Molecular evolution of patristacin genes in teleosts based on the genome survey.

During the evolution of astacin metalloprotease family genes, gene duplication occurred, especially in the lineage of teleosts, in which several types of astacins containing six conserved cysteines (c6ast) emerged. One of them is patristacin, originally found in syngnathid fishes, such as pipefishes and seahorses. Patristacin is expressed in the brood pouch and is present on the same chromosome as other c6ast (pactacin and nephrosin) genes. We first surveyed all the genes from 33 teleost species using a genome database, and characterized the genes by phylogenetic analysis. Pactacin and nephrosin gene homologs were found from all the examined species with only few exceptions, while patristacin gene homologs were found from only several lineages. The patristacin gene homologs were found as multicopy genes in most species of Percomorpha, one of the diverged groups in teleosts. Further diversification of the gene occurred during the evolution of Atherinomorphae, one of the groups in Percomorpha. Fishes of Atherinomorphae possess two types of patristacin, belonging to subclades 1 and 2. Among the Atherinomorpha, we chose the southern platyfish to examine the patristacin gene expression. Platyfish possess eight patristacin gene homologs, called XmPastn1, 2, 3, 4, 5, 7, 10, and 11. Of these genes, only XmPastn2 belongs to subclade 1, while the other seven belong to subclade 2. Only XmPastn2 showed strong expression in several organs of adult platyfish, as observed in reverse-transcription polymerase chain reaction of RNA extracts. Cells expressing XmPastn2 were predominantly mucus-secreting cells found in epidermis around the jaw, as revealed by in-situ hybridization. This result suggests that XmPastn2 is secreted and may contribute to mucus formation or secretion.

LGP2 virus sensor enhances apoptosis by upregulating apoptosis regulatory genes through TRBP-bound miRNAs during viral infection.

During viral infection, viral nucleic acids are detected by virus sensor proteins including toll-like receptor 3 or retinoic acid-inducible gene I-like receptors (RLRs) in mammalian cells. Activation of these virus sensor proteins induces type-I interferon production and represses viral replication. Recently, we reported that an RLR family member, laboratory of genetics and physiology 2 (LGP2), modulates RNA silencing by interacting with an RNA silencing enhancer, TAR-RNA binding protein (TRBP). However, the biological implications remained unclear. Here, we show that LGP2 enhances apoptosis by upregulating apoptosis regulatory genes during viral infection. Sendai virus (SeV) infection increased LGP2 expression approximately 900 times compared to that in non-virus-infected cells. Then, the induced LGP2 interacted with TRBP, resulting in the inhibition of maturation of the TRBP-bound microRNA (miRNA) and its subsequent RNA silencing activity. Gene expression profiling revealed that apoptosis regulatory genes were upregulated during SeV infection: caspases-2, -8, -3 and -7, four cysteine proteases with key roles in apoptosis, were upregulated directly or indirectly through the repression of a typical TRBP-bound miRNA, miR-106b. Our findings may shed light on the mechanism of apoptosis, induced by the TRBP-bound miRNAs through the interaction of TRBP with LGP2, as an antiviral defense system in mammalian cells.

[A Successful Case of Treatment by Lymphangiography for Chylothorax after Robotic Esophagectomy-A Case of Report].

A 50-year-old woman diagnosed with clinical Stage Ⅲ esophageal squamous cell carcinoma, received 2 cycles of docetaxel, cisplatin, 5-fluorouracil(DCF)therapy followed by robotic esophagectomy. In the 15th postoperative day, she suddenly had difficulty in breathing and a chest CT finding showed a large amount of right pleural effusion. Thoracocentesis yield a 1 L of chylous fluid confirmed diagnosis with postoperative chylothorax and conservative treatment was initiated. However, chylothorax was not improved. Therefore, a lipiodol lymphangiography from the inguinal lymph node was performed at the 20th postoperative day. Although the site of leakage could not identify, amount of drained pleural effusion was gradually decreased after lymphangiography, and drain was decannulated in the 28th postoperative day. Lipiodol lymphangiography may be a useful modality for both diagnosis and treatment for chylothorax after esophagectomy.

Standardization of tomato juice tastes using a taste sensor approach.

