ADAMTS17 variants. Here, we investigated a large family with WMS from Newfoundland, Canada. These patients displayed core WMS features, but with proportionate hands that were clinically equivocal for brachydactyly. Whole exome sequencing and autozygosity mapping unveiled a novel pathogenic missense ADAMTS17 variant . Sanger sequencing demonstrated variant co-segregation with WMS, and absence in 150 population matched controls. Given ADAMTS17 involvement, we performed deep phenotyping of the patients’ hands. Anthropometrics applied to hand roentgenograms showed that metacarpophalangeal measurements of affected patients were smaller than expected for their age and sex, and when compared to their unaffected sibling. Furthermore, we found a possible sub-clinical phenotype involving markedly shortened metacarpophalangeal bones with intrafamilial variability. Transfection of the variant ADAMTS17 into HEK293T cells revealed significantly reduced secretion into the extracellular medium compared to wild-type. This work expands understanding of the molecular pathogenesis of ADAMTS17, clarifies the variable hand phenotype, and underscores a role for anthropometrics in characterizing sub-clinical brachydactyly in these patients.Weill-Marchesani syndrome (WMS) is a rare disorder displaying short stature, brachydactyly and joint stiffness, and ocular features including microspherophakia and ectopia lentis. Brachydactyly and joint stiffness appear less commonly in patients with WMS4 caused by pathogenic  Patients may also have microspherophakia, severe myopia, ectopia lentis, glaucoma and cataracts. Occasionally, there is pulmonary or aortic valve stenosis. WMS is caused by highly penetrant pathogenic variants in fibrillin-1 (FBN1), that cause autosomal dominant WMS , while pathogenic variants in three other genes, ADAMTS10 , LTBP2  ADAMTS17  cause autosomal recessive WMS5. In addition to genetic heterogeneity, WMS also demonstrates subtle clinical heterogeneity, that together with its mechanistic basis, needs further clarification.Weill-Marchesani syndrome (WMS) is a rare heritable connective tissue disorder 7. The other genes mutated in WMS encode ECM proteins that either localize to fibrillin-1 microfibrils in the ECM or can be involved in the formation of microfibrils. LTBP2 encodes latent TGFβ binding protein-2, which directly binds fibrillin-1, and is required for stable assembly of microfibril bundles within the ciliary zonule9. ADAMTS10 and ADAMTS17 encode secreted metalloproteases, which require fibrillin-1 microfibrils for ECM localization and regulate assembly of microfibrils. ADAMTS10 accelerates microfibril assembly through direct interactions with fibrillin-110, whereas ADAMTS17 binds fibrillin-111, and may instead play a role in biogenesis and maturation of microfibrils, or act as a chaperone that facilitates fibrillin-1 secretion12.Fibrillin-1 forms tissue microfibrils that are key structural components of the extracellular matrix, also regulating TGFβ signaling and the biogenesis and homeostasis of elastic fibers, which support skin, ligaments, and blood vessels. Likewise, fibrillin microfibrils are the core structures of the ciliary zonule, which are compromised in WMS, and cause lens dislocationsADAMTS17 variants were first described in 20095. Review of the literature indicates there are eight pathogenic ADAMTS17 variants reported in six studies, which collectively describe 18 WMS4 patients  from Saudi Arabia. They reported four different pathogenic ADAMTS17 variants  in these families. Notably, affected individuals displayed core WMS features, but consistently had normal (i.e. proportionate) hands, without any evidence of brachydactyly or joint stiffness. This observation led the authors to hypothesize that pathogenic ADAMTS17 variants manifest as a ‘WMS-like’ syndrome  typified by the above.Family studies that characterize WMS patients are essential toward advancing our understanding of these molecular pathways within the extracellular matrix. Pathogenic et al.14 studied three families (i.e. four affected patients) with a complex phenotype involving both congenital ichthyosis and autosomal recessive WMS. They identified a contiguous 106.96 kb deletion involving CERS3 and ADAMTS17 (among other loci), in this unique 15q26.3 deletion syndrome. Notably, they described brachydactyly in three patients, and found joint stiffness in two patients. Unfortunately, given this causative deletion included multiple genes, it is difficult to correlate specific findings to ADAMTS17. Meanwhile, brachydactyly without joint stiffness was described by Shah et al.15, in the hands of one WMS patient with a pathogenic ADAMTS17 splice variant (c.873 + 1 G > T). Subsequently, Yi et al.16, studied three WMS siblings with brachydactyly and identified a nonsense variant (p.Lys351*) in ADAMTS17. Finally, Karoulias et al.12, reported a pathogenic ADAMTS17 missense variant (p.Thr343Ala) in a singleton WMS patient with no evidence of brachydactyly. There are inconsistencies however with how brachydactyly was ascertained among these studies . In 2007, family members gave written and informed consent to participate in an ocular genetics study. They consented to participate in the FORGE Canada Consortium study (now Care4Rare Canada Consortium) in 201219. The current study was approved by the Health Research Ethics Board of Newfoundland & Labrador (HREB#2011.060) and all experiments were performed in accordance with relevant guidelines and regulations.Family members live in a small rural community on the island of Newfoundland, Canada. Patients were first seen at an ophthalmology clinic by GJJ and were the subject of a case report in 198319. Briefly, 3 µg of genomic DNA from each affected individual  against the UCSC Genome hg19 was performed using BWA (v. 0.5.9)22. The Genome Analysis Toolkit (GATK)23 was used for local realignment around indels (short insertions and deletions), and for coverage assessment. Samtools (v. 0.1.17) mpileup24 and ANNOVAR25 were then used for variant calling and annotation, respectively. A custom filtering protocol was applied to the whole exome data and sequence variants were excluded in the event that: (1) <3 reads supported the alternative variant; (2) variant allele frequency was >0.05 in ExAC database and if variants were seen in >30 individuals in our database ; (3) variants were homozygous in ExAC database; (4) variants were synonymous; (5) variants were within 5’ UTRs or intronic variants outside of splice-site boundaries.Whole blood was drawn and genomic DNA was extracted from peripheral leukocytes by standard protocols. Whole exome sequencing (Illumina Hiseq. 2000) was performed according to protocols at the Genome Quebec Innovation Center, Montreal, CanadaFinally, hypothesizing autozygosity for our family, mapping was performed using in-house scripts written in Perl, to identify regions of homozygosity (ROH). An ROH was defined as a span of at least 25 SNPs where no more than two are called as heterozygous and the remainder were homozygous.ADAMTS17 variant, primers were designed using Primer326, capturing exon 21 . PCR conditions are available upon request. Sanger sequencing of PCR products was accomplished using an ABI 3130XL Genetic Analyzer, and chromatograms were interpreted using Sequencher 5.2.3.To validate the candidate 27 were screened for the ADAMTS17 variant.In this manner, familial segregation was then tested in individuals I-2, II-3, II-4, II-5 and II-8 Fig. . Finally28, taking each person’s age and sex into account. In this study, we defined brachydactyly as hands in affected patients that were appreciably smaller compared to unaffected siblings.Bilateral hand roentgenograms of an unaffected sibling (II-4) and two affected siblings  were obtained in 2016. To examine for dysmorphia, measurements of metacarpal bones, and proximal, middle and distal phalanges were taken. These measurements were compared against their expected proportions11. ADAMTS17-EA was used for initial analysis of the effect of the mutation on secretion, since the wild-type ADAMTS17 is autocatalytically processed and therefore difficult to detect in conditioned medium. To introduce the variant of interest, a synthetic DNA fragment spanning nucleotides 2799–3352 of ADAMTS17 (Ref.Seq. XM_017021975)  was ligated with the gel-purified 8335 bp SrfI x AfeI fragment obtained from the ADAMTS17-EAplasmid, using the NEBuilder® HiFi DNA Assembly Kit . Positive clones were screened via an additional ScaI restriction site introduced by the mutation and verified by DNA sequencing (Cleveland Clinic Lerner Research Institute Genomics Core).Mammalian expression plasmids for full-length human ADAMTS17 and its proteolytically inactive mutant ADAMTS17-EA were described previously2 atmosphere in a humidified incubator at 37 °C. HEK293T cells were seeded in 12-well plates  and transfected in triplicate with 1 μg plasmid DNA/well using Lipofectamine 3000 according to the manufacturer’s protocol. 16–24 h after transfection, the medium was removed and the cells were rinsed with 1 ml PBS  and incubated with 600 μl serum-free DMEM for 48 h. Conditioned medium was collected and cell debris were removed by centrifugation. The cell layer was rinsed with 1 ml PBS and cells were lysed in 0.1% NP40, 0.01% SDS, 0.05% Na-deoxycholate in PBS. Cell lysates were incubated for 5 min with rotation end-over-end and cleared by centrifugation .Human embryonic kidney cells  which were maintained in DMEM supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin in a 5% COEqual volumes of conditioned medium or cell lysate from each transfection were subjected to 7.5% SDS-PAGE under reducing conditions. Proteins were transferred onto PVDF membranes  for 1.5 h at 70 V at 4 °C in 25 mM Tris, 192 mM glycine, 20% methanol buffer. The membrane was blocked with 5% (w/v) milk in TBS  for 1 h at room temperature and anti-myc  was diluted in 5% (w/v) milk in TBS + 0.1% Tween 20 (TBST) and incubated overnight at 4 °C. Membranes were washed three times with TBST for 5 min at RT and incubated with fluorophore-labeled IRDye goat-anti-mouse secondary antibody   in 5% milk in TBST + 0.01% SDS for 1 h at room temperature. Membranes were washed three times with TBST for 5 min, once with TBS for 5 min at room temperature, and scanned wet on an Odyssey CLx scanner . The fluorescent signal was quantified using the Image Studio software.Five out of eight siblings were affected with WMS in this family Fig. . The pat17. Roentgenograms demonstrated shortened metacarpophalangeal bones. For example, sibling II-6 had bilateral shortening of 4th and 5th metacarpals, while sibling II-8 had bilateral shortening of 1st, 4th and 5th metacarpals . Likewise, we observed a unilaterally shortened 1st proximal phalanx (−5.3 standard deviations below expected) on the right hand of affected sibling II-3.We next analyzed the hand phenotype in these patients, given the incomplete clinical penetrance of brachydactyly in previous descriptions of individuals with WMS4. The hands of our patients appeared wide with stubby fingers, but clinically, were proportionate and equivocal for brachydactylyls Table . Anthrop 28 Fig. . In the ADAMTS17 (NM_139057.3: c.3068 G > A: p.C1023Y). GnomAD reports that p.C1023Y is rare, with a minor allele frequency (MAF) of 0.0016% . Sanger sequencing for p.C1023Y in 150 population matched controls from Newfoundland was negative. Familial co-segregation analysis corroborated the expected pattern of transmission for a fully penetrant, autosomal recessive disorder , PolyPhen-2 predicted damaging (score = 1.0), MutationTaster predicted disease-causing (score: 0.99), CADD score was 32, and GERP score was 5.23, suggesting a region of high evolutionary conservation. Positionally, p.C1023Y substitution resides in a thrombospondin type 1 repeat, which spans amino acids 976–1028 of the ADAMTS17 polypeptide  and p.E390A constructs (p = 0.00014). In cell lysates, there were no significant differences in wild type or p. E390A constructs harboring either wild type residue or the p.C1023Y substitution. Thus, ADAMTS17 expression was not significantly reduced inside HEK293T cells nor did it accumulate inside the cell, whereas secretion into extracellular medium was significantly impaired in p.C1023Y mutants.Thus, having identified a novel pathogenic 23Y