0.6 bar and ambient temperature (28–37 °C) regardless of the microalgal culture; however, the EPS varied in the range of 3.5 up to 231.3 mg/L depending on the culture. In another study, EPS from Porphyridium were successfully extracted using PES flat UF pilot membranes, obtaining 80% of the initial EPS content after ultrafiltration and diafiltration steps [Furthermore, the culture conditions  and the physiological state of microalgae affects the profile and concentration of EPS, a fact that contributes to make the extraction of microalgae EPS complex. The concentration of EPS in the culture medium typically reaches low values ranging from 0.1 to 1.0 g/L, which increases the downstream processing cost ,108. Thuon steps . AltogetParachlorella kessleri [Regarding microalgae lipids, they can be used not only as a feedstock for the production of biodiesel, but also in cosmetics ,109. In kessleri . The autScenedesmus acuminatus, Chlorella zofingiensis, C. pyrenoidosa, Dictyosphaerium sp., Nannochloropsis sp., Microcystis sp., along with shear sensitive species such as Dunaliella salina, Pavlova lutheri, among others. The average separation efficiency has been reported as high as 100%.This review paper denoted the ability of membrane techniques in the harvesting of a microalgae biomass, as well as key principles and factors involved for the successful operation of membranes in such a complex system (culture medium). By analyzing the ongoing advances on membrane-based harvesting processes, this paper identified the main strategies followed by the researchers in collecting the algae biomass overcoming the main bottlenecks in the operation. To date, plenty of microalgae strains have been successfully harvested using membrane-based operations, including In these applications, membrane fouling is still addressed as the most critical factor that compromises the performance of membranes and their continuous operation. Herein, the researchers are strongly working at modifying the membrane surfaces to reduce the membrane fouling, and it seems to be that a future direction will deal with the chemical modification of membranes to obtain highly hydrophilic membranes. As a recommendation to the new researchers in the field, even though that the recovery efficiencies and permeation fluxes seem to be high, future works should be aware of implementing membrane processes adapted with other technologies to guarantee long-term operation. In this regard, future works can give us an overview if such maintenance and operating costs can be reduced. Furthermore, the influence of operating parameters on the harvesting of microalgae at pilot scale deserves further attention.Finally, the application of membrane techniques not only deals with the harvesting of algae biomass, but also with the recovery of valuable molecules either contained or derived from algal biomass. Specific biomolecules, such as B-phycoerythrin, proteins , exopolysaccharides and lipids, can be separated from the culture media. As a concluding remark, the selection of the membrane types and operating parameters will mainly dictate the efficiency of the recovery process; however, the strategy proposed by the researchers will also contribute to the successful separation. In this way, depending on the target molecules, the membrane process design should be smartly proposed. Finally, it is of note that although membrane-based microalgae harvesting processes have become a promising technology, to date, however, it seems that the third-generation renewable energy production alone is uneconomical, but the development of microalgae biorefineries devoted to the co-production of biofuels and value-added fine chemicals could be a more sustainable and cost-effective approach. Thus, there is a gap in the design and evaluation of multi-objective approaches to simultaneously recover microalgal biomass and its related by-products by membrane technology, including its techno-economic assessment.


Xuebijing Injection have been found to improve the clinical symptoms of COVID-19 and alleviate disease severity, but the mechanisms are currently unclear. This study aimed to investigate the potential molecular targets and mechanisms of the Xuebijing injection in treating COVID-19 via network pharmacology and molecular docking analysis. The main active ingredients and therapeutic targets of the Xuebijing injection, and the pathogenic targets of COVID-19 were screened using the TCMSP, UniProt, and GeneCard databases. According to the ‘Drug-Ingredients-Targets-Disease’ network built by STRING and Cytoscape, AKT1 was identified as the core target, and baicalein, luteolin, and quercetin were identified as the active ingredients of the Xuebijing injection in connection with AKT1. R language was used for enrichment analysis that predict the mechanisms by which the Xuebijing injection may inhibit lipopolysaccharide-mediated inflammatory response, modulate NOS activity, and regulate the TNF signal pathway by affecting the role of AKT1. Based on the results of network pharmacology, a molecular docking was performed with AKT1 and the three active ingredients, the results indicated that all three active ingredients could stably bind with AKT1. These findings identify potential molecular mechanisms by which Xuebijing Injection inhibit COVID-19 by acting on AKT1.  It was first identified in December 2019 in Wuhan, Hubei, China, and has since spread around the world. The WHO has declared that the COVID-19 outbreak constitutes a Public Health Emergency of International Concern (PHEIC). This disease can be clinically classified as mild, severe, or critical. Fever, dry cough, and fatigue are the main manifestations, and patients classified as severe can rapidly progress to acute respiratory distress syndrome (ARDS) and multiple organ failure (MOF), amongst other conditions. Unfortunately, at present, there is no cure officially approved for this disease, creating a formidable challenge in its treatment, prognosis, and control. Traditional Chinese medicines (TCM), that are characterized as being anti-viral and affecting multiple pathways and targets, have been proven to be significantly effective in treating COVID-19.National Drug Catalog for Basic Medical Insurance, Work-related Injury Insurance and Maternity Insurance stated that XBJ injection should be used in the first aid treatment of critically ill patients. Furthermore, the Diagnosis and Treatment Protocol for COVID-19  states that it should be used to treat severe and critically ill patients with COVID-19. However, its mechanisms in the treatment of COVID-19 have not yet been elucidated.The Xuebijing (XBJ) injection is a traditional Chinese medicine that is based on the XueFuZhuYu Decoction (XFZYD). Its main herbal components are Carthami Flos, Paeoniae Radix Rubra, Chuanxiong Rhizoma, Salviae Miltiorrhizae Radix et Rhizoma, and Angelicae Sinensis Radix, and it can have several effects, such as anti-inflammation, anti-oxidation, immune regulation, improving blood stasis, anti-endotoxin, and anti-shock. Previously, it has been used to treat patients with diseases like severe pneumonia and sepsis. In 2019, the Highly pathogenic viruses should be handled under strict laboratory conditions and in compliance with specific biosafety procedures, this study has attempted to investigate the possible molecular mechanisms of XBJ injection when treating COVID-19 via computer simulation based on network pharmacology and molecular docking analyses; thereby avoiding potential laboratory biosafety hazards and improving the efficiency of research into infectious disease outbreaks and modern Chinese medicines. Recently, network pharmacology-based drug repositioning has become increasingly important for the research and development of drugs. Network pharmacology is an application tool based on systems biology theory and network analysis statistics, that conducts network analysis for specific biological systems, and designs multi-target drug molecules by selecting specific signal nodes and comparing signal pathways. Zhou et al. ,3.This study has improved upon the network pharmacology and molecular docking anlaysis methods, and determined how to identify the core target in the gene network using the maximum Degree value, which indicates the role of the nodes in network pharmacology. The cluster Profiler package in R language was then used to screen the latest Gene Ontology and KEGG pathways for the core target. The network pharmacology results were verified using protein-small molecule docking to simulate the binding between active ingredients and the core target. These analyses were used to predict the active ingredients, targets, and signaling pathways, provide a molecular basis to investigate the mechanisms by which the COVID-19 inflammatory stress response is inhibited by the XBJ injection, as well as enhance the available theoretical support for the wider application of this drug in the treatment of COVID-19.2.2.12.1.1http://tcmspw.com/tcmsp.php) according to oral bioavailability (OB) ≥ 30% and drug likeness (DL) ≥ 0.18. The drug targets of the main