It seems that nonspecific low back pain (NSLBP) cannot be successfully treated with a single intervention of any kind. However, a positive effect of an active lifestyle including physical activities in NSLBP is reported on pain and disability. Advising regular physical activity is one of the mainstays in physical therapy treatment, appealing to patients' adherence. Many patients with low back pain do not seem to adhere to their physiotherapist's advice. In this study, the influence of social support on adherence to an activity advice will be investigated.Objective of the study is to investigate if patients with low back pain randomised to receive social support adhere better to an activity advice than patients randomised to the control group.In a randomized controlled clinical trial, in private physiotherapy practices in the Netherlands, patients ≥18 years presenting with at least a second episode of nonspecific low back pain will be randomized over two groups: activity advice and social support as an add‐on to usual care versus usual care alone. The primary outcome measure is adherence to an activity advice measured by an activity monitor at 1, 6 and 12 weeks. All other objectives will be measured by questionnaires.This project is the first comprehensive research project to assess whether social support influences adherence to an activity advice outside the clinical setting without supervision of a healthcare provider. Where evidence of influence on social support is lacking, findings may have implications for the management of patients with low back pain. In addition, findings may enable physiotherapists to predict in which patients with low back pain an activity advice with social support makes sense.NCT02996955. Dutch people have access to their physiotherapist without a referral of their GP. People with an additional insurance are compensated for the costs. For this study physical inactive patients with low back pain are needed. It is hypothesized that patients having more than one episode of NSLBP are potentially more inactive compared to patients with a first episode, therefore patients with a first episode of low back pain will be excluded. Patients ≥18 years presenting with a self‐reported second episode (or more) of NSLBP are recruited for the study. Eligible patients will be recruited for the study together with their partner or friend. Patients will be excluded for the study if (1) they cannot bring a partner or friend, (2) they are unable to read, write or speak the Dutch Language, (3) they present red flags as mentioned in the Dutch low back pain guideline  being a certified graduate physiotherapist, (2) being instructed and updated on the content of the study.During the first meeting all physiotherapists will be informed about the Dutch guideline for low back pain  Appendix . The CIRIt is hypothesised that overweighted/obese patients are protentional physical inactive. BMI of the patient will be calculated and categorized according to the WHO criteria WHO, .It is hypothesised that the number of recurrences of low back pain influence adherence to an activity advice negatively, therefore, the number of recurrences of periods of low back pain will be noted.To assess whether perceived pain and/or disability influences adherence to the activity advice, the visual analogue scale (VAS) for pain and the Quebec Back Pain Disability Scale (QBPDS) for disability will be used at baseline, both of which have been validated for assessing pain and disability . During the assessment periods at one, six‐ and 12‐week patients are instructed to wear the Activ8 every day during one week. The number of days per week patients have walked or cycled according to the advice will be counted. Cut‐off for adherence will be set at a minimum of 5 times per week of at least 30 min walking and/or cycling. The Activ8 system is a valid instrument to quantify body postures and motions  assesses patients' and partner/friends' illness beliefs Appendix . Eight iThe module attitude and intention (MAI) of The Knowledge, Attitude, Self‐efficacy and Self‐regulation, Exercise and Social Support for Sports and Exercise Questionnaire will be used to assess attitude and intention to be physically active  of 80%, 142 patients are needed. With an expected overall drop‐out rate of 15%, a total of 163 patients is needed.2.6Recruitment is scheduled from October 2016 till December 2022. To achieve adequate patient enrolment, next to own consultation of patients experiencing non‐specific low back pain by participating physiotherapists, referring health care providers, such as general practitioners located in the city of the participating practices will be informed about the study, to send patients with non‐specific low back pain to those physiotherapists.2.7It is hypothesised that patients in the intervention group adhere better to the activity advice than patients in the control group. It is also hypothesized that patients having a BMI >25 and patients experiencing >3 episodes of NSLBP are potentially more inactive and non‐receptive to the interventions on adherence. To prevent influence of these factors on study outcomes, stratification is applied on these variables. Therefore, a concealed randomization sequence is prepared by the randomisation centre of the Maastricht University. Randomization will be performed with a block size of four, with strata for BMI  and number of previous episodes of low back pain .Investigators, patients, partners or friends and participating physiotherapists are blinded to the allocation and outcomes. To prevent that patient and their partners/friends of both groups will meet in the waiting room appointments of patients treated in the intervention or control group in the same private practice must be planned on different days or sections of the day. Physiotherapist are blinded for participating in the intervention group or control group. To ensure blinding of the physiotherapists, where their training is separate, it will be emphasized not to communicate about the interventions to each other during the investigation period.2.8Patient data will be coded and the key to the codes will be safeguarded by the secretary of the randomization centre of the Maastricht University. These codes are unknown and not accessible for the researchers. Outcomes of questionnaires and Activ8 will be collected by the assessing physiotherapist. Losses‐to‐follow‐up and respective reasons will be recorded. The researchers collect the data from the assessors every month and enter the data in an electronic database. Access to the data obtained in this research will be restricted to the researchers involved in the collection and analysis of the data. The handling of personal data complies with the General Data Protection Regulation (GDPR) EU, . Study f2.92 test will be used to investigate if there is a significant difference in the proportion of adherent patients to the activity advice between the intervention and control group after one, six and 12 weeks, and if there is a significant difference in the proportion of adherent patients between patients who experienced social support or not on the social support scale after 12 weeks. A Time Series Analysis will be used to present changes over time in perceived illness beliefs of patients and partner/friend. To understand whether illness beliefs influence adherence, Pearson's r between outcomes of the IPQ‐S at baseline and after 12 weeks and (non)adherence measured by the Active8, will be calculated, and linear regression will be used for modelling the relationship between these outcomes. To assess the correlation between attitude and intention to physical activity (MAI) and adherence at 12 weeks, Pearson's will be calculated, and linear regression will be used for modelling the relationship between these outcomes. Influence of age, gender, education level, comorbidity (CIRS), pain (VAS) and disability (QBPDS) on adherence, will be estimated by multiple regression analyses. A p value of <0.05 will be considered statistically significant.To analyse the data SPSS version 25.0  will be used. For demographic data descriptive statistics will be used and normality assumptions for the variables will be checked. An intention‐to‐treat analysis will be used for the main outcome. To correspond to the primary aim of the study, outcomes of the Activ8 will be dichotomized into adherent and not adherent patients in the intervention and control group. The Chi2.10Regular walking and cycling are not associated with adverse effects for NSLBP; there is no formal proof that any of these physical activities have harmful effects. The social support and treatment of illness beliefs described in this study are designed for better outcomes of physical activity levels and have no proven negative influence on pain and disability in NSLBP sufferers. Patients in both groups receive concomitant best evidence healthcare according to the Dutch guideline for low back pain  and has been approved by The Central Committee on Research Involving Human Subjects (CCMO), named METC Zuyderland‐Zuyd with Approval Number NL58005.096.16.All amendments, protocol deviations and SUSAR's will be notified to the METC Zuyderland‐Zuyd. The investigator will notify the METC Zuyderland‐Zuyd of the end of the study within a period of 8 weeks. The end of the study is defined as the last patient's last visit. In case the study is ended prematurely, the investigator will notify the METC Zuyderland‐Zuyd within 15 days, including the reasons for the premature termination. Within 1 year after the end of the study, the investigator will submit a final study report with the results of the study, including any publications/abstracts of the study, to the METC Zuyderland‐Zuyd.This research does not receive any specific grant from funding agencies in the public, commercial, or non‐for‐profit sectors. This paper presents independent research for which no conflicts of interest are to declare.3This project is the first comprehensive research project to assess whether social support influences adherence to an activity advice outside the clinical setting without supervision of a healthcare provider. Unique in this study is that intervening on illness beliefs will be provided to patient and partner to minimize the influence of maladaptive illness beliefs on adherence. Literature mentions that intervention on illness beliefs changes outcomes of the DIPQS, however, responsiveness of the questionnaire is lacking adherence are operationalized in existing literature resulting in variable outcomes (Essery et al., Strengths of the study are the concealed allocation and the randomization. To reduce the presence of observer bias, measurements will not be performed by the treating physiotherapist.Treatment of both groups in one physiotherapy practice is preferred to overcome selection bias. This implicates that despite the standardized protocol and the strict requirements to ensure blinding, it is possible that patients and treating physiotherapist exchange information about the interventions. This may influence outcomes and is therefore a limitation of the study.4When effectiveness of social support on adherence in NSLBP patients is demonstrated, in a patient centred approach, it can help to achieve patients' health goals.A simple low‐cost intervention, not requiring additional education, can be added as a treatment option to increase patient relevant outcomes.Ben van Koppen, is a first author, Pim Zandwijk is a second author and Henk van Mameren, Rob de‐Bie are guarantor of the study protocol.This paper presents independent research for which no conflicts of interest are to declare.All amendments, protocol deviations and SUSAR's will be notified to the METC Zuyderland‐Zuyd. The investigator will notify the METC Zuyderland‐Zuyd of the end of the study within a period of 8 weeks. The end of the study is defined as the last patient's last visit. In case the study is ended prematurely, the investigator will notify the METC Zuyderland‐Zuyd within 15 days, including the reasons for the premature termination. Within 1 year after the end of the study, the investigator will submit a final study report with the results of the study, including any publications/abstracts of the study, to the METC Zuyderland‐Zuyd.Not applicable.Not applicable.The study is registered at ClinicalTrial gov NCT02996955.Supplementary MaterialClick here for additional data file.


