Social protection programs issuing cash grants to caregivers of young children may influence fertility. Grant-related income could foster economic independence and/or increase access to job prospects, education, and health services, resulting in lower pregnancy rates. In the other direction, these programs may motivate family expansion in order to receive larger grants. Here, we estimate the net effect of these countervailing mechanisms among rural South African women.We constructed a retrospective cohort of 4845 women who first became eligible for the Child Support Grant with the birth of their first child between 1998 and 2008, with data originally collected by the Agincourt Health and Socio-Demographic Surveillance System in Mpumalanga province, South Africa. We fit Cox regression models to estimate the hazard of second pregnancy in women who reported grant receipt after birth of first child, relative to non-recipients. As a secondary analysis to explore the potential for grant loss to incentivize second pregnancy, we exploited a natural experiment created by a 2003 expansion of the program’s age eligibility criterion from age seven to nine. We compared second pregnancy rates between (i) women with children age seven or eight in 2002 (recently aged out of grant eligibility) to (ii) women with children age seven or eight in 2003 (remained grant-eligible).The adjusted hazard ratio for the association between grant exposure and second pregnancy was 0.66 . Women with first children who aged out of grant eligibility in 2002 had similar second pregnancy rates to women with first children who remained grant-eligible in 2003 [IRR (95% CI): 0.9 ].Across both primary and secondary analyses, we found no evidence that the Child Support Grant incentivizes pregnancy. In harmony with South African population policy, receipt of the Child Support Grant may result in longer spacing between pregnancies. Cash transfers are widely used in social protection programs for poverty alleviation. Many such programs focus on providing support for children by providing monthly cash transfers to families to help them meet basic needs. Typically, these programs increase support incrementally as number of children increases. Critics of such programs have argued that this condition can create perverse incentives for women to have more children to receive a larger monthly payment.–5. AlthoIt is also plausible that social protection programs may not influence fertility at all,–10 or maThe South African Child Support Grant (CSG) has not been found to be associated with increases in teenage pregnancy in previous studies.–20 HowevIn this study, we aim to establish for the first time the net fertility effects of the CSG using longitudinal, individual-level data. Understanding how, if at all, the CSG may influence fertility is critical to establish whether or not the grant unintentionally incentivizes pregnancy, and to better understand how economic inputs may influence fertility-related decisions.To explore the relationship between the Child Support Grant (CSG) and fertility, we conducted two distinct, but related, analyses. In our primary analysis, we used observational data from the Agincourt health and socio-demographic surveillance site to examine how receipt of the CSG after the birth of first child was associated with timing of second pregnancy. In our secondary analysis, we conducted a natural experiment using data from two socio-demographic surveillance sites (Agincourt and Africa Centre) to examine the influence of loss of the CSG due to age ineligibility on second pregnancy rates.The CSG was established in 1998, and, after initial low-uptake, is now SFor the primary analysis, we assembled a cohort of women from a health and socio-demographic surveillance site (HDSS) located in the Agincourt sub-district of Bushbuckridge in rural Mpumalanga Province, South Africa. The Agincourt HDSS, run by the Medical Research Council/Wits University Rural Public Health and Health Transitions Research Unit, is a longitudinal, population-based, full community cohort study that has continually monitored all vital events of all people living in the study area since 1992. The HDSS now covers over 110,000 people living in 21,000 households across 31 villages. The household census, updated annually, collects information on all births, deaths, and in- and out-migrations. Other important individual and household-level information is collected on a regular, but less frequent, basis. Details of the data collection methods have been described previously., 26We also performed a secondary analysis pooling Agincourt HDSS data with data from the Africa Centre Demographic Information System (ACDIS). Since 2000, ACDIS has collected longitudinal, population-based health and demographic data on a full community cohort located in the uMkhanyakude district of northern KwaZulu-Natal. Currently, ACDIS covers about 110,000 individuals in over 11,000 households., 28 In cCommunity, household, and individual consent have been obtained for all Agincourt HDSS research since its inception. Informed verbal consent is obtained at each household follow-up visit and participant consent records are retained on each household roster. Ethics approval for Agincourt HDSS research, including the consent procedures, was obtained from the University of the Witwatersrand’s Committee for Research on Human Subjects  and the Mpumalanga Province Health Research and Ethics Committee. Ethics approval for ACDIS data collection is obtained annually from the University of KwaZulu-Natal’s Biological Research Ethics Committee. Ethics approval for this analysis was obtained from the Office of Human Research Ethics at the University of North Carolina—Chapel Hill (#11–1605) and the Harvard Human Research Protection Program (#14–3909).second pregnancy. During each Agincourt HDSS and ACDIS census update, a household respondent identifies any currently or recently pregnant women living in the household. All recently pregnant women are then followed-up to collect a range of pregnancy-specific information, including the date of delivery. We back-calculated the estimated conception date of each second pregnancy by subtracting 40 weeks from the delivery date of the second child. Time to second pregnancy was defined as the interval, in days, between the delivery of the first child and the estimated conception date of the second child. Women who did not go on to have a second child during follow-up were censored at the end of the follow-up period.As the CSG can only be received after a woman has had her first child, we examined the individual impact of grant receipt on receipt of the CSG. The HDSS collected data regarding the CSG in 2002, 2005, and 2008. Respondents reported whether or not the grant was received and the date on which it was first received. We partitioned the dataset so that participants who became exposed contributed unexposed person-time up until CSG receipt and exposed person-time thereafter. In the secondary analysis, our exposure of interest was loss of CSG eligibility. To define loss of CSG, we exploited a natural experiment created by a 2003 age expansion of the grant. In April 2003, the age cut-off for CSG eligibility was extended from age seven to age nine. Thus, women with children age seven or eight in the year prior to the age expansion recently lost eligibility for the grant. Women with children age seven or eight in the year after the age expansion maintained eligibility for the grant.Our primary exposure of interest was age, at the time of first birth; household wealth, measured with an index of household assets; and 50th percentiles .The weighted KM curve supports the findings from the Cox models . Time toFinally, loss of CSG for first biological child was not associated with second pregnancy incidence . Women wIn this paper, we present evidence that receipt of the Child Support Grant was significantly associated with lower second pregnancy rates. In the context of South Africa, where fertility remains above replacement level among many socioeconomically disadvantaged groups, officialThere are several plausible mechanisms to explain how CSG receipt may lead to decreased pregnancy rates through an income effect. Economic independence facilitated by the grant may shift relationship power dynamics, leading to improved agency with respect to sexual and reproductive health decision-making, or decreasing the need for transactional-sex based relationships. SimilarlThe use of long-term, longitudinal, population-based HDSS data allowed us to draw stronger inferences over prior work. We conducted an individual-level analysis in a full community cohort, as opposed to the ecological analyses in many prior studies,–18 whichThis study examines some, but not all, of the ways the CSG could influence fertility. Specifically, we focus on receipt (and loss) of the grant as the exposure of interest. We assume grant receipt is related to both (i) awareness of grant and (ii) real-life experience with how much the costs associated with having children are (or are not) offset by the grant. Grant receipt is also clearly associated with increased income, which, as discussed above, is a plausible mechanism for the protective association we observed. The comparisons we make between grant recipients and non-recipients allow us to take advantage of the detailed, individual-level grant exposure data in the Agincourt HDSS. However, it restricts us to examining the effects of the grant on second order pregnancies or higher, as a first child is necessary to receive the grant. Whether or not the program incentivizes first pregnancies is not explored in this paper. Future studies, perhaps exploiting differences in the accessibility of the program in early years, could be conducted to examine this important question.Importantly, exposure to the grant was not randomly assigned in our study population. In our primary analysis, those who received the grant after the birth of their first child had significantly different covariate distributions than those who did not receive the grant. To account for the influence of potential confounders, we adjusted our models to close all identified confounding paths, updating the covariates whenever new information was available, and coding them flexibly with restricted cubic splines. Our point estimates were essentially identical before and after covariate adjustment. However, it is possible that the women who received the grant were different in other unmeasured ways than women who did not receive the grant (i.e. ability to navigate barriers to CSG access), which could confound our estimates.As the evidence continues to come in about the benefits provided by social protection programs in economic, health, and education realms , it is i


Dengue fever is one of the most important seasonal epidemics in Asia Case fatality rates vary from 1 to 5% ,3. The pThis was designed as a retrospective descriptive study in a tertiary-level pediatric ICU in South India. Case records of all children fulfilling the WHO case definition of severe dengue were included, those who received intravenous fluid for less than 12 hours were excluded. Primary parameters measured included fluid balance in the first 36 hours measured every 12 hours, durations of oxygen requirement, mechanical ventilation, ICU stay and total hospital stay. Outcomes measured were death and survival.P < 0.05) and fluid balance in the first 36 hours. Positive fluid balance (FO > 15%) in the first 36 hours was significantly higher in children who expired (P = 0.011). eGFR <90 ml/minute at admission had 100% sensitivity and 79% specificity to predict the possible occurrence of fluid overload >15% (area under curve = 0.882).Twenty-six children were enrolled, 14 boys and 12 girls. The median duration of ICU stay was 60 hours, and that of hospital stay 109 hours. eGFR was less than 60 ml/minute in six patients (83.3% expired and 16.7% survived). eGFR, measured by modified Schwartz's formula, at the time of admission correlated inversely with requirement of oxygen therapy and mechanical ventilation (Fluid balance in the first 36 hours had a significant positive correlation with mortality and negative correlation with eGFR. Children with admission eGFR <90 ml/minute may require restrictive fluid therapy to improve survival.


