Tumour-bearing Wistar rats were randomly distributed into a control group  and leucine group . After 20 days of tumour evolution, the animals underwent 18-fludeoxyglucose positron emission computed tomography (18F-FDG PET-CT) imaging, and after euthanasia, fresh tumour biopsy samples were taken for oxygen consumption rate measurements (Oroboros Oxygraph), electron microscopy analysis and RNA and protein extraction. Our main results from the LW group showed no tumour size change, lower tumour glucose (18F-FDG) uptake, and reduced metastatic sites. Furthermore, leucine stimulated a shift in tumour metabolism from glycolytic towards oxidative phosphorylation, higher mRNA and protein expression of oxidative phosphorylation components, and enhanced mitochondrial density/area even though the leucine-treated tumour had a higher number of apoptotic nuclei with increased oxidative stress. In summary, a leucine-rich diet directed Walker-256 tumour metabolism to a less glycolytic phenotype profile in which these metabolic alterations were associated with a decrease in tumour aggressiveness and reduction in the number of metastatic sites in rats fed a diet supplemented with this branched-chain amino acid.Leucine can stimulate protein synthesis in skeletal muscle, and recent studies have shown an increase in leucine-related mitochondrial biogenesis and oxidative phosphorylation capacity in muscle cells. However, leucine-related effects in tumour tissues are still poorly understood. Thus, we described the effects of leucine in both  Cachexia related to cancer, termed cancer cachexia, is a multifactorial syndrome that leads to an involuntary weight loss associated with poor quality of life, poor prognosis, and high mortality. The main cause of weight loss is skeletal muscle wasting2 that leads to muscle activity impairment. Thus, in order to prevent or to treat skeletal muscle wasting, some nutritional strategies have been used. In this regard, leucine supplementation has mainly been investigated due to its potential to increase protein synthesis in skeletal muscle. Alone or together with other branched-chain amino acids (BCAAs), including valine and isoleucine, leucine has stimulatory actions on skeletal muscle protein synthesis by acting as a cell signalling molecule leading to the activation of the mechanistic target of rapamycin (mTOR) pathway, the key cellular protein synthesis pathway4. Moreover, leucine can also act as an energetic molecule. Once in the cell, this amino acid is converted into α-ketoisocaproate by BCAA aminotransferase (BCAT) after which α-ketoisocaproate is converted to isovaleryl-CoA by BCAA α-ketoacid dehydrogenase (BCKD) and finally used in the citric acid cycle (TCA) to produce ATP5. Furthermore, leucine increases the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) expression leading to mitochondrial biogenesis and improving oxidative metabolism in skeletal muscle8.Cachexia affects up to 80% of advanced cancer patients and is responsible for almost 30% of all cancer-related deathsAlthough the effects of leucine are well-described in skeletal muscle, the effects of leucine on tumour tissues are still unknown, specifically with respect to the mechanism of tumour growth. Therefore, considering the potential benefits of leucine treatment of cancer-associated muscle wasting, we aimed to describe the effects of a leucine-rich diet in the cancer cachexia Walker-256 tumour model.max of 18F-fludeoxyglucose (FDG) decreased by 22.6% in the leucine-rich (LW) group (P ≤ 0.05) . We also found a reduction of 54% in metabolic tumour volume (MTV)  in the LW group  consumed less glucose  and produced less lactate  05) Fig. . Analysioup Fig.  and a deoup Fig. . Corrobo12) Fig. , showing12) Fig. .Figure 1Oxygen consumption supported by glutamate plus malate was evaluated in fresh tumour biopsies from both groups with the addition of adenosine diphosphate (ADP) and carboxyatractyloside (CAT) during the experiments. Figure in vitro study exposing the isolated Walker-256 cells to leucine (50 µM) for 24 h. Mitochondrial bioenergetics were assessed by measuring oxygen consumption rate using the Seahorse XF24 Analyser. Using the mitochondrial inhibitors oligomycin, rotenone, antimycin, and the protonophore CCCP, we assessed: basal respiration, ATP-linked respiration (portion of respiration associated with ATP production), maximum respiratory and spare capacities, which reflect OXPHOS integrity and electron transport chain capacity. The ATP-linked and basal oxygen consumption rates (OCR), proton leak, maximal respiration, and spare capacity were all significantly increased in leucine-treated Walker-256 cells, suggesting that leucine treatment also induced mitochondrial oxidative capacity in vitro  after treatment with leucine and whether leucine-treated cells would be more susceptible to an exogenous stressor, such as rotenone. However, different from tumour tissues, cells treated with leucine did not change their viability  in the presence of leucine and induction of a metabolic shift from glycolytic towards OXPHOS in both Warburg effect. This phenomenon is observed in the majority of cancer cells and is considered one of the hallmarks of cancer10. Even though it is inefficient regarding ATP production, aerobic glycolysis provides many advantages, such as a rapid and easily accessible source of glycolytic intermediaries that can be shunted into the pentose phosphate pathway and nucleotide synthesis, both of which are essential for cells with a higher proliferative rate. Moreover, this aerobic glycolysis causes an increase in lactate production and export, leading to acidification of the extracellular environment favouring metastasis formation11. In this study we found a decrease in 18F-fluorodeoxyglucose uptake and lactate exportation (lower expression of Ldha protein) in both the tumour biopsies and leucine-treated cells. Consequently, we found lower metastatic sites in tumour-bearing rats fed with a leucine-rich diet. This phenotype was followed by an increase in OXPHOS capacity of the tumours from the leucine-supplemented group.Most cancer cells present high rates of glucose uptake and aerobic glycolysis, a phenomenon known as the 12 cycle, leucine-treated cells favoured OXPHOS instead of glycolysis. The oxidative phenotype found in tumours biopsies from the leucine group was observed in both phosphorylating (ADP addition) and non-phosphorylating  states, which reflected an increase in oxidative capacity of these tumour biopsies. Accordingly, Walker-256 tumour biopsies and cells showed an increase in all bioenergetic parameters that were analysed in the functional respiratory assays.This increase in OXPHOS capacity could be stimulated by acetyl-CoA, which is one of the final catabolic products of leucine metabolism. Since acetyl-CoA is directly consumed by mitochondria through the TCAIn agreement with these results, leucine treatment caused an increase in the expression of mitochondrial genes  and proteins (CS and ATP synthase (Complex V) and UQCRC2 (Complex III)) associated with OXPHOS in the tumour biopsies. These molecular data agreed with higher mitochondrial density and area as observed by TEM images in the LW group.in vitro findings showed that leucine treatment boosts mitochondrial function as indicated by increased oxygen consumption rates and reduction in lactate levels. In line with these results, PGC-1α, NRF-1, COX5a, CS and CytC expressions, together with ATP synthase (Complex V) protein expression, were also significantly higher in leucine-treated cells, suggesting that leucine treatment also activated the mitochondrial oxidative phenotype in vitro.Interestingly, leucine treatment induced metabolic reliance on OXPHOS also in Walker-256 cells, suggesting a direct effect of leucine on this tumour metabolism. Notably, our 7. The study performed by Vaughan and colleagues8 evaluated the effects of leucine treatment on oxidative and glycolytic metabolism in human and murine skeletal muscle cells. The authors observed a significant reduction in glycolytic metabolism and also on lactate export in leucine-treated cells. Therefore, Vaughan and colleagues concluded that leucine could potentially induce an oxidative profile by increasing the oxidative capacity in skeletal muscle cells. The blockage of lactate export may explain the alteration to a more oxidative phenotype8. In a previous study13, we found that lactate serum concentration was increased in Walker-256 tumour-bearing rats in comparison to healthy rats. This increase reflects high lactate production and its release by tumour tissues. Interestingly, under leucine treatment, tumour cells produced less lactate. Thus, as observed by Vaughan and colleagues8 in skeletal muscle cells, leucine also caused a decrease in Ldha expression, favouring an OXPHOS metabolism in tumours in both in vivo and in vitro experiments performed in this study.Other studies have shown that leucine can increase OXPHOS and mitochondrial biogenesis in other types of cells, such as skeletal muscle, and change the metabolic profile in these cells14. In a glycolytic phenotype, the mitochondrial network appears interconnected, seems to be fragmented, or undergoes matrix expansion15. In fact, TEM images of Walker-256 tumour tissue showed a particular mitochondrial morphology, confirming to a glycolytic phenotype. On the other hand, the mitochondrial morphology of Walker-256 tumour tissue from leucine-treated rats was linked to an oxidative phenotype. The TEM images of mitochondrial morphology reinforce the hypothesis that leucine induces a metabolic shifting from glycolytic towards oxidative phosphorylation.It is also known that mitochondrial shape changes according to energy state16. We found higher MDA content in tumour biopsies from tumour-bearing rats fed with a leucine rich-diet. The increase in oxidative stress response was accompanied by a reduction in antioxidant response (decreased GST activity and higher MDA/GST ratio). Furthermore, a higher apoptosis index (a greater number of apoptotic nuclei) was observed in the LW group, in line with the increase in necrotic tumour mass observed by PET/CT images. Nakano and colleagues17 showed that BCAAs enhanced senescence in liver cancer, which was stimulated by DNA damage mediated through the mTORC1 pathway increasing the p21 translation, activated by p5318 leading to senescence and apoptosis. Accordingly, we observed an increase in protein expression of p53 in the tumour biopsies from the LW group. In fact, a study performed by Hagiwara and colleagues19 showed that BCAA supplementation decreased the incidence of liver cancer in both obese patients and diabetic model animals of carcinogenesis.Besides metabolic shifting, we observed that a leucine-rich diet stimulates oxidative stress and apoptosis in Walker-256 tumour biopsies. A higher rate of OXPHOS produces not only ATP but also increases the oxidative stressin vitro cells of Walker-256 tumours were used to elucidate this phenomenon. It was expected that cells treated with leucine would present higher cell death when exposed to exogenous oxidative stress, such as rotenone . However, cells treated with leucine (50 μM for 24 or 96 h) showed the same viability in comparison to non-treated cells and both groups had their viability diminished when the stressor agent rotenone was added. Thus, leucine treatment showed no effects on cell death or superoxide production within these in vitro experimental conditions. The fact that the results observed in tumour biopsies were not observed in isolated cells meant that probably other mechanisms underpinning this phenomenon could be acting differently from the in vivo study. Further studies should investigate the mechanism on how leucine could induce oxidative stress and apoptosis as seen in Walker-256 tumour tissue.Considering that leucine led to an increase in apoptotic nuclei and a decrease in GST levels in tumour biopsies, isolated 20 evaluated the effects of leucine supplementation on pancreatic cancer growth in lean and overweight mice and found that leucine increased tumour growth. Xiao and colleagues21 assessed the effects of leucine deprivation in breast cancer in in vitro and in vivo models and showed that the deprivation of leucine inhibits and induces apoptosis in the selected breast cancer