The relationship of sagittal spinal shape in the pathogenesis of adolescent idiopathic scoliosis (AIS) is recognised. What is not clear is the relationship between the sagittal shape of those without scoliosis and the potential development of AIS, including the greater prevalence in females. The use of a new parameter, the kyphosis–lordosis (KL) difference, was developed to explore this further.The KL difference was calculated for 117 males and 79 females over seven years with 831 measures made. For females, the KL difference, between the ages of 9 and 12 ½ years, decreases from 5° to nearly 0° until starting to climb again from the age of 14 years, back to 5° by the age of 16 ½ years. For males, there is a gradual decline from 9° at age 9 years to 5° at age 17 years. Both age and sex were statistically significant in the development of the parameter. When comparing to previously published data around the true, de-rotated, sagittal shape of the scoliotic spine, the KL difference has utility in explaining the female predominance in the prevalence of AIS. This adds to the weight of evidence behind understanding why AIS develops. The development of the sagittal shape of the spine is a hypothesis for the development of adolescent idiopathic scoliosis (AIS) –4. HowevOur group has described the development of kyphosis and lordosis in the developing spine without scoliosis in a longitudinal fashion over 5 years  using suThis is a re-analysis of prospectively collected longitudinal data of spinal and back shape following two further years of data collection. Ethical approval was via the NRES committee West Midlands—South Birmingham (11/H1207/10). The investigation was of children without spinal deformity, imaged using the ISIS2 surface topography system over seven years. ISIS2 is a radiation free method of gaining 3D information of the shape of the back and spine, with the individual standing unsupported in a comfortable upright stance. The development of kyphosis and lordosis of this group after 5 years of measures has been reported . ISIS2 mUsing a larger data set, the modelling of the development of kyphosis and lordosis was re-examined as previously described . The kypThe group consisted of 117 males and 79 females. Over the seven year period there were 831 measures. Not all were measured at every occasion, although 166 were measured on three or more occasions, comprising 787 of the total.2  for males and 19.7 kg/m2  for females.The age range of all participants was between 9 and 17 years. When all measurements were analysed individually, the mean height was 1.57 m  for males and 1.55 m  for females and the body mass index (BMI) was 20.4 kg/mThe participants were in the main Caucasian, with approximately 3% from a non-Caucasian heritage.Figure There is a paucity of understanding of the normal growth and development of children. Whilst growth standards are published for parameters that can be measured repeatedly without risk to the child (such as the WHO standards ), techniAIS is a condition with a 3D deformation of the shape of the spine, seen as an alteration of shape away from the norm in the coronal, sagittal and axial planes, with the development of scoliosis, a change of kyphosis and lordosis and the development of intervertebral rotation . There iAIS is more common in females . The reaConsequently, the KL difference is a method that examines the magnitude of both the kyphosis and lordosis as a whole. The analysis presented here demonstrates that sex is highly statistically significant in the development of the KL difference. In females between the ages of 12 and 14, the decrease seen shows that the mean value is only just positive with the 95% confidence interval crossing zero. As seen in Fig. It has recently become possible to create and manipulate a 3D model of the spine using orthogonal radiographs from EOS imaging . With a The lack of influence of the point of inflection in this analysis is expected given the work of Sanders  where, aThe results presented here add to the hypotheses describing the sagittal spinal shape as an initiating factor for the development of AIS –4. It isWhat this work does not do is differentiate between whether AIS is secondary to hypokyphosis, as popularised by Dickson , or to aIt is of note that the kyphosis–lordosis index is previously described as the internal dimensions of the thorax using an axial CT image . The KL This work presents the KL difference, a new parameter that describes how the overall sagittal shape of the spine in children without spinal deformity changes significantly with age and sex. This adds to the hypotheses around the development of the sagittal shape of the spine and the association to the development of AIS.The weakness of ISIS2 is the inability to image individual vertebral bodies or the pelvis to quantify any changes in pelvic rotation occurring during the study. This would require serial radiographs in growing children, causing ethical issues such that it would never be performed. Also, this study did not analyse the effects of handedness, weight of school bags or BMI with regards the KL difference. Handedness has been found not to be significant in the development of kyphosis and lordosis separately  and is u


With the advent of rapid multiplex PCR, physicians have been able to test for multiple viral pathogens when a patient presents with influenza-like illness. This has led to the discovery that many respiratory infections are caused by more than one virus. Antiviral treatment of viral coinfections can be complex because treatment of one virus will affect the time course of the other virus. Since effective antivirals are only available for some respiratory viruses, careful consideration needs to be given on the effect treating one virus will have on the dynamics of the other virus, which might not have available antiviral treatment. In this study, we use mathematical models of viral coinfections to assess the effect of antiviral treatment on coinfections. We examine the effect of the mechanism of action, relative growth rates of the viruses, and the assumptions underlying the interaction of the viruses. We find that high antiviral efficacy is needed to suppress both infections. If high doses of both antivirals are not achieved, then we run the risk of lengthening the duration of coinfection or even of allowing a suppressed virus to replicate to higher viral titers. The advent of multiplex PCR assays capable of detecting many different viruses in one patient sample  has led Given the variety in possible coinfection pairs, it has been difficult to determine the effect of coinfections on the severity of clinical outcomes, with some studies finding that coinfections are less severe than monoinfections ,18, otheUnfortunately, there is little guidance for physicians since studies of treatment of coinfections are limited. One retrospective study of influenza coinfections did not find any difference in outcomes between patients treated with an antiviral and those who were not . AnotherIn order to provide better clinical guidance, mathematical modeling can be used to investigate possible treatment strategies. Mathematical modeling with viral kinetics models has previously been used to help assess possible treatments for a variety of viral infections and has yielded actionable guidance for clinicians. Mathematical models have helped develop the triple-drug cocktail currently used for treatment of human immunodeficiency virus (HIV) ,40 and hIn this paper, we use mathematical models to study the effect of treatment on viral coinfections. We assume that antivirals are available for both infections, can be taken and dosed independently, and do not interact. We find that for certain combinations of antiviral drug efficacy, treatment can lead to longer lasting coinfections, suggesting that care must be taken in deciding whether or not to treat coinfected patients.We use two previously published mathematical models of viral coinfections ,58. The T) are infected by either virus at rate In the model, target cells  . This moThis model is similar to the previous model with the exception that we now include superinfected cells ε=εmp by In this study, we are not modeling specific antivirals, but choose to focus on two possible mechanisms of action that are used by several antivirals. We model a drug that blocks infection by multiplying nfluenza ,61, SARSnfluenza ,63, RSV nfluenza ,65, PIV nfluenza , and hMPnfluenza . We alsonfluenza , remdesinfluenza ,69, and nfluenza ,70.Since a previous study found that coinfections of viruses with similar growth rates result in little change in the viral time course of either virus, while coinfections of viruses with different growth rates lead to viral block, we study the two different cases using two different pairs of viruses. Influenza A virus (IAV) and respiratory syncytial virus (RSV) are paired together because their growth rates are similar, so there is a fairly equal sharing of target cells and little change in viral time course when the two occur in a coinfection. Parainfluenza virus (PIV) and human rhinovirus (hRV) are paired together because PIV’s growth rate is much slower than hRV, thus PIV growth will be inhibited in the presence of hRV. The parameter values for all four viruses are taken from  and are For the superinfection model, we assume that the infection rate for superinfecting a cell is the same as the infection rate for infecting target cells. For example, the infection rate for influenza is the same whether it is infecting an uninfected cell or a cell that is already infected with RSV. We also assume that the production rates for singly infected cells and superinfected cells are the same. For IAV/RSV simulations, we use the eclipse transition rate and infectious cell death rate for IAV as the eclipse transition rate and infectious cell death rate of superinfected cells. For PIV/hRV simulations, we use the eclipse transition rate and infectious cell death rate of hRV as the eclipse transition rate and infectious cell death rate of superinfected cells. We also set the cell regeneration rate to 3cells/d ,72, and We also explored the effect of mechanism of action of the antivirals. Since we are including two possible antiviral mechanisms, there are four combinations of treatments: (i) Both viruses are treated with a drug that blocks viral entry, (ii) both viruses are treated with a drug that blocks viral production, (iii & iv) one virus is treated with a drug that blocks viral entry, and the other with a drug that blocks viral production and the reverse.A is the characteristic being measured, such that a value of 1 indicates no change due to treatment, a value larger than 1 indicates an increase due to treatment, and a value less than 1 indicates a decrease due to treatment.The differential equations were solved using the odeint function in python’s scipy package. To assess how well the drugs treated coinfections, we measured the duration of each virus infection individually, the co-infection durations, and the maximum viral loads for each virus. We present these measurements as measurements relative to the untreated value, i.e., We use two previously published mathematical models of viral coinfection. The first model assumes the two viruses compete for the resource of target cells , while tWe first consider these two examples under the assumption that the viruses are competing for target cells (model Equation ). Viral There is a clear difference in the coinfection durations based on mechanism of action of the antiviral—the figures in the first and third rows assume the RSV antiviral reduces infection rate, while those in the second and fourth assume the RSV antiviral reduces viral production. The mechanism of action of the influenza antiviral also changes the predicted coinfection duration, however to a lesser degree. In all cases, it is possible for treatment to lengthen the duration of the coinfection; when the RSV antiviral reduces viral production, the increase in coinfection duration is slight (a maximum of 1.4 times longer than untreated), however when the RSV antiviral reduces infection, the increase in coinfection duration is significant (5.6 times longer than untreated). The longest coinfection durations occur when both antiviral efficacies are