Loading small molecular weight hydrophilic drugs into polymeric carriers is a challenging task. Metformin hydrochloride (MET) is a highly soluble oral antidiabetic drug of small size and high cationic charge. Hydrophobic ion pairing (HIP) is an approach for reversible modulation of solubility and hydrophilicity of water-soluble drugs via complexation with oppositely charged molecules. Herein, we prepared MET ion pairs and carefully studied and characterized MET interaction with different ligands, with the aim of increasing MET lipophilicity and loading efficiency. HIP was successful using three hydrophilic anionic ligands; sodium dodecyl sulphate (SDS) Carbopol (CB) and tannic acid (TA). Electrostatic interaction and hydrogen bonding drove the complexation per spectroscopic and thermal studies. Complexation efficiency depended on ligand type and charge ratio. While complexes had varying interaction strengths, the excessive stability of TA/MET resulted in unfavorable poor MET dissociation. Notably, HIP imparted a 450 and tenfold lipophilicity increase for SDS/MET and CB/MET, respectively. The latter showed favorable controlled, yet complete release of MET at pH 6.8 and was loaded into alginate beads. Complex bulkiness and decreased lipophilicity resulted in a dramatic 88% increase of MET loading, demonstrating the success of HIP as a simple, efficient and applicable approach for modulating drug’s properties. Frequent dosing, on top of MET’s gastrointestinal side effects, adversely affects patient compliance and therapy success. This challenge can be addressed through sustained-release formulations, where drug is given less frequently. Nevertheless, MET’s low molecular weight (165.65-g/mol), hydrophilic nature, and high-dose make its loading into oral polymeric carriers challenging.Metformin hydrochloride (MET) is a cationic biguanide drug widely used for chronic management of type-2 diabetes. However, its high-dose (500-mg), suboptimal bioavailability (40–60%), and short half-life (2–6 h)9. HIP is driven by non-covalent interactions; thus, the complex can rapidly dissociate in the presence of ions to the native drug. This approach has been used to increase encapsulation efficiency of various biomolecules, peptides, peptide antibiotics10, anticancer drugs11, and other water-soluble drugs13.Hydrophobic ion pairing (HIP) has emerged as an efficient approach to modify unfavorable physicochemical qualities of ionizable hydrophilic drugs. In HIP, ionizable water-soluble drugs are stoichiometrically complexed with counter ions driven by electrostatic interaction, resulting in a less soluble uncharged ion pair complexes14. Such ion-paired complex-in-beads would result in optimal pharmacokinetics, milder side effects less frequent dosing, and thus, higher patient compliance and therapy success.Here, HIP is examined as a strategy to modulate the MET properties and enable its efficient loading into a standard oral polymeric carrier . Such approach offers several advantages; (1) ion-paired MET complex will be less hydrophilic and bulkier than free MET, hence easier to load into beads, and (2) a sufficiently stable complex will sustain MET release from the beads. Alginate beads were selected as polymeric carrier because of their biocompatibility and extensive use as an oral carrier. Moreover, alginate acid-resistance would protect complexed MET from gastric pH, then release it in the intestineWe designed and optimized MET ion-paired complexes, testing five structurally diverse anionic ligands; Carbopol® 940 (CB), sodium dodecyl sulfate (SDS), tannic acid (TA), sodium deoxycholate (SDC), and low-methoxy low-amidated pectin (LMP). The interaction of each ligand with MET and the resulting ion pair, were carefully examined with the aims of (1) understanding how the two molecules interact at different ratios, (2) determining the strength of interaction and complex stability, and (3) evaluating how HIP modulated MET properties. A great discrepancy in ion pair properties and performance per ligand structure and nature of interaction with MET were observed. The most optimum complex was used to form MET-loaded alginate beads. Our complex-loaded beads were shown to have a dramatically higher loading efficiency than those loaded with free MET or polymer blend.16. It has two pKa values (2.8 and 11.5) thus it exists in a neutral, mono- or di-protonated form based on medium pH  exhibited lower %T levels at pH 3–10, showing interaction and formation of hydrophobic ion-paired complex, Fig. 20.CB is a crosslinked polyacrylic acid polymer with a molecular weight of 104,400-g/mole. It is rich in carboxylic acid groups with a pKa < 2)17. SDS was previously reported to slow down MET release from tablets at pH 2 due to its interaction with MET at low pH level17. In our study, SDS solution was prepared at concentrations below and above CMC  to inspect the effect of micelle formation on the association between SDS and MET. SDS solution CMC was verified using conductimetry 22. However, %T-pH profile of SDC/MET was not different from SDC alone at pH > 6.5 . LMP was previously reported to form nanoparticles with cationic debranched starch driven by electrostatic interaction24. When screened for interaction with MET, %T profile was no different from that of free LMP  mode of the interaction and (2) whether such interaction exists at various ligand/MET ratios. The interaction within the pair is likely a contribution of ionic and non-ionic interactions, such as hydrogen bonding and hydrophobic interactions26. As reported, the CB thermogram  spectroscopy.Finally, for TA samples Fig. C, TA its−1 corresponding to NH stretching of the amine group (expected site of complexation), NH stretching of the imido group, and C=N stretching, respectively30.FTIR spectra were obtained for free MET, free ligands, ligand/MET physical mixtures, and freeze-dried complexes at different charge ratios Fig. D–F. MET −1 corresponding to carboxylic O–H stretching, carboxylic C=O stretching, carbocyclic C–O stretching, =C–H alkenes bending, and –C=C–H alkyne bending, respectively31. Physical mixture indicated peaks of CB and MET, but CB/MET appeared differently; CB’s 1714 and 1270-cm−1 peaks disappeared for all ratios  reduced in intensity, especially at 1:1 (below CMC). This could be explained by ionic interaction and was observed in the remaining charge ratios. Similar behavior was reported with hydrophobic ion pairs of dioctyl sulfosuccinate and octreotide, where the vibration peaks were highly diminished and even disappeared due to the formation of the HIP complex33.For SDS/MET, Fig. −1 attributable to intermolecular and intra-molecular hydrogen bonding. Also, O–H bending appeared at 1440 cm−1, and ester C=O appeared at 1720 cm−129. Physical mixture showed TA and MET characteristic peaks with TA’s ester C=O stretching appearing as a shoulder. In TA/MET complex spectra, many changes were observed; (1) intensity of TA’s phenolic broad peak and C = O shoulder peak reduced, showing the availability of phenolic groups for hydrogen bonding with MET, and (2) intensity of MET sharp 3370-cm−1 peak and imido’s 3295.5-cm−1 peak reduced. The same pattern was observed with other complex ratios where TA and MET peaks intensity reduced with charge ratio increase. This pattern confirms predicted hydrogen bonding between TA’s phenolic OH and MET’s amine.When it comes to TA/MET complex Fig. F, the sp34.In summary, FTIR data is in accordance with thermal analysis; MET does interact with tested ligands in formed complexes at various ratios. CB/MET and SDS/MET complexes appear to be dominated by ionic interactions, while strong hydrogen bonding was noticed with TA/MET complexAfter complex formation was verified and the mode of interaction was elucidated, the efficiency with which MET was complexed, CE%, was measured Fig. . Mainly,For TA/MET complexes, only indirect method was used to determine CE%; it was not possible to free MET after complex formation because of extensive hydrogen bonding and high stability. Complex was indissociable and remained unaffected by heating or organic solvents. For all complexes, CE% was charge-ratio dependent. CE% increased upon increasing charge ratio, which was expected; more ligand is available to complex with MET, in non-stoichiometric hydrogen bonding contribution. This likely occurred with CB/MET and TA/MET; higher CE% were observed at ratios > 1:1. CE% attained a maximum of 55.6% at CB/MET 2:1 ratio, and 79% at TA/MET 3.3:1 ratio Fig. A,C.36. Similar findings were reported by Abdelhamid et al. for alendronate polyethyleneimine ion complexes37.For SDS/MET Fig. B, using For further tests, the charge ratio of the highest CE% from each complex was used. CB/MET, SDS/MET, and TA/MET at 2:1, 1:1 (> CMC) and 3.3:1 having CE% of 55.5, 61.5 and 78%, respectively, were selected.Visually, complex suspensions appeared turbid with a degree of translucency Fig. A. SuspenKp was equal to 29.1 ± 0.8, indicating a tremendous 450-fold increase in MET hydrophobicity, Supplementary Table 38. For CB/MET, a decent 10-folds increase in hydrophobicity was observed (Kp = 0.575 ± 0.079), Supplementary Table Kp was not measurable due to its poor dissociation. Nevertheless, MET hydrophobicity improvement via HIP with CB and SDS is appropriate for MET encapsulation into drug carriers and may modulate its permeability across biological barriers41.HIP's main aim is to increase the drug’s hydrophobicity facilitating its loading and delivery. The hydrophobicity of each MET complex, was measured by examining their partitioning between aqueous and organic phases. In counter ions absence, the partition coefficient of MET ranged from 0.059 to 0.064 at pH 8 and 2.7, respectively. HIP greatly increased MET’s hydrophobicity. For SDS/MET (1:1), From all results obtained, it is expected that TA/MET is the strongest complex. Stability is a crucial issue to examine when developing hydrophobic ion pairs; stability has to be optimized to