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Rationale: Myocardial infarction (MI) is one of the most dangerous adverse cardiovascular events. Our previous study found that lysophosphatidic acid (LPA) is increased in human peripheral blood after MI, and LPA has a protective effect on the survival and proliferation of various cell types. However, the role of LPA and its receptors in MI is less understood. Objectives: To study the unknown role of LPA and its receptors in heart during MI. Methods and Results: In this study, we found that mice also had elevated LPA level in peripheral blood, as well as increased cardiac expression of its receptor LPA 2 in the early stages after MI. With adult and neonate MI models in global Lpar2 knockout ( Lpar2 -KO) mice, we found Lpar2 deficiency increased vascular leak leading to disruption of its homeostasis, so as to impaired heart function and increased early mortality. Histological examination revealed larger scar size, increased fibrosis, and reduced vascular density in the heart of Lpar2 -KO mice. Furthermore, Lpar2 -KO also attenuated blood flow recovery after femoral artery ligation with decreased vascular density in gastrocnemius. Our study revealed that Lpar2 was mainly expressed and altered in cardiac endothelial cells during MI, and use of endothelial-specific Lpar2 knockout mice phenocopied the global knockout mice. Additionally, adenovirus- Lpar2 and pharmacologically activated LPA 2 significantly improved heart function, reduced scar size, increased vascular formation, and alleviated early mortality by maintaining vascular homeostasis owing to protecting vessels from leakage. Mechanistic studies demonstrated that LPA-LPA 2 signaling could promote endothelial cell proliferation through PI3K-Akt/PLC-Raf1-Erk pathway and enhanced endothelial cell tube formation via PKD1-CD36 signaling. Conclusions: Our results indicate that endothelial LPA-LPA 2 signaling promotes angiogenesis and maintains vascular homeostasis, which is vital for restoring blood flow and repairing tissue function in ischemic injuries. Targeting LPA-LPA 2 signal might have clinical therapeutic potential to protect the heart from ischemic injury.

BackgroundMuch of what we know about insulin resistance is based on studies from metabolically active tissues such as liver, adipose tissue, and skeletal muscle. Emerging evidence suggests that the vascular endothelium plays a crucial role in systemic insulin resistance, however, the underlying mechanisms remain incompletely understood. ADP ribosylation factor 6 (Arf6) is a small GTPase that plays a critical role in endothelial cell (EC) function. Here, we tested the hypothesis that the deletion of endothelial Arf6 will result in systemic insulin resistance.MethodsWe used mouse models of constitutive EC-specific Arf6 deletion (Arf6f/-Tie2Cre) and tamoxifen inducible Arf6 knockout (Arf6f/fCdh5Cre). Endothelium-dependent vasodilation was assessed using pressure myography. Metabolic function was assessed using a battery of metabolic assessments including glucose- and insulin-tolerance tests and hyperinsulinemic-euglycemic clamps. A fluorescence microsphere-based technique was used to measure tissue blood flow. Intravital microscopy was used to assess skeletal muscle capillary density.ResultsEndothelial Arf6 deletion impaired insulin-stimulated vasodilation in white adipose tissue (WAT) and skeletal muscle feed arteries. The impairment in vasodilation was primarily due to attenuated insulin-stimulated nitric oxide (NO) bioavailability but independent of altered acetylcholine- or sodium nitroprusside-mediated vasodilation. In vitro Arf6 inhibition resulted in suppressed insulin stimulated phosphorylation of Akt and endothelial NO synthase. Endothelial cell-specific deletion of Arf6 also resulted in systematic insulin resistance in normal chow fed mice and glucose intolerance in high fat diet fed obese mice. The underlying mechanisms of glucose intolerance were reductions in insulin-stimulated blood flow and glucose uptake in the skeletal muscle and were independent of changes in capillary density or vascular permeability.ConclusionResults from this study support the conclusion that endothelial Arf6 signaling is essential for maintaining insulin sensitivity. Reduced expression of endothelial Arf6 impairs insulin-mediated vasodilation and results in systemic insulin resistance. These results have therapeutic implications for diseases that are associated with endothelial cell dysfunction and insulin resistance such as diabetes.

Preventing or effectively treating metastatic uveal melanoma (UM) is critical because it occurs in about half of patients and confers a very poor prognosis. There is emerging evidence that hepatocyte growth factor (HGF) and insulin-like growth factor 1 (IGF-1) promote metastasis and contribute to the striking metastatic hepatotropism observed in UM metastasis. However, the molecular mechanisms by which HGF and IGF-1 promote UM liver metastasis have not been elucidated. ASAP1, which acts as an effector for the small GTPase ARF6, is highly expressed in the subset of uveal melanomas most likely to metastasize. Here, we found that HGF and IGF-1 hyperactivate ARF6, leading to its interaction with ASAP1, which then acts as an effector to induce nuclear localization and transcriptional activity of NFAT1. Inhibition of any component of this pathway impairs cellular invasiveness. Additionally, knocking down ASAP1 or inhibiting NFAT signaling reduces metastasis in a xenograft mouse model of UM. The discovery of this signaling pathway represents not only an advancement in our understanding of the biology of uveal melanoma metastasis but also identifies a novel pathway that could be targeted to treat or prevent metastatic uveal melanoma.

