Role of Wnt5b-Ror1 signaling in the proliferation of pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most refractory cancers with the worst prognosis. Although several molecules are known to be associated with the progression of PDAC, the molecular mechanisms underlying the progression of PDAC remain largely elusive. The Ror-family receptors, Ror1 and Ror2, which act as a receptor(s) for Wnt-family ligands, particularly Wnt5a, are involved in the progression of various types of cancers. Here, we show that higher expression of Ror1 and Wnt5b, but not Ror2, are associated with poorer prognosis of PDAC patients, and that Ror1 and Wnt5b are expressed highly in a type of PDAC cell lines, PANC-1 cells. Knockdown of either Ror1 or Wnt5b in PANC-1 cells inhibited their proliferation significantly in vitro, and knockout of Ror1 in PANC-1 cells resulted in a significant inhibition of tumor growth in vivo. Furthermore, we show that Wnt5b-Ror1 signaling in PANC-1 cells promotes their proliferation in a cell-autonomous manner by modulating our experimental setting in vitro. Collectively, these findings indicate that Wnt5b-Ror1 signaling might play an important role in the progression of some if not all of PDAC by promoting proliferation.

Flavivirus recruits the valosin-containing protein-NPL4 complex to induce stress granule disassembly for efficient viral genome replication.

Flaviviruses are human pathogens that can cause severe diseases, such as dengue fever and Japanese encephalitis, which can lead to death. Valosin-containing protein (VCP)/p97, a cellular ATPase associated with diverse cellular activities (AAA-ATPase), is reported to have multiple roles in flavivirus replication. Nevertheless, the importance of each role still has not been addressed. In this study, the functions of 17 VCP mutants that are reportedly unable to interact with the VCP cofactors were validated using the short-interfering RNA rescue experiments. Our findings of this study suggested that VCP exerts its functions in replication of the Japanese encephalitis virus by interacting with the VCP cofactor nuclear protein localization 4 (NPL4). We show that the depletion of NPL4 impaired the early stage of viral genome replication. In addition, we demonstrate that the direct interaction between NPL4 and viral nonstructural protein (NS4B) is critical for the translocation of NS4B to the sites of viral replication. Finally, we found that Japanese encephalitis virus and dengue virus promoted stress granule formation only in VCP inhibitor-treated cells and the expression of NS4B or VCP attenuated stress granule formation mediated by protein kinase R, which is generally known to be activated by type I interferon and viral genome RNA. These results suggest that the NS4B-mediated recruitment of VCP to the virus replication site inhibits cellular stress responses and consequently facilitates viral protein synthesis in the flavivirus-infected cells.

Ror1 is expressed inducibly by Notch and hypoxia signaling and regulates stem cell-like property of glioblastoma cells.

Ror1 plays a crucial role in cancer progression by regulating cell proliferation and migration. Ror1 is expressed abundantly in various types of cancer cells and cancer stem-like cells. However, the molecular mechanisms regulating expression of Ror1 in these cells remain largely unknown. Ror1 and its putative ligand Wnt5a are expressed highly in malignant gliomas, especially in glioblastomas, and the extents of Ror1 expression are correlated positively with poorer prognosis in patients with gliomas. We show that Ror1 expression can be upregulated in glioblastoma cells under spheroid culture, but not adherent culture conditions. Notch and hypoxia signaling pathways have been shown to be activated in spheroid-forming glioblastoma stem-like cells (GSCs), and Ror1 expression in glioblastoma cells is indeed suppressed by inhibiting either Notch or hypoxia signaling. Meanwhile, either forced expression of the Notch intracellular domain (NICD) in or hypoxic culture of glioblastoma cells result in enhanced expression of Ror1 in the cells. Consistently, we show that both NICD and hypoxia-inducible factor 1 alpha bind to upstream regions within the Ror1 gene more efficiently in GSCs under spheroid culture conditions. Furthermore, we provide evidence indicating that binding of Wnt5a to Ror1, upregulated by Notch and hypoxia signaling pathways in GSCs, might promote their spheroid-forming ability. Collectively, these findings indicate for the first time that Notch and hypoxia signaling pathways can elicit a Wnt5a-Ror1 axis through transcriptional activation of Ror1 in glioblastoma cells, thereby promoting their stem cell-like property.