ensure modulation of the drug’s lipophilicity yet allowing drug release. Conventional mathematical conversions using experimentally determined binding data were applied to evaluate complexes' stability. Following the standard addition (molar ratio) method . TA/MET is the most stable complex, and SDS/MET is the least stable with CB/MET in between.In-vitro release profiles of the three complexes were pH-and stability-dependent , while monoprotonated MET at pH 6.8 is not charged enough to maintain complex stability resulting in MET burst release17.In contrast, SDS/MET association complex exhibited an opposite trend Fig. B; only 2On the other hand, from TA/MET’s %T-pH profile, its complete dissociation was expected at pH 1.2 as TA is unionized. However, it did not release any MET, at either pH level, for the entire 24 h Fig. C. This w43. Therefore, CB/MET was selected as the optimum MET ion pair.Finally, revisiting the aim of this work, the intention is to increase MET lipophilicity, thus (1) enabling its efficient loading into polymeric carriers , and 2) sustaining its release upon oral administration. Hence, optimum MET ion pair should abide by certain criteria; it should: (1) increase MET’s partition coefficient, (2) be stable enough to withstand loading, (3) sustain MET release at intestinal pH, and (4) achieve complete dissociation and MET release. Based on these criteria, TA/MET was excluded; the complex was indissociable Fig. C. Compar sustaini44. Therefore, in our study, three different types of beads were incorporated  beted Fig. A.Figure 44. Additionally, even when EE% is calculated considering the MET lost during the complexation procedure ; CB/MET-loaded beads had far superior EE; 34% higher than MET + CB beads  was a kind gift from Chemical Industries Development company (Cairo Egypt), SDS was bought from Research lab industries , Carbopol 940 (CB) and sodium alginate were bought from the general chemical, and pharmaceutical Company Ltd. (England), tannic acid (TA), sodium deoxycholate (SDC), and octanol were bought from Sigma- Aldrich (Germany), low-methoxy low-amidated pectin  was bought from CP Kelco . The rest of the materials were bought from El-Nasr Pharmaceutical Chemicals Company .pKa at screened pH range. Resulting Ligand/MET solutions adjusted with 0.1 M sodium hydroxide and/or 0.1 N hydrochloric acid. %T was recorded using a UV spectrophotometer at 520-nm . Similarly, a change in %T for solutions containing only the ligand or MET was investigated. Optimum pH was defined as one that gave the lowest %T value in ligand/MET solution, indicating the formation of complexes10.Five anionic ligands  were screened for the formation of MET/ligand association complexes. Complexation was indicated by a change in the solution’s % transmittance (%T) upon pH change. Optimum pH for complex formation was determined first, then adapted for further studies. Equal volumes of aqueous solutions of MET, and each ligand, were mixed at a 1:1 charge ratio, Supplementary Table 46. For SDS/MET, complex aqueous solutions of SDS and MET were adjusted to pH 2.7 (optimum pH). SDS/MET charge ratios of 0.5:1 and 1:1 ) were used with 20 min stirring. SDS CMC was determined to explore the effect of micelle solvation on formed complex using conductimetry (Supplementary data). To achieve SDS concentration below and above CMC, the final SDS concentration in complex suspension was 0.087% and 0.174%, respectively. For TA/MET complex, phosphate buffer of pH 5.9 (200-mM) was used for complex preparation. TA/MET charge ratios of 1:1, 1.6:1, 3.3:1, and 6.6:1 were prepared by stirring for ten minutes.Based on screening performed, association complexes of MET with CB, SDS, and TA were prepared at corresponding optimum pH levels. MET solution was gradually added to the ligand solution of equal volume accompanied with stirring. For CB/MET complex, thepH of MET and CB solutions was adjusted to eight (using 0.1-M sodium hydroxide) and the complex solution was stirred for 20 min CB/MET charge ratios of 0.5:1, 1:1, 1.5:1, and 2:1 were prepared34. Dry samples were pressed using potassium bromide-disks, and the infra-red spectra were recorded from 400–4000-cm−1 at room temperature.DSC and FTIR spectroscopy were used to investigate the nature of interaction within complexes. Samples (4-mg) of MET powder, ligand, physical mixture, and freeze-dried complexes at different ratios were placed in aluminum pans sealed and heated at constant rate of 10 °C/min using computer-interfaced Shimadzu Calorimeter  in the range of 25–300 °C under a constant flow of nitrogen gas. For FTIR spectroscopy, samples were analyzed using Nicolet IS10 FTIR spectrometer max = 235 nm. CE% was determined according to Eq.  was determined either directly  or indirectly (TA/MET complex). In the direct method, the complex suspension was centrifuged to separate the complex from unentrapped MET. Samples were centrifuged for 15 min at 14,000 or 10,000-rpm for CB/MET and SDS/MET complexes, respectively. Then, obtained complexes were resuspended in a medium where the complex dissociates (0.1-M HCl for CB/MET and 0.2-M phosphate buffer (pH 6.8) for SDS/MET). Finally, samples were centrifuged a second time to separate precipitated ligand (SDS or CB) from supernatant containing freed MET. The supernatant was analyzed spectrophotometrically for MET content at ʎi.e., unentrapped MET. To avoid overestimation of CE% due to drug loss or adherence to tube. CE% was calculated for a standard MET solution (no TA was added) that underwent centrifugation and processed in parallel with a complex sample. Equation . Secondly, MET content was determined after centrifugation  and supernatant removal . CE% was calculated from Eq. . Alterna47. Second, complex samples (0.1-mL) were diluted to 0.5-ml with phosphate buffer (pH 5.9), and combined with 0.5-ml ninhydrin reagent. Samples were shaken, covered, placed in a boiling water bath for 20 min, then cooled and diluted with distilled water. The violet color developed was measured at λmax = 567 nm spectrophotometrically.For ninhydrin assay, first, ninhydrin reagent was prepared by dissolving 0.2-g ninhydrin and 0.03-g hydrindantin in 10-ml 3:1 dimethyl sulfoxide/sodium acetate buffer (pH 6.5)48.Particle size and PDI of freshly prepared CB/MET, SDS/MET, and TA/MET complexes of selected charge ratios were measured through dynamic light scattering using Malvern Zetasizer ZS90 (UK). Samples were measured in triplicates at 25 °C. For morphology, complexes were imaged using TEM. The complex suspension was diluted with the same medium used for complex association (to ensure stability). A sample drop was placed onto a carbon-coated copper grid then stained with uranyl acetate solution (2% w/v). Excess staining was removed using filter paper, and the sample was allowed to air-dry. Then, the sample was imaged using the TEM 49. Partition coefficient of TA/MET was not determined because it was indissociable. Ten mg of free MET or equivalent in complex was added to 10-ml water (at pH 8 for CB/MET and pH 2.7 for SDS/MET), and 10-ml octanol in a flask. Flask was shaken for 72 h, then the phases were separated using a separating funnel and centrifuged at 6000 rpm to ensure complete separation. Next, the aqueous phase was analyzed for MET content. For CB/MET, the pH was adjusted to 2 using hydrochloric acid to release free MET from complex, then filtered through 0.45-µm filter disk, and diluted (tenfold) using phosphate buffer (pH 6.8). For SDS/MET, fivefold dilution was perfomed with phosphate buffer (pH 6.8), followed by filtration. MET concentration was determined spectrophotometrically at 235-nm.Octanol/water partition coefficient was determined through shake flask method for ligand/MET complexes to determine the effect of the HIP on their hydrophobicityKp) and fold increase in lipophilicity were calculated from Eq.  concentrations and fixed MET concentration. In Job’s method, equimolar solutions of MET and SDS were prepared, then mixed at varying ratios. The absorbance of complexed MET was determined for both methods, and data were plotted to determine the stoichiometric ratio and stability constants. Method and calculations are detailed in the supplementary information.The stoichiometric ratios and stability constants of ligand/ MET complexes were estimated using either the standard addition or Job’s method of continuous variation. The latter was used for SDS/MET complex as its stoichiometry was expected to be in the range of 1:2–2:1, which is better assessed using Job’s methodIn-vitro release profiles of MET from freeze dried complexes were determined at pH levels resembling that of gastric fluid  and intestinal fluid . Complexes equivalent to 10 mg MET were added to each medium (100 mL), incubated in a thermostatically controlled shaking water bath  at 37 °C and 50 rpm. Samples were withdrawn at 1, 2, 4, 6, 12 and 24 h , and replaced with fresh medium. MET concentration was determined spectrophotometrically as mentioned before.The effect of MET HIP on its loading into calcium alginate beads was studied. Three types of beads were prepared; beads loaded with free MET, beads loaded with CB/MET complex, and beads loaded with a mixture of free MET and CB . For all, sodium alginate concentration and MET/sodium alginate ratio were kept at 5% and 1:20, respectively. Beads were prepared by ionotropic gelation method. MET or freeze-dried CB/MET of 2:1 charge ratio was dissolved or dispersed, respectively, in sodium alginate solution pH 8). Then, MET-sodium alginate bubble-free solution was dropped using a 22-gauge needle into a 2.5% calcium chloride solution under gentle stirring (9–11 drops/minute) at 25-cm height from the solution  from Eq. (For determination of encapsulation efficiency (EE%), a known weight of beads was crushed, dispersed in 0.2 M sodium citrate and sonicated for 30 min. MET concentration in filtered solution was measured at ʎET”, Eq.  was usedfrom Eq. .7\documeSupplementary Information.


