To evaluate the combined efficacy of multiparametric ultrasonography (mpUS) and multiparametric magnetic resonance imaging/transrectal ultrasound (mpMRI-TRUS) fusion for detecting clinically significant prostate cancer (csPCa).From November 2019 to September 2021, biopsy-naïve patients underwent mpMRI-TRUS fusion imaging combined with mpUS-guided targeted biopsies (TB) and systematic biopsies (SB). To further evaluate the additional diagnostic value of mpUS, the imaging features of 202 focus obtained from fusion imaging were assessed. The diagnostic accuracies of mpMRI-TRUS fusion imaging and the combination of mpMRI-TRUS fusion imaging with mpUS for csPCa were comparatively evaluated.p < 0.05). The imaging characteristics of mpUS in the PCa and non-PCa groups were significantly different (p < 0.001). When compared with mpMRI-TRUS fusion imaging, the positive predictive value, false positive rate, and area under the curve (AUC) of csPCa diagnosis by mpMRI-TRUS fusion imaging combined with mpUS increased by 11.30%, decreased by 19.58%, and increased from 0.719 to 0.770 (p < 0.05), respectively.A total of 202 prostate lesions (160 patients) were included in the final analysis, of which 105 were csPCa, 16 were ciPCa, and 81 were noncancerous. The median patient age was 69 (65–73) years and the median tPSA was 22.07 (11.22–62.80) ng/mL. For csPCa, the detection rate of TB was higher than that of SB (50.0% vs. 45.5%, TB can improve the detection rate of csPCa and hence can be effectively used in the diagnosis and risk assessment of csPCa. The mpUS-enriched valuable diagnostic information for mpMRI-TRUS fusion imaging and their combination showed a higher diagnostic value for csPCa, which can guide subsequent clinical treatment. The cancer-associated death of prostate cancer (PCa) was ranked the fifth highest among men in 2020 worldwide. In more than half the countries globally, PCa is the most common type of cancer . SeveralCompared with PCa, csPCa is more closely related to mortality, and hence increasing csPCa detection rates is the key to reducing mortality. At the same time, reducing clinically insignificant PCa (ciPCa) detection rates is important for improving patients’ quality of life. Hence, accurately identifying csPCa and providing more effective disease-related information to the clinics is crucial.New ultrasound technologies such as CEUS and TRES have a higher PCa detection rate than conventional TRUS. These new techniques are immensely helpful in diagnosing and treating PCa . PreviouBetween November 2019 and September 2020, biopsy naive men with PSA > 4 ng/mL underwent mpMRI a month before biopsies. All patients provided their signed informed consent. The exclusion criteria included contraindications for MpMRI and mpUS examination and biopsies.. The images were read jointly by 2 radiologists with 10 and 3 years of work experience, respectively. If the results provided by the two scholars were inconsistent, the result submitted by the scholar with greater work experience was used as the final result. The PCa location, size, prostate imaging reporting, Data System Version 2.1 (PI-RADS) score, and PCa invasiveness were described and documented for each patient. Patients with PI-RADS scores of 4 or 5 were considered positive [mpMRI consists of high-resolution T2-weighted imaging (T2WI), diffusion-weighted MR imaging (DWI), and dynamic contrast-enhanced MRI (DCE-MRI). A GE Signa HDxt 3.0 T superconducting MR scanner and an 8-channel phased-array coil were used. The parameters used in the mpMRI protocol are summarized in Table 1positive Table 1PampMRI Digital Imaging and Communications in Medicine (DICOM)-formatted images were uploaded in the UroNav  fusion system in Logiq GE20, which transmits the motion and orientation of the transrectal probe (IC5-9) to the fusion system through a triangulation registration method between the magnetic field generator and the electromagnetic tracking sensor on a probe. The motion of the ultrasound images presented by the probe and MRI images were synchronized to accurately guide the TB.The maximum cross-sectional CEUS and RTE imaging of the target area was performed in real-time and evaluated by the deputy chief physician and above (with more than 8 years of work experience) before the biopsy. CEUS was then