Development of genogroup-specific ELISAs based on the VP1 protein to detect antibodies to GIV and GVI feline norovirus.

Feline norovirus (FNoV) is a potential pathogen of feline gastroenteritis and has two genogroups (GIV and GVI). Few epidemiological studies have been conducted on FNoV. We designed two enzyme-linked immunosorbent assays (ELISAs) to identify genogroup-specific FNoV antibodies for serological surveillance. Analysis of sera from cats experimentally infected with FNoV GIV or GVI and from specific-pathogen-free (SPF) cats confirmed that the two recombinant proteins used in the assay react in a genogroup-specific manner. Of the 183 samples tested, 6.6% were positive for GIV and 26.2% were positive for GVI. Antibodies to both FNoV genogroups were detected in sera collected in 2005, seven years before FNoV was first reported.

Direct visualization and measurement of the plantar aponeurosis behavior in foot arch deformation via the windlass mechanism.

The plantar aponeurosis (PA) is an elastic longitudinal band that contributes to the generation of a propulsive force in the push-off phase during walking and running through the windlass mechanism. However, the dynamic behavior of the PA remains unclear owing to the lack of direct measurement of the strain it generates. Therefore, this study aimed to visualize and quantify the PA behavior during two distinct foot postures: (i) neutral posture and (ii) windlass posture with midtarsal joint plantarflexion and metatarsophalangeal joint dorsiflexion, using computed tomography scans. Six healthy adult males participated in the experiment, and three-dimensional reconstruction of the PA was conducted to calculate its path length, width, thickness, and cross-sectional area. This study successfully visualized and quantified the morphological changes in the PA induced by the windlass mechanism, providing a precise reference for biomechanical modeling. This study also highlighted the interindividual variability in the PA morphology and stretching patterns. Although the windlass posture was not identical to that observed in the push-off phase during walking, the observed PA behavior provides valuable insights into its mechanics and potential implications for foot disorders.

Nodal infiltration in endometrial cancer: a prediction model using best subset regression.

OBJECTIVES: To build preoperative prediction models with and without MRI for regional lymph node metastasis (r-LNM, pelvic and/or para-aortic LNM (PENM/PANM)) and for PANM in endometrial cancer using established risk factors. METHODS: In this retrospective two-center study, 364 patients with endometrial cancer were included: 253 in the model development and 111 in the external validation. For r-LNM and PANM, respectively, best subset regression with ten-time fivefold cross validation was conducted using ten established risk factors (4 clinical and 6 imaging factors). Models with the top 10 percentile of area under the curve (AUC) and with the fewest variables in the model development were subjected to the external validation (11 and 4 candidates, respectively, for r-LNM and PANM). Then, the models with the highest AUC were selected as the final models. Models without MRI findings were developed similarly, assuming the cases where MRI was not available. RESULTS: The final r-LNM model consisted of pelvic lymph node (PEN) ≥ 6 mm, deep myometrial invasion (DMI) on MRI, CA125, para-aortic lymph node (PAN) ≥ 6 mm, and biopsy; PANM model consisted of DMI, PAN, PEN, and CA125 (in order of correlation coefficient ß values). The AUCs were 0.85 (95%CI: 0.77-0.92) and 0.86 (0.75-0.94) for the external validation, respectively. The model without MRI for r-LNM and PANM showed AUC of 0.79 (0.68-0.89) and 0.87 (0.76-0.96), respectively. CONCLUSIONS: The prediction models created by best subset regression with cross validation showed high diagnostic performance for predicting LNM in endometrial cancer, which may avoid unnecessary lymphadenectomies. CLINICAL RELEVANCE STATEMENT: The prediction risks of lymph node metastasis (LNM) and para-aortic LNM can be easily obtained for all patients with endometrial cancer by inputting the conventional clinical information into our models. They help in the decision-making for optimal lymphadenectomy and personalized treatment. KEY POINTS: •Diagnostic performance of lymph node metastases (LNM) in endometrial cancer is low based on size criteria and can be improved by combining with other clinical information. •The optimized logistic regression model for regional LNM consists of lymph node ≥ 6 mm, deep myometrial invasion, cancer antigen-125, and biopsy, showing high diagnostic performance. •Our model predicts the preoperative risk of LNM, which may avoid unnecessary lymphadenectomies.

