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Ependymal cells have been suggested to act as neural stem cells and exert beneficial effects after spinal cord injury (SCI). However, the molecular mechanism underlying ependymal cell regulation after SCI remains unknown. To examine the possible effect of IL-17A on ependymal cell proliferation after SCI, we locally administrated IL-17A neutralizing antibody to the injured spinal cord of a contusion SCI mouse model, and revealed that IL-17A neutralization promoted ependymal cell proliferation, which was paralleled by functional recovery and axonal reorganization of both the corticospinal tract and the raphespinal tract. Further, to test whether ependymal cell-specific manipulation of IL-17A signaling is enough to affect the outcomes of SCI, we generated ependymal cell-specific conditional IL-17RA-knockout mice and analyzed their anatomical and functional response to SCI. As a result, conditional knockout of IL-17RA in ependymal cells enhanced both axonal growth and functional recovery, accompanied by an increase in mRNA expression of neurotrophic factors. Thus, Ependymal cells may enhance the regenerative process partially by secreting neurotrophic factors, and IL-17A stimulation negatively regulates this beneficial effect. Molecular manipulation of ependymal cells might be a viable strategy for improving functional recovery.

Stereocilia protrude up to 100 µm from the apical surface of vertebrate inner ear hair cells and are packed with cross-linked filamentous actin (F-actin). They function as mechanical switches to convert sound vibration into electrochemical neuronal signals transmitted to the brain. Several genes encode molecular components of stereocilia including actin monomers, actin regulatory and bundling proteins, motor proteins and the proteins of the mechanotransduction complex. A stereocilium F-actin core is a dynamic system, which is continuously being remodeled while maintaining an outwardly stable architecture under the regulation of F-actin barbed-end cappers, severing proteins and crosslinkers. The F-actin cores of stereocilia also provide a pathway for motor proteins to transport cargos including components of tip-link densities, scaffolding proteins and actin regulatory proteins. Deficiencies and mutations of stereocilia components that disturb this "dynamic equilibrium" in stereocilia can induce morphological changes and disrupt mechanotransduction causing sensorineural hearing loss, best studied in mouse and zebrafish models. Currently, at least 23 genes, associated with human syndromic and nonsyndromic hearing loss, encode proteins involved in the development and maintenance of stereocilia F-actin cores. However, it is challenging to predict how variants associated with sensorineural hearing loss segregating in families affect protein function. Here, we review the functions of several molecular components of stereocilia F-actin cores and provide new data from our experimental approach to directly evaluate the pathogenicity and functional impact of reported and novel variants of DIAPH1 in autosomal-dominant DFNA1 hearing loss using single-molecule fluorescence microscopy.

International audience; The liver is a complex organ composed of several cell types organized hierarchically. Among these, liver sinusoidal endothelial cells (LSECs) are specialized vascular cells known to interact with hepatocytes and hepatic stellate cells (HSCs), and to be involved in the regulation of important hepatic processes in healthy and pathological situations. Protocols for the differentiation of LSECs from human induced pluripotent stem cells, hiPSCs, have been proposed and in-depth analysis by transcriptomic profiling of those cells has been performed. In the present work, an extended analysis of those cells in terms of proteome and metabolome has been implemented. The proteomic analysis confirmed the expression of important endothelial markers and pathways. Among them, the expression of patterns typical of LSECs such as PECAM1, VWF, LYVE1, STAB1 (endothelial markers), CDH13, CDH5, CLDN5, ICAM1, MCAM-CD146, ICAM2, ESAM (endothelial cytoskeleton), NOSTRIN, NOS3 (Nitric Oxide endothelial ROS), ESM1, ENG, MMRN2, THBS1, ANGPT2 (angiogenesis), CD93, MRC1 (mannose receptor), CLEC14A (C-type lectin), CD40 (antigen), and ERG (transcription factor) was highlighted. Besides, the pathway analysis revealed the enrichment of the endocytosis, Toll-like receptor, Nod-like receptor, Wnt, Apelin, VEGF, cGMP-PCK, and PPAR related signaling pathways. Other important pathways such as vasopressin regulated water reabsorption, fluid shear stress, relaxin signaling, and renin secretion were also highlighted. At confluence, the metabolome profile appeared consistent with quiescent endothelial cell patterns. The integration of both proteome and metabolome datasets revealed a switch from fatty acid synthesis in undifferentiated hiPSCs to a fatty oxidation in LSECs and activation of the pentose phosphate pathway and polyamine metabolism in hiPSCs-derived LSECs. In conclusion, the comparison between the signature of LSECs differentiated following the protocol described in this work, and data found in the literature confirmed the particular relevance of these cells for future in vitro applications.

Zymbal's gland neoplasms are induced in rats through the administration of various carcinogens, but spontaneous neoplasia is rare. This report describes a spontaneous Zymbal's gland carcinoma with lung metastasis found in an aged male Fischer 344 rat. Macroscopically, the dome-like tumor nodule, approximately 30 mm in diameter with ulceration, was located near the ear canal of the rat. No healthy tissue or structure of Zymbal's gland was identified on the corresponding side, while the normal salivary glands and a lacrimal gland were observed. Histologically, a large part of the tumor mass was occupied by poorly differentiated neoplastic cells, the shapes of which were oval to polygonal or fusiform. Additionally, clusters of sebaceous-like foamy cells and squamous metaplasia with prominent keratinization were observed. Tumor cells were found to metastasize to the lung; these cells displayed histological similarities, including a sebaceous gland-like pattern, to those in the primary site. The tumor cells were immunohistochemically positive for cytokeratin AE1/AE3 or vimentin but negative for CD68, S100, α-smooth muscle actin, von Willebrand factor, and desmin. Our results indicate that the tumor was a poorly differentiated Zymbal's gland carcinoma with lung metastasis.

Oncolytic viruses (OVs) remodel the tumor microenvironment by switching a "cold" tumor into a "hot" tumor with high CD8+ T-cell infiltration. CD8+ T-cell activity plays an essential role in the antitumor efficacy of OVs. However, the activity of T cells is impaired by the programmed cell death protein-1/programmed cell death-ligand 1 (PD-1/PD-L1) interaction. To date, it remains unclear why OVs alone have a significant antitumor activity even when PD-L1 expression persists on tumor or immune cells. In this study, we found that canerpaturev (C-REV) treatment significantly suppressed tumor growth, even though it induced a significant increase in PD-L1 expression in tumors in vivo as well as persistence of high PD-L1 expression on antigen-presenting cells (macrophage and dendritic cells [DCs]). Surprisingly, we observed that C-REV treatment increased the abundance of activated CD8+ PD-1- tumor-infiltrating lymphocytes (TILs) in the tumor on both the injected and contralateral sides, although infiltration of CD8+ PD-1high TILs into the tumor was observed in the control group. Moreover, the difference in PD-1 expression was observed only in tumors after treatment with C-REV, whereas most CD8+ T cells in the spleen, tumor-draining lymph nodes and blood were PD-1-negative, and this did not change after C-REV treatment. In addition, changes in expression of T-cell immunoglobulin and mucin-domain containing-3 and T-cell immune-receptor with Ig and ITIM domains were not observed on CD8+ TILs after C-REV treatment. Taken together, our findings may reveal mechanisms that allow OVs to trigger an antitumor immune response, irrespective of a PD-L1-enriched tumor microenvironment, by recruitment of CD8+ PD-1- TILs.

