Liquid water is a critical component of habitability. However, the production and stability of surficial liquid water can be challenging on planets outside the Habitable Zone and devoid of adequate greenhouse warming. On such cold, icy exo-Earths, basal melting of regional/global ice sheets by geothermal heat provides an alternative means of forming liquid water. Here, we model the thermophysical evolution of ice sheets to ascertain the geophysical conditions that allow liquid water to be produced and maintained at temperatures above the pressure-controlled freezing point of water ice on exo-Earths. We show that even with a modest, Moon-like geothermal heat flow, subglacial oceans of liquid water can form at the base of and within the ice sheets on exo-Earths. Furthermore, subglacial oceans may persist on exo-Earths for a prolonged period due to the billion-year half-lives of heat-producing elements responsible for geothermal heat. These subglacial oceans, often in contact with the planet’s crust and shielded from the high energy radiation of their parent star by thick ice layers, may provide habitable conditions for an extended period. Liquid water is key for life as we know it. Here, the authors show even with a modest geothermal heat flow, subglacial oceans of liquid water can form at the base of and within the ice sheets on exo-Earths, which may provide habitable conditions for an extended period. However, key differences between the stellar and planetary environments between M-dwarf stars and the more luminous Sun-like stars have led to a long-standing debate around the habitability of M-dwarf orbiting exo-Earths3. In particular, the higher fraction of stellar luminosity emitted in the X-rays and ultraviolet (UV) by M-dwarfs, relative to sun-like stars4, as well as their exhibition of more flaring than sun-like stars5, presents challenges for the surface habitability of these planets. High surface radiation levels, which can present significant challenges for the stability of biological molecules, can be expected from the tendency of these planets to be depleted in X-ray/UV shielding ozone6, while flaring can lead to periodic, orders of magnitude higher surface X-ray fluxes7. Although such radiation only penetrates about 20 cm of liquid water8, this radiation still poses a challenge for near-surface aquatic environments where ocean circulation will periodically expose water from below this depth to the surface.The habitability potential of planets in the circumstellar habitable zone (CHZ) of M-dwarf stars are of great interest because M-dwarf stars make up 75% of the population of stars in the galaxy, and >40% of M-dwarfs stars are expected to harbor Earth-sized planets (exo-Earths) in their CHZTeq)  Fig. ; Table 1ion Fig. . In terrThe feasibility of basal melting depends on the thermal and phase evolution of an ice sheet which is primarily governed by a planet’s surface temperature and geothermal heat. Other factors, such as surface gravity and hydrosphere thickness, can also play a major role in the feasibility of basal melting, given the pressure-dependent melting temperature of water ice Fig. . Notably36,  coupled with a dynamic ocean GCM to show that even with an atmosphere consisting of 10,000 parts per million volume (ppmv) carbon dioxide (CO2) and 2000 ppmv methane (CH4), only a portion of the planet may be able to sustain liquid water38. Thus, if Proxima Centauri B is a water-rich planet, as its internal structure model suggests37, a significant fraction of the water may exist in the form of solid ice, where basal melting may be feasible.Numerous M-dwarf orbiting exo-Earths with a high Earth Similarity Index (ESI) value, a multiparameter index that compares attributes such as mass, radius, and temperature of an exoplanet to Earth, have been discovered in the last decade36, Fig. ; Table 141, although with notable uncertainty. Despite the strong far-UV photolysis of water (H2O) and large H2 escape rates expected from M-dwarf orbiting exo-Earths, models suggest that TRAPPIST-1 planets may have retained a significant amount of water42. While liquid water may be stable on the surface of TRAPPIST- 1 e with atmospheric H2O alone, the other two planets  require greenhouse gases such as CO2 and a thick atmosphere to sustain surface liquid water43. LHS 1140 B is a super Earth with an estimated mass 6 times than of Earth’s. A recent work44 utilized the internal structure model developed by45 to indicate that the water mass fraction (WMF) on LHS 1140 b could be as much as 4% (80 times more than Earth), leading to an average global ocean of 779 ± 650 km. That study’s 1% confidence level corresponds to a WMF = 0.007, still about 1.5 times more water than Earth44. The transmission spectrum of LHS 1140 b from the Hubble Space Telescope also shows tentative evidence of water, but future observations are needed to confirm this putative detection46. The exo-Earth GJ 1061 d receives a similar amount of energy as Earth receives from the Sun and may be a water-rich planet as well47. GJ 667 C e and GJ 667 C f lie also within the CHZ of its host star and may be water-rich; however, they may still require a thicker atmosphere than Earth and greenhouse gases like CO2 and CH4 for the liquid water to be stable on their surface48. Kepler-442 b is a potentially water-rich exo-Earth with a high ESI value and potential for habitability49. Both Kepler 1229 b and 186 f have extremely low Teq and thus would require a thick atmosphere with green houses gases to sustain liquid water50. In the absence of a thick atmosphere, a significant fraction of water on these two exo-Earths may exist in the form of ice. While there are significant uncertainties regarding the possible presence and the volume of the hydrosphere on these planets, even with an Earth-like WMF of 0.05%, these bodies could contain kilometers-thick global ice sheets  equals the estimated Teq . An example of a thermal profile and the time-dependent phase evolution of a 2-km thick ice sheet on Proxima Centauri B, assuming a Ts of 257 K and a basal heat flux of 30 mW m−2, is shown in Fig. We take a conservative approach and assume that the surface temperature   to undergo basal melting . In both cases, basal melting occurs within a few hundred thousand years post-deposition of the ice, and the thickness of basal melt increases until the system achieves thermal equilibrium. However, the basal melt resulting from a heat flow of 10 mW m−2 is notably thicker than basal melt from a heat flow of 5 mW m−2. Supplementary Fig. −2 to the current Mars-like heat flow of 30 mW m−2 within a billion years. In this rapid heat loss scenario basal melt would only be stable for approximately 750 million years. In the second, moderate heat loss scenario, the same amount of heat flow declines over a 4-billion-year period. As shown in Fig. All else being equal, the thickness of the basal melt increases with heat flow and time. For example, in Supplementary Fig. Teq is equal to the Ts, is an overestimate. The presence of greenhouse gases on any of these exo-Earths will raise the Ts, and therefore the heat flow required for basal melting would be notably lower than the estimates presented here.The primary goal of this paper is to demonstrate the relative ease by which basal melting may be attainable on M-dwarf orbiting exo-Earths. While there are notable uncertainties about the presence and the volume of hydrospheres on these bodies, if even a handful of potentially habitable exo-Earths discovered so far (or in the future) were to contain thick (>few km) hydrospheres, then liquid water via basal melting may be present on those bodies with relatively modest heat flow. Our heat flow constraint required for basal melting, under the assumption that the estimated Ts of exo-Earths can be placed based on the estimated Teq, heat flow on exo-Earths is entirely unconstrained. The main source of long-term heat in a planet after the initial stage of accretion and differentiation is the radiogenic decay of isotopes of heat-producing elements with billion-year half-lives such as 40K, 232Th, 235U, and 238U . For example, approximately 80% of the Earth’s present-day surface heat flow can be attributed to the decay of radioactive isotopes53. Radiogenic heat production as a function of age for cosmochemically Earth-like exoplanets suggests that exo-Earths similar in age to Earth should have a comparable heat production rate (H)54. However, there is a considerable degree of variations and uncertainties associated with the age of the M-dwarf systems considered in this study  which was marked by widespread glaciation20.The subsurface world of these exo-Earths might resemble the subsurface conditions found on Europa. In this class III habitat62. However, liquid aqueous solutions generated by basal melting at the rock/high-pressure ice mantle boundary will be buoyant and will permit the transportation of important life sustaining solutes/nutrients from the rocky core to the interglacial ocean. These bodies would classify as class IV habitat with liquid water layers between two ice layers66, such as the internal models of Ganymede and Callisto. In some scenarios, the melt is only observed within the ice layer and not at the base of the ice sheet 71 in conjunction with a heat transport model to investigate the temporal and depth-dependent evolution of the thermodynamic and elastic properties of water and ice polymorphs in the 0–2300 MPa and 220–500 K range.Underneath thick planetary glaciers, the melting point of ice varies due to both pressure and ice phase. Due to the estimated high surface gravity on LHS 1140 b, Proxima Centauri B, and some of the planets in the Trappist system, water-ice can experience extreme pressures and temperatures at depth and evolve into high-pressure ice phasesαv, g, cp are thermal expansivity, gravity, temperature, and specific heat at constant pressure, respectively. An example of an adiabatic temperature profile assuming a surface temperature of 235 K and associated temperature-dependent thermal parameters is shown in Fig. 71. Sea-Freeze does not model variations in thermal conductivity; thus, we use average thermal conductivities (Kavg) for each ice phase as listed in Supplementary Table Ice is compressive such that a large increase in pressure with depth can lead to a significant adiabatic increase of temperature with depth. The initial condition adiabatic temperature gradient implemented in all our simulations is given by the following:72. On Earth, widespread glaciation during the Neoproterozoic era  may have prevailed for over 200 million years20. For a given heat flow, it only takes a few million years for the ice sheet to achieve thermal equilibrium, thus for computational efficiency, we run the thermophysical evolution models for 5 million years. Given the relatively short timescale of our models, we assume a steady-state heat production rate and neglect the secular cooling of the planet. In some special cases, we also model the thermophysical evolution of icesheets for a billion years to ascertain the stability of basal melt. In these scenarios, we assume that the heat flow declines exponentially over a specified period.The duration of possible glaciation on these bodies is unknown, but ice sheets on the night side of tidally locked planets may exist in a near-equilibrium state with the atmosphere for an extended periodρ is density, cp is specific heat, T is temperature, t is time, z is the vertical spatial coordinate, and k is thermal conductivity53. The pressure and temperature dependence of density and specific heat are iteratively calculated using Sea-Freeze. The ice-phase dependency of thermal conductivity is updated based on