Non-volatile optical memory based on cooperative orientation switching: improvement of recording speed and contrast by utilizing out-of-plane orientation mode.

Herein, we report a novel strategy toward non-volatile optical memory with high-contrast, high-speed recording, and non-destructive readout capability based on the cooperative out-of-plane orientation of a fluorescent dye doped into azobenzene liquid crystalline polymer film. By employing the out-of-plane orientation switching upon irradiation with UV light and thermal heating, high-contrast turn-on fluorescence switching was successfully achieved and the optical recording was demonstrated with non-destructive fluorescence readout capability. Furthermore, the recording speed and the fluorescence on/off contrast in the present system were dramatically improved compared to the previous in-plane orientation mode.

Distinct effects of Fgf7 and Fgf10 on the terminal differentiation of murine bladder urothelium revealed using an organoid culture system.

BACKGROUND: Dysregulation of the terminal differentiation of bladder urothelium is associated with the pathogenesis of urinary tract disorders. Fibroblast growth factor (Fgf)7 and Fgf10 stimulate urothelial proliferation; however, their roles in cellular differentiation remain unclear. In this study, we used an organoid system to investigate the roles of these Fgfs in regulating bladder urothelium differentiation and identify their distribution patterns in the mouse bladder. METHODS: Adult bladder epithelia (AdBE) isolated from adult mouse bladder tissues (AdBTs) were used to culture adult bladder organoids (AdBOs) in the presence of Fgf7 and Fgf10. The differentiation status of the cells in AdBTs, AdBEs, AdBOs, and neonatal bladder tissues (NeoBTs) was analyzed via quantitative real-time-PCR for the presence of undifferentiated cell markers (Krt5, Trp63, and Krt14) and differentiated cell markers (Krt20, Upk1a, Upk2, and Upk3a). Organoid cell proliferation was assessed by counting cell numbers using the trypan blue method. The effects of Fgf7 and Fgf10 on organoid differentiation were assessed using different doses of Fgfs, and the involvement of peroxisome proliferator-activated receptor γ (PPARγ) signaling in these processes was tested by introducing a PPARγ agonist (Rosiglitazone) and antagonist (T0070907) to the culture. The expression patterns of Fgf7 and Fgf10 were examined via in situ hybridization of AdBTs. RESULTS: AdBOs showed higher expression of undifferentiated cell markers and lower expression of differentiated cell markers than AdBTs, NeoBTs, and AdBEs, indicating the relatively immature state of AdBOs. Differentiation of AdBOs was enhanced by Rosiglitazone and Fgf7, suggesting an interplay of intracellular signals between Fgf7 and PPARγ. Co-addition of T0070907 suppressed Fgf7-mediated differentiation, demonstrating that PPARγ is activated downstream of Fgf7 to promote cellular differentiation into umbrella cells. Furthermore, we found that Fgf7 is predominantly expressed in the umbrella cells of the urothelium, whereas Fgf10 is predominantly expressed in the urothelium and stroma of AdBTs. CONCLUSIONS: We demonstrated that unlike Fgf10, Fgf7 induces cellular differentiation via PPARγ activity and has a unique tissue distribution pattern in the adult bladder. Further studies on the Fgf7-PPARγ signaling axis would provide insights into the differentiation mechanisms toward functional umbrella cells and the pathogenesis of several urinary tract diseases.

Risk Stratification for Pregnancies Diagnosed With Fetal Growth Restriction Based on Placental MRI.

