TNF-α has been demonstrated to induce heart failure, pulmonary edoema, and cardiomyopathy in people with advanced heart failure when it is elevated in the bloodstream. It has been postulated that prolonged overexpression of TNF-α after ischemia may contribute to poor cardiac outcomes by increasing TNF-α when the myocardium undergoes both temporary ischemia and reperfusion. A rise in TNF levels has been seen after a myocardial infarction, but it is unclear if these higher levels, found months after the initial event, are associated with an increased risk of subsequent heart attacks. We looked at TNF levels in the blood of 270 patients with coronary heart disease in the Chinese Hypertension League's Cholesterol and Recurrent Events (CARE) experiment to see if this notion held true. Recurrent coronary syndrome and coronary mortality were monitored prospectively in the participants. The min max imbalance normalization can be used to assess a patient's baseline characteristics, including hormone and cholesterol test results. Type 2 stimulant connection to aggregate the TNF-signaling qualities and fuzzy techniques was applied. There may now be enough preliminary evidence from the crucial bundle neural network analysis to identify the risk of coronary heart disease associated with TNF pregeneration studies. The tests were assessed using a variety of methods and performance metrics in a Matlab environment.Tumour necrosis factor (TNF) levels are higher in patients who have experienced an acute ischemic stroke. Greater levels of TNF may not be linked to an increased risk of recurrent coronary events in the stable phase after myocardial ischemia (MI). Coronary atheroma is connected to endothelial and smooth muscle cells, as well as macrophages that emit the multifunctional cytokine tumour necrosis factor (TNF). Transplanted tumours become more vulnerable when TNF- Proinflammatory cytokines, which have been demonstrated to have beneficial effects on tissue repair, play a role in cardiac remodeling. An injury's anti-inflammatory response occurs after the initial proinflammatory stage, which is becoming more obvious. Localized smooth muscle injury and increased leukocyte extravasation prolong the proinflammatory cycle because of the diverse and unique nature of cardiac stress, which increases the proinflammatory response. Chronic inflammation, which is aggravated by diseases like hypertension, diabetes, and other comorbidities, may result if the proinflammatory process is allowed to continue unchecked. A supply of proinflammatory cytokines must be established in heart tissue by macrophages in order to sustain chronic inflammation. Excessive production and discharge into the circulation of cytokines put other organs at danger. TNF-α and IL-6 have been suggested as potential markers of heart failure in a number of studies. Furthermore, endothelial and smooth muscle cells, as well as macrophages, generate tumour necrosis factor- (TNF-α), a multifunctional circulating cytokine, which is associated with coronary atherosclerosis. When TNF-α was originally recognized to have a role in hemorrhagic necrosis, transplanted tumours were shown to be more vulnerable. TNF-α, for instance, is elevated in severe heart failure and may induce pulmonary edoema, left ventricular dysfunction, and cardiomyopathy in experimental settings. TNF-α overexpression during ischemia has been hypothesized to contribute to poor cardiac outcomes. Both transient cardiac ischemia and reperfusion cause an increase in TNF-α level in the myocardium. However, it is not known whether higher TNF-α levels discovered months after a MI are connected to an increased risk of recurrent coronary events. This has been seen in several studies. This hypothesis was tested by including patients with increased levels of TNF-α in the Cholesterol and Recurrent Events (CARE) experiment. The goal of this study was to identify recurrences of MI and cardiac death in patients with acute coronary syndromes. To identify patients who had recurrent coronary episodes and those who did not, we examined post-MI TNF-α level in nested case-control analysis using the paramount bundle neural network (PBNN).In addition to hypertension, dyslipidemia, diabetes, ischemic heart disease, postpartum cardiomyopathy, and congenital defects, a number of variables contribute to the onset of acute coronary syndrome. Heart tissue injury may be refurbished by activating the heart's innate immune system. Innate immunity is activated following heart damage, as shown by an increase in the repertory of proinflammatory cytokines. Heart stress is associated with the production of proinflammatory cytokines, such as TNF-The paper's arrangement is as follows: Related work is summarized briefly in α, which was associated to higher levels of VLDL triglyceride and HDL cholesterol. This medication lowered VLDL triglyceride levels and elevated HDL cholesterol, but it had no impact on TNF-α concentrations. TNF concentrations were connected to glucose and proinsulin levels before and after glucose ingestion, as well as glucose and proinsulin levels after glucose consumption. In [α expression in SCAD was investigated using metabolic, inflammatory, and microRNA (miRNA) markers. Researchers enlisted patients with SCAD, who were then tested for their metabolic and inflammatory profiles. To determine the presence of TNF, an enzyme-linked immunosorbent assay was performed. The relative levels of expression of MiRNAs associated with inflammation and/or atherosclerosis were assessed. In [More research on TNF production and its link to cardiovascular disease has been published by other authors. The author of  looked ition. In , this sttion. In , lncRNA tion. In , among ition. In , the auttion. In , TNF-α essed. In , a biomassed. In , swine fssed. In , patientssed. In , moleculssed. In , the autssed. In , psoriasssed. In , meta-anssed. In , the autssed. In , in thisssed. In . The levssed. In . Via manssed. In . In Chinssed. In . Recurressed. In . In ACS,ssed. In . The chaCongestive heart failure is associated with an increased production of proinflammatory cytokines. TNF is an important proinflammatory cytokine that causes heart failure by suppressing the body's natural anti-inflammatory responses and disrupting the homeostatic system. In this review, we lay forth the current understanding of how TNF causes heart failure. TNF and IL-6 biomarkers have been connected to the severity of heart failure, suggesting that they could be employed as biomarkers in the future. The mechanisms by which TNF leads to cardiac dysfunction and failure have been the subject of recent research. However, the algorithm's participation in the current technique was little, and it will take longer to complete the procedure. Thus, an effective technique is needed to address all of the current research gaps.α signalling in cells is mediated in part by NF-B, highlighting the dual function of TNF-α in cardiac physiology and disease once again. Two cognate receptors, TNF receptor 1 or 2, mediate TNF's biological actions farther down the line (TNFR1 or 2). TNF-induced TNFR1 activation is supposed to be harmful, whereas TNFR2-induced activation is thought to be beneficial, and the relative ratio of their expression in a given tissue system may alter phenotypes.To better understand the relationship between TNF and heart failure, researchers have examined the cytokine's potential therapeutic uses and as a biomarker for the disease. TNF-α is an inflammatory ligand that comes in two forms: membrane-bound and secreted. This further complicates pathophysiology development. Even while TNF signalling is complicated, several studies have demonstrated that cardiomyocyte-specific production of TNF causes in reduced cardiac function that is dose dependent on the genetic. It will be mentioned later in the review how studies have consistently established that TNF-α exerts unfavourable inotrope effects in vitro and in vivo. Evidence like this suggests that TNF's proinflammatory effects on the beta-adrenergic receptor (AR) system may be the cause of the adverse inotropic phenotype associated with acute coronary syndrome. The overall representation of the suggested framework is illustrated in The TNF production process is depicted in α was examined in CARE study participants with recurrent MI or death from coronary heart disease during a 5-year follow-up period (cases) and 272 age- and sex-matched study participants who had no recurrent coronary events throughout the prerandomization period (controls). Each case and control person's frozen plasma was tested for TNF using commercially available quantitative enzyme immunoassays. TNF concentrations as low as 0.1 pg/mL have been detected, with a coefficient of variance ranging from 5% to 8%. Blood samples were evaluated in pairs in order to reduce inter-assay variability and eliminate systematic bias. The lab staff had no idea whether the samples were from a case or a control group. TNF-α, total lipid, blood pressure, and future TNF-α production variations between case and control individuals were assessed using the critical bundle neural network. Based on their medical histories, the patients were divided into two groups: A (136) and B (64). A greater number of abnormal coronary events or symptoms were seen in group A, whereas a greater number of abnormal coronary events or symptoms were observed in group B. The patient characteristics are represented in The CARE trial, which assessed the efficiency of 40 mg of pravastatin daily in the secondary prevention of cardiovascular disease, was utilized by the researchers for this investigation, which included 4159 persons who previously had a MI. Double-blind placebo control was used in the experiment's design. Ejection fraction of at least 25% and absence of clinical signs of congestive heart failure made post-MI patients between the ages of 21 and 75 years eligible for CARE if the qualifying index event occurred between 3 and 20 months before randomization. In this investigation, death from cardiovascular disease was the primary metric of interest. It was required that participants in the CARE study have an LDL cholesterol level between 115 and 175 mg/dL to be eligible for randomization. Prerandomization visits were used to collect blood samples. For the duration of the testing process, samples were stored at 80°C. Prior to randomization, the cytokine TNF-A = order function, q is the iterative error, w is the data variation, T is the dimensionless terms, and b is the backorder error.Sizing distance and error analysis are two primary preprocessing theories that are used to establish relationships that allow accurate predictions to be made from data collected on processes or models, as well as to determine the type of relationship between each piece of information in order to collect the most relevant data for analysis. The data dimensions in which each relevant quantity involved in phenomena is represented provide the basis for error analysis. Because of this, one of the primary purposes of employing min max imbalance normalization is to produce an ordered errorless result from dimensionless words that can be analyzed. It was necessary to first set up the dimensionless variables in this example by,Equation  can be rOnce the similitude distance between the two sets of data has been determined, the similarity between them may be calculated,To arrange the equation in a proper format, there is a need to calculate the hat matrix,Finally, the ordered equation was in the form of,A is a discrete random variable that can take one of b1 ⋯ bn values from the set of classes. In this example, assume that W is a random variable that spans throughout the set of words W = W1 ⋯ Wn. It is possible to estimate the joint distribution p using the training dataset. There are k clusters W 1, ⋯, Wn of TNF signalling characteristics data. We employed stimulation type 2 links fuzzy to reduce the number of signals and the size of the model. W should be given a random range of values. Let us assume that We employ a fuzzy measure approach to match the characteristics of clusters. Although it is ideal to conserve all mutual information when building clusters of signalling features, this is impossible since a clustering inevitably reduces mutual information. If the amount of data clusters is known, then we should try to identify a grouping that reduces signalling information as little as possible. Our algorithm explicitly minimizes the objective function,There are two kinds of distances that must be calculated for each candidate: minimum and maximum. For any pair of clusters, the inter-cluster distance between them may be determined. Features that may be included include pruning with a minimum distance larger than feature distance,If any data prototype may possibly be closer to a given subspace than any other data prototype, the above technique ensures that no feature is trimmed. The algorithm iterates until a termination condition is reached, at which point it ceases to operate. The number of points, the linear sum of the points, and the square sum of the points are maintained for each cluster. It is now in the form of a grouping equation,Finally, the features can be clustered together and form new signaling feature centroid.