Multiplex Quantification of Exosomes via Multiple Types of Nanobeads Labeling Combined with Laser Scanning Detection.

In recent years, exosomes have attracted attention in many aspects from basic research to clinical application, including therapeutic reagents or biomarkers for liquid biopsy. The increasing understanding of exosome's heterogeneous properties is expected to lead to more advanced exosome research, and there is therefore a need for a multiplex system that can easily classify and analyze exosomes in complex biological samples according to their properties. In this study, we developed a simple and sensitive multiplexed exosome quantification system based on ExoCounter, an exosome quantification system utilizing optical disk technology, by introducing nanobeads made of different materials as exosome labeling substances. The refractive indices suitable for nanobead materials were analyzed by computer simulation of optical diffraction generated by nanobeads. The results showed that polymer (FG), Au, and Ag nanobeads exhibited superior discrimination capability in terms of the amplitude and polarity of detection pulses generated by each nanobead. The specificity and detection sensitivity of three types of nanobeads were confirmed by detecting HER2-positive exosomes with anti-HER2 antibody-conjugated nanobeads. Furthermore, CD147-positive, HER2-positive, and CD81-positive exosomes in 12.5 µL of serum were simultaneously quantified with high discrimination performance using the anti-CD147 antibody, anti-HER2 antibody, or anti-CD81 antibody conjugated for FG beads, Au nanobeads, or Ag nanobeads, respectively. A limit of detection was also evaluated as low as 210 exosomes/µL. This system is a promising tool for advanced exosome research because it enables multiplexed detection of heterogeneous exosomes in serum with high specificity, accuracy, and sensitivity without purification.

ExoCounter Assays Identify Women Who May Develop Early-Onset Preeclampsia From 12.5 µL First-Trimester Serum by Characterizing Placental Small Extracellular Vesicles.

BACKGROUND: Preeclampsia is a major cause of maternal and perinatal morbidity and mortality worldwide. Identifying women with high risk of developing preeclampsia in early pregnancy remains challenging. Extracellular vesicles released from the placenta offer an attractive biomarker but have been elusive to quantify. METHODS: Here, we tested ExoCounter, a novel device that immunophenotypes size-selected small extracellular vesicles <160 nm, for its ability to perform qualitative and quantitative placental small extracellular vesicles (psEV) analysis. To investigate disease-specific and gestational age-specific changes, we analyzed psEV counts in maternal plasma samples taken at each of the 3 trimesters from women who had (1) normal pregnancy (n=3); (2) women who developed early-onset preeclampsia (EOPE; n=3); and (3) women who developed late-onset preeclampsia (n=4) using 3 antibody pairs, CD10-placental alkaline phosphatase (PLAP), CD10-CD63, and CD63-PLAP. We further validated the findings in first-trimester serum samples among normal pregnancy (n=9), women who developed EOPE (n=7), and women who developed late-onset preeclampsia (n=8). RESULTS: We confirmed that CD63 was the major tetraspanin molecule coexpressed with PLAP-a known placental extracellular vesicles marker on psEV. Higher psEV counts for all 3 antibody pairs were detected in the plasma of women who developed EOPE than the other 2 groups in the first trimester, which persisted through the second and third trimesters. Significantly higher CD10-PLAP (P<0.01) and CD63-PLAP (P<0.01) psEV counts were validated in the serum of the first trimester of women who developed EOPE compared with normal pregnancy. CONCLUSIONS: Application of the ExoCounter assay developed here could identify patients at risk of developing EOPE in the first trimester, thereby providing a window of opportunity for early intervention.

Pneumococcal BgaA Promotes Host Organ Bleeding and Coagulation in a Mouse Sepsis Model.

Streptococcus pneumoniae is a major cause of invasive diseases such as pneumonia, meningitis, and sepsis, with high associated mortality. Our previous molecular evolutionary analysis revealed that the S. pneumoniae gene bgaA, encoding the enzyme ß-galactosidase (BgaA), had a high proportion of codons under negative selection among the examined pneumococcal genes and that deletion of bgaA significantly reduced host mortality in a mouse intravenous infection assay. BgaA is a multifunctional protein that plays a role in cleaving terminal galactose in N-linked glycans, resistance to human neutrophil-mediated opsonophagocytic killing, and bacterial adherence to human epithelial cells. In this study, we performed in vitro and in vivo assays to evaluate the precise role of bgaA as a virulence factor in sepsis. Our in vitro assays showed that the deletion of bgaA significantly reduced the bacterial association with human lung epithelial and vascular endothelial cells. The deletion of bgaA also reduced pneumococcal survival in human blood by promoting neutrophil-mediated killing, but did not affect pneumococcal survival in mouse blood. In a mouse sepsis model, mice infected with an S. pneumoniae bgaA-deleted mutant strain exhibited upregulated host innate immunity pathways, suppressed tissue damage, and blood coagulation compared with mice infected with the wild-type strain. These results suggest that BgaA functions as a multifunctional virulence factor whereby it induces host tissue damage and blood coagulation. Taken together, our results suggest that BgaA could be an attractive target for drug design and vaccine development to control pneumococcal infection.

