We propose that mechanical force drives fluid secretion through the tight junction complex via a “one-way check valve” that can be regulated. This pathway of filtration secretion complements chloride-coupled fluid secretion during high-volume fluid flow. The role of filtration secretion in the genesis of diarrhea in intact animals needs further study. Our findings may explain a potential linkage between intestinal motility and intestinal fluid dynamics.Studies of fluid secretion by the small intestine are dominated by the coupling with ATP-dependent generation of ion gradients, whereas the contribution of filtration secretion has been overlooked, possibly by the lack of a known mechanistic basis. We measured apical fluid flow and generation of hydrostatic pressure gradients by epithelia of cultured mouse enterocytes, Caco-2 and T-84 cells, and fibroblasts exposed to mechanical force provided by vigorous aeration and in response to ion gradients, inhibitors of ion channels and transporters and in vitro using intact mouse and rat small intestine. We describe herein a paracellular pathway for unidirectional filtration secretion that is driven by mechanical force, requires tight junctions, is independent of ionic and osmotic gradients, generates persistent hydrostatic pressure gradients, and would contribute to the fluid shifts that occur during digestion and diarrhea. Zinc inhibits the flow of fluid and the paracellular marker fluorescein isothyocyanate conjugated dextran (MW = 4 kD) across epithelia of cultured enterocytes (>95%;  The current understanding of the bidirectional movement of fluid into and out of the small intestine involves ATP-dependent generation of ion and solute gradients by the epithelium that drive osmotic flow through paracellular and transcellular pathways. The active uptake of glucose and other solutes ,2,3 creaAn alternative concept known as filtration secretion emerged from in vitro and in vivo studies conducted 40 to 50 years ago. Movement of fluid into the lumen was increased by imposing pressure on the serosal surface,  increasiWhile characterizing rheogenic glucose transport by low-resistance (“leaky”) epithelial monolayers prepared from freshly harvested murine enterocytes cultured on inserts with 3 µm pores , we wereAll phases of the research that used animals adhered to the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University of Memphis Institutional Animal Care and Use Committee. C57Bl/6 mice, 8–12 weeks of age, were obtained from a breeding colony maintained at the University of Memphis. Sprague–Dawley rats were purchased from a commercial supplier . The mice and rats were maintained under standard environmental conditions and fed a standard laboratory rodent chow.2 asphyxiation, the entire small intestine from the pyloric sphincter to the ileocolonic junction was removed, flushed with cold mammalian Ringer’s solution, and everted. The enterocytes were harvested following the method of Kimura et al. [®; HyClone; Logan, UT, USA) and an enzyme-free solution  for 15–30 min at 37 °C with gentle agitation to release enterocytes from the villus epithelium. The suspension was filtered through cheesecloth, and the cells were sedimented, and rinsed three times with cold DPBS. This approach typically yields 15–30 million enterocytes per small intestine that were suspended at a density of 10 million enterocytes per ml in cold low-glucose Dulbecco’s modified Eagle’s medium  with 10% sterile filtered fetal bovine serum (FBS), 1 mL of a combination of penicillin (100 U/mL) and streptomycin (100 μg/mL), 5 mL of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid , and a commercial combination of insulin, transferrin, and selenium . The enterocytes were seeded onto uncoated 0.6 cm2 polycarbonate membrane inserts  at a density of 1.5 million cells per insert and incubated for 7–9 h at 37 °C in 95% room air with 5% CO2.After a mouse was euthanized by COa et al. . Briefly2) and are capable of rheogenic glucose uptake.The enterocytes harvested from each mouse intestine were used to prepare 6 or more inserts. The plated cells rapidly settled and within 6 h were reorganized into a polarized epithelium with distinct tight junctions, and with microvilli restricted to the outer surface. All of the studies were performed after 7 to 9 h of incubation while the cells remain viable and carrier-mediated glucose transport is maximal . Like th® HTB-37™ Manassas, VA, USA) and T84 (ATCC® CCL-248™) as representative colonic cell lines. We also cultured fibroblasts  because they do not form tight junctions. The Caco-2 and T84 cells and fibroblasts were seeded on uncoated 0.6 cm2 polycarbonate membrane inserts with 12 μm pores and cultured using the same media and conditions. Confluence was confirmed visually and the formation of the tight junction complexes by the Caco-2 and T84 cell was verified by increased resistance (300–500 Ohms/cm2 for Caco-2 and 1000–3000 Ohms/cm2 for T84 cells).The majority of cell-based studies of intestinal chloride secretion use cell lines derived from the colon that form more extensive tight junctions, resulting in epithelia that have higher electrical resistance than the epithelia prepared from harvested enterocytes. We used Caco-2  with the epithelium, and hence the apical side, exposed to the right chamber and the underside of the insert, hence the basolateral side and membrane, exposed to the left chamber. Placing inserts in the reverse orientation simply reversed the direction of flow.2 1.2 KHsPO4, 1.2 MgSO4, and 20 NaHCO3; 290 mosm; pH = 7.3–7.4 when aerated with 95% O2 with 5% CO2. Fluid flow was measured at 5 min intervals as the increase in fluid level in the apical chamber during which the chambers were aerated with a gas mixture of 95% O2 and 5% CO2. After the gas was turned off, the change in fluid level in the apical chamber (mm) was recorded and converted to a volume (mL) using a constant that was determined by measuring the volume of fluid per mm change in the height of fluid in the chamber. The fluid levels in the two chambers were equilibrated before starting the next measurement by removing the excess that had accumulated in the apical chamber and adding fresh Ringer’s solution to the basolateral chamber.Clear plastic rulers were attached to both chambers to record the movement of fluid in 0.5 mm increments. Unless otherwise specified, the two chambers were filled to the same level with 37 °C mammalian Ringer’s solution with (in mmol) 128 NaCl, 4.7 KCl, 2.5 CaClDextran with a molecular weight of 4000 that was conjugated with fluoroscein isothiocyate (4 kD FITC-dextran) was added to either the apical or the basolateral chamber in some studies to visually track the movement of fluid.2.We considered it essential to determine whether the fluid flow by the cultured epithelial cells could be replicated using intact tissues. Additional C57/Bl6 mice from the same colony were used to prepare sacs of 4 to 6 cm length from small intestine segments that were either everted with the mucosa exposed or left non-everted with the mucosa internalized. The distal end was closed with a ligature and the entire length of intestine was filled using Ringer’s solution with 2 mg/mL 4 kD FITC-dextran until it expanded. Another silk ligature was used to close the proximal end. The sacs were gently blotted to remove adherent fluid, weighed, and then secured by the ligatures to metal rods. Throughout the preparation phase and after the sacs were attached to the rods, they were kept in cold (4 °C) Ringer’s solution aerated with a gas mixture of 95% oxygen and 5% COThe measurement of fluid flow and leakage of 4 kD FITC-dextran was started by positioning the rods in an incubation tube such that the sacs were completely immersed in 20 mL of Ringer’s solution (37 °C) that was aerated with the gas mixture using a hematocrit tube. Additional agitation was provided by a stir bar rotating at ~1200 rpm. Samples (200 µL) of the incubation solution were collected at the start and at 10, 20 and 30 min and loaded onto a microplate to measure 4 kD FITC-dextran by recording fluorescence. After 30 min, the sacs were removed from the rods, gently blotted, and the ending weights were recorded. The difference between beginning and ending weight was used to estimate fluid loss.2 asphyxiation followed by decapitation. The use of rats also addressed a need to verify whether the fluid flow was shared by other species and not unique to mice. After only the serosa was removed, segments of jejunum were mounted in sliders with a 12.7 mm diameter circular aperture, exposing 1.27 cm2 of mucosa, and placed in the Ussing system. Both chambers were filled to the same level with Ringer’s solution and vigorous aeration was applied to both chambers for 30 min. After the volume of Ringer’s solution moving from the basolateral to the apical chamber was measured, the chambers were rebalanced, zinc was added to both chambers , and the flow of fluid was again measured for 30 min. Both chambers were then drained, refilled with fresh Ringer’s solution, sodium citrate was added to both chambers , and the volume of fluid flow was again measured for 30 min.Other investigators using tissues mounted in Ussing systems remarked in discussions that they too had observed basolateral-to-apical fluid flow when aeration was mistakenly high . We verified this observation using intestinal tissue that was obtained from two rats that were used in a behavior experiment and were euthanized by COt-tests. A one-way ANOVA was used for studies involving multiple comparisons. When a significant treatment effect was detected, specific differences among treatments were identified by Duncan’s multiple range test. For all tests, p < 0.05 was accepted as the critical level of significance. Values in figures are the means and standard errors.Direct comparisons of paired values were made using The following sections present the series of eight studies we performed to confirm the existence and understand this pathway of fluid flow driven by mechanical force. For each study, we describe the experimental approach and the associated findings.To confirm our initial observation of fluid flow, mouse enterocytes were cultured on 3 µm pore inserts and exposed to three different intensities of aeration; none, low , and vigorous (~500 mL/min); the same intensity of aeration was provided to both chambers. Ussing chambers are designed for