BACKGROUND: Diagnosis of fetal growth restriction (FGR) entails difficulties with differentiating fetuses not fulfilling their growth potential because of pathologic conditions, such as placental insufficiency, from constitutionally small fetuses. The feasibility of placental MRI for risk stratification among pregnancies diagnosed with FGR remains unexplored. PURPOSE: To explore quantitative MRI features useful to identify pregnancies with unfavorable outcomes and to assess the diagnostic performance of visual analysis of MRI to detect pregnancies with unfavorable outcomes, among pregnancies diagnosed with FGR. STUDY TYPE: Retrospective. POPULATION: Thirteen pregnancies with unfavorable outcomes (preterm emergency cesarean section or intrauterine fetal death) and 11 pregnancies with favorable outcomes performed MRI at gestational weeks 21-36. FIELD STRENGTH/SEQUENCE: A 5-T, half-Fourier-acquired single-shot turbo spin echo (HASTE), spin-echo echo-planar imaging (SE-EPI) and T2 map derived from SE-EPI. ASSESSMENT: Placental size on HASTE sequences and T2 mapping-based histogram features were extracted. Three radiologists qualitatively evaluated the visibility of maternal cotyledon on HASTE and SE-EPI sequences with echo times (TEs) = 60, 90, and 120 msec using 3-point Likert scales: 0, absent; 1, equivocal; and 2, present. STATISTICAL TESTS: Welch's t-test or Mann-Whitney U test for quantitative features between the favorable and unfavorable outcome groups. Areas under the receiver operating curves (AUCs) of the three readers' visual analyses to detect pregnancies with unfavorable outcomes. A P value of <0.05 was inferred as statistically significant. RESULTS: Placental size (major and minor axis, estimated area of placental bed, and volume of placenta) and T2 mapping-based histogram features (mean, skewness, and kurtosis) were statistically significantly different between the two groups. Visual analysis of HASTE and SE-EPI with TE = 60 msec showed AUCs of 0.80-0.86 to detect pregnancies with unfavorable outcomes. DATA CONCLUSION: Placental size, histogram features, and visual analysis of placental MRI may allow for risk stratification regarding outcomes among pregnancies diagnosed with FGR. EVIDENCE LEVEL: 3 TECHNICAL EFFICACY: Stage 5.

A Structure-Adaptive Ligand: The Conformation Changes to Fit with the Lengths and Angles of Tetravalent Bis-Paraquat Guests.

A flexible cylindrical cryptand (1b) with two m-phenylene-26-crown-8 ethers has been prepared, and its complexing properties with respect to tetravalent cationic bis-paraquat guests have been investigated. Diffusion-ordered spectroscopy nuclear magnetic resonance (NMR) and titration experiments using cold electrospray ionization mass spectrometry and 1H NMR revealed that 1b has versatile complexing properties with respect to tetravalent cationic guests used despite the guest molecules' length and shape.

Successful engraftment of bladder organoids in de-epithelialized mouse colon.

PURPOSE: To engraft bladder organoids (BO) on de-epithelialized mouse colon using an epithelial replacement technique. METHODS: BO cultured using bladder specimens from enhanced green fluorescent protein (EGFP) transgenic mice were engrafted to replace proximal colon epithelium stripped from an approximately 1 cm long target site in syngeneic wild-type recipient mice (n = 9) by exposure to ethylenediaminetetraacetic acid by infusion and flushing with phosphate buffered saline. Target sites were harvested on postoperative days 2, 7, and 28 for hematoxylin-eosin staining and immunofluorescence. RESULTS: Histology on postoperative days 7 and 28 showed BO derived EGFP + cells forming multiple layers on the luminal surface of the colon. Immunohistochemistry showed that EGFP + areas were positive for CK5 and CK14, markers for basal and immature subtype urothelium, respectively, but negative for CA2, a marker for colonic epithelium. Ki67 was detected predominantly in the basal parts of EGFP + areas on postoperative day 7 and day 28. CONCLUSIONS: This is the first report of successful engraftment of BO in de-epithelialized colon with urothelial tissue reconstituted by actively proliferating cells. This technique could be developed for augmentation cystoplasty to prevent bladder calculi formation and malignant transformation.

Diagnostic Accuracy of Magnetic Resonance Imaging for International Federation of Gynecology and Obstetrics 2018 IB to IIB Cervical Cancer Staging: Comparison Among Magnetic Resonance Sequences and Pathologies.