active ingredients were then screened through the Targets information of TCMSP. Finally, gene symbol conversion was performed for the targets screened from the TCMSP according to UniProt [https://www.uniprot.org/). Additional information was added according to the literature, and 206 targets were obtained.The main active ingredients were screened from the 5 main herbal components of the XBJ injection, including Carthami Flos, Paeoniae Radix Rubra, Chuanxiong Rhizoma, Salviae Miltiorrhizae Radix et Rhizoma and Angelicae Sinensis Radix using the Traditional Chinese Medicine Systems Pharmacology Database  [In Genecards (ds.org/) , coronav2.32.3.1https://string-db.org/cgi/input.pl) [Basic Settings: ① Network type: full network ; ② meaning of network edges: evidence (line color indicates the type of interaction evidence); ③ active interaction sources: Textmining, Experiments, Databases, Co-expression, Neighborhood, Gene Fusion, and Co-occurrence; ④ minimum required interaction score: medium confidence (0.400); ⑤ max number of interactors to show: 1st shell: none/query proteins only, 2nd shell: none.Advanced Settings: ① network display mode: interactive svg; ② network display options: hide disconnected nodes in the network.Junction targets for the XBJ injection and COVID-19 were imported into the STRING database (nput.pl) , and theThe PPI network for the junction targets for the XBJ injection and COVID-19 was constructed using the STRING database, and the results were exported to a TSV file for further analysis using Cytoscape 3.7.2  to optimize the PPI network and screen for the core target with the maximum Degree value.2.3.2 GO enrichment analysis and KEGG pathway analysis for the core target for the XBJ injection and COVID-19Gene Ontology (GO) includes three aspects: biological process (BP), cellular component (CC) and molecular function (MF). GO enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted for the core target for the XBJ injection and COVID-19 using R (version 3.6.1)  package,2.4According to the network pharmacology results, AKT1 was identified as the core target of the XBJ injection when treating COVID-19, and the active ingredients of the XBJ injection, baicalein, luteolin, and quercetin were all connected with the core target AKT1. Consequently, further molecular docking verification was carried out based on these results.−1 indicates that the protein and small molecule can bind, and ≤−7.0 kcal·mol−1 indicates a strong binding ability.RCSB PDB databases were used to download the 3D structure PDB file of the core target AKT1 (PDB ID: 1UNP), and it was then imported into the Discovery Studio 2019 Client (DS) to remove solvent molecules and ligands. Solvents were removed from the structure, while polar hydrogen atoms were added to the 1UNP by clicking ‘Add Polar’, and a forcefield was applied to the 1UNP by selecting ‘Apply Forcefield’. The PubChem database was used to obtain the SDF files of the active ingredients for the core target AKT1 as small molecules, which were imported into the DS. Apply Forcefield was used to give the force field to the small molecules. Finally, the CDOCKER (Dock Ligands) was used for protein-small molecule docking, by adjusting the Top Hits-Pose Cluster Radius to 0.5, and the other parameters were their default values. CDOKER  is a hig3.This study aimed to investigate the molecular targets and mechanisms of the Xuebijing injection when used to treat COVID-19 using network pharmacology and molecular docking analyses. According to the ‘Drug-Ingredients-Targets-Disease’ network, AKT1 was identified as the core target, and baicalein, luteolin, and quercetin were identified as the active ingredients of the Xuebijing injection that were associated with AKT1. The results of the GO enrichment analysis showed that response to lipopolysaccharide was the most significant biological process in which AKT1 was involved, the cellular component was spindle, and its molecular function involoved nitric-oxide synthase regulator activity. The results of the KEGG pathway analysis showed that AKT1 was involved in the treatment of COVID-19, mainly by regulating TNF and other signaling pathways. Based on the results of the network pharmacology, molecular docking analysis was performed with AKT1 and the three active ingredients, and the results indicated that all three active ingredients could bind stably with AKT1.3.13.1.1The 5 herbal components were retrieved using TCMSP. According to the screening requirements of an OB ≥ 30% and DL ≥ 0.18, we identified 44 active ingredients, including 9 from Carthami Flos, 8 from Paeoniae Radix Rubra, 6 from Chuanxiong Rhizoma, 19 from Salviae Miltiorrhizae Radix et Rhizoma, and 2 from Angelicae Sinensis Radix. MOL000449-Stigmasterol and MOL000358-beta-sitosterol were the common active ingredients mapping to Carthami Flos, Paeoniae Radix Rubra, and Angelicae Sinensis Radix. MOL002714- baicalein was the common active ingredient mapping by Carthami Flos and Paeoniae Radix Rubra, while MOL000006-luteolin was the common active ingredient mapping to Carthami Flos and Salviae Miltiorrhizae Radix et Rhizoma. Following the removal of the duplicates, a total of 38 non-repeated active ingredients were identified. In addition, 206 non-repeated drug targets were obtained from the TCMSP, according to the active ingredients mentioned above.3.1.2The ingredient-target network for the XBJ injection was constructed using Cytoscape 3.7.2 and included 38 non-repeated active ingredients and 206 non-repeated potential targets see . The cor3.2GeneCards was used to identify 277 targets of COVID-19, and STRING and Cytoscape were used to screen out the free targets. These targets, such as TNF, IL6, GAPDH, TP53, and IL10, were then arranged in order of degree see .Table 23.3−16, avg. local clustering coefficient was 0.675). The PPI network was then optimized using the Cytoscape software , luteolin , and quercetin (Carthami Flos), and they were all connected with AKT1, suggesting that these three active ingredients may be effective for XBJ injection in the prevention and treatment of COVID-19 see .Figure 3.5 Verification of molecular docking for the XBJ injection when treating COVID-19 by the Results of Network Pharmacology−1, −31.5575 kcal·mol−1, and −31.53 kcal·mol−1, respectively. The binding models are shown in The activity of the three active ingredients obtained through the network pharmacology analysis were validated using protein-small molecule docking.The results showed stable binding between the three active ingredients  and AKT1, with binding energies of −25.5854 kcal·mol4.Diagnosis and Treatment Protocol for COVID-19 , critically ill patients are more likely to develop dyspnea and/or hypoxemia within a week, and severe patients, like those with SARS and MERS, may suffer from a cytokine storm (CS), which can rapidly develop into acute respiratory distress syndrome, septic shock, and multiple organ failure. The XBJ injection has been shown in previous studies to be effective at improving end-point outcomes for severe community-acquired pneumonia (CAP) and reduced the 28-day mortality in septic patients [According to the patients .In this study, 38 non-repeated active ingredients and 206 non-repeated potential targets of the XBJ injection, 277 potential targets of COVID-19, and 46 junction targets were screened using network pharmacology analysis, and the core target from the junction targets was identified to be AKT1. AKT1 is a member of the AKT kinase family that regulates metabolism, proliferation, cell survival, growth, and angiogenesis through a series of downstream substrates. AKT regulates the metabolism of fat, amino acids, and other substances by activating AS160 and PFKFB2, and also induces the TSC1/TSC2 complex and mTORC signal transduction after the stimulation of inflammatory factors, so as to regulate the growth of the endothelium, restore the endothelial barrier function, and promote proliferation of the lung fibroblasts . A previAccording to the results of the GO enrichment analysis, the response to lipopolysaccharide (LPS) was the most significant biological process that AKT1, the core target of junction targets to the XBJ injection and COVID-19, was involved. LPS is a major component in the cell walls of Gram-negative bacteria. It has been reported that LPS can damage alveolar epithelial cells and capillary endothelium cells, resulting in changes to the intercellular space and permeability . AKT couAccording to the results of the KEGG pathway analysis, the TNF signaling pathway is the most significant pathway in which the core target AKT1, that is the core target of junction targets to XBJ injection and COVID-19, was involved. Tumor necrosis factors (TNF) include TNF-α and TNF-β which can promote the expression of proinflammatory factors and participate in systemic inflammatory responses, while it can also produce an anti-infection effect and prevent early viral proteins from being synthesized to inhibit viral replication and kill infected cells. Therefore, the XBJ injection can regulate the TNF signal pathway through AKT1, so that it has