We hypothesised that the proportion of refractory tumour tissue (Oxy-R) would be a robust biomarker of tumour hypoxia across multiple models with different vascular and hypoxic phenotypes.There is need for non-invasive methods to identify, quantify and map tumour hypoxia. In this study we used an emerging technology – R2 were evaluated in well vascularised renal cancer 786-O xenografts. Dynamic sensitivity of proportional Oxy-R to acute changes in hypoxia was evaluated using hydralazine challenge. Relationship of proportional Oxy-R to tissue immunohistochemistry and gadolinium DCE-MRI were explored in parental and drug-resistant 786-O models and in SW620 xenografts.Methods: OE-MRI signal precision, stability and relationship to tissue pOin vivo experiments demonstrated the accuracy, precision and stability of R1 measurement. The proportion of tumour Oxy-R increased significantly following hydralazine challenge (p=0.045) relative to control. The proportion of tumour with perfused Oxy-R voxels was correlated to chronic hypoxia in well perfused 786-O-R xenografts and in relatively necrotic SW620 xenografts .Phantom and The proportion of tumour perfused Oxy-R is a robust biomarker of tumour hypoxia. Voxel-wise analysis of dual oxygen and gadolinium challenge has potential to quantify and map tumour hypoxia as prognostic, predictive and pharmacodynamic biomarkers that could facilitate personalised healthcare.


The purpose of this study was to investigate tumour thymidine concentrations and whether they correlated with [18F]FLT uptake across a broad spectrum of murine cancer models. A modified liquid chromatography-mass spectrometry (LC-MS/MS) method was used to determine endogenous thymidine concentrations in plasma and tissues of tumour-bearing and non-tumour bearing mice and rats. Thymidine concentrations were determined in 22 tumour models, including xenografts, syngeneic and spontaneous tumours, from six research centres, and a subset was compared for [18F]FLT uptake, described by the maximum and mean tumour-to-liver uptake ratio (TTL) and SUV.Recent studies have shown that 3′-deoxy-3′-[18F]FLT injection, determined in 14 of the 22 tumour models, ranged from 1.07 ± 0.16 to 5.22 ± 0.83 for the maximum and 0.67 ± 0.17 to 2.10 ± 0.18 for the mean uptake. TTL did not correlate with tumour thymidine concentrations.The LC-MS/MS method used to measure thymidine in plasma and tissue was modified to improve sensitivity and reproducibility. Thymidine concentrations determined in the plasma of 7 murine strains and one rat strain were between 0.61 ± 0.12 μM and 2.04 ± 0.64 μM, while the concentrations in 22 tumour models ranged from 0.54 ± 0.17 μM to 20.65 ± 3.65 μM. TTL at 60 min after [18F]FLT uptake in murine cancer models.Endogenous tumour thymidine concentrations alone are not predictive of [The online version of this article (doi:10.1186/s13550-016-0218-3) contains supplementary material, which is available to authorized users. However18F]FLT uptake [18F]FLT uptake and tumour thymidine concentrations, which was also observed by Schelhaas et al. [18F]FLT uptake, suggesting that thymidine and [18F]FLT compete as substrates for TK1. Measurement of plasma thymidine concentrations may, therefore, be a critical parameter in deciding whether a patient would be appropriate for [18F]FLT PET scanning.Previous studies have reported variability in tumour [T uptake –7 and haT uptake  measureds et al.  but not 18F]FLT uptake and tumour thymidine concentrations observed previously in smaller studies was valid across a wider range of tumour models. We also examined the variability of thymidine concentrations in plasma and other normal tissue between different murine strains and whether tumours themselves altered plasma thymidine concentration in their hosts.We describe here a modified thymidine assay, based on the method of Li et al. , which wPlasma and tumour samples were analysed from six centres, five participating centres in the IMI QuIC-ConCePT Consortium and one additional centre. The participating institutions were the CRUK Cambridge Institute, Cambridge, UK (CI); AstraZeneca, Alderley Park, Macclesfield, UK (AZ); Imperial College London, UK (IC); Westfälische Wilhelms-Universität, Münster, Germany (WWU) and Radboud University Medical Centre, Nijmegen, The Netherlands (Radboudumc). The additional centre was the Wolfson Molecular Imaging Centre Manchester, UK (WMIC). All procedures performed were carried out in compliance with the national laws on the use of animals in research in the UK  Act 1986), Germany and the Netherlands, and with local ethical approval, and also in compliance with the QuIC-ConCePT Ethical Guidelines, based on the NCRI Guidelines for the welfare and use of animals in cancer research FLT PET scans were conducted at four centres  on three different preclinical PET scanners . See Table 18F]FLT uptake. In murine liver, [18F]FLT is not metabolised and can therefore be used as a reference organ [18F]FLT injection. As the full range of parameters was not available for all 22 tumour models, data from only 14 models were used in the correlation of thymidine concentration with imaging data.[ce organ . Seconda18F]FLT was manufactured according to Ph.EU [[to Ph.EU  specifict test with Welch’s correction  and differences were considered significant if p ≤ 0.05. Mean [18F]FLT uptake value and mean thymidine concentration were determined in cohorts of mice, where each cohort was defined by tumour type and study centre. Correlations between cohorts were performed on mean data and assessed by the Spearman correlation coefficient .Data were expressed as mean ± one standard deviation (SD), unless stated otherwise. The significance of comparison between two data sets was determined using the unpaired, two-tailed Student n = 5) of quality control (QC) samples at four different concentrations. Recovery was assessed by comparing the peak response ratios of analytes spiked before and after extraction with those spiked at the same concentrations. Examples of chromatograms are shown in Fig. Assay performance was assessed by using the EMA Guidelines on bioanalytical method validation (EMEA/CHMP/EWP/192217/2009) as a guide to determining the linearity, precision, accuracy, matrix effects and recovery. Linearity was tested using eight non-zero standards with the back-calculated concentration of each standard value not exceeding ±15 % of the theoretical value (±20 % at the lower limit of quantification (LLOQ)). The precision and accuracy were assessed by replicate analysis . Any assay for which the accepted control criteria were not met was discarded and the assay repeated.p = 0.003). The variation in thymidine concentrations between strains at a single centre was not significant. The thymidine concentration in Wag/Rij rat plasma was within the range observed for mice.Plasma samples were collected from five mouse strains and one rat strain that are used routinely in preclinical studies. Plasma thymidine concentrations in non-tumour-bearing mice varied threefold between strains, from 0.61 ± 0.12 μM for CB17 SCID mice housed at the CI to 2.04 ± 0.64 μM for NMRI nude mice housed at WWU  and muscle (p = 0.03) but were comparable in plasma.Muscle, spleen and pancreas thymidine concentrations were measured in non-tumour-bearing mice in the PC and SCID mouse strains used at the CI Fig. . Plasma,p = 0.018). For A549, the WWU tumour thymidine concentration was 3.69 ± 3.96 μM, which was similar to the value of 5.25 ± 0.73 μM for the same tumours grown at WMIC. HCT116 tumours at IC and WMIC also had similar concentrations of 4.23 ± 0.97 and 3.27 ± 3.06 μM, respectively. There was no difference between the genetically engineered spontaneous KPC tumours and the syngeneic transplanted K8484 tumours.Tumour thymidine concentrations were measured in a panel of 22 different tumours, including three xenografts grown from the same cell line but in two different centres . The tumour thymidine concentrations ranged from between 0.54 ± 0.17 μM for the A431 xenograft to 20.04 ± 3.65 μM for the syngeneic rat metastasis model CC531  or MiaPaCa-2 tumours (p = 0.0016) than in non-tumour bearing mice, but higher in mice with KPC tumours than in the PC mice, which lack the KRAS mutation and do not develop spontaneous tumours (p = 0.006). The plasma thymidine concentration in tumour-bearing rats was also more than three times higher than that in naïve animals . Both sites obtained very similar maximum and mean tumour-to-liver uptake ratios (maxTTL 2.74 ± 0.36 (IC) and 3.1 ± 1.13 (WMIC); meanTTL 1.31 ± 0.36 (IC) and 1.31 ± 0.38 (WMIC)) and similar thymidine concentrations (4.23 ± 0.97 μM (IC) and 3.27 ± 3.06 μM (WMIC)).For 14 of the 22 tumour models studied, the ratio of TTL had been determined at a comparable time of 60 ± 5 min after [18F]FLT uptake than has been possible previously. Plasma thymidine concentrations of non-tumour-bearing animals varied threefold between different strains (from 0.61 ± 0.12 μM for SCID mice at the CI to 2.04 ± 0.64 μM for NMRI nude mice at WWU), while variation in thymidine concentrations between strains at a single centre was low  activity, dephosphorylation and inflammatory infiltration on [18F]FLT uptake all probably play a part to different degrees FLT uptake was inversely correlated with plasma thymidine concentration, which was delivered by a mini-pump. However, in the six tumour models where plasma samples were available, we found no correlation between endogenous plasma thymidine concentrations and [18F]FLT uptake


In the Funding section, the grant agreement name from the funder European Commission’s Seventh Framework Programme is listed incorrectly. The correct grant agreement name is as follows: The research leading to these results has received funding from the European Commission’s Seventh Framework Programme (FP7/2007-2013) under the grant agreement Lungtarget (project n° 259770).


