Concrete structures are often in different humidity conditions that have a significant impact on the elastic modulus of concrete, therefore, systematic research on the evolution of the law of concrete elastic modulus under different humidity conditions is needed. In this study, the variation laws of the water saturation of concrete specimens with strength grades C15, C20, and C30 were obtained, and then the influence laws of the water saturation on the concrete axial compressive strength were carried out, and the prediction model of elastic modulus of concrete with respect to water saturation was constructed. The results showed that the water saturation of concrete with strength grades C15, C20, and C30 increased with an extension of immersion time, and the water saturation showed an approximately linear rapid growth within three soaking hours, reaching 47.56%, 71.63%, and 47.29%, respectively. Note, the concrete reached saturation state when the soaking time was 240 h. The axial compressive strength with strength grades C15, C20, and C30 decreased with increased water saturation, and the axial compressive strength of saturated concrete decreased by 27.25%, 21.14%, and 20.76%, respectively, as compared with the dry state concrete. The elastic modulus of concrete with strength grades C15, C20, and C30 increased with increased water saturation, and the elastic modulus of saturated concrete was 1.18, 1.19, and 1.24 times higher than those of dry concrete, respectively. The elastic modulus and axial compressive strength of concrete is an important mechanical parameter of concrete, which has a significant impact on the structural distortion and the expansion of a concrete track ,2,3. It −6/s and increased by 30–63% when strain rate was 10−3/s, respectively. The study of Wang and Li [−6/s and increased by 35.5% when the strain rate was 10−3/s. The results of Rossi et al. [0.3/s, but decreased by 4.5% when the strain rate was 10−6/s.There are three main viewpoints throughout the research results related to elastic modulus of wet concrete. The first viewpoint was that a wet state has no effect on the elastic modulus of concrete; the experimental results of Bjerkeli et al.  showed tg and Li  showed ti et al.  showed tThe elastic modulus of single strength grade concrete has been researched in previous studies, however, the sensitivity of elastic modulus affected by humidity conditions of concrete with different strength grades was different.In the physical experiment of concrete elastic modulus, the research range of humidity was mostly dry and saturated humidity conditions, and there was no intermediate transient excessive humidity condition.The humidity level was expressed in terms of water content in previous studies, but the water content was an absolute quantity. The proportion of pores in concrete filled with water could be expressed by the moisture content.In conclusion, the physical experiment conclusion on the effect of humidity on the elastic modulus of concrete is still controversial, but the main conclusion is that the elastic modulus of concrete increases with an increase in humidity, and the larger the strain rate, the higher the increase of the elastic modulus of concrete. At present, research results still have the following three problems:This research was carried out on the evolution law of elastic modulus and axial compressive strength of concrete with three different strength grades, i.e., C15, C20, and C30, in six different humidity conditions to systematically research the influence law of humidity on elastic modulus of concrete. The concrete humidity conditions included completely dry, fully saturated, partially saturated, and intermediate humidity conditions. The humidity condition was expressed in terms of water saturation, and the proportion of pores filled with water was reflected through water saturation (range 0–100%). The prediction model of concrete elastic modulus with strength grades C15, C20, and C30 to water saturation was constructed. The research results on the variation law of concrete axial compressive strength and elastic modulus under different humidity conditions provides a foundation for the evaluation of the mechanical properties of concrete structures with different levels of humidity.3, and the accumulation density was 1540 kg/m3. The coarse aggregate was selected from pebbles, and the clay content of pebbles was 0.6%, the apparent density was 2650 kg/m3, the bulk density was 1563 kg/m3, and the maximum aggregate particle size was 40 mm. The strength grades of the concrete were C15, C20, and C30, which were cured for 28 days under standard conditions  according to the Hydraulic Concrete Mix Design Code DL/T5330-2005 [Composite Portland cement with P·C 32.5R grade was used in this test. The fine aggregate was selected from well-graded natural medium sand. The fineness modulus was 2.43, the clay content was 0.8%, the silt content was 0.8%, the apparent density was 2590 kg/m330-2005 .The temperature of an electric constant temperature blast drying oven ranged from room temperature to 300 °C, its working size was 800 × 800 × 1000 mm, its temperature uniformity was ±2.5%, and its temperature fluctuation was ±1 °C. The precision of electronic scales was 1 g. The maximum test force of the microcomputer-controlled electro-hydraulic servo pressure testing machine was 2000 kN and its test force accuracy was 1%. The component composition of the elastic modulus’ tester of the concrete is shown in The control factors of the experiment were the strength grade and the water saturation of the concrete. According to the preset factors, the concrete strength grade was set as C15, C20, and C30. The water saturation was obtained by soaking for different time periods; the immersion time periods were set as 0, 3, 10, 24, 72, and 240 h, and six humidity conditions from a completely dry state to a nearly saturated state were obtained, respectively. In this study, eighteen experimental groups were set, and six specimens were set in each experimental group, among which three specimens were used to measure the axial compressive strength of the concrete and the other three specimens were used to measure the elastic modulus of the concrete. A total of 108 specimens were tested. The specific experimental group setting is shown in 0, B0, and C0 were three completely dry groups. After standard curing for 28 days, the specimens were roasted using an electrothermal constant temperature and air-blown drying oven at 105 °C, until the vaporization mass per unit surface area was less than 0.002 kg/(h × m2) per unit time, which was deemed to have reached the completely dry state. The axial compressive strength and elastic modulus were measured, after natural cooling. A1–A5, B1–B5, and C1–C5 were the remaining fifteen experimental groups with different water saturations. First, the specimen was made to reach the dry state, and the dry process was the same as that of the completely dry group. After the specimen was cooled naturally, the mass 1–A5 were soaked for 3, 10, 24, 72, and 240 h, in turn, the specimens were taken out of the water tank and placed on an iron rack to control the water, for five minutes. Then, the surface was wiped with a wet cloth so that there was no visible water on the surface, but it still remained moist. The mass −3 kg/(m2 × h), the specimens were considered to have reached the approximate saturation state, and the mass of concrete specimens, in the saturated state, was 1–B5 and C1–C5 were determined by the same method as:AP was recorded. The axial compressive strength of each experimental group was the arithmetic average of the measured values of three specimens. If the difference between any measured value and the median value exceeded 15% of the median value, the median value was taken as the measured value. If the difference between the two measured values and the median value exceeded the above provisions, the test results of the group were invalidated. The axial compressive strength, fc, was calculated according to Equation (2) as follows:2), and The axial compressive strength was measured using a fully automatic microcomputer servo universal testing machine. Concrete specimens were placed on the underplate of the press with geometric alignment. The loading rate was 0.3 MPa/s and the loadings were uniform and continuous. When the specimen was close to failure and began to deform rapidly, the press accelerator was stopped and adjusted, until the specimen was damaged, and then the maximum load cf, which was the control load 2), For the determination of elastic modulus, first, each specimen was preloaded and the load was uniformly applied to 40% of the axial compressive strength The final test result was the arithmetic average of the measured values of the three specimens for elastic modulus. If the difference between the axial compressive strength of one specimen after the elastic modulus was measured and that of the three specimens was more than 20% of the latter, the measurement result of the elastic modulus of the specimen was discarded, and the average value of the remaining two specimens was taken as the final test result.The water saturation of each experimental group was the arithmetic mean value of the water saturation of six specimens. It can be seen from the When the concrete had soaked for 3–24 h, at this stage, the water mainly entered into the dry concrete pores through adsorption and surface diffusion. A water adsorption layer formed on the pore wall, therefore, the coefficient formed was mainly surface water absorption. The water saturation variation rule of C15 concrete soaked for 3–24 h was not exactly consistent with C20 and C30 concrete. The water saturation variation rule of C15 concrete soaked for 24–72 h was not exactly consistent with C20 and C30 concrete, and many repeated experiments were carried out in order to confirm the authenticity of this conclusion, indicating that this experimental conclusion was not accidental. When concrete was soaked for 24–72 h, as the concrete internal humidity continued to increase, water continued to be transmitted to the specimen in the form of a water film, and the form of water absorption was seepage. At this moment, the strength of seepage was closely related to the quantity and size of the concrete pores. The porosity of C15 concrete was higher than C20 and C30 concrete, and therefore the water saturation variation rule of C15 concrete soaked for 24–72 h was not exactly consistent with C20 and C30 concrete. Research on the change law of concrete water absorption rate influenced by porosity will be completed in future research work.2P, in the process of elasticity modulus measurement, the axial compressive strength of the cylinder specimens was measured. The axial compressive strength was tested by using a microcomputer-controlled electro-hydraulic servo pressure testing machine. The loading rate was 0.3 MPa/s. The maximum test force of the microcomputer-controlled electro-hydraulic servo pressure testing machine was 2000 kN, and its test force accuracy was 1%. The entire test procedure for axial compressive strength was strictly in accordance with the People’s Republic of China Industry Standard (SL 352-2006) Test Code for Hydraulic Concrete [In order to determine the compression load, Concrete . The curThe axial compressive strength of concrete under different water saturation tested values are shown in 5, B5, and C5) decreased by 27.25%, 21.14%, and 20.76%, respectively, as compared with the completely dry experimental group . Under the same water saturation conditions, the lower the strength grade of concrete was, the greater the axial compressive strength was reduced. For example, the axial compressive strength of concrete with strength grade C15 and water saturation of 80% decreased by 19% as compared with the completely dry state, while the axial compressive strength of concrete with strength grade C30 and water saturation of 80% decreased by 14% as compared with the completely dry state, therefore, the axial compressive strength of concrete with low-strength grade was more sensitive to water saturation.It can be seen from The elasticity modulus values under different water saturation values were tested according to the People’s Republic of China Industry Standard (SL 352-2006) Test Code for Hydraulic Concrete . The tesThe variations of elasticity modulus of concrete with strength grades C15, C20, and C30, from a completely dry state to an approximately saturated state, are shown in According to the experimental results of concrete elasticity modulus under different water saturation values, a two-parameter prediction model of concrete elasticity modulus with respect to water saturation was established, as shown in Equation (4). A comparative analysis of the predicted value and the experimental value in this research of concrete elasticity modulus is shown in Di, and the enhancement factors of the concrete elasticity modulus with strength grades C15, C20, and C30 affected by water saturation are defined, in accordance with Equation (5)  in CaW–OS and CaS–OS. Because CaW–OS, CaS–OS, and OS–HW had smaller bond lengths than CaW–OW, CaS–OW, and OW–HW, these chemical bonds decreased in length and increased in strength. The atoms were closely arranged and obviously disordered; the dislocation of atoms was caused during the process of compression, and the deformation capacity of the whole C-S-H molecular system was enhanced, therefore, the elastic modulus of concrete decreased. With an increase in water saturation of the concrete [w in the water molecules replaced part of the Os in the Caw–Os and Cas–Os (the oxygen in the C-S-H molecule) in the formation of Caw–Ow and Cas–Ow, and single Os–Hw turned into double layer or multilayer Os–Hw and Ow–Hw. The mezzanine area of the whole molecular system was increased, atoms that overlapped were significantly reduced, the distance between two atoms was increased, and it was difficult to cause atomic dislocation movement during the stage of compressive deformation. When the chemical bond breaks, the amount of deformation decreases, and the deformation capacity of the C-S-H molecular system weakens, thus, increasing the macroscopic elasticity modulus of saturated concrete [The molecular structure of C-S-H is one layer of calcium oxygen laminar structure sandwiched between two layers of silicon oxygen tetrahedron chain ,31,32. Tconcrete ,33, as sconcrete ,35.The water saturation of concrete increased with an extension in immersion time. The lower the strength grade of concrete, the faster its water absorption rate and the higher the water saturation reached under the same immersion time condition.The concrete axial compressive strength decreased with an increase in water saturation, and the effect of water saturation was significant. The axial compressive strengths of concrete with strength grades C15, C20, and C30, under approximate saturation, decreased by 27.2%, 21.1%, and 20.8%, respectively, as compared with the completely dry state. The concrete axial compressive strength with low-strength grade was more sensitive to the water saturation.The concrete elasticity modulus increased with an increase in water saturation. The elasticity moduli of concrete with strength grades C15, C20, and C30, under a saturated state, were 1.18, 1.19, and 1.24 times higher as compared with the completely dry state, respectively. The elasticity modulus of the concrete with high-strength grade was more sensitive to the water saturation, under the same water saturation condition.


