In the present study, an in vitro study model for tumour-induced DC dysfunction was established by culturing DCs with pooled sera from multiple non-small cell lung cancer (NSCLC) patients. The results demonstrated that tumour-induced human monocyte-derived DCs exhibited systematic functional deficiencies. Transcriptomics analysis revealed that the expression of major functional cluster genes, including the MHC class II family, cytokines, chemokines, and co-stimulatory molecules, was significantly altered in tumour-induced DCs compared to that in control cells. Further examination confirmed that both NF-κB and STAT3 signalling pathways were simultaneously repressed by cancer sera, suggesting that the attenuated NF-κB and STAT3 signalling could be the leading cause of DC dysfunction in cancer. Furthermore, reversing the deactivated NF-κB and STAT3 signalling could be a strategy for cancer immunotherapy.Tumour-induced dendritic cell (DC) dysfunction plays an important role in cancer immune escape. However, the underlying mechanisms are not yet fully understood, reflecting the lack of appropriate experimental models both  Lung cancer is one of the most common malignant tumours worldwide. More than 80% of lung cancer cases are non-small cell lung cancer (NSCLC). A high risk of metastasis in NSCLC indicates systemic anti-tumour immune deficiencyAs specialized antigen-presenting cells (APCs), dendritic cells are crucial for the initiation of adaptive immune responses468911126In vivo administration of VEGF in tumour-free mice can lead to impaired DC development19To date, several cytokines and growth factors involved in the abnormal differentiation and function of tumour-induced DCs, such as TGF-β, VEGF, and IL-10, have been identifiedin vivo model involves tumour-infiltrating DCs (TIDCs) obtained from clinical samples or tumour-bearing mice39in vitro models employ DCs generated from peripheral blood monocytes (MoDCs) or murine bone marrow progenitor cells (BMDCs), with tumour cell line conditional medium or specific factors added in cell culture, which may not well represent the complexity of the tumour environment. Therefore, building a proper experimental model of tumour-induced DC tolerance is urgently needed and may greatly accelerate mechanistic studies.Furthermore, current experimental models of tumour-induced DC dysfunction remain imperfect. The most commonly used in vitro model of tumour-induced DC dysfunction. In this model, the ability to initiate proper anti-tumour immune responses in DCs was systematically disrupted. Further transcriptomic analysis revealed that tumour-induced DCs harboured a unique gene profile. The disrupted upstream signalling in tumour cultured DCs, including the attenuated canonical NF-κB and STAT3 signalling pathways, may be the key reason. Taken together, these results indicate that the tumour environment manipulates DC functional deficiency by simultaneously attenuating canonical NF-κB and STAT3 signalling, leading to the abnormal transcription of downstream genes.Here, by using lung cancer patients’ sera, we generated an in vitro model of tumour-induced DC deficiency, we obtained the widely used MoDCs model, and sera from NSCLC patients were collected and pooled to represent the tumour environment. In this model, human monocytes separated from the peripheral blood of healthy donors were cultured with GM-CSF and IL-4 in the presence of sera from tumour patients or their healthy donor counterparts. Monocyte-derived dendritic cells (MoDCs) were subsequently collected 5–7 days later for further detection.To establish an in vitro generation of MoDCs, the cells were dual labelled with lineage cocktail 1 (lin-1)/CD11c for population analysis. Lin-1 contained antibodies against CD3, CD14, CD16, CD19, CD20, and CD56 to distinguish DCs from lymphocytes, monocytes, eosinophils, and neutrophils− CD11c+ population, suggesting typical MoDC generation in both cancer and healthy groups  class I and II molecules. When the cells were further stimulated with lipopolysaccharide (LPS), the expression of these surface markers was slightly increased but remained lower than that observed in the healthy controls . SimultaTo reveal the mechanism involved in tumour-induced DC deficiency, immature DCs from cancer or healthy groups were collected, and mRNA expression profiling experiments were performed (GEO Series GSE84720). Initially, we explored the microarray results using hierarchical clustering. Heatmap analysis showed similar gene expression patterns within parallel tumour sera pools, while the patterns considerably differed between cancer and healthy groups . In addiGene expression profiles analysis found 1419 up-regulated and 1108 down-regulated differentially expressed genes (DEGs) in both parallel samples of tumour-induced immature DCs , and a l23To assess the biological function of DEGs altered in tumour-induced DCs, gene ontology enrichment analyses were performed using DAVID bioinformatics resources25To further elucidate the crucial genes altered in tumour-induced DCs, the microarray data were analysed based on the immune-related gene sets obtained from the literatureTo further reveal the key signalling pathways altered in tumour-induced DCs that lead to functional deficiency, immune-related DEGs and major signalling pathway genes were identified to construct a protein-protein interaction (PPI) network using NetworkAnalyst28To identify high impact signalling pathways in the PPI network, the node degree and betweenness were sorted, and the top nodes are shown in http://www.bu.edu/nf-kb/the-gilmore-lab/ and http://bioinfo.lifl.fr/NF-KB/), matched with DEGs in tumour-induced immature DCs, and categorized according to gene function  decreased cell surface co-stimulatory molecules and MHC class I and II molecules, (II) impaired endocytosis by immature DCs, (III) reduced cytokine secretion from mature DCs, and (IV) suppressed DC-stimulated lymphocyte proliferation. In conclusion, MoDCs cultured with tumour sera lost their major functions as antigen-presenting cells, thereby preventing the initiation of proper immune responses against tumour cells. Moreover, we observed morphological changes in the cells in the tumour group, including cell elongation and decreased cellular aggregation , HLA-DM, and HLA-DO . Moreove41Signalling pathway enrichment analysis of tumour-induced DCs revealed that multiple signalling pathways associated with inflammation, growth, differentiation, proliferation, survival, and motility were diversely changed. We selected NF-κB and STAT3 from the high impact signalling pathways to perform further verification. The canonical NF-κB and STAT3 signalling in tumour-induced DCs was continuously disrupted and maintained at a low level during MoDC differentiation, while non-canonical NF-κB signalling was activated from day 3. These signalling alterations may be crucial for the transcriptional suppression of vital cytokines and antigen-presenting genes, leading to the functional deficiency of DCs. However, this signalling dysregulation was only partially described, reflecting the complicated mechanism involved, such as the disruption of upstream signalling, ubiquitin-mediated degradation4546The precise tumour sera-derived factors that induced DC dysfunction remain unknown. We detected several preliminary cytokines and growth factors using BD CBA technology and observed that both pro-inflammatory and anti-inflammatory cytokines, including IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17A, TNF, VEGF, and TGF-β, were expressed at higher levels in tumour sera. This finding is consistent with the understanding that the tumour environment is associated with chronic inflammation, and anti- and pro- inflammatory activities might eventually lead to DC dysfunction. Moreover, tumour sera also contained several chemokines and soluble ligands, such as sICAM-1 and sVCAM-1, which might also play roles in tumour-induced DC functional deficiency. Using the new techniques and methods in proteomics, more components might be identified in the tumour environment to obtain an understanding of their roles in tumour immune evasionThe other question that needs further discussion is how to define or describe tumour-induced DCs using the model in the present study. Although these cells remained a typical induced MoDC population, their antigen-presenting function was thoroughly disrupted. Moreover, the gene expression pattern of interleukins, chemokines, cell surface CD antigens, CAMs, ligands, and receptors were diversely altered. Whether these cells remained anergic or acquired tolerogenic and/or immunosuppressive activities48495152in vitro model of tumour-induced DC dysfunction using sera from NSCLC patients to represent the tumour environment. In this model, systematic functional deficiency of DCs was achieved, leading to failures in tumour antigen recognition and the initiation of proper immune responses against tumour cells. High-throughput microarray analysis revealed that tumour-induced DCs had abnormal functional gene expression, including cytokines, chemokines, immune receptors, and MHC class II genes. Further upstream analysis revealed that cell signalling was disrupted in DCs from the cancer group, including attenuated canonical NF-κB and STAT3 signalling, which might result in abnormal target gene transcription  and conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from all participants according to the guidelines established by the Institutional Review Board of Affiliated Hospital of Anhui Medical University. Patients in late stage NSCLC were selected and sampled prior to tumour treatment. There were a total of 56 patients, including 41 males and 15 females, aged 44–76 years. The patient sera were equally and randomly pooled into two groups to obtain a general view of its effects on DCs and to minimize individual variation. In the control group, blood plasma samples were collected from healthy donors at the Blood Centre of Anhui Province . All blood serum or plasma samples were incubated at 56 °C in a water bath for 30 min to deactivate complement activity prior to use in cell growth medium.2 for 5 days. To obtain mature DCs, the cells were cultured in the same cytokine mixtures in the presence of LPS  for an additional 2 days.Human peripheral blood mononuclear cell (PBMC) were isolated from the peripheral blood of healthy donors at the Blood Centre of Anhui Province using Ficoll-Hypaque density gradient centrifugation . Subsequent Percoll density gradient centrifugation  was performed for monocyte separation as previously reportedFor cell surface molecular detection, the cultured cells were harvested and coated with antibodies , PE-anti-CD11c, Alexa-488-anti-CD31, FITC-anti-CD18, PE-anti-CD54, and FITC-anti-CD106 , PerCP-Cy5.5-anti-CD40, PE-anti-CD80, and APC-anti-HLA-ABC , or their isotypes (BD Biosciences and BioLegend) and measured through flow cytometry using a BD FACSCalibur.5 cells/mL in RPMI 1640 medium containing 10% FBS. Then, 500 μg/mL FITC-labelled dextran (Sigma-Aldrich) was added, followed by incubation at 37 °C (or 4 °C for the control group) in 5% CO2 for 1 hour. The cells were washed with cold PBS and subsequently detected using a BD FACSCalibur.For cell endocytosis detection, the cells were collected, washed, and resuspended at 1 × 10For cell apoptosis detection, the cells were harvested and resuspended in apoptosis binding buffer, followed by incubation with FITC-labelled Annexin V (BioLegend) and PI  and subsequent detection using a BD FACSCalibur.3 cells/well onto 96-well plates in RPMI 1640 medium containing rhGM-CSF, rhIL-4, and 10% FBS for an additional 5 days to collect the supernatant. The cytokines were further measured using a BD CBA kit (Human Inflammatory Cytokines Kit and Human Th1/Th2/Th17 Cytokine Kit) and a BD FACSCalibur.For cell cytokine detection, the cells were collected, washed, and seeded at 3 × 10in vitro, human monocyte-derived