Hydration is a key aspect of the skin that influences its physical and mechanical properties. Here, we investigate the interplay between molecular and macroscopic properties of the outer skin layer – the stratum corneum (SC) and how this varies with hydration. It is shown that hydration leads to changes in the molecular arrangement of the peptides in the keratin filaments as well as dynamics of C-H bond reorientation of amino acids in the protruding terminals of keratin protein within the SC. The changes in molecular structure and dynamics occur at a threshold hydration corresponding to ca. 85% relative humidity (RH). The abrupt changes in SC molecular properties coincide with changes in SC macroscopic swelling properties as well as mechanical properties in the SC. The flexible terminals at the solid keratin filaments can be compared to flexible polymer brushes in colloidal systems, creating long-range repulsion and extensive swelling in water. We further show that the addition of urea to the SC at reduced RH leads to similar molecular and macroscopic responses as the increase in RH for SC without urea. The findings provide new molecular insights to deepen the understanding of how intermediate filament organization responds to changes in the surrounding environment. The healthy SC is a versatile material that combines the functional property of being an effective transport barrier, and material properties of being soft, strong and pliable to tolerate deformation from physical strain and stress. The SC is also a responding material, and its properties can be altered by changes in the skin environment5. Taken together, SC fulfills several essentially different requirements, and its special material properties can be related to the organization and dynamics of its molecular components. The SC is ca. 10–15 μm thick and consists of anucleated dead cells – corneocytes – that are filled with keratin filaments and wrapped by cornified envelope7. The keratin filaments have a rigid core with protruding terminals and they consist of bundles of protofilaments  and microtubules (∼24 nm)13. Intermediate filaments are ubiquitously present in the skin, hair and nail where it acts as a mechanical scaffold. Filament structures of similar size are also found in the neurofilaments in neuron cell14. The corneocytes are embedded in a multi-lamellar lipid matrix in a fashion often described as ‘brick and mortar’ structure15. In ambient conditions, the vast majority of the SC lipid and protein components exist in a solid state18, which is different from most other biological membranes. Still, the presence of a small fraction of fluid components can have huge impact on the SC macroscopic material properties19.The skin is a large interfacial film separating the human body and the outside environment. The outermost layer of the skin epidermis – the stratum corneum (SC) is responsible for the skin barrier function20, and they have been associated with SC mechanical properties. Overall, the corneocytes have a polar interior, and they may be obstacles for the diffusive transport of hydrophobic molecules, while they may provide an additional transport route for more polar compounds, for example, water. When the skin is exposed to a humid environment, the corneocytes take up substantial amounts of water21, which is apparent as swelling of the skin after, for example, taking a bath. Corneocytes can swell roughly 50% in height when fully hydrated22. The swelling of corneocytes is not uniform in all directions, and they swell more in the vertical direction compared to the parallel direction with respect to the skin surface23. The SC lipids are also affected by hydration by melting of a small portion of the lipid hydrocarbon chains25. Hydration as well leads to an increase in SC permeability at high relative humidity (RH)26, which can be associated with the presence of more mobile/fluid SC lipid and protein components27. This is taken advantage of in dermal and transdermal drug delivery, then called “occlusion”28. In occlusive condition, the skin penetration of chemicals beneath, for example, a skin patch or film of cream can be enhanced30.The keratin-filled corneocytes constitute ca. 85% of the total weight of dry SC32. Previous studies have reported that the water content in SC is indeed the primary factor that governs the SC flexibility33. A distinct brittle-to-supple transition has been observed in the SC sample when the moisture content was elevated to higher RH36. When hydrated SC, water is primarily taken up by the corneocytes. It has been hypothesized that the corneocytes control SC viscoelastic properties through the plasticization of the keratin filament macromolecules by water34. At low RH values, the keratin filaments are present in rigid state24, whereas recent studies have shown that there is a change in the molecular mobility of certain amino acids in the keratin filament upon hydration24. It is highly likely that these molecular changes in the keratin filaments strongly impact its swelling and mechanical properties, and this interplay is investigated in the present study. The molecular properties of the keratin can also be altered by the addition of other small polar molecules, e.g. urea and glycerol37. These molecules are naturally present in skin as part of the so-called “Natural Moisturizing Factor” (NMF), and they are commonly used in skin care products as “humectants”. Although the terminology is a bit misleading, the main function of these compounds in SC is not to increase the water content, but rather to replace water in dehydrated conditions and thereby retain fluidity in SC lipid and protein components37.The mechanical properties of the skin in terms of strength and elasticity can be affected by hydration38 and sorption microbalance. Based on the combination of results obtained from these techniques, we can correlate observed changes in SC macroscopic properties to molecular effects in terms of dynamics, structure and conformation, thereby deepening our understanding of SC hydration.In the present study, we aim at deepened understanding of the nature of hydration-induced changes in keratin filaments inside the corneocytes, and of how these molecular changes can lead to alterations in macroscopic material properties. The effect of hydration on molecular organization and dynamics is also compared to the effect of adding a so-called humectant molecule commonly used in moisturizing skin care formulations. We here present data for homogenous samples, which can be used to make predictions of the effects of water and humectant at different depth in SC. The samples investigated are composed of extracted isolated corneocytes or intact SC at different hydration conditions as determined by the RH of the surrounding air. We used a multi-technique approach, including wide angle X-ray diffraction (WAXD), Fourier transform infrared spectroscopy (FTIR), polarization transfer solid state NMR (PT ssNMR)2HPO4.2H2O, KH2PO4 and KNO3 were purchased from Merck. Water used to hydrate the SC and corneocyte samples and to prepare phosphate buffered saline (PBS) was of Millipore quality produced by MilliQ water filtration system with a resistivity of 18 MΩ·cm at 25 °C.Bovine pancreas trypsin (type III), urea, chloroform, methanol and deuterated water were purchased from Sigma-Aldrich Chemie GmbH . NaCl, NaPig ears were collected from a local slaughterhouse and stored at −80 °C until further used. Prior to dermatoming, the pig ears were thawed and rinsed in cold tap water. The hairs were shaved using a trimmer. The inner side of the pig ears was dermatomed  to a thickness of ca. 500 μm into small slices. The slices were placed on filter papers soaked in trypsin solution (0.2 wt% trypsin in MilliQ) and kept at 4 °C for about 20 h. The SC sheets were peeled off from the remaining epidermis using forceps and washed further in MilliQ five times to get rid off all the trypsin. The SC sheets were dried under vacuum in a desiccator and stored at −20 °C for subsequent use.39. Briefly, the SC sheets were made into small pieces and placed in a medium flask. Three extraction solutions with different chloroform:methanol mixtures of ratios 2:1, 1:1, and 1:2 v/v were used. For each extraction step, the solution with SC sample was kept at room temperature under gentle shaking for about 2 hours. The SC material was collected by filtration after each step. The whole extraction sequence was repeated once again with all three solvent mixtures for about 30 minutes for each extraction time. The filtered SC material was soaked in methanol overnight. In the final step, the methanol extracted corneocyte materials were rinsed in MilliQ several times and dried under vacuum in a desiccator.Corneocytes were extracted from the SC using the procedure described in−4%/min) at 32 °C and the desired RH as controlled in the sorption microbalance. The urea-containing samples were prepared as follows: ∼5 mg of dry SC or corneocytes powder was mixed with a small volume of aqueous solution containing the desired amount of urea (adjusted to give sample composition of 20 or 30 wt% urea with respect to the dry weight of SC or corneocyte samples). The mixing was followed by vigorous vortexing. The excess water was then evaporated in a desiccator under vacuum. The samples were subsequently placed in 80% RH chamber at 32 °C for 48 h to reach hydration equilibrium conditions. The SC and corneocyte samples were then transferred (within one minute) into the screw-tight sandwich cells with polyethylene films in-between to avoid dehydration. The samples were subsequently mounted on a sample holder in the X-ray device for measurements.Samples for X-ray diffraction studies were hydrated as follows; small pieces (∼5–10 mg) of dry SC or corneocytes were placed on a pan in the DVS  sorption microbalance with a stream of nitrogen to control the RH. The samples were hydrated for about 24–36 h to reach stable conditions (when the rate of weight change is <1040. The powder was used because the time needed to reach stable conditions (assumed equilibrium) is faster compared to SC sheets. Approximately 25 mg of dry corneocytes or SC sample was used for NMR sample preparation. For hydration at different RH, the samples were placed either in a DVS sorption microbalance with a nitrogen stream at controlled RH, or in a desiccator with a saturated salt solution to maintain the desired RH, e.g. KNO3 for 90% RH. The temperature was set to 32 °C in the sorption microbalance and desiccator. The samples were hydrated for about 24–36 h to reach stable condition and then transferred quickly in a minute into the NMR inserts (Bruker) for measurements.For NMR experiments, the SC and corneocyte samples were made into scaly powder by the aid of mortar and pestle. In previous studies, it was shown that there are no detectable differences in terms of molecular mobility in SC lipid and protein components between the SC sheet and pulverized SC observed with PT ssNMR2O for about 24 h at 32 °C prior measurements.For sorption measurements, ∼5–8 mg dry powder of SC and corneocyte samples was used in the DVS sorption pan. The FTIR experiments was also performed with ∼1–2 mg of powder samples and to measure in the fully hydrated condition, the powder samples were hydrated in DI) was measured as a function of scattering vector q . The latter is defined as θ is the scattering angle and λ is the wavelength of the incident beam, which is 1.54 Å in this case. The sample to detector distance was adjusted based on the q range selected. From the different position of q, the d– spacing was calculated using the equation WAXD studies were performed using in-house X-ray setup, GANESHA 300 XL SAXS system . The scattering intensity (41 and INEPT (insensitive nuclei enhanced by polarization transfer)42. All NMR experiments were performed on a Bruker Avance AVII 500 NMR spectrometer