Whilst some guests form 1:1 H⋅G complexes as we have seen before, an extensive family of bicyclic guests—including some substituted coumarins and various saturated analogues—form 1:2 2H⋅G complexes in the solid state, despite the fact that solution titrations are consistent with 1:1 complex formation, and the combined volume of the pair of guests significantly exceeds the Rebek 55±9 % packing for optimal guest binding, with packing coefficients of up to 87 %. Re‐examination of solution titration data for guest binding in two cases showed that, although conventional fluorescence titrations are consistent with 1:1 binding model, alternative forms of analysis—Job plot and an NMR titration—at higher concentrations do provide evidence for 1:2 2H⋅G complex formation. The observation of guests binding in pairs in some cases opens new possibilities for altered reactivity of bound guests, and also highlights the recently articulated difficulties associated with determining stoichiometry of supramolecular complexes in solution.A crystallographic investigation of a series of host–guest complexes in which small‐molecule organic guests occupy the central cavity of an approximately cubic M What a bind: Despite solution titrations showing binding of one guest inside the cavity of an octanuclear cubic coordination cage, in the solid‐state many guests  bind as pairs with up to 87 % of cavity volume being occupied—amongst the highest packing coefficients known. With this knowledge from crystallography, evidence for formation of 2H⋅G species can be found in solution under forcing conditions. The ability of self‐assembled coordination cages to bind small‐molecule guests in their central cavities remains a highly popular area of investigation in the general field of supramolecular chemistry8L12]X16  complex.H⋅G complexes and we have generally assumed this to be the case.Clearly, a detailed understanding of guest binding and the factors underpinning it are essential to underpin work on the properties of such host–guest systems. Guest binding is normally quantified by solution titration methods, often by H, with a wide range of guests, which substantially extends our understanding of the possibilities available for complex formation. In particular, the results of the combined crystallographic and solution binding study highlight important differences between the stoichiometry of a series of cage/guest complexes in solution and in the solid state. Of course there is nothing new in the idea that what is observed in a crystal structure is not necessarily the same as what happens in dilute solution, but the crystallographic studies reported here provide some quite unexpected results about possibilities for guest binding that nicely complement our observations from solution studies. The recent critical focus on methods used to determine the stoichiometry of supramolecular complexes (e.g. Job plots) and the erroneous conclusions that can be easily reached from careless application of unjustified assumptions makes this study particularly timely.We report in this paper the results of a detailed structural investigation of complexes based on our cubic host wH/H is shown in Scheme H) or aqueous (wH) solution as shown by NMR or fluorescence titrations;N‐oxide O atom) and a collection of convergent CH bonds (arising from methylene CH2 and naphthyl CH units) on the cage interior surface, which lie close to a metal ion and are therefore in a region of positive electrostatic potential.The series of guests that we investigated for binding in the cavity of cages Several examples of cage/guest complexes with larger guests that we have crystallographically characterised all demonstrate the same general behaviour, with the two H‐bond donor sites on the cage interior surface providing an anchoring point for H‐bond acceptor sites of the guest which help to position and orient the guest in the cavity. Thus the carbonyl group of a range of bulky aliphatic ketone guests is always anchored in this way,H with guests which could be taken up into the crystals without loss of crystallinity. This has proven to be a far more reliable method than growing crystals of the cage from solution in the presence of guest which usually afforded crystals of the guest‐free cage containing only solvent molecules. Good quality crystals of [Co8L12](BF4)16 (H) can be prepared from a solvothermal reaction of Co(BF4)2 with the edge‐bridging ligand L (2:3 ratio) in MeOH followed by slow cooling,We prepared the cage/guest complexes for crystallographic studies in this paper using the “crystalline sponge“ method,H⋅G complexes, both of which are aliphatic ketones but with either one (guest 1) or two (guest 2) ketone units for anchoring to the H‐bonding sites on the cage interior surface.In this section we describe two new examples of 1:1 H⋅1. The pseudo‐spherical skeleton of this guest is a good shape match for the host cavity and its molecular volume (162 Å3) is about 40 % of the volume of the host cavity , in accordance with the twofold symmetry of the host, it is not appropriate to over‐analyse these interactions. However it is clear that—in common with several previous examples—in complex H⋅1 there is one guest and one water molecule in the cavity, with the guest positioned such that its carbonyl group is docked into one of the H‐bond donor pockets, lying 5.59 Å from the nearby CoII centre at the back of the pocket ‐comparable to what we have seen in previous examples. This Co(cage)⋅⋅⋅O(guest) separation provides a convenient measure of the proximity of the H‐bond acceptor atom on the guest to the docking site on the host.In Figure r volume 2 Å3 is acis‐bicyclo[3.3.0]octane‐3,7‐dione (2) is shown in Figure 2 are located outside the cavity in the spaces between host cages, with these fractional site occupancies amounting to a total of 2.25 additional molecules per cage. Along with the guest 1,2,4,5‐tetracyanobenzene,The complex containing the diketone guest s‐bicyclo.3.0octan3), 4‐methyl‐coumarin (4), 4‐methoxycoumarin (5) and 7‐amino‐4‐methyl‐coumarin (6).In this section is described a set of related structures in which, unexpectedly, we observe a stacked pair of two planar bicyclic guests bound in the cage cavity. This came as a surprise as we had had no previous suggestions from solution binding studies that two guests could bind, with spectroscopic titrations in solution using guests of this type affording binding curves which could be fitted to 1:1 binding models from which the relevant association constants were derived. Systematic structural variations amongst the members of the guest series used have allowed us to determine any trends resulting in the structures observed. Initially we studied a series of coumarins of increasing molecular volume to examine any effects on complex structures in the solid state associated with guest size in the way that we have observed in solution. This set of guests consisted of coumarin (H⋅(3)2 is shown in Figure The structure of the complex 3, which is 36 % of the cage cavity volume. Thus, the volume of a pair of such guests exceeds the limit suggested by Rebek of 55±9 % for the guest volume which would give optimal binding.The molecular volume of coumarin is 149 Å4) and 4‐methoxy‐coumarin (5)  was used in solution experiments on guest binding in this family of hosts as a fluorescent reporter which was quenched on being taken up into the Co8 cage; displacement by competing guests restored its fluorescence, to an extent depending on the binding constant of the competing guests, and we have exploited this as the basis of a fluorescence displacement assay.6 on being taken up into the cage in water followed a 1:1 binding model and yet we see in the solid‐state, again, a stacked pair of guests in the 2H⋅(6) structure  with a CH proton of a pyrazole ring that is directly coordinated to a CoII ion, indicative of the N atom acting as a weak H‐bond acceptor. Hydrogen‐bonding interactions between 6 and the host surface at both termini of the guest will contribute to the high cavity occupancy observed of 87 %, which is the largest value for any 2H⋅G structure in this paper.4‐Methyl‐7‐aminocoumarin  we have a guest that is essentially isostructural with coumarin, apart from the cyclic O being replaced by NH, and unsurprisingly this complex shows the same features as the others which contain two planar guests and an exocyclic H‐bond‐acceptor atom lying in the H‐bond donor pocket at one corner of the cage. Saturation of the double bond of the guest to generate dihydroquinolinone (8) has little structural effect. The arrangement of the two guests across the inversion centre is such that the aromatic ring of one guest lies parallel to and stacked with the non‐aromatic (and now non‐planar) ring of the other guest which tends to suggest that aromatic stacking is not a particularly significant issue compared to steric factors in formation of these 2H⋅G complexes. This suggestion is further reinforced by the structure of the complex containing the wholly saturated guest cis‐octahydro‐benzimidazole‐2‐one (9). In this case the curvature of the guest arising from the syn arrangement of the two rings with respect to one another means that aromatic stacking interactions cannot be relevant in this case, and yet we still see a pair of guests occupying the cavity (packing coefficient 71 %) with each one docked into a different H‐bond donor pocket of the host, in the same way as the structures observed with the aromatic guest pairs.Starting with 2‐quinolinone , isoquinoline‐N‐oxide ,12; a quencher of the excited state in two isostructural cages which incorporate either metal‐based13). The disposition of the pair of guests in the cage cavity, and the main H‐bonding interactions with the H‐bond donor pockets on the cage interior surface, are summarised in Figure The similarity of all of the 12 as guest , although in this case the distance between two carbonyl groups is too short to allow the quinone unit to span the cavity and interact with both H‐bond donor pockets. However we note that one of the quinone O atoms of 12 docks with the H‐bond donor site at one of the fac tris‐chelate positions in the usual way, forming CH⋅⋅⋅O contacts as short as 2.51 Å with the convergent set of CH protons at that site , and again we see that pi‐stacked pair of guests astride an inversion centre such that the electron deficient part of one guest is parallel to and stacked with the electron rich part of the other. Packing coefficients for the cavity containing a pair of guests increase across this series from 71 % (guest 10) to 84 % (guest 13).Of these structures the one with t Figure  is notewe Figure  c; and tHaving seen several unexpected examples of 1:2 host–guest structures in the solid state, as reported above, we were interested to revisit some solution‐based measurements of binding constants to see if we could find evidence for formation of 1:2 complexes in solution, and if so under what conditions.2H⋅G complex was obtained from the NMR titration. In many other cases luminescence titrations have been used, and again—with a range of guests—binding curves that fit to a 1:1 binding model were obtained.2H⋅G complexes in solution. We note also that in some cases, titrations of the host cage with guests in solution showed by NMR spectroscopy that the guest binds in slow exchange, with separate signals observable for free and occupied cage.H⋅G binding: in these cases, a 1:1 model was assumed and K values were determined on that basis.In some of our earlier studies of guest binding, NMR titrations of the host cage with guests in solution sometimes showed that free and bound guest are in fast exchange on the NMR timescale: this results in a steady shift for the signal being observed as the titration proceeds, and the resulting binding curves could normally be satisfactorily fit to a 1:1 model.4) and 7‐amino‐4‐methyl‐coumarin (6). We reported the binding constant for 6 in the cavity of wH in aqueous solution a few years ago as 2.0(±0.2)×104 m−1, on the basis of a fluorescence titration in which the fluorescence of a fixed concentration of 6 was measured as wH was titrated in. In this experiment the normal roles of “host“ and “guest” in this titration are reversed for the spectroscopic convenience of being able to monitor quenching of a fixed amount of the guest; the fluorescence is steadily quenched by proximity to CoII ions as guest 6 is taken up into the cage cavity.The two guests whose binding in the cages in solution we have re‐examined are 4‐methyl‐coumarin ×104 m−1 and K2=3.6(±0.4)×103 m−1. The sum of residuals from the fit—a key element in a critical analysis of binding constantsA repeat measurement  are different.6 inside the cavity of wH this