Nonlinear optical properties of collagen type-I are investigated in thin tissue sections of pig tendon as a research model using a complete polarimetric second-harmonic generation (P-SHG) microscopy technique called double Stokes-Mueller polarimetry (DSMP). Three complex-valued molecular susceptibility tensor component ratios are extracted. A significant retardance is observed between the chiral susceptibility component and the achiral components, while the achiral components appear to be in phase with each other. The DSMP formalism and microscopy measurements are further used to explain and experimentally validate the conditions required for SHG circular dichroism (SHG-CD) of collagen to occur. The SHG-CD can be observed with the microscope when: (i) the chiral second-order susceptibility tensor component has a non-zero value, (ii) a phase retardance is present between the chiral and achiral components of the second-order susceptibility tensor and (iii) the collagen fibres are tilted out of the image plane. Both positive and negative areas of SHG-CD are observed in microscopy images, which relates to the anti-parallel arrangement of collagen fibres in different fascicles of the tendon. The theoretical formalism and experimental validation of DSMP imaging technique opens new opportunities for ultrastructural characterisation of chiral molecules, in particular collagen, and provides basis for the interpretation of SHG-CD signals. The nonlinear imaging of chiroptical parameters offers new possibilities to further improve the diagnostic sensitivity and/or specificity of nonlinear label-free histopathology. In addition, structural alterations of collagen have been the focus of many studies related to ECM remodelling due to tumorigenesis14. Collagen alteration includes degradation of collagen in basement membranes12 and remodelling of fibrillar collagen (mainly collagen type-I) throughout the connective tissue of different organs14. Hence, detailed understanding of the structural properties of collagen is required to elucidate the details of collagen function and its role in disease-related processes.Collagen is a major structural component of the extracellular matrix (ECM) in biological tissues and its structural organisation largely determines the mechanical and functional properties of tissues. For example, well-aligned collagen fibres provide the required viscoelastic properties of tendon and add elasticity to the composite material of bone. Further, the hierarchical structure of collagen determines differences in optical transparency of cornea and sclera16. The efficiency of SHG generated by a sample is characterised by the second-order nonlinear susceptibility tensor, χ(2), which contains information about the structure of the material and its spatial symmetry. Therefore, by extracting χ(2) values, the organisation of collagen fibres can be characterised and certain molecular details about the collagen structure can be inferred. In addition, detailed knowledge of the χ(2) tensor allows for better modelling of collagen molecular structure using ab initio calculations18. The ratio of independent components of the χ(2) tensor has been extracted using various polarisation-resolved SHG (P-SHG) microscopy techniques29. In most studies, it is commonly assumed that the χ(2) tensor components are real-valued when the fundamental and SHG signal frequencies are far from resonance frequencies in the material29. However, the χ(2) tensor components are generally complex-valued and their imaginary parts can be related to the near-resonance enhancement of electric-dipole contributions and/or retardation of the nonlinear responses related to magnetic-dipole and electric-quadrupole effects33.One promising technique to visualise and quantify the structural properties of collagen assembly is second-harmonic generation (SHG) microscopyχ(2) components, a recent technique called double Stokes-Mueller polarimetry (DSMP) can be used. The DSMP is a complete two-dimensional (2D) polarimetric SHG technique that is applicable for structural characterisation of optically-scattering samples35. In the DSMP formalism for SHG, the sample is represented by a 4 × 9 double Mueller matrix, which defines the relation between the polarisation states of incoming laser beam and the polarisation states of the outgoing SHG signal from the sample. Thirty-six measurements are needed to determine the Mueller matrix, from which the real and imaginary parts of six observable laboratory-frame χ(2) tensor components can be extracted36. These components can further be used to extract the real and imaginary parts of the molecular susceptibility tensor component ratios. This is an extra step compared to our previous works where only real parts of susceptibilities were extracted36. The presence of imaginary components in χ(2) has been related to chiroptical properties of collagen in thin films37.In order to extract both real and imaginary parts of the 39, in which the intensities of SHG for RCP and LCP were different. This response was considered as the equivalent of circular dichroism (CD) in linear optics. Hence, it is often referred to as SHG-CD. Since SHG is a surface-sensitive technique, its combination with CD provides an extremely sensitive tool for studying surface chirality. On the other hand, SHG-CD can also be useful in tissue imaging, such as for fast probing of nonlinear chiroptical properties of materials such as collagen, which is inherently chiral43. However, there are only a few studies on the origin of SHG-CD in biological tissues45 and, therefore, there is still a need to develop a model that relates the SHG-CD to the structural and organisational properties of collagen in tissue sections. Since the RCP and LCP incoming polarisation states are included in the DSMP formalism, the SHG-CD can be formulated using DSMP theory and can be deduced from a subset of a DSMP experiment. Using DSMP formalism and measurements, SHG-CD results can be related to the complex-valued chiral components of the susceptibility tensor and the 3D orientation of collagen fibres.Optical activity occurs in nonlinear optics when the nonlinear response of chiral molecules is different for right- and left-handed circularly polarised incoming laser light . The first demonstration of nonlinear optical activity was performed for SHG from chiral molecules deposited on surfacesIn this paper, DSMP is used to study the second-order nonlinear optical properties of collagen in thin tissue sections. Six observable complex-valued susceptibility tensor components are extracted. From these laboratory-frame components, three molecular-frame complex-valued susceptibility tensor component ratios are calculated for the first time. These reveal the presence of significant imaginary values of the chiral nonlinear susceptibility component, the phase and amplitude of which are also extracted. In addition, DSMP theory is used to formulate the SHG-CD response in terms of complex susceptibility tensor components and to determine the theoretical conditions under which SHG-CD has non-zero values. The SHG-CD response is deduced from a subset of DSMP measurements for a research model and the aforementioned theoretical conditions are experimentally validated.46. A previous modelling showed that this assumption results in less than 10% over estimation of susceptibility components ratio46. Under a plane-wave approximation, the polarisation orientations of the focused incoming laser beam remain in the image plane, and a 2D polarimetry such as DSMP can be performed to extract six laboratory-frame χ(2) tensor components29. In the DSMP formalism for SHG, the sample is represented by a 4 × 9 double Mueller matrix, χ(2) tensor components. A thin sample is assumed in this formalism (~5 μm) with negligible birefringence. For thicker samples, additional Mueller matrices can be introduced to account for the first order optical effects. The relation between the polarisation states of the incoming laser beam and the polarisation states of the outgoing SHG signal from the sample is defined using the nonlinear Stokes-Mueller polarimetry equation:s′(2ω) is a 4 × 1 Stokes vector representing the outgoing SHG radiation and S(ω) is a 9 × 1 double Stokes vector representing the polarisation state of the incoming laser beam34. S(ω) consists of nine polarisation states as follows: horizontally (0°) and vertically (90°) linearly polarised , diagonally linearly polarised (±45°), RCP and LCP, linearly polarised at −22.5° and right- and left-hand elliptically polarised . The Poincaré sphere coordinates of the states are given in previous publications35.For excitation objectives up to 0.8 NA, the polarization along the propagation axis is small and therefore a plane-wave approximation can be assumedγ = 1, 2, 336. By having S(ω) from the prepared incoming states and ω) from the measured SHG Stokes components, Eq. , via Xrec matrix as follows35:35. The derivation and properties of these two matrices are described by Samim et al.34. They are both constant invertible matrices, which do not depend on the sample or the experimental setup and, hence, they only need to be calculated for SHG once. The Xrec matrix is the second-order susceptibility product matrix which contains products of susceptibility tensor components and their complex conjugates, and defined as34:Xrec matrix can be obtained as:In a DSMP experiment, for each of the nine incoming polarisation states, four Stokes parameters of the outgoing SHG signal are recorded. To calculate the Mueller matrix elements, the scattering contribution from the dataset is removed by replacing the measured mentclass2pt{minimχ(2) tensor component, the elements of Xrec have to be unmixed. First, Xrec is reshaped to a 6 × 6 square coherency matrix, X:Hence, in order to recover an individual X elements (e.g. χ11) is real and positive, so thatχ (χ11 in this case) as follows:It can be assumed that one of the χ11. The off-diagonal elements can be complex-valued, indicating that the χ values can have a retardance with respect to each other. By taking the real and imaginary parts, the complex value of each observable χ component can be deduced. Consequently, the amplitudes of each susceptibility component as well as their relative phase differences can be calculated.Note that the amplitude and sign of the other elements are relative to χ(2) tensor components is performed in the laboratory coordinate system, as indicated by the uppercase IJK/XYZ indices. In this coordinate system, the sample is placed in the XZ-plane, the laser beam propagates along the Y-axis and the second-order nonlinear susceptibility tensor is indicated as XZ image plane, only six laboratory-frame χ(2) tensor components can be measured29: χ(2) tensor components in the laboratory coordinate system can be expressed in terms of the molecular second-order susceptibility tensor components, 29:α is the out-of-plane tilt angle with respect to the XZ image plane, and δ is the average in-plane fibre orientation angle measured from the Z-axis in the laboratory frame of reference. The terms containing 29. Equations (9) indicate that the susceptibility tensor components in laboratory coordinate system can be written as an achiral contribution plus a chiral contribution that depends on α. Consequently, for the collagen fibres that are in the image plane (α = 0) the contribution of the chiral terms is zero, so that the tensor is indistinguishable between a chiral and an achiral structure.The measurement of the IRCP−ILCP)/((IRCP + ILCP)/2). Since two of the nine incoming