the values reported in Supplementary Table The goal is to solve for the physical (melting) and thermal (heat transport) evolution of a thick, multilayered ice sheet containing Ice Ih, high-pressure ices, and any potential melt layers. To do so we solve a modified version of the 1-D thermal diffusion equation that accounts for the potential existence of three distinct heat transport regimes within the ice sheet. These regimes focus on representing the initial production of melt from heat transfer over the specified time intervals, and do not account for additional factors such as salinity prevalence and the buoyancy effects associated with viscosity contrasts at the resulting phase boundaries. Regime I is characterized by classic thermal diffusion in a solid and describes the heat transport in solid regions of the ice sheet that are not undergoing solid state convection. This includes ice layers that are too thin to undergo solid state convection as well as the conductive boundary layers of ice layers that are undergoing solid state convection . Conservation of energy in these regions is described by74,. To determine if any portion of an ice layer is undergoing solid state convection we check to see if the layer’s Rayleigh number (Ra) exceeds a preset critical Rayleigh number (Rac = 1000), a common approach in solid state mantle convection models76,. The Rayleigh number can be described asT is the temperature difference between the top and bottom of the ice layer, h is the thickness of the ice layer, and η is the viscosity of the top of the ice layer53. We additionally allow for the possibility of stagnant lid convection by testing whether any regions within each ice layer can undergo solid state convection. We assume ice viscosity follows a temperature dependent Arrhenius law77η0 is a characteristic viscosity .Regime II is characterized by solid ice layers that are undergoing solid state convection. If there exist significant temperature-dependent density differences between the top and bottom of an ice layer heated from below the layer is susceptible to solid state convectioner Rac = 000, a co78c)1/3)76.When a layer of ice is undergoing solid state convection it will retain transitional conductive boundary layers at both its upper and lower interface, the thicknesses of which can be described byentclass1pt{minimaO), and thus is appropriate in high viscosity ice layers undergoing solid state convection. However, in low viscosity liquid water layers where the convective time scale is much less than 10,000 years an alternate approach is needed. Given the significant length of our temporal discretization ), we assume that all liquid water layers will be well mixed throughout the duration of our simulations. To accomplish this, during each model time step we simulate thermal diffusion into and out of the liquid water layer governed by Eq. 26.Regime III describes the heat transport in melt (liquid water) layers. The amplified thermal diffusion approach of Eq. Hj and cj are the enthalpy and specific heat of the jth discretized layer. The enthalpies of a liquid layer are summed and then evenly redistributed throughout the liquid layer to simulate mixing. Enthalpy was employed, as opposed to temperature, to ensure conservation of energy during mixing given depth dependent specific heats. The temperature profile within the melt layer can easily be recovered by solving Eq. T.After the solution has stabilized, we calculate the enthalpy of each discretized layer within the liquid water layers The result is an energy conserving vertical temperature profile within the ice governed by Regimes I-III; thermal diffusion in solid ice layers not undergoing solid state convection (thin ice layers and conductive boundary layers), Nu-Ra parameterized convection within ice layers undergoing solid state convection, and thorough mixing of liquid water layers. After each temporal iteration of the thermal model the resulting temperature profile is fed to the SeaFreeze code to determine the new phase stratigraphy of the ice sheet , and the material properties of the ice sheet are updated prior to the next model time step accordingly. This process is then iterated for the duration of the five-million-year simulations.Q) can be estimated by multiplying the surface area of the planet (A) by the mean heat flow (q) required for basal melting. The mean heat generation per unit mass (H) is then given byM is the mass of the planet. Due to the lithophilic nature of heat-producing elements, they are assumed to be absent in the metallic core of a planet; thus, ideally, H should be calculated only for the silicate portion of a planet. However, since the size and mass of the core of most exo-Earths are unknown, we compute H for exo-Earths, assuming Earth-like core mass fraction of 30%. The H required for basal melting is compared with the age-dependent radiogenic heat production model for cosmochemically Earth-like planets54.The total heat flow from the interior of a planet (Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Acylated flavonoids are widely distributed natural metabolites in medicinal plants and foods with several health attributes. A large diversity of chemical structures of acylated flavonoids with interesting biological effects was reported from several plant species. Of these, 123 compounds with potential antimicrobial, antiparasitic, anti-inflammatory, anti-nociceptive, analgesic, and anti-complementary effects were selected from several databases including SCI-Finder, Scopus, Google Scholar, Science Direct, PubMed, and others. Some selected reported biologically active flavonoids were docked in the active binding sites of some natural enzymes, namely acetylcholinesterase, butyrylcholinesterase, α-amylase, α-glucosidase, aldose reductase, and HIV integrase, in an attempt to underline the key interactions that might be responsible for their biological activities. A clear SAR among the isolated flavonoids with regards to their inhibitory potential on aldose reductase failed to be revealed -7-O-α-rhamnopyranoside (O-[(6-caffeoyl)-β-glucopyranosyl (1→3)-α-rhamnopyranoside]-7-O-α-rhamnopyranoside  integrase enzyme. The inhibition of such an enzyme could have an efficient role in finding a new drug for the treatment of AIDS disease.+, K+ ATPase enzyme. Inhibitory actions on such an enzyme exerts lines of treatment for various cardiovascular disorders.Coumaroyl kaempferol glycosides exhibited inhibitory actions against the NaIn an attempt to increase blood flow, quercetin acetyl trisulphate and quercetin tetra sulphate showed antiplatelet activities; it is noted that the presence of quercetin acetylated residue with a lower number of sulphate groups markedly inhibited its antiplatelet aggregation activity, and it was concluded that there must be a balance between the lipophilicity as well as water solubility in order to exert antiplatelet aggregation effects.The conducted molecular docking study of the selected biologically active flavonoids in the binding pockets of different natural enzymes, namely acetylcholinesterase, butyrylcholinesterase, α-amylase, α-glucosidase, aldose reductase, and HIV integrase, highlighted the binding modes of these compounds with key amino acids and accordingly justified their mechanisms of action.Finally, a further elucidation of this interesting class of secondary metabolites could lead to the discovery of new drug, which is of clinical interest.


The similarity in antigenic structures, including the degree of correspondence between the amino acid sequence and the spatial structure, is not always reflected in the actual cross-reactivity of allergens. Inhibition tests appear to be an invaluable tool for assessing potential cross-reactivity between allergens under natural conditions. In this publication, we present two experimental models of inhibition tests: solid phase (SP-IT) and liquid phase (LP-IT). As an exemplary research model, we used the cross-reactivity between human PSA and anti-Can f 5 IgE antibodies. We performed an SP-IT model using a microplate coated with human PSA. The LP-IT model was based on mixing anti-Can f 5 IgE positive serum with a material containing human PSA. Anti-Can f 5 IgE and PSA concentrations before and after inhibition were measured to evaluate inhibition effectiveness. The results of the performed experiments showed that both proposed models of inhibition tests are an effective tools for studying cross-reactive relationships between antigens. In the SP-IT, the concentration of anti-Can f 5 decreased by 21.6% and, in the LP-IT, it decreased by 34.51%. In turn, the PSA concentration in the SP-IT decreased by 11.25% and, in the LP-IT, it decreased by 15.49%. In conclusion, both the SP-IT and the LP-IT seem to be effective tools for assessing the actual cross-reactivity between different allergens. Allergic cross-reactivity occurs when the body’s immune system identifies the proteins from different sources as being similar. Antibodies specific to one of these proteins may also recognize similar epitopes present in other proteins. In this situation, when an individual comes into contact with either of these proteins, the immune system can react in the same way, which in some cases can cause allergic symptoms. The immune system reacts in this way because these proteins are similar in structure and may have similarly structured epitopes that are recognized by antibodies specific for either of these proteins. The possibility of cross-reactivity can cause difficult diagnostics but also can lead to various unforeseen and unexpected allergic reactions in allergic individuals . This phThe clinical manifestation of cross-reactivity is not always proportional to the actual structural similarity of the proteins. It is also not always associated with the phylogenetic relationship of allergen sources. In some cases, we do not observe clinical manifestations proportional to the significant structural similarity of allergens. On the other hand, it is possible that, despite the slight antigenic similarity of allergens from different sources, the clinical manifestation of cross-reactivity after exposure to these allergens is significant ,3,4. CurIn the following research, two methods, based on the determination of the amino acid sequence as well as various techniques of spatial imaging of the protein structure, are proposed. These methods intend to help in estimating the likelihood of an allergic cross-reaction ,5.Canis familiaris prostatic kallikrein (Can f 5) is also known as arginine esterase. This is a 28 kDa protein found in the urine and fur of a dog. It is secreted in the prostate gland under the influence of androgens such as the prostate-specific antigen (PSA) in humans. Can f 5 is not found in female dogs. Its concentration is lower in young animals, and the castration of the animal causes a practically complete reduction in the secretion of this allergen [allergen .Prostate-specific antigen (PSA), a 33–34 kDa glycoprotein, is a serine protease member of the human kallikrein family. It is produced in both prostate tissue and secreted into seminal fluid. It is one of the proteolytic enzymes in male semen. Its physiologic function is to liquefy semen from its gel form ,8. It waIn our earlier publication , we demoThe aim of the study was to evaluate the usefulness of various models of inhibition tests in studies on cross-reactivity of allergens. We proposed two independent research models based on the inhibition assays we described before. The ability of anti-Can f 5 IgE antibodies to block human PSA had been the subject of our research previously .All results of the experiments performed in both experimental models are presented in Due