models. The different findings from these studies compared with the present one could be explained by some factors, such as different types of tumours and the presence or not of a cachexia state.Other studies have evaluated the effect of leucine on cancer cell growth and metabolism. The study performed by Liu and colleaguesIn conclusion, we describe for the first time the effects of a leucine-rich diet in Walker-256 tumour metabolism. Our data showed that leucine induced a metabolic shift from glycolytic towards OXPHOS in tumour biopsies and isolated cells. The metabolic shift was accompanied by enhanced mRNA and protein expressions of OXPHOS components, higher mitochondrial density, and area. Moreover, these metabolism alterations were associated with a decrease in tumour aggressiveness, suggesting that the leucine nutritional supplementation does not benefit this type of tumour. It is important to emphasize that the interpretation of our results considered only the Walker-256 tumour metabolism.22) with the following components: The control diet (W) contained 18% protein and was composed of 20% casein (as a protein source), 39.7% corn starch, 13.2% dextrin, and 10% sugar (as carbohydrate sources), 7% soy oil (as a fat source), 5% cellulose micro fibre (as a fibre source), 3.5% salt mix, 1.0% vitamin mix, 0.3% cysteine, and 0.25% choline. The leucine-rich diet (LW) also contained 18% protein and was composed of the same amounts of casein, fat, fibre, salt, vitamin mix, cysteine, and choline as the control diet in addition to 3% leucine, 38.7% corn starch, 12.2% dextrin, and 9% sugar (as carbohydrate sources). The diets were normoproteic, isocaloric, and normolipidic.Adult female Wistar rats  were housed in collective cages under controlled environmental conditions . The animals were monitored daily, weighed three times/week, and given food and water ad libitum. Semi-purified diets were made in accordance with the American Institute of Nutrition  were injected subcutaneously into the right flank of the rats. The general guidelines of the United Kingdom Co-ordinating Committee on Cancer Research, 1998 (UKCCCR)27 regarding animal welfare were followed, and the experimental protocol was approved by the Institutional Committee for Ethics in Animal Research .The Walker-256 tumour is a well-established experimental model of cancer cachexiaThe animals were randomly distributed into two experimental groups . The control group (W) was fed a control diet (18% protein), and the leucine group (LW) was fed a leucine-rich diet (18% protein + 3% leucine). At a pre-agonic moment (around twenty days following tumour inoculation), animals underwent 18F-FDG PET-CT imaging . After 20 days of tumour evolution, the rats were euthanased. The tumour was removed and weighed. Some tumour fragments were immediately placed on ice-cold buffer containing 10 mM Ca-EGTA buffer for oxygen consumption measurements. Other tumour fragments were frozen directly in liquid nitrogen and stored at −80 °C for real-time polymerase chain reaction (PCR) and western blotting analyses, and additional fragments of tumour were immediately fixed in 2.5% glutaraldehyde and 2.5% paraformaldehyde in sodium cacodylate buffer (0.1M) at pH 7.4 and CaCl2 (3 mM) for 24 h on ice before being processed for electron microscopy analysis.18F-FDG injection and image acquisition, rats were anesthetised via intraperitoneal injection of ketamine (100 mg/kg body weight) and xylazine (12.5 mg/kg body weight)18.F-FDG (37MBq [1mC]) in approximately 0.5 mL of NaCl 0.9% solution was injected via the caudal vein. Radioactive activity was measured prior to the injection. Sixty minutes after 18F-FDG injection, each rat was subject to PET/CT imaging in the prone position. The scans were performed on the PET/CT imaging system (Siemens – Biograph mCT40). CT acquisition conditions were set to 70 kV, 155 mA, and 0.5-mm slice thickness. Each bed was scanned for 20 min from head to tail. A three-dimensional (3D) reconstruction model was used for analyses: OSEM 3D with 24 subsets and two interactions. For this, PET and CT images were fused through True D software (Siemens). Regions of interest (ROI) were drawn using the semi-quantitative method (Isocontour), this way we could determine the maximal 18F-FDG uptake (SUVmax) in tumour areas and other tissues. SUV is defined as (A*W)/Ainj, in which A (Bq/mL) is the radioactivity measured in a ROI, W (g) is the animal weight, and Ainj (Bq) is the activity of injected 18F-FDG. SUVmax is more accurate for estimating true SUV than SUV mean for this kind of analysis.The animals were fasting for 6 h prior to PET/CT scans. For a precise 28. Briefly, Walker-256 tissues were harvested from Wistar rats and placed on ice-cold buffer containing 10 mM Ca-ethylene glycol-bis (B-aminoethyl ether)-N′N′N′N′-tetraacetic acid (EGTA) buffer , 20 mmol/L imidazole, 50 mmol/L K+/4-morpholinoethanesulfonic acid, 0.5 mmol/L dithiothreitol, 7 mmol/L MgCl2, 5 mmol/L ATP, 15 mmol/L phosphocreatine, pH 7.1. Individual bundles from eight to eleven mg of tumour tissue were separated with forceps. Samples were permeabilised in ice-cold buffer containing saponin (50 μg/mL) for 30 min, gently stirred, and washed three times with MiR05 medium  at 4 °C. Samples were dried with filter paper and weighed30.The oxygen consumption was made according to the previous study performed by Busanello and colleagues29 and28. Permeabilized tissues were added to a MiR05 medium containing EGTA (500 mM) at 37 °C supplemented with 10 mM glutamate plus 5 mM malate in an Oroboros oxygraph . ADP (1 mM) and carboxyatractyloside  were added during the experiments.Oxygen consumption was evaluated according toTissue samples were removed from animals and immersed in a fixative solution , pH 7.4, for 24 hours at 4 °C. Then, tissues were dehydrated in graded concentrations of alcohol, embedded in historesin  and sectioned at a width of 3 μm. The sections were mounted on slides and stained with haematoxylin and eosin. The sections were then examined for image analysis of apoptotic nuclei using a Nikon Eclipse E800 light microscope . The apoptotic nuclei were counted using Image Pro-Plus Premium software  after capturing the image on a Leica microscope  using 40× magnification. The number of apoptotic cells was determined by counting 20 fields on one slide from at least three samples per each group (W and LW).2 (3mM) for 24 h on ice. Tissue samples were then rinsed with cacodylate buffer/CaCl2 and were post-fixed in 1% OsO4 in sodium cacodylate buffer (0.1M), CaCl2 (3mM), and potassium ferrocyanide solution (0.8%) for 1 h on ice. Following that step, tissue samples were washed with milli-Q water and stained with uranyl acetate (2%) overnight at 4 °C. The next morning, tissue samples were washed in milli-Q water and dehydrated using an ethanol gradient. The samples were embedded in Epon 812 resin. Resin polymerisation was controlled in an incubator (60 °C) for 72 h. Ultra-thin sections were stained with uranyl acetate and lead citrate and then observed on a TEM LEO 906 (Zeiss), operated at 60 kV.For TEM, the tissue was immersed in a fixative solution consisting of 2.5% glutaraldehyde and 2.5% paraformaldehyde in a sodium cacodylate buffer (0.1M) at pH 7.4 and CaCl® reagent (Invitrogen) following the manufacturer’s instructions. The quality of the RNA samples was examined at 260/280 nm and 260/230 nm with a UV spectrophotometer . cDNA was produced using a high capacity cDNA reverse transcription kit  containing Multiscribetm Reverse Transcriptase. cDNA synthesis was performed on 1 μg of RNA at 42 °C. Finally, cDNA was diluted 1:2 before its use in qPCR. Real-time reactions were performed using standard methods  and qPCR analysis was normalised to beta-actin. The genes evaluated using qPCR were PGC-1α (forward primer 5′-GACCACAAACGATGACCCTC-3′ and reverse primer 5′-TGTTGCGACTGCGGTTGT-3′), COX5A (forward primer 5′-TGTTGGCTATGATCTGGTTCC-3′ and reverse primer 5′-TTATGAGGTCCTGCTTTGTCC-3′), NRF-1 (forward primer 5′-TGCCCAAGTGAATTACTCTGC-3′ and reverse primer 5′-TCGTCTGGATGGTCATTTCAC-3′), CS (forward primer 5′-TATGGCATGACGGAGATGAA-3′ and reverse primer 5′-CATGGACTTGGGCCTTTCTA-3′), ATP5a (forward primer 5′-TGTTGCTTACCGCCAGATGT-3′ and reverse primer 5′-AGCAGGCGAGAGTGTAGGTA-3′), Cytc (forward primer 5′-AGGCAAGCATAAGACTGGAC-3′ and reverse primer 5′-ACTCCATCAGGGTATCCTCTC-3′) and succinate dehydrogenase (SDH) (forward primer 5′-ACCCCTTCTCTCTCTACCG-3′ and reverse primer 5′-AATGCCCGCTTCTCCTTGTAG-3′).Total RNA from the Walker-256 tumour tissue and cells was extracted with TRIZOL®, Roche), and protein concentration was measured by the Bradford method3. The proteins were separated by electrophoresis, transferred to nitrocellulose membranes, and the membranes were then incubated with primary antibodies against Ldha , α-tubulin , OXPHOS (Abcam 1:1000), Citrate Synthase , p53 , and vinculin  as a loading control. After that, the membranes were probed with secondary antibodies conjugated with peroxidase, and bands were visualised using a chemiluminescent reagent (ThermoFisher Scientific). The membrane images were captured using an image system , and band intensity quantitation was done using the Gel Pro software.Samples of cultured tumour cells and tumour biopsies from Walker-256 tumour-bearing rats were lysed in RIPA buffer  supplemented with protease inhibitor cocktail (Complete−1 * µg protein−1) was determined following the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with glutathione31. The tumour lipid peroxidation product MDA was quantified using the substrate n-methyl-2-phenylindole (MPO)32. Once the GST activity and MDA content were determined, the MDA/GST ratio was calculated showing the intensity of oxidative process against the anti-oxidative response.Tumour tissue samples were weighed, homogenised in phosphate-buffered saline (PBS), and centrifuged for 15 min at 10,000 g. The supernatant was collected, maintained on ice, and assayed in duplicate. Tumour GST activity  supplemented with 10% bovine calf serum (Lonza) and 1% penicillin/streptomycin (Lonza) and maintained at 37 °C and 95% O2–5% CO2 atmosphere with 85% relative humidity. Lactate production and glucose consumption in vitro analyses were done as a measurement of the lactate concentration released in the medium, and the glucose consumed was measured accordingly to the manufacturer’s instructions . Briefly, cells were cultured in 12-well plates and treated with 50 µM L-leucine  for 24 h. The medium was removed and assayed for lactate production1 and glucose consumption2. Additional Walker-256 cells were seeded in 12-well plate and treated with 50 µM L-leucine  for 24 h for mitochondrial respiratory function analyses (Seahorse), protein extraction (western blotting), and RNA extraction (qPCR).For 33. The oxygen consumption rate (OCR) was measured in accordance with the manufacturer’s instructions. Oligomycin (1 µM) was used to inhibit ATP synthase, and protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (2 µM) was used in order to uncouple mitochondrial OXPHOS and rotenone (rot)/antimycin (AA) (1 µM) to block mitochondrial respiration and determine non-mitochondrial OCR. ATP-linked OCR was calculated by subtracting the uncoupled OCR (after the addition of oligomycin) from the basal OCR. Spare capacity was determined by subtracting basal OCR from the CCCP-induced OCR. Non-mitochondrial OCR values were subtracted from all data before being used for the analyses. All Seahorse measurements were normalised by protein quantified with respect to the Bradford assay.The Seahorse analysis  was performed according to the previous study performed by Lima and colleagues34. Briefly, Walker-256 cells were seeded in 96 well plates at 1 × 103 cells per well and allowed to attach overnight. On the next day, cells were treated with 50 µM L-leucine for 24 or 96 h, as indicated in the figure legends. Neutral red solution (40 µg × mL) was added and cells were incubated for additional 2 h. After that, the medium containing the staining solution was removed, and cells were washed twice with PBS. The stain was extracted from inside the cells by adding a solution of 50% ethanol, 49% H2O, and 1% glacial acetic acid. The absorbance was read at 540 nm.Cell viability was accessed using neutral red uptake as as described by Repetto and colleagues3 cells per well and allowed to attach overnight. On the next day, cells were treated with 50 µM L-leucine for 24 or 96 h, as indicated in the figure legends. DHE solution (10 µM) was added to the medium, and cells were incubated at 37 °C for 30 min, in the cell incubator. After that, Hoechst 33342 solution (1 µg × mL−1)  was added, and cells were incubated for 15 min in the cell incubator. Cells were then washed twice with PBS, and DHE fluorescence was measured using an excitation/emission 518/605 nm; HO fluorescence was measured using an excitation/emission 350/461 nm.Superoxide production was accessed using dihydroethidium . Walker-256 cells were seeded in 96 well plates at 1 × 10The statistical analyses were performed using the software Graph Pad Prism 6.0 . Data are expressed as mean ± standard deviation (s.d.) and analysed by t-test. P ≤ 0.05 was considered significant.Supplementary Figure 1 and 2