anti-inflammation and anti-virual effects, and thus the potential to reduce the inflammation caused by COVID-19.The protein-small molecule docking analysis revealed that all three active ingredients  had perfect CDOCKER Interaction Energy with AKT1 (1UNP), with the highest found in luteolin. This inferred that luteolin was the most important active ingredient contributing to the therapeutic effects of the XBJ injection on COVID-19. The analysis of the binding model showed that there were conventional hydrogen bonds between the luteolin and Gln104, Asp108, Lys111, and Gln59 of the AKT1 and van der Waals between luteolin and Cys60, Gln61, Cys77, and Leu78 of AKT1. It has been reported that luteolin can mitigate airway inflammation by regulating the body’s antioxidative stress response and the COX-2 signaling pathway. Luteolin is also shown to relieve myocardial ischemic injury by modulating the mitogen-activated protein kinase (MAPK)/NF-κB pathway and suppressing enterovirus type 71 infections by inhibiting cell apoptosis and inflammatory factor secretion, demonstrating its antiviral properties. Additionally, luteolin and quercetin are found to induce cell apoptosis by inhibiting the phosphorylation and activation of AKT . DespiteIn this study, potential targets were screened, a PPI network was constructed, and enrichment analysis was conducted to find potential pathways. The XBJ injection was shown to produce synergistic therapeutic effects on COVID-19 with ‘multi- active ingredients, multi-target and multi-pathway’, and molecular docking was performed to verify such results. However, the present study has some limitations: 1) The study results are based on existing data available in the given database, and differences in the results may arise if different databases were used. Therefore, future studies should focus on multidatabase analysis and databases that are updated regularly. 2) The biological network in this study was constructed using qualitative data, but TCM prescriptions usually have a complex composition, as well as intricate interactions, making it extremely difficult to perform an analysis based on the quantitative data. Consequently, the reliability of the conclusions from this study need to be further verified. Therefore, in vivo and in vitro experiments should be conducted to further examine the molecular mechanisms of the XBJ injection for the treatment of COVID-19.5.In conclusion, according to the network pharmacology and molecular docking analyses, the predicted active ingredients in the XBJ injection include baicalein, luteolin, and quercetin. The XBJ injection may inhibit the lipopolysaccharide-mediated inflammatory response, modulate NOS activity, and regulate the TNF signaling pathway by affecting the function of the AKT1, thereby ultimately suppressing the excessive inflammatory response associated with COVID-19. Due to the limitations of the computational methods for chemistry and biology, the results need to be verified with follow-up experiments to provide a basis for the treatment of COVID-19 with TCM.


The Korean Pregnancy Outcome Study (KPOS) was established to investigate the determinants of adverse pregnancy outcomes among Korean women.We recruited 4,537 pregnant women between 2013 and 2017 from two tertiary centers located in Seoul, Korea, and a total of 4,195 Korean women met inclusion criteria in the baseline analysis. A range of data on socio-demographics, past medical histories, reproductive information, health-related behaviors, psychological health and clinical information were obtained using interviewer-based questionnaires and clinical assessment at 12, 24, and 36 gestational weeks (GW), delivery and 6–8 weeks postpartum. Blood samplings were performed at 12, 24 and 36 GW, and placental tissues were obtained after delivery. The main outcome of this study was pregnancy-related complications including gestational diabetes mellitus (GDM), gestational hypertension, and screening positive for peripartum depression. Depression was assessed using the Korean version of the Edinburgh Postnatal Depression Scale, and a score of ≥10 indicated a positive screen for depression.Among 4,195 eligible pregnant women with a median age of 33.0 years, 3,565 (85.0%) pregnancy outcomes were available in this study, including 30 miscarriages, 16 stillbirths, and 3,519 deliveries. Mean gestational age was 38.8 GW, and mean birth weight was 3,236 gram. The prevalence of pregnancy complications of GDM, hypertensive disorders, and screening positive of depression during pregnancy and postpartum was 7.0%, 1.4%, 27.8%, and 16.6%, respectively.We designed KPOS to identify the determinants of pregnancy-related outcomes, and it may provide effective strategies for the prevention of pregnancy complications in Korean pregnant women. It has gone from being a high-reproductive society to one with the lowest of reproductive rates.9–22 Older pregnant women are at higher risk of having an adverse pregnancy-related outcome compared to younger women.9–14 Current evidence suggests a strong association between advanced maternal age and adverse obstetric outcomes, including chromosomal abnormalities, stillbirths, miscarriages, congenital anomalies, and perinatal deaths.12,15–19 Moreover, pregnancy induced complications, such as hypertensive disorders of pregnancy or gestational diabetes mellitus (GDM), may have lifelong adverse health outcomes for the pregnant women and their child, and they can lead to enormous burdens on families, communities, and society as a whole.9,23–25 However, there has been no well-organized study for the evaluation of pregnancy complications and perinatal outcomes in Korean pregnant women. Several authors reported adverse pregnancy outcomes in high-risk patients and related risk factors, but causal relationships remain undetermined because of the lack of prospective studies.26–29 Therefore, we developed the Korean Pregnancy Outcome Study (KPOS), which is a prospective study on general pregnant women in Korea, aiming to investigate the risk factors and distribution of adverse pregnancy outcomes, specifically GDM, hypertensive disorders of pregnancy, and screening positive for peripartum depression (during pregnancy or at 4 weeks postpartum). In addition, KPOS is aimed to determine the effect of well-known risk factors  and beneficial effect of health-related behaviour  to pregnancy complications and neonatal outcomes.It is well-established that advanced maternal age is associated with potentially adverse maternal and foetal outcomes.The aim of this article is to describe the design of the KPOS, present the characteristics of the study populations, and briefly report the results of the study.Between March 2013 and January 2017, all pregnant women who visited Cheil General Hospital & Women’s Healthcare Center and CHA Gangnam Medical Center for antenatal care during the first trimester were asked to participate in the KPOS. Trained research nurses explained the study in detail and obtained written informed consent from participants and assisted them in completing the interview-based questionnaires. Women were excluded from enrolment if they were not Korean or were pregnant with triplets or higher-order multiple gestations. Figure n = 3,364, 80.2%) and CHA Gangnam Medical Center  were included in the baseline analysis after excluding 10 foreigners, 218 individuals who declined to participate in the study, and 114 individuals who were missing key variables. In this study, we have defined ‘foreigners’ as people who are neither Korean citizens nor ethnically Korean. Gestational age was determined by the last menstrual period in naturally conceived women and was confirmed on first trimester ultrasound. If the difference between gestational age by last menstrual period and that by first trimester ultrasound exceeded 7 days, the gestational age according to ultrasound dating was used. In the case of in vitro fertilization pregnancies, the gestational age was determined based on the date of oocyte retrieval.During the baseline period, we recruited 4,537 participants. Both Cheil General Hospital and CHA Gangnam Medical Center are general hospital which focuses on obstetrics and gynaecology located in Seoul and approximately 5,000 and 2,500 deliveries, respectively, take place at these facilities per year, respectively. A total of 4,195 pregnant women from Cheil General Hospital  of Cheil General Hospital (IRB number: CGH-IRB-2013-10) and CHA Gangnam Medical Center (IRB number: 2013-14-KNC13-018), separately. It was emphasized to all participants that they were free to withdraw from any part of the study at any point in time.n = 3,000), we estimated the minimum detectable odds ratios of the exposure and the outcome as shown in To evaluate relevance of the required sample size . Dietary intake patterns were assessed with a specific questionnaire at visit 1.30,31 This scale was specially designed for detecting depression during the postpartum period and was also used to screen for depression during pregnancy.32 In addition, we investigated the perceived stress, satisfaction with