latter issue is avoided if we assume that all bound molecules of 6 are quenched by their proximity to CoII ions, such that binding the first and then the second guest result in an equal loss of luminescence intensity (Δ1=Δ2). The result of a Job plot experiment using different mole fractions of wH and 6 in water with a combined concentration of 0.2 mm is shown in Figure y‐axis shows the loss in luminescence intensity associated with guest binding. The result clearly shows a maximum at a guest mole fraction of two thirds, indicating that w⋅62H complex formation dominates in solution under these conditions. This is the first clear indication we have had that any guest of this size can form an 2H⋅G complex in solution with this cage.An alternative way of evaluating complex stoichiometry is the Job plot. Concerns about the validity of these have been expressed recently as it is easy to use them inappropriately and get misleading results.m guest, Figure m, Figure 2H⋅G complex formation can occur in solution—and the crystal structure confirms 1:2 2H⋅G complex formation in the solid state. The contradiction arises from the different conditions used for each experiment. For the fluorescence titration, addition of increasing amounts of cage wH to a fixed amount of fluorescent guest 6 means that the cage is in increasingly larger excess as the titration proceeds. This, plus the low concentration, results in conditions which will favour 1:1 complex formation. Given that binding of the second guest would result in >80 % cavity occupancy, it is reasonable to expect that K2≪K1. Moreover at low concentrations, simple simulations have shown that K2 can be appreciable compared to K1 and yet the binding curve, which is dominated by the first binding event, still apparently fits to a 1:1 model.m) with proportions of components optimised for 1:2 2H⋅G complex formation. Under these conditions it is clear that K2 is large enough to allow the 2H⋅G complex to dominate in the solution speciation, that is, K2>(1/concentration) which is 5000 m−1. The effects of different concentrations of the titration experiment versus Job plot experiment on the solution speciation are illustrated in Figures Thus we have a situation in which the fluorescence titration curve  under these conditions.We therefore performed an NMR titration at higher concentrations. The low solubility of the cage 6—we have two experiments performed under different conditions giving different results, with the fluorescence titrations indicating 1:1 binding but the Job plot (for 6) and the NMR experiment (for 4) indicating that 1:2 complex formation can also occur, in agreement with the crystallographic results. Together, these observations highlight the recently‐made points about the difficulties associated with determining stoichiometries of supramolecular complexes in solution and the benefits of using different techniques to probe this under different conditions—as well as incorporating in the analysis chemical information obtained about possible host–guest ratios from other sources .Again, therefore—as with guest H revealed a range of different guest binding motifs. 1:1 H⋅G complexes were formed in which a mono‐ketone  and a di‐ketone  were bound in the cage cavity, anchored by H‐bonding interactions between the ketone group on the guest and one or two hydrogen‐bond‐donor sites on the cage interior surface. In the latter case the distance between the two ketone groups in the guest is fortuitously appropriate to allow this guest to span the long diagonal of the cube host and interact with both H‐bond donor sites simultaneously.A crystallographic investigation of an extensive series of host–guest complexes based on the octanuclear cubic coordination cage host 3–13), including a family of substituted coumarins as well as some saturated bicyclic analogues, we unexpectedly observed in the crystal structures the inclusion of two guests in the cage cavity lying astride an inversion centre. In every case each guest showed the same type of hydrogen‐bonding interaction with the pockets on the cage interior surface that we saw in the 1:1 complexes, with each guest interacting with one of the two binding pockets. For the aromatic members of this series the two guests are separated by a graphitic π‐stacking distance; however, with saturated analogues the cavity still accommodates two guests, indicating that π‐stacking in the pair is not a prerequisite for both guests to fit in the cavity. The sum of the molecular volumes of the two guests—up to 87 % of the cavity volume, which are some of the highest packing coefficients knownWith many other bicyclic guests however , despite solution measurements indicating 1:1 host–guest binding.Given the knowledge that 1:2 H/wH cage systemThis is potentially interesting as encapsulation of two guests in the confined space of a cage cavity, even if this constitutes only a small proportion of the equilibrium speciation in solution, could potentially provide a pathway to new forms of catalytic behaviour or altered reactivity associated with a pair of molecules held in close proximity. There are many examples of light‐triggered reactions between two species held in close proximity in supramolecular assemblies—either cage‐type species,H/wH: and also highlight some of the recently expressed difficulties in determining stoichiometries of supramolecular complexes.Overall, these results provide substantially improved insights onto the guest binding and potential future catalysis‐based properties of our cage system H used for the X‐ray diffraction experiments were prepared solvothermally from a mixture of Co(BF4)2 and the ligand L in a 2:3 ratio in MeOH using the method previously published.Batches of single crystals of m detector. Each crystal was mounted on a MiTeGen micromount using a perfluoropolyether oil, and cooled for data collection by a Cryostream nitrogen‐gas stream.F2 values.3–13), most of the crystal structures refined successfully with each guest having 100 % site occupancy in the asymmetric unit, that is, there are two complete guests per cage. The exceptions are guest 9 which refined with a site occupancy of 0.63 per asymmetric unit (i.e. 1.26 guests per cage), and guest 12 which refined with a site occupancy of 0.86 per asymmetric unit (i.e. 1.72 guests per cage), presumably due to incomplete uptake of guest by the crystal used. However the total occupancy of >1 guest per cavity in each case confirms the possibility for two guests to be bound simultaneously.X‐ray crystallography measurements were performed in Experiment Hutch 1 of beamline I‐19 at the UK Diamond Light Source synchrotron facility,http://supramolecular.org.Software used: binding constants were calculated using the Bindfit software, and the simulations in Figure The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


IgG4-related disease (IgG4-RD) is a systemic fibro-inflammatory condition that predominantly involves exocrine organs. Concerning its thoracic presentation, it often manifests as interstitial lung disease or fibrosing mediastinitis. It is very rare for IgG4-RD to form a well-defined mass in the anterior mediastinum, mimicking an encapsulated thymoma.An 82-year-old man with autoimmune pancreatitis under treatment with oral corticosteroids was found to have peripancreatic lymphadenopathy on computed tomography. Subsequent positron emission tomography revealed abnormal uptake  by a thymic mass as well as the peripancreatic lymph nodes. Exacerbation of IgG4-RD was suspected, and we increased the oral steroid dosage. As a result, the peripancreatic lymph nodes, and the anterior mediastinal mass, decreased in size. The thymic mass was suspected to be an encapsulated thymoma because of its lobulated shape, degree of fluorodeoxyglucose accumulation, and response to steroids, and the patient was referred to our department. The serum anti-acetylcholine receptor antibody test was negative. A thoracoscopic tumor resection was performed as diagnostic therapy. Histopathological analysis revealed dense lymphoplasmacytic infiltration with sclerotic stroma within the tumor. Immunohistochemical analysis revealed abundant IgG4-positive plasma cell infiltrates and over 50% IgG4/IgG-positive plasma cells. We did not see either keratin-positive thymocytes or terminal deoxynucleotidyl transferase-positive lymphocytes. Furthermore, deviation in the kappa chain and lambda chain-positive plasma cells was not noted. Accordingly, IgG4-related sclerosing disease was diagnosed.IgG4-related sclerosing masses in the anterior mediastinum are very rare, and the effect of tumor resection on prognosis remains unclear. IgG4-RD had potentially been categorized as Castleman’s disease. IgG4-related disease (IgG4-RD) is a systemic disease characterized by an elevated serum IgG4 level and lymphoplasmacytic infiltration of multiple organs, such as the pancreas, salivary glands, and biliary tract . In the An 82-year-old man was referred to our department with an anterior mediastinal mass. Eleven years earlier, the patient had been diagnosed with IgG4-related disease (IgG4-RD) from autoimmune pancreatitis and had been taking oral steroid treatment. At the initial diagnosis of IgG4-RD, a well-defined homogeneous anterior mediastinal mass was detected on chest computed tomography (CT); however, it temporarily decreased in size after starting oral steroid therapy Fig. a, b. SerHis medical history was notable for a history of asthma, multiple pulmonary nodules, and mediastinal lymphadenopathy. The latter was suspected to be Castleman’s disease 22 years ago. His serum interleukin-6 level was within the normal limits at the time (details unknown).On this occasion, 5 months before surgery, peripancreatic lymphadenopathy without enlargement of pancreas was detected by CT, and subsequent positron emission tomography revealed abnormal uptake  by the thymic mass as well as peripancreatic lymph nodes Fig. c–e. ExacThe anterior mediastinal mass was suspected to be an encapsulated thymoma because of its lobulated shape, degree of fluorodeoxyglucose accumulation, response to steroids, moderate enhancement on contrast CT, and the gradual enlargement of the tumor. The patient was therefore referred to our department.He had no systemic symptoms, and his physical examination was normal. He had an elevated level of IgG4 at 715 mg/dL and soluble interleukin-2 receptor at 604 U/mL. The rest of his laboratory data were normal, including serum C-reactive protein and anti-acetylcholine receptor antibody. We performed tumor resection by video-assisted thoracic surgery. The tumor had not invaded the surrounding tissues. The operative findings were compatible with encapsulated thymoma, and the tumor was completely excised. His postoperative course was uneventful, and he was discharged on postoperative day 7. The levels of IgG4 and soluble interleukin-2 receptor after operation were 307 mg/dL and 403 U/mL, respectively.Macroscopically, the specimen was a lobulated, well-defined, white, solid mass Fig. a. The fiIgG4-RD was first reported as a systemic disease in 2001 when patients with sclerosing pancreatitis were observed to have elevated serum IgG4 concentrations . Since tThe differential diagnosis of an anterior mediastinal mass includes thymoma, germ cell tumor, and mucosa-associated lymphoid tissue (MALT) lymphoma . It is nIgG4-RD is diagnosed based on clinical and histopathological findings. Umehara H et al. proposed the following comprehensive diagnostic criteria for IgG4-RD: (1) organ involvement, (2) high serum IgG4 > 135 mg/dl), (3) histological features such as IgG4-positive plasma cells > 10/high power field and IgG4-positive/IgG-positive cells > 40% in the affected lesion . The pre5 mg/dl, Distinguishing IgG4-RD from MCD is important because their treatments are entirely different. IgG4-RD responds well to steroid therapy. Kamisawa et al. reported that the remission rate of patients with IgG4-related pancreatitis, receiving glucocorticoids was 98% . On the We described a case with an IgG4-related anterior mediastinal sclerosing mass. It is very rare for IgG4-RD to form a well-defined mass in the anterior mediastinum, and it is usually difficult to differentiate it from a malignant tumor before surgery. It is not clear why IgG4-RD formed the tumor-like swelling. There has been no study or review of the prognosis of intrathoracic lesions of IgG4-RD, and the impact of surgical resection is unknown.IgG4-RD may mimic MCD clinicopathologically, and this case was possibly earlier misdiagnosed as MCD. Therefore, we should be careful when diagnosing patients with IgG4-RD; it should be based on the detailed clinicopathological findings in addition to meeting the diagnostic criteria of IgG4-RD.


