A single gene determines allorecognition in hydrozoan jellyfish Cladonema radiatum inbred lines.

Allorecognition-the ability of an organism to discriminate between self and nonself-is crucial to colonial marine animals to avoid invasion by other individuals in the same habitat. The cnidarian hydroid Hydractinia has long been a major research model in studying invertebrate allorecognition, establishing a rich knowledge foundation. In this study, we introduce a new cnidarian model Cladonema radiatum (C. radiatum). C. radiatum is a hydroid jellyfish which also forms polyp colonies interconnected with stolons. Allorecognition responses-fusion or regression of stolons-are observed when stolons encounter each other. By transmission electron microscopy, we observe rapid tissue remodeling contributing to gastrovascular system connection in fusion. Meanwhile, rejection responses are regulated by reconstruction of the chitinous exoskeleton perisarc, and induction of necrotic and autophagic cellular responses at cells in contact with the opponent. Genetic analysis identifies allorecognition genes: six Alr genes located on the putative allorecognition complex and four immunoglobulin superfamily genes on a separate genome region. C. radiatum allorecognition genes show notable conservation with the Hydractinia Alr family. Remarkedly, stolon encounter assays of inbred lines reveal that genotypes of Alr1 solely determine allorecognition outcomes in C. radiatum.

Exercise changes the intrahepatic immune cell profile and inhibits the progression of nonalcoholic steatohepatitis in a mouse model.

BACKGROUND: NASH is an increasingly common cause of chronic liver disease and can progress to cirrhosis and HCC. Although exercise suppresses inflammation during acute hepatitis, its impact on the progression of chronic liver disease remains unclear. Here, we investigated the effects of exercise on disease progression and intrahepatic immune cell composition in a mouse model of NASH. METHOD: Mice were assigned to 4 groups: 2 control groups (normal diet) and 2 NASH groups (western diet and low-dose carbon tetrachloride injection). One of each group remained sedentary and one was exercised on a treadmill for 12 weeks (60 min/d, 5 times/wk). All mice were then analyzed for liver histomorphology, steatosis, inflammation, and fibrosis; liver, adipose tissue, and skeletal muscle expression of genes related to metabolism and inflammation; and intrahepatic immune cell composition. RESULT: Compared with the normal diet mice, NASH mice exhibited enhanced liver steatosis, inflammation, and fibrosis; upregulated expression of liver lipogenesis-related and inflammation-related genes; and increased frequencies of intrahepatic F4/80 int CD11b hi bone marrow-derived macrophages and programmed death receptor-1 (PD-1) + CD8 + T cells. Expression of inflammatory cytokines and the frequencies of bone marrow-derived macrophages and PD-1 + CD8 + T cells correlated positively with liver steatosis, inflammation, and fibrosis. Exercise was shown to reduce NASH-induced hepatic steatosis, liver inflammation, and fibrosis; induce alterations in metabolism-related genes and inflammatory cytokines in the liver; and suppress accumulation of liver bone marrow-derived macrophages and PD-1 + CD8 + T cells. In addition, we showed that exercise induced increased expression of IL-15 in muscle and its deficiency exacerbated the pathology of NASH. CONCLUSIONS: Exercise alters the intrahepatic immune cell profile and protects against disease progression in a mouse model of NASH.

Jaw1/LRMP is associated with the maintenance of Golgi ribbon structure.

Jaw1/LRMP is a membrane protein that is localized to the endoplasmic reticulum and outer nuclear membrane. Previously, we revealed that Jaw1 functions to maintain nuclear shape by interacting with microtubules as a Klarsicht/ANC-1/Syne/homology (KASH) protein. The loss of several KASH proteins causes defects in the position and shape of the Golgi apparatus as well as the nucleus, but the effects of Jaw1 depletion on the Golgi apparatus were poorly understood. Here, we found that siRNA-mediated Jaw1 depletion causes Golgi fragmentation with disordered ribbon structure in the melanoma cell, accompanied by the change in the localization of the Golgi-derived microtubule network. Thus, we suggest that Jaw1 is a novel protein to maintain the Golgi ribbon structure, associated with the microtubule network.

Deletion of skeletal muscle Akt1/2 causes osteosarcopenia and reduces lifespan in mice.