The purpose of the study was to uncover the role of tenascin X in modulation of healing in mouse corneas subjected to epithelium debridement. Healing in corneas with an epithelial defect was evaluated at the levels of gene and protein expression. Wound healing-related mediators and inflammatory cell infiltration were detected by histology, immunohistochemistry and real-time RT-PCR. Tenascin X protein was upregulated in the wounded wild-type (WT) corneal epithelium. The lack of tenascin X impaired closure of an epithelial defect and accelerated infiltration of neutrophils into the wound periphery as compared to the response in WT tissue. Expression of wound healing-related proinflammatory and reparative components, i.e., interleukin-6, transforming growth factor β, matrix metalloproteinases, were unaffected by the loss of tenascin X expression. Marked accumulation of malondialdehyde (a lipid peroxidation-derived product) was observed in KO healing epithelia as compared with its WT counterpart. Neutropenia induced by systemic administration of a specific antibody rescued the impairment of epithelial healing in KO corneas, with reduction of malondialdehyde levels in the epithelial cells. Finally, we showed that a chemical scavenging reactive oxygen species reversed the impairment of attenuation of epithelial repair with a reduction of tissue levels of malondialdehyde. In conclusion, loss of tenascin X prolonged corneal epithelial wound healing and increased neutrophilic inflammatory response to debridement in mice. Tenascin X contributes to the control of neutrophil infiltration needed to support the regenerative response to injury and prevent the oxidative stress mediators from rising to cytotoxic levels., Tenascin X-deficient mice exhibit impaired epithelial closure with accelerated infiltration of neutrophils into the tissue and an increased oxidative stress, when compared to wild-type mice. The impairment of epithelial healing in tenascin X-deficient mice was rescued by targeting neutrophil circulation and reducing reactive oxygen species using a chemical scavenger.

A 69-year-old woman was admitted to our hospital because of limb weakness. She was diagnosed to have chronic renal failure due to diabetes mellitus and had suffered from pericardial effusion at 67 years of age. She started taking colchicine 18 months before admission and thereafter gradually developed muscle weakness in her limbs and had become bedridden at the time of admission. The withdrawal of colchicine improved her limb weakness, and therefore we diagnosed her to have colchicine myopathy. Her muscle strength did not completely recover even after six months from cessation of colchicine. It was suggested that renal failure and muscle disuse had prevented the full recovery of her muscles in addition to the long-term use of colchicine. Typical colchicine myopathy improves rapidly, but the long-term use of colchicine is considered to cause muscle weakness. Although the CK level was elevated, the elevated CK and myopathy had been overlooked because the CK baseline was low due to the patient's small amount of muscle mass. Moreover, the estimated GFR was recorded to be higher than her actual renal function due to her small amount of muscle mass, therefore the risk of colchicine myopathy in this case remained unrecognized.

Introduction: Bacillus Calmette-Guérin instillation is an established therapy for the treatment of carcinoma in situ of the upper urinary tract. Case Presentation: A 72-year-old woman underwent radical cystectomy with cutaneous ureterostomy for invasive bladder cancer with carcinoma in situ of both ureters. For the treatment of upper urinary tract carcinoma in situ, an infusion of 40 mg bacillus Calmette-Guérin through each ureterostomy was performed once weekly for 5 times. Urine cytology of the left upper urinary tract became negative, with a recurrence-free survival of 4 months. On the other hand, the right upper urinary tract became temporarily negative but turned positive after 3 months. Conclusion: Bacillus Calmette-Guérin perfusion therapy via cutaneous ureterostomy is considered as one of the useful therapies for carcinoma in situ of the upper urinary tract. Furthermore, there are several factors to be discussed, including the treatment duration, dosage and secondary therapeutic options.

Background White globe appearance (WGA) is a small white lesion with a globular shape identified during magnifying endoscopy with narrow-band imaging. However, the association between WGA and synchronous multiple gastric cancer (SMGC) remains unclear. Methods Consecutive patients who underwent endoscopic submucosal dissection for gastric cancer (GC) between July 2013 and April 2015 at our institution were eligible for this study. We excluded patients with a history of gastric tumor or gastrectomy. Patients who had more than 2 GCs in their postoperative pathological evaluation were classified as SMGC-positive, and patients who had at least 1 WGA-positive GC were classified as WGA-positive patients. The primary outcome was a comparison of the prevalence of WGA in patients classified as SMGC-positive and SMGC-negative. Univariate and multivariate analyses were performed using the following variables: WGA, age, sex, atrophy, and Helicobacter pylori (H. pylori) status. Results There were 26 and 181 patients classified as SMGC-positive and SMGC-negative, respectively. Univariate analysis revealed that WGA-positive classification (50% vs. 23%, P=0.008) and male sex (88% vs. 66%, P=0.02) were significant factors associated with SMGC classification, while age ≥65 years (81% vs. 81%, P>0.99), severe atrophy (46% vs. 46%, P>0.99), and H. pylori positivity (69% vs. 65%, P=0.8) were not. In the multivariate analysis, only WGA-positive classification (odds ratio 2.78, 95% confidence interval 1.16-6.67; P=0.02) was a significant independent risk factor for SMGC. Conclusions Our exploratory study showed the possibility of WGA as a predictive factor for SMGC. In cases of WGA-positive gastric cancer, careful examination might be needed to diagnose SMGC.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid generated through sphingosine kinase1 (SPK1)-mediated phosphorylation of sphingosine. We show here that injury-induced S1P upregulation increases corneal neovascularization through stimulating S1PR3, a cognate receptor. since this response was suppressed in S1PR3-knockout mice. Furthermore, Cayman10444, a selective S1PR3 inhibitor, reduced this response in WT mice. Such reductions in neovascularization were associated with reduced vascular endothelial growth factor A (VEGF-A) mRNA expression levels in WT TKE2 corneal epithelial cells and macrophages treated with CAY10444 as well as macrophages isolated from S1PR3 KO mice. S1P increased tube-like vessel formation in human vascular endothelial cells (HUVEC) and human retinal microvascular endothelial cells (HRMECs) cells expressing S1PR3. In S1PR3 KO mice, TGFβ1-induced increases in αSMA gene expression levels were suppressed relative to those in the WT counterparts. In S1PR3 deficient macrophages, VEGF-A expression levels were lower than in WT macrophages. Transforming growth factor β1(TGFβ1) upregulated SPK1 expression levels in ocular fibroblasts and TKE2 corneal epithelial cells. CAY10444 blocked S1P-induced increases in VEGF-A mRNA expression levels in TKE2 corneal epithelial cells. Endogenous S1P signaling upregulated VEGF-A and VE-cadherin mRNA expression levels in HUVEC. Unlike in TKE2 cells, SIS3 failed to block TGFβ1-induced VEGF-A upregulation in ocular fibroblasts. Taken together, these results indicate that injury-induced TGFβ1 upregulation increases S1P generation through increases in SPK1 activity. The rise in S1P formation stimulates the S1PR3-linked signaling pathway, which in turn increases VEGF-A expression levels and angiogenesis in mouse corneas., Injury-induced transforming growth factor β1 increases sphingosine 1-phosphate (S1P) generation by upregulating in sphingosine kinase 1 activity. The high levels of S1P formation stimulate the S1PR3-linked signaling pathway, which in turn increases vascular endothelial growth factor-A expression levels and angiogenesis in mouse corneas.