Colombia has the fourth highest incidence rate of HIV/AIDS among all Latin American countries and it has been increasing since the 1980s. However, the number of studies that addresses this trend is limited. Here, we employed spatial and temporal trend analyses to study the behaviour of the epidemic in the Colombian territory.Our sample included 72,994 cases of HIV/AIDS and 21,898 AIDS-related deaths reported to the National Ministry of Health between 2008 and 2016. We employed the joinpoint regression model to analyse the annual HIV/AIDS incidence and AIDS mortality rates. In the spatial analysis, we used univariate autocorrelation techniques and the Kernel density estimator.While the HIV/AIDS incidence had an increasing trend in Colombia, the AIDS mortality rate was stable. HIV/AIDS incidence and AIDS mortality showed a downward trend in the 0–14 age group. An upward trend was observed for HIV/AIDS incidence in people older than 15 years and with the highest trend in the 65 years and above group. AIDS mortality showed an increasing trend among people aged 65 years or older. The comparison between the sexes showed an upward trend of HIV/AIDS incidence in all age groups and AIDS-mortality rates in 65 years and above in men, while in women, the incidence was upward among those aged 45 years and above, and concerning the AIDS-mortality rate in the 45–64 group. The high–high clusters of HIV/AIDS incidence and AIDS mortality were located in the Andean and Caribbean regions.Our study found an upward trend in HIV/AIDS incidence and a stable trend in the AIDS mortality rate in Colombia. The downward trend in HIV/AIDS incidence and AIDS mortality rate in the 0–14 age group reflects the downwards mother-to-child HIV transmission. The upward trend in HIV/AIDS incidence in older women and AIDS mortality in younger women rates, compared with men, may be due to late diagnosis and treatment. The Caribbean and the ‘coffee belt’ regions were the most impacted by the HIV epidemic, most likely due to sexual tourism. Our results provide crucial information that may help Colombian health authorities fight HIV transmission.The online version contains supplementary material available at 10.1186/s12889-021-10196-y. Since 1980, 74.9 million people have been infected by the Human Immunodeficiency Virus (HIV), and 32 million have died by Acquired Immunodeficiency Syndrome (AIDS) [The scarcity of public policies for combatting HIV and economic regional disparities are directly correlated with the increase in the incidence of HIV in Latin America (LA), where 1.9 million people live with HIV and AIDS (PLWHA). Between 2010 and 2018, a 7% increase in new HIV infections was recorded throughout LA .Among all LA countries, Colombia has the fourth highest HIV/AIDS prevalence rate. Its national HIV/AIDS prevalence is approximately 0.7%, but much higher among key populations – men who have sex with men, sex workers, injection drug users and prisoners. The Colombian Caribbean region has the second-highest prevalence of HIV/AIDS in the world after sub-Saharan Africa. By the end of 2019, there were 330,000 Colombian PLWHA, 6900 AIDS-related deaths and 13,000 new HIV infections recorded in the same year. Considering the progress towards 90–90–90 targets proposed by UNAIDS to end HIV by 2030, Colombia reached only 62% of that goal. For example, approximately 50% of Colombian people living with HIV still have not been diagnosed for the virus, and only 55% of PLWHA are on ART. Of those receiving treatment, only 68% are virally suppressed. All Colombians and legal immigrants have access to HIV tests and ART [Several factors contribute to the expansion of HIV in Colombia; such factors include social inequalities with the majority of its population living in poverty and with low access to the healthcare system, social discrimination and stigmatization of PLWHA, the forced displacement of the population by internal armed conflicts, the high level of violence against women and sexual tourism –8.The recent immigration flux of Venezuelans also contributes to the expansion of the HIV epidemic in Colombia by placing a substantial burden on the health care resources of Colombian border municipalities. Given the collapse of the healthcare system in Venezuela, Venezuelans living with HIV migrate to Colombia in search of treatment. Until 2018, approximately 2000 Venezuelans in Colombia were living with HIV. Although immigrants with Colombian Permits of Permanence have full access to the healthcare system, including free ART, the massive influx of Venezuelans into Colombia has led to an overload in administrative processing; many Venezuelan immigrants remain illegal , 10. ThiThe HIV epidemic can be addressed by describing its different dynamics in each Colombian territory given that the epidemic is influenced by sociopolitical and economic factors , 12. SpaTherefore, this study aimed to analyse the HIV/AIDS epidemic in Colombia by employing spatial and temporal trend analysis techniques. Here, we considered the incidence of HIV/AIDS and AIDS mortality rates.This ecological study used secondary data provided by the Colombian Ministry of Health.2 area. It is divided into five main political regions , 32 provinces and 1123 municipalities. Colombia borders Brazil and Venezuela in the east, Ecuador and Peru in the south, Panama in the northwest and the Caribbean Sea and the Pacific Ocean in the north and west borders, respectively  based on managed competition and with two kinds of insurance: a private contributory scheme and the state-subsidised one for low-income people. Also, 92.4% of the Colombian population is covered by the GHSI, with a larger proportion in the subsidized system. Both contribution schemes have different health provider companies, with the public one having a lower coverage of health services and lower accessibility than the private one. The states of the Amazon region have fewer health provider companies compared with other states , 16.This study population is comprised of all HIV/AIDS morbid cases and AIDS-related deaths, reported to the Public Health Surveillance System (PHSS) and to the Department of National Administration and Statistics (DNAS) from the Colombia National Institute of Health. Only information containing Colombian home addresses, occurring between 2008 and 2016, was included in this study.We collected the following variables: age, sex, year of diagnosis and death, province and city of residence. Data were double-checked to remove inconsistencies and redundancies.p-value. Upward and downward trends were considered only with positive or negative APC, respectively, and with p< 0.05. If these conditions were not met, it was considered a stationary trend.We employed joinpoint regression to analyse changes in the annual incidence of HIV/AIDS, as well as in AIDS mortality rates, during the period of the study. All analyses were done in the Joinpoint Trend Analysis software 4.8.0.1  and steps have been described previously . BrieflyAll data were grouped by year, sex, counties, region and age range. The age ranges (years) were categorised into four groups: 0–14, 15–44, 45–64 and 65 and over. These categories were made on the basis of available information in the DNAS database for these age groups. The annual incidence of HIV/AIDS and AIDS mortality was calculated on the basis of annual population projections for the whole country and regions, and for the specific age groups and sexes. Both rates were standardised by 100,000 inhabitants.We considered the incidence and mortality coefficients as the dependent variables. The study years were considered the independent variable.We analysed the spatial distribution of HIV/AIDS incidence and AIDS mortality rates and their spatial autocorrelation and Kernel intensity estimator. To avoid the annual variation in the reported cases of HIV/AIDS and AIDS-related deaths, we grouped the data in 3-year periods: 2008–2010, 2011–2013 and 2014–2016.Municipal HIV/AIDS incidence and AIDS mortality rates were analysed through choropleth maps. Both rates were calculated for each Colombian city on the basis of their average population projection for each 3-year period. The results were standardised for 100,000 inhabitants.p-value obtained from 999 permutations as the definition of ‘neighbourhood’. We only considered spatial dependency with Global’s I index (I) above 0 and with p< 0.05 [To assess the overall trend pattern of these variables, we analysed the spatial autocorrelation incidence and mortality rates using Global Moran’s I. The Global Moran’s l index ranges from − 1 to 1: an index of − 1 means dispersion, 0 is random behaviour and 1 means perfect association. However, to localize the clusters, we used the Local Indicator of Spatial Association (LISA) method. In LISA maps, we can identify four different spatial relationship groups of the analysed variable. High–high and low–low aggregations are areas with a high or low value of the variable under study surrounded by neighbouring areas, which have like values above or below average, respectively, of the specific variable. By contrast, high–low or low–high groups are areas with a high or low value of the analysed variable surrounded by neighbouring areas with opposite values below or above average of the same variable, respectively. We used the standardized first-order queen neighbours and the  p< 0.05 .The spatial distribution and the autocorrelation analyses were conducted using the software ArcGis 10.6.1 . The maps were constructed in the Universal Transverse Mercator Coordinate System (UTM), datum D_Bogota, on a scale of 1:12.000.000.To analyse the direction of expansion or contraction of the HIV epidemic in Colombia, we used the Kernel density estimator. This method allows estimation of a density of an event occurring in one area that can influence its neighbourhood. Events occurring in places closer to each other receive a higher weight than events occurring in distant places . Kernel From 2008 to 2016, Colombia recorded 72,994 cases of HIV/AIDS, and most of these cases were men . HIV was the preponderant diagnosis compared with AIDS in both men and women . During the same period of the study, 21,515 AIDS-related deaths were recorded by the Health Ministry with a higher frequency in men than in women . The Andean, Caribbean and Pacific Colombian regions had the highest recorded absolute frequency of HIV/AIDS cases .Table p= 0.006; 2013–2016: 3.2%, p=0.41). The same trend also occurred in the Caribbean region, where two periods were identified with different behaviours: between 2008 and 2011 the mortality rate was stable  and increased after 2011 .Different from the incidence, the AIDS mortality rate was stabilised in Colombia in both sexes and in all regions. The mortality rate had a decreasing trend in the 0–14 and 15–44 groups. However, in the 15–44 group, the joinpoint regression distinguished two different periods with a decreasing trend in the first period and stabilisation in the second one . The joinpoint regression detected