Fig. . HEK293TADAMTS17 variant (NM_139057.3: c.3068 G > A: p.C1023Y). Having been followed since 1983, this family provides us with a unique and longitudinal view of their WMS history (Supplementary Results). Patients demonstrated core WMS features such as short stature, early onset myopia, microspherophakia and ectopia lentis, but visually their hands were not strongly suggestive of brachydactyly  in these two family members. Thus, our findings further underscore the clinical heterogeneity and intrafamilial variability of the brachydactyly phenotype in WMS and emphasize that the overall clinical hand appearance can mask significant anomalies in individual metacarpals, which can be elicited by antropometry.Here, we investigated the molecular underpinnings for WMS in a large family from Newfoundland & Labrador and identified a novel homozygous pathogenic missense ADAMTS17 variants. For example, after excluding the 15q26.3 deletion syndrome, 10 out of 14 WMS patients with reported pathogenic variants had reportedly normal hands , two splice site mutations , two nonsense mutations , a contiguous gene deletion on 15q26.3, and now two missense variants , which occur in multiple regions of ADAMTS17  by Karoulias et al.12, and our current study respectively, further delineate the functional consequences of pathogenic ADAMTS17 variants. The p.Thr343Ala variant lies in a catalytic domain and results in decreased secretion of ADAMTS17 into the extracellular matrix, whereas the p.C1023Y variant resides in the thrombospondin-1 repeat domain of ADAMTS17 and results in comparable deficiency in secretion. Our analysis of ADAMTS17 p.C1023Y mutants in HEK293T cells revealed significantly reduced levels of ADAMTS17 in cellular medium as compared with wild-type ADAMTS17 (p = 0.0012) and ADAMTS17E390A (p = 0.00014) constructs  in Supplementary Information.


Two-phase flow with phase change in microstructure or nanostructure is an important issue in many fronts and critical applications nowadays, but with a lack of comprehensive understanding of the mechanism. This paper numerically investigates the transient behavior of two-phase flow with liquid phase change in the porous media, which consists of a series of connected pores at micro and nanoscale with the transient form of the semi-mixed model and self-compiled programs. Transient variation and spatial distribution of structure temperature, thermal non-equilibrium characteristic, phase change location and fluid-driven pressure are obtained and analyzed, and effects of initial system temperature, structure parameter and material property on the transient behaviors of two-phase flow and fluid-structure coupling heat transfer are discussed. The numerical simulations indicate that the two-phase flow with phase change in porous media is complex and ever-changing before reaching a steady state and affected by the above-mentioned three kinds of parameters significantly. Particularly, distinct phenomena of transient heat transfer deterioration and vapor block are discovered, and it is revealed that the transient heat transfer deterioration and vapor block are more serious in a porous matrix with smaller porosity and made of materials with higher heat capacity and density. Two-phase flow and heat transfer at the micro and nanoscale have attracted much attention from researchers in the fields of geothermal energy recovery ,2, fuel There is much research work on the steady problems of two-phase flow with liquid phase change in microstructure or nanostructure. A great number of experiments have been conducted to explore the wide range of two-phase flow regimes in different microstructures ,13,14. SHowever, there is a lack of comprehensive analysis and knowledge about the transient behavior of two-phase flow with phase change in the porous media so far. Different from the general immiscible gas-liquid two-phase flow, for example, oil-gas flow in rock layers and cracks, when phase change of liquid fluid occurs in pores and channels at micro or nanoscale, there are significant mass, momentum and energy transfers between liquid phase and vapor phase, huge amount of latent heat is released, and capillary force is generated. The resultant dramatic variations of fluid density and specific volume affect the transport characteristic of the two-phase flow and the fluid-solid coupling heat transfer greatly. Therefore, affected by the micro or nanoscales of the connected pores and the distributions of heat and pressure at structure boundaries, some special thermal responses and fluid transport characteristics would arise during the transient process. The experimental data from Huang et al.  indicateIn this paper, the transient form of the semi-mixed model under local thermal non-equilibrium condition is proposed to describe the transient two-phase flow with phase change in porous media, and a self-compiled program is developed as the numerical approach. Through the numerical simulation, the time-varying spatial distribution of important physical parameters, such as structure temperature, thermal non-equilibrium characteristic, phase change location and fluid-driven pressure are obtained and quantitatively analyzed, and the effects of system initial temperature, microstructure parameter and material property on the transient behaviors of two-phase flow and fluid-structure coupling heat transfer are systematically discussed.L. Heat flux According to fluid states, there are three regions in the porous matrix from bottom to top: the liquid region, the mixed region of liquid and vapor phases, and the vapor region. Some mathematical models have been developed specifically to describe the characteristics of fluid flow and heat transfer in the three regions. Our previous research  has compIn the single-phase region, the mass conservation equation has uniform expression, using a subscript “i” to represent liquid phase “l” and vapor phase “v”:In the two-phase region, the mass conservation equations are separately described for liquid and vapor phases by liquid saturation s:Similarly, the momentum conservation equation has uniform expression in the single-phase region, while it is separately described for liquid and vapor phases in the two-phase region.In the single-phase region:In the two-phase region:In the single-phase region, the conservation equation of fluid energy has uniform expression:In the two-phase region, the conservation equation of fluid energy is unitedly described for the mixture of liquid and vapor phases:s = 1, fluid is in a liquid state and s = 0, fluid is in vapor state and It can be found that when liquid saturation In all regions, energy conservation of a solid matrix is described by:After solving the mixed specific enthalpy, one can obtain the saturation and temperature of the fluid by the following algebraic relations:In the single-phase region, fluid in a vapor state is regarded as an ideal gas, while in the liquid state is considered to be incompressible. In the two-phase region, fluid is the mixture of liquid and vapor phases, of which the vapor phase maintains at saturation state, and the pressure of the vapor phase depends on the temperature:Then the pressure of the liquid phase can be obtained by Other constitutive relations used to close the solution are summarized in The transient problem that a porous matrix full of liquid fluid begins to be exposed to a high heat flux is considered in this paper. Hence, the initial conditions for the whole physical domain are set as:According to the discussion results in previous research , the mosAt cold side:At hot side:p between successive iterations are less than The transient SMM under local thermal non-equilibrium condition is solved by a self-compiled program at University of Science and Technology of China. The finite difference method is employed to discretize the governing equations: explicit scheme ,31 is us2 was applied to the upper surface of the porous test section, and liquid water with an initial temperature of 300 K was injected from the lower surface. The wall temperature along the flow direction was measured. Simulations under the completely identical operating condition with the experiments were carried out with our mathematical model and numerical approach, and the comparison results are shown in Due to the lack of proper transient experimental data in open literature, the mathematical model and numerical approach proposed in this work are validated with the steady-state experimental data from Hu et al. . In the 6 W/m2 and cooled by liquid water with a mass flow rate of 0.3 kg/m2s. It can be found that the fluid temperature at the hot side and the location of the interface between two-phase region and vapor region approach certain values at approximately 300 s and then maintain unchanged, which means the whole system reach the steady state by this time. From the beginning t = 0 s to t = 300 s, the system goes through an initial stage, in which physical parameters vary with time significantly.The two-phase flow with phase change in porous media will eventually reach a steady state when the thermal environment and fluid inject rate are unchanged. However, before that, the temperature of the porous structure, the location of phase change and the driven pressure of fluid change acutely with time, and this stage are known as initial stage. To study the two-phase fluid flow and fluid-solid heat transfer characteristics in the initial stage, Combining However, the initial temperature of the porous system has a great influence on the transient variation of the physical parameters in the initial stage. As shown in 0T = 300 K, the area of vapor region in the initial stage is always smaller than that in the steady state, but when 0T = 400 K and 500 K, the area of vapor region in the initial stage expands quickly and once exceeds the steady value. For the flow resistance of vapor is much higher than that of liquid, larger vapor region inevitably corresponds to larger fluid pressure at inlet. As shown in 0T = 400 K and 500 K, the fluid pressure at inlet in the initial stage likewise exceeds the steady value. This is a distinct phenomenon of transient vapor block, which is worthy of attention. The fluid-driven pressure is an extremely important parameter in the design of a two-phase flow system and is usually set according to steady-state operating conditions. However, based on the results obtained here, the driven force of fluid required in the initial stage could actually be much greater than that in the steady state. If lacking driven force in the initial stage, the fluid supply must break down, so in this situation, the cooling system may already fail in the initial stage.The magnitude of phase change and phase distribution of fluid also has great influence on the driven pressure of the fluid. From α = 6(1−ε)/pd, which indicates that fluid-to-solid heat transfer coefficient decreases with the porosity. Hence, more heat is stored in the solid matrix with larger porosity, resulting in a higher solid temperature. From s.effk. As the results, the temperature gradient within the solid matrix becomes larger and vapor region becomes smaller. Accordingly, the resistance of fluid flowing through the vapor region reduces, i.e., the driven pressure decreases. It should be particularly noted that the porosity not only affects the transient variation of physical parameters in the initial stage but also changes their steady values. Large porosity design could greatly reduce the fluid-driven force and impair the transient heat transfer deterioration and vapor block, but at the cost of increasing the system temperature. Therefore, porosity is an important structure parameter of the two-phase flow with liquid phase change in porous media and should be given special consideration in the practice of engineering design.(1)For the porous matrix with fixed microstructure, when the heat flux on the boundary and the fluid mass flow rate both keep unchanged, the two-phase flow with liquid phase change in porous media will eventually achieve equilibrium, which is known as the steady state. Before reaching steady state, the transient two-phase flow with liquid phase change in porous media goes through an initial stage, in which the physical parameters vary with time significantly;(2)The system initial temperature has no influence on the steady state of the two-phase flow with liquid phase change in porous media. However, when the initial system temperature is relatively high, distinct transient heat transfer deterioration and vapor block occur in the initial stage, which may cause unexpectedly premature cooling failure and structure ablation;(3)The porosity not only affects the transient variation of the physical parameter in the initial stage but also changes their steady values. Large porosity design could greatly reduce the fluid-driven force and impair the transient heat transfer deterioration and vapor block, but at the cost of increasing the system temperature;(4)As the heat capacity and the density of the solid material increases, the system spends more time reaching a steady state, and the transient heat transfer deterioration and vapor block effect in the initial stage are severer. Therefore, porous matrix made of a material with low heat capacity and density could reduce the transient heat transfer deterioration and the transient vapor block effect without affecting the steady state.This work is aimed at providing a comprehensive understanding of transient two-phase flow with a liquid phase change at the micro and nanoscale. A new mathematical model and corresponding numerical approach are developed, through which the dynamic evolution of phase change and transient behavior of fluid-structure coupling heat transfer are analyzed and the influences of initial system temperature, structure parameter and material property are discussed. From the simulations and analysis, the following conclusions can be drawn:


Fluid therapy is extensively used to treat traumatized patients as well as patients during surgery. The fluid therapy process is complex due to interpatient variability in response to therapy as well as other complicating factors such as comorbidities and general anesthesia. These complexities can result in under- or over-resuscitation. Given the complexity of the fluid management process as well as the increased capabilities in hemodynamic monitoring, closed-loop fluid management can reduce the workload of the overworked clinician while ensuring specific constraints on hemodynamic endpoints are met with higher accuracy. The goal of this paper is to provide an overview of closed-loop control systems for fluid management and highlight several key steps in transitioning such a technology from bench to the bedside. Fluid therapy is used extensively to treat traumatized patients as well as patients during surgery , 2. Fluiautopilot) have been used in various aircrafts over the past several decades. More recently, advances in autonomous driving holds the promise of using closed-loop technologies for the driverless operation of vehicles in the near future. Although closed-loop control technologies have been used in various industries for many years, it is not until recently that active control technology has transitioned into medical applications with the majority of applications focusing on pre-clinical or research settings.One industry that has witnessed a wide adoption of active  control technologies is the aerospace industry, where closed-loop control technologies ; (ii) an actuator , that changes the state of the system in order to drive the controlled variable of interest to a desired value; and (iii) a controller or processor, that uses the sensor measurements in order to compute an appropriate action that drives the actuator to execute the desired control action. The controller can be considered the central processing unit of the system (the “brain”) and, in general, is implemented using proprietary software on a computing device. The general architecture of a closed-loop control system is shown in In many systems related problems, a specific variable of interest (the controlled variable) needs to be maintained at a desired value. For example, in the classical inverted pendulum control problem consisting of a pendulum and a moving cart, the goal is to maintain the pendulum in a vertical position by moving the cart horizontally. Every closed-loop control system has three components; namely, (i) a 2). In order to design a feedback control system, we need a sensor  and an actuator  to regulate the trajectory of the controlled variable of interest over time. The control system can be implemented on a computing device such as a computer or a small and limited-resource computing device such as a microcontroller.Using this architecture, it is straightforward to apply this process to a clinical problem such as fluid management. In this case, the goal would be to maintain the controlled variable of interest at a desired value  as well as oscillatory system behavior or, in the worst case, divergent trajectories of the controlled variables. Lyapunov stability theory provides a powerful framework for guaranteeing system stability. Using this framework, an “energy-like” Lyapunov function is constructed and shown that its rate of change with respect to time is negative reflecting the fact that the “energy” of the closed-loop  system is decreasing over time . This is due to the complexities inherent in system physiology, wherein mathematical models capturing complex physiological mechanisms of action are not readily developed. Although there has been considerable progress in understanding the pharmacokinetics and pharmacodynamics of drug distribution and drug effect using compartmental models in pharmacology , the discompartments. A compartment is a macroscopic subsystem involving kinetic homogeneity, where it is assumed that any material entering the compartment is instantaneously mixed with the material in that compartment. Compartmental models have been successfully used to provide approximate models for capturing the behavior of drug and fluid distribution in the body. In addition, such models provide an opportunity for the use of model-based control design approaches that use mathematically rigorous closed-loop control techniques to solve specific problems related to drug and fluid distribution management.Compartmental models, which are characterized by conservation laws  involve several simplifying assumptions. One important assumption is that the drug or fluid distribution in the body can be approximated by using only a few structure of the biomedical system being modeled, whereas the model parameter values  are uncertain and vary from patient to patient; these model parameter values are critical in being able to design effective closed-loop, model-based control systems.Although compartmental models provide the opportunity to leverage model-based control designs, a critical limitation of this design approach is that the patient-specific parameters that define these models are vaguely known. More specifically, compartmental models capture the approximate physiological proportional-integral-derivative controller, also known as a PID controller) are “tuned” based on experimental data  are assumed to lie between upper and lower bounds or are unknown and estimated in real-time. Within this context, compartmental models that capture general fluid or drug distribution can be used to guide the control system design. Examples include using adaptive control technologies to deliver general anesthesia  and semi-automated fluid management for burn patients has been extensively studied by researchers at the US Army Institute of Surgical Research, San Antonio, TX , 28. TheThe authors in  investigAnother series of recent studies have investigated the use of closed-loop control for goal directed fluid therapy. Specifically, hemodynamic data obtained from patients has been used to develop an algorithm to administer a bolus of fluid . The perWhile the majority of currently designed closed-loop systems use heuristic approaches to determine fluid administration rates and volumes, a notable exception has been the employment of an adaptive control system predicated on a compartmental model to resuscitate dogs subject to absolute and relative hypovolemia . The stuin silico tests for 30 randomly generated patients was not investigated. It is also unclear whether the proposed architecture is robust to unmodeled dynamics not captured by the lumped parameter model.In a recent work, authors in  investigGiven the complexity of individual patient physiology under various clinical scenarios and issues associated with under- or over-resuscitation –5, desigClinical insight is critical in designing closed-loop systems for fluid management. The controlled system needs to administer fluids when appropriate, and needs to stop fluid administration when key physiological endpoints are maintained. In the case where the anticipated response to fluid administration is not achieved by the closed-loop system, the system needs to alert the clinician to take over the fluid administration  in much the same way wherein a well-designed autopilot alerts the pilot to take over control of an aircraft.in silico testing and hardware-in-the-loop simulation to pre-clinical and clinical testing. In addition, such tests need to assess the system performance in complex clinical scenarios to ensure safety and effectiveness. The clinical and workflow impact of using a closed-loop control system for fluid management in a clinical setting needs to be investigated in future studies.Prior to transitioning a closed-loop control system for fluid management to a clinical setting, such system needs to be comprehensively tested. This will involve a series of tests ranging from All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.BG has stock ownership in Autonomous Healthcare, Inc. BG was one of the inventors on a patent application  for a clinical decision support and closed-loop fluid resuscitation system and cardiovascular drug administration. WH has stock ownership in Autonomous Healthcare, Inc. JB has stock options and serves as the Chief Medical Officer in Autonomous Healthcare, Inc. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Thaumatotibia leucotreta, known as the false codling moth, is a pest of citrus and other crops in sub-Saharan Africa. As it is endemic to this region and as South Africa exports most of its citrus around the world, T. leucotreta has phytosanitary status for most markets. This means that there is zero tolerance for any infestation with live larvae in the market. Consequently, control measures prior to exporting must be exemplary. Certain markets require a standalone postharvest disinfestation treatment for T. leucotreta. However, the European Union accepts a systems approach, consisting of three measures and numerous components within these measures. Although effective preharvest control measures are important under all circumstances, they are most critical where a standalone postharvest disinfestation treatment is not applied, such as within a systems approach. Conventional wisdom may lead a belief that effective chemical control tools are imperative to achieve this end. However, we demonstrate that it is possible to effectively control T. leucotreta to a level acceptable for a phytosanitary market, using only biological control tools. This includes parasitoids, predators, microbial control, semiochemicals, and sterile insects. Simultaneously, on-farm and environmental safety is improved and compliance with the increasing stringency of chemical residue requirements imposed by markets is achieved. A phytosanitary or quarantine pest is a pest of potential economic importance to an area where it is not yet present . One of Phytosanitary protection is achieved through regulatory plant protection, which aims to safeguard agricultural crops from such pests and diseases . Such reThaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), known as the false codling moth, is one such phytosanitary organism [organism . It is aorganism . It is eorganism . This inorganism . ConsequT. leucotreta is controlled to as close to a nondetectable level as possible in the country of origin, before fruit are exported. All export markets that regulate T. leucotreta as a phytosanitary pest require a quarantine treatment of some sort [It is, therefore, imperative that ome sort . For mosome sort ,17. Coldome sort ,19 and aT. leucotreta consists of three measures: (1) preharvest controls and measurements and post-picking