During abdominal surgery, the use of protective ventilation with a low tidal volume, positive expiratory pressure (PEEP) and recruitment maneuvers (RMs) may limit the applicability of dynamic preload indices. The objective of the present study was to establish whether or not the variation in stroke volume (SV) during an RM could predict fluid responsiveness.−1, PEEP: 5–7 cmH2O; RMs). Hemodynamic variables, such as heart rate, arterial pressure, SV, cardiac output (CO), respiratory variation in SV (ΔrespSV) and pulse pressure (ΔrespPP), and the variation in SV (ΔrecSV) as well as pulse pressure (ΔrecPP) during an RM were measured at baseline, at the end of the RM, and after fluid expansion. Responders were defined as patients with an SV increase of at least 15% after infusion of 500 mL of crystalloid solution.We prospectively included patients receiving protective ventilation . A ΔrecSV value more than 16% predicted fluid responsiveness with an area under the receiver-operating characteristic curve (AU) of 0.95  and a narrow gray zone between 15% and 17%. The area under the curve values for ΔrecPP and ΔrespSV were, respectively, 0.81  and 0.80 . ΔrespPP did not predict fluid responsiveness.Thirty-seven (62%) of the 60 included patients were responders. Responders and nonresponders differed significantly in terms of the median ΔrecSV . ΔrecPP and ΔrespSV (but not ΔrespPP) were also predictive. Dynamic preload indices (such as the respiratory variation in pulse pressure (ΔrespPP) or stroke volume [ΔrespSV]) are based on the interaction between the cardiovascular and pulmonary systems during positive-pressure mechanical ventilation. Nevertheless, a low tidal volume (TV), heart rhythm disorders, and right heart failure decrease the reliability of these indices. Protective ventilation , and the use of a recruitment maneuver [RM]) appears to be associated with a shorter stay in hospital and a lower complication rate.–7 However, protective ventilation alters the predictability of dynamic preload indices and thus limits their use in the operating theatre. Other preload tests (such passive leg-raising and an end-expiratory maneuver) can be used but are less accurate and more difficult to implement. In this context, variations in the CO or stroke volume (SV) during the RM could also be monitored as a guide to preload dependency. RMs increases intrathoracic pressure, which in turn causes a transient decrease in CO and arterial pressure; this may depend on preload status. This approach has been studied and validated in septic patients, but not in patients with protective ventilation. The main objective of the present study was to establish whether or not the variation in SV during a RM (ΔrecSV) predicts a further increase in SV upon fluid expansion. We also assessed the predictive value of ΔrespPP, ΔrespSV and the variation in pulse pressure during a RM (ΔrecPP) in this context.Early goal-directed fluid therapy during surgery decreases the length of hospital stay and postoperative morbidity, and its use at the bedside is recommended.22.1Comité de Protection des Personnes Nord-Ouest II, Amiens, France; reference: 2014–79), we performed a prospective study in the operating theater at Amiens University Medical Center between January and September 2015. All patients received written information on the study's objectives and procedures, and gave their informed consent to participation before surgery. The present manuscript was drafted in compliance with the STROBE checklist for cohort studies.After approval by the local investigational review board , the use of protective ventilation, and fluid challenge in the operating theater. Patients with frequent ectopic beats, preoperative arrhythmia, right ventricular dysfunction, or spontaneous ventilation were not included. Fluid expansion was performed in patients with arterial hypotension  or an SV decrease of more than 10%. In our institution, fluid challenge consists of infusion of 500 mL of Ringer lactate via a pressure bag. Patients having undergone several RMs were included only once .2.3−1) or cisatracurium (0.15 mg kg−1). The TV was adjusted to the ideal body weight to obtain 6 mL kg−1, the ventilatory rate was set in order to maintain an end-tidal CO2 pressure of 35–37 cmH2O, and PEEP (5–7 cmH2O) was applied. Static pulmonary compliance was calculated as TV divided by . Epidural anesthesia was used after surgery, but was never used during surgery.Each patient was monitored with pulse oximetry, arterial invasive blood pressure monitoring, and 5-lead electrocardiography. Balanced general anesthesia was applied. All patients were intubated and ventilated in volume-controlled mode. The exact choice of drug was left to the anesthetist's discretion (either propofol or etomidate as hypnotics and either remifentanil or sufentanil as opioids). Anesthesia was maintained with either an inhaled hypnotic (desflurane or sevofurane) or propofol and the opioid used for induction. Neuromuscular blockade was systematically induced with rocuronium  intraobserver and interobserver reproducibility .The EDM probe  was positioned so as to obtain the optimum signal for blood velocity in the descending aorta. SV and CO were calculated continuously (beat-by-beat) from the aortic blood flow velocity by using EDM software, and the values were averaged over a 10-second moving window. Respiratory variations (Δresp) of EDM values were obtained as described previously, regardless of the respiratory cycle.2.5–7; immediately after intubation, every 30 minutes during the procedure, before extubation, and whenever the ventilatory circuit was disconnected. In the absence of a literature consensus on the type of intraoperative RM to be used in patients with normal  lung compliance, the RM consisted of the application of a continuous positive inspiratory pressure of 25 cmH2O for 25 seconds (as implemented in our center's department of anesthesia).RMs were performed as described in the literature2.6We recorded demographic data  score, medical history, and the type of operation), respiratory parameters , and hemodynamic parameters .2.7,11,13When a patient met the inclusion criteria, the investigating physicians collected a first set of demographic, ventilatory, and hemodynamic data (Base 1) before fluid expansion. After the RM had been performed, a second dataset (RM) was recorded. A third dataset was recorded 5 minutes after all the hemodynamic variables had returned to baseline values (Base 2). Next, fluid expansion (500 mL of Ringer lactate over 10 minutes via a pressure bag) was performed. On the basis of very sparse literature data on the maximum possible effect of fluid expansion on hemodynamic variables, we decided to acquire a fourth and last set of hemodynamic data (FE) 5 minutes after the fluid expansion. ΔrespSV was calculated as described previously: ΔrespSV = ([SVmax − SVmin]/[SVmax + SVmin])/2 × 100, where SVmin and SVmax are the minimum and maximum SV values over a single respiratory cycle, respectively. All values correspond to the mean of 3 measurements. ΔrecPP was calculated as follows: ΔrecPP = (PPbase1 – PPRM)/(PPbase1) × 100. ΔrecSV was calculated as follows: ΔrecSV = (SVbase1 – SVRM)/(SVbase1) × 100, where SVRM is the mean of the last 3 SVs at the end of the RM. ΔrecSV and ΔrecPP are expressed as absolute values.ΔrespPP was automatically calculated by the Philips monitoring system; this method has already been validated. A positive response (fluid responder) was defined as an SV increase of at least 15% between Base 2 and fluid expansion. This cut-off was chosen in accordance with the literature data on fluid expansion and because it is twice the value of the interobserver/intraobserver reproducibility of SV measurements using EDM.Nonresponders and responders were defined with regard to the change in SV (expressed as a percentage) after fluid expansion.2.8le) as appropriate. The nonparametric Wilcoxon rank sum test, Student's paired t test, Student's t test, the Mann–Whitney test, the Kruskal–Wallis test and an analysis of variance with Bonferroni post hoc correction were used to assess statistical significance, as appropriate. Categorical variables were compared in a chi-square test or Fisher exact test. Linear correlations were tested using Pearson rank method. A receiver-operating characteristic (ROC) curve was drawn for ΔrecSV, ΔrespSV, ΔrespPP, and ΔrecPP. We selected the threshold that gave the highest Youden index. The gray zone (corresponding to 2 cut-offs between which the prediction of fluid responsiveness remained uncertain) was calculated using 3 response classes . Inconclusive responses were cut-off values with a sensitivity and a specificity below 90% . The method described by DeLong et al was used to compare the areas under the ROC curve (AUC) associated with the variables. The association between the volume of fluid infused (mL kg−1), cardiovascular variables  and fluid responsiveness was assessed using a univariate logistic regression model. Variables with a P value less than 0.10 in the univariate model were included in a multivariate logistic regression model with backward selection. The threshold for statistical significance was set to P < 0.05. Statistical analyses were performed with SPSS software  and R software  with the ROCR package.Based on a pilot study of 15 patients, it was calculated that a sample size of 60 patients would be sufficient to demonstrate that (i) ΔrecSV can predict fluid responsiveness with an area under the curve (AUC) of between 0.8 and 0.87, a power of 80%, an alpha risk of 0.05 and a beta risk of0.2, and (ii) ΔrecSV is correlated with SV changes upon fluid expansion (with a ratio ranging from 0.5 to 0.7). The data distribution was assessed using a D’Agostino–Pearson test. Data are expressed as the number (percentage), the mean (SD) or the median . The baseline SV and CO were lower and ΔrespSV was higher in responders than in nonresponders (Table P < 0.05). The median ΔrecSV was greater in fluid responders than in nonresponders patients ; the same was true for ΔrecPP  of the 60 patients were classified as fluid responders, and thus the remaining 23 (38%) were classified as non-responders. The mean ± SD volume used in the fluid challenge was similar in the responders and nonresponders . Baseline ΔrespSV and ΔrespPP were also correlated with SV variations upon fluid expansion .