performed at a low mechanical index (0.12). SonoVue  and a 5-mL saline flush were intravenously injected subsequently. Continuous recording was performed for 180 s and then switched to the TRES mode followed by putting adequate pressurize on the prostate. When the square green pressure barcode in the upper left was close to full, it indicated that the technique was exact, after which the above images were saved and the lesions were evaluated., and none. The presence or absence of diffuse enhancement was evaluated  , and wheent area . Perfusient area –14.TB was performed using a Bard-MAGNUM  automatic biopsy gun and a disposable 18-gauge biopsy needle with a needle length of 20 cm. The target area was defined as follows: 1) for mpMRI or mpUS positive: if the target areas of mpMRI and mpUS were consistent, this area was selected for puncture. If the two target areas were inconsistent, they were punctured separately; 2) if both mpMRI and mpUS were negative, both visible nodules were selected for TB. One or two cores were performed per targeted area and then the fusion system was withdrawn for a sagittal 12-needle SB. Biopsy specimens were individually placed in 10% formalin solution and labeled accordingly. We selected the highest GS score of radical prostatectomy or needle biopsies as the final pathological diagnosis. We defined PCa with GS ≥ 3 + 4 as csPCa and GS = 6 as ciPCa [p < 0.05 was considered to indicate statistical significance.SPSS 20.0 software was used for statistical analysis. Enumeration data were represented by the number of cases (constituent ratio) and measurement data were represented by means ± standard deviation or median (interquartile range). McNemar Chi-square test was used to compare the csPCa detection rate between TB and SB. The remaining categorical variables were compared by using the Chi-square test. The diagnostic efficacy of mpMRI-TRUS fusion imaging, mpUS, and their combination was assessed by receiver operating characteristic (ROC) curve analysis. The test level α = 0.05 and A total of 193 patients agreed to participate in the study, of whom, 33 were not included in the analysis due to puncture failure (13) and imaging failure 20). A total of 160 patients (202 lesions) were finally included in the study, and their clinical data are shown in Table 0. A tota. Radical prostatectomy was performed in 20 patients, which included 2 cases with a GS score of 6, 15 cases with a GS score of 7, and 3 cases with a GS score of ≥ 8.There were 105 csPCa, 16 ciPCa, and 81 non-PCa among the 202 lesions. Their pathological results are shown in Table 3p < 0.05). Among these, TB detected 12 prostate cancers missed by SB, of which 10 were csPCa. SB detected 12 prostate cancers overlooked by TB, of which 9 were ciPCa. TB was upgraded to csPCa in 2 patients diagnosed with ciPCa by SB  B-mode: hypoechoic (61.16%), isoechoic (33.06%), irregular edges (38.02%), aspect ratio ≥ 1 (61.98%), signs of capsule bulge (71.07%), and an asymmetric blood flow distribution (56.20%).2) CEUS: fast-forward enhancement (23.14%) or fast forward and backward enhancement(41.32%). It showed hyperperfusion (45.45%), hypoperfusion/nonperfusion (18.18%), and diffuse enhancement (66.12%).3) TRES: measured as focal non-strain or asymmetric strain 72.73%) and measured as symmetric heterogeneous strain 17.36%)  B-mode: isoechoic (72.84%), hypoechoic (18.52%), regular edge (90.12%), aspect ratio < 1 (56.79%), no signs of capsule bulge (74.07%), and an symmetrical blood flow distribution (76.54%).2) CEUS: no fast forward or backward enhancement (62.96%), fast forward and backward (28.40%), normal perfusion (69.14%), and hyperperfusion (25.93%).3) TRES: homogeneous or symmetric heterogeneous strain (49.39%) as well as asymmetric strain 50.62%) (Table 0.62% (TaThe 81 non-PCa mpUS imaging features showed the following:p < 0.001).The proportions of the abovementioned characteristics in PCa patients were higher than those in the non-PCa subjects . When mpMRI-TRUS fusion imaging was used in combination with mpUS to diagnose csPCa, the sensitivity was 71.43%, specificity was 82.47%, positive predictive value was 81.50%, negative predictive value was 72.70%, and AUC was 0.770 . Please refer to Table According to the 2020 guidelines of the European Association of Urology, mpMRI