to the small numbers of the group and the non-parametric distribution of the analytes, the statistical analysis was performed using non-parametric tests.In the first experimental model (Experiment 1), we used the solid-phase inhibition test. We assumed that if there was cross-reactivity between canine and human kallikrein, IgE antibodies to Can f 5  would be bound by human PSA coated on the walls of the polystyrene plate. After performing a multistage experiment, we observed that human PSA partially binds anti-Can f 5 IgE antibodies. The concentration of anti-Can f 5 IgE in the tested sera decreased after incubation in a polystyrene plate coated with human PSA . The difThe effectiveness of this blocking model was confirmed by the fact that PSA measured by ELISA, PSA specific test, in the test plate used in the inhibition  was lower than in the calibration material used . The decThe presence of IgE was confirmed in the polystyrene plate used for blocking PSA-human/anti-Can f 5 . The optical density measured after performing an ELISA reaction using HRP-labeled anti-human IgE antibodies and PNPP as a substrate was higher with respect to the blank as the first negative control (test wells with anti-Can f 5 IgEnegative serum). The results were expressed as optical density (OD) units. A positive IgE result was observed in each of the test wells of the plate used during Experiment 1 .The second experimental model (Experiment 2) was based on the liquid phase inhibition test. We assumed that if IgE antibodies specific for canine kallikrein (anti-Can f 5 IgE) cross-react with human kallikrein, then mixing anti-Can f 5 positive serum with material containing PSA of a known concentration in the mixture will form complexes of “anti-Can f5 IgE/PSA human”. The formation of these complexes will cause both the PSA and anti-Can f 5 IgE concentrations in the mixture measured after incubation to be lower than the starting concentrations of both analytes, taking into account the material dilution related to the assay methodology.The analysis of the test results showed that the PSA concentration measured in the samples after the inhibition test  was lower than the initial PSA concentration in the calibration material used . The decIn branch B of this experimental model (Experiment 2B), we assumed that if, after mixing anti-Can f 5 IgE positive serum with PSA-containing material, “PSA/anti-Can f 5” complexes were formed, they could be detected by binding to the membrane coated with antibodies against human immunoglobulin E. We measured the PSA concentration  and the anti-Can f 5 concentration  in the material collected after the incubation of the reaction mixture from step 1 of this experiment with a membrane coated with anti-IgE antibodies. The PSA concentration in the collected filtrates was lower than that measured in the samples after the inhibition test  bound on the anti-human IgE coated membrane  was detected. The results were expressed as optical density (OD) units. The blank, which was the second negative control, was performed with a PSA calibrator and anti-Can f 5 IgE negative serum. The optical density of the filtrate after the reaction was higher than in the blank for all tested samples .Evaluation of both experimental models regarding the effect specific for the IgE anti-Can f 5/PSA (human) relationship, carried out by measuring the concentration or level of specific IgE with multiplex tests (ISAC and ALEX), showed statistically insignificant, multidirectional and non-reproducible changes in the concentration of IgE-specific for allergens other than Can f 5 in any experimental model. The changes in the level of anti-Can f 5 specific IgE antibodies in the ISAC test and IgE levels specific for the canine male urine extract , which is the equivalent of Can f 5 in the ALEX test, observed after the inhibition tests (SP-IT and LP-IT) were unidirectional and reproducible in both tests and both methods of inhibition .By comparing the two proposed experimental inhibition assay models (Experiment 1 and Experiment 2), we observed that both the use of the solid phase inhibition test and the liquid phase inhibition test were effective in demonstrating human kallikrein binding by canine kallikrein specific IgE antibodies. In the liquid phase inhibition test, a higher percentage decrease in the concentration of both PSA  and antiThe prediction of allergen cross-reactivity is now largely based on linear sequence data or homology information between epitopes exposed on the allergen surface. Determining structure and homology is sometimes insufficient to predict the true cross-reactivity between allergens. Tests based on direct inhibitory responses seem to be useful to evaluate the clinical response associated with cross-reactivity. Various experimental models that can be used to study this phenomenon are described. However, none of them are standardized. In our study, we proposed two different experimental models with which it is possible to study cross-reactivity between different allergens. This strategy also made it possible to check whether these models give comparable results and to evaluate their effectiveness .The suspicion of cross-reactivity between the human PSA and the Can f 5 canine allergen was postulated earlier ,15. In oInhibition tests are a useful tool for identifying primary allergens and cross-reacting allergens. Standardized protocols for conducting this type of research are not available. The most frequently described methods are inhibition reactions based on the ELISA or ImmunoCAP technique. These tests are successfully used to test both plant and animal allergens ,20,21. WOur research was based on two different test strategies to block allergen-specific antibodies with a protein with cross-reactivity potential. We used a model with the test allergen bound in the solid phase on the walls of the test wells of the ELISA plate and the model using this allergen left in the liquid phase. In both test systems, we achieved effective binding of the anti-Can f 5 IgE antibody to the human prostate antigen. Such a relationship, cross-reactivity between Can f 5 and human PSA, was previously described by Weidinger et al.  and KoflOn the other hand, the efficiency of the inhibition process in the experimental models proposed by us may also be determined by the structure of the PSA antigen contained in the calibration material used as a source of prostate antigen. The PSA in the experiment was derived from ROCHE calibration material . ROCHE total PSA CalSet II is a lyophilized human serum with added human PSA. Fung et al.  identifiSince two different blocking models were used in our experiment, it was possible to evaluate their effectiveness in the cross-reactivity study. Blocking of human PSA by anti-Can f 5 IgE was observed in both experimental models, but the effectiveness of this process was significantly higher for the liquid phase inhibition test compared to the results of the solid phase inhibition test . This result is in line with our expectations. This result is mainly influenced by the mutual availability of epitopes of the antigen and specific for my paratopes on antibody molecules. Both the liquid and solid phase inhibition methods have previously been described as useful methods for testing the cross-reactivity of various types of antigens ,30,31,32In the case of the experimental solid-phase inhibition model, the binding efficacy of PSA anti-Can f IgE was confirmed by detecting the presence of IgE on the walls of the polystyrene microplate after performing an inhibition test. For the liquid phase inhibition model, we expected that when mixing anti-Can f 5 IgE positive sera with PSA-containing calibration material, “anti-Can f 5 IgE/PSA human” immune complexes would form. After incubating the mixture with the ImmunCAP membrane coated with anti-human IgE antibodies, we checked the presence of PSA on this template by performing an ELISA reaction using HRP-labeled anti-PSA antibodies and TMB as the chromogen. The optical density for the test samples was higher than the optical density for the blank, which seems to confirm the binding of PSA on the ImmunCAP membrane coated with anti-IgE antibodies. This situation is only possible if PSA has been bound to anti-Can f 5 IgE during the earlier stages of the experiment. ImmunoCAP matrix factory coated with allergens have been successfully used earlier in inhibition tests to test cross-reactivity by our team  and otheIn our opinion, both the solid phase inhibition test method and the liquid phase inhibition test method appear to be useful for assessing the actual cross-reactivity of allergens. However, the results of the experiments conducted lead us to conclude that the method of the inhibition test in the liquid phase seems to be more effective, at least in the case of using non-standardized antigens for the tests.The aim of the study was to evaluate the usefulness of various models of inhibition tests in studies on cross-reactivity of allergens. We proposed two independent research models based on the inhibition assays we described before. The ability of anti-Can f 5 IgE antibodies to block human PSA had been the subject of our research previously .In the course of the experiment, we assessed the effectiveness and usefulness of two different, independent methods for studying the phenomenon of cross-reactivity between allergens on the example of the cross-reactivity assessment of canine kallikrein and human kallikrein.In both proposed experimental models, we used sera from 31 women allergic to dogs, whose serum concentration of anti-Can f 5 IgE antibodies was at least 0.35 kU/L (≥0.35 kU/L), in accordance with common practice in the field.At the beginning of the experiment, we measured the initial concentration of anti-Can f 5 IgE in all tested sera and the human-derived calibration material used , which we used in the experiment.The serum concentration of anti-Can f 5 IgE was determined by the fluoro-immuno-enzymatic method (FEIA) on ImmunCAP 100 system, using compatible reagents . ImmunoCAP is an in the vitro test system for the quantitative measurement of specific IgE in human serum or plasma. The allergen of interest, covalently coupled to ImmunoCAP, reacts with the specific IgE from the patient sample. After washing non-specific particles enzyme-labeled anti-IgE  are added to form the complex. Following incubation, unbound enzyme-anti-IgE is washed away and the bound complex is then incubated with a developing agent . After stopping the reaction (by sodium carbonate 4%), the fluorescence of the eluate is measured. The higher the response value, the more specific IgE is present in the sample. To evaluate the test results, the responses for the patient samples are transformed to concentration with the use of a calibration curve, with a calibrator range of 0–100 kUA/L.The overall limit of quantitation for allergen-specific IgE antibodies is 0.1 kUA/L. The cross-reactivity with other human immunoglobulins is not detectable at physiological concentrations of IgA, IgM, and IgG.The standard in vitro diagnostic (CE IVD) certified multiplex tests ImmunoCAP ISAC  and ALEX  were also used in the experiment.The first experimental model was based on a polystyrene microplate ELISA test , ALPCO Salem, NH, USA) coated with goat anti-human PSA antibody, and after the first two stages, it was carried out in two independent branches.As a source of human PSA with a known concentration in our experiment, we used the ROCHE calibration solution . Total PSA CalSet II is a lyophilized human serum with added human PSA. The PSA concentration declared by the manufacturer in this material is 58.5 ng/mL. The concentration of PSA in this calibrator as