Inner ear development in cyclostomes and evolution of the vertebrate semicircular canals.

Jawed vertebrates have inner ears with three semicircular canals, the presence of which has been used as a key to understanding evolutionary relationships. Ostracoderms, the jawless stem gnathostomes, had only two canals and lacked the lateral canal1-3. Lampreys, which are modern cyclostomes, are generally thought to possess two semicircular canals whereas the hagfishes-which are also cyclostomes-have only a single canal, which used to be regarded as a more primitive trait1,4. However, recent molecular and developmental analyses have strongly supported the monophyly of cyclostomes5-7, which has left the evolutionary trajectory of the vertebrate inner ear unclear8. Here we show the differentiation of the otic vesicle of the lamprey Lethenteron camtschaticum and inshore hagfish Eptatretus burgeri. This is the first time, to our knowledge, that the development of the hagfish inner ear is reported. We found that canal development in the lamprey starts with two depressions-which is reminiscent of the early developmental pattern of the inner ear in modern gnathostomes. These cyclostome otic vesicles show a pattern of expression of regulatory genes, including OTX genes, that is comparable to that of gnathosomes. Although two depressions appear in the lamprey vesicle, they subsequently fuse to form a single canal that is similar to that of hagfishes. Complete separation of the depressions results in anterior and posterior canals in gnathostomes. The single depression of the vesicle in hagfishes thus appears to be a secondarily derived trait. Furthermore, the lateral canal in crown gnathostomes was acquired secondarily-not by de novo acquisition of an OTX expression domain, but by the evolution of a developmental program downstream of the OTX genes.

Development of branchial ionocytes in embryonic and larval stages of cloudy catshark, Scyliorhinus torazame.

In teleost fish, branchial ionocytes are important sites for osmoregulation and acid-base regulation by maintaining ionic balance in the body fluid. During the early developmental stages before the formation of the gills, teleost ionocytes are localized in the yolk-sac membrane and body skin. By comparing with teleost fish, much less is known about ionocytes in developing embryos of elasmobranch fish. The present study investigated the development of ionocytes in the embryo and larva of cloudy catshark, Scyliorhinus torazame. We first observed ionocyte distribution by immunohistochemical staining with anti-Na+/K+-ATPase (NKA) and anti-vacuolar-type H+-ATPase (V-ATPase) antibodies. The NKA- and V-ATPase-rich ionocytes appeared as single cells in the gill filaments from stage 31, the stage of pre-hatching, while the ionocytes on the body skin and yolk-sac membrane were also observed. From stage 32, in addition to single ionocytes on the gill filaments, some outstanding follicular structures of NKA-immunoreactive cells were developed to fill the inter-filament region of the gill septa. The follicular ionocytes possess NKA in the basolateral membrane and Na+/H+ exchanger 3 in the apical membrane, indicating that they are involved in acid-base regulation like single NKA-rich ionocytes. Three-dimensional analysis and whole-mount immunohistochemistry revealed that the distribution of follicular ionocytes was limited to the rostral side of gill septum. The rostral sides of gill septum might be exposed to faster water flow than caudal side because the gills of sharks gently curved backward. This dissymmetric distribution of follicular ionocytes is considered to facilitate efficient body-fluid homeostasis of catshark embryo.

Molecular basis of the unique osmoregulatory strategy in the inshore hagfish, Eptatretus burgeri.