DCs were co-cultured with PBMCs obtained from another healthy donor at a 1:65 ratio in 96-well plates in RPMI 1640 medium containing rhGM-CSF, rhIL-4, and 10% FBS for an additional 5 days. Tritiated thymidine  was added at 1 μCi/well on day 4, and the cells were collected and measured using a TriCarb Liquid Scintillation Analyser (PerkinElmer) 12–18 hours later.To detect the function of DCs in stimulating allogeneic lymphocyte proliferation 3H-TdR was added on day 4 and measured using a TriCarb Liquid Scintillation Analyser (PerkinElmer) 12–18 hours later.In the MHC Class II antibody competitive blocking experiment, 5 μL/mL of purified mouse anti-human HLA-DR (BD Biosciences) or mouse IgG2a, κ isotype control (BD Biosciences) was added to the DC-PBMC co-culture system, and 1 μCi/well in vitro transcription in the presence of T7-RNA polymerase. The aRNA was subsequently purified and labelled with CY5  or CY3 (cancer group) followed by further purification, and gene expression profiling was performed based on the Operon Human Genome Array-Ready Oligo SetTM (Version 4.0).Immature DCs cultured with tumour sera or healthy controls were collected, washed and stored in TRIzol reagent  at −80 °C, and gene expression profiling was performed at Chipscreen Biosciences . Total RNA was isolated, and T7 oligo(dT) primer and reverse transcriptase were used to synthesize first-strand cDNA. The second cDNA strand was subsequently synthesized by using the first cDNA strand together with RNase H and DNA polymerase. The double-stranded cDNA was purified and used as the template for http://brb.nci.nih.gov/BRB-ArrayTools.html) with LOWESS normalization. Next, DAVID Bioinformatics Resources25http://david.abcc.ncifcrf.gov) were used to perform Gene Ontology (GO)-based functional enrichment and KEGG pathway enrichment analyses. For immune-related gene analysis, hierarchical clustering was performed by Cluster 3.0 based on complete linkage and visualized with TreeView software. Furthermore, a protein-protein interaction (PPI) network was constructed using NetworkAnalyst28http://www.networkanalyst.ca/NetworkAnalyst/faces/home.xhtml) based on the InnateDB interactome PPI databaseFor the microarray analysis, 22,484 unigene data were selected, and the fold-changes were calculated using BRB-ArrayTools , and cDNA was synthesized using M-MLV Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. Quantitative real-Time PCR was performed on a StepOne PCR System  with FastStart Universal SYBR Green Master  according to the manufacturer’s instructions, and the primer pairs  are listed in 4P2O7, and the protein concentration was measured using a BCA Protein Assay Kit . Subsequently, the samples were loaded onto SDS-PAGE gels and separated through electrophoresis. The proteins were transferred onto PVDF membranes and incubated with the corresponding primary Abs and HRP-conjugated secondary Abs. Chemiluminescent detection was performed using Alliance 4.7  with Luminata Forte Western HRP Substrate .The cells were harvested, washed, and lysed with RIPA lysis buffer containing a protease inhibitor cocktail  together with NaF and NaThe following antibodies were used: Phospho-IκBα (Ser32) , IκBα (CST#4812), Phospho-NF-κB p65 (Ser536) (CST#3033), NF-κB p65 (CST#3034), NIK (CST#4994), NF-κB2 p100/p52 (CST#4882), RelB (CST#4922), Phospho-Stat3 (Tyr705) (CST#9131), Phospho-Stat3 (Ser727) (CST#9134), SHP-1 (CST#3759), SHP-2 (CST#3397), Stat-3 , β-Actin (ACTB) , HRP Anti-rabbit IgG (CST#7074), and HRP anti-mouse IgG .A t-test was used to statistically compare the differences between the two groups using Prism software . The data are shown as the mean ± SEM. A p-value less than 0.05 was considered to be the threshold for statistical significance, and the p-value is indicated as either n.s., not significant; *P < 0.05; **P < 0.01; or ***P < 0.001.How to cite this article: Li, R. et al. STAT3 and NF-κB are Simultaneously Suppressed in Dendritic Cells in Lung Cancer. Sci. Rep.7, 45395; doi: 10.1038/srep45395 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Rhesus macaques are an important model in behavioral neuroscience due to their advanced cognitive abilities. To motivate animals to engage in complex tasks, fluid rewards, in conjunction with fluid control protocols, are often used. The impact of these protocols on animal welfare is controversial. We compared two fluid control protocols against a protocol providing free access to water and evaluated the impacts on physiological states of hydration, behavioral measures of welfare, and scientific output. Blood physiology did not significantly differ between any of the protocols, and urine measures were indicative of well functioning, healthy kidneys. Changes in behaviors were limited, the main one being an increase in motivation to drink on the stricter fluid control protocol, and improved task performance early in the week. Overall, fluid control protocols had little measurable impact on the welfare of rhesus macaques while ensuring that scientific data of high quality could be obtained. This study demonstrates that two fluid control protocols caused no detrimental effects to the physiology of rhesus macaques. Behavioral impacts were limited, and laboratory performance improved during protocols with stricter controls. Overall, fluid control has much less of an impact than widely proposed.Nonhuman primates are a widely used model in neuroscience, due to their close phylogenetic relationship to humans, and thus their relevance to understanding cognitive functions, brain disease, and aiding therapy . PrimateCognitive and neural processes in awake, behaving primates are often investigated by means of electrophysiological and neural imaging methods. These require a high number of consecutively performed trials to achieve adequate data quality, statistical reliability, and sample sizes suitable for complex model testing. To achieve adequate daily performance, laboratories often use fluid rewards as motivators for correct trial performance and restrict fluid access for the animals outside of the experiment . DespiteThere have been previous attempts to evaluate the use of these techniques on some aspects of animal welfare using physiological or behavioral measures. We investigated the validity of the concerns surrounding fluid control in a controlled, within-subject design experiment in four macaques used in electrophysiological studies over a 16 week period. During this time, all four animals experienced two different fluid control protocols, which are used in primate research . The phyGuidelines for the Care and Use of Laboratory Animals), the Society for Neurosciences Policies on the Use of Animals and Humans in Neuroscience Research, and the Animals (Scientific Procedures) Act 1986.All experimental animal procedures complied with European Union Directive 2010 (2010 63 EU), the National Institutes of Health (Macaca mulatta) with ages ranging between 7 and 9 years, and weighing between 9 and 15 kg. The monkeys were used for electrophysiology and/or MRI studies and were experienced in the experimental setup and behavioral tasks. Subjects were each pair housed with another male (not recruited for this particular study), in cages of either 2.1 × 3.0 × 2.4 m or 2.3 × 2.45 × 2.4 m, in a facility where individuals could have visual and auditory contact with many monkeys in other cages. Toys were given on a rotational basis as an environmental enrichment. Dry food mix was added to shavings that covered the floor to encourage foraging, as a stimulant and reward ] was determined over a period of at least 5 d (not necessarily consecutive days). Following this, starting at a minimum of 70% FAI, the performance of the animal in the experimental setup was determined over at least 3 d. The minimum was then decreased as necessary (in steps of 10-15% of FAI) until the animal was sufficiently motivated to work for fluid rewards in order to obtain scientifically useful data . After each reduction, the work rate of the animal was assessed for at least 3 working days to determine the current levels of motivation and performance. Further decreases were implemented only if the current minimum was insufficient to achieve the required number of daily trials.We assessed the following three different conditions: a control period of free access to water (hereafter “free access”); and both a 5 d and a 7 d fluid control protocol . The 5 d protocol consisted of 5 d of fluid control with free access to water on days 6 and 7. This is the standard protocol implemented in our laboratory. The second protocol consisted of 7 d of fluid control, where animals had access to their individually established minimum every day, which they could exceed by means of their work rate during experimental weekdays, but not on days 6 and 7. The 7 d protocol was tested to investigate whether it could result in improved scientific output, and what the potential welfare implications would be. On a 5 d protocol, work rates following the 2 d of free access are normally too low to allow for electrophysiology recordings, leaving at least 1 d/week where the animal is fluid restricted and works on the cognitive task in the laboratory without usable data being collected. If a 7 d protocol was more effective at motivating animals to perform the task on a Monday, data collection could be quicker and periods of fluid control could be reduced. Given these potential benefits, it was important to be able to compare welfare measures between protocols as well as to a control period of free access.On the 5 d protocol, the subjects received their minimum allowance amount either in the laboratory (Monday to Thursday) or in the home cage (Sunday). On Friday evenings and Saturdays, they were given free access to water in the home cage. On the 7 d protocol, the monkeys received their minimum fluid intake every day (Monday to Friday in the laboratory and Saturday and Sunday in the home cage), but were never given free access to water. Each protocol lasted for 4 weeks at a time and was repeated twice . The protocols were given either in a 5-7-5-7 d order (two monkeys) or a 7-5-7-5 d order (two monkeys). The monkeys were sampled for blood and urine on the last Friday morning of each protocol (detailed below). After sampling, they were given free access to water for Friday morning and afternoon before the next protocol began on Saturday.Animals worked 5 d/week (Monday to Friday) throughout the experiment. Within a daily experimental session, the monkey was allowed to work for as much fluid as he wanted, but in situations where the minimum daily allowance was not earned during the task, the monkey was supplemented (to its established minimum) with water in the laboratory after the session had finished. Therefore, monkeys received at least their minimum fluid allowance every working day. Two monkeys were rewarded with blackcurrant juice , one monkey was rewarded with water and one with diluted Coca-Cola (the Coca-Cola Company). For the 16 weeks of fluid restriction, monkeys were separated from their cage mates from Friday evening until Sunday afternoon. This was done to obtain accurate recordings of fluid intake for the monkey of interest and to ensure that the cage mate had adequate (unrestricted) access to water for that period.Prior to the fluid control protocols, the monkeys experienced a control period of 12 d, during which they had free access to water, and during which behavioral and physiological measures were obtained. A second control period of 12 d was conducted 6 months after the completion of the fluid control protocols, and physiological measures were obtained again and used with those from the first control period for analysis.For the duration of this study, each monkey was also involved in ongoing neuroscience experiments, in which they were performing tasks in relation to visually presented stimuli to obtain fluid rewards. Three subjects were engaged