Most of the pore-scale imaging and simulations of non-Newtonian fluid are based on the simplifying geometry of network modeling and overlook the fluid rheology and heat transfer. In the present paper, we developed a non-isothermal and non-Newtonian numerical model of the flow properties at pore-scale by simulation of the 3D micro-CT images using a Finite Volume Method (FVM). The numerical model is based on the resolution of the momentum and energy conservation equations. Owing to an adaptive mesh generation technique and appropriate boundary conditions, rock permeability and mobility are accurately computed. A temperature and concentration-dependent power-law viscosity model in line with the experimental measurement of the fluid rheology is adopted. The model is first applied at isothermal condition to 2 benchmark samples, namely Fontainebleau sandstone and Grosmont carbonate, and is found to be in good agreement with the Lattice Boltzmann method (LBM). Finally, at non-isothermal conditions, an effective mobility is introduced that enables to perform a numerical sensitivity study to fluid rheology, heat transfer, and operating conditions. While the mobility seems to evolve linearly with polymer concentration in agreement with a derived theoretical model, the effect of the temperature seems negligible by comparison. However, a sharp contrast is found between carbonate and sandstone under the effect of a constant temperature gradient. Besides concerning the flow index and consistency factor, a master curve is derived when normalizing the mobility for both the carbonate and the sandstone. Understanding reservoir rock and fluid properties is essential for applications such as oil and gas industry, water management, hydrology, and geosciences. Oil exploration and production in conjunction with the prospective impact on the environment is highly related to the fluid flowing inside the highly complex geometry of the porous media ,2. In orUsing the 3D micro-CT images, numerical simulation has been applied to perform the pore-scale imaging modelling ranging from (i) the widely used lattice Boltzmann method ,7; (ii) However, it is worth mentioned some interesting contributions of non-Newtonian fluids at Darcy scale both at isothermal and non-isothermal conditions. Using a rheological power-law fluid , a One-dPredicting special core analysis (SCAL) data by means of digital rock physics (DRP) has gained attention recently. Most of the DRP simulations focus on Newtonian fluids and overlook the rheology of the fluid, which can be controlled by both the temperature and the polymer concentration. However, in petroleum engineering many fluids such as heavy oil and polymer solutions used for enhanced oil recovery (EOR) are non-Newtonian while the flow is non-isothermal at reservoir conditions. In order to use Digital Rock Physics (DRP) as a future tool to generating accurate, fast, and cost effective SCAL properties to support reservoir characterization and simulation; pore-scale modeling of non-Newtonian fluid at non-isothermal conditions is essential. This work proposes to shed a light on that aspect of the DRP.In the present paper, we use a finite volume method (FVM) coupled with an adaptive mesh generation technique to perform the pore scale simulation from the micro-CT images of both a sandstone and a carbonate rocks from the literature. Besides, we performed simulations based on the LBM method for comparison and validation of the FVM on Newtonian fluid. Finally, we implement a power-law  non-Newtonian fluid at non-isothermal conditions to test the pore-scale model sensitivity to both the rheological and operating parameters. This paper, focused on modeling at pore-scale, can be considered as complemental to the large body of works on simulating non-Newtonian fluids at Darcy-scale ,18,20.The paper is organized as follows: in C) and temperature (T), can be written as function of the shear rate  formulation expresses as follows:It is worth noting that the flow within the porous rocks is considered very slow and laminar, Reynolds number Equation .In addition to the mass and momentum conservation, we solve the energy conservation equation, given below:C) for EOR at different shear rate is evidenced in [n denote the consistency factor and the flow index, respectively. In addition, we assume that the consistency factor T) and the polymer concentration (C). Besides The principle of polymer flooding is to increase water phase viscosity by adding polymer at a given temperature. For our modeling purposes, the resulting polymer solution is assumed homogeneous with a constant concentration which remains constant during the fluid flow through the porous media. In addition, polymer molecules’ size is assumed to be much smaller than the resolved pore-size μTWe can recast Equation  to introfound in ,19 in thC is the polymer concentration, n is the flow behavior index.Finally, by adopting a two-parameters Ostwald-DeWaele non-Newtonian fluid and combining Equations  and 8)  8) into Equation  can be uDue to the challenge to perform experiments at the scale of the micro-CT 3D images resolution and the characterization of fluid flow within the pore network, the present work will be based on numerical simulations to gain insight into the physics of modeling complex fluid through a rock.The workflow from the micro-CT image to the computation of petrophysical properties such as the permeability is summarized in One of the challenges in simulating flow in porous rocks is that experimentally, laboratory measurements cannot be performed at the scale in which pore phase is resolved. However, pore-scale modeling within the emerging digital rock physics (DRP) paradigm has gained attention recently going from understanding the physics within the rock to a predictive tools to support laboratory measurements ,26. The Before running the simulation, the segmented micro-CT image is meshed using SnappyHexMesh/ C++ code by employing an adaptive mesh generation technique, through refinement and adjustment to fit onto the provided geometries of the rock. Besides incorporation of boundary layers’ cells near the solid surface is also performed for better accuracy .The pore-scale model is simulated by imposing a non-slip boundary conditions at the sides of the micro-plug sample and a pressure gradient between the inlet and outlet of the digital rock . The detz-component   see  of the fBased on the homogenization theory in , Darcy eQ is the flow rate, L and A are the sample length and surface area, respectively. It accounts for all the relevant parameters for the non-Newtonian flow in the porous media, namely the porosity, temperature, concentration, intrinsic permeability. It is worth noting that for a Newtonian fluid, Due to the non-Newtonian nature of the fluid and the dependency of the viscosity to both temperature and concentration, and for convenience, we will adopt an effective fluid mobility (The governing equations Equations –9) are  are 9) aQ,We first validate the model for a Poiseuille-Hagen flow with both a Newtonian and non-Newtonian fluids flowing between two parallel plates. The non-Newtonian fluid viscosity is taken as a power-law and is given below:The comparison between analytical and numerical solutions is provided in For validation purpose of our model in porous media, we apply the FVM model to 2 rocks samples from the literature, the Fontainebleau sandstone and Grosmont carbonate . We perfterature ,5, the vThe difference is less than 2% suggesting that the implemented finite volume method (FVM) is capable to simulate accurately at the pore-scale. Unlike in the LBM, extension of our FVM model to non-Newtonian fluid can be handled without any numerical tuning parameters. Finally, although a good agreement is found with the implemented code, a full validation would required to perform pore-scale experiment within the same scanned rock, which experimentally is still to be addressed.A and L are fixed, we can simplifyThe mobility describes the ability of the porous rocks to transmit the fluid, in the present case a power-law non-Newtonian fluid. The high mobility would indicate a better transmissibility of the fluid as opposed to the low mobility. In order to investigate the concentration effect on the mobility, we simulated the fluid flow at pore-scale by varying the concentration at a fixed temperature using the proposed fluid rheology model given in Equation . The kinEquation  we have:C = n = 0.81. Therefore, by expanding Equation , which are not account for when the mobility or permeability is computed over the z-axis. The difference in the mobility over the temperature gradient range is less than 0.1% , there is however a slight difference on the behavior index (n). In addition, the effect of K on the mobility seems to be more pronounced than that of n. It is worth noting the challenge posed experimentally to discriminate the effect of these two parameters contribution on the mobility. Hence, the possibility to perform such a sensitivity study numerically can help in the formulation of polymer solutions for EOR.We observe that the mobility is reduced by an increase of both n, the flow behavior index. The non-linearity nature of the fluid flow within the porous media seems to be more dominant for the carbonate, of complex structure, than for the sandstone sample. This effect is more pronounced at higher pressure gradient and Q on the power of pressure gradient is in agreement with the recent founding experimentally at Darcy scale in [Here the model sensitivity under the effect of pressure gradient is investigated from the two samples, namely the Fontainebleau sandstone and the Grosmont carbonate. In scale in  for yielscale in  for HersA comprehensive numerical model based on the Finite Volume Method (FVM) and an adaptive meshing technique to describe the flow properties at pore-scale of a non-Newtonian fluid is presented. The fluid rheology is modeled by incorporating its dependency to the concentration and temperature into a generalized power-law viscosity fluid model. Based on Newtonian fluid through rock samples from the literature, the FVM algorithm is validated against a Lattice Boltzmann Method (LBM). After implementing the non-Newtonian and non-isothermal fluids, the model is validated by simulating the fluid flow between two parallel plates for which the analytic solution is known. Subsequently, the model sensitivity to heat transfer and fluid rheology is tested by evaluating the effect of polymer concentration on the mobility as well as the relationship between flow rate and pressure gradient. The effective mobility dependency on the polymer concentration follows a linear trend, in line with a derived theoretical model, while the flow rate displays a disparity between the carbonate and sandstone. Besides the fluid rheological properties such as the flow behavior index and consistency factor lead to a master curve when normalizing the mobility for both the carbonate and the sandstone. Overall, the present work serves to implement a new simulator capable of computing the permeability and mobility of non-Newtonian fluid based on its thermal and rheological properties. The solver which can be used for different rock types at pore-scale can provide valuable insight into predicting flow behavior at reservoir conditions as well to better understanding polymer flooding for enhanced oil recovery (EOR).


