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In response to external threatening signals, animals evolve a series of defensive behaviors that depend on heightened arousal. It is believed that arousal and defensive behaviors are coordinately regulated by specific neurocircuits in the central nervous system. The ventral tegmental area (VTA) is a key structure located in the ventral midbrain of mice. The activity of VTA glutamatergic neurons has recently been shown to be closely related to sleep–wake behavior. However, the specific role of VTA glutamatergic neurons in sleep–wake regulation, associated physiological functions, and underlying neural circuits remain unclear. In the current study, using an optogenetic approach and synchronous polysomnographic recording, we demonstrated that selective activation of VTA glutamatergic neurons induced immediate transition from sleep to wakefulness and obviously increased the amount of wakefulness in mice. Furthermore, optogenetic activation of VTA glutamatergic neurons induced multiple defensive behaviors, including burrowing, fleeing, avoidance and hiding. Finally, viral-mediated anterograde activation revealed that projections from the VTA to the central nucleus of the amygdala (CeA) mediated the wake- and defense-promoting effects of VTA glutamatergic neurons. Collectively, our results illustrate that the glutamatergic VTA is a key neural substrate regulating wakefulness and defensive behaviors that controls these behaviors through its projection into the CeA. We further discuss the possibility that the glutamatergic VTA-CeA pathway may be involved in psychiatric diseases featuring with excessive defense.

Due to the increasing prevalence of drug-resistant fungi and the limitations of current treatment strategies to fungal infections, exploration and development of new antifungal drugs or substituents are necessary. In the study, a novel antimicrobial peptide, named Sparamosin, was identified in the mud crab Scylla paramamosain, which contains a signal peptide of 22 amino acids and a mature peptide of 54 amino acids. The antimicrobial activity of its synthetic mature peptide and two truncated peptides (Sparamosin1–25 and Sparamosin26–54) were determined. The results showed that Sparamosin26–54 had the strongest activity against a variety of Gram-negative bacteria, Gram-positive bacteria and fungi, in particular had rapid fungicidal kinetics (killed 99% Cryptococcus neoformans within 10 min) and had potent anti-biofilm activity against C. neoformans, but had no cytotoxic effect on mammalian cells. The RNA-seq results showed that after Sparamosin26–54 treatment, the expression of genes involved in cell wall component biosynthesis, cell wall integrity signaling pathway, anti-oxidative stress, apoptosis and DNA repair were significantly up-regulated, indicating that Sparamosin26–54 might disrupt the cell wall of C. neoformans, causing oxidative stress, DNA damage and cell apoptosis. The underlying mechanism was further confirmed. Sparamosin26–54 could bind to several phospholipids in the cell membrane and effectively killed C. neoformans through disrupting the integrity of the cell wall and cell membrane observed by electron microscope and staining assay. In addition, it was found that the accumulation of reactive oxygen species (ROS) increased, the mitochondrial membrane potential (MMP) was disrupted, and DNA fragmentation was induced after Sparamosin26–54 treatment, which are all hallmarks of apoptosis. Taken together, Sparamosin26–54 has a good application prospect as an effective antimicrobial agent, especially for C. neoformans infections.

Scyreprocin is an antimicrobial peptide first identified in the mud crab Scylla paramamosain. Herein, we showed that its recombinant product (rScyreprocin) could significantly inhibit the growth of human lung cancer NCI-H460 cells (H460), but showed no cytotoxicity to human lung fibroblasts (HFL1). rScyreprocin was a membrane-active peptide that firstly induced the generation of reactive oxygen species (ROS) in H460, and led to endoplasmic reticulum stress and Ca2+ release, which resulted in mitochondrial dysfunction and subsequently activation of caspase-3 cascades, and ultimately led to apoptosis. The comprehensive results indicated that rScyreprocin exerted anticancer activity by disrupting cell membrane and inducing apoptosis. The in vivo efficacy test demonstrated that intratumoral injection of rScyreprocin significantly inhibited the growth of H460 xenografts, which was close to that of the cisplatin (inhibition rate: 69.94% vs. 80.76%). Therefore, rScyreprocin is expected to become a promising candidate for the treatment of lung cancer.