measured by the ALPCO assay that was used in the experiment was 84.75 ng/mL. We took this value into account in later calculations.Before starting the experiment, we checked that the serum from the women participating in the study did not contain a detectable concentration of PSA. The calibrator used was free of anti-Can f 5 IgE. There were also no IgE antibodies specific for any of the allergens represented in the ISAC and ALEX tests.A schematic diagram of this experiment model (Experiment 1) is shown in The results of this experiment model (Experiment 1) are shown in Experiment 1 (branch A and branch B)Step 1:In the first step of this experiment, the plate was coated with human PSA. For this purpose, 100 µL of the ROCHE calibration solution containing human PSA was added to all microplate wells. The plate, prepared in this way, was then incubated at 4 °C for 24 h to immobilize PSA from the calibrator on the walls of the test wells of the plate. After this time, the calibrator was removed and wells were washed 3 times with distilled water to remove any unbound residues and clean wells. This procedure produced microplate wells coated with human PSA. This microplate was then used, in the second step of this experiment, to bound Can f 5 specific IgE (anti-Can f 5 IgE) from patients’ serum with human PSA coated to the walls of microwells of the polystyrene plate.Step 2:One hundred microliters of the patient serum (with known anti-Can f 5 IgE concentration) was added (in duplicate) into the appropriate wells of the polystyrene plate, pre-coated with human PSA. The prepared plate was incubated at 4 °C for 24 h. We assumed that during this step, anti-Can f 5 IgE from patients’ serum would bind to the PSA coated on the microplate wells.Step 3:After incubation (step 3A), patients sera were collected from microwells to the clean polystyrene tubes and after collecting sera, the plate was washed 5 times with distilled water (step 3B). Collected sera were used in branch A (Experiment 1A) and microplate wells were used in branch B (Experiment 1B) in this experimental model.Experiment 1AStep 4A:In this branch of the experiment, we assumed that some of the anti-Can f 5 (IgE) antibodies from the test sera were bound by PSA immobilized on walls of microplate wells. We expected to reduce the concentration of anti-Can f 5 (IgE) in the test sera relative to the baseline value. To verify this assumption, during this step, the concentration of anti-Can f 5 IgE was determined in each serum collected from microplate wells at the end of Step 2. The results for this branch of the experiment are shown in Experiment 1BStep 4B:In this branch of the experiment, we assumed that a portion of the PSA bound to the walls of the microplate during the first stage (Step 1) of the experiment was blocked by anti-Can f 5 IgE. We expected that the PSA concentration measured at this stage would be lower than that measured baseline in the ROCHE calibrator used (84.75 ng/mL). To verify this assumption, the concentration of PSA bound in each test well of the plate was determined according to the procedure provided by the manufacturer of the test ). One hundred microliters of Enzyme Conjugate Reagent  conjugate) was dispensed to the washed plate into each well. The plate was incubated  and then washed again (deionized water) 5 times, and 100 µL of a TMB Reagent (tetramethyl benzidine) was dispensed into each well. After 20 min of incubation (18–25 °C)) in dark, the reaction was stopped by adding 100 µL of the Stop Solution (1N HCl) to each well. Then, the optical density (450 nm) was read and the PSA concentration was calculated based on the simultaneously prepared calibration curve .The results for this branch of the experiment are shown in Step 5B:In this step of the experiment, we assumed that if the anti-Can f 5 of the test sera was bound by PSA on the walls of the microplate, it was possible to detect total IgE in that plate. To test the validity of this hypothesis, we checked the presence of IgE in the test plate used in the experiment. To detect the presence of IgE on the walls of the microplate, we used reagents dedicated to the determination of human IgE in serum, which is part of the validated HYCOR kit . The kit uses mouse anti-human IgE antibodies conjugated with alkaline phosphatase and a dedicated substrate p-nitro-phenyl-phosphate (PNPP). We have manually followed all steps of the manufacturer’s recommended procedure for selected elements of the procedure.After step 4B, the plate was rinsed 3 times with a washing solution dedicated to the method used . An appropriate volume of enzyme-conjugated mouse anti-human IgE antibodies was then added to each test well. After incubation , the plate was washed again (as before) and substrate was added. After incubation with the substrate , the reaction was stopped (1N sodium hydroxide solution) and the absorbance was measured for each test well (450 nm/630 nm). Due to the lack of possibility of calibration, the concentration of IgE was not calculated. The results were considered positive if the measured absorbance (O.D.) was higher than the blank . The results for this branch of the experiment are shown in The second experimental model was based on the blocking of PSA-anti-Can f 5 (IgE) as a result of mixing patient sera with the PSA calibrator solution. The same calibration material  was used as in the previous experimental model.A schematic diagram of this experiment model (Experiment 2) is shown in The results of this experiment model (Experiment 2) are shown in Experiment 2 (branch A and branch B)Step 1—Experiment 2A and Experiment 2B:The first stage was common to all branches (2A and 2B) of this experimental model. The experiment started with mixing the sera of patients with a calibrator with a known concentration of PSA (84.75 ng/mL) in equal volumes . The samples prepared in this way were then subjected to a two-stage incubation: first at 37 °C for 30 min and then 24 h at 4 °C. After the incubation was completed, the material was divided into two branches of the experiment (Experiment 2A and Experiment 2B).Experiment 2AStep 2A:In this branch of the experiment, we assumed that serum anti-Can f 5 IgE would be bound to PSA derived from the calibration material. We expected a decrease in the concentration of both anti-Can f 5 and PSA about the baseline values of these parameters. To verify the validity of the assumed hypothesis, the concentrations of anti-Can f 5 IgE (ImmunoCAP) and PSA ) were measured in the sample after incubation. Both procedures were described previously.The material dilution was included in all calculations. At the beginning of the experiment, we also checked that the serum from the women participating in the study did not contain detectable concentrations of PSA (ALPCO procedure) and that the calibrator used was free of anti-Can f 5 IgE (ImmunoCAP procedure).Experiment 2BStep 2 (Experiment 2B)During this stage of the experiment, we assumed that if the “PSA-anti-Can f 5 IgE” complexes were formed in the first step of the experiment, it is possible to remove them from the mixture using antibodies specific for human immunoglobulin E. To check the soundness of our assumption, the mixture from the first stage of the experiment was applied to cellulose supports coated with mouse anti-human IgE monoclonal antibodies.g, 15 min). We decided to use that mixture volume (40 µL) because it is the standard volume of material to which the ImmunoCAP reaction cups are calibrated . The reaction cups with the mixture from the experiment in Step 1 applied in this way were closed in Eppendorf tubes (which prevented evaporation) and were incubated for 30 min at 37 °C and then 24 h at 4 °C. After incubation, the reaction cups sealed in the tubes were centrifuged  to collect the filtrate.ImmunoCAP reaction cups for the determination of total serum IgE concentration  were used in the experiment. These cups are part of the system used to determine the concentration of total IgE in human serum in the range of 2.0 to 5000 kU/L. During the experiment, 40 µL of each mixture from step 1 was applied to a reaction cup coated with anti-IgE antibodies. Before this, all cups were pre-rinsed with a dedicated washing buffer  and dried (centrifugation 5000× Step 3.1. (Experiment 2B)We measured the concentration of PSA ) in the collected filtrate. We expected that if “PSA-anti-Can f 5 IgE” immune complexes were formed in Step 1 of Experiment 2 (A/B) and they were bound by anti-IgE antibodies in Step 2 of Experiment 2B, the PSA concentration in the filtrate was below the value that we measured during Step 2.1. of Experiment 2A and the baseline PSA concentration were determined in the calibration material before all experiments.Step 3.2. (Experiment 2B)We measured the concentration of anti-Can f 5 IgE in the collected filtrates (ImmunoCAP). We expected that if “PSA-anti-Can f 5 IgE” immune complexes were formed in Step 1 of Experiment 2 and they were bound by anti-IgE antibodies in Step 2 of Experiment 2B, the anti-Can f 5 IgE concentration in the filtrate was below the value that we measured during Step 2.2. of Experiment 2A and the baseline anti-Can f 5 IgE concentration was measured in the sera at the beginning of the experiments.During all analyzes and calculations, we took into account the occurring material dilutions.Step 4 (Experiment 2B)g). The filtrate was discarded. To rinse the cups, 100 µL of TMB was added to each. After 20 min of incubation (18–25 °C) in dark, the reaction was stopped by adding 100 µL of Stop Solution (1N HCl) to each well. After stopping the enzymatic reaction, all cups were centrifuged  and the optical density (450 nm) was measured in the collected filtrate. A blank test was run simultaneously with a clean algE reaction cup. The optical density measured in the filters was compared with the blank value. We assumed that if PSA was bound on the aIgE-CAP membrane, the optical density of the test samples would be higher than that of the blank.In this part of the experiment, we assumed that if “anti-Can f 5 IgE/PSA” complexes were bound to the ImmunoCAP membrane coated with human anti-IgE antibodies, it was possible to detect PSA from these complexes on the membrane surface. To check whether our assumptions are true, in order to detect bound PSA, we performed an ELISA test using anti-PSA human antibodies labeled with HRP and TMB as a chromogen. For this, we 100 µL of the monoclonal anti-PSA-HRP conjugate was dispensed to the washed reaction cups. All cups were incubated . After incubation, each cup was washed with deionized water 5 times. For this purpose, 100 mL of water were poured into each cup and centrifuged , as a negative control (first negative control), we used sera in which no anti-Can f 5 IgE antibodies were detected (anti-Can f 5 IgE concentration were lower than 0.1 kUA/L). In experimental model 2 , as a negative control (second negative control), we used a mixture of sera in which no anti-Can f 5 IgE antibodies were detected (anti-Can f 5 IgE concentration were lower than 0.1 kUA/L) and the calibration material  that was the source of the human PSA used in the experiment. The same procedures were applied to both controls, which were used for all test samples, adequately to the particular model of experiment to which the control was referring.In order to control and evaluate the performed experiments and to exclude the unexpected blocking of IgE antibodies specific for non-Can f 5 alergens by PSA, the concentration or level of specific IgE was measured using two independent, standard, multiparameter platforms certified for in vitro diagnostics (CE IVD): the ALEX test  and ImmunoCAP ISAC  in pooled sera. The tests were performed in