The studied seep carbonates from Tsushima, Japan, are embedded within marine siliciclastics of the lower Miocene Taishu Group and represent the earliest evidence of hydrocarbon seepage in the Sea of Japan. In contrast to Miocene and Pliocene examples from Honshu, which are often found above anticlines, the seeps from Tsushima formed within a pull-apart basin before major anticlines had formed. The three carbonates from Fukuzaki, Kanoura and Tanohama are composed chiefly of calcite, with significant admixture of ankerite only at Kanoura. The stable carbon isotope composition of calcites (δ13C as low as −40.2 ‰ VPDB for Fukuzaki, −41.8 ‰ VPDB for Kanoura, and −52.8 ‰ VPDB for Tanohama) indicate methanogenic origin of the carbonates. Textures of these deposits, including radiaxial and yellow cements, are indicative of formation at a methane seep. The stable oxygen isotope composition of calcites (δ18O values as low as −14.4 ‰ VPDB for Fukuzaki, −14.5 ‰ VPDB for Kanoura and −13.9 ‰ VPDB for Tanohama) indicate that they were influenced by burial fluids. Burial diagenesis is also indicated by the stable isotopic compositions of ankerite (δ13C ranging from −19.1 ‰ to −7.1 ‰ VPDB, δ18O from −11.1 ‰ to −9.7 ‰ VPDB). Molecular fossils from Tanohama comprise n-alkanes with short-chain predominance, interpreted to have formed due to thermal cracking of organic matter. The carbonates yield a chemosynthesis-based community comprising vesicomyids Pleurophopsis chitanii, P. cf. hamuroi, the bathymodiolin ‘Bathymodiolus’ akanudaensis, the lucinid Lucinoma sp. and the provannid Provanna? sp., which have never been hitherto identified. ‘Bathymodiolus’ akanudaensis, Lucinoma sp. and Provanna? sp. are the oldest records of these taxa in the Sea of Japan.

Oncostatin M (OSM), a member of the IL-6 family of cytokines, has important roles in renal diseases. The relationship between OSM and kidney stone disease, however, remains unclear. To investigate the roles of OSM in the development of kidney stone disease, we generated a mouse model of renal crystal formation using OSM receptor β (OSMRβ)-deficient mice (OSMRβ−/− mice). There were fewer renal crystal deposits in OSMRβ−/− mice than in wild-type (WT) mice. Crystal-binding molecules (osteopontin, annexin A1, and annexin A2), inflammatory cytokines (TNF-α and IL-1β), and fibrosis markers (TGF-β, collagen 1a2, and α-smooth muscle actin) were also decreased in the kidneys of OSMRβ−/− mice compared with those in WT mice. Immunofluorescence staining showed that OSMRβ was expressed in renal tubular epithelial cells (RTECs) and renal fibroblasts in the model of renal crystal formation. In the cultured RTECs and renal fibroblasts, OSM directly induced the expression of crystal-binding molecules and fibrosis markers. Expressions of inflammatory cytokines were increased by stimulation with OSM in cultured renal fibroblasts. OSM may promote the formation of renal crystal deposits by directly acting on RTECs and renal fibroblasts to produce crystal-binding molecules and inflammatory cytokines.

The gut microbiome has garnered attention as an effective target to boost immunity and improve cancer immunotherapy. We found that B cell-defective (BCD) mice, such as µ-membrane targeted deletion (µMT) and activation-induced cytidine deaminase (AID) knockouts (KOs), have elevated antitumor immunity under specific pathogen-free but not germ-free conditions. Microbial dysbiosis in these BCD mice enriched the type I IFN (IFN) signature in mucosal CD8+ T cells, resulting in up-regulation of the type I IFN-inducible protein stem cell antigen-1 (Sca-1). Among CD8+ T cells, naive cells predominantly circulate from the gut to the periphery, and those that had migrated from the mesenteric lymph nodes (mLNs) to the periphery had significantly higher expression of Sca-1. The gut-educated Sca-1+ naive subset is endowed with enhanced mitochondrial activity and antitumor effector potential. The heterogeneity and functional versatility of the systemic naive CD8+ T cell compartment was revealed by single-cell analysis and functional assays of CD8+ T cell subpopulations. These results indicate one of the potential mechanisms through which microbial dysbiosis regulates antitumor immunity.

AimsAlthough frameshift variants in the microsatellite area of shugoshin 1 (SGO1) have been reported in the context of microsatellite instability-high (MSI-H)/deficient mismatch repair gastrointestinal cancer, most have been evaluated only in early stage I–III patients, and only two of its five microsatellite regions have been evaluated. Therefore, we investigated the frequency and MSI status of microsatellite frameshift variants in gastric cancer cases, including stage IV.MethodsIn a total of 55 cases, 30 gastric cancer resection and 25 non-resection cases, DNA was extracted from both tumour and normal parts and PCR was performed. The variant was confirmed by TA cloning, and MSI was evaluated using GeneMapper software.ResultsA frameshift variant of c.973delA was observed in 16 of the 45 evaluable cases. Its frequency was 35.6%. Of the 25 cases that could be assessed for MSI status, two cases of MSI-H were associated with the c.973delA SGO1 variant. However, c.973delA SGO1 variant was also observed in four cases of microsatellite stable.ConclusionOur study shows that SGO1 frameshift variants are not always associated with MSI status.

Highlights • 4-MeMBI, 5-MeMBI and MBI inhibit CYP3A2 activity in cultured rat hepatocytes. • 4-MeMBI and 5-MeMBI, not MBI, induce CYP1A1/2 activity in cultured rat hepatocytes. • Effects of the chemicals on CYP3A2 suggest metabolic drug-drug interaction potential. • 4-MeMBI and 5-MeMBI induce CYPs 3A4 and 1A1/2 activity in cultured human hepatocytes. • Primary cultured hepatocytes are useful in CYP experiments of benzimidazole compounds., Effects of 4-methyl-2-mercaptobenzimidazole (4-MeMBI) and 5-methyl-2- mercaptobenzimidazole (5-MeMBI) on cytochrome P450 (CYP) activity were examined in primary cultured rat hepatocytes. Hepatocytes from male Wistar rats were cultured in the presence of 4-MeMBI or 5-MeMBI (0−400 μM), and the activity of CYPs 3A2/4 (48 and 96 h) and 1A1/2 (48 h) was determined by measuring the activity of testosterone 6β-hydroxylation and 7-ethoxyresorufin O-deethylation, respectively. As a result, 4-MeMBI and 5-MeMBI (≥12.5 μM) inhibited CYP3A2 activity. On the other hand, 4-MeMBI (≥25 μM) and 5-MeMBI (≥100 μM) induced CYP1A1/2 activity, being consistent with the previous in vivo results. In a comparative metabolism study using primary cultured human hepatocytes from two Caucasian donors, 4-MeMBI and 5-MeMBI induced the activity of CYPs 3A4 and 1A1/2 with individual variability. It was concluded from these results that 4-MeMBI, 5-MeMBI and MBI caused inhibition of CYP3A2 activity in primary cultured rat hepatocytes, suggesting their potential for metabolic drug-drug interactions. Primary cultured rat and human hepatocytes were considered to be useful for the evaluation of effects of the benzimidazole compounds on their inducibility and inhibitory activities of cytochrome P450 forms.