marriage, and quality of life in this study. Detailed description of survey method were represented in Table Screening positive for depression during pregnancy and postpartum was evaluated using the Korean version of the Edinburgh Postnatal Depression Scale (K-EPDS), which is a validated questionnaire that includes 10 items.Clinical parameters during pregnancy included height, weight before and after pregnancy, and blood pressure. Laboratory information included haemoglobin, haematocrit, glucose, liver enzyme, blood urea nitrogen (BUN), creatinine, and cholesterol levels, which were recorded at visits 1 and 3. Urinary tests to evaluate for glycosuria and proteinuria were performed at each visit. The results of each serum screening parameter for aneuploidy were recorded at visit 2.First trimester complications including hyperemesis and threatened abortion were recorded at visit 1. In patients who had the hyperemesis and threatened abortion, management options were determined by obstetricians according to the degree of the diseases after clinical evaluation. The delivery outcomes included gestational age at delivery, type of labour (induced or spontaneous), type of delivery, indication for Cesarean delivery, and delivery complications. Neonatal outcomes included birthweight, Apgar score at 1 and 5 minutes, height, head circumference, admission to neonatal intensive care unit, intubation, seizure, phototherapy, and presence of congenital anomaly.33,34 The participants who were diagnosed GDM were sent to endocrinologist, and management methods were determined after clinical evaluation. The information regarding the diagnosis of GDM and insulin therapy were recorded at visit 3 and at delivery.The main outcomes of this study were GDM, hypertensive disorders of pregnancy, and screening positive of depression during pregnancy and postpartum. GDM was diagnosed using a two-step method. Universal screening with a 50-g glucose challenge test (GCT) was conducted between 24 and 28 weeks. If the result of the GCT was 140 mg/dL or more, a confirmatory oral glucose tolerance test (OGTT) was performed. Two different confirmatory tests were conducted. The Cheil General Hospital used the 75-g OGTT as the confirmatory test and used the new diagnostic criteria from the International Association of Diabetes and Pregnancy Study Group . The CHA Gangnam Medical Center used the 100-g OGTT and the Carpenter-Coustan criteria for GDM diagnosis .35 Gestational hypertension was defined by a systolic blood pressure ≥140 mm Hg and/or diastolic blood pressure ≥90 mm Hg without proteinuria (<0.3 g in a 24-hour urine collection) and the hypertension must have developed after 20 weeks of gestation. Preeclampsia was defined as gestational hypertension plus proteinuria (≥0.3 g in a 24-hour urine collection or ≥1+ on a semi-quantitative dipstick test), or with end-organ dysfunction based on a platelet count <100,000/mm3, a creatinine level >1.1 mg/dL, elevation of serum transaminase, pulmonary oedema, or neurologic symptoms, all occurring after 20 weeks of gestation. Eclampsia was defined as preeclampsia with seizures unrelated to other cerebral conditions. The diagnosis of superimposed preeclampsia was based on the development of symptoms of preeclampsia in a pregnant woman with chronic hypertension.Blood pressures were measured at every visit using the automatic oscillometric technique, but a diagnosis of hypertensive disorders of pregnancy was confirmed via manual measurements using blood pressure cuffs and auscultation. Blood pressure measurements were taken during visits 2 and 3 and at delivery to identify hypertensive disorders of pregnancy, which were classified into gestational hypertension, preeclampsia, eclampsia, and superimposed preeclampsia.31Screening positive for perinatal depression can be applied to the case that current or most recent episode of major depression occurs during pregnancy or in the 4 weeks following delivery. A score of ≥10 on K-EPDS indicated a positive screen for perinatal depression in this study according to previous report.3) were excised from underneath the membrane as close to the umbilical cord as possible at the time of delivery. The placenta sampling (1.0 cm2 × 3) procedure was carried out at the time of delivery. The aim of storing maternal blood and placenta is to identify biomarkers related to pregnancy complications, and to develop prediction tools to improve pregnancy outcomes in the future. Those specimens were stored in −70°C freezers at a controlled temperature and humidity. All biological samples were marked with barcodes and stored in the National Biobank of Korea, which was constructed by the Korea Centers for Disease Control and Prevention. We uploaded the data from all questionnaires and examinations to a web-based clinical data management system (iCReaT) managed by the Korea National Institute of Health. The data were periodically monitored by an independent data management team for continuous quality control.The blood samples (10 mL) were obtained a total of 3 times during pregnancy and once more at 6–8 weeks postpartum in GDM patients only. Three sections of the placenta  in the current pregnancy. Most of them were former drinkers (80.1%), but the prevalence of former smokers was very low (10.9%). The frequencies of current smokers and drinkers were very low (0.1%) since almost all of the pregnant women tried to stop smoking and drinking during their pregnancies. The participants who were underweight (body mass index [BMI] <18.5 kg/m2) or overt obesity (BMI ≥30.0 kg/m2) before pregnancy accounted for 15.0% and 1.5% of the participants, respectively.The baseline characteristics of the study participants are presented in Table 36Of the 4,195 pregnant women recruited into the KPOS, 3,565 (85.0%) pregnancy outcomes were observed in the cohort, including 30 miscarriages, 16 stillbirths, and 3,519 deliveries  and the births included 49 sets of twins. The mean anthropometric measurements for height, weight, and head circumference of the baby were 49.6 cm, 3.2 kg, and 34.5 cm, respectively. The mean Apgar scores were 7.9 and 8.8 at 1 and 5 minutes, respectively.st, 2nd, and 3rd trimesters, respectively. We’ve additionally present the result of age-specific and parity-specific perinatal pregnancy-related outcomes analyses in Table 37 According to the Korea National Health and Nutrition Examination Survey, the percentage of smoking and drinking history among Korean women were 7.5% and 73.6%, separately.38 In comparison with Korea national population data, the enrolled patients of KPOS pregnant women are thought to be adequate to represent the Korean general reproductive-aged women . However, socioeconomic variables such as educational status (college or university degree or more: 40.5% vs 91.4%) and household income (3 million Korean-Won or more: 51.1% vs 87.0%) were different with Korea national population data, presumably because both hospitals are located in the center of Seoul city.37–39To the best of our knowledge, the KPOS is the first large-scale prospective study to focus on the limited scientific understanding of risk factors and the frequency of pregnancy complications in Korea. Data derived from the Korean Statistical Information Service, the average maternal age in 2016 was 32.4 years.11,13,18,40–44 An increasing trend in GDM prevalence has been reported globally.41–44 In a previous meta-analysis, the pooled prevalence of GDM in Eastern and South-eastern Asia was 10.1%, and more specifically, a 64.0% higher prevalence was observed among lower or upper-middle income countries.43 According to data from the Korean National Health Insurance Review and Assessment database, the prevalence of GDM significantly increased from 5.7% in 2009 to 9.5% in 2011.42 Similar to a previous Korean study, the prevalence of GDM was 7.0% in the present study. In contrast, this study found a very low prevalence (1.4%) of hypertensive disorders of pregnancy, compared to the prevalence of other countries. Among the 3,565 participants, 16, 32, and 1 of the participants developed gestational hypertension, preeclampsia, and eclampsia, respectively, during pregnancy. A previous study from the Korean National Health Insurance Service–National Sample Cohort reported that the overall prevalence rates of hypertensive disorders of pregnancy requiring magnesium sulfate therapy were 5.7, 4.7, and 4.1 per 1,000 pregnant women for gestational hypertension, preeclampsia, and eclampsia, respectively.40 Epidemiologic studies showed that the most cited and accepted estimate of the occurrence of hypertensive disorders of pregnancy is 5–10%, and the prevalences of hypertensive disorders of pregnancy, gestational hypertension, and preeclampsia are 5.2–8.2%, 1.8–4.4%, and 0.2–9.2%, respectively.45,46 KPOS participants were also more likely to be well educated, with higher income, were more frequently full-time employee, had relatively lower BMIs, and had healthy lifestyle factors, all reflective of the characteristics of the urban middle class in Korea. High socioeconomic status and appropriate prenatal care among the