The existence of either eccentricity or slenderness has a significant effect on the mechanical properties of a structure or member. These properties can change the working mechanism, failure mode, and bearing capacity of the structure or member. A concrete-filled, glass fibre-reinforced, polymer tube composite column has the same problem. We carried out experiments on the influences of eccentricity and slenderness on the mechanical properties of concrete-filled, glass fibre-reinforced, polymer tube composite columns. The experimentally recorded stress–strain relationships are presented graphically, and the ultimate axial stresses and strains and the FRP tube hoop strains at rupture were tabulated. The results indicate that the influences of slenderness and eccentricity on the composite columns were significant with regard to the axial strain, hoop strain, ultimate bearing capacity, lateral displacement, and failure mode. Based on the existing research literature and the results reported in this paper, the bearing capacity formula of a composite slender column under an eccentric load was established. The theoretical results were in good agreement with the experimental results. Over the past couple of decades, fibre-reinforced polymer (FRP) composites have found increasingly more applications in civil engineering due to their high strength-to-weight ratios and high corrosion resistance ,2. ExistA number of experimental studies have been conducted to investigate the axial compressive behaviour of CFFTs over the last two decades. Under axial loads, the FRP tube confines the concrete by reducing its lateral expansion, and therefore it increases its ultimate strain and strength ,9,10.In addition, concrete compression columns almost do not exist in practical engineering. Several researchers have studied the structural behaviour of concrete-filled FRP tubes under eccentric loads. The lateral deflection caused by longitudinal bending under an eccentric load causes premature buckling and failure of composite columns. Therefore, the effects of the secondary moment caused by lateral deflection on the mechanical properties of composite columns cannot be ignored . The fleIt is well known that most columns in practice are subjected to combined axial compression and bending due to load eccentricities. Additionally, the effect of slenderness on the compressive behaviour of a column is an important issue to be addressed. Many experimental studies have been conducted on slender FRP-confined concrete columns. These studies have confirmed that FRP confinement can enhance the strength and ductility of slender columns. It has also been reported in these studies that an increase of column slenderness generally leads to a reduction in the load capacity of the column .Maha Hussein Abdallah et al. presented the test results of an experimental program to investigate the structural performance of slender, concrete-filled, fibre-reinforced polymer (FRP) tube (CFFTs) columns under pure axial compression loads. The experimental results showed that the axial compressive strength of CFRP-reinforced CFFT columns was reduced by 22% when the slenderness ratio was increased from 8 to 20. In general, the use of an FRP tube induces a confinement effect on the concrete column which enhances its strength and ductility. However, the confinement results in slenderness, which increases the possibility of buckling instability in CFFT columns . Jason FAs mentioned previously, a large number of experimental studies on FRP-reinforced columns have concentrated on the influences of slenderness and eccentricity. Even if the parameters were different in each study, the facts indicate that FRP can significantly improve the mechanical properties of columns . IncreasTo date, research related to quantitative evaluations of the influences of slenderness ratio and eccentricity on the bearing capacity of FRP-confined concrete columns is still very limited, especially for GFGT composite columns, which are considered the most likely to be used in engineering practice. One reason is there are many unsolved problems in FRP-confined concrete short columns; the other is that the effect of column slenderness  is not included in various design guidelines for the FRP strengthening of RC structures ; that is, all these design provisions are limited to designs of FRP jackets for short columns, for which the second-order effect is negligible. This limitation can be attributed to the limited amount of research on the behaviour of slender FRP-confined RC columns .An experimental study was recently completed by the authors on the mechanical properties of CFGT columns with various slenderness ratios and eccentricities. The detailed experimental program and the test results are presented and discussed in the following sections. The main objective of this study was to assess the general behaviour of CFGT columns with slenderness ratios ranging from 14 to 48 and eccentric ratios ranging from 0.1 to 1.0. Additionally, based on the experimental data and the data in the literature, the reduction coefficients of slenderness ratio and eccentricity were determined via parameter identification method. Furthermore, the bearing capacity formula of the CFGT slender columns was established under eccentric loading. A comparison between the test data and the theoretical results showed that the proposed formula was successful.In total, twelve CFGT circular columns were fabricated. All of the specimens were laterally confined within GFRP tubes. The specimens were divided into two series, series A and series B; six specimens in each series. For series A, the effect of the slenderness ratio was the main focus, and the specimens in were tested under axial loads. For series B, the effect of the eccentricity was the main focus, and the specimens were tested under eccentric loads. All of the specimens had the same height (h = 1000 mm) in series B, whereas in series A, the height of the specimens changed from 600 to 2400 mm. For both series A and series B, specimen 0 was a contrast specimen. The test matrix is presented in Three materials were used for fabricating the test specimens. These materials were concrete, the FRP tubes, and steel reinforcements (deformed steel bars and hoop steels). The following sections provide descriptions of the mechanical properties of the different materials used in this research.All of the specimens were constructed using ready-mixed high strength concrete (HSC). According to the Chinese Standard GB/T50081-2019, the actual compressive strength was determined from the testing of six concrete cylinders “150 mm × 300 mm” on the same day of the testing of the columns. The average compressive strength and tensile strength of the concrete were 64.30 and 6.32 MPa, respectively.According to the Chinese Standard GB50010–2019, six deformed steel bars  were used as longitudinal reinforcements, and steel bars with diameters of 6.5 mm were used as hoop reinforcements with 180 mm spacing in all of the CFGT columns. The GFRP tubes were fabricated using a filament-winding technique and E-glass fiber and epoxy resin, with different fibre angles of ±57.50° with respect to the longitudinal axis of each tube. The internal diameters were equal to 200 mm, and the thickness of every tube was 5 mm. All of the parameters of the GFRP tube were provided by the manufacturer and not tested in the laboratory. All of the specimens were loaded using a 5000 kN capacity-testing machine. Top and bottom adjustable steel roller bearings bolted to the top rigid plates of the steel end caps were used to attain the predesignated eccentricity and to replicate the case of a perfect pin-ended column: k = 1. For part of the linear stress–strain testing (up to 75% of the specimen’s estimated capacity), the test was conducted with a load controlled technique at a rate of 2.5 kN/s. Then, the testing continued under displacement control at a displacement rate of 0.002 mm/s until failure . During The key test results for the long CFGT composite columns are shown in The lateral displacement data of the specimens under axial compression were obtained with the displacement sensors arranged axially on the composite columns. The longitudinal and circumferential strains on each GFRP tube at five levels of height were recorded by strain gauges attached to the GFRP’s surface, as shown in The relationship between the axial load and lateral deformation is shown in Based on the experimental results and phenomena, the failure form of the CFGT column gradually changed from material failure to overall instability with the increase of the slenderness ratio and the eccentricity. As a result, the ultimate bearing capacity decreased rapidly, which was very different from the calculation formula of the bearing capacity for the CFGT short column. Therefore, the influences of the slenderness ratio and the eccentricity had to be considered in the calculation of the CFGT slenderness columns under an eccentric load.Due to the existence of defects such as the initial eccentricity and initial bending, the axial compression columns with larger slenderness ratios produced lateral deflection at the initial stage of loading, and then produced a second-order effect and an additional bending moment. Finally, the concrete column was destroyed by bending instability, and the ultimate bearing capacity was lower than that of the short column. The same problem still existed for the CFGT columns, but the mechanical properties of the CFGT columns were more complicated. The stability bearing capacity of the concrete long columns was analysed with the following two methods: one method was the Euler formula calculation model based on tangent modulus theory, and the other method was the empirical formula based on the regression of test results. In this research, the reduction coefficient formula of the slenderness ratio was obtained from parameter identification method of the experimental data and some literature ,28,29: or moderate (e/D = 30%) eccentric loads exhibited compression failure—specifically concrete cover spalling at peak load, followed by a significant drop in column carrying capacity. Additionally, separations were observed on the FRP tube, among the horizontal FRP layers, on the tension side, for the specimens under high eccentric loads. Compared with specimen BC0-H, specimens BC1-H, BC2-H, BC3-H, BC4-H, and BC5-H had 49.99%, 77.42%, 89.06%, 93.45%, and 94.44% lower peak loads for the CFGT columns. This was mainly because the GFRP tubes could only affect the strength of the core concrete. Therefore, the larger the eccentricity, the worse the constraint effect, and the lower the peak load.The slenderness ratio and the eccentricity had significant influences on the ultimate axial strain and the hoop strain, with increases in the H/D ratio and the eccentricity causing decreases in the strain enhancement ratio.Based on the experimental results and the database from the literature, a modified equation was proposed to predict the bearing capacity of this type of CFGT column, and the predicted values were found to be in good agreement with the experimental results. The errors of 13 specimens were less than 5%, accounting for 39% of the total number of specimens. The errors of 23 specimens were not greater than 10%, accounting for 69% of the total. Only one specimen’s error was over 20%, and that error was 36.5%. Therefore, it could be considered that the test error was not greater than 20%, which proved the validity and applicability of the formula.The conclusions in this paper were based on experimental investigations of twelve specimens. Thus, more experiments need to be conducted to fully validate the behaviour of the CFGT columns under different loading conditions.


