Prospective observational study comparing the perioperative outcomes of laparoscopic colectomy with or without epidural anesthesia: the Kanagawa Yokohama Colorectal Cancer Study Group (KYCC) 1806.

PURPOSE: There have been no adequate comparisons of the efficacy, safety, and efficiency of analgesia after laparoscopic colorectal resection (LAC), with and without epidural anesthesia (EDA). METHODS: This was a multicenter prospective observational study of patients undergoing LAC. The primary end point was the mean visual analog scale (VAS) score on postoperative days (PODs) 1-7. The secondary end points were the highest VAS, complication rate, days to first ambulation and fatigue, length of hospital stay, and time to commencement of surgery. RESULTS: We compared an EDA group (Group E, n = 48) and a no-EDA group (Group O, n = 48) after matching. The mean VAS was not significantly different between the groups (28.7 vs. 30.1, p = 0.288). On assessing the secondary end points, the highest VAS was not significantly different between the groups. In fact, the VAS was lower in Group E only on POD 2. There was no difference in the incidence of complications, the time to first postoperative evacuation was shorter in Group E, and postoperative hospitalization was similar. The time to surgery was shorter in Group O. CONCLUSION: These results suggest that LAC without EDA is a feasible option, but with the early and regular use of adjunctive measures to provide more stable analgesia.

Engineering tRNAs for the Ribosomal Translation of Non-proteinogenic Monomers.

Ribosome-dependent protein biosynthesis is an essential cellular process mediated by transfer RNAs (tRNAs). Generally, ribosomally synthesized proteins are limited to the 22 proteinogenic amino acids (pAAs: 20 l-α-amino acids present in the standard genetic code, selenocysteine, and pyrrolysine). However, engineering tRNAs for the ribosomal incorporation of non-proteinogenic monomers (npMs) as building blocks has led to the creation of unique polypeptides with broad applications in cellular biology, material science, spectroscopy, and pharmaceuticals. Ribosomal polymerization of these engineered polypeptides presents a variety of challenges for biochemists, as translation efficiency and fidelity is often insufficient when employing npMs. In this Review, we will focus on the methodologies for engineering tRNAs to overcome these issues and explore recent advances both in vitro and in vivo. These efforts include increasing orthogonality, recruiting essential translation factors, and creation of expanded genetic codes. After our review on the biochemical optimizations of tRNAs, we provide examples of their use in genetic code manipulation, with a focus on the in vitro discovery of bioactive macrocyclic peptides containing npMs. Finally, an analysis of the current state of tRNA engineering is presented, along with existing challenges and future perspectives for the field.

A Microphysiological HHT-on-a-Chip Platform Recapitulates Patient Vascular Lesions.

Hereditary Hemorrhagic Telangiectasia (HHT) is a rare congenital disease in which fragile vascular malformations focally develop in multiple organs. These can be small (telangiectasias) or large (arteriovenous malformations, AVMs) and may rupture leading to frequent, uncontrolled bleeding. There are few treatment options and no cure for HHT. Most HHT patients are heterozygous for loss-of-function mutations for Endoglin (ENG) or Alk1 (ACVRL1), however, why loss of these genes manifests as vascular malformations remains poorly understood. To complement ongoing work in animal models, we have developed a microphysiological system model of HHT. Based on our existing vessel-on-a-chip (VMO) platform, our fully human cell-based HHT-VMO recapitulates HHT patient vascular lesions. Using inducible ACVRL1 (Alk1)-knockdown, we control timing and extent of endogenous Alk1 expression in primary human endothelial cells (EC) in the HHT-VMO. HHT-VMO vascular lesions develop over several days, and are dependent upon timing of Alk1 knockdown. Interestingly, in chimera experiments AVM-like lesions can be comprised of both Alk1-intact and Alk1-deficient EC, suggesting possible cell non-autonomous effects. Single cell RNA sequencing data are consistent with microvessel pruning/regression as contributing to AVM formation, while loss of PDGFB expression implicates mural cell recruitment. Finally, lesion formation is blocked by the VEGFR inhibitor pazopanib, mirroring the positive effects of this drug in patients. In summary, we have developed a novel HHT-on-a-chip model that faithfully reproduces HHT patient lesions and that is sensitive to a treatment effective in patients. The VMO-HHT can be used to better understand HHT disease biology and identify potential new HHT drugs. Significance: This manuscript describes development of an organ-on-a-chip model of Hereditary Hemorrhagic Telangiectasia (HHT), a rare genetic disease involving development of vascular malformations. Our VMO-HHT model produces vascular malformations similar to those seen in human HHT patients, including small (telangiectasias) and large (arteriovenous malformations) lesions. We show that VMO-HHT lesions are sensitive to a drug, pazopanib, that appears to be effective in HHT human patients. We further use the VMO-HHT platform to demonstrate that there is a critical window during vessel formation in which the HHT gene, Alk1, is required to prevent vascular malformation. Lastly, we show that lesions in the VMO-HHT model are comprised of both Alk1-deficient and Alk1-intact endothelial cells.

The anxious-depressive attack severity scale: development and initial validation and reliability.