in covert top–down attentional tasks with individual trial times of 2000–4000 ms. The other monkey was performing a memory-guided saccade task, with individual trials taking up to 5000 ms. Experiments were performed in a dimly lit room. Monkeys were weighed daily before the start of the task, and then transferred between the housing unit and the laboratory using a custom-made trolley, onto which the primate chair was fitted. Performance in the laboratory was monitored via computer control; task performance  was recorded for each session. The criteria for determining when the monkey had stopped working  differed slightly between animals and experimenter, but they remained consistent for individual monkeys over the course of the study. When animals had stopped working according to these criteria, they were transferred back to the cage. Experimenters were blind to which fluid control protocol their animal was currently subject to.Physiological measures of the hydration state were collected at the end of the free-access periods  and on the last day of each 4 week block of the 5 and 7 d protocols . Animals were sedated with ketamine , and blood was collected from the saphenous vein for hematological and biochemical analysis. During the sedation following the control period, the bladder was located using ultrasound, and urine was extracted via cystocentesis. During the fluid control protocols, this was not possible because of the small size of the bladder; instead, urine was collected from the cage on the morning of sedation, when possible. Urine was collected at least once per fluid control protocol for each monkey.To compare results to a relevant baseline, blood samples were also obtained from the CfM, the UK rhesus macaque breeding facility. Fourteen male monkeys, with ages ranging from 4 to 15 years and weighing between 9 and 16 kg, were sedated as described above and blood was collected from the femoral vein. The CfM monkeys received free access to water at all times and were group housed. Due to sampling procedures, it was not possible to obtain urine samples from the monkeys at CfM.Animals were weighed on each weekday before being taken to the laboratory. This allowed for an evaluation of weight change over the course of a working week as well as a further assessment over the duration of a fluid control block (4 weeks). The dataset obtained was not complete, and the following number of weights was collected for each animal of a possible 76 d : Monkey 1, 65 weights; Monkey 2, 75 weights; Monkey 3, 67 weights; Monkey 4, 74 weights.In order to assess the potential psychological impact of different fluid control protocols, behavioral measures were collected while monkeys were in their home cages. Behavior was recorded using cameras  attached to the corridors of the primate housing facility, outside of each cage of interest. Data were collected three times per week, as follows: early week (Monday evenings and Tuesday mornings); late week (Thursday morning and evening); and Weekend (Saturday morning and evening). Using a range of days permitted the assessment of changes in behavior throughout the week. Morning recordings lasted from 7:00 to 9:00 A.M., and evening recordings lasted from 5:00 to 6:40 P.M. (to coincide with lighting times). These times reduced the number of personnel present in the primate facility, which could on its own affect animal behavior.t tests for each animal, no difference was found between analyzing the middle hour  and the full recording. The remaining observations and all analyses were therefore performed using the middle hour only to ensure that representative data were obtained, while keeping analysis times manageable. In total, 393 h of video material were observed and analyzed with the following distribution across animals: Monkey 1, 105 h; Monkey 2, 101 h; Monkey 3, 88 h; and Monkey 4, 99 h.An ethogram was designed to capture behaviors that were potentially associated with changes in the welfare state . BehavioIn order to gauge the motivational drive to drink under the different fluid control protocols, “latency to drink” was measured on Saturday and Sunday mornings. If the motivation to drink was increased on a stricter fluid control protocol, we would expect that latency to approach the bottle would be quicker on the 7 d protocol than on the 5 d protocol, and that the volumes consumed would be higher. A water bottle containing either the minimum allowance, or 1 L of water (depending on the fluid control protocol) was attached to the home cage, and the latency to start drinking was recorded. In circumstances where the monkey began to drink before the bottle was fully attached to the cage, the latency was scored as <1 s and given a value of 0.5 s for analysis. As the volumes of water offered on Saturdays differed between the two protocols, an additional test was performed on Saturday morning, whereby the amount of fluid consumed in the first 5 min was also measured.All analyses were performed using SPSS version 21  and R R. R softwAll data from the physiological measures were normally distributed and analyzed using a linear mixed model (LMM), with fluid control protocol  as a fixed factor, and monkey as a random factor. For blood urea and urine osmolality, the variance of the random effect was <0.001, and so the tests were performed with the random effect omitted. To compare blood results from the study to those obtained at the breeding facility, a linear mixed model was used, with monkey colony as a fixed factor and monkey as a random factor.t test. Overall weight change for a fluid control block (4 weeks) was evaluated by taking the start and end weights of the animals and calculating the percentage weight change. Finally, to assess the changes in weights over the weekend, the percentage weight change from Friday to Monday was calculated, and results from the two protocols were compared using a t test.We assessed weight loss in the following three ways: over a working week, over each 4 week fluid control protocol, and over a weekend. Weight change over the working week (Monday to Friday) was calculated in the following way: (weight in kilograms Friday/weight in kilograms Monday) − 1*100. The weight changes for the 5 d and 7 d protocols were normally distributed and were compared using a linear mixed model with the percentage of weight change as the fixed factor and monkey as the random factor. This allowed short-term weight change to be assessed. Additionally for each fluid control, weekly weight changes were compared to zero (no change in weight) using a one-sample To increase the power of analyses and to detect potentially subtle changes among fluid control protocols, behaviors with similar functions  were grouped together and analyzed in categories . We firsContinuously sampled behaviors occurred infrequently in the 30 s scan samples because of their rare or short nature and were therefore omitted from the scan sample data and analyzed separately. As described above, drinking behavior was captured separately as the latency to approach the bottle and the volume consumed in 5 min at weekend time points. Since animals were separated from their cage mate on Saturdays, behavioral repertoires were not directly comparable between weekdays and weekends. Therefore, separate analyses were performed for weekday data and Saturday data.Behaviors were analyzed by creating two models in R. The first was a linear mixed model with an underlying gamma distribution, with monkey identity as a random factor and fluid control as a fixed factor. A second model omitting the effect of fluid control was created and an ANOVA was applied to compare the two models, to assess the overall main effect of fluid control . Scan-saU test for Saturdays. Due to the low occurrence of social behavior and aggression, and the high prevalence of zeros in the data, these two categories were analyzed using a binary logistic regression, with a random factor of monkey identity and fluid control as a fixed factor.Some behaviors occurred at low frequency or were not performed by all animals and, so, were analyzed separately. Pacing was performed by only two individuals and did not follow a normal distribution. It was therefore analyzed separately for each animal using a Kruskal–Wallis test for weekdays and Mann–Whitney U test. In order to make Saturday consumption data comparable across the monkeys, volumes drunk were converted to a percentage of the minimum daily allowance for each animal. These data were not normally distributed and were analyzed using a Mann–Whitney U test.Latencies to approach the bottles were not normally distributed and were analyzed by using a Mann–Whitney U test for each monkey individually. To assess the effect of weekend water intake on Monday work performance, a Pearson correlation was calculated using the percentage weight change from Friday to Monday and the number of trials performed on a Monday. In addition, trials performed on Monday were compared to 1000 (an approximately acceptable laboratory performance) using a one-sample Wilcoxon sign rank test.Monkey 1 was excluded from the task performance analysis . This was due to the difficulty of his task increasing across the study, as was necessary for the electrophysiological data collection, and the varied setting in which he worked (electrophysiology laboratory and MRI scanner). Trial data for the remaining three monkeys were not normally distributed, and differences in the number of trials performed when on the 5 and 7 d fluid control protocols were assessed using a Mann–Whitney The four animals differed in their FAI and in the minima established  to ensure adequate work rates, as follows: Monkey 1, 645 ml FAI, minimum 200 ml (31% FAI or 15 ml/kg/d); Monkey 2, 880 ml FAI, minimum 150 ml (17% FAI or 14 ml/kg/d); Monkey 3, 910 ml FAI, 355 ml minimum (39% FAI or 26 ml/kg/d); and Monkey 4, 305 ml FAI, minimum 150 ml (49% FAI or 17 ml/kg/d).F < 2.98, p > 0.076a-c; urea: F = 0.894, p = 0.424d; F = 16.91, p < 0.001e; Cr: F = 7.32, p = 0.011f; SG: F = 24.30, p < 0.001g). All three urine measures were lower when monkeys had free access to water than during either the 5 d or the 7 d protocol , hematocrit (HCT), urea, and creatinine (Cr) in the blood did not significantly differ between the 5 d protocol and the 7 d protocol, and the free access period  = 8.36, p = 0.006h). Creatinine levels were also lower at the CfM  = 5.79, p = 0.035i). The remaining blood measures were not significantly different between colonies  = 0.004, p = 0.95j; HCT: F = 4.60; p = 0.06k).We found that the levels of blood urea were higher at the CfM breeding center  = 9.48, p = 0.003l; t test, test value = 0, t(29) = 3.39 p = 0.002m), while their body mass remained relatively constant  on the 7 d protocol (t(31) = 0.452, p = 0.655n). However, across a fluid control block (4 weeks), we found the opposite trend, with animals maintaining weight on the 5 d protocol , but losing weight on the 7 d protocol . However, neither change was significantly different from zero  = 0.208, p = 0.814o; 7 d protocol: t(7) = 2.18, p = 0.066p), nor from one another  = 2.12, p = 0.173q).To investigate weight loss, we measured daily changes in body weight across the working week, across the 16 weeks of fluid control. From Monday to Friday, weight loss occurred on the 5 d protocol but not on the 7 d protocol . Where there were differences among the three conditions, we then investigated whether the 5 d and 7 d protocols differed from the free access data, and from each other.2 (2) = 42.27, p < 0.001r); locomotion (χ2 (2) = 11.77, p = 0.0027s); self-groom (χ2 (2) = 37.35, p < 0.001t); body shake (χ2 (2) = 30.86, p < 0.001u); yawn (χ2 (2) = 101.32, p < 0.001v); self-directed (χ2 (2) = 17.09, p < 0.001w); abnormal (χ2 (2) = 10.07, p = 0.0065x); social (χ2 (2) = 8.72, p = 0.013y); and inactivity (χ2 (2) = 6.51, p = 0.039z; t(184) < 7.06, p < 0.006; 7 d protocol: all t(194) < 7.69, p < 0.001), with no difference in frequency between the two fluid control protocols  < 1.07, p > 0.28).Significant differences in the frequency of nine behaviors occurred across the free access and fluid control protocols, as follows: interaction (χt(184) = 2.68, p < 0.001) and the 7 d protocol (t(198) = 2.79, p = 0.005), but there was no difference between free access and the 7 d protocol (t(194) = 0.08, p = 0.940). However, inactivity was lower on the 7 d protocol compared with free access (t(194) = 2.55, p = 0.01), but was not different from the 5 d protocol (t(198) = 1.39, p = 0.166). There was also no difference between free access and the 5 d protocol (t(184) = 1.18, p = 0.237). Social behavior was lower on the 7 d protocol than on the 5 d protocol (t(198) = 2.13, p = 0.033) and free access (t(194) = 0.2.79, p = 0.005), but there was no difference between free access and the 5 d protocol (t(184) = 0.76, p = 0.45).Three of 13 behavioral categories differed between the two fluid control protocols. Abnormal behavior was lower in frequency in the 5 d protocol compared with free access (2 (2) < 2.99; aggression, χ2 (2) = 1.08; pacing, H2 < 3.36; all p > 0.16aa-ae; No other behaviors were affected by fluid control : χ2 (1) = 8.68, p = 0.003af). The second behavior was pacing, which was only sufficiently frequent to allow for quantitative analysis in two of the four animals. We found that pacing increased for one monkey on the 7 d protocol compared with the 5 d protocol  < 3.23, p > 0.07ai-ar for all; We found a significant effect of fluid control on two behaviors on weekends. The first was consumption , which was lower when animals were on the 7 d protocol compared with the 5 d protocol . The monkeys also drank more in 5 min on Saturdays while on the 7 d protocol , compared with while on the 5 d protocol . There was no effect of fluid control protocol on the latency to approach the water bottle on Sundays .On Saturdays, approach was significantly quicker on the 7 d protocol , than on the 5 d protocol . The performance on Monday is of particular importance, since animals often do not perform enough trials for scientific data to be collected. On Mondays, there was a significant correlation between the percentage weight change over the weekend (from Friday to Monday) and the number of trials performed: when weight decreased over the weekend, more trials were performed on the Monday  = 3.58, p = 0.001az). On average, monkeys gained 0.83% weight over the weekend on the 5 d fluid control and performed <1000 trials on Mondays . Conversely, on the 7 d protocol, monkeys lost 0.76% of body mass and completed an average of 981 trials on Mondays .Only three monkeys were included in the analysis . There was no overall increase in the daily numbers of trials performed in their respective cognitive tasks when they were subjected to the 7 d protocol, rather than the 5 d protocol  harm caused by monkeys being repeatedly subject to the 5 d protocol for fluid control, we cannot be certain that this is the case for the 7 d protocol for fluid control, as the 7 d fluid control regime has not been implemented for extended periods of time. Further long-term studies would be required to investigate this.Potential weight loss is a key welfare issue surrounding fluid control, with concerns that fluid control and the potential associated reduction in food intake  could leThere were some behavioral changes in our monkeys between the free access and fluid control conditions. While some behavioral changes may be indicative of reduced welfare during the two fluid control protocols, for example, increased stereotypic pacing in one animal , others There were also very few behavioral differences observed between the two fluid control protocols, and again, the results were inconsistent. For example, on weekdays while monkeys were on the 7 d protocol for fluid control, rare social behaviors  were lower and abnormal behaviors were higher, which is sometimes indicative of increased stress . HoweverSmall behavioral differences between protocols were also observed on Saturdays. Consumption  was lower on the 7 d protocol compared with the 5 d protocol. There are two possible explanations for this. One possible explanation is that because water is required to absorb and digest food, animals cannot eat as much on the 7 d protocol compared with the 5 d protocol. This voluntary reduction in consumption has been previously documented in rats and humans  and is oThe second change was in pacing behavior. Two of the animals in our group performed pacing behavior during all protocols. In one of the two, higher levels of pacing occurred over the weekend on the 7 d protocol compared with the 5 d protocol. Stereotypies in captive macaques are often used as indicators of suboptimal welfare and may indicate higher levels of stress in this individual . HoweverWhen given access to water on Saturdays, monkeys appeared more motivated to drink on the 7 d protocol than on the 5 d protocol. They approached the bottle more quickly and consumed a larger volume of water. This may be due to many reasons, including a dryness or unpleasant taste in the mouth, as has been shown in humans . HoweverAn important aspect of this study was to assess the scientific outcomes associated with the use of different fluid control protocols. Typically, on a 5 d fluid control regime, animals do not participate in a sufficient number of trials on Monday to collect a robust dataset . Consequently, data collection is not usually attempted on a Monday. The number of trials performed on a Monday in this study was too low on the 5 d fluid control protocol to attempt electrophysiological recordings, given the scientific requirements of the studies involved. The most likely reason for this is that monkeys were not motivated to drink after increased access to water over the weekend. However, when the monkeys were restricted over the weekend on the 7 d protocol, performance on Mondays increased to levels that would generally allow electrophysiology to be performed. This suggests that a 7 d fluid control protocol might enable scientific studies to be conducted 5 d/week , which could significantly reduce the duration of a study by at least 20%. This would mean that the time individual monkeys spend on a fluid control protocol would be similarly reduced.Male macaques physiologically cope with periods of fluid control, maintaining blood parameters within normal ranges by concentrating their urine in response to both protocols. There were no detectable short-term effects of either the 5 d or 7 d protocol, or any long-term (>4 years) effect of a 5 d protocol, on kidney function.There were relatively small changes in behavior detected by our in-depth analysis, with some behaviors indicative of poor welfare being associated with fluid control protocols, and others with free access to water.5 d and 7 d fluid control protocols do not lead to rapid and sustained weight loss that would be of immediate welfare concern. More data are required to assess the continuing impact of 7 d fluid control on weight changes.Animals are more motivated to drink in their home cage when on a 7 d protocol compared with a 5 d protocol, but the subjective experiences of the animals are unknown.Improved task performance on a 7 d protocol compared with a 5 d protocol could allow more rapid collection of sufficient scientific data and a reduced amount of time spent on fluid control protocols for experimental animals.As with any procedure, the consequences of fluid control on the state of the subject are important to know so that judgments about the appropriateness for their use can be made on knowing the likely impact on the results. This study addressed the need for scientific data on the impact of different fluid control protocols on the welfare and performance of laboratory primates used in neuroscience research . The debOur data mostly fail to show the significant detrimental effects on the welfare of laboratory macaques, which often have been predicted to arise from the use of fluid control protocols. Our study counters some and alleviates many of the widely held welfare concerns surrounding these methods.


Special emphasis is placed on evolution of the probes’ optical properties in the absence and presence of Hg2+. These compounds show drastic changes in their photoabsorption and photoemission properties when they react with Hg2+, indicating that they are excellent candidates for ratiometric and colorimetric fluorescent chemosensors. Most importantly, the energy donor moiety is found to play a dominant role in sensing performance of these probes. Two-photon absorption cross sections of the compounds are increased with the presence of Hg2+, which theoretically suggests the possibility of the probes to be two-photon fluorescent Hg2+ sensors. Moreover, analysis of molecular orbitals is presented to explore responsive mechanism of the probes, where the fluorescence resonant energy transfer process is theoretically demonstrated. Our results elucidate the available experimental measurements. This work provides guidance for designing efficient two-photon fluorescent probes that are geared towards biological and chemical applications.Nonlinear optical properties of a series of newly-synthesized molecular fluorescent probes for Hg Studial field ,4. Thus, samples ,6,7. Amo samples . In this samples ,10. As aThere are several signaling mechanisms that can be employed for designing sensitive fluorescent molecular probes ,12, such2+ probes that can be used in the fluorescent imaging technology [2+ (named hereafter as Pro1) based on an intramolecular FRET with an excellent selectivity over other metal ions [2+ ions on the ppb scale under physiological conditions [2+ selective ratiometric fluorescence probe (named hereafter as Pro3), composed of a fluorescein fluorophore linked to a rhodamine B hydrazide by a thiourea spacer, was designed and synthesized by Shang et al. [Over the last decade, many experimentalists have been involved in searching for efficient fluorescent FRET-based Hgchnology ,20. Verytal ions . By adopnditions . Moreoveg et al. .2+. However, which one is preferable remains an open question. Moreover, although the responsive mechanism for this probe is discussed in the experiments, the first principle studies and theoretical analyses on this issue are still insufficient and not satisfying. Above all, the measurements were performed by one-photon excitation, and these probes were investigated at one-photon level. As is well known, a one-photon fluorescent probe has disadvantages with interferences from self-absorption, auto-fluorescence and photodamage [2+. By discussing the optical properties of the probes, their sensing performances are compared. Special attention is paid to the analysis of responsive mechanisms of the probes by illustrating molecular orbital distributions of the compounds involved in the absorptive and emissive processes. The present research would be helpful to understand the relationships between the structure and the optical properties for these FRET-based fluorescent probes. Most of all, guidelines for designing more efficient two-photon fluorescent probes are provided based on the present theoretical studies.The experiments mentioned above have shown that the probes Pro1, Pro2 and Pro3 have high efficiency for detecting Hgtodamage ,25,26. If denotes the excitation energy of the excited state f, μα is the electric dipole moment operator, and the summation is performed over x, y, z axes.Details of the theoretical methods are referred to in . Here, wa0, α, c is the Bohr radius, the fine structure constant, and the light speed, respectively. ħω is the photon energy of the incident light. g(ω) is the spectral line profile, which is assumed to be a δ function here, and Γf is level broadening of the final state, which is taken as a typical value of 0.1 eV [tpa is the orientational averaging