that is equipped with a Bruker E-free 4 mm MAS (magic angle spinning) probe. The operating frequency of MAS is 5 kHz and the 1H and 13C resonance frequencies were 500 and 125 MHz, respectively. The temperature was chosen to be 32 °C to maintain physiological skin temperature and was calibrated by using methanol43. All NMR spectra were recorded under 68 kHz two-pulse phase modulation (TPPM) 1H decoupling44 and with a spectral width of 250 ppm. 1H and 13C hard pulses were given at ω1H/C/2π = 80 kHz. For CP experiments, the 13C nutation frequency was 80 kHz and the 1H nutation frequency linearly ramped from 72 to 88 kHz during 1 ms contact time. For INEPT, the delay times of τ = 1.8 ms and τ′ = 1.2 ms were used. A total of 2048 scans were recorded per experiment with an acquisition time and recycle delay of 0.05 and 5 s, respectively. This gives a total experimental time of ∼9 h for all three sets of measurements  for a given sample. The 13C chemical shift scale was externally referenced to the methylene signal of solid α-glycine at 43.7 ppm. The experimental time domain data was processed with line broadening of 20 Hz, zero filling from 1597 to 8192 time domain points, Fourier transformation, phase correction45 and baseline correction by using in-house Matlab code partially derived from matNMR46.The NMR method comprise three types of measurements: DP (direct polarization), CP (cross polarization)−4%/min. The sorption data are presented in terms of water content as a function of RH. The water content (wt%) is calculated as / ms, where ms is the total mass of the sample including water at a given time point and ms,dry is the weight of the dry sample at 0% RH. The differences observed in the sorption data are also reproduced. All the sorption measurements were performed at 32 °C.Sorption measurements were recorded for intact SC and isolated corneocytes using DVS sorption microbalance. To compare the sorption isotherm, SC and corneocyte samples were run at the same time in a single experiment. The dry samples were placed on two pans in the DVS microbalance and exposed to a stream of nitrogen with controlled RH. The sorption was continuously recorded by weighing the microbalance. The equilibrium at each RH was defined by the conditions where the rate of change in mass is less than 10−1. All the spectra were collected at 25 °C.The ATR-FTIR setup consists of PerkinElmer instrument equipped with deuterated triglycine sulphate (DTGS) detector. The FTIR data collection and reduction procedure was performed using the built-in software. A total of 128 scans was acquired for each spectrum. The FTIR spectral resolution was set to 4 cmThe datasets generated during and/or analyzed during the current study are available from the corresponding authors on reasonable request.aw), as controlled by the RH of the sample surrounding (RH = aw × 100%). The WAXD measurements provide information on the molecular organization of small structural unit in both SC lipids and keratin filament components. The ATR-FTIR results give insight regarding protein secondary structure and hydrocarbon ordering, supporting the WAXD data. The PT ssNMR method is sensitive to the presence of a small amount of mobile (fluid) fraction in the complex SC material, and it provides information on molecular dynamics in different SC components with close to atomic resolution. Sorption measurements were performed to monitor the water uptake in SC and isolated corneocytes at varying RH. From the combination of experimental studies, we obtain a detailed picture that makes it possible to link between molecular properties in SC components, and macroscopic properties in terms of SC water-holding capacity.In the present study, we aim at the characterization of hydration-induced changes on molecular organization and dynamics within the keratin filaments inside the corneocytes of SC at varying hydration conditions. We also investigate the effects of adding urea to slightly dehydrated SC and corneocyte samples. We monitor structure and dynamics in the extracellular SC lipids in the very same samples. The molecular changes are then correlated to macroscopic changes in terms of SC water swelling. We have investigated systems of intact SC and isolated corneocytes at varying water activity  in the high q = 0.66 Å−1, which corresponds to a d– spacing of 9.5 Å  in the spectra of SC and isolated corneocytes in the dry state  for the most prominent peaks are vary as a function of RH. From this we conclude that the keratin interchain distance is sensitive to changes in hydration at RH > 85%, while distance of the peak originating from the lateral packing of SC lipids as well as the β-sheet structure in the keratin filaments is unaffected by the variations in RH. The same behavior is shown both for isolated corneocytes and intact SC. To further probe the nature of the observed abrupt change in keratin filament structure around 85% RH, we investigated molecular dynamics in different amino acid segments in the keratin filaments by means of PT ssNMR.To summarize the results obtained from the WAXD experiments, Fig. 13C. A detailed description of the method is given in ref.58. In previous studies, the method was used on intact SC, showing that all amino acids in the keratin filaments are rigid in dry conditions, while the Ser and Gly residues in the terminal segments of the keratin protein become mobile at higher RH24. Here we systematically investigate this rigid-mobile transition at varying RH in samples composed of either isolated corneocytes or intact SC. The PT ssNMR method comprises DP, CP and INEPT experiments. The DP is used as a reference spectrum as it presents resonances from all carbons in the sample. The CP and INEPT experiments involve transfer of polarization from 1H nuclei to neighboring 13C nuclei, although they rely on different mechanisms of transfer. The CP scheme involves polarization transfer through space via dipolar couplings and is normally used to enhance signal in the solid state NMR41. For INEPT, the polarization transfer occurs through covalent bonds via scalar couplings and is efficient in enhancing signals in liquid state NMR42. The magnitude of CP and INEPT signal for a particular segment can vary depending on the rotational correlation time (τc) and the 13C−1H bond order parameter (SCH). The SCH and τc quantifies anisotropy and the rate of C-H motion. A schematic model of how the CP and INEPT signal depends on τc and SCH is presented in supplemental Fig. 58, CP is selectively enhancing signals for slow or anisotropic segments while INEPT is doing so for fast and isotropic segments. The CP signal is inefficient for fast isotropic reorientation whereas INEPT lacks the signal for slow motion. Comparing the signals of CP and INEPT with respect to the DP signals, atomically resolved information on the structure and dynamics of different molecular segments of the sample can be obtained, which can be described in terms of mobility and rigidity. We can define a molecular segment as ‘rigid’ when only a CP signal is detected whereas a signal from INEPT could define a molecular segment as ‘mobile’. Due to the non-linear response of CP and INEPT signals with respect to changes in molecular motions and anisotropy , the PT ssNMR method cannot be used for direct quantification of the mobile to solid 13C fractions for certain segments58.The effect of hydration on the molecular dynamics in different amino acid segments in the keratin filaments was investigated using PT ssNMR on natural abundance α and Ser Cβ at ∼57 and 62 ppm, respectively) and glycine (Gly Cα at ∼44 ppm) . The Leu and Lys amino acid residues are highly enriched in the coiled coil core of keratin filaments24. Similar behavior was also observed for the amino acids Ser, Gly, Leu and Lys in the keratin in samples of intact SC at varying RH . The samples of dry intact SC contain a minute fraction of mobile segments found in the terminal of lipid acyl chains (ωCH3 at ∼14.6 ppm and (ω − 1)CH2 at ∼23.3 ppm)  RH. The observed effects can be explained by the fact that polar humectant compounds, like urea, have low vapour pressure and therefore remain in the SC also at reduced RH when water evaporates. In this way, urea can substitute for water under dehydration in such a way that the properties of the system remain largely unchanged as compared to a more hydrated state37. This is an important role of NMF in the SC, and it can be related to the effects of osmolytes in other biological systems under osmotic stress.To treat dry skin conditions, it is common to apply a skin care product containing small polar molecules named humectants, for example, urea or glycerol. We here investigate the effect of urea on the corneocytes and SC at conditions of 80% RH Fig. and B. T1 Å Fig. . Increas1 Å Fig. . The lip1 Å Fig.  is not aThe observed changes in molecular properties of the keratin filaments with variations in RH have impact on the macroscopic properties of the corneocytes, and it may influence the material properties and water-holding capacity of the intact SC. Here, we aim to correlate the molecular changes described above to water uptake in corneocytes and intact SC at varying RH. The water sorption isotherms of isolated corneocytes and intact SC are shown in Fig. d– spacings obtained from the WAXD measurements (green curves). The color code in the figures illustrates the changes in the molecular mobility in Ser and Gly residues obtained from PT ssNMR at varying RH where blue signifies the absence of INEPT signal, and the gradual change in red color illustrates increased molecular mobility in these segments. When comparing these sorption isotherm, keratin d– spacing and PT ssNMR data in Fig. 36. Taken together, these observations illustrate a threshold hydration level associated with molecular changes in terms of structure and molecular dynamics in the keratin filament. The observed molecular changes coincide with changes in SC mechanical properties as well. The threshold limit lies around 85% RH for both intact SC and isolated corneocytes. The hydration-induced brittle to ductile transition in SC has previously been associated with the hydration-induced glass transition in the keratin molecules34. To our knowledge, there is no literature study report the precise determination of how the glass transition temperature varies with hydration for SC keratin. However, it has been demonstrated for both hair and wool keratin that upon hydration the glass transition temperature reduced dramatically60, implying the possibility of hydration-induced transition at physiological temperatures. In general, an increase in water activity can lead to phase transitions analogous to temperature-induced transitions for many self-assembled systems composed of, for example, proteins, lipids and surfactants64.In Fig. 37. Indeed, the total content of polar solvent in SC sample treated with urea at 80% RH was shown to be close to the water content in SC at 97% RH65, again showing similar response in SC properties to increased hydration by variation in RH and the addition of urea at constant (reduced) RH. The threshold RH where SC was characterized by low elastic modulus also shift to lower RH in the presence of urea31. Taken together, these examples illustrate that the addition of urea to SC at reduced RH leads to similar molecular and macroscopic responses as the increase in RH for SC without urea. These findings provide new molecular insight into how small polar molecules in NMF and skin care formulations act to protect the skin from drying. In most practical situations, the distribution of water, NMF and added molecules in SC cannot be considered uniform67. The upper layer of the skin generally has the lowest water activity, while the deeper layers are close to physiological water activity (corresponding to 99.6% RH).The addition of urea at slightly dehydrated conditions