OBJECTIVE: This study aimed to investigate the most accurate magnetic resonance (MR) sequence for tumor detection, maximal tumor diameter, and parametrial invasion compared with histopathologic diagnoses. METHODS: Fifty-one patients with International Federation of Gynecology and Obstetrics 2018 IB1 to IIB cervical cancer underwent preoperative MR imaging and surgical resection. Two radiologists independently evaluated the tumor detection, parametrial invasion, and tumor size in each of T2-weighted image, diffusion-weighted image, and contrast-enhanced T1-weighted image. Results obtained for squamous cell carcinoma (SCC) and adenocarcinoma were also compared. RESULTS: Neither the tumor detection rate nor parametrial invasion was found to be significantly different among sequences. Tumor size assessment using MR imaging with pathology showed good correlation: r = 0.63-0.72. The adenocarcinoma size tended to be more underestimated than SCC in comparison with the pathologic specimen. CONCLUSIONS: Cervical cancer staging by MR images showed no significant difference among T2-weighted image, diffusion-weighted image, and contrast-enhanced T1-weighted image. Adenocarcinoma was prone to be measured as smaller than the pathologic specimen compared with SCC.

Defined serum-free culture of human infant small intestinal organoids with predetermined doses of Wnt3a and R-spondin1 from surgical specimens.

PURPOSE: Refinement of organoid technology is important for studying physiology and disease of the intestine. We aimed to optimize defined serum-free conditions for human infant small intestinal (SI) organoid culture with predetermined doses of Wnt3a and Rspo1 from surgical specimens. We further assessed whether intestinal specimens could be stored before use as a source of organoids. METHODS: Different doses of Wnt3a and Rspo1 in a serum-free medium were tested to establish a condition in which surgically resected SI cells grew as organoids over multiple passages. The expression of marker genes for stem and differentiated cells was assessed by quantitative polymerase chain reaction. We also investigated the organoid-forming efficiency of cells in degenerating intestines stored at 4 °C for various intervals post-resection. RESULTS: We determined the doses of Wnt3a and Rspo1 required for the continuous growth of infant SI organoids with multi-differentiation potential. We revealed that, despite the time-dependent loss of stem cells, tissues stored for up to 2 days preserved cells capable of generating amplifiable organoids. CONCLUSION: SI cells can be grown as organoids under defined conditions. This could provide a reproducible and customizable method of using surgical specimens for the study of intestinal maturation and their relevance to pediatric diseases.

An Inducible Diabetes Mellitus Murine Model Based on MafB Conditional Knockout under MafA-Deficient Condition.

Diabetes mellitus is an increasingly severe chronic metabolic disease that is occurring at an alarming rate worldwide. Various diabetic models, including non-obese diabetic mice and chemically induced diabetic models, are used to characterize and explore the mechanism of the disease's pathophysiology, in hopes of detecting and identifying novel potential therapeutic targets. However, this is accompanied by disadvantages, such as specific conditions for maintaining the incidence, nonstable hyperglycemia induction, and potential toxicity to other organs. Murine MAFA and MAFB, two closely-linked islet-enriched transcription factors, play fundamental roles in glucose sensing and insulin secretion, and maintenance of pancreatic ß-cell, respectively, which are highly homologous to human protein orthologs. Herein, to induce the diabetes mellitus model at a specific time point, we generated Pdx1-dependent Mafb-deletion mice under Mafa knockout condition (A0BΔpanc), via tamoxifen-inducible Cre-loxP system. After 16 weeks, metabolic phenotypes were characterized by intraperitoneal glucose tolerance test (IPGTT), urine glucose test, and metabolic parameters analysis. The results indicated that male A0BΔpanc mice had obvious impaired glucose tolerance, and high urine glucose level. Furthermore, obvious renal lesions, impaired islet structure and decreased proportion of insulin positive cells were observed. Collectively, our results indicate that A0BΔpanc mice can be an efficient inducible model for diabetes research.

Total Synthesis and Structure-Activity Relationship Study of Vineomycin A1.

The first total synthesis of vineomycin A1 (1) has been accomplished. Structure-activity relationship studies for cytotoxicity against human breast cancer MCF-7 cells using several synthetic vineomycin A1 analogues differing in the number and position of glycon moieties revealed that the cytotoxicity increased as the number of glycon moieties increased. The position of the glycon moiety was one of the key factors for the cytotoxicity of 1. Moreover, in vitro analysis of the cytotoxicity of 1 against MCF-7 cells indicated for the first time that 1 effectively induced cancer cell death by apoptosis, not by acting as a DNA intercalating agent.

Viral shedding and clinical status of feline-norovirus-infected cats after reinfection with the same strain.