To enable the taste evaluation of many food samples at a time as well as the comparison of taste evaluation data acquired at different times, a standardization method for taste intensities was developed by a combination of a taste sensor system and a standard solution prepared with taste substances. In the case of tomato juices, citric acid, sucrose, and monosodium glutamate were used as standard taste substances for sourness, sweetness, and umami taste, respectively. Each standard point of the taste intensities was determined using only one standard solution including these standard substances. The taste intensity was described as a value on a scale based on discrimination thresholds of human gustation, where intensities of sourness, sweetness, and umami taste of the tomato juices were classified into multiple levels. Organoleptic evaluation supported these results. Validation for the present standardization method revealed that this approach has enough precision for practical tomato taste evaluation.

LGP2 virus sensor regulates gene expression network mediated by TRBP-bound microRNAs.

Here we show that laboratory of genetics and physiology 2 (LGP2) virus sensor protein regulates gene expression network of endogenous genes mediated by TAR-RNA binding protein (TRBP)-bound microRNAs (miRNAs). TRBP is an enhancer of RNA silencing, and functions to recruit precursor-miRNAs (pre-miRNAs) to Dicer that processes pre-miRNA into mature miRNA. Viral infection activates the antiviral innate immune response in mammalian cells. Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), including RIG-I, melanoma-differentiation-associated gene 5 (MDA5), and LGP2, function as cytoplasmic virus sensor proteins during viral infection. RIG-I and MDA5 can distinguish between different types of RNA viruses to produce antiviral cytokines, including type I interferon. However, the role of LGP2 is controversial. We found that LGP2 bound to the double-stranded RNA binding sites of TRBP, resulting in inhibition of pre-miRNA binding and recruitment by TRBP. Furthermore, although it is unclear whether TRBP binds to specific pre-miRNA, we found that TRBP bound to particular pre-miRNAs with common structural characteristics. Thus, LGP2 represses specific miRNA activities by interacting with TRBP, resulting in selective regulation of target genes. Our findings show that a novel function of LGP2 is to modulate RNA silencing, indicating the crosstalk between RNA silencing and RLR signaling in mammalian cells.

[Laparoscopic Local Resection for Duodenal Gastrointestinal Stromal Tumor Located in the Third Portion Using Kocher's Maneuver-A Case Report].

Duodenal gastrointestinal stromal tumor(GIST)are relatively rare. Here, we report a case of a duodenal GIST located in the third portion that was successfully treated via laparoscopic local resection using the Kocher maneuver. A 49-year-old woman with a high BMI of 43.4 kg/m2 was diagnosed with a 20 mm duodenal submucosal tumor in the third portion that was suspected to be a GIST; subsequently, she underwent laparoscopic local resection. After mobilization from the first to third portion of the duodenum using the Kocher maneuver, local resection using a linear stapler was completed. The surgery time was 152 minutes, and the estimated blood loss was approximately zero. The postoperative course was uneventful, and she was discharged on the 7th postoperative day. The pathological diagnosis was ultra-low-grade GIST. This procedure can be a useful option for obese patients with good operative field of view.

[Successful Conversion Therapy for Gastric Cancer with Paraaortic Lymph Node Metastasis after SP Therapy-A Case Report].

A 69-year-old woman, who complained of loss of appetite, was admitted to our hospital and diagnosed with clinical Stage Ⅳgastric cancer and paraaortic lymph node metastases(cT4aN3M1[#16b1LYM], cStage Ⅳ). She underwent 2 cycles of SP therapy(combination of S-1 and CDDP). A partial response of the primary tumor was noted, with no distant metastases, except for the paraaortic lymph nodes. She underwent robotic total gastrectomy with D2 plus paraaortic lymph node dissection. Histopathology showed no residual tumor cells in the stomach or lymph nodes. Postoperatively, the patient underwent 3 cycles of SOX therapy(combination of S-1 and oxaliplatin)and survived for over 6 postoperative months, with no recurrences. For advanced gastric cancers with paraaortic lymph node dissection with no evidences of other distant metastases, gastrectomy with paraaortic lymph node dissection combined with chemotherapy could be a therapeutic option to achieve R0 resection.

Enhancement of single guide RNA transcription for efficient CRISPR/Cas-based genomic engineering.