value of the TPA probability [The TPA cross section that can be compared directly with the experimental value is defined asf 0.1 eV . δtpa isbability .The geometries of all the compounds are optimized at the time-dependent hybrid density functional theory (TD-DFT)/Becke’s three parametrized Lee-Yang-Parr (B3LYP) exchange functional level utilizing the Gaussian09 program  . Frequen2+. With the presence of Hg2+, the energy acceptor (rhodamine moiety) exists in an open-ring form as shown in Pro1+Hg2+, Pro2+Hg2+ and Pro3+Hg2+. On the other hand, Pro1, Pro2 and Pro3 contains different energy donors, namely, naphthalimide derivative for Pro1, boron-dipyrromethene (BODIPY) derivative for Pro2, and fluorescein derivative for Pro3, respectively. The different energy donors would affect the OPA and TPA properties of the compounds [2O are shown in Structural formulas of the studied compounds are depicted in ompounds ,32,33,342+, new strong absorption peaks with wavelengths of significant redshifts were observed [2O, including the excitation energies, the corresponding wavelengths, oscillator strengths and transition natures are calculated, and the results together with the experimental measurements are listed in In the experiment, Pro1, Pro2 and Pro3 show featured absorption peaks at 405 nm, 501 nm and 490 nm, respectively. With the addition of Hgobserved ,22,23. I2+ is present, two main electronic transitions are allowed for Pro1+Hg2+, locating at 489 nm and 434 nm with respective oscillator strength of 0.88 and 0.37. Here, the two peaks appeared in the OPA spectrum of Pro1+Hg2+ origin from the HOMO-1 to LUMO and HOMO to LUMO+1 transitions, respectively. It is noted that the transition from HOMO to LUMO+1, resulting in the adsorption peak at 434 nm is exactly the same as the one of Pro1, corresponding to the transition inside the energy donor. On the other hand, the peak at 489 nm results from the transition inside the energy acceptor. For Pro2 and Pro3, absorption peaks of the compounds are located at 442 and 436 nm, respectively. After reacting with Hg2+, both Pro2+Hg2+ and Pro3+Hg2+ show two absorption bands, i.e., 491 (440) nm with the oscillator strength of 0.96 (0.58) for Pro2+Hg2+, and 495 (426) nm with the oscillator strength of 0.73 (0.35) for Pro3+Hg2+. Similar to the case in Pro1, both Pro2+Hg2+ and Pro3+Hg2+ show new adsorption peaks comparing to Pro2 and Pro3.It demonstrates that the maximum absorption peak of Pro1 is located at 434 nm with the oscillator strength of 0.35, which is attributed to the transition from the HOMO-2 to the LUMO . When Hg2+, Pro2+Hg2+ and Pro3+Hg2+ look like redshifts, which is well consistent with the corresponding experimental results in trends [2+, which can be attributed to the different energy donors of the probes [The newly appeared peaks of Pro1+Hgn trends ,22,23. Ie probes .2O solvent are collected in 2+ are 534.72 GM and 2502.89 GM, located at 868 nm and 802 nm, respectively. Moreover, the maximum TPA cross section values of the probes Pro2 and Pro3 are 108.08 GM and 89.22 GM, located at 899 nm and 873 nm, respectively. With the addition of Hg2+, the maximum TPA cross sections are, respectively, 225.73 GM and 142.91 GM for Pro2+Hg2+ and Pro3+Hg2+. It is seen that the TPA cross section values of the compounds are enlarged to different extents when the probes react with Hg2+, especially in Pro1. In addition, the maximum TPA cross sections follow the order of Pro1(+Hg2+) > Pro2(+Hg2+) > Pro3(+Hg2+).As we have mentioned above, the experiments are performed upon one-photon excitation. It is valuable to explore whether the probes can be used as two-photon fluorescent probes. As is known, an excellent two-photon fluorescent probe should show enhancement in the TPA cross section when it detects the analyte. Thus, the calculated results of TPA including the excitation energies, the corresponding two-photon wavelengths, and TPA cross sections of the lowest five excited states for all the compounds in H2+. Due to the compounds possessing the same energy acceptor but different energy donors, it can be concluded that the energy donor plays a dominant role in TPA performance of the probes [2+. Furthermore, Pro1 has the largest TPA cross section, indicating that it can be used as a preferable two-photon fluorescent probe.Note that the TPA cross section of Pro1 is much larger than that of Pro2 and Pro3 both in the absence and presence of Hge probes ,33. As a2+ was added [2+ into the probes-containing solution, the donor’s characteristic emission peak gradually disappeared, while the characteristic emission peak of rhodamine grew, indicating an efficient energy transfer process upon the reaction. Emission properties of Pro1, Pro1+Hg2+, Pro2, Pro2+Hg2+, Pro3 and Pro3+Hg2+, including the emission energies, the corresponding emission wavelengths, oscillator strengths, and transition natures in H2O solution are calculated based on the optimized first excited state geometries of the compounds.Great changes in the fluorescent spectra of the probes were experimentally observed when Hgas added ,22,23. W2+ is redshifted to 535 nm with a stronger oscillator strength of 1.14. For Pro2, upon the addition of Hg2+, the emission wavelength is redshifted from 489 nm to 538 nm, which appears to be in reasonable agreement with the experimental data [2+. Similarly, with the titration of Hg2+ into Pro3, the emission position is significantly redshifted and the emission intensity is greatly increased with respect to the free Pro3. These calculated results show that emission peak positions for the three compounds are obviously redshifted with the presence of Hg2+, which is in good agreement with the experimental observations [As is clearly seen in tal data . Except rvations ,22,23. T2+ are displayed to explore the responsive mechanism of the probes.The orbital distribution diagram is shown to be a useful tool for analyzing the responsive process of a molecule to the exciting light . Thus, tThe absorptive and emissive processes of Pro1 are presented in 2+, the responsive process of the molecule to the light changes a lot. Generally, there are two main allowed electronic transitions for Pro1+Hg2+ upon excitation, i.e., from the HOMO to the LUMO+1 (434 nm) and from the HOMO-1 to the LUMO (489 nm). Different from Pro1, these two transitions are localized on the energy donor and energy acceptor moiety, respectively  easily reacts with the detect target; (2) the geometric and electronic structure of the probe should be modified by the target to emerge new transitions; (3) new transitions should exhibit high oscillator strengths and discrepant wavelengths. In the presence of Hgvely see b. Obviou2+, it can be seen from the distributions of the molecular orbitals that the emissive process of Pro1+Hg2+ resulting from the LUMO to HOMO-2 transition is localized on the energy acceptor unit , which was first proposed by Förster [With respect to the emission of Pro1+Hg Förster .2+ are investigated in 2+, there are two mainly allowed transitions for both Pro2+Hg2+ and Pro3+Hg2+. Moreover, the two transitions are assigned as the energy donor and energy acceptor localized transitions, respectively. Thus, Pro2+Hg2+ and Pro3+Hg2+ can be stimulated by either exciting the energy donor moiety or the energy acceptor moiety. However, radiative transitions of these molecules are localized on the energy acceptor unit according to the calculations. The above results demonstrate that emission of the energy acceptor moiety of Pro2+Hg2+ and Pro3+Hg2+ could be obtained by exciting the energy donor moiety. This FRET process is intuitively shown in The molecular orbitals involved in the excitation and emission for Pro2 and Pro3 in the absence and presence of Hg2+. In particular, the variation of the energy gap between the two orbitals involved in the emission process for Pro1 and Pro1+Hg2+ (0.44 eV) is the largest compared with that of Pro2 and Pro2+Hg2+ (0.20 eV) as well as Pro3 and Pro3+Hg2+ (0.20 eV), leading the redshift of Pro1+Hg2+ versus Pro1 to be the maximum. In addition, by comparing the absorption and emission wavelength, we ascertain the Stokes shifts for the probes and their reaction products with Hg2+. All of the molecules present large Stokes shifts that range from 44 nm to 122 nm, with Pro3+Hg2+ showing the largest one. An obvious Stokes shift means the avoidance of interference between the absorption and emission spectra. Thus, this is crucial to improving the efficiency of detection. Last but not least, after reacting with Hg2+, the fluorescent intensity of Pro1 displays a three-fold enhancement, while the fluorescence enhancements of Pro2 and Pro3 are 2.4-fold and 1.6-fold, respectively, which indicates the preferable sensitivity of Pro1 towards Hg2+.On basis of the above results, it is interesting to make a comparison among the probes on their sensing performances. It is obvious that the fluorescent emission wavelengths of the probes exhibit great redshifts in the presence of Hg2+-induced changes in the excitation and emission features. In addition, the FRET process in Pron+Hg2+  is demonstrated from the theoretical point of view, suggesting the probes to be promising FRET-based ratiometric and colorimetric fluorescent chemosensors. In particular, Pro1+Hg2+ shows the largest redshift in the fluorescent spectrum compared with the fluorescent spectrum of Pro1, and the strongest fluorescence enhancement with obvious Stokes shift relative to Pro2+Hg2+ and Pro2+Hg2+. As a result, one can anticipate Pro1 to be the superior fluorescent probe in comparison with Pro2 and Pro3.To summarize, the probes Pro1, Pro2 and Pro3 exhibit clear Hg2+. The effect of the donor on the sensing performance for the studied fluorescent probes is discussed with emphasis. Both the absorption intensity and the fluorescent intensity are enhanced, and the fluorescent wavelengths show visible changes when the probes react with Hg2+. Importantly, the TPA cross sections of the compounds are increased with the presence of Hg2+, which theoretically suggests the possibility of the probes to be two-photon fluorescent sensors for Hg2+. Moreover, the calculated results show that Pro1 exhibits the most remarkable changes on fluorescence with the largest TPA cross section in comparison with Pro2 and Pro3, and the variation of TPA cross sections between Pro1 and Pro1+Hg2+ is more obvious. Thus, the energy donor is demonstrated to play a dominant role in the sensing performances on the probes. In addition, Pro1 with naphthalimide derivative as the energy donor is demonstrated to be a preferable chemosensor for Hg2+. In addition, the analysis of molecular orbitals is presented to explore the responsive mechanism of the probes, where the FRET processes are intuitively shown in the compounds when they react with Hg2+. Our results agree with the available experimental measurements and make a comparison among the probes at the same theoretical level. The present research would be helpful for understanding the relationships of structure with optical properties for the studied fluorescent probes, and would further provide guidelines for designing more efficient fluorescent probes.The density functional theory in combination with the polarizable continuum model is used to investigate the one- and two-photon absorptions as well as the emission properties of a series of FRET-based fluorescent probes in the absence and presence of Hg