high (about 0.98 or 0.99), indicating that treatment of coinfections with a slightly suboptimal dose could potentially lengthen the coinfections.While there are differences in coinfection duration of treated infections caused by different mechanisms of action, the broad treatment outcomes (suppression of infection) are independent of the mechanism of action  with higIn this case, we again see a change in the amount of coinfection duration increase that depends on the mechanism of action, however it now depends on the mechanism of action of the rhinovirus antiviral. When the hRV antiviral reduces infection, we see a much larger increase in coinfection duration (up to 12 times longer than untreated) than when the hRV antiviral reduces viral production (up to 8 times longer than untreated). The large increase in coinfection duration occurs because hRV blocks replication of PIV in an untreated infection. When we treat the hRV infection, PIV has access to more target cells, so the PIV infection duration and viral titer peak both increase . Since tThe possible treatment outcomes for this coinfection differ from the treatment outcomes for IAV/RSV infections. In the case of hRV/PIV coinfection, there is almost no antiviral treatment combination that will lead to the suppression of hRV and only a PIV infection remaining. We also see a much broader range of dose combinations that will lead to complete suppression of PIV, which is not surprising since PIV was already naturally suppressed by hRV. Note that both antivirals need to be essentially 100% effective in this case to achieve a full cure of both infections.We examine the same two cases of interacting viruses using the superinfection model (Equation ). The viSince the untreated coinfection is chronic, treatment does not run the risk of lengthening the infection, as was seen with the previous model. However, there is a wide range of antiviral drug efficacies that will not shorten the coinfection (white band starting from the bottom left corner of the duration graphs). There is some slight variation in the size of this region based on mechanism of action of the antiviral, however mechanism of action does not have as strong an effect in this model as it did in the previous model.Interestingly, the broad treatment outcomes , drug cofound in  and for found in ). Since As noted in the previous section, the broad treatment outcomes  here areWe used mathematical models to examine antiviral treatment of four different viral coinfection scenarios: Viruses with similar growth rates in acute or chronic coinfections, and viruses with very different growth rates in acute or chronic coinfections. We found that antiviral dose combinations that suppress one or both infections are independent of the mechanism of action of the antiviral and also do not depend on whether the coinfection is chronic or acute. Rather, suppression of one or both infections is determined by a reduction of the basic reproductive number below 1.Antiviral mechanism of action and the type of coinfection do, however, affect how infection dynamics change with antiviral treatment that does not fully suppress infections. In particular, we found that it is possible to lengthen the coinfection duration with suboptimal treatment, or even to reveal hidden coinfections if antiviral treatment reduces the blocking effect of a dominant virus. Lengthening of infection durations with suboptimal treatment has been noted in other studies ,55,68,73While this study suggests that the antiviral mechanism of action and dose need to be carefully considered when treating coinfections, there are practical limitations that will play into clinical treatment decisions. There are not necessarily antivirals available for all respiratory infections, and if there are antivirals, they might not use one of the mechanisms of action examined here. For example, there are a number of antivirals for influenza , howeverIt is important to remember that this study models an idealized scenario: Both infections are initiated at the same time with the same amount of virus and treatment is initiated at the time of infection. In reality, patients are likely infected with the two viruses sequentially, which changes the dynamics of the resulting coinfection ,30,57,58Despite the limited scope of the respiratory virus coinfection scenarios examined in this manuscript, our results highlight some of the factors that need to be considered when making treatment decisions for viral coinfections. In particular, we have found that lengthening of coinfection duration is dependent on the antiviral mechanism of action and that possible treatment outcomes depend on the details of which viruses are involved in the coinfection.


The research on the potential of cavitation exploitation is currently an extremely interesting topic. To reduce the costs and time of the cavitation reactor optimization, nowadays, experimental optimization is supplemented and even replaced using computational fluid dynamics (CFD). One of the approaches towards sustainable water treatment is the use of the cavitation reactor with bluff elements mounted on its stator and rotor. The experimental results show that, besides the rotational speed, the spacing of the rotor pins has the most significant effect on the cavitation intensity and effectiveness, while the pin diameter and the surface roughness are less significant design parameters. The present paper uses a simplified CFD approach to investigate the conditions in the reactor and to select the optimal among a number of geometry variations. The availability of water is becoming an increasing problem in the globalized world, both in developed and developing countries. Therefore, an efficient and clean disinfection technology, such as the optimized use of cavitation, would be very welcome as a replacement or in combination with the existing technologies.Cavitation—the appearance of vapour cavities inside an originally homogeneous liquid medium—occurs when the pressure is reduced below the vapour pressure. The liquid medium is then ‘broken’ at one or more points, and ‘cavities’ are formed whose shape depends strongly on the structure of the flow. The vapour structures are unstable, and when they reach a region of elevated pressure, they often collapse violently.1 Expl (ii) blow‐through reactors, and (iii) rotor‐stator reactors. Most of the advanced laboratory‐scale reactors are of the pump and constriction type, where the contaminated water  is forced by the pump through an orifice or venturi constriction where the sample cavitates. Blow‐through devices are essentially identical, but the sample is forced through compressed air. These devices have more controlled conditions and are suitable for scientific studies but cannot be used efficiently in industry. Finally, the most complex are the rotor‐stator devices, which are often already used in pilot tests.Nowadays, different types of cavitation reactors are being promoted by researchers. We can generally divide them into (i) pumping and constriction reactors,3, 4 Nowadays, in order to reduce the cost and time of the optimization process, experimental optimization is supplemented or even replaced by computational fluid dynamics (CFD). Simulation of the physics of cavitation dynamics, which simultaneously involves large density and compressibility fluctuations, turbulence effects, and instabilities at various scales, is still beyond the current state of the art. But in the last 20 years, engineers (mainly from the field of turbomachinery) have developed reliable methods based on CFD to predict the main features of cavitating flows.One of the advantages of hydrodynamic cavitation is its scalability and potential to be used on an industrial scale. However, it is important to realize that the scaling effects and optimization are not exactly easy and inexpensive.11 NowIn the following sections, we first describe the numerical simulation of the flow in a rotating generator with hydrodynamic cavitation (RGHC). We investigate the influence of varying geometrical parameters (number and spacing of rotor pins and the gap between rotor and stator pins) on the flow characteristics inside the RGHC. From these, we select the most suitable combination and test it in the laboratory and on a pilot‐scale device for water treatment.2The investigated RGHC is a centrifugal rotor‐stator type device with bluff elements arranged on the rotor and stator perimeter Figure . The flu where mechanisms of cavitation generation were experimentally determined by high‐speed visualization and pressure fluctuation measurements. Principally, cavitation was found to occur behind the element, presumably in the low‐pressure wake behind, and fluctuating vapour cloud dynamics was attributed to the passing rotor‐stator interaction. Pilot‐scale experimental limitations (expensive part manufacturing) led us to make a scaled down laboratory size RGHC, where experimental analysis analogues to the pilot‐scale experiment was performed. The latter was equipped with a flow meter and pressure transducers to determine the numerical model boundary conditions and with a hydrophone to measure pressure fluctuation used to validate the numerical analysis results. Moreover, the front cover of the reactor was translucent, allowing for visualization with a high‐speed camera. The RHGC in question is schematically shown in the figure below.This type of device was initially investigated on a pilot scale by Gostiša et al.10 whe pointed out the number of pins as one of the most influential geometric parameters on the mechanical cavitation effects such as particle size reduction. Therefore, the number of rotor pins was selected as the first geometrical parameter. Second, gap size was also observed to have a significant effect on cavitation characteristics. It could not be directly observed in the pilot‐scale experiment but via rotor and stator pin diameter variation. Therefore, it was chosen as the second parameter for the present numerical analysis. The parameters and accompanying cases are listed in the table below.The results of the studies on the pilot‐scale device10 poi3CFD software packages offer a wide range of computational models and settings that, along with the increasing computing power of an average computer, enable reliable numerical modelling of engineering problems, including the phenomenon of cavitation. Although the large‐Eddy simulation (LES) and detached‐Eddy simulation (DES) approaches are gaining importance in academia, the large computational resources required still force engineers to use a simpler Reynolds‐averaged Navier–Stokes (RANS) approach to model turbulence. Two‐phase flow is usually averaged to a homogeneous mixture flow and the mass transfer is modelled by a transport equation with source terms based on very rudimentary bubble dynamics physics. we focused our numerical study on the investigation of the dynamics of the interaction between the rotor and the stator (the gap between them) and the interaction among the rotor pins (the spacing between them)—the goal was to determine the details of the influences of the beforementioned parameters on the development of the cavitation cloud and the conditions in the cavitating zone. Since the focus of the interaction covers only a very small part of the device, a simplified 2D geometry was used for the simulations. This has another important consequence—since cavitation is an extremely dynamical phenomenon, time resolution is essential. Reduced computational load enabled simulations with very short time steps (0.5 μs), which further allowed the observation of shock wave dynamics inside the flow tract.Based on the experimental observations on a pilot‐scale device10 we 3.1m), as explained in the section on the two‐phase flow modelling  differential equations. Since the solution for this geometry is very hard to obtain by the direct numerical approach, a RANS modelling approach is used to tackle the closure problem. Therefore, the governing equations are proposed and written as the continuity equation: Section . Many au3.2k–ω) is a commonly used approach in cavitation simulations. In essence, the SST k–ω model proposed by Menter blends k–ω by Wilcox and Chambers and k–ε, with the use of a special blending function. The function switches between