Hagfishes are characterized by omo- and iono-conforming nature similar to marine invertebrates. Conventionally, hagfishes had been recognized as the most primitive living vertebrate that retains plesiomorphic features. However, some of the "ancestral" features of hagfishes, such as rudimentary eyes and the lack of vertebrae, have been proven to be deceptive. Similarly, by the principle of maximum parsimony, the unique body fluid regulatory strategy of hagfishes seems to be apomorphic, since the lamprey, another cyclostome, adopts osmo- and iono-regulatory mechanisms as in jawed vertebrates. Although hagfishes are unequivocally important in discussing the origin and evolution of the vertebrate osmoregulatory system, the molecular basis for the body fluid homeostasis in hagfishes has been poorly understood. In the present study, we explored this matter in the inshore hagfish, Eptatretus burgeri, by analyzing the transcriptomes obtained from the gill, kidney, and muscle of the animals acclimated to distinct environmental salinities. Together with the measurement of parameters in the muscular fluid compartment, our data indicate that the hagfish possesses an ability to conduct free amino acid (FAA)-based osmoregulation at a cellular level, which is in coordination with the renal and branchial FAA absorption. We also revealed that the hagfish does possess the orthologs of the known osmoregulatory genes and that the transepithelial movement of inorganic ions in the hagfish gill and kidney is more complex than previously thought. These observations pose a challenge to the conventional view that the physiological features of hagfishes have been inherited from the last common ancestor of the extant vertebrates.

Functional characterization of follicle-stimulating hormone and luteinizing hormone receptors in cloudy catshark, Scyliorhinus torazame.

The follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in cloudy catshark were cloned, and recombinant FSHR and LHR were expressed for characterization. Ventral lobe extract (VLE) from the pituitary contains homologous FSH and LH, and it stimulated the cAMP signaling of FSHR and LHR dose-dependently. Two transcript variants of LHR (LHR-L with exon 10 and LHR-S without) were identified, and LHR-S was the dominant form with higher basal cAMP activity without VLE stimulation. Among various developmental stages of follicles, FSHR expression was mainly associated with the pre-vitellogenic and early white follicles. When follicles were recruited into vitellogenesis, the expression of FSHR decreased while of LHR was upregulated reciprocally, suggesting that LHR may also be responsible for the control of vitellogenesis in chondrichthyans. The expression of LHR-L was upregulated among maturing follicles before ovulation, indicating LHR-L could have a specific role in receiving the LH surge signal for final maturation. Plasma LH-like activity was transiently increased prior to the progesterone (P4)-surge and testosterone-drop at the beginning of P4-phase, supporting a pituitary control of follicle-maturation via LH signaling in chondrichthyans. The expression of follicular LHR was downregulated during the P4-phase when LH-like activity was high, indicating that the LH-dependent downregulation of LHR is conserved in chondrichthyans as it is in other vertebrate lineages. (213 words).

Segment-Dependent Gene Expression Profiling of the Cartilaginous Fish Nephron Using Laser Microdissection for Functional Characterization of Nephron at Segment Levels.

For adaptation to a high salinity marine environment, cartilaginous fishes have evolved a ureosmotic strategy. They have a highly elaborate "four-loop nephron" in the kidney, which is considered to be important for reabsorption of urea from the glomerular filtrate to maintain a high concentration of urea in the body. However, the function and regulation, generally, of the "four-loop nephron" are still largely unknown due to the complicated configuration of the nephron and its many subdivided segments. Laser microdissection (LMD) followed by RNA-sequencing (RNA-seq) analysis is a powerful technique to obtain segment-dependent gene expression profiles. In the present study, using the kidney of cloudy catshark, Scyliorhinus torazame, we tested several formaldehyde-free and formaldehyde-based fixatives to optimize the fixation methods. Fixation by 1% neutral buffered formalin for 15 min resulted in sufficient RNA and structural integrities, which allowed LMD clipping of specific nephron segments and subsequent RNA-seq analysis. RNA-seq from the LMD samples of the second-loop, the fourth-loop, and the five tubular segments in the bundle zone revealed a number of specific membrane transporter genes that can characterize each segment. Among them, we examined expressions of the Na + -coupled cotransporters abundantly expressed in the second loop samples. Although the proximal II segment of the second loop is known for the elimination of excess solutes, the present results imply that the PII segment is also crucial for reabsorption of valuable solutes. Looking ahead to future studies, the segment-dependent gene expression profiling will be a powerful technique for unraveling the renal mechanisms and regulation in euryhaline elasmobranchs.

Phylogenetic and functional properties of hagfish neurohypophysial hormone receptors distinct from their jawed vertebrate counterparts.