ΔrecSV and ΔrecPP were correlated with SV variations upon fluid expansion (P < 0.0001). The gray zone ranged from 15% to 17%. ΔrecPP predicted fluid responsiveness with an AUC of 0.81 . The gray zone ranged from 6% to 26%. ΔrespSV predicted fluid responsiveness with an AUC of 0.80 . The gray zone ranged between 6% and 11%. ΔrespPP was not predictive of fluid responsiveness, and yielded an AUC of 0.65 (95%CI: 0.51–0.77) . The AUCs of ΔrecPP and ΔrespSV were not different.ΔrecSV was strongly predictive of fluid responsiveness, with an AUC of 0.95 .In a multivariate logistic regression analysis, ΔrecSV was found to be the only factor significantly associated with fluid responsiveness  predict fluid responsiveness in patients receiving protective ventilation during surgery. A ΔrecSV more than 16% during RM predicted fluid responsiveness and had a narrow gray zone (between 15% and 17%). The ΔrecPP and ΔrespSV were also predictive of fluid responsiveness in patients with protective ventilation. In contrast, ΔrespPP was not predictive of fluid responsiveness in this setting.2O−1), and so an increase in intrapulmonary pressure would have been transmitted to the adjacent compartments. Since patients with right ventricular dysfunction or lung disease were excluded from the study, one can assume that right ventricular function in our study population was normal. Secondly, the increase in intrathoracic pressure caused by continuous PEEP acts on the right ventricle in 2 ways.–21 Firstly, this increase is transmitted to abdominal compartment, which increases resistance to venous return due to collapse of the vena cava and the hepatosplanchnic venous circulation.,19 However, PEEP increases the right ventricle afterload (resulting from the increase in pulmonary vascular resistance) and right ventricle ejectional impedance. These effects on right ventricular preload and afterload are particularly large when the preload is low.,21To understand the impact of the RM on the cardiovascular system, several physiological aspects must be taken into account. Firstly, none of our patients had pulmonary disease. The mean static pulmonary compliance in our population was in the normal range . This topic is still subject to debate, with the publication of contradictory finding.,24 It is important to take account of the characteristics of the study population , the study setting (operating theater vs intensive care unit) and the monitor used to track CO or SV changes . As mentioned earlier, the magnitude of the pulmonary transmission index submit to the conditions of the effect of intrathoracic pressure on the preloads and afterloads of the right and left ventricles. This suggests that respiratory variations are related to preload dependency—even at low TVs. As the TV decreases, so do respiratory variations in SV and PP; this may reduce the sensitivity and the cut-offs associated with these indices. As mentioned in the literature, the analysis of SV and its changes must take account of the measurement site  and the measurement device.,27 It is likely that measurements at a distal artery  have a greater signal/noise ratio due to the damping of the signal along the vascular tree and the low respiratory variations (due to the decrease in TV). This reduces the index's ability to discriminate between fluid responders and non-responders  and may explain in part why ΔrespSV (measured with EDM at the descending thoracic aorta) was predictive of fluid responsiveness but ΔrespPP  was not.In 2005, De Backer et al initiated a debate on the relevance of dynamic preload indices when patients were ventilated with low TVs  in disease-free patients. A recent review by Güldner and colleagues reported great heterogeneity in the RMs and protective ventilation modes used in the operating theatre, and did not provide specific recommendations on RMs. Hence, the use of ΔrecSV at the bedside during hemodynamic optimization can be considered. Even though this parameter was most predictive, it should be used as an adjunct to other parameters of preload dependency . Given that repeated RMs can have harmful hemodynamic effects, the use of this approach should be avoided in hemodynamically unstable patients. At the bedside, preload dependency should be assessed as part of a multimodal approach based on a clinical examination, hemodynamic parameters, dynamic preload indices, and the SV change during fluid challenge. This approach may increase the accuracy of diagnosis of preload dependency, especially when the preload indices are in the gray zone. Given that we excluded patients with cardiac arrhythmia, right ventricular failure, spontaneous ventilation, and altered lung compliance, our present results cannot be extrapolated to other patient profiles. Furthermore, the CardioQ EDM system does not measure the aortic diameter, and CO is estimated from the aortic blood flow velocity-time integral. This bias has been discussed in the literature, and a number of studies have demonstrated that ΔrespSV can still be predicted in the absence of an aortic diameter measurement.,15 Lastly, one must consider the impact of possible EDM probe displacement during the RM on the measurement of SV. To avoid artifacts related to failure to identify the beginning and end of aortic flow with each ventricular beat, we ensured that laminar flow was present by using a narrow frequency range  At the bedside, we did not observe any significant changes of the envelope of aortic flow signal during RM. This type of change would have worsened the predictive value of ΔrecSV because of false positives. Since ΔrecSV was of value in predicting fluid responsiveness, we believe that the putative impact of RMs on displacement of the EDM probe was marginal.This study had several limitations. Our institution's choice of RM can be debated. In the past, we have found that high intrathoracic pressures have harmful effects on patients without lung disease. Several studies have demonstrated that lower levels of PEEP still provide satisfactory levels of arterial oxygenation, with better hemodynamic tolerance.5During abdominal surgery with protective ventilation , ΔrecSV was strongly predictive of fluid responsiveness and had a narrow gray zone. ΔrecPP and ΔrespSV were weakly predictive of fluid responsiveness, and ΔrespPP was not predictive.


Methylobacterium extorquens AM1, a strain serendipitously isolated half a century ago, has become the best-characterized model system for the study of aerobic methylotrophy (the ability to grow on reduced single-carbon compounds). However, with 5 replicons and 174 insertion sequence (IS) elements in the genome as well as a long history of domestication in the laboratory, genetic and genomic analysis of M. extorquens AM1 face several challenges. On the contrary, a recently isolated strain - M. extorquens PA1- is closely related to M. extorquens AM1 (100% 16S rRNA identity) and contains a streamlined genome with a single replicon and only 20 IS elements. With the exception of the methylamine dehydrogenase encoding gene cluster (mau), genes known to be involved in methylotrophy are well conserved between M. extorquens AM1 and M. extorquens PA1. In this paper we report four primary findings regarding methylotrophy in PA1. First, with a few notable exceptions, the repertoire of methylotrophy genes between PA1 and AM1 is extremely similar. Second, PA1 grows faster with higher yields compared to AM1 on C1 and multi-C substrates in minimal media, but AM1 grows faster in rich medium. Third, deletion mutants in PA1 throughout methylotrophy modules have the same C1 growth phenotypes observed in AM1. Finally, the precision of our growth assays revealed several unexpected growth phenotypes for various knockout mutants that serve as leads for future work in understanding their basis and generality across Methylobacterium strains. Since M. extorquens AM1 is genetically tractable 1 compounds (tD∼3–4 h on methanol and methylamine) and multi-carbon compounds (tD∼3 h on succinate) Methylotrophy is the ability of microorganisms to grow on reduced single-carbon (CM. extorquens species and genome sequence data is now available for six strains M. extorquens PA1 (hereafter PA1) as it has the most streamlined genome, with a single 5.47 Mb chromosome, and contains only 20 intact IS elements. These features can make the design and implementation of genetic screens more efficient, and prevent beneficial elimination of extra-chromosomal elements or IS-mediated events from dominating the spectrum of beneficial mutations. Recent isolation of PA1 from the leaves of Arabidopsis thalianaOf late, an increasing number of studies have been conducted with several members of the 1 compounds such as methanol or methylamine are oxidized by dedicated periplasmic dehydrogenases to generate formaldehyde. Once in the cytoplasm, formaldehyde is oxidized to formate via a tetrahydromethanopterin (H4MPT) dependent pathway 2 via a panel of formate dehydrogenases 4F) dependent pathway 1 unit from methylene-H4F (an intermediate of the H4F pathway) along with an equal amount of CO2There are some clear advantages of the genome composition and culturing history of PA1 over AM1. In order to ascertain how well the decades of characterization of methylotrophy in AM1 will apply directly to PA1, we identified the shared repertoire of methylotrophy genes and performed a broad genetic analysis of the role of various methylotrophy modules in PA1 . In AM1,M. extorquens AM1 is largely transferable to M. extorquens PA1: an alternate model system for the study of aerobic methylotrophy in the future. Additionally, our quantitative physiological analysis has also unveiled novel phenotypes for methylotrophy-specific genes that generate leads to uncover poorly understood aspects of regulation in future work.The genetic and phenotypic analysis in this work demonstrated that the vast body of knowledge pertaining to methylotrophy in cel mutant of the pink-pigmented ‘wildtype’ stock of AM1 (CM2720) and the Δcel mutant of the pink-pigmented ‘wildtype’ stock of PA1 (CM2730) used for growth comparisons are described elsewhere 2HPO4 plus 22.5 g Na2HPO4 in 1L deionized water), 100 mL sulfate salts solution (5 g of (NH4)2SO4 and 2 g of MgSO4 • 7 H2O in 1 L deionized water), 799 mL of deionized water, and 1 mL of trace metal solution The ΔM. extorquens strains were acclimated, grown in 48-well microtiter plates (CoStar-3548) in an incubation tower (Liconic USA LTX44 with custom fabricated cassettes) shaking