Aging is considered to be accelerated by insulin signaling in lower organisms, but it remained unclear whether this could hold true for mammals. Here we show that mice with skeletal muscle-specific double knockout of Akt1/2, key downstream molecules of insulin signaling, serve as a model of premature sarcopenia with insulin resistance. The knockout mice exhibit a progressive reduction in skeletal muscle mass, impairment of motor function and systemic insulin sensitivity. They also show osteopenia, and reduced lifespan largely due to death from debilitation on normal chow and death from tumor on high-fat diet. These phenotypes are almost reversed by additional knocking out of Foxo1/4, but only partially by additional knocking out of Tsc2 to activate the mTOR pathway. Overall, our data suggest that, unlike in lower organisms, suppression of Akt activity in skeletal muscle of mammals associated with insulin resistance and aging could accelerate osteosarcopenia and consequently reduce lifespan.

Sphingosine-1-phosphate promotes tumor development and liver fibrosis in mouse model of congestive hepatopathy.

BACKGROUND AND AIMS: Chronic liver congestion reflecting right-sided heart failure (RHF), Budd-Chiari syndrome, or Fontan-associated liver disease (FALD) is involved in liver fibrosis and HCC. However, molecular mechanisms of fibrosis and HCC in chronic liver congestion remain poorly understood. APPROACH AND RESULTS: Here, we first demonstrated that chronic liver congestion promoted HCC and metastatic liver tumor growth using murine model of chronic liver congestion by partial inferior vena cava ligation (pIVCL). As the initial step triggering HCC promotion and fibrosis, gut-derived lipopolysaccharide (LPS) appeared to induce LSECs capillarization in mice and in vitro. LSEC capillarization was also confirmed in patients with FALD. Mitogenic factor, sphingosine-1-phosphate (S1P), was increased in congestive liver and expression of sphingosine kinase 1, a major synthetase of S1P, was increased in capillarized LSECs after pIVCL. Inhibition of S1P receptor (S1PR) 1 (Ex26) and S1PR2 (JTE013) mitigated HCC development and liver fibrosis, respectively. Antimicrobial treatment lowered portal blood LPS concentration, LSEC capillarization, and liver S1P concentration accompanied by reduction of HCC development and fibrosis in the congestive liver. CONCLUSIONS: In conclusion, chronic liver congestion promotes HCC development and liver fibrosis by S1P production from LPS-induced capillarized LSECs. Careful treatment of both RHF and liver cancer might be necessary for patients with RHF with primary or metastatic liver cancer.

The N-terminal region of Jaw1 has a role to inhibit the formation of organized smooth endoplasmic reticulum as an intrinsically disordered region.

Jaw1/LRMP is a type II integral membrane protein that is localized at the endoplasmic reticulum (ER) and outer nuclear membrane. We previously reported that a function of Jaw1 is to maintain the nuclear shape as a KASH protein via its carboxyl terminal region, a component of linker of nucleoskeleton and cytoskeleton complex in the oligomeric state. Although the oligomerization of some KASH proteins via the cytosolic regions serves to stabilize protein-protein interactions, the issue of how the oligomerization of Jaw1 is regulated is not completely understood. Therefore, we focused on three distinct regions on the cytosolic face of Jaw1: the N-terminal region, the coiled-coil domain and the stem region, in terms of oligomerization. A co-immunoprecipitation assay showed that its coiled-coil domain is a candidate for the oligomerization site. Furthermore, our data indicated that the N-terminal region prevents the aberrant oligomerization of Jaw1 as an intrinsically disordered region (IDR). Importantly, the ectopic expression of an N-terminal region deleted mutant caused the formation of organized smooth ER (OSER), structures such as nuclear karmellae and whorls, in B16F10 cells. Furthermore, this OSER interfered with the localization of the oligomer and interactors such as the type III inositol 1,4,5-triphosphate receptor (IP3R3) and SUN2. In summary, the N-terminal region of Jaw1 inhibits the formation of OSER as an IDR to maintain the homeostatic localization of interactors on the ER membrane.

Experimental observation of microplastics invading the endoderm of anthozoan polyps.