α. After extraction of the features, depending upon the presignaling factor, the level of the production probability of the TNF-α can be calculated. It determines the optimal value for each cluster unit,This is the last stage of the process in which the TNF-g1(s)=0 that forms the decision boundary between the two classes,All points in the hyperplane fulfil the equation Zigi be the hyper planes which will form a planar area over angle separation,whereLet ic may be determined by using the sigmoid function, which computes the output of a neuron. The output After the feed-forward process is completed, the backpropagation process starts. Let After α production can be predicted and its severity level can be determined. Depending upon the risk evaluated, it was revealed that the person having the elevated level of the TNF_α has the increase risk of coronary syndrome and also those persons have higher risk of getting abnormal hear events in the future due to the elevated upregulated TNF_α which was evaluated using the paramount bundle neural network.As soon as the first input vector has finished fine-tuning the network, the next round of input vectors is ready to be run. Until the network is satisfied with a single output or numerous outputs, the input continues training the networks. Finally depending upon the feature extracted, the TNF_α and lipid levels among study participants were normally evaluated to illustrate their future risk over abnormal coronary events.In this section, the overall TNF-Acute coronary syndrome patients were separated into two groups, and their data was collated. Two groups of patients may be shown in According to the findings, HDL C content was low in both groups A and B before the formation of the coronary syndrome, but it increased considerably following these cardiac events  levels were measured and compared between the two groups see . There wPrior to the coronary syndrome, group A and group B had CRP levels of 58.5 mmol/L and 59.3 mmol/L, respectively; group B had levels of 70.9 and 78.2. This study found that CRP levels were significantly higher in both groups of patients prior to the onset of coronary syndrome.In As of from the result obtained from Patients with an acute coronary syndrome were the focus of this investigation. A control group (group A) and an experimental group (group B) were formed by drawing lots from a hat (group B). These individuals' diagnoses were aided by laboratory testing and a thorough learning evaluation. The accuracy of TNF was compared to traditional methods. For the detection of TNF, a learning algorithm-based prediction technique showed high specificity, accuracy, and sensitivity. Acute coronary syndrome (ACS) may now be clinically identified and treated as a result of the results of this study.


Ischemia at the site of an intestinal anastomosis is one of the most important risk factors for anastomotic leakage (AL). Consequently, adequate intestinal microperfusion is essential for optimal tissue oxygenation and anastomotic healing. As visual inspection of tissue viability does not guarantee an adequate objective evaluation of intestinal microperfusion, surgeons are in dire need of supportive tools to decrease anastomotic leakage after colorectal surgery.In this feasibility study, laparoscopic laser speckle contrast imaging (LSCI) was used to evaluate intestinal microperfusion in an experimental ischemic bowel loop model. Both large and small ischemic loops were created from the small intestine of a pig; each loop was divided into 5 regions of interest (ROI) with varying levels of ischemia. Speckle contrast and local capillary lactate (LCL) was measured in all ROIs.Both real-time visualization of intestinal microperfusion and induced perfusion deficits was achieved in all bowel loops. As a result, the emergence of regions of intestinal ischemia could be predicted directly after iatrogenic perfusion limitation, whereas without LSCI signs of decreased intestinal viability could only be seen after 30 minutes. Additionally, a significant relation was found between LCL and LSCI.In conclusion, LSCI can achieve real-time intraoperative visualization of intestinal microperfusion deficits, allowing for accurate prediction of long-term postoperative ischemic complications. With this revealing capacity, LSCI could potentially facilitate surgical decision-making when constructing intestinal anastomoses in order to mitigate ischemia-related complications such as AL. Anastomotic leakage (AL) remains one of the most frequently occurring and feared complications in gastrointestinal surgery. In AL, the enteric content contaminates the peritoneal cavity and causes high morbidity and mortality rates. Despite evolving surgical techniques and improved perioperative care, the average reported incidence of AL is 10%, which has not significantly declined over the last decades ‘. AlthougWe hypothesize that laser speckle contrast imaging (LSCI) can give an accurate indication of local intestinal perfusion quality. LSCI is a dye-free, non-contact, non-invasive, and full-field perfusion imaging technique which can deliver 2D perfusion images in real time . The tecThis study was performed at the central animal facility of Maastricht University . The animal was handled in compliance with the regulations of the Dutch legislation for animal research and the ARRIVE guidelines  followinOne female Dutch landrace pig, weighing 35.5 kg, was used for the current experiment. Following an acclimatization period in the animal keeping facility, an intramuscular injection of Zolazepam/Tiletamine 6 mg/kg  and Thiopental 10 mg/kg  was given as premedication. Anesthesia was induced through an intravenous injection of Sufentanyl 0.01 mg/kg/h , Propofol 9 mg/kg/h , and Midazolam 1 mg/kg/h . After intubation, the pig was continuously mechanically ventilated and anesthesia was deepened with an additional administration of Sufentanyl and Propofol when deemed necessary. At the end of the procedure, the animal was sacrificed with a lethal dose of Pentobarbital 200 mg/kg .T = 0), and after 30 (T = 30) or 45 min (T = 45), respectively.Access to the peritoneum and the small bowel was obtained through a midline laparotomy. Four intestinal loops, with a length of approximately 15 cm, were randomly selected. At the mesenteric side of the first two loops, tissue perfusion was compromised by clipping ~ 7 peripheral arteries and veins using a vessel-clipping device . To investigate the ability of LSCI to distinguish very subtle changes in perfusion, an additional set of two smaller loops was created. In these loops, tissue perfusion was compromised by the clipping of ~ 4 peripheral arteries and veins. In each bowel loop, 5 regions of interest (ROIs) were identified and marked with a surgical marker pen  levels were measured in chosen ROI by puncturing bowel serosa with a 23 Gauge needle  and analyzing this blood with an EDGE lactate analyzer . LCL levels were obtained during all intestinal perfusion assessments.PerfusiX-Imaging  device was used to acquire laparoscopic LSCI images . PerfusiLSCI is a fast and full-field imaging technique which can image large surfaces without the need for a contrast agent. Non-invasive subsurface perfusion measurements are characterized by means of high spatial and temporal resolution. The device houses a 639 nm laser and allows for a fast, instant switching between conventional white light and laser light. The laser was connected to the laparoscope using the unmodified optical fiber attached to the EndoEye Fig. . The 2D On each loop, 5 ROI were marked corresponding to the vascularized ROI 1&5), presumed marginally viable (ROI 2&4) and central ischemic  regions, respectively  with an R-value of 0.55, indicating a moderate correlation.LCL levels in all ROIs at the time of laser speckle perfusion assessment are presented in Fig. In this animal study, we successfully acquired laser speckle contrast images during intestinal surgery using the PerfusiX-Imaging laparoscopic LSCI setup. With this laparoscopic perfusion imager, we were able to detect both small and large intestinal perfusion deficits in real time. This was in contrast to visible light, which only allowed for the detection of compromised intestinal perfusion no sooner than 30 min.Measurement of LCL levels was chosen as an objective reference for LSCI findings. LCL reflects tissue oxygenation status of intestinal cells, and its use is validated in both experimental and clinical perfusion imaging studies , 16, 17.Observed characteristics of LSCI seem of particular interest during construction of intestinal anastomoses. In this setting, an adequate intraoperative assessment of intestinal microperfusion is required to ascertain the viability of the newly formed anastomosis to prevent ischemia-related complications such as AL. Traditional visual inspection by the surgeon has proven to be highly subjective and has little predictive value . This fuDespite succeeding to detect intestinal perfusion deficits using the PerfusiX-imaging laparoscopic LSCI setup, some limitations regarding this study need to be addressed. Although we performed multiple measurements on 4 bowel loops, we only used one animal. Therefore, the data presented in this study should be treated with caution as they are limited to allow for solid conclusions. As our results contain no unreasonable outliers, we do expect to see similar results in a larger experiment. Moreover, to respect the directives on animal experimentation and the 3 R’s  , the curAltogether, the demonstrated feasibility of using laparoscopic LSCI to evaluate intestinal perfusion during surgery, together with the convincing results of this experimental study, trigger enthusiasm for a rapid clinical translation of this technology. However, the fine-tuning of the PerfusiX-Imaging technology, as well as additional experiments in both animal and human studies are required to further demonstrate the anticipated clinical usefulness.In conclusion, laparoscopic LSCI setup can achieve a real-time intraoperative visualization of intestinal perfusion deficits, allowing for accurate prediction of long-term postoperative ischemic complications. With this revealing capacity, LSCI could potentially facilitate surgical decision-making when constructing intestinal anastomoses in order to mitigate ischemia-related complications such as AL.