Human ACTG1 mutations are associated with high-frequency hearing loss, and patients with mutations in this gene are good candidates for electric acoustic stimulation. To better understand the genetic etiology of hearing loss cases, massively parallel DNA sequencing was performed on 7,048 unrelated Japanese hearing loss probands. Among 1,336 autosomal dominant hearing loss patients, we identified 15 probands (1.1%) with 13 potentially pathogenic ACTG1 variants. Six variants were novel and seven were previously reported. We collected and analyzed the detailed clinical features of these patients. The average progression rate of hearing deterioration in pure-tone average for four frequencies was 1.7 dB/year from 0 to 50 years age, and all individuals over 60 years of age had severe hearing loss. To better understand the underlying disease-causing mechanism, intracellular localization of wild-type and mutant gamma-actins were examined using the NIH/3T3 fibroblast cell line. ACTG1 mutants p.I34M p.M82I, p.K118M and p.I165V formed small aggregates while p.R37H, p.G48R, p.E241K and p.H275Y mutant gamma-actins were distributed in a similar manner to the WT. From these results, we believe that some part of the pathogenesis of ACTG1 mutations may be driven by the inability of defective gamma-actin to be polymerized into F-actin.

Laminin is a family of basement membrane proteins, whose selective and spatiotemporal expression profiles are linked to their various functions in development, maintenance, and functional regulation of different tissues. In the liver, α1-and α5-containing laminin isoforms have been documented to be critically involved in the developmental process of the epithelial tissue of the bile duct. However, possible roles of other laminin isoforms in bile duct formation and function remain elusive. Here, we evaluated public single-cell RNA sequencing databases on human liver cells to reveal expression landscape of laminin genes, and found that genes for laminin-332 subunits were conjointly expressed in the EPCAM+ biliary epithelial cell population. Expression of the β3 and γ2 subunit genes was restricted to biliary epithelial cells in the liver and, remarkably, showed apparent heterogeneity among them. We confirmed the heterogeneous nature of the laminin-β3 expression in murine livers, which was firmly related to morphological substructures in the biliary epithelium. Finally, we generated the liver epithelial tissue-specific laminin- β3 knockout mice and found that this laminin subunit was dispensable under physiological conditions. Together, our present findings have identified the β3 subunit and the related laminin-332 isoform as useful markers and potentially important regulatory molecules for future understanding of pathophysiology in the hepatobiliary system.

PURPOSE: We investigated the effect of systemic fasudil hydrochloride and an inhibitor of nuclear translocation of myocardin-related transcription factor-A (MRTF-A) on capsular contraction in a puncture-injured lens in mice. MATERIALS AND METHODS: Lens injury of an anterior capsular break was achieved in male adult C57Bl/6 mice under general and topical anesthesia at 1 h after systemic fasudil hydrochloride (intraperitoneal, 10 mg/kg body weight) or vehicle administration. The mice were allowed to heal after instillation of ofloxacin ointment, for 5 and 10 days with daily administration of fasudil hydrochloride or vehicle. In another series of experiment, we examined the effect of systemic administration of an MRTF-A inhibitor (CCG-203971, 100 mg/kg twice a day) on fibrogenic reaction and tissue contraction in an injured lens on day 5 or 10. The eye was processed for histology and immunohistochemistry for SM22, proliferating cell nuclear antigen (PCNA), or MRTF-A. In hematoxylin and eosin - stained samples, the distance between each edge of the break of the anterior capsule was measured and statistically analyzed. RESULTS: A cluster of lens cell accumulation was formed adjacent to the edge of the capsular break on day 5. It contained cells labeled for SM22 and PCNA. The size of the cell cluster was larger in fasudil group of mice than in control mice on day 5. Systemic fasudil or CCG-203971 suppressed an excess contraction of the capsular break at certain time points. CONCLUSION: Systemic administration of fasudil hydrochloride could be a treatment strategy of postoperative capsular contraction following cataract-intraocular lens surgery.

Group 2 innate lymphoid cells (ILC2s) are type 2 cytokine-producing cells that have important roles in helminth infection and allergic inflammation. ILC2s are tissue-resident cells, and their phenotypes and roles are regulated by tissue-specific environmental factors. While the role of ILC2s in the lung, intestine and bone marrow has been elucidated in many studies, their role in adipose tissues is still unclear. Here, we report on the role of ILC2-derived bone morphogenetic protein 7 (BMP7) in adipocyte differentiation and lipid accumulation. Co-culture of fat-derived ILC2s with pluripotent mesenchymal C3H10T1/2 cells and committed white preadipocyte 3T3-L1 cells resulted in their differentiation to adipocytes and induced lipid accumulation. Co-culture experiments using BMP7-deficient ILC2s revealed that BMP7, produced by ILC2s, induces differentiation into brown adipocytes. Our results demonstrate that BMP7, produced by ILC2s, affects adipocyte differentiation, particularly in brown adipocytes.

Two new oleanane-type triterpenoids with an epoxy bridge between C-3 and C-25, named lantacamaric acids A and B, were isolated from the leaves and stems of Lantana camara L. (Verbenaceae). In addition, seven known triterpenoids, two known iridoid glycosides, four known phenylethanoid glycosides, one known flavonoid glycoside, and one known cyanogenic glycoside were isolated. Their structures were determined based on the spectroscopic data. Furthermore, the cytotoxic activities of the isolated triterpenoids toward HL-60 cells were examined. The IC50 values of all compounds were obtained and ranged from 1.16 to 68.4 μM, with three compounds exhibiting stronger activities than the positive control, cisplatin. In addition, the structure-activity relationship was investigated.

Background: CXCR5+ T follicular helper (TFH) cells primarily promote B cells to produce an antigen-specific antibody through germinal centers (GCs). TFH cells exist in circulation, and circulating(c) TFH2 cells, a subset of cTFH cells, are able to help naïve B cells produce IgE in healthy individuals. Conversely, IL-10-producing regulatory B (Breg) cells inhibit an accelerated immune response. Methods: We investigated the roles of cTFH cells and cBreg cells based on a TH2 response in patients with atopic asthma (AA). Thirty-two patients with AA and 35 healthy volunteers (HV) were enrolled. We examined cTFH cells including their subsets, their expression of ICOS and PD-1, and cBreg cells by flow cytometry and their associations with clinical biomarkers. Plasma levels of CXCL13, which is a counterpart of CXCR5, were also measured using ELISA. Results: In patients with AA, cTFH2 cells were increased and cTFH1 cells were decreased compared with those in HV. The expression levels of ICOS on cTFH and their subset cells were elevated and Breg cells were greatly decreased. The plasma levels of CXCL13 in patients with AA were significantly elevated and correlated well with the cTFH2/cBreg ratio. These cells were examined in 10 patients AA before and after inhaled corticosteroid (ICS) treatment. Interestingly, the percentages and numbers of TFH2 and ICOS+ cTFH cells declined after ICS treatment together with improvements in symptoms and clinical biomarkers. Conclusions: The percentages and numbers of cTFH2 and ICOS+ cTFH cells might be useful as biomarkers of TH2 typed airway inflammation in patients with AA. Keywords: CXCL13, ICS, IgE, Regulatory B cell, T follicular helper cell