Lung cancer is a major health concern worldwide because of its increasing incidence and mortality. This study aimed to clarify the association between mesenchymal-epithelial transition (MET) genomic alterations and clinical characteristics of lung cancer.We collected data from 5,008 patients with lung cancer diagnosed and treated between January 2017 and July 2021 at the Affiliated Hospital of Qingdao University. Genomic alterations in the MET gene, including the exon 14 skipping mutation and amplification, were detected using amplification refractory mutation system-polymerase chain reaction  and next-generation sequencing . Clinical characteristics such as age, sex, tumor location, tumor stage, smoking, pleural invasion, and histology were statistically analyzed for MET exon 14 skipping mutation and amplification. The DNA splicing sites causing the MET exon 14 skipping mutation at the mRNA level were also investigated.The incidence of the MET exon 14 skipping mutation was 0.90%  in adenocarcinoma, 1.02% (3/294) in squamous cell carcinoma, and 8.33% (1/12) in sarcomatoid carcinoma specimens. It was more frequently observed in patients over 60 years of age than the MET exon 14 skipping mutation wildtype. The MET exon 14 skipping mutation co-occurred with epidermal growth factor receptor (EGFR) L858R, EGFR 19-Del, and BRAF V600E mutations. At the DNA level, single nucleotide mutation and small fragment deletion (1–38 base pairs) upstream and downstream of MET exon 14 led to MET exon 14 skipping mutation at the mRNA level. MET amplification occurred in 0.78%  adenocarcinoma and 1.07% (2/187) squamous cell carcinoma specimens and was significantly associated with advanced tumor stages (III + IV) compared to the MET amplification wildtype. MET amplification primarily co-occurred with the EGFR mutation.Our study found that MET genomic alterations were statistically related to age and tumor stage and co-existed with mutations of other oncogenic driver genes, such as EGFR and BRAF. Moreover, various splicing site changes at the DNA level led to the exon 14 skipping mutation at the mRNA level. Further studies are required to clarify the association between MET genomic alterations and prognosis. Lung cancer is the leading cause of tumor-related deaths worldwide. In China, the incidence and mortality of lung cancer have increased rapidly in recent years , 2. SeveMET is a tyrosine kinase receptor for the hepatocyte growth factor ligand. Aberrant activation of the MET signaling pathway is closely associated with the development of several solid tumors and can be caused by MET exon 14 skipping mutation, MET amplification, and overexpression of hepatocyte growth factor or MET , 4. Somain situ hybridization and NGS  and used blood samples for gene testing (339 cases) were excluded from the study. In total, 5,008 lung cancer cases were analyzed. For patients who developed more than one lung tumor, the more advanced tumor was selected for this study. All patients provided written informed consent, and the Ethics Committee of Qingdao University approved the study .Tissues were fixed with 4% paraformaldehyde, and genomic DNA was extracted using a Tiangen paraffin-embedded tissue DNA extraction kit  following the manufacturer’s instructions. The DNA quality was assessed using a Qubit dsDNA assay kit  according to the manufacturer’s instructions. Libraries were prepared using the human 10 gene mutation detection kit  and captured using a gene probe pool. NGS was conducted on an Illumina MiniSeq System  using a MiniSeq High Output Reagent Kit (Illumina) following the manufacturer’s instructions. Gene mutations were analyzed using Crest, ionCOPY, FreeBay, and Annoval.For MET mutation, the probe covers all exons and intron 13 of the MET gene and could detect the single nucleotide variations (SNVs) and indel mutations in this interval. MET mutation was determined when the sequencing depth was ≥500X and the mutation frequency was ≥1%. For MET amplification, the probe was separated into 22 segments, which cover all 21 exons and a part of intron 2. Sequencing data was calculated using the copy number (CN) value for each segment; CN ≥ 3.48 was defined as positive for each segment. When more than 90% of the segments are positive, it is interpreted as MET amplification.The quality of the NGS method was controlled in the following four ways: (1) FFPE samples prepared within 2 years and the proportion of tumor cells over 20% were used. (2) The total amount of DNA extracted should be over 200 ng, and the main band of the extracted DNA detected by gel electrophoresis should be above 500 bp. (3) After the hybridization library is purified, library concentration should be between 1 and 10 ng/μl and the average fragment size should be within 300–500 bp; and (4) the average sequencing depth of each sample should be ≥500X and the coverage over 95%.260/OD280 of the extracted RNA should be within 1.8–2.1, and the concentration of RNA should be 20–500 ng/μl.RNA was extracted from paraffin-embedded lung cancer tissues using an FFPE DNA/RNA extraction kit . The MET exon 14 skipping mutation was detected using the human lung cancer multi-gene mutation detection kit (fluorescence PCR method) , which includes a probe covering a sequence connecting exons 13 and 15 splicing genes and exons. ARMS-PCR was conducted on an ABI 7500 platform , and the running protocol was set up as follows: 5 min at 42°C and 5 min at 95°C; 25 s at 95°C, 20 s at 72°C for 10 cycles; 25 s at 93°C, 20 s at 72°C for 36 cycles; and maintained at 4°C. The FAM signal represents whether the MET gene in the sample RNA is amplified, and the HEX signal represents the MET gene expression. MET mutation was determined when the FAM signal Ct value ≤27 and △Ct value (FAM Ct - HEX Ct) ≤6. The quality control of ARMS-PCR method includes two parts: (1) FFPE samples within 2 years and the presence of tumor cells; and (2) the ODversus MET amplification wildtype; MET exon 14 skipping versus MET exon 14 skipping mutation wildtype) was evaluated using the standard Chi-square test. Data that were not qualified for the Chi-square test were merged into the groups to reach the standard. Statistical significance was set at P < 0.05.All data were analyzed using SPSS 26.0.0 statistical analysis software . The association between different groups , and there was no measurable significance between MET exon 14 skipping and MET exon 14 skipping wildtype for sex, tumor stage, tumor location, smoking, histology type, and pleural invasion.NGS and ARMS-PCR detected the MET exon 14 skipping mutation. We found 45 patients with the MET exon 14 skipping mutation among the 5,008 cases, and the mutation rate was 0.91% . Of these 45 cases, 27 were detected using ARMS-PCR, and 18 were tested with NGS. For different histological types, the incidence was 0.90%  in adenocarcinoma, 1.02% (3/294) in squamous cell carcinoma, and 8.33% (1/12) in sarcomatoid carcinomas. The association between the MET exon 14 skipping mutation and clinical characteristics is summarized in versus phase III + IV, P = 0.000). There was no measurable difference between MET amplification and MET amplification wildtype for age, sex, smoking history, histology type, and pleural invasion. Moreover, MET amplification exhibited a trend of higher incidence in the right lobe of the lung .We used NGS to detect MET amplification and found that among the 2,951 cases, 23 patients developed MET amplification, with an occurrence rate of 0.79% . For different histological types, the mutation rate was 0.78%  adenocarcinoma and 1.07% (2/187) squamous cell carcinoma. The relationship between MET amplification and clinical characteristics is shown in MET amplification is an important resistance mechanism of TKIs in EGFR-mutant NSCLC. However, in our study, we found 11 patients with primary MET amplification and EGFR mutation. Unfortunately, all patients underwent surgical resection and remained stable, making it impossible to track the therapeutic effect of TKIs on these patients in the short term. Usually, MET exon 14 skipping is mutually exclusive with other oncogenic driver genes, such as EGFR . HoweverWe cross-checked the MET exon 14 skipping patients using ARMS-PCR and NGS to analyze the DNA splicing sites that could cause the MET exon 14 skipping mutation at the mRNA level. Among the 45 cases of MET exon 14 skipping mutation, 7 patients refused to test again, DNA extracted from 3 samples were not qualified for NGS detection requirement, and 35 cases were cross-checked. The DNA splicing sites that could cause mRNA level MET exon 14 skipping and the clinical information of each case are summarized in MET activation is a primary oncogenic driver gene in lung cancer. MET dysregulation, including gene mutation, amplification, and rearrangement, might cause sustained MET activation and oncogenesis. Therapeutics targeting MET have been developed by several agents, including MET TKI, anti-MET, anti-hepatocyte growth factor antibodies, and anti-MET antibody-drug conjugates . Among tP = 0.001), and there were no significant differences in sex, tumor stage, tumor location, smoke, pleural invasion, and histology type. These findings are consistent with those of a previous study  and showed a trend of higher mutation rate in the right lobe of the lung (P = 0.058), which might be due to the anatomical difference of this lobe of the lung that is trilobar and the left lobe is bilobar  and downstream of exon 14, such as c.3028G>A , c.3028G>C , and c.3028G>T . Deletion also occurred upstream and downstream of MET exon 14, such as c.2888-13_2908del and c.3027_3028+9del. Our findings provide an interpretation basis for the MET exon 14 skipping mutation using NGS.In this study, we used DNA-based NGS and RNA-based ARMS-PCR methods to analyze MET exon 14 skipping mutation. A previous study has demonstrated that compared to qRT-PCR and Sanger sequencing, NGS method could be the first choice for multiplex detection due to its high sensitivity and large panel of detection . The conThe role of MET in carcinogenesis and cancer development has been extensively studied. The current challenges are identifying molecular subtypes and subgroups of patients that would benefit most from MET inhibitors or the combination of other genomic target agents. Further extensive studies and clinical trials must be conducted, and clinicians should closely follow up and observe the conditions of specific subsets of patients. We believe that in the next 5 years, as the widespread implementation of high-throughput sequencing and in-depth investigation of the role of MET in cancer occurs, more rare mutation sites will be discovered and more target drugs will be developed to treat patients with more detailed classifications.There were several limitations to the present study: (1) a single-center study; (2) lack of survival data of the patients; (3) no track of clinical treatment data such as chemotherapy and radiotherapy applications; and (4) no cross-comparison with other mutation genes such as EGFR, KRAS, and HER2.In summary, we collected and analyzed the genomic alteration of MET in 5,008 lung cancer cases and reported the accurate incidence of MET exon 14 skipping mutation and MET amplification and their correlations with clinicopathological features in patients from east China. We found that the MET exon 14 skipping mutation was significantly associated with older ages, whereas MET amplification showed a higher incidence in advanced tumor stage, providing a valuable reference for clinicians from east China to study this uncommon molecular marker. Moreover, we reported several DNA mutation sites that could lead to the exon 14 skipping mutation at the mRNA level to assist other genetic testing agencies in interpreting their MET genetic testing results.http://www.gov.cn/zhengce/content/2019-06/10/content_5398829.htm, http://english.www.gov.cn/policies/latest_releases/2019/06/10/content_281476708945462.htm), no additional raw data is available at this time. Data of this project can be accessed after an approval application to the China National Genebank . Please refer to https://db.cngb.org/, or email: CNGBdb@cngb.org for detailed application guidance. The accession code CNP0002208 should be included in the application.According to national legislation/guidelines, specifically the Administrative Regulations of the People’s Republic of China on Human Genetic Resources .YS, GL, and XX conceived and designed the study. XX contributed reagents, protocols, and materials. GL, WR, HZ, GZ, and YC conducted the experiments and analyzed the raw experimental data. YS provided a pathological diagnosis. YS, KJ, MH, YH, and XL collected and statistically analyzed the data. YS wrote the paper. XX modified the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Clinical Medicine+X Foundation of the Affiliated Hospital of Qingdao University (YS).Author GZ was employed by Geneis Beijing Co., Ltd.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.