New York, United StatesInformations: Arthur J. Smerling, MDajs8@cumc.columbia.eduE-mail: register.columbiacme.org/conference.cgi?rm=view&conference_id=814481Site:Bethesda, United StatesInformations: American Association for Thoracic SurgeryPhone: 978-252-2200meetings@aats.orgE-mail: www.aats.org/grantwritingSite: Belfast, United KingdomInformations: Isabelle FernerPhone: +44 (0)7949211636sctsadmin@scts.orgE-mail: www.scts.orgSite: Prague, Czech RepublicInformations: Sue HesfordPhone: + 44 7841519795Fax: + 44 1392 430671sue@ests.org.ukE-mail: www.ests.orgSite: London, United KingdomInformations: Ileana PeterPhone: 702-508-5200cardiovascular@cardiologyconference.orgE-mail: cardiovascular.conferenceseries.comSite: Windsor, United KingdomInformations: EACTSPhone: + 44 7841519795Fax: + 44 1392 430671info@eacts.co.ukE-mail: www.eacts.org/academy/2017-programme/Site: Zürs am Arlberg, United KingdomInformations: Beatrix SecklPhone: +43 664 88 671 571Fax: +43 274 222 210 015office@conventive.atE-mail: www.cardiovascular.atSite: Paris, FranceInformations: EACTSinfo@eacts.co.ukE-mail: www.eacts.orgSite: Seoul, South KoreaInformations: Jiwon YoonPhone: +82-2-3452-0507Fax: +82-2-521-8683reg@ascvts2017.orgE-mail: www.ascvts2017.orgSite: Izmir, TurkeyInformations: Prof. Dr. Oztekin OtoPhone: 0090 532 701 4241Fax: 0090 4642470oztekinoto@oztekinoto.comE-mail: www.uccvs2017.orgSite: Informations: EACTSinfo@eacts.co.ukE-mail: www.eacts.org/academy/2017-programme/Site: Chicago, United StatesInformations: Michelle Taylorwww.sts.org/roboticsSite: Houston, United StatesInformations: CMEPhone: 713.441.4971cme@houstonmethodist.orgE-mail: events.houstonmethodist.org/cmriSite: La Jolla, United StatesInformations: Angela Kinnunenwww.ctsurgery.pitt.edu/E-mail: Cairo, EgyptInformations: Dr. Deepak PuriPhone: +919814332901drdeephearts@gmail.comE-mail: www.cardiomersion.comSite: Cairo, EgyptInformations: Sameh M. ElameenPhone: +201001413339sameh.elameen@gmail.comE-mail: www.escts2017.comSite: Houston, United StatesInformations: CMEPhone: 713-441-4971cme@houstonmethodist.orgE-mail: https://events.houstonmethodist.org/reevolutionSite: Toronto, CanadaInformations: Nancy Bushinfo-ANS1705@cpdtoronto.caE-mail: www.cpd.utoronto.ca/anesthesia/Site: London, United KingdomInformations: Sharon WilliamsPhone: 7025085200sw124143@gmail.comE-mail: pediatriccardiology.conferenceseries.com/europe/Site: Berlin, GermanyInformations: Randi Wilsoneacta@ics.dkE-mail: www.eacta.org/congress-events/calendar-of-events/Site: Copenhagen, DenmarkInformations: Sue HesfordPhone: + 44 7841519795Fax: + 44 1392 430671sue@ests.org.ukE-mail: www.ests.org/Site: Rio de Janeiro, BrazilInformations: Brazilian Society of Cardiovascular SurgeryPhone: +55 (11) 3849-0341sbccv@sbccv.org.brE-mail: sbccv.org.br/44congressoSite: Vienna, AustriaInformations: Marek EhrlichPhone: +4314040056200marek.ehrlich@meduniwien.ac.atE-mail: www.visar.atSite: New York, United StatesInformations: The American Association for Thoracic SurgeryPhone: 978-252-2200Fax: 978-522-8469meetings@aats.orgE-mail: aats.org/mitralSite: Boston, United StatesInformations: American Association for Thoracic SurgeryPhone: 978-252-2200meetings@aats.orgE-mail: www.aats.org/innovationSite: Boston, United StatesInformations: American Association for Thoracic SurgeryPhone: 978-252-2200meetings@aats.orgE-mail: http://www.aats.org/centennialSite: Lausanne, SwitzerlandInformations: Paragon GroupPhone: +41225330948Fax: +41 (0) 22 5802 953GTito@paragong.comE-mail: San Antonio, United StatesInformations: CMEPhone: 713.441.4971cme@houstonmethodist.orgE-mail: Cambridge, United StatesInformations: Harvard Medical SchoolPhone: (617) 384-8600ceprograms@hms.harvard.eduE-mail: Innsbruck, AustriaInformations: Sue HesfordPhone: + 44 1392 430671sue@ests.org.ukE-mail: www.ests.orgSite:


The central venous pressure (CVP) is the most frequently used variable to guide fluid resuscitation in critically ill patients, although its use has been challenged. In this viewpoint, we use a question and answer format to highlight the potential advantages and limitations of using CVP measurements to guide fluid resuscitation. The central venous pressure (CVP) remains the most frequently used variable to guide fluid resuscitation in critically ill patients . Use of We agree that the CVP is influenced by many factors, but this is also true for other variables such as blood pressure, heart rate, and cardiac output—does that mean we should not measure them? No, rather we need to understand which factors influence these measurements and how, in order to use them optimally.CVP is an indicator of right ventricular and, to a lesser extent, left ventricular preload. CVP also reflects the limit to venous return and informs about right ventricular function. As such, CVP measurements may be helpful to guide fluid management. However, CVP is also affected by thoracic, pericardial, and abdominal pressures, which makes its interpretation more complicated. Indeed, although the CVP measured in these conditions overestimates the transmural CVP and may thus fail to reflect the true loading conditions of the right ventricle, it does represent the limit to venous return and the back pressure of all extrathoracic organs. In particular, the risk of peripheral edema, ascites, renal, and liver impairment is related to the absolute CVP value , 5.According to the Frank-Starling relationship, stroke volume increases with CVP until a plateau is reached Fig. . It may The classical statistical approach to evaluating the predictive value of a test is often limited as it considers the risk of error independent of the clinical situation. Nevertheless, it is less risky to administer fluids in a nonresponsive patient with a low CVP than one with a high CVP since the risk of edema formation is obviously lower.The presence of an “extreme” CVP value may be more helpful to guide fluid administration than intermediate values. In a recent systematic review by Eskesen et al., including 1148 patients from 51 studies that evaluated the response to a fluid bolus and reported CVP, the overall predictive value of CVP was poor . HoweverThese data suggest that extreme CVP values can help to guide the response to fluids whereas intermediate values cannot Fig. . It may Yes and no! The increase in CVP does not reflect changes in cardiac output! According to Guyton, venous return is inversely related to the difference between mean systemic pressure (Pms) and CVP, suggesting that the higher the CVP, the lower the cardiac output. The role of the heart in this context is to try to keep CVP as low as possible to maintain the gradient between Pms and CVP. The goal of fluid resuscitation is to increase the gradient between Pms and CVP by increasing Pms, not by increasing CVP itself. Analyzing data published by Cecconi et al.  who measAccordingly, changes in CVP during fluid challenge should be analyzed together with changes in cardiac output; a large increase in CVP with minimal change in cardiac output indicates poor tolerance to fluids, whereas minimal change in CVP together with an increase in cardiac output indicates fluid responsiveness Fig. .Fig. 2ReConsidering changes in CVP without taking into account changes in cardiac output can be very misleading as one may erroneously consider that the greater the change in CVP the larger the increase in preload and thus potentially the greater the effect of fluids. However, the opposite effect may be true, i.e., cardiac tolerance may be poor and the increase in cardiac output may have been minimal. Without cardiac output measurements, the only conclusion that can be drawn from the increase in CVP is that preload effectively increased during fluid administration, but the patient’s response to fluids is unknown.Changes in CVP during a fluid bolus can be used to predict the response to further fluid administration. In 80 surgical patients, Hahn et al. observed in nonresponders that fluid response to a second fluid challenge was more likely when changes in CVP during the first fluid administration were minimal  if CVP increased, compared to when CVP did not increase) .Hence, if anything, changes in CVP should be minimal, but accompanied by an increase in cardiac output; otherwise it implies that preload was not affected.One way to look at changes in CVP during a fluid challenge is to consider CVP as a safety variable. After all, the decision to administer fluids is based on a benefit/risk analysis; the benefits are related to an increase in cardiac output, and the risks to an increase in hydrostatic pressure increasing edema formation. CVP is an excellent variable to estimate the risk associated with extrathoracic organ congestion. Limiting CVP in liver surgery is associated with less risk of bleeding and better perioperative outcomes . SimilarImportantly, the CVP may fail to reflect the risk of developing pulmonary edema, which depends on capillary pressure and hence on left atrial pressure. In these conditions, measurements of pulmonary artery occluded pressure may be preferred as safety variables for the lungs, and/or measurements of extravascular lung water to estimate the severity of lung edema.This is a difficult question, as no clear cut-off value can be identified. As mentioned earlier, an elevated CVP may perhaps be more a marker of severity of cardiovascular and/or respiratory function than a factor contributing to a poor outcome; thus, it would be illogical to prevent it from reaching high values. In a study by Boyd et al.  in patieThis concept is the basis of some resuscitation algorithms including that used by Rivers et al.  in theirPhysiologically, it does not seem reasonable to target a specific CVP value because the CVP value above which a patient would not respond to fluids is highly variable. As indicated earlier, each patient follows their own Frank-Starling curve, and only extreme CVP values carry some predictive value for fluid responsiveness. Hence, targeting a specific CVP value is only valid at the population level. Applying bootstrap analysis on data obtained from 564 critically ill patients submitted to a fluid challenge, Biais et al.  nicely dIn stabilized patients, it is clear that no attempt should be made to increase CVP to specific target values. In the FACT trial, patients with acute respiratory distress syndrome (ARDS)  were ranThe reliability of CVP measurements has been questioned, with errors related both to positioning of the zero level as well as reading errors. Although these potential errors should be acknowledged as a clear limitation, they are not restricted to CVP measurements and, more importantly, adequate training should limit the risk of such errors occurring.Indeed, measurements of end-diastolic volume by transpulmonary thermodilution or echocardiography reflect cardiac preload better than do intravascular pressures, including CVP. In healthy volunteers, changes in stroke volume during fluid loading correlated better with changes in cardiac volumes than with CVP . Of noteIn fact, this question is actually a bit ‘passé’ as there is no evidence that volumes are better than pressures for fluid management in critically ill patients. The only exception is probably the abdominal compartmental syndrome, in which CVP is markedly increased due to the increase in intrathoracic pressure, while cardiac volumes are markedly reduced.Dynamic indices of fluid responsiveness, such as pulse pressure variation (PPV) or stroke volume variation (SVV), or changes in cardiac output during a passive leg raise, reflect that the heart is on the ascending part of the Starling relationship. Given their excellent predictive capacities for fluid responsiveness, these tests are now included in recent guidelines , 22. UnfAlthough these tests may predict the increase in cardiac output after fluid administration, they do not provide information about the risks associated with fluid administration. Indeed, a high CVP, whatever its cause, reflects a high risk with fluid administration, with a high likelihood of further increasing capillary leak and back pressure on extrathoracic organs.As elevated CVP levels may be associated with organ dysfunction , 5, one CVP values provide important information about the cardiocirculatory status of the patient and should not be abandoned. Use of CVP to guide fluid resuscitation has many limitations, but we believe it is wiser to understand and take into account these limitations rather than to discard CVP completely.


