Amine-modified nanoplastics promote the procoagulant activation of isolated human red blood cells and thrombus formation in rats.

BACKGROUND: Microplastics (MPs) and nanoplastics (NPs) formed from decomposed plastic are increasing environmental threats. Although MPs and NPs exposed through various routes enter the systemic circulation, the potential toxicity of those is largely unknown. We investigated whether polystyrene NPs (PS-NPs) promote the coagulation activity of red blood cells (RBCs). RESULTS: We tested several types of PS-NPs using human RBCs and found that amine-modified 100 nm PS-NPs were the most potent. We measured the uptake of PS-NPs using flow cytometry and confocal microscopy. Electron microscopy revealed morphological changes of RBCs by PS-NPs. PS-NPs induced the externalization of phosphatidylserine, generation of microvesicles in RBCs, and perturbations in the intracellular microenvironment. PS-NPs increased the activity of scramblases responsible for phospholipid translocation in RBCs. PS-NPs modulated the functional interaction to adjacent tissues and coagulation cascade, enhancing RBC adhesion and thrombin generation. Our observations in human RBCs were consistent with those in isolated rat RBCs, showing no inter-species differences. In rat venous thrombosis models, the intravenous administration of PS-NPs enhanced thrombus formation. CONCLUSION: Amine-modified PS-NPs induce the prothrombotic activation of RBCs causing thrombus formation. We believe that our study will contribute to understanding the potential toxicity of amine-modified polystyrene particles in blood cells and cardiovascular systems.

MARTX toxin of Vibrio vulnificus induces RBC phosphatidylserine exposure that can contribute to thrombosis.

V. vulnificus-infected patients suffer from hemolytic anemia and circulatory lesions, often accompanied by venous thrombosis. However, the pathophysiological mechanism of venous thrombosis associated with V. vulnificus infection remains largely unknown. Herein, V. vulnificus infection at the sub-hemolytic level induced shape change of human red blood cells (RBCs) accompanied by phosphatidylserine exposure, and microvesicle generation, leading to the procoagulant activation of RBCs and ultimately, acquisition of prothrombotic activity. Of note, V. vulnificus exposed to RBCs substantially upregulated the rtxA gene encoding multifunctional autoprocessing repeats-in-toxin (MARTX) toxin. Mutant studies showed that V. vulnificus-induced RBC procoagulant activity was due to the pore forming region of the MARTX toxin causing intracellular Ca2+ influx in RBCs. In a rat venous thrombosis model triggered by tissue factor and stasis, the V. vulnificus wild type increased thrombosis while the ΔrtxA mutant failed to increase thrombosis, confirming that V. vulnificus induces thrombosis through the procoagulant activation of RBCs via the mediation of the MARTX toxin.

Titanium dioxide nanoparticles enhance thrombosis through triggering the phosphatidylserine exposure and procoagulant activation of red blood cells.

BACKGROUND: Expanding biomedical application of anatase titanium dioxide (TiO2) nanoparticles (NPs) is raising the public concern on its potential health hazards. Here, we demonstrated that TiO2 NPs can increase phosphatidylserine (PS) exposure and procoagulant activity of red blood cells (RBCs), which may contribute to thrombosis. RESULTS: We conducted in vitro studies using RBCs freshly isolated from healthy male volunteers. TiO2 NPs exposure (≦ 25 µg/mL) induced PS exposure and microvesicles (MV) generation accompanied by morphological changes of RBCs. While ROS generation was not observed following the exposure to TiO2 NPs, intracellular calcium increased and caspase-3 was activated, which up-regulated scramblase activity, leading to PS exposure. RBCs exposed to TiO2 NPs could increase procoagulant activity as measured by accelerated thrombin generation, and enhancement of RBC-endothelial cells adhesion and RBC-RBC aggregation. Confirming the procoagulant activation of RBC in vitro, exposure to TiO2 NPs (2 mg/kg intravenously injection) in rats increased thrombus formation in the venous thrombosis model. CONCLUSION: Collectively, these results suggest that anatase TiO2 NPs may harbor prothrombotic risks by promoting the procoagulant activity of RBCs, which needs attention for its biomedical application.