Vertebrate neurohypophysial hormones, i.e., vasopressin- and oxytocin-family peptides, exert versatile physiological actions via distinct G protein-coupled receptors. The neurohypophysial hormone receptor (NHR) family was classically categorized into four subtypes (V1aR, V1bR, V2R and OTR), while recent studies have identified seven subtypes (V1aR, V1bR, V2aR, V2bR, V2cR, V2dR and OTR; V2aR corresponds to the conventional V2R). The vertebrate NHR family were diversified via multiple gene duplication events at different scales. Despite intensive research effort in non-osteichthyes vertebrates such as cartilaginous fish and lamprey, the molecular phylogeny of the NHR family has not been fully understood. In the present study, we focused on the inshore hagfish (Eptatretus burgeri), another group of cyclostomes, and Arctic lamprey (Lethenteron camtschaticum) for comparison. Two putative NHR homologs, which were previously identified only in silico, were cloned from the hagfish and designated as ebV1R and ebV2R. In vitro, ebV1R, as well as two out of five Arctic lamprey NHRs, increased intracellular Ca2+ in response to exogenous neurohypophysial hormones. None of the examined cyclostome NHRs altered intracellular cAMP levels. Transcripts of ebV1R were detected in multiple tissues including the brain and gill, with intense hybridization signals in the hypothalamus and adenohypophysis, while ebV2R was predominantly expressed in the systemic heart. Similarly, Arctic lamprey NHRs showed distinct expression patterns, underscoring the multifunctionality of VT in the cyclostomes as in the gnathostomes. These results and exhaustive gene synteny comparisons provide new insights into the molecular and functional evolution of the neurohypophysial hormone system in vertebrates.

Thyroid and endostyle development in cyclostomes provides new insights into the evolutionary history of vertebrates.

BACKGROUND: The endostyle is an epithelial exocrine gland found in non-vertebrate chordates (amphioxi and tunicates) and the larvae of modern lampreys. It is generally considered to be an evolutionary precursor of the thyroid gland of vertebrates. Transformation of the endostyle into the thyroid gland during the metamorphosis of lampreys is thus deemed to be a recapitulation of a past event in vertebrate evolution. In 1906, Stockard reported that the thyroid gland in hagfish, the sister cyclostome group of lampreys, develops through an endostyle-like primordium, strongly supporting the plesiomorphy of the lamprey endostyle. However, the findings in hagfish thyroid development were solely based on this single study, and these have not been confirmed by modern molecular, genetic, and morphological data pertaining to hagfish thyroid development over the last century. RESULTS: Here, we showed that the thyroid gland of hagfish undergoes direct development from the ventrorostral pharyngeal endoderm, where the previously described endostyle-like primordium was not found. The developmental pattern of the hagfish thyroid, including histological features and regulatory gene expression profiles, closely resembles that found in modern jawed vertebrates (gnathostomes). Meanwhile, as opposed to gnathostomes but similar to non-vertebrate chordates, lamprey and hagfish share a broad expression domain of Nkx2-1/2-4, a key regulatory gene, in the pharyngeal epithelium during early developmental stages. CONCLUSIONS: Based on the direct development of the thyroid gland both in hagfish and gnathostomes, and the shared expression profile of thyroid-related transcription factors in the cyclostomes, we challenge the plesiomorphic status of the lamprey endostyle and propose an alternative hypothesis where the lamprey endostyle could be obtained secondarily in crown lampreys.

Evidence from cyclostomes for complex regionalization of the ancestral vertebrate brain.

The vertebrate brain is highly complex, but its evolutionary origin remains elusive. Because of the absence of certain developmental domains generally marked by the expression of regulatory genes, the embryonic brain of the lamprey, a jawless vertebrate, had been regarded as representing a less complex, ancestral state of the vertebrate brain. Specifically, the absence of a Hedgehog- and Nkx2.1-positive domain in the lamprey subpallium was thought to be similar to mouse mutants in which the suppression of Nkx2-1 leads to a loss of the medial ganglionic eminence. Here we show that the brain of the inshore hagfish (Eptatretus burgeri), another cyclostome group, develops domains equivalent to the medial ganglionic eminence and rhombic lip, resembling the gnathostome brain. Moreover, further investigation of lamprey larvae revealed that these domains are also present, ruling out the possibility of convergent evolution between hagfish and gnathostomes. Thus, brain regionalization as seen in crown gnathostomes is not an evolutionary innovation of this group, but dates back to the latest vertebrate ancestor before the divergence of cyclostomes and gnathostomes more than 500 million years ago.

