Therapeutic Outcome of Multidisciplinary Treatment in Unresectable Biliary Tract Cancer: A Multicenter Retrospective Analysis.

Background: There is little established evidence regarding treatment strategies for unresectable biliary tract cancer (BTC). This study aimed to clarify the situation of multidisciplinary treatment for unresectable BTC in the 2000s when there was no international standard first-line therapy. Methods: We retrospectively reviewed 315 consecutive patients with unresectable BTC who had been treated at seven tertiary institutions in Kanagawa Prefecture, Japan between 1999 and 2008. Results: The unresectable factors were as follows: locally advanced, 101 cases (32.1%); hematogenous metastases, 80 cases (25.4%); and peritoneal dissemination, 30 cases (9.5%). Chemotherapy or radiation therapy was administered to 218 patients (69.2%). The best supportive care was provided in 97 cases (30.8%). The most common regimen was gemcitabine monotherapy, followed by gemcitabine combination therapy and S-1 monotherapy. The 1- and 2-year survival rates of all patients were 34.6% and 12.2%, respectively. The median survival time (MST) was 8 months in all patients. The 1-year survival rate was 65%, and the MST was 12 months among the locally advanced patients, whereas patients with peritoneal dissemination had the worst outcome; the 1-year survival rate was 7%, and the MST was 5 months. Among treated 90 cases of perihilar cholangiocarcinoma, patients who received chemoradiotherapy (n = 24) had a significantly better outcome than those who received chemotherapy alone (MST: 20 vs. 11 months, P < 0.001). Conclusions: Unresectable BTC has heterogeneous treatment outcomes depending on the mode of tumor extension and location. Multidisciplinary treatment seems useful for patients with locally advanced BTC, whereas patients with metastatic disease still have a poor prognosis.

Onnamide A suppresses the severe acute respiratory syndrome-coronavirus 2 infection without inhibiting 3-chymotrypsin-like cysteine protease.

Given the continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the development of new inhibitors is necessary to enhance clinical efficacy and increase the options for combination therapy for the coronavirus disease 2019. Because marine organisms have been a resource for the discovery of numerous bioactive molecules, we constructed an extract library of marine invertebrates collected from the Okinawa Islands. In this study, the extracts were used to identify antiviral molecules against SARS-CoV-2. Using a cytopathic effect (CPE) assay in VeroE6/TMPRSS2 cells, an extract from the marine sponge Theonella swinhoei was found to reduce virus-induced CPE. Eventually, onnamide A was identified as an antiviral compound in the extract using column chromatography and NMR analysis. Onnamide A inhibited several SARS-CoV-2 variant-induced CPEs in VeroE6/TMPRSS2 cells as well as virus production in the supernatant of infected cells. Moreover, this compound blocked the entry of SARS-CoV-2 pseudo-virions. Taken together, these results demonstrate that onnamide A suppresses SARS-CoV-2 infection, which may be partially related to entry inhibition, and is expected to be a candidate lead compound for the development of anti-SARS-CoV-2 drugs.

Goji Berry Juice Prevents Tumor Necrosis Factor Alpha-Induced Xerostomia in Human Salivary Gland Cells.

Sjögren's syndrome (SS) is an autoimmune disorder characterized by oral dryness that is primarily attributed to tumor necrosis factor alpha (TNF-α)-mediated reduction in saliva production. In traditional Chinese medicine, goji berries are recognized for their hydrating effect and are considered suitable to address oral dryness associated with Yin deficiency. In the present study, we used goji berry juice (GBJ) to investigate the potential preventive effect of goji berries on oral dryness caused by SS. Pretreatment of human salivary gland cells with GBJ effectively prevented the decrease in aquaporin-5 (AQP-5) mRNA and protein levels induced by TNF-α. GBJ also inhibited histone H4 deacetylation and suppressed the generation of intracellular reactive oxygen species (ROS). Furthermore, GBJ pretreatment reserved mitochondrial membrane potential and suppressed the upregulation of Bax and caspase-3, indicating that GBJ exerted an antiapoptotic effect. These findings suggest that GBJ provides protection against TNF-α in human salivary gland cells and prevents the reduction of AQP-5 expression on the cell membrane. Altogether, these results highlight the potential role of GBJ in preventing oral dryness caused by SS.