polarisation states are RCP and LCP, DSMP data can be used to extract the SHG-CD of the sample. The SHG-CD measured using DSMP technique can be written in terms of the Stokes vector parameters, which in turn can be expressed in terms of Mueller matrix elements by using Eq. , (ii) the sample has to be chiral δ, is absα. It is interesting to note that Eq. 13), the that Eq.  predictsThe SHG-CD also depends on the interaction of the chiral and achiral terms through mentclasspt{minima47. The laser was coupled to a custom-built laser-scanning SHG microscope, as described previously36. A 20 × 0.75 numerical aperture (NA) air objective  was used to focus the beam onto the sample, and a custom 0.85 NA objective  was used to collect the SHG signal in the transmission geometry. The SHG signal was detected using a single-photon-counting photomultiplier tube . A BG 39 filter and a 510–520 nm band-pass interference filter  were used to separate the SHG from the incident light. The microscope polarimeter consisted of a polarisation-state generator (PSG) inserted before the excitation objective and a polarisation-state analyser (PSA) placed after the collection objective. The PSG consisted of a fixed linear polariser , a half-waveplate (HWP) and a quarter-waveplate (QWP). The PSA contains a HWP, a QWP  and a linear polarizer . The PSG was set to produce nine different incoming polarisation states , for each of which the PSA was set to measure the SHG signal at six different polarisation states  in order to determine the SHG Stokes vector.A custom-built diode-pumped ytterbium-ion-doped potassium gadolinium tungstate (Yb:KGW) crystal-based oscillator was used as the laser source. It operates at 1028 nm and it generates ~400 fs pulses at 14.3 MHz repetition rateα = 30° (oblique cut) and perpendicular to the tendon axis (transverse cut). The samples were embedded in paraffin and cut into 5-μm thick sections. The sections were mounted on glass slides and stained with hematoxylin and eosin (H&E) to provide anatomical reference recorded with bright-field whole-slide scanner (Aperio: Leica Biosystems). One slide per cut angle was studied.Collagen type-I from Achilles tendon of a 25 kg Yorkshire pig was studied. Pig tendon was chosen because it is a well-ordered structure and due to its size, it can be easily cut at various angles. The tendon was harvested from a healthy dead pig euthanized after an unrelated study with approval of the Animal Care Committee of the University Health Network, Toronto, Canada. The tendon was formalin fixed and cut along the tendon axis , at δ~0 and α~0) and it can be seen that the real part of δ~0 and α~0. The real values of the δ~0 and α~0. The imaginary parts of all susceptibility components are small at this cut angle, indicating that all the observable susceptibility components are in phase with each other and so real-valued.DSMP microscopy was used to extract the real and imaginary parts of the six observable susceptibility tensor components for each of the three cut angles in the tendon: Fig. (2) Fig.  is maxim(2) Fig. . In addi(2) Fig.  are simi(2) Fig.  when δ~0ent Fig.  when δ~0Z-axis, meaning that δ~0. This can also explain why the real parts of δ, while the achiral contribution scales as cos δ (see Eq. (Z-axis (δ~0), the achiral terms are larger than the chiral terms, which results in the same sign for δ, while achiral contributions scale as sin δ, so that the chiral contribution is larger than the achiral contribution, resulting in opposite signs for fibres oriented close to δ = 0. Further, large and anti-correlated imaginary contributions are observed for For the obliquely-cut sample, all real susceptibility components have significant values, with the largest occurring in (2) Fig.  and with(2) Fig. , indicat(see Eq. ). Therefα = 90°, so that the SHG signal decreases due to symmetry considerations, resulting in a lower signal-to-noise ratio (SNR). Hence, fibres deviating from α = 90° show larger signal, so that care should be taken when interpreting the results. The transverse cut shows similar properties to the oblique cut, with emerging islands of anti-correlated positive and negative areas in For the transverse cut, the collagen fibres are closer to cut Fig. .α values were estimated using only linear polarisation measurements, as previously described29. These values are: 0° for longitudinal cut, 30° for the oblique cut and 70° for the transverse cut. The fitting results are shown in Fig. 29. Re. The presence of imaginary values only for the chiral susceptibility suggests a phase retardation between chiral  and consequently the chiroptical properties of collagen.The observations demonstrate that, although the second-order susceptibility components of collagen fibres have a chiral contribution, they can only be observed when the fibres are tilted out of the image plane  indicate that collagen fibres have a significant The data for SHG-CD involving RCP and LCP incoming polarisations are contained in the full DSMP measurement, so that the calculated parameters from DSMP presented in the previous sections can be used to analyse the SHG-CD images. These images are shown in Fig. g at Eqs  and 13)44. The S50, piezoelectric force microscopy52, interferometric SHG microscopy54 and polarimetric SHG microscopy with linearly polarised light29. This interpretation is further supported by the binomial distribution of SHG-CD values lying symmetrically from zero. In the transverse cut the positive and negative values are organised into islands, indicating that fibres have dominant orientation either parallel or anti-parallel with neighbouring fibres. The change in the polarity of the collagen fibrils changes the sign of the achiral χ(2) tensor components21, while chiral tensor components preserve the same sign under rotation. Hence, the sign of SHG-CD can be used to determine the relative out-of-plane tilt angle of the fibres. On the other hand, the SHG-CD magnitude depends on the values of susceptibility components, the retardance between chiral and achiral components and the tilt angle α, so that interpretation is not straightforward and requires a-priori knowledge of 2 of the 3 aforementioned parameters.Equation  and 13)13) show zzz,zxx = Δxxz,zxx = 0. On the other hand, the chiral component is not in phase with the other components. Using zzz,xyz = Δzxx,xyz = Δxxz,xyz = Δ, Eqs . Further, there are two values of α (±30°) for which the absolute value of SHG-CD is maximum. In Fig. α is fixed at 30° and Eq.  laboratory-frame susceptibility components were extracted for collagen fibres in pig tendon cut at different orientation angles, and were further used to deduce the chiral, α ≠ 0, (ii) presence of chiral components, α, where positive and negative values represent opposite polarities.All complex-valued observable 14. Further, the SHG-CD results can reveal the polarity of collagen fibres in the tissue. The polarity information can be used to investigate the piezoelectric and pyroelectric properties of collagen that are relevant for studying various tissues including bone and tendon58. SHG-CD can also be used as a method to investigate the 3D orientation of collagen in the sample. In addition, since SHG-CD has been already used as a new tool to differentiate normal from diseased tissues23, a better understanding of the underlying physical mechanisms of SHG-CD signals can help with interpretation of the results.Extraction of the molecular-ratio parameters, including the achiral and chiral ratio, and chiral amplitude and phase, provides a sensitive tool for ultrastructural characterisation of collagen. The collagen alterations are known to occur with diseases such as tumour initiation, progression and metastatic spreadTherein lies the potential for translation of these findings into clinical impact as a means to further improve the diagnostic sensitivity and/or specificity of non-linear label-free histopathology in, for example, biopsied tissues.


The compositions of the surface were analyzed through an X-ray photoelectron spectroscopy (XPS). Also, the delamination issue between the corner of the heat sink cap and the epoxy resin was observed for delamination of the LFBGA packaging. Further, this study analyzed the PBGA packaging process through the finite element analysis simulation software ANSYS. To improve the heat sink cap delamination issue of the PBGA, a new chamfer design of the corner seat was streamlined to decrease the stress value and delamination. Besides, the simulation results demonstrated that the stress value reduced after increasing the shoulder length. The results implicate that the stress value is inversely proportional to the shoulder width and the chamfer radius. This study demonstrated that the optimization in design was able reduce the delamination phenomena in configuration material.The electronic package interconnects electronic signals from one area to another and package delamination is a serious problem in the configuration of materials. This study focused on decreasing the delamination of the low-profile fine pitch ball grid array (LFBGA) and plastic ball grid array (PBGA) packages in terms of polymer thermal issue, metal bonding and bonding mechanisms. PBGA and LFBGA are a very common type of packaging processes in the electronics industry. The present study dealt first with delamination of the LFBGA packaging, through characterization and determination of physical and chemical properties such as surface roughness, surface energy, and contact angle. The relationship between surface roughness and delamination was verified through various roughness bonding experiments. In addition, the surface energy was determined by measuring the contact angle after cleaning the metal surface of Cu, Ni and Cr with Ar + O Since Texas Instruments (TI) developed integrated circuits (IC), numerous and vigorous studies in the field of semiconductors have been performed. The global market share of the Taiwanese semiconductor industry is increasing annually due to government support and active academic studies. Taiwan plays an active and important role in the IC design and packaging industry. This has been a focus of research and development in the semiconductor industry over the past two decades. The entire process requirement for the industry is available, from wafer materials to IC design, manufacturing, packaging and system testing. Ball grid array (BGA) packaging represented an important milestone in IC packaging industry as it eliminated the disadvantage of the dual in-line package (DIP) method. Still, delamination is an issue with BGA packaging and merits the attention of researchers.Our research aims to eliminate the delamination of the low-profile fine pitch ball grid array (LFBGA) and plastic ball grid array (PBGA) package. LFBGA is a thin BGA meeting Joint Electron Device Engineering Council (JEDEC) specifications, with a structure similar to that of conventional BGA, but the diameter and centre of the solder ball are smaller and thinner. Thus, the package contains more I/O numbers within the same size, which signifies that the assembly density of the package is enhanced. Bismaleimide triazine resin or glass layers were used by researchers as a substrate for PBGA packages, due to their similar thermal expansion coefficients . Therefo2O at room temperature, transforming into a less hydrophobic substance with a contact angle of about 110° [In the development process, contact angle measurements are required to calculate the surface energy of the materials. Materials with high surface energy exhibit hydrophilic characteristics, i.e., the contact angle is small, thus, the materials tend to be highly adhesive. Water is known to spread out almost completely on the surface of noble metals such as gold, silver and copper . Therefoout 110° .2 gases were used as working gases. In another study [2 and O2 gas plasma cleaning of Au and PtIr was investigated by X-ray photoelectron spectroscopy (XPS). Moreover, Ar and Ar + O2 gases were used for plasma cleaning of Au surfaces [Gas plasma treatment is known to enhance the adhesive properties of materials. In a previous study , Cu treaer study , the effsurfaces .In this study, we prepared LFBGA packaging and PBGA packaging materials, performed characterization and determination of physical and chemical properties and analyzed the delamination process. Further, we tried to perform a design optimization which can reduce the delamination phenomena.To perform the experiments, we took a LFBGA packaged material and observed the SEM image of delamination which was occurring between the metal and the compound. The dark portions, as shown in For the case of PBGA packaging, when PBGA was sealed with an epoxy resin, the delamination occurred in the corner of the heat sink cap as shown in The surface morphology and roughness of LFBGA were examined by a SEM and a profilometer, and, the relationship between the delamination percentage and roughness was verified. The delamination in the samples was analyzed and the results were shown in delamination area percentage. The delamination area percentage was classified into 0% to 10% and larger than 10%. A delamination area smaller than 10% is generally acceptable for the clients. 