This prospective randomised clinical trial evaluated the effect of warmed irrigation fluid on body temperature in anaesthetised dogs undergoing arthroscopic elbow surgery. Nineteen dogs undergoing elbow arthroscopy were included in the study and were randomly allocated to one of two groups. Group RT received irrigation fluid at room temperature (RT) while dogs in group W received warmed (W) irrigation fluid (36°C). A standardised patient management and anaesthetic protocol was used and body temperature was measured at four time points; (T1) pre-anaesthetic examination, (T2) arrival into theatre, (T3) end of surgery and (T4) arrival into recovery. There was no significant difference in body temperature at any time point between the groups. The mean overall decrease in body temperature between pre-anaesthetic examination (T1) and return to the recovery suite (T4) was significant in both groups, with a fall of 1.06±0.58°C (p<0.001) in group RT and 1.53±0.76°C (p<0.001) group W. There was no significant difference between the groups. At the end of surgery (T3) 4/19 (21.1%) of dogs were hypothermic (<37°C). The addition of warmed irrigation fluids to a temperature management protocol in dogs undergoing elbow arthroscopy during general anaesthesia did not lead to decreased temperature losses. In human anaesthesia hypothermia is also a common problem , and even mild hypothermia [34.0-36.5°C ] is associated with increased rates of wound infection , adverse cardiac events  and mortality in man .Hypothermia during and after anaesthesia is common in dogs, with up to 83.5% of dogs developing hypothermia in a recent large study . However, the link between hypothermia and delayed recovery from anaesthesia is well established .There is limited evidence to document similar adverse events associated with hypothermia in dogs and a retrospective veterinary study showed no increase in wound infection rates in cats and dogs with mild perioperative hypothermia . In both dogs and humans, warming fluids used for irrigation of body cavities has generally been associated with a protective effect against peri-operative hypothermia  and this practice has been incorporated into UK national guidelines on peri-operative temperature management in man as a simple, low cost intervention .Increasing emphasis is being placed on prevention of peri-operative hypothermia in both man  and dog . However, Kelly et al. (2000) found no significant difference in mean percentage temperature decrease between patient groups treated with warmed and room temperature irrigation fluids undergoing knee arthroscopy. The use of warmed irrigation fluids for arthroscopy has not been previously investigated in dogs. It was hypothesised that the use of pre-warmed arthroscopic irrigation fluid would result in less heat loss during the surgical period compared to the use of room temperature irrigation fluid.The use of warmed arthroscopic irrigation fluids in human patients undergoing arthroscopic shoulder surgery has been associated with a smaller reduction in core body temperature compared to the use of fluids at room temperature with differences in maximum temperature drop as large as 1.34°C being reported between groups .A clinically significant difference in heat loss between groups was considered to be one degree centigrade and a power analysis based on this suggested that in order to achieve an alpha of 0.05% with power of 85% 8 dogs would be required in each group. In order to allow for potential data loss it was planned to recruit 10 dogs to each group . Due to a change of surgical policy in the hospital the number of arthroscopies performed decreased dramatically during the course of the study and it proved possible to recruit only 19 dogs with 10 in group RT and 9 in group W.-1 intramuscularly)  and buprenorphine (0.02mg kg-1 intramuscularly)  administered approximately 45 minutes prior to induction of anaesthesia. Anaesthesia was induced with alfaxalone  administered intravenously to effect.All dogs received a premedication combination of acepromazine (0.03mg kg-1 subcutaneously)  or an alternate NSAID [ or ] depending on existence of preoperative NSAID regimen and amoxicillin clavulanate (20mg kg-1 intravenously)  were administered after induction of general anaesthesia.Robenocoxib (2mg kg-1 hr-1) .Anaesthesia was maintained with isoflurane  delivered in 100% oxygen via a small animal circle breathing system. Intra-operative intravenous fluid therapy consisted of room temperature Hartmann’s solution   was administered intravenously.If additional fluids were required then boluses of 10ml kg®; Augustine Biomedical and Design, MN, USA) which was set to 37°C throughout the surgical duration.All dogs had a heat and moisture exchanger (HME)  placed between the endotracheal tube and breathing system after endotracheal intubation. Once moved into the operating theatre, dogs were placed on a thermostatically controlled blanket (Hot Dog-1)  or morphine (0.1mg kg-1)  or a combination of both were administered intra-articularly.At the end of surgery bupivacaine hydrochloride  or pre-warmed in a thermostatically controlled heating cabinet (36ºC) for a minimum of 24 hours.A 10ml sample of saline was withdrawn from a 1 litre test bag of saline kept in the incubator to ensure agreement (+/- 0.5°C) with the incubator thermostat prior to the start of each case. A separate 1 litre test bag of saline was stored in the theatre suite from which 10ml samples were withdrawn and used to verify fluid equilibration with room temperature (20-22°C) and also agreement between rectal and oesophageal temperature probes prior to the start of each case.Irrigation fluids were removed either from the heated cabinet or theatre suite fluid store and mounted in the arthroscopy tower immediately prior to the onset of joint irrigation by the attending theatre nurse. The fluid was delivered to the joint via 4.8m tubing at a variable rate. No insulation was applied to the irrigation fluid bags.Under experimental conditions designed to mimic ambient temperature conditions during the study, a 2l bag of pre-warmed irrigation fluid (36.4°C) was mounted in the arthroscopy tower at room temperature (20.5°C) and fluid temperature at the point of outflow from the delivery tubing was measured over 60 minutes.® Anaesthesia Monitor; GE Healthcare, UK) and positioned approximately at the level of the carina.Body temperature at time points T2 and T3 was measured using an oesophageal temperature probe connected to a multi-parameter monitor  was used to measure temperature at T1 and T4 time points. Temperature measurement of a 10ml volume of room temperature saline was used to verify agreement between the oesophageal temperature probe and rectal probe prior to the start of each case. The rectal and oesophageal probes recorded identical temperatures on each occasion.An assigned rectal temperature probe .A one-way ANOVA was used to test for differences between the two groups at each of the four time points and time periods. A one-way ANOVA was also performed to assess temperature change over time within each group. No statistically significant differences existed between the groups in baseline data .There was no statistically significant difference in temperature between the groups at any time point .Within each group, temperature recorded at T2, T3 and T4 differed significantly from T1 .Within each group temperature reduction over time was significant between T1 and T2 . TemperaOverall temperature reduction (T1-T4) was significant in each group representing overall heat loss from the time of pre-anaesthetic examination to transfer into recovery .(p<0.001) was recorded and in group W, a temperature reduction of 1.53±0.76 (p<0.001) was recorded between T1-T4  fluid temperature decreased by 0.6°C after passing through the delivery tubing. After 12 minutes of administration, the fluid temperature at the outflow point was 35.1°C and after 40 minutes the fluid temperature was 32.0°C. After 60mins, temperature of the irrigation fluid at the outflow point was 29.6°C. Mean arthroscopy duration in group W was 43.3±20.62 mins.et al. (2000) in man. However, Board and Srinivasan (2008) and Kim et al. (2009) demonstrated a positive association between the use of warmed irrigation fluid and body temperature during shoulder arthroscopy in man. These studies involved the use of much higher volumes of irrigation fluid over a longer period of surgical time (8.8 litres over 84 minutes and 10.05 litres over 92.8 minutes respectively) compared to the present study where irrigation fluid volume was less than 2 litres and mean surgery time 57.5 minutes. It may be that during a longer surgical period there is more time for heat loss to occur, potentially magnifying the difference between the groups.The results of the current study suggest that the use of pre-warmed irrigation fluid for elbow arthroscopic surgery in dogs does not confer a thermoregulatory advantage when additional heating methods are employed. This is the first such study in dogs, and the results agree with those of Kelly et al. (2000) failed to find a positive association between body temperature and irrigation fluid temperature during knee arthroscopy in man where the inclusion of spinal anaesthesia may have compromised thermoregulatory control  leading to further heat loss in these patients.Kelly et al., 2009). No such attempt was made in our study or the human study which did not detect a positive effect of warmed fluid and so the fluid delivered to the patient cooled as the procedure proceeded . Conditions designed to mimic the current study setting showed that pre-warmed irrigation fluid temperature cooled with time but remained above 31°C for the mean arthroscopy time in group W of 43.3 minutes.A further difference between the reported studies is that in at least one of the human papers which reported a positive effect of fluid temperature, efforts were made to maintain the temperature of the irrigation fluid once removed from the warming cabinet  carried out similar testing and found a fluid temperature reduction of 2°C over their 20 minute typical infusion period, however their pre-warmed saline was heated to 40°C rather than 36°C. This narrowing of the intended fluid temperature difference between the groups may have hindered the detection of a treatment effect and this represents a significant limitation in study design. To improve the current study design, measures should be taken to maintain the pre-warmed fluid temperature using fluid bag insulation or in-line warming devices.Kelly The efficacy of using pre-warmed fluids alone has been questioned due to the heat loss which occurs during administration to the patient.et al., 1998).Intravenous in-line warming devices have been shown to reduce perioperative hypothermia compared to room temperature intravenous fluids in patients also receiving convective heating via a warm air blanket .However, other studies have shown that pre-warmed fluid, administered shortly after removal from a warming cabinet, is as efficient at preventing peri-operative hypothermia as that delivered through an in-line warming system .Warming efficacy of the in-line devices may be variable depending on desired fluid flow  and laparoscopy . Utilisation of such a device would have benefitted the current study by preserving the intended warmed fluid temperature throughout the administration period. The development of peri-operative hypothermia is common and may be attributed to the combination of anaesthetic agent induced thermoregulatory disturbance, exposure of clipped areas of skin to a cool ambient environment  and increased evaporative losses from wounds  and the respiratory tract . Redistributive heat loss is influenced by the core to periphery temperature gradient  which may be increased by agents such as acepromazine and isoflurane which inhibit protective vasoconstrictive responses. The current study demonstrated an initial significant reduction in temperature in the preparatory phase before active warming was instituted; a finding which is consistent with redistribution of body heat and similar to earlier reports .Specifically designed warming devices for irrigation fluid rates have been shown to effectively maintain warmed fluid temperature during arthroscopy  the prevalence of slight post-anaesthetic hypothermia (36.50-38.49°C) in dogs undergoing orthopaedic procedures was 48%. If equivalent temperature boundaries were applied to the current study, 94.7% of dogs were slightly hypothermic in the post anaesthetic period.In a large retrospective study by Redondo One dog in the current study was moderately hypothermic (36.49-34.00°C) while 37% were in this temperature range in the retrospective study.et al. (2012) where the only form of warming was passive insulation provided by blankets.In the current study no dogs were severely hypothermic (<34.00°C) while 3.3% were reported to be so in the study by Redondo et al., 2004; Fanelli et al., 2009; Röder et al., 2011) and the addition of a HME to the breathing circuit, although the value of HMEs has been questioned .The small numbers of dogs with moderate and severe hypothermia in this study may be due to the relatively short procedure length, the use of a thermostatically controlled blanket during surgery  A study with an experimental dog population could be devised to test the effects of warmed arthroscopy fluids alone.The benefit to the patients in employing measures to limit heat loss may have been a limitation of this study, since by limiting heat loss it may be more difficult to detect any effect of warmed irrigation fluids .Variation in breed was not accounted for which may cause coat thickness and subcutaneous fat to act as confounding factors in terms of heat retention. The study design could have been improved by matching dogs according to hair length and breed to eliminate this confounding factor . The ability to maintain and monitor fluid temperature during administration must be incorporated into the study deign if further work is to be carried out.The temperature of arthroscopy irrigation fluid actually delivered to the patient was not measured in this study and this omission must be considered a weakness in light of the evidence from man highlighting the difficulty in maintain the temperature of such fluids .Mean delivered isoflurane concentration was not included in data analysis in this study but may potentially affect thermoregulation by virtue of its vasodilatory properties  and it would have been useful to compare the groups to ensure there was no disparity in delivered isoflurane concentrations. Power calculations were based upon a minimal detectable difference of 1°C in line with data reported in man and sample size in the current study was similar to that used in comparable human studies .The overall significant reduction in body temperature experienced by the dogs in the current study reiterates the requirement for measures to be taken to reduce peri-operative heat loss particularly in the preparatory phase. Whilst in this study there is no detectable benefit to the use of warmed arthroscopy fluids in dogs, the positive results reported in man remain intriguing (Board and Srinivasan, 2008; Kim Future studies should examine the effect of warmed irrigation fluids during longer surgeries and incorporate measures to maintain and monitor the temperature of fluids used for arthroscopy during the surgical procedure.The results of this study suggest that warmed arthroscopic lavage fluids delivered as described in this paper offer no clinically significant advantage in the maintenance of body temperature.The authors declare that they have no conflict of interest.


