It is found that refractive index and density gradually decrease with increasing BaF2 content from 0 to 9 mol.%, due to the generation of more non-bridging oxygens. Furthermore, OH− absorption coefficient (αOH) reduces monotonically from 3.4 to 2.2 cm−1 and thus emission intensity near 1.8 μm in gallium tellurite glass with 9 mol.% BaF2 is 1.6 times as large as that without BaF2 while the lifetime becomes 1.7 times as long as the one without BaF2. Relative energy transfer mechanism is proposed. The maximum emission cross section and gain coefficient at around 1.8 μm of gallium tellurite glass containing 9 mol.% BaF2 are 8.8 × 10−21 cm2 and 3.3 cm−1, respectively. These results indicate that Tm3+ doped gallium tellurite glasses containing BaF2 appear to be an excellent host material for efficient 2.0 μm fiber laser development.The effects of substitution of BaF TeO2, Ga2O3, BaO, BaF2 and Tm2O3 with 99.99% purity (Aladdin) were used as raw chemicals. Appropriate amounts of these chemicals (~20 g) were well mixed and then melted in an alumina crucible with an alumina lid at ~950°C for 30 min. Afterwards, the melts were poured onto a preheated graphite mold and further annealed at 330°C for 2 h, after which they were cooled slowly inside the furnace to room temperature. The annealed samples for the optical property measurements need to be double-sided polishing into 10 × 10 × 1.5 mm3 cylinders. Densities of glasses were determined by the Archimedes' principle using the distilled water as the medium. The refractive index of all the samples was measured by the prism coupling method (Metricon Model 2010) at 633, 1,309, and 1,533 nm with an error of ±5 × 10−4. The infrared transmittance spectra were obtained using Vector 33 Fourier transform infrared (FTIR) spectrophotometer . The Raman spectra were measured by Raman spectrometer  and 532 nm laser as the excitation source. Optical absorption spectra measurements were performed on a Perkin-Elmer Lambda 900/UV/VIS/NIR spectrophotometer. The fluorescence spectra were recorded by a computer-controlled Triax 320 type spectrofluorimeter (Jobin-Yvon Corp.) equipped with an InAs detector upon the excitation of an 808 nm LD. After exciting the samples with an 808 nm LD, InAs detector was used to detect the lifetime of Tm3+:3F4 energy level (1.8 μm) along with a digital phosphor oscilloscope  and signal generator. All of the measurements were carried out at room temperature.Tmn) and density (ρ) of TGB glasses with different BaF2 contents. It is found that the refractive index and density monotonously decrease when BaF2 content increases from 0 to 9 mol.% in step of 3 mol.%. This indicates that the addition of BaF2 makes glass network looser  in TeO4 trigonal bipyramid and the peak C is due to the symmetrical stretching vibrations of Te-O− and Te=O bonds with non-bridging oxygens (NBO) in TeO3 trigonal pyramid and TeO3+1 polyhedra   and is described by the following equations  of Tmr represents the radiative decay rate, WOH is the energy transfer rate between Tm3+ and OH−, Wmp is the multiphonon decay rate, WET is the energy transfer rate between Tm3+ ions, NTm is the total concentration of Tm3+ ions and αOH is OH− absorption coefficient. kOH−Tm is defined as the strength of interaction between Tm3+ and OH− and doesn't rely on the concentrations of Tm3+ and OH−. OH. From this fit, kOH−Tm is determined and equals to 2.82 × 10−18 cm4/s, which is larger than kOH−Er (1.9 × 10−19 cm4/s)  in germanate glasses  between two adjacent Tm3+ ions 3+ ions in the excited 3F4 state return to the 3H6 ground state, emitting fluorescence at 1.8 μm. Significantly, the residual OH− in TGB glasses can impair 1.8 μm emission via two OH− ions, indicating that it is essential to decrease the hydroxyl content for improving 1.8 μm emission.Based on above-mentioned results, 3+ ions are very crucial parameters to evaluate the potential of TGB glasses as 2 μm laser material. Based on the Beer-Lambert equation and Fuchtbauer-Ladenburg equation           of Tm3+ near 1.8 μm can be obtained by the following equation  and the total concentration that ranges from 0 to 1. 2. It is found that the gain peak shifts to shorter wavelength with increasing p, which is a typical feature of the quasi-three-level system. Moreover, gain coefficient starts to be greater than zero in the wavelength range from 1,824 to 2,100 nm when p ≥ 0.2 and the maximum value is 3.3 cm−1 at 1,814 nm, which is larger than that of silicate glass (2.57 cm−1)   (Tian et al., 2 is a promising host material for efficient 2.0 μm fiber laser development.where 2 contents on density, refractive index, Raman spectra, OH− absorption coefficient and 1.8 μm spectroscopic properties of Tm3+ doped gallium tellurite glasses are studied in detail. When BaF2 content increases from 0 to 9 mol.% in step of 3 mol.%, refractive index and density gradually reduce. Meanwhile, αOH monotonically decreases from 3.4 to 2.2 cm−1, which makes emission peak value near 1.8 μm in TGB glass with 9 mol.% BaF2 being 1.6 times as large as that without BaF2 while the lifetime becomes 1.7 times as long as the value without BaF2. The maximum emission cross section at around 1.8 μm of TGB glass with 9 mol.% BaF2 reaches 8.8 × 10−21 cm2. In addition, positive gain coefficient in the wavelength range from 1,824 to 2,100 nm is achieved when p ≥ 0.2 and the maximum gain coefficient is 3.3 cm−1 at 1,814 nm. As a result, TGB glass with 9 mol.% BaF2 appears to be a highly promising host material for efficient 2.0 μm fiber laser development.In summary, the effects of BaFThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.JY, PX, and WW conceived the idea. JY and PX wrote the paper. TD, YY, DO, JC, and SY advised the paper. All authors contributed to the article and approved the submitted version.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.


ATP) channel. In addition to diabetes, DEND patients present muscle weakness as one of the symptoms, and although the muscle weakness is considered to originate in the brain, the pathological effects of mutated KATP channels in skeletal muscle remain elusive. Here, we describe the local effects of the KATP channel on muscle by expressing the mutation present in the KATP channels of the DEND syndrome in the murine skeletal muscle cell line C2C12 in combination with computer simulation. The present study revealed that the DEND mutation can lead to a hyperpolarized state of the muscle cell membrane, and molecular dynamics simulations based on a recently reported high-resolution structure provide an explanation as to why the mutation reduces ATP sensitivity and reveal the changes in the local interactions between ATP molecules and the channel.Developmental delay, epilepsy, and neonatal diabetes (DEND) syndrome, the most severe end of neonatal diabetes mellitus, is caused by mutation in the ATP-sensitive potassium (K ATP) channel11. The most severe phenotype of ND is known as the DEND  syndrome which patients experience developmental delay, epilepsy, muscle weakness and hyperglycemia18. The muscle weakness observed in DEND syndrome patients is considered to originate in the brain19. However, because the KATP channel is also expressed in skeletal muscle, a mutated KATP channel may contribute to the development of muscle symptoms in DEND syndrome patients.Neonatal diabetes (ND) is characterized by the development of hyperglycemia within the first 6 months of life resulting from impaired insulin secretion caused by gain-of-function mutations in the ATP-sensitive potassium  channel and a sulfonylurea receptor 30. Pancreatic beta-cells and neurons are known to mainly express in a combination of Kir6.2/SUR1 and the KATP channels in skeletal muscle are composed of Kir6.2/SUR2A32. The tetrameric Kir6.2 is a K+-channel and together with the channel regulating SUR subunits, they form a heteromeric complex in the cell membranes28. The interface between Kir6.2 subunits forms an ATP binding pocket, and ATP recruited from the cytosol drives structural rearrangement that results in the closure of the channel. Therefore, mutated Kir6.2 channels may have direct effect on muscle function in DEND syndrome patients. Moreover, the physiological role of the sarcoKATP channel remains unclear34. In the present study, we constructed an adeno-associated viral (AAV) vector and transfected the mouse myoblast cell line C2C12 with mutation in the Kir6.2 subunit (Kir6.2-R50P) which results in DEND syndrome17, and we investigated its effects on cell differentiation, myogenic index, myotube width, membrane potential and glucose uptake activity. In combination with molecular dynamics simulations of the wild type as well as the single mutant R50P, based on the most recently resolved structure40, we discuss the structural implications in the function and physiological role of the sarcoKATP channel.The K41. Co-assembly of both subunits shows profound enhancement of plasma surface expression compared with each subunit expressed alone42. Therefore, without the expression of the SUR regulatory subunit, Kir6.2 is not expressed in the plasma membrane, and almost no channel activity is exhibited. We examined the effect of the Kir6.2/R50P mutation on C2C12 cells by transfecting wild-type Kir6.2 (Kir6.2/WT) or DEND inducing Kir6.2 (Kir6.2/R50P). Heterozygous expression of Kir6.2 (Kir6.2/R50P) is supposed to increase the population of Kir6.2 (Kir6.2/R50P)/SUR on the plasma membrane surface. The cells we employed should reflect the condition of the patients because DEND syndrome patients are heterozygous with a mutated KATP channel. C2C12 cells express endogenous SUR, and therefore Kir6.2 transfected cells should express functioning KATP channels in combination with Kir6.2 and SUR. The expression of Kir6.2 were examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemical analyses  was found to prevent surface expression of the Kir6.2 and SUR subunitses Table , S2. As es Table . We applude Fig. ; the memACh Fig. .Figure 1ATP channel exhibits gain-of-function properties with reduced ATP sensitivity18. Structural analyses from recently resolved structures40 (PDB IDs: 6BAA and 6JB1) show that the Arg50 residue is located at a solvent exposed loop in the intracellular region of the Kir6.2 subunit, and that this residue facilitates wrapping around the nearby Kir6.2 subunit  between the channel structure at a given time and the initial structure. Analysis of the alpha carbon atom RMSD for the entire tetramer, and the M1 (residues 67–97) and M2 (residues 142–172) helices versus time revealed a similar pattern in each of the simulations Fig. . The ini. reported that Arg50 interacts with only the gamma phosphate group 37, Chen et al.reported that the residue extensively interacts with the beta and gamma phosphate groups 40  to approximately − 60°, leading to loss of flexibility.While Martin et al)40 Fig. . In addi44. Although the simulations were carried out in the absence of the SUR subunits, altering these SUR-Kir interactions could potentially affect the function of the complex.During the simulations, interactions between ATP and nearby residues were recorded as a function of time. It was found that residues Lys185, Lys39 and Arg54 as well as Arg50 in the wild-type and Gln52 in the mutant are in close contact with the