T = 230 K. Interestingly, we find that the disordered mixing of rhombic and hexagonal ice leads to a favorable configurational entropy that stabilizes the critical nucleus, facilitating the non-classical pathway. In contrast, the favorable energetics promotes the formation of hexagonal ice, resulting in the classical pathway. Furthermore, we discover that, at elevated temperatures, the nucleation process prefers to proceed via the classical pathway, as opposed to the non-classical pathway, since the potential energy contributions override the configurational entropy compensation. This study provides insights into the mechanisms of heterogeneous ice nucleation and sheds light on the rational designs to control crystallization processes.Ice nucleation on the surface plays a vital role in diverse areas, ranging from physics and cryobiology to atmospheric science. Compared to ice nucleation in the bulk, the water-surface interactions present in heterogeneous ice nucleation complicate the nucleation process, making heterogeneous ice nucleation less comprehended, especially the relationship between the kinetics and the structures of the critical ice nucleus. Here we combine Markov State Models and transition path theory to elucidate the ensemble pathways of heterogeneous ice nucleation. Our Markov State Models reveal that the classical one-step and non-classical two-step nucleation pathways can surprisingly co-exist with comparable fluxes at  Classically, ice nucleation on foreign surfaces is considered a one-step process. Here, Li et al. uncover a two-barrier pathway which becomes competitive at lower temperatures, facilitated by synergistic, entropic effects of rhombic and hexagonal ice structures. As a result, crystal nucleation has a profound impact on our daily life. Therefore, understanding the thermodynamics and kinetics of the nucleation process is imperative and has become a topic under intense study12. Classical nucleation theory (CNT) provides a phenomenological description for nucleation, based on the thermodynamic framework of overcoming a single free energy barrier that is attributed to the balance between the energy gained from forming a nucleus and the energy lost from the creation of interfaces13. However, recent experiments17 and simulations21 have shown that CNT is often insufficient to describe complex nucleation processes. For this reason, non-classical nucleation theories based on kinetic perspectives  have been proposed, in which the nucleation process consists of two or even more steps separated by multiple free energy barriers24. Non-classical crystal nucleation has been reported in a wide range of systems, including the crystallization of nanoparticles25, proteins17, and ionic compounds19. Nevertheless, the atomistic mechanisms and comprehensive elucidation of the structural evolutions during nucleus formation in the non-classical two-step nucleation pathways are not well known; and it remains challenging for experimental techniques to elucidate the kinetic pathways and reveal intermediate states for the crystal nucleation processes.Crystal nucleation from melt1, shaping our world from climate mediation to glacier formation7. The unique properties of the hydrogen bond network in water also make ice nucleation an archetypal and intriguing system to study31, drawing vast attention from physicists, chemists, and material scientists35. Although various experimental techniques have been adopted to probe ice nucleation in a broad spatiotemporal scale39, it remains challenging for experiments to characterize ice nucleation at characteristic resolutions, i.e., temporal (pico- to micro-second) and spatial (atomistic). Alternatively, molecular dynamics (MD) simulations, a powerful tool with high temporal (up to femtoseconds) and spatial (atomistic) resolutions, have been extensively applied to study the dynamics of ice formation48. Interestingly, previous studies40 have found that, even though homogeneous ice nucleation follows a one-step nucleation pathway, the formation of stacking-disordered ice nuclei is preferred due to a favorable entropic stabilization. However, it remains unclear whether the mechanism of entropic stabilization exists in HIN, and how the interactions from the surface interfere with the nucleation process. Previous studies49 have found that a single-barrier CNT is not sufficient to describe HIN, in which pre-nucleation polymorphic structures found at an early stage suggest a two-step nucleation process. However, previous simulation studies of ice nucleation on the surface mostly focus on individual simulation trajectories44, or one reaction pathway51, which may not be sufficient to systematically identify an ensemble of highly parallel kinetic nucleation pathways. Furthermore, it is challenging to reveal the critical nucleus and the intermediate states, which are crucial when characterizing the mechanisms for either the classical one-step or non-classical multi-step nucleation pathways.Heterogeneous ice nucleation (HIN), i.e., the formation of ice nuclei on a foreign surface, is one of the most ubiquitous crystal nucleation processes on earth58 constructed from many short MD simulations provide a promising approach to elucidate the complicated kinetic pathways of ice nucleation. In an MSM, the continuous dynamics are modeled as Markovian transitions among metastable states at discrete time intervals66. Moreover, MSMs have become a popular and powerful tool to simulate biomolecular dynamics, such as RNA63 and protein72 folding, ligand-receptor binding75, and protein allostery76. In addition, researchers, including ourselves, have previously applied MSMs to clarify the dominant self-assembly pathways of nanoparticles78 and lipids79, where MSMs can automatically identify intermediate states and calculate their respective thermodynamic and kinetic properties at equilibrium. To our knowledge, MSMs have not been previously used to elucidate the kinetics of ice nucleation.Markov State Models (MSMs)82 (TPT) to elucidate the ensemble of kinetic pathways of HIN simulated by MD simulations on a wurtzite-structured surface. Strikingly, our MSMs show that when water is supercooled to 230 K, ice nucleation can proceed via either the classical one-step or the non-classical two-step nucleation pathway, in which the non-classical pathway differentiates remarkably from the classical pathway by containing two activation steps. Moreover, compared to the classical pathway, the increase in configurational entropy from the disordered mixing of rhombic and hexagonal ice stabilizes the critical nucleus in the non-classical pathway, making both pathways accessible with comparable flux. Furthermore, by elevating the temperature, we discover that the nucleation process shifts significantly towards the classical pathway, mainly because the potential energy difference, which favors the classical pathway, prevails over the configurational entropy compensation.In this work, we employed MSMs and transition path theory84 to automatically choose CVs that can describe the slowest dynamics relevant to the kinetics of HIN from a pool of 18 candidate CVs that were identified based on physical intuition. The Spectral-oASIS chose five representative CVs by utilizing the Nyström method to approximately reconstruct the time-lagged correlation matrix of all candidate CVs using only a subset of the CVs. As listed in Supplementary Table 85 to distinguish the local structures of each water molecule  that can precisely describe the dynamics of HIN simulated from MD . Then, the MSMs were validated by implied timescale analysis and the Chapman–Kolmogorov test89 based on the microstate level , and are separated by free energy barriers .Based on the five selected CVs, we constructed MSMs following a two-step procedure using the MSMbuilder PackagesT = 230 K. Representative kinetic trajectories corresponding to either one or two activation steps are displayed in Fig. 90, which allowed us to reveal the ensemble of transition paths68. Fig. 24 since they all involve the intermediate development of characteristic pre-nucleation structures, i.e., a mixture of rhombic and hexagonal ice, which is different from the classical description of the direct formation of hexagonal ice. After the conversion of rhombic ice into hexagonal ice, the fluxes coalesce and enter the growth stage, as shown by the further growth of hexagonal ice.Strikingly, our MSMs revealed that both the classical one-step and non-classical two-step HIN pathways coexist at Jhet = 3.5 × 1030 ± 1 s−1m−3 for our system at T = 230 K (see Supplementary Methods Jhom6 ± 1 s−1m−3)47. We also notice that a previous study50 reported a fast heterogeneous ice nucleation rate of Jhet = 1026 ± 2 s−1m−3 on a Kaolinite surface using the same water model at 230 K. These underline the relevance of studying heterogeneous ice nucleation as the major mode in competition with homogeneous ice nucleation when water is supercooled to T = 230 K.Furthermore, the mean first passage times (MFPTs) for each transition between the two macro-states are reported in Supplementary Fig. st barrier of the non-classical two-step pathway (see Methods for the identification of the TSs using TPT). Interestingly, the MFPTs for the transition from macro-states I to III are shorter than that from macro-states I to IV . This suggests that the 1st free energy barrier along the non-classical pathway is smaller than that for the classical pathway. However, the difference in average potential energy  for each water molecule between the critical nuclei in TS I and II is approximately 2.08 kJ mol−1  at the saddle points which govern the transitions, is illustrated in Fig. see Fig. . Conside40, where the critical ice nucleus is stabilized by the increase in entropy from the stacking-disordered cubic and hexagonal ice. However, unlike homogeneous ice nucleation, the classical HIN pathway, which forms mainly hexagonal ice, is also accessible with comparable flux due to the favorable potential energy for the critical nucleus (TS I). This can be partially attributed to the surface, which has stronger interactions with the first layer of hexagonal ice than rhombic ice, as shown in Supplementary Fig. 91. In addition, for the non-classical HIN pathway, the formation of a disordered mixture of rhombic and hexagonal ice facilitates the system to overcome the first free energy barrier, after which the rhombic ice is further converted into hexagonal ice via transitioning from states III → IV or III → V , as shown in Fig. The configurational entropy