In recent years, the incidence of gastrointestinal cancer has remained high. Currently, surgical resection is still the most effective method for treating gastrointestinal cancer. Traditionally, radical surgery depends on open surgery. However, traditional open surgery inflicts great trauma and is associated with a slow recovery. Minimally invasive surgery, which aims to reduce postoperative complications and accelerate postoperative recovery, has been rapidly developed in the last two decades; it is increasingly used in the field of gastrointestinal surgery and widely used in early-stage gastrointestinal cancer. Nevertheless, many operations for gastrointestinal cancer treatment are still performed by open surgery. One reason for this may be the challenges of minimally invasive technology, especially when operating in narrow spaces, such as within the pelvis or near the upper edge of the pancreas. Moreover, some of the current literature has questioned oncologic outcomes after minimally invasive surgery for gastrointestinal cancer. Overall, the current evidence suggests that minimally invasive techniques are safe and feasible in gastrointestinal cancer surgery, but most of the studies published in this field are retrospective studies and case-matched studies. Large-scale randomized prospective studies are needed to further support the application of minimally invasive surgery. In this review, we summarize several common minimally invasive methods used to treat gastrointestinal cancer and discuss the advances in the minimally invasive treatment of gastrointestinal cancer in detail.

Myocardial infarction (MI) is one of the most dangerous adverse cardiovascular events. Our previous study found that lysophosphatidic acid (LPA) is increased in human peripheral blood after MI, and LPA has a protective effect on the survival and proliferation of various cell types. However, the role of LPA and its receptors in MI is less understood.To study the unknown role of LPA and its receptors in heart during MI.In this study, we found that mice also had elevated LPA level in peripheral blood, as well as increased cardiac expression of its receptor LPAOur results indicate that endothelial LPA-LPA

Background Natural orifice specimen extraction surgery for colorectal cancer has been introduced in order to reduce the abdominal incision, demonstrating major development potential in minimally invasive surgery. We are conducting this randomized controlled trial to assess whether robotic NOSES is non-inferior to traditional robotic-assisted surgery for patients with colorectal cancer in terms of primary and secondary outcomes. Method/design Accordingly, a prospective, open-label, randomized controlled, parallel-group, multicenter, and non-inferiority trial will be conducted to discuss the safety and efficacy of robotic natural orifice extraction surgery compared to traditional robotic-assisted surgery. Here, 550 estimated participants will be enrolled to have 80% power to detect differences with a one-sided significance level of 0.025 in consideration of the non-inferiority margin of 10%. The primary outcome is the incidence of surgical complications, which will be classified using the Clavien-Dindo system. Discussion This trial is expected to reveal whether robotic NOSES is non-inferior to traditional robotic-assisted surgery, which is of great significance in regard to the development of robotic NOSES for patients with colorectal cancer in the minimally invasive era. Furthermore, robotic NOSES is expected to exhibit superiority to traditional robotic-assisted surgery in terms of both primary and secondary outcomes. Trial registration ClinicalTrials.govNCT04230772. Registered on January 15, 2020.

Background: ARF nucleotide-binding site opener (ARNO) is a guanine nucleotide-exchange factor for ADP-ribosylation factor proteins. ARF nucleotide-binding site opener also binds MyD88, and small-molecule inhibition of ARNO reduces inflammation in animal models of inflammatory arthritis and acute inflammation. However, whether genetic deletion of Arno in mice reduces pathologic inflammation has not yet been reported. Furthermore, its role in the nasal cavity has yet to be investigated. Objective: To generate Arno knockout mice and to determine whether genetic loss of ARNO reduces eosinophilic inflammation in the ovalbumin (OVA) murine model of rhinitis. Methods: Arno knockout mice were generated and wild type and knockout littermates were subjected to the OVA-induced mouse model of rhinosinutitis. Eosinophilic inflammation was assessed through immunofluorescent quantification of EMBP+ eosinophils in the septal mucosa and cytokine expression was assessed by quantitative polymerase chain reaction. Results: Arno knockout mice are viable and fertile without any noted deficits. Arno wild type and knockout mice subjected to the OVA-induced model of rhinitis demonstrated an average of 314.5 and 153.8 EMBP+ cells per mm2 septal tissue, respectively ( P < .05). Goblet cells per mm of basal lamina were assessed via Alcian blue and there was no statistically significant difference between Arno wild type and knockout mice. Ovalbumin-induced expression of interleukin-5 (IL-5) was significantly reduced in Arno knockout mice ( P < .05). There was no statistically significant reduction in IL-4, IL-13, or eotaxin-1 expression. Conclusions: These data demonstrate that deletion of Arno reduces eosinophilic inflammation and IL-5 expression in an OVA-induced model of rhinitis.