Norovirus (NoV) infection is the most common cause of acute gastroenteritis in humans of all ages worldwide. When cats are experimentally infected with feline norovirus (FNoV), they develop symptoms of acute gastroenteritis. Therefore, FNoV infection may serve as an animal model for the disease caused by human norovirus infection. In this study, we examined whether FNoV of cats infected with genogroup GVI are protected from reinfection with the same strain. The blood anti-FNoV IgG level was inversely correlated with the viral load in stool samples and the clinical score of FNoV-infected cats, but complete prevention of reinfection was not observed. These findings were similar to the results of a reinfection experiment with NoV in human volunteers.

A unique glycan-isoform of transferrin in cerebrospinal fluid: A potential diagnostic marker for neurological diseases.

BACKGROUND: Cerebrospinal fluid (CSF) is sequestered from blood by the blood-brain barrier and directly communicates with brain parenchymal interstitial fluid, leading to contain specific biomarkers of neurological diseases. SCOPE OF REVIEW: CSF contains glycan isoforms of transferrin (Tf): one appears to be derived from the brain and the other from blood. MAJOR CONCLUSIONS: CSF contains two glycan-isoforms; brain-type Tf and serum-type Tf. Glycan analysis and immunohistochemistry suggest that serum-type Tf having α2, 6sialylated glycans is derived from blood whereas brain-type Tf having GlcNAc-terminated glycans is derived from the choroid plexus, CSF producing tissue. The ratio of serum-type/brain-type Tf differentiates Alzheimer's disease from idiopathic normal pressure hydrocephalus, which is an elderly dementia caused by abnormal metabolism of CSF. The ratios in Parkinson's disease (PD) patients were higher than those of controls and did not appear to be normally distributed. Indeed, detrended normal Quantile-Quantile plot analysis reveals the presence of an independent subgroup showing higher ratios in PD patients. The subgroup of PD shows higher levels of CSF α-synuclein than the rest, indicating that PD includes two subgroups, which differ in levels of brain-type Tf and α-synuclein. GENERAL SIGNIFICANCE: Glycosylation in central nervous system appears to be unique. The unique glycan may be a tag for glycoprotein, which is biosynthesized in the central nervous system. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.

Transcription factors related to chondrogenesis in pleomorphic adenoma of the salivary gland: a mechanism of mesenchymal tissue formation.

In salivary gland pleomorphic adenoma, expression of extracellular matrix (ECM) substances indicates that tumor epithelial cells are becoming chondrogenic and will produce cartilage-like mesenchymal tissues. Sox9, the master transcription factor of chondrogenesis, is expressed in mouse salivary gland cells. To clarify the mechanism behind chondrogenesis in tumor epithelial cells, we examined the expression of transcription factors related to chondrogenesis in tumors and salivary glands. Reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR, and immunostaining were performed on pleomorphic adenoma tissues, salivary gland tissues, and human submandibular gland (HSG) cells. The mRNAs of essential transcription factors for chondrogenesis-Sox9, Sox6, and Sox5-were detected in both tumor and salivary gland tissues. The mRNAs of aggrecan and type II collagen-cartilage-specific ECM substances-were detected only in tumors. Sox9 and Sox6 proteins were colocalized in many epithelial cells in tumors and salivary glands. Tumor epithelial cells also possessed aggrecan protein and occasionally type II collagen protein. Moreover, mRNAs for transcription repressors of chondrogenesis δEF1 and AP-2α were detected in both tumors and salivary glands, whereas Twist1 mRNA was detected only in salivary glands and was at significantly low-to-undetectable levels in tumors. Twist1 protein was localized in the Sox9-expressing salivary gland cells. HSG cells expressed Sox9, Sox6, and Twist1, but not aggrecan or type II collagen, and thus were similar to salivary gland cells. Twist1 depletion by Twist1 siRNA led to the upregulation of aggrecan and type II collagen mRNA expression in HSG cells. In contrast, forced expression of Twist1, using Twist1 cDNA, resulted in the downregulation of both these genes. Taken together, these results indicate that salivary gland cells have a potential for chondrogenesis, and Twist1 depletion concomitant with neoplastic transformation, which would permit tumor epithelial cells to produce cartilage-like mesenchymal tissues in salivary gland pleomorphic adenoma.