three portions of pooled sera and in calibration material, which was the source of PSA . Sera were pooled at three experimental points: before the experiments, after incubation on the PSA coated plate (Experiment 1), and after incubation of the serum mixed with the PSA calibrator in a 1:1 volume ratio (Experiment 2). The sIgE results positive for allergens, which are presented in both tests (ISAC and ALEX), were used for the analysis. Material dilution was taken into account where necessary in the evaluation of the results.We are aware that our experimental models have some limitations related to the research tools used and the assumed inclusion criteria.The study group did not include men allergic to Can f 5. It cannot be ruled out that restricting the group to individuals of one sex may affect the results of the study. In our opinion, however, in the case of men, it would be necessary to take into account the effect of PSA, which is naturally present in male serum. This would certainly require the design of a different experimental model and seems to be an interesting direction for the development of the presented research.It would probably also be advisable to extend the presented experiment to anti-Can f 5 IgE inhibition assays with proteins other than PSA. Such a strategy would require the definition of unambiguous criteria for the selection of proteins, which are known for certain not to cross-react with the analytes tested in the research system used by us. It certainly requires further, more extensive research.We wanted to present simple models for studying the phenomenon of cross-reactivity between allergens. The presented experimental models certainly have their limitations and require further research. It seems, however, that in the absence of standardized methods to study this phenomenon, each research proposal may increase knowledge in this field.Statistical analysis: Shapiro-Wilk Test, Wilcoxon Signed Rank Test, and ANOVA Friedman Test were used. Analyses were prepared using the Statistica Software, version 13.3, and Microsoft Excel Software.Due to the lack of standardized methods for testing cross-reactivity between allergens, inhibition tests seem to be a useful and effective experimental tool for this purpose. They can be used in both solid and liquid phases. Inhibition in the liquid phase seems to be a more effective research tool. This is probably because leaving the components of the immune reaction in suspension increases the mutual availability of antibodies and antigenic epitopes. In the solid phase reaction model, antigens are bound by antibodies immobilized on the walls of a polystyrene plate, which limits the availability of certain antigenic epitopes to the antibodies and may reduce the efficiency of the reaction.The conducted experiments also seem to confirm the partial cross-reactivity between canine and human kallikrein, which may be of significant clinical importance in the case of seminal fluid hypersensitivity observed in some women allergic to dogs.


Due to the productivity shocks, there exist inherently systemic biases when using purchasing power parity as the equilibrium real exchange rate measure standard. In this paper, based on the characteristic of Chinese economic transformation, considering demographic and weakened Balassa–Samuelson effect, we reestimate the equilibrium level of the real exchange rate of RMB with the quarterly frequency data since 1994 and measure the misalignment of the RMB real exchange rate. The empirical results show that the improved convolutional neural network (CNN) model is a reasonable analytical framework for the RMB equilibrium real exchange rate, and in the sample period, the real exchange rate of RMB is robust, and demographic is an important variable affecting the RMB equilibrium exchange rate. The real exchange rate of RMB is closely related to China's economic development strategy. Since the people's bank of China implemented a managed floating exchange rate system for the RMB exchange rate in January 1994, many western countries have criticized China's manipulation of the exchange rate. The US Treasury Department has proposed several bills to the congress to label China as a “currency manipulator.” At present, the RMB has been included in the SDR and has been pegged to a basket of currencies instead of the US dollar. The trading range of the RMB against the US dollar has been expanded to 2%, and the RMB has really evolved into a managed floating exchange rate system determined by the market. The internationalization of RMB requires a strong exchange rate and strong purchasing power. In 2017, the trade dispute initiated by the Trump administration on China made scholars discuss whether the RMB exchange rate should be devalued to deal with trade barriers. All these problems need to determine the equilibrium level and dislocation of the RMB exchange rate.With the development of productive forces and the globalization of finance, economic exchanges among countries in the world are becoming more and more frequent, so as to further promote the integration of the global economy. Because of the internationalization of world finance, the depth and breadth of financial penetration are more obvious and prominent. The fluctuation of foreign exchange rate not only has an impact on national financial relations but also affects the economic development of many fields in various countries and plays a more and more important role. Uusivuori et al.  studied Due to the impact of productivity factors, the real exchange rate of catch-up economies tends to be underestimated compared with that of developed economies. The Balassa–Samuelson effect (later referred to as the BS effect) provides an analytical framework for studying the impact of productivity shocks on a country's real exchange rate. In recent literature, some scholars estimated the equilibrium real exchange rate of RMB by using the extended PPP model  , 13. FroThe change in the real exchange rate will be affected by different impact factors, and the transmission mechanism of different impact sources is also different. Considering that the currency is neutral in the long run when analyzing the long-term trend of the real exchange rate, the impact of the real shock on the real exchange rate is mainly investigated. Productivity impact is the main influencing factor. Balassa–Samuelson effect provides an analytical framework for studying the impact of productivity shocks on a country's real exchange rate. The transmission mechanism of Balassa–Samuelson effect is assuming that the traded goods satisfy the one price theorem, the prices of the traded goods of the two countries will converge, and the difference in the productivity level of the tradable sectors of the two countries will determine their wage rate difference, and the faster the productivity growth, the higher the wage level. If the labor force flows freely in China, the wage rates of the two sectors in the economy will converge, but the productivity of the nontradable sector is less than that of the tradable sector, and since the price level is mainly determined by the wage level, the price level of nontradable goods will be higher than that of tradable goods, and at this time, the real exchange rate of the country will appreciate.As for the actual situation in China, the classical assumption of full employment due to the limited labor supply in Balassa–Samuelson effect is quite different from the situation in China. Due to the existence of urban-rural dual structure in China's economy and hidden unemployment in rural areas, the labor force is not fully employed, and at the same time, due to the factors of structural transformation, the labor force will be almost equivalent to unlimited supply in a certain period of time. These factors will restrain the increase in the wage rate to some extent.As can be seen from The financial market can reflect the operation of economy and enterprises. In general, the traditional analysis method is to predict the future development through the current situation of social economy and enterprise development. There are two mainstream traditional analysis methods: event-driven analysis method and performance-driven analysis method as shown in The computer-based research method takes the algorithm as the core to process and analyzes the data in time, the convolutional neural network adopts an “end-to-end” idea, and the whole prediction process does not carry out the artificial division of subproblem; instead, the convolution neural network(CNN) model is used to realize the transformation from the original input to the desired output, and based on this idea, synergy can be realized and can get the global optimal solution of prediction under the condition of greater possibility.et = ′ represents an m dimensional random time series, q represents the lag order of the series, and ct = ′, which is an m dimensional random disturbance time series. Under nonlimiting conditions, it can be expressed asThere are many problems in the traditional econometric modeling theory. Combined with the latest achievements of current econometrics, first, an unstructured method is introduced to establish the relationship model between economic variables, namely vector autoregressive model (VAR). The model is divided into two types, namely nonrestrictive VAR and restrictive VAR. Suppose that D (U) represents the polynomial of lag operator.Furthermore, if the lag operator is introduced, it can be expressed aset is regarded as an m dimensional endogenous random time series, which is affected by the b dimensional exogenous time series ft = ′, and correspondingly, VAR can be expressed asUnder restrictive conditions, R can be expressed asIf the lag operator is introduced, it can be expressed aset = ′ represents an m dimensional random time series, t = 1, 2, ..., T, and et∼I(1), that is, corresponding to each data item eit∼I (1), i = 1, 2, ..., m, which is affected by b dimensional exogenous time series ft = ′, first, a vector autoregressive model (VAR) is established.Suppose that et has cointegration relationship, Det−1∼I(0) in formula (If  formula  can geneφ′et−1 can be regarded as the error correction term (errt−1), which reflects the long-term equilibrium relationship between variables, and formula  countries consider external economic fundamentals of the real exchange rate, and we chose the following economic fundamentals: relative labor productivity (XDSCL), foreign trade conditions (TOT), economic openness (OPEN), net worth ratio (NFAR) abroad, government spending ratio (GGR), broad money supply (M2), and RURAL population ratio (RURAL).K) and labor (L) as two factors of production to produce tradable and nontradable goods. Tradable sector (T) and nontradable sector (N) exist in the economy, respectively, accounting for the same proportion as foreign countries. ϑ1 − ϑ tradable goods satisfy the law of one price. The price of tradable goods is determined by the international market, while the price of nontradable goods is determined by the domestic market. Meet the assumption of full employment (limited supply of labor), that is, L = LT + LN. There is a functional relationship between the general price level and the real exchange rate ; here is to simplify the result; the two departments are drawn as c-d production functionP = PTθ + PNθ)(1 − q = (P/eP∗)YT = ATKTαLTα1−YN = ANKNβLNβ1−. In the optimal case of production, wage is equal to the marginal output of labor, and then,Suppose a country USES capital 11),(12)PṖ represents the rate of change of the variable.Formula  explainsYT∗=AT∗KT∗μLT∗1−μYN∗=AN∗KN∗νLN∗1−νLet the production functions of the two foreign departments be, and the same can be