has also been shown to affect the macroscopic properties of SC. It has previously been reported that urea influences the water uptake in SC68. When the polymer brushes on different particles approach each other, there is a reduction in the configurational freedom of the flexible chains, which generates a repulsive force of entropic origin69. In the dry state at lower RH values, the terminal chains in the keratin filament are rigid, forming a collapsed structure, and the repulsive force between the keratin rods is relatively short-ranged. Consequently, there is only moderate swelling of the keratin-filled corneocytes in these conditions. At higher RH values or in the presence of urea, there is a conformational change in the terminal segments to form more flexible and mobile structures– similar to extended polymer brushes- which leads to more long-ranged repulsion and swelling of the corneocytes. This conformational change is also consistent with recent modeling of interactions between keratin filament in the presence of ions or NMF compounds70. The structural changes in keratin can explain present observations that interchain distance and water uptake increase at conditions close to the threshold RH where the terminal segments become mobile. The analogy to polymer brushes at intermediate keratin filaments has previously also been made for the flanking terminal regions in neurofilament structural assembly72. The present study can therefore also provide novel insights to the more general problem of how intermediate filament organizes in response to variations in surrounding conditions73.A cartoon representation of our interpretation of hydration effect on the keratin filament is presented in Fig. At the molecular level, there is correlation between changes in the interchain distance of keratin intermediate filaments and the molecular dynamics in the Gly and Ser amino acid residues in its protruding terminal segments. There is response in both structure and dynamics at RH’s humidities above the threshold limit of 85% RH, while no detectable changes are shown for lower RH’s. The same conclusions are drawn for intact SC and isolated corneocytes.At the macroscopic level, we detect a response in water uptake to variations in RH for RH > 85%, while the swelling profile is rather shallow at lower RH’s. Comparisons with previous studies of SC mechanical properties also suggest abrupt changes in SC material properties around similar threshold RH’s.There is a correlation between the molecular and macroscopic properties with respect to hydration, where the abrupt changes in SC molecular organization and dynamics coincide with changes in SC macroscopic swelling properties. The flexible terminals at the solid keratin filaments can be compared to flexible polymer brushes, creating long-range repulsion and extensive swelling in water.The addition of the NMF compound urea to SC at reduced RH lead to similar molecular and macroscopic responses as the increase in RH for SC without urea. The same conclusions are drawn for intact SC and isolated corneocytes. These findings provide new molecular insight into how small polar molecules in NMF and skin care formulations act to protect the skin from drying.In the present study, we investigate the interplay between molecular and macroscopic properties of SC and how this varies with hydration conditions. We also examine the effect of NMF compound urea on the SC molecular changes at reduced hydration condition. We use complementary experimental techniques to characterize molecular organization and dynamics as well as water uptake in intact SC and isolated corneocytes. The main conclusions are:Supporting information


Employing self-interaction corrected density functional theory combined with the non-equilibrium Green’s function method, we study the quantum transport through molecules with different numbers of phenyl (donor) and pyrimidinyl (acceptor) rings in order to evaluate the effects of the molecular composition on the transport properties. Excellent agreement with the results of recent experiments addressing the rectification behavior of molecular junctions is obtained, which demonstrates the potential of quantum transport simulations for designing high performance junctions by tuning the molecular specifications. Furthermore, promising intrinsic device functionalities are available, such as the rectification in a molecular junction5. Being determined by the ability of a system to support current differently in foward and backward direction, molecular diodes were early analyzed by Aviram and Ratner6. They exploit the energy gap between the highest occupied molecular orbital (HOMO) of the donor and the lowest unoccupied molecular orbital (LUMO) of the acceptor. Various similar concepts later have been proposed theoretically and demonstrated experimentally12. In a single molecule rectifier typically a donor-acceptor molecule is placed between Au electrodes using a thiol group as linker because of its high flexibility under stress13. For example, such contacts can be achieved by mechanical break junction techniques15. The effects of solvents on molecular junctions and their rectification properties recently have been studied in refs 18.Organic electronics is expected to play a key role in future semiconductor industry, as it costs typically less to process an organic than an inorganic semiconductor20, finding a pronounced rectification in the latter case, with a larger current in the direction from dipyrimidinyl (acceptor) to diphenyl (donor). Since these observations suggest that the properties of the junction can be tuned by chemical substitution, we study the rectification behavior of a series of molecular diodes consisting of pyrimidinyl and phenyl rings, covalently bound to two electrodes.Recent experiments have studied the charge transport through tetraphenyl and dipyrimidinyl-diphenyl molecular diodessp hybridization, which agrees with previous ab-initio calculations21. Moreover, we note that the planar structures of the molecules do not develop significant distortions in the geometry optimizations.Figure We first study the transport properties of the 2N, 4N, and 6N configurations by analyzing the zero-bias transmission coefficients as function of the energy, see Fig. π-orbitals. The HOMO–1 transmission eigenchannel is localized on the phenyl region and the HOMO transmission eigenchannel on both ends of the molecule. On the other hand, the LUMO transmission eigenchannel is centered on the first pyrimidinyl ring after the acceptor-donor contact, similar to the LUMO itself.Figure I-V characteristics, similar for all three configurations. Comparison of our results for the 4N configuration to ref. I-V characteristics, which demonstrates the predictive power of the employed computational approach. Figure IF/IF, where IF and IR are the forward and reverse currents at the same voltage. The 4N configuration performs generally better than the 2N and 6N configurations as a consequence of enhanced forward current, which reflects different potential profiles across the molecular junctions due to different screening in the pyrimidinyl and phenyl rings.We now turn to finite bias transport, see Fig. I-V characteristics when the HOMO peak enters the bias window. Moreover, since the LUMO peak shows hardly any shift, the HOMO-LUMO gap is reduced. The transport is hole dominated at low bias, because the tail of the HOMO peak contributes at the Fermi energy much more than that of the LUMO peak, which starts playing a role only at higher bias, see Fig. I-V characteristics, as the overlap with the green bias window is larger for positive than for negative bias of the same magnitude. Finally, our data fit well to the argumentation in ref. The bias-dependent transmission coefficients displayed in Fig. σ-type C-C bond in the transport direction, see the tandem configuration shown in Fig. Starting from the 4N configuration, which is so far the best performing system, we aim at impoving the rectification behavior. Since a serial connection of diodes appears to be a promising route, we couple two dipyrimidinyl-diphenyl molecules by means of a In conclusion, we have investigated the dipyrimidinyl-diphenyl molecule and its derivatives in rectification applications. In particular, we have dealt with the roles of the acceptor and donor rings and the consequences when their ratio is changed. Serial connection of two molecules has been shown to improve the rectification behavior and threshold voltage as compared to a single molecule configuration. Rational design of molecular junctions by means of quantum transport simulations turns out to be a viable approach.23 implementation of density functional theory, which uses an atomic orbital basis set . All core electrons are described by normconserving pseudopotentials of Troullier-Martins type25, including scalar relativistic corrections26, and a cutoff energy of 600 Ry is used. The ASIC is employed for the exchange-correlation functional in order not to underestimate the electron localization. Results obtained within the LDA27 are given for comparison only. Generally, the ASIC corrects the occupied states by downshifting them in energy and improves the level alignment between molecule and electrode as well as the HOMO-LUMO gap10. The parameter controlling the amount of ASIC added to the LDA  is set to α = 0.7, a value that we have tested for the systems under consideration and that is known to give reliable results for related molecular systems28.We employ the Siesta29, which builds up on Siesta and for which we use the same convergence criteria as for the electronic structure calculations. In the transport calculations the complex part of the integral yielding the charge density is computed using 16 energy points on the complex semi-circle, 16 points along the real axis, and 16 poles. For finite bias the integral over the real energies is evaluated for at least 500 points. An electronic temperature of 300 K is used in all calculations.All structures are geometrically optimized with a force threshold of 0.02 eV/Å. Using the optimized geometries, we perform quantum electron transport calculations in the non-equilibrium Green’s function framework as implemented in the Smeagol package


