Carbon-Fibre-Reinforced Polymers (CFRPs) have seen a steady rise in modern industrial applications due to their high strength-to-weight ratio and corrosion resistance. However, their potential is being hindered by delamination which is induced on them during machining operations. This has led to the adoption of new and innovative techniques like cryogenic-assisted machining which could potentially help reduce delamination. This study is aimed at investigating the effect of cryogenic conditions on achieving better hole quality with reduced delamination. In this paper, the numerical analysis of the drilling of CFRP composites is presented. Drilling tests were performed experimentally for validation purposes. The effects of cooling conditions and their subsequent effect on the thrust force and delamination were evaluated using ABAQUS/CAE. The numerical models and experimental results both demonstrated a significant reduction in the delamination factor in CFRP under cryogenic drilling conditions. Composites are having a significant influence on today’s fields of engineering and science. This is evident from their extensive applications, ranging from their use in space industries for the construction of intricate technologies such as cryogenic tanks to their use in the more conceivable fields of sports and transportation. Of all such composite materials, carbon-fibre-reinforced polymers (CFRPs) have seen a rise in popularity. This is primarily due to their high strength-to-weight ratio. The advent of CFRPs has allowed industries to use lighter and more robust materials without any loss in performance. Besides having a great strength-to-weight ratio, these materials also possess a plethora of other desirable properties such as high stiffness, low density and an excellent damping capacity. CFRPs are also immune to corrosion; however, they are prone to failure by penetration, cracking or delamination due to their highly anisotropic and inhomogeneous nature .Drilling is widely used to produce holes on composite parts which are then assembled. However, due to the non-homogeneous, anisotropic and highly abradant nature of CFRPs, excessive tool wear occurs during drilling which causes fibre pullout, particle fracture and delamination . DelaminExtensive studies have been conducted by researchers to negate the effects of delamination. Tsao  conducteDespite numerous models available to predict delamination, the majority of them considered the crack length alone, while a few studies conducted by Davim et al.  mooted tVarious other ideas have been provided to minimise delamination, such as changing cutting speeds, drill bit geometries, feed rate, tool materials and tool coatings . KrishnaNumerous literatures are available on the machining of CFRPs concerning tool wear, the effect of drilling parameters and the variability of the machining conditions. Bhattacharyya et al.  conducteSimulation software such as Abaqus possesses high computing ability and is quite easy to perform. It also allows for the numerical validation of various proposed loading models . PhapaleA 3D CAD model of the tungsten carbide drill with a diameter of 8 mm was designed in SolidWorks with the specifications provided by the tool manufacturer. The drill had a helix angle of 30°. The tool geometry had to be very precise to reduce any errors and to obtain accurate simulation results. To reduce the computational time, the drill bit was cut by 40 mm from the lower tip. The 3D model was then imported in Abaqus and was later assembled with the workpiece model for the simulation.For this simulation, the tool was assumed to be a rigid and nondeformable body, while the workpiece was modelled as a 3D deformable and rigid body. This assumption is justified as the yield stress of tungsten carbide is considerably higher than that of the CFRP. No material property was assigned to the drill bit as it was treated as a rigid body for the simulation. This is shown in The three major aspects of modelling the simulation were the boundary conditions, the interaction properties and the meshing. The boundary conditions were given to simulate the experimental procedure with as much accuracy as possible. The drill was assembled in the system in such a way that the tip of the drill was 2 mm above the workpiece centre. The drill was made to move in the –Z direction as per the setup. The drill movement was restricted in the X and Y directions, and it was given translational velocity as well as rotational velocity in the –Z direction. All the boundary conditions for the drill were assigned using the velocity boundary conditions on a singular reference point on the drill. The total period of the entire simulation was given as 2.5 s to reduce the computational load. Long step times generally lead to excessively long calculations and at the same time provide erroneous outcomes and convergence issues.Two major interactions occurred in the model: viz. 1. the interaction between the composite workpiece layers and 2. the interaction between workpiece layers and the drill. The cohesive elements of the composite layers of the workpiece are the main ones to show delamination. The CSDMG parameter is used in the field output variables to see where the nodal damage represents the scalar stiffness degradation for cohesive surfaces. A wedge with a free structure arrangement was used for the meshing of the drill because of its delicate geometry. The element size was given as 1 mm to have a fine mesh that does not cause high distortions.The mechanical properties of the CFRP were implemented in the simulation using the property module in Abaqus. The 30 mm × 50 mm workpiece was modelled in Abaqus/CAE. The CFRP workpiece was modelled with two fibre orientations (0° and 90°) in a 21-layer setup by using the composite layup option in Abaqus. The properties of the 3D deformable solid in the extrusion mode were applied to the workpiece. To simulate the composite characteristics, cohesive elements were added to the model with the cohesive surface parameters. The CFRP ply stack plot is shown in The boundary condition for the CFRP was set with the purpose of clamping the workpiece from all directions. All movements were restricted for the workpiece using the encastre boundary conditions on the side faces of the CFRP layers. Different interactions were given for cryogenic and dry drilling. The cryogenic model takes the heat transfer interaction between the drill and the workpiece into consideration as the liquid nitrogen was sprayed on the tip of the drill and the uppermost layer of the CFRP for the entire duration of drilling. In the simulation, this boundary condition was given by giving the conductivity values of the CFRP in material properties. Further, the CFRP layers were given a predefined temperature of −196 °C to simulate the cooling due to liquid nitrogen. The boundary conditions and the meshed model are portrayed in Reduced-integrated 8-node shell elements were used for the meshing of the CFRP plate. Cohesive behaviour was given between the plies for the simulation. The mesh density around the drilled hole was considered up to the element size of 0.05 mm to reduce the computational cost and to optimise the results for a clearer picture of the delaminated area. The element deletion criterion was activated to delete any excessively damaged elements. This is an important step as undeleted elements cause computational errors and problems during visualization. A finer mesh was given to the area which was facing damage, while the ends of the workpiece were given a coarser mesh size. The mesh was a hex element shaped to mesh with a swept structure, which allowed the mesh to be even on both the front and backside of the workpiece. An advanced front meshing algorithm was used as per the default setting in the software. An SC8R mesh was used during the thermal analysis as the use of a thermal shell created problems in the convergence of the simulation. The detailed specifications of both the workpiece and tool meshing are listed in Hashin’s failure criteria were employed as the primary failure criteria as they assume four failure modes for composite materials; viz. 1. fibre tension, 2. fibre compression, 3. matrix tension and 4. matrix compression.σ11 ≥ 0Fibre tension σ11 < 0Fibre compression σ22 + σ33 > 0Matrix tension σ22 + σ33 > 0Matrix compression The damage evolution of the CFRP was based on the energy dissipation during the process and is linear according to the Abaqus built-in Hashin model. The various modes and their corresponding failure criteria for the fibre and matrix are presented by Equations (1)–(4). In Abaqus/CAE, the element is deleted when the damage variables reach the value of 1 or greater under the failure modes presented in the above equations. The value of the variables viz. DAMAGEFT (fibre tensile damage variable), DAMAGEFC (fibre compressive damage variable), DAMAGEMT (matrix tensile damage variable) and DAMAGEMC (matrix compressive damage variable) is checked, and if the value is greater than the threshold, the element will be deleted according to the Hashin model subroutine incorporated in Abaqus/Standard CAE. Hashin’s parameters are well established and widely adopted in the industries .Before machining or initiating any damage, the material property is assumed to be linear elastic for CFRP composite laminates. Thus, the orthotropic elastic material is modelled with the stress–strain relationship.Once the damage initiation criterion is satisfied, the material degradation will start taking place. The two coefficients of the damage will come into the equation (df and dm). The coefficients will change accordingly.A computer numerical controlled (CNC) vertical drilling machine  was used for conducting the experiments. The drill used was an 8 mm solid carbide drill (uncoated) from Kennametal with a point angle of 130°. Solid carbide drills have excellent thermal stability, which was of prime importance while drilling under cryogenic conditions. After each experiment, a new drill bit was used to minimise errors due to tool wear. The experimental setup adopted in this research is presented in The drilling procedure was performed conventionally, while the liquid nitrogen was sprayed at the tool–workpiece interaction. This was done to maximise the utilisation of the cryogenic fluid. The workpiece was clamped between custom-made fixtures, which had sufficient room for the cryogenic fluid to affect a substantial area. After the workpiece was firmly secured, holes were drilled through its entire depth. The dynamometer (Kistler 9257B) was fixed on the mild steel base and was insulated using foam to protect it from subzero temperatures. A nozzle was used to spray the liquid nitrogen on the workpiece at 1 bar pressure and −196 °C. Drilling was done both in a cryogenic and dry environment.f). The delamination at the exit and entry sides of the drilled hole is shown in Delamination is the most common form of damage in CFRPs. A lot of research has been done to quantify the damage caused by delamination. Generally, delamination is expressed by a parameter known as the delamination factor  to assess the delamination. The delamination at the tool exit area is primarily considered as it faces the most damage. The delamination as determined by the experimental, analytical and numerical approaches under a dry and cryogenic environment is presented in It is inferred from the experiments that the cutting speed and feed rate have a considerable influence on the delamination factor. The delamination factor was found to be least at 125 m/min and 0.03 mm/rev under