obtained as By combining  with 1313,(14)PTe=(PT/PT∗)e.When the one price theorem is established, there is the nominal exchange rate of two countries, and then, the real exchange rate q of the country is Formula  proves tConsidering the problem of surplus rural labor in China, the transition from the agricultural population to the manufacturing population will have an important impact on the rise of the wage rate and further restrict the appreciation of the real exchange rate, and this paper introduces the demographic factor into ICNN and sets the following benchmark framework for empirical analysis of the equilibrium exchange rate of RMB.LICNN represents the natural logarithm of the variable of the real effective exchange rate of RMB, and the latter variable is treated in the same way.As the relative price of currency, the exchange rate serves as the basis for conveying information to the market as a price variable. At the same time, from the perspective of the structural breakpoint in statistical research, the first reform of the RMB exchange rate system on January 1, 1994, resulted in a huge change in the nominal exchange rate, and the statistical results including this breakpoint were lack of robustness. Therefore, this paper chooses 1994 as the starting point of the time series. Given the relatively small number of annual data since 1994, the statistical results are not robust, and quarterly data, which are more frequent than annual data, may contain more important economic information. In order to reduce information leakage and provide sample data of sufficient time series, this paper adopts high-frequency quarterly data. The starting and ending points of samples are the first quarter of 1994 and the third quarter of 2018, respectively, so as to obtain 99 sample points. As the population data only have annual data, it is smoothed quarterly according to the annual growth rate. All data are from the WIND database.Relative labor productivity is an important basic factor affecting the real exchange rate, and labor productivity affects the real exchange rate through the Balassa–Samuelson effect (BS effect). Due to the special economic background of the economies in transition, there is a surplus labor force in rural areas, which inhibits the growth of wages, thus leading to the weakening of the BS effect. Therefore, the symbol of this variable in the model cannot be determined.The term of trade refers to the ratio of a country's export price index to its import price index. Generally speaking, the improvement of a country's terms of trade will lead to the appreciation of its currency and vice versa. However, due to the opposite effects of “income effect” and “substitution effect” on the exchange rate, the sign of this variable in the model cannot be determined.The improvement of economic openness means the reduction of trade barriers and the increase of imports, which will promote the growth of imports relative to exports, resulting in the reduction of trade balance. Therefore, the improvement of economic openness will lead to the depreciation of the real exchange rate; that is, the sign of this variable in the model is expected to be negative.Foreign net assets refer to the difference between a country's foreign assets and its foreign liabilities, which are mainly expressed in the form of foreign exchange reserves and gold. The decline in foreign net assets means that it is necessary to increase the trade surplus to improve the balance of payments, which generally requires the devaluation of the local currency. Moreover, for emerging economies (transition economies), growth requires foreign savings to finance it, and inflows of foreign capital put upward pressure on real exchange rates. Generally speaking, this indicator is expressed as the ratio of a country's net assets abroad to its GDP. Here, we expect the variable to have a positive sign.Generally speaking, the object of government purchasing expenditure is nontradable goods, so when the government expenditure increases, the demand for nontradable goods increases, and the price has upward pressure, and the real exchange rate will appreciate and vice versa. This index is expressed as the ratio of total government expenditure to GDP. The sign for this variable in the model is expected to be positive.When the broad money supply M2 expands, the inflation rate will rise, and the current account balance of the country will deteriorate. In this case, the exchange rate depreciation is required to maintain the sustainability of the external balance. Conversely, when M2 contracts, the exchange rate should appreciate. Therefore, the expectation is negatively correlated.Due to China's special urban-rural dual structure, the rural surplus labor force is more prominent than other countries, and there is hidden unemployment in rural areas, so labor force is not full employment. At the same time, due to the factors of structural transformation, in this process, labor force is almost infinite supply within a certain period of time. These factors will restrain the increase in the wage rate to some extent. Only when the surplus labor force is fully released from the countryside, hidden unemployment is reduced to a certain extent, and the labor force is no longer infinite supply, and the key wage transmission mechanism in the BS effect will play its due role. Therefore, the change in China's rural surplus labor force measured by the ratio of rural population variable is expected to be negatively correlated with the real exchange rate.Based on the above analysis, we express the theoretical model of the RMB behavioral equilibrium exchange rate as follows:ICNN stands for the RMB equilibrium exchange rate; XDSCL represents labor productivity; TOT denotes the terms of trade; OPEN denotes openness to the outside world; NFAR represents the ratio of foreign net assets; GGR represents government expenditure ratio; M2 is the broad money supply; RURAL represents the rural population ratio. The sign above the theoretical model expression is the sign of the first partial derivative of each variable, indicating the change direction of the RMB equilibrium exchange rate, namely the theoretical expectation of the change direction of the equilibrium exchange rate, when the basic economic factors increase. The question mark indicates that the relationship between the two cannot be determined temporarily.First, the stationarity of each sequence was analyzed by the ADF unit root test. The test results are shown in I(l) sequence. Therefore, the stationarity of data requires the analysis based on the cointegration framework. Here, the Johansen cointegration test method is adopted for testing.According to the results of the ADF test, the original sequence of all variables is not stable, but the first-order difference sequence is stable, which is the First, the lag order used in VAR analysis was selected. Considering the data frequency and sample length in this paper, when selecting the maximum lag order of the VAR system, 8 was selected. The VAR(8) system was finally selected according to the AIC criterion in Then, we will examine the rationality of the VAR(8) model. It has been tested that the eigenvalues of the difference equations of the VAR(8) model are all less than 1 and located in the unit circle, which indicates that the VAR(8) system is reasonable, as shown in Johansen cointegration test is conducted. Trace test and maximum eigenvalue test indicate the existence of 7 and 7 cointegration equations, respectively, indicating the existence of cointegration relations.Although the maximum eigenvalue statistic indicates that there are 7 cointegration equations, there is only one cointegration equation that contains all variables. The standardized equation is given in Thus, we can obtain the long-term equilibrium relationship between the real exchange rate of RMB and the economic fundamentals that determine its changes.As can be seen from From the cointegration equation, we can see that all the variable signs that determine the direction are consistent with what we expect. An increase in the ratio of government spending leads to an appreciation of the real exchange rate. An increase in openness, the ratio of difference equations broad money supply to difference equations rural population, would devalue the real exchange rate. The sign of relative labor productivity is positive, indicating that there is a BS effect in the sample period we discussed. The increase in China's relative labor productivity with foreign countries makes the real exchange rate of RMB appreciate. The negative sign of the terms of trade indicates that the substitution effect generated by the improvement of the terms of trade is greater than the income effect, resulting in the depreciation of the real exchange rate.The improved convolutional neural network model (ICNN) divides equilibrium exchange rate into short-term equilibrium real exchange rate (CERER) and long-term equilibrium real exchange rate (PERER). It uses cointegration technology to find the relationship between short, medium, and long-term economic factors affecting internal equilibrium and external equilibrium and the real exchange rate in reality and then determines the ERER level of the equilibrium real exchange rate. According to the definition of misalignment of the exchange rate by Williamson , misaligAfter obtaining the PERER estimate of the RMB long-term equilibrium real exchange rate, we use  to measuIn the above formula, TMIS represents the long-term dislocation of the real exchange rate, and PERER represents the long-term equilibrium real exchange rate. The long-term dislocation of the real exchange rate of RMB obtained from the  measure On the whole, the RMB real exchange rate has experienced four periods of undervaluation and one period of overvaluation. The following detailed analysis of the long-term dislocation of the real exchange rate of the RMB cyclical changes.From the first quarter of 1994 to the third quarter of 1997, the real exchange rate of RMB was undervalued. On January 1, 1994, China implemented the integration of the official exchange rate of RMB with the market price of foreign exchange adjustment. The official exchange rate of RMB depreciated from 5.8 RMB/usd in the previous trading day to 8.7 RMB/usd in that day. The substantial depreciation of the RMB nominal exchange rate resulted in the undervaluation of the RMB real exchange rate in the sample period. Subsequently, the RMB adopted the exchange rate policy of pegging to the US dollar, while the rising domestic price level gradually reduced the degree of RMB's real exchange rate undervaluation.From the third quarter of 1998 to the third quarter of 2001, the real exchange rate of RMB was undervalued. As the Asian financial crisis has receded, Asian currencies have slowly appreciated, and the real exchange rate of the renminbi has fallen. At the same time from 1998, China entered the stage of deflation. In 1998, the year-on-year increase of CPI in China was −0.8%. It was −1.4% in 1999.From the second quarter of 2002 to the first quarter of 2009, the real exchange rate of RMB was undervalued. After 2002, the US dollar continued to depreciate significantly, and the real exchange rate of RMB in China changed from overvaluation to undervaluation. With the second reform of the RMB exchange rate system in July 2005, the peg to the US dollar was changed to a basket of currencies, a managed floating exchange rate system was implemented, and the undervaluation of the RMB real exchange rate began to decrease after reaching the peak at the end of 2005.From the fourth quarter of 2009 to the fourth quarter of 2011, the real exchange rate of RMB