Genomic engineering using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein is a promising approach for targeting the genomic DNA of virtually any organism in a sequence-specific manner. Recent remarkable advances in CRISPR/Cas technology have made it a feasible system for use in therapeutic applications and biotechnology. In the CRISPR/Cas system, a guide RNA (gRNA), interacting with the Cas protein, recognizes a genomic region with sequence complementarity, and the double-stranded DNA at the target site is cleaved by the Cas protein. A widely used gRNA is an RNA polymerase III (pol III)-driven single gRNA (sgRNA), which is produced by artificial fusion of CRISPR RNA (crRNA) and trans-activation crRNA (tracrRNA). However, we identified a TTTT stretch, known as a termination signal of RNA pol III, in the scaffold region of the sgRNA. Here, we revealed that sgRNA carrying a TTTT stretch reduces the efficiency of sgRNA transcription due to premature transcriptional termination, and decreases the efficiency of genome editing. Unexpectedly, it was also shown that the premature terminated sgRNA may have an adverse effect of inducing RNA interference. Such disadvantageous effects were avoided by substituting one base in the TTTT stretch.

An evolutionary insight into the hatching strategies of pipefish and seahorse embryos.

Syngnathiform fishes carry their eggs in a brood structure found in males. The brood structure differs from species to species: seahorses carry eggs within enclosed brood pouch, messmate pipefish carry eggs in the semi-brood pouch, and alligator pipefish carry eggs in the egg compartment on abdomen. These egg protection strategies were established during syngnathiform evolution. In the present study, we compared the hatching mode of protected embryos of three species. Electron microscopic observations revealed that alligator pipefish and messmate pipefish egg envelopes were thicker than those of seahorses, suggesting that the seahorse produces a weaker envelope. Furthermore, molecular genetic analysis revealed that these two pipefishes possessed the egg envelope-digesting enzymes, high choriolytic enzyme (HCE), and low choriolytic enzyme (LCE), as do many euteleosts. In seahorses, however, only HCE gene expression was detected. When searching the entire seahorse genome by high-throughput DNA sequencing, we did not find a functional LCE gene and only a trace of the LCE gene exon was found, confirming that the seahorse LCE gene was pseudogenized during evolution. Finally, we estimated the size and number of hatching gland cells expressing hatching enzyme genes by whole-mount in situ hybridization. The seahorse cells were the smallest of the three species, while they had the greatest number. These results suggest that the isolation of eggs from the external environment by paternal bearing might bring the egg envelope thin, and then, the hatching enzyme genes became pseudogenized. J. Exp. Zool. (Mol. Dev. Evol.) 9999B:XX-XX, 2016. © 2016 Wiley Periodicals, Inc.

The siRNA Non-seed Region and Its Target Sequences Are Auxiliary Determinants of Off-Target Effects.

RNA interference (RNAi) is a powerful tool for post-transcriptional gene silencing. However, the siRNA guide strand may bind unintended off-target transcripts via partial sequence complementarity by a mechanism closely mirroring micro RNA (miRNA) silencing. To better understand these off-target effects, we investigated the correlation between sequence features within various subsections of siRNA guide strands, and its corresponding target sequences, with off-target activities. Our results confirm previous reports that strength of base-pairing in the siRNA seed region is the primary factor determining the efficiency of off-target silencing. However, the degree of downregulation of off-target transcripts with shared seed sequence is not necessarily similar, suggesting that there are additional auxiliary factors that influence the silencing potential. Here, we demonstrate that both the melting temperature (Tm) in a subsection of siRNA non-seed region, and the GC contents of its corresponding target sequences, are negatively correlated with the efficiency of off-target effect. Analysis of experimentally validated miRNA targets demonstrated a similar trend, indicating a putative conserved mechanistic feature of seed region-dependent targeting mechanism. These observations may prove useful as parameters for off-target prediction algorithms and improve siRNA 'specificity' design rules.

Adult skeletal Class II high-angle case treated with a fully customized lingual bracket appliance.