stabilization of the nucleus via the disordered mixing of rhombic and hexagonal ice makes the non-classical nucleation pathway accessible. Similar mechanisms occur in homogeneous ice nucleationT = 230 K , which agrees with CNT and previous work29. As a result, the potential energy difference between the two TSs along the two pathways will increase with nucleus size. In terms of configurational entropy, we anticipate that the difference between the two pathways may not increase much since the rhombic ice mainly forms in the first layer, and the increase in critical nucleus size  will not increase the configurational entropy enough to compensate for the potential energy difference. Therefore, the potential energy difference overrides the configurational entropy compensation, making the classical nucleation pathway more accessible than the non-classical pathway. As a result, during the early stage before the bifurcation of nucleation flux, the nucleation process becomes more likely to proceed via the classical pathway, with the dominant formation of hexagonal ice, which can also be verified by the dramatic decrease of the stationary population of macro-state III based on microstate-MSMs in Fig. Temperature affects the thermal fluctuations of water molecules, and consequently, the nucleation pathway. As previously discussed, the free energy barrier of the critical nucleus for the non-classical pathway is comparable to that of the classical pathway at 40, the presence of a foreign surface in HIN can, under certain conditions, give rise to parallel one-step and two-step pathways. The critical ice nuclei along the two pathways are stabilized by contrasting energetics, showing a delicate competition of the driving forces between intrinsic entropy stabilization and water-surface interactions. In addition, the temperature-dependent kinetics makes it feasible to tune the nucleation pathway, offering a possible explanation for the contrasting scenarios of both the classical and non-classical crystal nucleation observed in experiments.MSMs combined with the TPT analysis offer a powerful framework to study the structural evolutions of ice nucleation and might be extended to other crystal nucleation processes that are challenging to study when using direct or unbiased MD simulations. Furthermore, MSMs allow us to identify hidden transition states of the disordered ice mixture and compare multiple parallel pathways, uncovering the underlying mechanisms of the co-existence of the classical and non-classical HIN pathways. Different from homogeneous ice nucleation, which has one-step and one dominant pathwayT = 230 K. In particular, we show that the configurational entropy stabilization of the critical nucleus via the disordered mixing of rhombic and hexagonal ice makes the non-classical nucleation pathway as accessible as the classical pathway, whose critical nucleus mainly consists of potential energy-favored hexagonal ice. Furthermore, we compared the major pathways by altering the temperature; and found that as temperature increases, the dominant nucleation flux shifts to the classical nucleation pathway. This study shows that MSMs are promising tools when investigating crystal nucleation, and sheds light on the mechanisms of non-classical nucleation via entropy stabilization. Moreover, by altering temperatures, we are able to achieve modified nucleation kinetics, paving the way to control the crystallization process.In conclusion, we constructed MSMs to elucidate the kinetic pathways of HIN. Using MSMs along with TPT analysis, we were able to illustrate the competitive kinetics of the classical one-step and the non-classical two-step ice nucleation pathways, and uncover the underlying mechanism of comparable flux at NVT MD simulations were performed using LAMMPS packages93 with a time step of 2 fs. The simulation system consisted of 4940 water molecules sitting on top of a wurtzite-structured surface in a periodic box with dimensions: x = 5.43 nm, y = 5.89 nm, and z = 7.0 nm, as shown in Supplementary Fig. 94 water model was employed to simulate water molecules, and the surface atoms were fixed in the wurtzite lattice under unit cell parameters of a = 4.519 Å and c = 7.357 Å. The wurtzite structure is shared by various materials such as AgI43 and the unit cell parameters were chosen to match the hexagonal ice to minimize strain energy due to lattice mismatch. The water-surface interactions were simulated by the Lennard-Jones (LJ) potential, which was cut off at 1.0 nm. Initially, unbiased all-atom simulations were performed at T = 230 K and T = 240 K. Seeded unbiased simulations were shot for T = 250 K, with original seeds from T = 240 K. (see Supplementary Methods The all-atom 85, which calculates the local order of an atom with its neighbors as follows:i’s closest neighbors j, and j and its neighbor k, respectively. For bulk water, 49 from liquid water with 49, respectively, whose corresponding structures are presented in Fig. To characterize the local structures of a water molecule, we employed the average bond order parameterentclass1pt{minimaT = 230 K through the following procedures (see Supplementary Methods k-centers clustering algorithm87. The hyperparameter for clustering, i.e., the number of microstates, was determined by conducting variational cross-validation with the GMRQ88, as illustrated in Supplementary Fig. 89, as shown in Supplementary Fig. 62, as illustrated in Supplementary Fig. We constructed MSMs to study the kinetics of HIN at T = 240 and 250 K (see Supplementary Methods T = 230 K. The MSMs were also validated by the implied timescales and Chapman–Kolmogorov tests, as shown in Supplementary Fig. MSMs were also constructed to study HIN at T = 230 K, as shown in Fig. We also used Markov Chain Monte Carlo simulations based on the transition probability matrix of the 1000-microstate MSM to generate ten independent 2 ms-long trajectories (see Supplementary Methods 90. We computed the ensembled flux of the nucleation pathways along with their relative probabilities at the microstate level. For visualization purposes, the net flux of the transitions was assembled into coarse-grained macro-states68. To identify the critical ice nuclei along the classical and non-classical pathways, the saddle points along the two pathways were obtained using two independent TPT analyses based on the committor analysis at the microstate level (see Supplementary Methods The flux of the nucleation pathways were analysed based on a mathematical framework of TPTSupplementary informationPeer Review File


Trichomonas vaginalis is a unicellular parasite and responsible for one of the most common sexually transmittable infections worldwide, trichomoniasis. Carbonic anhydrases (CAs) are enzymes found in all lifeforms and are known to play a vital role in many biochemical processes in organisms including the maintenance of acid–base homeostasis. To date, eight evolutionarily divergent but functionally convergent forms of CAs  have been discovered. The human genome contains only α-CAs, whereas many clinically significant pathogens express only β-CAs and/or γ-CAs. The characterization of pathogenic β- and γ-CAs provides important knowledge for targeting these biomolecules to develop novel anti-invectives against trichomoniasis. Here, we report the recombinant production and characterization of the second β-CA of T. vaginalis (TvaCA2). Light scattering analysis revealed that TvaCA2 is a dimeric protein, which was further supported with in silico modeling, suggesting similar structures between TvaCA2 and the first β-CA of T. vaginalis (TvaCA1). TvaCA2 exhibited moderate catalytic activity with the following kinetic parameters: kcat of 3.8 × 105 s−1 and kcat/KM of 4.4 × 107 M−1 s−1. Enzyme activity inhibition was studied with a set of clinically used sulfonamides and sulfonamide derivates. Twenty-seven out of the 39 compounds resulted in inhibition with a nanomolar range. These initial results encourage for future work entailing the design of more potent inhibitors against TvaCA2, which may provide new assets to fight trichomoniasis.Trichomonas vaginalis has two β-carbonic anhydrases (TvaCA1/2).• Protozoan parasite • TvaCA1/TvaCA2 represents promising targets for antitrichomonal drug development.• TvaCA2 is a dimer of 20.3 kDa and possesses moderate catalytic activity.KI of 222.9 nM.• The most efficient inhibitor was clinical drug acetazolamide with • The 39 tested sulfonamides form the basis for the design of more potent inhibitors.The online version contains supplementary material available at 10.1007/s00109-021-02148-1. Leishmania donovani chagasi, Mycobacterium marinum, and vancomycin-resistant Enterococci [Today, we live in an era with antibiotic-resistant microbes, which will continue to cause devastating problems unless new drugs are found. One of the promising novel biomolecular drug targets is a group of metalloenzymes called carbonic anhydrases (CAs) which catalyze the interconversion between water and carbon dioxide to protons and bicarbonate ions. This chemical reaction is a fundamental part of acid–base balance in all living creatures. CAs have also been identified as a probable key factor of the molecular machinery involved in the metabolic pathways of clinically significant pathogens . CAs areerococci , 3, 4. Gerococci  and epilerococci .Trichomonas vaginalis is a single-cell opportunistic parasite infecting the urogenital area of men and women [T. vaginalis will pose a real threat unless novel medications are discovered. Additionally, mild symptoms result in a more effective transmittance of the infection, and individuals are further exposed to more severe infections and diseases, such as prostate cancer and human immunodeficiency virus (HIV) [T. vaginalis has revealed the presence of two β-CAs. We have previously reported the production and characterization of the first isoform (TvaCA1) [nd women . This dind women . Even thnd women , the harus (HIV) , 10. Trius (HIV) . Genetic(TvaCA1) –15, and TvaCA2) was retrieved from Universal Protein Resource Database UniProt (protein entry A2DLG4) [Escherichia coli expression system with a multipromoter vector, pBVboostFG (Online Resource g) after reaching an optical density of 0.4–0.6, and they were mechanically disrupted with an EmulsiFlex-C3 homogenizer  in 50 mM Na2HPO4, 0.5 M NaCl, and 50 mM imidazole binding buffer , and centrifuged . The supernatant was combined with Protino® nickel-nitrilotriacetic acid (Ni2+-NTA) agarose affinity chromatography resin  and BB , after which the suspension was incubated