Coral reefs are being degraded worldwide by land reclamation and environmental factors, such as high seawater temperature, resulting in mass bleaching events. In addition, microplastics disturb the formation of coral-algae symbiotic relationships in primary polyps. In our experiments, we observed this effect in the bleached primary polyp Seriatopora caliendrum that lost its symbiont Symbiodiniaceae as a result of high water temperature. There was a higher incorporation of microspheres into bleached corals than in healthy ones. To understand the interference in symbiosis, we used the sea anemone Exaiptasia (as an anthozoan model organism) and fed it with microspheres. TEM results suggested the incorporation of microspheres and symbionts from the same phagocytosis zones in the mesenterial filament and endocytosis by the cells. In the tentacles, microspheres were in the same cell layer as the symbionts. These results suggest that microplastics occupy the spaces inhabited by Symbiodiniaceae, thereby hindering their symbiotic association.

Abnormal keratinization and cutaneous inflammation in Mal de Meleda.

Mal de Meleda (MDM) is a rare, autosomal recessive form of palmoplantar keratoderma due to mutations in the gene, encoding for secreted lymphocyte antigen 6/urokinase-type plasminogen activator receptor related protein 1 (SLURP1). We report a four-year-old Taiwanese MDM female case whose biopsy specimen of hyperkeratotic lesions showed abnormal keratinization and cutaneous inflammation with characteristic transmission electron microscopic (TEM) findings and immunostaining results. The patient presented with pruritic and severely hyperkeratotic plaques on the bilateral palms and soles whichwere fringed with erythematous scaly areas. A homozygous c.256 G>A mutation, predicting a conversion of p.Gly86Arg, in SLURP1gene was detected. Histopathological examinations showed marked hyperkeratosis, acanthosis and hypergranulosis in the epidermis, accompanied by perivascular lymphocytic infiltrates in the dermis. The whole layers of the epidermis and perivascular infiltrates of the dermis were stained positive with anti-tumor necrosis factor alpha (TNFα) antibody in the biopsy specimen from the sole and the ankle. TEM examination of the biopsy specimen from the plantar hyperkeratotic plaque showed various-sized vacuoles surrounding nuclei of many keratinocytes in the spinous layer. In addition, there were numerous irregular keratohyaline granules in the granular layer. Several microorganisms and many lipid-like droplets were found in the thickened cornified layer. SLURP1 protein is known as a marker of late differentiation, predominantly expressed in the granular layer, and also known to have an inhibitory effect on TNFα release. Our results exhibited excessive TNFα expression in keratinocytes and perivascular infiltrates of the dermis, and several characteristic morphological observations of keratinocytes in MDM.

Fasting-Refeeding Impacts Immune Cell Dynamics and Mucosal Immune Responses.

Nutritional status potentially influences immune responses; however, how nutritional signals regulate cellular dynamics and functionality remains obscure. Herein, we report that temporary fasting drastically reduces the number of lymphocytes by ∼50% in Peyer's patches (PPs), the inductive site of the gut immune response. Subsequent refeeding seemingly restored the number of lymphocytes, but whose cellular composition was conspicuously altered. A large portion of germinal center and IgA+ B cells were lost via apoptosis during fasting. Meanwhile, naive B cells migrated from PPs to the bone marrow during fasting and then back to PPs during refeeding when stromal cells sensed nutritional signals and upregulated CXCL13 expression to recruit naive B cells. Furthermore, temporal fasting before oral immunization with ovalbumin abolished the induction of antigen-specific IgA, failed to induce oral tolerance, and eventually exacerbated food antigen-induced diarrhea. Thus, nutritional signals are critical in maintaining gut immune homeostasis.

Jaw1/LRMP has a role in maintaining nuclear shape via interaction with SUN proteins.

Jaw1/LRMP is characterized as a Type II integral membrane protein that is localized to endoplasmic reticulum, however, its physiological functions have been poorly understood. An alignment of amino acid sequence of Jaw1 with Klarsicht/ANC-1/Syne/homology (KASH) proteins, outer nuclear membrane proteins, revealed that Jaw1 has a partial homology to the KASH domain. Here, we show that the function of Jaw1 is to maintain nuclear shape in mouse melanoma cell line. The siRNA-mediated knockdown of Jaw1 caused a severe defect in nuclear shape, and the defect was rescued by ectopic expression of siRNA-resistant Jaw1. Since co-immunoprecipitation assay indicates that Jaw1 interacts with Sad-1/UNC-84 (SUN) proteins that are inner nuclear proteins and microtubules, this study suggests that Jaw1 has a role in maintaining nuclear shape via interactions with SUN proteins and microtubules.