Long-term monitoring of egg-laying cycle using ultrasonography reveals the reproductive dynamics of circulating sex steroids in an oviparous catshark, Scyliorhinus torazame.

The many diverse reproductive strategies of elasmobranchs (sharks, skates and rays) from lecithotrophic oviparity to matrotrophic viviparity have attracted significant research attention. However, the endocrine control of elasmobranch reproduction is less well-documented largely due to their reproductive characteristics, such as a long reproductive cycle, and/or repeated internal fertilization using stored sperm in oviparous species. In the present study, for the first time, we succeeded in non-invasive monitoring of the continuing egg-laying cycle of the cloudy catshark Scyliorhinus torazame using portable ultrasound devices. Furthermore, long-term simultaneous monitoring of the egg-laying cycle and measurement of plasma sex steroids revealed cycling patterns of estradiol-17ß (E2), testosterone (T) and progesterone (P4). In particular, a decline in T followed by a reciprocal surge in plasma P4 were consistently observed prior to the appearance of the capsulated eggs, implying that P4 is likely associated with the ovulation and/or egg-case formation. While the cycling pattern of E2 was not as apparent as those of T and P4, threshold levels of E2 (>5 ng/mL) and T (>1 ng/mL) appeared to be crucial in the continuation of egg-laying cycle. The possibility to trace the dynamics of plasma sex steroids in a single individual throughout the reproductive cycles makes the catshark a useful model for regulatory and mechanistic studies of elasmobranch reproduction.

Morphological and functional development of the spiral intestine in cloudy catshark (Scyliorhinus torazame).

Cartilaginous fish have a comparatively short intestine known as the spiral intestine that consists of a helical spiral of intestinal mucosa. However, morphological and functional development of the spiral intestine has not been fully described. Unlike teleosts, cartilaginous fish are characterized by an extremely long developmental period in ovo or in utero; for example, in the oviparous cloudy catshark (Scyliorhinus torazame), the developing fish remains inside the egg capsule for up to 6 months, suggesting that the embryonic intestine may become functional prior to hatching. In the present study, we describe the morphological and functional development of the spiral intestine in the developing catshark embryo. Spiral formation of embryonic intestine was completed at the middle of stage 31, prior to 'pre-hatching', which is a developmental event characterized by the opening of the egg case at the end of the first third of development. Within 48 h of the pre-hatching event, egg yolk began to flow from the external yolk sac into the embryonic intestine via the yolk stalk. At the same time, there was a rapid increase in mRNA expression of the peptide transporter pept1 and neutral amino acid transporter slc6a19 Secondary folds in the intestinal mucosa and microvilli on the apical membrane appeared after pre-hatching, further supporting the onset of nutrient absorption in the developing intestine at this time. We demonstrate the acquisition of intestinal nutrient absorption at the pre-hatching stage of an oviparous elasmobranch.

Facilitated NaCl Uptake in the Highly Developed Bundle of the Nephron in Japanese Red Stingray Hemitrygon akajei Revealed by Comparative Anatomy and Molecular Mapping.