Salmonella enterica serovar Typhimurium has the ability to use molecular hydrogen as a respiratory electron donor. This is facilitated by three [NiFe]‐hydrogenases termed Hyd‐1, Hyd‐2, and Hyd‐5. Hyd‐1 and Hyd‐5 are homologous oxygen‐tolerant [NiFe]‐hydrogenases. A critical step in the biosynthesis of a [NiFe]‐hydrogenase is the proteolytic processing of the catalytic subunit. In this work, the role of the maturation protease encoded within the Hyd‐5 operon, HydD, was found to be partially complemented by the maturation protease encoded in the Hyd‐1 operon, HyaD. In addition, both maturation proteases were shown to form stable complexes, in vivo and in vitro, with the catalytic subunit of Hyd‐5. The protein–protein interactions were not detectable in a strain that could not make the enzyme metallocofactor. HA, hemagglutininIMAC, immobilized metal ion affinity chromatographyRBS, ribosome‐binding site2 is important during the infection process followed by Salmonella enterica serovar Typhimurium S. enterica genome encodes four [NiFe]‐hydrogenases, termed Hyd‐1, Hyd‐2, Hyd‐3, and Hyd‐5. Hyd‐1 and Hyd‐2 are known or predicted to be involved in respiratory H2 oxidation S. enterica Hyd‐1 is closely related in terms of protein sequence and genetic structure to Escherichia coli Hyd‐1, which is an oxygen‐tolerant enzyme S. enterica Hyd‐5 isoenzyme shares structural similarities with Hyd‐1, but is synthesized under aerobic conditions S. enterica Hyd‐5 is expressed during infection The respiration of HhydABCDEFGHI (STM1539‐STM1531) hydABC encodes the complete enzyme. The core catalytic subunits comprise a large subunit , that carries the catalytic cofactor [NiFe(CN−)2CO], and a small subunit  containing three Fe‐S clusters The Hyd‐5 isoenzyme is encoded by 2CO active site cofactor, and its insertion into the precursor of the large subunit, requires the action of specialist accessory proteins Biosynthesis of the NiFe(CN)E. coli HybD, for example, contains cadmium ions in the structure E. coli HycI has a calcium ion binding site The crystal structures of processing proteases revealed metal‐binding enzymes. S. enterica Hyd‐5 system has been employed to further understand the relationship between a [NiFe]‐hydrogenase large subunit and its cognate maturation endopeptidase. For Hyd‐5, the maturation protease was predicted to be encoded by the hydD gene. Here, deletion of hydD is shown to hamper Hyd‐5 biosynthesis, but HydD function can be partially rescued by the HyaD protein encoded within the operon for Hyd‐1. Furthermore, new evidence is provided that HydD, and HyaD, can form unexpectedly stable complexes with the large subunit of Hyd‐5. Surprisingly, genetic experiments suggest the interactions do not require the presence of the C‐terminal extension on HydB. Indeed, stable HydB‐HydD and HydB‐HyaD complexes can be isolated where the C‐terminal extension of HydB remains completely unprocessed. These data suggest more elaborate roles for these important accessory proteins beyond transient proteolysis.In this work, the S. enterica strains were grown in ‘low salt’ LB (5 g·L−1 NaCl) media while E. coli strains were cultured in standard LB (10 g·L−1 NaCl) medium. Final antibiotic concentrations were used as follows: ampicillin, 125 μg·mL−1; chloramphenicol, 12.5 μg·mL−1 (for S. enterica) or 25 μg·mL−1 (for E. coli).Strains constructed in this work are listed in Table S1. hyaD and hydD genes were amplified by PCR and cloned in vector pBAD24 EcoRI‐SalI. Plasmids for bacterial two‐hybrid analysis were constructed using the pUT18 and pT25 vectors hyaD and hydD genes were PCR amplified, minus stop codons, and cloned into pUT18 using BamHI‐EcoRI sites. The hydB gene was PCR‐amplified and cloned into pT25 using PstI‐BamHI sites. The first truncated form, HydBT1, was amplified and cloned into pT25 vector using BamHI‐SmaI. The second truncated form, HydBT2, was cloned as a BamHI and KpnI fragment into the pT25 vector.Plasmids constructed and studied in this work are listed in Table S2. For construction of plasmids pBADHyaD and pBADHydD, the hydB gene, including the initiation codon but with the native UGA stop codon replaced by two consecutive UAA stop codons to prevent read‐through, was amplified using primers described in Table S3. The forward primer included an artificial ribosome‐binding site (RBS) and six‐base spacer to the initiation codon. The PCR product was digested with EcoRI and SphI and cloned into pQE80 resulting in pQE80‐HydB. Then, hyaD or hydD were PCR‐amplified using forward primers that included an artificial RBS and six‐base spacer to the initiation codon. The reverse primers included the sequence for the hexahistidine affinity tag. PCR products were separately cloned into the pQE80‐HydB vector using HindIII‐PstI sites, resulting in pQE80‐HydB‐HyaDHIS or pQE80‐HydB‐HydDHIS vectors.For copurification experiments, vectors were designed to allow HydB to be overproduced along with either HyaD or HydD His‐tagged variants. The hypD, hydD, and hyaD genes were constructed using pMAK705 + using BamHI‐EcoRI sites. Next, DNA covering 500 bp downstream of the gene, including the terminator site, was amplified and cloned into the new vector using EcoRI‐HindIII sites. The deletion alleles were moved into the pMAK705 vector as BamHI/HindIII fragments and onto the chromosome of the S. enterica LB03 strain by homologous recombination Plasmids constructed in this work are listed in Table S2. Oligonucleotides used as primers are listed in Table S3. In‐frame deletions of chromosomal hypD, ΔhyaD, ΔhydD, and ΔhyaD ΔhydD) encoding a hemagglutinin (HA) tag at the N terminus of HydB was performed as follows. A first round of PCR reactions amplified DNA covering the up and downstream of the hydB gene using primers 1HA tag HydB _SacI_FW/2HA tag HydB _RV and 3HA tag HydB _FW/4HA tag HydB_XbaI_RV primers, respectively. The two PCR products were linked via their overlapping sequence coding for the HA tag (YPYDVPDYA) fused in frame in the corresponding site of hydB using primers 1HA tag HydB _SacI_FW/4HA tag HydB_XbaI_RV. PCR products were cloned in pBluescript‐II KS+ as SacI‐XbaI fragments, moved to pMAK705 with SacI/XbaI and the HA‐tag sequence incorporated into the chromosome by homologous recombination The construction of LB03 derivative strains (ΔE. coli MAE01 (MG1655 ΔcyaA::ApraR) 500 bp upstream of cyaA was amplified and cloned into pKS vector as an EcoRI‐XbaI fragment. Next, 500 bp downstream was amplified and cloned into the new vector using XbaI‐HindIII. Then, the deletion allele was subcloned into pMAK705 leading to yield pFGM1. An apramycin resistance cassette was amplified from pIJ773 SpeI, and cloned into XbaI‐digested pFGM1 to give pFGM2 and the deletion was moved onto the MG1655 chromosome E. coli MG1655 (ΔcyaA::ApraR ΔhypF::KanR) strain (MAE02) was constructed by P1 transduction the ΔhypF::Kan allele from JW5433 For 2‐dependent reduction of benzyl viologen m Tris/HCl (pH 7.5). Protein concentration of cell suspensions was determined by a modified Lowry method Hydrogenase activity was measured by HRocket immunoelectrophoresis was performed as described Bacterial two‐hybrid analysis was performed as described E. coli FTD147 (pQE80HydB‐HydDHIS) or FTD147 (pQE80HydB‐HyaDHIS) grown anaerobically at 37 °C in Duran bottles filled to the top with low salt LB media. Protein production was induced with 2 mm  of isopropyl β‐d‐thiogalactopyranoside at the outset. After 16 h, cells were harvested and suspended in Buffer A  containing protease inhibitors , 50 μg·mL−1 of lysozyme and 10 μg·mL−1 of DNase I. Cells were broken using Emulsiflex‐C3 homogenizer and soluble fractions prepared by ultracentrifugation at 134 000 g at 4 °C. The soluble extract was loaded onto a 5 mL His Trap HP column  equilibrated with buffer A. Bound proteins were eluted with a 50‐mL linear gradient of the same buffer containing 500 mm imidazole. Fractions containing the His‐tagged proteins were pooled and concentrated in a Vivaspin 20  filtration device (10 kDa molecular weight cutoff).Protein purification was carried using 5 L cultures of Proteins were separated by standard SDS/PAGE S. enterica LT2a strain was previously modified by the addition of a T5 promoter upstream of the hyd operon Hyd‐5 is normally produced aerobically at very low levels hydD was generated in LB03 to generate MAS02. Somewhat surprisingly, when the ΔhydD MAS02 strain was assayed, total hydrogenase activity levels were found to be 66% of that normally recorded for the parent strain  was constructed (MAS04). The ΔhyaD, ΔhydD double mutant (MAS04) was found to be severely compromised in terms of Hyd‐5 activity and showed only basal activity levels, similar to that exhibited by the LT2a strain under these growth conditions  by introduction of either hyaD or hydD on a plasmid  and an in‐frame deletion in 536 and aHA . First, a Δ. First, hydD and ΔhyaD alleles were introduced into the hydBHA background (Table S1). The removal of HyaD had a minor effect on the assembly and activity of Hyd‐5 . This strain was found to be completely devoid of periplasmic Hyd‐5 activity  was generated (Table S1) before being cotransformed with pT25‐HydB and either pUT18‐HydD or pUT18‐HyaD. The transformants were then grown anaerobically in rich medium and subsequent quantification of cellular β‐galactosidase levels showed that the negative control sample (empty vectors) displayed only a basal level of activity . Interestingly, coexpression of truncated T25‐HydBT1 with either HydD‐T18 or HyaD‐T18 generated high levels of β‐galactosidase activity, indicating the protein–protein interactions remained intact . In this case, coexpression of T25‐HydBT2 with either HydD‐T18 or HyaD‐T18 lead to β‐galactosidase activity being greatly reduced closer to that observed for the negative control  was transformed with the plasmids encoding fusions to the Hyd‐5 components and the results of these experiments revealed that the levels of interactions detected were reduced to basal levels in all cases , that lacks the genes for the large subunits of Hyd‐1, Hyd‐2, and Hyd‐3, was transformed with pQE80‐HydB‐HydDHIS and grown under anaerobic induction conditions to allow co‐overproduction of HydB and HydDHIS. A soluble protein fraction was prepared and analyzed by immobilized metal ion affinity chromatography  with 96% overall sequence coverage . The mass spectrometry demonstrated that the C‐terminal assembly peptide was still present on HyaB .The HIS  with 96% overall sequence coverage . Interestingly, a strongly staining band at ~ 90 kDa was present as copurifying with HyaDHIS and it was observed to cross‐react with the anti‐His antibody used , together with HyaD . One interpretation of these data is that the strongly staining band at ~ 90 kDa represents a stable complex between the large subunit and the maturation protease. It could be that the ~ 90 kDa band is a covalent complex between HydD and HyaD, especially as HyaD has three nonconserved cysteines and the samples were not boiled before SDS/PAGE due to the presence of imidazole A similar approach was taken for HydB and HyaDHIS Fig. B. In thiHIS Fig. B, but nesed Fig. B. TryptihydD was the most likely candidate as a maturation protease E. coli Hyd‐3 processing demonstrated that protease gene deletions (ΔhycI or ΔhycHI) were sufficient to block Hyd‐3 biosynthesis hoxM gene product is the essential maturation protease for the Hyd‐5‐like membrane‐bound‐hydrogenase from Ralstonia eutrophahoxW, for its maturation S. enterica HydD and HyaD are at least partly interchangeable, it is unlikely that the two proteins would be coexpressed under physiological conditions In organisms that contain several [NiFe]‐hydrogenases, genetic analysis usually points to each large subunit gene being coexpressed with a gene coding for a specific maturation protease mThe two‐hybrid system used here has proven useful in dissection of metalloenzyme biosynthesis pathways and can detect positive interactions that have been subsequently quantified by biophysical techniques to have dissociation constants in the region of 1–1000 nE. coliFurthermore, the two‐hybrid assay clearly shows that the C‐terminal extension on the large subunit cannot be the key docking position for the maturation proteases Fig. . This obInterestingly, while HydD and HyaD retained the ability to bind to the mature form of HydB  this interaction was also found to be dependent on an intact [NiFe] cofactor biosynthesis pathway, in so far as the bacterial two‐hybrid data can be interpreted Fig. . This waHIS in an E. coli host strain resulted in the isolation of a stable complex between the two proteins (Fig. in vitro during a metal competition assay Co‐overproduction of the Hyd‐5 large subunit HydB with HydDins Fig. . These dins Fig. . MoreoveMA and FS conceived and coordinated the study and wrote the paper. MA constructed the majority of bacterial strains and plasmids. GB provided technical assistance and constructed a minority of bacterial strains and plasmids. MA and FS designed the experiments and analysed data. MA performed all of the experiments and prepared figures and tables for publication. FS supervised the work.Fig. S1. Identification of HydB copurified with HydDHIS or HyaDHIS.Fig. S2. Identification of a HydB‐HyaDHIS complex.Fig. S3. Sequence identity shared between Salmonella enterica maturation proteases.Table S1. Bacterial strains constructed and studied in this work.Table S2. Plasmids used in this work.Table S3. Oligonucleotides used in this work.Click here for additional data file.