dry drilling conditions. A visual representation of the delaminated holes in both dry and cryogenic conditions is presented in The influence of the feed rate and cutting velocity on the delamination factors under the dry drilling of CFRP is presented in The delamination factor values as predicted by the numerical model were found to be closer to the experimental values (within 15%). However, the values obtained using the analytical model showed greater deviation from the experimental values as compared to the numerical data. This is because f DmaxD0 .The influence of the feed rate and cutting velocity on the delamination factors under cryogenic-assisted CFRP drilling is presented in The values of the delamination factor predicted by the numerical model were very close to the experimental values (within 15%). The values obtained using the analytical model were relatively close to the experimental values. Under a cryogenic environment, a higher impact is established by ironment .From The experimental and numerical predicted thrust force for CFRP drilling in a dry environment under different operating parameters is shown in The experimental and numerical results of the thrust force for CFRP drilling under a cryogenic environment with varying feed rates and cutting speeds are shown in The surface morphology of the drilled holes at a higher cutting velocity (150 m/min) under dry and cryogenic conditions is shown in In dry drilling, the matrix gets decomposed and softened due to higher cutting temperatures, resulting in poor surface quality. Under cryogenic conditions, better surface quality with fewer fibre pullouts is seen. Even though the thrust force is higher under a cryogenic environment, the surface quality deterioration is significantly less when compared with dry drilling. This is mainly due to the increase in fibre brittleness caused by liquid nitrogen. Thus, the failure mode of the fibre under cryogenic drilling changes from rupture to shear fracture. As the feed rate increases, the surface quality deteriorates. With increasing feed, the uncut chip thickness increases, resulting in higher order cutting forces. A high magnitude cutting force increases the angle between the fibre orientation and cutting direction, thus increasing the fibre pullout on the drilled surface.The thrust force obtained in cryogenic drilling is found to be much larger than that in dry drilling. An increase in the feed rate leads to increased thrust forces; the same trend is observed under the simulation and the experimentation as well.The delamination factor in cryogenic drilling is found to be much lower than that in dry drilling. An increase in the feed rate results in an increase in the delamination factor. This is ensured by both the experimentation and simulation results.The delamination factor obtained via the numerical model is in good agreement with both the experimental and force-adjusted delamination factors for cryogenic drilling.Drilling in cryogenic conditions gives better results than dry drilling that is evident from the experimental and simulation results.An exhaustive investigation was carried out to study the effects of the cryogenic environment on the thrust force and delamination factor during CFRP drilling. The present study that combines the experimental, numerical and analytical modelling approaches stands as evidence for the success of cryogenic conditions in mitigating the drilling-induced delamination of CFRP. This study closely predicts the values of the delamination factor as well as its variation with the changes in operating parameters. The major outcomes of the present study are summarised and presented below:The experimental, numerical and analytical modelling exhibited the supremacy of cryogenic over the dry condition for CFRP drilling. The reduced delamination factor will reduce the rejection rates in aerospace and other industries, which utilise CFRP for manufacturing a wide variety of components. The present research work can be extended by the numerical and analytical modelling of CFRP drilling under sustainable environments such as chilled air and minimum quantity lubrication (MQL).


AML, frequently showing megakaryocytic differentiation, is known to arise in MGCT and has a dismal prognosis. Close inspection of MGCT is required to identify concurrent AML. AML, frequently showing megakaryocytic differentiation, is known to arise in MGCT and has a dismal prognosis. Close inspection of MGCT is required to identify concurrent AML. A 30‐year‐old man presented with hemoptysis. A 15 cm anterior mediastinal mass was noted on CT, and a diagnosis of mixed germ cell tumor (MGCT) was made based on core biopsy and elevated serum levels of AFP and β‐hCG. He subsequently received bleomycin, etoposide, and cisplatin at standard dose and schedule. Following the excision of the residual tumor, histologic examination showed a mononuclear proliferation within the MGCT (Figure The authors state that they have no conflicts of interest.AA and PWR wrote the manuscript. AA prepared the figure.IRB approval is deemed not necessary as only one patient is described.


It is an old Soviet joke that “The future is certain; it is only the past that is unpredictable,” but history in general is often reinterpreted, and often in the interest of those in power. This process is made easier by the inevitable existence of some sloppy scholarly work that will help unscrupulous “influencers” to spread uncertainty and allow radical misinterpretations. Science is of course supposed to be a rational evidence-based activity and should therefore not have such problems with its own history. However, sloppy referencing, not picked up by sloppy reviewing, does occur. When checking earlier work carefully, it turns out that not all generally accepted narratives concerning when and by whom discoveries were made are correct.Do we care about the history of our scientific research fields? Unlike those working in other areas of intellectual life, for example, the arts, philosophy, and politics, scientists in general do not seem to worry much about getting the past right. Typically, an original research article refers in its introduction to a review article when describing the scientific background. What is known about the history of a particular field can therefore effectively be determined by a single review article in a prominent journal. Whereas even minute details of recent findings in a field will be tested, confirmed, modified, or rejected, the broad history of a field will often not be revisited and therefore not revised.Physiological Reviews dealing with the exocrine pancreas. One of the sections in this review deals with Ca2+-activated Cl– channels, as these pores play a key role in the control of secretion.1 Surveying the general literature on this channel, which is found in many different tissues in the body and has many different functions, I read the following introductory statement in a highly cited review2: “Many cell types express a type of Cl− channel that is activated by cytosolic Ca2+ concentrations ([Ca2+]i) in the range of 0.2–5 µM. For example, in Xenopus oocytes, where these channels were first described in the early 1980s …” The two references in this introductory statement are to papers published in 1982 and 1983, respectively, in which there are no references to earlier descriptions of this channel. Unfortunately, the statement is misleading. As pointed out in my recent review article,1 this channel was discovered by the late Sir Michael Berridge and his colleagues in the early 1970s and most completely described in their 1975 article in the Journal of Physiology.3 In this article, there is even a clear diagram illustrating how these Cl– channels, activated by a rise in the cytosolic Ca2+ concentration, are essential for the process of fluid secretion in insect salivary glands.3 The Ca2+-activated Cl– channels were extensively characterized in mouse pancreatic acinar cells and described in many papers published several years before their apparent “discovery” in oocytes.1 It was shown that acetylcholine opens Cl– channels in the pancreatic acinar cell membrane and that intracellular Ca2+ injection can mimic this effect. The anionic selectivity of the Ca2+-activated Cl– channel in the acinar membrane was established with the following permeability sequence (in descending order of permeability): NO3– > I– > Br– > Cl–.1 This same sequence was (re)established many years later in studies on the Cl– channel in Xenopus oocytes and other cells, but again without reference to the original finding.2 The 2005 review article on Ca2+-activated Cl– channels2 has a short section dealing with fluid secretion by exocrine glands, but even in this section there are no references to the original work carried out in the 1970s. Instead, another review article is cited, in which none of the original papers are mentioned, referring—again—to other review articles. This problematic pattern, seen in many review articles in many journals, is the reason that Function insists that discoveries reported in the original research articles are cited in our Evidence Reviews.My interest in this problem was reawakened when I recently completed a review article for 2+-activated Cl– channels2 had consequences, as virtually all subsequent investigators and review writers continued to perpetuate the myth that these channels were discovered in oocytes and neurons in the 1980s, disregarding the reports of the much earlier discovery of these channels in the exocrine glands.The misleading information about the discovery of Ca2+-activated Cl– channels are now known to be TMEM16 proteins,4 and there is an extensive literature about their functions, particularly in the nervous system. In 2013, an interesting study reported activation of the TMEM16A channel  in nociceptive sensory neurons by local, oscillating, Ca2+ signals generated by Ca2+ release via IP3 receptors.5 However, this phenomenon was discovered, and described in detail, many years earlier in a study of pancreatic acinar cells.6Ca2+-activated Cl– channels in exocrine glands may be regarded as esoteric by neuroscientists, but may become central in the current COVID era. A very recently published paper shows that the lungs of COVID-19 patients contain pneumocytes with activated SARS-CoV-2 spike protein at the plasma membrane level. These cells have increased cytosolic Ca2+ oscillations leading to increased TMEM16 activity, potentially causing increased Cl– secretion, which could lead to alveolar edema.7The field of CaFor most human endeavors, we believe that an accurate knowledge of the past is important, and it is my personal belief that this must also be the case for science and, therefore, of course, also for physiology. If this is so, then we should at least give some priority to getting the history of our many different research fields right. Review writers and referees should therefore not only be concerned about the accuracy of the description of what is currently known in a particular field but also check carefully that the history of the field is correctly described.As always, rectification of a problem requires in the first instance recognition that there is a problem. The stark example I have given in this editorial is probably not unique and may inspire others to check whether their particular field has an accurate knowledge of its past. I would be interested to hear about other cases where errors, or perhaps even deliberate omissions of key references in a prominent review article, have distorted the general perception of what was shown by whom and when.