the aeration to circulate the fluid, thereby exposing the epithelium to a current. Vigorous aeration increases the circulation of fluid, hence the current and the mechanical force imposed on the epithelium.Two batches of enterocytes were mistakenly cultured on inserts with 0.4 µm pores, the size commonly used for culturing epithelia. Surprisingly, the resulting epithelia did not generate hydrostatic pressure gradients, even with 30 min of vigorous aeration. To determine whether there was an influence of insert pore sizes, we cultured mouse enterocytes on inserts with 0.4, 3, 8, and 12 µm pores and exposed them to the different intensities of aeration.To understand whether the fluid movement was simply an artifact of insert pore size, inserts with different pores sizes, but without cells, were exposed to 30 min of vigorous aeration, with both chambers starting with the same level of fluid. We also explored the potential role of insert pore size as a barrier for passive fluid movement using cell free inserts. Both chambers were equilibrated with Ringer’s solution before adding an additional 0.5 mL to one chamber and recording the time for the levels in both chambers to re-equilibrate without aeration.The rate of fluid movement through epithelia of cultured mouse enterocytes is directly related to the intensity of aeration and pore size . At low-R is the internal radius of the tube, which reflects pore size. ΔP is the pressure difference between the two ends, η is the dynamic fluid viscosity, and L is the length of the tube. The greater resistance of smaller tubes (pores) reduces flow. The hydrostatic pressure (ΔP) imposed by adding 0.5 mL to one chamber was equal for all inserts. L is represented by the thickness of the membrane and would be constant across the inserts, and viscosity (ƞ) was identical. If velocity of flow through the pores is assumed to be equal, then the volumetric flow rate (Φ) increases more than 6 orders of magnitude from 0.4 to 12 µm pores. This explains why the time required for the two chambers to re-equilibrate after the addition of 0.5 mL to one chamber decreased as pore size increased.The flow of Ringer’s solution through inserts without cells was directly related to pore size . This waFurther studies of fluid flow by epithelia prepared from enterocytes, Caco-2 and T84 cells, and fibroblasts used inserts with 12 µm pores and both chambers were aerated vigorously.The directionality of flow was ascertained before and after reversing the orientation of inserts with cultured mouse enterocytes and by aerating just the apical or just the basolateral chamber or both chambers. The fluid flow was visualized by adding 100 µL of a stock solution of 4 kD FITC-dextran (5 mg/mL), which is a marker of paracellular movement  to eitheWe observed that the hydrostatic pressure gradient caused by the movement of fluid into the apical chamber did not diminish during the 2–3 min the aeration was turned off while we recorded the levels of fluid in the two chambers. The persistence of the hydrostatic pressure gradient was verified by exposing epithelia of mouse enterocytes to 5 min of vigorous aeration to create a hydrostatic pressure gradient then turning off the aeration and recording changes in volume after 15 min. As another indicator of the unidirectional flow, Ringer’s solution was added to the basolateral chamber to create a 10 mm basolateral-to-apical hydrostatic pressure gradient. Volume changes in both chambers in the absence of aeration were recorded for the next for 15 min.Changing the orientation of the inserts did not change the basolateral-to-apical direction of flow. Vigorous aeration of only the apical compartment produced volume shifts similar to when both chambers were aerated, while aeration of only the basolateral chamber reduced the fluid shift by 50–60% . This isAfter adding the 4 kD FITC-dextran to the basolateral chamber, vigorous aeration caused rapid movement of both fluid and the FITC-dextran to the apical chamber A,B. Withp < 0.01) of the volume that moved during the 5 min of vigorous aeration. These findings provide further evidence the apical flow of fluid is unidirectional and is enhanced by aeration.The hydrostatic pressure gradients that were established by exposing epithelia of cultured enterocytes to vigorous aeration for 5 min  did not decline during the subsequent 15 min period with no aeration . The impTo ascertain the role of the tight junction, fluid flow was measured by Caco-2 and T84 cells that develop extensive tight junctions and are commonly used as models of intestinal epithelia and fibroblasts that form monolayers while lacking many tight junction proteins.Exposure of epithelia of Caco-2 and T84 cells to vigorous aeration confirmed the movement of fluid into the apical chamber is unidirectional, but the volume was <20% of what was measured using epithelia of harvested enterocytes  correspo3. Another study assessed the possible role of bicarbonate secretion by measuring fluid flow while aerating with 100% nitrogen.The accepted coupling of chloride and fluid secretion led us to use multiple approaches to determine whether the flow of fluid driven by aeration was coupled with ion secretion. We prepared chloride-free Ringer’s solution by substituting sodium gluconate and potassium gluconate for NaCl and KCl. The chloride-free Ringer’s solution was added to the basolateral chamber with the expectation a basolateral-directed chloride gradient would at least slow and perhaps even reverse the flow of fluid by epithelia of cultured mouse enterocytes and Caco-2 cells. A second prediction was that an apical-directed chloride gradient by adding the chloride-free Ringer’s solution to the apical chamber would increase the flow of fluid. We also exposed epithelia of mouse enterocytes to gradients for sodium and potassium that were established using choline chloride for NaCl and KCl and choline bicarbonate for NaHCOBecause cell viability and epithelial integrity might be affected by prolonged exposure to Ringer’s solution devoid of specific ions, fluid flow by cultured enterocytes was measured in the presence of various compounds known to alter membrane fluxes of ions . These wAs a definitive experiment, we created an osmotic gradient by filling the apical chamber with Ringer’s solution that had been diluted with water to 200 mOsm . The expectation was that this osmotic gradient would slow the flow of fluid into the apical chamber and possibly reverse the flow into the basolateral chamber.Even though the enterocytes used to prepare the epithelia are from the upper villus  and exprInterestingly, when Caco-2 cells that are able to secrete chloride  were expThe slight reversal of fluid flow when epithelia prepared from Caco-2 cells were exposed to chloride-free Ringer’s solution in both chambers led us to consider whether other ions may be involved. Establishing sodium or potassium gradients did not alter the flow of fluid by epithelia prepared from enterocytes, irrespective of whether the choline substituted solutions were added to the apical, basolateral, or both chambers A, nor diAn even more striking indicator of the ion and osmotic independence of filtration secretion is how flow from the basolateral to the apical chamber was not hindered by filling the apical chamber with diluted Ringer’s solution to impose a counter osmotic gradient .Active ion secretion requires energy and is temperature sensitive . This le2) was surprisingly higher (p < 0.05) than subsequent measurements made with the same inserts at 20 °C (0.48 ± 0.005 mL/ min·cm2) and 37 °C (0.41 ± 0.004 mL/min·cm2).Fluid movement at 10 °C  by citrate (50 μM) would restore flow. However, citrate also chelates calcium, though not as avidly as EDTA and this could impact the integrity of tight junctions. Therefore, we also determined whether citrate alone would affect fluid flow.4) caused an immediate inhibition of fluid movement that was dose dependent  was matched by adding zinc chloride and zinc nitrate, indicating the inhibition was by zinc, not sulfate. Of the other metal ions evaluated, only copper sulfate inhibited fluid movement, as previously reported [The magnitude of inhibition caused by ZnSOreported , and the4 and CuSO4 was restored by adding citrate .We next compared weight loss and 4 kD FITC-dextran leakage by sacs of everted mouse intestine that were incubated in vigorously aerated and stirred Ringer’s solution with and without ZnSOp = 0.11) than the decrease in weight by sacs prepared from the distal small intestine (20.7 ± 5.4%). The respective losses in weight coincided with an insignificant difference in the leakage of 4 kD FITC-dextran based on leakage into the bath solution . To avoid any potential regional influences all subsequent comparisons were made using sacs prepared from adjacent segments of small intestine.Everted sacs prepared from the proximal small intestine exposed to intense aeration and stirring lost 33.9 ± 5.5% of the original weight. This was not significantly different  linked the accumulation of fluid in isolated segments of small intestine with in vitro Ussing chamber measurements of increased flux of chloride into the apical  compartment . The couWith research focused on chloride-coupled fluid secretion, the contribution of filtration secretion has been overlooked. The ability of cultured enterocytes to generate hydrostatic pressure gradients when exposed to vigorous aeration was not prevented by inhibitors of ion channels and transporters nor was it diminished by imposing ion and osmotic gradients that should have reversed the flow if solely reliant on ion movements. Even low temperature did not hinder fluid flow, despite the temperature sensitivity of ion channels and transporters . Our finThe reversed apical-to-basolateral flow when epithelia of Caco-2 cells were exposed to a basolateral-directed chloride gradient and vigorous aeration reinforces that the phenomenon is not an artifact. If the unidirectional basolateral-to-apical flow was caused by the epithelium covering the pores and preventing backflow, the reversal of flow would not occur. The contrasting responses to chloride gradients by cultured enterocytes and Caco-2 cells derived from the colon may reflect the different origins and associated characteristics.There are several sources of anecdotal evidence supporting a role of mechanical force in causing fluid secretion by the intestine. While investigating the influence of mechanical vibrations on organisms and possible applications for medical therapies, Nicolas Tesla made the following observation.