ATP molecules at the beginning of the simulations. Some of these interactions are maintained during the entire simulation time and in some other instances, there is an exchanged between the pair of atoms of a given residue that interact with the ATP molecule Fig. . During ATP channel on skeletal muscle, myogenesis and glucose uptake measurements were performed. As shown in Fig. ATP channels are not involved in the myogenic index. At the same time, myotube width analyses show that both Kir6.2 (WT)-expressing and Kir6.2 (R50P)-expressing myotubes exhibit almost similar width distribution with a maximum peak at 27–30 μm. Taken together, these results indicate that KATP channels are not involved in myogenesis on a cell line basis. Subsequently, glucose uptake was evaluated with confocal laser-scanning microscopy. The mean average values of the green fluorescence were recorded for the glucose uptake in Kir6.2/WT- and Kir6.2/R50P-expressing murine skeletal myotubes. Figure ATP channels are not involved in glucose uptake on a cell line basis.To examine the physiological effects of the R50P mutation of the K19. Interestingly, there was no significant difference in muscle membrane potential between wild-type and m-V59M mice or wild-type and n-V59M mice, indicating that the gain-of-function mutation in the KATP channel does not directly affect muscle function itself. However, the present study shows that the R50P mutation, DEND syndrome inducing mutation with more severe symptoms than the V59M, induces a hyperpolarized membrane potential. Although the muscle weakness is mainly originated in the brain, our present data indicate that severely reduced ATP sensitivity in the muscle KATP channels may take part to develop muscle weakness in DEND syndrome patients. From the recent KATP channel structures solved at atomic resolution, it is inferred that Arg50 can play pivotal roles in the recognition of nucleotides by establishing electrostatic interactions with ATP, and it is indeed part of the ATP binding pocket in Kir6.240. It is well established that this mutation decreases ATP sensitivity without affecting the open probability of the channel (denoted “binding mutation”)17. Our present simulation results further confirmed that R50P mutation indeed contributes to reduce the ATP sensitivity. It is of interest because the simulation enabled us to see the serial interaction between ATP and its binding residues while released from the binding pocket (such as the K185Q mutation described above), which facilitates the understanding of molecular bases of ATP binding mechanism and development of neonatal diabetes/DEND syndrome. In contrast, Val59, which has been reported to affect ATP sensitivity with increased open probability (denoted “gating mutation”)19 is located at a distal place, > 18 Å away from the ATP location (PDB ID: 6JB1) and not contributing directly to ATP binding. Consequently, the effect on the activation mechanism of each of these two mutations seems to be rather different from an atomistic perspective.A previous study demonstrated that muscle dysfunction was of neuronal origin by comparing muscle- and nerve-specific expression of the Kir6.2/V59M mutation in transgenic mice , the most commonly known mutation inducing intermediate DEND syndrome45. In line with these observations, higher concentrations of ADP were required to activate KATP channels that contained more SUR2A than SUR1 or SUR2B regulatory domains46. As a consequence, the KATP channel with the SUR2A subunit tended to be found in a closed state. Therefore, it is possible that a “binding mutation”, such as R50P, has higher impact on ATP sensitivity in SUR2A-containing KATP channels compared to “gating mutations” such as V59M, which showed no difference in membrane potential in skeletal muscle. As suggested by the recently reported KATP channel atomic structure40, SUR1 adopts an inward-facing conformation which is an inactive form, with a folded lasso motif that facilitates ATP binding in Kir6.2. In contrast, the lasso motif transforms to an extended form when the complex adopts an outward-facing structure. Considering these observations, given that SUR2A adopts an inward-facing conformation more likely than SUR1, the R50P mutation may disrupt SUR2A regulation more drastically than the SUR1 regulation, which is consistent with the previous observations46. Consequently, the R50P “binding mutation” in Kir6.2 may have a higher impact on the membrane potential than the “gating mutation” in skeletal muscles, or even in cardiomyocytes50.Inside-out patch clamp experiments demonstrated that the C-terminal tail comprised by 42 a.a. residues in SUR2A possess an inhibitory effect on NBD2-mediated ADP-induced channel activation51, we consider that our system is reflecting the heterozygous state of the patients without excessive expression of KATP channels in cell surface.In this study, we used C2C12 cells with transfected WT and R50P-mutated Kir6.2. Since Kir6.2 and SUR are both required to be expressed in cell surface, transfected Kir6.2 channel surface expression should be under the regulation of endogenous SUR. Because except for the rare case, most of the reported neonatal diabetes/DEND syndrome patients are heterozygous with mutated channel subunitATP channel, approximately 33% channel current is unblockable17. However, high dose of MgATP was able to fully block KATP channel current in Kir6.2/R50P transfected C2C12 cells -expressing myotubes, whereas the application partly restores the hyperporalized state in Kir6.2 (R50P)-expressing myotubes. These results indicate that in skeletal muscle, wild-type KATP channels are closed in physiological state with very small contribution on membrane potential and also indicating the possible effects of glibenclamide on DEND syndrome patients. Also, we need to keep in mind that glibenclamide is reported to act on activate exchange protein directly activated by cAMP 2 (Epac2A) or carnitine palmitoyltransferase 1 (CPT-1)55. Epac2A is not expressed in skeletal muscle but CPT-1 is known to be expressed in skeletal muscle and functions as the key regulatory enzyme of mitochondrial long-chain fatty acid oxidation. Therefore, further study is required to clarify the targets of glibenclamide other than KATP channels and its function in skeletal muscle.In previous study, ATP sensitivity experiments show that in pancreatic Kir6.2/R50P-SUR1 heteromeric Klls Fig. , it can ATP channel in skeletal muscle56. Therefore, we have investigated whether opening of KATP channel induced by R50P mutation has any effect on the downstream signaling of the channel. The Baf60c-Deptor-AKT2 axis was found not to be affected by the R50P mutation -expressing mouse is required to clarify the mechanism of muscle weakness in the DEND syndrome patients.As for the limitation, data from the transgenic mice with Kir6.2 (R50P) or any other neonatal diabetes inducing mutation are not available in this study. However, the present study provided insight on the links between muscle weakness and sarcoK59. Briefly, the murine C2C12 (muscle myoblast) cell line (code: CRL-1772) was purchased from the American Type Culture Collection . Myoblasts were cultured at 37 °C in a humidified atmosphere of 5% CO2 in growth medium consisting of high-glucose Dulbecco’s modified Eagle’s medium , 10% (v/v) heat-inactivated fetal bovine serum (FBS) , 100 U/mL penicillin (Wako), and 100 μg/mL streptomycin (Wako). When the cells reached 70–80% confluence, the culture medium was changed to a differentiation medium to induce myogenesis. The differentiation medium consisted of high-glucose DMEM, 2% heat-inactivated horse serum , 100 U/mL penicillin (Wako), and 100 μg/mL streptomycin (Wako).The murine myoblasts and myotubes were prepared as described in the previous study2HPO4, 5 mM KCl, 6 mM D( +)-Glucose). After transfection for 72 h, cells were harvested by centrifugation , resuspended in TBS Buffer  and disrupted by freeze–thaw cycles (three times). After disruption, benzonase (Novagen Millipore) was added and incubated with DNaseI for 30 min at 37 °C. After centrifugation , the centrifugation tubes with CsCl2 dissolved in phosphate-buffered saline (PBS) and the supernatant were centrifuged  for 24 h at 16 °C. This CsCl2-based purification step was repeated twice with two CsCl2 concentrations (1.24 g/mL and 0.55 g/mL). After gravity flow purification, each fraction was confirmed by reverse transcription polymerase chain reaction (RT-PCR) using the WPRE sequence, and dialyzed three times to replace the Cs containing buffer with PBS buffer. After concentrated approximately to 100 μL, the virus was frozen at -80 °C. The genomic titer was determined by RT-PCR.The plasmid pAAV vector, which was provided by K. Kobayashi, encoded the EF1α promoter, 2A self-cleaving peptide, red fluorescent protein (RFP) and woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequences. The point mutant (R50P) of Kir6.2 was generated by using the wild-type Kir6.2 cDNA as templates and primers 5′-GGG AAC TGC AAC GTA GCC CAC AAG AAC ATA CCT GAG CAG GGT CGC TTC TTG CAG GAC GTG TTT-3′ and 5′-AAA CAC GTC CTG CAA GAA GCG ACC CTG CTC AGG TAT GTT CTT GTG GGC TAC GTT GCA GTT CCC-3′. Amplified fragments of Kir6.2 (WT) and Kir6.2 (R50P) were incorporated into the pAAV vectors described above. Homogeneous recombination was performed by using the in-fusion HD cloning kit . Around 50% of confluent HEK293T cells grew in high-glucose DMEM growth medium supplemented with 10%(v/v) FBS, 100 U/mL penicillin and 100 μg/mL streptomycin were triple transfected with a pHelper vector , a pRC2-mi342 vector , and the above-described pAAV vector encoding the Kir6.2 (WT) or Kir6.2 (R50P) gene plasmids, using a HBSP solution , PDB IDs: 6C3O; 6C3P; 6BAA; 5WUA; 5TWV; 5YKE; 5YKF; 5YKG; 5YWA; 5YWB; 5YWC; 5YW8; 5YW9; 6JB1, all of which were obtained by cryogenic electron microscopy40. Structures with resolution higher than 4 Å were selected: 6C3O (3.9 Å), 6BAA (3.6 Å), 6JB1 (3.3 Å). The Arg50 residue in Kir6.2 is only assigned in PDBs 6BAA (3.6 Å) and 6JB1 (3.3 Å), and thus, these two structures were selected for structural analyses using PyMol (http://pymol.sourceforge.net/). To begin to address the structural mechanisms by which the mutations considered in this study alter the action of ATP on the Kir6.2 +-channel, molecular dynamics simulations were carried out using the wild type channel and the mutant R50P. The crystal structure of Kir6.2 closed at its inner gate was retrieved from the Protein Data Bank (PDB ID 6JB1)40 and resolved residues 26–114 were used for subsequent modelling. This particular crystal structure of Kir6.2 was chosen for this study over other available structures of the channel because it has a good atomic resolution, the two residues focused of the study are resolved, and it was obtained in the presence of the SUR subunits. R50P mutant channels were generated using the Mutator plugin of VMD. N- and C-termini were acetylated and methylated, respectively. The VMD solvate plugin was used to solvate internal cavities of the protein. The structures were aligned perpendicular to the bilayer and inserted into a membrane, a neutral membrane containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), with x and y dimensions of 96 Å. The VMD solvate