Neoadjuvant radio(chemo)therapy is increasingly used in rectal cancer and induces a number of morphologic changes that affect prognostication after curative surgery, thereby creating new challenges for surgical pathologists, particularly in evaluating morphologic changes and tumour response to preoperative treatment. Surgical pathologists play an important role in determining the many facets of rectal carcinoma patient care after neoadjuvant treatment. These range from proper handling of macroscopic specimens to accurate microscopic evaluation of pathological features associated with patients' prognosis. This review presents the well-established pathological prognostic indicators and discusses challenging features in order to provide both surgical pathologists and treating physicians with a checklist that is useful in a neoadjuvant setting. Preoperative radiotherapy with or without chemotherapy (RCT) followed by total mesorectal excision (TME) has become a standard treatment for locally advanced rectal cancers –4. The iThe aim of this review is to present the well-established pathological prognostic indicators and discuss challenging features, especially those with clinical impact, in order to provide a checklist that is useful in a neoadjuvant setting, for both surgical pathologists and treating physicians.The TME technique is based on the sharp dissection of the avascular plane between autonomic nerve plexuses and the mesorectum. This operation results in the excision of the rectum enveloped by a mesorectal fat column of 2 to 3 cm. This part of the review presents the methods of assessment of mesorectum quality, circumferential resection margin (CRM), distal resection margin, and lymph nodes.TME specimen handling begins with assessment of the quality of the mesorectum  29]. In. In29]. http://www.kankerregister.org/), which was adapted from Quirke et al. [Quirke et al.  and Nagte et al.  and Nagte et al. , suggeste et al. . When one et al. . ObviousRecommended method for tumour sampling and examination in rectal carcinoma following radio(chemo)therapy and surgery is as follows.Take 5 blocks from the tumour or scarred area (assuming no obvious tumour was found grossly). These should include the superficial and deep part of the tumour as well as the relationship between the tumour and the CRM or peritoneal surface. If there are anymesorectal nodules, blocks containing their relation to the CRM should also be taken.If no viable microscopic tumour is identified within the initial 5 blocks, the entire scarred area should be sampled.If no residual tumour is found after examining sections from the additional blocks, three levels should be cut through each block. If no viable tumour cells are present even after rigorous examination of these sections, complete pathologic response or ypT0 can be reasonably and reliably reported.The distal margin, although less important than the CRM in terms of frequency of involvement and impact on recurrence, is still important to assess. There are two issues to keep in mind when considering the distal margin: the extent of intramural and extramural continuous tumour growth and the discontinuous distal mesorectal spread through lymphatics. In 20% of cases with positive nodes, there is lymphatic spread distal to the primary tumour. Furthermore, in many cases these positive distal nodes are located >2 cm away from the main tumour mass . By contOne final issue to keep in mind, when measuring the distal margin, is shrinkage artifact. Goldstein et al.  have shoLymph node status probably constitutes the single most important prognostic determinant in patients with rectal cancer whether treated preoperatively or not , 41. IntOnce again, it is important to stress that the lateral mesorectal surface closest to the suspect nodes should be sampled together with the nodes to allow accurate evaluation of the CRM . Thus, tIn the pathological reporting of colorectal cancer (CRC), the internationally accepted histological classification of colorectal carcinomas proposed by the World Health Organization  (WHO) isIn summary, based on current evidence, it can be concluded that the only histological types of CRC that are prognostically significant are signet-ring cell and small-cell carcinoma (poor prognosis) and medullary and MSI-related mucinous carcinoma (favorable prognosis).Grade 1: well differentiated (>95% glandular formation),Grade 2: moderately differentiated (50%–95% glandular formation),Grade 3: poorly differentiated (5%–50% glandular formation),Grade 4: undifferentiated (<5% glandular formation).Nevertheless, the most recent World Health Organization series on tumours of the digestive system recommends using the two-tier grading system (low versus high grade) in grading colorectal cancer [In the WHO classification , gradingl cancer . Despitel cancer –77. Aftel cancer , 78–80.The prognostic significance of lymphovascular (LVI) and perineural (PNI) invasion has been suggested and largely confirmed in a nonneoadjuvant setting , 81–84. Following preoperative RCT, the prognostic significance of LVI and PNI has been demonstrated in several studies, mostly by univariate analysis –98. A stTumour deposits (TDs) are discrete adenocarcinoma nodules encountered in the pericolonic and perirectal fat during routine histopathological examination of advanced CRC specimens. Their prevalence in the mesorectum ranges from 6% to 64% –101. TDsThe TNM5 introduced the 3 mm rule, resulting in a classification based exclusively on size, independent of histological features. Accordingly, discontinuous mesorectal tumour cell aggregates were considered as being primary tumour extensions (pT category) if measured ≤3 mm or as lymph node metastasis (pN category) if >3 mm .The TNM6 replaced the size criterion with the shape criterion. Based on this classification, discontinuous mesorectal tumour nodules were considered as venous invasion if they have an irregular contour and as regional lymph node metastasis if they have the shape and smooth contour of a lymph node .vascular-invasion type : pT category,non-vascular-invasion type : pN category,aggressive TDs : pM1a (in-transit metastasis).The TNM7 again introduced changes regarding the definition and classification of TDs. In the last edition, discrete foci of tumour found in the perivisceral fat or in adjacent mesentery away from the leading edge of the tumour and showing no evidence of residual lymph node tissue are considered to be TDs. If TDs are observed in lesions that would otherwise be classified as T1 or T2, then the primary tumour classification is not changed, but the nodule is classified in N1c category [These two classifications have limited value since the TD classifications are based on a single morphologic criterion . Moreover, the 3 mm rule was based on unpublished data, which were subsequently not confirmed , 107, ancategory .It seems that the different editions of TNM replace one subjective definition with another. Moreover, they do not appear to have prospectively tested this new TD classification and evaluated its reproducibility, since TNM7 states that a perivisceral nodule should be recorded as a positive lymph node if the nodule is considered by the pathologist to be a totally replaced lymph node  .In summary, although the existing classifications of TDs need further improvement in terms of reproducibility and prognostic stratification, results from most studies on patients not receiving preoperative RCT indicated a worse prognosis for patients with TDs: increased local recurrence rates, increased development of distant metastases, and decreased survival . In studPathological staging following complete resection has long been considered the most powerful prognostic indicator in CRC . The samFor accurate staging of treated rectal carcinoma, it is important to keep in mind that when microscopic remnants of tumour are not found, the scarred area must be entirely sampled , 120. MoIn routine microscopic examination of neoadjuvantly treated rectal carcinoma specimens, mucus pools can be encountered in up to one-third of cases , 123. WiThe tumour associated inflammatory infiltrate has long been considered a type of host response and an important prognostic factor in rectal cancer , 127. AfResponse to RCT ranges from minimal treatment effects to complete eradication of the primary tumour. Some authors used cellular-response grading which is based on the amount of residual viable tumour in relation to stromal fibrosis , 16, 131p = 0.06). However, in the same study, there was no difference in the rate of local recurrence between patients with a positive CRM due to positive nodes compared to those with a negative CRM. As previously described, TDs are a frequent phenomenon in the mesorectum. Nevertheless, to date, no study has examined the prognostic relevance of CRM measurement based on the distance from lateral resection margin to TDs. Therefore, to allow further investigations on the prognostic significance of these two new CRM measurement methods , it is recommended that, in cases with positive lymph nodes or TDs, practicing pathologists keep a record of the distance from the lateral resection margin to these perirectal tumour nodules in addition to reporting the classic CRM.On microscopic examination, the distance of the tumour to the CRM may be the single most critical factor in predicting local recurrence (LR) after RCT and surgery , 29, 37.Preoperative RCT induces changes in both gross appearance of the surgical specimen and its pathological features, which could have impact on patient management and outcome. First of all, the assessment of the mesorectum is necessary as it is an important indicator of the resection quality. Then, the resected specimen should be sampled and examined properly, as summarized in Steps