Robotic rectal cancer resection with natural orifice extraction is a recently developed minimally invasive surgery used in the treatment of patients with rectal cancer. However, its safety and feasibility remain undiscussed and controversial. This study reported the clinical outcomes and prognostic factors pertaining to traditional robotic assisted rectal cancer resection alone against that of robotic rectal cancer resection with natural orifice extraction to provide a discussion on this issue. 49 patients who underwent robotic rectal cancer resection with natural orifice extraction and 49 matched patients who underwent conventional robotic assisted rectal cancer resection were systematically analyzed in this study. Regarding the baseline characteristics, after matching, no significant differences were observed between the natural orifice specimen extraction (NOSE) group and the robotic assisted rectal cancer resection (RARC) group. Patients in the NOSE group had a reduced visual analog scale (p p = 0.002) and suffered less surgical stress than those in the RARC group. Moreover, 4 complications were observed in the NOSE group and 7 complications in the RARC group with no significant difference (p = 0.337) in terms of complications. The two groups had a similar survival outcomes, where the 3-year overall survival (p = 0.738) and 3-year progression-free survival (p = 0.986) were all comparable between the two groups. Histological differentiation and T stage could be regarded as independent prognostic factors for 3-year overall survival and 3-year progression-free survival. Robotic rectal cancer resection with natural orifice extraction is a safe and feasible minimally invasive surgery for patients suffering from rectal cancer as it encompasses considerable several advantages. Histological differentiation and T stage may serve as independent prognostic factors for 3-year overall survival and 3-year progression-free survival.

Robotic- vs laparoscopic-assisted proctectomy for locally advanced rectal cancer based on propensity score matching: Short-term outcomes at a colorectal center in China

The diminished level of platelet-activating factor acetylhydrolase (PAFAH) in milk causes an enhanced level of platelet activating factor (PAF) in the skin, leading to a severe hair loss phenotype during neonatal pup's lactation. The deletion of very-low-density-lipoprotein receptor (VLDLR) prevents the expression and secretion of PAFAH. Here we revealed that deletion of Roundabout 4 (ROBO4) in mice ameliorated hair loss phenotype via reducing PAF concentration in skin. As a consequence, the neonatal pups with ROBO4 deletion lactated by mother with VLDLR deletion showed normal hair phenotype during lactation. In details,ROBO4 deletion reduced the protein but not mRNA expression of two PAF synthetic enzymes LPCAT1/LPCAT2 in macrophage as well as the expression of PAF receptor in both macrophage and ocular tissue, but increased PAFAH protein in serum. On the other hand, RNA expression profile analysis in macrophages revealed that the genes involving in oxidative phosphorylation and ribosome obviously decreased their expression in response to ROBO4 deletion. Moreover, through High Performance Liquid Chromatography (HPLC) analysis, we found that ATP concentration also reduced in ROBO4 deletion macrophages. Because ribosome and energy are very important factors for the mRNA translation, we then tested whether ROBO4 deletion affects LPCAT1/LPCAT2 mRNA translation using polyribosome assay. As expected, the mRNA level of LPCAT1/LPCAT2 significantly decreased in polyribosome in ROBO4 deletion macrophage comparing to that of wild type. Additionally, mice with ROBO4 deletion suppressed LPS-induced IL-6 expression as well as the phosphorylation of p44/42 and p65, but enhanced the AKT phosphorylation. Collectively, ROBO4 deletion alleviates PAF- and LPS-mediated inflammation. And above results also indicate PAF signal might be a crosstalk point of ROBO4- and VLDLR-activated pathways.

Promoting intestinal epithelial regeneration remains a major medical challenge. Female patients taking nonsteroidal anti-inflammatory drugs are less likely to have upper gastrointestinal bleeding and ulcers than males. Using a nonsteroidal anti-inflammatory drug-induced intestinal damage mouse model, we verified that female mice recover faster than males following acute intestinal insult. Using ex vivo intestinal organoid cultures, we showed that estrogen is necessary and sufficient in enhancing the female organoid formation from breached isolated crypts via the estrogen receptor β receptor. Thus, estrogen promotes female intestinal epithelial organoid regeneration to lower the incidence of intestinal bleeding and ulceration. Animal studies were approved by University of Utah IACUC under protocol number 16-05012 and 18-02010. Key words: estrogen; non-steroidal anti-inflammatory drugs; regeneration; small bowel; stem cells; upper gastrointestinal bleeding