To achieve optimum occlusal and facial results in a patient with high-angle maxillary protrusion, it is important to move Point A back with retraction of the anterior teeth and prevent clockwise rotation of the mandible through good vertical control. A woman, aged 42 years 5 months, with a protrusive profile sought lingual orthodontic treatment. She had a skeletal Class II high-angle pattern with maxillary protrusion and mandibular retrusion. The extraction of the 4 first premolars was indicated to correct the problems. The vertical bowing effect, a side effect known to occur with conventional lingual bracket systems owing to torque loss, would preclude adequate retraction of Point A and compromise the facial results. To prevent this issue, a fully customized lingual bracket system with vertical slots for the anterior teeth using ribbon-wise archwires was selected. A midpalatal miniscrew was used to prevent molar extrusion. As a result, the bodily retraction of the maxillary incisors and Point A was achieved, obtaining an attractive facial profile.

Psychological impact of lifestyle-related disease disclosure at general checkup: a prospective cohort study.

BACKGROUND: Little is known about psychological impact of disclosing lifestyle-related diseases. Previous studies discussed the long-term psychological impact of disease disclosure, and a significant psychological impact was not observed. This study clarified the psychological impact on anxiety state of patients when lifestyle-related diseases are disclosed at general checkups for local residents. In particular, this study evaluated the short-term impact on patients, and how the notification of abnormal values and the disclosure of disease at general checkups affect patients' subsequent behavioral changes. METHODS: The study design was a prospective cohort study. We compared the anxiety state of participants using a self-administered anxiety assessment scale, State-Trait Anxiety Inventory (STAI), before and after Physician's explanation of abnormal values in markers of lifestyle-related diseases. The participants were those between the age of 40 and 75 years who underwent general checkups at two primary care facilities. In addition, we assessed the effects on lifestyle habits and the psychological impact caused by general checkup using STAI and a survey on behavioral changes one month after the checkup. RESULTS: The valid response rate at the survey of the general checkup was 92% (534/578). Of those who showed abnormal levels in markers of lifestyle-related diseases, anxiety was augmented significantly among those who responded that the physician had told them of their diagnosis compared to those who responded that the physician had not told them of their diagnosis (Wilcoxon rank-sum test, P < 0.007). The percentage of patients whose state anxiety scale of STAI increased ≥5 points was 30% in the disease disclosed group (33/111) and 17% in the disease undisclosed group (27/159), respectively. The risk ratio was 1.5 (95% CI: 1.1-2.0). One month after the general checkup, overall anxiety diminished regardless of whether diagnosis of lifestyle-related diseases was disclosed to patients notified of abnormal values. In addition, improvements in daily life behaviors as a result of notification of abnormalities or disclosure of diagnosis at general checkup were not observed. CONCLUSION: Even in a general checkup for the general population, disclosing non-critical diseases such as lifestyle-related diseases exacerbated anxiety as a short-term psychological impact.

Relieving pain and distress during venipuncture: Pilot study of the Japan Environment and Children's Study (JECS).

Pain management for needle-related procedures is poor in Japan. In many countries the use of lidocaine/prilocaine cream for the relief of pain associated with venipuncture has been approved. In children, a psychological approach has also been shown to be effective in reducing pain with venipuncture. We developed a multidisciplinary procedure that combines a cream (2.5% lidocaine and 2.5% prilocaine) and pharmacological approaches such as preparation, education, positioning and distraction. We evaluated the feasibility and acceptability of the procedure for young children. Among 132 pediatric participants, 58.3% did not cry during venipuncture. According to caregiver questionnaire, 71.9% felt that the multidisciplinary procedure eliminated the fear of needle-related procedures in the children; 90.9% were satisfied with it and 75.8% thought it should be applied to all children undergoing venipuncture. The present results suggest that the multidisciplinary procedure is feasible, acceptable and suitable for use in children undergoing venipuncture.

Long-Term Chronic Toxicity and Mesothelial Cell Reactions Induced by Potassium Octatitanate Fibers (TISMO) in the Left Thoracic Cavity in A/J Female Mice.