Cldn2/12 double knockout mice were generated. They display decreased intestinal calcium absorption and renal calcium wasting, resulting in hypocalcemia and markedly reduced bone mineralization. Claudin-2 and claudin-12 don’t physically interact in vitro, and coexpression has an additive effect on calcium permeability. Our work identifies claudin-2 and claudin-12 as important constituents of the paracellular Ca2+ pathway in intestine and kidney enabling calcium transport and highlights their important complementary roles in maintaining calcium homeostasis.Significant calcium absorption across renal and intestinal epithelia occurs via the paracellular pathway. However, the identity of the paracellular pore involved is unknown. Claudin-2 and claudin-12 contribute paracellular calcium permeability in cell models, but single knockout animals don’t have altered serum calcium or bone mineralization. To investigate this,  2+) homeostasis is maintained through coordination between intestinal absorption, renal reabsorption, and bone remodeling. Intestinal and renal (re)absorption occurs via transcellular and paracellular pathways. The latter contributes the bulk of (re)absorption under conditions of adequate intake. Epithelial paracellular permeability is conferred by tight-junction proteins called claudins. However, the molecular identity of the paracellular Ca2+ pore remains to be delineated. Claudins (Cldn)-2 and -12 confer Ca2+ permeability, but deletion of either claudin does not result in a negative Ca2+ balance or increased calciotropic hormone levels, suggesting the existence of additional transport pathways or parallel roles for the two claudins. To test this, we generated a Cldn2/12 double knockout mouse (DKO). These animals have reduced intestinal Ca2+ absorption. Colonic Ca2+ permeability is also reduced in DKO mice and significantly lower than single-null animals, while small intestine Ca2+ permeability is unaltered. The DKO mice display significantly greater urinary Ca2+ wasting than Cldn2 null animals. These perturbations lead to hypocalcemia and reduced bone mineral density, which was not observed in single-KO animals. Both claudins were localized to colonic epithelial crypts and renal proximal tubule cells, but they do not physically interact in vitro. Overexpression of either claudin increased Ca2+ permeability in cell models with endogenous expression of the other claudin. We find claudin-2 and claudin-12 form partially redundant, independent Ca2+ permeable pores in renal and colonic epithelia that enable paracellular Ca2+ (re)absorption in these segments, with either one sufficient to maintain Ca2+ balance.Calcium (Ca Serum Ca2+ is tightly regulated within a narrow range, and homeostasis maintained through coordinated regulation between the intestines, kidneys, and bones  knockout (KO) mice have decreased colonic Ca2+ permeability but unaltered small intestine permeability. They display decreased fecal Ca2+ excretion  and have unaltered bone mineral content and serum Ca2+, relative to wild-type (WT) mice (Cldn12) KO mice has not been fully described, although fecal Ca2+ and serum Ca2+ levels are unaltered relative to WT mice  mice , 14. The WT mice . To date2+ reabsorption occurs across the renal proximal tubule and thick ascending limb. Claudin-2 and claudin-12 are expressed in the proximal tubule, in which two-thirds of filtered Ca2+ is reabsorbed  or calcitriol  levels  mice. Unlike previous models, DKO mice were unable to maintain normal serum Ca2+. Decreased Ca2+ permeability across the colon but not small intestine resulted in reduced intestinal Ca2+ absorption in DKO mice, which also had severe hypercalciuria and markedly decreased bone mineral density. We found that while claudin-2 and -12 were expressed in the same cells, they did not physically interact and coexpression of each claudin had an additive effect on Ca2+ permeability in vitro. These results suggest that claudin-2 and -12 mediate paracellular Ca2+ absorption and reabsorption independently and that only one of these claudins is sufficient to maintain a normal Ca2+ balance.We hypothesized that claudin-2 and claudin-12 form independent cation-permeable pores across intestinal and renal epithelia, contributing independently to paracellular CaCldn2 and Cldn12 DKO mouse by crossing mice from Cldn2 KO and Cldn12 KO colonies. DKO mice were born to typically sized litters, and the only observed difference to WT mice was an 8% lower body weight in DKO males . We previously reported decreased colonic Ca2+ permeability in Cldn2 KO mice . These results are consistent with both claudin-2 and claudin-12 conferring Ca2+ permeability to the proximal colon. We repeated the experiment and found decreased Ca2+ permeability across the colon of DKO mice . To determine whether the loss of both Cldn2 and Cldn12 in the DKO mice results in an additive loss of Ca2+ permeability, we analyzed the results of each KO or DKO relative to their respective WT mice. Colonic Ca2+ permeability was decreased by 16.9% (±9.1%) in Cldn2 KO animals, 16.7% (±9.0%) in Cldn12 KO animals, and 31.4% (±11.8%) in DKO mice .The tosidase . Given ttosidase , 21, we  WT mice E and F. 2+ absorption from the small intestine. However, prior work found no difference in Ca2+ permeability between Cldn2 KO and WT mice across the small intestine .The paracellular pathway is proposed to contribute significant Cantestine . SimilarCldn2 KO mice exhibit hypercalciuria and increased net intestinal Ca2+ absorption, while Cldn12 KO mice do not have an overt Ca2+ phenotype. To ascertain whether DKO mice have a phenotype with altered Ca2+ homeostasis, we performed metabolic cage studies. Water and chow consumption, urine volume, and fecal mass were not different between genotypes . Blood analysis revealed significantly decreased ionized calcium (iCa) in DKO animals . These results suggest that the DKO mice have a phenotype with altered Ca2+ balance. animals A. No dif2+ excretion to assess intestinal absorption as a percentage of Ca2+ consumed . DKO mice had decreased Ca2+ bioavailability . Claudin-2 and claudin-12 are also expressed in the renal proximal tubule . No differences were observed in urinary Na+, K+, PO43−, Cl−, or Mg2+ excretion. However, we found an almost fourfold increase in Ca2+ excretion in the DKO compared to WT mice . To account for glomerular filtration and decreased plasma Ca2+, we calculated fractional excretion of Ca2+ (FECa). Relative to WT mice, the FECa in DKO mice was 4.3-fold greater  . Cyp27b1 and Cyp24a1, genes encoding enzymes that activate and deactivate calcitriol, respectively, were not significantly increased in DKO mice .We examined expression of renal genes involved in Ca S4 A–C) , 22. Exp2+ channels Trpv6 and Cacna1d were not changed, the expression of the intracellular Ca2+-binding protein S100g and basolateral extrusion pump Atp2b1 were increased by fivefold and 0.5-fold, respectively. We observed no change in Cldn3 or Cldn4 expression but found a slight decrease in the expression of Cldn15, a gene that encodes a cation-permeable pore . These results altogether suggest that the DKO mice have increased transcellular Ca2+ absorption from the colon, which is not stimulated by calcitriol. The intestinal expression of genes encoding Ca2+ transporters have not been reported for the Cldn12 KO mouse. We therefore measured them and found that the only difference was a twofold increase in S100g expression in the colon . Taken together, the results indicate that the DKO mice have impaired intestinal Ca2+ absorption and renal reabsorption with insufficient compensation to maintain serum iCa.In the colon, although the expression of the apical Cable pore . No chanCldn2 KO mice but has not been examined in Cldn12 KO mice . We next examined bone microarchitecture and mineral density in the DKO mice and found a more-than-fourfold decrease in trabecular bone mineral density and both decreased trabecular thickness and number in DKO mice. Similarly, we found decreased bone volume, thickness, and tissue mineral density in cortical bone of DKO mice  cells expressing epitope-tagged claudin-2 and claudin-12. As a positive control, we found that myc-tagged claudin-2 was able to immunoprecipitate hemagglutinin (HA)-tagged claudin-2. However, myc-tagged claudin-2 was unable to immunoprecipitate HA-tagged claudin-12 . Immunofluorescence of this cell line demonstrates colocalization of CLDN2 and CLDN12 at the plasma membrane . These results are consistent with claudin-2 and claudin-12 forming separate Ca2+-permeable pores.If claudin-2 and claudin-12 form separate Case cells C. After se cells C. Colonimembrane E–G. Simimembrane H 24). P. P2+ per2+ homeostasis and optimal bone mineralization. This conclusion is supported by Cldn2/Cldn12 DKO mice having decreased Ca2+ permeability across the colon, contributing decreased net intestinal Ca2+ absorption. Furthermore, DKO mice have even greater hypercalciuria than the single-Cldn2 KO animals. Intestinal and renal compensatory mechanisms in the DKO mice are inadequate to maintain serum ionized Ca2+ levels. Ultimately, the loss of both proteins results in decreased bone mineral density. Moreover, we did not find that claudin-2 and -12 physically interact, despite being expressed in the same epithelia. Our results are consistent with additive effects of their deletion and their formation of independent Ca2+ pores in the proximal colon and renal proximal tubule.Employing murine models, detailed balance studies, and ex vivo and in vitro techniques, we provide evidence that either claudin-2 or claudin-12 is necessary to maintain Ca2+ absorption is proposed as the predominant pathway when dietary Ca2+ is adequate .Cldn2 and Cldn12 global DKO mice (Cldn2/12 DKO) by cross-breeding Cldn2  with Cldn12 KO  were transiently transfected with pTRE2hyg vector expressing HA-tagged claudin-12, pTRE2hyg vector expressing myc-tagged claudin-2, pcDNA 3.1 vector expressing HA-tagged claudin-2, and/or empty pTRE2hyg vector as described in the Tissue was subjected to X-gal staining and, subsequently, immunohistochemistry, as described in detail previously . BrieflyP < 0.05 was considered statistically significant.Data were analyzed using GraphPad Prism 9.0. The Shapiro–Wilk test was used to evaluate for normal distribution and F test to compare variances. Data were analyzed and presented as indicated in figure and table legends. Supplementary File