Breakdown of the blood-central nervous system barrier (BCNSB) is a hallmark of many neuroinflammatory disorders, such as multiple sclerosis (MS). Using a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), we show that endothelial-to-mesenchymal transition (EndoMT) occurs in the CNS before the onset of clinical symptoms and plays a major role in the breakdown of BCNSB function. EndoMT can be induced by an IL-1β-stimulated signaling pathway in which activation of the small GTPase ADP ribosylation factor 6 (ARF6) leads to crosstalk with the activin receptor-like kinase (ALK)-SMAD1/5 pathway. Inhibiting the activation of ARF6 both prevents and reverses EndoMT, stabilizes BCNSB function, reduces demyelination, and attenuates symptoms even after the establishment of severe EAE, without immunocompromising the host. Pan-inhibition of ALKs also reduces disease severity in the EAE model. Therefore, multiple components of the IL-1β-ARF6-ALK-SMAD1/5 pathway could be targeted for the treatment of a variety of neuroinflammatory disorders.

Familial exudative vitreoretinopathy (FEVR) is a severe retinal vascular disease that causes blindness. FEVR has been linked to mutations in several genes associated with inactivation of the Norrin/β-catenin signaling pathway, but these account for only approximately 50% of cases. We report that mutations in CTNNA1 (α-catenin) cause FEVR by overactivating the β-catenin pathway and disrupting cell adherens junctions. Three heterozygous mutations in CTNNA1 (p.F72S, p.R376Cfs*27 and p.P893L) were identified by exome-sequencing. We further demon-strated that FEVR-associated mutations led to overactivation of Norrin/β-catenin signaling due to impaired protein interactions within the cadherin/catenin complex. The clinical features of FEVR were reproduced in mice lacking Ctnna1 in vascular endothelial cells (ECs) or with overactivat-ed β-catenin signaling by an EC-specific gain-of-function allele of Ctnnb1. In isolated mouse lung endothelial cells, both CTNNA1-P893L and F72S mutants failed to rescue either the dis-rupted F-ACTIN arrangement or VE-Cadherin and CTNNB1 distribution. Moreover, we discov-ered that compound heterozygous Ctnna1 F72S and a deletion allele could cause similar pheno-type. Furthermore, a LRP5 mutation, which activates Norrin/β-catenin signaling, was identified in a FEVR family and the corresponding knock-in mice exhibited partial FEVR-like phenotype. Our study demonstrates that precise regulation of β-catenin activation is critical for retinal vascu-lar development and provides new insights into the pathogenesis of FEVR.

SUMMARYThe newly emerged SARS-CoV-2 caused a global pandemic with astonishing mortality and morbidity. The mechanisms underpinning its highly infectious nature remain poorly understood. We report here that SARS-CoV-2 exploits cellular CTP synthetase 1 (CTPS1) to promote CTP synthesis and suppress interferon (IFN) induction. Screening a SARS-CoV-2 expression library identified ORF7b and ORF8 that suppressed IFN induction via inducing the deamidation of interferon regulatory factor 3 (IRF3). Deamidated IRF3 fails to bind the promoters of classic IRF3-responsible genes, thus muting IFN induction. Conversely, a shRNA-mediated screen focused on cellular glutamine amidotransferases corroborated that CTPS1 deamidates IRF3 to inhibit IFN induction. Functionally, ORF7b and ORF8 activate CTPS1 to promote de novo CTP synthesis while shutting down IFN induction. De novo synthesis of small-molecule inhibitors of CTPS1 enabled CTP depletion and IFN induction in SARS-CoV-2 infection, thus impeding SARS-CoV-2 replication. Our work uncovers a strategy that a viral pathogen couples immune evasion to metabolic activation to fuel viral replication. Inhibition of the cellular CTPS1 offers an attractive means for developing antiviral therapy that would be resistant to SARS-CoV-2 mutation.

ARF nucleotide-binding site opener (ARNO) is a guanine nucleotide-exchange factor for ADP-ribosylation factor proteins. ARF nucleotide-binding site opener also binds MyD88, and small-molecule inhibition of ARNO reduces inflammation in animal models of inflammatory arthritis and acute inflammation. However, whether genetic deletion ofTo generateThese data demonstrate that deletion of

The rise in multidrug-resistant (MDR) organisms portends a serious global threat to the health care system with nearly untreatable infectious diseases, including pneumonia and its often fatal sequelae, acute respiratory distress syndrome (ARDS) and sepsis. Gram-negative bacteria (GNB), including Acinetobacter baumannii, Pseudomonas aeruginosa, and carbapenemase-producing Klebsiella pneumoniae (CPKP), are among the World Health Organization’s and National Institutes of Health’s high-priority MDR pathogens for targeted development of new therapies. Here, we show that stabilizing the host’s vasculature by genetic deletion or pharmacological inhibition of the small GTPase ADP-ribosylation factor 6 (ARF6) increases survival rates of mice infected with A. baumannii, P. aeruginosa, and CPKP. We show that the pharmacological inhibition of ARF6-GTP phenocopies endothelium-specific Arf6 disruption in enhancing the survival of mice with A. baumannii pneumonia, suggesting that inhibition is on target. Finally, we show that the mechanism of protection elicited by these small-molecule inhibitors acts by the restoration of vascular integrity disrupted by GNB lipopolysaccharide (LPS) activation of the TLR4/MyD88/ARNO/ARF6 pathway. By targeting the host’s vasculature with small-molecule inhibitors of ARF6 activation, we circumvent microbial drug resistance and provide a potential alternative/adjunctive treatment for emerging and reemerging pathogens.