sampling, inspection, and packinghouse procedures, (2) post-packing sampling and inspection, and (3) shipping conditions. All in all, Moore et al. [A systems approach is defined as “the integration of different risk management measures, at least two of which act independently, and which cumulatively achieve the appropriate level of protection against regulated pests” . Aluja ae et al.  and Hatte et al.  listed 1e et al. ,17, i.e.e et al. ,22.T. leucotreta for export markets. The downside of employing this rationale is that, although chemical pesticide residues spell no risk to consumers, due to strict compliance with scientifically determined maximum residue levels [Although effective preharvest control measures for phytosanitary pests are important under all circumstances, they are most critical where a standalone postharvest disinfestation treatment, which would provide adequate postharvest risk reduction on its own, is not applied. However, in order for a systems approach to succeed, every single measure and step in the system must be optimally applied; otherwise, inadequate overall risk reduction may result. Consequently, preharvest control measures are an extremely important component of the systems approach and, thus, need to be highly effective. Conventional wisdom may lead a belief that effective chemical control tools are, thus, imperative, generally being considered more effective and faster acting than their biological alternatives, particularly considering the zero-tolerance status of e levels , there ie levels  and the e levels . Furthere levels .T. leucotreta to a level acceptable for a phytosanitary market, using only biological control tools, considering the further risk reduction added by the postharvest measures applied thereafter. Simultaneously, compliance with the increasing stringency of chemical residue requirements imposed by markets, from a regulatory perspective and even more stringently through arbitrary retailer standards, is achieved [T. leucotreta for export markets can be sufficiently mitigated.Confounding the belief that chemical pesticide tools may be unavoidable for control of a phytosanitary pest, for which there is no tolerance, we demonstrate here that it is possible to effectively control achieved . ConsequT. leucotreta is considered to consist of three tiers. The foundation is orchard sanitation, without which no control programme can be effective. Overlaid onto this is an areawide control technique, such as the sterile insect technique or mating disruption. Finally, orchard specific control measures are applied, such as parasitoid releases or insecticidal sprays. Preharvest monitoring is conducted using pheromone traps and by evaluating fruit infestation from designated data trees [T. leucotreta is now primarily a phytosanitary pest, rather than an economic pest, these monitoring tools are no longer used to determine whether intervention is warranted or not, i.e., threshold purposes [T. leucotreta is a regulated phytosanitary organism [Preharvest control of ta trees . Howeverpurposes . This ispurposes . Fruit iorganism .Orchard sanitation involves the regular collection and removal of infested and injured fruit from the orchard, both hanging and fallen fruit, as well as the destruction of this fruit outside of the orchard. Moore and Kirkman  demonstrHere, we provide the details and efficacy of specific biological control measures, be they areawide technologies or orchard-targeted technologies, that are overlaid onto the foundation of orchard sanitation. However, before we do so, it is important that we clarify what we define as biological control. Biological control has been defined as the introduction and manipulation of natural enemies by man to control pests , or the T. leucotreta, it has been determined to be a minimum of 10 sterile to one wild male [The IPPC categorizes sterile insects as beneficial organisms . Consequild male . If otheild male , the sizild male . T. leucotreta has been commercially implemented in the citrus and table grape industries in South Africa since 2007 [SIT for nce 2007 . It is nnce 2007 .T. leucotreta infestation in 35 ha of Washington Navel orange orchards by 95.2%, relative to an untreated control orchard [T. leucotreta within an integrated programme, initially in the Western Cape Province (since 2007) and more recently in various other production regions, and it is proving extremely effective [SIT was commercialized after a successful pilot trial in citrus. As a standalone treatment in a semicommercial trial, SIT reduced  orchard . These fffective , having ffective . ConsequMating disruption is the broadcasting of sex attractant pheromones within a crop so as to disrupt reproduction of insect pests and, thus, suppress population build up . Most ofT. leucotreta was introduced into South Africa in the 1990s. Products were initially based on pheromone from the oriental fruit moth, Grapholita molesta (Busck), which showed some efficacy against T. leucotreta, but insufficient to warrant commercialization of the products [T. leucotreta pheromone, in Navel orange orchards. Isomate, reduced T. leucotreta infestation of fruit from December to the end of April by 55% in an orchard considered to have high pest abundance, and by 75% in an orchard considered to have low pest pressure [Mating disruption for products . Subsequpressure . More impressure . T. leucotreta’s disruption profile closely follows the predictions of the hybrid disruption profile of Miller and Gut [T. leucotreta is likely disrupted competitively at low doses and noncompetitively at high doses. However, the pheromone dose required to induce this shift from competitive to noncompetitive disruption was 192 g per ha [T. leucotreta remains competitive in nature and, therefore, most effective when the pest population is at a low level [Steyn  showed t and Gut , suggestg per ha . Unfortug per ha ; thus, iow level .T. leucotreta control: Isomate FCM, Checkmate FCM-F , Splat-FCM , and X-Mate  [Four registered products are available for  Africa) ,48,49. IAttract-and-kill technology is similar to mating disruption in that it also makes use of semiochemicals for achieving the end of controlling the pest. However, in attract-and-kill, semiochemicals are used in combination with insecticides (most often), sterilants, or insect pathogens . The insT. leucotreta. The product, under the name Last Call FCM , consists of a blend of synthesized female sex pheromone and a pyrethroid, in the form of a gel. The combination attracts and kills male moths [Currently, only one product based on the attract-and-kill technology is commercially available for management of le moths . As withle moths .Thaumatotibia leucotreta has a range of natural enemies that suppress it in the field [T. leucotreta is the egg parasitoid, Trichogrammatoidea cryptophlebiae Nagaraja (Hymenoptera: Trichogrammatidae) [T. cryptophlebiae reached between 80% and 100%, causing from a 67% reduction in T. leucotreta infestation in Navel oranges from December to harvest to total elimination of detectable T. leucotreta infestation by harvest [T. cryptophlebiae in controlling T. leucotreta can be variable [he field . The mosmatidae) ,53. Wher harvest . Howevervariable . Additiovariable ,55.T. cryptophlebiae augmentation were conducted during the 1970s and 1980s [T. leucotreta larval infestation of fruit, by releasing more than 862,000 parasitoids per hectare over a 3-month period. Newton and Odendaal [T. leucotreta infestation by up to 60% [T. leucotreta infestation than those initiated during November and December [Initial studies on the effectiveness of nd 1980s ,57,58. SOdendaal  releasedOdendaal  referredOdendaal . As few p to 60% . ImperatT. cryptophlebiae have been conducted by Cederberg Insectary in Citrusdal in the Western Cape Province [T. cryptophlebiae can only be conducted over a fairly limited area; thus, conservation of the naturally occurring population remains an attractive, practical, and universally available option. If parasitoids are available for augmentation, release is easily conducted by growers in the form of parasitized eggs placed at various well-spaced intervals throughout orchards, on a monthly basis for 4 months; thus, it is not an onerous process. However, if this cannot be initiated early in the season (spring or early summer), possibly due to the detrimental impact of chemical sprays applied against other pests, then augmentation should not be considered a viable option. Historically, commercial mass rearing and augmentation of Province  and at ZProvince ,63. CurrT. leucotreta have been recorded, as listed by Newton [Agathis bishopi (Nixon) [Several other parasitoids of y Newton  and Moory Newton . Probabl (Nixon) ,65,66. HAnoplolepis custodiens (Smith), and the brown house ant, Pheidole megacephala (Fabricius) [Planococcus citri (Risso), soft scales (Coccus hesperidum L. and Pulvinaria aethiopica (De Lotto)), and waxy scales  [Aonidiella aurantii (Mask.), causing outbreaks of these pests [Ants are considered as important pests in citrus trees. The main problematic species are the pugnacious ant, bricius) , which dbricius) ,68. This Newst.) ,68. Furtse pests .Ceratitis spp. (Wied.), bollworm, Helicoverpa armigera (Hüb), and T. leucotreta [A. custodiens and P. megacephala had been bait-poisoned and in control plots. Pest survival was significantly lower for all three species in control plots, indicating significant predation by ants. Predation on T. leucotreta pupae was significantly higher than for the other two species. Consequently, the practice of barrier banding citrus trees to exclude ants, as opposed to poisoning, is strongly recommended to growers [This emphasizes the importance of keeping ants out of citrus trees. However, on the orchard floor, ants can act as an important and effective predator of arboreal pests that pupate in the soil, such as fruit flies, ucotreta . Bownes ucotreta  planted  growers ,71. ThisOrius sp. and Orius insiduosus [Rhynocorus albopunctatus (Stål), and the egg/larval mite predator, Pediculoides sp. [Other predators that have been recorded include the hemipteran egg predators, siduosus ,72, the ides sp. . Obviousides sp. .T. leucotreta larvae [Cryptophlebia to Thaumatotibia [12 occlusion bodies (OBs)/ha in the case of Cryptogran and 6.6 × 1011 OBs/ha in the case of the other two products.The Cryptophlebia leucotreta granulovirus (CrleGV) was first isolated from the Ivory Coast from infected a larvae . Althougatotibia , the oriatotibia . Other iatotibia ,79,80. Tatotibia ,81. Two atotibia . These aT. leucotreta population [CrleGV has been extensively tested in the field for many years with more than 50 trials completed, and it has been used commercially over tens of thousands of hectares annually for almost two decades ,77,81. Tpulation ; howeverT. leucotreta control, including the chemical insecticides, bearing testimony to not only their efficacy, but also their affordability and ease of application. The products are applied using conventional pesticide spray equipment and can be tank-mixed with other pesticides without risk of compromised efficacy.The granulovirus products are probably the most widely used of all products for Despite field performance generally being good, further improvement of field efficacy is being sought, through combining isolates of the same virus. As discussed by López-Ferber et al. , the use50  and LC90  were reduced 1226- and 563-fold [Probably the most important shortcoming of baculoviruses is their sensitivity to ultraviolet (UV) degradation and, hence, rapid breakdown in the presence of direct sunlight ,86,87. C563-fold ,89. This563-fold . The genCryptophlebia peltastica (Meyrick), an African pest species [Cydia pomonella (L.), and T. leucotreta as to C. peltastica and marginally more virulent than the homologous GVs of these species [Recently, the first nucleopolyhedrovirus (NPV) in the Grapholitini tribe of Tortricidae was discovered , being t species . The Cry species , but als species ,93.T. leucotreta in citrus at rates of between 5 × 1011 and 5 × 1013 OBs/ha [Preliminary field trials have already been conducted with CrpeNPV against 3 OBs/ha . Very pr3 OBs/ha .T. leucotreta, not only on citrus, but also even more so on crops where more than one grapholitinid is a pest, such as on macadamias, where