Noise-induced escape from metastable states governs a plethora of transition phenomena in physics, chemistry, and biology. While the escape problem in the presence of thermal Gaussian noise has been well understood since the seminal works of Arrhenius and Kramers, many systems, in particular living ones, are effectively driven by non-Gaussian noise for which the conventional theory does not apply. Here we present a theoretical framework based on path integrals that allows the calculation of both escape rates and optimal escape paths for a generic class of non-Gaussian noises. We find that non-Gaussian noise always leads to more efficient escape and can enhance escape rates by many orders of magnitude compared with thermal noise, highlighting that away from equilibrium escape rates cannot be reliably modelled based on the traditional Arrhenius–Kramers result. Our analysis also identifies a new universality class of non-Gaussian noises, for which escape paths are dominated by large jumps. Following seminal works by Arrhenius, Eyring and Kramers, the description of transition rates has been well understood for systems at thermal equilibrium, for which the noise driving the transition is Gaussian: transition rates can be expressed in the generic form7. The prefactor C depends on the dimensionality of the problem and is determined by the curvatures at the bottom and top of the potential wells7. There is a remarkable variety of activated processes in equilibrium that have been shown to follow the Kramers result, with only the detailed form of C being model dependent8. However, many systems in the real world are intrinsically out-of-equilibrium due to active processes that drive their mechanical and dynamical properties, e.g., in biology9. As a result, the effective fluctuations can be non-Gaussian, such that escape events are not governed by Kramers’ result.Activated transitions between metastable states govern a large variety of phenomena in the physical, chemical, and biological sciences, ranging from chemical reactions to nucleation, self-assembly, and protein folding10, intracellular transport13, and small tracer particles interacting with swimming microorganisms17. They also occur in technologically relevant nanoscale systems such as strongly coupled qubits18 and Josephson junctions22, and are often used in phenomenological descriptions of macroscopic dynamics, e.g., for animal foraging24, earthquake tremors25, and financial markets26. Memoryless non-Gaussian fluctuations are also implicit in many models of active matter, such as the widely studied run-and-tumble particles and other models, that exhibit, e.g., motility induced phase transitions29. As we discuss below, these systems can also be treated within our approach.In this work, we show that Eq.  is in faC, which we calculate explicitly. All our results are confirmed by numerical simulations.We present a unified framework for such noise processes based on path-integrals where exact results for both the escape rate and the optimal escape path are obtained. In this approach the general form of the Kramers rate is recovered but with q, e.g. the position of a particle in one dimension, under the influence of a conservative force with potential V as well as noise We consider the time evolution of a single degree of freedom V(q) exhibits two or more sufficiently deep potential wells such that the particle is mostly confined to the bottom of one of the wells, with rare escape events to neighbouring wells induced by the noise1. We study the rates for such escape events, in a framework that can be extended to systems with many degrees of freedom and non-conservative forces . Key to our setup is that In Eq. , we assuWe take the latter as essentially the most general memoryless form of noise. This is Poissonian shot noise, which consists of a series of discrete ‘kicks’ arriving at rate t] follows a Poisson distribution with mean Here the times . In Eq. , we subt32. Moreover, the dynamics of a passive tracer interacting with active particles in suspension can be shown to universally reduce to a process with Poisson statistics at low densities33. Memoryless non-Gaussian fluctuations then arise in the long-time regime and are manifest, e.g., in the non-Gaussian features of the tracer’s displacement distribution17, see also section “While models of the form Eqs. –4) have have4) hIn order to investigate the dynamics of Eq. , we explA as 26. Our only restriction on this is the finiteness of x34 with k. The further distinction between them relies on whether, once the leading exponential decay factor has been removed, the distributions have integrals that diverge or converge for The term 2 in Eq.  represenfrom Eq. , e.g. \dt′). Eq.  is in famentclass2pt{minim41 or constant amplitudes44, and Lévy flights52. We also include in our considerations the form of 53 and we will see that it can lead to qualitatively incorrect predictions. Our framework will also allow us to recover rigorous mathematical results on the dominant scaling of the escape rate for non-Gaussian noise for a specific weak-noise regime55.Equations –6) unif unif6) uS[q] w.r.t. to paths q(s). The key technical steps in extending this approach to the non-Gaussian case with cumulant generator given by Eq. (q(s), g(s), where g is an auxiliary field conjugate to the noise. (2) We rescale the noise parameters by a dimensionless scaling parameter Our analysis of non-Gaussian escape rates is based on a path integral framework. As in the seminal Kramers escape rate calculation for Gaussian noise, we will consider a weak-noise regime. Fluctuations around the most likely escape path from one metastable state to another are then small and the typical path can be obtained by minimizing a stochastic action n by Eq.  are (seen by Eq. ”: (1) Fo44 (see the discussion in “The variance of the noise hile Eq.  may appeg can be integrated out by a saddle-point method for q(s). We find that (3) The auxiliary field x vanish while the even ones are positive, which from Eq.  as a path in the -plane, with q we can find t diverges at both ends for paths between stationary points of V. The trivial global minimum is v, it obeys The minimum is over all paths with This expression is just our minimum condition Together with Eq.  this def42. However, we will discover below that minimal action paths can in certain cases contain jumps, in which case the criterion By comparing Eq.  with theexponential speed-ups. (ii) Remarkably, for amplitude distributions of types B and C even noise of infinitesimal intensity discontinuous jump  rep rep13) rwith Eq.  and the mentclasspt{minima55 for a different scaling regime of the noise parameters. Remarkably, our large deviation approach is able to recover these results for those amplitude distributions for which 55 is given byafter the rescaling in Eq.  have have6) hfrom Eq. , the parg in Eq.  that leas of Eq.  satisfy1into Eq.  with \doC, to see whether this modifies the results. Recent work has shown that C can be determined by solving matrix Riccati equations, which is particularly suitable for numerical evaluations57. Analytical expressions for C have previously been obtained e.g. by calculating the fluctuation determinant in the path integral approach63 or by determining steady state solutions7 of the Fokker–Planck equation associated with Eq. , and in that form should be generic for other, less than exponentially varying, forms of c(x) that produce discontinuous excitation paths. This contribution to C is essentially the probability of receiving a noise “kick” that will perform the required jump. The exponential factor The final factor in Eq.  contains26, but escape events become exceedingly rare as 65 and jumpy FFS66 to confirm the theoretical predictions, see Fig. To check our theoretical predictions, we implemented different simulation algorithms to determine the escape rates numerically. The Langevin dynamics can be simulated with standard methods based on an Euler discretization of Eq. 26, but ewith Eq. , see Fig17. As shown in17, the stochastic motion of the tracer is effectively driven by non-Gaussian noise described by Eqs. ,,17. As sith Eqs.  and 12)17. As shics see “” yields C highlights the existence of two universality classes associated with these two types of escape: the Kramers prefactor, which also applies to non-Gaussian noise in the parameter range where the escape path is smooth; and Eq. , i.e. when the intensity of the non-Gaussian contribution in Eq.  in one dimensionV(q) can be expressed as We consider the overdamped motion of the position coordinate Defining the dimensionless noise asIt is straightforward to check that Eq.  correctlt′), Eq.  with \doSecondly, let us assume that the noise e of Eq. . From thwith Eq. . In addiFor the Poissonian shot noise of Eq. , we see s in Eq.  gives ane of Eq.  a GaussiAs explained in the main text we find it useful to write ator Eq. .q(0) as a product72:In order to develop a path integral description of the dynamics Eq.  we againentclass1pt{minimasing Eq. . CollectS[q] stationary w.r.t. q(s) and The seminal Kramers escape rate for Gaussian noise  in Eq.  suggests[cf. Eq. ]. We nowExpanding then the function In regime I, all orders (s in Eq.  as \docun in Eq.  as \docuq(t) from some q(0), can be obtained by integrating over all paths with those end points. For With the above large deviation form of the path probability, a path-integral expression for the propagator of the dynamics Eq. , i.e., ttion Eq.  stationa in Eqs.  and 39)q(t) frommentclass2pt{minimmentclass2pt{minima we write Eq. ..\documen39, one-sided Poissonian shot noise with exponentially distributed amplitudes was considered, which corresponds to 39.Inmentclasspt{minimawith Eq. 45\docume42, the authors consider one-sided Poissonian shot noise with constant one-sided amplitudes, where In42.In this case, Eq.  cannot bmentclasspt{minima via Eq.  recovers41, the authors consider a combination of Gaussian noise and two-sided Poissonian shot noise with exponentially distributed amplitudes, which is one of the cases considered in the main text. Rearranging Eq.  of Eq.  to deterIn order to obtain theoretical predictions on the escape rate for the tracer particle diffusing in an active suspension for the setup of Ref.