Reports in the field of robotic surgery for gastric cancer are increasing. However, studies only on patients with advanced gastric cancer (AGC) are lacking. This retrospective study was to compare the short-term outcomes of robotic-assisted distal gastrectomy (RADG) and laparoscopic-assisted distal gastrectomy (LADG) with D2 lymphadenectomy for AGC. From December 2014 to November 2019, 683 consecutive patients with AGC underwent mini-invasive assisted distal gastrectomy. Propensity-score matching (PSM) analysis was conducted to reduce patient selection bias. Short-term outcomes were compared between the two groups. The clinical features were well matched in the PSM cohort. Compared with the LADG group, the RADG group was associated with less operative blood loss, a lower rate of postoperative blood transfusion, less volume of abdominal drainage, less time to remove abdominal drainage tube, retrieved more lymph node, and lower rates of surgical complications and pancreatic fistula (P P > 0.05). This study suggests that RADG is a safe and feasible technique with better short-term outcomes than LADG for AGC.

The rise in multidrug resistant (MDR) organisms portends a serious global threat to the healthcare system with nearly untreatable infectious diseases, including pneumonia and its often fatal sequelae, acute respiratory distress syndrome (ARDS) and sepsis. Gram-negative bacteria (GNB) including Acinetobacter baumannii, Pseudomonas aeruginosa, and carbapenemase-producing Klebsiella pneumoniae (CPKP), are among the World Health Organization and National Institutes of Health’s high priority MDR pathogens for targeted development of new therapies. Here we show that stabilizing the host’s vasculature by genetic deletion or pharmacological inhibition of the small GTPase ADP-ribosylation factor 6 (ARF6) increases survival rates of mice infected with A. baumannii, P. aeruginosa, CPKP pneumonia. We show that pharmacological inhibition of ARF6-GTP phenocopies endothelial-specific Arf6 disruption in enhancing survival of mice with A. baumannii pneumonia, suggesting that inhibition is on target. Finally, we show that the mechanism of protection elicited by these small molecule inhibitors is by restoration of vascular integrity disrupted by GNB lipopolysaccharide (LPS) activation of TLR4/MyD88/ARNO/ARF6 pathway. By targeting the host’s vasculature with small molecule inhibitors of ARF6 activation, we circumvent microbial drug resistance and provide a potential alternative/adjunctive treatment for emerging and re-emerging pathogens.

With the progress of digital and information technology, the rise and rapid development of big data technology has drawn great attention from all quarters. However, there is a general lack of overall planning in the field of catering O2O. Combined with the development and application of catering O2O, this paper analyzes and studies the different levels of the design of the catering O2O cloud platform system. A decision support system for dietary recommendation based on Chinese traditional Chinese medicine theory is described in this research. The theory and method of diet decision support system are analyzed in order to provide a reference for the new method of catering O2O modeling.

This study presents a model-based dynamic optimization strategy for a dual-mode CO2 heat pump coupled with hot and cold thermal storages, which was proposed as a high-efficiency smart grid enabling option in heating and cooling services for buildings or industry. Dynamic optimal control for simultaneously charging of hot and cold thermal storages is very delicate. The optimal control of compressor discharge pressure were commonly used for optimal control of heat pump systems. In this study, the outlet water temperatures of hot and cold tanks are used as indicators in the dynamic optimal strategy for charging of hot and cold storages using a dual-mode heat pump. The Modelica based dynamic model of the coupled system was developed and validated. To optimize the overall coefficient of performance (COP) during energy process, the transient total COP is optimized by genetic algorithm based on Modelica-based modeling of dynamic system. A dynamic optimal control strategy was developed and implemented into an experimental system. Test results show that this developed model-based dynamic optimal control strategy is able to search the optimal transient total COP and optimize the overall COP of such coupled systems during energy charging; and the optimal results is better than those obtained using another two experiment-based methods.