two periods of time in the 15–44 age group with high APC in the second period . Similarly, the incidence rate among women had a downward trend in the 0–14 age group, but a sole upward trend was detected in the 45 years and above age group . The incidence among women increased only in the Caribbean and Andean regions , as opposed to that among men.The temporal analysis of annual HIV/AIDS incidence and AIDS mortality age-adjusted rates for men and women are shown in Table p=0.003). However, although the general trend in the 15–44 age group was stable, the joinpoint regression identified two different periods with a decreasing trend in the first period  and a stabilization in the second one . Among women, a decreasing trend in the 0–14 age group  and increasing trend in the 45–64 age group  in the Caribbean and Pacific regions was detected .The only change detected in AIDS mortality rate in men was in the age group 65 years and above with a significantly increasing trend . Figure All univariate spatial autocorrelations of Global Moran’s I indexes were positive and statistically significant in terms of both HIV/AIDS incidence and AIDS mortality  of the entire database that did not compromise the analyses. Finally, this work was also limited by the employment of two particular datasets (PHSS and DNAS) without identifiers, thereby making it impossible to extend analyses between variables.Our study found an upward trend in HIV/AIDS incidence and a stable trend in the AIDS mortality rate in Colombia. The decreasing trend in HIV/AIDS incidence and AIDS mortality rate in the 0–14 age reflects downwards mother-to-child HIV transmission. The upward trend in HIV/AIDS incidence and AIDS mortality rates in older and younger women, respectively, compared with those in men, may be due to late diagnosis and treatment. The Caribbean and the ‘coffee belt’ regions were the most impacted by the HIV epidemic which may be due to the sexual tourism.Our study provides information to Colombian health authorities in order to fight HIV transmission. This effort is much more than just testing and making ART or PrEP universally available. It is also necessary to combat social discrimination against HIV, to promote gender equality, to guarantee human rights to sex workers as well as discriminalization of prostitution, and to promote universal access to healthcare services. Lastly, our study showed the necessity and the urgency of establishing STI prevention programs for elderly people.Additional file 1: Table S1. Number of reported cases of HIV/AIDS by the main possible forms of HIV transmission, 2008-2016.


IgG4-related disease (IgG4-RD) of the biliary tract and pancreas is often difficult to distinguish from pancreatobiliary cancer. The blood IgG4/IgG RNA ratio has been reported to discriminate IgG4-RD from primary sclerosing cholangitis/pancreatobiliary cancer with high accuracy. This study aimed to prospectively assess the diagnostic accuracy of the blood IgG4/IgG RNA ratio for distinguishing IgG4-RD from cancer in patients with a suspected pancreatobiliary malignancy.In this prospective, single center, observational study, patients presenting at a specialized multidisciplinary, hepato-pancreato-biliary clinic with suspicion of pancreatobiliary malignancy were included. The IgG4/IgG RNA ratio (threshold 5.0%) was determined by quantitative PCR in addition to standard diagnostic procedures. Clinical, biochemical, radiological, and histo-/cytopathological findings were analyzed. For the diagnosis of IgG4-RD, the HISORt criteria were used as a reference standard. Malignancy was defined by the presence of neoplastic tissue at histo-/cytopathological examination.Overall, 213 consecutive patients (mean age 68 years) with a suspected pancreatobiliary malignancy were analyzed, of whom 3 patients were diagnosed with IgG4-RD and 178 patients were diagnosed with malignancy . The IgG4/IgG RNA ratio was true positive in 3 patients and false positive in 87 (40.8%) patients. In 123 (57.7%) patients the test was true negative. The sensitivity of blood IgG4/IgG RNA ratio was 100%, the specificity 58.6%, the positive predictive value 3.3%.a priori risk of malignancy, an elevated IgG4/IgG RNA ratio did not accurately discriminate pancreatobiliary cancer from IgG4-RD as illustrated by low specificity and concordant low positive predictive value. We advise against the use of this test to discriminate IgG4-RD from pancreatobiliary malignancies.In the setting of a high IgG4-related disease is a benign inflammatory multiorgan disease which predominantly affects the pancreas and biliary tree. Clinical symptoms, laboratory and imaging finding are often difficult to distinguish from pancreatic or biliary tract cancer. This prospective trial indicates that the recently proposed blood IgG4/IgG RNA ratio does not accurately distinguish benign IgG4-RD from malignant pancreatobiliary disease. •IgG4-related disease and malignancy of bile ducts/pancreas can be indistinguishable.•Blood IgG4/IgG RNA ratio was prospectively tested as an IgG4-RD marker when malignancy was suspected.•IgG4/IgG RNA ratio >5% did not accurately discriminate IgG4-RD from pancreatobiliary cancer. Standard diagnostic procedures were performed including laboratory assessment , radiographic imaging  and histo-/cytopathological analysis. In addition, the blood IgG4/IgG RNA ratio was determined and considered positive if the ratio was >5.0%.In this prospective, observational, single center study, all consecutive patients of ≥18 years of age presenting at a specialized multidisciplinary hepato-pancreato-biliary clinic, at the Amsterdam UMC, with suspected or proven biliary or pancreatic cancer were included between February 21The exact method for determining the IgG4/IgG RNA ratio has been extensively described.Clinical, laboratory, imaging and histo-/cytopathological findings were prospectively collected from all patients investigated and treated. Patients were discussed in a weekly multidisciplinary meeting in the presence of at least 1 surgeon, gastroenterologist, oncologist, pathologist and radiologist. Histo-/cytopathological findings were analyzed in patients who underwent a biopsy, brush or surgical intervention of the biliary tract or pancreas. For the diagnosis of IgG4-RD, the HISORt criteria were used as a reference standard.Every year approximately 450–500 patients are seen at the specialized outpatient hepato-pancreato-biliary clinic. Based on the assumption that every year circa 5 new patients with IgG4-RD will present, 1,000 patients had to be screened in 2 years to identify 10 patients with IgG4-RD. In order to evaluate the predictive value of the clinical decision model to distinguish between IgG4-RD of the biliary tract or pancreas and cancer, a minimum of 10 patients with IgG4-RD had to be tested, to allow for 1 false negative result, based on a sensitivity of 94% and a specificity of 99% of the IgG4/IgG RNA ratio. Using the IgG4/IgG RNA ratio alone, we estimated to have 1 false negative and 10 false positive results. However, when combining the accuracy of blood IgG4/IgG RNA ratio for IgG4-RD with brush cytology for cholangiocarcinoma and pancreatic carcinoma , EUS-FNA for pancreatic carcinoma  and biopsies of presumed metastases, we aimed to lower the number of false positive test results from 10 to 3. On the other hand, 9 patients would be diagnosed correctly with IgG4-RD and would be saved from unnecessary surgery or chemotherapy.An interim analysis of this open, prospective, single center trial was performed in September 2019 and the study was subsequently stopped on September 10, 2019.p <0.05) using SPSS version 25 (IBM SPSS statistics).The predictive value of the blood IgG4/IgG RNA ratio was calculated with a true positive test defined as an elevated IgG4/IgG RNA ratio ≥5% in combination with response to prednisolone treatment. A false positive test was defined as an elevated IgG4/IgG RNA ratio ≥5% in combination with a histopathologically proven malignancy or clear evidence of a benign disease different from IgG4-RD. A false negative test was defined as an IgG4/IgG RNA ratio <5% and benign histo-/cytopathology with histological features of IgG4-RD (tissue infiltration of >10 IgG4+ plasma cells per high power field (HPF) in biopsy or >50 IgG4+ plasma cells per HPF in organ resection material, in combination with lymphoplasmacytic infiltration, storiform fibrosis and/or obliterative phlebitisThe study protocol was approved by the Medical Research and Ethics Committee of the Amsterdam UMC, location AMC (W18_263 # 18.328).A total of 213 consecutive patients presenting at a specialized multidisciplinary hepato-pancreato-biliary outpatient clinic for suspicion of pancreatobiliary malignancy were investigated between February and September, 2019. One hundred and four patients (48.8%) were male and 109 (51.2%) were female, with a mean (±SD) age of 68 ± 11 years. Demographic, clinical and biochemical characteristics are summarized in The blood IgG4/IgG RNA ratio was true positive in 3 patients with IgG4-RD (3.3%) and false positive  in 87 patients (40.8%) with other benign or malignant disease. In 123 patients (58.6%) the test was true negative . Receivep <0.01), but no difference in serum IgG4 or blood IgG4/IgG RNA ratio  between these groups was observed  to UB.Funding provided by the Based on CRediT. EV – Data curation, Formal analysis, Investigation, Project administration, Writing ; FT – Data curation, Formal analysis, Investigation, Project administration, Writing ; LH – Conceptualization, Funding acquisition, Writing (review & editing); JH – Data curation, Project administration, Software; NM – Software, Validation, Visualization, Writing (review & editing); JV – Investigation, Writing (review & editing); JvHo – Project administration, Resources, Writing (review & editing); MB – Writing (review & editing); PF – Resources, Writing (review & editing); NV – Conceptualization, Methodology, Supervision, Writing (review & editing); UB – Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing , Writing (review & editing).The authors declare no conflicts of interest that pertain to this work.Please refer to the accompanying