These channels convey a large Ca2+ influx despite extracellular Na+ being 70-fold more concentrated than Ca2+; such high selectivity is lost in low Ca2+, and Na+ can permeate. Since the permeation properties and molecular identity of sensory Ca2+ channels are debated, in this paper, we examine the Na+ current flowing through the L- and R-type Ca2+ channels of labyrinth hair cells. Ion currents and cytosolic free Ca2+ concentrations were simultaneously monitored in whole-cell recording synchronous to fast fluorescence imaging. L-type and R-type channels were present with different densities at selected sites. In 10 nM Ca2+, the activation and deactivation time constants of the L-type Na+ current were accelerated and its maximal amplitude increased by 6-fold compared to physiological Ca2+. The deactivation of the R-type Na+ current was not accelerated, and its current amplitude increased by 2.3-fold in low Ca2+; moreover, it was partially blocked by nifedipine in a voltage- and time-dependent manner. In conclusion, L channel gating is affected by the ion species permeating the channel, and its selectivity filter binds Ca2+ more strongly than that of R channel; furthermore, external Ca2+ prevents nifedipine from perturbing the R selectivity filter.Some hearing, vestibular, and vision disorders are imputable to voltage-gated Ca T2+ channels are classified according to the properties of the current they carry: the L-type currents originate from the Cav1 channel family; the P/Q-, N-, and R-type currents arise from the Cav2 family; and T-type currents are mediated by the Cav3 family. This classification is the outcome of immense interdisciplinary research lasting more than fifty years, primarily studying the effects of specific drugs on these channels in ex vivo tissues, in vivo, and expressed (native or mutated) in cell lines [2+ channels and in their modulatory proteins, and particularly in some hearing, vestibular, and vision disorders that are probably imputable to Ca2+ channels at the level of the sensory cells. For instance, mutations in the subunits of the CaV1.3 channel type and in its modulator protein CaBP2 originate the autosomal recessive deafness, while many mutations in CaV1.4 channels and in their modulator protein CaBP4 are associated with autosomal recessive congenital stationary night blindness  to 1–10 µM, and other mono- and divalent cations can permeate [2+ channel permeability to monovalent cations has been investigated in turtle cochlear hair cells [2+ channel [2+ channel types are endowing the different sensory cells, and in general, it is important to understand the permeation properties of different channel types. To this aim, the hair cells of the frog semi-circular canal are particularly significant, since three distinct Ca2+ channel types have been identified in these cells on the basis of their biophysical and pharmacological properties [2+ current flows through a nifedipine-sensitive L-type channel; the remaining fraction (after nifedipine application at saturating concentration) flows through two distinct R-type channels, termed R1 and R2 for simplicity’s sake, on the basis of the following evidence. It has been found that approximately 60% of the hair cells recorded in whole-cell mode exhibited a steady current, maximal at −20 mV, whereas the remaining 40% were characterised by an initial peak, followed by an exponential decay (inactivation) to a plateau level. Peak amplitude was maximal at −30 mV, and inactivation was Ca2+-dependent [v1), and the current formed by the peak phase and the residual (steady) current flows through two distinct R-type channels (Cav2), called R1 and R2 hereafter, on the basis of their resistance to dihydropyridines, ω-cono- and aga-toxins , withou2+ channel has been found in hair cells of the turtle cochlea [v1.3 channel types have been found in chicken cochlear hair cell [v1.2 types have been found in the latter vestibular ganglia [2+ channels differs from the other known L-type Ca2+ channels  in that they are activated by low rather than high voltages [v1.3 channels expressed in cell lines actually form low-voltage activated channels with low nifedipine sensitivity, resembling the hair cells L-type Ca2+ channels, which indeed require micromolar amounts of dihydropyridine to block them, instead of nanomolar amounts as in other L-type channels. Remarkably, bullfrog saccular hair cells possess two Ca2+ channels: L- and non-L-type [2+ currents in mammals is indicated in the chinchilla crista by labelling of the basolateral membrane in type I and type II hair cells with an antibody against the subunits of P/Q channels  channel has been found in the hair cell membrane in the mouse organ of Corti [2+ current component is also supported by the lack of vestibular deficits in the Cav1.3 null mice [2+ currents to be mediated via Cav2 channels were found in bullfrog saccular hair cells [v2.3 Ca2+ channels are mandatory for physiological auditory information processing [v3.1 one, given its fast inactivation kinetics [Clear evidence for L-type Ca cochlea  and in b cochlea ,15,16. Tair cell  and in v ganglia . The haivoltages , suggestvoltages  and/or svoltages . It has voltages ,23 that n-L-type ,15,16. E; moreovof Corti . The preull mice . Nimodipocessing . Therefoocessing , and poskinetics . In conc2+ channels and on their molecular identity, the present work examines the non-specific monovalent cation current flowing through the L- and putative R-type Ca2+ channels in hair cells of the frog labyrinth in the presence of very low external Ca2+, by focusing on their (i) activation and deactivation kinetics, (ii) voltage-dependent conductance properties, and (iii) selectivity and blockade. The results offer a preliminary step in understanding the basic properties and structure of Ca2+ channels in such specialised sensory cells. They will also be helpful in analysing the permeation properties of functionally different channel types that share the same ion carrier.The L and R2 currents, which activate at −60 mV and peak at −20 mV, may sustain spontaneous transmitter release at the cytoneural junction, whereas the R1 component, which activates at −40 mV and carries the maximal current at −30 mV, may be functionally important in evoking synchronous transmitter release in response to short, strong stimuli [2+-dependent [n = 9) was not slowed down by the massively reduced Ca2+ influx (see below), as indeed occurred when external Ca2+ was lowered [2+ in the presence of a physiological level of Ca2+, and no Na+ was flowing through any of these channels, as found instead in other Ca2+ channel types, such as the Cav3 ones  located mostly in the basal (synaptic) pole of the cell, where the Ca2+ channels are indeed clustered  to a plateau level. Peak amplitude was maximal at −30 mV, and inactivation was Caair cell A that coair cell B. This air cells ; otherwi ≈ 17 Nm ). Other dynamics D elicitelustered C. In Fig2+ to 10 nM, a large current was recorded in response to a depolarisation to −50 mV , were maximal at −20 mV in 4 mM Ca2+  to the cation currents at the same voltage minus ~30 mV. These results confirm once more that all the recorded currents were carried by monovalent cations flowing through the open Ca2+ channels. Moreover, the ~−30 mV shift was then mainly due to the increased net negative charge on the external side of the plasma membrane produced by the removal of Ca2+ positive charges adsorbed there.The steady-state current to voltage (I–V) curves C of the 2+ A and of 2+ B current2+ D, n = 21eftwards D; the ra2+-dependent inactivation waveform, being maximal for a −30 mV depolarisation , cells with perforated-patch currents and access resistance no larger than 1.6 nA and 10 MΩ, respectively, were considered. The depolarisations elicited steady currents in 10 nM Ca2+, because the R1 component did not inactivate. Consequently, it was not possible to distinguish the R1 from the R2 component in the low Ca2+ recordings, or to directly assess whether R1 was still present or if it had undergone run-down. Therefore, at the end of the 10 nM Ca2+ perfusion, the presence of R1 was once more checked by returning the cell to the 4 mM Ca2+ solution and repeating the depolarisation protocol of 2+ . The steady-state I–V curve (2+ (n = 9) times larger than the peak and the plateau Ca2+ currents in 4 mM Ca2+, respectively  between different peak current amplitudes in 4 mM Ca2+ (at −30 mV) and the corresponding maximal cation current amplitudes in 10 nM Ca2+ (at −50 mV) in different cells, showing that the cation current flowed also through the R1 channels.Cells possessing all three components (L + R1 + R2) responded to the depolarisation step with a characteristic Carisation E. Most ool of 2+ E and wit–V curve G and the–V curve H of the urve , specific for Cav1.1 channels in skeletal muscle [n = 4) specific for Cav1.2 channels [2+ current [In order to further determine the contribution of each channel type to the generation of current responses in 10 nM Cael types  and coull muscle , and calchannels , were no current , it was 2+, we examined first the current kinetics of cells having L + R2 components only and when the L component was removed with 10 µm of nifedipine.To better understand the contribution of L, R1, and R2 contributions to the current waveform and the properties of the cation current flowing through these channels in 10 nM Ca2+ currents (at −20 mV) in cells having L + R2 components only could be conveniently fitted by a single exponential, as exemplified in τon = 0.70 ± 0.06 ms, n = 21), while the deactivation phase . Moreover, the τon in nifedipine  comparable to the τoff2 in the control. This indicates that nifedipine indeed completely cancelled the L component of the current, whose activation and deactivation time constants were similar and faster than the R2  in cells possessing the L and R2 components only  because τon was relatively voltage-independent, and it even increased with hyperpolarisation, as found for T-type channels [τon in 10 nM Ca2+ was similar to the one in 4 mM Ca2+, but shifted by ~−30 mV. The deactivation phase of the maximal cation current was bi-exponential as the Ca2+ current ones, but τoff1  and τoff2  were three times faster and similar to the ones of the maximal Ca2+ current, respectively. The activation kinetics of the maximal cation current (at −50 mV) in nifedipine (which flows through the isolated R2 channels) was fitted by a single exponential  twice as fast as that found in 4 mM Ca2+ + nifedipine (at −20 mV). In 10 nM Ca2+, the deactivation kinetics in nifedipine were monoexponential as in 4 mM Ca2+, with a similar time constant . All these results indicate that 10 nM Ca2+ accelerated the activation time constant of both L and R2 components by a factor of ~2, but accelerated the deactivation time constant of the L component (by a factor of ~3) only.The nts only C was abochannels ; the volonential D, with aτon of the maximal Ca2+ current generated by the activation of L + R1 + R2 channel types  had fully developed, thus artificially shortening it. The deactivation phase was bi-exponential , and as in cells possessing the L and R2 components only, the τon in nifedipine  was similar to the one in the control, but the deactivation kinetics was monoexponential, with a time constant  comparable to the τff2 in the control. These results are consistent with the notion that the R1 component fully inactivates during the depolarisation, and the current deactivation was generated by the sum of the fast-deactivating L component and the slow-deactivating R2 component.The τon of the L + R1 + R2 maximal cation current  or not. Since the R1 component did not inactivate in 10 nM Ca2+, the deactivation kinetics of the cation current were generated by the deactivation of L, R1, and R2 components. The latter was still biexponential : the cation current τoff1  was about threefold faster than that of the Ca2+ current, while τoff2 was similar for both currents ; the τoff1 and τoff2 of cation current were similar to the ones of L + R2 cells. If τoff2 is generated by the R2 inactivation only (as discussed above for cells with R2 and L components only), then τoff1 is generated by the inactivation of L and R1 components, and since this inactivation was threefold smaller in 10 nM Ca2+, then the inactivation of both R1 and L components was accelerated in 10 nM Ca2+. This view is confirmed by perfusing with nifedipine the L + R1 + R2 cells: after cancelling the L component, the τon  was accelerated compared to 4 mM Ca2+, and the deactivation was biexponential  instead of monoexponential as in 4 mM Ca2+. These results indicate that, after cancelling the L-type component in nifedipine in 10 nM Ca2+, the biexponential kinetics of monovalent ion current deactivation are described by a τoff1, generated by the deactivation of the R1 component, and by τoff2, generated by the R2 component. It cannot, however, be ascertained if the deactivation of the R1 component was accelerated or not in 10 nM Ca2+.The 2+ channel types, distributed at different densities in distinct hotspots. This view is consistent with the experiments of The L, R1, and R2 channels are clustered in hotspots located mostly in the basal (synaptic) pole of the cell : nifedipine reduced the maximal cation current (at −50 mV) by a factor of 2.93 ± 0.49, which became 4.57 ± 0.39 at the plateau . Moreover, the nifedipine cation current blockade was stronger at more negative potentials  and voltage-dependent manner , it can2+ channels are very similar to sodium channels in structure and function, yet subtle differences in their amino acid sequences allow them to convey a Ca2+ influx of 106 ions/sec into the cell, despite extracellular Na+ being 70-fold more concentrated than Ca2+. In fact, Ca2+ channels have a Ca2+ selectivity much larger than the Na+ one, especially the ones analysed here, whose Na+ inflow in the presence of physiological external Ca2+ is not measurable . In the absence of Ca2+, monovalent cations loosely occupy this binding site and therefore can flow relatively freely through the channel, along their electrochemical gradient. As the Ca2+ concentration is raised, this binding site becomes occupied by Ca2+, since monovalent cations are not able to displace it, given the higher affinity of the site for Ca2+ than for Na+; consequently, the bound Ca2+ actually blocks current flux through the channel. As the concentration of Ca2+ is raised even further, multiple binding sites in the pore become occupied by Ca2+; the electrostatic repulsion between the multiple bound Ca2+ ions would provide the driving force to move Ca2+ through the pore (often referred to in the literature as the knock-off mechanism), despite its tight binding [+ channels [2+ selectivity filter constructed in a homotetrameric bacterial Na+ channel, it has been proposed that this filter is composed of three binding sites ; Site 2, in the channel core, has the highest affinity for Ca2+ (dark brown); Site 3, close to the intracellular side of the channel (Int.), has the lowest affinity (light brown). At physiological Ca2+ concentrations, ions can occupy both Site 1 and Site 3 (state Sa) given the strong repulsion between Ca2+ ions, or a single Ca2+ ion can occupy Site 2 (state Sb). Considering the unidirectional Ca2+ flux in physiological conditions, the selectivity filter switches alternatively between the states aS and bS (aS and bS occurs when Ca2+ jumps from Site 1 to Site 2 spontaneously (since Site 2 has a higher affinity for Ca2+ than Site 1) and/or by ionic repulsion with a third ion that enters on the extracellular side of the filter. Ca2+ ion can proceed from Site 2 to Site 3, which has the lowest affinity for Ca2+, again for ionic repulsion with another Ca2+ ion that rapidly occupies Site 1 for the high Ca2+ concentration in the extracellular solution, while the weak binding of Ca2+ to Site 3 allows the Ca2+ exit toward the low Ca2+ concentration of the cytosol. The high-affinity binding of Ca2+ to Sites 1 and 2 ensures that Na+ and other monovalent cations cannot permeate; if external Ca2+ is removed, then monovalent cations can move freely along the channel, giving the observed much larger current  and their charge, but not on their ionic radius , as the selectivity filter does for the K+ channel [2+ channel TRPV6 confirmed the knock-off mechanism for Ca2+ permeation along the Ca2+ binding sites of the channel, and confirmed that Na+ permeates freely without needing such a mechanism. However, this simulation predicts that in the presence of ions, no water binds to or crosses the pore constriction [2+ is yet to be fully explained.Voltage-gated Caasurable . This is decades ,10, but  decades ,39,40. Tchannels . More re Site 3; ). Site 1a and Sb . For ins current B. In conchannels , Ca2+ sed cation ,39, on t channel . A recenτon) and the fastest time constant of deactivation (τoff1) of the maximal cation current were about two and three times faster, respectively, than that of the Ca2+ current. This acceleration, which was relatively voltage-independent (τoff2) generated by R2 component deactivation. If channel gating is not affected by the kind of ion flowing through the channel, then acceleration of the cation current activation phase vs. that of Ca2+ indicates that Ca2+ binding to the pore sites  channel gating, as demonstrated for the voltage-activated Na+ channels [The activation time constant  in a voltage- and time-dependent manner. For instance, at −50 mV, this residual current was exponentially reduced (with a time constant of ~6 ms) by 34% in both cases. The blockade was progressively stronger at more hyperpolarised voltages, but peak and plateau were reduced by the same amount , Ca2+ blocks the L channel type more compared to the R1 or R2 ones, since upon removal of external Ca2+, the monovalent cation influx (carried mainly by Na+) increased more in the former case than in the latter. On the basis of the scheme of 2+ than that of the R1 or R2 channels.Assuming that in 10 nM CaRana esculenta, 25–30 g body weight) as previously described [2, 10 HEPES, 6 glucose; pH ≈ 7.2, ≈260 mOsmol/kg). The hair cells were mechanically dissociated from the ampullae by gently scraping the epithelium with fine forceps. The recorded cells were subjected to the external control  and test solutions in rapid succession . This was performed by using a computer to move a multi-barrelled perfusion pipette horizontally in front of the recorded cell. The low Ca2+ solution and the choline solutions were the same as the control solution, but the former was nominally Ca2+-free and contained 1 mM Cs-BAPTA and the latter had 100 mM NaCl substituted with 100 mM of choline chloride. The WinMax program  and of the salts used to prepare the solutions. Calciseptine (2 µM), calcicludine (20–100 nM), TTX (1 μM), and nifedipine (10 µM) were added to the perfusion solutions after being dissolved in dimethyl sulfoxide. In the imaging experiments, the solutions were focally pressure-applied through a pipette using a gated PicoPump , 0.1 GTP , 10 HEPES, 5 EGTA; pH ≈ 7.2, ≈235 mOsmol/kg). The dye used to perform fluorescence imaging of intracellular Ca2+ was Oregon green, dissolved in the intracellular solution used for the whole-cell experiment at a 100 µM concentration. This solution also contained 0.5 mM EGTA to chelate residual Ca2+ of distilled water and the Ca2+ released from the glassware used for solution preparation as well as from the patch pipette itself. All salts, buffers, and solvents were purchased from Sigma Chemical Co.  except for calcicludine and calciseptine , Oregon green , and Cs-BAPTA, which was synthesised by our colleagues at the Department of Physics, University of Parma, Italy.The experiments were performed on frogs  was use2+ (2+ (200 µs). Cells were viewed on a TV monitor connected to a contrast-enhanced video camera  coupled to an inverted microscope  equipped with a 40X Hoffman modulation contrast objective (Olympus). To facilitate gigaseal formation in the perforated-patch experiments, an amphotericin-containing solution was used to backfill the electrode while the pipette tip was filled with an amphotericin-free solution. The test stimuli consisted of 50 or 160 ms steps from holding potential  to −20 or −30 mV. The current vs. voltage curves (I–Vs) were recorded starting from hV and proceeding with 50 ms steps to −20 mV or to +40 mV in 10 mV increments ; there were no differences between the current waveforms elicited by the same depolarising step whether hV was −70 or −110 mV. Just before and after performing each protocol, leak resistance was measured with a −10 mV step starting from hV and lasting 15 ms. Data were acquired using a computer interface  and its managing software  running on a host PC. The recordings were filtered at 10 kHz and acquired at 40 kHz via an eight-pole Butterworth filter .Cells were recorded at room temperature (20–22 °C) using the whole-cell or perforated-patch technique and a patch amplifier . The linear leak current was routinely subtracted from all recordings; the capacitive transients were compensated or their digital points were removed from the recordings, since the artefacts were significantly faster than the current onset and offset. The speed of the voltage clamp was adequate to detect the rapid activation and deactivation kinetics of the currents, especially the ones in 10 nM Ca2+ . These r2+  Results.2+ was performed as previously described [2+ binding reaction (approximately 5 ms at room temperature). Data were analysed off-line using a commercial software package . In order to correct fluorescence measurements for possible dye concentration and/or optical path length anisotropy, fluorescence signals were computed, for each image pixel, as ratios:t is time, F(t) is fluorescence following a stimulus that raises the Ca2+ within the cell, and F(0) is the pre-stimulus fluorescence computed by averaging 10–20 images. Both F(t) and F(0) were corrected for mean background fluorescence computed from a 20 × 20 pixel rectangle devoid of obvious cellular structures. The fluorescence ratio magnitude was smoothed with a two-dimensional 3 × 3 median filter and encoded by 8-bit look-up tables to produce 256 pseudo-colour indexed images ; figures were prepared using the commercial plotting program Sigmaplot . Throughout the paper, values in both texts and figures are given as


Significance: Idiopathic pulmonary fibrosis (IPF) patients have a poor prognosis with short lifespan following diagnosis as there are limited effective treatment options. Despite matrix stiffening being the hallmark of the disease there remains a lack of knowledge surrounding the underlying collagen alterations in the disease. Specifically, while increased collagen crosslinking has been implicated, the resulting effects on collagen macro/supramolecular changes have not been explored.Aim: We sought to determine if second-harmonic generation (SHG) microscopy could characterize differences in the collagen architecture in 3D spheroid models of IPF grown under different crosslinking modulation conditions (promotion and inhibition).Approach: We used SHG metrics based on the fiber morphology, relative SHG brightness, and macro/supramolecular structure by SHG polarization analyses to compare the structure of the IPF spheroids.Results: Comparison of the fiber morphology of the spheroids showed that the control group had the longest, straightest, and thickest fibers. The spheroids with crosslink enhancement and inhibition had the highest and lowest SHG conversion efficiencies, respectively, consistent with the resulting harmonophore density. SHG polarization analyses showed that the peptide pitch angle, alignment of collagen molecules, and overall chirality were altered upon crosslink modulation and were also consistent with reduced organization relative to the control group.Conclusions: While no single SHG signature is associated with crosslinking, we show that the suite of metrics used here is effective in delineating alterations across the collagen architecture sizescales. The results largely mirror those of human tissues and demonstrate that the combination of 3D spheroid models and SHG analysis is a powerful approach for hypothesis testing the roles of operative cellular and molecular factors in IPF. While IPF etiology is poorly understood, it has been attributed to a non-specific combination of both genetic and environmental factors, which promote repetitive alveolar injuries.,–While the role of several species  and its counterparts, lysyl oxidase-like (LOXL) proteins, are responsible for crosslinking collagen and elastin in the ECM and are required for the structural integrity of many tissues.in vitro models can yield new insight into the disease progression as this process can be modulated through selective promotion and inhibition. For example, using long term spheroids (40 to 60 days), Jones recently showed that increased bone type pyridinoline collagen crosslinking, mediated by lysyl hydroxylase 2 (LH2/PLOD2), LOXL2, and LOXL3 altered nano-scale architecture.Examining the impact of crosslinking on the collagen architecture using well controlled While this study focused on the biomechanics aspects, initial SHG imaging also revealed alterations in the collagen fiber morphology. Here, we extend the characterization of the collagen architecture in similar IPF spheroid models with selective crosslinking modulation by further analysis of fiber morphology, examination of coherence aspects through SHG conversion efficiency, as well as probing macro/supramolecular changes through polarization-resolved SHG (P-SHG and SHG-CD) imaging.22.1in vitro model of fibrosis as previously described.IPF lung fibroblasts were cultured using the 3D The fibroblasts were seeded in Transwell inserts in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS. After 24 h, the media were replaced with DMEM/F12 containing 5% FBS, Samples for imaging analyses were stored at 4°C in phosphate-buffered saline (PBS) for conventional SHG imaging or optically cleared by immersion in 50% glycerol overnight to reduce scattering-induced depolarization effects for SHG polarization-resolved imaging. For imaging, samples were mounted on glass slides in PBS or glycerol with #1.5 coverslips, and nail polish was used to seal the slides. A total of 12 independent samples were imaged.Collagen crosslinks were calculated as previously reported.2.2,The details of the SHG microscope have been described elsewhereForward SHG emission was collected using a photon-counting detector . The SHG wavelength (445 and 390 nm) was isolated with the respective 10 nm wide bandpass filters  and the excitation wavelength was confirmed using a fiber-optic spectrometer . Fields of view were ,Linear polarization was obtained using a half-wave plate to define the state entering the microscope and the desired linear rotation at the focal plane was achieved using a liquid crystal rotator  mounted in the infinity space.2.3,,We determined the relative SHG conversion efficiency, which arises from the collagen concentration and organization, by measuring the forward attenuation, i.e., the rate of SHG intensity decrease with increasing depth into the tissue. We have shown that this response is a coupled effect of the relative conversion and primary filter effects  and in general Monte Carlo techniques are necessary to isolate the former.2.4in