for 2 h at room temperature with gentle agitation. After incubation, the resin was packed into a chromatography column with an EMD Millipore™ vacuum filtering flask . The protein was eluted with 50 mM Na2HPO4, 0.5 M NaCl, and 350 mM imidazole (pH 7.0). The 6xHis-tag was removed by thrombin  according to the Thrombin CleanCleave™ Kit manual  followed by Ni2+-NTA affinity chromatography. NanoDrop One  was used to determine the protein yield, and the quality of the purified protein was analyzed by SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) using a 12% polyacrylamide gel visualized with PageBlue Protein staining solution . The obtained polypeptide bands of the SDS-PAGE gel were identified using tandem mass spectrometry .The gene sequence of the second β-CA of T. vaginalis ( A2DLG4) . An EschMultiple sequence alignment was performed using the Clustal Omega tool from EMBL-EBI  and visu2 hydration activity was investigated with an Applied Photophysics stopped-flow instrument at 20 °C [2SO4 (for maintaining a constant ionic strength). Phenol red of 0.2 mM was used as a pH indicator, working at a maximum absorbance of 557 nm. The initial rates of the CA-catalyzed CO2 hydration reaction were followed for 10–100 s. CO2 concentrations of 1.7–17 mM were used to determine the kinetic parameters and inhibition constants. For each inhibitor, the initial velocity was determined by using ≥ 6 traces of the initial 5–10% of the reaction. The uncatalyzed rates were determined similarly and subtracted from the total observed rates. DSMO was used at 5% to make the stock solution, from which 0.1 mM inhibitor solutions were prepared in dH2O. Subsequently, dilutions up to 0.01 nM were prepared with dH2O. Inhibitor and enzyme solutions were preincubated together  prior to the assay to allow the formation of the enzyme–inhibitor complex. The inhibition constants were obtained by nonlinear least-squares methods using PRISM 3 and represent the means from ≥ 3 different measurements.CA-catalyzed COMw) of TvaCA2 without a His-tag. The instrumentation consisted of a Malvern Zetasizer (microV)  and a liquid chromatography instrument  equipped with an autosampler (SIL-20A) and UV–VIS (SPD-20A) and fluorescence detectors (RF-20Axs). The protein concentration was determined with the UV absorption intensity at 280 nm. Lab Solution Version 5.51 (Shimadzu Corporation) and OmniSec 4.7  software were used to process the acquired data. The TvaCA2 sample was injected into a Superdex 200 5/150 column  equilibrated with 50 mM Tris–HCl (pH 7.5) buffer. Measurements were performed within a thermostable chamber at 12 °C, with a flow rate of 0.1 mL/min. The Mw of TvaCA2 was determined in two independent ways: first, based on elution time by using a standard curve calculated according to the elution profiles of standard proteins  12 kDa, alcohol dehydrogenase 150 kDa (ADH), β-amylase 200 kDa, bovine serum albumin (BSA) 66 kDa, standard CA 29 kDa ) and second, by calibrating the light-scattering detector based on the monomeric peak of BSA and using the SLS intensity to determine the protein size.Static light scattering (SLS) combined with size-exclusion chromatography (SEC) was used to determine the molecular weight  The most efficient inhibition was established with AAZ, ethoxzolamide (EZA), 4-amino-6-chloro-1,3-benzenedisulfonamide (compound 12), and methazolamide (MZA) with inhibition constants of 222.9 nM, 362.1 nM, 382.2 nM, and 389.8 nM, respectively. From these, AAZ and EZA exhibited similarly strong inhibitions toward both TvaCA1 and TvaCA2.(ii) Notably, the compounds with a 1,3-benzenedisulfonamide structure  were all submicromolar inhibitors of TvaCA2, whereas they were not efficient against TvaCA1.(iii) The least efficient inhibitors were topiramate (TPM), indisulam (IND), zonisamide (ZNS), sulpride (SLP), celecoxib (CLX), valdecoxib (VLX), sulthiame (SLT), and saccharin (SAC) as well as 3-bromo-4-amino-benzenesulfonamide (compound 9), 1,3-dibenzenesulfonamide (compound 10), 4-(2-amino-pyrimidin-4-yl)-benzenesulfonamide (compound 19), and 4-(2-hydroxymethyl-4-nitrophenyl-sulfonamido)ethylbenzene-sulfonamide (compound 24) with inhibition constants over 10 μM in the assay system, likely reflecting their weak affinity toward TvaCA2.KIs > 10 μM), whereas 25 compounds did not inhibit TvaCA1. In both cases, simple/heterocyclic sulfonamides including 3-bromo-4-amino-benzenesulfonamide (compound 9) and 4-(2-hydroxymethyl-4-nitrophenyl-sulfonamido)ethylbenzene-sulfonamide (compound 24), and the clinical drugs TPM, ZNS, SLP, IND, VLX, CLX, SLT, and SAC exhibited very weak affinities.(iv) Out of the 39 tested sulfonamides 12 compounds resulted in ineffective TvaCA1 inhibition , 4-amino-6-trifluoromethyl-benzene-1,3-disulfonamide (compound 11), 4-amino-6-chloro-benzene-1,3-disulfonamide (compound 12), MZA, DZA, BRZ, BZA, and HCT, which showed inhibition profiles ranging from strong to moderate/weak TvaCA2 inhibition, whereas with TvaCA1, they resulted in moderate/weak or ineffective inhibition.(v) Some of the tested compounds resulted in notably different TvaCA2 and TvaCA1 KI values of AAZ, EZA, and MZA for TvaCA2 were somewhat similar, in the range of 222.9–389.8 nM. The results of these three classical CA inhibitors were fairly similar between TvaCA1 and TvaCA2, except for MZA which showed approximately 10 times higher inhibition constant (3827.9 nM versus 389.8 nM) for TvaCA1 than for TvaCA2. The other clinically used drugs weakly inhibited both the TvaCA1 and TvaCA2 enzymes with over micromolar inhibition constants. Among compounds 1–24, very few similarities were found in the inhibition profiles of TvaCA1 and TvaCA2. The most similar inhibition constants at micromolar levels were observed for compounds 1 (3-amino-benzenesulfonamide), 13 , 15 (4-amino-/4-hydroxymethyl-benzenesulfonamide), and 21 (4-(2-hydroxymethyl-4-nitrophenyl-sulfonamidoethyl)-benzenesulfonamide) with moderate–weak inhibition constants ranging from 1148 to 4155 nM. Compounds 1, 13, and 15 have structures that incorporate compact, simple scaffolds with only two substituents, and compound 21 is an elongated molecule typical of sulfonamylated sulfonamides. The inhibition profiles of TvaCA2 and hCAs are remarkably different, indicating major differences in the active site cavity of these enzymes. This is, in fact, desirable when designing novel drugs against β-CAs that do not have any major off-target effects toward α-CAs. Of the human CAs, only hCA I exhibited a similar inhibition constant toward AAZ as TvaCA2 .As seen in Table SEC-SLS was used to investigate the quaternary structure of the purified TvaCA2. A total of three different measurements were performed with TvaCA2 samples. All measurements were done in a thermostable chamber at 12 °C. Measured retention volumes and molecular weights of TvaCA2 and standard protein samples  are presented in Table Mw estimation of 46 kDa. Second, the SLS intensity was used to obtain an Mw estimation of 44.3 kDa . For comparison, MS/MS revealed a Mw of 20.45 kDa for the monomeric TvaCA2, making the dimeric form 40.9 kDa. Additionally, based on the primary sequence, Expasy [Mw values determined with analytical gel filtration and SEC-SLS differed to some extent from those obtained from MS/MS and Expasy, it can be concluded that TvaCA2 is dimeric in solution, similar to the first isoform [Figure , Expasy  gave an  isoform .Fig. 4LiHomology modeling was used to predict the structure of TvaCA2, using the TvaCA1 crystal structure (PDB 6Y04) as a template . Subsequ2O is present in each active site, interacting with Zn2+ and Asp39 . For instance, experiments involving rulence. , 40–44 TE. coli and purified with affinity chromatography. On SDS-PAGE gel, TvaCA2 was present as a single polypeptide with an Mw of 20.3 kDa (determined by MS/MS). LS analysis revealed that the native form of TvaCA2 is dimeric in solution, which was predicted since the first characterized β-CA of T. vaginalis (TvaCA1) was previously determined to be a dimer by X-ray crystallography [In this study, TvaCA2 was successfully expressed in lography . There wT. vaginalis enzymes. In particular, among compounds 1–24, very few similarities were found between the inhibition profiles of TvaCA1 and TvaCA2. The differing results may be due to different charge distributions and differences in the protein surface architecture near the active sites of TvaCA1 and TvaCA2. Overall, out of the 39 tested sulfonamides, 12 compounds remained ineffective against TvaCA2, and 25 remained ineffective against TvaCA1. Most of these inhibitors possess a bulky scaffold, with various substituents on which the sulfonamide or sulfamate moieties are attached. These features most likely interfere with their efficient binding within the long, channel-like active site of TvaCA1 or TvaCA2, explaining their poor inhibition capacity.Inhibition of TvaCA2 was studied with a set of simple/heterocyclic primary sulfonamides 1–24 and several clinically approved drugs, which showed CA inhibition properties. The most successful inhibition was established with AAZ, EZA, 4-amino-6-chloro-1,3-benzenedisulfonamide (compound 12), and MZA with inhibition constants of 222.9 nM, 362.1 nM, 382.2 nM, and 389.8 nM, respectively. The 1,3-benzenedisulfonamide structure present in 1,3-disulfamoyl benzene (compound 3), 4-amino-6-trifluoromethyl-benzene-1,3-disulfonamide (compound 11), and 4-amino-6-chloro-benzene-1,3-disulfonamide (compound 12) appeared to be a promising scaffold for drug design against TvaCA2, whereas these compounds were inefficient against TvaCA1. These inhibitors incorporate compact scaffolds, which should be able to enter the long and narrow active site of both 2O interacts with Zn2+ and Asp39. Inspection of the vicinity of the active site revealed features of interest, which may explain the differences in inhibition profiles. For example, TvCA2 showed different charge distributions and there were differences in the protein surface architecture near the active site, potentially reflecting inhibitor specificity and affinity. More detailed studies are needed to develop high-affinity inhibitors for TvaCAs, and the current study provides a good starting point for such work.To learn about the mechanisms behind the different inhibition profiles, we built a 3D model of TvCA2. In silico homology modeling suggested that TvaCA2 and TvaCA1 have very similar protein structures. Both enzymes have Cys37, His96, and Cys99 within the active cavity, where HSupplementary file1 (DOCX 1926 KB)Below is the link to the electronic supplementary material.