T. leucotreta, C. peltastica, and T. batrachopa (Meyrick) can be a problem.CrpeNPV has also been evaluated for use in combination with CrleGV, with some mixtures identified to have a synergistic effect, resulting in an increase in insecticidal activity ,96. HoweThe appearance of a CrpeNPV-based product on the market is imminent. It can be expected that its mode and ease of application and its affordability will be as for the granulovirus products.T. leucotreta identified three entomopathogenic fungal (EPF) isolates with the greatest pesticidal potential, two Metarhizium anisopliae (Metchnikoff) Sorokin  isolates and one Beauveria bassiana  Vuillemin  isolate [T. leucotreta of greater than 80% was achievable when applied at a rate of 1 × 106 conidia/g soil with average LC50 values of 6.26 × 105, 1.92 × 106, and 1.98 × 105 conidia/mL for the selected M. anisopliae and B. bassiana isolates [Laboratory bioassays against final instar  isolate ,98. All  isolate . Resultsisolates ,98. Thesisolates .12 to 1 × 1014 spores/ha, using a commercial tractor-drawn spray machine [T. leucotreta-infested fruit per tree per week and any percentage reduction in T. leucotreta infestation that resulted from fungal application. In comparison to the control treatments, all three isolates used were capable of reducing T. leucotreta infestation in the field. The B. bassiana isolate performed best, with an average reduction in T. leucotreta infestation of 82%, measured over a 32-week period after a single application in spring. Over time, the reduction in T. leucotreta infestation remained constant for the B. bassiana isolate throughout the trial period, but declined for both M. anisopliae isolates (54.01% to 35.02%) and (47.39% to 27.65%) from 10 weeks after treatment to 32 weeks after treatment [Thereafter, large-scale field trials were conducted with application of the three selected isolates to the soil surface underneath the canopy of citrus trees at rates ranging from 1 × 10T. leucotreta is underway. In the meantime, two other isolates of B. bassiana are already registered for use against T. leucotreta on citrus [T. leucotreta is not commonplace, due largely to their limited efficacy. Consequently, development of an effective formulation to overcome UV sensitivity and other environmental hurdles is desirable in order to achieve more consistent efficacy with foliar application. In the meantime, efficacy can be optimized through accurate timing of applications with peaks in egg laying and frequent reapplication [Commercial development of these isolates for application against the soil-dwelling life stages of n citrus . Howevern citrus . Althouglication .T. leucotreta will increase, as not only will they be more effective, but application through the irrigation (microsprinkler) system will also be even easier than conventional spraying.It is expected that, once the soil-targeted formulations become available, the use of EPF against T. leucotreta larvae at rates as low as 20 infective juveniles (IJs)/cm2. These were Steinernema sp., Heterorhabditis bacteriophora, S. yirgalemense, S. khoisanae, H. zealandica, Heterorhabditis sp., Heterorhabditis baujardi, and Steinernema litchii. These results provided strong evidence of noteworthy potential of the EPNs for the control of T. leucotreta in the field. Consequently, Malan and Moore [T. leucotreta larvae with EPNs in a citrus orchard environment. Heterorhabditis bacteriophora, H. zealandica, and S. khoisanae were applied at concentrations ranging from 5 to 80 IJs/cm2. The Heterorhabditis species were the most effective, with up to 90% infection of T. leucotreta larvae and detectable persistence for 49 days, albeit at a reduced level. Malan et al.  and Steynd Moore  conducteS. yirgalemense and S. jeffreyense against T. leucotreta in semi-field trials in other crops , using a similar methodology [These results were confirmed with significant efficacy recorded with hodology ,107.Heterorhabditis bacteriophora and S. feltiae were applied underneath tree canopies in large orchards, using tractor-drawn spray machinery or through the irrigation system. Successful control was recorded in several trials, with EPN infection of larvae frequently exceeding 80% and larval infestation of fruit being reduced by up to 88%, with persistence recorded for up to 5 months [Consequently, large commercial-scale field trials were conducted in citrus orchards in the Eastern Cape, Western Cape, and Mpumalanga provinces of South Africa from 2011 to 2013, geared toward directly testing the ability of EPNs to achieve pest reduction and crop protection in the orchards . Heteror5 months . HoweverH. zealandica) were shown to reduce fruit infestation by T. leucotreta by 59% in orchards where they were not suppressed by the nematicide, cadusafos, emphasizing the importance of EPN conservation in orchards [Furthermore, naturally occurring EPNs (orchards .H. bacteriophora-based product (Cryptonem)  was registered for use against T. leucotreta in citrus [S. feltiae-based product has also been approved. This is an extremely positive development, as it now provides registered and commercially available biological products for targeting of the previously ignored soil-borne life stage of T. leucotreta [As a result of these positive results, an n citrus . This ren citrus ,111. Howucotreta .T. leucotreta.It remains to be seen how extensively these products are used once they become commercially available. Their mode of application will be through the irrigation system as described for the EPF products and, thus, very user-friendly. However, efficacy, perception of efficacy, and cost will also affect use. Generally, our trials have shown more consistent efficacy with EPF than EPN against the soil-dwelling life stages of T. leucotreta on citrus in southern Africa , they aT. leucotreta control. Similarly, another virus, the nucleopolyhedrovirus, CrpeNPV, was able to reduce T. leucotreta infestation to a comparable extent, i.e., by up to 90% [T. leucotreta fruit infestation by 95% in a single trial [T. leucotreta by up to 95% shortly before harvest [Thaumatotibia leucotreta infestation was shown to be reduced by up to 75% through augmentation of an egg parasitoid [There is a general perception that biopesticides may not be as effective as their chemical alternatives . Howeverp to 90% . SIT hasle trial  and by 9le trial . Chemica harvest ,46. Thaurasitoid , by 82% rasitoid , and by rasitoid .T. leucotreta control has become far more sophisticated than was previously the case, more specifically entailing the combination of various control measures as opposed to relying on a single treatment option [These are obviously best-case scenarios, and efficacy with biologicals does tend to be more variable than with chemicals ,112. Howt option . The levt option . FurtherT. leucotreta control programme recommended for citrus in South Africa is a foundation of regular diligent orchard sanitation, overlaid with an areawide control technology, such as SIT or mating disruption, and sprays (preferably virus products) applied at appropriate intervals, usually coinciding with peaks in egg hatch, forecast by moth flight peaks [T. leucotreta for the following season should be initiated immediately on completion of harvesting of the previous season, by collecting, removing, and destroying all fruit remaining in the orchard, both those hanging and those dropped to the ground [T. leucotreta population from one season’s crop to the next, particularly if there is a period of at least 3 months between harvest and fruit set, the maximum development period for the soil-dwelling life stages of T. leucotreta during winter [T. leucotreta population is still low, after suppression by winter conditions, and before population numbers can start to build. This is because all of these technologies are negatively density-dependent for optimal efficacy to be achieved [T. leucotreta egg hatch, which can be fairly accurately estimated through monitoring of male adult activity in pheromone traps [T. leucotreta is multivoltine (at least six generations per year in southern African conditions) [Consequently, the standard ht peaks . Controle ground . This wig winter . The recachieved ,41. Apprachieved ,101,108.achieved . Howeverachieved ,86,87, ane traps . Furtherditions)  and doesditions) , generatT. leucotreta is regulated as a phytosanitary pest by most export markets, meaning that there is zero tolerance for the pest, there are no thresholds dictating when control measures should be applied and when it is not considered necessary. Pheromone trap-based thresholds were recommended several years ago when T. leucotreta was primarily considered an economic pest, but this is no longer the case [T. leucotreta systems approach protocols, if a very low fruit infestation threshold is surpassed in fruit from the designated data trees in an orchard during the final 12 weeks before harvest, an additional corrective control measure must be applied, regardless of what control measures were previously applied [As the case . However applied . If this applied .T. leucotreta management programme that a grower chooses will ultimately depend on several factors: the grower’s assessment of the severity of the problem, the sensitivity of the market to the presence of T. leucotreta in the fruit, the severity of the postharvest handling conditions available and selected, the value of the particular cultivar, and the anticipated prices in the market place. None of these biological methods of control are incompatible with one another, possibly, surprisingly, not even mating disruption and SIT. In fact, superior efficacy was recorded against the closely related apple pest, C. pomonella, by combining these technologies [The full nologies . ConsequT. leucotreta occurrence at harvest to be suitable for export of fruit to a market that regulates T. leucotreta as a phytosanitary organism and exercises zero tolerance for the occurrence of any live larvae in a consignment of fruit. This threshold tolerance at harvest (as opposed to zero tolerance) is acceptable, as the fruit will still be subjected to several further steps that together will reduce the occurrence of T. leucotreta-infested fruit to a negligible level [T. leucotreta infestation on delivery at the packinghouse, to determine subsequent handling requirements, (2) packinghouse grading of fruit on the packing line, involving the removal of any fruit appearing to be damaged or infested, (3) inspection of a 2% fruit sample per pallet of fruit packed for export and rejection of any pallet in which live T. leucotreta was detected, (4) prescription of shipping condition options for each export consignment according to compliance with preceding steps of the systems approach, and (5) official phytosanitary certification of compliant consignments.Adoption of a judiciously constructed biological control programme will lead to a sufficiently suppressed level of le level ,17. ThisT. leucotreta after application of the systems approach, determining that it was 6–38 times less than the proportion associated with the Probit 9 (p ≤ 3.2 × 10−5) standard for a standalone cold treatment, being three survivors in 100,000 at the 95% confidence level, proven by zero survivors in 93,613 insects, i.e., a 99.9968% mortality [T. leucotreta, still concluding that the maximum potential proportion of fruit that may be infested with live T. leucotreta after application of the improved systems approach is no greater than the proportion of fruit that may be infested after application of a Probit 9 efficacy postharvest disinfestation treatment to fruit with a 2% pretreatment infestation. Additionally, they determined that the probability of a mating pair surviving the systems approach, as a more biologically meaningful alternative to the quantification of a mortality level such as the Probit 9 reference, was far lower. This reasoning would be strengthened by the fact that T. leucotreta is a poor invader [T. leucotreta-susceptible produce being exported throughout the world for more than 100 years [Moore et al.  calculat invader ,117, hav invader ,118 and  invader . This is00 years .T. leucotreta management programme only has the option of biological tools. Consequently, if a grower is in a region where SIT is not available, they have a choice of overlaying a biological approach (most likely virus sprays) or a chemical approach onto mating disruption. The average cost per hectare of a virus spray is 80 USD, whereas the average cost of a chemical spray is 243 USD. This is not considering the older chemistry, such as pyrethroids and some IGRs, whose use is discouraged or even restricted through residue intolerance by markets. Although the duration of control can be expected to be relatively similar between virus and chemical treatments, on average, it can be expected that the efficacy of a chemical treatment might be more consistent than that of a virus spray. However, the chemical treatments are more likely to have some detrimental effect on natural enemies, such as the parasitoids of T. leucotreta [Another assessment that must be made is that of the cost of a biological programme versus a conventional programme, as this too will influence a grower’s decision on what products to use. The cost of the biological products range from 62–309 USD per hectare per application or per programme with the product, whereas the cost of the chemical products ranges from 73–382 USD per hectare . The areucotreta ,55, thusMoore and Jukes  argue thT. leucotreta, for which export markets exercise zero tolerance, using exclusively biological means of control, but also highly desirable and beneficial for human (pesticide application) and environmental safety and in order to maintain the exportability of the fruit in a market that is increasingly chemical residue-intolerant.Consequently, in conclusion, it is not only possible to control the phytosanitary pest,