Glioblastomas are highly malignant brain tumours. Mathematical models and their analysisprovide a tool to support the understanding of the development of these tumours as well asthe design of more effective treatment strategies. We have previously developed a multiscalemodel of glioblastoma progression that covers processes on the cellular and molecular scale. Here, we present a novel nutrient-dependent multiscale sensitivity analysis of this modelthat helps to identify those reaction parameters of the molecular interaction network that influencethe tumour progression on the cellular scale the most. In particular, those parametersare identified that essentially determine tumour expansion and could be therefore used as potentialtherapy targets. As indicators for the success of a potential therapy target, a decelerationof the tumour expansion and a reduction of the tumour volume are employed. From the results, it can be concluded that no single parameter variation results in a lessaggressive tumour. However, it can be shown that a few combined perturbations of twosystematically selected parameters cause a slow-down of the tumour expansion velocity accompaniedwith a decrease of the tumour volume. Those parameters are primarily linked tothe reactions that involve the microRNA-451 and the thereof regulated protein MO25. The glioblastoma (GB) still can be considered to be the most aggressive primary brain tumour  in human Go or Grow principle [In recent years, the significance of microRNAs and the thereof dependent signalling pathways for cancer has been discovered –8. Microrinciple .Mathematical modelling has evolved as a useful tool for biology and medicine in gaining a better understanding of diseases and biological processes , 14. ConAll mathematical models have in common that they contain parameters . These parameters might vary from patient to patient or even from cell to cell and need to be determined/estimated from experimental or patient data in the model development process . One imp in silico simulation results to in vitro experiments.In , we presIn the work at hand we analyse how the cellular scale of our model depends on the reaction parameters of the molecular interaction network. Motivated by the idea presented in , 40, parIn the following, we will first briefly recapitulate the major aspects of our multiscale model, before we introduce the method for the multiscale sensitivity analysis. Finally, we will present the results of the analysis and draw some conclusions for future research.To model the progression of GB in a region of a few square millimetres (to be exact 3 mm × 3 mm), a cross-scale model bridging the molecular and cellular scale has been previously developed , 42. In The molecular interaction model describes the influence of the extracellular glucose level on the proliferating versus migrating phenotype emergence of GB cells. The glucose level controls the concentration of miR-451 that in turn regulates the concentration of the active LKB1-MO25-STRAD complex . This coThe molecular interactions are modelled by mass-balance reactions and Michaelis-Menten equations for the enzyme kinetics. In total, the concentration development of the nine molecular species is mathematically represented by a nondimensionalised system of nine nonlinear ODEs. These ODEs involve 31 reaction parameters which are given together with the details on the system of ODEs in . The ODETo model cellular processes (in terms of migration and proliferation) and the influence of environmental factors (in terms of the available nutrient concentration) on a tumour cell, an agent based modelling (ABM) approach is employed. In this model, each agent represents a single tumour cell. in vitro experiments in a petri dish, in this computational model, tumour growth is investigated in a two-dimensional region. The region of interest (3 mm × 3 mm) is represented by a regular grid of 200 × 200 grid points. Each of these grid points either can hold a tumour cell (the grid spacing of 1.5 × 101 μm × 1.5 × 101 μm corresponds to the average size of a GB cell [Comparable to GB cell ) or is ex10(0)) is assumed to be either constant or randomly distributed across the whole 3 mm × 3 mm region. Throughout the simulation, glucose is consumed by the tumour cells, cannot pass through the boundaries of the grid, and diffuses across the grid. The last is modelled by the use of a PDE of the formD is the diffusion coefficient for glucose and α a factor that realizes slower diffusion in case a grid point is occupied by a tumour cell.Furthermore, each grid point holds information on the local nutrient level, in particular on the concentration of glucose. Initially, the glucose concentration .x6) and mTORC1 (x9). According to this phenotype, the cells are moved to a new location, a daughter cell is placed at a neighbouring site , or no interaction takes place.To incorporate information from the gene and protein scale into the cellular level, each cell/agent on the grid (as described in x10(0)∈{3 × 10−1 gL−1, 1.125 gL−1, 2.25 gL−1, 4.5 gL−1} is assumed (a setting comparable to in vitro experiments). Initially, 797 cells are placed in a circular shape in the center of the grid. All simulations were run until the first cell reached the boundary. As could be seen in [ in vitro experiments.The general model setup is depicted in  seen in , these sIn total, 31 parameters determine the behaviour of the tumour growth model by regulating nine ODEs. One important question one should consider is whether there exist one or more parameters that have a crucial influence on the whole system. In particular, when considering novel targets for therapeutic interventions, one searches for parameters that slow down the tumour expansion and decrease the tumour volume.Sensitivity analyses are a tool to investigate the dependence of a system  on individual model parameters. The general procedure is to vary one or more parameters and measure the impact on the system. This allows identifying parameters that significantly influence the whole system. Generally, local sensitivity analyses (LSAs) and global sensitivity analyses (GSAs) can be distinguished . In an LThe general idea of this sensitivity analysis was to investigate the effects of changes in the intracellular setting of the tumour cells on the cell behaviour and overall tumour expansion. If, for example, a certain parameter significantly slowed down the tumour expansion, the respective reaction could be an indicator for future therapy targets. To accommodate for such an analysis across different spatial scales, we followed the idea introduced in . The parM is recorded. Next, we multiplied each reaction parameter kl  with different factors bn ∈ R. For each of these combinations, klbn : = kl · bn of a parameter kl and a factor bn separate simulations are performed and the model output Mkl,bn is recorded. For each parameter-factor setting, the simulation is run three times and the mean of the model output is calculated to account for the random elements of the model. Finally based on the model outputs and the parameter changes sensitivity coefficient Skl,bn are calculated as follows :(2)SklSkl,bn.bn < 1  and Mkl,bn < M , then the respective sensitivity coefficient is positive . The same holds true if bn > 1 and Mkl,bn > M. If, on the other hand, bn < 1 and Mkl,bn > M or bn > 1 and Mkl,bn < M, this results in a negative sensitivity coefficient .If kl is separately multiplied with two different factors b1 and b2 for which b1 − 1 = 1 − b2 and if additionally the resulting outcomes Mkl,b1 and Mkl,b2 do not differ , then the resulting sensitivity coefficients are the same except for the sign .If the same parameter kl is separately multiplied with two different factors b1 and b2 that result in the same simulation outcome Mkl,b1 = Mkl,b2, then the smaller factor results in the larger respective sensitivity coefficient .If the same parameter Following this definition, the undermentioned observations can be made regarding the properties of the sensitivity coefficients Mkl,bne, we choose four different endpoints e that are representative for the respective tumour expansions and tumour characteristics. As a measure for the tumour expansion velocity, we recorded the number of simulation time steps Mkl,bntime necessary for the first tumour cell to reach the boundary of the simulation region. A high expansion velocity corresponds to a short time until the first cell reaches the boundary  and vice versa. The final tumour volume is estimated by the number of cells in the last time step Mkl,bntotal. A large tumour volume is associated with a large number of tumour cells and a small volume with a small number of tumour cells. To further get a better understanding of the tumour build-up, additionally, the number of migrating cells in the last time step Mkl,bnmig and the number of proliferating cells in the last time step Mkl,bnprolif were documented. For all these four model outputs Mkl,bne, the corresponding sensitivity coefficients were calculated and were named accordingly Skl,bntime, Skl,bntotal, Skl,bnmig, and Skl,bnprolif.For the model output This setup allows for the assessment of the effects of changes in the reaction parameters on the overall behaviour of the tumour (tumour expansion and tumour build-up) as well in absolute as in relative terms.The model is implemented in C++. For the sensitivity analysis, each of the 31 reaction parameters of the molecular interaction model was separately multiplied with the factors 0.01, 0.1, 0.5, 0.8, 0.9, 0.95, 0.98, 0.99, 1.01, 1.02, 1.05, 1.1, 1.2, 1.5, 1.9, 1.99, 5, 10, 50, and 100.x10(0). In particular, for each parameter-factor combination, the tumour growth simulation was run three times for each of the four initial glucose settings . Thus, in total, simulations for 2480 different settings were carried out.As could be seen in Mkl,bntime(x10(0)), the final number of all tumour cells Mkl,bntotal(x10(0)), and the number of migrating Mkl,bnmig(x10(0)) and proliferating Mkl,bnprolif(x10(0)) cells in the last time step were recorded, and the respective sensitivity coefficients Skl,bne(x10(0)) were calculated for all four endpoints e. For the ease of notation, the reference to the parameter kl, the factor bn, and the initial glucose concentration x10(0) will be omitted in the following, if the sensitivity coefficients are discussed in general; that is, they will be referred to as Se.Each simulation was run until the first cell reached the boundary, the necessary number of time steps k1) are shown in detail  tends to result in rather low sensitivity coefficients  for an initial glucose concentration of 1.125 gL−1, and the diamond (◆) and the plus sign (+) for an initial glucose level of 2.25 gL−1 and 4.5 gL−1, respectively.Sk1,bne from zero. However, if the parameter k1 is multiplied with factors bn close to zero or two, this results in low sensitivity coefficients. The absolute maximum value of all sensitivity coefficients Sk1,bne−1 gL−1. With an increasing initial glucose level, the amplitudes of the sensitivity coefficients tend to decrease.For all of the four initial glucose settings, factors close to one cause the largest deviation of the four sensitivity coefficients mkle is taken. Therefore, mkle together with the respective glucose values x10(0) that