“…some of us, who had stayed longer on the (vibrating) platform, felt an unspeakable and pressing necessity which had to be promptly satisfied.”.The “necessity” referred to by Tesla was diarrhea. What he did not understand is that the vibrating platform caused diarrhea by disturbing the carefully regulated dynamic balance between the counter processes of fluid secretion and absorption that determines the fluid volume in the gut lumen. From the present results, we infer the vibrations imposed an external mechanical force on the intestine that caused filtration secretion and a large volume of fluid to rapidly flow into the small intestine. The link between mechanical force and fluid secretion explains why whole-body vibration  and vibrContractions of the smooth muscle during the processing of a meal impose a physiological mechanical force on the intestinal mucosa and epithelium . The preAnother consideration are the energetic costs and oxygen demands for basal chloride secretion are significant , likely The relative contributions of ion secretion and motility for small intestine fluid secretion need to be established for normal and disease states. Our findings suggest that during periods of high-volume fluid secretion, such as during digestion and diarrhea, filtration secretion driven by the increased motility plays a major role, whereas CFTR and chloride-coupled fluid secretion will be important between meals when the small intestine is mostly quiescent. An important consideration is that smooth muscle cells express CFTR and the state of contraction is responsive to activation and inhibition of CFTR . CF patiBoth chloride secretion and smooth muscle contraction are stimulated by cholera toxin , by 5-hyOur findings provide an explanation for the fact that the most effective therapeutic agents for diarrhea and constipation are known to alter both chloride secretion and motility. These includes 5-hydroxytryptamine receptor 4 agonists and other prokinetic agents ,65, prosAlthough aquaporins are implicated in intestinal fluid dynamics , they arSalmonella infection [Our discovery that filtration secretion can be reversibly regulated is a key finding and is relevant scientifically and important clinically. The inhibition of fluid and 4 kD FITC-dextran movement caused by zinc, even with vigorous aeration and agitation, is consistent with increased epithelial resistance and tight junction barrier functions ,30,31,32nfection  and seminfection .The proportionally greater basolateral inhibition compared with apical exposure is consistent with other studies using cultured cells and tissues ,29,30,84The rapid restoration of fluid flow with citrate, though not immediate or complete, indicates that the metal ions did not disrupt the tight junctions. Specifically, if zinc and copper caused the loss of tight junction integrity, the epithelia would have been incapable of establishing and maintaining hydrostatic pressure gradients. Moreover, the rapid restoration of fluid flow by citrate suggests that this pathway of fluid flow into the intestine can be considered as a target for therapies to address diarrhea as well as constipation.We propose that filtration secretion and high-volume fluid flow into the lumen of the small intestine involve a “one-way check valve” as a component of the tight junction complex . SimilarAlthough our findings contribute to a better understanding of the linkage between intestinal motility and filtration secretion, additional research is needed to address unanswered questions. Of particular importance is understanding how in vitro fluid secretion pertains to intestinal fluid dynamics in normal and disease states. There is uncertainty about how the experimental paradigm of agitating the bath solution corresponds with the mechanical force imposed on the epithelium in vivo by intestinal motility. There is a need to ascertain the role of the tight junction in filtration secretion, identify associated tight junction components and obtain morphological and molecular evidence for the proposed model, and elucidate the regulatory mechanisms underlying how zinc and other modulators of filtration secretion alter the rate of fluid movement. An important consideration for future studies is determining the relative contributions of filtration secretion and chloride-coupled secretion to intestinal fluid dynamics at different intensities of motility.


The main components of the GeoMICROBE can be converted into a mobile pumping system (MPS) that is installed on the front basket of a submersible or remotely-operated-vehicle (ROV). Here, we provide details of a hydrothermal fluid-trap used on the MPS, through which a gastight sampler can withdraw fluids. We also applied the MPS to demonstrate the value of fixing samples at the seafloor in order to determine redox-sensitive dissolved iron concentrations and speciation measurements. To make the best use of the GeoMICROBE sleds, we describe a miniature and mobile version of the GeoMICROBE sled, which permits rapid turn-over and is relatively easy for preparation and operation. Similar to GeoMICROBE sleds, the Mobile GeoMICROBE (MGM) is capable of collecting fluid samples, filtration of suspended particles, and extraction of organics. We validate this approach by demonstrating the seafloor extraction of hydrophobic organics from a large volume (247L) of hydrothermal fluids.  Specifications tableThe uppermost 40–500 m of submarine ridge flank basement, with ages between 1 and 65 million-years-old, is fractured and permeable, holds ~2% of the ocean volume and accounts for 70% of the seafloor hydrothermal heat flux In the early deployments, CORKs were designed as “instrumented borehole seal,” and used to provide the opportunity for long-term hydrogeological or seismic monitoring Seafloor sampling systems were, subsequently, designed, and developed to collect high-quality large-volume basement fluid and suspended particles via CORK FDLs 3He) In this work, we focus on a modification to the MPS for dissolved gas sampling. Gastight samplers 2+) in situ.We also describe the application of the MPS to in situ experiments. With the MPS-bag system, even when the sampling bags were pre-evacuated, the plumbing materials allowed trace amounts of oxygen to diffuse into the sampling bags, leading to an increase in dissolved oxygen of up to 4 μM when the sample bags were recovered shipboard . This amount of oxygen is sufficient to oxidize reduced compounds such as dissolved iron and dissolved hydrogen sulfide present in the system, leading to difficulty in evaluating the redox status and the energy available for the deep-subsurface microbial biosphere. To circumvent this issue, we demonstrate a mean to pre-charge sample bags with fixatives in order to preserve a reduced compound  modified from a standard GeoMICROBE, and demonstrate its utility for investigating dissolved organic matter in the deep subseafloor Submarine ridge flank hydrothermal fluids were drawn from the basement through a CORK FDL by the MPS secured on the front basket of the ROV Jason  and 2. AThe Mega Pump pulled basement fluids at a flow rate of 5 L/min through the FDL. A sufficient pumping time allowed the flush of at least six times of the FDL volume to remove stagnant fluids in the FDL. The MPS required a 48 V power supply from ROV Jason to the MPS controller, which operated the Mega Pump and sensors. The McLane valve system in the ROV back basket required a 24 V power supply from ROV Jason. The control van powered the MPS control box, which communicated between the MPS controller in the front basket to a custom-build computer program. The Optode oxygen sensor also received power and communication through the ROV Jason junction box and the readout shown on a screen in the control van.The bag samplers were composed of custom-made bags inside protection boxes  and 3. TThe first version of a gastight sampling reservoir in 2010 was a custom-built polyvinyl chloride (PVC) funnel that had a height of 20 cm and a largest cross-section of 20 cm . WithoutA three-way-vale with 1/2 NPT fitting connected to 3/8’’ PVDF tubing allowed the flowing crustal fluid to be directed to a LVBS on the front basket next to the fluid-trap c. This c3+) into ferrous ion (Fe2+). The ferrozine-acetate solution was prepared by dissolving 0.4925 g of Ferrozine in 100ml MQ and mixed with a buffer prepared by mixing 8.2 g sodium acetate with 5.7ml acetic acid in 100ml MQ. A check valve installed in the fluid line before the sample bag prevented the reagent flowing back to the 25-port valve system. Acrylic McLane remote access sampler (RAS) tubes protected the 500 mL bags. Each of the bags was programmed to collect 400 mL of basement fluid samples. Samples containing ferrous ion formed a pink iron-ferrozine complex  to reduce ferric ion , Sterivex (0.2 μm pore size). The MGM also coupled with a custom-built Teflon column packed with hydrophobic XAD-8 or Bond Elut ENV resin to extract organics for characterization. The feed sample to the absorbent resin had passed pre-combusted 47 mm GFF filter, i.e., to obtain the dissolved organic fraction. A McLane pump instead of the Mega pump controlled the extraction flow rate of 50-125 mL/min to ensure a minimum contact time of 2 min between the hydrothermal fluid and the resin, provided a column size is of 125–500 mL  spectrometry  of a sam13C-HCO3−,15N-NH4+, and 15N-NO3−) with basement fluid samples collected in the 500 mL Tedlar bags. A seafloor incubation is particularly plausible for a low-temperature system such as the North Pond basement fluids  whereas the heating components may take too much power to maintain at an in situ hydrothermal temperature  for the warm ridge flank sites such as the eastern flank of the Juan de Fuca Ridge. The seafloor incubation can also include deep seawater samples as control.The MGM can be applied to initiate incubation experiments by mixing stable isotope labels  filter need to be installed. Otherwise, clogging inside the pump and the 25-port-valve system may occur.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Dr. Santora will provide an overview of grant mechanisms available at the National Institute on Aging, with an emphasis on mechanisms of value to early career researchers.