Adiponectin can be a good predictor of urodynamic detrusor underactivity: a prospective study in men with lower urinary tract symptoms.

PURPOSE: To investigate whether circulating adiponectin, which is considered a possible marker of anti-atherogenic effects, is a useful predictor of bladder function, especially detrusor underactivity (DU), in men with lower urinary tract symptoms (LUTS). METHODS: A total of 130 treatment-naïve men with non-neurogenic LUTS were prospectively stratified into two groups (the DU and non-DU groups) based on the presence or absence of DU, where DU is defined as a bladder contractility index < 100 and bladder outlet obstruction index (BOOI) < 40. The impact of serum adiponectin levels on urodynamic function, including DU, was assessed using univariate, binomial logistic regression, and receiver operating characteristic (ROC) curve analyses. RESULTS: In total, data from 118 men were analyzed; 39 (33.0%) had DU (DU group) and 79 (67.0%) did not have DU (non-DU group). The median serum adiponectin in the DU group was significantly lower than in the non-DU group (6.2 vs 12.6 µg/mL, p < 0.001). In the binomial logistic regression analysis, lower adiponectin, smaller intravesical prostatic protrusion, and lower bladder voiding efficiency were significant factors related to DU. In the ROC analyses, serum adiponectin had the highest area under the curve value for DU diagnosis (0.849). Additionally, a cutoff value of 7.9 µg/mL for serum adiponectin level was identified for DU, which yielded a sensitivity and specificity of 79% and 90%, respectively. CONCLUSIONS: The serum adiponectin level was significantly associated with bladder function and may be a useful marker for predicting DU in men with LUTS.

Adaptive optics with spatio-temporal lock-in detection for temporal focusing microscopy.

Wavefront distortion in temporal focusing microscopy (TFM) results in a distorted temporal profile of the excitation pulses owing to spatio-temporal coupling. Since the pulse duration is dramatically changed in the excitation volume, it is difficult to correct the temporal profile for a thick sample. Here, we demonstrate adaptive optics (AO) correction in a thick sample. We apply structured illumination microscopy (SIM) to an AO correction in wide-field TFM to decrease the change in the pulse duration in the signal detection volume. The AO correction with SIM was very successful in a thick sample for which AO correction with TFM failed.

Characterization of Prototheca CYP51/ERG11 as a possible target for therapeutic drugs.

Prototheca spp. are achlorophyllous algae, ubiquitous in nature. An increasing number of human and animal cases of Prototheca infection (protothecosis) are reported, and antifungal azoles, which inhibit sterol 14α-demethylase (CYP51/ERG11) involved in ergosterol biosynthesis, have empirically been used for the treatment of protothecosis. Although Prototheca, like fungi, has ergosterol in the cell membrane, efficacy of the antifungal azoles in the treatment of protothecosis is controversial. For investigating the interaction of azole drugs with Prototheca CYP51/ERG11, the CYP51/ERG11 genomic genes of four strains of P. wickerhamii and one strain each of P. cutis and P. miyajii were isolated and characterized in this study. Compared with the CYP51/ERG11 gene of chlorophyllous Auxenochlorella Protothecoides, it is possible that ProtothecaCYP51/ERG11 gene, whose exon-intron structure appeared to be species-specific, lost introns associated with the loss of photosynthetic activity. Analysis of the deduced amino acid sequences revealed that Prototheca CYP51/ERG11 and fungal CYP51/ERG11 are phylogenetically distant from each other although their overall structures are similar. Our basic in silico studies predicted that antifungal azoles could bind to the catalytic pocket of Prototheca CYP51/ERG11. It was also suggested that amino acid residues away from the catalytic pocket might affect the drug susceptibility. The results of this study may provide useful insights into the phylogenetic taxonomy of Prototheca spp. in relationship to the CYP51/ERG11 structure and development of novel therapeutic drugs for the treatment of protothecosis. LAY SUMMARY: Cases of infection by microalgae of Prototheca species are increasing. However, effective treatment has not been established yet. In this study, gene and structure of Prototheca's CYP51/ERG11, an enzyme which might serve as a target for therapeutic drugs, were characterized for the first time.