As the travel purpose of non-occupied taxies is to find new passengers rather than to arrive at the destination, large differences exist in the route choice behavior between the occupied and non-occupied taxies. With the assistance of geographic information system (GIS) and taxi-based floating car data (FCD), this paper investigates the behavior differences between occupied and non-occupied taxi drivers with the same origin and destination. Descriptive statistical indexes from the FCD in Shenzhen, China are explored to identify the route choice characteristics of occupied and non-occupied taxies. Then, a conditional logit model is proposed to model the quantitative relationship between drivers’ route choice and the related significant variables. Attributes of the variables related to non-occupied taxies’ observed routes are compared with the case of occupied ones. The results indicate that, compared with their counterparts, non-occupied taxi drivers generally pay more attention to choosing arterial roads and avoiding congested segments. Additionally, they are also found less sensitive to fewer traffic lights and shorter travel time. Findings from this research can assist to improve urban road network planning and traffic management.

A dual-mode CO2 heat pump coupled with hot and cold thermal storages was proposed as a high-efficiency smart grid enabling option for supporting higher penetration levels of intermittent renewables in the energy system. However, the optimal control of simultaneous hot and cold thermal storage systems in real time is very delicate. In this study, a novel system-based optimization method is proposed, in which the COP of such system is simplified with empirical correlation, and a solution for the optimal control vector that maximize the transient system COP is determined using genetic algorithm for global optimization. To evaluate the system-based optimization method, experimental tests were conducted, and a real-time near-optimal control strategy for charging of dual-model energy storage systems was developed and implemented. Practical operation results show that the near-optimal control strategy is able to search the near-optimal transient total COP in real time, and optimize the overall COP of such dual-mode coupled systems during energy charging.

The cost-efficiency of public transport systems is a helpful measure for regulators to establish an effective subsidy mechanism. This study presents a cost-efficiency measurement model of bus routes based on stochastic frontier analysis, taking into account not only heterogeneous outputs but also operating environment factors. In addition, four new operating environment factors that can affect the efficiency of bus routes are introduced. An empirical analysis, based on 39 routes operating in Jiangyin City, China, is presented. It was found that vehicle kilometres have a bigger impact on total operating costs than other parameters such as fixed inputs and fuel price. Operating environment factors such as average bus stop density and the percentage of bus lanes have significant influences on cost-inefficiency differentials. Furthermore, the measurement of cost-efficiency enables identification of the difference between actual total operating costs and efficient total operating costs at a bus route level. The proposed approach could support regulators in setting reasonable cost benchmarks based on efficient total operating costs.

This paper presents an experimental study of the performances of a dual-mode CO 2 heat pump system with thermal storage. This CO 2 system was tested at a variable frequency compressor, variable expansion valve opening as well as variable frequency cooling/heating water pumps. It is found that the performances of CO 2 heat pump with thermal storage can be optimized through optimizing thermal stratification of thermal storage tank coupled with heat pump system performances. High compressor frequency benefits the system performances. Expansion valve opening affects the thermal stratification of thermal storage tanks very slightly. The overall COP reaches a maximum when heating and cooling water flow rates were set as 0.1 m 3 /h and 0.2 m 3 /h respectively at compressor frequency of 50 Hz and expansion valve opening of 330 pulses.

The lack of distinct morphological features of cryptic species is a hard problem for taxonomy, especially when the taxa are closely related with considerable amounts of ancestral polymorphism. Lately, intensive coalescent-based analyses involving multiple loci have become the preferred method to assess the extent of genetic distinctiveness in otherwise phenotypically similar populations. Previously, phylogenetic studies on Pachyhynobius shangchengensis uncovered five extremely deeply divergent clades, which suggested that this species may be a cryptic species complex. In this study, we used the complete mitochondrial genome data and samples from the entire range of stout salamander (Pachyhynobius), as well as publicly available mitochondrial genomes to assess species boundaries within this genus using a suite of diverse methodologies (e.g. general mixed Yule coalescent model, Automatic Barcode Gap Discovery). The phylogenetic relationships recovered two major groups within P. shangchengensis, with one group formed by four of the six extant populations and corresponding to the central and eastern range of the Dabie mountains, while the other group encompassed two other lineages in the north west of the Dabie mountain range. The species delimitation comparison within Pachyhynobius supported the presence of recognized species within the genus, and consensus was observed across methods for the existence of up to five cryptic species within what has been traditionally considered to be P. shangchengensis. While this implies the existence of four taxa in addition to the described P. shangchengensis species, morphological data and life history information are further required to contribute to the species definition. The observed pattern of genetic variation is likely the outcome of a discontinuous habitat combined with niche conservatism, which produced the sky-island effect observed in Pachyhynobius, and which led to formation of a hidden species diversity in this genus.

Nanhai Shengli (NHSL) FPSO, which is serviced in the South China Sea, was dry-docking to upgrade its internal turret mooring system in the first half of 2018, and the mooring system was re-hooked up to the FPSO subsequently in July. The whole installation of the mooring system will last for more than a month. Based on the metocean data of the South China Sea and operation experience, the mooring system installation may encounter typhoon conditions. The NHSL mooring system consists a 10-legs catenary system. The installation sequence of anchor legs may affect the performance of the mooring system under different intensity typhoon conditions. Steps in the design process of anchor leg installation sequence are described in this paper. Based on the tension and offset derived from the time domain mooring analysis, an optimized installation sequence is obtained in order that the mooring system has a better ability to withstand typhoon conditions.