FYN as the most affected region in neurons. FYN expression, kinase activity and the phosphorylation of its target Tau are increased by heroin use in the post-mortem human striatum, as well as in rats trained to self-administer heroin and primary striatal neurons treated with chronic morphine in vitro. Pharmacological or genetic manipulation of FYN activity significantly attenuates heroin self-administration and responding for drug-paired cues in rodents. Our findings suggest that striatal FYN is an important driver of heroin-related neurodegenerative-like pathology and drug-taking behavior, making FYN a promising therapeutic target for heroin use disorder.The current opioid epidemic necessitates a better understanding of human addiction neurobiology to develop efficacious treatment approaches. Here, we perform genome-wide assessment of chromatin accessibility of the human striatum in heroin users and matched controls. Our study reveals distinct neuronal and non-neuronal epigenetic signatures, and identifies a locus in the proximity of the gene encoding tyrosine kinase  Epigenetic mechanisms have emerged as contributors to the molecular impairments caused by exposure to environmental factors such as abused substances. Here the authors perform epigenetic profiling of the striatum and identify the tyrosine kinase FYN is an important driver of neurodegenerative-like pathology and drug-taking behaviour. Therefore, further studies of post-mortem human brains2 are critical to identify molecular alterations in long-term heroin users compared to age- and gender-matched controls.Heroin-use disorder continues to impose tremendous suffering and financial costs on society. Therefore, a better understanding of neuronal maladaptations contributing to this disorder is imperative to drive medication development. Historically, the field has focused on animal models of addiction, and surprisingly little direct molecular information is available about the addicted human brain5. We previously showed that chronic heroin use leads to hyperacetylation of specific lysine residues of histone H3 (H3K27ac) at specific genomic loci2. These alterations contribute to transcriptional changes related to glutamatergic neurotransmission and have a significant role in mediating addiction-like behaviors2. Nonetheless, epigenetic regulation is extremely complex and studying a single post-translational histone modification provides limited insight into global chromatin alterations. Rather, transcriptional activity is influenced by an elaborate combination of epigenetic marks, and direct assessment of chromatin state may afford a less biased and a more informative measure of gene regulation.Epigenetic mechanisms are dynamic processes that regulate gene expression and have recently emerged as key contributors to the molecular impairments caused by exposure to environmental factors such as abused substances6. We focus on the striatum due to its essential role in regulating reward, inhibitory control, motivation and goal-directed behaviors, impairments of which are linked to substance use disorders7. Using an established protocol that combines FANS (fluorescence-assisted nuclei sorting) and ATAC-seq, we generate quantitative open chromatin profiles in neuronal and non-neuronal nuclei9 and compare genome-wide chromatin accessibility between heroin users and matched controls. We identify a region near the gene encoding for tyrosine kinase FYN as most significantly affected by heroin in neurons. We show that this locus is a putative regulatory element that enhances transcriptional activity in vitro. In vivo, FYN expression and activity are significantly induced by heroin. Consistent with FYN kinase’s implication in tauopathy disorders and substance abuse10, we detect elevated phosphorylation of Tau at a residue directly targeted by FYN. Strikingly, saracatinib, a FYN inhibitor currently in clinical trials for Alzheimer’s disease11, attenuates addiction-like behaviors in rats self-administering heroin, similar to effects observed with siRNA-mediated knock-down of Fyn in the dorsal striatum. Overall, our findings suggest that opioid exposure induces neurodegenerative cellular processes associated with epigenetic disturbances and that the use of FYN inhibitors could be promising for heroin medication development.Here, we study dorsal striatal chromatin accessibility directly using the assay for transposase accessible chromatin coupled with high-throughput sequencing (ATAC-seq)n = 10) and matched controls  of long-term heroin users n = 0 and mat13, we found that neuronal peaks from heroin users were specifically enriched in CpG islands, promoters and 5′UTRs, whereas centromeres, introns, gaps and intergenic areas were depleted across all groups  and EZH2 binding sites . Although no conclusive statements can be made without cell-type-specific transcriptomic analysis, these differences suggest that overall transcriptional activity may be affected in neurons of heroin users , gender and post-mortem interval (PMI). This analysis is of particular interest for smaller post-mortem human cohorts, where criteria for genome-wide significance are rarely met. Most of the variance in chromatin accessibility average 38.9%) was explained by cell type Fig.  and, acc8.9% was p < 0.01 and |logFC | >1.7 , transmembrane receptor activity , transcription factor activity , RNApolII activity , as well as regulation of nervous system development , neurogenesis  and morphogenesis of branching . Regions that were more accessible in heroin-sourced neuronal nuclei compared to control , neuronal projections , post-synaptic density , dendrite , glutamate receptor activity , as well as regulation of nervous system development , modulation of synaptic transmission  and regulation of synaptic plasticity . Emphasizing the specificity of these findings, matching GO analyses from non-neuronal cell populations highlighted enrichment for categories related to cell adhesion, signal transduction, and nucleotide exchange , which might contribute to the transcriptional regulation of long-term adaptations underlying heroin-use disorder.1. Intriguingly, the unbiased variance analysis identified a peak in the proximity of FYN , member of the SRC tyrosine kinase family, as the genomic locus most significantly affected by heroin use  overlapping the transcription start site of FYN. Interestingly, the promoter region of FYN was more accessible in neurons of heroin users compared to controls , we observed elevated FYN mRNA levels in the putamen (homogenate tissue) of human heroin users compared to matched controls  in heroin users but not in controls  of rats self-administering heroin -Fyn  and significantly decreased inactive p(Y529)-Fyn  protein levels  but not inactive p-Fyn  showed a positive correlation with active lever pressing  or inactive (Y529) phosphorylated forms of Src family kinasesels Fig. . In addiing Fig. , furthern = 6/8 control/heroin; Student’s t-test, t12 = 1.207 and p = 0.2508 for active p-Src, Student’s t-test, t12 = 0.6566 and p = 0.5238 for inactive p-Src; Supplementary Fig. Taken together, our data indicate that chronic heroin use leads to increased accessibility, gene expression and tyrosine kinase activity of FYN in the dorsal striatum. Of note, we found that other SRC family members were not affected in terms of chromatin accessibility , the primary neuropathological marker of Alzheimer’s disease. As we previously detected increased phosphorylation of Tau in the post-mortem brain of human heroin abusers29, we hypothesized that heroin-induced increases in FYN might be driving Tau phosphorylation in these individuals, especially at Tyrosine-18, a residue specifically targeted by the FYN kinase27. We first tested this hypothesis in vitro by treating primary striatal-cortical co-cultures with morphine (10 μM every other day for 6 days) and observed a significant increase of Tyrosine-18 phosphorylated Tau -associated intracellular signaling pathway as one of the most significantly altered , or vehicle , 30 min prior to self-administration sessions. This dose, which was previously shown to rescue memory impairments in mouse models of Alzheimer’s disease26, was sufficient to significantly decrease Fyn kinase activity in the dorsal striatum of rats  or 72 h after the last saracatinib injection , all of which emphasize the specificity and acuteness of saracatinib’s effect on heroin-taking behavior.In order to test n = 6/7 for vehicle/saracatinib, F1,132 = 7.376, p = 0.0075; Fig. 34. Interestingly, the saracatinib-induced decrease of active lever pressing during the three self-administration sessions when saracatinib was administered correlated with the decreased level of responding during the subsequent cue-induced test , animals were carried through an operant session with exposure to the previous heroin-paired cue light, but where lever pressing did not result in the delivery of heroin. Saracatinib significantly decreased responding for the heroin-paired cue  activity , these novel findings indicate that FYN inhibitors may be promising therapeutic tools for heroin-use disorder.Using a genome-wide direct assessment of chromatin state in neuronal versus non-neuronal cells in the human brain, we identified the SRC family tyrosine kinase FYN as the most significantly affected locus in dorsal striatal neurons of heroin users. Focused transcriptional profiling of the 35, is highly expressed in the human brain36 and has an important role in the central nervous system37. Intriguingly, FYN localizes to the post-synaptic density of glutamatergic synapses and associates with NR2B, a subunit of the NMDA receptor38, which is elevated in heroin abusers. This interaction is mediated by the scaffolding protein RACK1 (receptor of activated protein C kinase 1)40 and leads to phosphorylation of NR2B43. FYN-induced phosphorylation of NR2B in turn enhances NMDA receptor function43 by increasing surface expression in post-synaptic membranes46, a cellular pathway that may mechanistically explain heroin’s impact on striatal synaptic plasticity.FYN belongs to the SRC family of protein tyrosine kinases47. Furthermore, long-lasting activation of Fyn has been previously described by Ron and colleagues during alcohol use25. This and our work focused on the anterior, medial part of the striatum and, while we show no differences in the nucleus accumbens, further studies will be necessary to address brain region specificity.Glutamate receptor trafficking and function is well known to have a central role in neuronal adaptations that underlie the abuse of various substances48. Moreover, saracatinib decreases symptoms of physical withdrawal in mice implanted with morphine pellets49, further emphasizing its relevance for opioid-related pathologies. Other SRC kinase inhibitors also attenuate instrumental responding for cocaine50. None of these inhibitors are, however, specific to FYN so the contribution of distinct SRC family members remains to be elucidated. Intriguingly, chromatin accessibility alterations in the post-mortem human brain were specific to FYN among members of the SRC family , this relationship was strongly exacerbated by heroin, suggesting accelerated neurodegenerative-like pathology in heroin users that might contribute to drug-induced cognitive impairments. Our current in vivo and in vitro studies demonstrate a direct causal link between opioid exposure, enhanced activity of FYN and abnormal Tau phosphorylation. It is important to note that the heroin-related pTau immunohistochemical pathology we detected in heroin abusers29 has a unique signature given that other neurodegenerative marks studied did not mirror those documented in various neurodegenerative disorders or tauopathies. For example, there was a lack of α-synuclein  and TDP-43  immunoreactivities and no deposits of amyloid beta  were detected in the forebrain of