Parkinson's disease (PD) is characterized by intracellular inclusions of aggregated and misfolded α-Synuclein (α-Syn), and the loss of dopaminergic (DA) neurons in the brain. The resulting motor abnormalities mark the progression of PD, while non-motor symptoms can already be identified during early, prodromal stages of disease. Recent studies provide evidence that during this early prodromal phase, synaptic and axonal abnormalities occur before the degenerative loss of neuronal cell bodies. These early phenotypes can be attributed to synaptic accumulation of toxic α-Syn. Under physiological conditions, α-Syn functions in its native conformation as a soluble monomer. However, PD patient brains are characterized by intracellular inclusions of insoluble fibrils. Yet, oligomers and protofibrils of α-Syn have been identified to be the most toxic species, with their accumulation at presynaptic terminals affecting several steps of neurotransmitter release. First, high levels of α-Syn alter the size of synaptic vesicle pools and impair their trafficking. Second, α-Syn overexpression can either misregulate or redistribute proteins of the presynaptic SNARE complex. This leads to deficient tethering, docking, priming and fusion of synaptic vesicles at the active zone (AZ). Third, α-Syn inclusions are found within the presynaptic AZ, accompanied by a decrease in AZ protein levels. Furthermore, α-Syn overexpression reduces the endocytic retrieval of synaptic vesicle membranes during vesicle recycling. These presynaptic alterations mediated by accumulation of α-Syn, together impair neurotransmitter exocytosis and neuronal communication. Although α-Syn is expressed throughout the brain and enriched at presynaptic terminals, DA neurons are the most vulnerable in PD, likely because α-Syn directly regulates dopamine levels. Indeed, evidence suggests that α-Syn is a negative modulator of dopamine by inhibiting enzymes responsible for its synthesis. In addition, α-Syn is able to interact with and reduce the activity of VMAT2 and DAT. The resulting dysregulation of dopamine levels directly contributes to the formation of toxic α-Syn oligomers. Together these data suggest a vicious cycle of accumulating α-Syn and deregulated dopamine that triggers synaptic dysfunction and impaired neuronal communication, ultimately causing synaptopathy and progressive neurodegeneration in Parkinson's disease. Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease (AD) , SCARB2 and ATP13A2 established lysosomal storage dysfunction as a second pathogenic pathway that also contributes to PD etiology  domain and an acidic tail Figure . α-Syn tSNCA gene have been identified in families that develop early onset PD  , wild type α-Syn is available in its native conformation as soluble monomers. These are thought to mediate its physiological function in presynaptic terminals Burré, . AlthougAggregated and oligomeric α-Syn are also implicated in prion-like transmission of progressive PD pathology along distant but connected brain regions  channels open, thereby allowing influx of Ca2+ into the presynaptic terminal  complex, including VAMP-2, SNAP-25, and Syntaxin-1  exocytosis in response to an action potential. It is at the presynaptic terminal where α-Syn is hypothesized to play its physiological or pathological role Katz, . By rece Südhof, . This Ca Südhof, . Ultimat Südhof, , for sub Südhof, . In addi Südhof, .The AZ matrix is composed of several proteins including RIM, ELKS , Munc13, Bassoon/Piccolo, Rim-BP, and Liprin-α   and frontotemporal lobar degeneration with ubiquitin- and TDP-43-positive inclusions (FTLD-TDP)  derived from PD patients that harbor the A53T α-Syn mutation. Kouroupi and colleagues observed a significant downregulation of genes coding for presynptic proteins in comparison to controls  and the dopamine transporter (DAT), respectively. VMAT2 is responsible for dopamine uptake from the cytoplasm into SV (Lotharius and Brundin, Consistent with this notion, Bellucci et al. showed that mice expressing the truncated α-syn120 exhibited marked redistribution of DAT/α-Syn complexes in the striatum and the SN (Bellucci et al., in vivo and that disrupting the ability of dopamine to stabilize or modify α-Syn oligomers was sufficient to rescue dopamine-mediated toxicity (Mor et al., in vitro data revealing the ability of dopamine to trigger formation of α-Syn oligomers (Choi et al., C. elegans (Mor et al., This data suggests an intricate and potentially direct interplay between α-Syn and dopamine that directly contributes to functional deficits of DA neurons. In support of this, Mor et al. recently demonstrated that dopamine promotes α-Syn oligomerisation Studies in PD-related animal models and post-mortem patient material demonstrate that soluble oligomers and aggregating protofibrils of α-Syn accumulate in synapses and axons prior to the onset of disease symptoms. These accumulating toxic α-Syn species impair synaptic compartments and presynaptic processes required for neuronal communication, including maintenance of SV pools, SV trafficking, tethering, docking, priming and fusion of SV within the plasma membrane at the active zone. The resulting synaptopathy phenotypes manifest prior to dopaminergic neurodegeneration, hence reinforcing the hypothesis that onset and evolution of PD progress in a dying back-like manner, from initial dysfunction at the synapse and impairing axonal connections, to eventual neuronal cell body death. Although α-Syn is expressed throughout the brain and enriched at presynaptic terminals, DA neurons are most susceptible to toxic effects exerted by accumulating and dysfunctional α-Syn. This fatal attraction is likely related to α-Syn regulating dopamine levels in two distinct manners. First, α-Syn is a negative modulator of dopamine synthesis by interplay with the rate-limiting enzyme TH and AADC. Second, α-Syn disrupts VMAT2 and DAT activity and their function in regulating dopamine levels in the synaptic terminal. In turn, dysregulated dopamine levels fuel the formation of soluble α-Syn oligomers that are considered to be the most toxic species. This fatal interplay and its ensuing vicious cycle of dopamine and α-Syn interference is likely the starting point driving synaptic dysfunction and impaired neuronal communication observed in the early, prodromal stages of PD.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a shared affiliation, though no other collaboration, with the authors.


Individual prediction of tumour behaviour based on molecular markers may refine adjuvant treatment strategies in endometrial cancer (EC). As these molecular alterations are determined in a small tumour fraction, high intratumour heterogeneity may interfere with correct risk prediction. This study aimed to investigate to which extent intratumour heterogeneity exists for molecular markers and whether it affects the molecular risk assignment in EC.POLE (n=10), CTNNB1 (n=8), p53 (n=10), mismatch repair (n=11), L1CAM (n=10), and ECs without any of these markers (n=9). Nine ECs carried more than one molecular marker. All 147 blocks were analysed for POLE exonuclease domain and CTNNB1 exon 3 mutations, and for p53, mismatch repair and L1CAM protein expression. All blocks were assigned to a favourable, intermediate or unfavourable risk group, based on a molecular risk assignment.Forty-nine ECs (three tumour blocks/case) were selected with alterations in POLE and CTNNB1 mutational status, and p53, mismatch repair and L1CAM protein expression was found in 100% (48/48), 95.9% (47/49), 93.9% (46/49), 98.0% (48/49), and 91.8% (45/49) of tumours, respectively. These discordances were found in a total of nine cases (18.4%). The intratumour heterogeneity impacted the risk assignment in five cases (10.2%).Concordance between the three tumour blocks for Intratumour heterogeneity of prognostic molecular markers in EC without morphologic heterogeneity is uncommon among three tumour fractions, affecting the molecular risk allocation in a limited number of cases. This low intratumour heterogeneity facilitates the implementation of the molecular risk assignment, advocating its use in clinical decision making. Endometrial cancer (EC) is the fourth most common malignancy among women in the Western world , 2. SurgPOLE) exonuclease domain mutations, CTNNB1 mutations, mismatch repair (MMR) deficiency, and TP53 mutations HER2 in bIn conclusion, intratumour heterogeneity of prognostic molecular markers in EC without morphologic heterogeneity is uncommon among three tumour fractions from hysterectomy specimen. The low levels of intratumour heterogeneity affected the molecular risk allocation in a limited number of cases. Therefore, prognostic molecular markers to guide adjuvant treatment decisions can be determined on a single representative sample of EC, facilitating their use in routine diagnostics.POLE and CTNNB1, alterations in MMR, p53 and L1CAM protein expression, and without any of these alterations. With this approach, our study set will be enriched for POLE-mutant ECs (20% of cases) in comparison to a random EC population (7-12% of cases) . P–7. P5–7]


During the 1970s some investigators proposed that refined carbohydrates, especially sugar and a low intake of dietary fiber, were major factors in coronary heart disease (CHD). This suggestion was eclipsed by the belief that an excess intake of saturated fatty acids (SFA) was the key dietary factor, a view that prevailed from roughly 1974 to 2014. Findings that have accumulated since 1990 inform us that the role of SFA in the causation of CHD has been much exaggerated. A switch from SFA to refined carbohydrates does not lower the ratio of total cholesterol to HDL-cholesterol in the blood and therefore does not prevent CHD. A reduced intake of SFA combined with an increased intake of polyunsaturated fatty acids lowers the ratio of total cholesterol to HDL-cholesterol; this may reduce the risk of CHD. The evidence linking carbohydrate-rich foods with CHD has been steadily strengthening. Refined carbohydrates, especially sugar-sweetened beverages, increase the risk of CHD. Conversely, whole grains and cereal fiber are protective. An extra one or 2 servings per day of these foods increases or decreases risk by approximately 10% to 20%. During the 1970s various investigators proposed that the epidemic of coronary heart disease (CHD) is caused, at least in part, by refined carbohydrates. Particular attention focused on sugar and a low intake of dietary fiber. This hypothesis never gained widespread support (apart from a minor role for fiber). At the same time it became widely accepted that the dominant dietary factor involved in CHD was an excessive intake of saturated fatty acids (SFA). This viewpoint became the accepted orthodoxy for four decades (from roughly 1974 to 2014). The pendulum has now swung in the opposite direction: SFA is now believed to play a much smaller role in CHD than was previously believed while that of sugar, whole grains, and cereal fiber have moved in the opposite direction.This paper looks at the changing viewpoints of the roles played by SFA, sugar, whole grains, and cereal fiber in CHD over the period from the late 1960s to the present.In the late 1960s and through the 1970s several medical investigators, all from southern England, proposed that CHD was strongly related to refined carbohydrates, sugar, and a low intake of dietary fiber. The main supporting evidence was based on international comparisons of diet and incidence of CHD.Yudkin argued that sugar was implicated in several diseases, most notably CHD ,2. HowevWhile Yudkin focused narrowly on the role of sugar in CHD and other diseases, Cleave spread the blame more widely on refined carbohydrates  ,5. He blRefined cereals have a much lower content of dietary fiber and of numerous micronutrients compared with unprocessed cereals. White sugar, of course, is totally lacking in fiber and all micronutrients. Most attention has focused on fiber. Burkitt and Trowell were instrumental in formulating the hypothesis that a low intake of fiber plays a central role in several chronic diseases of lifestyle including CHD ,7. BurkiOne result of this interest in fiber was the discovery that fiber helps lower the blood TC level. However, this property