Although MrIC has no activating effect on α7 nAChR when applied by itself, it evokes a large response when co-applied with the type II positive allosteric modulator PNU-120596, which potentiates the α7 nAChR response by recovering it from a desensitized state. A lack of standalone activity, despite activation upon co-application with a positive allosteric modulator, was previously observed for molecules that bind to an extracellular domain allosteric activation (AA) site at the vestibule of the receptor. We hypothesized that MrIC may activate α7 nAChR allosterically through this site. We ran voltage-clamp electrophysiology experiments and in silico peptide docking calculations in order to gather evidence in support of α7 nAChR activation by MrIC through the AA site. The experiments with the wild-type α7 nAChR supported an allosteric mode of action, which was confirmed by the significantly increased MrIC + PNU-120596 responses of three α7 nAChR AA site mutants that were designed in silico to improve MrIC binding. Overall, our results shed light on the allosteric activation of α7 nAChR by MrIC and suggest the involvement of the AA site. The α7 nicotinic acetylcholine receptor (nAChR) is a homo-pentameric ligand-gated ion channel belonging to the Cys-loop receptor family . α7 nAChConus species consisting of 13–19 amino acids with multiple disulfide bonds that constrain their conformations [s state, resulting in large responses to orthosteric and allosteric agonists [A group of natural peptides targeting nAChR are α-conotoxins. α-conotoxins are peptides found in rmations . Severalrmations . α-conotrmations ,17,18. Tagonists ,21. Of tagonists . HoweverEndogenous ligands of the α7 receptor bind to a site under the C-loop called “the orthosteric site”, which then triggers a cascade of structural motions that result in the opening of the ion channel in the transmembrane domain (TMD) . HoweverMrIA, MrIB, and MrIC were docked into the AA site through Rosetta peptide docking. We first docked MrIA, MrIB, and MrIC into the ECD AA site of α7 nAChR and tested whether the three molecules could bind to this site. The ECD AA site ligands investigated so far in the literature interact with this site by burying their hydrophobic parts deep into the cavity defined by the vestibular loop residues 87–93 and 98–106 ,24,25,26Due to the fact that the vestibular loops and the peptide backbones can show structural flexibility upon binding, we followed a protocol that takes such changes in flexibility into consideration. The ToxDock protocol was developed to model α-conotoxin–nAChR interactions . A variaMrIA and α7R99  to the cleft between the subunits A and B. The other interaction with the AA site was formed between the subunits B and C through the anchoring of P1MrIC. The second strongest interaction was a hydrogen bond between α7Y15 and E15MrIC. α7D101 formed a hydrogen bond with H6MrIC at subunit A and a backbone–backbone hydrogen bond with E2MrIC at the subunit B. These observations suggest that the role of P1MrIC may be the placement of E2MrlC close to α7R99 in a stable manner rather than forming interactions within the AA site by itself (The α7 nAChR residues that had an interaction energy with the peptide that was greater than −1 Rosetta energy units (REU) were R99, Y15, D101, F104, A102, G83, P81, H105, and T106. Of these residues, R99 had the lowest interaction score, owing to a strong electrostatic interaction between this residue and E2y itself . MrIC in close contact with α7R99. Contrarily, the E1MrIA residue could not anchor to the AA site, and R1MrIB clashed with α7R99 at the mouth of the vestibular site. A lack of MrIA and MrIB binding at the AA site is consistent with the non-allosteric modes of action observed for the two peptides.Overall, our results demonstrated that the N-terminal proline residue of MrIC is a suitable anchor point for the AA site, placing E2MrIC, H10MrIC, and V11MrIC L119. H10MrIC formed hydrophobic interactions with α7(-)L119 and a hydrogen bond with α7(-)Q57. V11MrIC formed hydrophobic interactions with α7W149, α7(-)L109, and α7(-)L119. Based on these results, MrIC may bind to the orthosteric site of α7 nAChR in addition to the allosteric site of the receptor. Therefore, we first tested for MrIC competition with ACh at the concentration used for the allosteric activation experiments.In order to determine whether MrIC can bind to the orthosteric site of α7 nAChR in addition to the vestibular allosteric site, we docked MrIC into the orthosteric site of the receptor. The results showed consistent binding poses at this site, with an average binding score of −29.2 REU. However, different from most α-conotoxins that interact with α7 nAChR through their positively charged arginine/lysine and negatively charged aspartate/glutamate residues , the mai V11MrIC . P7MrIC We started our experimental investigation by testing the binding of MrIC to the α7 nAChR orthosteric site in experiments with the wild-type (WT) α7 nAChR. First, we co-applied 60 μM acetylcholine (ACh) and 50 μM MrIC to test whether MrIC could compete with ACh at the orthosteric site. An ACh concentration of 60 µM was chosen as the standard control for α7 because this yields a near maximum (93%) net charge response while still following the kinetics of the solution exchange . Additiot-value = 3.43, p-value = 0.00566)  applied to WT α7 nAChR evoked no response, consistent with the previous observations in SH-S5Y5 cells ,19. On t0.00566) . The lacBased on the results with the WT receptor, we tested MrIC activity at the α7C190 mutant. α7 nAChR mutants such as C190A are called “non orthosterically activatible receptors” (NOARs) because they cannot be activated by traditional agonists of α7 nAChR, including ACh. Although NOARs cannot be activated orthosterically, they can be activated by standalone applications of agonist-PAMs (ago-PAMs) or allosteric agonists plus type II PAMs ,25,31. Dt-value = 4.57, p-value = 0.000637)  . However, 1 μM GAT107 gave a large response, which was inhibited by the application of 100 μM (-)TMP-TQS  or decrease (loss-of-activity) the allosteric responses of α7 nAChR. AA site mutations such as D101A are known to diminish allosteric activation . HoweverMrIC and L16MrIC and blocks the complete entry of the peptide into the AA site formed between the α7 nAChR subunits A and B. This mutation is also of interest since it responds to standalone applications of PNU-120596 in a (-)TMP-TQS-sensitive manner, which is suggestive of a role in receptor desensitization [We focused on identifying mutants that could improve the binding of MrIC. Computational studies performed to improve α-conotoxin binding to the α7 orthosteric site demonstrated the importance of hydrophobic interactions between α-conotoxins and the α7 binding site . Based otization . TherefoMrIC, as observed for the WT calculations. The backbone of P1MrIC formed a hydrogen bond with the -OH group of α7T103, different from the WT calculations. α7D101 showed backbone–backbone hydrogen bonding with E2MrIC. The H6MrIC side chain formed a hydrogen bond with the α7T103 backbone in some poses and interacted with α7D101 in others. α7K87 moved closer to the peptide and formed a hydrogen bond with the backbone of A8MrIC. α7V83 formed weak hydrophobic interactions with P14MrIC and interacted with the side chain of E15MrIC. E15MrIC also formed a hydrogen bond with α7Q84.The α7G83V mutation resulted in changes in identity and strength of the peptide interactions with the protein. As a result of the increased bulk of the binding site at a region close to the C-terminus of the peptide, the peptide C-terminus extended away from the AA site, pointing toward the vestibular space A. The avMrIC and T5MrIC, which replaced the N-terminal residues of the peptide, although P1MrIC remained anchored at the vestibular site , but resulted in the formation of four additional interactions between the peptide and α7V102 . Of these residues, H6MrIC also formed hydrogen bonds with α7E98 and α7R99. In the absence of α7A102, V11MrIC formed new interactions with the side chains of α7D82 and α7T106. The average interface score of the top ten poses was 2.1 REU lower than the WT α7 nAChR score   and 42 times greater than the WT α7 nAChR response to the MrIC + PNU-120596 application.The α7T106A mutation resulted in an increased background net charge response to 60 μM PNU-120596 (~6-fold), contrary to the results with the WT α7 nAChR, α7G83V, and α7A102V receptors TMP-TQS-sensitivity, and the ability to activate the NOAR α7C190A, strongly suggest that the source of MrIC allosteric activity is the vestibular AA site. The unique allosteric behavior of MrIC, among other Mr1.7 variants, may be connected to its N-terminal proline, which can anchor to the AA site and induce electrostatic interactions between E2ref2015_cart score function [The α-conotoxin ImI-bound (PDB ID: 2C9T) , and lobfunction ,37 and ffunction ). talaris2013 score function [All three structures were modeled based on the structure of α-conotoxin OmIA (PDB ID: 2GCZ) . The mutfunction . The reltalaris2013 score function. A modified version of the ToxDock protocol  was usedAcetylcholine chloride (ACh) and buffer chemicals were purchased from Sigma-Aldrich Chemical Company . PNU-120596 was synthesized in Dr. Nicole A. Horenstein’s laboratory  by Dr. Kinga Chojnacka following the published procedure by Hurst et al. 2005 . GAT107 2O (2.5%): TIPSi (2.5%): DODt (2.5%) solution for 40 min at 40 °C. Linear peptide was precipitated and washed with cold ether, dissolved in 45% acetonitrile/0.05% TFA/H2O, filtered, and lyophilized on a freeze dryer. The crude linear peptide was purified by preparative RP-HPLC (Vydac C18). For the one-pot regioselective formation of the 1–3 and 2–4  disulfide bonds, the method has been described before [2 treatment in 90% AcOH/MeOH to form the Cys 2–4 or Cys 2–3 disulfide bond. An aliquot was analyzed by HPLC and HRMS to confirm the complete formation of the first pair disulfide bond and the integrity of the Cys 1,3 or Cys 1,4 Acm groups. In order to induce the removal of the Acm groups and form the Cys 1–3 or Cys 1–4 disulfide bond, 1% TFA/H2O was added, followed by additional I2, and the reaction was allowed to proceed for a further 90 min, giving globular Cys1–3, 2–4 MrIC. Then, the oxidized peptide was purified by HPLC.MrIC was synthesized by standard Fmoc chemistry with Cys side-chains protected as one pair ACM  and the other pair Trt , starting from polystyrene AM RAM resin on a 0.62 mmol scale. The deprotection of the side chain group and cleavage of the linear peptide from the resin were conducted with a TFA (92.5%): HThe human α7 nAChR clone was obtained from Dr. J. Lindstrom . The human resistance-to-cholinesterase 3 (RIC-3) clone was obtained from Dr. M. Treinin  and co-injected with α7 in order to improve the level and speed of α7 receptor expression without affecting the pharmacological properties of the receptors . SubsequXenopus laevis frogs . Frogs were maintained in the Animal Care Service facility of the University of Florida and all procedures were approved by the University of Florida Institutional Animal Care and Use Committee. In brief, the frog was first anesthetized for 15–20 min in 1.5 L frog tank water containing 1 g of 3-aminobenzoate methanesulfonate (MS-222) buffered with sodium bicarbonate. The harvested oocytes were treated with 1.4 mg/mL Type 1 collagenase  for 2–4 h at room temperature in calcium-free Barth’s solution  to remove the ovarian tissue and the follicular layers. Stage V oocytes were subsequently isolated and injected with 50 nL of 5–20 ng nAChR subunit cRNA. Oocytes were maintained in Barth’s solution with calcium 2 and 