In this study, a novel promising approach for the fabrication of Halloysite nanotube (HNT) nanocomposites, based on the amino acid named Methionine (Met), was investigated. For this purpose, Met layered on the outer silane functionalized surface of HNT for controlled release of Phenytoin sodium (PHT). The resulting nanocomposite (MNT-g-Met) was characterized by FTIR, XRD, Zeta potential, TGA, TEM and FE-SEM. The FT-IR results showed APTES and Met peaks, which proved the modification of the HNTs. The zeta-potential results showed the interaction between APTES (+53.30) and Met (+38.80) on the HNTs (−30.92). The FE-SEM micrographs have displayed the grafting of Met on the modified HNTs due to the nanotube conversion to a rough and indistinguishable form. The amount of encapsulation efficiency (EE) and loading efficiency (LE) of MNT-g-Met was 74.48% and 37.24%, while pure HNT was 57.5%, and 28.75%, respectively. In-vitro studies showed that HNT had a burst release (70% in 6 h) in phosphate buffer while MNT-g-Met has more controlled release profile (30.05 in 6 h) and it was found to be fitted with the Korsmeyer-Peppas model. Due to the loading efficiency and controlled release profile, the nanocomposite promote a good potential for drug delivery of PHT. Chronic wounds are among the most costly unresolved health issues that reduce quality of life, costs, and acute conditions ,2. The wOne of the essential drugs that recently showed improvement in wound healing is Phenytoin sodium (PHT), which has been prescribed to treat epilepsy since 1973 . A commoO [Halloysite nanotube (HNT) is a mineral nanoclay that can be a suitable PHT carrier due to its remarkable properties. Recent studies have implemented HNTs for the controlled release of topical and gastrointestinal drugs due to their biocompatibility, high mechanical strength, low cost, hollow and nano-sized tubular structure ,19,20,21H)4.nH2O . While, H)4.nH2O ,24,25. TH)4.nH2O ,27,28,29H)4.nH2O ,31. 2 group of APTES molecule, which grafted on the surface of HNT, and the resulting nanocomposite will have a positive effect on drug loading and release of PHT. The use of amino acids, such as Met, is a novel approach to modifying HNT surfaces [One of the agents that can be interesting in modifying of HNT outer surface is amino acids, which in addition to being biocompatible, are very effective in improving drug release ,33,34. Ssurfaces . -g-Met nanocomposite preparation as a PHT carrier for chronic wound healing was investigated. First, the HNT outer surface was modified by APTES, and then Met was grafted on it. Fourier-transform infrared spectroscopy (FT-IR), Zeta potential, scanning electron microscope (SEM), transmission electron microscopy (TEM), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray diffraction analysis (XRD), and drug loading tests were used to characterize the prepared nanocomposite. The in-vitro drug release of the nanocomposite was performed via the dialysis bag method. PHT release kinetics were investigated based on four mathematical models: Zero-order, first-order, Higuchi, and Korsmeyer-Peppas. The data obtained from the mentioned models were processed and evaluated based on the correlation coefficient R2.In this study, MNTHNT , (3-aminopropyl) triethoxysilane (APTES), L-Methionine , triethanolamine (TEA), sulfuric acid, toluene, and dimethylformamide (DMF) were purchased from Sigma Aldrich. PHT  was received from Safeway Research Center (S.R.C.). All chemicals used as received. The distilled water was used throughout the experiments.To remove HNT impurities, it must be purified. Briefly, to have an ideal dispersion of HNT in water, a drop of polysorbate-80 (a nonionic surfactant) was first added to 50 mL of deionized water at 60 °C. HNT (500 mg) was added to the solution while on a magnetic stirrer at 60 °C and 1000 rpm for 18 h. Then washed and centrifuged three times with distilled water, and the remaining residue was dried at 100 °C for 10 h ,39. The functionalization of HNT with APTES is one of the most widely used methods for linking polymers or other active functions on HNT outer and inner surfaces. As mentioned in previous articles , 1 g HNT-g-Met) was washed three times with distilled water and ethanol and dried in a vacuum oven (~1 bar) at 50 °C overnight [According to vernight .-g-Met or MNT were added to the PHT solution (1 mg/mL) separately. The mixture was sonicated for 30 min. It was then placed in a vacuum jar (0.08 bar) for 20 min and then exposed to atmospheric pressure for 10 min after the vacuum breaks down. This process was repeated three times to achieve more load efficiency. The amount of drug-loading was measured by HPLC chromatography at 220 nm. The encapsulation efficiency (EE) and loading efficiency (LE) were calculated using the amount of free PHT by Equations (1) and (2) [A vacuum technique was used to load the PHT into the HNT lumen. This technique is based on the difference between the vapor pressure of the solution in the lumen and the bulk solution, which pushes the drug solution into the lumen and subsequently provides hydrogen and electrostatic bonds between PHT’s negative charge, the positive charge of the inner surface of HNT. In this method, the nanocomposite was dried, then 100 mg of the HNT and (2) ,46,47: at 32.5 °C . In this–5 bar (0.01 torr) with KBr and were examined in the range of 400 to 4000 cm−1. Zeta-potential results of the prepared sample in ethanol at pH = 7.4 using the SZ-100z dynamic light scattering and voltage range of ±200 mV were used to investigate particle surface charge changes and the stability of colloidal dispersions. The thermal degradation behavior of samples was studied using thermogravimetric analysis (TGA) from room temperature to 800 °C with a rate of 20 °C/min in the nitrogen atmosphere. The particle’s appearance and morphology were studied using scanning electron microscopy (SEM), the TESCAN MIRA3 LMU model and transmission electron microscopy (TEM) test, using a ZEISS EM10C model with a voltage of 80 kW. The X-ray diffraction (XRD) is a rapid analytical technique to determine the samples’ crystal structure, which was performed using the Siemens D5000 (Germany) instrument at 25 °C.FT-IR was used to study the functional groups and possible interactions in the prepared samples. For this purpose, samples were pressed under 1.3 × 10p < 0.05. All data are presented as mean values with standard error (mean ± SD), n = 3 in each test.The statistical analysis for the determination of reproducibility in the loading, zeta potential, and release test was performed using one-way ANOVA statistical analysis. The obtained data were considered to be significantly different at In this study, HNT was used to prepare a new nanocarrier for wound dressing using PHT. HNT was considered for loading capacity, biocompatibility, and tubular structure. For the first time, a new approach was used to prepare HNT-based nanocomposites using amino acids grafted on the surface of HNT. In the following, the effect of Met on drug release was investigated.g-Met are shown in −1 and 3695 cm−1 are related to the inner, and interlayer hydroxyl groups’ vibration, respectively. The peak shown in 913 cm−1 indicates the vibration of Al2OH. Symmetric stretching vibration of Si–O–Si appeared at 1038 cm−1 [−1 related to the amine groups in APTES. The two peaks in 2926 cm−1 and 1388 cm−1 are related to the CH2 group, respectively. The C-N peak appeared in 1088 cm−1. Another peak shown at 1533 cm−1 in the HNT-APTES spectrum belongs to the NH2 group. After the Met graft on the HNT surface, new peaks appeared that indicated a covalent bond between Met and APTES. The peak shown in 2363 cm−1 corresponds to S–CH3 of Met. Another short peak at 1646 cm−1 indicates the carbonyl group in the nanocomposite structure. Due to covalent bonds forming during the nanocomposite preparation process, it is necessary to study the bond strengths and compare them. For this purpose, the FT-IR spectrum of HNT, MNT, and MNT-038 cm−1 . After s-g-Met. Pure HNT has a negative zeta potential of −30.92 mV. After modifying of HNT surface with APTES, its negative charge was naturalized to +53.3 mV. By grafting Met on MNT, the amount of zeta potential was changed to +38.8 mV, which is related to the NH3+ group of amino acid layering on the surface of the HNT. These results can prove that HNT was chemically layered with a positively charged amino acid.The surface modification of HNT was successfully proved by changing the surface charge during the process. -g-Met nanocomposite decomposes in several thermal events [-g-Met DTG diagram for better differentiation of the TGA diagram, which confirms the above studies. l events . The fir-g-Met presents in -g-Met. After modification of HNT by APTES (MNT), the reflection of pure HNT remained unchanged, indicating that the APTES surface grafting was not affected the basal spacing and crystalline structure. This result confirms that APTES was not inserted into the space between the HNT layers. As shown in the diffractogram of MNT-g-Met, since Met does not have a specific crystalline structure, Met grafting does not affect MNT structure.The XRD pattern of HNTs, MNT, and MNT-g-Met nanocomposite. As shown in According to the TEM images, the tubular morphology of pure-HNT with hallow lumen was observed in -g-Met. The results show that when functionalized HNT was grafted with Met, the PHT loading was increased. This was due to the positive charge of Met and APTES and the electrostatic attraction between the negatively charged PHT and the amine group of these molecules. Due to the PHT molecule structure, hydrogen bonds can also increase LE and EE, although this increase is not significant with the Met layering.The LE and EE of different samples were evaluated in PBS 7.4. Loading was performed based on the vacuum technique. The amount of drug-loaded was calculated based on the difference between the initial drug concentration and the free drug in the bulk solution. -g-Met nanocomposite in PBS at pH 7.4. According to the diagram, the cumulative release of PHT from pure HNT reached 70.7% after 6 h, related to the burst effect. This phenomenon