is not shared by all types of fiber: viscous fiber, which is found in oats, beans, and fruit, lowers the TC, whereas insoluble fiber, the type found in wholewheat, is ineffective . While tIn summary, only weak supporting evidence was available in the 1970s for the hypothesis that refined carbohydrates, sugar, and a low intake of dietary fiber play an important role in the causation of CHD.At around the same time that the above debate was taking place, other investigators were studying the relationship between dietary fats and CHD.n-6 (omega-6) or n-3 (omega-3). The predominant n-6 PUFA is linoleic acid which has 18 carbons and two double bonds. Vegetable oils such as corn oil, sunflower oil, and soybean oil are especially rich sources. The major type of n-3 PUFA is alpha-linolenic acid which has 18 carbons and three double bonds. It is found in vegetable oils including flaxseed oil (a rich source), soybean oil, and to a lesser extent canola oil. Smaller amounts of n-3 PUFA occur in seafood as very long-chain fatty acids (20 or 22 carbons) with 5 or more double bonds. Rich sources include fatty fish such as herring, salmon, mackerel, and sardines. All double bonds in PUFA are normally in the cis configuration, but in trans fatty acids one or more double bonds are in the trans configuration.Dietary fats consist mostly of triglycerides. These are formed by fatty acids attached to glycerol. The fatty acids have much chemical variation. SFA are fully saturated  and mostly have a chain length of 12 to 18 carbons. Major sources are foods obtained from animals, such as meat and cheese, and tropical oils. Monounsaturated fatty acids have one double bond. The most common type is oleic acid, which has 18 carbons. Rich sources of monounsaturated fatty acids include red meat, nuts, olive oil, and canola oil. Polyunsaturated fatty acids (PUFA) have two or more double bonds. Depending on the location of the first double bond they are classified as either The Framingham study, a long-term cohort study, was crucial as it demonstrated that the blood TC level is a major risk factor for CHD . It was In contrast to SFA, polyunsaturated fatty acids (PUFA) lower the TC ,12. ThesThe above evidence, taken as a whole, led to the conclusion that an excess intake of SFA is a major cause of CHD. This verdict became widely accepted after about 1974. However, there was never universal support for the hypothesis. Indeed, several investigators argued that the hypothesis was not consistent with the evidence ). For exSerious flaws with the SFA-CHD hypothesis have steadily emerged. Many cohort studies were published after 1990 that have provided a wealth of information on the relationship between diet and risk of CHD e.g., ,19,20,21A few years after the SFA-CHD hypothesis had been widely accepted, it was realized that the relationship between SFA, blood lipids, and risk of CHD was much more complex than had been generally believed. By the late 1970s evidence emerged that LDL-cholesterol is directly related to risk of CHD whereas HDL-cholesterol has an inverse association ). The stHowever, some controversy has emerged in recent years regarding the interpretation of RCTs that aimed to prevent CHD by lowering the TC. Hamley  argued tThe SFA-CHD hypothesis became widely accepted after the evidence reached a critical weight. This has now happened in reverse: the weight of evidence now indicates that SFA play only a minor role in CHD .At the same time as these serious flaws with the SFA-CHD hypothesis were emerging, other evidence also emerged indicating that refined carbohydrates, sugar, whole grains, and dietary fiber play important roles in CHD.Comparisons have been made between the impact of carbohydrates and of SFA on the risk of CHD. A meta-analysis of cohort studies reported that a 5% lower intake of SFA combined with a 5% higher intake of carbohydrates has little impact on risk of CHD . The hazThere is an almost complete absence of evidence from RCTs regarding the effect of replacing dietary fat with carbohydrates on the incidence of CHD. The Women’s Health Initiative was conducted in the USA on 49,000 postmenopausal women . The expEspecially insightful findings came from two cohort studies: the Nurses’ Health Study and the Health Professionals Follow-up Study. The combined results showed that intake of carbohydrates from refined starches and added sugars was positively associated with risk of CHD  . The resCereal fiber has served as the commonly used proxy measure for whole grains in most cohort studies. A meta-analysis of cohort studies reported that cereal fiber has a protective association with risk of CHD . The pooThe colon has an enormous content of microbiota, now commonly referred to as the microbiome. Findings in recent years have linked dysbiosis  with various diseases ,43, inclMany studies have been made concerning the relationship between sugar, sugar-sweetened beverages (SSBs), and CHD. A meta-analysis of cohort studies reported that intake of SSBs is associated with risk of CHD . A 22% gIn summary, replacing dietary intake of SFA with refined starches has little effect on the risk of CHD. However, consumption of added sugars, especially of SSBs, may have a stronger association with risk than either SFA or refined starches. When SFA are replaced with whole grains, risk of CHD is decreased. However, there is still uncertainty regarding the absolute and relative importance of these different components of the diet. A growing weight of authoritative opinion is emerging that supports these conclusions ,30,31.quantity of dietary fats and carbohydrates but not on their quality. It is now clear that the study design was seriously flawed which resulted in a failure to achieve any impact on risk of CHD.A notable lesson from the story told here is that carrying out research into the relationship between diet and CHD is very challenging. This is best exemplified by the Women’s Health Initiative , which was discussed earlier . The stuThe two types of research study that have consistently yielded our most valuable information are RCTs and cohort studies. Evidence coming from RCTs is usually placed above evidence from cohort studies in the research hierarchy. However, Temple  argued tIt must be stressed that many variables come into play when conducting research in the area of diet and health, including CHD. These include components of the diet and attributes of the subjects . Another important variable is the substances in the blood that may be related to the development of CHD. For example, a recent development is the recognition of lipoprotein subclasses ,51,52. RThe key points made in this review are summarized in Growing evidence points to refined carbohydrates, especially SSBs, as being linked to risk of CHD. Conversely, whole grains are protective. An extra one or 2 servings per day of these foods increases risk (in the case of refined carbohydrates/SSBs) or decreases risk (in the case of whole grains) by approximately 10% to 20%, which is similar to the strength of the associations seen for trans fats, fish, and fruit and vegetables. Most cohort studies have measured intake of cereal fiber rather than whole grains. It is probable that whole grains contain many substances, in addition to fiber, that exert a protective action against CHD.


Aseptic loosening of artificial hip joint is a major complication affecting the long-term use of the artificial hip joint, and is the main cause of joint replacement failure. However, the mechanism of aseptic loosening of THR has not yet cleared. The aim of this study was to investigate the underlying mechanism of DANCR in osteoblast differentiation (OD).We detected the expressions of DANCR and FOXO1 in clinical samples and mesenchymal stem cells (MSCs) by qRT-PCR and western blotting. The effects of polymethylmethacrylate (PMMA) on OD of MSCs were examined by alkaline phosphatase (ALP) activity and Alizarin Red S (ARS) staining. The expressions of OD markers were measured by qRT-PCR and western blotting. The mechanism of DANCR in OD was detected by RNA pull-down, RNA immunoprecipitation (RIP) assay and ubiquitination assays.Compared with the surrounding normal tissues, DANCR expression was up-regulated and FOXO1 expression was down-regulated in periprosthetic tissues. PMMA suppressed ALP activity, increased DANCR expression, and decreased the expressions of FOXO1, Runx2, Osterix (Ostx) and osteocalcin (OCN). ARS staining showed that PMMA inhibited the OD of MSCs. Knockdown of DANCR attenuated the inhibitory effect of PMMA on OD. Knockdown of FOXO1 could reverse the effect of si-DANC. RNA pull-down and RIP assay implicated that DANCR bound to FOXO1. Ubiquitination assay indicated that si-DANCR could repress Skp2-mediated ubiquitination of FOXO1.LncRNA DANCR could inhibit OD by regulating FOXO1 expression. Total hip replacement (THR) is one of the most common methods for the treatment of osteoarthritis, rheumatoid arthritis and femoral neck fracture in the elderly. It can effectively relieve joint pain, improve joint function and elevate the quality of life of patients . AsepticLong non-coding RNA (lncRNA) is a class of RNA greater than 200 nt in length that does not have the ability to translate into proteins . StudiesFork box transcription factor (FOXO) is a subclass of the fork box protein family, which plays a major regulatory role in many biological processes . StudiesIn this study, we investigated the underlying mechanism of DANCR in OD. We found that DANCR could inhibit OD by regulating FOXO1 expression, then participate in osteolysis after THR.From October 2013 to March 2017, we screened 20 patients  who were admitted in our hospital and underwent revision of THR because of prosthetic aseptic loosening. Their median age was 66 years. Inclusion criteria: patients have clinical manifestations such as pain and movement disorder, the imaging findings include clear line around the prosthesis and obvious shift of prosthesis. Periprosthetic wear particles and surrounding tissues were collected during revision surgery. The expressions of DANCR and FOXO1 in the clinical samples were measured by qRT-PCR and western blotting. All experimental protocols were approved by the Ethical Committee of Changzhou first people’s hospital.According to the manufacturer’s instructions, total RNA was extracted from tissues or cells using Trizol reagent . Then, cDNAs were synthesized and qRT-PCR for the mRNA levels of DANCR, FOXO1, Runx2, Osterix (Ostx) and osteocalcin (OCN) were conducted using SYBR Green Master Mix . GAPDH was used as the internal reference. The primers sequence was as follows:DANCR--F: 5’-GCCACTATGTAGAG GGTTTC-3’.DANCR--R: 5’-ACCTGCGCTAAGAA-CTGAGG-3’.FOXO1--F: 5’-GGCTGAGGGTTAGTGAGCAG-3’.FOXO1--R: 5’-AAAGGGAGTTGGTGAAAGACA-3’.Runx2--F: 5’-ACTTCCTGTGCTCGGTGCT-3’.Runx2--R: 5’-GACGGTTATGGTCAAGGTGAA-3’.Ostx--F: 5’-GGCACAAAGAAGCCGTACTC-3’.Ostx--R: 5’-GCCTTGTACCAGGAGCCATA-3’.OCN--F: 5’-GGCAGCGAGGTAGTGAAGAG-3’.OCN--R: 5’-CTGGAGAGGAGCAGAACTGG-3’.GAPDH--F: 5’-TCGACAGTCAGCCGCATCTTCTTT-3’.GAPDH--R: 5’-GCCCAATACGACCAAATCCGTTGA-3’.Total protein was extracted from tissues or cells using RIPA Lysis Buffer . The protein was then separated by 12% SDS-PAGE and transferred onto PVDF membranes . The membranes were blocked with 5% skim milk and incubated with primary antibodies, including anti-FOXO1 antibody , anti-ALP antibody, anti-Runx2 antibody , anti-Ostx antibody , anti-OCN antibody , anti-Skp2 antibody  and anti-GAPDH antibody . Then, the membranes were incubated with anti-rabbit horseradish peroxidase-conjugate secondary antibody  at room temperature for 1 h. ECL development solution  was used to visualize the proteins.2. Polymethylmethacrylate (PMMA) was purchased from MITSUBISHI CHEMICAL POLYMER NANTONG CO. Ltd. . Grouping methods of MSCs: (1) Control group and PMMA group. MSCs in PMMA group were treated with 0.3% v/v PMMA. (2) Control group, PMMA group, PMMA+siRNA group and PMMA+si-DANCR group. (3) pcDNA group and pcDNA-DANCR group. pcDNA-DANCR (Invitrogen) was used to over-express DANCR. (4) Control group, PMMA group, PMMA+siRNA group, PMMA+si-DANCR group and PMMA+si-DANCR+si-FOXO1 group. MSCs in each group were incubated in complete medium containing 200 ng/mL bone morphogenetic protein 2  for 14 d. PMMA stimulation was performed