2 was used to evaluate the effect of the plasma treatment on surface energy, where the gas mixture ratio and operating power were 4:1 and 800 W, respectively. We measured the contact angle after the plasma treatment, based on which, the surface energy and the adhesion can be obtained. The contact angle was used as the method to calculate surface energy. The angle between the solid phase and the liquid phase is defined as the contact angle (θ), as shown in 1), solid phase (γ2) and the interface of solid and liquid phase (γ12) was expressed as Equation (1) [The plasma gas of Ar + Otion (1) . Adhesivtion (1) . For thed phase γ was exprtion (1) ,16,17,18Three different metals with varying roughness was electroplated on the Cu substrate, which was functioning as packaging cover. Electroplating process is schematically illustrated in The experimental process of PBGA was predicted by using finite element analysis to simulate the effect of the stress distribution on delamination. The geometric parameters of PBGA were provided by Advanced Semiconductor Engineering Inc. . According to the sample information, the heat sink cap is shown in Tg temperature of the epoxy was ~127 °C. There were two thermal expansion coefficients, CTE1 and CTE2 for ANSYS. The boundary condition was set as the upper mold contacting with a circle of ring on the heat sink cap, and lower mold contacting four corners of the heat sink cap. The upper mold and the lower mold were tightly closed, then epoxy was injected into the mold, where the lower mold served as a fixed support. In addition, the simulation molding model was simplified into quarter to reduce the simulation time due to the symmetry, as shown in The heat sink cap was made of Cu and the material parameters are listed in The simulation parameter settings of the PBGA packaging are shown in The main parameters of molding simulation were shoulder width and chamfer radius. The shoulder width is the length next to ring, as schematically shown in The surface morphology of the LFBGA sample as seen by SEM is shown in When the roughness increased from Ra 3.2 μm to Ra 3.6 μm, the delamination percentage decreased from 2.8% to 0.0%. This decrease was due to the increasing roughness and subsequently enhanced physical adhesion and the bonding force.2 plasma to clean the surface and the results of XPS are shown in 2 gas and might produce CuO and Cu2O compounds. Three different metals were treated with Ar + O2 plasma treatment, the metal surface became hydrophilic, and the water was completely stuck on the surface because the oxide layer was removed. After 60 h of Ar + O2 plasma treatment, the contact angle of Cu was increased to about 52.6° since oxide layers were growing on the surface, which may make the surface gradually hydrophobic, and thus, cause an increase in the contact angle. It means that after being treated for a period of time, the treated sample could lose hydrophilicity. The contact angle of Ni was increased to 52° and Cr was increased to 56.8°, however, the error bars of three metals were overlapping. Therefore, the differences of contact angle in three metals were not statistically significant.Based on a previous study , it is sThe results of the various metals with different roughness of Ra 3.2 and Ra 3.6 is shown in 2 could get into the Cu substrate from the Ni layer and form CuO and Cu2O compounds simultaneously, causing an uneven stress distribution beneath the Ni layer. Delamination could occur between polymers and Ni after packaging. Epoxy adhesion with Ni was weaker than with those of Cr or Cu. Therefore, in order to decrease the delamination, the substrate should not be coated with Ni metal.When Cu metal was bonded with epoxy, the delamination percentage was ~0–2.8 % for Ra 3.6 and 3.2, respectively. However, the delamination was 60.33% when Ni metal plate had Ra 3.6, whereas the delamination increased to 100% when Ra was down to 3.2. The Cr metal exhibited similar results to the Cu samples. Almost no delamination took place. Through these results, epoxy showed good bonding force with Cu and Cr. However, Ni did not have adequate bonding force with epoxy, which led to the delamination. In other words, after electroplating, the surface of the Ni layer was supposed to prevent the occurrence of copper oxidation and increase the adhesion with polymer. Since the Ni layer after electroplating could be less adhesive than Cr layer, OThe molding simulation result of PBGA heat sink cap is shown in As shown in The original design with a sharp geometry of the heat sink cap corner was not an ideal and optimal case, which led to a significant stress concentration. Therefore, the new design has changed the sharp geometry to chamfer with various radii. The comparison result between original and chamfer design with radius 0.15 mm is shown in In addition, the chamfer design of four different radii combined with the various shoulder widths to simulate the stress value. The result shows that when the radius and the shoulder width increased, the stress values were decreased see . Therefo2 gas plasma could remove most of the oxide layer and increase the surface energy. On the other hand, it could also induce the oxide layer to grow. The original state of three kinds of metals had oxide layers on the surface, but after Ar + O2 plasma treatment, most of the oxide layers were removed and the contact angles were decreased, which is suitable for IC packaging. The reason why the delamination between Ni and epoxy occurred could be the weak chemical bond formed between Ni and the epoxy compound materials. The Ni layer after electroplating could be less adhesive than the Cr layer, and O2 could get into the Cu substrate from the Ni layer and form CuO and Cu2O compounds simultaneously, causing the uneven stress distribution beneath the Ni layer. The composition of the epoxy was not known well at this stage due to the secrecy of the supplier, which will be studied further in the near future. However, Cu and Cr metal bonded well with the epoxy. As the shoulder of the PBGA heat sink cap was increased from 200 to 800 μm, the stress was decreased from 107 to 78.57 GPa. Under the same conditions, the geometry design was further improved and adding the chamfer effectively improved the stress value, dropping it from 107.1 to 80.37 GPa. When the shoulder width of the heat sink cap was changed to 800 μm and chamfer radius to 0.45 mm, the stress value was decreased from the original stress value of 107 to 35.58 GPa. The reduction in stress value indicated that the delamination between metal and epoxy has been improved significantly.From the aspects of physical and chemical properties, the delamination problems of LFBGA and PBGA were discussed in this study. The roughness has a significant influence on delamination, and it is inversely proportional to delamination. The delamination was improved when Ra was changed from 3.2 to 3.6 μm. Based on the XPS results, it can be observed that Ar or N


Herein, we analyzed for the first time the Ca2+ response to the physiological autacoid ATP in native aortic endothelium of obese Zucker diabetic fatty (OZDF) rats and their lean controls, which are termed LZDF rats. By loading the endothelial monolayer with the Ca2+-sensitive fluorophore, Fura-2/AM, we found that the endothelial Ca2+ response to 20 µM and 300 µM ATP exhibited a higher plateau, a larger area under the curve and prolonged duration in OZDF rats. The “Ca2+ add-back” protocol revealed no difference in the inositol-1,4,5-trisphosphate-releasable endoplasmic reticulum (ER) Ca2+ pool, while store-operated Ca2+ entry was surprisingly down-regulated in OZDF aortae. Pharmacological manipulation disclosed that sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) activity was down-regulated by reactive oxygen species in native aortic endothelium of OZDF rats, thereby exaggerating the Ca2+ response to high agonist concentrations. These findings shed new light on the mechanisms by which type 2 diabetes mellitus may cause endothelial dysfunction by remodeling the intracellular Ca2+ toolkit.An increase in intracellular Ca Endothelial cells form a monolayer comprising the innermost lining of blood vessels and maintain cardiovascular (CV) homeostasis by regulating vascular tone, permeability, coagulation, immune response and by controlling vascular growth and repair ,2. There2+ concentration ([Ca2+]i) is crucial to fine tune endothelial cell functions i leads to the production of the vasoconstrictor prostanoids, thromboxane A2, prostaglandin H2 and endothelin-1 i after agonist stimulation is achieved by several Ca2+-transporting systems, such as plasma membrane Ca2+-ATPase (PMCA), Na+/Ca2+ exchanger (NCX) and sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) i is dramatically enhanced in association with the down-regulation of SERCA activity in native endothelium of excised rat aorta of OZDF rats. These findings shed novel light on the mechanisms whereby diabetes causes endothelial injury and hint at the endothelial Ca2+ toolkit as a novel target to prevent CV complications in diabetic patients.Obese Zucker Diabetic Fatty (OZDF) rats have been widely employed as a type 2 diabetes mellitus model, which arises when the animals are fed with a high-energy diet as a consequence of a homozygous mutation (fa/fa) in the leptin hormone receptor . OZDF raal cells ,37,38. Ip < 0.05). Indeed, the abdominal circumference and the body mass index (BMI) increased by 35% and 57%, respectively, in OZDF rats. In addition, we found that the epididymal fat weight from OZDF rats was 4 times higher than that obtained in LZDF rats. Taken together, these data prove the obese phenotype of the OZDF rat group.The somatic and biochemical parameters obtained from 2–3-month-old littermate LZDF and OZDF rats are shown in p < 0.05).The biochemical results, reported in p < 0.05). After glucose loading, significant differences also were observed in the glucose