To assess the accuracy of magnetic resonance imaging (MRI) measurements in locally advanced oestrogen receptor-positive and human epidermal growth factor receptor 2-negative breast tumours before, during and after neoadjuvant endocrine treatment (NET) for evaluation of tumour response in comparison with clinical and pathological assessments.This prospective study enrolled postmenopausal patients treated neoadjuvant with letrozole and exemestane given sequentially in an intra-patient cross-over regimen. Fifty-four patients were initially recruited, but only 35 fulfilled the inclusion criteria and confirmed to participate with a median age of 77. Tumours were scanned with MRI prior to treatment, during the eighth week of treatment and prior to surgery. Additionally, changes in longest diameter on clinical examination (CE) and tumour size at pathology were determined. Pre- and post-operative measurements of tumour size were compared in order to evaluate tumour response.r) 0.64 compared to the correlation between CE and pathology r = 0.25. Post-treatment MRI and clinical results had a negligible bias towards underestimation of lesion size. Tumour size on MRI and CE had 0.82 cm and 0.52 cm lower mean size than tumour size measured by pathology, respectively.The correlation between post-treatment MRI size and pathology was moderate and higher with a correlation coefficient  contains supplementary material, which is available to authorized users. Current response assessments of locally advanced breast cancer (LABC) in the neoadjuvant setting are not entirely accurate in determining pathological complete response, depending on the modality used, the measurement technique and varied response of different tumour subtypes , 2. TailPerformed by a trained physician, CE is a widely recognized assessment for detecting breast cancers and for tumour size monitoring . CE is aAlthough mammography and ultrasound are reliable methods to analyse tumour size at diagnosis, changes within the tumour after neoadjuvant therapy may be difficult to assess. Targeted ultrasound is mandatory for malignant breast tumours and of the axilla for clinical staging if neoadjuvant therapy is planned . UltrasoDynamic contrast-enhanced MRI is the most sensitive of available imaging modalities in characterization of breast malignant tumours as a functional technique that allows the evaluation of residual viable tumour after neoadjuvant therapy by detecting changes in tumour vascularity –14. MultResponse Evaluation Criteria in Solid Tumours (RECIST) guidelines recommend assessment of the largest tumour diameter . OverestNo previous research has investigated the performance of clinical and MRI assessment methods in the evaluation of residual tumour size in patients receiving combined neoadjuvant endocrine therapy with third generation aromatase inhibitors , 22. AroThe current lack of published studies to assess the accuracy of MRI in predicting residual size of breast tumours following NET opens opportunities for evidence-based studies . There hThe aim of the current study is to compare tumour size estimated by CE and MRI with the tumour size in surgical specimen, in order to determine which is the most accurate assessment to evaluate tumour response in patients with LABC treated neoadjuvant with aromatase inhibitor (NAAI) therapy.From February 2015 to November 2019, patients with biopsy-proven locally advanced oestrogen receptor (ER)-positive breast cancer and human epidermal growth factor receptor 2 (HER-2)-negative status suitable for NET were eligible for this prospective, open-label, randomized sub-study of the Neoletexe trial, .n = 35).The diagnosis of breast cancer was established after a digital mammography, accompanied by breast and axillary ultrasound, a stereotactic or ultrasound- guided breast biopsy and CE. Primary tumours were classified using clinical and radiological tumour, node and metastasis (TNM) staging system for breast cancer, according to the classification system proposed by the American Joint Committee on Cancer AJCC, 2017) and Union for International Cancer Control . All patients, as part of their clinical care, were screened for distant metastasis with thoracic, abdominal and pelvic computed tomography scan and bone scintigraphy. Only two patients presented with limited systemic metastasis at baseline were allowed to enrol. Table 7 and Unihttps://www.acr.org/Clinical-Resources/Reporting-and-Data-Systems/Bi-Rads).MRI scans were performed prior to the start of NAAI therapy (baseline), 8 weeks after the first cycle and prior to the second cycle of therapy (second examination), after the final administration of NAAI therapy and immediately before surgery (third examination) Fig. . ImagingClinical tumour assessments were conducted at the oncological outpatient clinic by the same medical breast cancer oncologist every 4 weeks during the entire study, but only evaluations performed at baseline, following 8 weeks and 16 weeks were included in this analysis. The NAAI intra-patient cross-over regimen consisted of one of the following treatment arms: letrozole 2.5 mg o.d. for at least 8 weeks thereafter continuing with exemestane 25 mg o.d. for another 8 weeks prior to surgery; and exemestane 25 mg o.d. for at least 8 weeks thereafter continuing with letrozole 2.5 mg o.d. for 8 weeks prior to surgery , and reviewed afterwards in a consensus interpretation. The radiologists were blinded to findings from clinical assessment and were not aware of the post-operative histopathology results. Table Pathology was used as the gold standard for comparing tumour size measurements after neoadjuvant treatment. Comprehensive pathological analysis of surgical specimens was performed by an experienced breast cancer pathologist. During the course of the study supplementary scientific incisional surgical biopsies of tumour tissue were obtained, after the MRI examinations, at baseline, after 8 weeks and after surgery Fig. . These sPathological response was defined as complete responder  or not complete responder . Total extent of residual disease was reported, measured as the greatest one- dimensional extent in centimetres of residual invasive cancer including intervening areas of fibrosis and/or necrosis. In situ components measurements were not included.p ≤ 0.05 was considered statistically significant.Descriptive statistics were used to compared two measurements of tumour response (changes in LD at MRI and CE) to predict pathologic outcome. Each predictor variable was measured at three time points. Missing data elements were not included in the analysis. The Spearman rank correlation was applied to study the statistical dependence between the classification of the tumour sizes estimated by CE and MRI, and pathological results. The Bland–Altman analysis was used to evaluate deviations from the mean of the measures by two methods  and to estimate a range of agreement, within which 95% of the differences between the measurement by CE or MRI and the pathological measurement. Box plots were conducted to visually summarized the distribution of the numerical data. Statistical analysis was performed using R software. A two-sided The Regional Committee for Medical and Health Research Ethics South-East Norway approved the study protocol. Informed consent was obtained from all individual participants included in the study. The imaging findings have not previously been reported.The clinical-based TNM classification was used to identify 29 cases of T4 tumours (82.9%) and 6 cases of T3 tumours 17.1%) (Table 7.1% (Tabr = 0.71), .The median tumour size (LD) at baseline on MRI was 3.4 cm (interquartile range 2.9–5.0 cm); on CE was 5.0 cm (interquartile range 4.0–6.0 cm). Spearman correlation showed that baseline tumour size on CE and MRI had a strong positive agreement (r = 0.53), .The median tumour size between regimens on MRI was 2.7 cm (interquartile range 1.7–3.6 cm); on CE was 3.0 cm (interquartile range 2.5–3.8 cm). Spearman correlation showed that at this time point tumour size on CE and MRI had a moderate positive agreement (3), Fig. c. The Bl3), Fig. d analysir = 0.46, .The median size of the tumours after completion of NAAI therapy on MRI was 1.3 cm (interquartile range 0.4–2.6 cm) and on CE 2.0 cm (interquartile range 1.5–2.5 cm), with a moderate agreement, r = 0.64, p = 0.001) compared to the correlation between CE and pathology , . The correlation between post-treatment MRI size and pathology was moderate and higher  . MRI meaWeatherall et al. found that the sampling error between pathological and MRI measurements decreased when the lesion diameter was obtained by averaging measurements made in three orthogonal dimensions . This apCE was found to have a less dispersed data, and like MRI, it frequently underestimated size in comparison with pathology in our population. Clinical assessment was found to be less accurate for assessing complete responses, with a lower correlation with pathological measurements and 3 false-positive interpretations. Although, CE is essential, it is inherently subjective and cannot be validated as a definitive and exclusive assessment method.This current study should be considered in the context of its strengths and limitations. The first limitation of our study was the small sample size. However, this cohort of patients represents a pilot sub-study of an ongoing trial. A second limitation was that the imaging-based measurements were finally reviewed in a consensus imaging interpretation, and thus, interobserver variability was not evaluated. The major strength is that there are no previous studies analysing MRI accuracy measuring treatment response in patients receiving NAAI therapy. Another strength is the consecutive prospective inclusion of patients. With an increasing use of NAAI therapy it is of particular interest to determine the most accurate of the current assessment methods to evaluate at the earliest the treatment effect.In conclusion, our results showed that in postmenopausal patients diagnosed with LABC treated neoadjuvant with NAAI, the correlation between post-treatment MRI size and pathology was higher compared to the correlation between CE and pathology. MRI was found to be more accurate for estimating complete responses then clinical assessments. The results of this study provide further support for the benefit of a clinico-imaging preoperative assessment for evaluation of response, residual disease and the importance in deciding patient’s eligibility for BCS. Guidelines to correlate imaging and clinicopathological assessments, and further studies with larger sample sizes are needed.Supplementary file1 (DOCX 13 kb)Below is the link to the electronic supplementary material.