Batoidea (rays and skates) is a monophyletic subgroup of elasmobranchs that diverged from the common ancestor with Selachii (sharks) about 270 Mya. A larger number of batoids can adapt to low-salinity environments, in contrast to sharks, which are mostly stenohaline marine species. Among osmoregulatory organs of elasmobranchs, the kidney is known to be dedicated to urea retention in ureosmotic cartilaginous fishes. However, we know little regarding urea reabsorbing mechanisms in the kidney of batoids. Here, we performed physiological and histological investigations on the nephrons in the red stingray (Hemitrygon akajei) and two shark species. We found that the urine/plasma ratios of salt and urea concentrations in the stingray are significantly lower than those in cloudy catshark (Scyliorhinus torazame) under natural seawater, indicating that the kidney of stingray more strongly reabsorbs these osmolytes. By comparing the three-dimensional images of nephrons between stingray and banded houndshark (Triakis scyllium), we showed that the tubular bundle of stingray has a more compact configuration. In the compact tubular bundle of stingray kidney, the distal diluting tubule was highly developed and frequently coiled around the proximal and collecting tubules. Furthermore, co-expression of NKAα1 (Na+/K +-ATPase) and NKCC2 (Na+- K+-2Cl- cotransporter 2) mRNAs was prominent in the coiled diluting segment. These findings imply that NaCl reabsorption is greatly facilitated in the stingray kidney, resulting in a higher reabsorption rate of urea. Lowering the loss of osmolytes in the glomerular filtrate is likely favorable to the adaptability of batoids to a wide range of environmental salinity.

Comprehensive analysis of genes contributing to euryhalinity in the bull shark, Carcharhinus leucas; Na+-Cl- co-transporter is one of the key renal factors upregulated in acclimation to low-salinity environment.

Most cartilaginous fishes live principally in seawater (SW) environments, but a limited number of species including the bull shark, Carcharhinus leucas, inhabit both SW and freshwater (FW) environments during their life cycle. Euryhaline elasmobranchs maintain high internal urea and ion levels even in FW environments, but little is known about the osmoregulatory mechanisms that enable them to maintain internal homeostasis in hypoosmotic environments. In the present study, we focused on the kidney because this is the only organ that can excrete excess water from the body in a hypoosmotic environment. We conducted a transfer experiment of bull sharks from SW to FW and performed differential gene expression analysis between the two conditions using RNA-sequencing. A search for genes upregulated in the FW-acclimated bull shark kidney indicated that the expression of the Na+-Cl- cotransporter (NCC; Slc12a3) was 10 times higher in the FW-acclimated sharks compared with that in SW sharks. In the kidney, apically located NCC was observed in the late distal tubule and in the anterior half of the collecting tubule, where basolateral Na+/K+-ATPase was also expressed, implying that these segments contribute to NaCl reabsorption from the filtrate for diluting the urine. This expression pattern was not observed in the houndshark, Triakis scyllium, which had been transferred to 30% SW; this species cannot survive in FW environments. The salinity transfer experiment combined with a comprehensive gene screening approach demonstrates that NCC is a key renal protein that contributes to the remarkable euryhaline ability of the bull shark.

Prospective histological evaluation of a 20G core trap with a forward-cutting bevel needle for EUS-FNA of pancreatic lesions.

BACKGROUND: Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) has been established as a method to obtain tissues of various organs. To obtain sufficient tissue has clinical impact to facilitate the diagnosis by clinical pathologists, the assessment and subtyping of various neoplasms, and for further immunohistochemical investigations of tumor type. Recently, a novel 20G core trap with a forward-cutting beveled FNA needle (ProC-F) has become available. The aim of this prospective study was to evaluate the feasibility and diagnostic yield of EUS-FNA for pancreatic lesions using this needle. PATIENTS AND METHOD: In this study, the first puncture was performed using the ProC-F. Only tissue obtained with the first puncture using the ProC-F was used to evaluate diagnostic yield of ProC-F. The second puncture was performed using a 22G standard FNA needle using the same technique as for the first puncture. Second puncture was performed if the endosonographer did not feel that sufficient tissue had not been obtained by first puncture. RESULTS: Fifty-three consecutive patients who underwent EUS-FNA for pancreatic lesions were prospectively enrolled. The technical success rate of EUS-FNA using the ProC-F was 98.1% (52/53). The rate of adequate tissue obtained by ProC-F was 96.2% (50/52). On the other hand, the rate of adequate tissue obtained by the standard needle was 71.1%. The sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), and accuracy of the ProC-F and the standard needle were 92.5, 100, 100, 76.9, and 94.0%, and 85.2, 100, 100, 55.6, and 87.5%, respectively. Diagnostic yield of ProC-F about sensitivity (P = 0.027), NPV (P = 0.035), and accuracy (P = 0.004) was significantly higher than of standard needle. Adverse events were not seen in any patients. CONCLUSIONS: Although only tissue obtained by the first puncture was evaluated, the rate of adequate tissue and the histologic diagnostic yield for pancreatic lesions were extremely high using the ProC-F.