study participants may have led to the relatively low incidence of hypertensive disorders of pregnancy in this study compared to that of other studies.The prevalence of pregnancy-related complications have increased as the number of women with advanced maternal age continues to grow.47 There were differences between different geographical regions; however, the prevalence rates were similar, regardless of the type of diagnostic tool used. The prevalence of screening positive of depression at least once during pregnancy was 27.8%, and the prevalence of having screened positive for peripartum depression was 16.6%. A previous epidemiologic study in Korea showed that the prevalence of peripartum depression using the K-EPDS was found to be 18.8% in the 1st trimester, 12.9% in the 2nd trimester, 12.6% in the 3rd trimester, and 15.7% at 1 month after delivery.48 This was very similar with our results, and psychiatric treatment would be recommended to help depressed pregnant women.Depression is a common complication of pregnancy and the postpartum period. Recent meta-analysis suggested that the incidence of postpartum depression was 12.0%, while the prevalence was 17.0%.The main strengths of the KPOS include the following: 1) a prospective design that aims to understand key determinants of adverse pregnant outcomes to assist in determining causal relationships between pregnancy exposures and complications; 2) we have collected abundant information from the interview-based questionnaires, biological specimens, and clinical data obtained at multiple time points during the pregnancies to assess the independent and interactive effects of various factors on pregnancy complications; and 3) our KPOS investigate team has provided optimal obstetric care to the participants at Cheil General Hospital and CHA Gangnam Medical Center, both well-known maternity hospitals in Korea, inclusive of both low- and high-risk pregnant women.There are some limitations to be considered in this study. First, our study population may not have been representative of all Korean pregnant women because the socioeconomic status of our study participants tend to be higher than those of Korean national population; thus, the results need to be interpreted with cautious. Second, some of the KPOS data were collected via maternal self-report, which could have led to potential bias. Particularly with respect to cigarette smoking and alcohol consumption during pregnancy, self-reports of substance use may have underestimated actual use due to the negative connotations and stigmatization. Third, despite the quite large sample size of the study population, our data lacked the power to investigate relatively rare outcomes, such as preeclampsia or eclampsia, in Korea. Fourth, the extent of detailed and comprehensive questionnaires conducted in each trimester may have reduced the follow-up rate. Finally, the follow-up duration was relatively short because long-term follow-up was practically limited as pregnant women after delivery were scattered into each community. Although time was short to observe the transition from pregnancy complications to chronic diseases, it is expected that research using KPOS data will be actively conducted in the future.Given the increasing trend of advanced maternal age in Korea, we designed KPOS to provide evidence for preventing pregnancy complications. An understanding of the pregnancy-associated risk indicators may help to identify women who are at increased risk of pregnancy-related adverse outcomes later in life. Future studies on the KPOS data will provide information on the determinants of pregnancy-related disorders among pregnant women in Korea. The results will contribute to management of pregnancy-related complications, and thus improve overall maternal and neonatal health.


Cladosiphon okamuranus) on natural killer (NK) cell activity and to assess its safety in healthy adults via a randomized, double-blind, parallel-group, placebo-controlled pilot study. Subjects were randomly divided into two groups—a placebo group ) and a fucoidan group ; after 12 weeks, blood, biochemical, and immunological tests were performed. Clinically adverse events were not observed in any of the tests during the study period. In addition, adverse events due to the test food were not observed. In the immunological tests, NK cell activity was significantly enhanced at 8 weeks in the fucoidan group, compared to before ingestion (0 weeks). In addition, a significantly enhanced NK cell activity was observed in male subjects at 8 weeks, compared with the placebo group. These results confirm that Okinawa mozuku-derived fucoidan enhances NK cell activity and suggest that it is a safe food material.The aim of this study was to evaluate the effects of ingesting fucoidan derived from Okinawa mozuku ( Kjellmaniella crassifolia), Wakame (Undaria pinnatifida), and Okinawa mozuku (Cladosiphon okamuranus) are the main raw materials for fucoidan in Japan. In this study, fucoidan was extracted from Okinawa mozuku, which is an endemic species that grows only in the Okinawa Islands of Japan. Previous studies of Okinawa mozuku-derived fucoidan have reported its anticoagulant [Helicobacter pylori antibody titers. Previous studies have also reported the immune effects of fucoidan. For example, we reported that Okinawa mozuku-derived fucoidan has an immune cell proliferative effect in mice, enhances macrophage phagocytosis, increases IgM, IgG, and IgA production, and suppresses IgE production [Fucoidan is a general term for the sulfated polysaccharides contained in brown algae, the chemical structure of which differs depending on the seaweed species . Edible oagulant , anti-inoagulant , antiviroagulant ,4,5, antoagulant ,7, antitoagulant ,9, antihoagulant , and antoagulant  effects.oagulant . Althougoagulant ,14,15. Ioagulant , we repooduction . Nagaminoduction  also repIn the present pilot study, we conducted a clinical trial using a placebo-controlled, randomized, double-blind, parallel-group comparison method. The primary outcome was to evaluate the effects of ingesting Okinawa mozuku-derived fucoidan on NK cells derived from healthy humans. The secondary outcome was to evaluate the safety of fucoidan consumption in healthy subjects under the observation of a medical doctor. In summary, adverse events due to the test food were not observed and Okinawa mozuku-derived fucoidan enhanced NK cell activity, especially in male subjects.The background of the 39 subjects that completed the study is shown in In the fucoidan group, a significant increase in NK cell activity was observed at 8 weeks after ingestion when compared with NK cell activity at week 0 a. In malIn tests of plasma IFN-γ concentrations, the number of fucoidan group subjects in which IFN-γ was detected  during the ingestion period tended to increase, whereas the number of placebo group subjects in which IFN-γ was detected  tended to decrease . On the In addition, the number of detections in IFN-γ results were graphed in There were no significant differences in white blood cell count, hemoglobin, platelet count, or hematocrit between the placebo and fucoidan groups during the study period . In addiThe results of the biochemical tests showed no abnormalities . In the To evaluate the safety of the test food, adverse events during the ingestion period were recorded. Across both groups, 28 adverse events were observed during the ingestion period. However, following interviews and medical examinations by a doctor, it was concluded that serious adverse events had not occurred, and causal relationships with the test food did not exist. In addition, subjects recovered from all adverse events without further problems .C. okamuranus) was used as a test sample. In previous studies, fucoidan has been reported to have effects on immunity in animals [The raw materials for fucoidan, i.e., edible seaweeds such as kombu, wakame, and mozuku, have a long history of being consumed as food in Japan. In the present study, fucoidan derived from mozuku  for 4 weeks showed a significant suppression in the decrease of IFN-γ and IL-2 production. In a previous study in which fucoidan was administered to mice, NK cell activation significantly enhanced the production of IFN-γ [In general, the activation of NK cells requires the secretion of cytokines, such as IFN-γ, IL-2, and IL-12, by macrophages and helper T cells. In the present study, IFN-γ in the blood tended to increase over time in fucoidan group subjects, whereas IL-2 concentrations did not change significantly in any subject in either group during the ingestion and washout periods. Previously, Takahashi et al.  conducteof IFN-γ . Similarof IFN-γ  reportedPrevious studies have reported that macrophages are involved in the activation of NK cells by fucoidan. For example, we previously revealed that fucoidan significantly enhances IL-2 and IFN-γ levels in mice as well as macrophage phagocytosis . Addition = 11) was significantly higher than that of female subjects . In a previous ex vivo study using ovariectomized rats [Innate and adaptive immunity tends to be