Ultrasensitive detection of SARS-CoV-2 nucleocapsid protein using large gold nanoparticle-enhanced surface plasmon resonance.

The COVID-19 pandemic has created urgent demand for rapid detection of the SARS-CoV-2 coronavirus. Herein, we report highly sensitive detection of SARS-CoV-2 nucleocapsid protein (N protein) using nanoparticle-enhanced surface plasmon resonance (SPR) techniques. A crucial plasmonic role in significantly enhancing the limit of detection (LOD) is revealed for exceptionally large gold nanoparticles (AuNPs) with diameters of hundreds of nm. SPR enhanced by these large nanoparticles lowered the LOD of SARS-CoV-2 N protein to 85 fM, resulting in the highest SPR detection sensitivity ever obtained for SARS-CoV-2 N protein.

Epidemiological analysis of pneumococcal strains isolated at Yangon Children's Hospital in Myanmar via whole-genome sequencing-based methods.

Streptococcus pneumoniae causes over one million deaths from lower respiratory infections per annum worldwide. Although mortality is very high in Southeast Asian countries, molecular epidemiological information remains unavailable for some countries. In this study, we report, for the first time, the whole-genome sequences and genetic profiles of pneumococcal strains isolated in Myanmar. We isolated 60 streptococcal strains from 300 children with acute respiratory infection at Yangon Children's Hospital in Myanmar. We obtained whole-genome sequences and identified the species, serotypes, sequence types, antimicrobial resistance (AMR) profiles, virulence factor profiles and pangenome structure using sequencing-based analysis. Average nucleotide identity analysis indicated that 58 strains were S. pneumoniae and the other 2 strains were Streptococcus mitis. The major serotype was 19F (11 strains), followed by 6E (6B genetic variant; 7 strains) and 15 other serotypes; 5 untypable strains were also detected. Multilocus sequence typing analysis revealed 39 different sequence types, including 11 novel ones. In addition, genetic profiling indicated that AMR genes and mutations spread among pneumococcal strains in Myanmar. A minimum inhibitory concentration assay indicated that several pneumococcal strains had acquired azithromycin and tetracycline resistance, whereas no strains were found to be resistant against levofloxacin and high-dose penicillin G. Phylogenetic and pangenome analysis showed various pneumococcal lineages and that the pneumococcal strains contain a rich and mobile gene pool, providing them with the ability to adapt to selective pressures. This molecular epidemiological information can help in tracking global infection and supporting AMR control in addition to public health interventions in Myanmar.

O-Glycan-Altered Extracellular Vesicles: A Specific Serum Marker Elevated in Pancreatic Cancer.

Pancreatic cancer (PC) is among the most lethal malignancies due to an often delayed and difficult initial diagnosis. Therefore, the development of a novel, early stage, diagnostic PC marker in liquid biopsies is of great significance. In this study, we analyzed the differential glycomic profiling of extracellular vesicles (EVs) derived from serum (two cohorts including 117 PC patients and 98 normal controls) using lectin microarray. The glyco-candidates of PC-specific EVs were quantified using a high-sensitive exosome-counting system, ExoCounter. An absolute quantification system for altered glycan-containing EVs elevated in PC serum was established. EVs recognized by O-glycan-binding lectins ABA or ACA were identified as candidate markers by lectin microarray. Quantitative analyses using ExoCounter revealed that the ABA- or ACA-positive EVs were significantly increased in the culture of PC cell lines or in the serum of PC patients including carbohydrate antigen 19-9 negative patients with high area under curve values. The elevated numbers of EVs in PC serum returned to normal levels after pancreatectomy. Histological examination confirmed that the tumors stained with ABA/ACA. These specific EVs with O-glycans recognized by ABA/ACA are elevated in PC sera and can act as potential biomarkers in a liquid biopsy for PC patients screening.

Streptococcus pneumoniae Evades Host Cell Phagocytosis and Limits Host Mortality Through Its Cell Wall Anchoring Protein PfbA.