Antimicrobial peptides (AMPs) identified in the reproductive system of animals have been widely studied for their antimicrobial activity, but only a few studies have focused on their physiological roles. Our previous studies have revealed the in vitro antimicrobial activity of two male gonadal AMPs, SCY2 and scyreprocin, from mud crab Scylla paramamosain. Their physiological functions, however, remain a mystery. In this study, the two AMPs were found co-localized on the sperm apical cap. Meanwhile, progesterone was confirmed to induce acrosome reaction (AR) of mud crab sperm in vitro, which intrigued us to explore the roles of the AMPs and progesterone in AR. Results showed that the specific antibody blockade of scyreprocin inhibited the progesterone-induced AR without affecting intracellular Ca2+ homeostasis, while the blockade of SCY2 hindered the influx of Ca2+. We further showed that SCY2 could directly bind to Ca2+. Moreover, progesterone failed to induce AR when either scyreprocin or SCY2 function was deprived. Taken together, scyreprocin and SCY2 played a dual role in reproductive immunity and sperm AR. To our knowledge, this is the first report on the direct involvement of AMPs in sperm AR, which would expand the current understanding of the roles of AMPs in reproduction.

Natural antimicrobial peptides (AMPs) are potential antibiotic alternatives. Marine crustaceans are thought to generate more powerful and various AMPs to protect themselves from infections caused by pathogenic microorganisms in their complex aquatic habitat, thus becoming one of the most promising sources of AMPs or other bioactive substances. In the study, a novel protein was identified as an interacting partner of male-specific AMP SCY2 in Scylla paramamosain and named scyreprocin. The recombinant product of scyreprocin (rScyreprocin) was successfully expressed in Escherichia coli. rScyreprocin exerted potent, broad-spectrum antifungal, antibacterial, and anti-biofilm activity (minimum inhibitory concentrations from 0.5 to 32 μM) through differential modes of action, including disruption of cell membrane integrity and induction of cell apoptosis, and has rapid bactericidal (in 0.5–2 h) and fungicidal (in 8–10 h) kinetics. In addition to its fungicidal activity against planktonic fungi, rScyreprocin also prevented the adhesion of fungal cells, inhibited biofilm formation, and eradicated the mature biofilms. Moreover, rScyreprocin showed a profound inhibitory effect on spore germination of Aspergillus spp. (minimum inhibitory concentrations from 4 to 8 μM). This peptide was not cytotoxic to murine and mammalian cells and could increase the survival rate of Oryzias melastigma under the challenge of Vibrio harveyi. Taken together, the novel AMP scyreprocin would be a promising alternative to antibiotics used in aquaculture and medicine.

Pubertal molt is a vital stage in the cultivation of mature female crabs in the aquacultural industry of Scylla paramamosain. Since fasting occurs during molting, which requires a large supply of energy, internal energy reserves are critical. However, the dynamics of energy supply during pubertal molt is not clear. This study focuses on the variations of carbohydrates and lipids in serum during the pubertal molt of S. paramamosain via a metabolomics approach. Eleven lipid or carbohydrate metabolic pathways were significantly influenced postmolt. A remarkable decrease in carbohydrates in serum suggested that free sugars were consumed for energy. A significant decrease in glucose and alpha-d-glucosamine 1-phosphate showed that chitin synthesis exhausted glycogen, resulting in insufficient glucose supply. An increase in l-carnitine and acetylcarnitine, and a significant decrease in 15 fatty acyls and 8 glycerophosphocholines in serum indicated that carnitine shuttle was stimulated, and β-oxidation was upregulated postmolt. In addition, astaxanthin, ponasterone A, and riboflavin in serum were significantly decreased postmolt. Eleven potential metabolite biomarkers were identified for pubertal molt. Taken together, carbohydrates and lipids were possibly major energy reserves in pubertal molt. This study suggests that an increase in carbohydrate and lipid levels in crab feed may alleviate the effects of fasting during molt and improve farm productivity in mature female crabs.