tolerance in the OZDF group at 30, 60, 90 and 120 min corresponding to increases of 52%, 70%, 107%, and 97%, respectively  was not statistically different in OZDF  vs LZDF rats  p > 0.05  than low (20 µM) ATP concentration, and was mainly due to SOCE activation i in both animal groups  (1 µM), which is another structurally unrelated SERCA inhibitor . As obsel groups A,C. In tau level B, right au level D in both2+ signal in OZDF rats. There was no difference in the amplitude of the initial Ca2+ peak , a selective PMCA blocker i, thereby increasing the driving force for extracellular Ca2+ entry ); (2) the Ca2+-transporting system is affected in intact aortic endothelium of OZDF rats; and (3) this impairment is fully unmasked by stimulating the cells with the highest ATP dose, i.e., 300 µM. Therefore, we will first describe how SERCA, PMCA and NCX interact to remove cytosolic Ca2+ under physiological conditions, i.e., in native endothelium of excised aorta of LZDF rats, and then discuss the remodeling of the Ca2+ handling machinery in OZDF rats.Earlier studies revealed that high glucose may enhance endothelial SOCE by up-regulating its molecular components ,52, nameectively ,53. Furtectively . Neverth2+ response in native rat aortic endothelium which consists in an initial Ca2+ peak, due to InsP3-dependent ER Ca2+ release, followed by SOCE activation i from declining to the baseline, thereby enhancing the plateau phase and prolonging the duration of the Ca2+ response, which collectively lead to an increase in the AUC. CPA is also known to stimulate ER Ca2+ release and activate SOCE in intact aortic endothelium i i ,60.2+ response to ATP is more puzzling to decipher. The blockade of NCX activity with two structurally distinct drugs, i.e., KBR and SEA, decreased the duration of the Ca2+ response to 20 µM ATP, thereby causing a significant reduction in the AUC. This effect was detected in the absence of extracellular Ca2+ and cannot be ascribed to the inhibition of the reverse-mode NCX activity and is unlikely to reflect off-target effects. It has, however, long been known that NCX and SERCA compete for clearing Ca2+ during the decay phase of a Ca2+ transient and that the clearing rate of SERCA activity may be remarkably faster as compared to NCX ) and dampens the inhibitory effect of CPA and thapsigargin. Third, blocking PMCA activity with carboxyeosin exerted a similar impact on the decay phase of the Ca2+ signal induced by 20 µM and 300 µM ATP in LZDF and OZDF rats . This observation indicates that endothelial PMCA activity is not altered by type 2 diabetes mellitus. Fourth, NCX inhibition with KBR or SEA only weakly affected the Ca2+ response to 20 µM ATP  in OZDF rats, while it exerted a similar effect on the Ca2+ signal evoked by 300 µM ATP  in LZDF and OZDF rats.The Ca2+ release are not different between LZFD and OZDF aortae. This finding indicates that: (1) the releasable ER Ca2+ content; and (2) NCX and PMCX activities, which are the major Ca2+-clearing mechanisms acting under such conditions, are not heavily altered in the intact endothelium of OZDF rats. Taken together, these observations strongly suggest that SERCA is the major Ca2+-transporting mechanism to be affected in native endothelium of excised aorta from OZDF rats. When the cells are stimulated with a low [ATP], i.e., 20 µM ATP, SERCA2B is still able to cope with the amount of Ca2+ introduced into the cytosol by InsP3-induced Ca2+ release and SOCE in both LZDF and OZDF rats. However, its impaired activity causes [Ca2+]i to decay slower to the baseline, thereby slightly enhancing the AUC of the Ca2+ signal. When ATP concentration is increased up to 300 µM, the higher amount of Ca2+ delivered into the cytosol of OZDF aortae through InsP3 receptors and SOCE results in an excessive influx of Ca2+ that cannot be entirely sequestered into ER lumen by the impaired SERCA2B and is mainly extruded by PMCA.It should also be pointed out that the amplitude, AUC and duration of the CPA-endogenous Ca2+ sequestration was significantly dampened i causes the up-regulation of the Ca2+-dependent xanthine oxidase and induces ROS overproduction in human umbilical vein endothelial cells i in intact endothelium has been previously described i was monitored by measuring, for each regions of interest, the ratio of the mean fluorescence emitted at 510 nm when exciting alternatively at 340 and 380 nm (termed Ratio (F340/F380)). An increase in [Ca2+]i causes an increase in the Ratio (F340/F380). Ratio (F340/F380) measurements were performed and plotted on-line every 3 s. Ratio (F340/F380) values are expressed as arbitrary units (A.U.). The experiments were performed at controlled room temperature (22–23 °C) to limit time-dependent decreases in the intensity of the fluorescence signal.This technique for evaluating changes in [Caescribed ,36,39,832+ response to ATP was measured as the difference between the ratio at the peak and the mean ratio of 1 min baseline before the peak. The amplitude of the plateau phase was measured as the difference between the ratio at 600 s after the application of ATP 20 µM and ATP 300 µM, respectively, and the mean ratio of 1 min baseline before the peak. The duration of the Ca2+ response to ATP was measured as the time it takes the Ca2+ signal to be reduced at 90% (early), 60% (intermediate) and 30% (late) of the initial Ca2+ peak amplitude. The area under the curve (AUC) was measured by calculating the integral of each Ca2+ tracing from when the ATP is applied until it is removed. Data are expressed as mean ± standard error (SE). Non-Gaussian data ), were statistically analyzed using nonparametric Mann-Whitney test. For normal data an unpaired Student’s t-test was used. A p value < 0.05 was considered statistically significant.For each protocol, data were collected from at least three rats under each condition. The amplitude of the peak CaFura-2/AM was obtained from Molecular Probes . SEA0400 was obtained from Tocris Bioscience . All other chemicals were purchased from Sigma .To detect SERCA expression, aortic tissues were extracted from OZDF or LZDF rats and were fixed in 4% paraformaldehyde solution, washed and incubated with PBS 0.25% Triton X-100 and blocked with PBS 3% bovine serum albumin followed by incubation at 4 °C overnight with 2 mg/mL Anti-SERCA2 ATPase antibody ; washed and incubated with 2 mg/mL Alexa Fluor488-conjugated anti-mouse IgG antibody  for 1 h at room temperature. Additionally, the cell nuclei were counter-stained with 0.001% Hoescht 33342 solution. The tissues were mounted in slides and fluorescence was detected in 2 μm sections in a Zeiss Observed Z1 inverted microscope equipped with an Axiocam MRm camera and an Apotome illumination system with a 63X oil immersion objective .2+ handling machinery is altered in native endothelium of excised aorta from OZDF rats. The Ca2+ response to physiological stimuli is remarkably enhanced when agonist concentration is elevated within the high micromolar range. Rewiring of the endothelial Ca2+ toolkit involves the ROS-dependent down-regulation of SERCA activity, which results in the increase of the amplitude of plateau phase, thereby prolonging the duration of the Ca2+ signal. This remodeling of the endothelial Ca2+ machinery could contribute to explaining how type 2 diabetes leads to endothelial dysfunction.In conclusion, this report demonstrated for the first time that the Ca


We show that complete loss or increased abundance of H3K56ac in these mutants allows timely entry into meiosis and sporulation and does not impair S phase progression, first and second meiotic cell divisions, and spore viability. In the asf1Δ, rtt109Δ, hst3/4Δ mutants, DSBs and crossovers form normal levels with a short (60-min) delay at the HIS4-LEU2 artificial recombination hotspot, however, DSB formation shows a ∼threefold decrease in the H3K56A mutant at the natural BUD23-ARE1 hotspot. The latter DSB phenotype, showing significant DSB reduction in the H3K56A mutant, was also observed at DSB sites using genome-wide mapping of Rfa1-coated single-stranded DNA flanking DSBs (RPA ChIP). Parallel mapping of H3K56-acetylated histones in wild type cells revealed strong depletion of the H3K56ac ChIP signal over Spo11-oligo DSBs, albeit most H3K56-acetylated histones were enriched adjacent to the identified RPA ChIP binding sites. Taken together, these associations demonstrate a prominent role of H3 lysine 56 acetylation in the formation of DNA breaks within recombination hotspot regions.Meiotic recombination is initiated by Spo11-catalyzed DNA double-strand breaks (DSBs) that are promoted by histone modifications and histone modifying enzymes. Herein we investigated the role of histone H3 lysine 56 acetylation (H3K56ac) located near the entry/exit points of the DNA in the globular H3 domain. We generated a series of mutant cells ( S. cerevisiae, meiotic DSBs are controlled by the elements of chromatin structure. This involves a complex interplay between DNA sequence composition, local chromatin status, nucleosome occupancy, and transcription factor binding  catalyzed by Spo11 and accessory factors . In S. c binding . Among t binding . A simil binding , however binding .The widely localized H3K4me3 mark has the virtue of initiating recombination at numerous places in the genome, however, other histone modifications are also needed to keep recombination flexible for the diversity of recombinant haplotypes . These “S cerevisiae cells to modify the natural levels of H3K56ac to study the biochemical phases of meiosis. Our results demonstrate that H3K56ac is necessary for formation of normal levels of DSBs within recombination hotspot regions.In the current study, we focused on the role of histone H3 lysine 56 acetylation located near the entry/exit points of the DNA in the globular H3 domain, predicted to destabilize the histone/DNA contact . H3K56acAll methods are available as asf1Δ and rtt109Δ deletion mutants during the meiotic time course (∼fourfold reduction compared to wild type), in agreement with the crucial role of Asf1 and Rtt109 in the deposition of this epigenetic tag . DSB levels were monitored at the HIS4-LEU2 hotspot . Importantly, meiotic DSBs detected in the mutants were highly recombinogenic  and unmodifiable H3K56A as the only source of histone H3 . Like wiand RFA1 , which wand RFA1 .dmc1Δ and H3K56A dmc1Δ cells that accumulate unrepaired DSB ends. DSB frequencies were determined by Southern blot within the natural hotspot region BUD23-ARE1   , consist3 (ctrl) . In addi3 (ctrl)  were pos3 (ctrl) , whereas3 (ctrl) . These rBUD23 and ERG1 hotspot regions were flanked by H3K56ac at the time of DSB formation as measured by H3K56ac ChIP-qPCR and ChIP-chip (asf1Δ cells that are deficient in H3K56 acetylation (rDNA region (JBrowse link), suggesting that the presence of H3K56 residue and/or deposition of H3K56-acetylated nucleosomes near DSB hotspots is required for complete DSB formation. Our H3K56ac ChIP results show that H3K56 acetylation is preferentially associated with protein-coding ORFs , as opposed to chromosome axis sites  is currently not known. We obtained complementary results that are fully consistent with recent data identifying differences in the RNAPII binding profile of  mutants . Based o mutants . H3K56ac mutants . We assu mutants . Future http://geneart.med.unideb.hu/pub/h3k56ac). Raw data are availabe at GEO (GSE37487).Datasets generated for this study can be accessed in ZK and LH analyzed the genomic data. LS performed the experiments and wrote the manuscript.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.