A proportion of patients with immunogloblin G (IgG) 4-related disease (IgG4-RD) have hypocomplementemia. We aimed to identify characteristics of such patients.We analyzed the demographic and clinical data and complement levels of 85 patients with IgG4-RD. We defined hypocomplementemia as serum C3 and/or C4 levels below the lower limit of normal at diagnosis. We also compared the characteristics of patients with and without IgG4-RD.Thirty-two (38%) patients had hypocomplementemia at diagnosis. Patients with hypocomplementemia had more lymph node (p < 0.01), lung (p < 0.01), and kidney (p = 0.02) involvement and a higher IgG4-RD responder index than those without (p = 0.05). Additionally, patients with hypocomplementemia had significantly higher IgG (p < 0.01), IgG4 (p < 0.01), and soluble interleukin 2-receptor (sIL-2R) (p < 0.01) levels and total IgG minus IgG4 (p < 0.01). C3 and C4 levels negatively correlated with IgG, IgG4, and sIL-2R levels, total IgG minus IgG4, and number of IgG4-RD responder index: a measure of the disease activity in IgG4-RD. Patients with hypocomplementemia at diagnosis had a significantly higher frequency of relapse (p = 0.024), as determined using the log-rank test. A multivariate logistic regression analysis showed the presence of hypocomplementemia was independently associated with relapse .Patients with IgG4-RD with hypocomplementemia have a more active clinical phenotype, suggesting contributions of the complement system in the pathophysiology of IgG4-RD. Involved tissues exhibit IgG4-positive lymphoplasmacytic infiltrates and fibrosis on histopathology . IgG is phocytes .Hypocomplementemia, defined as having C3 or C4 complement levels below the lower limits of normal, is one of the serological features of IgG4-RD. More than one-third of Japanese patients with IgG4-RD have hypocomplementemia . In a smA total of 107 patients diagnosed with IgG4-RD between December 2008 and September 2021 were recruited from the Department of Immunology and Rheumatology of Nagasaki University Hospital, the Department of Rheumatology of Fukushima Medical University Hospital, and affiliated hospitals. Twenty-two IgG4-RD patients whose complement levels were not measured at diagnosis were excluded. All patients fulfilled the 2020 revised comprehensive diagnostic criteria for IgG4-RD . Disease activity was determined based on the IgG4-RD responder index (IgG4-RD RI) . We obtath–75th centiles . Spearman’s correlation coefficient was used to evaluate correlations. The chi-square test was used to compare categorical variables represented as frequencies. The relapse-free survival analysis was assessed using Kaplan–Meier analysis, with the significance based on the log-rank test.Data were analyzed with SPSS Statistics software version 22.0 . Results were nonnormally distributed and compared with the Mann–Whitney U test and are presented throughout the manuscript with median and 25We compared the characteristics of patients with IgG4-RD with hypocomplementemia (n = 32) to those without (n = 53). Categorical variables are described as frequencies, whereas quantitative variables are described as medians and IQR. Associations between the variables were assessed using Fisher’s exact test for categorical variables and Wilcoxon’s rank-sum test for quantitative variables. Relationships between variables were analyzed using Spearman’s rank correlation. The prognostic factors for relapse were identified using a stepwise multiple logistic regression model. All tests were two-sided, and p-values of <0.05 were considered significant.The study was approved by the Institutional Review Board of Nagasaki University Hospital (17091109), Fukushima Medical University (29317), and affiliated hospitals. Informed consent for data use was obtained from some of the patients, and an opt-out procedure was used for the rest.The demographic and clinical characteristics of all 85 patients with and without hypocomplementemia at diagnosis are summarized in Thirty-two patients (38%) were classified in the hypocomplementemia group. In the hypocomplementemia group, 22 patients (69%) had C3 and C4 reduction, 3 (9%) had only C3 reduction, and 7 (22%) had only C4 reduction.The median age at diagnosis was 66 years; 62 patients were men (73%). There were no significant differences in age at diagnosis, sex, body mass index, and history of allergy between the two groups.The hypocomplementemia group had significantly higher IgG  , lung , and kidney  involvement. Patients with hypocomplementemia also had a significantly higher IgG4-RD responder index  Table 1.The correlations between C3 levels, C4 levels, and other biological measures are presented in Kaplan–Meier survival analysis on disease relapse is shown in Hypocomplementemia was previously reported to occur concomitantly with IgG4-RD , 11. Thivia the classical pathway  . C5a recIgG4-RD is generally treated using corticosteroid, but the treatment other than corticosteroid has not been established. Recent report has shown that C5a receptor inhibitor is effective in ANCA-associated vasculitis . HoweverBased on these considerations, the hypothesis of pathophysiology in IgG4-RD is shown in Our study had some limitations. First, it was a retrospective study of a relatively small number of patients, thereby the number of explanatory variables in multivariable analysis may be limited. In addition, the other complement factor including C5a could not be measured because our sample were stored at -20°C. which is unstable condition for complement factors.Second, the proportion of patients with pancreas and kidney involvement was small; the patients had been referred from the Department of Rheumatology, thereby potentially leading to selection bias regarding the involved organs. Third, evaluating the extent of IgG4-RD only by the number of affected organs and IgG4-RD RI may be insufficient to assess disease activity because of the differentiation of radiological modality in each patient. The measurement of other biomarkers such as plasmablasts  and CC-cIn conclusion, our study revealed that patients with IgG4-RD with hypocomplementemia had more serious disease course than those with normal complement levels. Furthermore, the strong treatments may be considered for IgG4-RD patients with hypocomplementemia.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Institutional Review Board of Nagasaki University Hospital. The patients/participants provided their written informed consent to participate in this studyYF made substantial contributions to the study concept and design with the assistance of SF. YF conducted the statistical analyses and drafted the manuscript with the assistance and supervision of KM and AK. YF, MU, ST, NI, YN, YH, TS, AO, TA, YU, AM, TO, and HW treated the patients and collected the primary data. AK critically revised the manuscript, supervised the entire study, and gave final approval to the article. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Patients and Methods. Forty-five patients with advanced gastric cancer who underwent neoadjuvant chemotherapy and surgical treatment were enrolled in this study. All patients received neoadjuvant chemotherapy with paclitaxel + S-1 + oxaliplatin (PSOX) regimen, and CTCs and cfDNA of the peripheral blood were detected before and after neoadjuvant therapy. Relationships between the number/type of CTC or cfDNA and the efficacy of neoadjuvant chemotherapy were analyzed.  To explore the application value of circulating tumor cells (CTCs) and circulating free DNA (cfDNA) from peripheral blood in the prognosis of advanced gastric cancer (AGC). Here, we measured CTCs and cfDNA quantity for predicting the outcome of patients. P < 0.05), and the larger N stage will have the higher proportion of mesenchymal CTC. Patients with a small number of mesenchymal CTC before neoadjuvant chemotherapy were more likely to achieve partial response (PR) with neoadjuvant therapy. Patients with positive CA-199 were more likely to achieve PR with neoadjuvant therapy (P < 0.05). Patients in the PR group were more likely to have decreased/unchanged cfDNA concentration after neoadjuvant therapy (P=0.119). After neoadjuvant therapy (before surgery), the cfDNA concentration was higher and the efficacy of neoadjuvant therapy (SD or PD) was lower (P=0.045).  Among 45 patients, 43 (95.6%) were positive, and the positive rate of mesenchymal CTC was increased with the increase in the T stage. The proportion of mesenchymal CTC was positively correlated with the N stage ( Peripheral blood CTC, especially interstitial CTC and cfDNA, has a certain value in predicting the efficacy and prognosis of neoadjuvant chemotherapy in advanced gastric cancer. Gastric cancer (GC) is one of the most common malignant tumors of the digestive tract according to World Health Organization (WHO) data . WorldwiThe patients with advanced gastric cancer (AGC) who had undergone neoadjuvant chemotherapy and surgery in the Department of Gastrointestinal Surgery of the Affiliated Hospital of Qinghai University between September 2019 and October 2020 were enrolled in this study. Enrollment criteria were as follows: (1) a total of 45 cases were diagnosed with gastric cancer by endoscopy and tumor tissues by biopsy; (2) TNM staging of all patients was T3-4N × M0 ; (3) physical status score of eastern cooperative oncology group (ECOG) ≤2 points and could tolerate chemotherapy; (4) newly diagnosed patients with no previous radical or palliative surgery, radiotherapy, and chemotherapy history; (5) the functions of liver and kidney were in the normal range; and (6) age was between 18 and 80 years old. Exclusion criteria were as follows: (1) patients with pyloric obstruction, upper gastrointestinal hemorrhage, gastrointestinal perforation, severe infection, and other complications; (2) existed history of radical or palliative surgery, radiotherapy, and chemotherapy or biological therapy; (3) history of allergy to chemotherapy drugs; (4) pregnant or breastfeeding; (5) patients with distant metastases; and (6) other malignant tumors. In this study, 45 patients with advanced gastric cancer were enrolled, including 40 men, 5 women, and age of 29–69 years old with an average of 54.90 (±10.89) years old.All enrolled patients received 3 cycles of PSOX neoadjuvant chemotherapy. The peripheral blood for number and subtypes of CTC and cfDNA measurement were collected before (baseline) and after neoadjuvant therapy (postoperative) on the 10th day after surgery.2 and oxaliplatin of 85 mg/m2 were injected in the vein on day 1. Tiggio was orally taken based on the patient's body surface area (BSA) from day 1 to day 14, twice a day (2 tablets in the morning and 3 tablets in the evening). 21 days was defined as a chemotherapy cycle. The clinical efficacy and toxicity of neoadjuvant chemotherapy were judged after at least 2 cycles. If the disease progresses during chemotherapy, it will be evaluated after 1 cycle. All cases were confirmed efficacy after 4 weeks.PSOX regimen was as follows: paclitaxel of 135 mg/mA total of 10 milliliters (mL) of venous blood from patients before and after chemotherapy and surgery was collected and placed in an ethylenediaminetetraacetic acid (EDTA) anticoagulant tube as a test sample. The samples were centrifugated at 1,500 r/m for 5 minutes within 4 hours, and the plasma phase was removed. CanPatrol® CTC enrichment counting was used to further separate CTC. Furthermore, multiple RNA in situ hybridization technology was used to perform CTC typing detection, and the epithelial type-specific genes  and the mesenchymal-specific genes (vimentin and twist) were detected, respectively. The amplification probe was hybridized with the above-mentioned type-labeled probe labeled with a fluorescent group to generate a fluorescent signal, the fluorescent signal was read by an automatic identification system, and the CTC typing detection result was automatically judged through the fluorescent signal of different colors.The CTC results were analyzed. The epithelial CTC was displayed as red fluorescent signal points, and the mesenchymal type was displayed as green fluorescent signal points. The red and green signal points in one cell were displayed as a mixed type.