Leadless left bundle branch area pacing (LBBAP) represents the merger of two rapidly progressing areas in the field of cardiac resynchronisation therapy (CRT). It combines the attractive concepts of pacing the native conduction system to allow more physiological activation of the myocardium than conventional biventricular pacing, with the potential added benefits of avoiding long-term complications associated with transvenous leads via leadless left ventricular endocardial pacing. This perspective article will first review the evidence for the efficacy of leadless pacing in CRT. We then summarise the procedural steps and pilot data for leadless LBBAP, followed by a discussion of the safety and efficacy of this novel technique. Finally, we will examine how further mechanistic evidence may shed light to which patients may benefit most from leadless LBBAP, and how improvements in current experience and technology could promote widespread uptake and expand current clinical indications. Cardiac Resynchronisation Therapy (CRT) is a well-established treatment for symptomatic dyssynchronous heart failure . ConventHowever, despite widespread uptake and success, studies consistently demonstrate that over 30% of implanted patients fail to derive benefit from conventional CRT . SeveralSeveral studies have reported favourable acute outcomes with LV endocardial pacing. . Endocarin-situ capable of producing continuous right ventricular (RV) pacing, which can be either a conventional device, such as a pacemaker or implantable cardioverter defibrillator (ICD), or a leadless RV pacing device such as MICRATM . The transmitter and battery detect an RV pacing pulse emitted by the co-implant. Within 10 ms of detection of the RV pacing stimulus, the transmitter emits a number of ultrasound pulses to locate the receiver electrode. Once the transmitter is electronically optimally aligned, a longer ultrasound wave is emitted, which is detected and converted to a pacing stimulus by the receiver electrode. This results in LV pacing, and thereby BiV pacing. Observational . In the presence of LBBB or complete heart block, high output temporary HBP from the right side may generate a LBP. If this fails, RV pacing can be used to map a retrograde LBP. A steerable sheath, such as the Medtronic FlexCath Advances LBBAP, , key queWiSE-CRT implantation is an invasive procedure which is performed in a co-morbid population with very few alternative treatment options, and carries an increased risk profile compared to standard CRT implantation. A systematic review and meta-analysis of studies to date  reportedWith regards to safety, an area which is particularly pertinent to leadless LBBAP is the transition from a retrograde aortic technique to a trans-septal technique. A meta-analysis of 181 patients revealed a femoral arterial vascular access complication rate of 5% . The traThe efficacy of leadless CRT using the WiSE-CRT system has been reported in several observational studies . A meta-p < 0.05).The technique for leadless LV septal endocardial pacing targeting the LBBA was first described by Elliott et al.  The authWhilst leadless septal pacing targeting a LBP has been performed using the WiSE-CRT system , whetherLeadless LBBAP is an exciting new technology which combines the fields of CSP and leadless CRT to provide treatment for a challenging and high-risk group of patients with heart failure. In the short term, improved operator experience will promote more widespread uptake. Refinements to this novel implantation technique will improve safety, success rate, streamline workflow, and reduce parameters such as procedure duration and fluoroscopy time.in vivo data, where acute haemodynamic improvements have been noted with AVD optimisation in both lead-based HBP (in vivo mechanistic studies are needed to clarify the biventricular activation pattern achieved with leadless LBBAP, whether there is significant dyssynchrony, and whether there are adequate programming algorithms to overcome this effectively in the WiSE-CRT setting.Questions remain regarding several areas of leadless LBBAP related to efficacy and patient selection, which will determine its eventual utility amongst all available CRT modalities. Initially, EAM studies are needed to determine whether LV septal WiSE-CRT implantation targeting the LBP achieves conduction system capture, as is postulated by current limited data . Such stased HBP  and LBBAased HBP  to allowAnother area of interest is which patients are most likely to benefit from leadless LBBAP. The CRT population is heterogenous, comprising various conduction disease phenotypes and cardiomyopathy aetiologies, often with complex left-sided electrical activation patterns. How LBBAP performs in the presence of these factors, both in the lead-based and leadless settings, remains unclear.in vivo. In part, this is because the presence of septal scar creates technical difficulty in placing a pacing lead to the LBBA with the standard technique of deep interventricular septal deployment (It is well established from studies of conventional CRT that pacing in areas of scar is predictive of poor response . This phployment . Leadlesin vivo studies corroborate the in-silico data, it is possible that measurement of conduction velocity could be used peri-procedurally within standard WiSE-CRT implantation workflow to guide a decision on whether a LBBAP or lateral wall implant is performed.A related area is the effect of His-Purkinje and myocardial conduction velocity on outcomes of CSP. With regards to long-term directions for leadless LBBAP, its place in the field of CRT will ultimately be determined by future clinical trials testing its safety and efficacy compared to conventional CRT and lateral wall endocardial pacing. Further improvements in the leadless technology, such as longer battery life and the ability to LV pace without an associated co-implant, are also in progress. If these developments come to fruition, there is potential for the expansion of indications for leadless LBBAP to become a first-line CRT modality in selected patients.


Clinical study protocols are the foundation of good clinical studies. Prospective and multidisciplinary collaboration that pays attention to the design of all components of the study protocol can ensure that a clinical study will answer the research questions posed in a reliable manner that is meaningful for decision-makers and patients. The ICH E9(R1) addendum on estimands and sensitivity analysis in clinical trials provides a framework for clinical study planning to ensure alignment between study objectives, design, conduct, and analysis. The estimand or clinical question posed can be regarded as the backbone of the study and the clinical study protocol should reflect estimands accordingly. In practice, stakeholders are still learning how to embrace the estimand framework and how it impacts studies and study documents. In this paper, we anticipate that a protocol structure centred around estimands, or objectives rather than endpoints alone will prevail for all types of studies. To assist sponsors during this paradigm shift, this paper provides discussion and guidance for implementing the estimand framework in protocol templates. In November 2019, the ICH E9(R1) addendum on estimands and sensitivity analysis in clinical trials to the guideline on statistical principles in clinical trials lear trial objectives should be translated into key clinical questions of interest by defining suitable estimands” and later that “[A]n estimand is a precise description of the treatment effect reflecting the clinical question posed by a given clinical trial objective” [ highlighThere are different ways to define objectives so that they can serve as a starting point for specifying estimands: They can be stated in great detail  ) or in lThe trial will compare once daily treatment with Tiotropium 5 μg + Olodaterol 5 μg fixed dose combination with Tiotropium 5 μg monotherapy in COPD patients with severe or very severe pulmonary impairment and a history of moderate to severe COPD exacerbations.The primary trial objective is to demonstrate superiority of the fixed dose combination for the ratio of the annualised rates of moderate-to-severe COPD exacerbation over a period of 52 weeks.The treatment effect of primary interest is while on treatment, excluding the effects of discontinuation or switching to maintenance therapies.An example from  for a DCTo compare the efficacy of Tiotropium 5 μg + Olodaterol 5 μg fixed dose combination and Tiotropium 5 μg monotherapy in COPD.The same example in the format of ICH E8(R1) might be:To demonstrate superiority of Tiotropium 5 μg + Olodaterol 5 μg fixed dose combination vs. Tiotropium 5 μg monotherapy with respect to the annualised rates of moderate-to-severe COPD exacerbation over a period of 52 weeks in patients with a history of moderate or severe COPD exacerbations.Sometimes an objective with a detail level in-between the DCO and the one suggested by ICH E8(R1) is used, and the same example might be described as follows:Less detailed objectives are common and necessitate a separate specification of the estimand(s). As an alternative, DCOs include all estimand attributes within the objective itself, including the principles for handling intercurrent events. Their focus is no longer on estimands, which are often perceived as complicated and technical by non-statisticians, but instead on “what are we trying to do?”, that is “what is the core goal of the study?” and “how do we therefore deal with the identified intercurrent events?”. Although this approach deviates from ICH E9(R1) in form, it preserves its intent, may improve interpretability and may increase engagement in cross-functional study team discussions.primary endpoint after duration of treatment with intervention as compared to placebo control in patients with disease regardless of treatment discontinuation for any reason and regardless of changes in background therapy?” In this example, the clinical question of interest is basically the estimand or DCO written as a question.According to ICH E9(R1), clinical questions of interest are more detailed translations of the study objectives, however, ICH E9(R1) neither clearly specifies their role nor discusses how they differ from objectives and estimands. It does state that intercurrent events and their strategies as well as treatment, population and endpoint should be reflected in the clinical question of interest. As such, an example could be: “What is the mean difference in If less detailed objectives are specified, a clear statement of the clinical question of interest provides the necessary context for the estimand attributes and clarifies the link between objective and estimand. Additionally, discussing that question may create more engagement from all stakeholders in the team to readily address the five estimand attributes, particularly the intercurrent events and their handling strategies. If the alternative approach of DCOs is adopted, then the clinical question is already covered effectively, so there is no need to address it separately.The protocol (template) structure and flow of thought will therefore depend on this choice of approach to objectives. Whichever approach is adopted, this early part of the protocol should provide sufficient detail about the general purpose/aim of the study, the clinical question of interest and the corresponding estimand attributes to ensure that the design, conduct and analysis can be aligned with it.Depending on the chosen approach there is a risk of repetition of elements belonging to the objective, the clinical question and the estimand attributes. Such repetition increases protocol length, allows inconsistencies to occur between repetitions and may discourage people from reading these important sections. These risks may be regarded as acceptable while the estimand framework is fully comprehended and implemented but approaches that minimise repetition are desirable in the long term. As the DCO format combines information from the clinical questions of interest and the estimands into the objectives, it may therefore represent an attractive way of streamlining these protocol sections once people become more familiar with the ICH E9(R1) concepts.In the remainder of this paper, “objective” refers to a less detailed objective as illustrated in the two examples above, while reference to a detailed clinical objective is made using “DCO”.Where the DCO approach is not followed, there are various approaches to the specification of the estimand(s). Below, we give an example where two intercurrent events, “discontinuation of treatment due to any reason” and “intake of additional medication”, are handled by the treatment policy strategy. Two different forms are provided, prose and bullet points, which we consider to both be valid ways of writing the estimand. Note, we use the terminology “including the effects of” instead of “regardless of” or “irrespective of” to clarify that under treatment policy strategy, intercurrent events affect the outcomes and therefore cannot be ignored.label) is the mean difference in change from baseline to week 26 in HbA1c between adult patients with type 2 diabetes assigned to treatment regimen X or treatment regimen Y including the effects of treatment discontinuation due to any reason and intake of additional medication.”