the two turbulence models depending on how close to the wall of the domain the flow is. Near wall k–ω is enabled, on the other hand, in the outer region, k–ε model is enabled since it performs better away from the walls. A detailed formulation of blending functions and transport equations for extra variables of turbulent kinetic energy (k), turbulent frequency (ω) and turbulence eddy dissipation (ε) can be found in the literature.The two‐equation turbulent model . Since rotor pins are mounted on a rotating disc, a rotational velocity of 7000 rpm was prescribed with the position of the rotation axis in the centre of the disc . The boundary conditions are schematically shown in Figure 3.6 against the average pressure difference Δp  algorithm was used to couple the pressure and velocity. Generally, we used the second‐order upwind scheme, for spatial discretization of partial differential equations, which gives more accurate results with slightly higher consumption of computer resources compared to the first‐order upwind scheme. Only in the case of the volume fraction equation, the first‐order upwind scheme was used to achieve better stability. The PREssure STaggering Option (PRESTO)! interpolation scheme was used to discretize the pressure, and for the discretization of the volume fraction first‐order upwind scheme was used. For transient solutions, time integration was calculated using the Bounded second‐order implicit transient formulation.Numerical simulations were performed using time‐dependent Reynolds‐averaged Navier–Stokes equations implemented in Ansys Fluent 2021R1 solver. A homogeneous mixture of water and water vapour was considered, and a Schnerr‐Sauer cavitation model24 wit−4 of the iterative numerical solution of the individual equations in each time step of the simulation. The iteration error of less than 0.2% was estimated. The size of the time step of 0.5 μs was used, and results were saved with a 1 μs increment to allow for the shockwave propagation evaluation. After initial calculation for the period of multiple rotor rotations, a period of a single rotation (duration of 8 ms) was found to be sufficient for the cavitation cloud dynamics description.The convergence criterion was determined by observing the evolution of various flow parameters, such as inlet and outlet velocities, absolute pressure, and mass‐flow balance in the computational domain. The monitored flow parameters were always converged after the sum of the imbalance of the transport equations between iterations over all cells in the computational domain fell below 104 Then, a closer look at the local shear is provided, again due to the recent reports on it possibly being a needed condition for contaminant destruction. Finally, a short discussion on the guidelines for further optimization of the rotor‐stator geometry is given. We consider the first case—rotor with 8 pins and a 1.75 mm gap between rotor and stator pins as the representative geometry against which the other geometries are compared.We show and discuss the results of simulations for the five cases, highlighted in Table 30 The4.1First, we take a closer look at the vapour cloud dynamics during the rotor‐stator pin pass. The cavitating conditions can already be roughly evaluated based on integral vapour area across the entire domain over the entire calculation time as listed in the Table  and the fact that this is the most commonly used value in the studies . Also shown is the local pressure field. The rotor pins rotate in the counter‐clockwise direction.To get a deeper insight into cavitation generation mechanisms, a part of the domain was isolated, and the vapour cloud dynamics was investigated Figure . For eac32 andA general  observation of a single, representative, rotor‐stator pin pass Figure , we can In the case of an enlarged gap between the rotor and the stator (case of 4.25 gap), cloud stretching in the wake of the rotor pin was not observed. This is due to the less pronounced streamline pattern and thus the less abrupt pressure change at the SP passing, as described above. Similarly, the case of 16 pins also exhibits less pronounced cloud stretching and, in some instances, even cloud throttling, which is attributed to the short spacing between the rotor pins. Because of that, a sufficiently low‐pressure wake is not able to form and hence the cavitation cloud growth is restricted. At a smaller size, the cavitation dynamics in the growth phase, is still comparable to the 8 and 12 pin cases, while the stretching and detaching of the cloud are less likely to occur. Finally, we can observe that increasing the number of pins from 8 to 12 does not change the topology or the dynamics of cavitation significantly. On the other hand, decreasing the gap from 1.75 mm to only 0.25 mm causes more abrupt pressure change at the passing of the rotor and consequently distinctly prolonged vapour clouds.Further on, temporal pressure and volume fraction dynamics was investigated in the gap area between the rotor and stator. The diagrams in Figure We can notice that the pressure peaks at the moment when the vapour cloud collapses (when the vapour volume fraction drops to zero), which indicates the emission of the shockwave at this moment. Sometimes, a delay occurs between the point where the vapour fraction reaches zero and the pressure peak. The delay is attributed to the collapse happening downstream of the observed point, such as in the case of 8 pin at 0.0015 s. In this instance, the cloud has not yet fully collapsed when the observed vapour reaches zero as the centre of the collapse is not aligned with the observed point. Since the pressure peak occurs at the collapse, the delay between the vapour fraction zero and the pressure peak can be observed. The cloud sometimes even sticks to the stator pin and collapses after being extensively prolonged. In this case, an even longer delay can be observed, as in the case of 8 pin at a time of 0.004 s.The pressure peaks, indicating shockwave emissions, are most pronounced in the case of 8 and 12 pin rotor and even more in the case of the smallest rotor stator gap (0.25 mm). Not only the number but also the peak pressure values are higher in these three cases. Interestingly, regarding the number of pressure peaks, the case with 8 pins seems to be favourable over the case of 12 pins. As previously shown in Table 4.2Since liquid and vapour phases were both modelled as compressible fluids, pressure wave formation and propagation could be investigated. As previously mentioned, pressure waves were attributed to cloud collapse and were indicated by discrete pressure peaks in the time series of pressure averaged in the small area between the rotor and stator pins. Further on, the pressure wave is visually evaluated as shown in Figure One can see that a shockwave forms when the vapour cloud collapses and propagates with the sonic velocity (roughly 1500 m/s) across the domain. Two types of collapse can be distinguished, that is, the collapse of spherical and elongated clouds. Figure An interesting observation can be seen in the case of a 0.25 mm gap, where a spherical cavity collapse triggered a shockwave that collapsed a secondary cavity , which again emitted a shock wave.Since a field image cannot cover the whole amplitude of the pressure peak, an average pressure in a small area is plotted in a diagram form and shown in Figure Looking at both Figures 4.3 The shear is most simply shown by the strain rate magnitude field. The strain rate tensor definition is written as:Recent experimental and numerical studies on the interaction between bubbles on one side and a solid object, liposome, or bacterium on the other side point to the high shear as the dominant mechanism of contaminant destruction.36 TheThe strain rate field exhibits areas of increased strain magnitude behind both stator and rotor pin edges, on the ‘edge’ of the low‐pressure wake behind the pin. The ‘hair‐like’ shaped area of high strain is oriented in the direction opposite to the pin movement. Typical for all cases, except the 4.25 gap is higher strain between the rotor and stator pins, affected by the interaction of the two. Regarding the number of the pins, the hair‐like area of increased strain occurs on every rotor and stator pin, and based on that, we could conclude that the area of the increased strain is higher in the cases of 12 and 16 pin compared to 8 pin. As expected, one observes higher strain rate magnitude in the case of the smallest gap between the rotor and the stator discs (0.25 gap case). Still, the other cases, apart from the one with the largest gap, show the strain to be in the same order of magnitude. Interesting is also a closer observation of the strain rate evolution in a small area between the rotor and the stator discs, and the influence of cavitation itself on its evolution Figure .A correlation between the local vapour volume fraction and the strain rate magnitude can be seen. This is most apparent for the cases of the smallest gap (0.25 gap case) and the 8 pin rotor case. We see that the strain rate peaks as cavitation appears (denoted by the high vapour volume fraction). This particular result shows that cavitation triggers high strain. And it indirectly confirms our previous experimental and numerical observations that cavitation‐triggered high strain rate causes the destruction of contaminants.36Based on the results interpretation, the optimization should proceed in the direction of shortening the rotor‐stator gap and not increasing the rotor pin number or shortening the distance between to consequent rotor elements. The cases with 8/12 pins and the shortest rotor‐stator gap exhibit more cavitation events, namely, vapour cloud onset, growth, and collapse, indicated by larger overall vapour fraction in the cases. Moreover, cavitation cloud collapses are shown to cause high pressure pulses, indicated as discrete pressure peaks in the graph in Figure 5Experimentally obtained data was first used to determine the calculation boundary conditions, and second, validation was performed based on the pressure fluctuation measurement and cavitation phenomena visualization.5.1p), the inlet pressure with an absolute pressure transducer  and the flow rate with an electromagnetic flow metre  is higher than the one measured in the case of 0.25 gap. The latter could indicate the significance of not only pin proximity but also cavitation cloud collapse dynamics in the pressure fluctuation.6A simplified numerical model of the RGHC has been developed and verified with experimentally obtained data. Vapour fraction, pressure, and strain rate dynamics were used to quantify the cavitation intensity and shockwave magnitudes were related to vapour cloud collapse types. Geometrical characteristics number of rotor pins and rotor‐stator gap size were investigated, where lower pin number and smaller gap size were found to have favourable effects on cavitation properties.A future study investigating the link between cavitation properties and effects on the contaminated samples, such as particle size reduction, chemical properties, settleability, etc., is planned. The newly developed tool will be used to evaluate further RGHC improvements in the direction of increased pressure fluctuation level and the number of cavitation events.One of the immediate next steps will be the stator pin number and rotor pin shape effect investigation, while triangular shapes returned promising experimental results. Solely, shape variation might be promising, but it is not likely to increase the number of cavitation events for a factor. That could be achieved by a multiple rotor‐stator cascade device type Figure . PrelimiJurij Gostiša: Conceptualization; investigation; methodology; visualization; writing – original draft; writing – review and editing. Primož Drešar: Methodology; writing – original draft. Marko Hočevar: Conceptualization; methodology; supervision; writing – original draft. Matevž Dular: Conceptualization; funding acquisition; investigation; methodology; supervision; writing – original draft; writing – review and editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.https://publons.com/publon/10.1002/cjce.24572.The peer review history for this article is available at Video S1 Supplementary VideosClick here for additional data file.