results in a lack of controlled drug release, which is not desirable for a drug delivery system. After 48 h, the cumulative release of PHT reached an almost constant concentration. The burst effect in the initial stage is due to the dissolution of drug molecules adsorbed on the outer surface and layer of the HNT. In the next step, PHT release from the HNT lumen was slower due to the strong absorption of drug molecules. It is expected that this burst release problem will be partially remedied by modifying the HNT surfaces with Met. The PHT release profile from MNT-g-Met progressed more rapidly in the early hours (after 6 h: 30.05%) of release, but no burst effect was observed. This rapid release was due to drugs adsorbed on the Met surface. This rapid release was due to drugs adsorbed on the Met surface. When Met is grafted onto the outer surface of functionalized HNT (MNT), the amino acid molecule blocks the HNT lumen’s output to such an extent that it leads to a controlled and sustained release of the PHT. The results show that the fabricated nanocomposite can act as a carrier for drug delivery to chronic wounds. -g-Met are presented in -g-Met in PBS fitted by Korsmeyer-Peppas equation, which is expressed as follows [The release kinetics of PHT from MNT follows :(3)MtM∞=Mt/M∞ is the fractional amount of PHT that release at time t, k is a constant value, and n is the release exponent. -g-Met. According to the obtained results, the n values for HNT and nanocomposite were about 0.47 and 0.5, respectively. An n value less than 0.5 indicates that the release follows a Fickian diffusion from polydisperse systems. The results also show that the nanocomposite particles possess a smaller k value than the pure HNT, which indicates a controlled and sustained release in the same condition [ondition .-g-Met nanocomposite was fabricated via functionalizing HNT with APTES, followed by Met grafting on its outer surfaces. The prepared nanocomposite was presented as a suitable carrier for PHT for use in wound dressing. The nanocomposite’s physicochemical properties were studied by FT-IR, FESEM, TGA, XRD, and zeta potential, which all confirmed the success of the composite preparation. The morphological results presented the structure of uniform nanotube particles. The results indicated that the prepared nanocomposite of MNT-g-Met could improve the amount of PHT from 28.75% to 52.61 wt% compared with the pure HNTs. In-vitro studies showed that PHT release from HNT occurs very rapidly (k = 26.57/h), while this process is controlled in MNT-g-Met (k = 9.22/h). The Korsmeyer-Peppas model fitted the release kinetics, which indicated the Fickian diffusion mechanism from cylindrical multilayer structure. Finally, all the results show that the prepared nanocomposite can be a suitable carrier for PHT or similar drugs to heal chronic wounds.In this study, a novel approach for the preparation of HNT nanocomposites, based on the amino acid graft on the outer surface of HNT, was proposed. For this purpose, HNTs and Met were selected as the encapsulating agents for improving the PHT loading and release, and APTES was selected as a linker for making a chemical bond between the HNTs and Met. The MNT


Symptoms and all the hematologic presentation improved rapidly in both patients after steroids initiation. These two cases illustrate the rare bone marrow involvement in active IgG4-RD accompanied by other hematologic syndromes. The significance of disease pathogenesis is worthy of further study.IgG4-related disease (IgG4-RD) is a systemic fibro-inflammatory disease, histopathologically characterized by dense lymphoplasmacytic infiltration rich in IgG4-positive plasma cells in affected organs. Classic hematologic presentations including lymphadenopathy, eosinophilia and polyclonal hypergammaglobulinemia are common, whereas bone marrow involvement of IgG4-RD is rarely reported. Here we present two patients of multi-organ IgG4-RD with bone marrow involvement, one on bone marrow biopsy, and the other on PET/CT. Presentations of other organ involvement included biopsy-proven IgG4-related tubulointerstitial nephritis, lymphadenopathies, submaxillary glands, arteritis, asthma, dysosmia, and constitutional symptoms. Bone marrow involvement was initially suspected due to leukopenia, anemia and thrombocytopenia in case#1, and was finally confirmed by histological evidence of significant IgG4-positive plasma cells infiltration in bone marrow. In case#2, we incidentally observed high uptakes of multi-bone marrow on  IgG4-related disease (IgG4-RD) is a fibro-inflammatory condition recognized for decades, characterized by multi-organ enlargement with histopathological dense lymphoplasmacytic infiltration rich in IgG4-positive plasma cells, storiform fibrosis and obliterative phlebitis. IgG4-RD was first reported in patients with autoimmune pancreatitis (AIP) and it was gradually recognized as a systemic immune disease over the following years , 2. Usua18F-FDG-PET/CT), showing high uptakes in multi-bone marrow. These reports suggest the possibility of hematologic pathogenesis of IgG4-RD.Here, we present two patients of IgG4-RD with bone marrow involvement. One patient had significant IgG4-positive plasma cells infiltration in bone marrow aspiration and biopsy samples. In the other patient, hematologic involvement was detected by 18 fluorodeoxyglucose positron emission tomography  was insidiously elevated from 270 to a peak of 410 μmol/L, and went back to normal range after supportive treatment. However, symptoms relapsed 2 weeks before his hospital admission to Peking University First Hospital. The abdominal enhanced-CT scan revealed swelling kidneys with renal parenchyma patchy filling defects, and dilated small bowel loops. Thickening of the abdominal aorta, superior mesenteric and splenic artery walls could also be recognized  with mild mesangial proliferation and typical IgG4-related tubulointerstitial nephritis. The ratio of IgG4/IgG was 65% Fig.  in immunCase 2 was a 38-year-old male referred to the nephrology clinic for investigation of renal insufficiency as his serum creatinine was 146 μmol/L. He presented severe fatigue, dysosmia and conspicuous weight loss of 11 pounds during the last 2 months. He had a history of anaphylactic rhinitis and bronchial asthma. On admission, blood pressure was 150/90 mmHg, physical examination revealed bilateral submandibular gland swelling with left supraclavicular lymphadenopathy, and these were later confirmed by ultrasonography and FDG-PET/CT scan.18F-FDG-PET/CT showed high uptakes in bilateral submandibular glands, kidneys and bilateral scapula, spine, sternum, ribs, pelvis, and upper femur  showing IgG4-dominated plasma cells infiltration in BM, with no hypermetabolic lesion found in FDG-PET/CT. For another patient (#2), we identified elevated FDG uptake lesions involving multi-bone marrow in the context of the active IgG4-RD. Reported cases of definite IgG4-related bone marrow involvement were rare as listed in Table low/CD38+/CD138dim/CD56−/CD45+) and in the BM  [IgG4-RD is a systemic disease characterized by largely consistent pathomorphological manifestations across a wide range of organ systems. We call it a pathologically fibro-inflammatory pattern with enlarged organs or tissues. With the increasing number of reports and research in this field, clinicians have gradually become familiar with this syndrome. IgG4-related bone marrow presentation has rarely been mentioned before , since f in 2003 . Moreove in 2003 . As repo in 2003 , and wasG-PET/CT , was finG-PET/CT  declaredCD45low) . As an aLymphadenopathy is the common presentation in IgG4-RD. Involved lymph nodes typically have > 100 IgG4 + plasma cells/HPF with an IgG4/IgG ratio > 40%. In addition, some pathologists have noticed ectopic germinal centers formed in IgG4-RD affected tissues and their possible relationship with dense deposits along tubular basement membrane , 14, as Eosinophilia and tissue eosinophils infiltration are common in IgG4-RD. Eosinophilic angiocentric fibrosis is now part of IgG4-RD, usually affecting orbits and the upper respiratory tract. In the current report, renal eosinophils aggregation was obvious in both patients, even with an absence of eosinophilia as in case#1. A previous study presented similar findings that eosinophils infiltration was evident in 30.8% of IgG4-RD patients compared with 9.5% in non-IgG4 tubulointerstitial nephritis . Regulat18F-FDG-PET/CT has been considered a promising diagnostic tool to aid IgG4-RD diagnosis in recent years. As a whole-body imaging technique, it is reported to detect more lesions in 71.4% of IgG4-RD patients than the conventional imaging methods, providing a more comprehensive evaluation of disease distribution as an entity [18F-FDG-PET/CT had a sensitivity of 85.7% and specificity of 66.1% for diagnosing IgG4-RD [18F-FDG uptake in IgG4-RD changes along with disease remission and relapse [18F-FDG-PET/CT did not show high uptake in both sites. It was speculated that 18F-FDG might not be able to visualize chronic fibrotic lesions. At present, more novel PET probes have been exploited to better target fibrotic lesions  [18F-fluoroarabinofuranosyl cytosine) [Disease diagnosis and monitoring based on clinical syndromes and general imaging findings are currently required criteria. Pathological examination of involved tissue remains irreplaceable in the diagnosis of IgG4-RD. While in the absence of pathological evidence, it is difficult to accurately distinguish inflammatory from fibrotic activity in human immune-mediated disease in vivo. n entity . In anot IgG4-RD . The int relapse . Althoug relapse , such as 68FAPI)  or to deytosine) . This woSteroids are still the mainstay for IgG4-RD therapy. However, its long-term use is not recommended. B cells depletion strategy is superior to steroids, especially for refractory and recurrent cases. Rituximab has shown a response rate of 97% in one prospective trial  and a loThese two cases illustrate the rare bone marrow involvement in active IgG4-RD accompanied by other hematologic syndromes. The significance of disease pathogenesis is worthy of further study.