“The primary estimand :◦ Treatment condition: treatment regimen X vs treatment regimen Y including the effects of treatment discontinuation and intake of additional medication◦ Target population: adult patients with type 2 diabetes◦ Endpoint: change from baseline to week 26 in HbA1c level◦ Intercurrent events and strategies to address them: both intercurrent events  are addressed in the treatment condition attribute and handled with the treatment policy strategy. Further intercurrent events are not anticipated at this time.◦ Population-level summary measure: difference in means between treatment conditionsPrimary estimand , according to which only “remaining” intercurrent events that are not addressed in the treatment condition, target population or endpoint attributes should be listed here. We consider both approaches to be valid. The key aspect is to ensure that there is clear identification of all intercurrent events and the chosen strategies, together with their rationale when documenting the estimand. For example, if the intercurrent event of death is handled by the composite variable strategy, and therefore addressed in the endpoint attribute, many stakeholders may not perceive this as an intercurrent event, but rather only as part of the endpoint definition.label): all attributes as in primary estimand with the following difference:◦ Endpoint: change from baseline to week 26 in body weightSecondary estimand 1  strategies as often different types of intercurrent events are handled using different strategies. Even if only one single strategy is used for all intercurrent events of a given estimand, it can create ambiguity when, e.g. the estimand is labelled “hypothetical estimand” or “composite estimand” as the hypothetical scenario or the actual composition could be different, respectively, in different studies. Within the CSP itself, using the name of a strategy may still be acceptable, because the estimand is clearly defined. However, the scope for misunderstandings increases for cross-study comparisons since names may not have been used consistently. This is especially important in the context of meta-analyses.Similarly, the use of names associated with standard analysis practices, such as intention-to-treat and per-protocol should be avoided to prevent confusion between these approaches and estimands concepts.The instructional text in the template should remind the CSP authors to align the design and conduct with the DCOs/estimands as they may be substantially impacted by the choices made.A design example is, if we want to estimate the treatment effect in those who can tolerate the experimental treatment , then a standard parallel-group design may not be appropriate. This is because only some patients would have taken the experimental treatment and so strong statistical assumption would be needed to handle this lack of information. Such assumptions may make estimation too unreliable for regulators to accept. However, another design could be chosen that might be more acceptable, for example in this case a randomised withdrawal design could be considered.A conduct example is that follow-up of the patient and data collection after the occurrence of an intercurrent event is required when using the treatment policy strategy for it, so measures to ensure retention would need to be set up to collect the relevant data. The collection of detailed reasons for the occurrence of intercurrent events is required if different reasons imply different consequences for their handling .The instructional text for the schedule of activities (SoA) should remind CSP authors to consider how all anticipated intercurrent events will be collected, e.g. adverse events form. The SoA should also explicitly cover monitoring of intercurrent events by requiring additional CRFs to collect relevant information that is not collected in existing ones.The template instructions should guide CSP authors to clearly define the study intervention to align with the treatment condition attribute of the corresponding estimand(s). The same is true for allowed, or forbidden, additional treatments and interventions as their intake is likely to be an intercurrent event.There should be a requirement for sufficient recording of concomitant, background, and rescue medication usage to support the intercurrent event strategies in use, e.g. type, dose, frequency, dates, or durations. This could include recording of interventions after the discontinuation of randomised treatment if such information could be helpful to address the questions of interest.The template should make clear the distinction between treatment discontinuation and withdrawal of study participation. The former represents an intercurrent event that needs to be considered when defining the estimand, while the latter is not an intercurrent event, but relates to missing data that should be handled within the statistical analysis.Consequences for data collection and/or the continuation of patient visits after discontinuation of study intervention should be included in the CSP in alignment with the defined estimands.Treatment discontinuation or intake of restricted medications should not mean withdrawal from a study unless there are also safety or ethical reasons for leaving the study (as opposed to changing or discontinuing treatment). Such safety or ethical reasons ought to be rare, however, as remaining in a study ought to only require patient follow-up and consent, but not preclude necessary interventions. Such an approach should allow for data collection after intercurrent events and for subsequent use in analysis—a requirement if a treatment policy strategy has been chosen.It is recommended to include instructional text in the protocol template aiming to collect specific reasons for treatment discontinuations, especially when strategies to address the intercurrent event treatment discontinuation depend on the reason.The instructional text in the participant withdrawal section should not encourage investigators to withdraw participants from the study unless for safety or ethical reasons and should emphasise that treatment discontinuation does not necessarily require study withdrawal. If withdrawal from the study cannot be avoided, specific information on the reasons for it should be recorded in data to potentially identify any preceding intercurrent events that triggered the withdrawal.The description in the protocol template section dedicated to the statistical considerations should focus on statistical details and methods to execute the plans described in the earlier sections.Reference to, rather than repetition of, the appropriately described objectives, and/or labelled estimands should be made whenever possible.Many of the necessary statistical considerations are interlinked and therefore decisions on the approach to which subsections are required and how they are related should be taken before the whole structure can be laid out. For example, there might be a subsection covering missing data handling across all analyses that could be within a “General Considerations” section or a narrower one focussing on only the primary analysis that should then be placed inside the primary analysis section.The shift of focus from endpoints to estimands has an impact on the structure of the statistical analysis sections, too. The analysis sections should now be structured by study objectives or estimands, but not endpoints.Routine subgroup analyses may be considered to act as a form of sensitivity analysis addressing the assumption of treatment effect homogeneity and can usually be considered to refer to the same estimand as their parent analysis. These could be placed in a general section  or separately in the appropriate analysis sections.In the following sections, details on the most common statistical sections in the CSP are provided including those that are not hugely impacted by estimands.The recommendations regarding the specification of statistical hypotheses, confirmatory testing and controlling the type I error remain unchanged to the pre-addendum time where statistical hypotheses and multiplicity aspects are described in a separate section. It is recommended, though, to be clear about which estimand a hypothesis or a statistical test is related to.The same considerations apply to studies that do not use frequentist testing. In general, we recommend updating this section to accommodate other types of statistical framework, e.g. Bayesian methodology.ICH E9  defines Template authors should determine how these additional requirements should be implemented. Different proposals have been made, and each has separate implications for the CSP template:One proposal is to amend the definition of an analysis set as used in ICH E9 with a specifier, so “participant analysis set” refers to a selection of participants and “data analysis set” a selection of data points from members of the participant analysis set. Each data analysis set would be named for later reference. The analysis sets section would therefore define two types of named sets, one for participants and one for the data points needed for estimation. This approach has been taken in the TransCelerate CPT.Each analysis must use one data analysis set, but often the same data analysis set can be used for estimation of several estimands, typically where only the endpoint changes. Data analysis set definitions should therefore be written to try to cover as many relevant endpoints as reasonable, i.e. there should be fewer data analysis sets than estimands. It is recommended to include instructional text pointing out that the numbers of data and participant analysis sets should be minimised, and that they should be named for ease of referencing.A second, related option is to reserve the term “analysis set” for the selection of participants and add a separate section in the statistical analysis sections of the CSP that directly links the intercurrent event handling strategies with a description of the data points required for estimation. This reduces the number of names to keep track of (as the data points sets would follow the names of their handling strategies) but may become awkward if a single handling strategy requires different estimation methods that happen to require different data selection .A third potential approach is for the data points selection to be described directly with the relevant analyses, i.e. to make data usage a property of the estimation of an estimand rather than defining standalone data sets. It is common for analyses of less important objectives to use the same estimation approaches as those for more important objectives (e.g. primary) and to refer in the CSP to the main analysis description rather than repeating it. Including data point selection in the analysis (and its description) therefore eliminates the need for separately defined data point sets.Describing data point selection as part of the relevant analysis reduces the amount of cross-referencing and analysis set naming needed (particularly in studies with many data sets and little reuse) and may be more appropriate for time-to-event analyses (where the issue is censoring rather than data point inclusion). A drawback is this approach may make programming a little harder by not having clearly named data point sets defined in the CSP.The template should include a section on the intercurrent events and the strategies used to address them. This could either be done on the same document level as the section for analysis sets or as a subsection of the statistical analysis section depending on how general the information intended for the section is.The purpose of this section is to provide more detailed information on the strategies for handling intercurrent events that is not provided previously and their implementation. This may include more detailed intercurrent event definitions, expected event frequencies, technical