μL). About 2 μL of the extracted nucleic acid was taken and added into the aliquoted working solution, shaken, and mixed well. The standard nucleic acid working solution was used to formulate the standard curve of the Qbit quantifier, and the fluorescence value of the standard curve was about 15,000, and then, the concentration of each nucleic acid was detected. The concentration of each sample was recorded.A total of 10 mL of peripheral venous blood was collected with an EDTA anticoagulation tube. KminTrak plasma extractor was used to extract plasma DNA. Qbit was used to determine the calculated concentration of cfDNA samples. Briefly, the Qbit quantifier reagent and the corresponding amount of DNA quantitative working solution were prepared according to the manufacturer's introduction. Qbit quantitative working solution was divided into QB tubes, and each tube contained 198 microliters : (1) complete response (CR): all lesions disappeared and were maintained for 4 weeks; (2) partial response: reduced by 30% in tumor size and were maintained for 4 weeks; (3) progressive disease (PD): 20% increase in tumor size; (4) non-CR/PR/stable disease (SD) before lesions increase; new lesions appear; and (5) SD: based on the minimum sum of the longest diameters after the start of treatment, the reduction was less than the standard for PR, and the increase was not to the standard of PD.t-test was used for comparison. Categorical data were expressed as examples (%), and the chi-square test was used for comparison. Spearman's correlation coefficient was used to analyze the correlation between CTC number change, cfDNA number change, and chemotherapy effect, and the Kaplan–Meier method was used for survival analysis. P < 0.05 indicated that the difference was statistically significant.All data were input into spss 20.0 software for statistical analysis. Continuous data were expressed as mean ± standard deviation (Mean ± SD), and an independent sample Basic information of 45 patients with AGC is shown in To investigate the CTC number and subtypes of 45 patients, we identified different CTC characteristics. The result is shown in N stage was larger; the ratio of mesenchymal CTCs was higher. It was consistent with the positive correlation between mesenchymal CTCs and disease stage and prognosis proposed in the current related literature. However, there was no significant correlation between other clinicopathological indicators and the number/type of CTCs.The clinical information of the enrolled patients with gastric cancer mainly included the following parameters: age, gender, pathological type, tumor location, tumor diameter, degree of differentiation, cTNM staging, T staging, N staging, Lauren type, Borrmann type, whether CA19-9 was normal or not, and so on. The CTC value before neoadjuvant chemotherapy was the baseline CTC. The relationship between CTC and various clinical indicators of gastric cancer was mainly to analyze the total number of CTCs, whether the total number of CTCs >7, the number of each type, and the relationship between the positive/proportion of mesenchymal CTCs and the above parameters. The results of baseline CTC testing are summarized in CTCs before neoadjuvant chemotherapy were used as the baseline. We compared CTC number and subtype changes after three cycles of neoadjuvant chemotherapy. The efficacy of neoadjuvant treatment was performed according to the RECIST1.1 evaluation standard. There were 27 patients  with neoadjuvant efficacy evaluation results, including 10 cases of PR, 16 cases of SD, and 1 case of PD. The results are indicated in P < 0.05). The level of CA19-9 was related to the efficacy of chemotherapy, and patients with positive CA19-9 were more likely to achieve PR with neoadjuvant therapy. Correlation between the end of neoadjuvant therapy (preoperative) CTC and neoadjuvant efficacy was seen that the number/type of mesenchymal CTC after neoadjuvant treatment was significantly related to the efficacy (As shown in efficacy .P=0.119). The cfDNA concentration at baseline (before neoadjuvant therapy) was not significantly correlated with CTCs and neoadjuvant efficacy; however, the relationship could be seen that the number/type of CTCs after neoadjuvant treatment was not significantly related to the efficacy.A total of 25 patients in this study were tested for CTCs before neoadjuvant and preoperative chemotherapy, and some patients were tested for cfDNA. Correlation between changes in the number/type of CTCs and changes in cfDNA concentration and neoadjuvant efficacy were analyzed, indicating that the changes in the total number of total CTCs and mesenchymal CTCs were similar in the PR and SD/PD groups, and there was no significant difference . As for Worldwide, cancer is the second cause of death affecting global residents after cardiovascular disease . Of no dWith the continuous development of molecular technology, liquid biopsy is widely used in the field of tumors. In 2013, it was used as an early cancer detection method , which hN staging of gastric cancer [N staging were significantly correlated (P=0.034) and positively correlated. The larger the N staging, the proportion of interstitial CTC was the higher. The higher N stage indicates that there is cancer cell infiltration in the lymph nodes or lymph vessels around the tumor, and these cancer cells are more likely to enter the peripheral blood system through the lymphatic circulation, which may be the reason why the higher the N stage, the easier it is to detect interstitial CTC in the peripheral blood. Therefore, the detection of interstitial CTC at the first diagnosis (before neoadjuvant therapy) may be another indicator for predicting N staging. In TNM staging, the T stage indicates the depth of primary tumor invasion. In the traditional TNM staging method, T staging is of great significance, but T staging reflects the local condition of the tumor, and CTC reflects the peripheral circulation. This may be the reason why T staging is not related to the number of CTCs, and it may also be related to the proportion of T stage in the enrolled patients.In this study, the total positive rate of CTCs (43/45) in this study was 95.6%, of which the positive rate of mesenchymal type (23/45) was 51.1%. In the study of patients with advanced gastric cancer, the CanPatrol® system monitored the detection rate of CTC capture in the peripheral blood of advanced gastric cancer >80%. The N staging in TNM staging indicates regional lymph node metastasis, but it is difficult to accurately evaluate the N stage before surgery. Generally, high-quality enhanced CT combined with invasive ultrasound gastroscopy is required for assessment, which increases the patient's radiation risk, economic burden, and physical pain. It has been reported that the number of mesenchymal CTC is closely related to the TNM staging and c cancer . This stP=0.086), but after grouping according to PR and SD/PD, the total number of interstitial CTC before and after neoadjuvant therapy in each group showed no significant difference. It can be seen that the total number of interstitial CTCs was related to the efficacy of neoadjuvant therapy, which was consistent with the conclusions of related studies. The less CTC before neoadjuvant therapy indicated the better effect of neoadjuvant chemotherapy. Therefore, interstitial CTC can be used as an index to predict the efficacy of chemotherapy. This result is consistent with the other reports [This study found that patients with a small number of intermediate CTC types before neoadjuvant therapy were more likely to achieve PR with neoadjuvant therapy. After analyzing the relationship between the number and classification of CTC before and after neoadjuvant chemotherapy and the efficacy of chemotherapy, the total number of CTCs before and after neoadjuvant therapy was changed, and there was no significant difference in CTC before and after neoadjuvant therapy in patients in the PR, SD/PD, PR, or SD/PD groups. Comparison of changes in the total number of interstitial CTC before and after neoadjuvant therapy is as follows: patients with high interstitial CTC were more likely to have SD/PD ( reports –43 and cP=0.045). Patients in the PR group were more likely to have a decreased/unchanged cfDNA concentration after neoadjuvant therapy (P=0.119). Chemotherapy acts on tumors with different cell cycles to prevent tumor cell replication and reduce tumor burden. PR after chemotherapy suggests a reduction in tumor burden in this group of patients through chemotherapy. So the cfDNA concentration of patients was decreased, cfDNA reflected the condition of circulating free DNA, and the concentration did not change, indicating that chemotherapy was effective. On the other hand, the cfDNA before neoadjuvant in the PR group was higher (P=0.073), and the cfDNA after neoadjuvant in the SD/PD group was higher. This conclusion was similar to CA-199 and interstitial CTC, and it also reflected from the side that cfDNA concentration before neoadjuvant therapy can predict sensitivity and efficacy of chemotherapy. Comparing the cfDNA concentration before and after neoadjuvant, cfDNA before and after neoadjuvant treatment in the PR group showed a downward trend, but there was no significant difference. The concentration of cfDNA after neoadjuvant in the SD/PD group was significantly increased (P=0.008), suggesting that the increase in cfDNA after chemotherapy reflected the poor efficacy of chemotherapy. In summary, the cfDNA concentration before neoadjuvant therapy can predict the efficacy of chemotherapy, and the higher cfDNA concentration before neoadjuvant therapy was relatively sensitive to chemotherapy and easy to achieve PR. If the cfDNA concentration remained elevated after neoadjuvant therapy, it indicated poor chemotherapy efficacy. It was worth noting that the total number of CTC in most patients showed a downward trend 10 days after surgery, but there were also some patients with PR who had an increase in the number of cfDNA after surgery. Follow-up of these patients should be strengthened because their risk of metastasis and recurrence may be higher.Normal cell apoptosis will produce cfDNA . The accCTC and cfDNA are safe and minimally invasive detection techniques compared to surgery and endoscopic biopsy. This study suggested that the level of mesenchymal CTC was positively correlated with tumor T staging and N staging, and patients with higher cfDNA concentration before neoadjuvant chemotherapy were easier to achieve PR, indicating that CTC and cfDNA had a certain value in evaluating the efficacy of neoadjuvant therapy for advanced gastric cancer. However, due to the small number of cases currently enrolled in this study, the follow-up has not yet been completed. After a large sample and follow-up study, it may better reflect the role of CTC and cfDNA in the perioperative treatment, recurrence risk assessment, and prediction of the recovery of advanced gastric cancer.