Mooring system life extension for offshore floating production installation has become a hot topic for offshore industry as more and more floating units approach the end of their design lives. Some mooring system life extension projects have already been completed successfully, there are still some issues upon which the mooring industry has not reached consensus, such as field inspection capability/accuracy, uninspected items assessment, component degradation assessment, and acceptance criteria for numerical evaluation. This paper demonstrates two mooring system life extension processes located at Liuhua (LH) 11-1 oil field, following ABS rules as well as industry best practices. It also presents the challenging issues met during this long period of operation, initiated in 2016 and to be completed by end of 2019. Nanhai Shengli (NHSL) FPSO and Nanhai Tiaozhan (NHTZ) FPS are located in Liuhua 11-1 oil field. Both of the mooring systems were installed in 1995 with an originally designed life of 10 years, and already upgraded/replaced partially to extend their lives for continual safety operation. Thus, this is the second life extension for them, and some special challenges are presented. The upgrade of the mooring system is proposed based on inspection, testing, and analysis results for the existing mooring components. This paper focuses on some of the work that has been performed. These experiences may be helpful for the life extension of other similar mooring systems.

This study develops an independent efficiency measurement model framework of bus routes, so as to treat preferably the heterogeneous outputs in a data envelopment analysis (DEA)-based efficiency measurement. It then introduces four new exogenous operating environment factors at the microscopic level of bus routes, and applies the three-stage DEA approach to incorporate operating environment effects and statistical noise into the efficiency measurement framework. The main purpose is to measure impartially the managerial efficiency of bus routes, purged of operating environment effects and statistical noise, and additionally to investigate the operating environment effects on efficiency. An empirical analysis, based on 39 routes operated in Jiangyin City, China, is presented. The principal finding is that the adoption of the independent efficiency measurement model framework is reasonable and has many virtues. Moreover, the proposed approach could substantially provide decision support both for regulators and for producers of bus services. In addition, the operating environment does indeed significantly affect operating efficiency and quality efficiency, especially the latter.

Purpose This study aims to develop an impedance-based drug screening platform that will help identify drugs that can enhance the vascular barrier function by stabilizing vascular endothelial cell junctions. Methods Changes in permeability of cultured human retinal microvascular endothelial cells (HRMECs) monolayer were monitored in real-time with the xCELLigence RTCA system. Using this platform, we performed a primary screen of 2100 known drugs and confirmed hits using two additional secondary permeability assays: the transwell permeability assay and the XPerT assay. The cellular and molecular mechanisms of action and in vivo therapeutic efficacy were also assessed. Results Eleven compounds blocked interleukin 1 beta (IL-1β) induced hyperpermeability in the primary screen. Two of 11 compounds, apigenin and ethaverine hydrochloride, reproducibly blocked multiple cytokines induced hyperpermeability. In addition to HRMEC monolayers, the two compounds stabilized three other types of primary vascular endothelial cell monolayers. Preliminary mechanistic studies suggest that the two compounds stabilize the endothelium by blocking ADP-ribosylation factor 6 (ARF6) activation, which results in enhanced VE-cadherin membrane localization. The two compounds showed in vivo efficacy in an animal model of retinal permeability. Conclusions We developed an impedance-based cellular phenotypic drug screening platform that can identify drugs that enhance vascular barrier function. We found apigenin and ethaverine hydrochloride stabilize endothelial cell junctions and enhance the vascular barrier by blocking ARF6 activation and increasing VE-cadherin membrane localization. Translational relevance The drugs identified from the phenotypic screen would have potential therapeutic efficacy in retinal vascular diseases regardless of the underlying mechanisms that promote vascular leak.

Primary open-angle glaucoma (POAG) is the leading cause of irreversible blindness worldwide and mutations in known genes can only explain 5–6% of POAG. This study was conducted to identify novel POAG-causing genes and explore the pathogenesis of this disease. Exome sequencing was performed in a Han Chinese cohort comprising 398 sporadic cases with POAG and 2010 controls, followed by replication studies by Sanger sequencing. A heterozygous Ramp2 knockout mouse model was generated for in vivo functional study. Using exome sequencing analysis and replication studies, we identified pathogenic variants in receptor activity-modifying protein 2 (RAMP2) within three genetically diverse populations (Han Chinese, German, and Indian). Six heterozygous RAMP2 pathogenic variants (Glu39Asp, Glu54Lys, Phe103Ser, Asn113Lysfs*10, Glu143Lys, and Ser171Arg) were identified among 16 of 4763 POAG patients, whereas no variants were detected in any exon of RAMP2 in 10,953 control individuals. Mutant RAMP2s aggregated in transfected cells and resulted in damage to the AM-RAMP2/CRLR-cAMP signaling pathway. Ablation of one Ramp2 allele led to cAMP reduction and retinal ganglion cell death in mice. This study demonstrated that disruption of RAMP2/CRLR-cAMP axis could cause POAG and identified a potential therapeutic intervention for POAG.