heroin users29. Heroin users exhibit both transient and long-lasting cognitive impairments58, but it is currently unknown whether this is accompanied by striatal or cortical neurodegeneration. Intriguingly, a recent study reported neurodegenerative-like mRNA expression profiles in blood samples from over 800 heroin-dependent subjects59. Whether observed pTau pathology represents an early sign of vulnerability to neurodegenerative disorders later in life or reflects the lower end of the Tau-related spectrum of cognitive impairment is unknown. Intriguingly, saracatinib attenuated neurodegenerative-like pathology in rats, although no complete reversal was observed due to the short duration of treatment. The mechanistic link we demonstrated between opioid exposure and dysregulated FYN and Tau pathology could also have marked implications for the large number of individuals exposed chronically to potent opioid analgesics during the past decade. Future experiments overexpressing Fyn will be important to further corroborate its direct role in Tau phosphorylation related to heroin-associated behavioral phenotypes.We and others have previously observed hyperphosphorylated Tau in specific brain areas, including the striatum, of autopsy specimens from human heroin abusers9. Although the current study focused on neuronal impairments, non-neuronal populations were also strongly affected by heroin and potentially contribute to addiction pathology. For example, SLITRK1, the most significantly altered gene in the non-neuronal population, is closely linked to synaptic plasticity60.The ATAC-seq of neurons and non-neuronal cells offered direct insights into the heroin-addicted human brain. Intriguingly, we revealed distinct perturbations of the chromatin landscape in the two cellular populations. This duality is consistent with our recent findings in the healthy human brain, indicating that chromatin state varies markedly by cell typeOverall, we report several lines of converging evidence with direct insights from the addicted human brain that was translatable to animal models of this disease. Such orthogonal approaches are imperative to improve current treatment strategies for neuropsychiatric disorders. Direct interrogation of the chromatin state of different cell types in the brains of heroin abusers provided evidence of discrete epigenetic dysregulation in the genome with greater impairment within striatal neurons emphasizing the potentially greater vulnerability of those cells to heroin. Identifying FYN as the most affected locus was surprising but provides a logical molecular mechanism to account for the unexpected hyper-pTau pathology we previously detected in heroin abusers and could presently link to the causal exposure to opioids in our in vivo and in vitro rodent models. Importantly, the fact that the FYN inhibitor saracatinib potently decreased addiction-like behaviors strongly suggests that FYN, and related network, is a key target for future therapeutic development. Although the potential efficacy of FYN inhibitors through current clinical trials for Alzheimer’s disease remains to be determined, most drugs targeting FYN appear to be well tolerated and therefore could be excellent candidates to repurpose for accelerated clinical studies focused on substance use disorders. As other members of the SRC family of kinases do not have a similar role, more selective inhibitors of Fyn could also be developed based on the chemical structure of saracatinib, and used with perhaps even greater efficacy in heroin users. The development of such non-addictive and non-opioid therapies for opioid addiction is critically needed to help combat the current opioid epidemic.61 were collected at autopsy within 24 h of time of death at the Department of Forensic and Insurance Medicine, Semmelweis University, Hungary62. Informed consent from legally authorized representatives was obtained at the time of autopsy. Cause of death was determined by the forensic pathologist performing the autopsy  were explored and significant variables were included in the final model. Ventral striatal (nucleus accumbens) and dorsal striatal (putamen) tissue punches were obtained from the same coronal plane 8. Briefly, 50 mg of frozen post-mortem brain tissue was homogenized in cold lysis buffer 2, 0.1 mM, EDTA, 10 mM Tris-HCl (pH = 8), 1 mM DTT, 0.1% Triton X-100) and filtered through a 40 µm cell strainer. The flow-through was underlaid with sucrose solution 2, 1 mM DTT, 10 mM Tris-HCl, pH8) and subjected to ultracentrifugation at 24,000 rpm for 1 h at 4 °C. Pellets were thoroughly washed and resuspended in 500 µl DPBS, then incubated in BSA  and anti-NeuN antibody  under rotation for 1 h, at 4 °C, in the dark. Prior to FANS sorting, DAPI (ThermoScientific) was added to a final concentration of 1 µg/ml. Neuronal (NeuN+) and non-neuronal (NeuN−) nuclei, counter-stained with DAPI, were sorted using a FACSAria flow cytometer (BD Biosciences).We used an established protocol that combines FANS and ATAC-seq to generate open chromatin profiles in neuronal and non-neuronal nuclei63 (Illumina Cat #FC-121-1030) and the resulting libraries amplified using the Nextera index kit (Illumina Cat #FC-121-1011). Optimal library amplification was determined by visualization with Bioanalyzer High Sensitivity DNA Chips (Agilent technologies Cat#5067-4626). Libraries were amplified for a total of 12–17 cycles and were quantified by Qubit HS DNA kit (Life technologies) and by quantitative PCR (KAPA Biosystems Cat#KK4873) prior to sequencing. Finally, ATAC-seq libraries were sequenced on Hi-Seq2500 (Illumina) obtaining 50 bp paired-end reads. All ATAC libraries met or exceeded ENCODE guidelines  (Supplementary Data ATAC-seq (assay for transposase accessible chromatin) reactions were performed using an established protocolFYN (Hs00941600_m1 for human and Rn00562616_m1 for rat), GRIA1 (Hs00181348_m1), GRM5 (Hs00168275_m1), HOMER1 (Hs00188676_m1), and DLG4 (Hs00176354_m1) were obtained from Applied Biosystems . Eukaryotic 18S rRNA  was included in each multiplex PCR as an internal control. Real-time PCR and subsequent analysis were performed with a Roche LightCycler 480 Real-Time PCR system . Quantification of target gene expression in all samples, performed in triplicate, was normalized to 18S rRNA by the equation CT(target) – CT (18S) = ΔCT, where CT is the threshold cycle number. Differences between control and heroin subjects, including individual variation, were calculated by the equation ΔCT  – ΔCT (mean control) = ΔΔCT. Changes in target gene expression (n-fold) in each sample were calculated by 2CT)−(ΔΔ from which the means and standard errors of the mean (SEM) were derived.Total RNA was extracted from approximately 10 mg of pulverized putamen tissue using the RNAqueous Micro Kit (Ambion) and following the manufacturer’s protocol. First-strand cDNA was synthesized using qScript cDNA SuperMix  and subjected to qPCR analysis using TaqMan-based probes. Primers and probes against 65. In brief, pregnant Long-Evans dams were euthanized with continuous CO2 and embryos were carefully removed by Cesarean section at embryonic day 18.5 (E18.5). Striatal and cortical tissues were dissected, separately trypsinized for 15 min at 37 °C, washed three times with room temperature HBSS for 5 min, and passed through a 40 µm cell strainer to remove debris. Primary cells were plated 3:2  or striatal only at a density of 2 × 105 cells per glass coverslip, which were coated with poly-l-lysine-coated and embedded on a 35 mm2 dish. Cells were plated in plating media . On the next day, plating media was replaced with maintenance neurobasal media . Cells were treated with a mitotic inhibitor  on the fourth day in vitro (DIV4) and half the maintenance media was exchanged with fresh media every 3–4 days until completion of the experiment. Cells were treated with 10 or 100 µM morphine every other day for one week (from DIV8 to DIV14) and fixed 24 h after the last dose with 4% PFA in DPBS (with 4% sucrose) for 10 min at room temperature. These levels of morphine are physiological and routinely used in the literature. Importantly, concentrations in this range are sufficient to induce alterations in synaptic plasticity consistent with known effects observed in human heroin abusers and reported in animal models of heroin exposure68. Primary striatal:cortical co-cultures were used for immunocytochemistry; primary striatal cultures were used for real-time quantitative polymerase chain reaction.Primary rat striatal cultures and striatal-cortical co-cultures were generated from Long-Evans embryosAfter fixation, cells were washed with DPBS, permeabilized in 0.25% Triton X-100 in PBS for 5 min, and blocked in 10% BSA in DPBS for 1 h. Cells were incubated overnight at 4 °C with primary antibodies recognizing pTau-Y18  and DARPP-32  diluted in DPBS with 3% BSA. Cells were washed three times with DPBS and AlexaFluor-488 secondary antibodies (Thermo Fisher A32731 and A32723) were diluted 1:1000 in DPBS with 3% BSA for subsequent 1-h incubation at room temperature. Finally, cells were washed three times with DPBS and briefly rinsed with water before mounting in hard-set media containing DAPI . Striatal medium spiny neurons were selected on the basis of DARPP-32-immunoreactivity then imaged on a Zeiss LSM780 inverted confocal microscope at ×40 magnification. Quantification of images was performed by a researcher blind to the treatment condition using Fiji (2017) for processing and MATLAB9.7 to generate the average intensity.Approximately 15 mg of pulverized dorsal striatum (putamen) punches per subject were used to generate histone extracts using 100 µl 0.2 N HCl containing protease  and phosphatase inhibitors  and 5 mmol/l Na-butyrate. The protein was diluted in Laemmli buffer, denatured (for 5 min at 95 °C), subjected to electrophoresis, and transferred onto nitrocellulose membranes. The membranes were blocked in Odyssey blocking buffer  and incubated at 4 °C overnight with primary antibodies. Rabbit polyclonal antibody was used against p(Ser199)-Tau  and Fyn  and mouse monoclonal antibody was used against p(Y18)-Tau . Membranes were subsequently incubated with goat anti-rabbit or goat anti-mouse IRDye 680 , or IRDye 800  secondary antibodies diluted 1:5000 at room temperature for 1 h. Membranes were developed with the LICOR infrared imaging system (LICOR) and images quantified using average integrated density values. GAPDH  levels were used to control for total protein content. All uncropped western blots are shown in Supplementary Figs. Rat dorsal striatum tissue was homogenized using BioRuptor in 1× IP buffer  containing protease and phosphatase inhibitors. Homogenates were pre-cleared using protein G Dynabeads for 1 h at 4 °C. For immunoprecipitation, 1 mg of pre-cleared protein was incubated overnight at 4 °C with 7.5 µg of total Fyn  or 5 µg of total Src  antibody. Immunocomplexes were pulled down with protein G Dynabeads for 4 h at 4 °C and eluted into Laemmli buffer at 55 °C for 10 min. 50 µg of immunoprecipitated protein was used for western blotting. To determine kinase activity, antibodies against active p(Y418)-Src  and inactive p(Y529)-Src  were used. In addition, dorsal striatal protein tyrosine kinase activity was also measured using a commercially available kit following the manufacturer’s instructions .FYN regulatory region (hg19_dna range=chr6:112232247-112232896) were designed using Primer3 version 0.4.0. Restriction enzymes sites for KpnI and MluI were chosen from the multiple cloning site on Promega’s pGL3 promoter vector (cat no. E1761). Forward and reverse primers with KpnI (GGTACC) and MluI (TGCGCA) restriction sequences, and leader sequences (TAAGCA/ATTCGT) were as follows: Forward 5′-TAAGCAGGTACCCATGTAAAAAGTGATTATCAGAAGAGG-3′. Reverse: 5′-TGCTTAACGCGTCTTGTTGCTCTACTCTCCTCAGC-3′. The human putative Fyn regulatory region was amplified using human cerebellar genomic DNA. The PCR conditions were as follows for 20 μl reactions: 250 nM pooled primers, 1 μl of human gDNA (~100 ng), 0.5 μl MilliQ water, and 18 μl of Invitrogen’s Platinum PCR Supermix High Fidelity. PCR cycles were 94 C for 2 min, 39× , 4 °C for infinite hold. The insert sequence was verified by Sanger sequencing. 