Considering the paradigmatic case of random piles of spherical beads, fluid front morphologies emerging during slow immiscible displacement are investigated in real time by X-ray micro–tomography and quantitatively compared with model predictions. Controlled by the wettability of the bead matrix two distinct displacement patterns are found. A compact front morphology emerges if the invading fluid wets the beads while a fingered morphology is found for non–wetting invading fluids, causing the residual amount of defending fluid to differ by one order of magnitude. The corresponding crossover between these two regimes in terms of the advancing contact angle is governed by an interplay of wettability and pore geometry and can be predicted on the basis of a purely quasi–static consideration of local instabilities that control the progression of the invading interface.Wettability is an important factor which controls the displacement of immiscible fluids in permeable media, with far reaching implications for storage of CO Despite its enormous importance and a substantial body of previous research10, there is as yet no conclusive understanding of this process at pore–scale25.Immiscible displacement of one fluid by another within the pore space of a random permeable medium, also called fluid invasion, is of utmost importance in many settings in nature and technology, such as the penetration of soils by water or oil spills, storage of CO27. Experiments using dense random piles of spherical particles, the widely used paradigmatic model system24, have revealed that if the invading fluid wets the solid completely , the front stays rather smooth and expels almost all of the defending phase, while a non-wetting front (θ ≈ 180°) acquires a ramified morphology, leading to a large residual fluid saturation25.The residual saturation with defending fluid during slow immiscible displacement is known to be influenced by the wettability of the pore walls29, who considered a two-dimensional ‘fluid’ front invading an arrangement of circular disks. The centers of the disks were placed on a regular lattice, but their radii were varied in a random fashion in order to mimic the random nature of a porous medium. The sizes of the disks were such that neighboring disks were not in contact. The invading liquid front consisted of circular arcs of equal curvature, extending between disk perimeters and intersecting the latter at a prescribed angle, θ. The numerical calculations showed that if θ was small, the front remained rather smooth, while a large θ resulted in a very ramified front, in qualitative agreement with recent experiments in quasi–two–dimensional Hele-Shaw cells31 as well as full scale30 and coarse-grained32 fluid dynamics simulations.A similar cross–over has been found in a numerical model by Cieplak and RobbinspL = 2γκ, where γ is the surface tension of the fluid–fluid interface, and κ its mean curvature. During the progression of the meniscus, the Laplace pressure varies as a consequence of the complex geometry of the medium. If pL decreases with progressing fluid, the meniscus will burst into the next pore in an accelerated manner. This instability was originally described in a seminal work by Haines2 and is henceforth referred to as ‘Haines jump’ in the remainder of this paper. For the mentioned geometry of circular disks, it takes only elementary geometry to show that such jumps are quite unlikely if θ is small, but become very probable if the contact angle is large29. In the numerical calculations performed for many different values of θ, it was generally found that whenever Haines jumps were occurring frequently, a ramified fluid front was observed in two dimensions. The frequent occurrence of Haines jumps, however, is tantamount to a reduction of cooperative pore filling events where the menisci of at least two throats are involved. As θ was gradually decreased, the frequency of Haines jumps displays a pronounced decrease while approaching a critical contact angle where the morphology of the front changed from rough to smooth29. We shall call this the Cieplak–Robbins cross–over.A qualitative explanation of this finding can be given as follows. The porous solid matrix divides the invading front into a large number of intruding fluid menisci, each of which has a certain Laplace pressure, 29 also exist in three dimensions. The fluid–fluid interface of the invading phase in the two–dimensional model of Cieplak and Robbins is a collection of well separated, circular menisci. Considering random piles of spherical beads as a model system in three dimensions, each bead has only a few point contacts with its neighbors in a stable mechanical equilibrium, and one generically encounters continuous interfaces between the fluid phases in the pore space. In view of the potentially strong cross–talk between interfacial equilibrium shapes in adjacent pores, it appears rather questionable that the highly idealized two–dimensional model of Cieplak and Robbins can be applied to a ‘real’ three–dimensional porous media consisting of a dense bead pack.So far, such a steep wettability controlled cross–over of fluid displacement patterns in a fully three–dimensional porous matrix was found neither in experiments nor in full–scale fluid dynamic simulations. This is because a systematic variation of stable and reliable contact angles is hard to achieve in experiments. Fluid dynamics simulations that allow to study partial wetting conditions are computationally expensive because they demand a high spatial resolution close to moving three phase contact lines. Thus, it is not clear whether the pronounced difference of front morphologies that were found in a regular two–dimensional lattice of circular discs by Cieplak & Robbinsin situ X–ray micro-tomography that the interface of a non-wetting fluid invading a random bead pile prefers to penetrate through larger throats while it develops a ramified morphology. A wetting fluid invading a random bead pile obeys no such preference in its flow path and the emerging front morphology remains compact. These observations strongly support the assumption that the invasion process is indeed quasi–static and dominated by non–cooperative interfacial instabilities for high contact angles6. Using simple geometric arguments and the local geometry of the bead pack, we are able to compute the contact angle at the cross-over between the two displacement regimes where either cooperative or non-cooperative pore filling events dominate the local front advance. Not only the precise location of the cross–over, but also the relative frequency of non–cooperative Haines jumps as a function of the contact angle can be employed to predict the residual saturation of the defending phase. A strong correlation between the experimentally determined residual saturation and the relative frequency of Haines jumps corroborates once more that pore–scale processes during slow invasion are fully captured in the quasi–static picture put forward in this article.In fact, we show by θ with respect to the invading phase. The interstitial space of the samples was first filled with an oil (defending fluid), which was then expelled by an invading aqueous phase. The morphology of the fluid fronts and the residual saturation with the defending fluid were observed during invasion by X–ray micro-tomography. Volume flux controlled experiments were conduced at an average front velocity of U ≈ 3 μm/s. Employing the dynamic viscosity η and density ρ of the invading aqueous phase, as well as the interfacial tension γ between the fluid phases, this velocity corresponds to a capillary number Ca = ηU/γ = 1.6 × 10−7, and to a Reynolds number of in situ, without any restrictions from the data acquisition rate. It was carefully checked that no systematic change, neither in the front morphology nor in the residual saturation of the pore space with the defending fluid, occurred when the displacement velocity was varied even by more than one order of magnitude. Buoyancy effects in the evolution of the fluid interfaces can be ruled out by the very small Bond number ρ denotes the density difference between the invading and defending phase, and g the acceleration of gravity. Given the small value of the Bond number, being two orders of magnitude smaller than unity, capillary pressure variations caused by hydrostatic pressure gradients across the imaged field of view can be safely neglected33. This is supported by repeats of the experiments with the sample cell turned upside down and thus with inverted flow direction that gave no noticeable changes of the displacement patterns. We finally note that the viscosities of the aqueous and the oil phase were similar, thus ruling out a Saffmann-Taylor–type instability as responsible for shaping the fluid front. Moreover, no changes in the morphology of the displacement patterns were observed when the viscosity of the oil phase was increased by one order of magnitude.Dense piles of spheres are employed as three–dimensional model porous media, with controlled surface contact angle θ of the invading phase is well below 90°, the front is observed to be smooth, with the total interfacial area of the invading fluid remaining less than two times the sample cross–section throughout, c.f. the circle symbols in Fig. θ of the invading phase is larger than θ ≈ 110° , we clearly see that the interface of the invading fluid is selectively arrested in the smaller throats, and advances preferentially through the larger ones. For small contact angles , this selectivity is absent, as the corresponding curve shows. This observation is in line with the expected presence of cooperative effects and explains the smooth fronts observed for small contact angles.To further explore the evolution of the interface morphology, we analyzed the progression of the invading fluid at the level of individual pores and throats. A typical result observed for large contact angles is shown in Fig. 34, or whether they conform to the preconditions of a quasi–static description of the invasion process. The quasi–static approach is justified whenever the interface stays close to a stable mechanical equilibrium for most of the time. However, in the course of an interfacial instability, i.e. after mechanical stability has been lost, the motion of the fluids in the quasi–static picture is governed by a fully over–damped dynamics. Under this condition, the fluid interfaces move exclusively into the downhill direction of the free energy landscape and evolve into the next local energy minimum.Returning to the long invading ‘finger’ structure from Fig. U ≈ 0.5 mm/s and Re ≈ 0.5, respectively. However, the duration of a filling event can be shorter leading to even larger local velocities36.An estimate for the local interfacial velocity during a pore–filling event can be obtained from the sequence of fast X–ray tomography images presented in Fig. 6.Even though we cannot rule out inertial effects completely, a statistical analysis of the filling events observed within the entire sample volume demonstrates that inertia does not govern the progression of the front. Both the average radius of throats invaded in a primary step and the average radius of throats invaded in a secondary step, which could not time resolved, are larger than the average throat radius, Fig. R) in mutual contact, which we call an ideal tetrahedral pore, c.f. Fig. α = 60° with respect to its center.In order to predict the contact angles at which Haines jumps are to be expected in three dimensions, we consider the geometry of pore invasion in a (random) pile of spheres in a quasi–static setting. Note that in three-dimensions, a densely packed pile of spheres is both porous and permeable, as opposed to dense two–dimensional packing of disks. As a most simple model of a pore, we start by considering four spheres of equal size  bulge as a function of the position of its center along that line. It is obvious that if the top sphere were absent, pL would at some point start to decrease. If this happens before the bulge touches the surface of the top sphere, a Haines jump occurs. This sudden decrease of the Laplace pressure during invasion of a non–wetting fluid into a pore was first described by Haines as a per saltem motion of a fluid–fluid interface1.If the contact angle θ ≈ 110°, which is displayed in Fig. The condition for a Haines jump in a tetrahedral pore is that We can compute the probability for Haines jumps by considering the statistical weights for the various