Liver Sinusoidal Endothelial Cells (LSECs) are an important component of the liver as they compose the microvasculature which allows the supply of oxygen, blood, and nutrients. However, maintenance of these cells in vitro remains challenging as they tend to rapidly lose some of their characteristics such as fenestration or as their immortalized counterparts present poor characteristics. In this work, human induced pluripotent stem cells (hiPSCs) have been differentiated toward an LSEC phenotype. After differentiation, the RNA quantification allowed demonstration of high expression of specific vascular markers (CD31, CD144, and STAB2). Immunostaining performed on the cells was found to be positive for both Stabilin-1 and Stabilin-2. Whole transcriptome analysis performed with the nanoCAGE method further confirmed the overall vascular commitment of the cells. The gene expression profile revealed the upregulation of the APLN, LYVE1, VWF, ESAM and ANGPT2 genes while VEGFA appeared to be downregulated. Analysis of promoter motif activities highlighted several transcription factors (TFs) of interest in LSECs (IRF2, ERG, MEIS2, SPI1, IRF7, WRNIP1, HIC2, NFIX_NFIB, BATF, and PATZ1). Based on this investigation, we compiled the regulatory network involving the relevant TFs, their target genes as well as their related signaling pathways. The proposed hiPSC-derived LSEC model and its regulatory network were then confirmed by comparing the experimental data to primary human LSEC reference datasets. Thus, the presented model appears as a promising tool to generate more complex in vitro liver multi-cellular tissues.

SummaryThe Amazonas was one of the most heavily affected Brazilian states by the COVID-19 epidemic. Despite a large number of infected people, particularly during the second wave associated with the spread of the Variant of Concern (VOC) Gamma (lineage P.1), SARS-CoV-2 continues to circulate in the Amazonas. To understand how SARS-CoV-2 persisted in a human population with a high immunity barrier, we generated 1,188 SARS-CoV-2 whole-genome sequences from individuals diagnosed in the Amazonas state from 1st January to 6th July 2021, of which 38 were vaccine breakthrough infections. Our study reveals a sharp increase in the relative prevalence of Gamma plus (P.1+) variants, designated as Pango Lineages P.1.3 to P.1.6, harboring two types of additional Spike changes: deletions in the N-terminal (NTD) domain (particularly Δ144 or Δ141-144) associated with resistance to anti-NTD neutralizing antibodies or mutations at the S1/S2 junction (N679K or P681H) that probably enhance the binding affinity to the furin cleavage site, as suggested by our molecular dynamics simulations. As lineages P.1.4 (S:N679K) and P.1.6 (S:P681H) expanded (Re > 1) from March to July 2021, the lineage P.1 declined (Re < 1) and the median Ct value of SARS-CoV-2 positive cases in Amazonas significantly decreases. Still, we found no overrepresentation of P.1+ variants among breakthrough cases of fully vaccinated patients (71%) in comparison to unvaccinated individuals (93%). This evidence supports that the ongoing endemic transmission of SARS-CoV-2 in the Amazonas is driven by the spread of new local Gamma/P.1 sub-lineages that are more transmissible, although not more efficient to evade vaccine-elicited immunity than the parental VOC. Finally, as SARS-CoV-2 continues to spread in human populations with a declining density of susceptible hosts, the risk of selecting new variants with higher infectivity are expected to increase.

Vaccines against severe acute respiratory syndrome coronavirus-2 have been introduced. To investigate the relationship between vaccine-induced humoral immunity and patient age, we measured antibody levels and neutralization in vaccinated sera. Sera from 13 to 17 days after the second dose of the BNT162b2 vaccine were collected from health care workers at the University of Toyama (n = 740). Antibody levels were measured by the anti-receptor binding domain antibody test (anti-RBD test), and neutralization against wild-type (WT), α- and β-variant pseudotyped viruses were assayed using a high-throughput chemiluminescent reduction neutralizing test (htCRNT; positivity cutoff, 50% neutralization at serum dilution 1:100). Basic clinical characteristics were obtained from questionnaires. Antibodies were confirmed in all participants in both the anti-RBD test (median, 2,112 U/ml; interquartile range [IQR], 1,275 to 3,390 U/ml) and the htCRNT against WT (median % inhibition, >99.9; IQR, >99.9 to >99.9). For randomly selected sera (n = 61), 100.0% had positive htCRNT values against the α- and β-derived variants. Among those who answered the questionnaire (n = 237), the values of the anti-RBD test were negatively correlated with age in females (P < 0.01). An age-dependent decline in neutralization was observed against the variants but not against the wild-type virus (wild type, P = 0.09; α, P < 0.01; β, P < 0.01). The neutralizing activity induced by BNT162b2 was obtained not only against the wild-type virus, but also against the variants; however, there was an age-dependent decrease in the latter. Age-related heterogeneity of vaccine-acquired immunity is a concern in preventive strategies in the era dominated by variants. IMPORTANCE Since mRNA vaccines utilize wild-type SARS-CoV-2 spike protein as an antigen, there are potential concerns about acquiring immunity to variants of this virus. The neutralizing activity in BNT162b2-vaccinated individuals was higher against the wild-type virus than against its variants; this effect was more apparent in older age groups. This finding suggests that one of the weaknesses of the mRNA vaccine is the high risk of variant infection in the elderly population. Because the elderly are at a higher risk of SARS-CoV-2 infection, the age-dependent decline of neutralization against viral variants should be considered while planning vaccination programs that include boosters.

Serological tests are beneficial for recognizing the immune response against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). To identify protective immunity, optimization of the chemiluminescent reduction neutralizing test (CRNT) is critical. Whether commercial antibody tests have comparable accuracy is unknown. Serum samples were obtained from COVID-19 patients (n = 74), SARS-CoV-2 PCR-negative (n = 179), and suspected healthy individuals (n = 229) before SARS-CoV-2 variants had been detected locally. The convalescent phase was defined as the period after day 10 from disease onset or the episode of close contact. The CRNT using pseudotyped viruses displaying the wild-type (WT) spike protein and a commercial anti-receptor-binding domain (RBD) antibody test were assayed. Serology for the B.1.1.7 and B.1.351 variants was also assayed. Both tests concurred for symptomatic COVID-19 patients in the convalescent phase. They clearly differentiated between patients and suspected healthy individuals (sensitivity: 95.8% and 100%, respectively; specificity: 99.1% and 100%, respectively). Anti-RBD antibody test results correlated with neutralizing titers (r = 0.31, 95% confidence interval [CI] 0.22–0.38). Compared with the WT, lower CRNT values were observed for the variants. Of the samples with ≥100 U/mL by the anti-RBD antibody test, 77.8% and 88.9% showed ≥50% neutralization against the B.1.1.7 and the B.1.351 variants, respectively. Exceeding 100 U/mL in the anti-RBD antibody test was associated with neutralization of variants (P < 0.01). The CRNT and commercial anti-RBD antibody test effectively classified convalescent COVID-19 patients. Strong positive results with the anti-RBD antibody test can reflect neutralizing activity against emerging variants. IMPORTANCE This study provides a diagnostic evidence of test validity, which can lead to vaccine efficacy and proof of recovery after COVID-19. It is not easy to know neutralization against SARS-CoV-2 in the clinical laboratory because of technical and biohazard issues. The correlation of the quantitative anti-receptor-binding domain antibody test, which is widely available, with neutralizing test indicates that we can know indirectly the state of acquisition of functional immunity against wild and variant-type viruses in the clinical laboratory.