The extracellular matrix (ECM) dynamics in tumour tissue are deregulated compared to the ECM in healthy tissue along with disorganized architecture and irregular behaviour of the residing cells. Nitric oxide (NO) as a pleiotropic molecule exerts different effects on the components of the ECM driving or inhibiting augmented angiogenesis and tumour progression and tumour cell proliferation and metastasis. These effects rely on the concentration of NO within the tumour tissue, the nature of the surrounding microenvironment and the sensitivity of resident cells to NO. In this review article, we summarize the recent findings on the correlation between the levels of NO and the ECM components towards the modulation of tumour angiogenesis in different types of cancers. These are discussed principally in the context of how NO modulates the expression of ECM proteins resulting in either the promotion or inhibition of tumour growth via tumour angiogenesis. Furthermore, the regulatory effects of individual ECM components on the expression of the NO synthase enzymes and NO production were reviewed. These findings support the current efforts for developing effective therapeutics for cancers. Nitric oxide (NO) exerts multiple effects on tumour biology, where it can be both pro- and anti-tumorigenic depending on the NO dose and duration of exposure. These effects are mediated by the different components of the tumour tissue including the different cells residing in the tumour tissue, which produce and are sensed by NO and the multiple constituents of the extracellular matrix (ECM). In human, NO is mainly synthesized in the tumour tissue via three nitric oxide synthase (NOS) enzymes: endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS), all of which can play a role in tumorigenesis including tumour proliferation, angiogenesis and tumour metastasis . TumoursCancer tissues are composed of (i) cellular components including tumour cells, stroma which includes endothelial, smooth muscle, immune cells, epithelial fibroblasts and myofibroblasts along with different types of mesenchymal cells and (ii) non-cellular part comprising of the ECM proteins ,16. The In this review, we examine the role of NO in the modulation of ECM and its implications for tumour angiogenesis. Furthermore, the recent studies on the effects of the ECM components on NOS expression and NO production in tumours are comprehensively analysed with highlighting the required research for the complete understanding of these interactions. This will help get a more in-depth idea on how the tumour tissues develop and facilitate the screening and development of more effective treatments for cancers.For the metastasis of cancer cells, degradation of the stromal ECM and basement membrane is mediated by MMPs to facilitates tumour access to the vascular and lymphatic systems ,31. MMPsin vitro [Vβ3 integrin [The regulation of MMPs by NO also takes place at the level of the ECs and modulation of angiogenesis. For example, Membrane Type 1-MMP (MT1-MMP) degradation of type I collagen at the cell membrane of HUVECs was activated by DETA-NONOate and accompanied with enhanced cell migration and tube formation in vitro . Similarin vitro . Additiointegrin . Besidesintegrin . These oFor sustained tumour growth, the demands for nutrients and oxygen, as well as for waste exchange, makes it essential for angiogenesis and lymphangiogenesis to be activated, so the tumour can grow, and metastasize to facilitate cancer progression ,54. The Furthermore, NO may have different effects on the ECs, which can be either proangiogenic or anti-angiogenic depending on its concentration and duration of exposure via cGMP signalling pathway . The treMoreover, NO-stimulated HUVEC chemotaxis at the low DETA-NONOate concentrations was inhibited after treatment with TSP-1, mediated via CD36 . Similarex vivo study, where the treatment of both explanted WT and TSP1−/− muscle biopsies in collagen matrices with DETA-NONOate (10 µM) caused enhanced relative vascular outgrowth in the later biopsies owing to the inhibited expression of TSP-1. However, the binding peptides to CD36, CD47 or heparin sulfate proteoglycans derived from TSP-1 inhibited this outgrowth, but NoC1 enhanced the vascular outgrowth [−/− muscle biopsies treated with DETA-NONOate and TSP-1, and the non-sensitivity of angiogenesis in CD47−/− muscle biopsies exposed to NO to the TSP-1 treatment refers to the necessity of CD47 as a receptor for the inhibitory functions of TSP-1. These results were supported by the studies performed on CD47-null aortic smooth muscle cells (ASMCs) and HUVECs, where the ligation of CD47 was found to be essential for inhibiting the NO-induced cell proliferation and NO-stimulation of cGMP  with NO from 10 µM DEA-NONOate induced myristate-stimulated cells adhesion to collagen . The VSMutgrowth . Further of cGMP . However2 shear stress resulted in phosphorylation of the Ser1177 residue on eNOS demonstrating one mechanism by which shear stress rapidly controls the release of NO [NO has been considered one of the important shear stress-responsive signalling molecules in ECs ,75,76. Fse of NO . Moreovese of NO .Relying on the properties of the ECM within the tumour microenvironment, particularly the alignment of the protein/polymer fibres ,88, dimeS-nitroso-glutathione caused enhanced cell proliferation along with increased synthesis and deposition of elastin, HA, and glycosaminoglycans by the SMCs within the cell matrix layers [The incubation of human umbilical artery ECs with κ-elastin caused a significant increase in eNOS mRNA expression (31%) only at the elastin concentration of 2.5 µg/ml. In comparison, the concentration of 0.5% did not affect it . Howeverx layers . The trex layers . Further2 for 6 h showed almost no change, moderate, and twofold increase, respectively [in vivo [BAECs monolayers exposed to physiological shear stress exhibited alignment in the direction of flow with elongation following the treatment with intact glycocalyx . Howeverectively . This wa[in vivo .Furthermore, laminin I added to cells under static conditions did not change eNOS expression, referring to its modulating roles for the response of a vessel to the applied mechanical force mediated by shear stress sensitivity of cells in terms of eNOS expression involved in tumour invasion. Moreover, the treatment of isolated canine femoral arteries with hyaluronidase resulted in a significant decrease in the flow-induced NO production, but this flow was not affected in the case where heat-inactivated hyaluronidase was used  Figure . This bein vivo mainly via the induction of PI3K-specific pathway [For metastasis of cancer cells to occur, degradation of ECM is an essential step, which was found to be facilitated by invadopodia formation, with the functions of these membrane protrusions found to be dependent on its lipid rafts and caveolae components ,102. The pathway .Interestingly, a negative regulation of NO synthesis was observed in cells expressing caveolin-1, where a particular caveolin-1 scaffolding domain was found to interact with eNOS causing inhibition of its enzymatic activity . FurtherThe exposure of B16 melanoma explants to NO from 10 µM DETA-NONOate stimulated the vascular cell outgrowth from the explants in the 3D collagen matrix. However, the TSP-1 analogue ABT-510 inhibited this stimulation . FurtherNO also plays an essential role in the promotion of colorectal cancer growth and angiogenesis. iNOS mRNA, protein and activity are elevated in colorectal cancer compared to the healthy tissues and were higher in metastatic than in non-metastatic tumours with a corresponding increased cGMP production ,113. TheThe treatment of LS174T colonic adenocarcinoma cells with SNP (10 mM) caused enhanced mucin secretion and increased levels of nitrite, indicating released NO . A signiAs with its effects on the other cancers and based on the tumour microenvironment and its concentration, NO may act as either an inducer for tumour cell metastasis and tumour progression or may ameliorate it ,122. TheExposure of non-malignant MCF10A breast cells to laminin (LN5) resulted in increased P-53 (pSer20-p53) and phosphorylated NOS-1 (Ser1417 phosphorylation) , which win situ, in IrECM gel caused reduced production of NO and nitrite along with reduced levels of the cytosolic SNOC compared to MCF10A cells [Furthermore, in a study by Ricca et al. 2018 , a short0A cells . However- ions from the intracellularly generated NO and O2−. molecular fragments, correlated with the nitrosation of tyrosine.The IHC analysis of 185 invasive breast cancer lesions done by Karihtala et al. 2007  had the in vivo, where the treatment of rats with L-NAME caused a reduction in mechanically-induced bone formation in the tibiae [In spite of the recent progress in studying the effects of the mechanical properties of the ECM on NO production in different diseases, limited progress has been achieved in addressing its specific role in cancer metastasis. For instance, the role of NO in bone mechano-transduction has been demonstrated e tibiae . Furthere tibiae . More stImportance of the field: Tumour angiogenesis and growth are multifactorial processes, with a significant role played by the ECM proteins and enzymes in their regulation. NO, through its dichotomous effects, regulates these processes either through activating or inhibiting specific ECM proteins, with mutual effects of these proteins on NO production within different tumours.Summary of the current thinking: NO's modulation of tumour growth is dependent on NO concentration & NOS activity level, duration of exposure, physical localization of NOS enzymes. This, together with the composition of the tumour ECM, mediates the effects of NO on tumour progression.Future directions: Studying the interactions between NO and ECM components will improve our understanding of relevant changes within the tumour microenvironment, which drive further angiogenesis and tumour growth. Moving forward, these findings will guide the development of new and more effective therapeutics for cancers, and increase our understanding of the biology of aggressive tumours.