at 48 h after transfection with si-DANCR (5’-GGAGCTAGAGCAGTGACAATG-3′) or si-FOXO1 (5’-GAGGAUUGAACCAGUAUAATTUUAUACUGGUUCAAUCCUCTT-3′). The cell transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. After these treatments, cells were harvested for qRT-PCR or western blot analysis.MSCs were purchased from Shanghai Jining Biological Technology Co. Ltd. MSCs were incubated in complete medium supplemented with 10% fetal bovine serum  and 100 U/mL penicillin-streptomycin (Invitrogen) at 37 °C in 5% CO2. Grouping methods of HEK-293 cells: (1) S-phase kinase-associated protein 2 (Skp2) group and Skp2 + si-DANCR group. Skp2 and si-DANCR plasmids were transiently transfected into cells using jetPRIME  according to the directions of reagent. Twenty-four hours after transfection, 10 μg/mL cycloheximide (CHX) was added into DMEM medium. Cells were incubated in this medium for 0, 3, 6 and 9 h. (2) Ubiquitin (UB) + Skp2 + pcDNA-FOXO1 + MG13 group and UB + Skp2 + pcDNA-FOXO1 + MG132 + DANCR group. A ubiquitination assay was performed in the 2 groups.HEK-293 cells were purchased from Shanghai Jining Biological Technology Co. Ltd. HEK-293 cells were incubated in DMEM medium  containing 10% FBS and 100 U/mL penicillin-streptomycin (Invitrogen) at 37 °C in 5% COUb , Skp2 and DANCR plasmids, and pcDNA-FoxO1  were transiently transfected into cells using jetPRIME (Polyplus Transfection). After 36 h, 10 nm MG132  was added into DMEM medium. Cells were incubated in this medium for 8 h. After these treatments, cells were harvested for western blot analysis. The cell lysates were immunoprecipitated (IP) with the labeled antibodies at 4 °C overnight. Western blotting was used to detect the eluted proteins.According to manufacturer’s instructions, ALP activity detection kit (Yeasen) was used to measure the ALP activity. Briefly, MSCs were lysed with Triton X-100 lysis buffer (Yeasen), and centrifuged at 1000×g for 20 min. ALP activity in the cell supernatant was then measured according to manufacturer’s instructions.ARS staining was used to detect mineralization. MSCs were fixed with 4% paraformaldehyde for 10 min, and stained with 1% ARS solution  at room temperature for 30 min. MSCs were then washed with PBS. The stained cells and mineral nodules were observed at 450 nm in a microplate reader (Thermo).RNA pull-down was performed using RNA-Protein Pull-Down Kit (Thermo) according to the manufacturer’s instructions. Briefly, T4 RNA polymerase (Roche) was used to label DANCR. Biotinylated RNAs were then mixed with streptavidin magnetic beads (Invitrogen) at 4 °C overnight. The cell lysates were interacted with biotinylated RNA on ice for 1 h. Finally, The RNA-protein binding mixtures were identified by western blot assay.According to the manufacturer’s instructions, RIP assay kit  was used for RIP assay. Briefly, HEK-293 cell suspension was prepared in RIP buffer. Anti-FOXO1 antibody  was incubated with the cell suspension at 4 °C overnight. Then, the precipitated RNA was purified and analyzed by qRT-PCR. Isotype-matched IgG (5 μg) was used as a negative control.t test or one-way analysis of variance (ANOVA) was used to compare quantitative variables. Differences between groups were considered statistically significant if P < 0.05.SPSS 20.0 software was used to calculate the statistical analysis. All data were expressed as means ± standard deviation (SD). Student To explore the role of DANCR and FOXO1 in osteolysis, we first evaluated their expression levels in periprosthetic wear particles and surrounding tissues. Compared with the surrounding normal tissues, the expression of DANCR Fig.  was incrTo investigate the effect of PMMA on the OD of MSCs, MSCs were divided into 2 groups (control and PMMA group). The degree of OD and the levels of OD markers were measured at day 0, 1, 3, 7 and 14. Results showed that ALP activity was lower in PMMA group than in control group at each time point Fig. . qRT-PCRTo detect the effect of DANCR on OD, MSCs were divided into 4 groups . Results showed that PMMA could inhibit ALP activity, and si-DANCR reversed the inhibitory effect Fig. . MoreoveTo investigate the relationship between DANCR and FOXO1, we performed RNA pull-down assay Fig. . MoreoveFirstly, we carried out a CHX chase assay to detect the effect of DANCR on the stability of FOXO1 protein. Compared with cells transfected with Skp2 alone, FOXO1 expression was more stable in cells transfected with both Skp2 and si-DANCR Fig. . Then, wTo detect the effect of DANCR silencing on FOXO1 expression, MSCs were divided into control group, PMMA group, PMMA+siRNA group, and PMMA+si-DANCR group. FOXO1 expression was measured by qRT-PCR and western blotting. Results showed that PMMA repressed the expression of FOXO1 and increased DANCR expression, while si-DANCR could enhance the mRAN and protein expression of FOXO1, and reduce DANCR expression Fig. . Then, MTo investigate the mechanism of PMMA on the OD of MSCs, MSCs were divided into control group, PMMA group, PMMA+siRNA group, PMMA+si-DANCR group and PMMA+si-DANCR+si-FOXO1 group. Results revealed that PMMA repressed ALP activity, and knockdown of DANCR could enhance ALP activity. However, knockdown of FOXO1 could reverse the effect of DANCR silencing Fig. . MoreoveNowadays, THR is the major method for the treatment of end-stage hip joint diseases , which cLncRNAs have been reported to be related with OD and bone formation . Xu et aThe further research demonstrated that DANCR could bind to FOXO1, and knockout of DANCR could inhibit Skp2-mediated ubiquitination of FOXO1. Skp2 can specifically recognize phosphorylated substrates and mediate their ubiquitination degradation . Skp2 haIn summary, the present study demonstrates that DANCR could inhibit OD by regulating FOXO1 expression. The findings suggested that DANCR might be a potential therapeutic target in osteolysis after THR.


The most common human sex chromosomal disorder is Klinefelter syndrome . Adult patients with KS display a diverse phenotype but are nearly always infertile, due to testicular degeneration at puberty. To identify mechanisms causing the selective destruction of the seminiferous epithelium, we performed RNA-sequencing of 24 fixed paraffin-embedded testicular tissue samples. Analysis of informative transcriptomes revealed 235 differentially expressed transcripts (DETs) in the adult KS testis showing enrichment of long non-coding RNAs, but surprisingly not of X-chromosomal transcripts. Comparison to 46,XY samples with complete spermatogenesis and Sertoli cell-only-syndrome allowed prediction of the cellular origin of 71 of the DETs. DACH2 and FAM9A were validated by immunohistochemistry and found to mark apparently undifferentiated somatic cell populations in the KS testes. Moreover, transcriptomes from fetal, pre-pubertal, and adult KS testes showed a limited overlap, indicating that different mechanisms are likely to operate at each developmental stage. Based on our data, we propose that testicular degeneration in men with KS is a consequence of germ cells loss initiated during early development in combination with disturbed maturation of Sertoli- and Leydig cells. There is a huge variation in the phenotype of men with KS. In adulthood, the classical symptoms are tall stature, gynecomastia, reduced virilization, hypergonadotropic hypogonadism, and small testes5. Many men with KS, however, first discover their condition when they want to have a child as the most pervasive symptom is azoospermia or, rarely, severe oligozoospermia.Klinefelter syndrome (KS) is caused by the presence of at least one additional X-chromosome in men. KS is the most common sex chromosomal disorder with an estimated prevalence of 1 in 660 newborn boys, but only one fourth of the expected cases are diagnosed throughout life6. In some men with KS, focal spermatogenesis is nevertheless observed and is considered the result of elimination of the supernumerary X-chromosome in a subset of spermatogonia10. Presence of focal spermatogenesis opens the possibility to recover sperm in the ejaculate or to perform surgical microdissection of the seminiferous tubules and isolate areas with focal spermatogenesis (micro-TESE procedures) that subsequently can be used for intracytoplasmic sperm injection. This allows such patients to become fathers of children with apparently normal karyotype (reviewed in refs. 11).The histology of a typical testis from an adult KS man shows absence of germ cells, high amounts of degenerated, hyalinized tubules, and Leydig cell hyperplasiaSHOX gene, which accounts at least in part for the tall stature observed in men with KS12. Recently, several studies have reported transcriptome analysis of peripheral blood samples15 and of cultured lymphocytes isolated from blood16 from KS patients compared to controls. The only overlap between the transcriptome profiles was upregulation of the long non-coding RNA (lncRNA) XIST16. Upregulation of XIST is expected and can be used as a quality control, as XIST is involved in X-chromosome inactivation (XCI)18. Only 12 transcripts were differentially expressed in more than one study16, highlighting the diverse phenotype.The molecular mechanisms responsible for the KS phenotype are poorly understood. Best described is the increased dosage of the X-chromosomal 19. However, our recent study indicated that despite the supposedly normal pre-pubertal testicular development, the fetal KS testis displays impairment of fetal gonocyte differentiation resulting in loss of pre-spermatogonia20. This effect is most likely caused by the extra X-chromosome because the fetal KS testis transcriptome revealed enrichment of X-chromosomal transcripts as well as lncRNAs20. Later on, initiation of spermatogenesis at pubertal onset fails in the KS testis and results in testicular degeneration19.Pre-pubertal testicular development in KS has been described as similar to boys with normal karyotypes, albeit with smaller testis volume and reduced germ cell numbers21. In another study, D’Aurora et al. performed microarray analysis of bilateral testicular biopsies from six non-mosaic KS patients with absence of germ cells compared to a pool of three biopsies from patients with obstructive azoospermia but complete spermatogenesis22. While the material used in the two studies is unique and very precious, the huge cellularity differences are expected to drive most of the changes in the transcriptome and mask transcripts involved in the actual testicular phenotype of KS. In another study by D’Aurora et al., testicular biopsies from three atypical adult KS patients with focal spermatogenesis were compared to the controls used in their former study23. The cellularity differences were thereby less pronounced but the transcriptome now reflected the atypical situation of KS testis with focal spermatogenesis.Gene expression analysis of human KS testes is challenging, as available material is scarce because testicular biopsies are not performed in the routine clinical work-up. But three recent studies used leftover testis tissue from TESE and micro-TESE. In one study, expression of metabolic components was examined in six KS and six controls with azoospermia but conserved spermatogenesisTo describe the adult KS testicular transcriptome and to account for cellularity differences, we performed RNA-sequencing of fixed, paraffin-embedded testicular biopsies from adult KS patients and cellularity-matched controls (CMC), which had Sertoli-cell-only (SCO) pattern and Leydig cell hyperplasia. The cellular origin of the differentially expressed transcripts (DETs) was determined by including samples with complete spermatogenesis. Moreover, to describe developmental changes in the testicular KS transcriptome, we included testis samples from pre-pubertal KS boys along with our published data on the fetal KS testis.XIST in the KS samples , DACH2 was expressed in a subset of Sertoli cell nuclei of type A (mature) and type B (immature) SCO tubules (identified according to ref. 25)   Fig. . Sertoli 3) Fig. , which c 3) Fig. . However 3) Fig. .Fig. 2LoIn a fetal testis aged gestational week (Gw)10, a large fraction of the Sertoli cells indeed showed expression of DACH2  (data not shown). A double fluorescent staining with FAM9A and insulin-like 3  showed that FAM9A and INSL3 