Cell-free synthetic biology is a maturing field that aims to assemble biomolecular reactions outside cells for compelling applications in drug discovery, metabolic engineering, biomanufacturing, diagnostics, and education. Cell-free systems have several key features. They circumvent mechanisms that have evolved to facilitate species survival, bypass limitations on molecular transport across the cell wall, enable high-yielding and rapid synthesis of proteins without creating recombinant cells, and provide high tolerance towards toxic substrates or products. Here, we analyze ~750 published patents and ~2000 peer-reviewed manuscripts in the field of cell-free systems. Three hallmarks emerged. First, we found that both patent filings and manuscript publications per year are significantly increasing . Second, we observed that the innovation landscape has changed. Patent applications were dominated by Japan in the early 2000s before shifting to China and the USA in recent years. Finally, we discovered an increasing prevalence of biotechnology companies using cell-free systems. Our analysis has broad implications on the future development of cell-free synthetic biology for commercial and industrial applications. Escherichia coli S30 extract to decipher the genetic code, which later earned them the 1968 Nobel prize in Physiology or Medicine [In 1961, Heinrich Matthaei and Marshall Nirenberg used Medicine ,5,6,7,8.Medicine , or lipoMedicine   PATENTSCOPE search engine was used . The croM2. Curation and categorization of patents and publications: Curation of the patents and publications included elimination of duplicates of documents that shared the same title. Then documents missing publication dates or authors were removed. After these steps, 5225 patents and 2637 publications remained. The patent data was further curated by restricting the patents to those filed by the following countries: World-wide (WO), United States (US), Japan (JP), Europe (EP), China (CN), and Canada (CA). Patents from these countries included 2098 patents. Publications were assigned countries based on the affiliation of the corresponding author. These publications were manually categorized into the following categories: Improve CFPS, Protein modification, Specific proteins, and Applications. In both the patent and publication search results, there were numerous documents that were returned that did not pertain to the technology discussed in this article. Numerous patents were removed as they referred to topics including cancer therapeutics and fuel cells. The eliminated publications included the use of a cell-free protein synthesis assay where cells were lysed and tested for inhibition of protein synthesis, mostly for the study of antibiotics. Review articles were also eliminated. This curation left 762 patents and 1723 publications that were relevant to the CFPS field. The applicants of the patents were also curated to group the applicants regardless of the variation of the name used. All the patents and publications used in this study, including all metadata and categorization, are available in the To investigate whether the recent interest in CFPS systems is backed by tangible progress, the total counts of patents and publications were calculated over the period of 1990 to 2020 a,b. In tInvestigating the types of advances being made across the patent and publication landscape is essential to understanding innovation trends in cell-free synthetic biology. To do this, we manually categorized all documents into subtopics: ‘Improved CFPS’, ‘Protein Modifications’, ‘Specific Proteins’, and ‘Applications’ (refer to Methods section M2). The ‘Improved CFPS’ category includes system-level improvements of CFPS . These improvements can include increasing accessibility to the technology with commercial CFPS kits, generation of extracts from non-model organisms, such as wheat-germ  and XenoIn the 1990s, nearly all the publications pertained to the synthesis of ‘specific proteins’ . These eOn the other hand, the patent space has seen less dramatic changes over time, excluding the patent spike of 2005, and potential recent rise. While very few patents were filed in the 1990s, most of the early patents were focused on improving CFPS. Looking closely, more than 40% of all patents filed during the 2001–2008 ‘patent spike’ pertained to ‘Improved CFPS’ , with a To better understand how innovation has changed over time, we categorized patent innovation into three groups based on affiliations of patent filers: Academia (university and government labs), Company, and Unaffiliated. Companies appear to have driven the ‘patent spike’ we see in the mid-2000s a. From 2We next sought to reveal whether a handful of entities dominated CFPS developments or if innovation was more distributed. To do this, we documented the number of patent filings per group over the 30-year period from 1990 to 2020. We found that more than half of all groups filed a single patent, with 95% of all groups filing less than 10 patents c. While The current growth of both commercial and academic patents correlates with the diversification of cell-free systems beyond the production of ‘specific proteins’ that dominated early commercial efforts. However, therapeutic protein production maintains significant commercial interest as researchers continue to optimize post-translational modifications, protein yields, and reaction scales of cell-free systems ,40. The To understand the distribution of effort in this field across the globe, each document was assigned a country. The documents were then aggregated based on location  to illusThe publication trends of the United States and Europe follow the global trend of a decrease in publications until about 2005 and then a continuous increase. Japan, however, showed the opposite trend where they begin with few publications and gradually built to 45 publications per year in 2010 before decreasing by an average of three publications per year. Chinese publications followed a similar trend as their patent filings. From 2018 to 2020, cell-free researchers in China nearly doubled all previous publications since 1990.Here, we analyzed the emergence of the cell-free synthetic biology field based on publication and patent frequency. The picture that emerges is that system development and applications are coming of age, with an increasing rate of research and potential. There are a few caveats in analyzing the progress and impact of a field using the patent and publication data. This study focuses on the total count of published patents and peer-reviewed manuscripts and their separation into various groups based on relevant metadata, such as their topic and authors. It does not investigate the long-term impact of these documents. Few highly influential advancements could impact the field far more than numerous others. We did not investigate the revenue generated by each patent, but our data suggest that various stakeholders were willing to invest the resources and effort necessary to file patents on the technology. It is also important to note that the observed changes in publications or patent filings are influenced by a variety of economic and societal factors that the current data set is unable to distinguish between, making the establishment of causal connections challenging.Despite these caveats, we can make several conclusions. The number of patents and publications have been steadily increasing in cell-free synthetic biology over the last decade . BetweenBased on these conclusions, we envision a few developments that could enhance investment and research interest in CFPS technology. Recent work has investigated the robust quality control of CFPS . The foc


The effect of the temperature used during guestuptake and the influence of changing the host framework ZnX2 nodes on guest encapsulation were investigated. Additionally, threecompounds containing chemical fragments similar to those of metalaxyl-Mand S-metolachlor  were also encapsulated.This allowed for the effect of guest size on the position that guestsoccupy within the host frameworks to be examined. The disorder experiencedby the guest compounds was documented, and an analysis of the intermolecularhost–guest interactions (CH···π and π···π) used for guest ordering within the hostframeworks was also undertaken in this study.Thecrystalline sponge method is a technique that provides theability to elucidate the absolute structure of noncrystalline or hardto crystallize compounds through single-crystal X-ray diffractionby removing the need to obtain crystals of the target compound. Inthis study the crystalline sponges {.x(solvent)}n  producing novel inclusion complexes is reported. Thetarget compounds included the first agrochemical fungicide activeingredient  to have their X-ray structure determinedby the crystalline sponge method. The influence of guest size, inclusiontemperature, and the change in the ZnX2 nodes on the positionsof the host pores the guests occupy were also analyzed.The inclusion of five target compounds into the crystallinesponges {[(ZnX The ability to determinethe absolute structure of chemical compoundsis essential for the progression of chemical research. Traditionalmethods of characterization such as nuclear magnetic resonance (NMR)spectroscopy, infrared spectroscopy, and mass spectrometry routinelyprovide the basic structure of a target compound. The determinationof the absolute stereochemical structure requires the use of a differenttechnique: e.g., single-crystal X-ray diffraction (SCXRD). The requirementfor the sample to be a single crystal is a fundamental limitationof the SCXRD technique; the formation of good-quality single crystalscan be a difficult and time-consuming process and is not always possiblefor all liquid, powder, and amorphous solid compounds.1 The technique,known as the crystalline sponge method (CSM), involves soaking presynthesizedcrystals of a metal–organic framework (MOF) in a solution ofthe target compound (referred to as the guest after encapsulationinto the MOF pores). Intermolecular interactions between the hostMOF and guest compound allow for the guest to become ordered withinthe pores of the host, resulting in the postcrystallization of theguest within the host framework.2 The MOFcrystal, with guest encapsulated, can then be analyzed by SCXRD toelucidate the structure of the guest molecule. Due care and attentionmust be paid to the quality of the crystallographic data obtained.This was highlighted by Fujita et al. in their attempts to assignthe absolute stereochemistry of miyakosyne A. After “ambiguitiesin the crystallographic data” were found, a correction hadto be published.3 The success of this techniquehas been demonstrated by the ability of the CSM to determine a varietyof structures such as ozonides,4 volatilearomatic isomers such as cis- and trans-asarone,5 druglike nucleophilic compounds,6 natural products,8 and metabolites.10 The CSM has also been used in several mechanistic studies by elucidatingthe structures of reaction intermediates. If the reaction intermediatesmatch those in a speculated reaction mechanism, they could be usedas confirmatory evidence.15 An example of this is the confirmation of the syn-addition mechanism for metal-free diboration.13A procedurethat had claimed to overcome this limitation was reportedin 2013 by Fujita et al.2)3(TPT)2].x(solvent)}n -1,3,5-trazine).16 The organic linker molecule, TPT, is highlyaromatic and electron deficient. This allows for the formation ofintermolecular π···π and CH···πinteractions between the host and electron-rich guest molecules.17 The intermolecular interactions formed allowfor the guest molecules to become regularly ordered within the MOFpores. The size of the guest molecules that can be encapsulated withinthe MOF is determined by its pore size; in this case 1 has a pore size of 8 × 5 Å2.18 Clardy et al. investigated different variants of MOF 1, {[(ZnX2)3(TPT)2].x(solvent)}n (X = Br (2), Cl (3)), for use in the CSM. These MOF variantsdisplayed a reduction in the relative scattering contribution fromthe host framework which allowed for more guest and solvent electrondensity peaks to be observed.19 It hasalso been noted in other studies that these MOF variants are towardnucleophilic compounds more robust than 1; this is dueto the electron-withdrawing nature of the Br and Cl atoms, which strengthensthe pyridyl–zinc bond.6 The strongerpyridyl–zinc bond reduces the ability of nucleophilic compoundsto exchange with the TPT linker molecule through bonding to the ZnX2 nodes.6The most widely used MOF for the CSM is {.x(solvent)}n . Theencapsulation of Metalaxyl-M, which to the best of our knowledge isthe first fungicide active ingredient to have its X-ray structuredetermined via the CSM, further establishes the potential of the CSMfor the characterization of noncrystalline or hard to crystallizecompounds in agrochemical research and product development. In particularthe CSM can be seen as a possible option for the structural elucidationof the metabolites of crop protection active ingredients.The work presented here highlights both the possibilities and thecurrent limitations of the crystalline sponge method. The X-ray structuresof four liquid guest molecules, including the agrochemical fungicideactive ingredient Metalaxyl-M, have been successfully determined byencapsulation into the pores of the host frameworks {[ and 2,4,6-tris(4-pyridyl)-1,3,5-triazine. This mixture was placed into a sonication bathfor 10 min to allow the 2,4,6-tris(4-pyridyl)-1,3,5-triazine to dissolve.After sonication the mixture was filtered through a glass pasturepipet plugged with cotton wool into another test tube. A 0.5 mL layerof MeOH was then carefully layered on top using a 1 mL glass syringecreating a clear interface between the two layers. Then 1 mL of a0.03 M of ZnBrTable S1 in the Supporting Information) beforegood-quality block or rod-shaped crystals were chosen for SCXRD analysis.A smallnumber of crystals was placed into a 14 mL screw-cappedvial. The chloroform storage solvent was carefully removed using aglass pipet immediately followed by the addition of 1 mL of neat guestinto the vial, submerging the MOF crystals. The vial was then sealedand placed in an incubator at a specific temperature and time  equipped with Cu Kα radiation (λ= 1.5418 Å) was used to perform the X-ray diffraction analysisat 150 K. The program CrysAlisPro