The formation of most multicomponent crystals relies on the interaction of hydrogen bonds between the components, so rational crystal design based on the expected hydrogen-bonded supramolecular synthons was employed to establish supramolecular compounds with desirable properties. This theory was put into practice for metformin to participate in more therapeutic fields to search for a fast and simple approach for the screening of candidate crystal co-formers. The prediction of intermolecular synthons facilitated the successful synthesis of a new multicomponent crystal of metformin (Met) and barbital (Bar) through an anion exchange reaction and cooling crystallization method. The single crystal X-ray diffraction analysis demonstrated the hydrogen bond-based ureide/ureide and guanidine/ureide synthons were responsible for the self-assembly of the primary structural motif and extended into infinite supramolecular heterocatemeric structures. Metformin (Met), a first-line oral hypoglycemic drug, is widely used in type 2 diabetes ; the cheIn recent years, more and more attention has been given to the field of multicomponent crystals including salts or cocrystals ,8 becausMulticomponent crystals represent the application of supramolecular concepts in the pharmaceutical industry . In suprBased on the synthon theory, Met can introduce a CCF to be a new entry with intellectual property rights used in the central nervous system. Therefore, a set of multicomponent crystal structures of metformin was extracted from the Cambridge Structural Database (CSD). As drawn in Barbiturates, derivatives of the barbituric acid, were commonly used as sedative–hypnotics in the mid-twentieth century . In 1903We, therefore, postulated that guanidine/ureide-based supramolecular assembly would employ supramolecular homo- or heterosynthons for constructing hydrogen bonds with other supramolecular reagents. Consequently, by allowing APIs containing guanidino groups to associate with a co-former involving the –(CONHCO)– group, we surmised that guanidino and acylurea moieties would organize supramolecular synthon assemblies formed via N–H···O and N–H···N hydrogen bonds into extended architectures. It was expected, therefore, that Met and/or Bar were able to form a homodimer through synthon A and I, respectively, and then linked with each other via synthon B or synthon III to form a 1D chain.1H NMR spectroscopy analysis. Dynamic vapor sorption (DVS) was conducted to measure the moisture sorption behavior of multicomponent crystals.To test our design strategy, the anion exchange reaction was employed, and the resulting crystal was determined primarily by single crystal X-ray diffraction (SCXRD). The other characterization methods included powder X-ray diffraction (PXRD), Fourier-transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and Single crystalline materials of 1:1 Met–Bar isolated by cooling crystallization of 1,1-dimethyl-biguanidhydrochloride and 5,5-diethylbarbituric acid sodium (1:1) from methanol were suitable for analysis by SCXRD. The corresponding crystallographic data and refinement details are summarized in As shown in Compared with the pure crystal structure of Bar extracted from the CSD in 1H NMR spectroscopy analysis. Unfortunately, the single crystal of the desolvate could not be obtained.The thermal behavior was analyzed using the TGA and DSC techniques. We obtained the weight change values of the substance converted to the percentage by weight within a specific heating temperature range of 25–500 °C, as drawn in As drawn in 1H NMR spectroscopy analysis to characterize molecular structures of Met·HCl, Bar·Na, and the solvate and desolvate samples of Met–Bar. In d6, the active hydrogen atom with a chemical shift of 9.70 on Bar disappeared. Additionally, the methanol peaks were shown at 3.16 in the solvate crystal in We further conducted 1H NMR data of Met·HCl, Bar·Na, multicomponent crystal of Met–Bar, and the solvate crystal, respectively, were:The 1H NMR  δ 7.22 , 6.79 , 2.93 .1H NMR  δ 9.70 , 1.64 , 0.63 .1H NMR  δ 7.28 , 2.92 , 1.64 , 0.63 .1H NMR  δ 7.26 , 2.92 , 1.64 , 0.63 .Powder X-ray diffraction had much broader applications than single crystal X-ray diffraction, which could quickly identify the structural difference between our raw materials and Met–Bar, and the results are shown in By means of Fourier-transform infrared spectroscopy analysis, we observed the shifts in the location and intensity of absorption peaks of the characteristic functional groups in the molecules of Met·HCl, Bar·Na, and Met–Bar, testifying whether there were new bonds in the multicomponent crystal of Met–Bar based on their differences, as shown in −1 and 1667 cm−1, which corresponded to the absorption peaks of the multicomponent crystal of Met–Bar at 1715 cm−1 and 1668 cm−1, suggesting the formation of the hydrogen bond between N–H in Met and C=O in Bar. In the fingerprint region of the infrared absorption spectrum, 1303 cm−1 and 653 cm−1 were the absorption peaks of the C–N stretching vibration in the structure of Bar·Na, and the positions in the multicomponent crystal were 1305 cm−1 and 652 cm−1, almost unchanged (not involved in hydrogen bonding).The C=O stretching vibration, the carbonyl group of Bar·Na, showed characteristic absorption peaks at 1697 cm−1, 3294 cm−1, and 3173 cm−1, which were very similar to the vibrational frequencies of the newly formed compound at 3369 cm−1, 3299 cm−1, and 3177 cm−1, indicating that the new compound may also be a salt-type compound with proton transfer like Met·HCl. It agreed with the single crystal X-ray diffraction data. The absorption signal of the C=N stretching vibration was 1625 cm−1 in Met·HCl and 1644 cm−1 in the structure of the multicomponent crystal, which was shifted to high frequency.The absorption frequencies of the N–H stretching vibration in Met·HCl were 3371 cmDVS was used to evaluate hygroscopicity of Met–Bar. Barbiturate drugs with low solubility are mainly made into alkali metal salts to increase solubility, such as barbital sodium, phenobarbital sodium. However, the aqueous solution of sodium salts is usually unstable, easy to hydrolyze when placed at room temperature, so it is often added to powder needles for injection use, limiting the diversity of formulation of this class of drugs . BesidesHowever, Met and Bar were combined into a multicomponent crystal, and even when the relative humidity reached 80%, the weight change of the sample was only 0.25%, which well overcame the shortcoming of Met’s high moisture sorption behavior, suggesting that the new compound could be stored stably at ambient RH.In this experiment, successful simple synthesis of a multicomponent crystal of Met-Bar demonstrated that the method of designing crystals based on supramolecular synthons with valence bond strength is trustworthy. The crystal structure-based predicted synthon II formed a homodimer and assembled into a larger architecture through the expected one-point synthon III. Additionally, the hydrogen bonds network established by Met and Bar changed the molecular arrangement of individual components and generated a new unit cell packing form, which structurally affected the physicochemical properties of the crystal, reduced the moisture sorption of Met, and increased the stability of Bar.However, systemic studies of the structure–properties relationship of multicomponent crystals are only rarely conducted at present. Accordingly, the mechanism of multicomponent crystals formation should be discussed in depth to improve crystal screening efficiency. In particular, the main challenge that the emerging research field of multicomponent crystals faces is that the interaction and stacking arrangement between drug molecules can be controlled through other rational and precise crystal designs to achieve targeted changes in the drug melting point, dissolution rate, solubility, bioavailability, biological activity, and other desirable properties to increase the speed of drug development.Barbital sodium  was used as received. Metformin hydrochloride and other solvents and chemicals were purchased from Adamas Reagent Company  and used without further purification unless otherwise stated.Met·HCl (0.165 g) and Bar·Na (0.206 g) were dissolved in 20 mL boiling methanol and stirred for 1 h. The filtrate was transferred to a 50 mL Petri dish sealed with a parafilm perforated with a few small holes, and finally left undisturbed in a refrigerator at 4 °C to allow cooling crystallization. Crystals were obtained within a week, which were suitable for single crystal X-ray diffraction analysis. For preparing the bulk sample of Met–Bar, 1.650 g Met·HCl and 2.060 g Bar·Na were transferred to 100 mL boiling methanol in a beaker and heated to 90 °C while continuously stirring for 2 h. The filtrate was left to cool at 4 °C with the beaker covered by the parafilm. Large crystals of Met–Bar grew overnight. The crystals were harvested by filtration and dried at 55 °C in a vacuum oven overnight. The crystals were milled for further characterization and formulation.SCXRD data were collected using a Bruker SMART CCD diffractometer , with Cu-Kα radiation (λ = 1.54184 Å) at 293 K. Integration and scaling of the intensity data were corrected for empirical absorption effects with the spherical harmonics implemented in the SCALE3 ABSPACK scaling algorithm. The structure was solved with the SHELXS-97 program by direct methods and refined with the least squares minimization using the SHELXL-2014 refinement package. All the non-hydrogen atoms were refined with anisotropic displacement parameters, and the hydrogen atoms were located in the differential Fourier map and refined with isotropic displacement parameters.The samples (2.307 mg) were heated in a hermetically sealed aluminum pan containing a pinhole on a thermogravimetry analyzer  from 25 °C to 500 °C at 10 °C/min under 100 mL/min nitrogen gas.−1 under nitrogen gas.Differential scanning calorimetry (DSC) was performed with a TA DSC25 . Sample weights of 1.20 mg were heated in Tzero aluminum pans at the scan rate of 10 °C minPXRD data were collected using a Bruker D8 Advance X-ray diffractometer  coupled with Cu-Kα radiation . The data for each sample were recorded over the 2θ range from 5° to 50°  at ambient temperature with a scan speed of 5°/min.−1 range at the resolution of 4 cm−1 with 12 accumulative scans under ambient conditions.Each sample was compressed into a tablet made using the KBr pellet technique and collected by a Nicolet Spectrum FT-IR spectrometer  in the 4000–500 cm1H NMR data were collected on a Bruker AVANCE III NMR spectrometer operating at 600 MHz and using DMSO-d6 as the NMR solvent.The materials’ water sorption and desorption profiles were measured using an automated vapor sorption analyzer  at 25 °C. The samples of Met–Bar, 26.86 of the mass at the end of the first 0% RH stage, were studied at each step size of 5% RH, following the 0%–80%–0% sorption and desorption cycle. If the weight change within 10 min was smaller than 0.02%, relative humanity (RH) was changed to the next step, with the maximum hold time of 3 h.