The characterisation and clinical relevance of tumour-infiltrating lymphocytes (TILs) in leiomyosarcoma (LMS), a subtype of soft tissue sarcoma that exhibits histological heterogeneity, is not established. The use of tissue microarrays (TMA) in studies that profile TIL burden is attractive but given the potential for intra-tumoural heterogeneity to introduce sampling errors, the adequacy of this approach is undetermined. In this study, we assessed the histological inter- and intra-tumoural heterogeneity in TIL burden within a retrospective cohort of primary LMS specimens. Using a virtual TMA approach, we also analysed the optimal number of TMA cores required to provide an accurate representation of TIL burden in a full tissue section. We establish that LMS have generally low and spatially homogenous TIL burdens, although a small proportion exhibit higher levels and more heterogeneous distribution of TILs. We show that a conventional and practical number (e.g. ≤3) of TMA cores is adequate for correct ordinal categorisation of tumours with high or low TIL burden, but that many more cores (≥11) are required to accurately estimate absolute TIL numbers. Our findings provide a benchmark for the design of future studies aiming to define the clinical relevance of the immune microenvironments of LMS and other sarcoma subtypes. This approach offers several advantages over conventional examination of full tissue sections by minimising consumption of often limited tissue while providing various efficiencies in downstream sample processing and analysis. However, an inherent limitation to the use of TMAs is that, for each included specimen, only a small amount of tissue is sampled and arrayed, meaning that sampling error may lead to a distorted representation of the full tissue section. This limitation is of particular relevance in the study of tumour specimens, where intra-tumour spatial heterogeneity in terms of morphology and underlying molecular pathology is now well established in many cancer types2. Multiple studies have been undertaken to validate the TMA methodology in the assessment of various cancer biomarkers, with the aim of demonstrating that biomarker levels reported by TMAs are representative of results obtained when full sections are assessed. In this manner, it has been shown that expression levels of a diverse repertoire of tumour biomarkers are accurately reported through the assessment of TMAs, typically with the provision that between 1 and 3 replicate cores from each included tumour are assessed and aggregated6.Tissue microarrays (TMAs) are useful diagnostic and research tools that permit high-throughput histological and molecular studies of up to several hundred tissue specimens simultaneously by arraying them into a paraffin block10. TMAs continue to be frequently employed in studies that seek to investigate the potential role of TILs as putative biomarkers15. The success of such studies is dependent on the ability of the TMA approach to capture a sufficiently representative picture of the immune phenotypes present within the wider tumour. Observations of quantitative and qualitative spatial heterogeneity in the immune microenvironment of individual tumours of various cancer types call into question how well TMAs can provide such representation and whether the scope for sampling error renders them inappropriate for studies of tumour immunity20. There is currently little published evidence addressing this question23.Investigating the immune microenvironment as a potential source of cancer biomarkers is an area of renewed research interest. It is now known that the presence or absence of immune-related factors such as tumour-infiltrating lymphocytes (TIL) can serve as powerful prognostic and/or predictive biomarkers across a range of cancer types24. As with other STS subtypes, the immune microenvironment and its potential prognostic value is not well characterised in LMS. There is accumulating evidence that LMS is a disease that harbours extensive inter- and intra-tumoural genetic and morphological heterogeneity. For instance, recent genomic profiling analyses demonstrate that LMS is characterised by inter-tumour variability in somatic copy number alterations, a molecular characteristic found to have negative correlation with active anti-tumour immune response in a number of other cancers26. Furthermore, clinical evidence suggest that a small minority of LMS patients respond to immune checkpoint inhibitor therapy29. As such, inter-patient differences in the immune microenvironment of LMS may be useful for predicting response to therapy and prognosis. LMS often present with large primary tumours that exhibit intra-tumoural morphological heterogeneity of tumour cells and associated stroma, and consequently may also display intra-tumoural TIL heterogeneity30. To assess the suitability of TMAs for profiling TIL burden in LMS, we sought to address two questions in this study: 1) What is the extent of inter- and intra-tumour heterogeneity of TIL burden in LMS, by comparing related tumour blocks from spatially distinct areas of primary tumours and 2) how many TMA cores are required to provide sufficient representation of the TIL burden of the full tissue section?In this study, we assessed a cohort of leiomyosarcoma (LMS) tumour specimens to investigate the extent of inter- and intra-tumoural heterogeneity of TIL burden and how accurately TIL burden are represented by the TMA methodology, compared to full tumour sections. LMS are tumours of smooth muscle lineage that can arise from the uterus or from other anatomic sites including vessel walls. LMS are one of the more common soft tissue sarcoma (STS) subtypes, representing 10–20% of all STSSurgical resection specimens of primary LMS (n = 47) and accompanying annotation of baseline clinicopathological variables were identified and retrieved through retrospective review of departmental database and medical notes at a single specialist cancer centre. Histological diagnosis was confirmed by a specialist sarcoma histopathologist . Where available, 5 blocks containing formalin-fixed paraffin-embedded (FFPE) viable tumour from spatially distinct areas (at least 2 blocks each from tumour margins and core) of the same primary tumour were selected. Newly prepared haematoxylin and eosin (H&E) slides from each block were assessed to confirm presence of viable tumour material. Immunohistochemical (IHC) staining of T lymphocyte markers  was performed on consecutive 4 µm sections from each block . Human tonsillar tissue was used for positive control for expression of CD3, CD4 and CD8, with omission of primary antibody for negative control and use of Mouse IgG1  or Rabbit IgG  for CD4/CD8 or CD3 isotype controls respectively. IHC staining for B lymphocytes  was performed on all blocks from an initial set of 19 tumours – this was not expanded to all tumours due to uniformly low numbers of infiltrating B cells in this initial set.2) was manually counted by direct brightfield microscopy for each stained slide.The number of CD3, CD4, CD8 and CD20 IHC-positive lymphocytes in ten non-adjacent, tumour-containing high-power fields (HPF) . To generate a virtual TMA (vTMA), 1 mm diameter circular areas were selected from viable-tumour areas on each H&E image. For assessment of optimal number of cores, 20 × 1 mm areas were selected at random, while to assess for intra-tumoral variance between central and peripheral tumour regions, 20 × 1 mm areas were selected at both the tumour periphery (defined as within 3 mm of inked resection margin) and central (defined as ≥10 mm from nearest resection margin) regions. The corresponding areas were then selected on CD3 and CD8 digital slide images. Images of these areas at x10 magnification were exported as.tif files that were cropped to uniform 0.785 mmTo generate the physical TMA (pTMA), triplicate 1 mm diameter cores were sampled from areas of viable tumour within donor blocks from 44/47 LMS and re-embedded in an arrayed recipient paraffin block. Consecutive 4 µm sections from the arrayed block were stained for H&E, CD3 and CD8. After assessment of H&E slides to confirm viable tumour content, all CD3+ and CD8+ TILs were counted under direct brightfield microscopy. Average TIL number per 1 mm core (referred to herein as ‘TIL/core’) was calculated from triplicate cores for each tumour.To assess the extent of TIL burden in each of 47 LMS cases, an average number of infiltrating CD3+, CD4+ and CD8+ TIL per HPF (referred to herein as ‘TIL/HPF’) was calculated from 50 HPF per tumour (10 HPF from each of 5 related tumour blocks). Comparison of TIL burden of tumours from different anatomical sites of origin and grade was performed using 1-way ANOVA of Log2-transformed average TIL/HPF values with Prism v7.0 (GraphPad Software Inc).To assess the variability in TIL burden between different blocks from the same surgical specimen, we assessed the relative contribution of inter-block variation (block effect) and inter-tumour variation (tumour effect) on the total amount of variance in TIL numbers within the 47 LMS cohort by (i) Log2 transformation of all raw TIL/HPF count values (ii) calculation of average TIL/HPF with 95% confidence interval for each tumour block (average of 10 HPF), and across all 5 related blocks from each primary tumour (average of 50 HPF), and (iii) Ordinary 2 way ANOVA (Prism v7.0) to assess the percentage of total variability attributable to block effect, tumour effect, interaction between the two effects and residual variation. Correlation between Log2-transformed values for tumour average TIL/HPF and the standard deviation of average TIL/HPF of constituent blocks was assessed through calculation of Pearson correlation coefficients.n cores that fell within the following prescribed boundaries were then calculated: a) within +/− 20% of true TIL burden; b) within correct (i.e. same as true TIL burden) side of dichotomised ‘high/low’ boundary set at median of true TIL burdens from 47 LMS cohort; c) within correct quartile of 47 LMS cohort.Automated counts of infiltrating CD3+ and CD8+ TIL in all 20 × 1 mm vTMA cores for each tumour were used to calculate average TIL/core – this value was taken as representing the ‘true’ TIL burden of each tumour. Estimates of these true TIL burdens were then derived from the average TIL/core from all possible combinations of between 2 and 19 randomly-selected cores. The percentage of estimates generated from Automated counts of infiltrating CD3+ and CD8+ TIL in 20 × 1 mm vTMA cores were Log10-transformed and then used to derive sample frequency distributions, average TIL/core and standard deviation values from matched peripheral and central tumour areas in a subset of 6 tumours. Paired T tests were then performed to assess for differences between average TIL/core values from margin and core areas using Prism v7.0 (GraphPad Software Inc).The differences between Log2-transformed values of the estimated average TIL/core values derived from the pTMA and the true TIL burdens derived from the vTMA were calculated and plotted against the average of the two values in a Bland Altman plot along with 95% levels of agreement (Prism v7.0).Average of TIL/core values derived from triplicate cores within pTMA were used to identify each included LMS as having a ‘high’ or ‘low’ TIL burden, relative to the cohort median of true TIL burdens, as defined in the vTMA experiment. This high/low identification was then compared to a ‘gold standard’ high/low allocation, defined as the ‘true’ TIL burden of that tumour as derived from all 20 vTMA cores. Accuracy (%) of pTMA was defined as 100*(True Positive + True Negative)/Use of archival FFPE tumour samples and linked anonymised patient was approved by Institutional Review Board as part of the PROSPECTUS study, a Royal Marsden-sponsored non-interventional translational protocol .Adequate tumour material was identified for 47 patients with a confirmed diagnosis of LMS who had undergone radical resection of primary tumour  taken to represent the overall tumour TIL burden. Exemplar IHC images showing different degrees of CD3+ lymphocyte infiltration are shown in Fig. 36.For each case in the cohort, 5 tissue blocks that sampled spatially distinct tumour areas were assessed for TIL burden , indicated the near-absence of infiltrating B cells in a subset of 19 tumours. Across the different T lymphocyte markers, a