Mesenchymal stromal cells (MSCs) showcase remarkable immunoregulatory capabilities in vitro, positioning them as promising candidates for cellular therapeutics. However, the process of administering MSCs and the dynamic in vivo environment may impact the cell–cell and cell–matrix interactions of MSCs, consequently influencing their survival, engraftment, and their immunomodulatory efficacy. Addressing these concerns, hydrogel encapsulation emerges as a promising solution to enhance the therapeutic effectiveness of MSCs in vivo. Hydrogel, a highly flexible crosslinked hydrophilic polymer with a substantial water content, serves as a versatile platform for MSC encapsulation. Demonstrating improved engraftment and heightened immunomodulatory functions in vivo, MSCs encapsulated by hydrogel are at the forefront of advancing therapeutic outcomes. This review delves into current advancements in the field, with a focus on tuning various hydrogel parameters to elucidate mechanistic insights and elevate functional outcomes. Explored parameters encompass hydrogel composition, involving monomer type, functional modification, and co-encapsulation, along with biomechanical and physical properties like stiffness, viscoelasticity, topology, and porosity. The impact of these parameters on MSC behaviors and immunomodulatory functions is examined. Additionally, we discuss potential future research directions, aiming to kindle sustained interest in the exploration of hydrogel-encapsulated MSCs in the realm of immunomodulation.

Allograft rejection is one of the obstacles in achieving a successful vascularized composite allotransplantation (VCA). Treatments of graft rejection with lifelong immunosuppression (IS) subject the recipients to a lifelong risk of cancer development and opportunistic infections. Cell therapy has recently emerged as a promising strategy to modulate the immune system, minimize immunosuppressant drug dosages, and induce allograft tolerance. In this review, the recent works regarding the use of cell therapy to improve allograft outcomes are discussed. The current data supports the safety of cell therapy. The suitable type of cell therapy in allotransplantation is clinically dependent. Bone marrow cell therapy is more suitable for the induction phase, while other cell therapies are more feasible in either the induction or maintenance phase, or for salvage of allograft rejection. Immune cell therapy focuses on modulating the immune response, whereas stem cells may have an additional role in promoting structural regenerations, such as nerve regeneration. Source, frequency, dosage, and route of cell therapy delivery are also dependent on the specific need in the clinical setting.

Solid organ and composite tissue allotransplanation have been widely applied to treat end-stage organ failure and massive tissue defects, respectively. Currently there are a lot of research endeavors focusing on induction of transplantation tolerance, to relieve the burden derived from long-term immunosuppressant uptake. The mesenchymal stromal cells (MSCs) have been demonstrated with potent immunomodulatory capacities and applied as promising cellular therapeutics to promote allograft survival and induce tolerance. As a rich source of adult MSCs, adipose tissue provides additional advantages of easy accessibility and good safety profile. In recent years, the stromal vascular fraction (SVF) isolated from adipose tissues following enzymatic or mechanical processing without in vitro culture and expansion has demonstrated immunomodulatory and proangiogenic properties. Furthermore, the secretome of AD-MSCs has been utilized in transplantation field as a potential “cell-free” therapeutics. This article reviews recent studies that employ these adipose-derived therapeutics, including AD-MSCs, SVF, and secretome, in various aspects of organ and tissue allotransplantation. Most reports validate their efficacies in prolonging allograft survival. Specifically, the SVF and secretome have performed well for graft preservation and pretreatment, potentially through their proangiogenic and antioxidative capacities. In contrast, AD-MSCs were suitable for peri-transplantation immunosuppression. The proper combination of AD-MSCs, lymphodepletion and conventional immunosuppressants could consistently induce donor-specific tolerance to vascularized composite allotransplants (VCA). For each type of transplantation, optimizing the choice of therapeutics, timing, dose, and frequency of administration may be required. Future progress in the application of adipose-derived therapeutics to induce transplantation tolerance will be further benefited by continued research into their mechanisms of action and the development of standardized protocols for isolation methodologies, cell culture, and efficacy evaluation.