Concrete, as one of the essential construction materials, is responsible for a vast amount of emissions. Using recycled materials and gray water can considerably contribute to the sustainability aspect of concrete production. Thus, finding a proper replacement for fresh water in the production of concrete is significant. The usage of industrial wastewater instead of water in concrete is considered in this paper. In this study, 450 concrete samples are produced with different amounts of wastewater. The mechanical parameters, such as slump, compressive strength, water absorption, tensile strength, electrical resistivity, rapid freezing, half-cell potential and appearance, are investigated, and a specific concentration and impurities of wastewater that cause a 10% compressive strength reduction were found. The results showed that the usage of industrial wastewater does not significantly change the main characteristics of concrete. Although increasing the concentration of wastewater can decrease the durability and strength features of concrete nonlinearly, the negative effects on durability tests are more conspicuous, as utilizing concentrated wastewaters disrupt the formation of appropriate air voids, pore connectivity and pore-size distribution in the concrete. In the modern era, concrete is one of the most used materials in the construction industry. In fact, the only other substance that humans use more than concrete is water, which indicates the importance of concrete and the water used for it ,2,3,4. S3), which compose the slag. Their results indicated that the existence of steel slags results in more shear potential than the traditional RC beams, crack widths are smaller and the basic ductility is augmented. Bahraman et al. [In this research, help is provided to find the level of wastewater refinement to be used in concrete production. This can help to keep the wastewater infrastructures well maintained due to the massive amount of caustic materials in industrial wastewater. It defines what amount of impurity in a sample can cause a less than 10% loss of compressive strength, compared with a control sample; this is a crucial factor because it can help with the approval of the use of different types of wastewater as appropriate replacements for drinkable water. Al-Ghusain et al.  reportedn et al.  carried n et al.  studied n et al.  assessed2 production. Therefore, it is vital to substitute new promising ways in which components are replaced by other materials, yet rapt attention should be paid to recycled keys [Undeniably, concrete production is the main reason for a considerable amount of energy consumption as well as COled keys . ClearlyIn the present research, different concentrations of industrial wastewater were used for producing concrete specimens. Subsequently, the durability and strength of concrete specimens within 365 days were assessed and compared with the control specimen, which was produced with drinkable water. Eventually, a statistical analysis is presented to augment the level of strength prediction in concrete based on the obtained results.The wastewaters were gathered from Toos industrial town, Mashhad, Iran and within a maximum of three hours, they were analyzed in the laboratory. The analyses were performed on industrial primary wastewater, treated wastewater, diluted treated wastewater, and concentrated treated wastewaters. The control specimen was produced with drinkable water from Mashhad City, which is a standard water. Altogether, 430 specimens were created, pouring concrete ten times and fourteen skilled operators participated in producing them, which took two hours in total. The number of completed tests on specimens were as follows: slump 10 samples, compressive strength 240 samples, electrical resistivity 20 samples, water absorption of thirty minutes 10 samples, mass water absorption 10 samples, capillary water absorption 30 samples, tensile strength 40 samples, rapid freezing and thawing 40 samples and half-cell 30 samples. All of the tables, results, and tests were carried out exclusively for this research, and no archive data were used. The Technical and Vocational University (TVU), Mashhad, Iran, provided the researchers with testing facilities. The used standards are shown in The methodology consisted of several stages of operation and processing. In this research, ten different groups of specimens were produced with different wastewater concentrations. All groups of specimens had the same mix design, and no additive was used in order to figure out the exact effect of wastewater concentration on concrete durability and strength features. In this study, one of the targets was to find the optimum concentration of treated wastewater that may cause a less than 10% compressive strength loss compared with the control sample (made with drinkable standard water). Technically, 10% compressive strength reduction could still be counted as an acceptable substitution for the water in a concrete mix design . The maiFor producing concrete with wastewaters, the amount of distilled water was considered, based on the quality of the control specimen, and all other extra substances were subtracted. The majority of the time, there is an allowable limit for the water of mix design; within those restrictions, the impurity can be harmless and acceptable. Nevertheless, there is no limitation for organic materials in concrete and it is assumed that only wastewater impurities are the reasons for negative effects on the water in concrete mix design.Treated wastewater is also known as output wastewater and goes through three steps of refinement including filters, aeration and chlorination. Treated wastewater was used as the main replacement for drinkable water and for producing distilled and concentrated specimens as well. TWW was used for curing the specimens if they were intended to be cured by wastewater separately. The characteristics of TWW are presented in Diluted specimens were produced by TWW plus mixing with distilled water. They contained 75% wastewater (75%TW), 50% wastewater (50%TW), and 25% wastewater (25%TW), respectively, and the remainder was distilled water. These water percentages of the mix designs were selected in order to investigate the existence of linear or non-linear relationships in strength and durability features by diluting treated wastewater as the water of the mix design. Based on the laboratory results, the number of parameters was reduced correctly by dilution percentages. In order to obtain the number of parameters in diluted specimens, the characteristics of treated wastewater  should bConcentrated specimens were produced from TWW by evaporation; concentrating percentages were 20% (TW + 20%C), 25% (TW + 25%C), 30% (TW + 30%C), and 35% (TW + 35%C), respectively. Based on laboratory results, the parameters of the thickened specimens were increased by the concentration percentages. So, concentrated specimens had the same parameters as the treated wastewater  but theiThe initial discharge of industrial wastewater is primary wastewater, which is from a collection of several polluting industries such as pharmaceutical, food, ironmaking and chemical. It contains many organic materials and caustic heavy metals such as Cadmium and Chromium because it does not go through any refinement process and, technically, this is the TWW before the refinement procedure. PWW contains a huge amount of organic materials, microorganisms and heavy metals, which are mostly harmful and caustic for both the environment and concrete. For producing the control sample and curing all groups with normal water, the potable water of Mashhad, Iran, was used. In order to conduct this research, concrete cubic samples (100 × 100 × 100 mm), including 400 kg cement per cubic meter, were made and tested; for each test, the required standard, specification and introduction were fully considered. The Portland cement type ΙΙ, produced by Mashhad cement factory, Mashhad (Iran), was chosen and its quality was tested according to the ASTM-C150. The strength class of the Portland cement was 42.5R, and the desired strength of the concrete was 35 Mpa, which was achieved based on breaking cylinder specimens and regarding the average result. Moreover, the concrete grade was M35, which was commensurate with the achieved results. The desired workability based on NS 8500 EN 165 was S3. The quality of the required material in the production of concrete, the chemical and physical analysis of water and wastewaters, and the sieve analysis of the aggregates were experimentally assessed. The resistance of degradation in large size coarse aggregate to abrasion and impact was carried out using a Los Angeles machine. In addition, the soundness of the aggregates was evaluated using the sodium sulfate or magnesium sulfate and sulfate content of the aggregates. In this research, the following parameters were also checked: the hydraulic cement autoclave expansion, the amount of water essential for an ordinary consistency of hydraulic cement, and the setting time of hydraulic cement using a Vicat needle. The ASTM C33 standard was adopted for checking the coarse and sand sizes using a sieve assessment and the precise percentage passing through sieve number 200 was determined. To reach the optimum mix design, the ACI method of concrete mix design was used based on the water–cement ratio of 0.42 and, for the mechanical mixing of the cement, ASTM-C305 [The slump shall be consistent with the placement and consolidation methods, equipment, and site conditions and shall be identified by the contractor and concrete supplier prior to construction. According to the achieved results, TWW had less workability than the control sample. Diluted specimens reacted like TWW, which shows that the existence of the wastewater can affect the workability even at low percentages. The concentrated specimens followed the same method of treated TWW, but TW + 25%C had a reduction and stayed in the next specimens too. The TWW had 13.3% lower workability than the control specimen and by increasing the concentration of treated wastewater TW + 25%C by 25%, the workability declined 20% more than the control. It clearly showed that wastewater has a subtractive effect on workability and it is dependent on wastewater concentration. So, it is highly recommended, for projects with a high required workability, that the additives should be considered to increase the slump, especially when more concentrated wastewaters are used as the water of the mix design. No linear relationship was observed in any specimen when their concentration was increased or decreased. The concentrated specimens were more viscous and greasy, which is one of the reasons why concentrated specimens had less workability; it was obvious in the PWW sample, which had the highest impurities. The compressive strength results at different ages and days are shown for specimens cured by drinkable water  and cureBy aging, concentrated specimens had less compressive strength growth than the control sample and, by increasing the specimens’ concentration, the reduction increased. One of the most important intentions of this study was to find the impurity and concentration of wastewater which causes a 10% reduction in the compressive strength of concrete in comparison to the control after 28 days. Based on The level of permeability of concrete has a direct effect on the electrical resistivity of specimens. This test indicates