Clinicopathological significance of cystatin A expression in progression of esophageal squamous cell carcinoma.

We previously conducted transcriptome analysis of a paired specimen of normal and esophageal squamous cell carcinoma (ESCC) tissues and found that mRNA expression of cystatin A (CSTA), a member of the cystatin superfamily, was perturbed in tumors compared with that in the background mucosa. However, little is known about the significance of CSTA expression in ESCC.The mRNA expression of CSTA was evaluated by qRT-PCR using 28 paired frozen samples of tumor and nontumor mucosae. The protein expression of CSTA was evaluated by the immunostaining of formalin-fixed, paraffin-embedded sections of ESCC samples from 59 patients who underwent surgery, and its relationship with clinical features was analyzed.The mRNA expression of CSTA was significantly decreased in ESCC compared with that in matched normal mucosa (P < .0001). The protein expression of CSTA was limited in stratum granulosum and stratum spinosum but not in stratum basal in normal esophageal mucosa. It was reduced in all ESCC tissue samples compared with normal tissues; however, CSTA expression levels in tumors showed considerable variation. Of the 59 samples, 20 did not express CSTA, whereas 39 clearly expressed it. The expression of CSTA in tumors was significantly associated with pT classification (deeper tumor invasions) (P = .0118) and advanced TNM stages (P = .0497). In CSTA-positive tumor samples, CSTA-expressing cancer cells often expressed Ki67, a proliferation marker, which was in sharp contrast to normal mucosa, where Ki67-expressing cells were limited to the basal layer and did not express CSTA. Furthermore, CSTA expression was observed in all 22 lymph node metastases analyzed.Relatively high levels of CSTA expression in tumors were correlated with tumor progression and advanced cancer stage, including lymph node metastasis.

Disruption of the TWEAK/Fn14 pathway prevents 5-fluorouracil-induced diarrhea in mice.

AIM: To clarify the roles of TWEAK and its receptor Fn14 in 5-fluorouracil (5-FU)-induced diarrhea. METHODS: Diarrhea was induced in wild-type (WT), Fn14 knockout (KO), and IL-13 receptor (IL-13R)α1 KO BALB/c mice using a single injection of 5-FU. Histological analysis, cytokine analysis, and flow cytometry was performed on ileal tissues and cells. Murine colon carcinoma-bearing mice were co-treated with an anti-TWEAK antibody and 5-FU. Embryonic fibroblast response to cytokines was also analyzed. RESULTS: 5-FU induced high Fn14 expression in epithelial cells. The severity of 5-FU-induced diarrhea was lower in Fn14 KO mice compared with WT mice. Administration of anti-TWEAK antibody reduced 5-FU-induced diarrhea without affecting the antitumor effects of 5-FU in vivo. 5-FU-induced expression of IL-13, IL-17A, TNF-α, and IFN-γ in the ileum was Fn14 dependent. The severity of 5-FU-induced diarrhea was lower in IL-13Rα1 KO mice, indicating major role for IL-13 signaling via IL-13Rα1 in pathogenesis. We found that IL-13Rα2, an IL-13 neutralizing/cell protective receptor, was strongly induced by IL-33 in vitro and in vivo. IL-13Rα2 was upregulated in the ileum of 5-FU-treated Fn14 KO mice. Thus, the deletion of Fn14 upregulated IL-13Rα2 expression, which reduced IL-13 expression and activity. CONCLUSION: Disruption of the TWEAK/Fn14 pathway affects several interconnected pathways, including those associated with IL-13, IL-33, and IL-13Rα2, to attenuate 5-FU-induced intestinal side effects.

Fatty acids in a high-fat diet potentially induce gastric parietal-cell damage and metaplasia in mice.