were only partially overlapping with distinct FAM9A-positive/INSL3-negative and FAM9A-negative/INSL3-positive populations visible  enrichment of long, intergenic ncRNAs (lincRNAs) among the upregulated transcripts. Also, protein-coding transcripts among the upregulated  and antisense transcripts among the downregulated transcripts were significantly enriched   Fig. .Fig. 4EnTo gain insight into the potential function of the DETs, we searched for enrichment of particular gene ontologies (GO). Among the most interesting results were those involved in reproduction , development , as well as “response to hormone” Fig. .In order to identify the potential cellular origin of the 235 DETs, we used a comparative approach, and profiled the transcriptome of five adult testis biopsies with SCO and normal amounts of Leydig cells, and four adult testis biopsies with full spermatogenesis and normal amounts of Leydig cells (these samples are termed Norm) Table . We exclFirst, we compared the expression of known cell type-specific transcripts in the four groups Fig. . As expeWe also evaluated the cellularity of each sample by measuring the area of the cellular compartments . One transcript was downregulated in pre-pubertal and adult KS (GLI1), and the remaining transcripts showed different directions of regulation between two time points . and the Human Protein Atlas , PNMA is expressed exclusively in the testis and brain. Yet another pro-apoptotic protein upregulated in the KS samples was AIFM331. Increased apoptosis could be a very relevant mechanism responsible for degeneration of the KS testis but when KS samples were stained with cleaved PARP (cPARP), a known marker of apoptotic cells, we only observed very few positively stained cells (data not shown).Another interesting upregulated KS transcript was 16 and the remaining nine were identified in studies performed on adult testis biopsies23. Since the control used in the testicular studies23 included complete spermatogenesis, the overlap with our study, using CMC, was expected to be limited. Moreover, the microarray approach used in these studies would not detect all types of transcripts  contrary to the RNA-sequencing approach we used. In line with our previous study of fetal KS samples20, we indeed observed an enrichment of lincRNAs among the upregulated transcripts in the adult KS testis. This indicates that dysregulation of lincRNAs may play a central role in the KS testis.When we compared our 235 KS DETs with previous transcriptome studies of KS samples, the overlap consisted of only 12 transcripts. Three DETs, were identified in studies performed on peripheral bloodDLK1, FAM9A and FBOXO43, which all have been described previously in relation to testicular function. In the mouse, Fbxo43 is expressed solely in the testis and ovary32, and was shown to be involved in meiosis I progression in rodent spermatocytes34. DLK1 is a marker of immature Leydig cells26 and its upregulation detected in the previous study23 and in our present study is consistent with the presence of more immature Leydig cells in the KS testis. In line with this, expression of FAM9A, which has been shown to be testis-specific35, was also found in less mature Leydig cells in our present study. Taken together, these results indicate that the somatic compartment in the adult KS testis is less mature than that in CMC.Among the protein-coding DETs, three overlapped with previous studies; JAK3 and GLI1. JAK3 was upregulated in the pre-pubertal samples but downregulated in the adult samples, and GLI1 was downregulated both in pre-pubertal and adult samples. A relationship between GLI1 and JAK3 was identified in acute T-cell lymphoblastic leukemia, where their expression levels showed a positive correlation36. As GLI1 is a component of Hedgehog signaling, which regulates development and cancer progression37, downregulation of this transcript in both pre-pubertal and adult samples, as well as downregulation of JAK3 in the adult samples, may indicate a disturbance in the development of the KS testis. Indeed, Hedgehog signaling within the primary cilia of Sertoli cells from adult KS samples has previously been described to be different to that of Sertoli cells in tubules with ongoing spermatogenesis38. In our previous study of the fetal KS testis transcriptome20, we found enrichment of X-chromosomal DETs, which we could not recapitulate in the adult  KS testis. This indicates that the effect of the extra X-chromosome is more direct in fetal life than in the adult. The adult KS testis consequently seems a consequence of the disturbed fetal gonadal development caused directly by the extra X-chromosome.When comparing the KS DETs in fetal, pre-pubertal and adult testes, the identified overlap was limited, indicating that different mechanisms are responsible for the testicular architecture and cellular composition at the different developmental stages. We did see an overlap in dysregulation of Based on our results we hypothesize that the overexpressed genes from the extra X-chromosome in patients with KS are directly causing changes in the testicular transcriptome primarily in fetal life, leading to the initiation of the germ cell loss, gonadal dysgenesis, and subsequent failure of the differentiation of the somatic cells, which consequently appears immature in the adult KS testis. Clearly our study was limited by lack of power due to compromised quality of the isolated RNA. Hopefully, technical advancements in the field will allow future studies to isolate RNA of better quality. Future studies should also focus on including more samples, as the individual variation in testicular development besides variations in cellularity is quite substantial. The present study nevertheless provided reference data for further mechanistic studies, and could also have some clinical implications concerning management of patients with KS. Our results indicate that intervention by supporting or stimulating development of the testicular somatic niche already during childhood could increase chances of fertility in adulthood. Before puberty, many boys with KS still have germ cells in the testis that with proper support by somatic cells might enter spermatogenesis. Such interventions, however, need to be investigated before they can be considered.In conclusion, we were able to analyze the transcriptome of a total of 24 adult KS testis and controls. Enrichment analysis and protein expression patterns showed disturbed maturation of Sertoli-  and Leydig cells. We identified enrichment of lincRNAs, but not of X-chromosomal transcripts, indicating an indirect effect of the supernumerary X-chromosome. Furthermore, the adult testicular KS transcriptome showed a very limited overlap with that of pre-pubertal and fetal KS testis, indicating that distinct processes occur at these stages of development. Our study indicates that Sertoli-  and Leydig cells in the testis of adult men with KS are immature and that impairment of testicular function has its origin early in development.The use of testicular tissue for this project was approved by a regional medical and research ethics committee (permit no. H-2-2014-103) and the Danish Data Protection Agency . The testis samples were collected between 1976 and 2014. Following surgical excision, the samples were immediately fixed in different fixatives, dehydrated, and embedded in paraffin , and four with full spermatogenesis  was applied for 30 min followed by development with diaminobenzidin DAB (ImmPress detection kit) and nuclear counterstaining with Mayer’s Hematoxylin. Control sections with omission of primary antibody were all negative (data not shown).Staining with anti-DACH2 and anti-FAM9A were performed on testes from two adult men with KS, three with full spermatogenesis and three with CMC testicular histology, and staining with anti-AMH was performed on testes from two adult men with KS  BSA. The anti-FAM9A antibody was applied and the slides were left overnight at +4 °C and 1 h at room temperature. The section was incubated with peroxidase-conjugated chicken anti-rabbit secondary antibody  for 30 min followed by incubation with Tyramide Cy3  for 10 min. Antigen retrieval with pressure cooker, blockade with horse serum, and incubation with primary antibody was repeated for the anti-INSL3 antibody. The section was then incubated with peroxidase-conjugated chicken anti-rabbit secondary antibody followed by incubation with Tyramide Fluorescien (Perkin Elmer) for 10 min. Finally, the section was counterstained with DAPI (Sigma-Aldrich). The section was imaged with an Olympus BX61 microscope, captured using the Cell Sense Dimensions V1.6 software  and processed in Adobe Photoshop 6.0.Details of antibodies and detection reagents are shown in Supplementary Table Adobe Photoshop CC 2014  was used to assess the cellularity of each sample. The area of tubules with germ cells was evaluated on the basis of the MAGE-A4-staining and was quantified with the “lasso” tool. SCO tubules, areas of Leydig cells, blood vessels, hyalinized tubules and connective tissue were identified by visual inspection also measured with the “lasso” tool.Fixed, paraffin-embedded tissue was sectioned on a microtome at a thickness of 10 μm and placed in Eppendorf tubes at +4 °C until RNA extraction. A total of 20–50 μm of tissue was used. Serial sections of each tissue were collected before and after the collection on membranes and stored at −20 °C for subsequent histological assessment of cellularity.Total RNA was extracted using the Recover All Total Nucleic Acid Isolation Kit for FFPE  according to the manufacturer’s recommendations. RNA was eluted in LoBind tubes using nuclease-free water. To assess the quantity and quality of the samples, the Qubit RNA HS Assay Kit (Thermo Fisher Scientific) and the Agilent RNA 6000 Pico Kit , respectively, were used.The Ovation Human FFPE RNA-Seq Multiplex Systems  were used for library preparation from 150–200 ng of total RNA according to the manufacturer’s recommendations with the following modifications: The cDNA samples were fragmented to yield sizes of approximately 150 bp using the Covaris E210 sonicator . For the final library amplification, 20 cycles of amplification were used. An extra DNA purification was performed with a 1.4 times overload of Agencourt AMPure XP beads  to get rid of adapters and primers. Samples were pooled in groups of five with equal amount of each. We performed 150 nt paired-end read sequencing on the Illumina HiSeq 2000 sequencing platform .41. Reads were mapped to the human reference genome (GRCh38) in which the pseudoautosomal region (PAR) on the Y-chromosome  were masked using STAR v. 2.5.2b42. Read counts were summarized using HTseq v. 0.6.043.Reads were demultiplexed using a custom perl script and adapter sequences trimmed using AdapterRemoval v1.5http://www.r-project.org). Briefly, the count data was loaded into the limma/edgeR package45 and heatmaps were generated with the package gplots46 using the heatmap.2 function with the “complete” hierarchical clustering method. Based on this analysis, seven KS samples and two CMC were excluded  ≥ 1.5 or ≤ 1.5 was used as cutoff. Enrichment analyses were performed using mroast and camera functions. XCI status was acquired from ref. 24 and GENCODE transcript biotypes were obtained from Ensembl using the biomaRt package in R49. A hypergeometric test using the phyper function in R was used to test for enrichment of transcript biotypes. Expression of known Sertoli, Leydig, germ cell, and peritubular/blood cell markers in KS, CMC, SCO, and Norm were plotted with GraphPad Prism version 4 .The R package limmahttp://www.interactivenn.net/, accessed 2 Sept 2017). Overrepresentation of GO terms50  was tested using the DAVID Bioinformatics Resources52.DETs were identified between KS, CMC, SCO, and Norm as performed with the KS vs. CMC, and the overlap was identified in R. The relative expression level of transcripts that overlapped was visualized with the heatmap.2 function using the “manhattan” distance matrix and the “ward.D2” hierarchical clustering method. The overlap was visualized using the interactive venn website (Data has been deposited in the GEO public database with the accession number GSE103905.Supplementary TablesSupplementary Figures