at 650 rpm, in a room that was constantly maintained at 30°C and 80% humidity 600 for strains grown in 48-well microtiter plates was measured using an automated, robotic culturing and monitoring system 600) measurements. The dynamics and specific growth rate of cultures were calculated from the log-linear growth phase using an open source, custom-designed growth analysis software called CurveFitter available at http://www.evolvedmicrobe.com/CurveFitter/ using the allelic exchange vector pCM433 NotI cut site in pCM433. The reverse primer for the upstream flank was designed to have a 30 bp sequence at the 5′ end homologous to the first 30 bp of the downstream flank. The reverse primer for the downstream flank was designed to have a 30 bp long sequence at the 5′ end homologous to the sequence downstream of the NotI cut site in pCM433. The PCR products representing the upstream and downstream flank were ligated on the pCM433 vector cut with NotI using the Gibson assembly protocol described elsewhere fae, ftfL, glyA, mptG, the mxa operon, fhcBACD, mch, and hprA in pCM433 resulted in pDN50, pDN56, pDN66, pDN68, pDN94, pDN108, pDN109, and pDN125, respectively  containing the donor construct, an E. coli strain containing the conjugative plasmid pRK2073  or hprA , Table 1ectively . Mutant rrn operons, and their GC contents are quite similar (68.2% versus 68.5%). Of the identified 5333 coding sequences in the PA1 genome, 4260 are shared with AM1 (amino acid identity >30%). Of the 90 genes known to be involved in methylotrophy, 62 have >99% identity and the remaining 28 have at least 95% identity at the amino acid level between AM1 and PA1  is missing in PA1. The mau cluster in AM1 and M. extorquens CM4 is flanked by IS elements, and is also missing in another sequenced strain, M. extorquens DM4. These data further support the hypothesis that the mau cluster was acquired by horizontal gene transfer As a first step to compare methylotrophy in PA1 and AM1, we considered the content, similarity and organization of genes in each genome. Apart from 100% identity at the16S rRNA locus  and PA1 . This rene cycle . The arrne cycle . There i1- or multi-C substrates. In order to rigorously compare the growth capabilities of the two strains, we took advantage of the recent development of an automated, robotic platform for high-throughput, quantitative measurements of M. extorquens growth 600 ratio was observed between PA1 and AM1  of our growth measurement platform. At a genomic level, such a striking difference in ethanol growth rates might be due to specific genes downstream of primary oxidation, such as an aldehyde dehydrogeanse (Mext_1295), present in PA1 but absent in AM1. On multi-C organic acids, PA1 grew faster than AM1 by 5–25% with 12–25% higher yield.With a few notable exceptions, PA1 grew faster, with significantly higher yields, than AM1 on Cbstrates . PA1 grebstrates ; we were1 substrates: methylamine and betaine . A small but significant increase in OD600  key genes involved each methylotrophy-specific module and examined the resulting growth phenotypes on C1, multi-C, and a combination of C1 and multi-C substrates  for each of these substrates exist in the PA1 genome.There are 15 methanol oxidation genes in AM1, as well as in PA1. In AM1, 14 of these genes are co-transcribed methanol  demonstr as well –S8. This4MPT) dependent pathway is the sole route for the oxidation of formaldehyde to formate in AM1 4MPT dependent pathway is required for formaldehyde oxidation in PA1, we individually deleted two key genes of this pathway: mptG  and the Δfae mutant grew 7% slower (p<0.01) than WT. These results are consistent with, and build upon, previous work in AM1 that qualitatively demonstrated that the ΔmptG mutant has a growth defect on succinate Genetic and biochemical analyses have determined that the tetrahydromethanopterin  ftfL mutant in PA1 could not grow on methanol or formate, most likely because of a lesion in the first dedicated step toward assimilation of C1 compounds ftfL PA1 mutant, unlike the ΔftfL AM1 mutant In order to determine the role of the H1 substrates is converted to various components of biomass through the serine cycle in AM1 1 assimilation in PA1, we deleted glyA (serine hydroxymethyltransferase), and hprA (hydroxypyruvate reductase)  in growth rate on succinate compared to WT   [15]. Asbstrates . While ted to WT –S8. Thes1- and multi-C metabolism in PA1 to that established for AM1. In addition, however, we also uncovered a number of unexpected growth phenotypes, especially on the combination of C1- and multi-C substrates. In contrast to a previous study The major goal of this study was to compare the metabolic network involved in C1 and multi-C substrates. Since the Δmxa mutant is incapable of methanol growth, we anticipated that its growth rate on a combination of methanol and succinate would be the same as that on succinate, but with 50% lower yield since the combination contains half the concentration of succinate and methanol. While the observed yield on the combination matched the expected value, the Δmxa mutant grew 11% faster (p<0.001) on the combination than on succinate alone  might be able to grow faster on a combination of methanol and succinate because these mutants are capable of completely oxidizing methanol to generate reducing power. Furthermore, the ability to generate additional reducing power from methanol might also result in a proportional increase in the yield. Although yields on the combination of methanol and succinate were significantly elevated for the ΔglyA and ΔhprA strains, growth rate was compromised relative to succinate growth  C1 intermediates and/or a regulatory mismatch between C1 dissimilation and succinate assimilation. Future work will be required to understand the physiological basis for these effects, and to determine whether they hold for other strains of Methylobacterium.Based on the growth characteristics of mutants in methylotrophy specific modules, we hypothesized likely phenotypes during growth on the combination of Cte alone . One posSince extremely closely-related strains sometimes occupy distinct niches 1 growth are the same as described in AM1. In addition, owing to the quantitative nature of our analyses, several knockout mutants also revealed unexpected phenotypes on multi-C compounds as well as a combination of C1 and multi-C compounds. These phenotypes point towards yet-undiscovered aspects of metabolism in these facultative methylotrophs in terms of regulation that allows cells to switch between C1 metabolism and multi-C growth, as well as to establish balanced growth on multiple substrates simultaneously.Our genetic analysis of methylotrophy in PA1 establishes that the roles of the various methylotrophy specific modules during CFigure S1A line plot of strand conservation (in purple) and strand inversion (in blue) between the chromosome of PA1 and the main chromosome of AM1 (bottom).(PDF)Click here for additional data file.Figure S2Growth of three biological replicates of the Δcel ‘wild-type’ strain of PA1 (gray) and the Δcel ‘wild-type’ strain of AM1 (black) in nutrient broth. The inset shows the semi-log plot of the growth curves to emphasize the deceleration in growth.(PDF)Click here for additional data file.Figure S3Growth curves of three replicates of the Δcel strain of PA1 (WT) (in green), the Δmxa mutant of WT (in red), and the ΔglyA mutant of WT (in blue) on a combination of 7.5 mM methanol and 1.75 mM succinate as seen in the open-source growth curve fitter software, Curve Fitter.(PDF)Click here for additional data file.Table S1A list of methylotrophy-specific genes shared between M. extorquens strains AM1 and PA1. Green: genes involved in methanol oxidation, pink: genes involved in the H4MPT dependent formaldehyde oxidation pathway; purple: genes involved in the H4F-dependent formate reduction pathway; orange: genes encoding each of the four formate dehydrogenases; and gray: C1 assimilation genes.(PDF)Click here for additional data file.Table S2Mean FSC (Forward Scatter) and SSC (Side Scatter) of 50,000 cells of M. extorquens AM1 and PA1 (grown in 3.5 mM succinate) using a flow cytometer. FSC is an estimate for the relative size of the cell and SSC is an estimate for the granularity or the biomass/OD600 ratio of the cell. Values are reported as mean±95% confidence interval of the mean for three independent flow cytometer runs with 50,000 cells each.(PDF)Click here for additional data file.Table S3−1) and the standard error of the mean growth rates on C1 substrates M (15 mM methanol), MA (15 mM methylamine), F (15 mM formate) for AM1 and PA1 (both lacking the Mean growth rates (in hcel locus), as well as the mutants strains of Δcel PA1.(PDF)Click here for additional data file.Table S4−1) and the standard srror of the mean growth rates on multi-C substrates S (3.5 mM succinate), P (5 mM pyruvate), E (7.5 mM ethanol) for AM1 and PA1 (both lacking the Mean growth rates (in hcel locus), as well as the mutants strains of Δcel PA1.(PDF)Click here for additional data file.Table S5−1) and the standard error of the mean growth rates on a joint C1 and multi-C substrate B (15 mM betaine), or a combination of C1 and multi-C substrates ½M+½S (7.5 mM methanol and 1.75 mM succinate) for AM1 and PA1 (both lacking the Mean growth rates (in hcel locus), as well as the mutants strains of Δcel PA1.(PDF)Click here for additional data file.Table S6600 and the standard error of the max OD600 on C1 substrates M (15 mM methanol), MA (15 mM methylamine), F (15 mM formate) for AM1 and PA1 (both lacking the Max ODcel locus), as well as the mutants strains of Δcel PA1.(PDF)Click here for additional data file.Table S7600 and the standard error of the max OD600 on multi-C substrates S (3.5 mM succinate), P (5 mM pyruvate), E (7.5 mM ethanol) for AM1 and PA1 (both lacking the Mean max ODcel locus), as well as the mutants strains of Δcel PA1.(PDF)Click here for additional data file.Table S8600 and the standard error of the max OD600 on a joint C1 and multi-C substrate B (15 mM betaine), or a combination of C1 and multi-C substrates ½M+½S (7.5 mM methanol and 1.75 mM succinate) for AM1 and PA1 (both lacking the Mean max ODcel locus), as well as the mutants strains of Δcel PA1.(PDF)Click here for additional data file.Table S9List of primers used for deleting methylotrophy-specific genes in M. extorquens PA1.(PDF)Click here for additional data file.