higher in women than in men, but the number of NK cells in men is higher than that in women . Specifized rats , it was In the present study, fucoidan did not cause problematic adverse events when ingested at 3 g per day for 12 weeks. In addition, abnormalities were not detected in blood and biochemical tests. Although adverse events were occasionally observed during the study period, all were judged by the medical doctor to be unrelated to fucoidan ingestion, and patients recovered after the study. These results are consistent with mozuku, the raw material of fucoidan, being a type of seaweed that is often eaten in Japan and considered highly safe as a food item. Similar to the present results, Abe et al.  reportedIn conclusion, we have shown that Okinawa mozuku-derived fucoidan is safe as a food item and that it enhances NK cell activity, especially in males. In future research, we intend to investigate the effects of fucoidan on dendritic cells, macrophages, and cytokines (as examples) to elucidate the immunomodulatory action of these sulfated polysaccharides.l-fucose, 18.8% sulfate ions, 14.4% uronic acid; mean molecular weight: 73.4 kDa). For the placebo group, citric acid and sucralose were blended in a beverage with caramel, which was used to ensure that the appearance of the placebo beverage did not differ from that of the fucoidan beverage.A beverage containing 1.5 g/50 mL of fucoidan derived from Okinawa mozuku  was used as a sample for the fucoidan treatment group; citric acid and sucralose were added to the raw materials , who also gave final approval to conduct the study. Moreover, the study was conducted under the guidance of a doctor at the Shinagawa Season Terrace Healthcare Clinic, Tokyo, Japan. The subjects were healthy men and women between the ages of 20 and 65 years old; each subject gave written consent after being provided with a sufficient explanation of the study. The target number of participants was set as the number required for statistical analysis. Subjects were recruited by KSO Corporation  and were assigned by block randomization to ensure that independent variables, e.g., age, gender, etc., did not differ significantly.The controller assigned subjects to two groups—the fucoidan and placebo groups—in a randomized, double-blind, parallel-group, placebo-controlled study . Both grAll specimen measurements were performed by LSI Medience Corporation .51Cr using a cytotoxicity test. The NK cell activity test used subject blood on the day of first ingestion (week 0) and after 4, 8, 12, and 16 weeks. Blood samples were collected into heparinized tubes. After centrifugation of the blood samples with a lymphocyte separation medium, interface mononuclear cells were collected and suspended in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). The samples were centrifuged again, and mononuclear cells were collected and mixed with 51Cr-labeled target cells (K562) at a ratio of 50:1. The cell mixture was cultured at 37 °C and 5% CO2 for 4 h. The 51Cr released from the target cells by NK cell cytotoxicity was determined using a gamma counter . The percentage of cytotoxicity was calculated as follows: cytotoxicity (%) = / × 100.NK cell activity was measured in the K562 cell line  labeled with IFN-γ  and IL-2  concentrations in the plasma of subjects were measured using sandwich ELISA on the day of first ingestion (week 0) and after 4, 8, 12, and 16 weeks.Blood tests  and biochemical tests  were performed on the day of first ingestion (week 0) and 4, 8, 12, and 16 weeks later. In addition, during the same period, a doctor interviewed and examined the subjects as a safety evaluation.t-test between groups. p < 0.05 was considered to indicate statistical significance. Statcel version 4 software  was used to conduct all statistical analyses.Data are presented as means ± standard deviation. Data were analyzed using Dunnett’s multiple-test within groups and Welch’s


Fasters also had lower intakes of vitamins A, B1, B2, B6, B12, D, folate, pantothenate, sodium, calcium, zinc, and phosphorus. Most participants (in both groups) did not meet the recommended dietary allowances of most vitamins and elements. Most serum biochemical parameters did not reflect the differences in nutrient intakes between groups, and none exhibited a correlation coefficient above 0.5 with nutrient intakes. Our findings suggest that COC fasting is associated with reduced intake of many nutrients, although this does not seem to have an impact on the blood biochemical profile.The aim of the present study was to examine differences and correlations in nutrient intakes and serum parameters related to nutrient intake  in 200 lifelong Christian Orthodox Church (COC) fasters with periodic abstinence from certain foods  for approximately half of the year and 200 non-fasting controls, all of whom did not take dietary supplements. Nutrient intakes were assessed through three-day dietary recalls. Blood samples were drawn for the analysis of potential biomarkers of nutrient intake. Fasters had lower energy intake, due to lower fat and protein intake, compared to non-fasters ( Abstinence from certain foods, permanently or periodically, has been practiced by humans for millennia. Examples of permanent abstinence include vegetarianism (in its various nuances), whereas examples of periodic abstinence include religious fasting, as that practiced by faithful followers of the Christian Orthodox Church (COC). These abstain from certain foods, predominantly of animal origin, for periods totaling approximately half of the year . FastingWe recently completed a large project examining the impact of lifelong COC fasting on nutrient intake and various health indicators. We showed that (a) men and women fully adhering to COC fasting for decades, whether being older  or youngThe aim of the present report is to (a) compare nutrient intakes and serum biochemical parameters between the lifelong COC fasters and non-fasters and (b) examine possible associations between these variables that might provide new information on how diet affects metabolic health.This is a cross-sectional study that included 200 fasters (131 women and 69 men) and 200 non-fasters (126 women and 74 men). The fasters had been adhering to religious fasting for a median of 15 years, ranging from 10 to 32 years and with 15 (10–26) years as the starting age of fasting. Details of the study design and the participants have been presented previously [COC fasting involves abstaining from certain foods—predominantly of animal origin , except seafood and snails—during five main periods, three important religious days, Wednesdays, and Fridays, totaling 159 to 197  days per year depending on when Easter falls and does not involve abstaining from food consumption during certain hours of the day, which characterizes intermittent fasting. The COC has set two periods, comprising a total of 47 days, of no food restriction (not even on Wednesdays or Fridays) to avoid nutrients deficiency in the body due to prolonged fasting. Between these extremes (fasting periods and periods of no food restriction) lie 17 to 23 weeks of moderate fasting, that is, only on Wednesdays and Fridays. Therefore, to make the data regarding fasters as representative of the entire year as possible, all measurements, interviews, and blood sampling were performed during these weeks.Body weight was measured to the nearest 0.1 kg and height to the nearest 0.01 m on a digital scale with a built-in stadiometer, and body mass index (BMI) was calculated from the measurements. Waist-to-hip ratio was measured using a stretch-resistant tape.Nutrient intakes were assessed through interviewer-based recalls of food consumed over three days, which included a Wednesday or Friday (during which the fasters obeyed fasting), another weekday, and a weekend day. Participants were interviewed about all foods and liquids consumed during those days and the means of the three days are presented. None of the participants in the study received any dietary supplements. Food intake records were analyzed using the Food Processor Nutrition Analysis software . Vitamin and element intakes were compared to their recommended dietary allowances (RDAs) or adequate intakes (AIs), as reported by the Food and Nutrition Board of the Institute of Medicine, National Academy of Sciences .12, calcium, magnesium, iron, and phosphate were measured in two automatic analyzers, an Abbott Architect i2000SR and a Mindray BS-300, with manufacturers’ kits. The coefficients of variation for all parameters ranged from 1 to 5%, and the laboratory carrying out the analyses participated in a nationwide external quality control program.Fasting venous blood samples were drawn and treated as described . Serum gThe study was approved by the Bioethics Committee of the then Alexander Technological Educational Institute of Thessaloniki, presently International Hellenic University (31.5/5679/17-12-2013), and all procedures were in accordance with the Declaration of Helsinki. Each participant was informed about the aims, benefits, and potential risks of the study and provided