BACKGROUND: Anxious-depressive attack (ADA) is a symptom complex that comprises sudden intense feelings of anxiety or depression, intrusive rumination of regretful memories or future worries, emotional distress due to painful thoughts, and coping behaviors to manage emotional distress. ADA has been observed trans-diagnostically across various psychiatric disorders. Although the importance of ADA treatment has been indicated, a scale to measure the severity of ADA has not been developed. This study aimed to develop an Anxious-Depressive Attack Severity Scale (ADAS) to measure the severity of ADA symptoms and examine its reliability and validity. METHODS: A total of 242 outpatients responded to a questionnaire and participated in an interview, which were designed to measure the severity of ADA, depressive, anxiety, anxious depression, and social anxiety symptoms. Based on the diagnostic criteria for ADA, 54 patients were confirmed to have ADA and were included in the main study analyses. RESULTS: The exploratory factor analysis of the ADAS identified a two factor structure: severity of ADA symptoms and ADA frequency and coping behaviors. McDonald's ωt coefficients were high for the overall scale and the first factor (ωt = .78 and ωt = .83, respectively) but low for the second factor (ωt = .49). The ADAS score was significantly positively correlated with clinical symptoms related to anxiety and depression. CONCLUSION: The present study demonstrated that the ADAS has sufficient reliability and validity; however, internal consistency was insufficient for the second factor. Overall, the ADAS has potential to be a valuable tool for use in clinical trials of ADA.

Investigation of the hepatic respiration and liver zonation on rat hepatocytes using an integrated oxygen biosensor in a microscale device.

The development of an in vitro functional liver zonation model is a major issue to reproduce physiological liver features. Oxygen concentration is one of the potential explanations of a primary regulating factor of zonation. In this frame, we investigated the oxygen gradient inside a microfluidic device containing rat hepatocyte cultures. The device integrated a platinum (Pt) (II) octaethylporphyrin sensor, allowing a 2D mapping of the oxygen concentration. After 3 hr adhesion of the hepatocytes, the sensor indicated an intense oxygen depletion, leading to an oxygen shortage in the center of the device. After a 30 min perfusion of the culture medium, we monitored the formation of the oxygen gradient along the culture due to cellular respiration. The profile of the oxygen gradient was modulated and controlled by increasing either the perfusion flow rate or the device thickness. In addition, the oxygen gradient was time dependent as far as it decreased with the time of culture. Perivenous and periportal liver patterns were characterized by the immunostaining of the hepatic markers. We put in evidence a spatio temporal hepatic organization. We observed the overexpression since 24 hr of perfusion of the APC and PCK1 proteins upstream in the oxygen-rich area of the device. The overexpression of GS, GCK, CYP1A, and HIFα proteins were observed downstream in the oxygen-poor area. Then, CYP3A2 and ß-catenin spatial reorganization was achieved after 48 hr of culture. The results presented a partial zonation-like pattern that was superimposed with an oxygen gradient profile.

Genome-wide association study and genomic prediction in citrus: Potential of genomics-assisted breeding for fruit quality traits.

Novel genomics-based approaches such as genome-wide association studies (GWAS) and genomic selection (GS) are expected to be useful in fruit tree breeding, which requires much time from the cross to the release of a cultivar because of the long generation time. In this study, a citrus parental population (111 varieties) and a breeding population (676 individuals from 35 full-sib families) were genotyped for 1,841 single nucleotide polymorphisms (SNPs) and phenotyped for 17 fruit quality traits. GWAS power and prediction accuracy were increased by combining the parental and breeding populations. A multi-kernel model considering both additive and dominance effects improved prediction accuracy for acidity and juiciness, implying that the effects of both types are important for these traits. Genomic best linear unbiased prediction (GBLUP) with linear ridge kernel regression (RR) was more robust and accurate than GBLUP with non-linear Gaussian kernel regression (GAUSS) in the tails of the phenotypic distribution. The results of this study suggest that both GWAS and GS are effective for genetic improvement of citrus fruit traits. Furthermore, the data collected from breeding populations are beneficial for increasing the detection power of GWAS and the prediction accuracy of GS.

Dysfunction of paracellin-1 by dephosphorylation in Dahl salt-sensitive hypertensive rats.

A high-salt diet reduced the levels of renal cAMP content and serine-phosphorylated paracellin-1 in Dahl salt-sensitive hypertensive rats. In MDCK cells expressing paracellin-1, protein kinase A inhibitor reduced the serine-phosphorylated paracellin-1 and transepithelial Mg(2+) transport, suggesting that a dephosphorylation of paracellin-1 induces the reduction of Mg(2+) reabsorption in salt-sensitive hypertension.

Phosphorylation of paracellin-1 at Ser217 by protein kinase A is essential for localization in tight junctions.

Although paracellin-1 (PCLN-1) is known to have a crucial role in the control of Mg2+ reabsorption in the kidney, the molecular pathways involved in the regulation of PCLN-1 have not been clarified. We used FLAG-tagged PCLN-1 to investigate these pathways further, and found that PCLN-1 is phosphorylated at Ser217 by protein kinase A (PKA) under physiological conditions in Madin-Darby canine kidney (MDCK) cells. PCLN-1 expression decreased Na+ permeability, resulting in a decrease in the transepithelial electrical resistance (TER). By contrast, PCLN-1 enhanced transepithelial Mg2+ transport. PKA inhibitors, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) and myristoylated protein kinase A inhibitor 14-22 amide PKI, and an adenylate cyclase inhibitor, 2',5'-dideoxy adenosine (DDA), reduced the phosphoserine level of PCLN-1. The inhibitory effect of DDA was rescued by 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br-cAMP). PKA and adenylate cyclase inhibitors decreased transepithelial Mg2+ transport and TER. Dephosphorylated PCLN-1 moved from detergent-insoluble to soluble fractions and was dissociated from ZO-1. A fusion protein of PCLN-1 with glutathione-S-transferase revealed that Ser217 was phosphorylated by PKA. Phosphorylated PCLN-1 was localized in the tight junction (TJ) along with ZO-1, whereas dephosphorylated PCLN-1 and the S217A mutant were translocated into the lysosome. The degradation of dephosphorylated PCLN-1 and S217A mutant was inhibited by chloroquine, a specific lysosome inhibitor. Thus, the PKA-dependent phosphorylation of Ser217 in PCLN-1 is essential for its localization in the TJ and transepithelial Mg2+ transport.