result in these maximal values for all parameters. In contrast to The graphs of the sensitivity coefficients for the other parameters look similar to the ones explained above. The main differences affect the range of the sensitivity coefficients and the initial glucose concentration for which the maximum Skl,bne, the maxima of the absolute values mkle are primarily reached for the lowest glucose level of x10(0) = 3 × 10−1 gL−1 for all parameters. Only a few parameters exist for the absolute maximum mkle for a medium glucose level (1.125 gL−1 or 2.25 gL−1). The absolute maxima vary between 3.71–17.94 and 8.52–38.42 for Stime and Stotal, respectively. For Smig, the absolute maxima are in the range 9.44–43.66 and the sensitivity coefficients for the number of proliferating cells in the last time step Sprolif take maximum values between 8.54 and 34.89. The maximum over all parameters of all maximal sensitivity coefficients mkle (maxkl(mkle)) is taken for the parameter k4. Except for the endpoint e = time, the parameter k17m2 results in the lowest mkle.From the table, it can be seen that, for all sensitivity coefficients Stime, Stotal, Smig, and Sprolif, we also analysed the correlation between changes in Mtime and Mtotal. Thus, we examined the correlation between tumour expansion velocity and tumour volume for different parameter variations. This is motivated by the fact that the number of time steps necessary for the first tumour cell to reach the boundary Mtime can be considered as a measure for the velocity of the tumour expansion and the total number of tumour cells in the last time step Mtotal is a direct measure for the tumour volume.Besides investigating the sensitivity coefficients Mkl,bnj and M as in δMkl,bntotal < 0. Furthermore, if the variation of parameter kl by multiplication with a factor bn causes an increase in the number of time steps necessary for a simulation to terminate, this comes along with δMkl,bntime > 0. In general, δMkl,bnj = a can be translated into Mkl,bnj = (a + 1) · M.We defineδMkl,bntotal on the x-axis and δMkl,bntime on the y-axis. Each marker corresponds to a simulation with a different parameter scaling. As before, the simulations were run for four different glucose settings and the crosses  represent the data for an initial glucose level of 3 × 10−1 gL−1, the circles  for an initial glucose concentration of 1.125 gL−1, and the diamond  and the plus sign  for an initial glucose level of 2.25 gL−1 and 4.5 gL−1, respectively.Mtime corresponds to a decrease of Mtotal. The increases are most prominent for a low initial glucose level of 3 × 10−1 gL−1. For medium glucose levels (1.125 gL−1 and 2.25 gL−1), one mainly observes an increase in the number of time steps  and a decrease of the number of tumour cells . Assuming a high glucose level (4.5 gL−1), primarily a decrease of the number of time steps and a decrease of the final total number of tumour cells become apparent. None of the parameter scalings results in a significant decrease of the final total number of tumour cells at the same time with an increase in the number of time steps.For each glucose setting, an increase in the number of time steps comes along with an increase in the number of tumour cells. A decrease of δMtime > 9 for an initial glucose value of 3 × 10−1 gL−1 , and Factor− refers to those factors whose multiplication with a given parameter resulted in δMtotal < −0.8 (for 4.5 gL−1 glucose concentration). It can be noted that δMtime > 9 (for 3 × 10−1 gL−1 glucose concentration) and δMtotal < −0.8 (for 4.5 gL−1 glucose concentration) by multiplication with different factors. Out of all 20 factors (from 0.01 to 100), the highest and lowest scaling factors  predominantly resulted in a high number of time steps for a low glucose value or a low total number of cells for a high glucose value. No factors close to one appear in The respective parameters and factors are given in kl1, kl2 are modified: one parameter (kl1) with an associated factor (bn1) from the Factor+ group and another parameter (kl2) with a corresponding factor (bn2) out of the Factor− group. Obviously, there are some parameter-factor pairs that cannot be combined, since, for example, it is impossible to vary parameter k1 at the same time by the factors 10 and 0.01. In total, 603 combinations are possible and, for each of these combinations, three simulations are carried out for each of the four glucose settings. For each of the simulations, the necessary number of time steps for the first cell to reach the boundary and the total number of cells in the last time step are evaluated and δMtime and δMtotal are calculated based on the mean values. The results are shown in Mtime and Mtotal was previously presented in Next, we explore all possible combinations of two parameter-factor pairs (that are listed in −1 gL−1), most variations resulted in a significant increase of the number of time steps (δMtime > 0) and of the number of tumour cells . For the medium and high glucose levels , some combined parameter variations exist that yield a positive δMtime > 0 and at the same time a negative δMtotal < 0. These combined parameter variations, therefore, cause a decrease of the tumour expansion velocity and of the tumour volume. For the low initial glucose concentration (3 × 10−1 gL−1), no such combination of parameter changes exists.For a low initial glucose concentration  that resulted in an increase of the number of time steps (δMtime > 0.01) along with a decrease of the total number of tumour cells . Instead of using exactly zero as the threshold, an additional margin of 0.01 is applied. Thus, combinations are excluded that influence the system output only very slightly.For initial glucose levels of 1.125 gL−1, 2.25 gL−1, and 4.5 gL−1), in particular, as follows:k2 · 50 combined with k9 · 100 or k10 · 0.01,k11c2 · 50 combined with k8 · 100.A few combined parameter scalings result in a decrease of the tumour expansion velocity and the tumour volume for all medium and high glucose levels  could be combined with a quite large selection of other parameter variations .On the one hand, some of the parameter scalings caused a decrease of the tumour expansion velocity and the tumour volume only in combination with a few (1–3) other parameter modifications across all initial glucose concentrations. On the other hand, four parameter scalings , the maximum values among all parameter variations are taken for parameters that are relevant for the control of the level of miR-451 and MO25 .Godlewski et al.  discoverSkl,bn, , small −1 gL−1). Since tumours growing in a low glucose environment expand very fast , any changes that only slightly slow down or speed up the expansion are considerably noticeable. The same holds true for the dependence of the tumour volume (corresponding to the number of tumour cells) on the parameter variations.Nevertheless, on the basis of δMtime > 0) goes along with an increase of the total number of tumour cells  and vice versa. An increase of the number of tumour cells by a parameter variation is ultimately caused by an increase of the number of proliferating cells. Proliferation, in turn, is a process that happens much slower than migration and therefore the tumour expansion is slowed down. Thus, this relationship was to be expected in a more or less explicit form. The extreme changes for a low initial glucose condition (3 × 10−1 gL−1) are due to the fact that, in the original setting, the tumour expands very fast and is only very loosely packed, that is, consisting of rather few cells. Therefore, the potential for an increase of the number of time steps and the total number of tumour cells due to an increase of the number of proliferating cells is very high. Consequently, δMtime and δMtotal reach high values. On the contrary, under a high glucose level (4.5 gL−1), the number of time steps and tumour cells is already rather high and it is hardly possible to increase these. However, also, the decrease due to the parameter variations turns out to be only modest. Unfortunately, none of the parameter perturbations resulted in a slower growing tumour that consists of fewer cells  of the model output . For a low initial glucose condition (3 × 10−1 gl−1), none of the variations resulted in the desirable effect. The combined scaling of two selected parameters see  resultedFor a previously developed multiscale model of glioblastoma growth, we could identify a few combinations of reaction parameter modifications that result in a less aggressive tumour progression. Therefore, these parameters can also be thought of as potential targets for therapies. The parameters are primarily part of reactions that involve the microRNA-451 and the thereof dependent protein MO25 that are responsible for the further downstream signalling. Thus, the analysis supports the relevance of these two molecules for future therapy research.The supplementary material provides the complete results of the sensitivity coefficient calculations for all model parameters. For each parameter modification and each of the four system endpoints the corresponding sensitivity coefficients are presented in form of figures comparable to Figure 4 of the article.Click here for additional data file.


We recently identified RIG-I-like helicases (RLH) as therapeutic targets of pancreatic cancer for counteracting immunosuppressive mechanisms and apoptosis induction. Here, we investigated immunogenic consequences of RLH-induced tumour cell death.Murine pancreatic cancer cells (Panc02) were treated with RLH ligands to induce apoptosis and were then cocultured with primary dendritic cells (DC). DC maturation marker expression, antigen uptake and antigen cross-presentation were assessed.+ DC effectively engulfed apoptotic tumour material and cross-presented tumour-associated antigen to naïve CD8+ T cells. In comparison, tumour cell death mediated by oxaliplatin, staurosporine or mechanical disruption failed to induce DC activation, antigen uptake or cross-presentation. Moreover, tumour cells treated with sublethal doses of RLH ligands upregulated MHC-I and Fas expression and were sensitised towards CTL- and Fas-mediated killing.RLH ligands induced production of type I IFN, HMGB1 and Hsp70 and translocation of calreticulin to the outer cell membrane of tumour cells. In cocultures, DC upregulated B7 expression, which was mediated by tumour-derived type I IFN, whereas TLR, RAGE or inflammasome signaling was dispensable. CD8aRLH ligands induce a highly immunogenic form of tumour cell death linking innate and adaptive immunity.