written informed consent before data collection and blood sampling.U test. Correlation analysis between all variables in the entire sample was performed by determining Spearman’s ρ correlation coefficient. Statistical analysis was performed using the SPSS, version 27 . All tests and corresponding p values were two-sided, and the level of statistical significance was set at α = 0.05.The Kolmogorov–Smirnov test and histogram charts were used to assess normality of distribution. The distribution of almost all variables differed significantly from the normal. Thus, we report all variables as median (interquartile range) and compared groups (fasters vs. non-fasters) by using the non-parametric Mann–Whitney p > 0.05). The respective values were 45 (27–58) years vs. 46 (24–57) years, 72.9 (62.9–81.4) kg vs. 71.3 (60.5–84.0) kg, 1.66 (1.60–1.72) m vs. 1.66 (1.61–1.74) m, 26.6 (23.0–29.5) kg/m2 vs. 25.6 (22.7–29.1) kg/m2, and 0.89 (0.82–0.97) vs. 0.89 (0.81–0.98).Fasters and non-fasters did not differ in age, body weight, height, BMI, and waist-to-hip ratio (p = 0.027). Macronutrient intakes by the participants are presented in Total daily energy intake was lower in fasters than in non-fasters, that is, 1496 (1256–1825) kcal vs. 1611 (1303–1903) kcal in non-fasters  and percentage coverage of the corresponding RDAs were significantly lower in fasters compared to non-fasters  were detected between the study parameters. To focus on the meaningful ones, we only considered correlations that were moderate or high (ρ > 0.5). Regarding correlations between the serum parameters, phosphate correlated negatively with insulin and folate , whereas no correlation between serum parameters and dietary intakes had a ρ above 0.5. Additionally, there were many moderate or high correlations between dietary intakes, most of which could be explained by the concomitant abundance of certain nutrients in certain foods. For example, protein intake correlated with cholesterol intake , apparently as a result of the abundance of both in meat products, and monounsaturated fatty acids correlated with vitamin E , apparently due to the abundance of both in olive oil. Finally, all macronutrient intakes, some vitamins , and most of the elements  were strongly (ρ between 0.52 and 0.84) correlated with total energy intake.A plethora of significant correlations  for decades, according to the dictates of COC fasting, impacts nutrient intakes and the biochemical profile in relation to metabolic health. When comparing the group of fasters to that of non-fasters, a first observation was that, although the former had lower daily energy intake, they did not differ in indices of fatness (such as BMI and waist-to-hip ratio) from the latter. This could be explained by a difference in energy expenditure, although we have shown that the two groups did not differ in exercise patterns ,2. HowevIt is interesting to note that the lower energy intake by fasters was not due to lower carbohydrate intake but, rather, to lower fat and protein intakes, compared to non-fasters . This caA striking preponderance of fat as an energy source , since most participants had values within the corresponding reference intervals. By contrast, most participants had a serum concentration of folate below its lower reference limit (3.1 ng/mL), apparently as a consequence of its low intake by both groups.It is noteworthy that vitamin and element intakes were generally low, a problem that other studies have also highlighted ,15,16,17The absence of an effect of nutritional inadequacies on most of the biochemical parameters tested is in accordance with our previous findings of no effect of inadequate calcium intake on bone health in the same sample ,2. Possi1, vitamin B2, vitamin B12, Na, and Fe in fasters; and vitamin B6 and Fe in non-fasters) in which it was previously below 100%. This, however, does not change the conclusion that the intakes of most micronutrients were below their RDAs. A problem frequently encountered in nutritional studies that are based on self-reported food intake is misreporting , which is considered unavoidable . To asseOf interest is the negative correlation of serum phosphate with insulin found in the present study, since it agrees with the finding that, in healthy subjects, low serum phosphate was associated with reduced insulin sensitivity . As is tThe dearth of correlations between serum biochemical parameters and nutrient intakes in our work is in accord with the few studies that have addressed the same issue or the correlation of biochemical parameters with dietary habits ,22,23. IFaithful followers of COC fasting had lower intakes of total daily energy, fat, and protein during periods of moderate fasting , periods that we consider representative of the whole year, compared to non-fasting controls. Fasters had also lower intakes of most vitamins and some elements. Most participants (in both groups) did not meet most of the vitamin and element RDAs. Most serum biochemical parameters did not reflect the differences in nutrient intakes between groups, and none exhibited a meaningful correlation with nutrient intakes.


As a preliminary experiment to explore the possibility of clinical application as a future reading assist, we present CNNs for the diagnosis of ovarian carcinomas and borderline tumors on MRI, including T2WI, DWI, ADC map, and CE-T1WI, and compare their diagnostic performance with interpretations by experienced radiologists. CNNs were trained using 1798 images from 146 patients and 1865 images from 219 patients with malignant tumors, including borderline tumors, and non-malignant lesions, respectively, for each MRI sequence and tested with 48 and 52 images of patients with malignant and non-malignant lesions. The CNN of each sequence had a sensitivity of 0.77–0.85, specificity of 0.77–0.92, accuracy of 0.81–0.87, and an AUC of 0.83–0.89, demonstrating diagnostic performances that were non-inferior to those of experienced radiologists, and the CNN showed the highest diagnostic performance on the ADC map for each sequence .Background: This study aimed to compare deep learning with radiologists’ assessments for diagnosing ovarian carcinoma using MRI. Methods: This retrospective study included 194 patients with pathologically confirmed ovarian carcinomas or borderline tumors and 271 patients with non-malignant lesions who underwent MRI between January 2015 and December 2020. T2WI, DWI, ADC map, and fat-saturated contrast-enhanced T1WI were used for the analysis. A deep learning model based on a convolutional neural network (CNN) was trained using 1798 images from 146 patients with malignant tumors and 1865 images from 219 patients with non-malignant lesions for each sequence, and we tested with 48 and 52 images of patients with malignant and non-malignant lesions, respectively. The sensitivity, specificity, accuracy, and AUC were compared between the CNN and interpretations of three experienced radiologists. Results: The CNN of each sequence had a sensitivity of 0.77–0.85, specificity of 0.77–0.92, accuracy of 0.81–0.87, and an AUC of 0.83–0.89, and it achieved a diagnostic performance equivalent to the radiologists. The CNN showed the highest diagnostic performance on the ADC map among all sequences . Conclusion: The CNNs provided a diagnostic performance that was non-inferior to the radiologists for diagnosing ovarian carcinomas on MRI. Ovarian cancer is the eighth most common cancer diagnosis and cause of cancer death in women; there were approximately 313,959 cases and 207,252 deaths worldwide in 2020 .MRI is a better radiologic method for differentiating between malignant and benign ovarian tumors ,5,6. HowConvolutional neural networks (CNNs) are a class of deep learning models that combine imaging filters with artificial neural networks through a series of successive linear and nonlinear layers. It is considered a promising tool for diagnostic imaging and, recently, several CNNs for diagnostic imaging have been constructed and achieved excellent performance in image classification using radiography, US, CT, and MRI , and CNNAs a preliminary experiment to explore their possibility of clinical application as a future reading assist, we present CNNs for the diagnosis of ovarian carcinomas and borderline tumors on MRI, including T2WI, DWI, ADC map, and fat-saturated contrast-enhanced T1-weighted imaging (CE-T1WI), and compared their diagnostic performance with interpretations by experienced radiologists.This retrospective study was approved by our local institutional review board, which waived the need for written informed consent . The inclusion criteria were as follows: (a) aged above 20 years for ethical reasons; (b) pelvic MRI scan obtained as per the protocol followed at our hospital between January 2015 and December 2020; (c) pathologically proven malignant epithelial tumors  or borderline tumors of the ovary for the malignant group; (d) pathologically proven or clinically apparent benign lesions in the non-malignant group. The exclusion criteria were as follows: (a) malignant tumors in the pelvis other than the ovary; (b) past history of surgery of the uterus or ovaries other than caesarean section, chemotherapy, or radiation therapy of the pelvis; (c) malignant ovarian epithelial tumors mixed with non-epithelial components. A flowchart of the patient selection process is shown in Tumor stage was comprehensively determined based on the International Federation of Gynecology and Obstetrics (FIGO) 2014 classification using pretreatment MRI, CT, surgical, and pathological findings. For operated cases, tumor type was determined by pathological diagnosis, and mixed carcinomas were classified according to the most dominant histological type.