The ability of recombinant antibodies to adequately penetrate into tumours is a key factor in achieving therapeutic effect; however, the behaviour of antibodies at a cellular level in tumours is poorly understood. The purpose of this study was to investigate those factors that influence the macroscopic and microscopic intratumoural distribution of an IgG1-humanized antibody, huA33, in colorectal tumours.Twelve patients were infused with radiolabelled huA33 at 7 days prior to elective surgery for colorectal carcinoma. Macroscopic huA33 uptake was determined by both gamma well counter and autoradiography measurements of the resected tumour specimens. Microscopic uptake was then quantitated at a cellular level and compared to vascular penetrance. The impact of variation in tumour antigen (GPA33) expression, tumour size, specimen type (primary vs metastatic), presence of macroscopic necrosis, and tumour vasculature on huA33 uptake were examined.−3% injected dose per gram (ID/g). GPA33 was expressed in all viable tumour cells, and huA33 uptake was excellent regardless of tumour size and specimen type. In tumours with macroscopically evident central necrosis (n = 5), huA33 uptake in tumour necrotic centres was lower than in viable peripheries . However, when corrected for low cell viability in necrotic centres, uptake of huA33 at the cellular level was highly comparable to that in the more viable tumour periphery . In the five patients who exhibited macroscopic necrosis in their tumours, huA33 showed excellent tissue penetration, with a maximum penetration distance of 26 μm in peripheral tumour regions and 118 μm in central regions. No correlation was observed between 131I-huA33 uptake in tumour on a cellular basis and tumour vascularity.The I-huA33 uptake in whole tumour sections was (mean ± SD) 5.13 ± 2.71 × 10In patients with colorectal carcinoma, monoclonal antibody huA33 effectively targets viable tumour cells in all cellular milieus examined, including effective penetration into necrotic tumour centres, a novel and therapeutically important finding. There is increasing interest in the use of humanized monoclonal antibodies to treat epithelial cancers. This technique is attractive owing to limited chemotherapy options for many such cancers and the relatively low toxicity of using tumour-specific radiolabeled monoclonal antibodies. Many unlabeled monoclonal antibodies (mAbs) do not demonstrate clinically significant anti-cancer activity, despite induction of complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Hence, attempts have been made to utilize the targeting ability of mAbs to deliver radioactive isotopes or other cytotoxic molecules to individual cancer cells ,2.In order for antibody therapy approaches to be effective, it is necessary for the antibody to adequately penetrate within tumours after intravenous administration. Previous studies have found variable uptake in epithelial tumours depending on tumour size, histological type, vascularity, degree of necrosis, antigen expression, antibody size and affinity, antibody internalization, and other factors that are less well understood -10. SuboOne of the most promising targets in colorectal cancer is the A33 antigen (hereafter referred to as GPA33): a transmembrane glycoprotein of the immunoglobulin superfamily with a molecular weight of 43 kDa -14. GPA3131I and 124I have shown selective and rapid localization of huA33 to colorectal carcinoma on imaging [in vivo, with particular focus on the quantitative cellular uptake and vascular penetrance of huA33 in viable and necrotic tumours.HuA33 is a humanized monoclonal antibody (mAb) directed against GPA33 and has been studied in phase I trials in patients with colorectal cancer -25. Biod imaging ,22,23,26 imaging ,22,23. A9/L, platelets > 100 × 1012/L, prothrombin time <1.3× control. All patients gave written informed consent to participate in this study. The protocol was approved by the Human Research Ethics Committee of the Austin Hospital, Melbourne, Australia and the Protocol Review Committee, Ludwig Cancer Research, New York, USA.Patient eligibility and treatment has previously been described . In brie131I− and 37 to 74 MBq (1 to 2 mCi) of 125I− conjugated to huA33 (IgG1) over a 30-min period, at antibody dose levels of 0.25, 1, 5, and 10 mg/m2. 125I− and 131I− were used to assist with both imaging and autoradiography studies. Seven days later, surgery was performed and tumour samples were obtained for histology and autoradiography. Surgery comprised complete tumour resection (of either the primary tumour or metastases) or biopsy of accessible tissue for patients having surgery only for placement of intrahepatic artery catheter.Eligible patients received a single intravenous infusion of 296 to 370 MBq (8 to 10 mCi ) of 131I-huA33 and on at least three further occasions up to day 7 following infusion. Single photon emission computed tomography (SPECT) images of a region of the body with known tumour were also obtained on at least one occasion during this period. All gamma camera images were acquired on a dual-headed gamma camera . Whole body images were performed as sweeps in a 1,024 × 256 bit matrix, and a standard of known 131I activity was included in the field of view to allow dosimetry calculations. In selected patients, resected tumours were also imaged on a gamma camera to aid in determination of regional localisation of 131I-huA33.All patients underwent gamma camera imaging on day 0 after infusion of 131I-huA33 uptake in whole tumour was assessed using gamma well counter measurements  in all 12 patients. Each tumour was divided into subsections which were weighed individually and counted in a gamma counter (set to the 131I window) together with the appropriate counting standards. Results were expressed as percent injected dose per gram (%ID/g) tissue.The magnitude of 131I-huA33 uptake was confirmed by whole tumour autoradiography in four patients whose tumours were suitable for evaluation using this technique ; two other patients (patients 1 and 2) could not be assessed due to technical problems and six patients  had tumours which were too large to allow whole tumour radiography. For autoradiography, the resected tissue was weighed and bisected with a portion fixed in 10% neutral buffered formalin, embedded in paraffin, and stored at room temperature. Tissue sections were cut at a thickness of 5 μm and mounted onto SuperFrost® Plus slides . Paraffin sections were exposed to x-ray film  for 4 to 21 days. X-ray film was then developed in D19 developer  and fixed in Ilford Rapidfix . A set of radioactivity standards was prepared by applying known amounts of 125I/131I to discs of tissue sections 3 mm in diameter and 5-μm thick mounted onto SuperFrost® Plus slides. These standards were exposed simultaneously with the radioactive biopsy sections to allow quantitation of antigen density by computerized densitometry using an image analysis system . The grey value of the pixels on x-ray film was converted to %ID/g for quantitation of antibody uptake. High-power images of haematoxylin and eosin (H&E) sections were captured using a microscope and attached digital camera .The magnitude of The GPA33 expression on cells was probed using murine anti-A33 antibody  in fresh frozen sections. The concentrations used were optimized for GPA33 detection  and bound antibody detected by application of a biotinylated sheep-anti-mouse IgG  for 30 min followed by streptavidin peroxidase complex . Negative controls for both antibodies were either secondary antibody alone or isotype control antibodies.2O2 for 10 min to eliminate endogenous peroxidase activity, used 0.1% avidin and 0.01% biotin for 10 min to block endogenous biotin and avidin activity respectively, and treated with protein blocking agent  at a 1:1 dilution for 10 min to reduce non-specific binding. JC70A was used at 3.5 μg/ml for 18 h at room temperature, and bound antibody was detected using the LSAB®2 Peroxidase System . Staining was then visualized using chromogen 3-amino-9-ethylcarbazole  for 20 min and counterstained with Mayer’s haematoxylin. Digital images of the sections were captured using an Aperio Scanscope  and the images were viewed using the Aperio ImageScope software . Using these images, microvessel density (MVD) was determined by manually counting CD31-positive vessels in five ×0.118 mm2 randomly selected fields at ×200 magnification and results expressed as mean number of vessels per square millimetres. Then, intercapillary distance (ICD) was determined using the Aperio ImageScope ruler tool and by measuring the distance between a randomly selected index vessel and the four nearest vessels in each quadrant of a randomly selected field, with results expressed as the mean of these distances.CD31-positive blood vessels were determined by staining for CD31 expression  in paraffin sections which were deparaffinized and rehydrated by microwaving in a citrate buffer (pH 6.0) for 10 min. Sections were then treated with 3% HA detailed analysis was undertaken in patients  whose tumours contained macroscopic areas of necrosis by comparing five representative fields in viable peripheral tumour tissue with five fields in central necrotic tissue. Mean percent injected dose per cell was determined by dividing the huA33 uptake (%ID/g), calculated using the autoradiographic images, by the total number of cells in each field, determined by manual cell counting in digitized high-power H&E images. Subsequently, images of sections stained for CD31-positive blood vessels were superimposed on their autoradiographic images and five representative sections from the viable peripheral tissues were compared with a similar number of sections from the necrotic central tissue. The mean antibody penetration was determined using Aperio ImageScope Ruler Tool and measuring the distance between five random areas of huA33 uptake as detected by autoradiography and the nearest CD31-positive blood vessel as determined by the superimposed image.U test. All statistical tests were conducted using a two-sided alpha level of 0.05.All statistical analyses were performed with the statistical