specimens’ permeability and specifies existing voids and cracks in the concrete structure, which have a significant effect on concrete durability . Figure The control sample had better resistance when it was cured by drinkable water and a reduction was observed when cured by treated wastewater. By increasing the concentration, electrical homogeneity decreased. The diluted specimens’ behavior was inclined towards the TWW results and not the control, even when insignificant amounts of treated wastewater were involved. For example, 25%TW, which is 75% drinkable water, followed the TWW’s resistance and not that of the control, cured by either drinkable water or treated wastewater. It showed that whenever wastewater parameters are involved in the specimens, they could exceedingly influence the concrete’s structure and create voids and porosity in specimens. So, diluting the concentration has an insignificant effect on electrical resistivity enhancement. TWW and specimens with concentrations close to that of TWW had better resistivity when they were cured by treated wastewater at lower ages; however, with aging the positive effect declined even on them, as if being cured by treated wastewater in the long term has caustic effects on concrete structure and causes more avenues of penetration. Nevertheless, in specimens with a lower concentration at an early age, a resistance growth was observed which again supported the positive effect of homogeneity features, as well as the negative effect of being cured by treated wastewater in the long term.At the age of 28 days, TW + 30%C, cured by drinkable and treated wastewater, had 15% and 14% less electrical resistivity than the control sample, respectively. This indicated that using wastewater in concrete has more negative effects on the concrete’s durability features than on its strength, because it had 10% reductions in the compressive strength test but a 15% reduction in electrical resistivity. Therefore, it is recommended that treated wastewater should not be used for projects with high contact with caustic material or marine projects.For this test, specimens are dried using an industrial oven. In the drying process, heating and cooling at a special set of temperatures in both the oven and desiccator over a determined period are used. At every step, the materials are weighed. The workflow of a water absorption test is based on drying specimens in a laboratory or industrial oven according to BS1881-122 . The timThe capillary test evaluates the process of non-saturated concrete water absorption by capillary suction while it is in touch with water. The tensile strength of concrete is a prominent property when it is to be utilized for making prestressed concrete structures, roads and runways. Not only did the tensile strength decline by increasing the concentration, but the rate of tensile strength growth was also lower than in the control sample. For instance, within days 7 to 28, the control sample cured by drinkable water and treated wastewater had 85.3% and 84.4% growth, but TW + 30%C had 78% and 77% tensile strength growth and TW + 35%C had 77% and 75% growth, respectively.The recommended bubble distribution is less than 0.2 mm, but by using wastewater instead of drinkable water, the pores become harmful for the concrete . The betResults indicated that the reduction rate in the control and TWW samples was almost the same until 100 cycles, but then TWW demonstrated different behavior and declined more than the control sample. For instance, until 100 cycles, in comparison to day 28 compressive strength, the control and TWW had an almost 3% strength reduction, whereas in 150 and 200 cycles, the control sample had 4% and 5% reductions while TWW had 6% and 7.6% compressive strength reduction, respectively. It was observed that the strength of the specimens was negatively affected by the lack of proper pore connectivity and air voids in the specimens produced by wastewater, especially when the smooth exterior layer of the specimens was gone due to initial cycles of the test, and the inner pore structures were the main effective factor. This illustrated the destructive effect of harmful pores and tiny air voids on the concrete structure caused by wastewater impurities. Interestingly, 75%TW also had the same reaction as TWW and the rate of reduction rose after cycle 100. No significant difference was observed in 25%TW; its concentration was fairly close to that of the control specimen and it supported the negative impact of larger pores in the concrete structure even after using diluted wastewater. Compared to the control sample, concentrated specimens had more compressive strength reduction in all cycles, and by increasing the concentration and cycles, the rate of reduction increased. Clearly, PWW, which contained the highest impurity, had the lowest strength due to having harmful pores. It should be noted that the exterior layer of PWW was already honeycombing with vulnerability, yet cracks and flaws intensified the volume expansion and failure in this specimen.Using wastewater causes larger pores and more voids in the concrete’s structure and these specimens can contain water and, subsequently, more frozen water inside of the concrete and more corrosion are expected in the rapid freezing and thawing test. TW + 30%C at 50, 100, 150 and 200 cycles had 12%, 13.9%, 17.4% and 21.4% less compressive strength than the control. The rate of compressive strength reduction was higher in the specimens with higher concentrations. Therefore, the improper pore structures and clogged air voids caused by the total dissolved and all impurities in the more concentrated wastewaters can be interpreted as the primary factors of strength reduction in the concrete specimens produced by wastewater. Based on the achieved results, void parameters, air voids and size-distribution have more prominent effects on the resistance than the air-void spacing. Therefore, based on the obtained results, air void structure in air-entrained concretes utilizing a Protected Paste Volume (PPV) parameter is recommended as it protects the paste area with air voids in the total paste area.σ, N, ix and µ are the population standard deviation, the size of the population, each value from the population and the population mean, respectively.In scientific research, researchers seek to present results as practically and, of course, as easily as possible. One of the most important ways that other researchers can make practical use of research is to provide mathematical models for use in future experiments and research . In thisIn the next step, the normality of the data should be checked. The normality of data is generally determined by examining the Skewness and Kurtosis coefficients ,51,52. Ai is the independent variable (inputs of the model), and ai are the regression coefficients of the model. The models obtained from the analysis performed with SPSS software are presented in 2, standard error and standard deviation were used.Once the correlation coefficients have been determined, it is time to determine the estimation models for research outputs. Multivariate linear regression is used for this purpose. The general form of multivariate linear regression is as follows ,55,56:Y The equations presented in In sum, the achieved results were commensurate with other tests with data integrity, and no significant contradiction was observed. The negative impact of the wastewater was conspicuous in durability results and curing with wastewater is not recommended. This research indicated that using industrial wastewater could decrease the quality of produced concrete according to its concentration but, based on the results, a proper understanding of the use of different concentrations was presented, which helps to reach the economic level of refinement in industrial towns for using their wastewater in concrete.In this paper, ten groups of concrete specimens with different industrial wastewater concentrations were produced and cured by drinkable water and treated wastewater separately according to tests and standards. Using wastewater as the water for mix design reduces the strength and durability, but industrial TWW can be a good and acceptable replacement for the water in mix design, having insignificant strength and durability reduction effects on concrete in all tests. By concentrating the treated wastewater properties by up to 30% in the TW + 30%C specimen, the compressive strength declined by almost 10% after 28 days, which showed the optimum level of concentration for an industrial town’s refinement. However, concentration had more adverse effects on durability tests, which showed that using wastewater causes more negative impacts on the durability than on the strength features of concrete. PWW did not have acceptable behavior in any tests, and it was rejected. Although in concentrated and diluted specimens the percentage of wastewater was increased or decreased in order, no linear relationship in the strength and durability features of the tests was observed. Control specimens showed better strength and durability when they were cured by drinkable water, which proved homogeneity and similarity features; nonetheless, wastewater specimens showed better strength and durability when they were cured by wastewater only at lower ages, whereas the good effect disappeared at late ages; as if being in touch with treated wastewater for curing deteriorates the specimens’ strength and causes corrosion. All of the specimens had less growth in terms of strength and durability when they were cured by treated wastewater in comparison to being cured by drinkable water. Using wastewater reduced the electrical resistivity and increased the water absorption of samples, yet diluting the treated wastewater could not correct the negative effects on concrete durability. Diluted specimens’ results were closer to those of TWW, not of the control and, by increasing the concentration, negative effects became more conspicuous. Using wastewater in the concrete had a negative impact on the rapid freezing and thawing results. It is concluded that the improper pore-size distribution, lack of pore connectivity and air voids caused by total dissolved and impurities of the wastewaters can be counted as the main reasons of strength reduction in the specimens produced by wastewater. Despite dilution, it was not an adequate solution for resolving the strength reduction of specimens in the freezing and thawing test, and even low concentrations of wastewater disrupt the formation of appropriate air voids and pore structure void parameters in the wastewater specimens in comparison with the control. By concentrating industrial wastewater, not only were the freezing–thawing properties negatively affected, but also the strength reduction rate increased, especially after 100 cycles. In the half-cell potential test, using wastewater insignificantly damaged the reinforcement, but specimens with more concentrated wastewater started to corrode faster. The specimens’ appearances had insignificant differences, except for PWW, which had more discontinuity and a distinguishable lack of hydration; however, by increasing the concentration, uneven and small cracks on the exterior layers were observed. Using wastewater increased water absorption and decreased workability. Therefore, it is highly recommended that it not be used in projects with caustic materials, exposed surfaces, or when a high slump is needed.