vitro systems (unpublished). Here, spheroids were sliced into sections of about While the spheroids are not highly scattering, we have shown even one scattering length gives rise to significant depolarization in collagenous tissues.2.4.1Polarization-dependent measurements were performed as previously described,The SHG signal polarization was further determined on a pixel basis and as a function of laser polarization. The anisotropy, 2.4.2SHG-CD analysis was used to interrogate the overall chirality of the collagen where the method has been described previously.2.5One-way analysis of variance with post hoc two-way Student’s 33.1,We compared the collagen fiber morphology of 42- and 60-day spheroids for the four treatment groups: (i) Control (Ctrl); (ii) PXS-5120 (Representative SHG images for the four groups are shown in We employed curvelet transform-fiber extraction (CT-FIRE)First, we examined collagen fiber/fiber bundle straightness in the four groups. The Ctrl and IOX2 (enhanced crosslinking) spheroids had the straightest fibers . AdditioTo further characterize the effects of collagen crosslink modulation on the fiber architecture, we determined the relative normalized SHG conversion efficiency of each group by measuring the forward attenuation see Sec. . The aveThese results are consistent with our expectations as increased crosslinking should increase SHG brightness due to collagen molecules being more densely packed in fibers. This is because in the limiting case of complete alignment, the SHG efficiency scales as the square of the collagen concentration. Conversely, crosslinking inhibition should result in weaker SHG due to decreased harmonophore density. We note that we performed this analysis on 3.23.2.1–By reconstructing the pixel-based SHG intensity as a function of excitation polarization angle using the generic model  and then3.2.2We also examined the SHG signal anisotropy, [t groups . As prev3.2.3,We probed the net chirality of the collagen using the SHG-CD approach described previously.4,,,,,While previous efforts have probed structural changes in IPF and normal lung architecture via immunofluorescence, AFM, and other techniques,–,–SHG has great potential for this task as we have shown that sub-resolution structural information can be extracted.in vitro models with selective promotion and inhibition. This work builds on the investigations of Jones and coworkers, where using spheroid models and mechanical and biochemical measures, they identified the importance of dysregulated crosslinking in IPF in terms of collagen structure-function.,ex vivo human tissues, and further, no representative animal models of IPF exist. Thus, the combined use of detailed SHG imaging analyses with well-defined crosslink modulation allows systematic hypothesis testing of biochemical factors that affect structure. Identifying these features in a known in vitro system is a critical step for eventual translation to in vivo imaging diagnostics. Below we describe the effects on different levels of collagen architecture.As a new approach to examining the effects of crosslinking modulation on collagen architecture, we implemented SHG microscopy using several metrics based on analysis of fiber morphology, conversion efficiency (based on coherence), and polarization responses using The relative conversion efficiency arises in part from the phase-mismatch, ,,None of the three polarization responses  are uniquely associated with crosslinking. However, these metrics for the control spheroids (Ctrl) are similar to those seen in normal Col I, for example in tendon and collagen gels.We also note a possibly unexpected difference between the decreased anisotropy and increased alignment in the IOX2 treated group. The apparent discrepancy arises from different operative size scales in collagen architecture. The SHG signal anisotropy measurement describes the dipole moment alignment angle within collagen fibrils . Collagen molecules are almost perfectly aligned on the long axis in a normal collagen fibril,We also note that SHG-CD  is relatThe decreased chirality along with large changes in the effective pitch angle  are cons,in vitro models and ex vivo tissues. This analysis can then provide insight into disease etiology and progression.We can place our findings in the context of several human studies. First, we point out that these models of crosslink modulation are relevant to human IPF, where for example, Tschumperlin and co-workers found that LOXL1 and LOXL2 gene and protein levels were increased in IPF samples.5in vitro models of IPF with enhanced and inhibited crosslinking. These models have relevance to human IPF as it has been established that LOX expression is up-regulated in human disease, resulting in increased crosslinking and corresponding matrix stiffness. Moreover, the use of multiple SHG metrics can provide structural information not possible by other methods. While there are no SHG metrics uniquely related to crosslinking, using a suite of SHG tools, we found changes in structure consistent with those expected by LOX modulation. Moreover, these findings using in vitro models produced under well-controlled conditions were analogous to those in our previous work on ex vivo IPF tissues. Collectively, these measurements using 3D models and SHG analysis provide great promise for understanding disease etiology of IPF as the approach permits extensive hypothesis testing of cellular and molecular factors relevant to this disease. Identifying these features in a known in vitro system is a critical step for eventual translation to in vivo imaging diagnostics.We have shown that SHG metrics based on coherence and polarization analysis are powerful for probing the collagen macro/supramolecular alterations in 3D


There are few epidemiological studies of urticaria, published in the indexed literature (PubMed/Medline).The study aimed to evaluate the epidemiological and clinical data among patients with urticaria/angioedema attending a reference clinic in Brazil.Two hundred sixty-seven patients were evaluated retrospectively considering demographic data, time course of the disease, triggering symptoms, the presence of angioedema, complementary laboratory tests including total blood count, reactive-C protein, erythrocyte sedimentation rate, IgE serum levels, and other, as necessary.The most commonly diagnosed type of urticaria was chronic spontaneous urticaria (56.93%). Angioedema was associated with chronic urticaria in 108 patients (40.08%).Unicentered and retrospective.Some relevant findings in this study are the observation of a female prevalence of cases , a result more elevated than demonstrated in previous studies in Europe and Asia, the median age was 43-years old and the delay of time between the diagnosis of urticaria and the admission for treatment in a specialized center was approximately 2-years. Other multicenter studies can better establish these differences in Brazilian patients. Urticaria can be classified as acute, if it lasts less than 6-weeks, or chronic if it lasts more than 6-weeks.In the Brazilian population, there are few epidemiological studies in urticaria conducted in the last decade, retrospectively or prospectively, published in the indexed literature (PubMed/Medline).7The authors described the profile of 267 patients registered in the Urticaria Unit of Dermatology Department during the period from January 2011 to December 2017. This study was approved by the local Ethical Committee.A retrospective study was performed based on the evaluation of the medical registration of 336 patients of the Urticaria Unit of the Department of Dermatology of Faculdade de Medicina do ABC. Unfortunately, 69 patients were excluded from the study due to incomplete or uncollected exams during their follow-up. The authors collected epidemiological information such as age, gender, time of follow-up, time to seek for medical evaluation, and clinical data as type of urticaria , laboratory exams including blood cells counts , C-reactive protein , Erythrocyte Sedimentation Rate (ESR) and total IgE serum levels . Other procedures were performed if indicated by the clinical setting, such as the Autologous Serum Skin Test (ASST) if there was a familial or personal history of autoimmune thyroid disease, following the technical recommendations of Konstantinou et al.,The data for the patients enrolled in the Urticaria Unit of FMABC was collected from the medical charts. The authors have included all the patients that had the information required as described above. Patients that did not have the age, gender, time to seek medical care, time of follow-up for the urticaria, presence or absence of angioedema, and the laboratory exams were excluded from this study. The study excluded all cases of Angioedema without urticaria suspicious of hereditary or acquired bradykinin angioedema.The limitations of this study include the retrospective design, a tertiary medical center population, and low statistical power due to the small sample size.For the analysis of the data collected, it was established a statistical significance of 95% (p < 0.05). The qualitative values were analyzed by their mean and relative value. The quantitative data were tested for their normality in the Shapiro-Wilk test. For p < 0.05, the distribution was considered non-normal, and it was analyzed by the interquartile range and median. For p > 0.05, the data was analyzed by the mean and standard deviation. The