Fresh Crab Plays an Important Role as a Nutrient Reservoir for the Rapid Propagation of Vibrio vulnificus.

Vibrio vulnificus is a well-known opportunistic pathogen causing food-borne illnesses by ingestion of contaminated seafood. A new strain of V. vulnificus FORC_016 was isolated from a patient's blood sample in South Korea. The genome consists of two circular DNA chromosomes: chromosome I (3,234,424 bp with a G + C contents of 46.60% containing 2,889 ORFs, 106 tRNA genes, and 31 rRNA genes) and chromosome II (1,837,945 bp with a GC content of 47.00% containing 1,572 ORFs, 13 tRNA genes, and 3 rRNA genes). In addition, chromosome I has a super integron (SI) containing 209 ORFs, which is probably associated with various additional functions including antibiotic resistance and pathogenicity. Pan-genome analysis with other V. vulnificus genomes revealed that core genome regions contain most of the important virulence factors. However, accessory genome regions are located in the SI region and contain unique genes regarding cell wall biosynthesis and generation of host cell protecting capsule, suggesting possible resistance ability against environmental stresses. Comparative RNA-Seq analysis of samples between contact and no contact to the crab conditions showed that expressions of amino acid/peptide and carbohydrate transport and utilization genes were down-regulated, but expressions of cell division and growth-related genes were up-regulated, suggesting that the crab may be a nutrition reservoir for rapid propagation of V. vulnificus. Therefore, consumption of the contaminated fresh crab would provide a large number of V. vulnificus to humans, which may be more dangerous. Consequently, biocontrol of V. vulnificus may be critical to ensure the safety in seafood consumption.

Molecular interaction between methicillin-resistant Staphylococcus aureus (MRSA) and chicken breast reveals enhancement of pathogenesis and toxicity for food-borne outbreak.

To study pathogenesis and toxicity of Staphylococcus aureus in foods, FORC_062 was isolated from a human blood sample and complete genome sequence has a type II SCCmec gene cluster and a type II toxin-antitoxin system, indicating an MRSA strain. Its mobile gene elements has many pathogenic genes involved in host infection, biofilm formation, and various enterotoxin and hemolysin genes. Clinical MRSA is often found in animal foods and ingestion of MRSA-contaminated foods causes human infection. Therefore, it is very important to understand the role of contaminated foods. To elucidate the interaction between clinical MRSA FORC_062 and raw chicken breast, transcriptome analysis was conducted, showing that gene expressions of amino acid biosynthesis and metabolism were specifically down-regulated, suggesting that the strain may import and utilize amino acids from the chicken breast, but not able to synthesize them. However, toxin gene expressions were up-regulated, suggesting that human infection of S. aureus via contaminated food may be more fatal. In addition, the contaminated foods enhance multiple-antibiotic resistance activities and virulence factors in this clinical MRSA. Consequently, MRSA-contaminated food may play a role as a nutritional reservoir as well as in enhancing factor for pathogenesis and toxicity of clinical MRSA for severe food-borne outbreaks.

Comparative genomic analysis reveals genetic features related to the virulence of Bacillus cereus FORC_013.

BACKGROUND: Bacillus cereus is well known as a gastrointestinal pathogen that causes food-borne illness. In the present study, we sequenced the complete genome of B. cereus FORC_013 isolated from fried eel in South Korea. To extend our understanding of the genomic characteristics of FORC_013, we conducted a comparative analysis with the published genomes of other B. cereus strains. RESULTS: We fully assembled the single circular chromosome (5,418,913 bp) and one plasmid (259,749 bp); 5511 open reading frames (ORFs) and 283 ORFs were predicted for the chromosome and plasmid, respectively. Moreover, we detected that the enterotoxin (NHE, HBL, CytK) induces food-borne illness with diarrheal symptom, and that the pleiotropic regulator, along with other virulence factors, plays a role in surviving and biofilm formation. Through comparative analysis using the complete genome sequence of B. cereus FORC_013, we identified both positively selected genes related to virulence regulation and 224 strain-specific genes of FORC_013. CONCLUSIONS: Through genome analysis of B. cereus FORC_013, we identified multiple virulence factors that may contribute to pathogenicity. These results will provide insight into further studies regarding B. cereus pathogenesis mechanism at the genomic level.