dynamic range of 2–3 orders of magnitude (e.g. CD3 range 1–124 TIL/HPF) was seen in the extent of TIL burden between individual tumours Fig. . No signHaving established that overall TIL burdens can vary between individual LMS tumours, we assessed the extent of heterogeneity in average TIL/HPF between blocks taken from different regions from the same LMS specimen Fig. .Average TIL burden (stated as TIL/HPF) from each of 5 sampled blocks from the 47 cases are shown aligned with overall tumour average values in Fig. We performed 2-way ANOVA to objectively assess the relative extent that intra-tumoural heterogeneity (i.e. variation between blocks from the same tumour – ‘block effect’) and inter-tumoural heterogeneity  contributed to the overall amount of variation in TIL burden within the cohort Fig. . We founTaken together, these results indicate that while intra-tumoural heterogeneity was observed in a subset of LMS cases with higher overall TIL levels, intra-tumoural heterogeneity in TIL burden across the cohort was outweighed by the extent of inter-tumoural heterogeneity.in silico ‘virtual TMA’ (vTMA) that would allow for the iterative sampling of a number of cores that would be impractical for a physical TMA. We then assessed how many cores were required to produce an estimate of TIL burden that either (i) accurately recapitulated the true TIL burden of a tumour, or (ii) was sufficiently accurate to identify whether a tumour had high or low TIL burden, relative to the median or quartile TIL values of the entire cohort– this second approach was based on the observation that, in many published studies that have demonstrated clinical relevance of TIL numbers, similar rank-based categorisation was used, often based on dichotomisation around cohort median value36.To address the question of how many TMA cores must be sampled from a tumour to provide adequate representation of the overall TIL burden of a tumour, we devised an 2 - equivalent to approximately 50 HPF) were selected on H&E images, with the number of TILs within the corresponding areas (TIL/core) on CD3 and CD8 stained slides digitally counted  was required for >80% of estimated TIL burdens to fall within 20% of the ‘true’ CD3+ or CD8+ TIL burden for the corresponding tumour Fig. . However36. We found that sampling only 1 core was sufficient to correctly identify a majority of tumours as has having a ‘high’ or ‘low’ degree of infiltration, while 2–5 cores was adequate to correctly identify a majority of tumours as having ‘very low’, ‘low’, ‘high’ or ‘very high’ degree of TIL infiltration, based on categorical cut-offs at cohort quartiles. These data demonstrate that TMAs that include a practical number of replicate cores as used in previously reported studies (e.g. 3 or fewer) would be sufficiently representative when ordinal categorisation of TIL burden is planned. However, should precise quantification of the absolute value of true TIL burden be desired, a conventional TMA approach is unlikely to provide an adequate representation.Taken together, these data indicate that a large and likely impractical number of TMA cores (11 cores) must be sampled in order to accurately recapitulate the true burden of infiltrating T lymphocytes in LMS. However, many studies that have described an association between TILs and clinical outcome ultimately applied cut-off thresholds to assign TIL counts into ordinal categories (e.g. ‘high’ or ‘low’ infiltration) that reflect relative rather than absolute degree of infiltration33. In order to assess the degree of variability in TILs between the tumour periphery and central regions in our study, a subset of 6 LMS cases were subjected to a vTMA workflow  that included triplicate 1 mm cores from sampled tumours. Forty-four out of 47 LMS cases were included in this TMA. In 11/44 (25%) tumours, the same block was used for pTMA construction as was used for the vTMA model. In 33/44 (75%) tumours, due to insufficient tissue depth remaining in blocks used for the vTMA, a different tumour block from the same specimen was used for core sampling for the pTMA.In the vTMA model, a median of 11 cores were required to accurately estimate the absolute value for true TIL burden. We thus assessed if the triplicate cores used in pTMA were similarly inaccurate in estimating absolute true TIL burdens  was itself independently associated with worse prognosis. This suggests that spatial uniformity of TIL burden may vary between different cancer types and within different molecular and histological subtypes, and that spatial distribution itself may provide clinically relevant information, both findings that warrant caution when adopting TMA methods for assessing TIL burden. Immune microenvironment-based biomarkers that have been described in colorectal and breast cancers depend upon intra-tumoural spatial distinction of TIL, where comparison of infiltrates between the tumour core or invading margin, or between tumour nests and stromal components, have been shown to be important considerations8. In our study, we find that across the 6 cases analysed, there was no significant difference in TIL burden between tumour periphery and central regions. Comparative analysis indicates variation in TIL levels with some cases showing higher TIL levels in the periphery versus the core and others presenting the opposite observation. Cohen et al., have recently shown in a small cohort of 11 LMS cases that CD3+ TILs were present in both the central (11/11) and periphery (9/9) regions of the tumour. In their study, CD8+ TILs were found in a subset of cases in the central (10/11) and periphery (7/9) of the tumour41. Given the small number of cases assessed in both studies, further evidence in larger cohorts is required to determine if these observations are generally applicable to LMS and other STS subtypes.The applicability of our findings to other STS or epithelial cancers remains to be determined. Intra-tumoural heterogeneity in the immune microenvironment has been described in numerous epithelial cancer types, both within primary lesions and between different metastatic sites42. Our study does not provide any direct information on the use of TMAs to assess non-TIL immune factors and the extrapolation of our findings beyond TILs remains to be investigated.While TILs are accepted as central mediators of anti-tumour immune responses, the immune-tumour microenvironment constitutes a broad and complex range of cellular and protein factors that actively determine the nature and clinical consequence of any tumour-related immune responseOur data indicate that TMAs can provide a degree of representation of overall tumour TIL burden which is adequate for ordinal categorisation into high or low subgroups. The design of future studies of the immune microenvironment of tumours should acknowledge the inherent limitations of TMA methods and consider the incorporation of additional orthogonal approaches such as gene expression analysis and flow cytometry methodologies that are capable of providing complementary information regarding the composition of immune subsets leading to a more comprehensive and accurate representation of tumour immune microenvironment.Supplemental info


Thaumatotibia leucotreta is a key pest of citrus orange and other plants causing fruit loss through larval feeding. Although this pest is native to sub-Saharan Africa little is known on its performance on orange and vegetables in Kenya and Tanzania. Our objective was to assess the incidence, oviposition preference and offspring performance of FCM on orange and vegetables, namely, okra, African eggplant, chili and sweet peppers. A higher percentage of orange with FCM damage symptoms was recorded from the ground than from the tree sampled fruit. However, FCM larval incidence was higher for the latter (tree sampled fruit). The highest FCM larval incidence amongst the vegetables was recorded on African eggplant (12%) while the lowest was on okra (3%). Orange was the most while African eggplant was the least preferred for oviposition by FCM. Among the vegetables tested, strong oviposition preference was found for sweet pepper; however, larval survival was lowest (62%) on this crop. Highest larval survival (77%) was recorded on orange. Most demographic parameters  were comparable among the studied host plants. The results are discussed in line of FCM management.False codling moth (FCM),  Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), one of the pests of these crops, is native to sub-Saharan Africa [Persea americana (Mill) (Lauraceae) [Macadamia spp. (Proteaceae) [Gossypium spp.  [Thaumatotibia leucotreta is a multivoltine pest [T. leucotreta vary with the host plant. For example, scull on avocado [T. leucotreta infestations also cause financial losses due to quarantine restrictions imposed on exporting countries and detection of a single larva can result in rejection of an entire consignment [Fruit and vegetable production are important source of income for East African growers. Production of these crops is, however, constrained by insect pests and diseases resulting in yield loss and poor quality. The false codling moth (FCM), n Africa  and has n Africa . It is an Africa ,4, avocauraceae)  macadamiteaceae)  and cottlvaceae) . Thaumatine pest  which doine pest . The femine pest . The hat avocado  and a ye avocado . In addisignment .T. leucotreta has been reported in Kenya and Tanzania [T. leucotreta infestations on avocado grown at different altitudinal gradients in Kenya and Tanzania. Knowledge on the effect of altitude on T. leucotreta infestation on orange will contribute to management of the pest. The incidence of the pest on other crops which may serve as alternative host crops between successive orange fruiting seasons is not well known.Although South Africa and Egypt are the largest citrus producing countries in Africa, Tanzania and Kenya are considered as the leading countries in citrus production in East Africa . Citrus Tanzania , little Tanzania ,15,16, wTanzania . Odanga Tanzania  reportedT. leucotreta on orange in a laboratory study was reported by Love et al. [T. leucotreta reared on artificial diet have been reported [T. leucotreta on other key host plants has been reported. The field dynamics of T. leucotreta in a mixed cropping system, a common practice in sub-Saharan Africa, need to be investigated to develop better management strategies. The aims of this study were therefore to determine T. leucotreta larval incidence on ripe orange as well as on mature vegetables of okra (Abelmoschus esculentus (L.) Moench var. Clemson), African eggplant , chili pepper  and sweet pepper . These vegetables are mainly grown near or within orange orchards. The developmental performance and life table parameters of the pest on three solanaceous vegetables viz. chili, sweet pepper and African eggplant were also determined and compared to that of orange, the most common host.The ovipositional preference and offspring performance of e et al. . The ovie et al. , the plareported ,20,21,22reported . HoweverField surveys were carried out between June and September 2017. In Kenya, the survey was conducted in three citrus producing areas namely Kilifi , Makueni  and Machakos  representing low (0–500 masl), mid (501–1200 masl) and high (1201 masl and above) altitudes, respectively . RainfalThaumatotibia leucotreta damage symptoms were recorded for each fruit, washed using a non-caustic liquid dish washing soap and incubated for three weeks. The incubated fruit were dissected and the T. leucotreta larvae or pupae counted. The last instar larvae collected were placed in plastic containers (Kenpoly) (2 L) with a thin layer of soft sand covering the bottoms. Smaller larvae were transferred to glass jars containing an artificial diet developed by Moore et al. [T. leucotreta adults enclosing from the pupae were confirmed using standard keys. The percentage of orange fruit with T. leucotreta damage symptoms as well as percentage fruit infested with T. leucotreta larvae were calculated.Ripe orange fruit sampling was conducted from June to September 2017 in the three rainfed citrus producing areas of Kenya and Tanzania . The frue et al.  and rearT. leucotreta larvae or pupae. Percentage T. leucotreta larval incidence was calculated only. Since T. leucotreta damage symptoms on vegetables were not as clear as on oranges, assessment of the percentage of