The influence of multiple oral administration on the pharmacokinetics and distribution profile of dalcetrapib in rats.

We investigated the influence of multiple oral administration on the accumulation of dalcetrapib (JTT-705/RO4607381), a novel cholesteryl ester transfer protein inhibitor, in rats. It is well known that orally administered dalcetrapib is rapidly hydrolysed to its active form, which has a sulfhydryl group, in the body. The active form then binds covalently to endogenous thiols via mixed disulfide bonds. Following multiple once daily oral administration of 14C-dalcetrapib for seven days to rats, the concentration of radioactivity in the plasma and almost all tissues reached the steady state by day 4. At 24 h after the last dose, there was a relatively high concentration of radioactivity in the mesenteric lymph nodes, liver, adrenal glands and fat. After the last dose to rats, the radioactivity was almost completely recovered in the urine and faeces, indicating that dalcetrapib is not retained in the body, probably due to the reversibility of the disulfide bonds despite being covalent bonds.

Vertical Ridge Augmentation With a Honeycomb Structure Titanium Membrane: A Technical Note for a 3-Dimensional Curvature Bending Method.

Guided bone regeneration is the most commonly used technique for vertical ridge augmentation (VRA), and it is popular because it is less invasive and highly formative. Since the augmented site is exposed to external pressure, it is preferable to support the membrane using a framework to maintain the shape of the VRA. Recently, a titanium framework-reinforced ultrafine titanium membrane was developed by laser processing technology. The technique allows microperforations to be made (φ20 µm) into a titanium membrane, which is expected to prevent fibrous tissue ingrowth from outside the membrane. In addition, significant bone regeneration was confirmed on ridge defects in previous animal studies. However, the membrane tends to crumple during the bending process, because it is very thin (20 nµm); thus, the bending procedures are technically sensitive. Since this titanium honeycomb membrane was first approved for clinical use in Japan, no international clinical reports have been published. The purpose of this case report is to describe a technical note for a 3-dimensional curvature bending method in VRA using the newly developed honeycomb structure titanium membrane.

The clinical benefit of sequential therapy with androgen receptor axis-targeted agents alone in patients with castration-resistant prostate cancer: A propensity score-matched comparison study.

BACKGROUND: The optimal sequential therapy for castration-resistant prostate cancer (CRPC) remains unknown. In recent years, some doubts have emerged regarding the clinical benefit of sequential therapy with androgen receptor axis-targeted agents (ART) such as abiraterone (ABI) or enzalutamide (ENZ) for patients with CRPC. We compared the effect of ART-to-ART (AA) sequential therapy after castration resistance with that of docetaxel (DTX)-combined sequential therapy (ART to DTX or DTX to ART) in patients with CRPC. METHODS: We retrospectively identified and analyzed the data of 315 patients with CRPC treated in our seven affiliated institutions between 2009 and 2019. All patients received either DTX or ART (ABI or ENZ) as the first- or second-line therapy after castration resistance. We compared the overall survival (OS) and the second progression-free survival (PFS2), calculated from the initiation of first-line therapy after castration resistance, between the AA sequence group and the DTX-combined sequence group. PFS2 was defined as the period from the start of first-line treatment after castration resistance to progression on second-line treatment. To minimize selection bias from possible confounders, we performed propensity score matching using one-to-one nearest neighbor matching without replacement. RESULTS: Overall, 106 and 209 patients were administered the AA sequential therapy and DTX-combined sequential therapy, respectively. The clinicopathological variables of patients were well balanced after propensity score matching, and there were no significant differences between the two groups. In the propensity score-matched cohort, OS was not significantly different between the two groups (median, 37.9 vs. 45.4 months; hazard ratio [HR], 1.10; 95% confidence interval [CI], 0.68-1.79; p = .701), while PFS2 was significantly shorter in the AA group than in the DTX-combined group (median, 12.9 vs. 21.6 months; HR, 1.70; 95% CI, 1.16-2.48; p = .007). CONCLUSIONS: Certain patients with CRPC can benefit from ART-to-ART sequential therapy in a daily clinical setting.