Early detection of nonperitoneal recurrence may contribute to survival benefit after curative gastrectomy for gastric cancer.

BACKGROUND: There is no evidence that strict follow-up using cross-sectional imaging after curative gastrectomy benefits survival; however, nonperitoneal recurrence detected early might be treated with additional surgery. The present study examined whether early detection of recurrence by imaging modalities could increase survival, particularly in patients with nonperitoneal recurrence. METHODS: We retrospectively analyzed 218 patients with recurrent gastric cancer after curative gastrectomy performed from 2002 to 2014. The patients were divided into an asymptomatic group (n = 117) and a symptomatic group (n = 101), according to the presence of symptoms at the time of recurrence, to compare clinicopathological characteristics and long-term survival. RESULTS: Peritoneal recurrence was less frequent in the asymptomatic group (22.2%) than in the symptomatic group (62.4%), the median time to recurrence was shorter (12.7 months vs 18.9 months; P < 0.001), and the median survival time after recurrence was longer (18.7 months vs 7.5 months; P < 0.001). In the asymptomatic group, 10 of 117 patients (8.5%) received additional curative surgery after recurrence. Median overall survival after gastrectomy was not significantly different between the groups (30.1 months for the asymptomatic group vs 30.0 months for the symptomatic group; P = 0.132); however, it was significantly longer among asymptomatic patients with nonperitoneal recurrence compared with symptomatic patients (35.9 months vs 24.0 months; P = 0.039). CONCLUSIONS: The presence of symptoms at recurrence did not affect survival in patients with recurrent gastric cancer. However, detection of nonperitoneal recurrence before the appearance of symptoms may provide survival benefit. Therefore, regular follow-up, including use of imaging modalities, is recommended.

Prospective clinical study of endoscopic ultrasound-guided biliary drainage using novel balloon catheter (with video).

BACKGROUND AND AIM: On endoscopic ultrasound (EUS)-guided biliary drainage, fistula dilation may be accompanied by leakage of bile juice, potentially along with bacteria or air, so this procedure should be kept as simple and short as possible. To date, various techniques to dilate the fistula have been reported. This prospective, single-center, single-arm study evaluated the technical feasibility and safety of EUS-guided biliary drainage using novel balloon catheters. METHODS: Tip of novel balloon catheters is only 3 Fr and tapered, and the catheters also show favorable push ability. First, biliary tract was punctured using 19-G fine needle aspiration needle. Next, we immediately inserted the novel balloon catheter, and the biliary tract and intestinal wall were dilated. Then, we performed metallic stent placement without any dilation fistula. RESULTS: A total of 20 patients were prospectively enrolled in this study. Technical success rate was 100%. Eleven patients underwent EUS-guided choledochoduodenostomy, and nine patients underwent EUS-guided hepaticogastrostomy. Median procedure time was only 11 min (range, 8-16 min) for EUS-guided choledochoduodenostomy and 14 min (range, 11-18 min) for EUS-guided hepaticogastrostomy. Adverse events were seen in 15% (3/20; self-limited abdominal pain n = 2, peritonitis n = 1). CONCLUSIONS: Although additional cases and a randomized controlled comparison with another dilation technique such as the graded dilation or cystotome dilation technique are needed, our technique may be helpful for EUS-guided biliary drainage.

Clinical utility of endoscopic ultrasound-guided biliary drainage as a rescue of re-intervention procedure for high-grade hilar stricture.