Introduction: New oral anticoagulants (NOACs) have shown comparable efficacy to warfarin in the treatment of patients with venous thromboembolism (VTE), stroke and atrial fibrillation (AF). Various studies on quality-of-life improvement in rural patients following the switch from vitamin K antagonists (VKAs) to NOACs have produced inconclusive results. The aim of the study is to assess the impact of switching from warfarin to NOACs on remotely living patients’ quality of life and the burden of travel.Methods: A questionnaire was provided to the patient by their pharmacists. The questionnaire assessed their travel burden and their level of satisfaction with their treatment.Results: The switch from warfarin to NOACs reduced the burden of travel in 75% of patients. A total of 66% of patients were hesitant about the efficacy of their warfarin treatment. The inconvenience caused due to international normalized ratio (INR) monitoring was reduced in 83% of patients; 70% and 72% of patients strongly agreed that NOACs improved their adherence and treatment satisfaction, respectively. The average number of patients’ travels for INR testing for warfarin monitoring was 7.27 trips/year. The average number of trips made by the patient to obtain their NOACs and warfarin scripts was 2.1 and 4.81 trips/year, respectively.Conclusion: The switch from warfarin, a VKA, to NOACs in patients who live in remote areas without medical services improved their quality of life, decreased their travel burden, and increased their treatment satisfaction and adherence. Switching to NOACs reduced the number of trips travelled by patients to obtain their anticoagulation scripts and/or to adjust their doses. Warfarin, a vitamin K antagonist (VKA), was the main oral anticoagulant used for the prevention of venous thromboembolism (VTE) for decades. Recently, the new oral anticoagulants (NOACs) were released to the market and showed potential as a therapy for VTE . In AustVKAs reduce the risk of VTE coagulation through the inhibition of the action of vitamin K in the coagulation cascade. On the other hand, dabigatran acts as a direct thrombin inhibitor while rivaroxaban and apixaban act as competitive inhibitors of factor Xa . The usuGebler-Hughes et al. assessed patients’ perception of long-term treatment with NOACs versus warfarin and whether their residential status might affect the transition from warfarin to NOACs. The study revealed that 82% of rural patients and 50.3% of urban patients had not heard about the NOACs. The transition from warfarin to NOACs showed fewer benefits in these cohorts of patients .Keita et al. used three validated questionnaires to evaluate satisfaction, adherence, and quality of life in VTE patients who were using VKAs or who were transferred from VKAs to NOACs and vice versa. The study showed that NOACs are non-superior to VKAs in patients’ satisfaction. Patients who switched from VKAs to NOACs and who used NOACs for at least three months showed the highest adherence. In relation to the quality of life, NOACs were superior to VKAs .Bellinge et al., in a retrospective study, assessed whether or not the distance from the medical services impacted the determination of the anticoagulation treatment. The study found that 29% versus 36% P = 0.08) had been prescribed warfarin versus NOACs for patients who do not live locally. Moreover, the study showed that NOACs are superior to warfarin in relation to bleeding events in the patients while they are on the anticoagulation regimen (nine events for NOAC therapy vs 20 events for warfarin therapy). The study found that only one-third of the patients eligible for anticoagulation had received NOACs [ had beenThe purpose of our study is to assess the impact of the switching from warfarin to NOACs on patients who are living in remote Australian areas where medical amenities are not available in their suburbs. Our goal in this research is to evaluate the outcome of this switch on the burden of travel incurred by those patients and their satisfaction after the switch. We expect that the switch from warfarin to NOACs will reduce the travel burden and increase patients’ satisfaction with their treatments. Thus, the present state of knowledge would benefit from further evaluation of the relevance of NOACs for rural patients.This was a survey-based study, and the Oceania University of Medicine (OUM) Institutional Review Board committee granted an approval (no. 20-0619SK). The participants of the study were selected from remote places in Australia where there was no medical practitioner and no pathology services were available to patients to undergo the INR testing. The remote locations included were Ariah Park, New South Wales (NSW); Ardlethan, NSW; and Stanley, Tasmania, Australia.We included patients who initially received warfarin as an anticoagulant and then switched to NOACs in the last 24 months and had used warfarin for at least six months. Patients were excluded if they have not used warfarin before and were currently taking NOACs. We also excluded patients who were taking warfarin and had never taken NOACs before and patients who switched from warfarin to NOACs more than 24 months ago.The questionnaire was offered to patients by their attending pharmacist. Informed consent was obtained from the interviewed patients and their anonymity was guaranteed. The pharmacist explained to the patients that there were two sets of questions to be answered. In the first set of questions, patients were asked to choose the answer that best described their perceptions, while in the second set of questions, they were asked to give a numerical answer to the questions. The pharmacists did not intervene with the answers of the patients and the patients were offered a private area in which to answer the questionnaire.The questionnaire (provided in the Appendix) was designed to assess our hypothesis in the study; it included six Likert style questions and five numerical questions ,10. The The total number of participants was 53, with 25 male participants (47%) and 28 female participants (53%). The average age of the patients was 69.88 years. After the switch from warfarin, 21 patients received rivaroxaban 20 mg once daily, 30 patients received apixaban 5 mg twice daily, and 2 patients received dabigatran 150 mg twice daily.The study utilized a questionnaire; therefore, the results are based on patient perceptions. Self-evaluation is always a potential concern or limitation for studies that utilize questionnaires for data collection. In order to further evaluate the patients’ responses against a more quantitative measure, we included the second set of numerical questions. For example, patients were asked whether they felt travel was burdensome owing to duration or frequency in the Likert format questions. The numerical questions obtain data on the actual number and duration of trips. If patients answered that questions about dosage led them to doubt the efficacy of their warfarin treatments, this perception could be compared with the actual number of trips they had to make in order to measure or adjust the warfarin dose. Figure In relation to the switching from warfarin to NOACs and their effect on the reduction of the travel burden, 75% versus 25% of the patients strongly agreed and agreed, respectively, that switching to NOACs had reduced their burden of travel.The data regarding the inconvenience incurred by the travel to adjust the dose of warfarin based on the INR measurement showed that 83% and 17% of the patients strongly agreed and agreed, respectively, that the travel to measure the INR was inconvenient. Patients were asked whether the uncertainty about correct warfarin dosing caused them to question the efficacy of their treatment. We observed that 66% and 34% of the patients strongly agreed and agreed, respectively, that the dose uncertainty caused them to question the treatment efficacy.Assessing the effect of the switch from warfarin to NOACs on the patient’s adherence, 70% of the patients strongly agreed that their adherence was improved by the switch and a further 30% agreed that the switch to NOACs had improved their adherence. We noted that 72% and 28% of the participants strongly agreed and agreed, respectively, that the treatment with a fixed dose had increased their satisfaction with their treatment. Regarding the effect of the NOACs on the improvement of the participant’s quality of life, 72% of the patients strongly agreed that the NOACs had improved their quality of life and 28% of the participants agreed that the new treatment had improved their quality of life. Figure On average, patients travelled 7.41 times to measure their INR per year and the range of the number of travels was 3-12 times per year. Comparing the number of trips travelled by the patients to obtain warfarin scripts versus NOAC scripts per year, the average number of the trips was 4.9 versus 2.2 trips, respectively. The mean number of times that the patients found that the warfarin dose needed to be changed was five times per year with a range of 2-10 times. The average number of hours spent on travel by different participants per visit was 3.81 hours with a range of two to seven hours per trip.In rural Australia, where there is a shortage in medical services in a patients’ vicinity, patients need to travel to have their INR measured. Travelling may be considered a burden on the patients and may affect their quality of life. The assessment of whether switching from warfarin to NOACs can reduce the burden of travel showed that 75% of the participants strongly agreed that their burden of travel had been reduced by switching their treatment to NOACs. The remaining 25% of the patients agreed that switching had reduced their burden of travel. Patients used to travel an average of 7.41 trips/year to measure their INR where the minimum number of trips was 3 trips/year, and the maximum number of trips was 12 trips/year. Considering the NOAC dosage does not require such INR monitoring, the high number of patients’ trips/year to monitor their INR can reflect why the switch from warfarin to NOACs reduced the patients’ burden of travel. That finding is augmented by the fact that 83% of the patients strongly agreed that the travel to test for their INR was inconvenient and the remaining patients (17%) agreed that the travel to adjust their INR was inconvenient.Mearns et al. found that about 61% of the time, the patients treated with VKAs were found out of the optimal range of the INR. The poorer the INR control was, the greater the chances of VTE recurrence were . We expeGebler-Hughes et al. concluded that there were no benefits from the transition from warfarin to NOACs. However, 82% of the rural recruited patients had not heard about the NOACs at the time of the study . In our Keita et al. found that the switch from VKAs to NOACs did not improve patients’ satisfaction . HoweverIn relation to the assessment of patients’ adherence and quality of life upon switching to NOACs, NOACs were found to improve the quality of life of the patients and increase their adherence. A total of 70% and 72% of the participants strongly agreed that the switch to NOACs has improved their adherence and quality of life, respectively. Our findings for the improvement of adherence and quality of life were similar to the finding of Keita et al. [In Australia, Medicare coverage greatly contributes to the cost of the medications; for example, the cost of NOACs and warfarin on the national Pharmaceutical Benefits Scheme (PBS) is the same for pensioners . In our Our study’s main limitation was the small population in the remote areas of Australia. Future studies on more locations may include a larger number of participants. Moreover, studies assessing the effectiveness of a treatment strategy or plan in the future might need to account for travel burdens in remote populations.The switch from warfarin to NOACs by rural Australians who did not have medical services in their vicinity improved their quality of life, adherence to their medication and increased their satisfaction with their treatment. Patients who were treated with warfarin showed high hesitancy about the efficacy of their treatment and they needed to adjust their warfarin dose frequently per year. Switching to NOACs reduced the number of trips made by patients by eliminating the need for INR measurement and by reducing the frequency of trips to obtain scripts for anticoagulants and/or their dose adjustment. Patients' perceptions of the switch were found to be unanimously positive. The numerical data both support patient perceptions and provide a plausible explanation.