Propensity score-matched comparison of docetaxel and androgen receptor axis-targeted agents in patients with castration-resistant intraductal carcinoma of the prostate.

OBJECTIVE: To evaluate the efficacy of docetaxel and androgen receptor axis-targeted (ARAT) agents in patients with castration-resistant prostate cancer (CRPC) with intraductal carcinoma of the prostate (IDC-P) using a propensity score-matched analysis. PATIENTS AND METHODS: We retrospectively identified 309 patients with CRPC from February 2007 to February 2016 at Nagoya University and its affiliated hospitals. All patients received initial androgen-deprivation therapy (ADT). After progression to CRPC, they received docetaxel or ARAT (abiraterone or enzalutamide) as first-line life-prolonging therapy. Docetaxel (70-75 mg/m2 ) every 3 weeks vs enzalutamide (160 mg) once daily orally or abiraterone (1 g) once daily plus prednisone (5 mg) twice daily orally was administered. The primary outcome of interest was overall survival (OS) from the time of CRPC diagnosis. A propensity score analysis with a 1:1 ratio using an optimal matching algorithm was used to adjust for confounding factors. RESULTS: Overall, 234 patients were analysed. Propensity score-matching identified 85 patients in each group. There were no significant differences in patient characteristics between the groups. The median OS in the docetaxel group was 38.2 vs 58.3 months in the ARAT group (P = 0.03). For patients with IDC-P, OS was significantly longer in the ARAT group than the docetaxel group (P = 0.01), and there was no significant difference in each group, as in patients without IDC-P (P = 0.67). A multivariate analysis showed that the presence of IDC-P, duration of primary ADT, visceral metastasis, and administration of ARAT as the first-line treatment for CRPC were independent prognostic factors for OS. CONCLUSION: Administration of ARAT as the first-line treatment for CRPC may prolong OS more than that of docetaxel, especially in patients with IDC-P.

Matrix metalloproteinase-7 induces homotypic tumor cell aggregation via proteolytic cleavage of the membrane-bound Kunitz-type inhibitor HAI-1.

Matrix metalloproteinase-7 (MMP-7) plays important roles in tumor progression and metastasis. Our previous studies have demonstrated that MMP-7 binds to colon cancer cells via cell surface-bound cholesterol sulfate and induces significant cell aggregation by cleaving cell-surface protein(s). These aggregated cells exhibit a dramatically enhanced metastatic potential. However, the molecular mechanism inducing this cell-cell adhesion through the proteolytic action of MMP-7 remained to be clarified. Here, we explored MMP-7 substrates on the cell surface; the proteins on the cell surface were first biotinylated, and a labeled protein fragment specifically released from the cells after MMP-7 treatment was analyzed using LC-MS/MS. We found that hepatocyte growth factor activator inhibitor type 1 (HAI-1), a membrane-bound Kunitz-type serine protease inhibitor, is an MMP-7 substrate. We also found that the cell-bound MMP-7 cleaves HAI-1 mainly between Gly451 and Leu452 and thereby releases the extracellular region as soluble HAI-1 (sHAI-1). We further demonstrated that this sHAI-1 can induce cancer cell aggregation and determined that the HAI-1 region corresponding to amino acids 141-249, which does not include the serine protease inhibitor domain, has the cell aggregation-inducing activity. Interestingly, a cell-surface cholesterol sulfate-independent proteolytic action of MMP-7 is critical for the sHAI-1-mediated induction of cell aggregation, whereas cholesterol sulfate is needed for the MMP-7-catalyzed generation of sHAI-1. Considering that MMP-7-induced cancer cell aggregation is an important mechanism in cancer metastasis, we propose that sHAI-1 is an essential component of MMP-7-induced stimulation of cancer metastasis and may therefore represent a suitable target for antimetastatic therapeutic strategies.