IntroductionVascularized bone marrow (VBM) is essential in tolerance induction through chimerism. We hypothesized that the inclusion of VBM contributes to the induction of mystacial pad allotransplantation tolerance.MethodIn this study, 19 VBM, nine mystacial pad, and six sequential VBM and mystacial pad allografts were transplanted from Brown Norway (BN) rats to Lewis (LEW) rats to test our hypothesis. The VBM recipients were divided into antilymphocyte serum (ALS) monotherapy group (two doses of ALS on day 3 pretransplantation and day 1 posttransplantation), immunosuppressant group [a week of 2 mg/kg/day tacrolimus (Tac) and 3 weeks of 3 mg/kg/day rapamycin (RPM)], and combined therapy group. The mystacial pad recipients were divided into VBM and non-VBM transplantation groups, and both groups were treated with an immunosuppression regimen that consists of ALS, Tac, and RPM. For the recipients of sequential VBM and mystacial pad allotransplantations, additional Tac was given 1 week after mystacial pad transplantation. Allograft survival, donor-specific tolerance, and chimerism level were evaluated.ResultsWith the administration of ALS and short-term Tac and RPM treatments, VBM recipients demonstrated long-term graft survival (>120 days) with persistent chimerism for 30 days. CD3+ T cells from tolerant rats showed donor-specific hyporesponsiveness and tolerance to donor skin grafts but not to third-party counterparts. Furthermore, mystacial pad graft recipients with VBM transplantation exhibited a higher allograft survival rate than those without VBM transplantation [median survival time (MST) >90 days vs. 70 days, p < 0.05].ConclusionThis study demonstrated that VBM transplantation is an efficient strategy to induce and maintain donor-specific tolerance for an osseous-free allograft.

Summary: Background: Visceral fat is considered important in the pathogenesis of metabolic syndrome (MS). Here, we developed a novel method for determining visceral fat by measuring liver–kidney space (LKS) on abdominal sonography and expanded its utilization in the elderly to predict MS. Methods: To assess the correlation between the LKS and MS, 317 consecutive outpatients scheduled for health evaluation were retrospective analyzed. Anthropometric measurements, blood pressure, fasting blood glucose levels, and lipid profiles were obtained following standard protocols. On sonography, the thickness of visceral fat between the liver and right kidney was measured. We also compared its accuracy to predict MS with sonographic fatty liver changes. A total of 72 elderly patients older than 65 years were evaluated (mean age: 66.02 [65–83]). Results: In the current study, LKS = 4 mm enabled a better prediction of MS. The area under the receiver operating characteristic curve was 0.626. The sensitivity and specificity for the presence of visceral fat to predict MS in the elderly were 0.58 and 0.73, respectively. The accuracy to predict MS was 68.1% for the measurement of visceral fat compared with 59.6% for sonographic fatty liver change in the elderly. Conclusion: Measuring LKS by sonography may be a practical method for evaluating visceral fat in the elderly and for predicting MS better than sonographic fatty liver changes. LKS was more associated with abdominal girth and BMI in the elderly from the study supporting the observation that LKS are well correlated with general adiposity. Keywords: obesity, body mass index, metabolic syndrome, sonography, visceral fat