XAB2 is a multi-functional protein participating processes including transcription, splicing, DNA repair and mRNA export. Here, we report POLR2A, the largest catalytic subunit of RNA polymerase II, as a major target gene down-regulated after XAB2 depletion. XAB2 depletion led to severe splicing defects of POLR2A with significant intron retention. Such defects resulted in substantial loss of POLR2A at RNA and protein levels, which further impaired global transcription. Treatment of splicing inhibitor madrasin induced similar reduction of POLR2A. Screen using TMT-based quantitative proteomics identified several proteins involved in mRNA surveillance including Dom34 with elevated expression. Inhibition of translation or depletion of Dom34 rescued the expression of POLR2A by stabilizing its mRNA. Immuno-precipitation further confirmed that XAB2 associated with spliceosome components important to POLR2A expression. Domain mapping revealed that TPR motifs 2–4 and 11 of XAB2 were critical for POLR2A expression by interacting with SNW1. Finally, we showed POLR2A mediated cell senescence caused by XAB2 deficiency. Depletion of XAB2 or POLR2A induced cell senescence by up-regulation of p53 and p21, re-expression of POLR2A after XAB2 depletion alleviated cellular senescence. These data together support that XAB2 serves as a guardian of POLR2A expression to ensure global gene expression and antagonize cell senescence. Gene expression is a fundamental and highly complex process that includes many steps, such as transcription, RNA splicing, RNA export, RNA degradation, translation and protein degradation . RegulatIn eukaryotes, the transcription of all mRNAs as well as several noncoding RNAs, including some snRNAs, snoRNAs, siRNAs and all miRNAs, is achieved by RNA polymerase II (pol II) . RNA polact complex (activated spliceosome), to B* complex , to C complex , to C* complex , to post-splicing complex, and to the intron-lariat complex transition, a large number of proteins are involved in spliceosome assembly and activation, such as SR proteins, hnRNP proteins, Prp19-related complex, and the exon junction complex  and numerous proteins . The spl complex . Therefo complex .act -binding protein 2 (XAB2) is a multifunctional protein involved in transcription ,28, tranact , C 37),,act (37)act ,39 complact ,28. Likeact  and tranact . In Sacccription ,42. In acription , howeverBased on our microarray data, POLR2A mRNA was significantly down-regulated after XAB2 knockdown , suggestTo generate the XAB2 plasmid with HA tag in pcDNA5/FRT/TO vector (Invitrogen), previously constructed plasmid containing XAB2 cDNA  was usedRabbit polyclonal antibody against XAB2 , Dom34 , SNW1 , SNRNP200 , PLRG1 , PRPF8 , EFTUD2 , AQR , CDC5L , RNPS1 , P53 , Rabbit monoclonal antibody against POLR2A-CTD , POLR2A-pSer2 , POLR2A-pSer5 , CSTF2T , p21 , mouse monoclonal antibodies against POLR2A-NTD , HA tag , CPSF7 , α-tubulin  were used in western blots. Antibodies for ALY, Prp19, UAP56 and SC35 were gifts from Reed Lab in Harvard Medical School. Antibody against POLR2A-CTD was used in western blot for POLR2A expression in this study unless otherwise indicated. Rabbit monoclonal antibody against POLR2A  was used in immunofluorescence.HeLa and FRT HeLa cells were gifts from Reed Lab in Harvard Medical School, 293T, MDA-MB-231 and HFF1 cells were purchased from the American Type Culture Collection (USA). All the cells were cultured in DMEM medium supplemented with 10% FBS except for HFF1 with 15% FBS.To inhibit pre-mRNA splicing, HeLa cells were treated with 30 uM madrasin (MCE) for 24 h. To inhibit translation, HeLa cells were treated with 50 ug/ml cycloheximide (CHX) (MCE) or 10 ug/ml emetine (Merck) for 24 h. For mRNA stability assay, HeLa cells were treated with 5 ug/ml actinomycin D (Act.D) (MCE) to inhibit transcription and then harvested at the indicated time points following addition of Act.D.siRNA or shRNA mediated gene knockdown were performed as described before . All siRTo establish doxorubicin (Dox)-inducible XAB2 knockdown stable cell clone used in Figure To establish Dox-inducible XAB2 stable cell clone used in Figures To establish Dox-inducible XAB2 full-length and truncation mutants stable cell clone used in Figure Cells were grown in 35 mm cell culture dish with glass bottom , fixed with 4% paraformaldehyde (Sigma) for 30 min, followed by permeabilization with 0.5% Triton X-100 for 10 min at room temperature. Cells were then incubated with primary antibody diluted in PBS with 10% calf serum at 4°C overnight. DAPI staining was performed after incubation with Alexa-Fluor-488 or Alexa-Fluor-555-conjugated secondary antibodies (Invitrogen) diluted in the same buffer at room temperature for 30 min, images were captured using DMI8 microscope (Leica).®488 In Vitro Imaging Kit  according to the manufacturer's protocol. Briefly, cells were grown in 35 mm cell culture dish with glass bottom , incubated with 500 uM ethynyl uridine (EU) in DMEM complete culture medium for 2 h. After EU labeling, cells were fixed with 4% paraformaldehyde (Sigma) for 30 min, permeabilized with 0.5% Triton X-100 for 10 min, and then stained with Apollo 488 at room temperature for 30 min, followed by DAPI staining. Fluorescence was detected using DMI8 microscope (Leica).Nascent RNA detection assay was performed using Cell-Light™ EU ApolloP-value of 0.05 was used as the statistical cutoff for differentially spliced AS events. RNA-seq data have been deposited at the NCBI Gene Expression Omnibus under the accession number GSE130087.HeLa cells were transfected with control or XAB2 siRNA for 48 h. Total RNA was then purified using TRIzol, treated with DNase I and Ribo-Zero rRNA Removal kit to generate sequencing libraries, and subjected to strand-specific paired-end RNA-seq analyses using Illumina Hiseq X Ten platform by Novogene company (China). We used Integrative Genomics Viewer (IGV) to visualize sequence alignments  and junc32P-UTP. Nuclear extract from XAB2 overexpression or knockdown HeLa cells was prepared as described before  in reaction mixtures containing d before . FollowiHeLa cells were plated in six-well plate and transfected with control or XAB2 siRNA-1 for 48 h. Then cells were harvested and subjected to TMT-based quantitative proteome analysis by Jingjie PTM Biolabs (China). In brief, cell pellets were sonicated in lysis buffer , and the extracted protein was quantified with BCA kit. Then the protein solution was digested with trypsin for two times and labeled with TMT kit (Thermo) according to the manufacturer's instructions. The tryptic peptides were fractionated into 60 fractions by high pH reverse-phase HPLC, combined into 9 fractions, and then analyzed by EASY-nLC 1000 UPLC system and Orbitrap Fusion Lumos mass spectrometer (Thermo). Raw MS files were analyzed using MaxQuant software (version 1.5.2.8). Tandem mass spectra were searched against SwissProt Human database . TMT-6plex was specified as fixed modifications, and the false discovery rate was set at 0.01 for both protein and peptide identification. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner FRT HeLa cell line stably expressing HA-tagged XAB2 was generated using Flp-In System (Invitrogen) according to the manufacturer's protocol. To immuno-precipitate XAB2, HA antibody (Biolegend) was crosslinked to protein G Sepharose beads  with DMP. The cells were harvested 24 h after Dox induction for nucleo-cytoplasmic separation to prepare nuclear extract as previous described . Nuclearhttp://amp.pharm.mssm.edu/Enrichr/) (For gene list enrichment (Gene Ontology and KEGG) analysis, genes were applied to Enrichr web application (nrichr/) ,48.Senescence-associated β-galactosidase  was detected using SA-β-gal Staining Kit  following the manufacturer's protocol. Cells were then photographed using Ti-S microscope (Nikon).t-test with statistical significance defined as: *P < 0.05, **P < 0.01 and ***P < 0.001.Data were presented as mean ± SD (standard deviation of the mean). Statistical analyses between two groups were performed using Student's Previous studies indicated that XAB2 may function in transcription regulation ,28, howeNext, IF staining of POLR2A was performed in XAB2 depleted HeLa cells, consistent with the decreased RNA and protein levels of POLR2A, POLR2A IF signal was substantially weakened compared to the control Figure . InteresTo further determine whether down-regulation of POLR2A after XAB2 depletion will have an impact on global transcription, we depleted XAB2 in HeLa cells and analyzed the expression of newly transcribed RNA using fluorescent labeled EU incorporation assay. As shown in Figure To investigate how XAB2 regulates POLR2A expression, we first did time points assay using Dox-inducible XAB2 knockdown stable cell clone. As shown in We next tested whether the decrease of POLR2A mRNA is regulated at transcription level. To do this, POLR2A or CENPE promoter reporter was transfected to inducible HeLa cell line expressing shRNA against XAB2, and luciferase activities were assayed after Dox induction. As shown in Next, we performed total POLR2A (detected with 8WG16) ChIP on the POLR2A gene using Dox-inducible XAB2 knockdown stable cell clone at 36 h (XAB2 was depleted but POLR2A protein was not down-regulated) and 72 h  of induction respectively . As showTo further investigate whether down-regulation of POLR2A at RNA level induced by XAB2 knockdown is due to reduced transcription or not, we examined the level of POLR2A nascent RNA transcripts after XAB2 depletion. Western blot using specific markers to each fraction indicated clean separation of chromatin from other fractions . As showSince XAB2 is a Prp19 complex component, we then tested whether down-regulation of POLR2A is due to splicing defects induced by XAB2 depletion. We designed two sets of primers to assay intron retention of POLR2A intron 1 and intron 10  genes and increased expression of 5778 and 5298 (4179 overlapped) genes respectively . Among them, about 50% was intron retention (IR) events. Strikingly, among 15,150 and 13,762 IR events induced by siXAB2-1 or siXAB2-2, 14,691 (97.0%) and 13,362 (97.1%) were increased levels of IR respectively, five of which were verified by RT-PCR . Among them, 243 proteins were up-regulated and 460 proteins were down-regulated (In an attempt to identify such factors, we performed TMT-based quantitative proteome analysis on HeLa cells after XAB2 depletion. This analysis revealed a total of 703 proteins with significant change in expression (>1.2-fold, egulated . KEGG anegulated . Accordiegulated , includiegulated . Specifiegulated  and protegulated . Analysiegulated  and L. TTo identify proteins interacting with XAB2, we performed immuno-precipitation followed by mass spectrometry. Consistent with previous study , proteinTo determine if these proteins have a similar impact on POLR2A expression, we treated HeLa cells with siRNA against some of these factors. Depletion of SNRNP200, EFTUD2, SNW1, PRPF8, PLRG1 and AQR also caused a reduction at POLR2A RNA and protein levels, but depletion of Prp19 or CDC5L showed no apparent effect on POLR2A protein expression Figure –F. TogetTo investigate the domain requirement for XAB2-mediated POLR2A expression, we constructed Dox inducible HeLa cells stably expressing siRNA-resistant full-length human XAB2 or a series of truncation mutants Figure . Dox indP < 0.001, Figure To gain deep insight into the physiological role of XAB2, we checked whether XAB2 could modulate cellular senescence. First, we depleted XAB2 in the human skin fibroblast cell line HFF1, SA-β-gal staining showed that XAB2 knockdown