vegetables with damage symptoms caused by this pest, was excluded from the study.Vegetable sampling was conducted at Mlali ward in Morogoro rural , Table 1icipe), Nairobi, Kenya. This colony was established from field-collected larvae from citrus orange, Citrus sinensis L. var Washington Navel fruit in Wote, Makueni county  and had been maintained continuously for over 33 generations. The colonies were maintained on artificial diet described by Moore et al. [icipe. A wax paper sheet was inserted into the cage through a slit in the floor to serve as an oviposition substrate for the moths. Wet cotton wool as a source of water for the moths, was plugged into a hole cut at one side of the cage. The oviposition sheet was removed daily, cut into equal pieces, surface-sterilized with 25% formaldehyde  and placed onto the diet for egg hatching. The neonates fed on the diet and pupated in the cotton wool plug, which was then transferred into a wooden moth emergence box (40 × 40 × 40 cm) made at icipe. Eclosing moths were attracted through an exit hole on the side of the emergence box by light, entering through glass vials (32.6 cm3). The colony was maintained at 25.0 ± 2.0 °C, 60% relative humidity and 12-h photoperiod (L12:D12).Insects were obtained from the mass rearing room at the Animal and Quarantine Containment Unit at the International Centre of Insect Physiology and Ecology  closed with moist cotton wool to serve as a source of water [icipe and had two openings (diameter 10 cm) on two opposite sides. One of the openings was covered with netting cloth; and the other was fitted with a fine netting sleeve for easy handling of the insects and fruit [Newly emerged naive n et al. . One malof water  and keptnd fruit . Eight mThe no-choice test was done similar to the choice test but with only one type of fruit placed separately per Perspex cage (20 × 15 × 15 cm). Four moths were released in each cage. A replicate consisted of four cages with one fruit per cage. This no-choice test was replicated 12 times.Eggs obtained from the rearing colony were incubated in 2 L plastic containers (Kenpoly) until hatching. Two neonate larvae were transferred onto individual fresh fruit of orange, var Washington Navel, African eggplant, chili and sweet peppers of similar cultivars as above, collected from farmers’ fields at Wote, Makueni and Kangundo, Machakos. The inoculated fruit were incubated until final instar larvae exited. The larvae from each fruit type were transferred into separate ventilated plastic containers (Kenpoly) (2 L) with a thin layer of sterilized soft sand for pupation. The containers with the pupae were placed in separate emergence boxes. Adult collection was done following the procedures described above.T. leucotreta eggs from moths of which larvae were reared on the respective host plants, viz. orange, African eggplant, chili and sweet peppers. Newly emerged moths were transferred to separate cage containing wax paper sheet for oviposition. Wax paper sheets with eggs were removed and cut into four strips containing at least 100 eggs. The eggs were counted under a stereomicroscope (Leica WILD M3Z). Each strip of wax paper with eggs from moths for each host plant was individually placed in plastic containers (Kenpoly) (2 L) to allow for hatching. The eggs were checked under a microscope twice a day for emergence of neonates. Egg incubation period was recorded as time in days taken for 50% of eggs to hatch as described by Mkiga and Mwatawala [Four Perspex oviposition cages (37 × 22 × 6 cm) were used to obtain watawala . The expT. leucotreta larval development was determined on each host fruit type. Upon hatching, a cohort of 100 one day old neonates originating from eggs laid by T. leucotreta reared from orange, African eggplant, chili and sweet pepper were inoculated onto a fruit placed in a plastic container following the procedures described by Love et al. [T. leucotreta larval development was recorded as time in days taken for 50% of final instar larvae to exit from the fruit for pupation. The experiment was replicated four times.Duration of e et al. . A totalT. leucotreta larvae from each host plant were weighed individually using a digital analytical balance . Ten larvae were weighed per replicate for each host plant and the experiment was replicated four times.To determine the effect of the rearing host on larval mass, recently exited T. leucotreta larvae from each host, 300 neonates originating from eggs laid by T. leucotreta reared from orange, African eggplant, chili and sweet pepper were transferred onto orange fruit and the respective vegetables. The neonates were collected following the procedures described above. A cohort of 100 final instar larvae exited from each fruit type on the same day were introduced into the four ventilated plastic containers (25 larvae/container) containing a thin layer of soft sterilized sand as pupation media. The larvae were monitored every 12 h until cocoon formation. Duration of the pre-pupal stage was recorded as time in days taken for 50% of final instar larvae to form cocoons. The experiment was replicated four times and each replicate contained 100 larvae.To obtain final instar icipe and monitored daily for adult eclosion. Pupal duration was recorded as time in days taken for T. leucotreta adults to eclose from 50% of the cocoons.Pupae were obtained by rearing larvae, inoculated as neonates onto the fruit of each host until cocoon formation following the procedures above. A cohort of 100 cocoons were carefully collected and placed in a Perspex cage (15 cm × 15 cm × 10 cm) manufactured at Thaumatotibia leucotreta stage specific survival was determined using the cohorts from the developmental duration bioassays above to calculate the percentage of eggs laid on the wax paper strips for each plant species that successfully hatched, percentage of neonates that successfully developed into final instars larvae, percentage of cocoons formed from the final instar larvae and percentage of adults that eclosed from cocoons.T. leucotetra life history was assessed using a cohort of newly emerged moths from larvae reared on orange, sweet pepper, African eggplant and chili pepper. The sex ratio was determined by counting the number of males and females from a sample of 100 moths emerging from pupae from which the larvae were reared on each host plant species. One male-female pair of moths was kept in a vial (32.6 cm3) corked with wet cotton wool serving as a source of water. A total of 15 vials were used for each host. The pre-oviposition period was recorded as the number of days from adult eclosion until the first eggs were laid. The pair of moths were carefully removed from the vial and transferred into a new vial using the protocol described by Opoku-Debrah et al. [Adult h et al. . The numThe net reproductive rate (R0), intrinsic rate of natural increase (rm), mean generation time (T), doubling time (DT) and finite rate of increase (λ) were calculated using the equations from Carey .T. leucotreta damage symptoms as well as fruit containing T. leucotreta larvae were arcsine transformed before analysis. Data on percentage of orange fruit with T. leucotreta damage symptoms as well as fruit with larvae of the pest were analysed with a three-way ANOVA followed by Tukey’s HSD test to determine statistically significant differences between countries, altitudes and sampling methods. Data on percentage of infested vegetable fruit of the respective host plants were analysed by means of one-way ANOVA followed by Tukey’s HSD test. Thaumatotibia leucotreta development time, larval mass and adult life history parameters were analysed by means of one-way ANOVA followed by Tukey’s HSD test. Data on T. leucotreta survival in the developmental experiments were analysed using logistic regression models followed by Tukey’s HSD test. Likelihood ratio test on a GLM  was used to analyse the number of eggs laid per fruit in the oviposition preference assays as well as the number of eggs laid per day by each female reared from the respective hosts. Pairwise comparison of Least Squares Means  of number of eggs laid on choice and no-choice tests and daily oviposition of moths reared on the respective hosts was done with Tukey’s HSD (α = 0.05). All statistical analyses were performed using R-version (3.5.2) statistical software packages  with ore Team ).T. leucotreta damaged fruit  and higher larval incidence were recorded in Kenya than in Tanzania  . Significantly more fruit with symptoms were sampled from the ground compared to those sampled from the tree by branch shaking or without shaking . Significantly fewer larvae were found in damaged fruit sampled from the ground at low compared to high altitudes in Kenya. In Tanzania, the percentage of larval infestation per sampling method was similar, regardless of altitude (Significantly more ˂ 0.001) . The per shaking . The per shaking . There win Kenya . Signifialtitude . The peraltitude .T. leucotetra differed significantly among the vegetables . There was no significant difference in percentage T. leucotetra larval infested African eggplant, chili and sweet pepper. Significantly fewer okra was, however, infested compared to the other vegetables (The incidence of getables .p = 0.001)  A. The hip = 0.001).The highest number of eggs were also laid on orange in the no-choice test with African eggplant and chili pepper being the least preferred for oviposition B. The nuT. leucotreta . The larval development time  differed significantly among the respective solanaceous vegetables   but duration of the pupae originated from larvae reared on these solanaceous vegetables differed significantly  (The host plant on which a specific generation was reared had no significant effect on the egg incubation period of < 0.001) A. Develo< 0.040) B. Pupal < 0.040) B. There F = 5.84; df = 3, 12; p = 0.011). It did, however not differ significantly between the vegetables  . The percentage of larvae that survived differed significantly between the rearing hosts . A significantly higher percentage of larvae reared on orange survived compared to sweet and chili peppers  as well as the percentage of adults that eclosed from these pupae .The percentage fertility of eggs laid by moths of which larvae were reared on orange, sweet pepper, chili pepper and African eggplant in the previous generation did not differ significantly , oviposition period , female longevity  and male longevity  . Moths originating from larvae that were reared on orange laid significantly more eggs than moths from larvae reared on African eggplant as well as on sweet and chili peppers. Moths from African eggplant reared larvae were also more fecund than those reared on sweet peppers but fecundity of moths reared on African eggplant and chili pepper was similar.The host plant on which the larvae were reared did not significantly affect the pre-oviposition period (= 0.990) . The fecp = 0.205) between moth age and host plant in terms of daily oviposition (p ˂ 0.001). Moths from larvae reared on orange laid the most eggs per day. The number of eggs laid per day varied considerably with female age . The most eggs were laid by moths from larvae reared on orange, five days after moth eclosion. Egg laying for females reared on the other host plants peaked on the fourth day after moth eclosion. On the days where oviposition was high, females from the larvae reared in orange laid more egg followed by females from larvae reared from African eggplant, chili and sweet peppers.There was no significant interaction  , intrinsic rate of increase , doubling time  and finite rate of increase  were similar for females reared from larvae on the respective host plants.Net reproductive rate of ˂ 0.001) . The higThaumatotibia leucotreta occurred in all surveyed sites of Kenya and Tanzania from low to high altitudes. The higher percentage of damaged fruit recorded in Kenya compared to Tanzania could be attributed to the different farming practices within each country. In Kenya, most citrus trees are intercropped with peppers and maize which are also recorded as crop hosts of T. leucotreta [T. leucotreta damage symptoms than those sampled from trees (with or without branch shaking). Higher T. leucotreta larval incidence was, however, recorded from fruit sampled after shaking of the tree branches. This could be ascribed to T. leucotreta laying eggs mainly on fruit