The COVID-19 epidemic in Brazil experienced two major country-wide lineage replacements, the first driven by the lineage P.2, formerly classified as variant of interest (VOI) Zeta in late 2020 and the second by the variant of concern (VOC) Gamma in early 2021. To better understand how these SARS-CoV-2 lineage turnovers occurred in Brazil, we analyzed 11,724 high-quality SARS-CoV-2 whole genomes of samples collected in different country regions between September 2020 and April 2021. Our findings indicate that the spatial dispersion of both variants in Brazil was driven by short and long-distance viral transmission. The lineage P.2 harboring Spike mutation E484K probably emerged around late July 2020 in the Rio de Janeiro (RJ) state, which contributed with most (∼50%) inter-state viral disseminations, and only became locally established in most Brazilian states by October 2020. The VOC Gamma probably arose in November 2020 in the Amazonas (AM) state, which was responsible for 60-70% of the inter-state viral dissemination, and the earliest timing of community transmission of this VOC in many Brazilian states was already traced to December 2020. We estimate that variant Gamma was 1.56-3.06 more transmissible than variant P.2 co-circulating in RJ and that the median effective reproductive number (Re) of Gamma in RJ and SP states (Re = 1.59-1.91) was lower than in AM (Re = 3.55). In summary, although the epicenter of the lineage P.2 dissemination in Brazil was the heavily interconnected Southeastern region, it displayed a slower rate of spatial spread than the VOC Gamma originated in the more isolated Northern Brazilian region. Our findings also support that the VOC Gamma was more transmissible than lineage P.2, although the viral Re of the VOC varied according to the geographic context.

The SARS-CoV-2 has infected almost 200 million people worldwide by July 2021 and the pandemic has been characterized by infection waves of viral lineages showing distinct fitness profiles. The simultaneous infection of a single individual by two distinct SARS-CoV-2 lineages provides a window of opportunity for viral recombination and the emergence of new lineages with differential phenotype. Several hundred SARS-CoV-2 lineages are currently well characterized but two main factors have precluded major coinfection/codetection analysis thus far: i) the low diversity of SARS-CoV-2 lineages during the first year of the pandemic which limited the identification of lineage defining mutations necessary to distinguish coinfecting viral lineages; and the ii) limited availability of raw sequencing data where abundance and distribution of intrasample/intrahost variability can be accessed. Here, we have put together a large sequencing dataset from Brazilian samples covering a period of 18 May 2020 to 30 April 2021 and probed it for unexpected patterns of high intrasample/intrahost variability. It enabled us to detect nine cases of SARS-CoV-2 coinfection with well characterized lineage-defining mutations. In addition, we matched these SARS-CoV-2 coinfections with spatio-temporal epidemiological data confirming their plausibility with the co-circulating lineages at the timeframe investigated. These coinfections represent around 0.61% of all samples investigated. Although our data suggests that coinfection with distinct SARS-CoV-2 lineages is a rare phenomenon, it is likely an underestimation and coinfection rates warrants further investigation.DATA SUMMARYThe raw fastq data of codetection cases are deposited on gisaid.org and correlated to gisaid codes: EPI_ISL_1068258, EPI_ISL_2491769, EPI_ISL_2491781, EPI_ISL_2645599, EPI_ISL_2661789, EPI_ISL_2661931, EPI_ISL_2677092, EPI_ISL_2777552, EPI_ISL_3869215. Supplementary data are available on https://doi.org/10.6084/m9.figshare.16570602.v1. The workflow code used in this study is publicly available on: https://github.com/dezordi/IAM_SARSCOV2.

Adaptive immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) dynamics remain largely unknown. The neutralizing antibody (NAb) levels in patients with coronavirus disease 2019 (COVID-19) are helpful for understanding the pathology. Using SARS-CoV-2 pseudotyped virus, serum sample neutralization values in symptomatic COVID-19 patients were measured using the chemiluminescence reduction neutralization test (CRNT). At least two sequential serum samples collected during hospitalization were analyzed to assess NAbs neutralizing activity dynamics at different time points. Of the 11 patients, four (36.4%), six (54.5%), and one (9.1%) had moderate, severe, and critical disease, respectively. Fifty percent neutralization (N50%-CRNT) was observed upon admission in 90.9% (10/11); all patients acquired neutralizing activity 2–12 days after onset. In patients with moderate disease, neutralization was observed at earliest within two days after symptom onset. In patients with severe-to-critical disease, neutralization activity increased, plateauing 9–16 days after onset. Neutralization activity on admission was significantly higher in patients with moderate disease than in patients with severe-to-critical disease (relative % of infectivity, 6.4% vs. 41.1%; P = .011). Neutralization activity on admission inversely correlated with disease severity. The rapid NAb response may play a crucial role in preventing the progression of COVID-19.

Number of liver organoids have been reported, though it is not clearly shown whether the functional connection between hepatocytes and cholangiocytes is recapitulated in those organoids. Here, we report generation of a hepatobiliary tubular organoid (HBTO) using mouse hepatocyte progenitors called small hepatocytes (SHs) and cholangiocytes. SHs differentiate and form the bile canalicular network in HBTOs and secret metabolites into the canaliculi, which are then transported into the biliary structure. Hepatocytes in the organoid acquire and maintain metabolic functions including albumin secretion and cytochrome P450 activities, over the long term. We provide the step-by-step protocol for induction of HBTO including isolation of cholangiocytes and SHs and co-culture of these two types of cell to generate functional connections between hepatocytes and cholangiocytes.

Niemann−Pick disease type C (NPC) is a rare neurodegenerative disorder caused by a recessive mutation in the NPC1 or NPC2 gene, in which patients exhibit lysosomal accumulation of unesterified cholesterol and glycolipids. Most of the research on NPC has been done in patient-derived skin fibroblasts or mouse models. Therefore, we developed NPC patient neurons derived from induced pluripotent stem cells (iPSCs) to investigate the neuropathological cause of the disease. Although an accumulation of cholesterol and glycolipids, which is characteristic of NPC, was observed in both undifferentiated iPSCs and derived neural stem cells (NSCs), we could not observed the abnormalities in differentiation potential and autophagic activity in such immature cells. However, definite neuropathological features were detected in mature neuronal cells generated from NPC patient-derived iPSCs. Abnormal accumulation of cholesterol and other lipids identified by lipid droplets and number of enlarged lysosomes was more prominent in mature neuronal cells rather than in iPSCs and/or NSCs. Thin-sectioning electron microscopic analysis also demonstrated numerous typical membranous cytoplasmic bodies in mature neuronal cells. Furthermore, TUJ1-positive neurite density was significantly reduced in NPC patient-derived neuronal cells. In addition, disruption of the p62/SQSTM1−KEAP1−NRF2 axis occurred in neurons differentiated from NPC patient-derived iPSCs. These data indicate the impairment of neuronal network formation associated with neurodegeneration in mature neuronal cells derived from patients with NPC., Highlights • Niemann−Pick disease type C patient-derived neurons showed pathological features • Lipid droplets and lysosomes accumulated at high levels in patient's cells • Patient-derived neurons showed defective neuronal network formation • Disruption of the p62/SQSTM1−KEAP1−NRF2 axis occurred in patient-derived neurons

DNA aneuploidy, the altered DNA content of a cell, is a common feature of canine tumors. However, it is unclear whether aneuploid DNA in canine tumor cells show centrosome amplification (CA), which contributes to numerical and structural chromosome aberrations that result in DNA aneuploidy. Here, we evaluated whether DNA aneuploidy and CA occur concurrently in canine tumor cell lines. Centrosome numbers were evaluated in 18 canine tumor cell lines by immunocytochemistry with anti-γ-tubulin antibody, and DNA content was evaluated by flow cytometry using propidium iodide. A total of 15 cell lines showed DNA aneuploidy, and CA was observed in 5 of these 15 cell lines. Together, our results suggest that DNA aneuploidy in canine tumor cells might be explained at least in part by CA. In addition, cell lines with CA may be useful tools to examine the detailed relationship between CA and DNA aneuploidy and the molecular mechanism of CA in canine tumor.