A number of recent studies report enhancement of chemical reactions on water microdroplets or inside nanobubbles in water. This finding promises exciting applications, although the mechanism of the reaction acceleration is still not clear. Specifically, the spontaneous combustion of hydrogen and oxygen in nanobubbles opens the way to fabricate truly microscopic engines. An example is an electrochemical membrane actuator with all three dimensions in the micrometer range. The actuator is driven by short voltage pulses of alternating polarity, which generate only nanobubbles. The device operation is, however, restricted by a fast degradation of the electrodes related to a high current density. Here it is demonstrated that the actuator with ruthenium electrodes does not show signs of degradation in the long-term operation. It is the only material able to withstand the extreme conditions of the alternating polarity electrolysis. This property is due to combination of a high mechanical hardness and metallic conductivity of ruthenium oxide. The actuator combines two features considered impossible: on-water catalysis and combustion in a microscopic volume. It provides an exceptional opportunity to drive autonomous microdevices especially for medical or biological applications. Acceleration of organic reactions has been revealed in microdroplets in air5. It has been reported also that hydrogen peroxide can be formed spontaneously in microdroplets with a size from 1 to 20 μm6. These findings open new possibilities for biological and environmental applications, although the acceleration mechanism is still unclear. Moreover, unexplained chemical processes have been observed in bulk nanobubbles (NBs) with a size smaller than 1 μm. Shrinking air microbubbles are able to produce OH radicals without any external stimuli as was observed with the electron spin resonance spectroscopy8. It has been confirmed independently with probe molecules that OH radicals are produced by NBs filled with air, O9. The formation of free radicals is a puzzling phenomenon since no high-energy sources in the system.Today, there are a number of reports on unusual chemical activity of aqueous interfaces for objects with a high surface-to-volume ratio11. The heat produced by the reaction has been measured using microfluidic devices13. The normal combustion reactions in such a small volume cannot be supported, because the heat escapes too fast via the bubble walls15. The smallest bubble, where it was possible to ignite the normal combustion, had a size of 2 mm16. Nevertheless, the combustion proceeds spontaneously in NBs without a significant increase in the local temperature (see review17 for details).Spontaneous combustion of H12; such an actuator can be small , fast, and strong. The most advanced piezo actuators cannot be smaller than a few millimeters to produce a reasonable stroke21. They also need a high voltage to drive them. The actuators using the electrostatic forces are weak24, but those using the thermal principle are slow26. The electrochemical actuators are notoriously slow32, since the gas can be produced fast in a closed chamber, but one needs minutes to get rid of this gas even using the electrodes with catalytic properties34. We demonstrated the electrochemical actuator that uses the spontaneous combustion of gases in NBs and has the response time comparable with that for the piezo actuators35. However, the principal problem of such devices is a fast degradation of electrodes. On one side, the energy deposition from exploding NBs provides significant local stresses36 on chemically inert electrodes such as Pt. On the other side, harder materials are oxidised resulting in the reduction of the current37 as it occurs for Ti.The spontaneous combustion has been proposed to use as a basic principle for a new actuator to drive microdevicesIn this paper we present an electrochemical microactuator that produces hydrogen and oxygen NBs in a tiny closed chamber and consumes these gases during milliseconds via the spontaneous combustion process in NBs. The device uses ruthenium electrodes, which do not show signs of degradation because of high hardness and metallic conductivity of ruthenium oxide.The actuator is fabricated on a 460 μm thick silicon wafer covered by a thermally grown SiOmentclass2pt{minim35. Finally, the block with the membrane is bonded to the SU-8 layer using the method proposed in38.The side walls of the chamber and filling channels (100 μm wide) are made in 16 μm thick layer of SU-8 3005 photoresist. The chamber is sealed by a flexible polydimethylsiloxane (PDMS) membrane with a thickness of 30 μm. To do the sealing a separate Si wafer is covered by a 30 μm thick PDMS layer. PDMS Sylgard 184 by Dow Corning is used with a base and curing agent mixed at 10:1 ratio. A thick (4.7 mm) auxiliary block of PDMS with a circular opening (8 mm in diameter) located in the center is bonded to the 30 μm layer of PDMS. Due to weak adhesion between Si and PDMS the block covered by the 30 μm membrane can be easily separated from the wafer. The procedure has been described in detailTo test degradation of different electrode materials, special samples containing the concentric electrodes have been fabricated. These samples did not include the chamber and they were tested in a Petri dish with a sufficient amount of electrolyte. For Ru, Ti, and Au a 100 nm thick top layer has been deposited on 500 nm Al layer. For Cu the electrodes had a thickness of 500 nm (deposited on 10 nm Ti adhesive layer) and did not include the Al sublayer.A molar solution of sodium sulfate in distilled water is used as an electrolyte. The conductivity of the electrolyte measured with Mettler Toledo SevenMulty is 39. The driving voltage and current flowing through the electrodes are registered by a 4-channel USB oscilloscope 5444D. Two other channels are used to capture the output signals of the homodyne quadrature interferometer that is used to measure the membrane deflection. The optical scheme of the interferometer and the way to calculate the deflection from the raw signal are described in35. For the interferometric measurements a 20 nm thick layer of Al is deposited onto the membrane to increase its reflectivity.The actuator is driven by a homemade computer-controlled generator. The voltage pulses applied to the contact pads are generated by STM32F051R8T6 microcontroller using direct digital synthesis method and amplified 20 times by a built-in class AB power amplifier described previouslyDegradation of the electrodes is inspected visually using an optical microscope equipped by a camera Moticam 1SP. Chemical composition is determined using an energy dispersive X-ray spectrometer Oxford Instruments INCA x-ACT mounted into the scanning electron microscope (SEM) Zeiss Supra 40. The measurements are performed at an accelerating voltage of 6 kV. The surface of electrodes is analysed with SEM and atomic force microscope (AFM) Smart-SPM 1000 (AIST-NT). The latter is used in the tapping mode.10. In contrast with the normal electrolysis, the high density Faraday current generates H17. The size of NBs has been measured with the dynamic light scattering39 as 60–80 nm.The voltage applied to the electrochemical cell in the AP process is shown in Fig. mentclass2pt{minimThe chip prepared for testing is shown in Fig. mentclass2pt{minimT is the current temperature, 12. The same reaction is responsible for a rapid reduction of pressure in the chamber when the pulses are switched off.The current increase with time is explained by heating of the solution. If the electrolyte in the near electrode region is heated, the Faraday current grows owing to the increase of the conductivity as mentclass2pt{minim40, but still they merge slowly by the Ostwald ripening mechanism. When two bubbles are separated by a liquid membrane, they can exchange the content by diffusion with a rate, which is proportional to the concentration difference of one and the same gas in the bubbles. For the bubbles with the same gas this difference is small, but for the bubbles with different gases the difference is maximum and oxygen from one bubble will diffuse to the bubble with hydrogen and vice versa. As the result both bubbles will disappear in the combustion reaction.Figure 17.All the gas produced in 20 ms disappears from the chamber in 80 ms. Thus, for given geometrical characteristics of the chamber and parameters of the driving pulses the device can perform an up-and-down cycle in 100 ms. The stroke of the membrane increases with the amplitude of the pulses as shown in Fig. f) are applied in series repeated with the frequency f alternating polarity pulses, then during the passive time For practical applications the actuation has to work in a cyclic regime when the membrane is moving up and down with a certain cyclic frequency entclass1pt{minima39. Taking into account the Laplace pressure in a NB one finds the number of molecules in a bubble as 10. The concentration of the survived NBs averaged over the volume Simple estimates based on the Faraday and ideal gas laws support the description presented above . After 30 min processing Ti electrodes becomes darker due to oxide formation43. The dissolution occurs also for W44 but at a lower rate. This property makes impossible to use these materials as electrodes.It is possible to separate the third class of electrode materials. One could expect that the hardest materials like Cr or W will suit the best for the electrodes, however, it is definitely not the case. Chromium is going very fast (in seconds) to the solution because of anodic dissolutionFrom the analysis above one can conclude that Ru is the most promising material for the electrodes, which is able to withstand the high current density of the AP electrochemical process.45, but stability of this material in the AP electrochemical process has not yet been tackled. In this relation we analyse the long-time performance of the actuator with ruthenium electrodes.Ruthenium is a somewhat harder material on the Mohs scale than titanium, but in contrast with TiOThe actuator has been tested in a 5 h run for cyclic operation at mentclass2pt{minimComparison of the electrode surfaces before and after 5 h operation at 17. The current during the 4 h test is shown in Fig. The situation is different for the lower driving frequency 39, where the average bubble size has changed from 80 nm at U needed to reach the critical density.The reduction of the bubble size with the increase of the driving frequency agrees well with both expectations and observations. This dependence has been observed with the dynamic light scattering methodmentclass2pt{minim12. Thus, we believe that the cyclic frequency of the actuator with Ru electrodes can be significantly increased without offset of the membrane.Operation of the actuator at high cyclic frequencies is very important for practical applications. For the investigated device the preferable frequency is not more than 10 Hz; otherwise, the middle position of the membrane will be lifted up that is not always possible in normal operation. For fast recombination of HThe acceleration of chemical reactions on water microdroplets and formation of radicals in nanobubbles have been discovered in the last decade. The observed chemical processes demonstrate unusual activity of the gas-water interface for objects with a high surface-to-volume ratio. Although the reason for this activity is not clear, it is possible to use the phenomenon for practical purposes as demonstrated in this paper. We fabricated an electrochemical actuator that generates NBs producing pressure in a closed chamber and annihilate the gases in the spontaneous combustion of hydrogen and oxygen in NBs relieving the pressure in milliseconds. In comparison with the existing electrochemical actuators the response time is reduced orders of magnitude. However, the main problem of such an actuator is the degradation of electrodes. All but one tested materials degrade in a minute or so of continuous operation of the actuator because of a high current density in the AP electrochemical process. It was found that only ruthenium electrodes have no signs of degradation in the long-term test. The longevity of Ru is due to a unique combination of high hardness and metallic conductivity of ruthenium oxide. Nevertheless, even this material started to degrade, when the frequency of the driving pulses was reduced to 12.The actuator breaks prejudices that the gas-water has no catalytic properties and that the combustion of hydrogen-oxygen mixture is not possible in volumes smaller than a few microliters. In this sense, the realization of the actuator has a fundamental significance. On the other hand, thanks to its small size the actuator can be used for practical purposes as an engine driving autonomous microfluidic devices in medical and biological applications. Moreover, the dimensions of the actuator can be scaled down further, if one will use thinner and stiffer membrane, for example, made of silicon nitride as that used inSupplementary Information.