0.41 mM CaCl2), and recordings were carried out 2–14 days after injection. Oocytes were surgically removed from mature female 2, 10 mM HEPES, and 1 μM atropine, pH 7.2) at 2 mL/min. In order to evaluate the effects of experimental compounds compared to ACh-evoked responses of α7 nAChR subtypes expressed in oocytes, control responses were defined as the average of two initial applications of ACh made before test applications. Solutions were applied from 96-well plates via disposable tips. Drug applications were 12 s in duration, followed by 181 s washout periods. A typical recording for each set of oocytes constituted two initial control applications of ACh, one or more experimental compound applications, and then a follow-up control application(s) of ACh. The control ACh concentration was 60 μM for the wild-type (WT) and the three AA site mutant receptor experiments, and the average of independent 1 μM GAT107 responses were used as the control for the NOAR experiments. The responses were calculated as both peak current amplitudes and net charge, as previously described [Experiments were conducted using OpusXpress 6000A  . Both thescribed , and thep-values [p-value > 0.05, the measurements were considered to fall within the normal distribution, and comparisons of results were made using t-tests between the pairs of experimental measurements. For the cases where both the test and the control had Shapiro p-values < 0.05, Mann–Whitney U-test (MTW) [p < 0.05 was used to constitute the level of significance for both the t-tests and the u-tests at N-1 degrees of freedom. The statistics were calculated using an Excel template provided in Microsoft Office or with the scipy.stats modules ttest_ind and mannwhitneyu. For some experiments, statistical comparisons were not calculated but the data was plotted as the point-by-point averages (±SEM), permitting qualitative evaluation of the data by visual inspection.In order to determine whether the sets of measurements fall within normal distribution, the Shapiro–Wilk p-values  were calst (MTW)  was usedType II PAMs produce extremely large increases  in the single-channel currents of a small fraction of the receptors (≤1%), so they are intrinsically variable in amplitude and duration , making


A translationally silent single nucleotide mutation in exon 44 (E44) of the von Willebrand factor (VWF) gene is associated with inefficient removal of intron 44 in a von Willebrand disease (VWD) patient. This intron retention (IR) event was previously attributed to reordered E44 secondary structure that sequesters the normal splice donor site. We propose an alternative mechanism: the mutation introduces a cryptic splice donor site that interferes with the function of the annotated site to favor IR. We evaluated both models using minigene splicing reporters engineered to vary in secondary structure and/or cryptic splice site content. Analysis of splicing efficiency in transfected K562 cells suggested that the mutation-generated cryptic splice site in E44 was sufficient to induce substantial IR. Mutations predicted to vary secondary structure at the annotated site also had modest effects on IR and shifted the balance of residual splicing between the cryptic site and annotated site, supporting competition among the sites. Further studies demonstrated that introduction of cryptic splice donor motifs at other positions in E44 did not promote IR, indicating that interference with the annotated site is context dependent. We conclude that mutant deep exon splice sites can interfere with proper splicing by inducing IR. Aberrant splicing is a common cause of human genetic disease with various studies estimating that 15–60% of disease-causing mutations disrupt pre-mRNA splicing  is a major form of alternative splicing that regulates expression of many genes during normal differentiation ,16,17, cThe current study examined an alternative model for intron retention in this VWF patient, based on the observation that the mutation generates a strong 5′ splice site motif deep within E44. The presence of cryptic splice site motifs near annotated exon/intron boundaries has been shown in several contexts to inhibit use of neighboring annotated sites, leading to intron retention ,33,34. HVWF E44 normally possesses a fairly weak 5′ splice site, AGT/gtaggt, which has a score of 3.31 according to a commonly used algorithm for estimating splice site strength . InspectTo investigate the mechanism(s) responsible for IR in the VWF gene, we generated minigene splicing reporters with which splicing phenotype could be assessed following transfection into K562 erythroleukemia cells. SF3B1-mut35 is a splicing reporter used to study intron 4 retention in the SF3B1 gene . This coTwo series of VWF splicing reporters were engineered into the mut35 base construct. VWFwt-short was generated by inserting into mut35, at the site previously occupied by decoy exon 3e , a 314 nGel analysis of spliced products amplified from transfected K562 cells is shown in Having reproduced the VWF patient’s IR phenotype, we then generated a series of splicing reporters to explore the mechanism(s) by which mutation m1 promotes IR. These reporters were designed to alter either of the features proposed to induce IR: (a) secondary structure at the annotated E44-5′ss that might impact its ability to interact with the splicing machinery  and/or , and strong stabilization of stem 3. When tested in K562 cells, m1m5 exhibited strong IR , right, Finally, we asked whether the ability of a strong internal 5′ss motif to promote IR is position dependent. We generated splicing reporters containing strong 5′ splice site motifs, identical to that of m1 (AAG/gtgagt), at two different locations within E44. One was 48 nt upstream of the annotated E44-5′ss (m48) and the other was 70 nt upstream (m70). The splicing phenotypes of these pre-mRNAs are shown in Von Willebrand disease is one of the most common inherited bleeding disorders, caused by any one of hundreds of mutations in the VWF gene ,39. The Two major hypotheses have been proposed to explain how the m1 mutation interferes with function of the annotated 5′ splice site. First, the splice site sequestration model predicts that mutation m1 would alter long range RNA folding across exon 44 so as to increase base-pairing at the annotated 5′ splice site. According to this model, RNA–RNA interactions promote IR by sequestering that splice site away from the splicing machinery . This moOne instructive finding was the difference in splicing behavior between mutants m1 and m5. Both were predicted to stabilize stem 3  to the sA potential limitation of these interpretations is that secondary structure has not been probed directly. However, using splice site usage as a proxy for accessibility, we observed that residual splicing switched from the cryptic site in m1 to the annotated site in m1m2, consistent with a more accessible conformation of the annotated site in the double mutant. Interestingly, the double mutant m1m5 shifts residual splicing in the opposite direction back to the annotated 5′ splice site. Although folding models suggest that m1 and m1m5 have equivalent potential to sequester the annotated site, the strong cryptic site in m1m5 may be disfavored for residual splicing, either because it is slightly weaker than m1 or because m1m5 itself becomes partially sequestered in stem 3 . A caveaAnother limitation to this study is that we have not directly measured spliceosomal binding to the cryptic site. For that reason, the possibility that a non-spliceosomal complex may bind at the cryptic site region to induce IR cannot be formally excluded. However, several observations support involvement of a spliceosomal component. First, cryptic site m1 does operate as a functional splice site as shown by sequence analysis of residual spliced products. Second, variant substrates bearing a strong cryptic splice site were associated with strong IR , while those having a weak cryptic site were correlated with low IR . Finally, there is precedence for the idea that cryptic splice sites can interfere with annotated splice sites to promote IR .In the Drosophila P element gene, binding of spliceosomal components to an exonic decoy splice site modulates retention of the downstream intron in a tissue-specific manner . The splGiven these precedents, we propose that the splice site introduced by the VWF patient’s mutation interacts in a nonproductive manner with the annotated 5′ splice site located 86 nt downstream. If the downstream intron 44 is recognized by an intron definition mechanism , then blMore globally, relatively little is known about the incidence of IR due to the deep exon mutations and what might be their contribution to human disease. Methodological and conceptual considerations may have limited the identification of such mutations in previous exome and RNA-seq studies. Detecting association between deep exon SNVs and intron retention events requires reads of sufficient length to overlap both features, sequencing depth sufficient to identify allele-specific association of such features, and analytical strategies focused on finding these associations. Earlier work has, in fact, revealed that many exonic SNVs are associated with IR , but thiConstruction of splicing reporters: Reporters diagrammed in The lower case regions provided overlap sequences to facilitate fusion  of this The second wild-type reporter (long) encompassed a 2.65 kb region of the VWF gene spanning across a terminal portion of intron 43, exon 44, intron 44, exon 45, and a proximal region of intron 45.The fragment amplified with these primers was cloned into VWFwt short using In-Fusion methods  with 15 Mutations in VWF E44 were introduced by amplifying the wild-type VWF reporter with primers carrying the desired mutations. The various 5′ splice site motifs (boxed) and their immediate flanking sequences are shown below. Construct m1 displays the patient’s sequence. Construct m5 was mutated to stabilize stem 3 equivalently to m1 but without generating a strong 5′ splice site motif see . m1m5 coConstruct m2 contained 6 nucleotide changes near the annotated 5′ splice site, designed to disrupt secondary structure in this region. The linear sequence of the annotated 5′ splice site region for the wild-type (“closed conformation”) and m2 (‘open conformation’) constructs is given below. Predicted secondary structures are shown in Analysis of splicing: Splicing reporters were transfected into K562 using FuGENE according to the manufacturer’s instructions . RNA was isolated after 48 h using RNeasy columns as per instructions from the manufacturer  with the inclusion of a DNase step to minimize contamination by genomic DNA. RNA was reverse transcribed with Superscript III (Invitrogen) into cDNA using the BGH reverse primer in the vector (5′-tagaaggcacagtcgagg-3′). Spliced products were amplified using a forward primer in exon 3 (5′-catcatctacgagtttgcttgg-3′) and a reverse primer in the vector (5′-atttaggtgacactatagaatagggc-3′). This strategy amplified minigene-derived transcripts but not endogenous mRNA as confirmed using RNA from untransfected or empty vector-transfected cells. When assaying IR products, PCR reaction conditions were adjusted to allow for amplification of DNA bands ≥ 3 kb in length  using KOD polymerase in the presence of betaine. PCR products were analyzed on either 2% agarose gels or 4.5% acrylamide gels. Identity of the PCR products was confirmed by DNA sequencing, and all splicing reporters were assayed at least three times. The splicing phenotype of test constructs relative to control constructs was highly reproducible, despite inevitable variations in baseline intron retention.