while they are still on the trees [T. leucotreta infested fruit do often not show damage symptoms, final instar larvae may be present in the fruit mainly in the centre cores. This may have implications for T. leucotreta management especially in terms of orchard sanitation and post-harvest treatment. Proper orange orchard sanitation and harvesting should therefore also take in consideration infested fruit that is still on the tree by shaking of the branches. Higher T. leucotreta larval incidence on orange fruit was recorded in high than low altitudes of both countries. In contrast, Odanga et al. [ucotreta ,6 while ucotreta , excessihe trees  and the he trees . When tha et al.  reportedT. leucotreta infested fruit was on African eggplant compared to okra, sweet and chili peppers. This could be due to the lower moisture content of the fruit of this host plant, since infested African eggplant produced very little fluid under field and laboratory conditions. According to Jaenike [T. leucotreta when grown in the vicinity or in mixed cropping with orange orchards. These vegetables are also grown throughout the year in irrigated fields which provides a continuous availability of host plants for this pest.Amongst the vegetables sampled, the highest percentage of  Jaenike , the suiT. leucotreta, oranges were the most preferred host plant for oviposition in both choice and no-choice tests. The T. leucotreta moths do prefer certain orange varieties above others for oviposition. For example, Love et al. [T. leucotreta for different citrus varieties and guava under laboratory and field conditions [T. leucotreta ovipositional preference for and performance on solanaceous vegetables, with chili and sweet pepper more preferred for oviposition than African eggplant.Although vegetables can serve as host plants for e et al.  reportede et al. . Most ofnditions ,33. ThisT. leucotreta did not mirror host suitability for development of the immatures and survival on these host plants. The sweet and chili peppers which were more preferred than African eggplant for oviposition were less suitable in terms of developmental time, larval mass and survival of immature stages (larvae and pupae) as well as female fecundity and net reproductive rate. The mismatch between preferred vegetable hosts for oviposition and host suitability of peppers could be explored for use as trap crop. A similar finding was reported for the butterfly, Anthocharis cardamines (L) (Lepidoptera: Pieridae). The oviposition preference of females did not correlate with the fitness parameter of the offspring reared on 51 populations of two ploidy types of the perennial herb Cardamine pratensis L.  [T. leucotreta could be explained by the higher protein and lower moisture content of African eggplant compared to sweet and chili pepper [Maruca vitrata (Fabricius)  reared on cowpea flowers to have a short developmental time compared to those reared on the other plant parts. The effect of host plants on the life table parameters is dependent on the quality of certain components, such as carbon, nitrogen and defensive metabolites [In general, insects are reported to oviposit on hosts that maximize the performance of their progeny, a hypothesis referred to as “mother knows best” ,32,34,35icaceae) . The suii pepper ,38,39. Ri pepper  reportedabolites .T. leucotreta ranged from a total of 341 to 415 eggs per female for those reared on sweet pepper and orange, respectively. Both these values are within the range reported for fecundity of T. leucotreta from larvae reared on artificial diet [Thaumatotibia leucotreta oviposition from larvae reared on artificial diet reached a peak, 2 and 3 days earlier than for the females reared on solanaceous vegetables and orange, respectively [In this study, adult life table parameters for moths from larvae reared on the respective host plants are similar except for fecundity and net reproductive rate. Fecundity of ial diet ,25. Femaectively . GeneralT. leucotreta is equal on orange, sweet and chili pepper as well as African eggplant. This has implications for management strategies of the pest. According to Birch [The intrinsic rate of natural increase, mean generation time, doubling time and finite rate of increase are important biotic parameters of insect performance ,43. Consto Birch  the intrto Birch .T. leucotreta damage symptoms compared to fruit sampled from the trees, the latter hosted a higher number of T. leucotreta larvae. It is therefore recommended that citrus trees should be subjected to gentle shaking prior to fruit harvesting to reduce citrus orange post-harvest fruit decay and minimize indirect losses due to quarantine measures. Infested orange fruit from both ground and tree after branch shaking should be collected and removed using sustainable sanitation measures such as the use of augumentorio which sequesters the moth while conserving any parasitoids. This control tactic should be integrated with other T. leucotreta control measures such as the use of biopesticides viz. Cryptophlebia leucotreta granulovirus (CrleGV), entomopathogenic nematode, Heterorhabditis bacteriophora, entomopathogenic fungi, Metarhizium anisopliae, Beauveria bassiana, attract and kill through the use of Lastcall® and the Sterile Insect Technique. The potential of solanaceous vegetables to act as T. leucotreta reservoirs between successive citrus orange seasons for rainfed orange production, the common cropping systems in Kenya and Tanzania were determined. The strong preference of T. leucotreta to chili and sweet peppers for oviposition and relatively higher mortality of the larvae on these crops could be explored for management of this pest using attract and kill approaches. Although there is currently a very potent male moth attractant commercially available, a need to identify semio-chemicals that can attract female T. leucotreta moths for both monitoring and suppression of the pest, is needed.Although oranges sampled from the ground had more T. leucotreta damage symptoms are on the ground but the larvae are abundant in the fruit still hanging on the tree. Orange is strongly preferred for oviposition and highly suitable for T. leucotreta larval survival when compared to solanaceous vegetables. Thaumatotibia leucotreta has a strong oviposition preference to sweet and chili pepper compared to African eggplant. African eggplant is, however, highly suitable for larval survival. Fecundity is the only affected adult T. leucotreta fitness trait from larvae reared from orange and solanaceous vegetables. The findings from this study will contribute to sustainable management of this pest.More ripe orange fruit with


Three dimensional (3D) printing technology is rapidly evolving such that printing speed is now a crucial factor in technological developments and future applications. For printing heads based on the inkjet concept, the number of nozzles on the print head is a limiting factor of printing speed. This paper offers a method to practically increase the number of nozzles unlimitedly, and thus to dramatically ramp up printing speed. Fluid reservoirs are used in inkjet print heads to supply fluid through a manifold to the jetting chambers. The pressure in the reservoir’s outlet is important and influences device performance. Many efforts have been made to regulate pressure inside the fluid reservoirs so as to obtain a constant pressure in the chambers. When the number of nozzles is increased too much, the regulation of uniform pressure among all the nozzles becomes too complicated. In this paper, a different approach is taken. The reservoir is divided into an array of many micro-reservoirs. Each micro-reservoir supports one or a few chambers, and has a unique structure with auto-pressure regulation, where the outlet pressure is independent of the fluid level. The regulation is based on auto-compensation of the gravity force and a capillary force having the same dependence on the fluid level; this feature is obtained by adding a wedge in the reservoir with a unique shape. When the fluid level drops, the gravitational force and the capillary force decrease with it, but at similar rates. Terms for the force balance are derived and, consequently, a constant pressure in the fluid micro-reservoir segment is obtained automatically, with each segment being autonomous. This micro reservoir array is suggested for the enlargement of an inkjet print head and the achievement of high-speed 3D printing. Inkjet technology has made continuous developments since the 1950s  quickly A general limiting factor for the entire 3D printing technologies is the printing speed. Although there are different concepts of 3D printing, model printing still takes too much time for many applications. A breakthrough to obtain a high-speed printer that can form a printed model on the scale of seconds is needed.The DOD technology is generally divided into thermal inkjet . The micro reservoir has a unique structure allowing a constant pressure at its base regardless of the fluid level it contains. Therefore, no external means for pressure regulation are needed and the surrounding system can be simplified. Moreover, adding segments is practically unlimited. The print head can be sized as a large liquid crystal display (LCD) screen, having nozzles numbered similar to those of light-emitting diodes (LEDs) on the screen. Therefore, printing speed can be dramatically increased. With such a large print head, objects of significant size can be printed in seconds instead of hours, as current technology allows. The arrangement of such a 2D print head is described in Reference , where tLet us first assume a regular fluid reservoir filled up to a certain level . The prem is the fluid mass, g is the gravitational constant and TUP is the capillary force portion directed upwards, and is calculated as:T is the total capillary force and is calculated as the surface tension σ multiplied by the contact line length between the fluid and the chamber L. The direction of T is along the contact angle α. Taking into account the chamber cross-section S, the specific gravity of the fluid ρ and fluid level h, Equation (1) becomes:V is the fluid volume. As seen, the force is dependent on the fluid level h. Therefore, the pressure in the chamber base P is also dependent on the fluid level:In large reservoirs, the capillary force is much smaller and can safely be neglected. However, as the reservoir becomes smaller (or porous) this force becomes relatively stronger and can even be the dominant one. In the following derivation, the capillary force will be taken into account. The total force operating on the fluid is :F = mg As seen in Equation (4), the pressure decreases together with the fluid level. In light of the above, a critical question forms: is there a possibility to develop a structure where the pressure is independent of the fluid level?Such a structure, if possible, would clearly have superior properties regarding inkjet and micro-pump applications.Looking at Equation (4), if the structure of the reservoir could be delicately chosen to obtain a unique and desired influence on the magnitude of the capillary force, it may be possible to compensate the change in the gravitational force. Since the gravitational force on the fluid decreases as the fluid level drops, a reservoir structure might be found in which a similar dependence of the capillary force on the fluid level occurs. This, in turn, means that the capillary force also drops at the same rate as the gravitational force. Thus, the changes in the two forces compensate each other and the level change does not influence the pressure at the base. Such a solution is presented in the next paragraph.H where the wedge begins, and h is the fluid level above this height. The wedge width is b, and β is the angle of the wedge. The fluid level determines a which is the width of the wedge that is covered with fluid.Let us insert a wedge into the chamber as seen in S is the base area and H, h, a, b, and β are as noted in b is kept small enough, the volume (and gravity) does not change much due to the wedge, but the capillary force is strongly affected. Calculating the capillary force, we get three components:b is the contact angle in the wedge slope (here L is the contact line length between the fluid and the chamber without the wedge). As can be seen, the first component is due to the reservoir