Genomic Insights and Its Comparative Analysis with Yersinia enterocolitica Reveals the Potential Virulence Determinants and Further Pathogenicity for Foodborne Outbreaks.

Yersinia enterocolitica is a well-known foodborne pathogen causing gastrointestinal infections worldwide. The strain Y. enterocolitica FORC_002 was isolated from the gill of flatfish (plaice) and its genome was sequenced. The genomic DNA consists of 4,837,317 bp with a GC content of 47.1%, and is predicted to contain 4,221 open reading frames, 81 tRNA genes, and 26 rRNA genes. Interestingly, genomic analysis revealed pathogenesis and host immune evasion-associated genes encoding guanylate cyclase (Yst), invasin (Ail and Inv), outer membrane protein (Yops), autotransporter adhesin A (YadA), RTX-like toxins, and a type III secretion system. In particular, guanylate cyclase is a heat-stable enterotoxin causing Yersinia-associated diarrhea, and RTX-like toxins are responsible for attachment to integrin on the target cell for cytotoxic action. This genome can be used to identify virulence factors that can be applied for the development of novel biomarkers for the rapid detection of this pathogen in foods.

Complete genome of Vibrio parahaemolyticus FORC014 isolated from the toothfish.

BACKGROUND: Foodborne illness can occur due to various pathogenic bacteria such as Staphylococcus aureus, Escherichia coli and Vibrio parahaemolyticus, and can cause severe gastroenteritis symptoms. In this study, we completed the genome sequence of a foodborne pathogen V. parahaemolyticus FORC_014, which was isolated from suspected contaminated toothfish from South Korea. Additionally, we extended our knowledge of genomic characteristics of the FORC_014 strain through comparative analysis using the complete sequences of other V. parahaemolyticus strains whose complete genomes have previously been reported. RESULTS: The complete genome sequence of V. parahaemolyticus FORC_014 was generated using the PacBio RS platform with single molecule, real-time (SMRT) sequencing. The FORC_014 strain consists of two circular chromosomes (3,241,330 bp for chromosome 1 and 1,997,247 bp for chromosome 2), one plasmid (51,383 bp), and one putative phage sequence (96,896 bp). The genome contains a total of 4274 putative protein coding sequences, 126 tRNA genes and 34 rRNA genes. Furthermore, we found 33 type III secretion system 1 (T3SS1) related proteins and 15 type III secretion system 2 (T3SS2) related proteins on chromosome 1. This is the first reported result of Type III secretion system 2 located on chromosome 1 of V. parahaemolyticus without thermostable direct hemolysin (tdh) and thermostable direct hemolysin-related hemolysin (trh). CONCLUSIONS: Through investigation of the complete genome sequence of V. parahaemolyticus FORC_014, which differs from previously reported strains, we revealed two type III secretion systems (T3SS1, T3SS2) located on chromosome 1 which do not include tdh and trh genes. We also identified several virulence factors carried by our strain, including iron uptake system, hemolysin and secretion system. This result suggests that the FORC_014 strain may be one pathogen responsible for foodborne illness outbreak. Our results provide significant genomic clues which will assist in future understanding of virulence at the genomic level and help distinguish between clinical and non-clinical isolates.

Genome Sequence of Bacillus cereus FORC_021, a Food-Borne Pathogen Isolated from a Knife at a Sashimi Restaurant.