Generation of salivary glands derived from pluripotent stem cells via conditional blastocyst complementation.

Various patients suffer from dry mouth due to salivary gland dysfunction. Whole salivary gland generation and transplantation is a potential therapy to resolve this issue. However, the lineage permissible to design the entire salivary gland generation has been enigmatic. Here, we discovered Foxa2 as a lineage critical for generating a salivary gland via conditional blastocyst complementation (CBC). Foxa2 linage, but not Shh nor Pitx2, initiated to label between the boundary region of the endodermal and the ectodermal oral mucosa before primordial salivary gland formation, resulting in marking the entire salivary gland. The salivary gland was agenesis by depleting Fgfr2 under the Foxa2 lineage in the mice. We rescued this phenotype by injecting donor pluripotent stem cells into the mouse blastocysts. Those mice survived until adulthood with normal salivary glands compatible in size compared with littermate controls. These results indicated that CBC-based salivary gland generation is promising for next-generation cell-based therapy.

Conditional blastocyst complementation of a defective Foxa2 lineage efficiently promotes the generation of the whole lung.

Millions suffer from incurable lung diseases, and the donor lung shortage hampers organ transplants. Generating the whole organ in conjunction with the thymus is a significant milestone for organ transplantation because the thymus is the central organ to educate immune cells. Using lineage-tracing mice and human pluripotent stem cell (PSC)-derived lung-directed differentiation, we revealed that gastrulating Foxa2 lineage contributed to both lung mesenchyme and epithelium formation. Interestingly, Foxa2 lineage-derived cells in the lung mesenchyme progressively increased and occupied more than half of the mesenchyme niche, including endothelial cells, during lung development. Foxa2 promoter-driven, conditional Fgfr2 gene depletion caused the lung and thymus agenesis phenotype in mice. Wild-type donor mouse PSCs injected into their blastocysts rescued this phenotype by complementing the Fgfr2-defective niche in the lung epithelium and mesenchyme and thymic epithelium. Donor cell is shown to replace the entire lung epithelial and robust mesenchymal niche during lung development, efficiently complementing the nearly entire lung niche. Importantly, those mice survived until adulthood with normal lung function. These results suggest that our Foxa2 lineage-based model is unique for the progressive mobilization of donor cells into both epithelial and mesenchymal lung niches and thymus generation, which can provide critical insights into studying lung transplantation post-transplantation shortly.

Association Between eHealth Literacy and Health Education Experiences and Confidence Regarding Online Health Information Among Nurses: A Cross-sectional Study.

This cross-sectional study examined nurses' eHealth literacy, health education experiences, and confidence in health education regarding online health information and explored their association. A self-administered questionnaire was distributed among 442 nurses in Japan from September 2020 to March 2021. The survey items were the Japanese version of the eHealth Literacy Scale, health education experiences and confidence in health education regarding online health information, and sociodemographic variables. The final analysis comprised 263 responses. Nurses' mean eHealth literacy was 21.89. Most nurses had never received questions regarding online health information in search (66.9%), evaluation (85.2%), and utilization (81.0%) from their patients. Further, most nurses lacked experience (84.0%-89.7%) and confidence (94.7%-97.3%) in health education regarding online health information. The factor associated with having health education experience regarding online health information was eHealth literacy (adjusted odds ratio, 1.08; 95% confidence interval, 1.02-1.15). Factors associated with having confidence in health education regarding online health information were eHealth literacy (adjusted odds ratio, 1.10; 95% confidence interval, 1.10-1.43) and having learning experiences regarding eHealth literacy (adjusted odds ratio, 7.36; 95% confidence interval, 2.06-26.39). Our findings suggest the importance of enhancing eHealth literacy among nurses and a proactive approach by nurses to improve patients' eHealth literacy.

Relationship between perfusion index and central temperature before and after induction of anesthesia in laparoscopic gastrointestinal surgery: A prospective cohort study.