significantly induced cellular senescence (36.4% and 36.2% in XAB2 siRNA-1 and siRNA-2 transfected cells respectively compared to 3.8% in control, P < 0.001, Figure Similarly, depletion of POLR2A in HFF1 cells resulted in striking increase of SA-β-gal positive cells (49.7% in POLR2A siRNA transfected cells compared to 3.1% in control, To further confirm whether XAB2 regulates cellular senescence via POLR2A, HFF1 cells were treated with XAB2 siRNA-1 followed by over-expression of POLR2A. As shown in Figure Gene expression is a fundamental cellular process that is essential for life. Although XAB2 has been reported to function in several regulatory processes, such as transcription, pre-mRNA splicing and mRNA export, detailed mechanism on regulation of gene expression by XAB2 remains unclear. In this study, we reported POLR2A as a major target gene down-regulated after XAB2 depletion, XAB2 depletion resulted in substantial loss of POLR2A and triggered cascades on global transcription and cell senescence.We previously reported that knockdown XAB2 led to cell death, cell cycle blockage and mitotic defects , in an aAs SYF1, the homolog of XAB2 in yeast, has been reported to interact with RNA pol II and function in transcription elongation , we firsSeveral studies reported that knockdown of proteins functioning in pre-mRNA splicing led to reduction of POLR2A. For example, depletion of splicing factor CWC22 caused POLR2A mRNA down-regulation , knockdoAt late time points of XAB2 depletion, POLR2A protein lost, which should have a significant impact on global transcription and indeed we observed substantial decrease of nascent transcripts using EU incorporation assay. The impact on global transcription was further supported by RNA-seq with a large number of genes showing decreased expression with the other large number of genes showing elevated expression. Consistently, treatment of the cells with splicing inhibitor madrasin led to similar loss of POLR2A and reduction of nascent transcripts. Although it should be noted that madrasin treatment also led to decreased XAB2 level and thus the observed effect of madrasin on POLR2A might be partially due to reduced XAB2 activity.It has been documented that nuclear speckles may act as assembly/modification/storage/recycling compartments that can provide splicing factors to active transcription sites ,54, and It is surprising to mention that inhibition of translation can restore POLR2A mRNA level, whereas depletion of Dom34 restores POLR2A at both RNA and protein levels, this effect is due to increased stability of POLR2A mRNA. Thus in addition to the splicing defects in POLR2A induced by XAB2 deficiency, the level of Dom34 and other proteins in mRNA surveillance may also have an impact on POLR2A mRNA expression. Previous studies revealed that Dom34 and HBS1 joined exosome-Ski complex to function in non-stop decay  and no-gOur previous data showed that XAB2 knockdown led to cell cycle arrest and DNA damage , XAB2 waBased on these data, we propose a model for the role of XAB2 in gene expression Figure . At the While the current study expands our understanding of how XAB2 is involved in gene expression, it raises more interesting questions for future investigation. Detailed function of XAB2 in splicing remains to be dissected, common features of introns with retention are still elusive. Further study will also be needed to gain deep insight into how Dom34 or other proteins in mRNA surveillance function in POLR2A mRNA degradation. Finally, it is fascinating to explore whether POLR2A plays a more general role in different types of senescence or causes human diseases at its deficiency.RNA-seq data have been deposited at the NCBI Gene Expression Omnibus under the accession number GSE130087.gkz532_Supplemental_FilesClick here for additional data file.


Mentoring is a required component of health systems strengthening technical assistance interventions in low- and middle-income countries (LMICs). Mentoring is useful because it does not necessarily compromise service delivery and promotes the sharing of newly acquired knowledge and skills. However, there is a lack of research on the implementation of mentoring in the context of the HIV epidemic in southern Africa.This qualitative evaluation focussed on understanding the implementation process of targeted mentoring for clinical practice, data management and pharmacy management, at public health care facilities in South Africa; and on identifying critical factors influencing the effectiveness of mentoring as a technical assistance intervention in this context.Purposive sampling was used to select participants from public health facilities in three South African Provinces. Participants were invited to take part in structured interviews. Datawere analysed using thematic analysis, and two core themes were identified: mentoring as knowledge and skills transfer; and mentoring as psychosocial support.In terms of knowledge and skills transfer, the sequential implementation of proactive and reactive mentoring was critical. Initial proactive mentoring involved mentors initiating training and developing professional relationships with mentees. Thereafter, a reactive mentoring phase allowed mentees to request support when required. This enabled mentors to leverage real-world problems faced by health workers to support their implementation of new knowledge and skills. The availability and accessibility of mentors alongside the relationships between mentors and mentees provided psychosocial support for health care workers which facilitated their self-efficacy in implementing new knowledge and skills.These findings suggest that the success of mentoring programmes in LMICs may require specific attention to both knowledge transfer and the management of interpersonal relationships. Using mentors to support local staff enables implementing agencies and governments to target specific cadres of health workers to develop and entrench the skills and knowledge required to successfully implement interventions.3While mentoring has the potential to significantly improve service provision, there are a number of important features of the mentor-mentee relationship that may affect the relative success of such programmes. Straus et al. for example, found that successful mentoring relationships were characterised by reciprocity, mutual respect, clear expectations, a personal connection and shared values.2Factors related to the context of local health systems may also affect the success or failure of mentoring initiatives. In Kenya, for example, Ndwiga et al. found that the effectiveness of mentoring was affected by erratic supplies of medication and commodities, high client caseloads and staff shortages.6 While these programmes have shown impressive results, their focus on either clinical or administrative processes may lead to constraints in their effectiveness. Therefore, we explore the role of targeted mentoring using a needs-based approach to support both clinical and some administrative processes as part of a technical assistance package targeting HIV and tuberculosis (TB) services at public healthcare facilities in three provinces of South Africa.Mentoring programmes supporting the scale-up of HIV treatment in sub-Saharan Africa (SSA) have frequently focussed either on mentoring clinical staff or on supporting administrative and logistical processes.The study was conducted at selected health facilities in Limpopo, Gauteng and the Western Cape. Facilities in Limpopo and the Western Cape were situated in rural districts, while the Gauteng facilities were located in peri-urban townships. All facilities had been receiving technical assistance from Anova Health Institute (hereafter referred to as ‘the implementing agency’) since 2010, and most had previously been sites where the agency had directly provided HIV-related services.7 Hence, the focus of the implementing agency’s work between 2009 and 2014 was on HSS and the provision of technical support to the South African Government’s HIV response, with staff mentoring forming a central aspect of this support. Mentoring was provided to nurses to assist with the implementation of nurse initiated and managed ART (NIMART); to data capturers and administrators to assist with managing the ART  programmes reporting requirements; and to pharmacy staff to support efficient stock control and pharmacy management.The implementing agency’s HIV and TB-related health systems strengthening (HSS) support activities were primarily funded by USAID/PEPFAR. In 2009, PEPFAR’s focus shifted away from direct service provision towards technical assistance and HSS.9 as well as research focussing more specifically on mentoring in the context of technical assistance programmes in LMICs.10 This literature was reviewed in reference to the World Health Organization’s 200611 recommendations on the use of clinical mentoring to support HIV programme implementation in resource-limited settings.This study was informed by literature focussing on mentoring as a broad approach to individual and organisational development in various contexts,This study is a qualitative evaluation of the role of mentoring as part of a technical assistance package targeting HIV and TB services as provided by the implementing agency at public healthcare facilities in three South African health districts.Structured interviews to explore health workers’ perceptions of the implementing agency’s mentoring programme were conducted by independent consultants with 74 participants  from government health facilities in Limpopo Province, Gauteng and the Western Cape. Interviews were conducted in person, in private rooms at individual facilities, between October 2014 and July 2015. Refusal rates were very low, with only five participants refusing to participate in interviews as a result of repeatedly being unavailable or having to attend to urgent medical problems in their facilities. Participants were purposively selected based on their involvement in the technical assistance programme. Interviews were conducted with facility managers and their deputies, nurses, pharmacists and data staff. While the evaluation included questions relating to the entire technical assistance package, this article focusses on understanding healthcare workers’ perceptions of the mentoring component of the package.Signed informed consent was obtained from all participants prior to their participation. To maintain anonymity of respondents, no identifying information was collected. The study protocol and materials were approved by the University of the Witwatersrand’s Human Research Ethics Committee (M120625).12 and were analysed using a thematic analysis approach.13 Firstly, interview transcripts were read carefully while paying particular attention to discussion related to the mentoring programme. Once the two authors responsible for data analysis were familiar with the data set, initial coding was conducted independently by each author to identify units of text with relevance to the research topic. Text units dealing with the same issue were grouped together in an initial set of themes and 48 basic themes were interpreted from the data in this stage. The same unit of text could be included in more than one theme.Interviews were audio-recorded and transcribed verbatim. Data were managed using NVIVO 11These themes were then discussed and refined in relation to both the coded content of the basic themes, and the data set as a whole, in order to create a coherent set of organising themes. Five organising themes were produced in this stage of analysis. Finally, these organising themes were analysed to develop two main themes related to the role of the implementing agency’s mentoring programme in implementing HIV-related services. These were: (1) targeted mentoring as knowledge and skills transfer and (2) mentoring as psychosocial support.Signed informed consent was obtained from all participants. The study protocol was approved by the University of the Witwatersrand’s Human Research Ethics Committee (reference number: M120625).14The targeted mentoring programme focussed on supporting three main aspects of the South African Department of Health’s (DoH) antiretroviral therapy (ART) scale up. These were: NIMART, pharmacy management and data management. The mentoring programme adopted a two-stage approach, which we classified as proactive and reactive mentoring.Proactive mentoring involved scheduled visits to health facilities by mentors. During these visits, several activities took place, which were noted as important by the DoH staff. For professional nurses being trained to initiate ART, an important feature of the mentoring visits was having nurse-mentors accompany them during consultations with patients. In this role, mentors were able to provide bridging support between off-site training courses and the actual implementation of the skills learnt on these courses. This was particularly important because initiating and managing ART was a significant increase in nurses’ responsibilities in the context of HIV treatment and care:‘I think the mentoring system is good and it is needed … ART initiation is difficult so the mentoring has helped.’ Supporting nurses in learning to initiate ART, and to manage the care of patients on ART, was a critical feature of participants’ discussions about the role of the mentoring programme, and several participants discussed the knowledge they had gained through the training and mentoring process as empowering them to take on a more active role in treating HIV-positive patients required by the NIMART programme:‘… before I was scared of patients who had HIV/AIDS, I was scared and immediately after I was trained I had power and now I can do it and even critically ill patients are living healthy lives.’ Several participants also discussed support from the implementing agency in terms of their mentors’ roles in assessing and evaluating their practice. In this context, the mentors’ presence facilitated the application of the skills learnt through NIMART training:mentors] come for mentoring us for treatment and checking if we have difficulties. If I have a patient and want to initiate and I am taking the baseline to find the patient is in bad condition they check if we are coping.’ ‘And  come to check how we pack treatment and make sure that if over-stocking arrange the drugs so we use the old ones first, and they come and check the data capturer …’ ‘… mostly [Name] helps to ensure the stock in the pharmacy is arranged logically.’ ‘They do the stock checks of ARVs and the other stock. They also available on the phone to help with stock control. [the implementing agency]. When they come and check the computers and check the data and check the data is clear.’ ‘We get support from  does the work but does not explain what she is doing … she does more clinical work than a mentor should do.’ ‘ gives technical support whenever I have a problem, I call or they visit and whatever challenges I face they come.’ ‘ and she consulted with another doctor and came back to me to do in-service training and teach us about the HIV virus and how deal with the patients.’ ‘Last week there was a patient here and we called [Name] is my mentor and she regularly comes to check my defaulter list – every problem I come across she is there to help … there was a duplication of patients and she helped.’ ‘ can phone [the implementing agency].’‘With complicated cases I like to know that [‘I’m being mentored at the moment, and it really helps to know the doctor is there.’ Participants’ willingness to contact their mentors when they experienced problems was also a result of trust and support that mentors built up when interacting with the DoH staff when they were asked for help:‘And when my mentors come and evaluate me they don’t complain, they just guide.’ ‘They give us a listening ear … they do not come to find mistakes but to give us support and information to have team work spirit.’ These quotes also point to the importance of developing personal relationships between mentors and mentees which in turn meant that participants felt comfortable enough to contact their mentors when they needed help.A second important way in which the mentoring programme provided psychosocial support to the DoH staff was by facilitating an improvement in health workers’ self-efficacy for implementing their training and managing the initiation of ART without having doctors present:‘I am like a doctor in the clinic as I can initiate.’ ‘I feel it empowered me through what I learnt and learn every day.’ Finally, several nurses discussed how their mentors provided an emotional outlet, which allowed them to deal with the stress of taking on the responsibility of initiating and managing patients on ART:‘And the mentors come and encourage us as sometimes we get depressed when patients stop taking drugs.’ ‘Their support is helpful, and if we are feeling stressed we offload.’ The use of mentoring as an integral part of the implementing agency’s technical assistance programme appears to be successful in facilitating healthcare workers’ implementation of knowledge and skills gained through training. Based on our interpretation of evaluation interviews conducted with healthcare workers, we suggest several attributes of the programme contributed to this success.15 Our findings suggest that health facility staff value these broader approaches to mentoring that include non-clinical staff as they may facilitate an overall improvement in services available to patients while simultaneously limiting constraints to the impact of clinical mentoring resulting from administrative or pharmacy-related problems. However, this approach does not address broader health system management issues, which Edwards et al. suggest may further improve patient outcomes.6Firstly, the mentoring programme used a multifaceted approach to supporting the health system. The implementing agency’s prior service delivery in the three provinces led them to identify key bottlenecks to ART implementation, and the mentoring programme was therefore designed to support the clinical, administrative and pharmacy facets of the health system simultaneously. Chien et al. studied a similar approach to mentoring in Malawi and found that supplementing clinical mentoring with support for facility and district-level systems improved the overall impact of the mentoring programme.18 In the context of HSS in SSA, Edwards et al.6 found that mutual trust between implementing agency and Mozambique DoH staff based on an open and collaborative relationship was a critical enabling factor supporting the success of health management mentoring. The importance of developing trusting mentoring relationships during proactive mentoring phases of programme implementation should therefore be emphasised to mentors during the preparatory stages of these programmes.A second important attribute of the mentoring programme was the fact that the programme adopted a sequential proactive and reactive approach, which allowed mentors to leverage real-world problems faced by health workers to support their implementation of new knowledge and skills. The sequence of implementing the proactive and reactive aspects of the programme directly affected the effectiveness of mentoring. It was important that initial proactive mentoring occurred to build trust and relationships between mentors and mentees. Based on these relationships, health workers were comfortable enough to ask for help (i.e. to initiate reactive mentoring) when they needed it. Where mentors did not build relationships with mentees sufficiently, there appeared to be less willingness to ask for support. The development of trusting relationships between mentors and mentees as a prerequisite for successful mentoring relationships is a common finding in the mentoring literature.8 More broadly, however, Eller et al. found that a ‘caring personal relationship’ was an important aspect of effective mentoring relationships among nurses in the US, and the emotional support provided by mentors in other contexts has been found to improve mentees’ self-efficacy and emotional well-being.19 Our findings support the value of the mentor-mentee relationship as a source of psychosocial support. This suggests that the effectiveness of ART scale-up using mentoring programmes could potentially be improved by paying particular attention to the development of supportive relationships within these interventions.Finally, mentors played an important role in providing psychosocial support to nurse mentees in particular. Psychosocial support was important because it helped nurses to develop the self-confidence to apply their NIMART training and to take responsibility for managing the ongoing care of patients on ART without direct supervision. There were several ways in which the mentoring programme was able to provide this type of support. Simply having the knowledge that mentors were available if they needed them enabled nurses to initiate patients onto ART without excessive anxiety about managing side effects or complications. Mentors also played an important role in providing psychosocial support by providing a means for nurses to deal with the stress of managing patients on ART. Here, the personal relationships between mentors and mentees were critical, and several participants noted how important their mentors’ caring and supportive attitudes were in helping them to gain confidence in implementing their new skills and knowledge. Although psychosocial support has long been recognised as a critical aspect of mentoring relationships, there is relatively little research conducted on the role of psychosocial support for mentees in health system-related interventions in LMICs.There are some limitations to our study that may have affected our interpretation of the data. Firstly, our data only included interviews with the DoH staff; as such it is possible that we would have reached different conclusions if interview data were available from mentors and other implementing agency staff. Secondly, the authors are employed by the implementing agency, and while we made a conscious effort to remain objective, it is possible that our interpretations of the data were skewed by our personal investment in the mentoring programme.In spite of these limitations, this study is among the first to examine the role of mentoring in strengthening HIV and TB programmes in South Africa. By identifying key factors supporting and limiting the success of the mentoring programme, this study may provide important lessons for the implementation of similar programmes elsewhere.6 However, the likelihood of success of mentoring programmes depends on the dual role of mentors as supporting the proactive and reactive transfer of knowledge and skills to mentees, while simultaneously providing the psychosocial support necessary for mentees to develop the confidence to apply these in practice. Where this balance in mentors’ dual role is successfully achieved, mentoring is likely to be a powerful tool to improve healthcare globally.Mentoring is a well-recognised and frequently used means of providing technical assistance in health-related interventions around the world.