Background: Although studies have examined the influence of materialism on adolescent well-being, there are several methodological limitations: studies examining the influence of materialism on adolescent delinquency are almost non-existent; researchers commonly used cross-sectional designs; the sample size in some studies was not large; validated measures on materialism in non-Western contexts are rare; there are very few Chinese studies. Besides, no study has examined the hypothesis that egocentrism is the mediator in the influence of materialism on adolescent delinquency. Methods: Using a short-term longitudinal design, two waves of data were collected from 2648 early adolescents in mainland China. At each wave, students completed validated measures of materialism, egocentrism and delinquent behavior. Results: Materialism and egocentrism positively predicted adolescent delinquency at Wave 1 and Wave 2 and over time. While materialism at Wave 1 positively predicted increase in delinquency over time, egocentrism did not. However, PROCESS analysis showed that egocentrism mediated the longitudinal influence of materialism on adolescent delinquent behavior. Conclusions: Materialism and egocentrism are predictors of adolescent delinquency, with egocentrism serving as a mediator in the influence of materialism on adolescent delinquency. In his book entitled “An Inquiry into the Nature and Causes of the Wealth of Nations” , Adam Smith  examinedThe focus on accumulation of wealth, consumption of goods and making money is closely related to the concept of “materialism”, which is commonly understood as over-emphasis on material possessions such as money. In the scientific literature, there are different conceptions of materialism in terms of values, needs and motives ,3. In a What is the influence of materialism on human well-being? Based on 259 studies regarding how materialism is associated with personal well-being, Dittmar et al. , althougThe literature review shows several methodological problems in the studies on the relationship between materialism and well-being. The first limitation is that there are few studies based on adolescents, although adolescence coincides with a substantial increase in materialism . In the jun zi ai cai, qu zhi you dao” (gentlemen love fortune but in a proper way). In Buddhist thoughts, the emphasis is the transcendence of the material world as reflected in the saying of “kan po hong chen” . On the other hand, there is a strong emphasis on materialism in Chinese folk culture. For example, while the concept of “feng yi zu shi” (have ample food and clothing) was proposed more than two thousand years ago, it is still a dominant Chinese belief. One clear indication is that people commonly say “gong xi fa cai” (may you make much money) to each other during Lunar Chinese New Year.The second methodological limitation is that existing studies are mostly Western studies and Chinese studies are almost non-existent. Amongst the 259 studies in the review conducted by Dittmar et al. . Hence, The third limitation surrounds the assessment of materialism. As Kasser  commenteThe final limitation is that most studies in this field are cross-sectional studies with some of them having small samples. In his systematic review on materialism and personal well-being, Dittmar et al.  reportedIn addition to the above-mentioned methodological limitations, there are two conceptual issues intrinsic to the existing studies on how materialism is linked to well-being. The first conceptual issue is the definition of well-being and the related indicators. According to Dittmar et al.’s review , well-beThere are three reasons why we should examine materialism and antisocial behavior, particularly in adolescents. First, researchers have regarded some delinquent behavior  as externalizing behavior ,11, whicThe second conceptual issue is on the mediating processes involved in the influence of materialism on adolescent well-being. Dittmar et al.  remarkedBesides, Donnelly et al.  argued tBased on the above discussion, a materialistic individual is likely to have low self-esteem, which may trigger and be reinforced by materialism. According to Awanis, Schlegelmilch and Cui , materiaWhile materialism may predict ecocentrism, there is also direct and indirect evidence showing a positive association between egocentrism and adolescent delinquency. For example, Chandler  showed tIn view of the above conceptual and methodological gaps, we examined the concurrent and longitudinal predictive effects of materialism and egocentrism on adolescent delinquency using a short-term longitudinal study in which Chinese mainland high school students responded to validated measures of materialism, egocentrism and delinquency at two time points separated by one year. Specifically, we attempted to address four research questions as follows:Research Question 1: Does materialism predict adolescent delinquency concurrently and longitudinally? We expected that materialism would have a concurrent and longitudinal positive prediction on adolescent delinquency (Hypotheses 1a and 1b).Research Question 2: Does egocentrism predict adolescent delinquency concurrently and longitudinally? In accordance with the scientific literature, we expected that egocentrism would have a concurrent and longitudinal positive prediction on adolescent delinquency (Hypotheses 2a and 2b).Research Question 3: Does materialism predict egocentrism at the same time and over time? Based on the notion of cognitive deconstruction and the above discussion, we expected that materialism positively predicted egocentrism at the same time and over time (Hypotheses 3a and 3b).Research Question 4: Does egocentrism mediate the influence of materialism on adolescent delinquency? Based on the above discussion, we expected that egocentrism would mediate the influence of materialism on adolescent delinquency (Hypothesis 4).A longitudinal research design was used in this study, with data collected at two time points with one year apart. We recruited four high schools in mainland China from the schools participating in the “Tin Ka Ping P.A.T.H.S. Project” ,27,28 toThere were 2648 students in the final matched sample . Parents of most respondents (N = 2225) were in their first marriage . In contrast, parents of 401 students were separated, divorced or re-married .In both waves of data collection, a questionnaire assessing psychosocial development of adolescents in mainland China was used. In this study, we focused on the influence of materialism on adolescent delinquency via the mediating effect of egocentrism.Assessment of materialism. Using the responses of 1658 adolescents, Shek, Ma and Lin  developeAssessment of egocentrism. Using 1658 adolescents as participants, Shek, Yu and Siu  developeAssessment of Delinquency. We used 12 items to assess the occurrence of delinquent behaviors in the participants in the previous 12 months. These behaviors include stealing things, cheating at examinations, truancy, running away from home, damaging the properties of other people, attacking other people physically, having sex with others, fighting in gangs, speaking foul language, not returning home without parental permission, bullying or harassing other people and trespassing. For each item, there is a six-point scale . This scale has been validated and widely used among Chinese adolescents ,32. ReliConsistent with the analytical approach adopted in previous studies ,27,33,34t = 4.40, p < 0.001, Cohen’s d = 0.27).As far as sample attrition over time is concerned, results showed that student dropout at Wave 2 was not high. Besides, the background socio-demographic characteristics of those who dropped out of the study and those who did not drop out were basically similar. Furthermore, the matched sample and the dropouts did not have a significant difference regarding their delinquency and materialism levels at Wave 1. The only significant difference was that egocentrism was lower in the study sample than the dropouts at Wave 1 , materialism had a positive relationship with egocentrism at Wave 1 . Similar observations were found for Wave 2 . Results also showed that Wave 1 materialism and egocentrism were positively associated with Wave 2 adolescent delinquency scores .Concurrent and longitudinal correlation coefficients on the inter-relationships amongst materialism, egocentrism and adolescent delinquency are shown in β = 0.25, p < 0.001, Cohen’s f2 = 0.06 and β = 0.11, p < 0.001, Cohen’s f2 = 0.02, respectively). Similarly, Wave 2 materialism and egocentrism predicted Wave 2 delinquency . For longitudinal prediction analyses shown in β = 0.15, p < 0.001, Cohen’s f2 = 0.02 and β = 0.07, p < 0.01, Cohen’s f2 = 0.01, respectively). These findings provided support for Hypotheses 1a, 1b, 2a, 2b, 3a and 3b.For all multiple regression analyses, the control variables  were entered at Step 1, followed by the relevant predictor variable(s). For Wave 1 analyses, β = 0.05, p < 0.01, Cohen’s f2 = 0.003), egocentrism at Wave 1 did not predict change in delinquency at Wave 2 .To examine the influence of materialism or egocentrism on the change in delinquency over time, after entering the control variables at Step 1 and Wave 1 delinquency scores at Step 2, Wave 1 materialism or egocentrism scores were included in Step 3. Results showed that while Wave 1 materialism positively predicted increase in Wave 2 delinquency . The results provided support for Hypothesis 4.Results on the mediating analyses can be seen in There are several constructive responses to the methodological problems outlined in the Introduction. First, in view of the paucity of related research findings based on adolescents, we employed high school students as participants in this study. Second, as there are very few Chinese studies in this field, we collected data from mainland Chinese high schools. Third, we used validated Chinese measures in this study ,30,32. FBesides, we also addressed two conceptual issues discussed above in this paper. First, as there are few research studies examining materialism and antisocial behavior, the present study examined this relationship. Computer search based on PsycINFO on 6 September 2020 showed that there were 20 citations when we used “materialism” and “antisocial behavior” (or delinquency) as the search terms. Second, although Donnelly et al. [Consistent with our expectation, materialism significantly predicted adolescent delinquency at each wave. Materialism at Wave 1 also predicted delinquency and its change at Wave 2. Although the effect size of the findings is small, these novel findings suggest that apart from those frequently used indicators of well-being  , materiaRegarding the influence of egocentrism on delinquency, the findings are also consistent with our expectation and the findings that egocentrism  and lackBesides, we also found that materialism predicted egocentrism at Wave 1 and Wave 2. Over time, Wave 1 materialism also predicted Wave 2 egocentrism and its change, although the effect size was small. The findings expand the theoretical formulation of Donnelly et al.  that matFinally, the findings provide support for the hypothesis that egocentrism is a mediator of the influence of materialism on delinquency. There are two contributions of the present findings. First, they underscore the dynamic relationships amongst materialism, egocentrism and adolescent antisocial behaviors. Second, they demonstrate the important role of self-representation (as indexed by egocentrism in this study) regarding the impact of materialism on delinquency. In the future, as there are several “steps” in the model of Donnelly et al. , longituIn addition to the theoretical implications mentioned above, the present findings have two practical implications. First, changing materialistic beliefs of adolescents would help to reduce adolescent antisocial behavior. With reference to the major theories in the positive youth development (PYD) literature, helping adolescents to develop positive values is an important adolescent developmental task. Positive values are an important internal asset in the model of developmental assets . CharactAlthough this study is pioneering in this field, it has several limitations. First, we should realize the limitation of a short-term longitudinal study which has only two time points. Ideally speaking, we should design a longitudinal study with more time points which can help us further understand the long-term inter-relationships amongst materialism, egocentrism and adolescent delinquency. This is particularly important when we test the framework proposed by Donnelly et al.  that theThe present study addressed several conceptual and methodological limitations in the scientific literature on the association between materialism and well-being. Conceptually, we examined the influence of materialism on well-being indexed by adolescent delinquency, investigated the influence of materialism on egocentrism and tested egocentrism as a mediator of the linkage between materialism and adolescent delinquency. Methodologically, we recruited mainland Chinese high school students and used validated measures in a short-term longitudinal study with a large sample. Consistent with our expectations, the present study showed that materialism positively predicted egocentrism and adolescent delinquency over time, with egocentrism serving as a mediator. In the future, we will further examine the findings of this study in other cultures, in particular, we would like to explore whether the cultural differences between collective and individualistic societies would influence the impacts of materialism and egocentrism on adolescent delinquency.