1 μg of insert and pGL3 vector were double digested with KpnI and MluI for 15 min at 37 °C, followed by purification with Qiagen’s PCR Purification kit. The human Fyn insert was ligated into the pGL3 promoter vector with Invitrogen’s Stabl3 E. coli following manufacturer’s instructions under 3:1 insert to vector ratio. The bacterial cells were plated on 100 μg/ml ampicillin Agar plates overnight for 16 h at 37 °C. The next day, single colonies were selected with a pipette tip and inoculated into 5 ml LB broth and 100 μg/ml ampicillin and shook overnight at 250 rpm for 16 h. Plasmid DNA was isolated using Qiagen’s Miniprep kit. The isolated plasmid DNA was then verified by Sanger Sequencing for presence of the human Fyn insert. Maxipreps were prepared by combining 250 ml LB Broth with 100 μg/ml ampicillin and 500 μl of starter culture, followed by shaking at 250 rpm at 37 °C for 16 h. Plasmid DNA was isolated using Qiagen’s Endotoxin-free Maxiprep kit and the hFyn-pGL3 vector was verified by Sanger sequencing.Primers for amplifying the putative human 2 flasks and maintained in high glucose DMEM with 10% FBS and without Penicillin–Streptomycin. The day before transfection, cells were trypsinized and seeded into 24-well plates at 25,000 cells per well. On the day of transfection, cells were co-transfected with pGL3 control vector and pRL-SV40 vector  or hFyn-pGL3 vector and pRL-SV40 vector at a 10:1 ratio of pRL-SV40 vector to pGL3 vector in 200 ng total DNA per well. 1.5 μl of Lipofectamine 2000 reagent was used per well. Cells were then placed in a 37 C with 5% CO2 incubator for 48 h.HEK293T cells were passaged into 75 cmPromega Dual-Glo Luciferase Assay reagents were prepared as per manufacturer’s instructions (cat. no. E2920). 48 h post transfection, growth media was removed. 200 μl of crude cell lysis buffer  was added into each well. After mechanical titration with a pipette, the plate incubated on a shaker at room temperature for 10 min. 50 μl of cell lysate were transferred into a 96 well plate, followed by 50 μl of Dual-Glo Luciferase Reagent. Plate incubated at room temperature for 10 min on a shaker, followed by Firefly luciferase luminescence reading on a luminometer. 50 μl of Dual-Glo Stop & Glo Reagent was then added to each well. Plate incubated at room temperature for 10 min on a shaker, followed by Renilla luciferase luminescence reading on a luminometer.Adult male Long-Evans rats were maintained on reversed 12 h dark/light cycle and underwent jugular vein catheterization. They were subsequently food restricted  and trained to self-administer heroin via the jugular catheter to control their own drug intake. Animals were placed in an operant chamber (29.5 cm × 32.5 cm × 23.5 cm) housed in sound-attenuating boxes  with two levers; depression of one (designated the active lever) resulted in the delivery of 30 μg/kg heroin under a fixed-ratio 1 (FR1) schedule of reinforcement, whereas depression of the other (designated the inactive lever) had no programmed consequences. Each self-administration session was 3 h long and performed in the morning (8 am to 11 am). Group sizes for each cohort are reported throughout the text. Each cohort underwent 7 FR1 sessions, followed by 4 additional FR5 sessions. The only exceptions were the animals shown in Fig. In rats previously trained to self-administer heroin, guide cannulae were implanted at a 10° angle at AP + 1.7 mm, ML + 3.5 mm and DV + 2 mm from bregma  or Prism8 . Data were tested for normality and normalized by natural log transformation, if not normally distributed. Univariate statistical analyses were used to study the effect of each independent demographic variable  and toxicology data  on mRNA expression or protein levels. Variables with a p-value cutoff 4.7 × 10−6 based on Irreproducibility Discovery Rate (IDR) analysis69 on technical replicates. Overall QC included: more than 10 million unique, non-redundant reads; <5% of reads mapping to the mitochondrial genome; more than 0.5 for PCR bottleneck coefficient, which is an approximate measure of library complexity estimated as /(uniquely mapped reads); more than 0.3 for relative strand cross-correlation coefficient, which is a metric that uses cross-correlation of stranded read density profiles to measure enrichment independently of peak calling; more than 5% of reads in peaks, which is the fraction of reads that fall in detected peaks; more than 10,000 detected peaks; no outliers or admixture of cellular consistency based on principal component analysis (PCA). Overall, we excluded four samples due to low number of called peaks , cellular admixture and outlier based on PCA. Open chromatin regions were defined in each sample using the MACS2 algorithm as described above. Artifact regions (defined as “Blacklist” regions by ENCODE) with excessive unstructured anomalous reads mapping including regions at centromeres, telomeres and satellite repeats were excluded. Peak sets across cell types  and disease group (heroin and control) were consolidated into four single lists by union operations of peaks that were present in at least two libraries. The final consensus set of 143,003 peaks was generated by combining the four sets of peaks .Briefly, reads were mapped to the gender appropriate reference genome hg19 using STAR aligner version 2.5.0, by disabling spliced alignments  and prohibiting soft-clipping of the reads . Reads that (1) mapped to more than one locus, (2) were duplicated, and/or (3) mapped to the mitochondrial genome were excluded. Model-based Analysis of ChIP-seq (MACS) v2.1 was used to call peaks without building the shifting model (setting --nomodel). We retained peaks passing a 70 to generate a sample-by-peak read count matrix . We counted fragments (defined from pair-end reads), instead of individual reads, that overlapped with the final consensus set of peaks. Scaling normalization to remove composition biases in sequencing data was applied to log(CPM)  using the trimmed mean of M-values method71.We used the featureCounts function from the Rsubread packageWe performed PCA of the normalized read count matrix and examined which variables were significantly correlated with the high-variance components (explaining > 1% of the variance) of the data. This identified four covariates, which encompassed seven statistical degrees of freedom (df): Cell type , Gender , Disease (control and heroin) and post-mortem interval (PMI). Gender and PMI clearly affect chromatin accessibility and were included as covariates in the differential analysis that examined disease and cell-type effects.72. The input to variance partition is counts per million (log2 CPM) matrix. The categorical and continuous variables were modeled as random and fixed effects, respectively. We initially fitted a model considering all variables (model 1). Because the majority of variance is attributed to cell type, we then modeled within/between Disease variance separately for each Cell type (model 2). We finally model the variance of Disease Group, Subject, Gender and PMI in separate models that considered only neuronal (model 3) and non-neuronal (model 4) samples. Each peak was considered separately and the results for all peaks were aggregated afterwards. Results were summarized in terms of the fraction of total variation explained by each variable for each peak.For each open chromatin region, the variance was decomposed into variation attributable to Cell type, Disease Group, Subject, Gender, PMI, plus the residual variation based on linear mixed models implemented in variancePartition v1.4.1 package71 to model the normalized read counts using negative binomial distributions. The estimateDisp function was used to estimate an abundance-dependent trend73. To normalize for compositional biases, the effective library size for each sample was estimated using the trimmed mean of M-values approach as described above. We performed separate analyses for neuronal and non-neuronal samples. For each open chromatin region, we applied the following model for the effect on chromatin accessibility of each variable on the right-hand side: chromatin accessibility ~ Disease + Gender + PMI. Then, for each open chromatin region, the disease coefficient (heroin and control) was statistically tested for being non-vanishing, implying an estimated effect for disease, above and beyond any other effect from the covariates (Gender and PMI). A quasi-likelihood F-test was conducted using the glmQLFTest function74 from the edgeR package, with robust estimation of the prior degrees of freedom. p-values were then adjusted for multiple hypothesis testing using false discovery rate estimation.We used the edgeR package75. p-values of enrichment analysis were then adjusted by Bioconductor qvalue package76. Genomic regions were collected from the following data sources. Transcription factor binding sites (TFBS) were downloaded from ENCODE77, CODEX78, and Cistrome79. Enhancer segments and repressed segments were downloaded from ENCODE. Tissue clustered DNase hypersensitive sites were downloaded from Sheffield et al80. CpG islands, microsatellites, genome repeat regions etc. were obtained from UCSC Genome Browser database. Transcriptional factor motifs were obtained from JASPAR81. Epigenomics modification regions of various conditions were downloaded from Cistrome and ROADMAP82. Genome enrichment/depletion analysis was performed using Homer13.Enrichment analysis of peaks with genomic regions was done using R package LOLA  with Fisher’s exact testFurther information on experimental design is available in the Nature Supplementary InformationPeer Review FileDescription of Additional Supplementary InformationSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Supplementary Data 11Supplementary Data 12Supplementary Data 13Supplementary Data 14Reporting Summary