Su(var)3–9, Enhancer-of-zeste, and Trithorax (SET) domain-containing protein 8 (SET8) is the sole enzyme that monomethylates Lys-20 of histone H4 (H4K20). SET8 has been implicated in the regulation of multiple biological processes, such as gene transcription, the cell cycle, and senescence. SET8 quickly undergoes ubiquitination and degradation by several E3 ubiquitin ligases; however, the enzyme that deubiquitinates SET8 has not yet been identified. Here we demonstrated that ubiquitin-specific peptidase 17–like family member (USP17) deubiquitinates and therefore stabilizes the SET8 protein. We observed that USP17 interacts with SET8 and removes polyubiquitin chains from SET8. USP17 knockdown not only decreased SET8 protein levels and H4K20 monomethylation but also increased the levels of the cyclin-dependent kinase inhibitor p21. As a consequence, USP17 knockdown suppressed cell proliferation. We noted that USP17 was down-regulated in replicative senescence and that USP17 inhibition alone was sufficient to trigger cellular senescence. These results reveal a regulatory mechanism whereby USP17 prevents cellular senescence by removing ubiquitin marks from and stabilizing SET8 and transcriptionally repressing p21.

Pointed gourd (TrichosanthesdioicaRoxb.) is a dioecious, cucurbit summer vegetable, and green fruit is the main edible part at 15-18 days after pollination. However, consumer preference goes to fruit without seed since seeds are unpalatable to have after cooking due to their hard seed coat. To overcome this problem by parthenocarpy induction, six types of plant growth regulators [2,4-dichlorophenoxyacetic acid (2,4-D); naphthaleneacetic acid (NAA); N-(2-chloro-4 pyridyl)-N-phenylurea (CPPU); forchlorophenuron (Fulmet); gibberellic acid (GA3) and 2,3,5-triiodobenzoic acid (TIBA) with four concentrations (25, 50, 100 and 200ppm) were sprayed to the unpollinated ovaries of three different female accessions of pointed gourd at anthesis. Results revealed successful parthenocarpy induction by plant growth regulators in all the studied accessions while progressive variation observed in fruit length, diameter and weight according to the differences of plant growth regulators concentrations. The length, diameter and weight of parthenocarpic fruit induced by GA3at 200ppm, CPPU and TIBA at 100ppm in three studied accessions of pointed gourd were comparable with those of their respective hand-pollinated fruits. In most of the parthenocarpic fruits, seeds were found as empty at ripening stage apart from NAA and GA3at 200ppm treated fruits had few abnormal seeds. The results confirmed the useful technique of producing seedless fruits in pointed gourd through the application of GA3at 200ppm, CPPU and TIBA at 100ppm regardless of accessions.

In diagnosing onychomycosis, diseases with similar features must be excluded by demonstrating the presence of fungal infection and identifying the fungal species. However, fungal culture of onychomycosis-derived samples usually takes many weeks to yield species identification results, and is associated with a low successful culture rate. Loop-mediated isothermal amplification (LAMP) is a highly sensitive and specific molecular biological method that can amplify DNA at a constant temperature, allowing for a simpler testing procedure, shorter detection time and less cost than conventional techniques including quantitative polymerase chain reaction. We have developed a new LAMP method specifically to detect Trichophyton rubrum (T. rubrum) and Trichophyton interdigitale (T. interdigitale), major causative dermatophytes for onychomycosis, and analyzed the correlation between LAMP results and those of the existing fungal culture method for the detection and identification of Trichophyton species from onychomycosis-derived samples. The results showed that all 59 specimens in which T. rubrum or T. interdigitale was identified by fungal culture also tested positive by LAMP, giving a 100% positivity concordance rate between the two methods. Moreover, all 55 and four specimens in which T. rubrum and T. interdigitale were identified by fungal culture, respectively, also tested positive for each species by LAMP, again giving a 100% species-identification concordance rate. The high correlation demonstrated between LAMP and fungal culture results in detection and identification of Trichophyton species from onychomycosis-derived samples suggests high reliability of LAMP as a promising, alternative mycological detection and identification technique which can serve as an alternative to the fungal culture method.

Backgrounds/Aims: Vonoprazan (VPZ) is the first clinically available potassium competitive acid blocker. This class of agents provides faster and more potent acid inhibition than proton pump inhibitors. Most strains of Helicobacter pylori are sensitive to amoxicillin. We hypothesized that dual therapy with VPZ and amoxicillin would provide the sufficient eradication rate for H. pylori infection. To evaluate this, we compared the eradication rate by the dual VPZ/amoxicillin therapy with that by the standard triple VPZ/amoxicillin/clarithromycin therapy. Methods: Non-inferiority of the eradication rate of H. pylori by the dual therapy with VPZ 20 mg twice daily (bid) and amoxicillin 500 mg 3 times daily (tid) for 1 week to that by the triple therapy with VPZ 20 mg bid, amoxicillin 750 mg bid and clarithromycin 200 mg bid for 1 week was retrospectively studied. Propensity score matching was performed to improve comparability between 2 regimen groups. Successful eradication was diagnosed using the [13C]-urea breath test at 1–2 months after the end of eradication therapy. Results: The intention-to-treat analysis demonstrated that the eradication rate by the dual therapy (92.9%; 95% CI 82.7–98.0%, 52/56) was not inferior to that of the triple therapy (91.9%; 95% CI 80.4–97.0%, 51/56; OR 1.275, 95% CI 0.324–5.017%, p = 0.728). There were no statistically significant differences in incidences of adverse events between 2 regimens. Conclusion: VPZ-based dual therapy (VPZ 20 mg bid and amoxicillin 500 mg tid for 1 week) provides an acceptable eradication rate of H. pylori infection without the need for second antimicrobial agents, such as clarithromycin.