plugin60 was then used to create a rectangular water box around the membrane-protein system. The overlapping water and lipid molecules around the ion channel structure were removed with the cut-off distance (1.2 Å). Potassium and chloride ions were added using Autoionize Plugin of VMD to neutralise the systems and obtain a concentration of 150 mM. The final system size was approximately 183,000 atoms.The Khttps://doi.org/10.5281/zenodo.3562512). Four replicates of 500 ns were run for the wild type and R50P mutant amounting to 4 µs in total. CHARMM3661 parameters were used for the protein, ions, ATP and lipids, and the TIP3P model was used for water. The particle mesh Ewald method was used for the treatment of periodic electrostatic interactions62, with an upper threshold of 1 Å for grid spacing. Electrostatic and van der Waals forces were calculated every time step. A cut-off distance of 12 10 Å was used for van der Waals forces. A switching distance of 10 Å was chosen to smoothly truncate the non-bonded interactions. Only atoms in a Verlet pair list with a cut-off distance of 13.5 Å reassigned every 20 steps were considered. The SETTLE LINCS algorithm63 was used to constrain all bonds involving hydrogen atoms, to allow the use of a 2 fs time step throughout the simulation. MD simulations were performed in the NPT ensemble. The Parrinello–Rahman barostat64 was employed to control the pressure with a 50 fs period, and the desired value of 1 atmosphere. The system was coupled to a Nose–Hoover thermostat66 to sustain a temperature of 310.15 K throughout.MD simulations were performed with the gromacs v2020.1 software (2), the ATP , and lipid headgroups . Harmonic restraints were gradually reduced to a force constant of 2 kcal/mol/Å2 and were removed in consecutive steps from the lipid headgroups, protein sidechains and protein backbone over the course of a 10 ns trajectory. Production runs followed and analysis of the simulations was performed with python v3.7.3 scripts using the MDTraj v1.9.4 python package.The same equilibration protocol was applied to all the systems. The systems were subjected to 10,000 steps of minimization, with harmonic constraints on protein backbone atoms and protein sidechains  using an EPC-800 patch clamp amplifier  and pCLAMP 10 software . The membrane potentials were recorded in the current clamp mode in standard whole-cell technique68 and the effects of 10 mM ACh and 0.1 mM glibenclamide were evaluated. The pipette solution contained (in mM): 107 KCl, 2 MgCl2, 1 CaCl2, 10 EGTA, and 10 HEPES at pH 7.2 with KOH. The extracellular solution contained (in mM): 138 NaCl, 5.6 KCl, 1 MgCl2, 10 HEPES, and 2.6 CaCl2 at pH 7.4 with NaOH. Standard patch clamp techniques with inside-out membrane patch was used to record KATP channel currents for MgATP sensitivity measurements. The pipette solution contained (in mM): 140 KCl, 2.0 CaCl2, 5.0 HEPES at pH 7.4 with KOH. The composition of bathing solution was (in mM): 110 KCl, 11 HEPES, 2 MgCl2, 11 EGTA, 1 CaCl2 at pH 7.2 with KOH. For the KATP channel recording, patch membrane potential was held at − 60 mV and various concentration of MgATP was applied. Relative channel activity was plotted as a function of MgATP concentrations, and each points were fitted in following equation.50 is the [MgATP] at which inhibition is half-maximal and h is the slope factor (Hill coefficient). Data were analyzed using Origin Graphing and Data Analysis Software .Electrophysiological analyses were performed according to the previous study2+]i was measured as previously reported69. Mouse skeletal myotubes were incubated with 2 μmol/L Fura-2/AM  for 30 min at room temperature, mounted in a chamber and superfused with KRB at 1 mL/min at 37 °C. Fluorescence images due to excitation at 340 and 380 nm were captured and the ratio (F340/F380) images were produced by an Argus-50 system . Amplitudes of [Ca2+]i responses to ACh were calculated by subtracting the prestimulatory basal [Ca2+]i ratio from the peak [Ca2+]i ratio. A response was considered when increases in [Ca2+]i took place within 5 min after the addition of agents. For calculation of 10 mM of ACh, the basal [Ca2+]i ratio from peak [Ca2+]i ratio for 5 min within 10 min from onset of measurement was analyzed.-2-deoxy-d-glucose (2-NBDG) uptake. After confirming the differentiation, the culture medium was changed to modified Krebs Ringer Buffer without glucose  and incubated for two hours. A final concentration of 50 μM 2-NBDG was added to the media, and incubated for two hours. After washing three times with PBS buffer, the dishes were covered with cover glasses. We confirmed that the results of 2-NBDG uptake showed a good correlation with 2-[18F]-2-deoxy-d-glucose (2-FDG) uptake, a well-established method for measurement of glucose uptake. A total of 60–90 myotubes (30 fields for each independent experiment) were evaluated for each treatment group.For quantitative analyses, the glucose uptake was evaluated by fluorescent glucose analog, 2-[Polymerase chain reaction amplification was performed by using a StepOne Real-time PCR system  under the following conditions: one cycle of 95 °C for 30 s; 45 cycles of 95 °C for 5 s and 56 °C for 10 s and 72 °C for 15 s. The amplified fragments were applied to 2% (w/v) agarose gel, and visualized by staining with SYBR Safe DNA staining reagent . In immunostaining experiments, the glass bottom dishes, which were cultured transfected and non-transfected myotubes, were washed in phosphate buffered saline (PBS) and incubated in blocking solution containing 2% bovine serum albumin (BSA) and 5% normal goat serum for 1 h. Then, the dishes were incubated with rabbit polyclonal Kir6.2 antibody  for 48 h at 4 °C. Dishes were washed in PBS and incubated with Alexa 488-labelled anti-rabbit IgG  for 30 min and mounted with a DAPI-containing mount medium and covered with a cover glass.71, the data are presented as the means ± standard error of the mean (SEM) unless otherwise indicated. One-way analysis of variance (ANOVA) was used, and the treatment groups were compared with Tukey’s post hoc test for honest significant difference. All statistical analyses were performed using GraphPad Prism 8.2.1  for MacOS, version 10.14.6. A p value of < 0.05 was considered statistically significant.As previously reportedSupplementary Information


Total collagen deposition was measured colorimetrically after Sirius red staining of fixed cell layers, and type I, III, and VI collagen biosynthesis and degradation were specifically determined by the biomarkers PINP, PRO-C3, PRO-C6, and C3M in conditioned media by competitive enzyme-linked immunosorbent assays. Total collagen deposition by fibroblasts from the colon and rectum did not significantly differ. TGF-β1 treatment increased PINP, PRO-C6, and total collagen deposition. Mechanistically, TGF-β1 treatment increased COL1A1 and ACTA2 (encoding α-smooth muscle actin), and decreased COL6A1 and MMP2 mRNA levels in colorectal fibroblasts. In conclusion, we found no effect of anatomic localization on collagen production by fibroblasts derived from the large intestine. TGF-β1 represents a potential therapeutic agent for the prevention of AL by increasing type I collagen synthesis and collagen deposition.Anastomotic leakage (AL) is a devastating complication after colorectal surgery, possibly due to the loss of stabilizing collagen fibers in the submucosa. Our aim was to assess the formation of collagen in the colon versus the rectum with or without transforming growth factor (TGF)-β1 exposure in a human cellular model of colorectal repair. Primary fibroblasts were isolated by an explant procedure from clinically resected tissue rings during anastomosis construction in 19 consecutive colorectal patients who underwent laparoscopy. The cells, identified as fibroblasts by morphologic characteristics and flow cytometry analysis (CD90 Anastomotic leakage (AL) is the most feared complication after colorectal surgery ,2,3. AL The extracellular matrix (ECM) is key for maintaining homeostasis of the large bowel ,11. In pCOL1A1 mRNA levels in an experimental model of anastomotic wound healing [Fibroblasts are key cells in the production of type I, III, and VI collagens . Growth  healing . In anot healing . Finally healing . Thus, TCOL1A1 and COL3A1 mRNA levels in the rectum compared to colon, which may explain the higher risk of AL after rectal resection compared to colonic resection [We recently demonstrated decreased esection . The priesection , and speesection ,37,38. Mesection .Thirty-three eligible patients scheduled for laparoscopic sigmoid resection (SR) or low anterior resection (LAR) were approached for participation the day before their operations from 2 October 2017 to 18 January 2018, and 31 patients were included after providing their written consent. Tissue samples from 10 patients were excluded for various reasons as indicated in We applied an explant procedure to obtain primary fibroblasts . This meCells had the typical spindle-shaped appearance of fibroblasts, as shown in Fibroblasts of the third passage at a mean of 45 days (range: 33–72 days) from the harvest of the tissues were used for assays in 6-well tissue culture plates . The amop = 0.361), measured by crystal violet-stained fixed cells, derived from proximal (OD590nm: 0.90 ± 0.18/well) or distal tissue rings (OD590nm: 0.70 ± 0.072/well).Furthermore, we observed no difference in fibroblast density , type III (PRO-C3 and C3M), and type VI (PRO-C6) collagens released into the culture medium. The lack of difference in the amount of deposited insoluble collagen between the proximal and distal locations agrees with the collagen biomarkers released into the conditioned media, except for accumulated PRO-C6, which was higher in rectal versus colonic fibroblasts, as shown in AL occurs more often in male versus female patients . Therefop = 0.244) between CoReFs isolated from male (OD540nm: 0.18 ± 0.012/well) and female (OD540nm: 0.20 ± 0.020/well) patients. The corresponding cell densities of the CoReFs were OD590nm: 0.78 ± 0.10/well for males and OD590nm: 0.82 ± 0.12/well for females (p = 0.784).Collagen deposition did not differ  (p < 0.0001) compared to the control . Representative staining results are shown in Concomitantly, we observed significantly increased numbers of CoReFs treated with TGF-β1  and ACTA2 (8.3-fold) mRNA levels, while the normalized COL6A1 and MMP2 mRNA levels decreased in response to TGF-β1 treatment, as shown in To elucidate the mode of action of TGF-β1, we measured levels of key genes by RT-qPCR. TGF-β1 treatment increased Immunofluorescence analyses of CoReFs indicated a low level of basal α-smooth muscle actin (α-SMA) expression, which increased considerably in response to TGF-β1, as shown in The dose-dependency of TGF-β1 on collagen deposition, cell density, and MMP-2 was studied in six different CoReF strains.p = 0.797) of TGF-β1 on MMP-2 levels in the conditioned media on Day 8. Results from three of the six tested CoReF strains are shown in TGF-β1 treatment increased collagen deposition and cell growth in a dose-dependent manner, as shown in ACTA2 (α-SMA) with TGF-β1 presupposes that the original cells are fibroblasts [AL is a serious complication following colorectal surgery and has been attributed to altered