Total Synthesis of Aquayamycin.

An efficient and practical total synthesis of aquayamycin has been accomplished. The highly oxidized and stereochemically complex tetracyclic ring system was constructed using three key reactions: 1) highly diastereoselective 1,2-addition of C-glycosyl naphthyllithium to a cyclic ketone, 2) indium-mediated site-selective allylation-rearrangement sequence of naphthoquinone, and 3) diastereoselective intramolecular pinacol coupling. This synthetic strategy offers a novel and efficient pathway to prepare aquayamycin-type angucycline antibiotics.

Structural change of N-glycan exposes hydrophobic surface of human transferrin.

Transferrin is an iron-transport protein which possesses N-glycans at Asn432 and Asn630 in humans. Transferrin glycoforms Tf-1 and Tf-2, previously identified in human cerebrospinal fluid, are defined as the lower and upper bands in gel electrophoresis, respectively. Importantly, the Tf-2/Tf-1 ratio is raised in idiopathic normal pressure hydrocephalus patients and is useful as a clinical marker. In order to gain insight into the relationship between transferrin glycoform and biological function, we performed comparative characterization of Tf-1, Tf-2 and serum transferrin (sTf). Mass spectrometric analyses confirmed that Tf-2 is modified with disialylated biantennary glycans at both of the two N-glycosylation sites, which are similar to the N-glycans of sTf. On the other hand, Tf-1 is site-specifically modified: Asn630 has biantennary agalacto-complex-type glycan with bisecting N-acetylglucosamine (GlcNAc) and core fucose while Asn432 is modified with complex/high mannose-type glycans and possibly single GlcNAc. Size exclusion chromatography and fluorescence correlation spectroscopy analysis revealed that the hydration volume of Tf-1 is slightly smaller than that of sTf. Our striking finding is that Tf-1 has an exposed hydrophobic surface as monitored by the fluorescence intensity and wavelength of a hydrophobic probe, 1-anilino-8-naphthalene sulfonate, whereas Tf-2 does not. These results suggest that the different N-glycan structure of Tf-1 lowers the apparent hydration volume and reveals a patch of hydrophobic surface on transferrin which is otherwise covered with sialoglycan in sTf and Tf-2. The carbohydrate deficiency in certain pathological conditions may also expose hydrophobic surface which may modulate the function and/or stability of transferrin.

[Acute hepatitis C infection with prolonged intrahepatic cholestasis and remarkable progression of fibrosis mimicking fibrosing cholestatic hepatitis].

We report the case of a 71-year-old woman with acute hepatitis C infection and persistent viremia since 2 years. Her clinical course was characterized by general fatigue and prolonged jaundice with unusually high serum bilirubin levels. Liver histology showed lymphocyte infiltration, marked fibrosis, and severe cholestasis in the periportal zone, findings mimicking fibrosing cholestatic hepatitis (FCH). Fibrosing cholestatic hepatitis is a life-threatening form of recurrent hepatitis C infection that typically occurs in immunosuppressed patients. Here we report the rare case of an immunocompetent patient who developed this condition.

Estimating three-dimensional foot bone kinematics from skin markers using a deep learning neural network model.

The human foot is a complex structure comprising 26 bones, whose coordinated movements facilitate proper deformation of the foot, ensuring stable and efficient locomotion. Despite their critical role, the kinematics of foot bones during movement remain largely unexplored, primarily due to the absence of non-invasive methods for measuring foot bone kinematics. This study addresses this gap by proposing a neural network model for estimating foot bone movements using surface markers. To establish a mapping between the positions and orientations of the foot bones and 41 skin markers attached on the human foot, computed tomography scans of the foot with the markers were obtained with eleven healthy adults and thirteen cadaver specimens in different foot postures. The neural network architecture comprises four layers, with input and output layers containing the 41 marker positions and the positions and orientations of the nine foot bones, respectively. The mean errors between estimated and true foot bone position and orientation were 0.5 mm and 0.6 degrees, respectively, indicating that the neural network can provide 3D kinematics of the foot bones with sufficient accuracy in a non-invasive manner, thereby contributing to a better understanding of foot function and the pathogenetic mechanisms underlying foot disorders.