This allows IP3Rs to evoke regenerative Ca2+ signals, the smallest of which are Ca2+ puffs that reflect the coordinated opening of a few clustered IP3Rs. We use total internal reflection microscopy (TIRF) microscopy to record Ca2+ signals in HEK cells expressing all three IP3R subtypes or a single native subtype. Ca2+ puffs are less frequent in cells expressing one IP3R subtype, commensurate with them expressing fewer IP3Rs than wild-type cells. However, all three IP3R subtypes generate broadly similar Ca2+ puffs with similar numbers of IP3Rs contributing to each. This suggests that IP3R clusters may be assembled by conserved mechanisms that generate similarly sized clusters across different IP3R expression levels. The Ca2+ puffs evoked by IP3R2 had slower kinetics and more prolonged durations, which may be due to IP3 binding with greater affinity to IP3R2. We conclude that Ca2+ puffs are the building blocks for the Ca2+ signals evoked by all IP3Rs.All three subtypes of inositol 1,4,5-trisphosphate receptor (IP Summary: All IP3 receptor subtypes can generate Ca2+ puffs, suggesting that these coordinated openings of clustered IP3Rs are the building blocks of all IP3-evoked Ca2+ signals. IP3 binding to IP3R initiates its activation by promoting the Ca2+ binding that leads to channel opening . Each forms a Ca2+ channel that is co-regulated by IP3 and Ca2+, but the subtypes differ in their expression patterns  in human embryonic kidney 293 (HEK) cells expressing one or all three IP3R subtypes. We demonstrate that all three IP3R subtypes generate Ca2+ puffs with broadly similar properties. We conclude that Ca2+ puffs are the fundamental building blocks for Ca2+ signals evoked by all IP3Rs.Here, we use total internal reflection fluorescence microscopy (TIRFM) to record Ca3R subtypes  .We used wild-type (WT) HEK cells, which express three IPsubtypes , and HEKsubtypes . Identicine-IP3) . We conf3R subtype (Ab1–3) confirmed that WT HEK cells express three IP3R subtypes , we established that overall IP3R expression levels were similar in each cell line expressing a single IP3R subtype , photolysis of ci-IP3 evoked Ca2+ puffs after a short latency. The Ca2+ signal then propagated after a few seconds to produce a global increase in cytosolic free Ca2+ concentration ([Ca2+]c)  A–D. Thes[Ca2+]c) . However[Ca2+]c) , the sitcordings .Fig. 2.3 also evoked Ca2+ puffs in HEK cells expressing single IP3R subtypes. In these cell lines, all of which were exposed to the same stimulus, the latency before the first Ca2+ puff was longer than in WT cells, Ca2+ puffs were less frequent, we detected fewer Ca2+-release sites and it was rare for Ca2+ signals to propagate into a global increase in [Ca2+]c . Fortuitously, the average number of Ca2+ puffs detected during the entire recording interval was not significantly different between the four cell lines, but we detected significantly more Ca2+ release sites in WT HEK cells . The number of new sites identified in each successive interval declined mono-exponentially with half-times (t1/2=3.76–5.53 s)  that were fast enough to ensure that, within the 40-s recording interval , we should identify >94% of all Ca2+ release sites. The analysis of WT cells used a shorter time-bin (2 s) to accommodate the increased frequency of Ca2+ puffs (2+ release sites (t1/2=2.01 s), indicating that we should again detect >93% of sites . Hence, for all cell lines our analyses are likely to detect most Ca2+ release sites. The fewer sites identified in cells with single IP3R subtypes are, therefore, not due to inadequate sampling. We conclude that the cell lines with single IP3R subtypes express about one-third the total number of IP3Rs of WT cells   before the Ca2+ signal invaded the cell, whereas – for the other cell lines – it entailed recording less frequent Ca2+ puffs for longer periods (40 s) .There is considerable variation in the shape and duration of Caig. S1E) . For Ca2baseline C. To secs (40 s) F,G. To v2+ puffs in the four cell lines . The rise times (∼20 ms), decay times (∼30 ms) and durations at half-maximal amplitude (∼30 ms) were similar in WT cells and in cells expressing only IP3R1 or IP3R3. However, the rise and decay times  were significantly longer in cells expressing only IP3R2, as was the mean duration of Ca2+ puffs (62.6±5.6 ms) . We conclude that all three IP3R subtypes generate Ca2+ puffs with broadly similar characteristics, although Ca2+ puffs evoked by IP3R2 typically last about twice as long as those in cells expressing IP3R1, IP3R3 or all three subtypes.We compared mean rise times, decay times and durations of the Call lines A–C and t±5.6 ms) . The fre2+ puff, it was sometimes possible to observe stepwise closures of individual IP3Rs   and the peak amplitude of each Ca2+ puff, to estimate the number of IP3Rs contributing to a Ca2+ puff . In WT cells, the mean number of IP3Rs per puff was 2.77±0.10  . The ampig. S1E) B, suggesductance . We useda2+ puff . The res.77±0.10 C,D. Our a2+ puff . Collectht IP3Rs . The meaWT cells C,D, but ectively D. We con subtype D, Ca2+ pell line .Fig. 4.2+ puffs occurring in the most-active cells differed in their properties. There were no clear differences in the frequency distribution for rise time, decay time or Ca2+ puff duration, or in the estimated number of IP3Rs per puff for the more-active and less-active cells . We also compared the mean peak amplitude of Ca2+ puffs evoked in each cell with mean rise and fall times. Again, there was no significant correlation between puff amplitude and kinetics; the only exception was a positive correlation between amplitude and decay time for cells expressing IP3R2 .We considered whether Ca3R subtypes generate Ca2+ puffs with broadly similar properties. Ca2+ puffs have hitherto been observed only in cells that express all three IP3R subtypes, albeit with one subtype usually predominating. Ca2+ puffs originate at fixed intracellular sites, at each of which a loose cluster of a few IP3Rs is anchored near the plasma membrane, where an additional signal licenses the cluster to respond to IP3 . These results suggest that, first, for each subtype, IP3Rs are similarly distributed across the ∼90-nm depth of the TIRF field, consistent with each coming close to the plasma membrane. Second, across a range of IP3R expression levels, IP3Rs are assembled into similarly sized clusters, but with more clusters in cells with more IP3Rs . We conclude that the clusters of IP3Rs that generate Ca2+ puffs are probably not random associations. Instead, IP3Rs appear to be assembled through mechanisms that are conserved for all IP3R subtypes, and which ensure that similarly sized clusters are assembled irrespective of IP3R expression levels.We show, for the first time, that all three IPd to IP3 . Neithersubtypes D, yet ea2+ puffs evoked by cells expressing all IP3R subtypes or by those expressing only a single subtype are striking. However, Ca2+ puffs evoked by IP3R2 were more long-lasting than those evoked by other subtypes. We have not determined the mechanisms responsible for the slower kinetics of Ca2+ puffs evoked by IP3R2. One possibility is that the greater affinity of IP3 for IP3R2  and Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 (50:50) supplemented with GlutaMAX were from ThermoFisher . Pluronic acid F-127, dimethyl sulfoxide (DMSO), poly-l-lysine and foetal bovine serum were from Sigma-Aldrich . EGTA-AM was from Merck Millipore . Caged-IP3-PM  was from SiChem GmbH . Custom-made rabbit anti-peptide antibodies against IP3R2  and to a sequence conserved in all three IP3R subtypes  were provided by Poccono Rabbit Farm and Laboratory Inc. . Additional primary antibodies were: IP3R1 , IP3R3  and β-actin . Secondary antibodies conjugated to horseradish peroxidase  were from Insight Biotechnology Ltd .Cal520-AM was from Stratech Scientific . Bovine serum albumin (BSA) was from Europa Bio-Products . Gibco TrypLE Express, NP-EGTA-AM   were grown on poly-l-lysine-coated (10 µg/ml) glass-bottomed dishes . They were used after 48 h, when they were sub-confluent.WT HEK cells, from which the gene-edited lines were generated, were provided by Dr D. Yule . HEK cells in which CRISPR/Cas9 had been used to generate lines expressing a single IP(HEK-KO)  were frog, 4 min, 4°C) were separated  and transferred to a polyvinyl difluoride (PVDF) membrane using an iBLOT gel-transfer system (ThermoFisher). Membranes were blocked in Tris-buffered saline  and incubated  with primary antibody. After three 5-min washes in TBS, membranes were incubated  with secondary antibody and washed (3×5 min). Bands were visualised with ECL Primer Western blotting detection reagent  and a Syngene PXi chemiluminescence detection system . We confirmed that, within the range of loadings used for analysis, IP3R band intensities scaled linearly with the amount of protein loaded.Cells were lysed in cold RIPA buffer  with protease inhibitors . Proteins in the supernatant  relative to the common antibody (e.g. Ab1:AbC for IP3R1). These ratios were used to convert intensities of IP3R subtype-selective bands in WT cells to AbC intensities, from which we estimated the relative expression of the three IP3R subtypes  and ci-IP3. Cells were washed with HEPES-buffered saline  and then incubated (20°C in darkness) in HBS containing pluronic acid F-127 (0.02% w/v), Cal520-AM (5 µM) and ci-IP3-PM (1 µM). After 1 h, cells were washed and incubated in HBS containing pluronic acid F-127 and EGTA-AM (5 µM). After 45 min, the medium was replaced with HBS. After a further 30 min, the cells in HBS at 20°C, were used for imaging. The same method, but with NP-EGTA-AM (1 μM) replacing ci-IP3 PM, was used to load cells with caged EGTA .To record local Caiffusion , a Ca2+ 2+ signals by using total internal reflection fluorescence microscopy (TIRFM). Excitation (488 nm) was provided by a diode-pumped solid-state laser  and a band-pass filter . The angle of the excitation beam was adjusted to achieve TIRF with a penetration depth of ∼90 nm. Emitted light was captured through a band-pass filter  using an Andor iXon 897 EMCCD camera . To achieve high temporal resolution, images were captured (188 Hz) from only a part of each cell  by streaming directly into RAM. After visualisation using Metamorph , images were exported as tif files. Images were captured for 40 s before and after flash photolysis of ci-IP3.An Olympus IX83 inverted microscope equipped with 100× oil-immersion TIRF objective  was used for measurements of local Ca3 or NP-EGTA within the entire imaging field was achieved using a light-emitting diode , with the duration of exposure  determined by a shutter controlled within Metamorph. The conditions used for flash photolysis of ci-IP3 were optimised to allow identical stimulation of all cell lines, with each line providing sufficient Ca2+ puffs for quantitative analysis within a practicable recording interval (≤40 s).Photolysis of ci-IPFig. S2C). Images were processed in Fiji  that identifies clusters of pixels within which fluorescence changes exceed a critical threshold. From each recording, the last 500 frames (∼2.7 s) before photolysis were averaged to provide an F0 value and its standard deviation (s.d.) for each pixel. Image stacks of [(F/F0 )−1]/s.d. were then created and Gaussian-filtered, and pixels that exceeded a critical value  were located. A threshold-cluster algorithm , we measured the unitary steps during the decay phase of 40 Ca2+ puffs , where ΔF = Fpeak−Fpre. Fpeak and Fpre are, respectively, the F/F0 values determined at the peak of the Ca2+ puff and the average from the same region for ten frames before and after the puff.To estimate the number of active IPn usually referring to the number of cells analysed. Statistical analyses used two-tailed Mann–Whitney tests or, for multiple comparison, the Kruskal–Wallis test with Dunn's correction for multiple comparisons . Statistical significance is denoted by: *P<0.05, **P<0.01, ***P<0.001.Results are presented as means±s.e.m. unless stated otherwise, with