The perfusion index (PI) cutoff value before anesthesia induction and the ratio of PI variation after anesthesia induction remain unclear. This study aimed to clarify the relationship between PI and central temperature during anesthesia induction, and the potential of PI in individualized and effective control of redistribution hypothermia. This prospective observational single center study analyzed 100 gastrointestinal surgeries performed under general anesthesia from August 2021 to February 2022. The PI was measured as peripheral perfusion, and the relationship between central and peripheral temperature values was investigated. Receiver operating characteristic curve analysis was performed to identify baseline PI before anesthesia, which predicts a decrease in central temperature 30 minutes after anesthesia induction, and the rate of change in PI that predicts the decrease in central temperature 60 minutes after anesthesia induction. In cases with a central temperature decrease of ≥ 0.6°C after 30 minutes, the area under the curve was 0.744, Youden index was 0.456, and the cutoff value of baseline PI was 2.30. In cases with a central temperature decrease of ≥ 0.6°C after 60 minutes, the area under curve was 0.857, Youden index was 0.693, and the cutoff value of the PI ratio of variation after 30 minutes of anesthesia induction was 1.58. If the baseline PI is ≤ 2.30 and the PI 30 minutes after anesthesia induction is at least 1.58-fold the PI ratio of variation, there is a high probability of a central temperature decrease of at least 0.6°C within 30 minutes after 2 time points.

Towards human organ generation using interspecies blastocyst complementation: Challenges and perspectives for therapy.

Millions of people suffer from end-stage refractory diseases. The ideal treatment option for terminally ill patients is organ transplantation. However, donor organs are in absolute shortage, and sadly, most patients die while waiting for a donor organ. To date, no technology has achieved long-term sustainable patient-derived organ generation. In this regard, emerging technologies of chimeric human organ production via blastocyst complementation (BC) holds great promise. To take human organ generation via BC and transplantation to the next step, we reviewed current emerging organ generation technologies and the associated efficiency of chimera formation in human cells from the standpoint of developmental biology.

Role of Snai2 and Notch signaling in salivary gland myoepithelial cell fate.

Myoepithelial (ME) cells in exocrine glands exhibit both epithelial and mesenchymal features, contributing to fluid secretion through contraction. However, the regulation mechanism of behind this unique phenotype in salivary glands remains unclear. We established a flow cytometry-based purification method using cell surface molecules, epithelial cell adhesion molecule (EpCAM) and alpha 6 integrin (CD49f), to characterize ME cells. EpCAM+CD49fhigh cells showed relatively high expression of ME cell-marker genes, such as alpha-smooth muscle actin (α-SMA). For lineage tracing and strict isolation, tdTomato+EpCAM+CD49fhigh-ME cells were obtained from myosin heavy chain 11 (Myh11) -CreERT2/tdTomato mice. Transcriptome analysis revealed that expression of genes involved in the epithelial-mesenchymal transition, including Snai2, were upregulated in the ME cell-enriched subset. Snai2 suppression in stable ME cells decreased α-SMA and increased Krt14 expression, suggesting that ME cell features may be controlled by the epithelial-mesenchymal balance regulated by Snai2. In contrast, ME cells showed reduced ME properties and expressed the ductal markers Krt18/19 under sphere culture conditions. Notch signaling was activated under sphere culture conditions; excessive activation of Notch signaling accelerated Krt18/19 expression, but reduced α-SMA and Snai2 expression, suggesting that the behavior of Snai2-expressing ME cells may be controlled by Notch signaling.

Induction of salivary gland-like cells from epithelial tissues transdifferentiated from mouse embryonic fibroblasts.

Salivary gland hypofunction due to radiation therapy for head and neck cancer or Sjögren syndrome may cause various oral diseases, which can lead to a decline in the quality of life. Cell therapy using salivary gland stem cells is a promising method for restoring hypofunction. Herein, we show that salivary gland-like cells can be induced from epithelial tissues that were transdifferentiated from mouse embryonic fibroblasts (MEFs). We introduced four genes, Dnp63a, Tfap2a, Grhl2, and Myc (PTMG) that are known to transdifferentiate fibroblasts into oral mucosa-like epithelium in vivo into MEFs. MEFs overexpressing these genes showed epithelial cell characteristics, such as cobblestone appearance and E-cadherin positivity, and formed oral epithelial-like tissue under air-liquid interface culture conditions. The epithelial sheet detached from the culture dish was infected with adenoviruses encoding Sox9 and Foxc1, which we previously identified as essential factors to induce salivary gland formation. The cells detached from the cell sheet formed spheres 10 days after infection and showed a branching morphology. The spheres expressed genes encoding basal/myoepithelial markers, cytokeratin 5, cytokeratin 14, acinar cell marker, aquaporin 5, and the myoepithelial marker α-smooth muscle actin. The dissociated cells of these primary spheres had the ability to form secondary spheres. Taken together, our results provide a new strategy for cell therapy of salivary glands and hold implications in treating patients with dry mouth.