Compact OXC architecture, design and prototype development for flexible waveband routing optical networks.

A novel compact OXC node architecture that combines WSSs and arrays of small scale optical delivery-coupling type switches ("DCSWs") is proposed. Unlike conventional OXC nodes, the WSSs are only responsible for dynamic path bundling ("flexible waveband") while the small scale optical switches route bundled path groups. A network design algorithm that is aware of the routing scheme is also proposed, and numerical experiments elucidate that the necessary number of WSSs and amplifiers can be significantly reduced. A prototype of the proposed OXC is also developed using monolithic arrayed DCSWs. Transmission experiments on the prototype verify the proposal's technical feasibility.

Prototheca miyajii sp. nov., isolated from a patient with systemic protothecosis.

Species of the genus Prototheca are achlorophyllous algae and ubiquitous in nature, and so far, six species have been listed in this genus: Prototheca wickerhamii, Prototheca zopfii, Prototheca blaschkeae, Prototheca cutis, Prototheca stagnora and Prototheca ulmea. A strain of the genus Prototheca, IFM 53848T, was isolated in Japan from a patient with systemic protothecosis and had been designated P. wickerhamii. Our previous study, by using PCR analysis, revealed that its SSU rRNA gene (rDNA) was distinctively larger than that of P. wickerhamii and other species of the genus Prototheca. In this study, molecular analysis showed that the exceptionally large SSU rDNA of IFM 53848T contains four group I introns. The morphology of IFM 53848T was indistinguishable from those of P. wickerhamii or P. cutis, and phylogenetic analyses, based on the sequences of the SSU rDNA exons and the D1/D2 region of the large subunit rDNA, indicated that IFM 53848T was closely related to P. cutis. On the other hand, unlike P. cutis, IFM 53848T failed to assimilate fructose or lysine and grew well at higher temperatures of up to 42 °C. In addition, the nucleotide sequence of the ribosomal internal transcribed spacer and the matrix assisted laser desorption ionization time-of-flight mass spectrometry profile of IFM 53848T were clearly distinct from those of P. cutis. The results strongly suggest that IFM 53848T represents a novel species, and so the seventh member of the genus Prototheca, which we have named Prototheca miyajii sp. nov. The unique characteristics of the strain may provide useful insights into the systematic taxonomy of the genus Prototheca.

Production of single-round infectious chimeric flaviviruses with DNA-based Japanese encephalitis virus replicon.

A method for rapid production of single-round infectious particles (SRIPs) of flavivirus would be useful for viral mutagenesis studies. Here, we established a DNA-based production system for SRIPs of flavivirus. We constructed a Japanese encephalitis virus (JEV) subgenomic replicon plasmid, which lacked the C-prM-E (capsid-pre-membrane-envelope) coding region, under the control of the cytomegalovirus promoter. When the JEV replicon plasmid was transiently co-transfected with a JEV C-prM-E expression plasmid into 293T cells, SRIPs were produced, indicating successful trans-complementation with JEV structural proteins. Equivalent production levels were observed when C and prM-E proteins were provided separately. Furthermore, dengue types 1-4, West Nile, yellow fever or tick-borne encephalitis virus prM-E proteins could be utilized for production of chimaeric flavivirus SRIPs, although the production was less efficient for dengue and yellow fever viruses. These results indicated that our plasmid-based system is suitable for investigating the life cycles of flaviviruses, diagnostic applications and development of safer vaccine candidates.