Malignant musculoskeletal tumour may cause considerable burden to general health. The fast growth combined with the tumour characteristics and its invasion capability resulted in the poor prognosis of malignant musculoskeletal tumour. Malignant musculoskeletal tumour may cause significant disability by destroying normal tissue that plays important role in body kinematics. Thromboembolism, including deep vein thrombosis, pulmonary embolism, and other kinds of venous thromboembolism, is one of the most underestimated complications of musculoskeletal tumour. Normally, thrombosis ensues when pathologic factors overcame the body hemostatic regulatory capabilities, which will predispose the body to the formation of thrombus. Venous thromboembolism in musculoskeletal tumour may develop as a result of interaction between the tumour pathologic capabilities and its interaction with normal bodily functions. In this study, we reviewed the burden of musculoskeletal tumour and its complication on global health. Then, the review will focus on the pathologic and clinical aspect of thromboembolism in malignant musculoskeletal tumour, including pathophysiology, diagnosis, and treatment based on recent findings and literature. In recent years, malignant cancers are currently on the rise with an estimated incidence of 15.2 million of new cases of cancer with a death count of 8.8 million for year 2015. In terms of incidence, musculoskeletal tumours are relatively rare compared to other types of cancer in adults and are more often seen in pediatric and adolescents. Musculoskeletal tumour only makes up for around 0.2–0.5% of all tumour cases in adults, and the distribution varies greatly across the globe. Musculoskeletal tumour can be differentiated into benign and malignant tumours based on the tumour invasiveness, rate of growth, and many other different factors. Compared to its benign counterpart, malignant musculoskeletal tumour generally grows much more extensively in a faster rate. If not adequately treated as soon as possible, malignant musculoskeletal tumour mainly has fatal outcome. Osteosarcoma and Ewing sarcoma are two of the most encountered types of musculoskeletal tumour, and both types are malignant. The mainstay treatment for malignant musculoskeletal tumour is complete excision of the tumour followed by reconstruction of tumour-associated defects. Limb-salvaging treatment options are much more preferable than ablative treatments [Some complications are associated with malignant musculoskeletal tumour, ranging from surgical site infection after tumour excision to loss of limb function and even mortality. One of the most concerning complication associated with malignant musculoskeletal tumour is thromboembolism, including pulmonary embolism and deep vein thrombosis. Thromboembolism is deeply correlated with both orthopedic surgery and orthopedic tumour. Literature studies reported the incidence of thromboembolism in orthopedic cases ranging from <1 to 22% . MorbidiComprehensive literature search was done by searching PubMed, Scopus, EBSCOHost, ProQuest, ScienceDirect, SpringerLink, and Wiley on November 2020. The literature search included all articles regarding thromboembolism in malignant musculoskeletal tumour, including systematic reviews, clinical trials, cohorts, case controls, and case reports. Articles on different aspects of thromboembolism in malignant musculoskeletal tumour including pathophysiology, diagnosis, and treatment modalities are included in this study in order to provide comprehensive review about thromboembolism in malignant musculoskeletal tumours. Letters, editor opinion, and commentary studies are excluded. Analysis of the articles obtained from the search is done on December 2020. Findings are screened and extracted by a single reviewer. The result of the literature search is described and summarized in The exact mechanism triggering clotting in patients with thromboembolism is not fully understood. It is thought that arterial thrombus may be incited by disruption of endothelial integrity, while venous thromboembolism was found to be preceded by venous stasis. It is agreed that intact vasculature microstructure is vital in preventing thromboembolism, and disturbance of the vessel wall integrity may initiate pathophysiologic cascade that resulted in the formation of thrombus, which may get unattached to form emboli that travel and block vessels smaller in size compared to the emboli distant to the thrombus formation location. Hemostasis is the balancing process that maintains the blood flow system inside blood vessels. When pathologic factors exceeded the protective and regulatory features of hemostasis, blood coagulation will be initiated. Blood coagulation is a very complex process that happens as a result of interplay between many different intravascular elements, including shear stress on the inner lining of the vessel wall, blood coagulation factors, and other contributing agents, which ends in the generation of thrombus. Physiologically, thrombosis is actually an important protective sequence that protects the body from excessive blood loss , 5.2, adenosine diphosphate, and serotonin that will activate other platelets and enhance thrombus formation [In general, thromboembolism may happen as a combination of dysfunctional vascular endothelium, activity of an intravascular tissue factor, the existence of anticoagulant endovascular proteins, and the inhibition of fibrinolysis. Normally, endothelium prevents the formation of thrombus by producing thromboregulator agents, including nitric oxide, prostacyclin, and ectonucleotidase CD39. Following endothelial disruption, exposure of collagen and intravascular tissue factor to blood will initiate the generation of thrombus by accumulation and activation of accumulated thrombocytes. Collagen and intravascular tissue participated in the thrombus regulatory equilibrium by acting as hemostatic barrier in order to maintain the integrity of the circulatory system. Intravascular tissue factor, a transmembrane glycoprotein, is expressed in vessel wall tissue and functions as a receptor and cofactor of factor VII that ultimately plays vital role in the activation of factor IX . Intravaormation . Anticoaormation , 9.The risk of venous thromboembolism in cancer patients is almost five-fold higher compared to general population . In cancβ which were found to induce the hypercoagulable state by increasing the expression of intravascular tissue factor, and increases platelet aggregation on the vessel [Some literature studies found that the musculoskeletal tumour itself may contribute to the formation of thrombus. Tumour tissue can induce hypercoagulable state by increasing the activation of coagulative factors and inactivating the anticoagulant factors at the same time. Musculoskeletal tumour also hampers the fibrinolytic cascade by upregulating plasminogen activator inhibitor-1 (PAI-1), producing proinflammatory cytokines including IL-6 and TGF-e vessel . Modalite vessel . Yoshimue vessel . Groot ee vessel .Thrombosis may be one of the first signs of undetected neoplasm. The likelihood of the existence of tumour in patients with bilateral deep vein thrombosis is almost five times higher than in general population. Patients with metastatic cancers were especially predisposed for venous thromboembolism even when compared to other cancer patients without metastases. The Vienna Cancer and Thrombosis Study found an increased cumulative probability of venous thromboembolism in metastasized cancer patients , 17.μg/mL [Thrombus in cancer patients is mainly diagnosed by imaging modalities. Imaging modalities that can be used include CT, MRI, Doppler ultrasound, and positron emission tomography. Contrast-enhanced MRI may be used to find intraluminal soft tissue enhancement along the suspected vessel . Ultrasoμg/mL . Howeverμg/mL .Vascular surgery is one of the main treatments for thromboembolism in musculoskeletal tumour patients. Venous thrombectomy is one of the most used procedures, where the intravascular thrombus is taken out from the blood vessel. However, recent advances in medical science noted the availability of pharmacological agent as treatment and prevention modality for thromboembolism in cancer. Haase et al. found that the usage of tranexamic acid in cancer patients undergoing endoprosthetic reconstruction decreased the number of patients with venous thromboembolism postoperatively. However, the result was statistically insignificant, and the main purpose of tranexamic acid is to decrease blood loss and not to decrease or change the risk of postoperative venous thromboembolism in musculoskeletal cancer patients . ThromboAnother widely known agent that may be used as thromboprophylaxis is heparin, particularly low molecular weight heparins (LMWH). LMWH has been widely known as a pharmacological agent with beneficial role in preventing and treating thromboembolism. Medical guidelines have stated the utilization of LMWH injections to prevent postoperative thromboembolism in patients suffering from neoplasms with a risk of long-term immobilization . Past clMalignant musculoskeletal tumour poses a threat to public health since it may cause significant morbidity and mortality. One of the most concerning complication associated with musculoskeletal tumour is the incidence of thromboembolism, which may happen as result of disturbance in the hemostatic equilibrium. Timely diagnosis using accurate diagnostic methods, while also considering significant predisposing factors together with right treatment, is beneficial in preventing disabilities caused by cancer-associated thromboembolism.