program Stata 11.0 was used.rd, 4th and 5th decades . The gender of most of the patients was female . The age of the youngest patient was 8-years old, and the oldest, 83-years old (median 43-years old) (IR: 33–54), and most patients (59.93%) were in the 3 decades . The aveChronic urticaria was the most diagnosed type of urticaria (spontaneous 56.93% and 35.95% of induced-urticaria). Eighty-two patients (33.06%) with chronic urticaria presented both spontaneous induced-urticaria. Among chronic induced-urticaria patients, 50% of the cases were classified as symptomatic dermographism (48 patients), 22.91% delayed-pressure urticaria (22 patients), 4.16% solar urticaria (4 patients), 15.62% as cholinergic urticaria (15 patients), and 7 patients presented symptomatic dermographism and delayed pressure urticaria (7.29%). One hundred and eight patients with chronic spontaneous urticaria (CSU) - chronic inducible urticaria (CINDU) (40.44%) had angioedema.Only 6 patients registered showed acute spontaneous urticaria (2.24%). Eleven (4.13%) patients were diagnosed with urticarial vasculitis after cutaneous biopsies, and in most of these cases, nine patients did not show hyperchromic residual lesions or purpura on the description of the physical exam. Two cases (0.75%) of acquired autoinflammatory diseases were diagnosed due to recurrent fever episodes, arthralgia, urticarial lesions, and persistent laboratory exams suggesting systemic inflammation, and these cases were classified as Adult-Onset Still Disease (AOSD) and the other as Neutrophilic Urticarial with Systemic Inflammation (NUSI), as previously described by Mehrpoor et al. and Belani et al. and referred to the Rheumatology Department of the Institution .10, 11 TMost of the patients had normal blood cells counts and CRP. In the blood cell count, the mean of hemoglobin was 14.04 (SD = 2.04), and hematocrit test 41.23 (SD = 5.13). The mean of IgE was 262.05 (SD = 424.9). The IgE was elevated in 82 patients (32.2%), and distributed under the following ranges, 101–150 kU/L , 151–500 kU/L , and ≥ 500 kU/L , but there were no significant differences between the patients with isolated CSU and CSU-CINDU. The Erythrocyte Sedimentation Rate (ESR) mean was 22.1 (SD = 15.75), therefore, abnormal in 41.13% of the patients , and CRP was elevated  in 35 patients (13.1%) .Results of Hemoglobin (Hb) and Hematocrit (Hct) are demonstrated in The prevalence of urticaria in the dermatology department was 1.71% during the seven years of this retrospective study. Balp et al.,In the present study, the mean age of patients with urticarial was 43 years old. Dias et al. studied the impact of Chronic Urticaria (CU) on quality of life in a Brazilian University hospital in 2016.Maurer et al.Also, in a review of physical urticaria, Trevisonno et al.Kolkhir et al.16Total IgE serum levels were elevated in 86 of our patients (32.2%) with a mean of 262.05 kU/L (± SD 42.49) in all patients with urticaria. Chang et al. in 2013, studied acute and chronic urticaria in a total of 165 patients (104 with acute and 61 with chronic urticaria) in Taiwan.In 2018, Eun et al published an interesting study about the natural course of new-onset urticaria, as a result of a 10-year follow-up, nationwide, population-based study in South Korea.It is undeniable that women are more likely to have urticaria than men.In the present study, the mean follow-up time was approximately two and a half years, and the time the disease lasted at the Institution was from 1 to 20 years showing that CU is a long lasting disease.The diagnosis of urticaria is mainly clinical; only rarely auxiliary exams or skin tests are performed to clarify a possible etiology, such as Autologous Serum Skin Test (ASST). ASST is correlated to disease severity by some authors.Regarding the biopsy in the urticarial lesions in the patients, it was performed only because of suspicion of Urticaria Vasculitis (UV), chronic urticaria refractory to conventional therapy, and when the diagnosis of urticaria pigmentosa was suspected. This explains why only 42 patients (15.73%) had the results of the biopsy in their medical chart and it led to a diagnosis of urticarial vasculitis in eleven patients . Usually urticaria is not complicated and is susceptible to conventional treatment.21This retrospective study of 7-years in an Urticaria Unit of a tertiary center in Brazil showed limitations proper to this kind of study design (retrospective), where more detailed data often is missing. However, the authors are adding new data to the Latin American profile of patients suffering from urticaria in the last decade.The study demonstrated that there is a clear predominance of the female gender with urticaria, more than is observed in Europe and some countries of Asia. Most urticaria patients in the outpatient clinic are middle-aged women, with chronic urticaria without angioedema, and a few alterations in their laboratory exams, with almost one-third of patients, having IgE and ESR serum levels elevated. The median time to obtain a specialized consultation in an Urticaria Reference Center was 24-months, and most of the patients were followed in the Urticaria Unit for a period of 30-months. More efforts are needed to recognize the disease and to obtain medical assistance for urticaria patients in specialized units.10.13039/501100002322CAPES) – Código Financeiro 001.This study was funded in part by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (Roberta Fachini Jardim Criado: Design and planning of the study; drafting and editing of the manuscript; collection, analysis, and interpretation of data; effective participation in research orientation; intellectual participation in the propaedeutic and/or therapeutic conduct of the studied cases; critical review of the literature.Paulo Ricardo Criado: Approval of the final version of the manuscript; design and planning of the study; drafting and editing of the manuscript; effective participation in research orientation; critical review of the literature; critical review of the manuscript.Nathalia Baldavira: Design and planning of the study; drafting and editing of the manuscript; collection, analysis, and interpretation of data.Debora Cardial: Design and planning of the study; drafting and editing of the manuscript; collection, analysis, and interpretation of data.Ana Maria Gimenez-Arnau: Effective participation in research orientation; critical review of the literature; critical review of the manuscript.Carlos D'Apparecida Machado Filho: Effective participation in research orientation; intellectual participation in the propaedeutic and/or therapeutic conduct of the studied cases; critical review of the literature.RFC and PRC are medical consultants to Novartis and Takeda. AMG-A is a medical consultant to Uriach Pharma, Genentech and Novartis. She is the recipient of research grants from Intendis-Bayer, Uriach Pharma and Novartis, and participated in educational activities sponsored by Uriach Pharma, Novartis, Genentech, Menarini, Glaxo Smith & Kline, Merck MSD, Almirall and Leo Pharma.


ABSTRACT IMPACT: This work will help to understand a novel therapeutic approach to a common type of acute myeloid leukemia. OBJECTIVES/GOALS: FMS-like tyrosine kinase 3 (or FLT3) mutations occur in ˜30% of acute myeloid leukemia (AML) cases. FLT3 tyrosine kinase domain (TKD) mutations are particularly important in relapsed/refractory FLT3 mutant AML, which portends poor prognosis. This study describes a therapeutic approach to overcoming resistance conferred by FLT3-TKD mutations. METHODS/STUDY POPULATION: To understand the efficacy of a novel type 1 FLT3 inhibitor (NCGC1481), as a monotherapy and combination therapy, several assays were utilized to interrogate functionality of these therapies. Cell lines and patient samples containing aspartate 835 to tyrosine mutations  and phenylalanine 691 to leucine (F691L) were utilized to examine the effects of NCGC1481 with and without other targeted therapies like MEK inhibitors. Specifically, assays measuring viability, cell death using flow cytometry, in vitro clonogenicity, cellular signaling, and xenograft survival were examined in these FLT3-TKD AML models. Synergy was also measured using well described methods, which also allowed for appropriate dose finding for drug combination experiments. RESULTS/ANTICIPATED RESULTS: Our novel type 1 FLT3 inhibitor (NCGC1481) was particularly effective in the most common FLT3 TKD mutant, D835Y. NCGC1481 reduced viability and cell signaling, while also inducing cell death and prolonging xenograft survival in the FLT3-D835Y model system. In contrast, clinically approved FLT3 inhibitors were less effective at suppressing AML cells expressing FLT3-D835Y. In the case of FLT3-F691L, most of the FLT3 inhibitors tested, including NCGC1481, suppressed canonical FLT3 signaling, but did not significantly reduce viability or leukemic clonogenicity. However, when NCGC1481 was combined with other targeted agents like MEK inhibitors, at synergistic doses, eradication of the FLT3-F691L AML clone was substantially increased. DISCUSSION/SIGNIFICANCE OF FINDINGS: In AML, response to FLT3 inhibitor therapy is often short-lived, with resistance sometimes occurring via FLT3-TKD mutations. Given the dismal prognosis of relapsed FLT3 mutant AML, novel therapies are necessary. This study describes efficacy of a novel FLT3 inhibitor, along with its synergistic activity when combined with other targeted agents.