IgG4 is associated with two emerging groups of rare diseases: 1) IgG4 autoimmune diseases (IgG4-AID) and 2) IgG4-related diseases (IgG4-RLD). Anti-neuronal IgG4-AID include MuSK myasthenia gravis, LGI1- and Caspr2-encephalitis and autoimmune nodo-/paranodopathies (CNTN1/Caspr1 or NF155 antibodies). IgG4-RLD is a multiorgan disease hallmarked by tissue-destructive fibrotic lesions with lymphocyte and IgG4 plasma cell infiltrates and increased serum IgG4 concentrations. It is unclear whether IgG4-AID and IgG4-RLD share relevant clinical and immunopathological features.We collected and analyzed clinical, serological, and histopathological data in 50 patients with anti-neuronal IgG4-AID and 19 patients with IgG4-RLD.p = .004). Moreover, those IgG4-AID patients with elevated IgG4 did not meet the diagnostic criteria of IgG4-RLD, and their autoantibody titers did not correlate with their serum IgG4 concentrations. In addition, patients with IgG4-RLD were negative for anti-neuronal/neuromuscular autoantibodies and among these patients, men showed a significantly higher propensity for IgG4 elevation, when compared to women (p = .005). Last, a kidney biopsy from a patient with autoimmune paranodopathy due to CNTN1/Caspr1-complex IgG4 autoantibodies and concomitant nephrotic syndrome did not show fibrosis or IgG4+ plasma cells, which are diagnostic hallmarks of IgG4-RLD.A significantly higher proportion of IgG4-RLD patients had serum IgG4 elevation when compared to IgG4-AID patients (52.63% vs. 16%, Our observations suggest that anti-neuronal IgG4-AID and IgG4-RLD are most likely distinct disease entities. TheySera of 50 patients  with a clinical diagnosis of anti-neuronal IgG4 autoimmune disease  or Caspr2 (n=9) autoantibodies, chronic inflammatory demyelinating polyneuropathy (CIDP) associated with anti-NF155 (n=2) or pan anti-NF155/140/186 , anti-CNTN1/Caspr1-complex (n=2), anti-CNTN1 (n=5) or anti-Caspr1 (n=1) autoantibodies and MuSK myasthenia gravis (n=15)) taken at the time of diagnosis or at the earliest time point available where a clear autoantibody titer was present, and sera of 53 patients with suspected neurometabolic diseases  and from 13 healthy controls  were selected. The cohort of “suspected neurometabolic diseases” includes samples from patients with non-inflammatory diseases that were sent for diagnostic testing of metabolic diseases including Tay-Sachs disease, Sandhoff´s disease, X-linked adrenoleukodystrophy, Gaucher disease, and Fabry disease. 49/53 patients had no biochemical evidence for any type of these diseases, while 4 patients had the diagnosis of Gaucher disease. Sera of the 50 IgG4-AID patients, 53 suspected neurometabolic disease patients, and 13 healthy control patients were derived from archival blood samples that were sent for diagnostic purposes and stored at the biobank of the Division of Neuropathology and Neurochemistry, Department of Neurology, Medical University of Vienna, Austria (EK1123-2015). Archival nephelometry serum samples from the biobank of the Division of Rheumatology, Department of Internal Medicine III, Medical University of Vienna, Austria from 19 patients with IgG4-related diseases  were analyzed retrospectively (EK559/2005). The samples were processed and stored according to standard operating procedures at the Medical University of Vienna biobank in an ISO 9001-certified environment .6 cells. After 24 hours, cells were transfected at 70 to 80% cell confluence with plasmids encoding relevant neuronal/neuromuscular antigens [LGI1-ADAM23 – courtesy of Prof. Francesc Graus, Barcelona, Spain; Caspr1 – pCMV6-Entry OriGene RC218019; CNTN1 – pCMV GeneCopoeia EXA1153-MO29; Caspr2 – courtesy of Prof. Francesc Graus, Barcelona, Spain; NF155 – pCMV6-Entry OriGene RC228652; MuSK - pIRES2-AcGFP1-MuSK (Human embryonic kidney (HEK293T) cells were cultured in Dulbecco´s modified Eagle´s medium (DMEM) – high glucose supplemented with 10% fetal calf serum , 200 mM L-glutamine (Gibco), 1x penicillin-streptomycin (Sigma) and 1x non-essential amino acids (Sigma). For transfection, cells were seeded onto poly-D lysine  coated coverslips in tissue culture plates (p60) at a density of 1 x 10FP1-MuSK  and NF15FP1-MuSK  the cell® secondary antibodies against human (AF594) and rabbit IgGs (AF488)  or cultured medium supplemented with 1% BSA (MuSK) for 30 min  or 45 min (MuSK) at RT in the dark). For nuclear staining, 4´,6-diamidino-2-phenylindole (DAPI) was used. Cells were mounted with aqua polymount (Polysciences) onto glass slides. After drying the slides over night at 4°C in the dark, antibody binding was analyzed using an OLYMPUS BX63 fluorescence microscope.Briefly, live HEK293T cells were incubated with patients’ sera starting at a dilution of 1:40 and followed by serial dilutions, diluted in cultured medium  or cultured medium supplemented with 1% bovine serum albumin (BSA) (MuSK) for 30  or 60 minutes (min) (MuSK) at 37°C. Afterwards, cells were fixed with 4% chilled paraformaldehyde  for 10 min, permeabilized with 0.3% Triton X-100 (Merck) for 5 min  and incubated with a commercial antibody  for 60 min at room temperature (RT). HEK293T cells were then immunolabeled with the appropriate fluorescent-conjugated Alexa Fluor® secondary antibodies against human (AF594) and mouse/rabbit IgGs (AF488) (both diluted 1:750 in 1xPBS). To stain the nuclei, DAPI was used and finally, the cells were mounted with aqua polymount onto glass slides. Microscopic examination and fluorescent images were performed using an OLYMPUS BX63 fluorescence microscope.Cultured HEK293T cells were first fixed with 4% chilled PFA for 10 min, permeabilized with 0.3% Triton X-100 for 5 min and blocked for 1.5 hours with 1% BSA to prevent unspecific protein binding. Following the blocking step, cells were incubated with patients’ sera at a dilution of 1:40, followed by serial dilutions (diluted in 1% BSA) over night at 4°C. Cells were then immunolabeled with commercial antibodies  for 30 min at RT, followed by the corresponding Alexa FluorNeuropathologic analysis was performed on formalin-fixed paraffin-embedded (FFPE) tissue sections of human brain and kidney biopsy material. The brain biopsy was available from a patient with IgG4-RLD, whereas the kidney biopsy from a patient with IgG4-AID exhibiting CNTN1/Caspr1-complex autoantibodies. Tissue sections were stained with hematoxylin and eosin (H&E), Periodic acid Schiff (PAS) and silver impregnation. According to the manufacturer´s protocol, immunohistochemical stainings for the following primary antibodies were performed on an automated platform Autostainer Link 48 using the EnVision™ FLEX+ kit (Dako/Agilent) as a secondary system: CD138 , IgG , IgG4 . Heat-induced epitope retrieval (HIER) was performed either with target-retrieval solution low pH (Dako/Agilent) for IgG or with target-retrieval solution high pH (Dako/Agilent) for CD138 and IgG4.Image acquisition was performed using a NanoZoomer 2.0-HT digital slide scanner C9600 .For conventional transmission electron microscopy (EM) small samples of kidney tissue immediately were prefixed resting in toto at room temperature under fixative for a proper preservation of ultrastructure. Tissue processing was accomplished by adhering to routine standardized methods for EM, in brief fixation, dehydration and finally embedded in epoxide resin.Human total serum IgG and subclass IgG4 concentrations were determined using particle-enhanced immune nephelometry with the BN II System . The internal reference values for IgG4 were 0.03 - 2.01 g/L, and for total IgG 7-16 g/L. The published upper threshold for IgG4 in IgG4-RLD is 1.35 g/L. Serum concentrations ≥1.35 g/L were considered as elevated.Due to the heteroscedastic distribution of the data and small sample size of some groups, statistical analysis with ANOVA to compare serum IgG4 concentrations between the different disease groups was considered as inappropriate. Instead, we report the mean and 95% confidence interval for each group. Nonparametric Spearman correlation was used to analyze the relationship between total IgG and IgG4 concentrations per group and between autoantibody titers and serum IgG4 concentrations. In order to test gender- and disease-dependent proportions of IgG4 elevation in patients, Fisher’s exact tests were applied. Statistical analysis was conducted using GraphPad Prism 9 and IBM SPSS Version 27.Anti-neuronal/neuromuscular autoantibodies were identified in all 50 patients with anti-neuronal IgG4 autoimmune disease by cell-based assays Figure 1p = .004).Elevated serum IgG4 concentrations are frequently observed in patients with IgG4-RLD, but it is unknown if it is the same for patients with anti-neuronal IgG4-AID. Total serum IgG and IgG4 concentrations were measured using nephelometry, and sera with concentrations reaching the published cut-off value of ≥1.35g/L IgG4  were conThe highest absolute serum IgG4 concentrations . Elevated IgG4 was also observed rather in male patients with IgG4-AID (21.21%) than in females (5.88%) . The absolute IgG4 concentrations in all disease groups were higher in males than in females  had elevated serum IgG4 concentrations, but all IgG4-RLD patients were negative for anti-neuronal/neuromuscular autoantibodies. In a tissue-based assay, which represents a broad screening method for autoantibodies against a variety of neuronal and glial epitopes, no reactivity was found in the sera from patients with IgG4-RLD. However, we did not test for the presence of non-neuronal IgG4 autoantibodies, which therefore cannot be ruled out.In the healthy and neurometabolic controls as well as in the IgG4-AID patient group, few individuals showed elevated serum IgG4 concentrations. This has to be expected, since IgG4 concentrations vary and may temporarily/seasonally increase due to a change in immune status, e.g. due to infections or allergy. IgG4 levels are known to be higher in males than in females , 29, whiOur patients fulfilled clinical, serological and/or histopathological characteristics of IgG4-RLD . In geneImportantly, antigen-specific IgG4 directly cause neurological symptoms of IgG4-AID , while t+ plasma cells, fibrosis and sometimes elevated serum IgG4 levels  associated vasculitis . GPA is characterized by antigen-specific IgG4, IgG3 or IgG1 against proteinase 3  or myeloperoxidase  in neutrophils and monocytes, increased levels of IgG44 levels –52. The 4 levels –55, but 4 levels , which a4 levels . Neverthvia altered IL-10 production. MuSK myasthenia gravis patients with HLA-DRB1*14 presented with increased serum levels of IgG4 autoantibodies and IL-10 concentrations  and interleukin-13 (IL-13), but additional stimulation with interleukin-10 (IL-10) is considered as decisive factor for IgG4 class switch –61. IL-1trations , and IL-trations –72 and Mtrations . Anothertrations –76. Perhtrations . Overalltrations , 41.IgG4-AID and IgG4-RLD are most likely distinct disease groups. Due to their low disease prevalences, comparative data to characterize these diseases are limited. In our study we provide three relevant findings, 1) a significantly higher proportion of IgG4-RLD patients (52.63%) had elevated serum IgG4 concentrations compared to IgG4-AID (16%), 2) IgG4-AID patients with elevated IgG4 did not meet the diagnostic criteria of IgG4-RLD and their autoantibody titers/scores did not correlate with serum IgG4 concentrations, while 3) patients with IgG4-RLD were negative for anti-neuronal/neuromuscular IgG4 autoantibodies. Furthermore, male IgG4-RLD patients presented significantly more frequently with elevated serum IgG4 compared to female patients.In summary, our data do not support clinical or histopathological commonalities between IgG4-AID and IgG4-RLD, suggesting that they are in fact unrelated. Further studies on IgG4-AID and IgG4-RLD will lead to a better understanding of these diseases.The original contributions presented in the study are included in the article/The study was approved by the Institutional Review Boards of the Medical University of Vienna, Austria (EK 1442/2017). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.VE: Data Curation, Formal Analysis, Investigation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing. CT, LE, AR, RW, LW, T-YY, AF, TP, HH, CS, GR, SK, LD, CL, DdS, K-NP, GM, SP, WP, AA, MV, MM, MG, RR, DS, MP, KK, and GA: Investigation, Writing – Review & Editing. NK: Investigation, Visualization, Writing – Review & Editing. SH and FF: Data Curation, Formal Analysis, Investigation, Writing – Review & Editing. RH: Conceptualization, Funding Acquisition, Investigation, Project Administration, Resources, Supervision, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing. IK: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Project Administration, Resources, Supervision, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing. All authors have approved the final version of the manuscript.This work was supported by grants from the Austrian Science Fund (FWF), project number T996-B30, SYNABS project number I4685-B, DOC33-B27 and the Austrian Society of Neurology (Österreichische Gesellschaft für Neurologie). LD received a grant from Fleury Laboratory for the Brazilian Autoimmune Encephalitis Project without personal compensation. CL served as a consultant for Roche. K-NP received a travel grant from Merck. RH reports speakers’ honoraria from Novartis and Biogen. The Medical University of Vienna  receives payment for antibody assays and for antibody validation experiments organized by Euroimmun .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Few studies have been done on inpatient antibiotic use in Japan and antibiotic stewardship programs with dedicated full-time equivalents are rare. We sought to better understand inpatient antibiotic use in Okinawa, Japan. We applied the World Health Organization (WHO) Access, Watch and Reserve (AWaRe) Classification to compare our findings to international literature. Access antibiotics are common front-line antibiotics, Watch antibiotics are high-priority antibiotics with toxicity or resistance concerns, and Reserve antibiotics are last-line treatments for multi-drug resistant infections.A point prevalence study was conducted in five hospitals in Okinawa, Japan on Oct 1, 2020. Physicians conducted chart reviews of all patients receiving intravenous antibiotics. Type of antibiotic, reason for use, duration, and microbiologic data was collected. The primary aim was to evaluate the proportion of patients who received antibiotics on the assessment date; secondary aims were to categorize antibiotics according to indication, class and AWaRe classification. Descriptive statistics were used to derive the distribution of AWaRe Classifications and drug class.≥1 antibiotic on the assessment date. A total of 559 antibiotics were used for 504 patients and 22.0% (n=123) were for prophylaxis. Of those receiving antibiotics for treatment (N=436), 385 (88.3%) patients had a documented infection source. The most common indications for antibiotic use were pneumonia (24.2% n=93), urinary tract infection (19.7% n=76), and intraabdominal (17.9% n=69). Overall, 43.1% (n=241) of the antibiotics were categorized Access and 54.4% (n=304) Watch [Figure 1]. Cephalosporins were the most common antibiotic class (56% n=313), followed by β-lactam inhibitors (18% n=106) and narrow penicillins (8.2% n=46) [Figure 2].1,728 unique patients were included and 504 (29%) received 29% of inpatients in these 5 Okinawan hospitals were prescribed an antibiotic on the survey date. A majority of antibiotics used fall under the WHO AWaRe Watch classification which are antibiotics that may be more likely to cause resistance. Understanding appropriateness of antibiotics used in this population could inform antibiotic stewardship strategies and reduce antibiotic resistance.Figure 1. Antibiotic Distribution According to World Health Organization (WHO) Access, Watch and Reserve (AWaRe) ClassificationFigure 2. Antibiotic Distribution by Class in Okinawan HospitalsAll Authors: No reported disclosures


Dear editor,It is with great interest that we have read the recent article on agreement between intermittent and continuous pulmonary artery (PA) thermodilution measurements by Kouz et al. . HoweverFirst, the authors consider intermittent cardiac output (CO) measurements the Gold Standard. Although historically correct, such approach is clearly debatable. Intermittent PA CO measurements are by definition dependent on injected volume, temperature, and rate. Inadequate timing during the respiratory cycle further influences its accuracy. All of us who have regularly done this by hand, know how variable these CO measurements are, even when executed by one well-trained person within the shortest possible timeframe . GeneralSecondly, the authors state that “continuous” should actually be “semi-continuous” and that there is a delay up to several minutes. This is partially incorrect, since the thermal signal is measured continuously, but updated every 30 to 60 s as ‘STAT CCO’. In case of major changes an update of the CO will be displayed after 270 s . The cla We thank de Lange et al. for their interest in our meta-analysis of clinical studies comparing continuous with intermittent pulmonary artery thermodilution cardiac output measurements . De Langcontinuous—or rather a semi-continuous—cardiac output monitoring method. Although its usually called “continuous” pulmonary artery thermodilution we would prefer calling it automated semi-continuous pulmonary artery thermodilution. For automated semi-continuous pulmonary artery thermodilution a filament attached to the catheter heats up the blood in a random sequence (in contrast to cold fluid boluses used for intermittent pulmonary artery thermodilution) [The authors further stimulate a discussion on whether automated continuous pulmonary artery thermodilution is actually a ilution) , 7. The


Concrete is a heterogeneous material with a disordered material morphology that strongly governs the behaviour of the material. In this contribution, we present a computational tool called the Concrete Mesostructure Generator (CMG) for the generation of ultra-realistic virtual concrete morphologies for mesoscale and multiscale computational modelling and the simulation of concrete. Given an aggregate size distribution, realistic generic concrete aggregates are generated by a sequential reduction of a cuboid to generate a polyhedron with multiple faces. Thereafter, concave depressions are introduced in the polyhedron using Gaussian surfaces. The generated aggregates are assembled into the mesostructure using a hierarchic random sequential adsorption algorithm. The virtual mesostructures are first calibrated using laboratory measurements of aggregate distributions. The model is validated by comparing the elastic properties obtained from laboratory testing of concrete specimens with the elastic properties obtained using computational homogenisation of virtual concrete mesostructures. Finally, a 3D-convolutional neural network is trained to directly generate elastic properties from voxel data. Concrete is a highly heterogeneous composite material with a random microstructure across the length scales. This includes the material topology, size distribution, as well as their spatial configuration. Variations in the aggregate size distribution and the pore-size distribution from the nanometer scale to the decimeter scale manifest as variations in the behaviour of concrete at the macroscopic scale. The macroscopic properties of concrete such as strength, stiffness, permeability, diffusivity, etc. are completely determined by the heterogeneities in the material . To enabHowever, the process of virtually generating the mesostructure is much faster and cheaper as compared to extracting the microstructure using CT scans, provided that the virtually-generated mesostructure provides the same level of detail and information. Moreover, a method to generate realistic virtual mesostructures would significantly accelerate and support simulation models that integrate CT data into their pipelines. The aim of this paper is to present a tool to generate realistic virtual concrete mesostructures. In order to generate a virtual mesostructure, inclusions are first generated using a variety of computational methods and then assembled onto the main mesostructure volume using certain packing algorithms. Several optimised algorithms have been proposed in the last two decades for the random packing algorithm, constituting the most basic packing algorithm. Applications include integrated spherical particle kinetics models , moleculhttps://pycmg.readthedocs.io/en/latest/ (accessed on 9 June 2021). The proposed methodology is calibrated and validated using a variety of data obtained from laboratory measurements of real concrete specimens. Finally, as an add-on, and one of the many possible potential applications of CMG, we develop an artificial neural network (ANN) model for directly predicting the elastic properties from voxel data of concrete mesostructures generated by CMG.In this contribution, a computational tool called Concrete Mesostructure Generator (CMG) that allows an efficient generation of ultra-realistic concrete mesostructure, is developed. A Python implementation is available here: The procedure for generating a virtual concrete mesostructure consists of two steps: (a) The generation of realistic concrete aggregate inclusions using irregular polyhedron geometries with concave depressions and (b) the packing of aggregates into the cementitious mortar . The final mesostructure morphology is represented in terms of discrete voxels.Coarse concrete aggregates are characterised by multiple faces, sharp corners, and irregular surfaces. Even though the shape of aggregates has a strong effect on the stress concentration, crack initiation, and propagation in concrete , often oh [mm/voxel] is the resolution of the virtual mesostructure generated by the CMG. h is used to convert physical dimensions in SI units into voxels, its value can be set depending on the