was undervalued. With the passing of the inflation peak in 2008, China entered a slight deflation stage in 2009. At the same time, the real exchange rate of RMB also experienced a temporary decline, which made the real exchange rate of RMB gradually become undervalued.Major undervaluation stages are as follows:From the fourth quarter of 1997 to the third quarter of 1998, the real exchange rate of RMB was overvalued. Due to the most serious inflation since China's reform and opening up in 1995, the general price level soared sharply, and the real exchange rate converted to the overvaluation stage from 1997 to 1998 and reached the peak in the first quarter of 1998, with an overvaluation of 9.5%. An important factor that caused such a serious misalignment of the RMB's real exchange rate was the Chinese government's commitment not to devalue the RMB during the Asian financial crisis in 1997. Because of the pegging policy of the RMB to the US dollar at that time, the RMB appreciated sharply with the US dollar, resulting in the overvaluation of the RMB real exchange rate.From the third quarter of 2001 to the second quarter of 2002, the real exchange rate of RMB was overvalued. Since 2000, China's economy has gradually stepped out of deflation, and the US dollar began to appreciate sharply from the first quarter of 2000 until 2002 when the real exchange rate of the US dollar rose by a cumulative rate of 8%. These factors make the real exchange rate of RMB overvalued at this stage.From the first quarter of 2009 to the fourth quarter of 2009, the real exchange rate of RMB was overvalued. Affected by the financial crisis in developed countries caused by the subprime crisis in the United States at the end of 2008, China's steady economic growth relative to the western developed countries makes the expectation of RMB appreciation strong.From the fourth quarter of 2011 to the first quarter of 2017, the real exchange rate of RMB was overvalued. With the continuous and steady growth of China's economy and the promotion of economic transformation, the strategies of “going global” and “One Belt And One Road” were successively proposed, and the RMB was planned to be included in the SDR of the IMF. All these require the strong exchange rate.Major overvaluation stages are as follows:Yuan undervalued real exchange rate stage is closely related to China's export-led economic model because there is a great quantity of recessive unemployment in rural China, mainly labor-intensive industries in the early years of the development of the rise of manufacturing can quickly absorb the surplus labour, in order to obtain an export competitive advantage, and the RMB exchange rate level is low. Therefore, from 1994 to 2012, except for a few intermittent overvaluation, the RMB's real exchange rate was basically undervalued. An important supply-side factor that determines the real exchange rate level is the Balassa–Samuelson effect. When using the ICNN model to calculate the exchange rate of emerging market economies, the country characteristics factor is an important consideration in the modeling. Due to the special economic background of China's economies in transition, there is a surplus labor force in rural areas, and the growth of wages is suppressed, which leads to the weakening of the Balassa–Samuelson effect, and then, the real exchange rate is suppressed. After the gradual disappearance of China's demographic dividend and the basic consumption of surplus rural labor force, the rise of the wage rate will be further reflected in the appreciation of the real exchange rate.


Calcium ions play a pivotal role in cell proliferation, differentiation, and migration. Under basal conditions, the calcium level is tightly regulated; however, cellular activation by growth factors increase the ion level through calcium pumps in the plasma membrane and endoplasmic reticulum for calcium signaling. Orai1 is a major calcium channel in the cell membrane of non-excitable cells, and its activity depends on the stromal interaction molecule 1 (Stim1). Several groups reported that the store-operated calcium entry (SOCE) can be modulated through phosphorylation of Stim1 by protein kinases such as extracellular signal-regulated kinase (ERK), protein kinase A (PKA), and p21-activated kinase (PAK). PKC is a protein kinase that is activated by calcium and diacylglycerol (DAG), but it remains unclear what role activated PKC plays in controlling the intracellular calcium pool.Here, we investigated whether PKC-β controls intracellular calcium dynamics through Stim1.Several biochemical methods such as immune-precipitation, site directed mutagenesis, in vitro kinase assay were employed to investigate PKC interaction with and phosphorylation of Stim1. Intracellular calcium mobilization, via Stim1 mediated SOCE channel, were studied using in the presence of PKC activator or inhibitor under a confocal microscope.Our data demonstrate that PKC interacts with and phosphorylates Stim1 in vitro. phosphorylation of Stim1 at its C-terminal end appears to be important in the regulation of SOCE activity in HEK293 and HeLa cells. Additionally, transient intracellular calcium mobilization assays demonstrate that the SOCE activity was inhibited by PKC activators or activated by PKC inhibitors.In sum, our data suggest a repressive role of PKC in regulating calcium entry through SOCE.The online version contains supplementary material available at 10.1007/s13258-022-01230-3. The activation of store-operated calcium entry (SOCE) can be initiated by various ligands, such as growth factors, neurotransmitters or Thapsigargin (TG), and is propagated by phospholipase C (PLC) through the production of inositol triphosphate (IP3) and IP3 binding to ER-resident calcium channels, resulting in the release of Ca2+ from the ER store , both of which are downstream effectors of PLC  activity as a feedback mechanism. This question led us to study the role of PKC in the regulation of intracellular Ca2+ mobilization in two human cell lines (HeLa and HEK293). In an initial screening study for any interaction between PKC and Stim1, we included both classical and novel forms of PKCs, but subsequently, focused on a classical PKC-β since it is widely reported to play diverse roles in many different cellular contexts  is a family of serine/threonine kinases that regulate both positive and negative signaling pathways involved in the immune response, platelet activation, and migration of cancer cells  PKC-β interacts with and phosphorylates the C-terminal region of Stim1, whereby residue Ser660 represents an important target site, (2) mutation of Ser660 to Ala restores SOCE activity in response to PKC activation, (3) PKC activity is inversely related to SOCE activity, and (4) Stim1 oligomerization induced by ionomycin is affected by either chemical activators or inhibitors of PKC isozymes. Taken together, our data suggest that PKC regulates SOCE through phosphorylation of the C-terminal region and de-oligomerization of Stim1.Using immunoprecipitation and Ca2 at 37 ℃. TG and fluorescent dyes, such as Alexa488 and Alexa594, were purchased for calcium assay from Invitrogen . The Ca2+-sensitive dye Fluo-4 was provided by Thermo Fischer Sci . Flip-in T-REx™ 293 cell line was purchased from Invitrogen. Hygromycin B and Ionomycin were obtained from Sigma , and PKC modulators  were provided by Calbiochem . The active form of purified PKC-βII was provided by Echelon Biosciences, Salt Lake City, UT, USA. The PCR reaction kit (TOYOBO blend tag) was purchased from Toyobo .HeLa  cells and human embryonic kidney (HEK)293 cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and 1 mg/ml penicillin/streptomycin  under 5% COStim1-5’-Hind3-For, 5’- CGC-AAGCTT-GCCATGGATGTATGCGTCCGTCTT -3’,CTACTTCTTAAGAGGCTT -3’Stim1-3’-XhoI-Rev, 5’- CGC-CTCGAG-The cDNAs of Stim1 (NM_003156.4) and PKC-βII (NM_001316672) were amplified by PCR using gene-specific primers and cloned into myc-tagged or FLAG-tagged pCS4 expression vectors. Site-directed mutagenesis of the phosphorylation sites was performed by the overlap extension PCR protocol .E. coli as reported before  for 15 min at 30 ℃. The reaction mixtures were separated on 10% SDS–polyacrylamide gels, transferred onto nylon membranes and autoradiographed by exposure to X-ray film.The C-terminal region of Stim1 cDNA was divided into 3 sub-regions, which were amplified by PCR with the primers summarized in Table 3 cells/well and incubated at 37℃, overnight. After incubation, the cells were transfected with Stim1 and/or PKC-β cDNA plasmids as described above, followed by chemical treatment , PBS washings, fixation, permeabilization, and PBS washings as described before Jeon et al. (HeLa cells were plated in a 4-well dish (NUNC) at a density of 5 × 10n et al. . To dete2+ indicator dye, for 30 min, washed once with Krebs–Ringer-HEPES buffer, and incubated with Krebs–Ringer-HEPES high K+ buffer. Then, EGTA (5 mM), TG (2 µM), and Ca2+ (2 mM) were added in sequence to the culture medium, and the fluctuation in intracellular Ca2+ mobilization was imaged by confocal microscopy (Carl Zeiss LSM710).HeLa and HEK293 cells were incubated with 1 µM Fluo-4 , a fluorescent CaFor our initial study for PKC-Stim1 interaction, we obtained a set of constitutively active and truncated forms of classical (PKC-α and -β) and novel (PKC-δ and –ε) PKCs from Dr. Soh, Inha Univ., . As shown in Fig. We then attempted to narrow down further the domain of amino acid residues targeted by PKC phosphorylation by liquid chromatography-tandem mass spectrometry (LC–MS/MS). The C-terminal region of Stim1 was (co-)expressed with or without PKC-β in HEK293 cells and treated with either PBS buffer or the DAG analog PMA (Phorbol 12-myristate 13-acetate) (Supplementary data 1). The Stim1 C-terminal region was immuno-precipitated (with or without PKC-β and PMA) and subjected to LC–MS/MS analysis. Although the overall signal level was low, the analysis revealed 10 potential phosphorylation sites, mainly located in the C3 sub-region, as summarized in Fig. S3.To confirm this result, we conducted site-directed mutagenesis to identify the potential phosphorylation sites by mutating Ser and Thr residues to Ala. Through laborious in vitro kinase assays, we narrowed down the potential target sites to Thr626, Ser628, and Ser660 positions. Finally, Ser660 was identified as the most important phosphorylation site of PKC Fig. . The ser2+ store depletion, Stim1 undergoes self-oligomerization and forms puncta, which led us to question whether PKC-β has an effect on this process. For this purpose, HeLa cells were transfected with PKC-β- and Stim1-expressing plasmids and treated with phosphate-buffered saline , TG, PMA + TG, or PMA + TG + extracellular Ca2+. Confocal imaging showed that TG stimulated Stim1 self-oligomerization (2nd column), whereas PMA, a PKC activator, interfered with this oligomerization process in PMA + TG (3rd column) or PMA + TG + Ca condition (4th column) as Stim1 was diffusely distributed in the cytosol after PMA treatment and co-localized with PKC-β at the cell periphery (marked by open arrows)  decreased SOCE activity by approximately 18% and 12%, respectively, compared to that in the DMSO control group. In contrast, the PKC inhibitors Go6983 and bisindolylmaleimide I increased SOCE activity by 14% and 8%, respectively, compared to that in the DMSO control group  showed the highest level, which is about 60% more SOCE activity than that of control vector cell line  and inhibitors  of PKC seem to decrease and increase the extracellular CaConfocal imaging revealed that TG induced Stim1 self-oligomerization  as reported elsewhere Below is the link to the electronic supplementary material.