Chimerism has been associated with the induction and maintenance of tolerance to vascularized composite allotransplants (VCA). Although most VCA studies have examined chimerism using flow cytometry, we proposed that precision in the measurement of chimerism may be better approximated when complimentary polymerase chain reaction (PCR) is applied to a specific short tandem repeat (STR). We identified a STR, D10Rat25, which exhibited a ~20 bp difference in length between two rat strains (BN and LEW) often utilized as the donor and recipient in many allotransplantation studies. D10Rat25 was PCR-amplified and quantified with capillary electrophoresis. With pure LEW and BN DNA, a standard curve was constructed to measure chimerism with good linearity. When applied to rat VCA, the relationship between systematic (in peripheral blood) or local (at specific organ/tissues) chimerism to allograft outcomes was noted. We found that peripheral chimerism was elevated by up to ~9% postoperative month 1 (POM 1) but then reduced regardless of the final VCA outcome. However, differences in VCA skin chimerism between early rejection and POM 1 (shown as ΔChimerismPOM1-ER) were notable with respect to VCA outcomes. ROC analysis identified the optimum cutoff value as 17.7%. In summary, we have developed a reliable method to quantify the percentage of BN cells/DNA in BN-LEW chimeras. The detection limit was characterized, and the acquired data were comparable with flow cytometry. This method can be applied to solid organ and composite tissue allotransplantation studies.

An innovative immunosuppressant with a minimally invasive delivery system has emerged in the biomedical field. The application of biodegradable and biocompatible polymer forms, such as hydrogels, scaffolds, microspheres, and nanoparticles, in transplant recipients to control the release of immunosuppressants can minimize the risk of developing unfavorable conditions. In this review, we summarized several studies that have used implantable immunosuppressant delivery to release therapeutic agents to prolong allograft survival. We also compared their applications, efficacy, efficiency, and safety/side effects with conventional therapeutic-agent administration. Finally, challenges and the future prospective were discussed. Collectively, this review will help relevant readers understand the different approaches to prevent transplant rejection in a new era of therapeutic agent delivery.

Mesenchymal stromal cells (MSCs) are tissue-derived progenitor cells with immunomodulatory as well as multilineage differentiation capacities, and have been widely applied as cellular therapeutics in different disease systems in both preclinical models and clinical studies. Although many studies have applied MSCs in different types of allotransplantation, the efficacy varies. It has been demonstrated that preconditioning MSCs prior to in vivo administration may enhance their efficacy. In the field of organ/tissue allotransplantation, many recent studies have shown that preconditioning of MSCs with (1) pretreatment with bioactive factors or reagents such as cytokines, or (2) specific gene transfection, could prolong allotransplant survival and improve allotransplant function. Herein, we review these preconditioning strategies and discuss potential directions for further improvement.

BACKGROUND:Tipping the balance toward regulatory T cells (Tregs) through adoptive cell therapy has shown promise to induce transplantation tolerance. Although such strategy has been explored in many mice organ transplantation studies, less knowledge was available in rat systems. Furthermore, the behaviors of the transferred cells have not been well studied in real-time fashion. METHODS:Tregs from naïve LEW rats were purified in two steps with the autoMACS system. Immunosuppression potential of these cells was examined with mixed lymphocyte reaction. Following stimulation by the alloantigen in vitro, the purified Tregs were infused into the recipients of vascularized composite allotransplantation (VCA). Secondary allogeneic skin grafting challenge was performed on the recipients with long-term survived VCA. Live optical imaging was performed to track luciferase-expressing Tregs following infusion to the VCA recipients. Expression of relevant molecules was studied by flow cytometry or quantitative RT-PCR. RESULTS:Rat Tregs were enriched following two-step cell sorting and showed immunosuppressive capacity. Upon infusion into the VCA recipients that have been treated with antilymphocyte serum and short-term Cyclosporin A, the antigen-stimulated Tregs significantly prolonged VCA survival and induced donor-specific tolerance. Tracking of the infused bioluminescent Tregs showed their specific homing to lymph nodes, and then to the VCAs. Following secondary skin grafting, Tregs specifically gathered at the donor-derived skin that was not rejected by the recipient. The in vivo migratory pattern coincided with the altered expression of cell surface molecules of CD62L, CD103, CD134, and CD278, following donor-antigen stimulation. Elevated expression of CCR4 and CCL22 in allograft may also participate in recruiting Tregs for maintenance of VCA survival and promoting donor-specific tolerance. CONCLUSION:Sorted Tregs induced donor-specific tolerance to VCA in rats. Live cell tracking demonstrated that activated CD4+CD25+FoxP3+ Tregs targeted primarily to the lymph nodes and VCA. The Tregs migrated to the secondary grafted donor skin and contributed to the maintenance of donor-specific tolerance. These behaviors were associated with phenotypic changes induced by donor antigen stimulation. Increased expression of CCR4 and CCL22 in VCA skin may also be relevant.