BACKGROUND AND AIM: Re-intervention after stent placement for malignant hepatic hilum obstruction (HBO) is challenging. endoscopic ultrasound-guided biliary drainage (EUS-BD) has been developed as an alternative method for failed endoscopic retrograde cholangiopancreatography (ERCP). In this retrospective study, the clinical utility of EUS-BD as a rescue drainage technique for HBO patients who failed re-intervention under ERCP guidance was evaluated. METHODS: Between April 2012 and August 2015, patients with HBO were enrolled. Patients' characteristics, kinds of metallic stents, configuration of stent placement, overall survival, stent patency, and results of re-intervention were reviewed, along with the re-intervention technical success rate. RESULTS: The biliary stricture type was Bismuth type IV in 59% (23/39) and types IIIa and b in 20.5% (8/39) each. Biliary metallic stent placement was bilateral in 38.5% (15/39), and unilateral in 61.5% (24/39). Primary stent dysfunction was observed in 71.8% (28/39) of cases. Planned re-intervention under ERCP guidance was attempted in 26 patients. The technical success rate was 62% (16/26). For the 10 patients who failed re-intervention under ERCP guidance, EUS-BD was planned. The technical success rate was 100% (10/10). The procedure time was 25.8 min. Adverse events were not seen in any patients. Stent patency after re-intervention was not significantly different between ERCP (165 days) guidance and EUS-BD (152 days) guidance (P = 0.463) CONCLUSIONS: In conclusion, EUS-BD as a re-intervention method for metallic stent obstruction was safe and feasible. If ERCP was failed, EUS-BD may be one of option as re-intervention method for high-grade hilar stricture.

Circulating adipocyte fatty acid-binding protein is a predictor of cardiovascular events in patients with stable angina undergoing percutaneous coronary intervention.

BACKGROUND: Adipocyte fatty acid-binding protein (A-FABP) is expressed in both adipocytes and macrophages. Recent studies have shown that A-FABP is secreted by adipocytes and that the A-FABP concentration is associated with obesity, insulin resistance, and atherosclerosis. We have reported that the coronary atherosclerotic burden is associated with the serum A-FABP concentration. In the present study, we investigated whether the serum A-FABP concentration is associated with prognosis in patients with stable angina pectoris who have undergone percutaneous coronary intervention (PCI). METHODS: This was a prospective single-center trial. In total, 130 patients with stable angina pectoris undergoing their first PCI were enrolled from August 2008 to July 2010 at Kagawa Prefectural Central Hospital. The primary endpoints were cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, revascularization, and hospitalization for heart failure. RESULTS: During the follow-up (median, 50 months; interquartile range, 23-66 months), 49 cardiovascular events occurred. Kaplan-Meier analysis showed that the cumulative incidence of the primary endpoints in the high A-FABP group (median A-FABP concentration of ≥ 18.6 ng/ml) was greater than that in the low A-FABP group. Cox analysis showed that the A-FABP concentration was an independent predictor of cardiovascular events adjusted for age and the presence of multi-vessel disease (hazard ratio, 1.03; 95% confidence interval, 1.01-1.04; p = 0.01). CONCLUSION: The serum A-FABP concentration is associated with prognosis in patients with stable angina undergoing PCI, suggesting that the serum A-FABP concentration could be useful for risk assessment of secondary prevention. TRIAL REGISTRATION: UMIN Clinical Trials Registry UMIN000029283 (registration date: September 25, 2017), retrospectively registered.

Distributional shift of urea production site from the extraembryonic yolk sac membrane to the embryonic liver during the development of cloudy catshark (Scyliorhinus torazame).