orientations of the pore with respect to the invading front. Each pore has, by its very definition, four throats through which it can be entered. We know for sure that the pore under consideration is still empty, and it is located at the front. Hence neither all four throats are being invaded at once , nor do they all remain empty (then the pore would still be ahead of the front). Consequently, we have to distinguish three cases: either the pore is invaded by one bulge in which case the Haines jump may be precluded by touching the opposite pore wall, or by two or three at the same time. In the two latter cases, the Haines jump is precluded by two or three bulges coalescing. As the pore under consideration is located at the invading front, we may assume that the probability that a neighboring pore is already filled is 1/2 (for each neighboring pore separately). Hence if the orientation of the pore with respect to the invading front is random, the statistical weight of the pore being filled by one bulge invading from the ‘bottom’ or by three bulges from ‘top’ cf. Fig.  is 1/4 eα = 60° with respect to the center of the respective sphere . For this geometry, the evaluation of the critical angles for occurrence (or preclusion) of Haines jumps is more tedious, but still analytically straightforward (see Supplementary Information). Instead of a single critical angle, we now obtain, for every orientation of the pore with respect to the invading front, a distribution of critical contact angles which derives directly from the distribution of α depicted in the inset of Fig. Now we can go one step further and consider a more realistic pore model. In a real random pile of spheres, not all spheres of a tetrahedral pore are in direct contact, thus enclosing a smooth distribution of angles around As a final step, we take the distributions of critical contact angles precluding Haines jumps for the three possible orientations of the pore, multiply them by the corresponding statistical weights, and add them all up. By virtue of this calculation, we can derive the probability of Haines jumps to occur in a random pile of spheres as a function of the contact angle. The result is plotted as the solid curve in Fig. 34. These morphologies can be seen in the tomography images shown in Fig. θ ≈ 125°, but are absent in corresponding packings of basalt beads with θ ≈ 75°, c.f. Fig. 38, such that our reasoning is not restricted to the considered spherical bead packs but should be more general provided the beads have convex contacts.What is still missing to complete the picture of fluid invasion at different wetting conditions is a connection of the probability of Haines jumps to what is presumably the most important parameter in fluid invasion, the residual saturation. Recall that significant residual saturation is found at contact angles above the cross–over. This is, in particular, at angles a = A/V of surface area A to volume V of the defending phase. Figure a of the percolated subset of the defending phase normalized by the value a0 for a random close pack of beads fully saturated with the defending phase. For the smaller contact angle of the invading phase  the surface–to–volume ratio a remains small at a maximum value corresponding to about twice the surface–to–volume ratio a0 as expected for compact morphologies in a random close packing of beads38. In the case of large contact angle , we find an asymptotic value of a∞ ≈ 3.3a0 of the surface–to–volume ratio for the percolated oil phase after injecting several pore volumes of the non–wetting invading phase  percolation threshold. Upon further liquid injection, small liquid ganglia of the defending phase detach from the percolated network and remain behind the front  drops below 13% of the pore volume (PV). As a consequence, we expect that whenever the front is ramified the maximum RSD is about 13% of the PV. Because ramified fronts and the corresponding regular morphologies of the residual defending phase only emerge globally or locally for large contact angles where Haines jumps occur, it suggests itself to link the residual saturation of the defending fluid to the probability of Haines jumps. For a rough estimate of the residual saturation as a function of the contact angle, we multiply the probability of Haines jumps from Fig. The residual distribution of oil ganglia for invading phases with large contact angle In conclusion, our experiments clearly demonstrate that smooth fronts emerge upon invasion of a wetting fluid into a random pack of spherical beads, while ramified fronts emerge when non–wetting liquids are injected. Ramified fronts lead to residual saturations of the defending phase that are about one order of magnitude above the values found for smooth fronts. The build up of ramified fronts is the result of the selectivity of the flow path following the largest throats that progresses mainly by Haines jumps following the largest throats. During each Haines jump the local Laplace pressure of the respective liquid meniscus suddenly drops upon progression causing the fluid to invade the neighboring pore in an accelerated manner. As a result of these fluid ‘bursts’, an intertwined network of the invading and the defending phase emerges. The network of the defending phase slowly drains during further invasion and forms characteristic morphologies upon de–percolation which remain behind the ramified front.θ ≈ 100° above which Haines jumps are the dominating local instability. Given the known generality of liquid morphologies in wet granular piles38, our results are expected to remain valid to some extend for unconsolidated and consolidated granular piles consisting of a large class of convex grains. By establishing the probability of Haines jumps as the main mechanism which governs fluid invasion, our results also clarify why pore–network models are often found to have very limited predictive power for quasi-static fluid invasion, even if throat and pore size distributions are well in line with the sample characteristics41. It is the assumption of constant cross–section of the throats inherent in these models, which inhibits any non–monotone variation of Laplace pressure with front progression. Hence such simple pore network models have a blind spot which precisely eclipses the relevant mechanism in the quasi-stationary case. Our results may be instrumental for adopting pore network model appropriately to the porous media they are meant to model.Important features of fluid invasion into a three–dimensional porous matrix observed in the presented X–ray tomography data can be well understood on the basis of a strikingly simple, analytically tractable geometrical consideration, invoking only quasi–static concepts. The residual saturation with defending fluid can be predicted quantitatively. The overall behavior is found to be governed by a cross–over in the front morphology which occurs at a contact angle μm. The average bead diameter was determined by X–ray tomography to (383 ± 25) μm and (414 ± 9) μm, respectively. The basalt beads were cleaned with ethanol resulting in a water contact angle in a surrounding dodecane phase of θ = (75 ± 15)°. To achieve very small contact angles of (20 ± 10)° with the aqueous phase, glass beads were sonicated with organic solvents , cleaned with piranha etch (50:50 mixture of H2O2 and H2SO4) and carefully rinsed with hot Millipore waterTM. For very large contact angles of θ = (165 ± 15)° the thus cleaned glass beads were additionally coated with a self-assembled monolayer of octadecyl-trichlorosilane molecules42. Intermediate contact angles of (60–140)° were achieved by various combinations of beads, cleaning procedures (sonication only in water or ethanol) and different oils like dodecane or silicon oil , as summarized in Table Permeable media employed in our experiments consist of dense random packs of glass or basalt beads  with sieve fraction of (355–425)  μm and 44 ± 9 μm,2  with a density of 1080 kg/m3 and a viscosity of η ≈ 1 mPa · s, was used as the invading phase. The ZnI2 dopant guarantees a sufficient X-ray attenuation to distinguish the aqueous phase from other phases present in the system whereas the viscosity is hardly affected43. A mixture of bromododecane - 8% by volume - in dodecane , in the text termed as ‘dodecane’, is used as the oil phase having a density of 761 kg/m3 and a viscosity of 1.383 mPa · s44. The bromododecane also increases the X–ray attenuation and allows to resolve potentially remaining or emerging gas bubbles. Dodecane was filtered three times through a column of aluminum oxide powder45 to remove impurities and to obtain a stable water-oil interfacial tension of γ = (46.7 ± 0.2) mN/m as determined by the pendant drop method. A similar purification or dopant was not required for the used silicon oils. The advancing contact angle at the beads’ surfaces, as given in Table Ultrapure water doped with ZnIϕ = 0.61 ± 0.01 and average throat radii of (37 ± 17) μm, average throat length of (380 ± 130) μm, and average pore radii of (70 ± 25) μm. For all experiments the aqueous phase is injected into the bead matrix using home built computer controlled syringe pumps at constant volumetric flow rate of 61 nL/s leading to an average front velocity of 3 μm/s.The fluid invasion experiments were conducted in cylindrical polycarbonate cells . The top and the bottom of the cell consist of cylindrical caps with a 4 mm and 1.2 mm diameter hole, respectively. A Whatman #40ashless filter paper was placed on the top of the base inlet of the cell to inject a homogeneous front at the start of the experiment. The reservoir in the top cap is sufficiently wide so that the fluid level in the reservoir is about constant during fluid invasion and the hydrostatic pressure only increases by replacing the lighter oil phase with the heavier aqueous phase. The inner side of the cell is additionally coated with cross-linked polydimethylsiloxane that swells in contact with the oil phase and prevents bypassing of the aqueous phase through the larger throats along the cell wall. The cell was first filled with the oil phase, and the beads were dropped in portions from the top and tapped between each layer to guarantee a random close packing. Then the top cap was pushed into the cell to firmly arrest the bead matrix. The resulting homogeneous bead packs have packing fractions of in situ during continuous flow by X–ray micro-tomography using the available setup at beamline ID15A of the European Synchrotron Radiation Facility in Grenoble, France. X–ray photon energies in our experiments ranged from 47 to 54 keV, providing the best results with respect to signal to noise ratio and X-ray dose/damage. The sample was placed on the rotation stage of the tomography setup and the aqueous phase was pumped from below towards the top. The fluid invasion was recorded within an image height of 6 mm close to the bottom of the sample cell. A series of absorption projections (radiographs) was collected by rotating the sample by 180°. An exposure time of about 0.35 ms was used to record each of the radiographs. The three–dimensional tomographic images (tomogram) were reconstructed from the radiographs using a filtered back projection algorithm. To minimize the X–ray dose while recording time series, X–ray radiations between two consecutive tomograms were blocked with a fast shutter and no visible X-ray damage was detected.The fluid invasion was observed μm and typically 4000 radiographs were recorded during a total data acquisition time of 1.4 s. All radiographs belonging to one tomogram were downloaded from the CMOS camera (PCO Dimax) after acquisition and saved to a hard drive providing a nearly unlimited number of tomograms. The downloading time however, required an interval of at least 30 s between consecutive tomograms. To record tomograms at higher time resolution as acquired for Fig. The voxel resolution was 11 46, including edge preserving anisotropic diffusion filtering. In a first step, the bead phase recorded prior to an experiment was subtracted from the subsequently recorded time series. In a second step, the remaining two fluid phases during fluid invasion were segmented. The segmented data were used both for visualization purposes using Drishti volume rendering software47 and for the quantitative analysis.All reconstructed tomograms were processed for phase identification using the Mango software toolkitThe Role of Local Instabilities in Fluid Invasion into Permeable MediaS1 Basalt bead packS2 Glass bead packS3 Fast-tomos