A Case of Mandibular Cancer Involving Almost Entire Attached Gingiva.

Here we describe a rare case of mandibular cancer involving almost the entire attached gingiva in a 71-year-old man. First, marginal resection of the entire mandible was performed, followed by one-stage reconstruction comprising application of a split-thickness skin graft onto the wound. This resulted in good alveolar ridge morphology, allowing for a mandibular prosthesis to be installed soon postoperatively. Histopathological analysis revealed a well-differentiated squamous cell carcinoma extending throughout most of the resected attached gingiva, but no malignant features in the stumps. Furthermore, no infiltration into the jawbone was observed, and no vascular or lymphatic invasion or perineural infiltration. At 3 years postoperatively, the patient's clinical course has remained uneventful, with no recurrence or problems arising in the remaining mandible. The patient is also able to eat regularly using the mandibular prosthesis provided.

Oxy-Polybrominated Diphenyl Ethers from the Indonesian Marine Sponge, Lamellodysidea herbacea: X-ray, SAR, and Computational Studies.

Polybrominated diphenyl ether (PBDE) compounds, derived from marine organisms, originate from symbiosis between marine sponges and cyanobacteria or bacteria. PBDEs have broad biological spectra; therefore, we analyzed structure and activity relationships of PBDEs to determine their potential as anticancer or antibacterial lead structures, through reactions and computational studies. Six known PBDEs (1-6) were isolated from the sponge, Lamellodysdiea herbacea; 13C NMR data for compound 6 are reported for the first time and their assignments are confirmed by their theoretical 13C NMR chemical shifts (RMSE < 4.0 ppm). Methylation and acetylation of 1 (2, 3, 4, 5-tetrabromo-6-(3', 5'-dibromo-2'-hydroxyphenoxy) phenol) at the phenol functional group gave seven molecules (7-13), of which 10, 12, and 13 were new. New crystal structures for 8 and 9 are also reported. Debromination carried out on 1 produced nine compounds (1, 2, 14, 16-18, 20, 23, and 26) of which 18 was new. Debromination product 16 showed a significant IC50 8.65 ± 1.11; 8.11 ± 1.43 µM against human embryonic kidney (HEK293T) cells. Compounds 1 and 16 exhibited antibacterial activity against Gram-positive Staphylococcus aureus and Gram-negative Klebsiella pneumoniae with MID 0.078 µg/disk. The number of four bromine atoms and two phenol functional groups are important for antibacterial activity (S. aureus and K. pneumoniae) and cytotoxicity (HEK293T). The result was supported by analysis of frontier molecular orbitals (FMOs). We also propose possible products of acetylation and debromination using analysis of FMOs and electrostatic charges and we confirm the experimental result.

Aging-associated stem/progenitor cell dysfunction in the salivary glands of mice.

Although stem cell aging leads to a decline in tissue homeostasis and regenerative capacity, it remains unclear whether salivary gland stem cell function changes during this process. However, the salivary glands are gradually replaced by connective tissue during aging. Here, we show a decline in the stem cell ability of CD133-positive stem/progenitor cells in the salivary glands of aged mice. The CD133-positive cells were isolated from young, adult, and aged mice. The number of CD133-positive cells was significantly decreased in aged mice. They also showed a lower sphere formation capacity compared to young and adult mice. RNA sequencing revealed that CD133-positive cells in aged mice exhibited lower gene expression of several aging-related genes, including FoxO3a, than those in young and adult mice. Salivary gland cells infected with a recombinant lentivirus encoding the FoxO3a gene showed a reduction in oxidative stress induced by hydrogen peroxide compared with those infected with a control virus. Thus, FoxO3a may inhibit stem cell aging via oxidative stress.

Application of regenerative medicine to salivary gland hypofunction.