ECM metabolism, especially collagen metabolism . In the roblasts ,30. Otheroblasts . NeverthCOL1A1 and COL3A1, as indicators of reduced type I collagen and type III collagen synthesis in the rectum versus colon, were not translated into fibroblasts in the present study [The accumulated collagen was the main outcome in this study. No significant differences in the capacity to form insoluble collagen by fibroblasts isolated from the rectum or colon were found, which corroborates earlier in vivo measurements . This cont study . One posWe estimated collagen deposition using the dye Sirius red to be approximately 20 µg in control cultures. Because Sirius red also binds, albeit weakly, to noncollagenous proteins and stronger to type I than type III collagen, this is most likely an overestimation of the collagen deposited by the fibroblasts in our culture system .AL rates are higher in male versus female colorectal patients, possibly due to reduced collagen biosynthesis . We did Interestingly, rectal fibroblasts appeared to synthesize more type VI collagen than colonic fibroblasts. Although type VI collagen is important for the architecture of the ECM, the effect of type VI collagen on cell motility is striking. The rate of migration of dermal fibroblasts is increased on fibroblast-generated ECM substrates deficient in type VI collagen . Thus, wTGF-β1 is central in wound healing and fibrosis. TGF-β1 regulates collagen synthesis at several levels ,27,29. ICOL1A1 mRNA and the biomarker PINP but not type III collagen synthesis measured by COL3A1 mRNA or the biomarker PRO-C3. Treatment of normal human dermal fibroblasts with TGF-β1 also resulted in increased type I collagen but not type III secretion or deposition [COL6A1 mRNA levels in the CoReFs. Juhl et al. [COL6A1 in normal human dermal fibroblasts.Specifically, TGF-β1 increased the synthesis of type I collagen measured by position . Althougl et al.  found thIn another study, TGF-β did not increase relative collagen synthesis by colonic fibroblasts and reduced the relative collagen synthesis when cultured in the presence of 10% serum  ,52. ThesMMP-2 mRNA and protein levels [MMP-2 mRNA and protein levels as well as MMP activity measured by the C3M biomarker on a cell basis.MMP-2 degrades type I collagen and several other ECM components, and is induced during wound healing . The roln levels ,56. The In an animal model, the TGF-β1 gene was administered locally to the colonic anastomosis via a viral vector. The application of TGF-β1 on postoperative Day 3, but not on Day 0, resulted in improved anastomotic wound healing on postoperative Day 6 . Thus, tThe microbiome of the gastrointestinal tract influences the outcome of colorectal surgery and AL has been associated with specific microorganisms ,58,59. TCollagen formation during anastomotic wound healing is important not only to reestablish the collagen network but also to contribute to the restitution of the epithelial layer .In conclusion, we found no effect of anatomic localization on collagen production by fibroblasts derived from the large bowel. TGF-β1 represents a potential therapeutic agent for the prevention of AL by increasing collagen synthesis and collagen deposition. It would be extremely valuable to systematically examine the impact of the secretome of the microorganisms causing AL in our cellular model. The usefulness of the biomarkers examined here to predict AL and monitor the effect of novel treatments would also be worthy of exploration.This study was approved by The Committee on Biomedical Research Ethics for the Capital Region of Denmark (H-17011833), registered by The Danish Data Protection Agency (BFH-2017-058), and conducted at the Digestive Disease Center, Bispebjerg Hospital, University of Copenhagen, Copenhagen, Denmark.Patients older than 18 years with colorectal cancer who underwent elective laparoscopic sigmoid resection (SR) or low anterior resection (LAR) with primary anastomosis using a circular stapler were invited to participate . PatientChemicals and reagents were purchased from Sigma-Aldrich  unless stated otherwise.2+, 0.80 Mg2+, 5.6 mM glucose, and antibiotics-antimycotics . The mucosa was scraped off with a knife, including the lamina propria with subepithelial myofibroblasts [®, Greiner Bio-One, Frickenhausen, Germany), air-dried for 20 min, and incubated in 5 mL of complete culture medium  in a humidified atmosphere of 5% CO2/air at 37 °C, as shown in Proximal and distal tissue rings were collected during the laparoscopic surgical procedure . Macroscroblasts , and the2/air. Detached cells were resuspended in 5 mL complete culture medium, and the cell count was performed using a Bürker-Türk hemocytometer or Countess™. Approximately 0.5 × 105 cells were transferred to a new T75 flask. Cells at passage 3 were used for the assays.Cells were washed in PBS and incubated with 1 mL 0.05% trypsin-0.02% EDTA  for 5–6 min at 37 °C in a humidified atmosphere of 5% CO6 cells) in complete culture medium were centrifuged, and the pellet was suspended in 1 mL cryopreservation medium  and frozen in a Mr. Frosty™ container (Thermo Fisher Scientific) at −80 °C. Cryopreserved cells were kept at −140 °C for long-term storage.Freshly trypsinized cell strains (0.5 × 105) were incubated with a (1) FITC-conjugated mouse monoclonal anti-human CD90 (Thy-1) antibody  diluted 1:50 in PBS [g for 5 min), resuspended in PBS, and added to microtiter plates for flow cytometry analysis . Data were analyzed using Kaluza software (Beckman Coulter).Cells were grown in a T75-flask to 80% confluence, trypsinized, suspended in PBS, and counted. Cells  4 cells/well) in 6-well tissue culture plates . Cells were incubated to 70–80% confluence in complete culture medium (3 mL/well), which took a mean of 5 days (range: 3–7 days) in culture. The medium was then removed and replaced with assay culture medium . Cells were then incubated for a total of 8 days with collection of conditioned media with replacement with fresh assay culture medium after 4 days. After 8 days, conditioned media were again collected and stored at −80 °C until analysis. Cells from the last 12 analyzed patients were treated with or without 1 ng/mL rhTGF-β1 . rhTGF-β1 reconstituted in 1 mg bovine serum albumin (A3803)/mL 4 mM HCl was added to 2 separate wells (30 µL of 100 ng/mL TGF-β1/well), and the remaining 4 wells received control alone (30 µL of 1 mg BSA/mL 4 mM HCl/well). The dose-response effect of TGF-β1 was studied in 6 of 12 patients. Fibroblasts in two 6-well tissue culture plates from each patient were used; in each plate, 3 wells received control, while the other 3 wells received 0.01, 0.1, or 1 ng/mL TGF-β1. To estimate the amount of collagen deposited by the fibroblasts, neutralized type I collagen (0.5 mg/mL) from rat tails  was dispensed into an empty 6-well plate, incubated in a humidified atmosphere of 5% CO2/air at 37 °C for 2 h and dried in a laminar flow bench. The plate was then processed identically to the plates with cultured cells. The wells were washed in PBS, fixed in 2.5 mL Bouin’s solution for 1 h, washed in running tap water, and air-dried overnight.We adopted the method of Trackman et al.  to measu540nm) was measured in 200 µL duplicate aliquots in a microtiter plate against the blank 0.1 M NaOH. Cells were then washed with PBS and stained with 3 mL crystal violet (Merck 1.01408) in deionized water at 1 mg/mL for 30 min on an orbital shaker. The superfluous dye was washed away in running tap water, and the stained cells were air-dried overnight. The stain was eluted with methanol (5 mL/well) and the OD590nm measured in 200 µL duplicate aliquots with methanol as blank in a 96-well microtiter plate. The mean OD540nm and OD590nm measurements of the 200 µL aliquots are reported.The fixed cells/collagen were stained with 2 mL Direct Red 80 dye (Fluka 43665) at 1 mg/mL water-saturated picric acid (1.3%) for 1 h on an orbital shaker at 100 rpm. The nonbound dye was removed, and the wells were washed 4 times with 0.01 M HCl. The bound dye was eluted in 1.0 mL 0.1 M NaOH for 30 min on the orbital shaker. The optical density  grown for 6 days to confluence in 12-well tissue culture plates  in complete culture medium. The medium was removed and the CoReFs were treated for 48 h with or without 1 ng/mL TGF-β1 in assay culture medium. Total RNA was extracted from the treated cells with 1 mL TriReagent® . Bromochloropropane (100 µL) was added to isolate the aqueous phase containing the RNA, which was precipitated using isopropanol. The RNA pellet was then washed in ethanol and subsequently dissolved in 10 μL RNAse-free water. Total RNA concentrations were determined with the RiboGreen assay . Total RNA (500 ng) was converted into cDNA in 20 μL using OmniScript reverse transcriptase  and 1 μM poly-dT (Thermo Fisher Scientific) according to the manufacturer’s protocol (Qiagen). For each target mRNA, 0.5 μL cDNA was amplified in 25 μL SYBR Green polymerase chain reaction (PCR) containing 1× Quantitect SYBR Green Master Mix  and 100 nM of each primer, as shown in GAPDH mRNA was chosen as the internal control.The effect of TGF-β1 on ®, Trasadingen, Switzerland) at 1 × 104 cells per well in complete culture medium and incubated for 4 days. The medium was then replaced with 200 μL assay culture medium/well with or without 1 ng/mL TGF-β1, and cells were incubated for another 48 h.Cells were seeded into 96-well plates (TPP450nm-OD630nm) was measured on a microplate reader .WST-1 reagent  was added (20 µL/well) 0.5 h before the end of the 48 h treatment period, and ΔOD (OD2SO4 and the ΔOD (OD450nm–OD630nm) was measured on a microplate reader.BrdU in DMEM with 1% FBS was added to all wells (20 μL per well) to a final concentration of 10 μM, except for the background control, to which 20 μL of DMEM with 1% FBS alone was added, and was present during the final 4 h of incubation. The cells were then washed three times with PBS, fixed, and incubated with a mouse monoclonal anti-BrdU antibody conjugated with peroxidase (1:50 dilution) according to the manufacturer’s instructions . The immune complex was detected by the tetramethyl-benzidine substrate , and the reaction stopped after 20 min with 0.2 M H2/well) chamber glass slides essentially following the procedures described by Smith-Clerc and Hinz [2 for 1 h before CoReFs (1 × 104 cells/well) in 0.5 mL of DMEM with 2% or 10% FBS were seeded. After one day, TGF-β1 (1 ng/mL) was added, and the cells were incubated for 4 days. The media were removed, and the cells were washed 3 times in PBS and then fixed in 3% paraformaldehyde/PBS (1 mL/well) for 10 min at room temperature (RT). Fixed cells were permeabilized with 0.2% Triton X-100 (TX-100) in PBS for 5 min at RT (2 mL/well) and incubated with a mouse monoclonal antibody against human α-SMA  diluted in PBS/0.02% TX-100 to 2 µg/mL (250 μL/slide) overnight at 4 °C [® 488-conjugated goat anti-mouse IgG  secondary antibody diluted in PBS/0.02% TX-100 to 1 µg/mL (250 µL/slide). Cells were washed with PBS/0.02% TX-100 and deionized water at RT and then incubated with Hoechst 33342  at 2.5 µg/mL at RT for 5 min in the dark (250 μL/slide). After PBS washes, cells were cover-slipped with Fluorescence Mounting Medium .Immunofluorescence was performed in Nunc™ Lab-Tek™ 4-well (1.8 cmand Hinz . The wel at 4 °C . Cells w®) standard was run in parallel with the samples. The gelatin-copolymerized gel was renatured in 25 μL/mL TX-100 for 30 min at ambient temperature and incubated at 37 °C for 18 h in developing buffer composed of 50 mM Tris-HCl (pH 7.5), 10 mM CaCl2, 1 μM ZnCl2, 1 μL/mL TX-100, and 0.2 mg/mL NaN3. The gel was stained with the Colloidal Blue Staining Kit (Thermo Fisher Scientific), destained, mounted, and scanned. The amounts of MMP-2 were estimated by densitometry using ImageJ software  and presented as pg [Gelatin zymography was performed with 15 wells using a 1.0 mm thick 10% Tris–glycine SDS-PAGE gel containing 1 mg/mL gelatin. Conditioned medium samples (5 µL) were mixed with 5 μL of 2× Laemmli sample buffer  and electrophoresed at a constant voltage of 125 V for 105 min. The rhMMP-2 . The statistical significance level was defined as p < 0.05. Data are presented as the mean ± SEM or median (interquartile range).The effect of localization, sex, and TGF-β1 on collagen deposition and cell density was analyzed by a paired or unpaired Student’s