Antibiotic resistance is increasing among urinary pathogens, resulting in worse clinical and economic outcomes. We analysed factors associated with antibiotic-resistant bacteria (ARB) in patients hospitalized for urinary tract infection, using the comprehensive French national claims database.Hospitalized urinary tract infections were identified from 2015 to 2017. Cases (due to ARB) were matched to controls (without ARB) according to year, age, sex, infection, and bacterium. Healthcare-associated (HCAI) and community-acquired (CAI) infections were analysed separately; logistic regressions were stratified by sex.From 9460 cases identified, 6468 CAIs and 2855 HCAIs were matched with controls. Over a 12-months window, the risk increased when exposure occurred within the last 3 months. The following risk factors were identified: antibiotic exposure, with an OR reaching 3.6 [2.8–4.5] for men with CAI, mostly associated with broad-spectrum antibiotics; surgical procedure on urinary tract (OR 2.0 [1.5–2.6] for women with HCAI and 1.3 [1.1–1.6] for men with CAI); stay in intensive care unit > 7 days (OR 1.7 [1.2–2.6] for men with HCAI). Studied co-morbidities had no impact on ARB.This study points out the critical window of 3 months for antibiotic exposure, confirms the impact of broad-spectrum antibiotic consumption on ARB, and supports the importance of prevention during urological procedures, and long intensive care unit stays.The online version contains supplementary material available at 10.1186/s12879-021-06287-1. Urinary tract infections (UTIs) are one of the most common infections. In the United States, UTIs are estimated to account for 8.6 million healthcare visits annually with associated costs of 1.6 billion dollars in 2007  and 0,93 [0.88–0.98] , 34. ThaThis study confirms the importance of broad-spectrum antibiotic consumption on the risk of UTI with ARB in the last 3 months, and the necessity of controlling antibiotic prescription. It also highlights the importance of prevention during surgical procedure on the urinary tract, and prolonged ICU stays.Additional file 1 Includes all supplementary material. Supplement 1. Describes the database and patient selection. Table S1. Shows the ICD-10 codes used. Table S2. Describes the selected bacteria included and the associated resistance markers. Supplement S2. Describes the algorithms for identification of risk factors. Table S3. Shows the code lists used for the definition of risk factors. Table S4. Shows the conditional univariate logistic regressions of risk factors of having a community-acquired or healthcare-associated urinary tract infection caused by a resistant bacterium compared with a susceptible one, by sex. Table S5. Shows the number of antibiotic dispensing during the previous 3 months for each antibiotic class, by type of infection and sex. Table S6. Shows the characteristics of patients excluded and included for the analysis of association between antibiotic classes and resistant-bacterial acquisition, according to type of infection and sex.


The intensity ratio between the delayed fluorescence and phosphorescence was tuned by the guest species and concentration. Molecular dynamics simulations were used to simulate the molecular conformation of guest molecules in the host matrix and the interaction between the host and guest molecules. Therefore, the photophysical properties were calculated using the QM/MM model. This work provides a new concept for the study of molecular packing of guest molecules in the host matrix.Organic host–guest doped materials exhibiting the room temperature phosphorescence (RTP) phenomenon have attracted considerable attention. However, it is still challenging to investigate their corresponding luminescence mechanism, because for host–guest systems, it is very difficult to obtain single crystals compared to single-component or co-crystal component materials. Herein, we developed a series of organic doped materials with triphenylamine (TPA) as the host and TPA derivatives with different electron-donating groups as guests. The doped materials showed strong fluorescence, thermally activated delayed fluorescence ( Molecular dynamics simulations were used to simulate the molecular conformation and interaction between hosts and guests. This work provides a new concept for the study of molecular packing for the investigation of the luminescence mechanism. In general, the RTP properties of pure organic molecules are highly dependent on intermolecular interactions in the solid state. Strong intermolecular interactions can overcome the spin-forbidden transitions between the excited singlet and triplet states to realize an efficient intersystem crossing (ISC) process and can stabilize excited triplet states, promoting the persistent RTP effect.13–15 Various building blocks including carbazole, dibenzothiophene, and 9,9-dimethylxanthene have been successfully utilized as core units in the construction of single-component RTP luminogens.16–24 The inherent mechanism and the relationship between the molecular structure and packing performance can be easily investigated, since their crystal structures are easily obtained and molecular packing can be tuned via molecular design.Organic room temperature phosphorescent (RTP) materials with long afterglow emission have attracted much attention owing to their unique advantages including structural diversity, multiple functionality, and biocompatibility.25–31 The ease of the preparation process facilitates the ever expansive library of RTP materials and provides insights into the host–guest system. However, exploration of host–guest RTP systems for application as organic opto-electronic materials is still in its infancy due to the lack of a clear understanding of the luminescence mechanism. Wang et al. showed that the host matrix of the doped system only restricts the motion of guest molecules, thus decreasing the non-radiative decay and promoting radiation transition of triplet excitons.20 Adachi et al. found that the rigid host matrix can act as an oxygen barrier to avoid triplet quenching. Recent studies also demonstrated that the host matrix plays a role in energy synergy in phosphorescence emission.25–29 However, because of trace amounts of guest molecules (<1%) in the doped system, traditional characterization methods, such as single crystal X-ray diffraction, scanning electron microscopy (SEM), and transmission electron microscopy (TEM), are considered unsuitable for accurate investigation of the specific molecular conformation of the guest molecules in the host matrix. As a result, researchers have been unable to perform exact theoretical calculations of the corresponding photophysical properties of the guest molecules in the doped systems, limiting further development of host–guest RTP materials.Compared with single-component RTP materials, the host–guest doped system or co-crystal system can convert two non-phosphorescent components into excellent RTP materials.In this work, we provide an approach to investigate molecular packing in host–guest systems. A series of organic RTP materials with triphenylamine (TPA) bearing perfect symmetry and high crystallinity as the host and four triphenylamine derivatives  with different electron-donating groups as the guests  are deve32–35 Therefore, four TPA derivatives  with electron donating groups were designed and synthesized as the guests  of the four doped materials are all greater than 70%, which are much higher than the fluorescence intensities of the guests in tetrahydrofuran (THF) solvent , indicating the existence of the RISC of excitons,36–38 and then decreased with increasing temperature from −40 °C to 100 °C due to elevated thermal intramolecular vibrational motions   . The phory (DFT) .32–35 Asolecules .32–35 Moaterials , Table 1ecreases . This is39 Compared with the conformations of the host molecule TPA in a single crystal, the corresponding conformations of the host molecules adjacent to the MADBA molecule doped in the MADBA/TPA system are different, and the twist angles show an obvious increase after doping (marked in red, †40 (ESI Fig. S741 BMK,42 CAM-B3LYP,43 M06-2X44 and ωB97XD,45 were chosen to perform benchmark tests and select the most appropriate functional to deal with the current system. The B3LYP functional shows the optimal performance. Here the doped MADBA molecule was selected as the QM region and calculated at the B3LYP/6-31G** level, whereas the other TPA molecules were treated as the MM region by the universal force field.46 During QM/MM geometry optimization, the QM region was fixed and the MM region was allowed to move. The results show that after doping the MADBA molecule into the TPA host matrix, the structural changes of MADBA from S0 to T1 in the doped state are obviously smaller than those in the solution state, as shown in Fig. S9, Tables S1 and S2.47,48 were performed using the Multiwfn program.49,50 As shown in 1 and low-lying triplet states also support the easier ISC process of the doped system than that in the pure MADBA crystal. This is due to the smaller energy gaps and larger SOCs in the doped system than in the MADBA crystal, see 0 and T1 of the doped system is 0.55 cm−1, and the corresponding value of the MADBA crystal is only 0.22 cm−1. The corresponding SOC between S1 and T1 for the doped system, 0.29 cm−1, reduces to only 0.08 cm−1 in the MADBA system. Therefore, doping trace amounts of MADBA is beneficial to promote ISC of excitons, thereby leading to phosphorescence emission in the host matrix.The analysis of the single crystal structure of materials by using an X-ray diffractometer is currently the available method to observe organic compounds at the molecular level. The high molar ratio of guest to host molecules in doped materials prevents obtainment of single crystals that contain both host and guest molecules. Therefore, although researchers have developed a variety of doped systems, the specific configuration and molecular packing of the guest molecules in the host matrix are still unknown. In order to address the inherent issues in this research field, further theoretical calculations were performed to simulate the doped configurations and spatial molecular packing of the guest molecule in the host matrix using MD simulations. MADBA/TPA with the best RTP performance was selected. The initial MADBA/TPA model was set up based on the TPA crystal. As shown in In this work, a series of organic doped materials with TPA as the host and TPA derivatives with different electron-donating groups as the guests were developed. The doped materials showed strong fluorescence, TADF, and RTP emission. The intensity ratio between TADF and RTP could be adjusted by either the guest species or concentration. Moreover, molecular dynamics simulations were employed to simulate the molecular conformation and spatial location of guest molecules in the host matrix. To the best of our knowledge, this is the first use of molecular dynamics simulations to study doped materials. The photophysical properties of the guest molecules were calculated using the QM/MM model. This work provides a new concept for studying the specific molecular packing of guest molecules in the host matrix by molecular dynamics simulations.Y. D., Z. C. and X. Z. designed the research work and revised the manuscript. Y. L. synthesized the materials and wrote the manuscript. J. Y. carried out density functional theory calculations and wrote the manuscript. Y. L., W. D., J. Y. and Y. L. carried out photophysical property measurements. J. S. and B. T. edited the manuscript. All authors discussed the results and commented on the manuscript.There are no conflicts to declare.SC-012-D1SC01175H-s001