Dear editors,Incarcerated screws pose a challenging task to many junior orthopaedic surgeons who hone their skills through experience. One must first have sufficient knowledge regarding orthopaedic implants and the methods of their removal, and should have at their disposal a variety of techniques should one come across difficulty intra-operatively. We recently encountered a case of removal of femoral nail which was complicated with incarcerated screws. The screws were eventually removed using a technique of creating two straight and parallel planes on the screw head with a high speed diamond-tipped burr and stable grip of a locking plier.The patient was a 26-year-old gentleman who was referred to our trauma unit for removal of implant in view of left knee instability with suspicion of left posterior cruciate ligament injury requiring confirmation of diagnosis by magnetic resonance imaging (MRI). He had a history of left femur fracture in which a titanium retrograde nail was inserted four years ago which had achieved union. During the surgery, the most proximal locking screw and distal two locking screws were found to be well integrated with bony overgrowth over the screw heads. Upon clearing the bone within and around the screw heads, attempt at removal of the screws using the conventional way failed and repeated attempts led to stripped screws .1. We also attempted to grasp the screw heads with locking plier, but failed due to the round shape of the screw heads. To improve the grip of the locking plier, the surgeon proceeded to form two straight and parallel planes on the screw head by using a high speed diamond-tipped burr (Further attempts using the conical extraction screw from the universal screw removal set were futile as it did not engage within the screw heads. We believe this was because the screw was tightly jammed due to osteointegrationped burr . The flaped burr .2. The procedure should never be underestimated and thorough pre-operative planning with regards to types of implants and removal instruments should be prepared. Surgeons should equip themselves with broad knowledge and should have at their disposal various methods of removal of implant should they come across any difficulties intra-operatively. The above technique would be a handy method to be added into a trauma surgeon’s armamentarium in removing incarcerated screws.Incarcerated screws pose a challenging and delicate situation in removal of an implant


Pumilio homology domain with specific short motifs in the 3’-UTR of an mRNA and thereby influence the stability, localization and/or efficiency of translation of the bound transcript. In our prior unbiased proteome-wide screen for targets of the TORC2-stimulated protein kinase Ypk1, we identified the paralogs Puf1/Jsn1 and Puf2 as high-confidence substrates. Earlier work by others had demonstrated that Puf1 and Puf2 exhibit a marked preference for interaction with mRNAs encoding plasma membrane-associated proteins, consistent with our previous studies documenting that a primary physiological role of TORC2-Ypk1 signaling is maintenance of plasma membrane homeostasis. Here, we show, first, that both Puf1 and Puf2 are authentic Ypk1 substrates both in vitro and in vivo. Fluorescently tagged Puf1 localizes constitutively in cortical puncta closely apposed to the plasma membrane, whereas Puf2 does so in the absence of its Ypk1 phosphorylation, but is dispersed in the cytosol when phosphorylated. We further demonstrate that Ypk1-mediated phosphorylation of Puf1 and Puf2 upregulates production of the protein products of the transcripts to which they bind, with a concomitant increase in the level of the cognate mRNAs. Thus, Ypk1 phosphorylation relieves Puf1- and Puf2-mediated post-transcriptional repression mainly by counteracting their negative effect on transcript stability. Using a heterologous protein-RNA tethering and fluorescent protein reporter assay, the consequence of Ypk1 phosphorylation in vivo was recapitulated for full-length Puf1 and even for N-terminal fragments (residues 1-340 and 143-295) corresponding to the region upstream of its dimerization domain (an RNA-recognition motif fold) encompassing its two Ypk1 phosphorylation sites . This latter result suggests that alleviation of Puf1-imposed transcript destabilization does not obligatorily require dissociation of Ypk1-phosphorylated Puf1 from a transcript. Our findings add new insight about how the TORC2-Ypk1 signaling axis regulates the content of plasma membrane-associated proteins to promote maintenance of the integrity of the cell envelope.Members of the Puf family of RNA-binding proteins typically associate via their  Signaling pathways can enable rapid responses to changing external conditions via phosphorylation of proteins that directly influences their activity, localization and/or stability. Accompanying these more immediate regulatory events, signaling can concomitantly elicit longer term adaptation through effects on gene expression because transcription factors are often among the targets of phosphorylation. Further fine-tuning of stimulus-evoked changes in protein expression also can be achieved via phosphorylation of RNA-binding proteins that regulate the packaging, transport, sequestration, localization, translation efficiency, and rate of decay of mRNAs ,2,3,4,5.Saccharomyces cerevisiae), there are approaching 700 gene products annotated as RNA-associated proteins ,2, 0.1 mM EDTA, 50 mM Tris-Cl pH 7.5) with 100 µM ATP containing 2 μCi [γ-32P]ATP at 30 °C in the presence or absence of 10 μM 3-MB-PP1 in a total volume of 20 μL for 30 min. Reactions were terminated by the addition of SDS/PAGE sample buffer containing 6% SDS followed by boiling for 5 min. Labeled proteins were resolved by SDS-PAGE and analyzed by Coomassie blue staining and autoradiography using Phosphorimager™ plates  and a Typhoon™ imaging system .Ypk1-as, purified as described in the preceding section, was incubated with the purified GST-fusion protein of interest in 1X kinase assay buffer  with 18 μL of 1 M Tris base and 40 μL of 10% SDS and 2% β-mercaptoethanol. The solubilized protein was then diluted with 900 μL of 50 mM Tris-Cl (pH 8.5), to which was added 90 units of calf intestinal phosphatase , and the resulting mixture incubated for 2 h at 30 °C in the absence or presence of phosphatase inhibitor (4 mM Na3VO4). Proteins were reprecipitated again, and then resuspended and resolved by SDS-PAGE, as above.Yeast cells were grown to mid-exponential phase, harvested by centrifugation and then stored at −80 °C before use. Cell pellets were thawed on ice and incubated in 2 vol of lysis buffer . To precipitate the proteins released from the lysed cells, a one-tenth volume of ice-cold 50% TCA was added. After incubation on ice for 20 min, the precipitated protein was collected by centrifugation at maximum rpm in a microfuge. After removal of the supernatant solution, the protein pellet was washed twice with chilled (−20 °C) acetone. To resuspend the precipitated protein and ensure neutralization of any residual TCA, the pellets were resuspended in 5% SDS in 0.1 M Tris base and then samples were diluted into SDS-PAGE sample buffer and analyzed by SDS-PAGE. For phosphatase treatment, TCA-precipitated proteins were solubilized in a solution constituted by mixing 42 μL of solubilization buffer  for 1 h at room temperature, prior to immunoblotting. Membranes were then incubated with appropriate antibodies diluted in OdysseyTM buffer . Antibodies for detection of specific proteins used in this study were: rabbit polyclonal anti-tRFP (Evrogen AB233) (1:1000); mouse monoclonal anti-HA (6E2)  (1:1000); mouse monoclonal anti-FLAG (Sigma-Aldrich F1804) (1:5000); rabbit monoclonal anti-GFP (D5.1)  (1:500); rabbit polyclonal anti-Jsn1/Puf1  a second mRNA encoding a red fluorescent protein (mCherry) with multiple copies of a different (irrelevant) hairpin (PP7) in its 3′-UTR. Cells expressing the chimeric proteins are interrogated using flow cytometry/fluorescence-activated cell sorting (FACS) to monitor the relative levels of YFP and mCherry expression in individual cells, and compared to the YFP and mCherry expression levels in otherwise identical cells expressing the phage protein fused to an irrelevant protein . The control phage protein fusion and the yeast protein-phage protein chimeras are tagged with the blue fluorescent protein (BFP), which allows for gating of the FACS so that only BFP-positive cells are examined.The high-throughput yeast cell-based assay for genome-wide characterization of eviously ,48 and wS. cerevisiae phosphoproteome ATP and purified Ypk1-as in the absence and presence of the inhibitor 3-MB-PP1  displayed the same phenotype as the Puf1AA-6HA mutant , indicating that, in the case of this particular protein and these particular sites, Ser/Thr-to-Glu mutations are not an adequate mimic for authentic phosphate groups, as has been observed for other proteins whose function is controlled by phosphorylation both by us [A Puf1th by us  and otheth by us . WT and Puf2WT-6HA or Puf1AA and Puf2AA-6HA. Puf1 mRNA itself is reportedly a target for Puf1 and Puf2 [LEU2 marker just after the stop codon of the PUF1 and PUF2 ORFs to sever the native 3′-UTRs from the resulting PUF1 and PUF2 coding sequences, and confirmed by immunoblotting that both the WT and AA versions of both proteins were expressed at an equivalent level in the two strains (top and bottom panels). To further explore at the biochemical level the above genetic findings suggesting that phosphorylation at their Ypk1 sites negatively regulates Puf1 and Puf2 function, we examined the steady-state level of protein products of transcripts characterized as direct targets of Puf1 and Puf2. Toward this end, we constructed a pair of strains that express from their native promoters at their normal chromosomal loci either Puf1and Puf2 ,17. HencPMP3 mRNA was bound by Puf1 and Puf2 [left panel). We corroborated this conclusion using a genetic approach. It has been shown that cells lacking Pmp3 are hypersensitive to the growth inhibitory action of the ergosterol-binding polyene antibiotic amphotericin B (AmB) [We used this pair of strains to look, first, at Pmp3, a highly conserved, very small protein (55 residues) with two, strongly predicted, hydrophobic transmembrane segments  because and Puf2 ,63,64. Tand Puf2 ,66. We f B (AmB) ,68. We rright panel).We next examined Pdr5, another integral PM protein whose transcript is a Puf1 and Puf2 target ,17. Pdr5These results indicate that, when Puf1 and Puf2 cannot be phosphorylated by Ypk1, production of the protein products of the transcripts to which they bind is reduced. This post-transcriptional repression could be exerted by impeding the efficiency of translation of each existing mRNA to which Puf1 and/or Puf2 is bound with no change in its level, or could be due to a reduction in the level of the cognate mRNA because binding of Puf1 and/or Puf2 is destabilizing. Indeed, there is evidence that association of Puf family proteins, including yeast Puf1 ,70 and PPMP3 and PDR5 transcripts. We found that, indeed, relative to the cells expressing WT Puf1 and Puf2, the cells expressing Puf1 and Puf2 lacking their Ypk1 sites reproducibly contained a lower level of both transcripts  cells, the level of the ZEO1 mRNA was reproducibly lower in the cells expressing Puf1 and Puf2 lacking their Ypk1 sites -based screen for proteins and protein fragments able to exert post-transcriptional repression in yeast was devised and implemented ,48. As dleft panel) and equivalent amounts of RFP were produced in the BFP-positive cells from all three cultures (middle panel), but, compared to the control cells, cells expressing the Puf1WT(1-340)-phage protein fusion produced slightly less YFP, whereas the decrease in YFP was more pronounced in the cells expressing the Puf1AA(1-340)-phage protein fusion (right panel). We next tested fusions of full-length Puf1, without and with its Ypk1 sites mutated to Ala. Once again, the same trends were observed, but were even more obvious. Equivalent number of BFP-positive cells were present in all three cultures (left panel) and equivalent amounts of RFP were produced in the BFP-positive cells from all three cultures (middle panel), but, compared to the control cells, cells expressing the Puf1WT-phage protein fusion clearly produced less YFP, and the decrease in YFP was even more pronounced in the cells expressing the Puf1AA-phage protein fusion (right panel). Presumably, as we observed for the PMP3, PDR5 and ZEO1 transcripts, the lower level of YFP produced in the Puf1AA cells is due to a lower level of the corresponding transcript because, without Ypk1 phosphorylation, Puf1 is better able to promote its decay, although we did not extract RNA from these cultures and test that supposition directly.Using the method just described, a fragment of Puf1 (residues 143-295) A was recWT is phosphorylated at its Ypk1 sites). Because cells expressing Puf1WT produced more YFP than cells expressing Puf1AA in this assay context, we suggest that Ypk1 phosphorylation need not obligatorily cause dissociation of Puf1 to reduce its ability to promote decay of the mRNA to which it is bound. More likely, Puf1 recruits other factors that execute the steps needed for mRNA turnover and Ypk1-mediated phosphorylation may interfere with the ability of Puf1 to recruit those factors.Thus, as with the endogenous Pmp3 and Pdr5 proteins, YFP  was expressed at a distinctly lowered level when Puf1 could not be phosphorylated by Ypk1. In this tethering assay system, the Puf1 protein or its fragments are tightly bound via the stem-loop binding protein-RNA interaction at multiple hairpins, making it unlikely that, in this context, Puf1 is able to dissociate fully from the transcript, even when phosphorylated . The same has been observed for Puf1-GFP [AA-mKate2 displayed an indistinguishable pattern . Moreover, Puf1WT-mKate2 and Puf1AA-mKate2 were expressed at an equivalent level (lower panel). Thus, the absence or presence of Ypk1 phosphorylation does not influence the stability or localization of Puf1. To further explore how Ypk1-mediated phosphorylation affects Puf1 and Puf2, we tagged both WT and AA derivatives of each protein with a fluorescent marker protein and expressed them from their native promoters at their endogenous chromosomal loci to examine their level and distribution in live cells. In agreement with previous work using fixed cells and indirect immunofluorescence with anti-Puf1 antibodies , we founPuf1-GFP , providiWT-mKate2, we found that Puf2WT-GFP was largely confined to the cytoplasm (upper left panel), as has been reported before [AA-GFP localized to peripheral puncta closely associated with the PM (upper right panel), resembling those observed for Puf1-mKate2. As judged by immunoblotting, the steady-state level of Puf2AA-GFP was similar to that for Puf2WT-GFP (lower panel). Thus, Ypk1 phosphorylation appears to cause dramatic dispersal of Puf2 from cortical sites without markedly affecting its overall level.In marked contrast to Puf1d before . StrikinWT-GFP and those expressing Puf2AA-GFP with AbA. Under this condition, in both cultures, the bulk of both Puf2WT-GFP and Puf2AA-GFP accumulated in both small and large intracellular bodies  that resemble P-bodies, stress granules, and other classes of protein-RNA condensates formed by liquid-like phase separation that have been observed in yeast [WT-GFP and Puf2AA-GFP, this shift cannot be attributed to direct phosphorylation of Puf2 by Ypk1 per se. However, this behavior could reflect loss of Fpk1-mediated phosphorylation in both Puf2WT-GFP and Puf2AA-GFP, given that stimulation of TORC2-Ypk1 signaling inhibits Fpk1 function.To acutely elevate TORC2-Ypk1 signaling, we treated cells expressing Puf2in yeast ,75. BecaIn any event, these structures do not appear to be canonical P-bodies because when we co-expressed an mCherry-tagged derivative of a known P-body component Dcp2  and treaAs we have documented here, Puf1 and Puf2 are authentic and physiologically relevant cellular targets of the protein kinase Ypk1. We have shown further here that the effect of Ypk1-mediated phosphorylation is to negatively regulate Puf1 and Puf2 and to enhance production of the protein products encoded by the transcripts to which they bind. Finally, we have demonstrated that Ypk1 seems to achieve this up-regulation by interfering with the ability of Puf1 and Puf2 to promote turnover of the mRNAs to which they bind, consistent with other evidence that association of Puf1 and Puf2 with their client transcripts enhances their rate of decay ,70.S. pombe Puf1 and Puf2 orthologs from crystal structures [S. cerevisiae Puf1 and Puf2 [Pumilio homology domain binds a UAAU motif, whereas that in other Puf family members typically recognizes a UGUA motif. Second, their upstream RNA recognition motif (RRM)-like fold, despite that descriptor, does not bind RNA, but rather serves as a dimerization domain. Third, the preferred binding site for Puf1 in its target mRNAs is actually a tandem UAAU-x3-4-UAAU motif because the RRM-mediated dimerization of Puf1 allows the Pumilio homology domain in each subunit to bind to one of the two UAAU sites, thus greatly favoring binding to such dual UAAU motifs over a single UAAU. It is noteworthy, in this regard, that the two Ypk1 sites (and the one Ypk1-regulated Fpk1 site) conserved in both Puf1 and Puf2 all lie just upstream of their RRM. Hence, it is possible that one way that changes in Ypk1-mediated phosphorylation at these sites negatively regulate Puf1 and Puf2 is by weakening dimer formation, thereby reducing binding affinity for UAAU-x3-4-UAAU-containing transcripts. However, using the protein tethering and dual fluorescent reporter assay, we found that N-terminal fragments of Puf1 that contain the Ypk1 (and Fpk1) sites that it shares with Puf2, but do not contain its RRM (residues 340-441), were sufficient to exert down-regulation of protein expression and were more potent in doing so when their Ypk1 phosphorylation sites were absent, suggesting that interference with dimerization is not (or not the only) mechanism by which phosphorylation negatively regulates Puf1 function.Among the yeast Puf family members, Puf1 and Puf2 have certain unique features. First, as determined for the ructures  and fromand Puf2 ,64,80, tFor these reasons, we favor a model in which Ypk1-mediated phosphorylation interferes with the ability of Puf1 and Puf2 to associate with factors responsible for executing reactions necessary to initiate mRNA decay, consistent with substantial evidence that other Puf family members in yeast and other organisms recruit the CCR4–POP2–NOT deadenylase complex for poly(A) tail removal (reviewed in ). IndeedS. cerevisiae cells lacking Pmp3 are hypersensitive to the growth inhibitory action of AmB [PMP3 transcript, it increases the sensitivity of cells to AmB, as expected. Another means to prevent efficient Ypk1-mediated Puf1 and Puf2 phosphorylation and, thus, would be expected to lower Pmp3, is to eliminate Ypk1 itself. Indeed, such a reduction in Pmp3 may be a contributing factor to our previous observation that ypk1∆ mutants are significantly more sensitive to AmB than otherwise isogenic WT cells [11A, that is immune to phosphorylation by another protein kinase (Gin4) that, like Ypk1, negatively regulates Fpk1 function [enhance Puf1- and Puf2-mediated translational repression. Given this suggestion that the effects of Ypk1 and Fpk1 phosphorylation have opposing effects on the translation-repressing functions of Puf1 and Puf2, it makes physiological sense that stimulation of TORC2-Ypk1 signaling would promote Ypk1-mediated phosphorylation of Puf1 and Puf2 and, simultaneously (through Ypk1-imposed inhibition of Fpk1) reduce Fpk1-mediated phosphorylation of Puf1 and Puf2. In this way, activation of TORC2-Ypk1 signaling would more efficiently upregulate production of the protein products of the transcripts under Puf1 and Puf2 control. At least for Puf2, the lack of Ypk1 phosphorylation has a dramatic effect on its subcellular localization, whereas the same was not observed for Puf1. In subsequent studies, it will be interesting to explore the molecular basis for why Puf1 and Puf2 differ in this respect. A related question is the Ypk1 site-independent mechanism by which depletion of complex sphingolipids in AbA-treated cells elicits the formation of the intracellular Puf2 aggregates we observed. This behavior is reminiscent of the finding that, when hypophosphorylated, yeast Puf3 becomes trapped in intracellular RNA-containing foci under conditions of glucose starvation [Pmp3, one of the Puf1 and Puf2 targets we analyzed, is a small highly hydrophobic protein that shows remarkable cross-kingdom conservation, from bacteria to plants ,84,85. Sn of AmB ,68. As wWT cells . Likewisfunction , are alsarvation .puf1∆, puf2∆, and even puf1∆ puf2∆ mutants.Because Puf1 and Puf2 control such an extended network of PM components, it might be anticipated that alterations in the functions of these proteins would impact PM structure and function in unanticipated ways. For example, we showed here, as have others , that ceThe most important conclusion of the work presented here is that phosphorylation of Puf1 and Puf2 by the protein kinase Ypk1 is yet another mechanism by which the TORC2-Ypk1 signaling axis controls the composition and function of the PM. In this sense, the regulatory circuitry we have described here joins a growing list of examples which illustrate that phosphorylation is a widely used means for regulating the function of Puf family members ,89,90 an