Dry mouth results from hypofunction of the salivary glands due to Sjögren's syndrome (SS), various medications, and radiation therapy for head and neck cancer. In severe cases of salivary gland hypofunction, sialagogues are not always effective due to the loss of salivary parenchyma. Therefore, regenerative medicine using stem cell therapy is a promising treatment for severe cases. Stem cells are classified into three groups: tissue stem cells, embryonic stem cells, and induced pluripotent stem cells. Tissue stem cells, such as hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and salivary stem/progenitor cells, could rescue irradiation-induced salivary gland hypofunction. Both HSCs and MSCs can rescue salivary gland hypofunction through soluble factors in a paracrine manner, while salivary stem/progenitor cells can reconstitute the damaged salivary glands. In fact, we clarified that CD133-positive cells in mouse submandibular glands showed stem cell features, which reconstituted the damaged salivary glands. Furthermore, we focused on the challenge of producing functional salivary glands that are three-dimensionally induced from mouse ES cells.

Sox9 function in salivary gland development.

BACKGROUND: Organogenesis is regulated by morphogen signaling and transcription networks. These networks differ between organs, and identifying the organ-specific network is important to clarify the molecular mechanisms of development and regeneration of organs. Several studies have been conducted to identify salivary gland-specific networks using a mouse submandibular gland model. The submandibular glands (SMGs) of mice manifest as a thickening of the oral epithelium at embryonic day 11.5 and invaginate into the underlying mesenchyme. The network between Fgf10 and Sox9 is involved in SMG development in mice. HIGHLIGHT: Sox9, a member of the Sox family, is expressed in the SMG in mice from the embryonic stage to the adult stage, although the distribution changes during development. A null mutation of mouse Sox9 is lethal during the neonatal period due to respiratory failure, whereas deletion of Sox9 in the oral epithelium using the Cre/lox P system, can lead to smaller initial buds of SMGs in conditional knockout (cKO) mice than in normal mice. In addition, we showed that adenoviral transduction of Sox9 and Foxc1 genes into mouse embryonic stem cell-derived oral ectoderm could induce salivary gland rudiment in an organoid culture system. ChIP-sequencing revealed that Sox9 possibly regulates several tube- and branching-formation-related genes. CONCLUSION: Sox9 may serve as an essential transcription factor for salivary gland development. The Sox9-mediated pathway can be a promising candidate for regenerating damaged salivary glands.

Sangiangols A and B, Two New Dolabellanes from an Indonesian Marine Soft Coral, Anthelia sp.

A new, rare trinor-dolabellane diterpenoid, sangiangol A (1), and one new dolabellane diterpenoid, sangiangol B (2), together with known cembranes and dolabellanes (3-8), were isolated from the ethyl acetate layer of an extract of an Indonesian marine soft coral, Anthelia sp. Compounds 1-8 exhibited moderate cytotoxicity against an NBT-T2 cell line (0.5-10 µg/mL). The structures of the new compounds were determined by analyzing their spectra and a molecular modelling study. A possible biosynthetic pathway for sangiangols A (1) and B (2) is presented. Cytotoxicity requires two epoxide rings or a chlorine atom, as in 4 (stolonidiol) and 5 (clavinflol B).

Cystitis-Related Bladder Pain Involves ATP-Dependent HMGB1 Release from Macrophages and Its Downstream H2S/Cav3.2 Signaling in Mice.

Cystitis-related bladder pain involves RAGE activation by HMGB1, and increased Cav3.2 T-type Ca2+ channel activity by H2S, generated by upregulated cystathionine-γ-lyase (CSE) in mice treated with cyclophosphamide (CPA). We, thus, investigated possible crosstalk between the HMGB1/RAGE and CSE/H2S/Cav3.2 pathways in the bladder pain development. Bladder pain (nociceptive behavior/referred hyperalgesia) and immuno-reactive CSE expression in the bladder were determined in CPA-treated female mice. Cell signaling was analyzed in urothelial T24 and macrophage-like RAW264.7 cells. The CPA-induced bladder pain was abolished by pharmacological inhibition of T-type Ca2+ channels or CSE, and genetic deletion of Cav3.2. The CPA-induced CSE upregulation, as well as bladder pain was prevented by HMGB1 inactivation, inhibition of HMGB1 release from macrophages, antagonists of RAGE or P2X4/P2X7 receptors, and N-acetylcysteine, an antioxidant. Acrolein, a metabolite of CPA, triggered ATP release from T24 cells. Adenosine triphosphate (ATP) stimulated cell migration via P2X7/P2X4, and caused HMGB1 release via P2X7 in RAW264.7 cells, which was dependent on p38MAPK/NF-κB signaling and reactive oxygen species (ROS) accumulation. Together, our data suggest that CPA, once metabolized to acrolein, causes urothelial ATP-mediated, redox-dependent HMGB1 release from macrophages, which in turn causes RAGE-mediated CSE upregulation and subsequent H2S-targeted Cav3.2-dependent nociceptor excitation, resulting in bladder pain.