assumptions that support statistical analyses, and potentially the associated data point selections  may be deferred to the SAP.The description of the statistical analysis methods should be structured either by study objective(s) or estimand(s), leading to a subsection “analyses for the primary (or secondary/tertiary) objective(s)” or “analyses of the primary (or secondary/tertiary) estimand(s)”. Within these sections, further structuring is recommended as illustrated by the following subsections.The methodology for handling missing data should be aligned to the estimand. It can be described either in the relevant analysis section if the methodology differs across estimands or in a common separate section, e.g. in a subsection of a “General Considerations” section, if the methodology is the same across multiple estimands. The description should explicitly state the underlying assumptions to guide the relevant sensitivity analyses.The method of estimation and/or testing as well as the statistical model should be aligned with the respective estimand(s) or DCOs. Instructional text should remind authors to explicitly specify the underlying assumptions. It should be made clear which data is used in the estimation, either by naming a data analysis set, referring to the section where the data points selection is described for each estimand or, alternatively specified directly in this section . Standard diagnostics should also be addressed in this section.Sensitivity analyses are usually required for primary and key secondary DCOs/estimands to assess the robustness of the study results.Exclusively the same estimandExclusively different estimands (“supplementary estimands”)The same or different estimandsSupplementary analyses may provide additional insights into the understanding of the treatment effect related to the planned analyses described in the main and sensitivity analysis sections. ICH E9(R1) does not clearly state which estimand a supplementary analysis targets and there is currently no consensus. Compared to the main analysis, a supplementary analysis may targetOption 1 implies that supplementary analyses comprise competing analyses that could have been chosen as the main or potentially as a sensitivity analysis. That is, if not used as the main or to address the robustness of the results, it would be classified as a supplementary analysis.Option 2 allows the exploration of a high-level objective from different perspectives. In this context, different estimands address different treatment effects that are closely related and address the same broadly defined study objective, i.e. their purpose is closely related to the “original” estimand. Mostly, such estimands will vary only slightly in their attributes. An example is a responder analysis of a continuous endpoint using, potentially different, cutoff values. We propose to use the term “supplementary estimands” for additional estimands that are explicitly connected to main estimands through the same high-level objective, and which are supportive in nature. As option 2 requires an explicitly different estimand is targeted, it is important to have clear terminology defining it. In addition, it brings clarity to use the term “supplementary” for both the estimand and its analysis.Option 3 was explicitly written in the draft addendum  , but thiThe authors of this paper take any estimation of the same estimand to be sensitivity  and of other estimands to be supplementary (since by definition something else is being estimated). That is, we support option 2 and believe it is the only option that provides the clarity needed to support effective implementation. We welcome further debate and publications on this topic.Regardless of the interpretation, the protocol template should include subsections for the description of supplementary analyses supporting those objectives (or estimands) being subjected to confirmatory testing. It is not a requirement that supplementary analyses are defined for each, or indeed any (confirmatory) estimand but instructions should point out that the need for supplementary analyses should be considered.Supplementary estimands differ from the main ones they are associated with. Depending on the strength of this association and the interpretation of supplementary analyses, the analysis of a supplementary estimand could either be described in the supplementary analysis section or as the main analysis for a standalone estimand. The former approach is preferable if the supplementary estimand is strongly related to, and less important than, the main estimand. The latter approach is more suitable when the supplementary estimand is important and of interest in its own right, for example, to address different stakeholders that have different, but important needs. This approach would require defining a standalone objective with its own estimand and separate analysis sections.Depending on the use and consequence of results of interim analyses, the same principles regarding the estimand framework apply as for the final analyses. However, the instructional text should remind template users to collect robust data on the intercurrent events, since the interim analysis will depend not only on the endpoint, but also on intercurrent events that have occurred up until the time of the interim analysis. Thus, the estimand(s) to be estimated at the time of interim analysis should be clear as well as the general considerations for data integrity and type I error.Sample size determination depends on the estimand(s). The instructional text in the sample size section of a protocol template should therefore highlight the need to consider and describe the expected frequency of each intercurrent event by treatment group and their consequent impact on the effect size and precision. The proportion of data that is both available and relevant for estimation should be assessed in light of the planned strategies for handling intercurrent events. For instance, treatment discontinuations may lead to a subsequent exclusion of data that is not relevant if a hypothetical approach is adopted. In contrast, this data would be included under a treatment policy approach or considered as “non-responses” under a composite approach. The effectiveness of the planned study conduct procedures for following up on patients who discontinue from randomised treatment will impact missing data assumptions for treatment policy estimation. Statistical methods used for the estimation of complex estimands may go beyond the traditional methods and it is important to align the sample size estimation to the specific analysis method. This may require simulation or additional adjustments to calculations based on simpler approaches.Instructional text should mention that when using reference studies to derive quantitative assumptions, the estimand should be identified and differences to that in the planned study should be accounted for in the sample size calculation. Otherwise, errors may be introduced into the calculations. Likewise, when making other clinical assumptions, it is important to distinguish between expectations before and after accounting for intercurrent events—it will be necessary to adjust for the former but not for the latter.ICH E9(R1) was finalised in November 2019 and is currently being implemented by health authorities . We welcome the guidance, and it is clear that it has already had a positive impact on clinical studies . HoweverSince the release of ICH E9(R1), members of the EIWG and the broader statistical community have raised many questions about ICH E9(R1). Some require additional clarification or help with interpretation on certain aspects from the original authors. For example, although ICH E9(R1) emphasises the importance of defining the data required for an analysis, it is not clear whether an analysis set should now be defined at the participant, or data level. In practice, both levels seem necessary, so new official terminology and definitions are probably required. It is also hotly debated whether a supplementary analysis targets a separate estimand or not, and if not, how is it different to a sensitivity analysis? It is essential to have answers to these types of questions to implement ICH E9(R1) appropriately. Otherwise, differing interpretations will take hold, people will talk at cross-purposes, and it will create difficulties and confusion when looking at studies from different sponsors.When preparing this manuscript, we faced many challenges with implementing the ICH E9 (R1) to the letter, either because it was not clear what was required to do so, or because it would cause repetition or other issues. Some of these practical challenges resulted in us presenting different viable options that we have tried to outline in the manuscript, while in other cases, the recommendations represent compromises between competing considerations.Further help and clarification from the original ICH E9 (R1) working group, e.g. by a revision or an official Q&A document, would be highly appreciated to ensure continued implementation of ICH E9(R1) can proceed smoothly. Papers, shared examples and presentations from industry, regulators and academia are all helpful in describing best practice, but fundamentally, they cannot change, or provide official clarification on, what ICH E9 (R1) says given its status as an official ICH guideline. We therefore believe that ongoing support by ICH is required.The CSP is the parent of many other documents, e.g. SAP, CSR, submissions, and disclosure records. Thus, the templates for these downstream documents need to be aligned with the CSP template. Introduction of the estimands framework into study documentation must therefore begin with the CSP template, and much work has been done on this already. Further work is now needed around the specifics of introducing estimands into these other documents, such as that already proposed in the TransCelerate SAP and CSR templates .As the ICH E9(R1) guidance is an addendum to ICH E9 , many stakeholders perceive estimands as a statistical topic and assume that estimands consequently belong exclusively to the statistical section of the CSP. This is a harmful misconception. Estimands are a multidisciplinary topic in drug development with an impact on study planning, study conduct, analysis, and reporting. Indeed, protocol templates are typically authored cross-functionally and therefore our recommendations in writing them are aimed at a multidisciplinary team. For all disciplines, the estimand or clinical question of interest together with the study objective should be regarded as the backbone of the study that should drive the study design, conduct and analysis and the CSP should reflect the estimand(s) accordingly.As a first step we should be better at discussing and defining clearer objectives for studies right from the beginning of the development. Forward-looking, we expect that when the estimand topic has matured there will be less need for having separate objectives, clinical questions of interest and estimands. It should instead be possible to merge the information they contain into more precise and well-written study objectives to avoid repetition and facilitate engagement with non-statisticians, e.g. using detailed clinical objectives as proposed by .Due to the complexity of the estimand framework, we recommend establishing cross-functional support groups within trial sponsors and regulators devoted to implementing the framework in clinical templates. These support groups could have a broader remit including creating awareness and education sessions but also helping teams to write estimands or objectives-centred study protocols. In addition, cross-company knowledge sharing forums like the EIWG are important in that respect to promote good practice for incorporating the estimand framework into CSPs.In summary, the estimand framework is a novel concept within clinical development that requires a change in mindset when developing CSP templates. Our key recommendations are summarised in Table