Urea is an essential osmolyte for marine cartilaginous fishes. Adult elasmobranchs and holocephalans are known to actively produce urea in the liver, muscle and other extrahepatic organs; however, osmoregulatory mechanisms in the developing cartilaginous fish embryo with an undeveloped urea-producing organ are poorly understood. We recently described the contribution of extraembryonic yolk sac membranes (YSM) to embryonic urea synthesis during the early developmental period of the oviparous holocephalan elephant fish (Callorhinchus milii). In the present study, to test whether urea production in the YSM is a general phenomenon among oviparous Chondrichthyes, we investigated gene expression and activities of ornithine urea cycle (OUC) enzymes together with urea concentrations in embryos of the elasmobranch cloudy catshark (Scyliorhinus torazame). The intracapsular fluid, in which the catshark embryo develops, had a similar osmolality to seawater, and embryos maintained a high concentration of urea at levels similar to that of adult plasma throughout development. Relative mRNA expressions and activities of catshark OUC enzymes were significantly higher in YSM than in embryos until stage 32. Concomitant with the development of the embryonic liver, the expression levels and activities of OUC enzymes were markedly increased in the embryo from stage 33, while those of the YSM decreased from stage 32. The present study provides further evidence that the YSM contributes to embryonic urea homeostasis until the liver and other extrahepatic organs become fully functional, and that urea-producing tissue shifts from the YSM to the embryonic liver in the late developmental period of oviparous marine cartilaginous fishes.

Evaluation of the safety of endoscopic radiofrequency ablation for malignant biliary stricture using a digital peroral cholangioscope (with videos).

BACKGROUND AND AIM: The clinical impact of catheter-based radiofrequency ablation (RFA) under endoscopic retrograde cholangiopancreatography (ERCP) guidance has recently been reported; however, severe adverse events have also been noted. If tumor is not present in the biliary tract, severe adverse events such as perforation or bleeding as a result of vessel injury around the biliary tract may occur. In addition, the effectiveness of RFA may not be sufficient based solely on radiographic guidance. The aim of the present study was to evaluate the actual feasibility of intraductal RFA by peroral cholangioscope (POCS) evaluation before/after RFA. METHODS: In this retrospective study carried out between July and September 2016, consecutive patients who underwent RFA for malignant biliary stricture and POCS evaluation before/after RFA were enrolled. Primary endpoint of this study was technical feasibility of RFA, which was evaluated by POCS. Secondary endpoints were rates and types of adverse event. RESULTS: A total of 12 consecutive patients were retrospectively enrolled in this study. Stent placement using uncovered metal stents had been previously done in six patients before RFA. Tumor was seen in the biliary tract in all patients. RFA was technically successful in all patients, and clinical success was confirmed in all patients by POCS imaging. Adverse events were seen in only one patient. Median stent patency was 154 days. CONCLUSIONS: RFA for malignant biliary stricture may be safe. To confirm the feasibility and efficacy of RFA, additional cases, prospective studies, and a comparison study between with and without endobiliary RFA are needed.

Prospective evaluation of digital single-operator cholangioscope for diagnostic and therapeutic procedures (with videos).

BACKGROUND AND AIM: Recently, the digital single-operator cholangioscope (SPY-DS) has become available. This system may allow diagnosis by direct visualization and allow performance of various therapeutic procedures. The aim of the present study was to prospectively evaluate the clinical utility of DSOCS for diagnostic and therapeutic procedures for biliary disease. METHODS: Technical success was defined as successful visualization of target lesions in the biliary tract and carrying out forceps biopsy as a diagnostic procedure, and successfully carrying out treatment such as guidewire insertion for the area of interest, electrohydraulic lithotripsy (EHL), or migrated stent removal. Also, the present study aimed at investigating diagnostic yield of the cholangioscopic findings and biopsy specimens. RESULTS: A total of 55 consecutive patients were prospectively enrolled in this study; a diagnostic procedure was done in 33 patients, and a therapeutic procedure was done in 22 patients. Overall accuracy of visual findings was 93%, with a sensitivity of 83%, a specificity of 89%, positive predictive value (PPV) of 83%, and negative predictive value (NPV) of 100%. However, the overall accuracy of forceps biopsy was 89%, with a sensitivity, specificity, and PPV of 100%, and NPV of 90%. Overall technical success rate of therapeutic procedures such as selective guidewire insertion, EHL or migrated stent removal was 91% (20/22). Finally, adverse events were seen in two cases in the diagnostic group, but were not seen in the therapeutic group. CONCLUSION: Although additional cases and a randomized, controlled study with another cholangioscope are needed, diagnostic and therapeutic procedures using SPY-DS appear to be feasible and safe.