The present study was conducted to examine the chronic effects of potassium octatitanate fibers (trade name TISMO; chemical formula K2O·6TiO2) on the mouse lung and thoracic cavity. This method of infusion was employed to examine the direct effects of the fibers to the pleura. In the present study, 52- and 65-week experiments were employed to examine the long-term chronic effects after infusion of fiber-shaped TISMO into the thoracic cavities of A/J mice. Following this infusion, TISMO fibers were observed in the alveoli, indicating penetration through the visceral pleura. The additional histopathological detection of TISMO fibers in the liver, spleen, kidneys, ovary, heart, bone marrow, and brain of TISMO-infused mice indicated migration of the fibers out from the thoracic cavity. Atypical mesothelial cells with severe pleural proliferation were observed, but malignant mesotheliomas were not detected. This study demonstrated that intrathoracic infusion of TISMO fiber did not cause malignant mesothelioma but did cause severe chronic inflammation and proliferation of pleural mesothelial cells.

Computed tomographic features of canine nonparenchymal hemangiosarcoma.

The purpose of this retrospective study was to describe pre- and postcontrast computed tomographic (CT) characteristics of confirmed nonparenchymal hemangiosarcoma in a group of dogs. Medical records were searched during the period of July 2003 and October 2011 and dogs with histologically confirmed nonparenchymal hemangiosarcoma and pre- and postcontrast CT images were recruited. Two observers recorded a consensus opinion for the following CT characteristics for each dog: largest transverse tumor diameter, number of masses, general tumor shape, character of the tumor margin, precontrast appearance, presence of dystrophic calcification, presence of postcontrast enhancement, pattern of postcontrast enhancement, presence of regional lymphadenopathy, and presence of associated cavitary fluid. A total of 17 dogs met inclusion criteria. Tumors were located in the nasal cavity, muscle, mandible, mesentery, subcutaneous tissue, and retroperitoneal space. Computed tomographic features of nonparenchymal hemangiosarcoma were similar to those of other soft tissue sarcomas, with most tumors being heterogeneous in precontrast images, invasive into adjacent tissue, and heterogeneously contrast enhancing. One unexpected finding was the presence of intense foci of contrast enhancement in 13 of the 17 tumors (76%). This appearance, which is not typical of other soft tissue sarcomas, was consistent with contrast medium residing in vascular channels. Findings indicated that there were no unique distinguishing CT characteristics for nonparenchymal hemangiosarcoma in dogs; however, the presence of highly attenuating foci of contrast enhancement may warrant further investigation in prospective diagnostic sensitivity and treatment outcome studies.

Immunohistochemical characteristics of surfactant proteins a, B, C and d in inflammatory and tumorigenic lung lesions of f344 rats.

Surfactant proteins (SPs), originally known as human lung surfactants, are essential to respiratory structure and function. There are 4 subtypes, SP-A, SP-B, SP-C and SP-D, with SP-A and SP-D having immunological functions, and SP-B and SP-C having physicochemical properties that reduce the surface tension at biological interfaces. In this experiment, the expressions of SP-A, SP-B, SP-C and SP-D in lung neoplastic lesions induced by N-bis (2-hydroxypropyl) nitrosamine (DHPN) and inflammatory lesions due to quartz instillation were examined and compared immunohistochemically. Formalin fixed paraffin embedded (FFPE) lung samples featuring inflammation were obtained with a rat quartz instillation model, and neoplastic lesions, hyperplasias and adenomas, were obtained with the rat DHPN-induced lung carcinogenesis model. In the rat quartz instillation model, male 10-week old F344 rats were exposed by intratracheal instillation (IT) to quartz at a dose of 2 mg/rat suspended in saline (0.2 ml) on day 0, and sacrificed on day 28. Lung tumorigenesis in F344 male rats was initiated by DHPN in drinking water for 2 weeks, and the animals were then sacrificed in week 30. Lung proliferative lesions, hyperplasias and adenomas, were observed with DHPN, and inflammation was observed with quartz. The expressions of SP-A, SP-B, SP-C and SP-D were examined immunohistochemically. SP-B and SP-C showed strong expression in lung hyperplasias and adenomas, while SP-A and SP-D were observed in mucus or exudates in inflammatory alveoli. These results suggest the possibility that SP-B and SP-C are related to lung tumorigenesis.

Validation of continuous and noninvasive hemoglobin monitoring by pulse CO-oximetry in Japanese surgical patients.