Despite a high level of sequence identity between cow's, goat's, and sheep's milk  proteins, some patients tolerant to CM are allergic to GM and SM. In most cases, this specificity is due to the presence of IgE antibodies that bind only to caprine and ovine caseins. The patients may then develop severe allergic reactions after ingestion of CM products contaminated with low amounts of GM or SM. We thus aimed to develop an assay able to detect traces of caprine/ovine β-caseins in different food matrices, irrespective of the presence of the bovine homolog. We produced monoclonal antibodies (mAbs) specific to caprine caseins in mice tolerized to the bovine whole casein then sensitized to the caprine whole casein. In order to develop a two-site immunometric assay, we selected mAbs that could discriminate the caprine β-casein from its bovine homolog. Characteristics and performances of two tests were determined with various dairy products. Results were analyzed in relation with the IgE-immunoreactivity of the food matrices, thanks to sera from CM, GM/SM allergic patients. Our two-site immunometric assays demonstrated a high sensitivity with a detection limit of 1.6–3.2 ng/mL of caprine and ovine β-caseins. The tests were able to detect contaminations of GM in CM at the ppm level. Heat-treatment, ripening and coagulation processes, usually applied to dairy products that exhibit a very high IgE-immunoreactivity, did not impair the test sensitivity. These quantitative assays could then be useful for the risk assessment of food products potentially contaminated with GM and SM in order to prevent adverse reactions in patients specifically allergic to these milks. Milk is one of the foods causing the most frequently an adverse reaction , 2. CrosThe development of this specific allergy to GSM does not always rely on earlier sensitization to CM proteins as only 19% of the GSM-allergic/CM-tolerant patients had suffered from an outgrown CM allergy . MoreoveWhole Casein (CN) constitutes up to 80% of the GSM proteins and is present in various dairy products as well as ingredients in processed food matrices. Among the four components of CN, i.e., αS1, αS2, β, and κ-caseins, the β-casein (β-CN) constitutes ~36% of the whole bovine CN and up to 50–60% of the whole CN from GM and SM, depending on genetic polymorphisms of the small ruminants , 23. TheIn the present work, we aimed to develop an ELISA for the specific detection of the caprine β-CN, irrespective of the presence of CM proteins in the food matrix. The test was evaluated for its performances (sensitivity and specificity) with various processed dairy foods, in relation to their IgE reactivity. We finally assessed the assay sensitivity for the detection of GM contaminations in CM.Antibodies specific to caprine caseins were produced in mice tolerized to whole bovine CN through the oral route and then sensitized to whole caprine CN as previously described . In paraSpleen cells from mice producing the highest levels of IgG1 to CN were fused with NS1 mouse myeloma cells. The antibody-secreting cells (hybridomas) were screened by analyzing cell supernatants on microtiter plates coated with either bovine or caprine β-CN and using acetylcholinesterase (AChE)-labeled goat anti-mouse antibodies  as tracer .Selected hybridoma cells were cloned and expanded as ascitic fluids in BALB/c mice. Specificity of the mAb purified from ascitic fluids by protein A affinity chromatography was fully characterized using recombinant, plasmin-derived, and synthetic peptides of bovine and caprine β-CN as previously described for human IgE-binding studies , 25. Briblank + 3 × SDblank and meanblank + 10 × SDblank, respectively. Precision was characterized by determining intra- and inter-assay coefficients of variation CV. Intra-assay variation was estimated by measuring different concentrations of native caprine β-CN standards eight times on a single plate (range from 0.156 to 200 ng/mL). The working range was then defined as the concentration range for which an intra-assay CV <10% was obtained. The inter-assay CV was calculated with two concentrations of standard inside the working range routinely assayed on different plates. Assay specificity was assessed by measuring signals in the concentration range from 0.1 to 10 μg/mL of purified proteins from CM, GM, and SM  at 5 μg/mL in 50 mM phosphate buffer, pH 7.4, for 24 h at 4°C. Plates were then washed and saturated for at least 4 h at RT using EIA buffer. Diluted samples or internal standards were added (50 μL/well) and incubated for 2 h at RT under agitation. Wells were washed and 50 μL of biotinylated anti- β-CN mAb  were dispatched to each well at a concentration of 100 ng/mL in EIA buffer. After 2 h at RT, wells were washed and 50 μL of AChE-labeled streptavidin were dispensed for 20 min at RT. After washing, Ellman's reagent was added and absorbance was measured at 414 nm. Mean blank and SD blank were estimated by measuring eight replicates of signals obtained with EIA buffer. The limit of detection (LoD) and of quantification (LoQ) were estimated as mean, and SM , 24. CroDairy products classically found in France were purchased from retail outlets. They comprised semi-skimmed UHT milks (Lactel), yogurts , soft goat cheese (Soignon) and semi-soft cheeses (Mini-Babybel cow cheese and Mini-Babybel goat cheese containing 10% of GM). Raw milks from one cow and one goat were included as control.Yogurt or cheese (1 g) were resuspended in 10 mL of 50 mM ammonium carbonate buffer, 10 mM EDTA, 1% Octyl-beta-Glucoside using DT-20 dispersing tube (IKA® ULTRA-TURRAX®). After homogenization, samples were mixed during 1 h at 4°c, aliquoted and stored at −80°C. Samples were diluted in the ammonium carbonate buffer at 1 mg of protein/mL by taking into account the initial protein content of the dairy product.IgE binding capacity was performed in a reverse EAST inhibition assay using sera from retrospective studies , 25. OneFor reverse EAST inhibition, plates were coated with anti-human IgE mAb LE27. Fifty μL/well of serum from each patient at appropriate dilutions (1:100 to 1:400) were incubated overnight at 4°C. After washing, 25 μL of inhibitors  and 25 μL of AChE-labeled caprine β-CN were mixed and incubated for 4 h at RT. Results were expressed as B/B0, where B0 and B represent the amount of labeled caprine β-CN bound to immobilized IgE antibodies in the absence or presence of a known concentration of inhibitor, respectively.One mL of raw GM was diluted in 10 mL of raw CM corresponding to a contamination of 100,000 ppm. Ten-fold serial dilutions (1 in 10 mL) were then performed in CM to finally obtain a solution of 10 ppm of GM in CM. This last dilution was used to generate a CM contaminated at 2 and 1 ppm of GM. Spiking experiment was similarly performed with UHT CM spiked with UHT GM.For two-site immunometric assays, the samples were two-fold diluted in EIA buffer with 0.1% Tween® 20 Detergent before analysis. Mean blank and SD blank were estimated by measuring replicates of signals obtained with EIA buffer, 0.1% Tween® 20. The signal-to-noise ratio was evaluated in comparison with CM milk.Four hybridomas producing mAb specific to the caprine β-CN and not reacting to the bovine homolog were selected, cloned, expanded and purified. The mAb CC1 and CC7B recognized the C-terminal part (f108-207) of the caprine β-CN . The solTwo cross-reactive mAb, CC11 and VB1, were produced from mice sensitized to the bovine CN. The mAb CC1 bound to the fragments (f106–209) and (f108–207) covering the C-terminal part of the bovine and caprine β-CN, respectively. The mAb VB1 recognized the fragment (f29–105) of the bovine β-CN and the peptides (f29–107) and (f80–107) from the caprine β-CN.A matched antibody pairs test was performed using each of the non-cross reactive mAb SCB1/SCB4 or CC1/CC7 as immobilized mAb to capture caprine β-CN. For the detection, in addition to a mAb specific to the caprine β-CN, we also tested cross-reactive mAb, i.e., mAb CC11 and VB1 that recognized both caprine and bovine β-CN.As shown in Development of two-site immunometric assays was then pursued only with SCB1 as the capture mAb and VB1 or CC11 as the detection mAb. The two assays displayed very similar sensitivity and specificity . A LoD bWe first evaluated the IgE-reactivity of β-CN present in protein extracts from different dairy products, including raw and heat-treated milk, yogurts, and cheeses . Using aConversely, a very low IgE cross-reactivity between CM and GSM products was observed when using sera from two GM-allergic/CM-tolerant patients . The proWe then analyzed the processed dairy products using the two-site immunoassays with the matched mAb pair SCB1/VB1 or SCB1/CC11 . No signFor both two-site immunoassays, signals measured with raw CM were below the LoD  while thDetection of allergen contaminations in food matrices is one of the keystones of allergen risk management. Unintentional cross-contamination with milks from different ruminants may in particular arise during the processing of dairy products. Various methods including biochemical, immunochemical and molecular biology techniques have been developed to discriminate between milk proteins from different ruminants –30. ThesWe then chose to target specifically the β-CN for its high IgE immunoreactivity and because it is generally the most abundant CN in GSM , 33. MorThe mAb recognizing the caprine β-CN, with or without cross-reactivity with the bovine β-CN, were directed against four different domains. Although the pair of non-cross-reactive mAb CC7 and SCB1 was functional to detect caprine β-CN, a higher sensitivity without decrease of specificity was obtained by using for detection a cross-reactive mAb, i.e., mAb VB1 or CC11. Interestingly, the mAbs SCB1/VB1 pair recognized the short domain 49-107 of β-CN, which encompasses major IgE-binding epitopes . As obseConsidering a LoD of 10 ng/mL of caprine β-CN in dairy products resuspended with a mass-to-volume ratio of 1:10, a LoD of 0.1 ppm (100 ng of β-CN in 1 g of dairy matrix) was reached without significant interference from bovine milk. The LoD of our immunoassays was then at least as sensitive as the ones developed to assess CM protein contamination, in particular to detect bovine casein , 47. UnfIn the present work, we developed two immunoassays allowing the detection of caprine β-CN with a LoD lower than 4 ng/mL and without any cross-reactivity with the bovine β-CN. These in-house immunoassays were both able to detect GM contamination in CM at a level as low as 1–2 ppm. They are also effective for the detection of caprine β-CN in dairy products whatever the processes used, i.e., pasteurization, UHT treatment, fermentation, casein coagulation, and cheese ripening, in line with IgE-immunoreactivity of the tested products.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the French Veterinary Services and CEA agreement D-91-272-106 from the Veterinary Inspection Department of Essonne (France). All experiments were performed in compliance with the French and European regulations on care and protection of Laboratory Animals  with permission 91–493 of French Veterinary Services.HB and SH designed the whole study, performed the experiments, interpreted the data, and wrote the manuscript. NG help to perform some experiments. KA-P managed the study and critically revised the manuscript. All authors approved the submitted version.This study obtained a financial support from INRAE .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.