BACKGROUND: Obesity is associated with risk of adenocarcinoma in the proximal stomach. We aimed to identify the links between dietary fat and gastric premalignant lesions. METHODS: C57BL/6 mice were fed high fat diet (HFD), and gastric mucosa was histologically analysed. Morphological changes were also analysed using an electron microscope. Transcriptome analysis of purified parietal cells was performed, and non-parietal gastric corpus epithelial cells were subjected to single-cell gene-expression profiling. Composition of gastric contents of HFD-fed mice was compared with that of the HFD itself. Lipotoxicity of free fatty acids (FFA) was examined in primary culture and organoid culture of mouse gastric epithelial cells in vitro, as well as in vivo, feeding FFA-rich diets. RESULTS: During ~8-20 weeks of HFD feeding, the parietal cells of the stomach displayed mitochondrial damage, and a total of 23% of the mice developed macroscopically distinct metaplastic lesions in the gastric corpus mucosa. Transcriptome analysis of parietal cells indicated that feeding HFD enhanced pathways related to cell death. Histological analysis and gene-expression profiling indicated that the lesions were similar to previously reported precancerous lesions identified as spasmolytic polypeptide-expressing metaplasia. FFAs, including linoleic acid with refluxed bile acids were detected in the stomachs of the HFD-fed mice. In vitro, FFAs impaired mitochondrial function and decreased the viability of parietal cells. In vivo, linoleic acid-rich diet, but not stearic acid-rich diet induced parietal-cell loss and metaplastic changes in mice. CONCLUSIONS: Dietary lipids induce parietal-cell damage and may lead to the development of precancerous metaplasia.

Aberrant DNA hypermethylation reduces the expression of the desmosome-related molecule periplakin in esophageal squamous cell carcinoma.

Periplakin (PPL), a member of the plakin family of proteins that localizes to desmosomes and intermediate filaments, is downregulated in human esophageal squamous cell carcinoma (ESCC). Little is known, however, about the molecular mechanism underlying the regulation of PPL expression and the contribution of PPL loss to the malignant property of the cancer is unclear. We demonstrated that PPL mRNA expression was significantly reduced in ESCC tissues compared with that in normal tissues. Therefore, we hypothesized that CpG hypermethylation is the cause of the downregulation of PPL. Bisulfite-pyrosequencing of 17 cases demonstrated that the frequency of PPL methylation was higher in ESCC tissues than in normal tissues. When human ESCC cell lines were treated with 5-aza-2'-deoxycytidine (5-aza-dC), a DNA-methyltransferase inhibitor, PPL transcription was induced. Human KYSE270 ESCC cells do not stratify under ordinary culture conditions and rarely produce desmosomes; however, the forced expression of PPL promoted cell stratification. PPL induction also promoted adhesion to extracellular matrix but delayed cell migration. The abundance of desmosome-like structures was greatly increased in PPL transfectant as determined by transmission electron microscopy. Very low expression of another desmosome protein EVPL in ESCC, even in PPL transfectant, also supported the significant role of PPL in desmosome formation and cell stratification. Our results first indicate that the downregulation of PPL mediated by DNA hypermethylation, which may play an important role in the loss of ESCC stratification and likely in metastatic phenotype.

Integrative omics analysis reveals differentially distributed proteins in dimorphic euspermatozoa of the squid, Loligo bleekeri.

In the coastal squid Loligo bleekeri, each male produces one of two types of fertilization-competent spermatozoa (eusperm) that exhibit morphological and behavioral differences. Large "consort" males produce short-tailed spermatozoa that display free-swimming behavior when ejaculated into seawater. Small "sneaker" males, on the other hand, produce long-tailed spermatozoa that exhibit a self-swarming trait after ejaculation. To understand the molecular basis for adaptive traits employed by alternative male mating tactics, we performed the transcriptome deep sequencing (RNA-seq) and proteome analyses to search for differences in testicular mRNAs and sperm proteins, respectively. From mature male testes we identified a total of 236,455 contigs (FPKM ≧1) where 3789 and 2789 were preferentially (≧10-fold) expressed in consort and sneaker testes, respectively. A proteomic analysis detected 4302 proteins in the mature sperm as post-translational products. A strongly biased (≧10-fold) distribution occurred in 55 consort proteins and 61 sneaker proteins. There was no clear mRNA-protein correlation, making a ballpark estimate impossible for not only overall protein abundance but also the degree of biased sperm type expressed in the spermatozoa. A family encoding dynein heavy chain gene, however, was found to be biased towards sneakers, whereas many enzymes involving energy metabolism were heavily biased towards consort spermatozoa. The difference in flagellar length matched exactly the different amount of tubulins. From these results we hypothesize that discrete differential traits in dimorphic eusperm arose from a series of innovative alterations in the intracellular components of spermatozoa.