Hypoxia-induced alternative splicing is a potent driving force in tumour pathogenesis and progression. In this review, we update currents concepts of hypoxia-induced alternative splicing and how it influences tumour biology. Following brief descriptions of tumour-associated hypoxia and the pre-mRNA splicing process, we review the many ways hypoxia regulates alternative splicing and how hypoxia-induced alternative splicing impacts each individual hallmark of cancer. Hypoxia-induced alternative splicing integrates chemical and cellular tumour microenvironments, underpins continuous adaptation of the tumour cellular microenvironment responsible for metastatic progression and plays clear roles in oncogene activation and autonomous tumour growth, tumor suppressor inactivation, tumour cell immortalization, angiogenesis, tumour cell evasion of programmed cell death and the anti-tumour immune response, a tumour-promoting inflammatory response, adaptive metabolic re-programming, epithelial to mesenchymal transition, invasion and genetic instability, all of which combine to promote metastatic disease. The impressive number of hypoxia-induced alternative spliced protein isoforms that characterize tumour progression, classifies hypoxia-induced alternative splicing as the 11th hallmark of cancer, and offers a fertile source of potential diagnostic/prognostic markers and therapeutic targets. Tumour chemical and cellular microenvironments interact continually to select survival-adapted tumour cell and tumour-associated normal cell populations, and underpins both metastatic progression and therapeutic resistance. The tumour cellular microenvironment is comprised of “normal”  and neoplastic components that co-exist within a poorly defined and poorly organized extracellular matrix, characterized by heterogeneous niches created by a highly abnormal vasculature and episodes of microenvironmental hypoxic, nutrient, metabolic and redox stress, which elicit cellular hypoxic, nutrient, oxidative and metabolic stress responses. Tumour hypoxia promotes glycolytic metabolic adaptation by tumour cellular components, combined with oncogene-promoted metabolic changes, result in the malignant tumour-associated “Warburg” metabo-type –3. The m2] concentrations ≤2.5 mmHg, significantly below those of normal vascular tissues, as a result of an imbalance between oxygen consumption and supply, e.g. [pO2] of 10–16 mmHg in cervical tumour tissues is significantly lower than the [pO2] 40–42 mmHg of normal cervical tissues ..99].Hypoxia-induced EMT also associates with repression of ESRP1 splice factor expression, resulting in α6B integrin subunit alternative splicing and the generation of α6Bβ1 integrin receptors that characterize cancer stem cell phenotypes , 212. AtEMT also associates with a progressively more cancer stem cell-like phenotype –216, repIn head and neck cancers, hypoxia-induced laminin α3 chain alternative splicing and expression of the splice variant LAMA3 isoform has been implicated in tumour invasion and metastatic progression  as has hIL1-RAP alternative exon 13 inclusion, membrane associated mIL1-RAP expression and IL1β/IL1R1/mIL1RAP-dependent expression of CD47, the “don’t eat me” inhibitor of macrophage phagocytosis, identifying a UPR/SRSF10/mIL1RAP/CD47-dependent tumour-promoting axis  biogenesis factors and by SRSF1 and Slu7 splicing factors. Hypoxia promotes SRSF1 and Slu7 cytoplasmic stress granules sequestration, reducing nuclear levels and resulting in mitotic aberrations, R-loop formation and genomic instability, characterized by DNA-damage, mitotic derangement and sister chromatid cohesion, dependent upon aberrant SRSF1 (ASF/SF2) splicing factor activity, alternative SRSF3 truncated SRp20 and -TR isoforms expression –240. HypIn breast cancer cells, hypoxia also triggers alternative BRCA1-IRIS splicing in hypoxic/necrotic niches, promoting tumour progression by de-regulating wtBRCA1 function –247, andFinally, expression of the hypoxia-regulated alternative TrkAIII splice variant in neuroblastoma cells augments sister chromatid exchanges and re-localizes to centrosomes in active form, inducing polo kinase 4 activation, centrosome amplification, enhanced tubulin polymerization and chromosomal instability Fig. b 221, 2.Between 10 and 30% of solid tumours are characterized by fluctuating acute and chronic hypoxia, resulting in cellular hypoxic responses that include alternative pre-mRNA splicing and the expression of novel protein isoforms that promote tumour progression and impact therapeutic efficacy. Hypoxic regions of tumours are populated by slowly dividing tumour cells that escape death induced by cytotoxic agents that target proliferating cells and are infiltrated by immature tortuous permeable blood and lymphatic vasculatures that increase tumour interstitial hypertension, a potent force for drug expulsion. Tumour glycolytic adaptation renders the hypoxic tumour microenvironment acidic and reducing, further de-regulating inflammatory and immune cell recruitment and function, enhancing multidrug resistance through elevated expression of p-glycoprotein multidrug transporter, which combined with mechanisms to evade programmed cell death, greatly reduce therapeutic efficacy. Considering the many roles of hypoxia-induced alternative splicing in tumour pathogenesis and progression, targeting tumour microenvironmental hypoxia, the tumour microvasculature, hypoxic responses, hypoxia-induced alternative splicing and tumour promoting alternative splice protein isoforms, are all of potential therapeutic importance –257.Therapeutic efficacy can be enhanced by interfering with or reprogramming the hypoxic tumour niche to improve drug efficacy . Tumour “Normalization” of the aberrant tumour vasculature is also emerging as an alternative way to improve tumour oxygenation, reduce tumour progression and therapeutic efficacy. This stems from observations that vascular destruction by anti-angiogenic agents promotes tumour hypoxia, reduces therapeutic efficacy and facilitates metastatic progression. The tumour microvasculature is immature, permeable, tortuous, haphazard, exhibits aberrant basement membranes and lacks a complete repertoire of cellular and matrix components required for vascular maturation and function. This flawed system increases interstitial hypertension resulting in drug expulsion, inducing selection of more aggressive phenotypes through adaptation to hypoxia, which is facilitated by hypoxia-induced alternative splicing. Vascular “Normalization” requires the delicate rebalancing of angiogenic factor/inhibitor equilibria and can be achieved by careful selection and dosage of antiangiogenic agents. This has been demonstrated by down regulating VEGFA expression in a human tumour mouse xenograft model, resulting in the pruning immature permeable vessels, re-modeling a less-permeable, less-tortuous vasculature with more pericytes and near-normal basement membrane, responsible for increasing tumour oxygenation, decreasing tumour interstitial pressure and improving drug penetration . ConsistTumour promoting subversion of pre-mRNA splicing by hypoxia, resulting in oncogene activation, tumour suppressor inactivation, immortalization, metabolic adaptation, evasion of programmed cell death and anti-tumour immunity, angiogenesis, tumour-promoting inflammation and genetic instability, not only depends upon specific alternatively spliced protein isoforms but also specific spliceosome components, splicing factors, splice factor kinases and splicing, all of which represent potential therapeutic targets.HIF transcription factors are activated by tumour hypoxia, promote HIF-target expression and alternative splicing of HIF-target and non-HIF target genes . HypoxiaHIF inhibitors fall into different categories and include inhibitors of HIF mRNA and protein expression, inhibitors of HIF dimerization, DNA-binding and transcriptional activity and promoters of HIF degradation, with some inhibitors exhibiting more than one function. Inhibitors of HIF-1 mRNA and protein expression, include: lncRNA PIN1-v2 , S-TRPM2Inhibitors of Hif-1α accumulation and transcriptional activity, include the Hsp90 inhibitors GA, 17-AAG, 17DMAG and EC154 that promote VHL-dependent HIF-α degradation , 272, thInhibitors of HIF dimerization include cyclic CLLFVY that binds the HIF-1α PAS-B domain disrupting dimerization, transcriptional function and hypoxic response in tumour cells , TC-S700HIF transcriptional Inhibitors, include: chetomin dithio-diketopiperizine that impedes HIF-1α interaction with its transcriptional activating histone acetyltransferase p300 co-factor and increases the radiosensitivity of human fibrosarcoma cells ; idenopyTargeting the spliceosome is an alternative way to inhibit tumour-promoting, hypoxia-induced alternative splicing.50 concentrations, making them more suitable for therapy [Bacterial products that bind the SF3B component of U2 snRNP and interrupt spliceosome assembly, include: the