This study aimed to validate the accuracy of major-event risk models created in the multicenter J-ACCESS prognostic study in a new cohort of patients with chronic kidney disease (CKD).n = 526, major events 11.2%). Survival analysis of low (< 3%/3y), intermediate (3% to 9%/3y), and high (> 9%/3y)-risk groups showed good stratification by all three models . However, actual event rates were equally high across all risk groups of patients with eGFR < 15 mL/min/1.73 m2.Three multivariable J-ACCESS risk models were created to predict major cardiac events : Model 1, four variables of age, summed stress score, left ventricular ejection fraction and diabetes; Model 2 with five variables including estimated glomerular filtration rate ; and Model 3 with categorical eGFR. The validation data used three-year (3y) cohort of patients with CKD  contains supplementary material, which is available to authorized users. The present study also aimed to determine the effect of eGFR on associations between risk models and outcomes.99mTc-tetrofosmin SPECT MPI. The inclusion criteria were ≥ 20 years of age and undergoing stress and rest MPI due to suspected or known ischemic heart diseases. Patients with the onset of MI or unstable angina pectoris within 3 months, cardiomyopathy, and heart failure with class III or higher New York Heart Association classification were excluded. Perfusion defects were semi-quantified in a 17-segment model using a scale from 0  to 4 (absent) for each segment and then summed stress/rest/difference scores (SSS/SRS/SDS) were calculated.Table n = 54; 1.3%), non-fatal MI  and severe HF requiring hospitalization .During 3 years of follow-up, a total of 175 (4.3%) of the patients developed the major events of cardiac death  was calculated using the multivariable logistic model:Multivariable logistic analysis of the 4,031 individuals showed that SSS , LVEF, age, and diabetes were significant variables  clarified the importance of including CKD or eGFR. Therefore, all coefficients were determined based on similar logistic analysis with five variables  classified as 1 (< 30), 2 , 3 , 4 , and 5 (≥ 90); and SSS was classified as 0 (≤ 3), 1 (4-7), 2 (8-11), and 3 (≥ 12).Model 3 was used in Heart Risk View software , which was a revision of Model 2 that included categorical variables and was intended for practical application to clinical situations. The model included eGFR . Chronic kidney disease was defined based on the Japanese equation for eGFR (mL/min/1.73 m2).The principal design and results of J-ACCESS-3 are described elsewhere.99mTc-tetrofosmin and Anger-type cameras. Pharmacological stress studies proceeded using adenosine (91%), dipyridamole, or adenosine triphosphate. The average administered doses of 99mTc-tetrofosmin for the initial and second studies were 312 and 689 MBq, respectively. Chest pain , ST depression , atrioventricular block (the second degree or worse) , and serious arrhythmia  developed during the pharmacological stress study. Both LVEF and LV volumes were quantified using QGS software .Either 1-day (96%) or 2-day (4%) protocols were applied in pharmacological stress MPI studies using a standard protocol of An interpretation committee comprising seven nuclear cardiology experts objectively analyzed all data from standard SPECT slices including SSS, SRS, and SDS. Thresholds for defect scoring were based on a normal database derived from the Japanese Society of Nuclear Medicine working group database.16The endpoints of the J-ACCESS-3 study were major cardiac events comprising cardiac death, sudden cardiac death, non-fatal MI, and hospitalization to treat HF. Sudden death was defined as death of unknown cause at 24 hours after occurrence. These major cardiac events were confirmed at each institution based mainly on medical charts, along with written questionnaires and telephone interviews. During the 3-year follow-up, 4.8% and 3.6% of the patients were lost to the J-ACCESS and J-ACCESS 3 studies, respectively.The Institutional Review Boards of all participating hospitals approved both the J-ACCESS and J-ACCESS-3 studies, both of which also complied with the Ethical Guidelines for Epidemiological Research in Japan.T tests and analyses of variance. Categorical data between groups were compared using χ2 tests. Since C-reactive protein (CRP) was one of the major independent prognostic factors at a threshold of 0.3 mg/mL,P value of < 0.05 was considered to indicate a significant difference.Data are expressed as mean ± standard deviation (SD) or median and ranges for non-normal distribution. Continuous variables were compared using 2 (P < .0001), respectively. The SSS, SRS, and SDS were lower in J-ACCESS-3 than in the Model 2 derivation populations .Table n = 42) sudden death (n = 6), cardiac death (n = 3), death due to HF upon admission (n = 3), non-fatal MI (n = 2), non-fatal MI and HF upon admission (n = 2), and non-fatal MI and subsequent cardiac death (n = 1). We found lower values for LVEF  and eGFR , and higher values for EDV , ESV , SSS , and SRS , in a group that developed HF as the first event (n = 45) compared with a group that did not develop events (n = 466). The SDS did not significantly differ between these groups .Out of 526 patients 59 (11.2%) patients developed cardiac events, and 13 (2.5%) had cardiac or sudden death. The events in these 59 patients comprised HF treated upon admission (2 (n = 99) showed more frequent renal events including 40 (40.4%) with hemodialysis and 4 (4.0%) with peritoneal dialysis.Renal events of hemodialysis and peritoneal dialysis developed in 45 (8.6%) and 4 (0.8%) patients, respectively. Patients with eGFR < 15 mL/min/1.73 mP < .0001; Model 2 vs 3 P = .0041). High correlation was observed for risk values between Models 1, 2, and 3 showing Risk by Model 2 = 1.5 + 1.21 × Risk by Model 1  and Risk by Model 2 = 0.40 + 1.03 × Risk by Model 3 .The median risk values were 3.3% (range 0.2%-36.6%), 5.3% (0.25%-44.2%), and 5.0% (0.25%-44.5%) for Models 1, 2, and 3, respectively, and were highest for Model 2  were 0.66 , 0.67 (0.59-0.73), and 0.66 (0.58-0.73) for Models 1, 2, and 3, respectively, indicating that the models similarly predicted low to high major cardiac event. Thresholds were determined from the highest value for sensitivity + specificity − 1 at 4.3, 5.2, and 4.5% risk for Models 1, 2, and 3, respectively.P = .0001), 2 (P = .0014), and 3 (P = .0047).Figure 2, each risk group was clearly separated by the three models among those with eGFR ≥ 15 mL/min/1.73 m2. The actual event rate was the lowest among the low-risk (< 3%) patients in Model 2 (3.1%). In contrast, none of the three models could stratify patients with eGFR < 15 mL/min/1.73 m2 (n = 99) into three risk groups . Figure 2 depending on risk for all models . However, the frequency of elevated CRP was high in all groups of patients with eGFR < 15 mL/min/1.73 m2 regardless of model type.The CRP values ranged from 0 to 10.1  mg/dL. Analyses of ROC curves showed that the optimal cutoff to obtain the highest sensitivity + specificity − 1 was 0.3 . The risk of events predicted by Model 1 was underestimated compared with actual event rates (1%-3% vs 5.4% and 3%-9% vs 14.9%), whereas Model 2 including eGFR predicted 1%-3% and 3%-9% risk of events, compared with the actual rates of 3.1% and 9.9%, respectively. These outcomes were still slightly higher than the upper limit of the predicted values with eGFR. Although Models 2 and 3 tended to be similar, the risk of events predicted by Model 2 was 3.1%, which was lower than the 4.5% predicted by Model 3 for low-risk patients. Therefore, although the J-ACCESS risk model including eGFR improved risk estimation (%/3 years), the actual outcomes for patients with CKD remained underestimated to some degree.2 (stage 5), which was selected because this threshold is a significant predictor of survival in patients with CKD.2, the rates were 3%-5% and 9%-13% for low- and intermediate-risk patients, respectively, whose predicted risk was apparently underestimated when eGFR was < 15 mL/min/1.73 m2. A comparison of Models 2 and 3 showed that Model 2 estimated lower risk for low-risk patients, and that the difference in outcome events among the risk groups tended to be larger than that of Model 3. This could be explained by differences between the numerical and categorical applications of variables; eGFR < 30 mL/min/1.73 m2 was categorized into one category in Model 3, but continuous in Model 2. These model structures might have caused the larger difference in calculated risk at lower eGFR.Predictive accuracy was quite different depending on eGFR > 15  or < 15 mL/min/1.73 m2) is an important limitation of the present risk model. Since few patients (3%) had stage 5 CKD (end-stage) in the model-derivation group, the predictive accuracy of the model might be less accurate with respect to this validation group. Although the present study could not determine the pathophysiological reasons for this phenomenon, high CRP or an inflammatory reaction might have been associated, which has also been noted in previous studies. Potentially critical elements in the initiation, progression, and rupture of plaque have become evident, and anti-inflammatory and antioxidant therapies have been sought.123I-metaiodobenzylguanidine uptake was low among patients with low eGFR, and this contributed to the high cardiac mortality risk among patients with chronic HF.23The underestimated risk among patients with CKD stage 5 , a wide range of risk was evaluated. A recent validation of J-ACCESS Model 2 also found that an event risk of 10% separated the prognosis of patients with a mean eGFR of 67.4 ± 24.3 mL/min/1.73 m2), whereas patients with stage 5 CKD (eGFR < 15 mL/min/1.73 m2) are potentially at high risk across all estimated risk values.Three J-ACCESS models were applied to assess risk of major cardiac events among CKD patients in the J-ACCESS-3 study. Although all risk models for major cardiac events could stratify outcomes of patients with CKD, the model that included eGFR was more appropriate for these patients. Risk stratification was effective for patients with stages 3a, 3b, and 4 CKD Below is the link to the electronic supplementary material.