Bacillus cereus causes food-borne illness through contaminated foods; therefore, its pathogenicity and genome sequences have been analyzed in several studies. We sequenced and analyzed B. cereus strain FORC_021 isolated from a sashimi restaurant. The genome sequence consists of 5,373,294 bp with 35.36% GC contents, 5,350 predicted CDSs, 42 rRNA genes, and 107 tRNA genes. Based on in silico DNA-DNA hybridization values, B. cereus ATCC 14579T was closest to FORC_021 among the complete genome-sequenced strains. Three major enterotoxins were detected in FORC_021. Comparative genomic analysis of FORC_021 with ATCC 14579T revealed that FORC_021 harbored an additional genomic region encoding virulence factors, such as putative ADP-ribosylating toxin, spore germination protein, internalin, and sortase. Furthermore, in vitro cytotoxicity testing showed that FORC_021 exhibited a high level of cytotoxicity toward INT-407 human epithelial cells. This genomic information of FORC_021 will help us to understand its pathogenesis and assist in managing food contamination.

Analysis of microbiota on abalone (Haliotis discus hannai) in South Korea for improved product management.

Abalone is a popular seafood in South Korea; however, because it contains various microorganisms, its ingestion can cause food poisoning. Therefore, analysis of the microbiota on abalone can improve understanding of outbreaks and causes of food poisoning and help to better manage seafood products. In this study, we collected a total of 40 abalones from four different regions in March and July, which are known as the maximum abalone production areas in Korea. The microbiota were analyzed using high-throughput sequencing, and bacterial loads on abalone were quantified by real-time PCR. Over 2700 species were detected in the samples, and Alpha- and Gammaproteobacteria were the predominant classes. The differences in microbiota among regions and at each sampling time were also investigated. Although Psychrobacter was the dominant genus detected on abalone in both March and July, the species compositions were different between the two sampling times. Five potential pathogens (Lactococcus garvieae, Yersinia kristensenii, Staphylococcus saprophyticus, Staphylococcus warneri, and Staphylococcus epidermidis) were detected among the abalone microbiota. In addition, we analyzed the influence of Vibrio parahaemolyticus infection on shifts in abalone microbiota during storage at different temperatures. Although the proportion of Vibrio increased over time in infected and non-infected abalone, the shifts of microbiota were more dynamic in infected abalone. These results can be used to better understand the potential of food poisoning caused by abalone consumption and manage abalone products according to the microbiota composition.

Complete genome sequence of Vibrio vulnificus FORC_017 isolated from a patient with a hemorrhagic rash after consuming raw dotted gizzard shad.

BACKGROUND: Vibrio vulnificus, a resident in the human gut, is frequently found in seafood, causing food-borne illnesses including gastroenteritis and severe septicemia. While V. vulnificus has been known to be one of the major food-borne pathogens, pathogenicity and virulence factors are not fully understood yet. To extend our understanding of the pathogenesis of V. vulnificus at the genomic level, the genome of V. vulnificus FORC_017 isolated from a female patient experiencing a hemorrhagic rash was completely sequenced and analyzed. RESULTS: Three discontinuous contigs were generated from a hybrid assembly using Illumina MiSeq and PacBio platforms, revealing that the genome of the FORC_017 consists of two circular chromosomes and a plasmid. Chromosome I consists of 3,253,417-bp (GC content 46.49 %) containing 2943 predicted open reading frames (ORFs) and chromosome II of 1,905,745-bp (GC content 46.90 %) containing 1638 ORFs. The plasmid pFORC17 consists of 70,069-bp (GC content 43.77 %) containing 84 ORFs. The average nucleotide identity (ANI) value of the FORC_017 and CMCP6 strains was 98.53, suggesting that they are closely related. CONCLUSIONS: Pathogenesis-associated genes including vvhA, rtx gene cluster, and various hemolysin genes were present in FORC_017. In addition, three complete secretion systems (Type I, II and VI) as well as iron uptake-related genes for virulence of the FORC_017 were detected, suggesting that this strain is pathogenic. Further comparative genome analysis revealed that FORC_017 and CMCP6 share major toxin genes including vvhA and rtx for pathogenesis activities. The genome information of the FORC_017 provides novel insights into pathogenicity and virulence factors of V. vulnificus.

Complete genome sequence of Vibrio parahaemolyticus strain FORC_008, a foodborne pathogen from a flounder fish in South Korea.