We evaluated the accuracy of noninvasive and continuous total hemoglobin (SpHb) monitoring with the Radical-7(®) Pulse CO-Oximeter in Japanese surgical patients before and after an in vivo adjustment of the first SpHb value to match the first reference value from a satellite laboratory CO-Oximeter. Twenty patients undergoing surgical procedures with general anesthesia were monitored with Pulse CO-Oximetry for SpHb. Laboratory CO-Oximeter values (tHb) were compared to SpHb at the time of the blood draws. Bias, precision, limits of agreement and correlation coefficient of SpHb compared to tHb were calculated before and after SpHb values were adjusted by subtracting the difference between the first SpHb and tHb value from all subsequent SpHb values. Trending of SpHb to tHb and the effect of perfusion index (PI) on the agreement of SpHb to tHb were also analyzed. Ninety-two tHb values were compared to the SpHb. Bias ± 1SD was 0.2 ± 1.5 g/dL before in vivo adjustment and -0.7 ± 1.0 g/dL after in vivo adjustment. Bland-Altman analysis showed limits of agreement of -2.8 to 3.1 g/dL before in vivo adjustment and -2.8 to 1.4 g/dL after in vivo adjustment. The correlation coefficient was 0.76 prior to in vivo adjustment and 0.87 after in vivo adjustment. In patients with adequate perfusion (PI ≥1.4) the correlation coefficient was 0.89. In vivo adjustment of SpHb significantly improved the accuracy in our cohort of Japanese surgical patients. The strongest correlation between SpHb and tHb values was observed in patients with adequate peripheral perfusion suggesting that low perfusion may affect the accuracy of SpHb monitoring.

Prognosis of primary pulmonary adenocarcinoma after surgical resection in small-breed dogs: 52 cases (2005-2021).

BACKGROUND: Tumor size is an important prognostic factor in lung cancer in dogs, and the canine lung carcinoma stage classification (CLCSC) recently has been proposed to subdivide tumor sizes. It is unclear if the same classification scheme can be used for small-breed dogs. OBJECTIVES: To investigate whether the tumor size classification of CLCS is prognostic for survival and progression outcomes in small-breed dogs with surgically resected pulmonary adenocarcinomas (PACs). ANIMALS: Fifty-two client-owned small-breed dogs with PAC. METHODS: Single-center retrospective cohort study conducted between 2005 and 2021. Medical records of dogs weighing <15 kg with surgically resected lung masses histologically diagnosed as PAC were examined. RESULTS: The numbers of dogs with tumor size ≤3 cm, >3 cm to ≤5 cm, >5 cm to ≤7 cm, or >7 cm were 15, 18, 14, and 5, respectively. The median progression-free interval (PFI) and overall survival time (OST) were 754 and 716 days, respectively. In univariable analysis, clinical signs, lymph node metastasis, margin, and histologic grade were associated with PFI, and age, clinical signs, margin, and lymph node metastasis were associated with OST. Tumor size classification of CLCS was associated with PFI in all categories, and tumor size >7 cm was associated with OST. In multivariable analysis, tumor size >5 cm to ≤7 cm and margin were associated with PFI, and age was associated with OST. CONCLUSIONS AND CLINICAL IMPORTANCE: The tumor size classification of CLCS would be an important prognostic factor in small-breed dogs with surgically resected PACs.

Intake levels and main sources of nutrients for Japanese children with motor or intellectual disabilities.

Proper nutritional management is important for the growth and development of children with motor or intellectual disabilities; however, few studies have investigated the nutrient intake of children with disabilities. This study aimed to investigate the nutrient intake and food groups that are the main sources of nutrients for children with disabilities. This cross-sectional observational study included twenty-five children (mean age, 11⋅8 years) from five hospitals in Japan. Using a 3-d weighed dietary record, we estimated the daily nutrient intake and food and beverage sources that contributed to nutrient intake. The mean values of calcium, magnesium, iron, vitamin A, thiamine, riboflavin, and vitamin C intake were below the recommended dietary allowance, and those of dietary fiber and potassium were below the levels recommended by the Tentative Dietary Goal for Preventing Lifestyle-related Diseases (DG). In contrast, the mean intake values of fat, saturated fatty acids, and sodium were above the DG levels. Dairy products, meat, vegetables, and cereals were found to be the major contributors of nutrients. Increased intake of vegetables may help alleviate insufficient micronutrient intake in children with disabilities.