The activation of the small GTPase ARF6 has been implicated in promoting several pathological processes related to vascular instability and tumor formation, growth, and metastasis. ARF6 also plays a vital role during embryonic development. Recent studies have suggested that ARF6 carries out these disparate functions primarily by controlling protein trafficking within the cell. ARF6 helps direct proteins to intracellular or extracellular locations where they function in normal cellular responses during development and in pathological processes later in life. This transport of proteins is accomplished through a variety of mechanisms, including endocytosis and recycling, microvesicle release, and as yet uncharacterized processes. This Commentary will explore the functions of ARF6, while focusing on the role of this small GTPase in development and postnatal physiology, regulating barrier function and diseases associated with its loss, and tumor formation, growth, and metastasis.

Introduction: Uveal melanoma is the most common primary ocular malignancy and there is currently no effective treatment for metastatic uveal melanoma largely because of the lack of understanding of the molecular mechanisms underlying this cancer. Although activating oncogenic mutations in GNAQ and GNA11 are present in 95% of uveal melanoma tumors, virtually nothing is known about the molecular mechanisms that drive oncogenesis in the remaining 5% of uveal melanomas that possess only wild type Gαq alleles and the molecular and cellular mechanisms that promote metastasis have not yet been discovered. This latter deficit in knowledge is extremely vexing, given that metastatic disease causes death in most uveal melanoma patients. We have recently shown that ADP-ribosylation factor 6 (ARF6), a small GTPase, is an immediate downstream effector of oncogenic Gαq and controls the entire major signaling pathways known to drive Gαq-mediated tumor formation and growth in uveal melanoma cells that have an activating mutation in Gαq. Moreover, in cutaneous melanoma, stimulation of the receptor by WNT5A activates ARF6 and controls metastasis via the release of β-catenin from N-cadherin and β-catenin's subsequent nuclear transport. Previous studies have also demonstrated that ARF6 plays a role in the internalization of various classes of membrane receptors including receptor tyrosine kinase (RTK) and G protein coupled receptor (GPCR) to control signal transduction, and WNT5A and its receptor promote tumor invasion and metastasis via receptor internalization in human cancers. Based on these intriguing findings, we postulate that ARF6 might be functioning to control tumor invasion and metastasis in uveal melanomas. Experimental Procedures: To test our hypothesis, we conducted cell invasion assays on uveal melanoma cell lines while targeting ARF6 expression by siRNA or ARF6 activity by pharmacological inhibition. We then proceeded with cell fractionation to look for any differences in the properties of subcellular components in treated cells compared to untreated cells. Unpublished findings: Here we show that WNT5A is expressed at high levels in Gαq wild type uveal melanoma cells and that WNT5A activates ARF6 through ROR2 receptor. When we block ARF6 expression by knockdown or ARF6 activity with a small molecule inhibitor, uveal melanoma invasion is significantly reduced. Notably, ARF6 is necessary for WNT5A-mediated ROR2 internalization. Conclusions: This work indicates that ARF6 functions in the internalization of WNT5A receptor to activate the signaling pathways that drive invasion and metastasis in uveal melanoma. These results would suggest that targeting ARF6 activation might be an effective therapy for the treatment of all uveal melanomas, regardless of their mutational status for Gαq. Citation Format: Donghan Shin, Coulson P. Rich, Lehi Acosta, Jackson R. Richards, Jae Hyuk Yoo, Allie H. Grossmann, Zongzhong Tong, Alan L. Mueller, Weiquan Zhu, Dean Y. Li, Shannon J. Odelberg. ARF6 controls WNT5A receptor internalization to promote uveal melanoma invasion and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5157.

Cerebral cavernous malformation (CCM) is a common vascular dysplasia that affects both systemic and central nervous system blood vessels. Loss of function mutations in the CCM2 gene cause CCM. Here we show that targeted disruption of Ccm2 in mice results in failed lumen formation and early embryonic death through an endothelial cell autonomous mechanism. We show that CCM2 regulates endothelial cytoskeletal architecture, cell-to-cell interactions and lumen formation. Heterozygosity at Ccm2, a genotype equivalent to that in human CCM, results in impaired endothelial barrier function. On the basis of our biochemical studies indicating that loss of CCM2 results in activation of RHOA GTPase, we rescued the cellular phenotype and barrier function in heterozygous mice with simvastatin, a drug known to inhibit Rho GTPases. These data offer the prospect for pharmacological treatment of a human vascular dysplasia with a widely available and safe drug.