We previously showed that JAK1/JAK2 inhibitors reversed autoimmune insulitis in non-obese diabetic (NOD) mice and also blocked IFN-γ mediated MHC class I upregulation on β cells. Blocking interferons on their own does not prevent diabetes in knockout NOD mice, so we tested whether JAK inhibitor action on signaling downstream of common γ chain cytokines, including IL-2, IL-7 IL-15, and IL-21, may also affect the progression of diabetes in NOD mice. Common γ chain cytokines activate JAK1 and JAK3 to regulate T cell proliferation. We used a JAK1-selective inhibitor, ABT 317, to better understand the specific role of JAK1 signaling in autoimmune diabetes. ABT 317 reduced IL-21, IL-2, IL-15 and IL-7 signaling in T cells and IFN-γ signaling in β cells, but ABT 317 did not affect GM-CSF signaling in granulocytes. When given in vivo to NOD mice, ABT 317 reduced CD8+ T cell proliferation as well as the number of KLRG+ effector and CD44hiCD62Llo effector memory CD8+ T cells in spleen. ABT 317 also prevented MHC class I upregulation on β cells. Newly diagnosed diabetes was reversed in 94% NOD mice treated twice daily with ABT 317 while still on treatment at 40 days and 44% remained normoglycemic after a further 60 days from discontinuing the drug. Our results indicate that ABT 317 blocks common γ chain cytokines in lymphocytes and interferons in lymphocytes and β cells and are thus more effective against diabetes pathogenesis than IFN-γ receptor deficiency alone. Our studies suggest use of this class of drug for the treatment of type 1 diabetes.Cytokines that signal through the JAK-STAT pathway, such as interferon-γ (IFN-γ) and common γ chain cytokines, contribute to the destruction of insulin-secreting β cells by CD8 Two cytd humans –5. IFNs d humans . Common d humans , 8. IL-7d humans . Treatmed humans . Similard humans , 12.in vivo, it is also likely that they affect the progression of diabetes in NOD mice by blocking common γ chain cytokine signaling to inhibit T cell proliferation and survival.Both interferons and the common γ chain cytokines signal through the JAK-STAT  pathway. Our lab previously demonstrated that the JAK1/JAK2 inhibitor AZD1480 blocked IFN-γ mediated MHC class I upregulation on β cells, reduced immune cell infiltration into pancreatic islets, and reversed established autoimmune insulitis in NOD mice . HoweverJAK inhibitors have been investigated for autoimmune and inflammatory diseases because they block many cytokines that function in the immune system. Currently, three JAK inhibitors, baricitinib, tofacitinib, and upadacitinib, have been approved for clinical use for rheumatoid arthritis and ruxolitinib for myelofibrosis. The JAK1 selective inhibitor upadacitinib (ABT-494), has 74- and 58-fold greater selectivity for JAK1 over JAK2 and JAK3, respectively , 19. BecIn the present study, we used ABT 317, a JAK1-selective inhibitor, at exposures that are pharmacologically relevant to better understand the mechanism by which JAK inhibitors reverse autoimmune diabetes in NOD mice. The effect of ABT 317 on the function of common γ chain cytokines and T cell proliferation as well as the possible sides effects of ABT 317 on blood were characterized.NOD/Lt and C57BL/6 mice were bred and maintained at St. Vincent’s Institute. All animal studies were approved by the institutional animal ethics committee.50 of 0.016 µM) and IL-2-stimulated pSTAT5 . In contrast, ABT 317 showed reduced potency against cytokine signaling pathways mediated by JAK2  or TYK2 . These cellular potency data . In cells , 22, ABTncy data Table S1in vitro, ABT 317 was resuspended at 10 mM in DMSO and diluted in media to a final concentration of 2 µM. Splenocytes and whole blood cells were incubated with 2 µM ABT 317 for 1 h at 37°C. For use in vivo, 3 mg/kg or 10 mg/kg of drug was delivered twice daily by oral gavage in the vehicle 0.5% hydroxypropyl methylcellulose in sterile H2O. The JAK1/JAK2 inhibitor baricitinib was purchased from SelleckChem and used at a concentration of 3 mg/kg in vivo made up in a vehicle of 0.5% methyl cellulose.For use via orbital bleed at 0.5, 1, 3, 7, 12, and 24 h after gavage and analyzed for concentration of ABT 317.For pharmacokinetics, non-fasted male NOD mice were divided into three groups (n = 2/group) for 3, 10, or 30 mg/kg oral gavage of ABT 317. Plasma was collected To isolate pancreatic islets, the bile duct was injected with 1.3 U/ml collagenase P  in complete HBSS (Lonza), removed and digested for 17 min at 37°C in a water bath, followed by vigorous shaking for 1 min. The digested tissue was filtered through a 500 μm mesh, then washed with RPMI (Invitrogen). Islets were purified using a histopaque-1077 density gradient (Sigma-Aldrich) and dispersed into single cells using bovine trypsin (342 U/ml) and 2 mM EDTA in PBS. Finally, the cells were washed and rested in complete RPMI for at least 1 h at 37°C.Single-cell suspensions from the spleens were prepared by mechanical tissue disruption through a 70 μm cell strainer followed by red cell lysis. Cells were resuspended in FACS buffer (0.5% FCS in PBS) and filtered through a nylon mesh.4Cl, 10 mmol/L Tris-HCl, pH 7.5). Leukocytes were analyzed by flow cytometry.Whole islets were treated with vehicle or ABT 317 for 1 h before culture with 10 ng/ml IFN-γ for 72 h, and analysis by flow cytometry. Splenocytes and whole blood were stimulated with 100 U/ml IL-2, 50 ng/ml IL-7, 30 ng/ml IL-15, 25 ng/ml IL-21, or 40 ng/ml GM-CSF for 30 min at 37°C. Red blood cells were lysed (155 mmol/L NHBromo-deoxyuridine (BrdU) was diluted to 10 mg/ml in sterile PBS. Mice were injected i.p. with 200 µl (2 mg) of BrdU solution. Mice were harvested 24 h after injection.Mouse islet cells were stained with anti-CD45 and biotinylated anti-H2Kd (BD Pharmingen) followed by streptavidin-allophycocyanin (BioLegend). β‐cells were identified by autofluorescence . Dead cePancreases were Bouins fixed and paraffin embedded, 5 µm sections were cut from three levels (200 µm apart) and stained with hematoxylin and eosin (H&E). Insulitis was scored using the following scale: 0 = no infiltrate, 1 = peri-islet infiltrate, 2 = extensive (>50%) peri-islet infiltrate, 3 = intraislet infiltrate, and 4 = extensive intraislet infiltrate (>80%) or total β cells loss. The percentage of islets with each score per pancreas was calculated by addition of the scores for the three sections.Spleen cells were cultured with 2 µM ABT 317 for 1 h prior to the addition of 25 ng/ml IL-21 for 15 min. Western blot for pSTAT3 and actin were performed using standard procedures.For anti-CD3 monoclonal antibody (mAb) treatment, NOD mice were treated with ABT 317 or vehicle by oral gavage for 2 days. On day 2, mice were injected intraperitoneally with 10 µg anti-mouse CD3 Ab . Islets were isolated on day 3 and analyzed by flow cytometry.Mice were monitored for diabetes by measurement of blood glucose levels with Advantage II Glucose Strips (Roche) twice a week. Mice suspected of hyperglycemia were tested on the following day, and two consecutive blood glucose readings >13.9 mmol/L was considered diabetic.When female NOD mice were diagnosed with diabetes, they were implanted with a LinBit insulin pellet  the following day . At the Statistical analysis was performed using GraphPad Prism 8 Software . All data shown as bar graphs are represented as the mean ± SEM. A p value of <0.05 was considered to be significant.The pharmacokinetic (PK) profile of ABT 317 was assessed in NOD mice to confirm that sufficient drug exposure could be achieved and that the corresponding drug level was sufficient to test the JAK1 selectivity. The pharmacokinetic behavior of ABT 317 in NOD mice was evaluated following escalating single oral doses. The compound exhibited rapid absorption, with plasma concentrations reaching their mean maximum concentration between 0.5 and 1 h. The exposure increased less than dose proportionally between 3 and 30 mg/kg, and the mean elimination half-life ranged from 1.9 to 2.6 h Table 1.in vitro , granulocyte-macrophage colony-stimulating factor (GM-CSF), and thrombopoietin (TPO), neutropenia has been found in clinical trials with the JAK inhibitors baricitinib , 26 and In vitro, ABT 317 did not affect GM-CSF signaling in granulocytes   that mediate β cell destruction. These cells acquire effector function in the islets, a process dependent on cytokine action  , while Iof STAT3 Figure 1versus JAK3.Although common γ chain cytokines function through JAK1 and JAK3, a dominant role of JAK1 over JAK3 has been reported. It was shown that to maintain IL-7 signaling, continuous synthesis of new JAK1 protein is required . In contAfter the marketing of the first two pan-JAK inhibitors, tofacitinib and baricitinib, more recent research has been focused toward the development of more selective drugs with the ability to modulate the activity of only one JAK family member. JAK1 selective inhibitors were developed to improve the safety profile by minimizing the effects on JAK3 and JAK2. ABT 317 is an ATP competitive inhibitor selective for JAK1. Our findings indicate that ABT 317 is sufficient for preventing JAK1-dependent cytokine-dependent signal transduction. However, we saw a slight reduction in blood white blood cell numbers at 10 mg/kg ABT 317 compared to control, suggesting that high concentration ABT 317 may also inhibit cytokines other than those primarily dependent upon JAK1 at the concentration we used in NOD mice. Other research indicates that upadacitinib also inhibits the JAK2-dependent cytokines IL-3 and GM-CSF . The 10 In conclusion, we show that treatment of NOD mice with a JAK1 selective inhibitor, ABT 317, blocked MHC class 1 upregulation on β cells and inhibited common γ chain cytokine effects on T-cell proliferation and generation of effector and memory T cell to reverse autoimmune diabetes. Our research provides a basis for additional testing of JAK1 inhibitors to prevent immune-mediated β cell death in T1D.