Vibrio parahaemolyticus is a Gram-negative, motile, nonspore-forming pathogen that causes foodborne illness associated with the consumption of contaminated seafoods. Although many cases of foodborne outbreaks caused by V. parahaemolyticus have been reported, the genomes of only five strains have been completely sequenced and analyzed using bioinformatics. In order to characterize overall virulence factors and pathogenesis of V. parahaemolyticus associated with foodborne outbreak in South Korea, a new strain FORC_008 was isolated from flounder fish and its genome was completely sequenced. The genomic analysis revealed that the genome of FORC_008 consists of two circular DNA chromosomes of 3266 132 bp (chromosome I) and 1772 036 bp (chromosome II) with a GC content of 45.36% and 45.53%, respectively. The entire genome contains 4494 predicted open reading frames, 129 tRNAs and 31 rRNA genes. While the strain FORC_008 does not have genes encoding thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), its genome encodes many other virulence factors including hemolysins, pathogenesis-associated secretion systems and iron acquisition systems, suggesting that it may be a potential pathogen. This report provides an extended understanding on V. parahaemolyticus in genomic level and would be helpful for rapid detection, epidemiological investigation and prevention of foodborne outbreak in South Korea.

Complete genome sequence of Vibrio parahaemolyticus FORC_023 isolated from raw fish storage water.

Vibrio parahaemolyticusis a Gram-negative halophilic bacterium that causes food-borne gastroenteritis in humans who consumeV. parahaemolyticus-contaminated seafood.The FORC_023 strain was isolated from raw fish storage water, containing live fish at a sashimi restaurant. Here, we aimed to sequence and characterize the genome of the FORC_023 strain. The genome of the FORC_023 strain showed two circular chromosomes, which contained 4227 open reading frames (ORFs), 131 tRNA genes and 37 rRNA genes. Although the genome of FORC_023 did not include major virulence genes, such as genes encoding thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), it contained genes encoding other hemolysins, secretion systems, iron uptake-related proteins and severalV. parahaemolyticusislands. The highest average nucleotide identity value was obtained between the FORC_023 strain and UCM-V493 (CP007004-6). Comparative genomic analysis of FORC_023 with UCM-V493 revealed that FORC_023 carried an additional genomic region encoding virulence factors, such as repeats-in-toxin and type II secretion factors. Furthermore,in vitrocytotoxicity testing showed that FORC_023 exhibited a high level of cytotoxicity toward INT-407 human epithelial cells. These results suggested that the FORC_023 strain may be a food-borne pathogen.

Complete genome sequence of Bacillus cereus FORC_005, a food-borne pathogen from the soy sauce braised fish-cake with quail-egg.

Due to abundant contamination in various foods, the pathogenesis of Bacillus cereus has been widely studied in physiological and molecular level. B. cereus FORC_005 was isolated from a Korean side dish, soy sauce braised fish-cake with quail-egg in South Korea. While 21 complete genome sequences of B. cereus has been announced to date, this strain was completely sequenced, analyzed, and compared with other complete genome sequences of B. cereus to elucidate the distinct pathogenic features of a strain isolated in South Korea. The genomic DNA containing a circular chromosome consists of 5,349,617-bp with a GC content of 35.29 %. It was predicted to have 5170 open reading frames, 106 tRNA genes, and 42 rRNA genes. Among the predicted ORFs, 3892 ORFs were annotated to encode functional proteins (75.28 %) and 1278 ORFs were predicted to encode hypothetical proteins (748 conserved and 530 non-conserved hypothetical proteins). This genome information of B. cereus FORC_005 would extend our understanding of its pathogenesis in genomic level for efficient control of its contamination in foods and further food poisoning.

Inhibitory effects of black soybean on platelet activation mediated through its active component of adenosine.