A novel approach of simulating hydromechanical coupling in pore-scale models of porous media is presented in this paper. Parameters of the sandstone samples, such as the stress-strain curve, Poisson's ratio, and permeability under different pore pressure and confining pressure, are tested in laboratory scale. The micro-CT scanner is employed to scan the samples for three-dimensional images, as input to construct the model. Accordingly, four physical models possessing the same pore and rock matrix characteristics as the natural sandstones are developed. Based on the micro-CT images, the three-dimensional finite element models of both rock matrix and pore space are established by MIMICS and ICEM software platform. Navier-Stokes equation and elastic constitutive equation are used as the mathematical model for simulation. A hydromechanical coupling analysis in pore-scale finite element model of porous media is simulated by ANSYS and CFX software. Hereby, permeability of sandstone samples under different pore pressure and confining pressure has been predicted. The simulation results agree well with the benchmark data. Through reproducing its stress state underground, the prediction accuracy of the porous rock permeability in pore-scale simulation is promoted. Consequently, the effects of pore pressure and confining pressure on permeability are revealed from the microscopic view. Above all fluid transport properties of porous media at in situ conditions, permeability has been conducted as a critical topic in many research fields such as oil and gas production, carbon dioxide storage, underground gas storage, coal mining, and fuel cell –4. Wide In recent years, pore-scale modeling representing microstructures of porous media has been widely used for fluid transport properties prediction. Breakthrough in this field can be summarized as two aspects: lattice Boltzmann method (LBM) and finite element method (FEM). In LBM, the discrete form of Boltzmann equation, which is solved over a regular lattice grid, is used to conduct the simulation of fluid flow in the porous media , 11. WhiThis paper proposes a novel approach of reconstructing the 3D finite elements model from natural porous media images employing the commercial software MIMICS  and ICEMSandstone core samples used in this paper are provided by the Daqing Oilfield in China. As is shown in In the imaging process, small cylinders are drilled out of larger core samples for scanning. Then, the pore space is evacuated to inject a mixed resin of Epoxy resin and Struers Caldofix, which is typical for embedding of materialographic specimens. The resin is impregnated into the samples to maintain the integrity of sandstones during the physically destructive drilling to obtain small size samples. The small samples are imaged by the Micro X CT scanner in the State Key Laboratory of Oil and Gas Reservoir Geology and Exploitation in Southwest Petroleum University. One cross-section of micro-CT images is shown in raw suffix to the MIMICS software. Then surface meshes are transformed into tetrahedral elements wrapping the solid and the pore models. Based on surface meshes, mapping algorithm is adopted to generate 4-node tetrahedral element meshes. To ensure the accuracy of the simulation, the rock matrix and pore boundaries must be fitted to each other. Thus, nonmanifold assembly function in MIMICS is used when generating the volume meshes, which means the volume meshes of rock matrix and pore space are generated as assembly. The reconstructed model as well as the finite element models of S2 is shown in A challenging problem in numerical simulation of fluid properties in porous medium is how to build mesh models for pore space, as well as for the rock matrix. Moreover, both of the models must match each other perfectly to ensure the accuracy of hydromechanical simulation. Here, MIMICS software is employed to establish the 3D finite elements models based on the micro-CT images. The rock matrix and pore space are distinguished by the threshold of different color, after importing the micro-CT image data with a  .v represents the velocity of fluid flow. The fluid is assumed to be continuous and incompressible.The continuity equation represents conservation of mass for fluid flow, which can be written as ,(1)Δ·v=Navier-Stokes equation, representing conservation of momentum for the fluid flow, can be written as ,(2)ρ[∂vv is velocity of fluid flow, ρ is density of fluid, μ is dynamic viscosity of fluid, pp is fluid pressure, and g is acceleration due to gravity. Fluid is assumed to be incompressible and Newtonian.Here Q can be acquired. Then the absolute permeability will be calculated in the following term:L is the length of the cubic sandstone finite element model.By the CFX software, the outlet flow rate pc is the confining pressure, and α is the coefficient, ranging from 0 to 1. In the simulation, it is defined as 1.The pressure on the fluid-solid interface is transferred to each other by the following equation, which is called principle of effective stress firstly proposed by Terzaghi :(4)fi=pσij is the stress tensor, and fi is the body force.The three-dimensional equilibrium differential equation is (5)∑j∂σiεx, εy, and εz are the normal strain, γxy, γyz, and γzx are the shear strain, and u,v, and w are the displacement components.The three-dimensional geometric equations of the rock matrix are (6)εx=∂uE is modulus of elasticity and μ is Poisson's ratio.The elastic physical equations are (7)εx=1Epc) for structural analysis is added to the four sides of the cubic matrix models. The data of the stress-strain curve shown in z direction. Water is used as the fluid medium in the simulation. Fluid field will be solved by CFX solver at the beginning of simulation. After the fluid pressure on the fluid-solid interface is transferred to ANSYS structural solver, the meshes on the fluid-solid interface of both rock matrix and pore model will be remeshed according to the deformation data. The iteration of the fluid field and deformation of rock matrix is continued until the models converge. The brief schematic of boundary conditions in the simulation is shown in The structural analysis for matrix model and fluid flow for pore model are solved by ANSYS solver and CFX solver, respectively. As a result, boundary conditions of the matrix model and pore model should be set in ANSYS workbench separately. Firstly, confining pressure  is adapted to simulation for its effect on the rock permeability. The images of fluid pressure contours and velocity vectors along pc − pp = 3 MPa are plotted. It is found that the rock permeability changes at the fixed effective pressure level. The rock permeability increases along with the increase of pore pressure at the same effective pressure initially and curves become relatively flatter when pore pressure exceeds 15~20 MPa. It is a common sense that permeability of porous media depends on the size and connectivity of throats (the narrow openings connecting the pores). In fact, when fluid is injected into the porous media, the pore space will be extendedly driven by the pore pressure. Moreover, stress concentration usually occurs because of the irregular matrix shape of the porous media. As is shown in In this paper, the same boundary conditions are adopted for both simulation and experiment. As is shown in pp = 2 MPa are plotted in pc − pp = 20 MPa, the rock matrix is compressed and stress concentration occurs in the same parts as Meanwhile, curves of rock permeability versus confining pressure at the condition of Based on micro-CT images scanned by the State Key Laboratory of Oil and Gas Reservoir Geology and Exploitation in the Southwest Petroleum University, four sandstone 3D finite element models are generated using MIMICS and ICEM software. Through the hydromechanical coupling simulation on fluid transport properties in pore-scale models of porous media by ANSYS and CFX software, permeability of sandstone samples under different pore pressure and confining pressure is predicted. Good agreements are acquired on simulation result against the benchmark data. This indicates that the hydromechanical coupling analysis is able to promote the accuracy of permeability prediction of rock through reproducing its stress state underground.It should be noted that this study provides a preliminary study for microfluids in deformable porous media using finite element methods. Though examples used in this study mainly focus on the petroleum industry, it can also be widely applied to hydromechanical coupling analysis in other relevant area.


The presence of a metastable fluid-fluid critical point is thought to dramatically influence the crystallization pathway, increasing the nucleation rate by many orders of magnitude over the predictions of classical nucleation theory. We use molecular dynamics simulations to study the kinetics of crystallization in the vicinity of this metastable critical point and throughout the metastable fluid-fluid phase diagram. To quantitatively understand how the fluid-fluid phase separation affects the crystal nucleation, we evaluate accurately the kinetics and reconstruct the thermodynamic free-energy landscape of crystal formation. Contrary to expectations, we find no special advantage of the proximity of the metastable critical point on the crystallization rates. However, we find that the ultrafast formation of a dense liquid phase causes the crystallization to accelerate both near the metastable critical point and almost everywhere below the fluid-fluid spinodal line. These results unveil three different scenarios for crystallization that could guide the optimization of the process in experiments Crystallization is a very important phenomenon that usually proceeds via the nucleation and subsequent growth of nanometer-sized crystallites that form spontaneously out of a supersaturated solution or undercooled melt1234This initial nucleation step determines many properties of the emerging phase that are crucial in scientific and technological applications that range from material science, to medicine, or food scienceThis is particularly important in the case of protein crystallization because it is associated with many diseases, e.g., cataracts, sickle cell anemia, and Alzheimer’sThe main challenges in protein crystallization stem from the fact that, unlike simple molecules and atoms, proteins can be much larger than the interactions between them. As a consequence, the phase diagram of protein solutions, especially globular proteins, differ substantially from the phase diagrams of simple liquids, by having the gas-liquid critical point, and the entire gas-liquid coexistence region, below the melting line8In recent years, simulation and theoretical studies have suggested that the presence of this metastable fluid-fluid critical point can open up new pathways for nucleation because it allows a “two-step mechanism”1112131419It is thus not clear from current experimental and theoretical work whether there is a general way to optimize crystallization in systems with a metastable fluid-fluid phase transition. Here we perform a comprehensive analysis of the kinetics and thermodynamics of crystal nucleation in a wide region encompassing the metastable fluid-fluid critical point. We will show that there are three different scenarios for crystallization and that the formation of a metastable dense “liquid” patch does indeed speed up nucleation rates considerably. However, the enhancement of crystallization is associated to the whole metastable phase transition rather than to the metastable critical point itself. These results clarify the different mechanisms of crystal formation and open the door to a better experimental control and optimization of protein crystallization.We perform this study using MD simulations of crystal nucleation in a coarse-grained model for globular proteins with a short-range attractive interaction potential16a is the hard-core diameter, b = 1.06a the attractive well diameter, and U0 the attraction energy. The ratio b/a determines whether the system has a metastable liquid phase821Tc = (0.3916 ± 0.0005)U0/kB, ρc = (0.523 ± 0.005)(1/a3), and Pc = (0.0519 ± 0.0005)U0/a3, with Tc/Tm ≃ 0.64 at ρc, where Tm is the temperature of sublimationwhere Tc = 0.39 ± 0.0005 ρc = 0.5 ± 0.005, the rate of crystallization κ is a kinetic pre-factor and ΔG* is the nucleation barrier . Following the assumptions