Understanding changes in extreme compound hazard events is important for climate mitigation and policy. By definition, such events are rare so robust quantification of their future changes is challenging. An approach is presented, for probabilistic modelling and simulation of climate model data, which is invariant to the event definition since it models the underlying weather variables. The approach is based on the idea of a ‘moving window’ in conjunction with Generalised Additive Models (GAMs) and Bayesian inference. As such, it is robust to the data size and completely parallelizable, while it fully quantifies uncertainty allowing also for comprehensive model checking. Lastly, Gaussian anamorphosis is used to capture dependency across weather variables. The approach results in probabilistic simulations to enable extrapolation beyond the original data range and thus robust quantification of future changes of rare events. We illustrate by application to daily temperature, humidity and precipitation from a regional climate model. For instance, efforts such as the Coupled Model Intercomparison Project Phase 6 or CMIP6 makes use of 134 climate models from 53 modelling centres . To bettinvolved . One exaInterest usually lies in events that are rare and therefore in some sense extreme. Despite the amount of available data, say in CMIP6, quantifying the occurrence of rare events may result in very high uncertainty — depending of course on the rarity of the event. The frequency of warm–dry days for instance might not cause an issue, however the frequency of rocesses . This emAn appealing way of remedying this problem is by statistical (probabilistic) modelling of the climate model output. Assuming that such probabilistic models can reliably capture the statistical properties of the climate model data, they can then be used to stochastically simulate as many realisations as necessary of the original raw variables. Then, quantification of rare and/or compound events is performed by computing the associated frequency of such events from the many realisations e.g. . Such anFor such an approach to be of practical use however, certain requirements have to be taken into account. Firstly, the method needs to be scalable, i.e. robust to the size of the data. This is extremely important with the continuously increasing resolution of climate models and the multivariate nature of compound events. Second but equally important, the approach needs to be interpretable otherwise the utility of its outputs cannot realistically be quantified. We classify interpretability in two ways: (a) the method must be transparent and therefore reproducible and comprehensible by its user and (b) uncertainty must be fully quantified and so the method needs to be probabilistic. If we are to simulate events, we must be more uncertain about ones further away from the range of the data. We must be more uncertain about future changes, when for instance we compare 10-year versus 20-year periods since in the latter case there are more data. Third, we must be able to fully assess the ability of the approach to capture the nature of the data in terms of statistical properties such as tail behaviour, spatial structure, temporal correlation and so on. We argue that this can only be done rigorously if the simulations are probabilistic. Fourth, the approach must be flexible enough to be applicable across a range of meteorological variables with potentially very different statistical properties/distributions.In this paper, we present an approach motivated by these requirements. The method is based on using the well-established ideas of a moving window and of Generalised Additive Models utilising penalised smoothing splines. A Bayesian interpretation of the method provides full quantification of uncertainty and therefore posterior predictive model checking. We demonstrate that the method is both scalable and paralleliseable and we present it in quantifying changes in regional climate model output over the UK. Due to the simplicity of its components, it can be argued that the approach is highly interpretable and robust to the choice of variables, and we illustrate this using temperature, relative humidity and precipitation — variables which are known to have very different statistical behaviour. Furthermore, we use the concept of Gaussian anamorphosis to capture dependence across climate variables and show this by quantifying future changes in compound events such as warm–dry days in the UK.1Statistical analysis and modelling of climate model output is a well-established concept and includes methods for downscaling , bias co1.1Hazard events often have very different temporal and spatial scales compared to the variables they are functions of. For instance, from daily mean temperature ght risk  as well ght risk .The procedure has the added benefit of being invariant to the definition of the hazard, since the simulation is of the original raw variable. We could for example change the definition  process .This simulation approach is often termed Monte Carlo simulation, since in practice it is akin to the method of Monte Carlo numerical integration . The ideNote that the idea of using stochastic weather generators for quantifying hazard events and the associated effects of climate change is well-established. For instance, 1.2Statistical modelling can be used to learn the appropriate probability distribution that describes the nature of the data. The primary difficulty lies in finding a statistical model that compromises complexity and scalability. Complexity is necessary as it provides the appropriate flexibility to adequately capture the nature of the data, albeit at computational cost. Moreover, even simple methods such as linear regression have limits to how well they scale with really big data sets, typical of those being outputted by climate models today.There are a few things to consider when defining a statistical model for weather data. Firstly, for a single variable  we require that the spatial, temporal and also space–time structure are adequately captured. This requirement alone is enough to warrant rather expensive and complex statistical methods, such as Gaussian Process models or Markov Random Field based approaches . The sitA further challenge is to have a unifying method with which to assess that the above properties of the data have been adequately captured, preferably without adding to the computational burden. Model checking is an important part of any data modelling approach, but here extra emphasis needs to be placed on this aspect. We must have confidence that the stochastically simulated data closely resemble the original data, otherwise the estimated impact is biased. E.g, if spatial structure is not well-captured because spatial correlation has been underestimated, then the extremes of any spatially aggregated hazard event will be underestimated. In the following section we describe an approach to deal with these challenges.22.1The biggest challenge with big data is scalability. One simple but arguably effective way to deal with big data is to subset them. Given the space–time structures in climate model output, we propose here to use a moving window approach over subspaces of the space–time domain. A moving window concept is by definition very scalable — it lies at the heart of modern machine learning methods such as random forests . In its The size of the window is important: too small leads to over-fitting (the extreme being a single data point per window) and too large might not be flexible enough to capture structure in the data. le e.g.,  albeit aHere, we consider using moving windows in a way that is robust to window size. The idea is to have a data model inside each window that can flexibly capture structure in the data (in each window) at low computational cost. Flexibility is crucial, for if the window size is too large, then data points deemed too far (in covariate space) from the prediction point will have less influence in the prediction. We propose the use of Generalised Additive Models (GAMs) that utilise penalised splines as an option.2.2 where GAMs are semi-parametric regression models  where thage mgcv  also inc where As an example, we implement the following GAM in mgcv to the data in Penalised maximum likelihood is used to estimate coefficients ve skill . In pracHowever, recall that the goal here is to be able to simulate values of 2.3The fact that in GAMs we assume a-priori that all unknown functions of the covariates are smooth, can be viewed as a constraint on the values that matrix S . When thmatrix S  and we ccan show  that the ϕ and λ . The R pWith In practice, we use Monte Carlo to approximate 2.4The ability to simulate from the PPD of checking . The ideDoing this for a sequence of quantiles values provides the equivalent of a Q–Q plot that compares the  probability distribution of the data with ones simulated from the model. 2.5We now go back to the idea of a moving window over space and time, within which a GAM is implemented and used to predict the value of the response. We start with the time dimension To investigate robustness to window size, We also consider the behaviour of the approach when discontinuities are present. A discontinuity, such as the one shown in ngle GAM  to the dIn other words, although we know that by increasing the window size we obtain a more optimal estimate of the underlying trend, we now have the potential problem that a too large window size might “under-smooth” the data if discontinuities are present. We suggest that a way to remedy this issue is to ensure that the auto-correlation structure in the data is adequately captured. Auto-correlation  and where (e.g. near the coast). However some dependence may still exist . In our approach, we suggest using Gaussian anamorphosis  from 1545 land-based gridboxes over the UK mainland. The data come from a single ensemble member (number 1) of a regional climate model (RCM) of 12 km horizontal resolution used in the latest UK Climate Projections (UKCP) assessment . The RCMThe idea is to statistically model the projection data over two 11-year periods (1985–1995 and 2065–2075) and compare changes in compound events in the two periods, which are 80 years apart. We will model four variables: mean daily temperature, minimum daily temperature, daily precipitation total and daily mean relative humidity. For each 11-year period, there are 1,529,550 data points. To fully investigate changes to the climate, ideally 30-year length periods would be examined so that multi-decadal variability would be ‘averaged out’ to some extent, but here we are focusing on presenting the method using 11-year blocks of illustrative data.The compound events that we are interested in are1.Mean daily temperature exceeding 24 °C for 3 consecutive days ;3.Seasonal average number of warm–dry days, defined as days where mean temperature exceeds its historical period mean and precipitation is below its historical period mean (index of heat and drought risk ).3.2 where For minimum and mean daily temperature we assume the Normal distribution so that in grid cell 3.3Relative humidity  and temporal (e.g. 15 days) “neighbours”, and also number of knots for  Section  we check Section  is adequFollowing this procedure, we decided upon 5 days and 30 grid cells as a good choice of neighbourhood and 90 knots for 3.6We start by visual inspection of model predictions compared with the data. For brevity we only show this for precipitation but note that similar checks were performed for the other variables. Moving window approaches will generally capture trends well so it is also important to check whether the distribution was appropriate. We can use the Q–Q plots suggested in To check whether the amount of smoothing was appropriate, we check temporal and spatial autocorrelation. Lastly, we also perform out-of-sample predictive checks to more stringently test the model. For instance, we can leave out the grid cell with the highest precipitation value  and check how well its values are predicted. 