Endolaser probes have been designed and sold for single-use only. However, in Brazil, they are not included in the list of single-use medical products that are prohibited from being reprocessed and could potentially be reused if safety requirements are accomplished. Therefore, this study aimed to determine and compare the quality, safety and costs of reprocessed versus original single-use endolaser probes of a specific brand and model.The study, conducted at a university hospital in Sao Paulo, Brazil, was divided in two phases. The first one tested the feasibility, sterility and physical integrity of ten reprocessed laser probes. In the second phase, all vitrectomy procedures using endolaser probes  from August 2017 to October 2019 were evaluated. The operated cases were followed for any signs of infection and number of defective probes for each group were counted. The cost of acquiring a new probe and for all reprocessing stages were evaluated and quantified in US dollars($).Microbiologic, residual ethilen oxide and microscopic evaluation of integrity of reprocessed laser probes were all within acceptable range. The second phase of this study included 590 endolaser probes, of which 375 were original and 215 were reprocessed. Functionality rates differed significantly between groups. Among the original probes, 373 (99.47%) were functioning and 2 (0.53%) were non-functioning. Among the reprocessed ones, 201 (93.5%) were functioning and 14 (6.5%) were non-functioning (p < .001). The average cost of one reprocessing was $3.00, and the average cost of an original probe was $150.00. Considering the loss rates, potential savings were $147.60 for each once-reprocessed probe. The frequency of infectious endophthalmitis was null in both groups.Our study showed that a single cycle endolaser probe reprocessing was safe and efficient, not associated with increase in endophthalmitis rate and proved to be significantly cost-effective, even considering a greater malfunction rate when compared to the original devices. First reported in 1981 . EndolasDespite having no lumen, endolaser probes have been designed and sold for single-use only. In Brazil, the National Health Surveillance Agency (ANVISA) has been responsible for the regulation of medical products reprocessing . AccordiThe present study aimed to establish safety, functionality and cost-effectiveness of reprocessing laser probes of a specific brand and model. Therefore, a two-phase study was conducted; the first phase focused on microbiologic and integrity tests; the second phase prospectively evaluated and compared patients submitted to pars plana vitrectomy with either original or reprocessed endolaser probes.This study was conducted at the University of Sao Paulo Medical School Clinics Hospital (HCFMUSP), located in Sao Paulo-SP, Brazil, and it was approved by the Institutional Review Board, under registration number 31251520.1.0000.0068. All vitrectomy procedures from August 2017 to October 2019 in which surgeon used endolaser probes were included in the study.At HCFMUSP, the endolaser probes used (23 Gauge Straight Laser Probe) are compatible with the Constellation vitrectomy system and belong to the Purepoint 532 nm thin disc laser system . They are terminated at one side with a connector that attaches the fiber end to the laser source. At the other side, they are terminated with a handpiece and a 23-gauge stainless steel tubing (without lumen) that holds the straight tip of the fiber. A flexible plastic jacket covers and protects the length of the fiber Fig. .Fig. 123As these probes are marketed for single use and there is no formal reprocessing guidance, a protocol for endolaser probes reprocessing was implemented after microbiological, residual, and physical integrity tests. In the first phase of the study, ten endolaser probes initially used in vitreoretinal procedures that would be discarded were collected and evaluated for bacterial, yeast, and hyphae count before and after a cleaning process. In addition, they were submitted to a sterilization cycle with 90/10 percent ethylene oxide mixture and sterility was tested. The presence and amount of pyrogen was assessed with a bacterial endotoxin test and measured in units of endotoxin per milliliter (UE/mL). As for the physical–chemical analyzes, the residual ethylene oxide (ETO), ethylene chlorohydrin (ETC), and ethylene glycol (ETG) were quantified by gas chromatography in parts per million (PPM). Integrity assessment was performed using an electron microscope with an increase of up to 200 times.The second phase of the study was designed in conjunction with the Hospital Infection Control Committee of our institution. According to the sterilization protocol accorded at the institution, each probe could be resterilized only once. Hence, already reprocessed probes had to be discarded after their intraoperative use.After each surgery, information such as the use of sterilized or new probes, as well as any malfunction of the probe, was collected. Laser malfunction was defined as the non-visualization of the burns during the intraoperative use that required the use of another probe. The sheet with the information above was filled by the surgeon right after the procedure, and it had the patient identification on it in order to track for any adverse events. All operated cases were carefully followed for any signs of infection for the post-operative period of one year, and any suspicious or confirmed event would have to be reported immediately to the Infection Control Committee of the Hospital.The number of defective probes for each group  were counted and statistically analyzed. The loss rates were calculated by the ratio between the number of non-functioning probes and the total number of probes used in each group. Moreover, the hospital costs of acquiring a new endolaser probe and the cost of all stages of probe reprocessing were evaluated and compared for the whole period. All costs were presented in US dollars ($).Data analysis included chi-square test for categorical variables. Level of significance was set to p < 0.05. All the analyses were performed by the Statistical Package for Social Sciences  software.The results of the first phase of the study are presented in the Table The second phase of this study included 590 endolaser probes used during vitrectomy surgeries performed in our institution between August 2017 and October 2019, of which 375 (63.56%) were original and 215 (36.44%) were reprocessed. Functionality rates differed significantly between the groups . Among the original probes, 373 (99.47%) were functioning and 2 (0.53%) presented malfunction, while among the reprocessed ones, 201 (93.5%) were properly functioning and 14 (6.5%) were not  . AlthougIn the present study, between August 2017 to October 2019, the overall incidence of presumed endophthalmitis following vitrectomy procedures was null in both groups. Thus, the use of reprocessed endolaser probes has not been associated with an additional risk of endophthalmitis in this cohort and based on our findings and following the methods described, one single reprocessing cycle can be safely recommended.Another Brazilian study evaluated the microbial growth on single-use reprocessed vitrectomy probes (including aspiration lines with lumen) . WithoutRegarding the cost analysis, the instruments’ functionality is an important factor. In the group of reprocessed probes, there was a higher loss of function rate (6.50%) when compared to the group of original probes (0.53%), OR = 12.99, p < 0.001. We believe this functionality loss is related to the strict cleaning process implemented in the sterilization process, that may damage the optic fibers responsible for laser transmission. Even though, reprocessed probes exhibited great cost-effectiveness, with savings of $147.60 for each reprocessed probe. In the context of public health, these savings can be socially impactful, as they allow investment in other devices and even in an increasing number of procedures . MoreoveIn summary, our study showed that the one-cycle reprocessing of endolaser probes was safe and efficient, since it has not contributed to an increase in endophthalmitis cases and it was significantly cost-effective, even with greater functionality loss rates.