software IBM® SPSS® Statistics version 18.0 . All groups were verified for normal distribution before statistic calculations. All comparisons across groups were determined using a non-parametric Mann-Whitney 131I-huA33 was sensitive and specific for tumour . As a subset of patients exhibited macroscopic necrosis in their tumours, these results were stratified by the presence of tumour necrosis , whereas patients whose tumours had necrotic centres showed a trend towards less uptake in the tumour centres than the tumour periphery, though this was not significantly different . However, the number of viable tumour cells was also found to be significantly lower in central necrotic areas compared to peripheral viable areas (Table p < 0.001). Given the significantly different numbers of viable cells in necrotic versus viable regions, huA33 uptake was corrected for the differing cell count by calculating the percent injected dose per cell (%ID/cell). Interestingly, cellular uptake of huA33 in tumour central necrotic areas was not significantly different from that in peripheral viable areas .Table 2, p < 0.001). Interestingly, we observed that MVD in stromal tissue was approximately threefold higher than in adjacent peripheral viable tumour. The mean ICD was also significantly wider in the necrotic tumour than areas of viable tumour, being 207.4 ± 121.7 μm and 69.4 ± 34.5 μm, respectively (p < 0.001).We next examined whether there were differences in MVD between these tumours Table . There wp < 0.001). The greatest absolute distance the antibody penetrated was 118 μm in the centre of necrotic tumours and 26 μm in the periphery.Given that that huA33 uptake was preserved despite the relative hypovascularity of the central necrotic regions, we examined whether antibody penetration was different in central necrotic versus peripheral viable tumour regions. We found that huA33 was able to penetrate further in necrotic areas rather than viable areas , suggesting that intratumoural interstitial pressure may reduce antibody penetration. However, detailed histologic and IHC analysis demonstrated that necrotic tumour centres had significantly lower number of viable tumour cells compared to viable peripheral regions and when corrected for the number of viable tumour cells, uptake of huA33 in necrotic areas was comparable to that in peripheral viable tumour areas . Whilst most published studies have shown reduced antibody uptake in necrotic areas, Boxer et al. found that, in some cases, patients infused with antibody to CEA had increased concentrations of antibody in necrotic more than viable areas of tumour [Initial histologic and quantitative autoradiography analysis of huA33 showed that uptake in necrotic areas was moderate  and the mAb with a moderate monovalent affinity penetrated to an average distance of 59.7 μm. It is therefore likely that the low internalization rate of huA33 [Interestingly, the ability of huA33 to penetrate into the necrotic centres of large tumours and localize to viable tumour cells was not reduced despite the demonstrably poorer perfusion of these necrotic central areas (long ICD and low MVD) compared to the periphery (short ICD and high MVD). Other studies have not found consistent relationships between degree of vascularity and antibody uptake in tumours ,8,9,27. argeting -32. Simi124I-huA33 uptake and antigen concentration in tumour, with estimated binding site occupancy for both tumour and normal colon of 20% to 50% [in vivo[131I-huA33 uptake. This is highly relevant to the design of future studies investigating the therapeutic potential of huA33, particularly for payload delivery strategies. One obvious implication of our results is that patients with large or necrotic tumours should not be excluded from potential treatment with huA33 because of concerns of poor tumour cell targeting. Another is that combination therapy with antivascular agents will also require careful design given their potential effects on antibody penetration, as also shown in other studies [Other intratumoural factors may contribute to the long penetration distance of huA33, such as an enhanced permeability and retention (EPR) effect which is known to lead to a prolonged retention of drugs into tumour interstitium ,35. Whil% to 50% . Interes% to 50% . It is i [in vivo. Previou [in vivo,16. Over studies ,41 whereThe limit of our study is the small patient numbers, so further studies are needed to confirm the present findings.In summary, targeting GPA33 with huA33 presents a very attractive option given the universal and uniform expression of this antigen in colorectal cancers, and, within the limit of the study, the data presented here shows excellent uptake in primary and metastatic tumours of varying size and locations. Tumour cellular uptake is very high, even in the necrotic centres of tumours where tumour cell numbers and microvessel densities are very low, an unusual and therapeutically important finding for an intact IgG. These findings provide an important mechanistic support for ongoing clinical trials of huA33 in patients with metastatic colorectal carcinoma and provide data to rationally design them.The authors declare that they have no competing interests.All authors have met the criteria for authorship. MC and GC collected, analyzed, and interpreted the data, and wrote the paper. HG interpreted the data and revised the manuscript critically. CM and FL analyzed the data and revised the manuscript. AC collected and analyzed data. DM collected and interpreted data. TJ collected and interpreted data. JH analyzed data and contributed to the manuscript writing. ME analyzed data and contributed to the manuscript writing. AB analyzed data and contributed to the manuscript writing. AS designed the study, interpreted the data, and revised the manuscript critically. All authors read and approved the final manuscript.


Most microfluidic chips utilize off-chip hardware  to control fluid flow on-chip. This expensive, bulky, and power-consuming hardware severely limits the utility of microfluidic instruments in resource-limited or point-of-care contexts, where the cost, size, and power consumption of the instrument must be limited. In this work, we present a technique for on-chip fluid control that requires no off-chip hardware. We accomplish this by using inert compounds to change the density of one fluid in the chip. If one fluid is made 2% more dense than a second fluid, when the fluids flow together under laminar flow the interface between the fluids quickly reorients to be orthogonal to Earth’s gravitational force. If the channel containing the fluids then splits into two channels, the amount of each fluid flowing into each channel is precisely determined by the angle of the channels relative to gravity. Thus, any fluid can be routed in any direction and mixed in any desired ratio on-chip simply by holding the chip at a certain angle. This approach allows for sophisticated control of on-chip fluids with no off-chip control hardware, significantly reducing the cost of microfluidic instruments in point-of-care or resource-limited settings. Consequently, many microfluidic instruments remain unsuitable for use in point-of-care or resource-limited settings, where instrument cost, power consumption, and size must be minimized.The advantages of microfluidics over conventional lab-scale techniques—reduced reagent consumption, faster reactions, smaller instrument size, enhanced automation, higher throughput, and so on—have enabled applications for microfluidic instruments in fields as diverse as health care, environmental monitoring, and space exploration. The ability to control fluid flow on a microfluidic chip is a fundamental need in these instruments. However, most existing techniques for controlling on-chip fluid flow rely on off-chip hardware. For example, to control the mixing ratio of two fluids in a microfluidic chip, two off-chip pumps (pressure regulators or syringe pumps) are typically required. Valves and pumps can be moved on-chip , 2, but no off chip hardware. In this method, by simply orientating a microfluidic chip at a certain angle, on-chip fluids can be routed and mixed in any desired ratios. We accomplish this by adding inert compounds that slightly increase the density of certain fluids in the chip. When two fluids of different densities flow together under laminar flow on-chip, the interface between the two fluids quickly reorients itself to be orthogonal to Earth’s gravitational force. If the channel containing the fluids then splits into two channels, the amount of each fluid flowing into each channel is a function of the angle of the channels relative to gravity. In this manner, different amounts of different fluids can be routed on-chip simply by changing the orientation of the microfluidic chip as shown in Here we describe a method for precisely controlling fluid flow inside a microfluidic chip that requires any desired concentration or mixing ratio without redesigning the chip. Additionally, mixers containing microfluidic valves and pumps still rely on off-chip hardware for controlling these valves and pumps. Finally, existing valve- and pump-based or split-and-combine mixers consume a relatively large amount of fluid to generate a relatively small amount of the desired mixture. In summary, there is an unmet need for simple, equipment-free methods for generating arbitrary mixtures of fluids in microfluidic chips.As a proof-of-concept demonstration, we used the principle of orientation-based microfluidic control in a simple mixer chip that is capable of generating any desired mixing ratio of two fluids. We chose to create a mixer because of the importance of mixing in microfluidic devices. Microfluidic mixers automate the time-consuming process of preparing arbitrary concentrations and mixtures of solutions by hand. Most existing microfluidic mixers utilize either microfluidic valves and pumps , 2 or aru is the velocity vector, μ is the fluid viscosity, P is the pressure applied to the upstream end of the fluid, ρ is the density of the fluid, and g is the gravitational acceleration of objects on Earth (9.8 