®, Achieva®; Philips Medical Systems, Amsterdam, Netherlands). The protocol employed to obtain the image along the uterine short axis included T2WI, DWI with a b value of 0 and 1000, and CE-T1WI with gadopentetate dimeglumine 5 mmol . The bolus intravenous contrast injection rate was 4 mL (2 mmol)/s . Further details of these parameters are provided in MRI was performed using 3 T or 1.5 T equipment . For benign lesions other than ovarian tumors, the slices in which the uterus and ovaries were visualized were extracted. The same cross-section was extracted for all sequences.A total of 465 patients were randomly assigned to the training and testing groups. In the training phase, 3663 images for each sequence from 365 patients  were used. In the test phase, based on the consensus of the two radiologists , one slice indicating a solid component, a central slice for each ovarian tumor containing no solid components, or an image showing a normal caudal ovary in the absence of an ovarian tumor was selected. According to O–RADS MRI, solid tissue was defined as a lesion component that conformed to one of these morphologies: papillary projection, mural nodule, irregular septation/wall, or other larger solid portions. Then, for each sequence, 100 images from 100 patients  were used.In this study, the software used was unable to handle the Digital Imaging and Communications in Medicine (DICOM) images; therefore, the DICOM images were converted into Joint Photographic Experts Group (JPEG) images that employ a lossless compression method using a Centricity Universal Viewer . Next, the margins containing the patient information were automatically trimmed and resized to 240 × 240 pixels using XnConvert .Deep learning was performed on a deep station entry  with a GeForce RTX 2080Ti graphics processing unit , a Core i7-8700 central processing unit , and the deep learning software Deep Analyzer .The conditions were optimized based on the ablation and comparative studies of the previous research as follows: a CNN with Xception architecture was used for deep learning. Xception is characterized as depth-wise separable convolutions that enable the use of model parameters more efficiently than the previous CNN architecture . ImageNeThree experienced radiologists , with 26, 12, and 8 years of experience in interpreting pelvic MRIs independently, reviewed the 100 test images of each sequence from 100 patients in random order. They evaluated each image by assigning confidence levels to the diagnosis of malignant tumors, including borderline tumors, using a 6 point scale . The radiologists were blinded to the pathological and clinical findings. A duration of one week or more was observed between each sequence interpretation.The age and histological type for each group were compared using the Mann–Whitney U test and the chi-square test.The test data set was used to calculate the sensitivity, specificity, and accuracy of diagnosing malignant tumors. With the radiologists, 1.0–0.6 was treated as malignant, while 0.4–0.0 was treated as non-malignant. In the CNN, the classification into malignant and non-malignant groups was output as a continuous number from 1 to 0: 1.00–0.50 was considered malignant, while 0.49–0 was considered non-malignant.Receiver operating characteristic (ROC) curve analysis was performed to assess diagnostic performance. Moreover, the area under the receiver operating characteristic curve (AUC) was compared between the CNN and the radiologists with their 95% confidence intervals, and significant differences were estimated .p < 0.05.Inter-observer agreement for the two choices of malignancy was also assessed using kappa (κ) statistics. The κ-statistic interpreted the agreement as follows: 1.00–0.81, almost perfect; 0.80–0.61, substantial; 0.60–0.41, moderate; 0.40–0.21, fair; 0.20–0, slight; less than 0, none . All stap < 0.001), there was no significant difference between the training and testing data in patient age or histological tumor type. In the malignant group, 116  patients were scanned at 1.5 T, and 78  patients were scanned at 3 T. In the non-malignant group, 114  patients were scanned at 1.5 T, and 157  patients were scanned at 3 T. All tumors in the malignant group were confirmed pathologically. Only 57  lesions in the non-malignant group were not pathologically confirmed but were clinically apparent benign lesions including imaging findings such as small myoma and nabothian cyst.A total of 465 women  were evaluated across the data sets. For the selection of the validation ratio and epochs, among the CNNs with a sensitivity and specificity above 0.75, a model with a validation ratio of 0.1 and 100 epochs was adopted for T2WI, a model with a validation ratio of 0.2 and 50 epochs was adopted for DWI, and a model with a validation ratio of 0.1 and 50 epochs was adopted for the ADC map , while aThe CNN correctly diagnosed 28 of 37 carcinomas and 9 of 11 borderline tumors on T2WI; 31 of 37 carcinomas and 10 of 11 borderline tumors on the DWI and ADC map; 31 of 37 carcinomas and 8 of 11 borderline tumors on the CE-T1WI as malignant tumors. There was one case each in which the CNN showed false negative and false positive in all sequences. Reader 1 had three cases with false negatives, while readers 2 and 3 had no cases with false negatives or false positives in all sequences.This preliminary study presented CNNs for diagnosing ovarian carcinomas, including borderline tumors, using several MRI sequences, demonstrating that the diagnostic performance was not inferior to that of the experienced radiologists, under the limited conditions.Few studies have used deep learning in the field of gynecological imaging. Urushibara et al. recently constructed a CNN that showed good diagnostic performance for identifying the presence of cervical cancer on T2WI . AramendEpithelial ovarian tumors can be classified into serous, mucinous, endometrioid, clear cell, Brenner, seromucinous, and undifferentiated types. Furthermore, each is subdivided into benign, borderline, or malignant . One of The image interpretation of ovarian tumors is complicated as mentioned above. However, although our CNNs included less than 500 cases and the images used were not cropped images of the ovarian lesions, they showed diagnostic performances equivalent to those of experienced radiologists in our study. In addition, there was no inferiority in diagnostic performance even for borderline tumors, which are generally difficult to diagnose as benign or malignant. The fact that the CNN showed the highest diagnostic performance with the ADC map is consistent with a previous study of the diagnosis of prostate cancer , indicatThe inter-observer agreement between the CNNs and the radiologists was lower than that between the radiologists; however, the agreement between the radiologists was not high. Radiologists usually make a comprehensive judgment by referring to multiple sequences; therefore, making a diagnosis using only a single sequence is difficult. For example, in the case of ovarian endometriosis, the contrast between the background of the cyst content showing T2 shading and the solid components is poor, and the solid components might be noticed only on CE-T1WI. For endometriosis showing hyperintensity on T1WI, it can be difficult to recognize the contrast enhancement of the solid components even on CE-T1WI, and the use of a subtracted image is required. Therefore, the use of a single sequence without multiple sequences may have undermined the overall agreement among radiologists.Our study had several limitations. First, each sequence was evaluated individually, which is quite different from clinical practice in which all sequences are referenced and comprehensively diagnosed and did not meet the definition of O-RADS MRI. It also differed from O-RADS in that it was scored by confidence level rather than malignancy for comparison with the diagnosis made by the CNN. In addition, the lack of interpretation in the series images is believed to be another reason why the radiologists’ performance was worse than in past O-RADS reports. Using a combination of several sequences may show superior diagnostic performance to the current models as reported for the CNN of prostate cancer using fused images ,29, and Although diagnostic imaging of ovarian tumors is complex, deep learning has shown good diagnostic performance for diagnosing ovarian carcinomas, including borderline tumors, on MRIs under the limited conditions of this study.