Pharmacokinetics and disposition of dalcetrapib in rats and monkeys.

1. The pharmacokinetics and metabolism of dalcetrapib (JTT-705/RO4607381), a novel cholesteryl ester transfer protein inhibitor, were investigated in rats and monkeys. 2. In in vitro stability studies, dalcetrapib was extremely unstable in plasma, liver S9 and small intestinal mucosa, and the pharmacologically active form (dalcetrapib thiol) was detected as major component. Most of the active form in plasma was covalently bound to plasma proteins via mixed disulfide bond formation. 3. Following oral administration of (14)C-dalcetrapib to rats and monkeys, active form was detected in plasma. The active form was mainly metabolized to the glucuronide conjugate and the methyl conjugate at the thiol group. Several minor metabolites including mono- and di-oxidized forms of the glucuronide are also detected in the plasma and urine. 4. The administered radioactivity was widely distributed to all tissues and mainly excreted into the feces (85.7 and 62.7% of the dose in rats and monkeys, respectively). Most of the radioactivity was recovered by 168 h. Although the absorbed dalcetrapib was hydrolyzed to the active form and was bound to endogenous thiol via formation of disulfide bond, it was relatively rapidly eliminated from the body and was not retained.

Esthetic management of anterior implants in advanced periodontal defects. A case report

Achieving optimal implant prosthodontic outcomes in the esthetically demanding anterior region requires sufficient hard and soft tissue volume to provide adequate support and coverage to ensure that the implant restorations are functional and yield predictable, long-lasting treatment results. A comprehensive biologic understanding of the 3D relationships between hard and soft tissue is crucial when treating esthetically demanding areas. Various techniques, notably guided bone regeneration, have been developed and are well documented as being reliable methods for larger 3D bone augmentation procedures. Additionally, dental modification and tooth repositioning in proximity to prospective implant placement sites has been extensively discussed. Recently, orthodontic extrusion with deferred extraction has emerged as a predictable treatment strategy for gaining additional vertical hard and soft tissue. Implementing treatment sequences and the timing of combined treatment methodologies have also been subjects of discussion. Combining orthodontic treatment with staged or delayed tooth extractions has been shown to be beneficial in providing the necessary osseous foundation for implant sites that may not be as amenable to more conventional augmentation techniques. These augmentation techniques and treatment methods require adequately stable and predictable periodontal health since uncontrolled periodontal disease poses a significant challenge and is detrimental to successful outcomes. The purpose of the present clinical report is to demonstrate the staging and sequencing of vertical hard and soft tissue management techniques for a severe anterior periodontal defect to achieve an esthetically functional implant treatment result.

Intentional Replantation in Combination with Fibroblast Growth Factor-2 Application and Orthodontic Tooth Extrusion for Periodontal Regeneration in the Treatment of Severe Endodontic-Periodontal Lesions.

Endodontic-periodontal lesions are characterized by the involvement of the pulp and periodontal disease in the same tooth. Despite successful root canal treatment, if the majority of bone support has been lost from periodontitis, the tooth may have a poor prognosis. In severe endodontic-periodontal lesions, the periodontal tissue regenerates poorly because of the significant loss of the periodontal ligament and cementum, poor tooth stability, and bone defect morphology unfavorable for bone regeneration. To overcome these difficult situations, in this case, osteotomy of the replantation bed and tooth replantation with horizontal rotation and deep placement were performed. To improve periodontal regeneration, fibroblast growth factor (FGF) 2 was applied to the artificially made periodontal defect. In addition, orthodontic extrusion of the deeply replaced tooth was performed for potential coronal migration of the periodontal tissue. This case presents a unique multidisciplinary method of treating severe endodontic-periodontal lesions using intentional replantation combined with FGF 2 application and orthodontic extrusion.

Dissemination of the Flavivirus Subgenomic Replicon Genome and Viral Proteins by Extracellular Vesicles.