of Turnbull and FisherI is determined by the degree of supercooling Tm − T, with a CNT nucleation barrier that is constant along the (iso-CNT) lines where the quantitywhere is constant116χ = 35.7) but that does not cross the fluid-fluid spinodal line. The details of the simulations and the methodology used to analyze the nucleation rates, the critical cluster sizes, the nucleation barriers, and the pathways for crystallization are described in the Methods section.In order to analyze the effect of the fluid-fluid transition on the crystallization rate, we perform simulations along all the iso-CNT lines indicated in Tm, but rather by the kinetic pre-factor. The iso-CNT line at χ = 35.7  does not show any special behavior with respect to the other lines crossing the spinodal line, even at densities more than 50% higher or lower than ρc, at variance with what suggested earlierχ = 35.7 and 33.7, that show a minimum barrier at the point of closest approach to the spinodal line, rather than specifically at the critical point.The second important observation is that the iso-CNT line crossing the critical point  a location at high ρ outside the coexistence region.Consistently with these observations, we have also found that there are essentially three different pathways towards crystallization, illustrated by the three points  indicated by squares in In case (a) , for a vbefore the crystal cluster emerges , located below the spinodal line, a large liquid droplet forms rges see . The pors 2.5kBT . This rencrys and nliq seem uncorrelated in this case (In case (c), located above the binodal line, the dense liquid phase is unstable. However at high densities, transient liquid droplets are continuously forming and disappearing by thermal fluctuations, as indicated by the strong fluctuations in his case .Our results show that there are three different scenarios for crystallization depending on where is the state point with respect to the metastable spinodal line. (a) Between the binodal and the spinodal line at subcritical conditions we observe (effectively) direct crystallization from the low-density phase. (b) Within the spinodal region we find that the pathway proceeds through a “two-step mechanism”: (i) spinodal decomposition and then (ii) nucleation-and-growth inside the high-density fluid with a very low residual barrier, much smaller than the case outside the spinodal line. Indeed, this “spinodal-assisted” mechanism could be so fast that could lead to imperfect and amorphous aggregates rather than perfect crystals. Finally, (c) above the binodal line, specially at supercritical densities, crystallization is triggered by the transient appearance of liquid droplets that are unstable but with size and life-time that are large enough to allow the stochastic formation of a crystal critical cluster. For all the pathways we find that the crystallization free-energy barriers are substantially lowered by the formation of dense liquid patches where crystallization takes place easily. This was suggested already in Ref. any density by setting T above the spinodal line at an appropriate distance. Even though it might seem tempting to aim for conditions within the spinodal region directly, it is more likely that crystals with many defects form under these extreme conditions. The results of our study open up many more and potentially easier-to-realize pathways for the experiments and might have a very important impact on the handcrafted art of protein crystallization.We conclude that crystallization in systems exhibiting a metastable coexistence region behaves dramatically different from the prediction of CNT. In particular, the existence of a high-dense fluid phase lowers the free-energy barrier and hence accelerates the subsequent crystallization considerably; in our case by more than three orders of magnitude. This effect is not a singularity caused by the proximity of the critical point. Any state point of the system below the metastable spinodal line will give rise to a much faster crystallization because of the ultrafast formation of the dense liquid phase through spinodal decomposition. This is of particular interest for experiments that aim to leverage this effect for systems that are notoriously difficult to crystallize. Rather than focusing on just one single point, our findings suggest that there is an entire region for optimal crystallization  within the attractive range (r < b), and the size nrysc of the largest crystal cluster, defined by molecules having more than 11 n.n. at r < b and a local bond order parameter ≥ 0.475 as described in Ref. We study crystallization without any bias or constraint, by rapidly quenching the fluid to a I with high accuracy using the method of mean first-passage times (MFPT) τmax(n), defined as the average time at which the largest crystal cluster in the simulation reaches or exceeds the size n for the first timen*, the Zeldovich factor Z and the characteristic time τI related to the nucleation rate I = (τIV)−1, by fitting the simulation data to the functionWe obtain the crystallization rate How to cite this article: Wedekind, J. et al. Optimization of crystal nucleation close to a metastable fluid-fluid phase transition. Sci. Rep. 5, 11260; doi: 10.1038/srep11260 (2015).


While behavior of equilibrium systems is well understood, evolution of nonequilibrium ones is much less clear. Yet, many researches have suggested that the principle of the maximum entropy production is of key importance in complex systems away from equilibrium. Here, we present a quantitative study of large ensembles of carbon nanotubes suspended in a non-conducting non-polar fluid subject to a strong electric field. Being driven out of equilibrium, the suspension spontaneously organizes into an electrically conducting state under a wide range of parameters. Such self-assembly allows the Joule heating and, therefore, the entropy production in the fluid, to be maximized. Curiously, we find that emerging self-assembled structures can start to wiggle. The wiggling takes place only until the entropy production in the suspension reaches its maximum, at which time the wiggling stops and the structure becomes quasi-stable. Thus, we provide strong evidence that maximum entropy production principle plays an essential role in the evolution of self-organizing systems far from equilibrium. Classical thermodynamics offers elaborated theoretical instruments to study behavior of systems that are in or near equilibrium. But often objects and processes which we deal with in everyday life, such as living biological organisms or weather events, are far from equilibrium. Therefore, the search for principia governing nonequilibrium systems is of great interest.th century45689111314151718192021The notion of entropy in equilibrium states and its production in nonequilibrium processes not only form the basis of modern thermodynamics and statistical physics, but have also been at the core of various philosophical discussions concerned with the evolution of the world, the course of time, etc. There is a general agreement on how the entropy of a nonequilibrium system should evolve, postulated by the second law of thermodynamics. At the same time, the question how entropy production changes with time is still extensively discussed. So-called the maximum entropy production principle (MEPP)12reduces the oil entropy. At the same time, convective flows associated with the RB structures increase the heat transfer rate, leading to a greater entropy production of the entire system, which includes the hot plate and the environment. It is unclear under which conditions nonequilibrium systems develop a new order. Yet, it is plausible to hypothesize that MEPP might be one of the main guiding principles for not only the evolution of biological species, but their appearance on Earth due to the strongly nonequilibrium energy distribution created by the Sun32Frequently, thermodynamic systems driven out of equilibrium have a tendency for symmetry breaking. This includes pattern formation252628quantitatively. In particular, it enables precise measurements of generated Joule heating . As a result, entropy production in the fluid, which is directly proportional to the Joule heating, can be correlated with the process of self-assembly of carbon nanotube (CNT) chains occurring under the effect of the applied E-field35363738In this work, we investigate possible evolution paths of an electrorheological (ER) fluidwiggle, i.e. exhibit quasi-periodic motion and deformation  or multi-wall carbon nanotubes  suspended in an insulating non-polar solvent (toluene). The field is generated between a pair of metallic electrodes immersed in the ER fluid and connected to a voltage source in series with a commercial resistor. The measurement setup involves a voltage source connected in series with a resistor, ammeter, and two cylindrical stainless steel electrodes submerged into the sample , can reach and even exceed sU/2R, where sR is the series resistance and U is the applied voltage. Since the current depends on the presence of continuous nanotube chains and electro-convection, we can use it as a measure of the order in the suspension.The fluid is comprised of electrically conducting either of single- , exceeds sR by at least 3 orders of magnitude. The time t is measured from the moment when the voltage is turned on. During the measurement, the value of the series resistor is fixed, while the resistance of the fluid is changing owing to the formation of CNT chains. We use a precision electrometer (Keithley 6517B) to set the voltage and to measure the current in the circuit. The power dissipated in the fluid, fP, can be deduced from the equation fP(t) = I(t)[U-I(t)sR], where I(t)sR is the voltage drop on the resistor.Prior to each experiment, the nanotubes in the fluid are dispersed by means of sonication, ensuring that the initial resistance of the suspension, i.e. the chains can get destroyed, what represents an avalanche. This evolution stage is called the “avalanche” phase due to the fact that they frequently, but not necessary, occur here. The reasons for the avalanches have been clarified in the model presented in Ref. Snapshots of the system evolution under the influence of a strong electric field are demonstrated in 0t and continues as long as the experiment goes on. During that phase, the chain breaking events can no longer be detected visually. Quite the contrary, more and more nanotubes are attracted to the electrodes, making the chain network larger and denser, while the rest of the fluid becomes clearer . Once the maximum is achieved, the evolution processes slow down, giving rise to the peak in the vicinity of <f/PmaxP> = 1. Note that if we consider the entire system that includes the fluid and the resistor, then the maximum of the entropy production is never achieved, since it would correspond to fR = 0.The tendency of the system to maintain dissipation that is close to its maximum is demonstrated in Occasionally, towards the end of the avalanche phase, nanotubes form a dissipative pattern that exhibits quasi-periodic collective motion and deformation. f/PmaxP(t) starts to jitter near the maximum, but does not actually reach it ) = maxP, and incomplete, max(fP(t)) < maxP. In the course of complete evolution, the total resistance of self-assembled carbon nanotube structure tends to a value which maximizes energy dissipation in the structure. This fact reflects the realization of the maximum entropy production principle. Moreover, the self-assembled dissipative structures can start to wiggle, thus resembling a self-created living organism. We find that the system is dynamic if its resistance is greater than the series resistor, and it becomes static when the impedance matching condition, fR = sR, is achieved. Our conclusions apply to systems subjected to external forces and gradients, which are sufficiently strong so that the behavior is nonlinear and the self-assembly cannot be prevented by external or internal perturbations. On the other hand, the applied forces and/or gradients should not be too strong. Otherwise the generated heating might destroy self-assembled patterns.To sum up, we have studied the behavior of the electrorheological fluid under the influence of a strong electric field. Two types of the evolution have been observed: complete, max(A.H. wrote the grant that made this work possible. A. Bez. and A. Bel. designed the experiment. A. Bel. performed the measurements. All authors contributed to the analysis and the writing of the manuscript.Motion of the CNT