Tacrolimus is an immunosuppressive agent for acute rejection after allotransplantation. However, the low aqueous solubility of tacrolimus poses difficulties in formulating an injection dosage. Polypeptide thermosensitive hydrogels can maintain a sustained release depot to deliver tacrolimus. The copolymers, which consist of poloxamer and poly(l-alanine) with l-lysine segments at both ends (P−Lys−Ala−PLX), are able to carry tacrolimus in an in situ gelled form with acceptable biocompatibility, biodegradability, and low gelling concentrations from 3 to 7 wt %. By adding Pluronic F-127 to formulate a mixed hydrogel system, the drug release rate can be adjusted to maintain suitable drug levels in animals with transplants. Under this formulation, the in vitro release of tacrolimus was stable for more than 100 days, while in vivo release of tacrolimus in mouse model showed that rejection from skin allotransplantation was prevented for at least three weeks with one single administration. Using these mixed hydrogel systems for sustaining delivery of tacrolimus demonstrates advancement in immunosuppressive therapy.

Background/purpose: Nonalcoholic fatty liver disease (NAFLD) is a common condition comprising a wide spectrum of liver damage strongly associated with type 2 diabetes, obesity, and hyperlipidemia. The pathogenesis of fatty liver is multifactorial, and it has been suggested that the presence of insulin resistance (IR) is an essential requirement for the accumulation of hepatocellular fat. Although NAFLD may affect people of any age, in general, increasing age is associated with increasing prevalence. The aim of this study was to determine the prevalence of fatty liver and its influence on age and sex; and to assess the association of different degrees of fatty liver to IR and metabolic syndrome. Materials and methods: The study was performed in 8350 alcohol- and virus-negative individuals who underwent routine physical check-up at the health evaluation centre of Mackay Memorial Hospital, from February 2004 to May 2009. They underwent clinical examination, anthropometry, biochemical tests including serum fasting insulin, and routine liver ultrasonography. Steatosis was graded as absent, mild, moderate, or severe. Results: The overall prevalence of fatty liver was 34.40% with the prevalence of fatty liver being significantly higher in males than in females (22.34 vs. 12.06%, p = 0.015). A progressive increase in the means of a homeostasis model assessment of IR (HOMA-IR), body mass index, systolic blood pressure, plasma triglyceride, alanine aminotransferase, low-density lipoprotein-cholesterol and glucose level and decrease in high-density lipoprotein-cholesterol (p

Background: Nonalcoholic fatty liver disease (NAFLD), which represents metabolic abnormality and reflects extra visceral fat deposition, has been shown to be a threat to public health and associated with cardiovascular risks. However, data regarding the differences in clinical presentation and related metabolic abnormalities in the aging group population remained scarce in the Taiwanese population. Methods: We subsequently examined 8,658 subjects participating general health evaluation in Mackay Memorial hospital from 2003 to 2007. Baseline characteristics, anthropometrics, medical history, and biochemical markers were all collected. Abdominal ultrasonography was performed in all subjects. Two estimated cardiovascular risk scores were calculated according to criteria of the U.S. National Cholesterol Education Program Adult Treatment Panel III as metabolic score and Framingham risk score. Univariate logistic regression model was used to examine whether the baseline characteristics, anthropometrics, histories, and biochemical markers were independently associated with NAFLD from various age groups (young vs. aging groups) classified by 60 years of age. Respective receiver operating characteristic curves (ROC) with area under the curve were generated to test the capability of both cardiovascular risk scores in NAFLD discrimination from different age groups. Results: Totally 7,204 subjects (mean age: 44.5±11 years, 36% female) were finally enrolled in our study. Subjects with NAFLD were observed to have high body weight, body mass index, and circumferential waist and significantly abnormal biochemical markers accompanying worsening lipid profiles when compared with those without NAFLD in both the young and aging group populations (all p