m s−2). The solute concentration follows the equation of conservation of mass and Fick’s Law of Diffusion:D is the diffusivity of the solute and c is the concentration of the solute. Eqs ρ is the density of the solution, ρwater is the density of pure water (1.00 g/mL), c is the solute concentration, and B is an experimentally-obtained, solute-specific constant that correlates a solution’s density with its solute concentration . Eqs To explore the effects of chip orientation on fluid flow inside a microfluidic chip, we used finite element analysis software to simulate the behavior of a simple microfluidic chip held at different angles relative to gravity. Our model combines the Navier-Stokes equations, Fick’s law of diffusion, and a function that describes the density of a solution as a function of the concentration of a solute . COMSOL ∇·u=0ρu·∇u=-∇c=∇·(D∇c)where D ∇·u=0ρu·∇u=-∇ter(1+Bc)where ρ ρ = 1.00 g/mL, and Inlet 2 contains a slightly-more-dense blue fluid (ρ = 1.07 g/mL). In θ between Inlet 2 and gravity is 0°). In this orientation, the force of gravity keeps the more-dense blue fluid flowing along the bottom of the channel and the less-dense yellow fluid flowing along the top of the channel. Consequently, when the channel splits into two outlets, the fluids leave in the same directions they came from: the more-dense blue fluid exits through Outlet 2 in the direction of gravity, and the less-dense yellow fluid exits through Outlet 1.The H-shaped microfluidic channel network simulated in opposite direction they came from: the more-dense blue fluid entered from Inlet 2 but exits through Outlet 1, and the less-dense yellow fluid entered from Inlet 1 but exits through Outlet 2. In this manner two different fluids can be routed to two different destinations on-chip by orienting the chip at either 0° relative to gravity  and the exit channels each contain the same mixture (50% yellow and 50% blue).Finally, in θ. If the less-dense fluid at Inlet 1 contains a solute A with a concentration [A0], and the more-dense fluid at Inlet 2 contains a solute B with concentration [B0], the concentrations [A] and [B] in each of the outlet channels are:The results in θ. These equations only apply to a microfluidic chip with circular cross-section channels, although similar equations may be derived for other channel shapes.Using the above equations, we can predict the concentrations [A] and [B] flowing in a chip held at any desired angle To demonstrate the principle of orientation-based control of fluid flow in a microfluidic chip, we fabricated microfluidic chips similar to the one simulated in ρ = 1.00 g/mL, and Inlet 2 contains more-dense sucrose solution, ρ = 1.07 g/mL. Sucrose solutions of precisely known densities were prepared using our software NaCl.py (available for download at http://groverlab.org). FD&C Blue #1 and Yellow #6 were used specifically for visual characterizations. The inlets were connected by tubing to fluid reservoirs that were held 3 cm above the chip. Since the inlet reservoirs were higher than the outlet reservoirs, a head pressure P = ρgh developed that pumped fluid flow from the input reservoirs through the chip and to the output reservoirs (g = 9.8 m s−2 and h = height difference between input and output reservoirs). The inlet and outlet reservoirs were maintained at the same height regardless of the tilt of the chip. Consequently, the orientation of the chip does not affect the flow rate. No off-chip fluid control hardware like pumps or valves were used.Pe, is used to determine whether convection or diffusion is dominant in a system:L is the characteristic length, u is the flow velocity, and D is the solute’s diffusion coefficient. The calculated Peclet number for our flow rate  is much greater than one, indicating that convection is dominant and diffusional mixing between the different fluid streams is negligible.The phenomenon of orientation-based control depends upon convection, not diffusion, being dominant in the microfluidic system. A dimensionless parameter called the Peclet number, Chips of different channel geometries were held at different angles. The average residence time of fluid in the chip was calculated to be 0.7 seconds. To make sure that the system reaches steady state, samples were collected from the chip after 1 minute of flow. Samples of 5 mL were collected from each outlet for each orientation and chip geometry. To quantify the resulting mixing ratios, fluids from both outlets were collected and analyzed using an UV-Vis-NIR spectrophotometer .θ = 0°), the more-dense blue fluid remains on the bottom of the horizontal channel and the less-dense yellow fluid remains on the top of the channel. Consequently, the fluids exit the mixer chip in the same directions from which they entered (yellow fluid at Outlet 1 and blue fluid at Outlet 2). This case is identical to the simulation shown in θ = 90°), the two fluids reorient relative to gravity (rotating 90° clockwise) to place the more-dense blue fluid at the bottom of the horizontal channel and the less-dense yellow fluid on the top of the channel. When the horizontal channel splits, both output channels have the same contents . This situation is identical to the simulation shown in other edge in θ = 180°), the yellow and blue fluids swap places in the horizontal channel to place the more-dense blue fluid on the bottom and the less-dense yellow fluid on the top. Consequently, the fluids leave the Outlet channels opposite from where they entered, with yellow fluid at Outlet 2 and blue fluid at Outlet 1.When the chip is oriented on its edge relative to gravity in θ is varied from 0° to 180°, the concentration of blue fluid rises and yellow fluid drops in Outlet 1, and an opposite trend is observed in Outlet 2. The relationship of concentration on angle of rotation is roughly linear as predicted by μm. This could result in a microfluidic channel that is not perfectly circular and requires a more complex model than the one shown in θ to within a few percentage points.To demonstrate orientation-based control of fluid flow over a wide range of angles . We also examined the behavior of two fluids of different densities flowing in rectangular channels with much higher aspect ratios . The experimental results further support the assertion that orientation-based control of microfluidics is most practical in channels with cross-sectional aspect ratios near one (circular and square channels).To explore the role of channel cross-section shape in orientation-controlled microfluidics, we designed and 3D printed mixer chips with square (1 × 1 mm) and rectangular (1 × 1.25 mm) cross-section channels. We then repeated the experiments from In addition to channel geometry, fluid density has an effect on our phenomenon. The speed at which rotation occurs is influenced by the density difference. However, in practice we found that all rotations we observed were nearly instantaneous. The channel length was kept excessively long for characterization purposes. However, for a channel diameter of 1 mm, a minimum length of 10 mm is required for the rotation to occur. The larger the difference between fluid densities, the quicker the rotation occurs. Rotation speed could be increased by further increasing the difference between fluid densities; however, the resulting increase in concentration gradients will result in an increase in diffusive flux, potentially allowing for more mixing between the two layers.To determine how much of a difference in density between the two fluids is necessary to employ orientation-based control, sucrose solutions with different concentrations (and thus different densities) were prepared as shown in We demonstrated that the orientation of a microfluidic chip can be used to precisely control the flow of fluids inside the chip. By using the orientation of a chip to control fluid flow instead of on-chip valves or off-chip pumps and regulators, our technique can eliminate a substantial portion of the cost, size, and power-consumption of a microfluidic assay or instrument. Thus, this technique should facilitate the spread of microfluidic technologies to new applications in resource-limited and point-of-care settings.does need to be controlled, a number of compounds have been developed that increase a fluid’s density without affecting its osmotic strength. These include Ficoll, a high molecular weight polysaccharide used in density gradient centrifugation [Orientation-based control of microfluidics does depend upon different fluids in the chip having different densities. However, the amount of density difference necessary to use orientation-based control is very small (only ∼2%), and there are many different substances that can be added to a fluid to adjust its density. For applications that do not require precise control of the osmotic strength of fluids, small amounts of solutes like sucrose (as used here) and sodium chloride can be easily and inexpensively added to a fluid to enable orientation-based control. For applications in which the osmotic strength of the fluid fugation ; Percollfugation ; Visipaqfugation  and metaet al. [A density difference of ∼2% is required for fluid reorientation to occur. Increasing the density of a solution by ∼2% may change the viscosity of the solution. For example, increasing the density of a sucrose solution by 2% increases the effective viscosity of the solution by 17%. However, this viscosity difference is actually too low to affect the concentration of the output fluid. Karst et al.  studied Finally, is orientation-based control powerful enough to control real-world microfluidic chips? Certainly chips with hundreds of computer-controlled valves offer a level of fluid control that may be unattainable by orientation-based control (though this level of control comes with a significant cost). However, many real-world microfluidic devices require simpler fluid control and could be controlled using chip orientation. For example, the proof-of-concept mixer chip shown here could be used in a drug toxicity screening assay by exposing cells downstream of the mixer to various concentrations of a drug . This co