The foundation of recommended diarrhea management in young children is increased fluids and continued feeding. This increase in fluids is necessary to replace those lost during diarrhea and ultimately prevent dehydration. There may be an opportunity to prevent deaths in children under five by discouraging the practice of reducing or curtailing fluids during diarrhea episodes across different settings worldwide.We quantify and describe the extent of fluid curtailment in children with diarrhea in a selection of countries  with high burden of diarrhea-related mortality with national cross sectional survey data. We examine the practice of fluid curtailment in these countries and its relationship to child and household traits and to characteristics of diarrhea management.The prevalence of fluid curtailment among children under five with diarrhea is strikingly high in these countries: 55 % in Nigeria, 49 % in Ethiopia, 44 % in Uganda, 37 % in Tanzania, 36 % in DR Congo and 32 % in Burkina Faso. Fluid curtailment is associated with giving less food, potentially worsening the impact of this harmful practice. Children who were reported to have had fluids curtailed during diarrhea episodes were also 3.51  times more likely to be reported to have food withheld . Children who received care from non-governmental providers, and those who were breastfed were more likely to have their fluids curtailed, as were children with an unimproved water source. Children of poorer or less educated mothers and those living in rural areas are more likely to have curtailed fluids, compared to children of less poor or more educated mothers, or those living in urban areas.The harmful practice of curtailing fluids for a child with diarrhea is highly prevalent, representing an increased risk of dehydration and complications due to diarrhea, including death, especially for children in specific subgroups.The online version of this article (doi:10.1186/s12889-015-1878-z) contains supplementary material, which is available to authorized users. The Countdown to 2015 for Maternal, Newborn and Child Health (“Countdown”) is a global initiative that tracks progress in achieving high, sustained and equitable coverage for interventions of proven effectiveness in preventing unnecessary deaths among women and children in 75 priority countries . Countdooffered to a child with diarrhea, others about fluids given to a child or actually ingested, and unfortunately many do not report on actual formulation of the question posed to caregivers. The review concludes that previous research on the extent of fluid curtailment suggests that this harmful practice is highly prevalent in some settings, but that high quality, recent, and comparable data are scarce.A recent systematic review found that there have been few previous studies of fluid curtailment . The evidence base is also difficult to summarize succinctly, because a variety of methods have been used, and even among those using population-based surveys, survey questionnaires used to assess fluid curtailment are not standardized. For example, some surveys ask about fluids In this study we use data from recent, standardized, nationally-representative household surveys to examine the practice of fluid curtailment during episodes of childhood diarrhea in six African countries that are among those with the highest burden of diarrhea mortality globally.We selected the six African countries identified in 2012 in the Global Action Plan for the Prevention and Control of Pneumonia and Diarrhea (GAPPD) , as majoMICS and DHS both collect data on fluid intake during childhood diarrhea episodes from nationally-representative samples of mothers or caretakers, using a standardized approach . These qWe examined the distribution of fluids given during diarrhea using these six categories, while accounting for the stratification and clustering designs for each survey. We then collapsed the responses into two summary categories for further analysis: 1) “fluids curtailed”, defined as children for whom caregivers reported that no fluids were given or that the fluids given were either much less than usual or somewhat less than usual; and 2) “fluids not curtailed”, defined as children for whom caregivers reported that either the same amount or more fluids were given. We compare these weighted distributions across the six countries.In order to inform activities related to advocacy and behavior change interventions, we consider a selection of factors possibly related to fluid curtailment with a priori interest. We classify these into two groups: first, those related to diarrhea management, to clarify if fluid curtailment is compounded with other indicators of treatment. Second, in order to identify those most at risk, we examine factors related to each child and describe factors determined for each household.For associations between fluid curtailment and other diarrhea case management practices, we investigated the relationships between fluid curtailment and how much food was given to the child during diarrhea (food), ORS administration, reported use of antibiotics and antimotilics and care-seeking for the diarrhea episode. Response options for care-seeking were summarized as: 1) no care-seeking outside the home; 2) care-seeking from government (public) providers; and 3) care-seeking from all other sources, as defined by survey documentation. We would have liked to include the administration of zinc, but this treatment was reported in only 181 of the 12,012 (1.5 %) of the childhood diarrhea cases included in the analysis.For relationships between fluid curtailment and child and household characteristics, child variables included the age and sex of the child, as well as whether the child was still breastfeeding (yes/no) at the time of the survey. Household characteristics included the mother’s educational level , the number of births or parity of the mother, household wealth (socioeconomic quintile from poorest to least poor as defined in the surveys by the presence of country-specific sets of household assets), urban versus rural residence, and whether the household had an improved water source (yes/no).We described these relationships by country with tabulations using weights determined by each survey’s design, and estimate bivariate statistical differences in fluid curtailment between levels of each factor using Pearson’s design-adjusted F test, which accounts for survey related variability , 13. PriFinally, we will summarize the results of observed factor relationships with fluid curtailment for each factor of interest, addressing the potential generalizability to other settings. We estimated the relative risk of fluid curtailment in each country, and meta-analyzed the results to estimate an overall comparison of the rates of fluid curtailment between different groups Fig. . These rWeighted tabulations of responses to the question on fluid intake during childhood diarrhea episodes are shown in Table The percent of children with diarrhea whose fluids were curtailed is shown in Table Children with diarrhea whose mothers report curtailing fluids are also more likely to be given less food Table . This ret tests [Care-seeking for diarrhea was found to be significantly related to fluid curtailment only in Uganda. Pairwise Student’s t tests  comparinIn the meta-analyses of diarrhea management practices Fig. , giving The descriptive results on the percent of children for whom caregivers report curtailing fluids during diarrhea episodes are presented by child and household characteristics in each country in Table The results show that children under the age of six months are more likely to have their fluids curtailed than older children in all countries but Nigeria, with statistical significance in Tanzania (p < .01), Burkina Faso (p = 0.03) and Ethiopia (p < .01). In each of these three countries, the highest fluid curtailment was in the youngest age group, 0–6 months, and the lowest was in the oldest, 24–59 months. No consistent pattern of associations was found between fluid curtailment and the sex of the child or the total number of children born to the mother. In the meta-analyses of child characteristics, breastfeeding was associated with increased fluid curtailment across countries, with those who were breastfed at more risk of having their fluids curtailed .Caregivers in rural areas were more likely than caregivers in urban areas to curtail fluids in all countries, but this difference was not significant in Nigeria or Uganda. Estimated differences in rates of fluid curtailment between urban and rural households, in descending order by the magnitude of the difference, are Tanzania (26 % and 41 %), Ethiopia (38 % and 50 %), DRC (28 % and 39 %) and Burkina Faso (23 % and 34 %).Relationships between water source and fluid curtailment are mixed. Households with an improved water source had lower rates of fluid curtailment in Tanzania (p < .01), however, there was not sufficient evidence to support this relationship in Burkina Faso, DR Congo, or Ethiopia. In Nigeria and Uganda, the estimated difference in fluid curtailment rates by water source was negligible.Mothers with less education generally reported giving their children less to drink, especially in Nigeria (p = .04), Burkina Faso (p < .01), and DRC (p < .01). The greatest observed difference in rates of fluid curtailment by education is in DRC, followed by Burkina Faso with the smallest difference in Nigeria. Household wealth is significantly related to fluid curtailment in Tanzania (p = .02), Burkina Faso (p < .01), and DRC (p < .01). In all three of these countries, the least poor are substantially less likely to curtail fluids than poorer households, although patterns differ.In the meta-analyses of household characteristics, residence location was associated with fluid curtailment, with rural households being more likely to curtail fluids than urban households . Improved water is associated with less fluid curtailment , and mothers with more education tend to curtail fluids less often ; relative risk for secondary vs no education, 0.75 ). Wealth quintile, the final household characteristic considered, is also related to fluid curtailment across countries. Households in the least poor wealth quintile were less likely to curtail fluids than those in the poorest quintile  , standardized data on fluid intake practices from MICS and DHS surveys to describe fluid curtailment practices in six countries in sub-Saharan Africa with high burden of diarrhea-attributable mortality.The results show clearly that the curtailment of fluids among children reporting recent diarrhea is a widespread practice in these countries. Between 32 % (Burkina Faso) and 55 % (Nigeria) of child caregivers report that they reduced the amount of fluid they gave to their child during the most recent diarrhea episode.Normative standards for correct home management of diarrhea, including those reflected in the WHO/UNICEF guidelines for both the integrated management of childhood illness  and the The findings presented here suggest that there is an important missed opportunity to prevent child deaths due to diarrhea by reaching caregivers with effective behavior change messages about the need to increase, not stop or decrease, giving fluids during diarrhea episodes. These messages must be reinforced in health worker training  and supervision, but may also warrant direct behavior change interventions at community level.The findings also reveal patterns that may be useful in targeting behavior change programs which aim to build capacity at the community and household level. Although there is variation across countries, the results suggest that fluid curtailment is more common among children who live in rural areas, who live in the poorest households, or who have mothers with no education.Some aspects of fluid curtailment practices were not able to be investigated thoroughly using the available survey data sets. Sample sizes were too small to support definitive analyses in some areas, notably patterns of fluid curtailment by antimotilic practices and treatment with zinc. In addition, the role of local and cultural beliefs and practices in determining caregiver understandings of diarrhea and appropriate responses would require further