Background: Abdominal wall, one of the most commonly transplanted composite tissues, is less researched and lacking animal models. Its clinical necessities were emphasized in multiple case series to reconstruct large abdominal defects. Previous animal models have only studied components of the abdominal wall transplant. We describe findings from a new model that more likely reflect clinical transplantation. Methods: Full-thickness hemiabdominal wall flap was procured from Brown Norway (BN) rats and transplanted to an orthotopic defect on Lewis rats. Three groups were studied: group 1: Lewis to Lewis syngeneic; group 2: BN to Lewis control; and group 3: BN to Lewis with postoperative cyclosporine. Vascular imaging and cross vessel section were performed along with full-thickness abdominal wall. Immune cell profiling with flow cytometry at different time points was studied in all groups. Results: Syngeneic group had no rejection. Control group consistently showed rejection around postoperative day 6. With cyclosporine treatment, however, transplant and recipient tissue integration was observed. Flow cytometry revealed that innate immunity is responsible for the initial inflammatory events following abdominal wall engraftment. Adaptive immunity cells, specifically interferon-γ-producing T helper (Th) 1 and interleukin-17-producing Th17 cells, dramatically and positively correlate with rejection progression of abdominal wall transplants. Conclusions: Technical, histological, and immunological aspects of a new rat model are described. These results give clues to what occurs in human abdominal wall transplantation. In addition, Th1, a proinflammatory cell, was found to be a potential biomarker for allograft rejection.

Abstract A new method was used to study the performance of pile across cave. This paper investigated the vertical bearing characteristics of piles cross caves using centrifugal model tests and a theoretical model of sensitivity. Twelve pile scenarios were selected, the first was a conventional pile, 24 cm long and 2.5 cm in diameter, with no karst cave as a control. In the other eleven scenarios the piles passed through karst caves of four different heights, of four different spans, and three different numbers of caves. The results reveal that increasing the height, span, and number of caves all are negative for vertical ultimate bearing capacity of piles. The axial force and unit shaft resistance of piles are great different. According to the ratios of the tip and shaft resistance, caves change the type of piles. The sensitivity of vertical ultimate bearing capacity to these factors from high to low is height, number, and span of caves. Importantly, the bearing characteristics of piles decrease faster once the height of the prototype karst cave is higher than 9 m, but decreases slowly when the cave’s span is greater than 9 m × 9 m.

Piston prover is widely used as a standard gas flow device for its advantages of high accuracy in standard volume, flow stability and repeatability. The applications of the conventional piston provers are limited by the maximum calibration flow generated by the piston cylinder volume. In the current paper, a reciprocating double-pistons gas prover was proposed, which can provide a continuous standard flow to calibrate meters like critical nozzles and so on. There are two pistons within the proposed prover, which could be operated in three modes: single-piston mode, double-pistons parallel mode and double-pistons reciprocating mode. The working mechanisms of all three modes were presented and the mathematical model was also introduced, especially for the double-pistons reciprocating mode. Then experiments were carried out to validate the new calibrating method; it is shown that the proposed reciprocating double-pistons gas prover is capable of precisely calibrating gas flow meters. Comparisons between the experimental results and data obtained using the piston prover based on the balance adjustment principle are also presented. [ABSTRACT FROM AUTHOR]