3.7Having modelled each of the four variables separately, we use the method in Section 3.8After ensuring that the models adequately capture the nature of the climate data, we can now investigate the future changes of the compound events of Section 4We proposed a practical approach based on statistical modelling, for quantifying future changes in compound events from climate model output. The approach is based on well-established statistical concepts and is thus transparent and interpretable. Predictions and estimates from this model can be thoroughly assessed, with straightforward uncertainty estimation of the predicted quantities. In addition, the fact that it is the raw weather variables that are being modelled, the approach is applicable to practically any compound event. Computationally the approach is very scaleable and parallelisable, so that the resolution of the model output is not a barrier.We demonstrated the approach on data from a single ensemble member of a particular regional model, but for a variety of weather variables illustrating its flexibility and thus potential applicability to output from other physical models such as dispersion models. Having simulation-based predictions implies that the method can be applied in the same way to other ensemble members so that all simulations can be blended together. In this way, ensemble variability is also captured in the resulting predictions.The same can be performed for multi-model climate data, where all simulations are put “in the same pot”. Given the Bayesian nature of the simulations however, one can additionally weigh the simulated predictions from each climate model on the basis of prior information regarding how “good” each climate model is. If the weights sum to one, then this is equivalent to Bayesian model averaging and in practice amounts to weighting the percentage of simulations to be included in the pot by the prior weights.The approach does require certain user input, specifically in choosing a-priori the window size. We have presented a prescriptive way of choosing the window size, the uncertainty of which can always be propagated to the final simulations by repeating the modelling for various window-size choices and blending the simulations together. Nevertheless, future efforts will be focused on more elegant ways of choosing the window from the data — preferably during model fitting rather than a-priori.Theodoros Economou: Conceptualization, Formal analysis, Methodology, Software, Writing – original draft. Freya Garry: Conceptualization, Data curation, Writing – review & editing.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Theo Economou reports financial support and article publishing charges were provided by The Cyprus Institute.


Microscopy techniques and image segmentation algorithms have improved dramatically this decade, leading to an ever increasing amount of biological images and a greater reliance on imaging to investigate biological questions. This has created a need for methods to extract the relevant information on the behaviors of cells and their interactions, while reducing the amount of computing power required to organize this information.This task can be performed by using a network representation in which the cells and their properties are encoded in the nodes, while the neighborhood interactions are encoded by the links. Here, we introduce Griottes, an open-source tool to build the “network twin” of 2D and 3D tissues from segmented microscopy images. We show how the library can provide a wide range of biologically relevant metrics on individual cells and their neighborhoods, with the objective of providing multi-scale biological insights. The library’s capacities are demonstrated on different image and data types.This library is provided as an open-source tool that can be integrated into common image analysis workflows to increase their capacities. The effect of the cellular heterogeneity on the spatial organization and function of biological tissues is emerging as a major paradigm to understand complex collective behaviors. This is the case for example in the study of how spatial structure leads to the emergence of germ layers during embryonic development  or in deIn this context fluorescence microscopy has emerged as an invaluable tool to generate large and high-resolution images of two-dimensional 2D) or three-dimensional (3D) tissues. These images have a typical resolution below one micron, with several fluorescence channels being acquired in parallel, often on several planes. As a result it is now common to generate thousands or more multi-channel images that represent a trove of data on the tissues being imaged . This tD or threGeometric networks constitute a natural representation of spatially encoded information that can be used to describe the structure and interactions in biological tissues. The network representation has already been used in a range of settings, ranging from city structure and railways  to flowsHere we describe Griottes, an open-source tool to construct a network representation from imaging data. The routine creates a “network twin” of the imaged sample by extracting individual cell information from segmented images and building a network representation of the tissue. This new object makes it easier to address quantitative questions on complex tissues with single-cell precision. Below we begin by describing the code and its functions, followed by a few examples on how biologically relevant information can be obtained from typical images.A network is a set of objects that are related to each other. Each object is described by a node, which can have several attributes, and the nodes are connected by edges or links that represent the relationship between them. The network is represented by a graph that shows these objects as nodes and their relationships as links. In Griottes we choose to represent each cell by a node that is placed on its geometric center in 2D or 3D. The links in the network represent neighborhood, either through contact or through distance, as described below. Different attributes  can be assigned to the nodes, while the links can contain information about the distance between cells or the length of the common border, as summarized in Table Griottes takes as an input segmented tissue images and builds the graph to represent them. The input to Griottes can consist of data tables, as well as labeled images of nuclei and/or cytoplasms. The inputs can be 2D or 3D, without any impact on the program and a single line of code is enough to generate the graph representation of the tissue.The Griottes analysis pipeline contains two main steps: individual cell attribute measurements step and then the network construction step. These processes and the program inputs and outputs, described in Fig. program documentation).Griottes takes as input the output of standard cell segmentation codes. This can be in either as a labeled image (2D or 3D) or as a data table. The labeled image has at least one channel composed of a zero-padded matrix in which the area occupied by each cell is filled with an integer label that is unique to this cell. A data table containing single cell information can also be used as an input , or graph neural networks (GNNs) to investigate the neighborhood influence on biological properties such as cell division . Once thThe input image has to contain at least a labeled image that contains the information on the segmented cells. This allows the conversion of pixel-based information on the different cell types to the node-based representation of the tissue. In the following paragraphs we summarize the different features that can be extracted using Griottes.The geometric properties can be of particular interest as it is known that cell morphology can be a significant feature of tissues and their characterization can shed light on morphogenesis or mechanical constraints within the tissue . To be aA or B?), or phenotype information (what is this cell’s production of molecule C?). The method is particularly reliable when the cell masks are built from the membranes and the full volume of the cell is recovered. If only the nucleus is stained, then only the information from the pixels inside the nucleus mask is taken into account.If the input image contains supplementary channels containing the fluorescence information, Griottes can measure the mean fluorescence levels inside each cell mask and attribute this number to the cell. This allows for downstream cell classification  in the adult zebrafish brain requires a deeper understanding of the connections between individual cells in the tissue. High-quality in vivo images have recently been obtained by Dray et al. , as showThe image shown in Fig. From the input image, Griottes can also extract the fluorescence in the PCNA channel of the individual cells in the tissue. The distribution of fluorescence intensities is shown in Fig. In images where the whole cells are segmented, as is the case here, it is possible to include the individual cell-cell contact surfaces in the network representation of the tissue. Indeed, cells can have different shapes leading to a high heterogeneity of cell contact surfaces within the tissue Fig. g. Cell-cThe network representation can also be applied to 3D cultures, in the form of spheroids or organoids. These model systems recapitulate many aspects of the in vivo tissue structure, as shown, e.g., by comparing cryo-sections of organs with organoids , 23. In The MSC spheroids, with CD146 and DNA staining  for each cell within the tissue. By repeating the measurement on k0〉=8.0±2.4 neighbors per cell in the outer layer (layer 0) to 〈k6〉=13.3±2.9 neighbors in the inner layers (layer 6 in this case). In contrast to the increase in the number of neighbors as a function of the layer, the cell-cell distance remains stable across the spheroid depth. Indeed, the mean distance, defined by length from one nuclei barycenter to another, varies between 〈l3〉=22.6±2.3 μm and 〈l0〉=23.8±2.6 μm  derived from the Wharton’s jelly of the umbilical cord  were obtained at passage 2. The cells were maintained as previously described . To isolLight sheet imaging has been performed using a commercial version of the Dual Inverted Single Plane Imaging Microscope (DiSPIM)  . Fast single plane acquisition was performed by objective translation via a piezo-electric element over a 150-μm by 0.5-μm increment. This provided a final cubic volume of 248 × 248 × 150 μm of the acquired spheroid.In order to excite the labeled samples, each plane was excited with 100 ms (Camera exposure time) sequential illumination of three discrete wavelengths: 488, 561, and 640 nm corresponding to GFP, RFP, and Sir-DNA, respectively. Chromatic fluorescence selection is done via a set of quad-band filters and dichroics (Semrock DA/FI/TR/Cy5-4X-B) on the CMOS Camera.