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by St Vincent’s Hospital Animal Ethics Committee.TG, GJ, SF, SA, SL, EP, TC, IV, and AL performed experiments, analyzed data, and revised the manuscript. TG, GJ, LO, BK, TK, and HT designed the study and wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was funded by the Juvenile Diabetes Research Foundation , the National Health and Medical Research Council of Australia (GNT1126237 and GNT1150425) and fellowships from the JDRF and Manpei Suzuki Diabetes Foundation (SA). The St Vincent’s Institute receives support from the Operational Infrastructure Support Scheme of the Government of Victoria.At the time of the study, AL and LO were both employees of Abbvie.The remaining 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 blood–saliva barrier (BSB) consists of the sum of the epithelial cell layers of the oral mucosa and salivary glands. In vitro models of the BSB are inevitable to investigate and understand the transport of salivary biomarkers from blood to saliva. Up to now, standardized, cell line-based models of the epithelium of the submandibular salivary gland are still missing for this purpose. Therefore, we established epithelial barrier models of the submandibular gland derived from human cell line HTB-41 (A-253). Single clone isolation resulted in five different clones . Clones were compared to the parental cell line HTB-41 using measurements of the transepithelial electrical resistance (TEER), paracellular marker permeability assays and analysis of marker expression for acinar, ductal, and myoepithelial cells. Two clones  were characterized to be of acinar origin, one clone (F11) to be of myoepithelial origin and one isolation (B4) derived from two cells, to be presumably a mixture of acinar and ductal origin. Clone B2, presumably of ductal origin, showed a significantly higher paracellular barrier compared to other clones and parental HTB-41. The distinct molecular identity of clone B2 was confirmed by immunofluorescent staining, qPCR, and flow cytometry. Experiments with ferritin, a biomarker for iron storage, demonstrated the applicability of the selected model based on clone B2 for transport studies. In conclusion, five different clones originating from the submandibular gland cell line HTB-41 were successfully characterized and established as epithelial barrier models. Studies with the model based on the tightest clone B2 confirmed its suitability for transport studies in biomarker research. Since saliva offers the possibility of non-invasive sample collection accompanied with minimal contamination risk, efforts have been made to validate salivary biomarkers in the past decades. Up to now, several salivary biomarkers for cancer, infectious, or autoimmune diseases are already in use ,2,3,4. FWhile a significant number of cell line based in vitro models for the oral mucosa epithelium have already been described, only a few salivary gland epithelium models were reported, and most of them are based on cells from the largest major salivary gland, namely the parotid gland.In this study, we focused on the establishment of human in vitro models for the submandibular salivary gland. Up to now, most studies with submandibular salivary gland models used rat  or mice (CSG 120/7) derived cell lines ,16,17,18Even though models of salivary glands based on primary human cells have been described to form a distinct paracellular barrier previously, the restricted availability of biopsies of salivary glands limit their applicability. Moreover, donor variations also result in decreased reproducibility of the experiments . Hence, Here, we describe the ability of the submandibular salivary gland epithelium cell line HTB-41 (A-253) to form a significant paracellular barrier. Single cell cloning resulted in further optimization of the model. Transport studies with ferritin across the optimized HTB-41 model were compared to studies with a human oral mucosa model based on cell line TR146 and confirmed the feasibility of these models for salivary biomarker assessment.3 cells/cm2 for HTB-41 or 9.33 × 103 cells/cm2 for TR146 in 5 mL medium in T25 TC-treated cell culture flasks  once a week and cultivated at 37 °C, 5% CO2, 95% air atmosphere and 95% humidity. Media change was performed every 2–3 days.The submandibular salivary gland cell line HTB-41, first isolated and described in 1973 by Giard et al. , was purw/v) gelatin  in H2O bidest. per well for 30 min at room temperature prior to seeding. Cell seeding was performed with a cell concentration of 10 cells/mL and 20 cells/mL (100 µL/well) at passage 8. Conditioned media from T25 flasks with parental HTB-41 cells was collected with every media change of the flasks and stored at 4 °C until usage. Media change of 96-well plates for single cloning was performed with conditioned media diluted 1:2 with fresh McCoy media. As soon as the clones from the wells B2, B4, B9, D3, and F11 reached confluency in the 96-well plates, they were propagated on 24-well plates  pre-coated with 0.5% gelatin (300 µL/well) and were further cultivated with media change every 2–3 days using fresh McCoy media. Cells were transferred to T25 flasks after reaching confluency on 24-well plates and propagated using the cell seeding density as described above.To isolate single clones from cell line HTB-41, transparent 96-well plates  were coated with 50 µL 0.5% , whereby 900 µL media was provided in the basolateral wells. Parental HTB-41 cells were seeded in McCoy media at passages 5–12, isolated clones from cell line HTB-41 were seeded at passage 5–12 after single cell cloning at passage 8. Seeding procedure of TR146 was described in detail previously [For transepithelial electrical resistance (TEER) experiments HTB-41 as well as isolated clones were seeded at a cell density of 8 × 10eviously .® inserts, cultivation condition was either switched to airlift for designated inserts or stayed under submerged condition. For inserts cultivated under airlift conditions, apical media was added for TEER measurements and removed after the measurement. TEER measurement was performed after media change using the Milicell® ERS-1 Voltohmeter . The chopstick electrode  was sterilized for a maximum of 10 min in 70% sterile EtOH and incubated in McCoy media for at least 10 min before the measurement. The cells were equilibrated at room temperature for at least 30 min prior to TEER measurement. The output of the measurement (Ω) was multiplied with the surface area of the Transwell® insert after subtraction of the mean value of three blank inserts.As soon as cells reached confluency on the TranswellUpon reaching maximum TEER values, the permeability assay using the paracellular marker carboxyfluorescein was performed. For this, apical media was replaced with media containing 10 µM carboxyfluorescein . Cells were incubated for 2 h in the dark at 37 °C in the incubator. Samples were collected and stored at 4 °C in the dark until measurement. Fluorescence of carboxyfluorescein samples was measured at 488 nm excitation/520 nm emission wavelength using an Enspire Multimode Plate Reader . The permeability coefficient (µm/min) was calculated according to Neuhaus et al. (2008) .Zonula occludens-1 (ZO-1) were selected as markers for the submandibular gland epithelium [For the characterization of the parental cell line and the clones claudin-1 (CLDN1), E-cadherin (E-cad), and ithelium ,25,26 anithelium . Additioithelium ,14,27. Aithelium ,12,13,14ithelium , shown i® inserts were lysed with 350 µL lysis buffer  supplemented with 1% β-mercaptoethanol) and pooled together as one biological sample. RNA was isolated using the NucleoSpin RNA kit  according to the manufacturer’s instruction and each sample was eluted with 40 µL nuclease-free water . For cDNA synthesis with the Multiscribe Reverse Transcriptase Kit  350, 650, 700, or 1000 ng RNA were applied to synthesize 20 µL cDNA solution. QPCR was performed in white 96-well plates  with 20 µL reaction volume containing 4 µL diluted  cDNA solution equivalent to 20 ng cDNA, 2.8 µL of 3 µM primer dilutions, 10 µL PowerUp Sybr Green Kit  and 3.2 µL nuclease-free H2O. The samples were amplified for 40 cycles (3 s at 95 °C and for 30 s at 60 °C) after starting the reaction at 95 °C for 20 s. The melting stage to obtain the melting curves was performed at 95° for 15 s, 60° for 1 min and 95 °C for 15 s with the LightCycler® 480 II . Data analysis was performed with the LightCycler® 480 Software 1.5 . Ct values of the markers were referred to the corresponding Ct value of the endogenous control, 18sRNA, of respective samples and normalized to ∆Ct values of the parental HTB-41. The 2−ΔCt values were further normalized against the expression levels of the parental HTB-41 and displayed as x-fold values. Primer sequences are listed in the Two 24-well Transwell3 cells/cm2 for the parental HTB-41 at passage 6 or for the clones at passage 8–11 after isolation, with media changes performed every 2–3 days. Staining was accomplished on day 10 after seeding.For staining upon cultivation on microscopic slides , the slides were first disinfected in 24-well plates with 500 µL 70% EtOH per well for 45 min, washed twice with 500 µL PBS  per well and coated with 300 µL 0.5% gelatin/well for 30 min at room temperature. Then, cells were seeded at a density of 8 × 102+/Ca2+  following fixation and permeabilization with 300 µL pre-cooled (at −20 °C) methanol at −20 °C for 20 min. After washing twice with 300 µL PBS per well, cells were kept in PBS for 15 min for rehydratization. Then 250 µL primary antibody solutions of α-amylase, cytokeratin 5/8, vimentin or ZO-1 diluted in PBS containing 1% BSA  were added over night at 4 °C  by qPCR. For analysis of ferritin in the media samples with ELISA, high-binding microtiter plates  were coated with 100 µL/well of 2 µg/mL ferritin antibody  dissolved in PBS sealed with an aluminum foil over night at 4 °C under gentle orbital shaking conditions. On the next day, wells were washed three times with 100 µL/well blocking buffer  0.1% Tween 20 . Afterwards 300 µL/well of blocking buffer was applied and incubated on an orbital plate shaker at room temperature for 1.5 h. A washing step with 300 µL blocking buffer was performed immediately prior to filling of 100 µL sample in duplicates. Stock solutions of the apical and basolateral side and used stock concentrations of ferritin were additionally diluted 1:100 in basal McCoy media or HBSS prior to addition.TM ELISA Pico Substrate  consisting of 50:50 Super Signal ELISA Pico Luminal/Enhancer Solution and SuperSignal ELISA Pico Stable Peroxide Solution, was added per well. The plate was sealed with aluminum foil and incubated in the dark at room temperature for up to 15 min prior to the measurement of the chemiluminescent signal with the EnSpire Multimode Plate Reader . The permeated concentration of ferritin was calculated as apparent permeability (Papp) using the formula below with crec as the measured concentration in the receiving chamber (ng/mL), Vrec as the volume of the receiving compartment (mL), dt as the time period of the transport in (s), A giving the surface area of the insert membrane (cm2) and c0 as the measured concentration of the applied stock solution (ng/mL).A standard curve with ferritin was freshly prepared for each plate as a threefold dilution series  in blocking buffer. HBSS and basal McCoy media without ferritin were used for background control. After incubation at room temperature on the orbital shaker sealed with aluminum foil for one hour, the wells were washed with 100 µL blocking buffer for