spliceostatins, spliceostatin A, FD-895 and the derivatives FR901463, FR901464 and FR901465; sudemycins, from pseudomonas ; herboxi therapy –295. The therapy . Recentl therapy , have be therapy , 299.RNA-based therapeutics have the potential to target any mRNA and, therefore, any protein, including proteins that lack catalytic activity, cannot be targeted by small molecular inhibitors or that are unamenable to antibody targeting.Antisense oligonucleotides (ASOs) are the mainstay of RNA-based therapeutics. ASOs are 15-39mer chemically-modified RNA or DNA molecules that either redirect specific splicing events to prevent pathology-promoting splice isoforms or to generate isoforms that inhibit pathology. The first proof of concept for therapeutic ASOs efficacy in preventing aberrant alternative splicing has come from the FDA-approved ASO “Spiranza”, that targets survival motor neuron 2 (SMN2) pre-mRNA to promote exon 7 inclusion and full length SMN2 protein expression and has been successfully employed to treat spinal muscular atrophy , 301. TaCamptothecin and topotecan topoisomerase inhibitors, doxorubicin and cisplatin have all been reported to induce splicing changes in genes involved in DNA damage-repair, genetic stability and immortality , 306. Docis enhancer or silencer elements in introns and exons (e.g Spiranza which binds SMN2 mRNA to promote exon 7 inclusion by inhibiting an ISS) [Splicing factors are divided into the serine/arginine-containing SR proteins: SRSFs 1–12, SC35, SRp20, SRp30c, SRp38, SRp40, SRp54, SRp55, SRp75, HTra2α, HTra2β1, 9G8, SF2/ASF and SRm160; nhRNPs: hnRNPA0, A1, A2/B1, A3, C, C1, C2, D, D0, DL, E1, E2, F, G, H1, H2, I (PTB), J, K, L, LL, M, Q, U, nPTB; and others: RBFox-1, RBFox-2,, DAZAP1, PSF, TDP43, RBM4, RBM5, RBM 10, RBM25, CUG-BP1, ESRP1, ESRP2, ETR-3, HuB, HuC, HuD, HuR, TIA-1, TIAL1, QK1, Sam68, SLM-1, SLM-2, SF1, SRSF2, SRSF3B1, U2AF1, FMRP, Nova-1, Nova-2, PRPF40B, KSRP, ZRANB2, MBNL1, YB-1, SAP155 ZRSR2 . Splicin an ISS) , 301. Fu an ISS) , 312 andThe oral selective inhibitor of nuclear export KPT-330 (Selinexor) , impairsThe ubiquitin proteasome pathway inhibitor Bortezomib reduces the proliferation CA46 and Daudi Burkitt lymphoma cells by down regulating the expression of high molecular weight sumoylated hnRNP K splicing factor and cMyc and up-regulating the expression of low molecular weight de-sumoylated hnRNP K, implicating sumoylated hnRNP K and cMyc repression in Bortezomib inhibition of Burkitt Lymphoma cell proliferation . SiRNA hTherapeutic targeting of splice factor kinases, which modulate splice factor involvement in spliceosome assembly, splice factor binding to splice sites and subsequently alternative splicing may also reduce the expression and activity of hypoxia-induced alternatively spliced tumour promoting protein isoforms . SelectiTargeted therapies for the hypoxia-induced alternatively spliced EGFRvIII driver oncogene, include 1st generation , 2nd generation (Afatinib and Dacomitinib) and 3rd generation (Rociletinib and AZD9291) tyrosine kinase inhibitors; EGFR L2 domain targeting antibodies , the EGFRvIII-specific antibody mAB806 or antibodies conjugated with a toxins or radioisotopes (125ImAB425) and the double specificity (bis) antibody bscEGFRvIIIxCD3. A vaccine against EGFRvIII (Rindopepimut CDX110), however, has failed in phase III trials. CAR-T cells that target EGFRvIII are another promising option and are in phase I clinical trials, and RNA-based anti-EGFRvIII therapeutics are being evaluated in animal models , 327. ThIt is clear that tumour hypoxia-induced alternative splicing plays a fundamental role in all 10 cancer hallmarks, from initiation to metastatic growth, and is also a critical determinant of therapeutic resistance. The influence of hypoxia upon the basic mechanisms of pre-mRNA splicing including spliceosome assembly, splicing factor expression, activity and intracellular localization, miRNA synthesis and maturation, and the added influence of mRNA structure and elongation rate, results in an impressive number of alternatively spliced protein isoforms that promote tumor pathogenesis, metastatic progression and therapeutic resistance, warranting classification of hypoxia-induced alternative splicing as the 11th hallmark of cancer. Tumour hypoxia-induced alternative splicing provides a plethora of biomarkers of prognostic potential and therapeutic targets with potential to slow tumour progression and enhance therapeutic efficacy.


The hyper-activation of innate immunity caused by genetic factors sometimes results in skin inflammatory diseases with hyperkeratosis. Such cutaneous inflammatory disorders whose hyperkeratosis has genetic autoinflammatory pathomechanisms are termed “autoinflammatory keratinization diseases” (AiKDs) , 2. AiKDAiKDs involve significant genetic factors causing the hyper-activation of innate immunity (autoinflammation), primarily in the epidermis, and the upper dermis. The hyper-activation of innate immunity in the epidermis and the superficial dermis results in aberrantly up-regulated keratinization, leading to the additional inflammatory symptoms of AiKDs.The shared clinical characteristics of AiKDs are hyperkeratotic inflammatory cutaneous lesions, although the clinical features of AiKDs vary and some AiKDs have unique clinical features. The majority of AiKD patients have recurrent and persistent cutaneous manifestations which are intractable against treatment with standard therapeutics for inflammatory keratinization disorders.We believe that the idea of AiKDs provides us with clues to further understand the pathomechanisms behind various inflammatory keratinization diseases. The present Research Topic article aims to assess the debate over the pathomechanisms behind AiKDs, to propose avenues of investigation into therapeutic strategies for these diseases, and to deliver broad, divergent visions of how autoinflammation contributes to various cutaneous diseases.Kanazawa proposed that AiDs present various skin manifestations and that not a few AiDs were described as clinical entities originally from the skin symptoms. One example is cryopyrin-associated periodic fever syndrome (CAPS), which was originally called familial cold urticaria.The term “autoinflammatory diseases” (AiDs) was first introduced in 1999 when germline mutations in tumor necrosis factor receptor superfamily 1 (TNFRSF1) were reported to underlie tumor necrosis factor receptor-associated periodic syndrome (TRAPS) . Dramatic advances in the understanding of the mechanisms behind innate immunity have enabled us to designate inflammation caused by the genetic hyper-activation of innate immunity as “autoinflammation.” Monogenic disorders with systemic autoinflammatory symptoms are the principal AiDs. Kanazawa introduced various AiDs that show skin manifestations, such as CAPS, Blau syndrome and pyogenic arthritis, acne and pyoderma gangrenosum (PAPA) syndrome. Information on the skin symptoms of AiDs shows the similarities and differences between AiDs and AiKDs, and lets us recognize that AiKDs are unique clinical entities distinct from AiDs.To elucidate the major skin symptoms of AiDs, IL36RN encoding the IL-36 receptor antagonist (IL-36Ra)  caused by CARD14 mutations/variants , impetigo herpetiformis and acrodermatitis continua due to mutations in utations .Akiyama described the rapid expansion of disorders categorized as AiKDs in recent years. Indeed, patients with hidradenitis suppurativa (HS), also called acne inversa, especially familial cases, have been proposed to have an AiKD  have been reported to be associated with HS ( an AiKD . HS is and PSEN1 . HoweverTakeichi and Akiyama). KLICK syndrome is a rare genodermatosis that clinically shows generalized ichthyosiform scaling, diffuse palmoplantar keratoderma with constriction rings on the fingers/toes, and hyperkeratotic plaques and papules distributed linearly on the arm folds and the wrists. Thickening of the spinous, granular and cornified cell layers in the epidermis and mild inflammatory cell infiltration are seen in affected skin. KLICK syndrome is due to a mutation in POMP which encodes proteasome maturation protein (POMP)  syndrome as an AiKD (n (POMP) . POMP plMVK, MVD, PMVK and FDPS) is also suggested to be an AiKD (In addition to the diseases discussed above, porokeratosis with mutations in four mevalonate pathway genes ( an AiKD .Akiyama expects that increasing numbers of inflammatory keratinization diseases will be included in AiKDs in the future as the pathogenic mechanisms of inflammatory keratinization diseases in the skin are successively elucidated. AiKD as a novel clinical entity has provided us with a novel concept of inflammatory keratinization diseases. In addition, advances in the understanding of the aberrant hyper-activation of innate immunity in the skin as pathomechanisms behind AiKDs could lead us to innovations in more effective, targeted, causal treatments for various AiKDs.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.