Streptococcus pneumoniae is a Gram-positive bacterium belonging to the oral streptococcus species, mitis group. This pathogen is a leading cause of community-acquired pneumonia, which often evades host immunity and causes systemic diseases, such as sepsis and meningitis. Previously, we reported that PfbA is a ß-helical cell surface protein contributing to pneumococcal adhesion to and invasion of human epithelial cells in addition to its survival in blood. In the present study, we investigated the role of PfbA in pneumococcal pathogenesis. Phylogenetic analysis indicated that the pfbA gene is highly conserved in S. pneumoniae and Streptococcus pseudopneumoniae within the mitis group. Our in vitro assays showed that PfbA inhibits neutrophil phagocytosis, leading to pneumococcal survival. We found that PfbA activates NF-κB through TLR2, but not TLR4. In addition, TLR2/4 inhibitor peptide treatment of neutrophils enhanced the survival of the S. pneumoniae ΔpfbA strain as compared to a control peptide treatment, whereas the treatment did not affect survival of a wild-type strain. In a mouse pneumonia model, the host mortality and level of TNF-α in bronchoalveolar lavage fluid were comparable between wild-type and ΔpfbA-infected mice, while deletion of pfbA decreased the bacterial burden in bronchoalveolar lavage fluid. In a mouse sepsis model, the ΔpfbA strain demonstrated significantly increased host mortality and TNF-α levels in plasma, but showed reduced bacterial burden in lung and liver. These results indicate that PfbA may contribute to the success of S. pneumoniae species by inhibiting host cell phagocytosis, excess inflammation, and mortality by interacting with TLR2.

Secretory Carcinoma of Minor Salivary Gland in Buccal Mucosa: A Case Report and Review of the Literature.

Secretory carcinoma (SC) of the salivary gland was recently added to the fourth edition of the World Health Organization classification of head and neck tumors. Some salivary tumors, including acinic cell carcinoma, have been reclassified as SC. Most of these tumors are located on the parotid gland with very few cases reported in the minor salivary glands of the buccal mucosa. Herein, we present a case of SC of buccal mucosa, which appeared clinically as a benign lesion in a 54-year-old Japanese female patient. Histopathologically, the tumor cells presented with an eosinophilic cytoplasm with microcytic structure along with eosinophilic secretory material and hemosiderin deposit. Immunohistochemical staining revealed strongly positive staining for S100, vimentin, and mammaglobin and negative staining for DOG-1. The tumor was finally diagnosed as secretory carcinoma of the buccal mucosa. We present a review of the medical literature of SC arising from minor salivary glands. We found only 15 cases of SC of buccal mucosa out of 63 cases of SC in the minor salivary glands. They showed good prognoses and only one case of SC in the buccal mucosa exhibited local recurrence and lymph node metastases.

Development of a Highly Sensitive Device for Counting the Number of Disease-Specific Exosomes in Human Sera.

BACKGROUND: Although circulating exosomes in blood play crucial roles in cancer development and progression, difficulties in quantifying exosomes hamper their application for reliable clinical testing. By combining the properties of nanobeads with optical disc technology, we have developed a novel device named the ExoCounter to determine the exact number of exosomes in the sera of patients with various types of cancer. METHOD: In this system, individual exosomes were captured in the groove of an optical disc coated with antibodies against exosome surface antigens. The captured exosomes were labeled with antibody-conjugated magnetic nanobeads, and the number of the labeled exosomes was counted with an optical disc drive. RESULTS: We showed that the ExoCounter could detect specific exosomes derived from cells or human serum without any enrichment procedures. The detection sensitivity and linearity with this system were higher than those with conventional detection methods such as ELISA or flow cytometry. In addition to the ubiquitous exosome markers CD9 and CD63, the cancer-related antigens CD147, carcinoembryonic antigen, and human epidermal growth factor receptor 2 (HER2) were also used to quantify cancer cell line-derived exosomes. Furthermore, analyses of a cross-sectional cohort of sera samples revealed that HER2-positive exosomes were significantly increased in patients with breast cancer or ovarian cancer compared with healthy individuals and those with noncancer diseases. CONCLUSIONS: The ExoCounter system exhibits high performance in the direct detection of exosomes in cell culture and human sera. This method may enable reliable analysis of liquid biopsies.

[Clinical investigation of bisphosphonate-related osteonecrosis of the jaws].

We examined the clinical features of bisphosphonate(BP)-related osteonecrosis of the jaws(BRONJ), a serious complication resulting from intravenous BP treatment for multiple myeloma and malignant tumors with bone metastasis. We retrospectively reviewed the medical records of 36 patients who received intravenous BP therapy for the above-mentioned conditions, at Sapporo Medical University Hospital between July 2006 and October 2008. BP therapy caused BRONJ in 7 of 24 patients, but did not affect the bones of the other 17 patients. The other 12 of the 36 patients involved in the study were prescribed BP only after they had undergone an oral examination and treatment for dental inflammation. Of these patients, 7 developed BRONJ with BP treatment, after tooth extraction or acute dental inflammation. Treating dental inflammation before prescribing BP prevented the development of BRONJ. BRONJ is highly intractable and does not resolve with the standard treatment for osteomyelitis. Therefore, preventive therapy, which can be achieved by cooperation between medical doctors and dentists, is currently the most effective strategy for BRONJ. Conservative treatment with antibiotics may also be useful for maintaining or improving the quality of life of BRONJ patients.