three times. For measurement, 100 µL SuperSignalt-test, one-way ANOVA or two-way ANOVA with Holm–Sidak or Dunn´s method as post-hoc tests with α = 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. Heatmap was illustrated with Qlucore Omics Explorer 3.6 .Results are shown as mean ± SEM, if not otherwise indicated. Statistical analysis was performed with SigmaPlot 14.0  as Student’s ® inserts either under submerged or airlift conditions showed a significant increase of TEER over time under submerged condition starting on day 10 with 70.4 ± 16.14 Ω × cm2  compared to the airlift condition with 28.16 ± 4.70 Ω × cm2  was measured under submerged condition, reaching a plateau on day 15 with TEER values of 106.31 ± 18.4 Ω × cm2. TEER values were calculated from measured ohmic values of 658 ± 54.04 Ω for the cells before subtraction of blank average (338 ± 6.17 Ω) and multiplication with the surface area of 0.336 cm2 . Morphological characterization by hematoxylin and eosin staining of cells cultivated under both conditions confirmed confluent HTB-41 cell layers  and day 22 (161.28 ± 3.32 Ω × m2), clone B4 reached the highest resistance value on day 10 (79.68 ± 22.44 Ω × cm2), clone B9 on day 15 (44.62 ± 4.20 Ω × cm2), clone D3 on day 23 (101.53 ± 3.92 Ω × cm2) and clone F11 on day 22 (112.22 ± 20.84 Ω × cm2). Strikingly, clone B2 showed significantly higher TEER values compared to the parental HTB-41 line starting on day 8  with a maximum 4.5-fold higher TEER value on day 22 (732.14 ± 73.37 Ω × cm2). The subsequent permeability studies with the paracellular marker carboxyfluorescein reflected the results of TEER experiments showing the significantly lowest permeability coefficient for clone B2 with 2.16 ± 0.26 µm/min (3.60 ± 0.43 × 10−6 cm/s) compared to the parental HTB-41 with 3.96 ± 0.13 µm/min. Clone B4 and clone B9 showed the highest permeability (7.01 ± 0.66 µm/min and 6.77 ± 0.42 µm/min), while clone F11 and clone D3 displayed permeability coefficient values of 3.81 ± 0.24 µm/min and 4.88 ± 0.51 µm/min, confirming the ranking of TEER values   .p < 0.001), while displaying a significant upregulation of CK18 (p < 0.001) as well as of ZO-1 and CLDN1 (p < 0.001), proteins important for tight junction formation. On the other hand, prospective markers for myoepithelial cells CK14, Vim and S100A4 were downregulated in comparison to the parental HTB-41, while showing a significant upregulation for the ductal marker CK7 (p < 0.001).First, the expression of several marker molecules at the mRNA level was investigated. Data are summarized in p < 0.05–p < 0.01) as well as the acinar marker α-amylase, while displaying a downregulation of the myoepithelial cell markers. As clone B4 also showed a significant upregulation for the ductal marker CK7 (p < 0.05), it was assumed for this clone to derive from two cells of acinar and ductal origin. Clone B9, D3, and F11 showed a significant downregulation for the ductal marker CK7. In addition, clone B9 and D3 also exhibited a weaker expression of vimentin and a significant downregulation of S100A4 in comparison to the parental HTB-41. Hence both clones  were classified to be of acinar origin. Clone F11 showed an upregulation of the myoepithelial markers vimentin and S100A4, and hence was classified to be of myoepithelial origin based on mRNA data.Isolated clone B4 showed a significant upregulation for E-cadherin, ZO-1 and CLDN1 (® inserts confirmed the presence of ZO-1 on cell-cell boarders (data not shown).Immunofluorescence staining was accomplished in order to evaluate some of the qPCR results on the protein level. Images for α-amylase A confirmapp showed a higher, but not statistically significant permeation of ferritin from the apical (blood) to the basolateral  side (A/B) in comparison to the corresponding transport from the basolateral to the apical side (B/A). In detail, Papp for 300 ng/mL ferritin was 0.021 ± 0.007 × 10−6 cm/s (A/B) versus 0.013 ± 0.002 × 10−6 cm/s (B/A), and for 1000 ng/mL ferritin Papp was 0.016 ± 0.002 × 10−6 cm/s (A/B) versus 0.012 ± 0.001 × 10−6 cm/s (B/A) (app of 0.038 ± 0.004 × 10−6 cm/s (B/A) versus 0.021 ± 0.002 × 10−6 cm/s (A/B). For 1000 ng/mL ferritin, the permeability was similar in both directions . In summary, it was shown that the novel salivary gland epithelium model based on clone B2 was suitable to study the transport of the salivary biomarker ferritin. Although the differences of the transport of ferritin from A/B to B/A across HTB-41 clone B2 were not significant, the same trend for enhanced ferritin transport in the direction from the blood to the saliva compartment was found in the oral mucosa epithelium model.The optimized barrier model with the highest paracellular barrier based on clone B2 was used to investigate the transport of the biomarker ferritin across a salivary gland epithelium model. Ferritin was applied at two different concentrations (300 and 1000 ng/mL) either at the apical or the basolateral compartment. After 24 h the calculated apparent permeability P/s (B/A) A. Result/s (B/A) B. Data sSeveral transport systems are present in salivary glands epithelia indicating that active transport processes take place between blood and saliva. To investigate these transport processes, we aimed to establish a human submandibular salivary gland in vitro model. As only few human submandibular salivary gland models have been published, varying significantly in TEER values, we decided to develop a standardizable model with distinct paracellular barrier properties. We selected to test cell line HTB-41, one of few available cell lines of the human submandibular gland. Until now, only few studies were published describing cell line HTB-41, and no data about its paracellular barrier were reported ,30,31.® experiments with HTB-41 proved the ability of HTB-41 to form confluent layers on Transwell® inserts under submerged conditions. Haematoxylin and eosin staining confirmed the morphology of a continuous cell layer. TEER values indicated the formation of a significant paracellular barrier sufficient for subsequent transport studies.First Transwell2 depending on the type of Transwell® inserts, or HSDEC , an immortalized cell line, achieved TEER values of about 1700 Ω × cm2 [However, single cell isolation experiments were performed to obtain single clones with even more distinct barrier properties. It is known that cancer cells can exhibit inhomogeneity, this was already described in the late 1950s . Isolati Ω × cm2 ,36.According to these high TEER values, it was assumed that clone B2 might be also of ductal origin. While the integrity of the paracellular barrier could be used as a first characteristic for cell type classification, it is crucial to further characterize the clones at the mRNA and protein level applying several different markers for each cell type. In this context, Azevedo et al. (2008) highlighted the importance of using several markers for one cell type, as the expression of multiple cytokeratins was not restricted to one cell type, while the diverse patterns of the expressed cytokeratins enabled a proper classification . With reThe remaining four isolated clones B4, B9, D3, and F11 were classified being from acinar or myoepithelial cell origin based on the TEER data and marker expression. While clone B4 expressed significantly more α-amylase than the parental cell line, the ductal cell marker (CK7) was also upregulated, but both myoepithelial markers were downregulated. Hence, clone B4 was classified to be a mixture of acinar and ductal cells. In clone B9 and clone D3 myoepithelial markers vimentin, S1004 as well as the ductal marker CK7 were downregulated, whereas acinar marker α-amylase was upregulated. Thus, both clones were classified as acinar cells. Clone F11 showed as the only clone an upregulation of vimentin and a weak paracellular barrier. Therefore, clone F11 was classified as being of myoepithelial origin.To further investigate the differences between the parental HTB-41 cell line and the isolated clone B2, flow cytometry experiments were performed with α-amylase, cytokeratin 5/8, vimentin, and ZO-1 . Interes2, followed by a study of Hegyesi et al. in 2015 showing that an adaption of the protocol led to TEER values of 622 ± 117 Ω × cm2 (mean ± SEM) on day seven of cultivation [Only few studies based on primary epithelial salivary gland cells have been published until now, perhaps because the accessibility of biopsy samples of salivary glands is limited. In 2005, Tran et al. described a primary cell model of the human submandibular gland exhibiting TEER values of 250–300 Ω × cmtivation ,25. Furttivation ,38,39.2 being in a similar TEER range as the optimized primary cell model of Hegyesi et al. (2015) [n = 22, median) in healthy individuals while subjects with chronic periodontitis showed elevated ferritin levels of 265.1 ng/mL in serum and 16.74 ng/mL in saliva  [In this context, clone B2 was chosen for the transport experiments as this clone showed the tightest paracellular barrier reaching TEER values of 732 ± 73 Ω × cm. (2015) . In biom. (2015) . Elevate. (2015) ,42. For  median) . While f median) , levels  median) . Decreas median) .To study the transport of ferritin in our models, 300 or 1000 ng/mL were applied either on the apical (= blood compartment) or basolateral  side. After 24 h incubation sufficient amounts of ferritin were detected on each opposite side, underlining the suitability of the established model for transport studies with high-molecular weight biomarkers. Although no significant concentration or permeation direction dependent effects were found in the salivary gland model, the tendency to permeate faster into the saliva compartment was detected for ferritin. Corresponding to this, ferritin (300 ng/mL applied) permeated significantly faster from the blood  to the saliva side compared to the other direction in the oral mucosa model. Studies across both BSB models indicated the general tendency for ferritin to be transported faster from blood to saliva and suggested the suitability of ferritin as salivary biomarker. In 2009, the transport mechanism of ferritin mediated by the endocytosing cell-surface receptor transferrin receptor-1 (TfR-1) was described comprehensively . With TfThe ability ofthe human submandibular salivary gland cell line HTB-41 to form distinct paracellular barrier properties was described for the first time. Isolation of single clones of the heterogeneous cell line HTB-41 resulted in five clones with distinct different properties and cell type characteristics , and led to a clone with significantly increased barrier properties compared to the parental cell line. Transport studies with the potential salivary biomarker ferritin showed the applicability of the optimized model for biomarker studies.