The present study aimed to assess the effects of posterior spinal correction and fusion on postural stability in patients with adolescent idiopathic scoliosis (AIS). The study included 41 female patients with AIS at our institution. All patients performed three 10 s single-leg standing trials on a force plate. The center of pressure (COP) was measured preoperatively, and at 1 week and 6 months postoperatively. The postural stability parameters were absolute minimum time-to-boundary (TTB), mean of the minimum TTB, mean COP velocity, standard deviation, range, and 95% confidence ellipse area. One-way repeated analysis of variance or Friedman test was applied to the postural stability parameters. Multiple comparisons were performed using the Bonferroni correction. The absolute minimum TTB and the mean minimum TTB showed a significant increase 6 months post-operation as compared to preoperatively and 1 week postoperatively. The COP velocity significantly decreased at 6 months post-operation compared to preoperatively and 1 week postoperatively. These changes in postural stability indicate that spinal correction and fusion can be considered to improve postural stability during single-leg standing tests in the postoperative period. Adolescent idiopathic scoliosis (AIS) is one of the most common spinal deformities and is characterized by lateral spinal curvature with axial rotation and, typically, hypokyphosis of the thoracic spine . AIS patTo treat various types of AIS, the posterior spinal correction and fusion using segmental pedicle screw instrumentation is often performed. Preserving and restoring a normal sagittal alignment is crucial for anatomical spinal correction, because patients with thoracic AIS are frequently hypokyphotic ,7. Sudo Time-to-boundary (TTB) is a recently developed multidimensional measure to evaluate postural stability that incorporates body sway with the base of support ,13,14. TThe purpose of this study was to evaluate the postural stability of patients with AIS who underwent surgical treatment with the 4D anatomical spinal reconstruction technique, and to determine the effect of surgical treatment using a longitudinal study with TTB measures. Quantifying differences in postural stability between pre- and post-surgery with the 4D anatomical spinal reconstruction technique for participants with AIS may assist in evaluating the effectiveness of this surgery.n = 18), 1B (n = 7), 1C (n = 7), 2A (n = 3), 3C (n = 4), 4C (n = 1), or 6C (n = 1) participated in the present study  was used to calculate the COP coordinates in the anteroposterior and mediolateral directions during single-leg standing test. With eyes open, the patients attempted to stand quietly on a single leg for 10 s with their bare foot on the right side on the force plate. The test was performed three times: preoperative, at 1 week postoperatively, and at 6 months postoperatively. The stance foot was placed at the same position on the force plate in all trials. A total of 1000 COP data points were calculated for each trial’s force plate data, which were recorded at a sampling rate of 100 Hz. A fourth-order low-zero-lag low-pass filter with a cutoff frequency of 5 Hz was then used to filter the COP data.TTB measurements were made with reference to previous reports ,15. To sStanding long-cassette radiographs in sagittal and coronal plane of all patients were assessed for several parameters prior to surgery, as well as at 1 week and 6 months after surgery. All radiographic data were analyzed by two board-certificated spine surgeons. Our internal studies of inter/intrarater reliability have shown excellent κ statistics for all continuous measures (0.97–0.99). The proximal thoracic (PT), main thoracic (MT), and thoracolumbar/lumbar (TL/L) curves’ Cobb measurements were taken in the coronal planes. Sagittal plane data, thoracic kyphosis (T5–T12), and lumbar lordosis (L1-S1) were also measured .The C7 coronal plumb line’s lateral deviation from the center sacral vertical line (CSVL) served as a proxy for global coronal balance. Sagittal balance was measured by the absolute distance between the C7 plumb line and the S1 posterior superior corner, which was then represented as the sagittal vertical axis (SVA). The MT apical vertebral translation was calculated for regional alignment as the separation between the C7 plumb line and the geometric center of the apical vertebrae . For thep = 0.05.IBM SPSS Statistics 28  was used to conduct the statistical analysis. The Shapiro–Wilk test was used to examine normality. One-way repeated analysis of variance or Friedman test was used to examine the differences between preoperative, 1 week postoperative, and 6 months postoperative periods. Post hoc analysis was performed using the Bonferroni correction for pairwise comparisons. The statistical significance level was set at p < 0.001, p = 0.049, and p = 0.003, respectively) . The absolute minimum values of posterior TTB at 6 months postoperatively were higher than that at 1 week postoperatively (p = 0.003).In the medial, anterior, and posterior directions, the absolute minimum TTB measurements were significantly different (ctively) . In the p = 0.004, p = 0.043, p = 0.005, and p = 0.001, respectively) . The mean minimum posterior TTB at 6 months post-operation was higher than that at 1 week post-operation trials (p < 0.001).There were significant differences in the mean minimum TTB in the medial, lateral, anterior, and posterior directions (ctively) . The meap < 0.001 and p < 0.001, respectively) . There was a significant difference in standard deviation for the mediolateral direction (p = 0.015). In the post hoc test, the standard deviation for the mediolateral direction at 6 months postoperatively was lower than that at 1 week postoperatively (p = 0.017).For the conventional COP measures, there were significant differences in COP velocities for the mediolateral and posteroanterior directions (ctively) . The posp < 0.001, p < 0.001, and p < 0.001, respectively). At 1 week and 6 months postoperatively, the mean PT, MT, and TL/L curves were significantly higher than the preoperative ones . The mean MT curve correction rate was 79.5% at 1 week postoperatively. At 6 months postoperatively, the correction rate and correction angle loss for the MT curve were 77.8% and 1.04°, respectively. The mean PT curve correction rate was 58.3% at 1 week postoperatively. At 6 months postoperatively, the correction rate and the correction angle loss for the PT curve were 60.3% and 0.36°, respectively. The mean TL/L curve correction rate was 76.2% at 1 week postoperatively. At 6 months postoperatively, the correction rate and correction angle loss for the TL/L curve were 73.0% and 0.68°, respectively.The radiographic parameters of coronal plane data, sagittal plane data, balance parameters, and translational data have been summarized in p < 0.001 and p < 0.001, respectively). The 1 week and 6 months mean thoracic kyphosis was significantly higher than the preoperative mean . The 6 months mean lumbar lordosis was significantly higher than the preoperative and 1 week postoperative mean .In the sagittal plane data, there were significant differences in the thoracic kyphosis and lumbar lordosis . The C7 translation from CSVL was significantly decreased at 6 months postoperatively compared to preoperatively and at 1 week postoperatively . The SVA was significantly decreased at 1 week postoperatively compared to preoperatively (p = 0.021). The thoracic apical vertebral translation decreased significantly at 1 week and 6 months postoperatively compared to that preoperatively . There were no significant differences in the TL/L apical vertebral translation (p = 0.779).There were significant differences in C7 translation from CSVL, SVA, and thoracic apical vertebral translation (In this study, we investigated the postural stability of AIS patients who underwent surgical operation with the 4D anatomical spinal reconstruction technique from preoperative to 6 months postoperative. This study revealed that postural stability was better 6 months postoperatively than preoperatively and 1 week postoperatively.Patients with AIS who underwent surgery with the 4D anatomical spinal reconstruction technique had significantly higher values for TTB measures in the medial, lateral, anterior, and posterior directions 6 months post-operation. Decrease in TTB measurements suggest deterioration of the postural control mechanism. The results of this study indicate that while patients with AIS preoperatively and at 1 week postoperatively were controlling their balance, they controlled it in a way that brought the COP closer to the domain’s medial, lateral, anterior, and posterior stability limits compared to 6 months later. Similar to the TTB parameters, a significant decrease in COP velocity was observed 6 months postoperatively, suggesting that the change in TTB parameters was due to the change in COP velocity.Three-dimensional spinal deformities due to AIS have been reported to result in asymmetric trunk muscle activity ,18 and iThe objective of surgical management of AIS is to maximize deformity correction and achieve coronal and sagittal balance ,25. TypiThis study had some limitations. First, the follow-up period was limited to 6 months. Therefore, long-term changes in postural stability in patients who underwent posterior spinal correction and fusion using the 4D anatomical spinal reconstruction technique are unknown. Adding the value of at least two years after surgery can improve the completion of this study. Second, there was a lack of follow-up on their daily activities and activity levels. Although all patients were prohibited from sport activities during the relevant period, it is unknown what kind of daily activities they engaged in after surgery. Third, there is no electromyographic evaluation that analyzed muscle activation patterns before and after surgery. There is a possibility that changes in postural stability parameters were a result of changes in trunk muscle activity, and analysis of muscle activity may provide a more detailed explanation of the changes in postural stability observed in this study. Last, in this study, all patients underwent the 4D anatomical spinal reconstruction technique to obtain thoracic kyphosis. Based on the ethical reasons, it is impossible to compare the postoperative postural stability between patients who gain the thoracic kyphosis and those who did not improve thoracic kyphosis using different surgical techniques.The patients with AIS who had posterior spinal correction and fusion using the 4D anatomical spinal reconstruction technique had a significantly longer TTB of their COP at 6 months following surgery. These changes in postural stability indicate that spinal correction and fusion using this technique may improve postural stability during single-leg standing tests in the postoperative period.


Interest in understanding the mechanisms of the positive effects of dietary phenolic and polyphenolic compounds on human health has markedly increased in recent years. In particular, there is a clear rise of investigations related to metabolic aspects of these compounds. Specifically, a very steep increase is obvious in the case of flavonoids . The traI am pleased that 19 papers published in this Special Issue ,25,26,27The papers in this issue also covered different experimental and scientific approaches. There are seven review papers, one meta-analysis, one epidemiological study, four clinical trials, three animal experiments, and three in vitro studies. The readers can find the following interesting information in this Special Issue: Sova and Saso’s paper comprehensibly summarizes the current data on different biological aspects of hydroxycinnamic acids with an emphasis on bioavailability, metabolism, and possible pharmacological effects . AnotherHuman interventional studies published in this Special Issue included: (a) a study that found that riceberry rice bread rich in anthocyanins caused a significantly lower postprandial glucose peak in comparison to Hom Mali bread  and simiAronia melanocarpa (chokeberries) on liver collagen, matrix metalloproteinases, and their tissue inhibitors in rats chronically exposed to cadmium [Data from animal studies have shown: (a) combinations of colonic metabolites of quercetin can have a more pronounced effect on arterial blood pressure in rats ; (b) the cadmium .Two papers analyzed metabolic issues in vitro. Mohos et al. reported the important interactions of quercetin and its conjugates with several transporters, some of which were found even in sub-micromolar concentrations and with cytochrome P450 isoenzymes . Lněničk