Objectives MCL1 is an anti-apoptotic BCL2 family member that is highly expressed in various malignant tumors. However, little is known about the role of MCL1 in KRAS-mutant lung adenocarcinomas. In this study, we aimed to clarify whether MCL1 could be a therapeutic target in KRAS-mutant lung adenocarcinomas for which no effective molecular targeted drugs are available. Materials and methods We examined to what extent MCL1 knockdown either alone or in combination with MEK inhibitor trametinib suppressed growth or induced apoptosis in the KRAS-mutant lung adenocarcinoma cell line H441 and EGFR-mutant lung adenocarcinoma cell line H1975. Furthermore, we investigated the therapeutic effects of dual inhibition of MCL1 and Bcl-xL, another anti-apoptotic BCL2 family member, in these two cell lines. Results MCL1 knockdown alone did not induce apoptosis in H441 or H1975 cells. However, MCL1-depleted H441 and H1975 cells underwent apoptosis and decreased in number in the presence of trametinib. We also confirmed that combined therapy by MCL1 knockdown and trametinib almost completely suppressed the growth of H441 cells in vivo. Moreover, dual knockdown of MCL1 and Bcl-xL induced extensive apoptosis in H441 and H1975 cells. Conclusion These findings suggest that combined treatments of MCL1 knockdown and trametinib or dual inhibition of MCL1 and Bcl-xL would be effective therapies for lung adenocarcinomas including the KRAS-mutant subtype.

Summary: Zygotic genome activation (ZGA) begins after fertilization and is essential for establishing pluripotency and genome stability. However, it is unclear how ZGA genes prevent mitotic errors. Here we show that knockout of the ZGA gene Zscan5b, which encodes a SCAN domain with C2H2 zinc fingers, causes a high incidence of chromosomal abnormalities in embryonic stem cells (ESCs), and leads to the development of early-stage cancers. After irradiation, Zscan5b-deficient ESCs displayed significantly increased levels of γ-H2AX despite increased expression of the DNA repair genes Rad51l3 and Bard. Re-expression of Zscan5b reduced γ-H2AX content, implying a role for Zscan5b in DNA damage repair processes. A co-immunoprecipitation analysis showed that Zscan5b bound to the linker histone H1, suggesting that Zscan5b may protect chromosomal architecture. Our report demonstrates that the ZGA gene Zscan5b is involved in genomic integrity and acts to promote DNA damage repair and regulate chromatin dynamics during mitosis. : In this article, Yamada and colleagues show that Zscan5b deficiency increases DNA stress, compromises chromosomal structure during mitosis, and leads to the development of early-stage cancers. Zscan5b deficiency may offer a murine model of human chromosomal breakage syndromes. Keywords: Zscan5b, genome integrity, mitosis, postreplicative DNA damage repair, embryonic stem cell, knockout mouse, chromosome instability syndrome, mosaicism

Lysosomes are an essential organ for cellular metabolism and play an important role in autophagy. We examined the association between methylation and autophagy in a severely affected female patient with Fabry disease, which is caused by mutation of the GLA gene on the X chromosome, and her two sisters, who had few symptoms. We confirmed autophagic flux by LC3 turnover assay using fibroblasts from each sister. In the severe female patient, autophagic flux showed abnormal while her two sisters with few symptoms had normal autophagic flux, revealing the direct relationship between symptoms and autophagic flux. Furthermore, we observed the levels of p62, which is a substrate for autophagy, and lysosome morphology. In the severe patient of this family, lysosomes were enlarged and p62 was accumulated. The methylated allele of the GLA gene in the severe patient had a high proportion of wild alleles; conversely, the sisters' methylated allele had a high proportion of mutant alleles. Therefore, we examined the mRNA expression level of the mutant allele by allele-specific PCR. It was high in the severe patient and low in the siblings with few symptoms. That is, the correlation between the mRNA expression level of the mutant allele and disease severity was confirmed. We showed a correlation between severe symptoms, dysfunction of autophagy and methylation of wild alleles in Fabry disease. It was suggested that allele-specific PCR may lead to a diagnosis and help to determine the prognosis of female patients with Fabry disease.

This paper demonstrates the effects of terrigenous sediment load reduction by watershed managements on coastal turbidity in Banate Bay, Iloilo located in central Philippines, using field observations and numerical simulations. Measurements of the total suspended solid and particulate organic carbon indicated that the bulk component of the coastal turbidity comprised terrigenous mineral particles rather than phytoplankton at the rise of the river after heavy rain. The suspended sediment concentration and underwater light intensity were simulated by an atmosphere-watershed-coastal ocean model to investigate the contribution of the terrigenous sediment load to the coastal turbidity in rainy season. The coastal sediment simulation indicated that the turbidity in Banate Bay is highly impacted by terrigenous sediment inputs from distant watersheds, which are transported to the bay by coastal currents. In contrast, the contributions of sediment loads from the adjacent watersheds to the bay turbidity were limited. The simulation also indicated that the majority of the bay is not inhabitable for seagrasses due to limited light availability caused by the high turbidity. Scenario analysis of the sediment load reduction demonstrated that significant reduction of turbidity and improvement of light penetration are conditionally expected only when the remediation is implemented with cooperative management of a series of neighboring watersheds because of the significant contributions of sediment loads from multiple basins.

In this study, rats were fed a high-fat diet containing calcium alginate (Ca-Alg) for 5 weeks to examine the effects of Ca-Alg on lipid metabolism including triglyceride (TG) levels in the blood. We also investigated the mechanism of the TG-reducing effect of Alg in vitro. Rats were randomized into 5 groups: high-fat diet group (14% (w/w) lard, HF); three Ca-Alg-containing diet groups (2.5, 5 or 10% (w/w) Ca-Alg) and a resistant maltodextrin (RMD) diet group as a positive control (with 5% (w/w) RMD). The 10% Ca-Alg group showed a significant reduction of body weight increase from the 7th day. In addition, the increase of TG in blood was significantly suppressed, and the amount of TG excreted in feces was increased. Increase of body fat mass was in the order HF > RMD > Ca-Alg 2.5% > Ca-Alg 5% > Ca-Alg 10%, while the total weight of the extracted fat tissues was significantly reduced in the RMD, 5% and 10% Ca-Alg groups. Hepatic pathology showed clear circular vacuoles apparently representing TG accumulation in the HF group, while fewer vacuoles were seen in the Ca-Alg groups. The results of in vitro experiments indicated that Ca-Alg does not directly inhibit lipase activity, but may suppress absorption of TG by forming non-absorbable macromolecular micelles containing TG. These results suggest that Ca-Alg promotes excretion and suppresses absorption of TG, leading to reduced blood TG levels, and decreased hepatic and total body accumulation of fat. The findings should be helpful for designing future clinical trials.

In order to understand the pathobiology of neurotrophic keratopathy, we established a mouse model by coagulating the first branch of the trigeminal nerve (V1 nerve). In our model, the sensory nerve in the central cornea disappeared and remaining fibers were sparse in the peripheral limbal region. Impaired corneal epithelial healing in the mouse model was associated with suppression of both cell proliferation and expression of stem cell markers in peripheral/limbal epithelium as well as a reduction of transient receptor potential vanilloid 4 (TRPV4) expression in tissue. TRPV4 gene knockout also suppressed epithelial repair in mouse cornea, although it did not seem to directly modulate migration of epithelium. In a co-culture experiment, TRPV4-introduced KO trigeminal ganglion upregulated nerve growth factor (NGF) in cultured corneal epithelial cells, but ganglion with a control vector did not. TRPV4 gene introduction into a damaged V1 nerve rescues the impairment of epithelial healing in association with partial recovery of the stem/progenitor cell markers and upregulation of cell proliferation and of NGF expression in the peripheral/limbal epithelium. Gene transfer of TRPV4 did not accelerate the regeneration of nerve fibers. Sensory nerve TRPV4 is critical to maintain stemness of peripheral/limbal basal cells, and is one of the major mechanisms of homeostasis maintenance of corneal epithelium.