available computational resources. If l is an even number, then l is incremented by one. This is to ensure that the array contains a mid-point voxel. The voxel at the mid-point of the array at position Given the maximum aggregate size l, we first inscribe an imaginary ellipsoid with dimensions N on the polyhedron representing the aggregate is specified. Subsequently, N points, denoted by the position vector i is defined using a planar equation, which is a function of the point i. The rotation angles In order to model flat and elongated aggregates, we introduce the aspect-ratio mined as:Xi=P·R·QiIn order to perform the imaginary cutting operation , all voxels with position vectors kth voxel is denoted as Here, M denotes the total number of concave depressions centered at w, the depth is d, and Certain aggregate geometries are characterised by concave depressions, e.g., from crushing the aggregates during the production process. So far, we have designed a polyhedron whose geometry is described by voxel value = 1 embedded in a matrix material with voxel value = 0. Certain volumes of the voxel can be sculpted out to introduce concave depressions. This translates to replacing certain voxels with value 1 that are inside the volume to be sculpted out to 0. Concave depressions are introduced on the inscribed ellipsoid using the Gaussian surface equation see , right. M number of positions d and w, which control the depth and width of the concavity from the ellipsoid surface. The voxel values of the points which lie inside the polyhedron and below the Gaussian surface are changed from zero to one as shown in The procedure is as follows: First, Equation ) are genA summary of all parameters for the generation of realistic generic aggregates are provided in t of the ITZ is the only input. The algorithm uses the same technique as that of the polyhedron and the Gaussian surface, but with a larger concentric imaginary ellipsoid. The thickness t is added to the polyhedron ellipsoid axes to obtain the coating surface,In order to include the ITZ as a coating around the aggregate, the thickness Hence, there will be two sets of concentric polyhedron and Gaussian equations. The final algorithm checks for all points in the domain first for the outer surface followed by the inner surface. Voxels at the ITZ are represented with voxel value 2. This methodology can also be used to enforce a minimum spacing between the aggregates by setting these voxel values equal to that of the matrix (host) material.N and the elongation  are The random sequential adsorption (RSA) algorithm  is used As the hosting domain is incrementally populated with aggregates, the probability of finding free space to embed a new aggregate becomes increasingly small. In the original  MATLAB implementation of the model, the RSA algorithm, even though it generates statistically equivalent mesostructures by randomly distributing the aggregates in the mesostructure, achieving a packing density of more than 30%, requires a significant time for computations since the assembly procedure is completely random. Hence, to achieve a higher packing density in less computation time, the algorithm was modified from a random to a semi-random assembly algorithm (SRSA). According to SRSA, if the aggregate to be assembled does not find free space in the current random position, instead of finding a new random position, the current aggregate is incrementally translated along an orthogonal plane  of different sizes, embedded in the cementitious matrix. One of the most conventional aggregate size distribution curves is that of standard DIN-1045 or the Fuller curves. As a result, a realistic virtual concrete sample should statistically represent such a distribution. To this end, the concrete standard AB16 (according to DIN-1045) with the largest aggregate size of 16 mm is considered. The voxel size is chosen to be 0.5 mm, i.e., the parameter corresponding to the resolutions A detailed quantification of aggregate size distribution for AB16, together with the measurement of elastic properties of the investigated concrete samples was performed in the laboratory. The aggregates size distribution obtained from the measurements is summarised in N. As it was found that the aggregates of a larger size exhibit a wider range of aspect ratio, the aspect ratio of aggregates greater than 8 mm is randomly chosen from 1.75 to 3.25. Similarly, the aspect ratio range of aggregate sizes from 4 mm to 8 mm is within 2 to 3 and for a size of 3 mm, it is within 1 to 2. The number N for each size is determined by multiplying the aggregate size (in terms of voxel) by a factor whose value is either 0.5 or 0.625. It is to be noted that this range of values was chosen after performing several trials to obtain a realistic geometry. With regards to the concave depression in the aggregates, only aggregates larger than 12 mm are assumed to exhibit such a feature, with the prescribed values of 5 concave surfaces, 3 mm in width, and 2.5 mm in depth per aggregates particle. The variance parameter of the surface was chosen as The procedure for the calibration of the parameters defining the morphology of the aggregates is as follows: For each particle, the two most important parameters are the aspect ratio and the number of faces of the polyhedron In order to test the capability of the method to generate virtual AB16 mesostructures using the data from the previous section, three sample sizes of 5, 10, and 20 cm were considered. From each sample size, three specimens are generated to capture the possible stochastic fluctuation of the samples, analogous to experimental practice. The visualisation of each size is shown in The time required for generating a mesostructure of resolution In this section we compute the elastic properties of the virtual AB16 mesostructure using computational homogenisation and validate the results with data from the laboratory. First we describe the data for model validation and then we compare this data with model predictions.In order to validate CMG, a series of tests were conducted to determine the effective properties of concrete and aggregates. The investigated concrete samples are made of Portland cement of type CEM I 52.5 R with w/c = 0.45 and one type of aggregates (Quartz) with a maximum size of 16 mm. The Young’s modulus and Poisson ratio of Quartz aggregates are measured as 84.6 GPa and 0.12. The measurement of the average Young’s modulus for the concrete samples is obtained from an uniaxial compression test. Two samples of size 10 cm were loaded using displacement control with a displacement rate The elastic properties of the virtual concrete mesostructures are computed by directly using voxelised data from the CMG in a Lippmann–Schwinger-based computational homogenisation scheme (LS-FFT)  and a fienerated . The aggThe elastic material parameters for the voxels representing the aggregate material are set to In this final section we explore the potential of training an artificial neural network using data generated from the numerical simulations for predicting stiffness and the poisson ratio of concrete. The aim is to directly predict the elastic properties using virtual mesostructure data i.e., directly from CMG. Machine learning (ML) techniques have successfully established linkages between the microstructure and the macroscopic property of several materials based on the data acquired from experiments and numerical simulations. Once trained, the ML model can predict the material properties at speeds comparable to that of analytical methods. Hashemi et al.  used artp and the value of the matrix = 1. Thus, for a mesostructure with phase contrast 4, voxel values at the aggregate positions are represented by the value 4 and voxels corresponding to the mortar phase are set to 1.Assuming concrete to be an isotropic material, a 3D-convolutional neural network is trained to predict the elastic modulus  depending on the application. The requirement at hand involves building a link between volumetric features of the mesostructure, such as volume fraction, shape, and distribution of aggregates, along with phase contrast information, to the elastic concrete properties.It can be argued that 2D slices can be used to train a 2D-CNN for generating the structure-property relationship, as this would be much cheaper. However, a 2D image analysis of virtual concrete mesostructures generated by CMG  shows, tIn order to choose the optimal parameters for the model, we performed a preliminary study, based on which we finally selected two 3D convolutional layers with two max-pooling layers. The architecture of the layers is given in Training of the 3D-CNN model was performed on 80 Intel R Xeon R Gold 6148 CPUs. The total time for training the model was approximately 22 hours for 500 epochs with a batch size of 32. The total dataset of 1440 mesostructures with the corresponding labels (the elastic properties) is split into 1080, 180, and 180 batches for training, validation, and testing, respectively. The testing batch is used for testing the performance of the trained model since these mesostructures are completely unseen by the CNN model during training and validation. Once the model was trained, the macroscopic properties of the new RVEs from the training set are predicted, and results are compared with actual values from FCH simulations. The mean squared error (MSE) calculated between the actual results from FCH simulations and the predicted values from the 3D-CNN are found to be 0.0048, showing an excellent prediction accuracy. It can also be seen in In this paper, we presented a computational tool, denoted as the Concrete Mesostructure Generator (CMG) for generating realistic virtual mesostructures for application in computational mesoscale simulations of concrete. CMG is open-source, implemented in Python, and available for all users working on mesoscale analysis of concrete structures. The results of the virtual RVE’s using the CMG have been validated by comparing the elastic properties obtained from laboratory measurements with results from two different methods of computational homogenisation (finite-cell homogenisation and Lippmann–Schwinger-based scheme) of the virtual mesostructures. An excellent agreement was obtained for both homogenisation methods. Finally, we developed a 3D-convolutional neural network model that is able to generate the elastic properties directly from virtual mesostructure images in a voxel format. The output from the trained CNN model shows an excellent agreement with results from computational homogenisation. It is concluded that the tool can be used to rapidly estimate the elastic properties of a real concrete mesostructure given either the design data from specific concrete designs, such a the grading curve, or directly using data obtained from CT scans in a voxel format. The application and use of CMG in mesoscale models that simulate distributed damage and damage identification using diffuse ultrasonic waves will be presented in a subsequent publication.