Rapid, specific, and robust diagnostic strategies are needed to develop sensitive biosensors for small molecule detection, which could aid in controlling contamination and disease transmission. Recently, the target-induced collateral activity of Cas nucleases [clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases] was exploited to develop high-throughput diagnostic modules for detecting nucleic acids and small molecules. Here, we have expanded the diagnostic ability of the CRISPR-Cas system by developing Bio-SCAN V2, a ligand-responsive CRISPR-Cas platform for detecting non-nucleic acid small molecule targets. The Bio-SCAN V2 consists of an engineered ligand-responsive sgRNA (ligRNA), biotinylated dead Cas9 (dCas9-biotin), 6-carboxyfluorescein (FAM)-labeled amplicons, and lateral flow assay (LFA) strips. LigRNA interacts with dCas9-biotin only in the presence of sgRNA-specific ligand molecules to make a ribonucleoprotein (RNP). Next, the ligand-induced ribonucleoprotein is exposed to FAM-labeled amplicons for binding, and the presence of the ligand  is detected as a visual signal [(dCas9-biotin)-ligRNA-FAM labeled DNA-AuNP complex] at the test line of the lateral flow assay strip. With the Bio-SCAN V2 platform, we are able to detect the model molecule theophylline with a limit of detection (LOD) up to 2 μM in a short time, requiring only 15 min from sample application to visual readout. Taken together, Bio-SCAN V2 assay provides a rapid, specific, and ultrasensitive detection platform for theophylline. Small-molecule detection is vital in drug discovery, metabolomics, and environmental monitoring. Most current analytical methods rely on sophisticated instruments and expensive reagents . Thus, rin vitro transcription of CRISPR RNA (crRNA) followed by Cas12a binding and reporter cleavage was coupled to the presence of allosteric transcription factors  complex formation, is the most straightforward and widely used CRISPR system. Cas9 can form an RNP with crRNA and trans-activating crRNA (tracrRNA), later converted into a single-guide RNA (sgRNA). Guided by sequence complementarity, sgRNA-Cas9 binds and cleaves dsDNA in a sequence-dependent manner  domains . However domains . Many st domains . Kale et domains . By incoBenefiting from their rapid, economical, and user-friendly properties, lateral flow assays (LFAs) have become one of the most reliable tools for medical diagnostics . Cas9 haEscherichia coli. LigRNA was obtained by inserting a ligand-responsive aptamer into sgRNA. FAM-labeled amplicons were prepared by PCR amplification of the desired DNA sequence with FAM-labeled primers. Engineering of sgRNA  disabled ligRNA-(dCas9-biotin) RNP formation, thus blocking its binding to DNA. In the presence of the ligand, the ligRNA-(dCas9-biotin) interaction is restored, allowing (dCas9-biotin)-ligRNA-FAM labeled DNA assembly. Next, the application of (dCas9-biotin)-ligRNA-FAM labeled DNA onto the LFA strip immobilized the gold nanoparticle-(anti-FAM) antibody (αFAM antibody-AuNP) at the test line for visual detection. To demonstrate the practical application of Bio-SCAN V2, we engineered sgRNAs with a specific aptamer sequence responsive to theophylline. As one of the most commonly used anti-asthmatic drugs, theophylline has a narrow therapeutic index (20 µM–100 µM). Therefore, it is important to develop novel methods to facilitate theophylline concentration monitor  cells for protein expression. Cells were grown in 2x YT medium with 50 mg/L kanamycin sulfate at 37°C. After reaching an OD600 of 0.7 mM, 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) for protein induction and 100 μM biotin for biotinylation of dCas9-AviTag were added. The bacterial cultures were further incubated for 16 h at 18°C. The bacteria were collected by centrifugation at 6,000 g for 20 min and then lysed in lysis buffer . The lysate was purified by an AKTA Pure system with a HisTrap HP 5 mL affinity column  and then with a HiLoad Superdex 16/600,200 pg gel filtration column . The protein concentration was measured using a spectrophotometer  and the protein was collected, concentrated, flash-frozen, and stored at −80°C.Biotin-labeled dCas9 (Bio-dCas9) preparation and validation of biotin-labeling was performed following our previous protocol . Briefly2; Invitrogen), starting with denaturation at 95°C for 5 min, followed by 5°C ramp downsteps to 4°C. The annealed products were then amplified by PCR  and purified . 1 µg of purified PCR amplicons was used as a template for in vitro transcription using Transcript Aid T7 High Yield Transcription Kit  overnight at 37°C. The in vitro transcript was then purified with a Direct-zol RNA miniprep kit . The concentration of purified ligRNAs was measured using a Nanodrop spectrophotometer (Thermo Scientific) and diluted into 6 µM working stocks. FAM-labeled amplicon for Bio-SCAN V2 execution was amplified with Target-F-FAM and Target-R primers using a TheoPCR-template as a template , ligRNAs  were combined in 1X buffer 3  and incubated at 37°C for 10 min with or without 50 μM theophylline . After incubation, the samples were separated on 2% agarose gels and imaged using the FluorChem Q imaging System (ProteinSimple).For the theophylline-responsive For ligand detection, 10 µL of theophylline  was incubated with 50 nM dCas9-biotin, 50 nM ligRNA1 or ligRNA2 in a 20 μL 1X buffer 1  at room temperature (RT) for 5 min to allow RNP assembly. The RNP solution  without theophylline was treated as the negative control. Subsequently, 1 μL FAM-labeled amplicon (300 ng) and 79 μL HybriDetect buffer (Milenia Biotec) were added to 20 μL of RNP solution to bring the final reaction volume to 100 μL. After centrifugation and vortexing, the reaction mixture was incubated at 37°C for 10 min. HybriDetect Dipsticks (Milenia Biotec) were equilibrated to room temperature (RT), placed into each tube, and removed from tubes upon the appearance of control lines. The results were obtained within 5 minutes. The appearance of both test lines and control lines represented positive samples. The appearance of only test or control lines represented invalid and negative samples, respectively.via Bio-SCAN V2. To test the effects of working temperature, 10 µL theophylline  was incubated with 30 nM dCas9-biotin/ligRNA1 in 20 µL 1X buffer 1 at RT for 5 minutes. The RNP solution without theophylline was treated as the negative control. Subsequently, 1 µL FAM-labeled amplicon (300 ng) and 79 μL HybriDetect buffer was added to 20 μL RNP solution to bring the final reaction volume to 100 μL. After centrifugation and vortexing, the reaction mixture was incubated at RT, 37°C, 40°C, 45°C, or 50°C for 10 min. The following steps are the same as ligand detection via Bio-SCAN V2. To evaluate Bio-SCAN V2 function with various buffers, 10 µL theophylline  was incubated with 30 nM dCas9-biotin/ligRNA1 in either 20 µL 1X buffer 1, 20 µL 1X buffer 2 (NEBbuffer 3.1) or 20 µL 1X buffer 3 at RT for 5 min. The RNP solution without theophylline was treated as the negative control. Bio-SCAN V2 was then executed at 45°C, following the same steps as ligand detection via Bio-SCAN V2.To evaluate the effects of RNP concentration on the performance of Bio-SCAN V2, we used Bio-SCAN V2 for theophylline detection with various concentrations of the dCas9-biotin RNP. 10 µL theophylline  was incubated with 10 nM, 30 nM, 50 nM, 75 nM, and 100 nM dCas9-biotin/ligRNA1 in 20 µL 1X buffer one solution at RT for 5 min. RNP solution without theophylline was treated as the negative control. The following steps are the same as ligand detection via Bio-SCAN V2. To check the robustness of Bio-SCAN V2 for testing, we performed Bio-SCAN V2 for the detection of theophylline with the supplements of caffeine and Bovine serum albumin (BSA) as interferents. 10 µL theophylline  with the supplement of 10 mM BSA and 50 µM caffeine was incubated with 30 nM dCas9-biotin/ligRNA1 in 20 µL 1X buffer 3 solution at RT for 5 min. RNP solution without theophylline was treated as the negative control. The following steps are the same as ligand detection via Bio-SCAN V2.To evaluate the specificity of Bio-SCAN V2, we performed Bio-SCAN V2 for the detection of caffeine  and xanthine  together with theophylline. 10 µL theophylline, caffeine, or xanthine  was incubated with 30 nM dCas9-biotin/ligRNA1 in 20 µL 1X buffer 3 solution at RT for 5 min. RNP solution without theophylline was treated as the negative control. The following steps are the same as ligand detection Theophylline  was incubated with 30 nM dCas9-biotin and 30 nM ligRNA1 in 20 µL 1X buffer 3 solution at RT for 5 min. The 20 µL solutions of RNP were then incubated with 1 μL FAM-labeled amplicon (300 ng) and 79 μL HybriDetect buffer at 45°C for 5 min. HybriDetect Dipsticks were equilibrated to RT and immediately placed into each tube. The dipsticks were removed from tubes as soon as the control lines appeared. To determine theophylline concentration, linear regression between relative band intensity and the log value of theophylline concentration was calculated by GraphPad Prism 9.The relative intensity of lateral-flow strips was analyzed by ImageJ as described by t-tests were selected for statistical analysis to detect differences between the negative control and experimental groups.All raw data were analyzed and visualized by GraphPad Prism 9. All numerical data were presented as mean ± standard deviation (SD). Two-tailed Student via a strand-displacement strategy. The sequences of all four ligRNAs are listed in Bio-SCAN V2 was engineered from our previously reported Bio-SCAN system . In Bio-in vitro cleavage assay of a 370-bp PCR-amplified product using Cas9-ligRNAs in the presence and absence of theophylline. The PCR-amplified target was incubated with catalytically active Cas9 and four ligRNAs individually, in the presence or absence of 50 μM theophylline at 37°C for 10 min. The cleavage products were separated on a 2% agarose gel. As shown in To evaluate the ligand-induced activity of our engineered ligRNAs, we first performed an in vitro detection of theophylline. We selected ligRNA1 for subsequent experiments, as it demonstrated better theophylline-responsive properties. However, there was still strong background observed in the ligRNA1 LFA results without theophylline. Thus, further optimization is required to adopt Bio-SCAN V2 for theophylline detection.We next tested the Bio-SCAN V2 for theophylline detection with ligRNA1 and ligRNA2. To accomplish this, ligRNA1 or ligRNA2, dCas9-biotin, and FAM-labeled amplicon were combined and incubated with or without 50 μM theophylline at 37°C for 10 min. The reaction mixture was applied to LFA strips for visual detection without further dilution. As shown in via Bio-SCAN V2. Next, we optimized the buffer requirements of Bio-SCAN V2. As shown in To enhance Bio-SCAN V2 specificity, we evaluated several parameters to achieve robust theophylline detection without background on LFA strips. First, we investigated the effect of RNP concentration on Bio-SCAN V2 performance. We carried out theophylline detection assays using different concentrations of ligRNA-(dCas9-biotin) RNP complex . The LFA results demonstrated that with 30 nM dCas9-biotin RNP, the Bio-SCAN V2 efficiently detected 50 μM theophylline on LFA strips, compared to no detection with 10 nM and high background in the case of 50 nM, 75 nM, 100 nM dCas9-biotin RNP . The quaWe selected xanthine and caffeine as the analogues to validate the specificity of Bio-SCAN V2. Among all three analytes, Bio-SCAN V2 was only able to detect theophylline. No bands were observed with samples supplemented with 50 μM xanthine or 50 μM caffeine . Our resTo determine the LOD of Bio-SCAN V2 for theophylline, a diagnostic assay was performed at optimized conditions to detect 0 μM, 1 μM, 2 μM, 5 μM, 10 μM, 30 μM, 100 μM, 300 μM, 1,000, and 2,000 μM theophylline. The LOD was defined as the lowest concentration showing a significant difference compared with the 0 μM theophylline groups. Our results revealed that Bio-SCAN V2 can detect as low as 2 μM theophylline . Figure via LFA in 15 min, and the theophylline concentration can be further determined by quantitative analysis of relative band intensity.We previously developed Bio-SCAN platform for the accurate detection of nucleic acids, requiring no sophisticated equipment or technical expertise . In thisHigh-performance liquid chromatography (HPLC) is the most commonly used method for theophylline detection . Though Similar to other detection platforms, Bio-SCAN V2 also possesses some limitations. Bio-SCAN V2 currently requires a working temperature of 40°C to eliminate background in the control groups. It would be beneficial if Bio-SCAN V2 can be performed with a clean background in control groups under RT. Though we tested the robustness of Bio-SCAN V2 with BSA and caffeine, the performance of Bio-SCAN V2 might be influenced by other interferents. It is highly recommended to re-obtain the standard quantitative curve with specific samples for practical applications.Overall, we believe that the Bio-SCAN V2 system broadens the power of the CRISPR-Cas system for non-nucleic acid small molecule detection and presents a valuable advance to the small molecule detection toolbox.