study using qualitative methodologies . ExplanaThese results are also likely to be affected by the difficulty of obtaining accurate responses from caregivers about the amount of fluid ingested by a child during a diarrhea episode. First, the recommended treatment for dehydrating diarrhea is ORS, which is administered by mixing the salts with water. Mothers may consider ORS solution as a medicine, rather than a fluid, affecting whether or not they report curtailing or giving increased fluids during the episode. This may explain our failure to find an association between reports of ORS administration and fluid curtailment. Second, although standardized questionnaire wording was used in asking mothers and caregivers about the fluids they gave the child, the term “gave” could have been ambiguous to respondents or the meaning may have shifted after translation into local languages. In addition, the definition of “fluids”, particularly in relation to breastfeeding, may be subject to variable interpretation across countries or languages, or even among responses from the same survey, with the potential for additional error in our estimates of fluid curtailment. However, the standardization of the survey tools plays an important role in minimizing potential biases. For instance, all surveys in this analysis used the same wording (“gave”) in the core question on fluid intake. In addition, the reported prevalence of fluid curtailment is sufficiently high to warrant attention even if some mothers/caretakers interpreted the question differently.Despite these limitations, then, these results should alert the public health community to this harmful practice and the need to strengthen program strategies. Of particular importance is the need to determine how contacts with the health system can be used effectively to counsel caregivers on the dangers of dehydration together with the need to increase fluids and continue feeding when their child has diarrhea. Continuing analyses by Countdown can examine whether programs are put in place to reduce this harmful practice and can reduce its overall prevalence as well as inequalities among specific subpopulations.Diarrhea deaths are preventable. These findings show that the harmful practice of curtailing fluids when a child has diarrhea is highly prevalent, and that current strategies to promote correct management of childhood diarrhea have not been effective to date in changing this behavior. New and reinforced efforts to reach caregivers, including those not currently utilizing public health services, are needed urgently. No one solution will be enough, and efforts to strengthen counseling during sick child contacts must be buttressed by direct efforts to reach mothers and their communities to ensure that dehydration is prevented in children with diarrhea and that they receive appropriate treatment during the diarrhea episode.The survey data analyzed in this research was collected and managed by local government entities with technical support from MICS and DHS. In each country, these government entities managed and approved the informed consent process and other ethical issues. Prior to public release, MICS and DHS make their data anonymous. All data used in this research are anonymous and not identifiable, and so we sought no further ethical approval.www.dhsprogram.com).This research uses national survey data that is publicly available for research collected by MICS (mics.unicef.org) and DHS (http:/


Capsule: We observed that first trimester pregnancy loss is associated with an altered expression profile of the three isoforms of the NK receptor NKp30 expressed by NKs in PBMC and placental tissue.In this study, we aimed to investigate whether first trimester pregnancy loss is associated with differences in expression of NKp30 splice variants (isoforms) in maternal peripheral blood or placental tissue. We conducted a prospective case–control study; a total of 33 women undergoing dilation and curettage due to first trimester pregnancy loss were further subdivided into groups with sporadic or recurrent pregnancy loss. The control group comprises women undergoing elective termination of pregnancy. The qPCR approach was employed to assess the relative expression of NKp30 isoforms as well as the total expression of NKp30 and NKp46 receptors between the selected groups. Results show that in both PBMC and placental tissue, NKp46 and NKp30 expressions were mildly elevated in the pregnancy loss groups compared with the elective group. In particular, NKp46 elevation was significant. Moreover, expression analysis of NKp30 isoforms manifested a different profile between PBMC and the placenta. NKp30-a and NKp30-b isoforms in the placental tissue, but not in PBMC, showed a significant increase in the pregnancy loss groups compared with the elective group. Placental expression of NKp30 activating isoforms-a and -b in the pregnancy loss groups was negatively correlated with PLGF expression. By contrast, placental expression of these isoforms in the elective group was positively correlated with TNFα, IL-10, and VEGF-A expression. The altered expression of NKp30 activating isoforms in placental tissue from patients with pregnancy loss compared to the elective group and the different correlations with cytokine expression point to the involvement of NKp30-mediated function in pregnancy loss. Miscarriage, defined as the spontaneous loss of a fetus up to 20 weeks of gestation, is the most common complication of pregnancy. The prevalence of miscarriage is around 15% in clinically proven pregnancies, and up to 30% in “chemical pregnancies” . RecurreNKp30 is a 30-kDa triggering receptor expressed on resting and activated natural killer (NK) cells . This reRecently, it was shown that the human NKp30-encoding gene (NCR3) is transcribed in six different splice variants, among which the most highly expressed are NKp30-a, NKp30-b, and NKp30-c . ZitvogeThe reported function of NKp30 in pregnancy and pregnancy disorders and the differential expression of NKp30 activating and suppressive isoforms in cancer and HIV motivated the current study. We compared (i) patients with RPLs (>2 spontaneous miscarriages), (ii) patients with sporadic pregnancy loss (≤2 spontaneous miscarriages), and (iii) patients undergoing elective termination of pregnancy (TOP) in the first trimester due to medical or social considerations or fetal malformations as a control group. The aims of this prospective case–control study were to investigate whether first trimester pregnancy losses are associated with (A) differences in mRNA expression of the expression profile of NKp30 isoforms, as well as the total NKp30 and NKp46 expression in maternal blood and placental tissue; and (B) the correlation of NKp30, NKp46, and isoform expression with early pregnancy-related cytokines.A single center prospective case–control study was conducted in the Department of Obstetrics and Gynecology at the Soroka University Medical Center, Beer Sheva, Israel in collaboration with the Clinical Biochemistry Laboratory at Ben-Gurion University. The study was approved by the ethical institutional review board .n = 10, sporadic n = 16, recurrent n = 7, and Sp + Re n = 23, and the placental tissue samples were elective n = 7, sporadic n = 16, recurrent n = 7, and Sp + Re n = 23.The study population comprises women who presented to the Soroka Medical Center for dilation and curettage (D&C) during the first trimester of pregnancy (<14 weeks of gestation), who consented to participate in the study. The combined study group comprises women undergoing D&C due to first trimester pregnancy loss, who were subdivided into groups with sporadic or RPL (>2 previous spontaneous miscarriages). The control group comprises women undergoing elective TOP. Women with known uterine anomalies, multiple gestations, and women undergoing elective curettage who had a history of ≥2 spontaneous pregnancy loss were excluded from the study. The PBMC samples were elective ® CPT™ Cell Preparation Tube with sodium heparin (REF 362753). PBMC samples were isolated according to the manufacturer’s instructions. Placental tissue samples were collected from the retrieved specimen, just after curettage, and placed in sterile ice-cold 1× PBS until RNA extraction was performed.Collection of blood samples was performed using a BD Vacutainer® Mini Kit  according to the manufacturer’s instructions and was stored at −80°C until further use. RNA concentration and purity were assessed by NanoDrop . Reverse transcription PCR (RT-PCR) was performed using a High Capacity cDNA Reverse Transcription Kit . cDNA synthesis was performed according to the manufacturer’s instructions using 1 μg of total RNA and was performed for all of the samples at the same time. cDNA samples were stored at −20°C until further use.Total RNA was extracted from fresh placenta tissue and isolated PBMC using the RNeasy® Green PCR Master Mix . Twenty microliters of reactions were set-up in duplicate for each sample according to the manufacturer’s instructions. Each reaction contained 30 ng of cDNA. qPCR experiments were performed using the Applied Biosystems 7500 Real Time PCR system, software v2.0.5. Reaction cycling protocol was 95°C for 10 min, then 40 cycles with a 2-step program . Collection of data was performed during the annealing step. Expression levels of NKp46, NKp30, NKp30-a, NKp30-b, NKp30-c, IFNγ, TNFα, IL-10, PLGF, and VEGF (target genes) were normalized to β-actin (reference gene). For each gene, all samples per tissue (blood or placenta) were checked in the same plate. Plate normalization was performed by checking the β-actin of two fixed samples in each plate and adjusting Ct values accordingly. Calculation of gene expression was performed using the 2−ΔCt = 2−(Target gene Ct) − (Reference gene Ct) method. Ratio analysis of NKp30 isoforms was calculated using the NKp30x/NKp30y = 2(ΔΔCt NKp30y−ΔΔCt NKp30x) formula, as previously described , NKp30: Fw primer: 5′-CATGGTCCATCCAGGATCC-′3, Rev primer: 5′-GTTCCATTCCTCACCTCCTTC-′3, , NKp30-a: Fw primer: 5′-GGTGGTGGAGAAAGAACATC-′3, Rev primer: 5′-CTTTCCAGGTCAGACATTTGC-′3, , NKp30-b: Fw primer: 5′-GGTGGTGGAGAAAGAACATC-′3, Rev primer: 5′-GAGAGTAGATTTGGCATATTTGC-′3, , NKp30-c: Fw primer: 5′-GGTGGTGGAGAAAGAACATC-′3, Rev primer: 5′-CATGTGACAGTGGCATTTGC-′3, , β-actin: Fw primer: 5′-GCATTGTTACCAACTGGGAC-′3, Rev primer: 5′-GGTCTCAAACATGATCTGGG-′3, , IFNγ: Fw primer: 5′-GCCAGGACCCATATGTAAAAGA-′3, Rev Primer: 5′-TTCTGTCACTCTCCTCTTTCCAA-′3, , TNFα: Fw primer: 5′-GACAAGCCTGTAGCCCATGT-′3, Rev Primer: 5′-TTATCTCTCAGCTCCACGCC-′3, , IL-10: Fw primer: 5′-TGCTGGAGGACTTTAAGGGTTA-′3, Rev Primer: 5′-ACAGGGAAGAAATCGATGACAG-′3, , PLGF: Fw primer: 5′-GATGAGCATGGTGGTTTTCC-′3, Rev Primer: 5′-AGACACAGGATGGGCTGAAC-′3, , VEGF-A: Fw primer: 5′-GCAGAATCATCACGAAGTGGT-′3, Rev Primer: 5′-ATCAGGGTACTCCTGGAAGATGT-′3, . Target and reference human gene sequences were taken from the National Center for Biotechnology Information .p < 0.05 was considered statistically significant.Graphics and statistical analysis were performed using GraphPad/Prism5 software. Statistical significance was tested using the two-tailed Mann–Whitney test. Correlation was tested using Spearman’s rank correlation coefficient. Thirty-three women consented to participate in the study. These included 16 patients with sporadic pregnancy loss (“sporadic” group), 7 patients with recurrent (>2) pregnancy loss (“recurrent” group), and 10 patients with elective TOP (“control” group). Demographic and clinical characteristics of the three groups are summarized in Table p = 0.0425, Figure p = 0.049, Figure We first evaluated the expression of NKp30 and NKp46 in PBMC, as these receptors are known to be highly expressed on peripheral blood NK cells. NKp46 expression was significantly enhanced in the sporadic group compared to the control group , which in turn may mediate cytokine secretion through NKp30, as was shown by El Costa et al. , 16. It Overall, our observations correlate with previous publications regarding the enhancement of placenta NKp46 expression during spontaneous pregnancy loss. In addition, our results shed new light on the contribution of NKp30 by demonstrating (i) changes in NKp30 isoform status between the peripheral blood and placenta in pregnancy loss, and (ii) changes between elective and spontaneous pregnancy loss in regard to NKp30 isoform status.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.