These dates appeared as 2012 to 2013 but should have appeared as 2011 to 2013. This article has been corrected.1In the Original Investigation titled “Prevalence of Diabetes Medication Intensifications in Older Adults Discharged From US Veterans Health Administration Hospitals,”


The paper presents a concept of thermal-to-electrical energy conversion by using the oscillatory motion of magnetic fluid slugs which has potential to be applied in the field of sensors. A pulsating heat pipe (PHP) is introduced to produce vapor-magnetic fluid plug–slug flow in a snake-shaped capillary tube. As the magnetic fluid is magnetized by the permanent magnet, the slugs of magnetic fluid passing through the copper coils make the magnetic flux vary and produce the electromotive force. The peak values of induced voltage observed in our tests are from 0.1 mV to 4.4 mV. The effects of the slug velocity, heat input and magnetic particle volume concentration on the electromotive force are discussed. Furthermore, a theoretical model considering the fluid velocity of the working fluid, the inner radius of the PHP and the contact angle between the working fluid and the pipe wall is established. At the same time, the theoretical and experimental results are compared, and the influences of tube inner radius, working fluid velocity and contact angle on the induced electromotive force are analyzed. The pulsating heat pipe (PHP) is a new type of high-performance heat exchanger, which was first proposed by Akachi  and consFlow visualization shows that the flow inside a PHP can be random, oscillatory and circulatory ,6. Seol When it comes to some applications, the shape of the PHP will be more complex, and the structural strength and thermal conductivity of the PHP will be more demanding. Burban et al.  designedWhen the experiment is carried out with a copper tube rather than a PHP which is made of transparent materials such as glass, the movement of the working fluid (liquid slug and vapor plug) in the tube cannot be captured and observed with a camera. Besides, compared with the PHP made of glass, the experimental method with the PHP made of copper is limited. At present, whether the PHP has started can only be judged by the fluctuation of the wall temperature . To solvThere are two effects induced by two-phase flow, the magnetic force effect and the magnetic electricity effect, and Kamiyama and Ishimoto  proposedThe electromagnetic induction is conventionally produced by the motion of an electrical conductor in an external magnetic field to cause changes in magnetic flux over time. Since the magnetic fluid is a stable colloidal suspension of single-domain magnetic nanoparticles, which maintains a liquid state with magnetism like liquid oxygen, nitric oxide and platinum ,4, it isA PHP possesses the abovementioned property since that it is a kind of heat exchange equipment that relies on the phase change of working fluid and transfers heat in the form of latent heat and sensible heat. If the temperature difference between the condenser section and the evaporator section is large enough, continuous vapor–liquid two-phase flow can be generated in the PHP. It can be determined whether the PHP has started according to whether there is an induced electromotive force in the coil. Compared with the method of using a thermocouple to determine whether the PHP is started or not, the time lag of this method is shorter.Besides, the velocity of the working fluid will affect the value of the induced electromotive force. According to a precise theoretical model, the velocity of working fluid in the tube and the length of working fluid (liquid slug and vapor plug) can be obtained by the induced electromotive force and the time difference between adjacent induced electromotive force peaks. This method will become a new experimental method besides the visual experimental method.Besides, many pieces of production equipment have strict requirements for the temperature difference of materials. For example, in the field of chemical production, there are often strict requirements for the temperature difference of various reactants involved in the reaction, and the magnetic fluid PHP generator possesses a promising prospect for application in this field. The magnetic fluid PHP is applied to the sensor field without increasing energy externally, which can greatly improve the reliability of the sensor.Obviously, when the magnetic fluid is used as the working fluid, the magnetic fluid itself will affect the heat transfer performance of the PHP. Maziar et al.  used magIn this paper, we demonstrated that the thermally driven magnetic fluid slug flow can lead to the variation of magnetic flux through the induction coils and generate a considerable electromotive force. We also discussed the influence of velocity of vapor plug, heat input and magnetic particle volume concentration on the voltage. An alternating induced voltage with a peak value from 0.1 mV to 4.4 mV was obtained.The waveform of induced voltage can reflect the flow states inside the pipes, and the influences of two-phase flow patterns and heat input on the waveform, amplitude and frequency of induced voltage are investigated experimentally. Thus, a theoretical model considering the fluid velocity of the working fluid, the inner radius of the PHP and the contact angle between the working fluid and the pipe wall is established, and the influence of the inner radius of the PHP and contact angle between the PHP wall and working fluid on the electromotive force are also discussed.ϕ in the coils is:B is the magnetic induction, S is the cross-section area, μ0 is the vacuum permeability (4π × 10−7 H/m) and d is the diameter of the tube. If there is no other phase in the magnetic fluid under a constant magnetic field, the magnetic field intensity H and the magnetization M are constant. In this situation, there is no electromotive force due to the magnetic flux on the derivative of the time dϕ/dt, which is zero.If there is no vapor bubble or plug in the magnetic fluid, the magnetic flux M of magnetic fluid can be calculated by the classical law of Langevin:3O4 nanoparticles, dM is the saturation moment of the bulk magnetic solid (4.46 × 102 kA·m−1), Bk is the Boltzmann constant (1.381 × 10−23 J·K−1) and T is the absolute temperature.In Equation (1), the magnetization α, which represents the ratio of the liquid area to the total cross-sectional area. Thus, the expression of the magnetic flux in this case is:As the vapor bubbles/plugs pass through the coil, the magnetic flux will no longer remain constant because a portion of space is occupied by non-magnetic vapor. We defined the cross-section fraction α changes with time, its derivative of time could be nonzero. According to Faraday’s law of induction, the expression of induced electromotive force ε can be written as:n is the number of turns of the coils.Since α in our study.From Equation (4), we know that the electromotive force is proportional to the number of turns of the coils, the magnetization of magnetic fluid and the cross-section fraction on the derivative of time. Additionally, the magnetization is the function of temperature under the non-adiabatic condition. Therefore, the electromotive force is mainly determined by the change in the cross-section fraction As shown in α. When the gas–liquid contact surface enters the coil, an induced electromotive force will be generated in the coil. However, due to different lengths of the liquid column, three typical cases for the liquid slug passing through the coil appear in the experiment, as shown in Since the PHP is made of copper, the oxidation of the inner wall of the tube is hydrophilic with the water-based magnetic fluid. Based on this, we assume that the gas–liquid contact surface is an arc. As mentioned above, the induced electromotive force is mainly determined by the change in the cross-section fraction α can be given by Equation (5) as:This paper establishes a theoretical model for case 1, as the liquid slug passes through a single coil, as shown in S1 is the section area of liquid working fluid in the cross-section, S2 is the cross-section area of the PHP.R, r, r0 are the radius of the liquid–liquid contact surface, PHP and the wire, respectively, and V is the speed at which the liquid slug moves toward the coil. We can see from Equation (6) that the electromotive force t (t), when t = 0, L = r), when the liquid slug moves at a constant speed.Substituting Equation (5) into Equation (4), the electromotive force can be written as:For multiple coils, we assume that each coil is arranged in a square and, as the liquid slug moves, the coil in the red box generates electromotive force, as shown in The schematic of the experimental set-up is shown in There are 11 elbows in the PHP. The upper, middle and lower parts of the PHP are heating, adiabatic and cooling zones, respectively. The outer wall of the heating section is intertwined with the heating wires. The data collection system collects the parameters, such as the temperature of the cooling water and environment, pressure and current of the heating wire.Before starting the experiment, the PHP is vacuumed into 400 Pa (absolute pressure). Then the valve is closed before the vacuum pump and a certain amount of magnetic fluid is injected into the PHP. The angle of inclination is maintained at 60 degrees, and the filling ratio is 40% (the heat transfer performance is at a high level with a filling ratio of 30%~40%). The heat input range is from 60 W to 180W with an interval of 30 W.The inlet temperature and flow rate of cooled water are maintained at 20 degrees and 0.1 kg/min. The PHP is partially filled with magnetic particle volume concentrations of 1.91% and 3.83% water-based magnetic fluids, and the open-circuit voltage of each power generation unit is recorded in the test.There are 10 power generation units composed of two Nd-Fe-B permanent magnet rings and an induction coil, and the structure is shown in the inset of ® SCXI-1000 series) data collection system. The magnetic fluids used in the experiment are made by the chemical coprecipitation preparation method [3O4 nanoparticles are 1.91% and 3.83%, respectively.The values of induced voltage are measured by a National Instruments , and the peak value of the induced voltage is also improved. At 150 W heat input, the maximum peak value is 4.4 mV.Voltage waveforms of magnetic liquids with magnetic fluid of 1.91% and 3.83% were similar. However, the magnetic susceptibility of 3.83% magnetic fluid is doubled  inside the tube on the induced electromotive force, there are 0.5 s of data to analyze the waveform. As shown in the right inset of Through further analysis of the theoretical model, the influence of contact angle and pipe diameter on the PHP generator was found, and the electromotive force limit of the generator was studied. As a proof-of-concept demonstration, a PHP which is filled with magnetic fluid with different concentrations has been tested. The device can generate induced voltage with a peak value of 0.1 mV to 4.4 mV. Only when heat input reaches a critical value, could the pulsation phenomenon be triggered. The higher heat input could produce more intense pulsation and stronger induced electromagnetic force. However, dry-out will appear gradually with massive heat input, which may lead to a reduction of induced voltage. The induced voltage waveform can reflect the flow situation in the PHP.By comparing the experimental data, the theoretical model possesses reliable accuracy, and can accurately calculate the induced electromotive force corresponding to different fluid velocities, pipe diameters and contact angles.The calculation results indicate that as the inner radius of the PHP increases, the induced electromotive force increases significantly. When the inner radius is 1.25 mm and the fluid velocity is 0.8 m/s, the maximum electromotive force can reach 13.191 mV, which indicates that the magnetic fluid PHP can generate an objective electromotive force under certain conditions.At a certain fluid velocity, the induced electromotive force decreases as the contact angle increases. However, the size of the contact angle exerts a very limited influence on the induced electromotive force. When the fluid velocity is 0.8 m/s, the induced electromotive force only decreases by 5.14% when the contact angle increases from 40° to 70°.