walls and is similar to the case of a standard reservoir. The second one is negligible when b is small enough and does not contribute much. The last component is the new important part. As seen, this component is dependent on h and can potentially compensate for the dependence of the gravitational force on h. If all the components contributing to the base pressure in the modified chamber are collected and organized according to the dependence on h, the pressure is obtained as follows:K is a constant containing all the components that are not dependent on h (both from gravity as well as the capillary force):Repeating the derivation for this chamber, the volume of the fluid is almost the same, calculated for a cube structure, subtracting the wedge volume:K1 is the coefficient of h:2K is the coefficient of h2:h2, but it is also dependent on b, which is the wedge width. If we enforce a small b, as small as the limit of practical production in microelectromechanical systems (MEMS) technology, we can neglect this term.The last term in Equation (7) contains a dependence on h, which still varies as the fluid level drops. However, with proper choice of the constants in this term, it is possible to obtain a zero coefficient meaning: K1 = 0. The condition for that will be:The second term in Equation (7) contains a dependence on P becomes:h. Therefore, a chamber with a constant pressure regardless of its fluid level is obtained.As can be seen, if the above conditions are fulfilled, the final expression for the pressure at the base of the modified chamber σ = 0.072 N/m3ρ = 997 kg/mL = 2.5 mmS = 6.25 mm2g = 9.81 m/s2α = 24.45°β = 65°For example, assuming a squared modified chamber filled with water has the following parameters:K1 (Equation (9)) practically cancel each other out:The two terms in And cancellation of the dependence on the fluid level is obtained.K can be done in order to choose the constant pressure; positive as well as negative values are possible with proper determination of the constants.Also, determining The calculation is repeated for other values of surface tension typical for inks and the results are detailed in In order to obtain the predicted stability of the pressure as the level drops, a calculation was done according to Equation (7) (without any negations). As a basis pressure, a value of −100 Pa was taken. The wedge width b is taken to be 100 μm in this calculation. The results are shown in In this paper, an inkjet micro-reservoir with inherent structure-based pressure regulation is presented. The structure-based regulation is obtained by inserting a wedge that adds a capillary force which is dependent on the fluid level. This dependent capillary force is equal in magnitude to, and opposite in direction from, the gravitational force at any fluid level. Therefore, a base pressure that is independent of the fluid level is obtained. An example is given to demonstrate the concept.An important feature of this solution is related to a case where a plurality of reservoirs is in use. In such a case, with standard reservoirs, the pressure within will not be uniform among the reservoirs. However, with the suggested reservoir, even when the level of the fluid is not uniform in the reservoirs, the pressure would still be uniform and constant—an important solution for a segmented system. This reservoir can be built with MEMS technology to obtain a large area of similar reservoirs. A huge array can be built where each reservoir is autonomous and independent of its surrounding reservoirs. As a result, this reservoir enables the enlargement of a print head to include an enormous number of nozzles and thus achieve a high-speed 3D print head where an entire layer is printed simultaneously without scanning.An example for such a system (with standard reservoirs) is presented in References ,20, wherAlong with the suggested design, several aspects should be addressed. As seen, the pressure is greatly dependent on the geometry. How will imperfections influence the performance? Clearly, the answer is dependent on the technology used to form the reservoirs. However, from the graph in Another aspect is the influence of dust and pollution that may interfere and clog the system. This is a well-known problem in inkjet systems and many means have been used to attempt pollution removal. Usually this task is difficult because the system is closed and the only way out is through the nozzle which is rather small. Clearly, such pollution can interfere in unexpected ways. However, in the suggested reservoir there is a unique remedy for this problem. This reservoir can be reached from the upper side, which is wide. Therefore, a back-priming can be done; i.e., sucking out all the fluid from the wide side (with the pollution) and replenishing a new clean fluid.A unique property of the presented micro reservoir is its quasi-static operation. Since the reservoir feeds only one (or a few) nozzles, replenishing can be done rarely and most of this operation is done in a static manner, where no fluid is added. As a result, dynamic effects can be minor and negligible. It should be noted that cross talk between neighboring nozzles is also a dynamic phenomenon, which would still be present if the proposed reservoir would support multiple nozzles. Nevertheless, the problem is significantly reduced for a low number of nozzles connected to the same reservoir, and totally eliminated when every nozzle has its own reservoir.Another limitation related to a high speed inkjet 3D printer is the ultraviolet (UV) curing which is done after each layer in most inkjet 3D printers. This process might be the next bottle neck towards high speed printing. A solution for this issue may come from chemical research and development of advanced fluids with a fast curing mechanism that can be integrated to a fast inkjet print head.The same solution is applicable for micro-pumps and other MEMS devices utilizing fluids where constant pressure is required. It should be noted that with proper tuning of the parameters, both positive and negative constant pressure could be obtained for various applications.


Solitary fibrous tumour (SFT) is a mesenchymal tumour of fibroblastic type, and it develops in almost any part of the human body. However, according to previous studies, the occurrence of intravascular SFTs is extremely rare.We reported a case of intravascular SFT in a 67-year-old woman who has been experiencing swelling and pain in the right leg for 2 months. Computed tomography venography scan revealed a well-defined mass obstructing the inferior vena cava (IVC). Surgical resection was performed, and histopathologic and immunohistochemical results were consistent with SFT. Further, next-generation sequencing (NGS) analysis was performed, and results revealed two tumour-related gene mutations (deletion of PMS2 and variation of ESR1 [L536P]). The patient did not receive any adjuvant therapy, and no signs of tumour progression were observed during the 6-month follow-up.To the best of our knowledge, this study first presented about SFT arising from the IVC and carried out an NGS analysis to validate the molecular mechanism of such condition. Solitary fibrous tumour (SFT) is a mesenchymal tumour of the fibroblastic type, typically arising from the pleura . This tyA 67-year-old woman was admitted to our hospital. She complained of swelling and pain in the right leg. She claimed that the swelling was not apparent during onset, which was approximately 2 months back, and it subsided over the past days. Moreover, she had developed persistent pain for 7 days before hospitalization. The patient denied any other symptoms, including physical disturbance, weight loss, or fever. She was not a smoker, did not present with hypertension or diabetes and had undergone complete surgical resection of the uterus in 2005. Colour Doppler ultrasonography indicated that the veins in the right lower limb were completely occluded. Computed tomography venography scan of the abdomen and both lower extremities revealed that a well-defined mass obstructed the inferior vena cava (IVC), with segmental filling defect of the right iliac vein and occlusions in the right femoral, popliteal and calf veins Fig. .Fig. 1CoFor further diagnosis and treatment, surgical excision of the intravascular mass was performed. Under blood blocking, a transverse incision was made in the wall of the inferior vena cava. Next, the 5 × 4 × 3-cm mass with white capsule was cautiously separated from the venous wall. The mass was resected along with part of the venous wall because some tumour tissues were connected to the intravascular neoplasm. Because the tumour adhered tightly to the right common iliac vein and could not be completely resected, a partial resection was carried out. In addition, intraoperative observation revealed that the collateral branch veins from the posterior wall had abundant blood supply, whereas the distal vena cava was completely embolized. Thus, vena cava resection was conducted.The cut surface was similar to that of other mesenchymal neoplasms, with a fish flesh soft grey-white appearance Fig. a, b. TheThe patient presented with intestinal obstruction after surgery, which was relieved with pharmacologic and symptomatic treatments for 4 weeks. She was discharged on day 30 after surgery and did not undergo chemotherapy or radiotherapy. During the 6-month follow-up, ultrasonography was conducted, and progression of the disease was not observed.Formalin-fixed paraffin-embedded (FFPE) tissues were manually macrodissected. DNA was extracted from the original tumour, and blood samples were collected using the QIAamp DNA FFPE Tissue Kit (Qiagen). Next-generation sequencing (NGS) was conducted on the MiSeq platform using a TruSeq Amplicon Cancer Panel, covering 520 tumour-related genes. Then, the results were analysed using the MiSeq Reporter software.NGS results indicated two tumour-related mutations (deletion of PMS2 and variation of ESR1 (L536P)), and the tumour mutation burden was approximately < 0.8 Muts/Mb. In addition, the microsatellite stability was assessed, and the tumour was classified as microsatellite stable.Solitary fibrous tumour (SFT) is a rare type of neoplasm of mesenchymal origin, mostly arising from the submesothelial connective tissue beneath the pleura . HoweverLike most SFTs, the initial presentation of intravascular SFT depends on tumour size, location, and adjacent anatomic structures. Based on our review, intravascular SFT can be asymptomatic and incidentally diagnosed on imaging examinations, or it can cause venous obstruction at advanced stage. In addition, some patients with SFTs may present with constitutional symptoms, such as hypoglycaemia  due to sAs the symptoms and imaging results are not typical, the preoperative diagnosis of such disease is extremely challenging. In such location, it can be misdiagnosed as phlebothrombosis or other more common tumours, such as leiomyomas, leiomyosarcoma , well-diThe patient presented with a well circumscribed spindle cell tumour with a complete capsule. The cellular area was hypervascular, with classical patternless pattern of proliferation. Cellularity ranged from hypercellular to laxis areas surrounding varying amounts of hyalinized collagen bundles . Based oHowever, histologically benign SFT can also have aggressive clinical features. The study of Gold, J. S. et al. have revealed that the rate of recurrence and metastasis of benign SFT were 6.7 and 5.3%, respectively . Regardlp = 0.0236) [In this report, we presented two tumour-related gene mutations according to the NGS analysis. PMS2 is one of the mismatch repair (MMR) genes , which c 0.0236) . However 0.0236) , and itsAs for the treatment, there is consensus showing that surgical resection is the most appropriate treatment for SFTs. However, the most intractable problems are recurrence and metastases after surgery . In the Metastases are usually blood borne and have been observed in sites, such as the liver , lung 5 and vertIn summary, intravascular SFTs are extremely rare and generally present with luminal defects and changes in haemodynamics . The imaIn this study, we first reported a case of solitary fibrous tumour located in the inferior vena cava. Moreover, NGS analysis was conducted, and two tumour-related mutations were found. We believe that our results might be useful in future studies of such disease.