The Trial Innovation Network has established an infrastructure for single IRB review in response to federal policies. The Network’s single IRB (sIRBs) have successfully supported over 70 multisite studies via more than 800 reliance arrangements. This has generated several lessons learned that can benefit the national clinical research enterprise, as we work to improve the conduct of clinical trials. These lessons include distinguishing the roles of the single IRB from institutional Human Research Protections programs, establishing a consistent sIRB review model, standardizing collection of local context and supplemental, study-specific information, and educating and empowering lead study teams to support their sites. Challenges to implementation of a single IRB (sIRB) model in multisite research have been posited in the recent literature, since the mandate for use of a sIRB has been issued by both the National Institutes of Health (NIH) and the US Department of Health and Human Services (DHHS) effective in 2018 and 2020, respectively−6. The TWhile an sIRB may take responsibility for the review and approval of research using criteria that are well defined in the federal regulations, the research institution must retain responsibility for the other elements of institutional Human Research Protection (HRP) and oversight Fig. . This inThe use of a reliance agreement that clearly distinguishes the roles of the sIRB from the relying institution is also critical for addressing this challenge. The TIN SIRBs utilize the SMART IRB Agreement  as the bDue to national inexperience using an sIRB for multisite research, there was a great need to create a consistent, step-wise sIRB review model. The TIN sIRBs recognized that having a consistent model would help to set expectations for relying institutions and generate routine familiarity when working with the TIN sIRBs, in addition to setting a standard other sIRBs could follow. By reducing confusion and increasing familiarity, the goal was to improve the study initiation process and timelines.The TIN sIRB review model includes the following steps Table : reliancDespite success in establishing a clear process, some challenges remain. Some sites are still not prepared to function in relationships with sIRBs. Sites new to research – such as private practices, nonprofit and community organizations, and businesses – must secure a federal-wide assurance, delaying the reliance relationship. These sites new to research also have difficulty performing all aspects of the site HRP review, which requires additional support from more experienced sites or the sIRB. There are also sites that are uncomfortable documenting their willingness to rely on the sIRB before the sIRB has performed initial review of their site; however, without the commitment to rely and provide initial local context information, it is difficult or impossible for the sIRB to commit to conduct a meaningful review of the site. Further education is needed to facilitate relationships with inexperienced research sites; distinguish the implications of a reliance decision, sIRB approval for a site, and site HRP review completion, and resolve the different expectations of the sIRB and institution for the initial sIRB review steps and timing.To identify early which sites may be inexperienced and require additional support to navigate the sIRB review model, it is recommended that each site’s sIRB experience be assessed as part of site selection and feasibility questionnaires, which will enable lead teams and coordinating centers to identify and budget for additional resources that may be required to support both experienced and inexperienced sites. It is further recommended that site HRPPs publicize their HRP review components and processes, such that investigators can easily access this information and prepare for the process.Through IREx, TIN sIRBs standardized and centralized collection of local context information via the institutional profile, an HRP survey, and an investigator survey . The insLocal context considerations ended up being more complex than originally anticipated, which required revisions to the local context collection tools in IREx to reach their present state described above. It also required use of supplemental, study-specific local context forms to capture information requested by the sIRB about the relying sites as part of their review. Traditional views of local context have centered on an IRB’s understanding of the social, cultural, and political attitudes and norms of the community where an institution will draw participants . While tWhile the TIN sIRBs have established consistent processes to collect local information at study onset, the quality of the information received varies, delaying site onboarding. Moreover, the ability to collect relevant local information throughout the study rather than at a single time point is limited. Lastly, there continues to be a struggle with institutional preference and policy differing from the way the protocol, consent, and other materials are drafted, such as preferences over how statements in the consent document are written. This leads to delays as desired changes are negotiated. To identify early which sites may be inexperienced and require additional support to navigate the sIRB review model, the TIN sIRBs recommend that sIRB experience be standardly assessed as part of site selection and feasibility questionnaires which will enable lead teams and coordinating centers to identify and budget for additional resources that may be required to support such sites.Study teams, accustomed to interactions and support from their local IRBs, are now required to interact with external IRBs where the same level of communication and tailored support may not be forthcoming or even feasible. In response to this shortcoming, TIN sIRBs have developed a robust model for investigator engagement. This begins in consultation with the project’s lead study team. Expectations for sIRB review processes are established. Tailored training is offered for site study personnel via webinar and phone, or in-person where feasible. TIN sIRB coordinating center teams help sites navigate the onboarding process, providing individual support for the review of local information, and preparing materials for sIRB review. Some TIN sIRBs also enhanced their electronic submission systems to enable direct communication with site personnel. However, practical communication challenges still exist between site investigators and the TIN sIRBs, such as the time needed to build meaningful working relationships, as well as the sIRB staff needed to engage in conversations with many site study personnel.Outside of the TIN, many sIRB offices do not have the staff capacity to support the needs of researchers at other sites. Lead study teams unaffiliated with the TIN may be unfamiliar with the demands of sIRB site coordination or they are similarly understaffed to assume the responsibilities. While TIN sIRB resources and IREx exist to help standardize and support sIRB coordination, additional consideration of how researchers coordinate the sIRB process is needed.The TIN sIRB experience has uncovered additional fundamental challenges related to sIRB review. These include lack of preparedness of research teams and organizations to operate under an sIRB model, inadequate attention to the true cost of sIRB review, and misplaced emphasis solely on measurements of efficiency to evaluate the sIRB model.Prior to the rollout of the NIH policy, little focus was given to the critical importance of a robust educational program surrounding sIRB. Despite improved relationships, robust tools, and standardized communications, researchers and organizations remain confused about the process and purpose of sIRB review. As a result, TIN sIRBs have dedicated many hours developing educational materials and discussing the fundamentals of reliance and the sIRB review process with researchers and HRPPs. One advantage that the TIN sIRBs found was the ability to work with the same lead teams or coordinating centers over time. As there is generally a steep up-front learning curve for those responsible for submitting to the sIRB and coordinating local site completion of HRP review, the TIN sIRBs saw a measurable improvement in navigation of sIRB processes for lead teams/coordinating centers that had been through the process before. This experience highlights the importance of these roles in sIRB implementation and suggests that efficiencies may be gained when lead teams/coordinating centers become adept in managing these responsibilities.Additionally, there has been limited public discourse on the true costs of the sIRB model. Where local IRB review systems were relatively well established, sIRB review has brought new infrastructure costs associated with system enhancement and expansion, tool development, and growth in IRB staff and membership. Research teams must hire new staff or coordinating centers to manage sIRB communications and must account for this in the direct costs of their budgets. These expenses have not been well documented, nor have they been evaluated against the cost of a traditional local review model. Additionally, budgets must include sIRB review fees, and investigators need to seek quotes for these fees up-front to include in funding applications. Though sIRB fees vary depending on the organization performing the sIRB review, fee structures commonly charge for initial and ongoing protocol and document reviews, as well as additional fees on a per-site basis. Until true costs are determined, sIRBs, relying sites, and researchers will likely remain under-resourced for sIRB review.Finally, a dominant misconception remains that centralized IRB review is and should be faster than the use of local IRB review at each site. Not only does this emphasis on speed pose potential ethical challenges for human subject protections, it dismisses the true steps required to effectuate sIRB review. While sIRB review avoids duplicative “IRB reviews,” it does not alleviate the need for site HRP involvement with each study. Thus, the question becomes: does separating the responsibility for the IRB review and site HRP review streamline the process? While this may take years to evaluate, we must ensure our metrics capture the full time to site activation – including the site HRP review and finalization steps that lead to activation. Importantly, we must also ensure that our measurements of the success of the sIRB mandate are not simply based on time, but on the quality of human subject protections. Arguably, our greatest remaining challenge is how to keep that mission at the forefront of research considerations.In view of the challenges and costs uncovered by the experience in the TIN, it may be reasonable to reconsider the current federal policy that requires sIRB review for studies with two or more sites , allowinThe TIN sIRBs have successfully overcome many challenges while implementing a sIRB review model. These lessons learned provide a solid foundation that the research enterprise can build upon to create greater innovation in this arena. As further work is performed, it will be important for all involved in sIRB implementation to report on their successes and challenges not only to strengthen the base of knowledge but also to fuel additional improvements to clinical research.