Owing to the beneficial health effects on human cardiovascular system, soybeans and soy-related products have been a focus of intensive research. Soy isoflavones are known to be primarily responsible for the soy-related biological effects including anti-platelet activity but its in vivo relevancy has not been fully verified. Here we compared the role of adenosine, an active ingredient abundant in black soybean (BB) extract, in the anti-platelet effects of BB, to that of soy isoflavones. At the concentrations existing in BB, isoflavones such as genistein and daidzein could not attenuate collagen-induced platelet aggregation, however, adenosine significantly inhibited platelet aggregation with an equivalent potency to BB, suggesting that adenosine may be the major bioactive component. Consistently, the anti-aggregatory effects of BB disappeared after treatment of adenosine receptor antagonists. The effects of BB are mediated by adenosine through intracellular cAMP and subsequent attenuation of calcium mobilization. Of note, adenosine and BB significantly reduced platelet fibrinogen binding and platelet adhesion, other critical events for platelet activation, which were not affected by isoflavones. Taken together, we demonstrated that adenosine might be the major active ingredient for BB-induced anti-platelet activity, which will shed new light on the roles of adenosine as a bioactive compound in soybeans and soy-related food.

Loss of Integrity: Impairment of the Blood-brain Barrier in Heavy Metal-associated Ischemic Stroke.

Although stroke is one of the leading causes of death and disability worldwide, preventive or therapeutic options are still limited. Therefore, a better understanding of the pathophysiological characteristics of this life-threatening disease is urgently needed. The incidence and prevalence of ischemic stroke are increased by exposure to certain types of xenobiotics, including heavy metals, suggesting the possible toxicological contribution of these compounds to the onset or aggravation of stroke. Among the potential targets, we have focused on alterations to cerebral endothelial cells (CECs), which play important roles in maintaining the functional integrity of brain tissue.

Rapid, simultaneous and nanomolar determination of pyroglutamic acid and cis-/trans-urocanic acid in human stratum corneum by hydrophilic interaction liquid chromatography (HILIC)-electrospray ionization tandem mass spectrometry.

A rapid, sensitive and specific hydrophilic interaction liquid chromatography coupled to tandem mass spectrometric (HILIC-MS/MS) method for the simultaneous determination of pyroglutamic acid, cis- and trans-urocanic acid in human skin stratum corneum (SC) were developed and validated. This method was carried out without derivatization or addition of ion-pair additives in mobile phase. The analytes were extracted by PBS buffer solution and analyzed using an electrospray positive ionization mass spectrometry in the multiple reaction monitoring (MRM) mode. Chromatographic separation was performed on an AQUITY UPLC amide column using gradient elution with the mobile phase of water and acetonitrile. The standard curves were linear over the concentration range of 1.0-250 ng/mL with a correlation coefficient higher than 0.999 with an LLOQ of 0.5 ng/mL. The lower limits of detection (LLOD) of these analytes were lower than 0.2 ng/mL. The intra- and inter-day precisions were measured to be below 7.7% and accuracies were within the range of 94.3-102.6%. The validated method was successfully applied to determine the level of pyroglutamic acid and cis-/trans-urocanic acid in the SC samples from forearm and forehead region of 19 human volunteers.

Factors affecting the apparent solubility of ursodeoxycholic acid in the grinding process.

Ursodeoxycholic acid (UDCA) was ground by a vibrating mill. Apparent solubility of the ground sample was determined by Coulter counter method. The samples were characterized by pore size distribution measurement, powder X-ray diffraction (PXRD) measurement, near infra-red (NIR) spectroscopy and contact angle measurement. The dispersive and polar components of surface free energy were calculated from the contact angle data determined by a contact angle analyzer. Surface polarity was calculated from the surface free energy components. The apparent solubility of UDCA was increased by the grinding with vibrating mill, however, the particle size of ground sample was not decreased. An amorphization was observed in the PXRD pattern of the ground sample, and the crystallinity of sample was decreased with increasing the grinding time. During the initial grinding, the dispersive component of surface free energy was decreased, whereas the polar part of surface free energy was increased. The surface polarity of the sample was increased in the same manner. Relationship between the solubility and the factors changing in the grinding process was evaluated. There was a significant correlation between the apparent solubility and the surface polarity and crystallinity of the sample. The NIR spectra confirmed the appearance of [bond]OH group on the sample surface. The apparent solubility increase of the ground sample was closely related to the improvement of the surface polarity and the destruction of crystalline structure.