Tubulin-Based Nanotubes as Delivery Platform for Microtubule-Targeting Agents.

Tubulin-based nanotubes (TNTs) to deliver microtubule-targeting agents (MTAs) for clinical oncology are reported. Three MTAs, docetaxel (DTX), laulimalide (LMD), and monomethyl auristatin E (MMAE), which attach to different binding sites in a tubulin, are loaded onto TNTs and cause structural changes in them, including shape anisotropy and tubulin layering. This drug-driven carrier transformation leads to changes in the drug-loading efficiency and stability characteristics of the carrier. TNTs coloaded with DTX and LMD efficiently deliver dual drug cargoes to cellular tubulins by the endolysosomal pathway, and results in synergistic anticancer and antiangiogenic action of the drugs in vitro. In in vivo tests, TNTs loaded with a microtubule-destabilizing agent MMAE suppress the growth of tumors with much higher efficacy than free MMAE did. This work suggests a new concept of using a drug's target protein as a carrier. The findings demonstrate that the TNTs developed here can be used universally as a delivery platform for many MTAs.

In vitro three-dimensional culture systems of salivary glands.

Dry mouth can be caused by salivary gland hypofunction due to Sjögren's syndrome (SS) or radiation therapy for head and neck cancer, and it can also be a side effect of medications. The use of sialagogues effectively increases saliva secretion in patients with dry mouth. However, the application of sialagogues is not always satisfactory because of their side effects, such as sweating, nausea, runny nose and diarrhea. Two-dimensional (2D) cell cultures have been used not only for drug screening and discovery but also to clarify disease mechanisms. However, three-dimensional (3D) cell cultures are expected to be even more advantageous than 2D cell cultures. Therefore, we have tried to develop an in vitro cell culture system that can reconstitute 3D salivary glands. Sox9 and Foxc1 were identified as important genes that differentiate mouse embryonic stem cell-derived oral ectoderm into salivary gland placode. Using these genes and organoid culture systems, we succeeded in generating salivary gland organoids that exhibited a morphology and gene expression profile that were similar to those of the embryonic rudiment from which salivary glands arise in normal mice. These organoids are expected to be a promising tool for disease modeling, drug discovery and regenerative medicine in salivary glands.

PLAG1 enhances the stemness profiles of acinar cells in normal human salivary glands in a cell type-specific manner.

OBJECTIVES: Details of the histogenesis of salivary gland tumors are largely unknown. The oncogenic role of PLAG1 in the salivary gland has been demonstrated in vivo. Herein, we demonstrate PLAG1 roles in the acinar and ductal cells of normal human salivary glands to clarify the early events that occur during the histogenesis of salivary gland tumors. METHODS: Normal salivary gland cells with acinar and ductal phenotypes were transfected with PLAG1 plasmid DNA. Subsequently, PLAG1 overexpressed and mock cells were examined by cell proliferation, transwell migration, and salisphere formation assays. Differentiation and salivary and pluripotent stem cell marker expression levels were evaluated by quantitative reverse transcription-polymerase chain reaction and immunofluorescence. Alterations in transcriptional expressions were investigated via cap analysis of gene expression with gene-enrichment and functional annotation analysis. RESULTS: PLAG1 promoted cell proliferation and transwell migration in the acinar and ductal cells, and markedly enhanced the stemness profiles and luminal cell-like profiles in acinar cells; the stemness profiles were partially increased in the ductal cells. CONCLUSION: PLAG1 enhanced the stemness profiles in the acinar cells of normal human salivary glands in a cell type-specific manner. Thus, it may be involved in salivary gland tumorigenesis by increasing the stemness character of the normal salivary gland cells.