Extracellular vesicles (EVs) such as exosomes have been shown to play physiological roles in cell-to-cell communication by delivering various proteins and nucleic acids. In addition, several studies revealed that the EVs derived from the cells that are infected with certain viruses could transfer the full-length viral genomes, resulting in EVs-mediated virus propagation. However, the possibility cannot be excluded that the prepared EVs were contaminated with infectious viral particles. In this study, the cells that harbor subgenomic replicon derived from the Japanese encephalitis virus and dengue virus without producing any replication-competent viruses were employed as the EV donor. It was demonstrated that the EVs in the culture supernatants of those cells were able to transfer the replicon genome to other cells of various types. It was also shown that the EVs were incorporated by the recipient cells primarily through macropinocytosis after interaction with CD33 and Tim-1/Tim-4 on HeLa and K562 cells, respectively. Since the methods used in this study are free from contamination with infectious viral particles, it is unequivocally indicated that the flavivirus genome can be transferred by EVs from cell to cell, suggesting that this pathway, in addition to the classical receptor-mediated infection, may play some roles in the viral propagation and pathogenesis.

Photoacoustic microscopy for real-time monitoring of near-infrared optical absorbers inside biological tissue.

Significance: We developed a high-speed optical-resolution photoacoustic microscopy (OR-PAM) system using a high-repetition-rate supercontinuum (SC) light source and a two-axes Galvano scanner. The OR-PAM system enabled real-time imaging of optical absorbers inside biological tissues with excellent excitation wavelength tunability. Aim: In the near-infrared (NIR) wavelength range, high-speed OR-PAM faces limitations due to the lack of wavelength-tunable light sources. Our study aimed to enable high-speed OR-PAM imaging of various optical absorbers, including NIR contrast agents, and validate the performance of high-speed OR-PAM in the detection of circulating tumor cells (CTCs). Approach: A high-repetition nanosecond pulsed SC light source was used for OR-PAM. The excitation wavelength was adjusted by bandpass filtering of broadband light pulses produced by an SC light source. Phantom and in vivo experiments were performed to detect tumor cells stained with an NIR contrast agent within flowing blood samples. Results: The newly developed high-speed OR-PAM successfully detected stained cells both in the phantom and in vivo. The phantom experiment confirmed the correlation between the tumor cell detection rate and tumor cell concentration in the blood sample. Conclusions: The high-speed OR-PAM effectively detected stained tumor cells. Combining high-speed OR-PAM with molecular probes that stain tumor cells in vivo enables in vivo CTC detection.

Characterization of genotype V Japanese encephalitis virus isolates from Republic of Korea.

Japanese encephalitis (JE), caused by the Japanese encephalitis virus (JEV) infection, continues to pose significant public health challenges worldwide despite efficient vaccines. The virus is classified into five genotypes, among which genotype V (GV) was not detected for a long period after its initial isolation in 1952, until reports emerged from China and the Republic of Korea (ROK) since 2009. The characteristics of the virus are crucial in estimating its potential epidemiological impact. However, characterization of GV JEVs has so far been limited to two strains: Muar, the original isolate, and XZ0934, isolated in China. Two additional ROK GV JEV isolates, NCCP 43279 and NCCP 43413, are currently available, but their characteristics have not been explored. Our phylogenetic analysis revealed that GV virus sequences from the ROK segregate into two clades. NCCP 43279 and NCCP 43413 belong to different clades and exhibit distinct in vitro phenotypes. NCCP 43279 forms larger plaques but demonstrates inefficient propagation in cell culture compared to NCCP 43413. In vivo, NCCP 43279 induces higher morbidity and mortality in mice than NCCP 43413. Notably, NCCP 43279 shows more severe blood-brain barrier damage, suggesting superior brain invasion capabilities. Consistent with its higher virulence, NCCP 43279 displays more pronounced histopathological and immunopathological outcomes. In conclusion, our study confirms that the two ROK isolates are not only classified into different clades but also exhibit distinct in vitro and in vivo characteristics.