should be used as a routine application before prostate biopsy . MeanwhiOver-diagnosis of PCa is a common problem in the medical profession. One of the reasons is that SB based on abnormal PSA and DRE has the disadvantage of over-detecting ciPCa, missing the diagnosis of csPCa, and underestimating the invasion of cancer foci . To avoip < 0.001), which is similar to the observations reported by Shinohara, Sano, et al. [The Cochrane database of systematic reviews suggests that the MRI pathway is superior to SB in making a precise diagnosis of csPCa, albeit it still misses some csPCa. Therefore, it is imperative to conduct further research to resolve this concern . To impr, et al. , 21. Wit, et al. . The inw, et al. , which m, et al. . When co, et al. . BPH tisIn this study, we performed TB of 31 lesions that were reported negative by MRI but positive by mpUS imaging features, with pathological findings of 13 csPCa and 18 non-PCa. The additional use of mpUS in the targeted region increased the detection of csPCa by 12.4% (13/105). The additional use of mpUS examinations in the targeted area improved the detection of csPCa and provided more efficient diagnostic information for fusion imaging.To the best of our knowledge, there are only a few studies on the combined application of MpMRI-TRUS fusion imaging and mpUS. Maxeiner et al. investigate whether mpUS can further characterize mpMRI-suspected lesions through fusion and found that mpUS are strong predictors of PCa detection and PI-RADS 5 prediction; therefore, mpUS can be used as an additional tool as well as to reassure the PI-RADS score . Howeverp < 0.05).Our results showed that, when compared with mpMRI-TRUS fusion imaging alone, the combined diagnosis of csPCa increased the positive predictive value by 11.30% and reduced the false positive rate by 19.58%. When mpMRI-TRUS fusion imaging was used alone, the false positive rate was 37.11% (36/97). The additional use of mpUS in mpMRI-TRUS fusion imaging can reduce the false positive rate of diagnosing csPCa to 17.53% (17/97), indicating that if mpMRI-TRUS is negative and mpUS does not show suspicious signs, the probability of excluding non-csPCa is 82.47%. Therefore, when compared with mpMRI-TRUS fusion imaging alone, the combined method can more effectively diagnose csPCa (AUC 0.719–0.770, A single imaging method demonstrates limited value in diagnosing cancers. It needs to be combined to maximize the diagnosis of csPCa, which can give patients the most accurate disease information and further guide the clinical treatment. mpUS can be expected to detect plausible characteristics of PCa and provide additional information for patients that can be helpful for their treatment. In the future, further research is required to determine the role of fusion imaging in these patients and imaging at the molecular imaging level to determine which patients may benefit the most from this diagnostic procedure.The disadvantages of this study are as follows: First, only 20 patients underwent radical prostatectomy and for the remaining 140 patients, we used puncture results as the gold standard. All patients involved underwent biopsies for the first time. Therefore, if the patient’s biopsy result was negative, radical prostatectomy was not performed, leading to the missed diagnosis of low-grade and some advanced PCa to some extent. However, radical prostatectomy is not always prescribed for every patient owing to more complications and elaborate surgical criteria. Second, the high dependence of the PI-RADS distribution on the prevalence of csPCa is the reason for the lack of consensus on PI-RADS category 3 or 4 as positive. In European biopsy-naïve patients, the percentage of PI-RADS 3 potentially indicates the “certainty” of diagnosis and thus that of image quality and reading. However, according to our results, compared with PI-RADS category 3, PI-RADS category 4 produced a better result. Another study reported that the best cutoff for differentiating benign lesions from PCa was PI-RADS category 3. The best cutoff for differentiating low-risk PCa from csPCa was PI-RADS category 4. Therefore, for csPCa evaluation, we used PI-RADS category 4 or more as positive .However, unlike some previous studies, we performed pre-biopsy MRI for patients without contraindications to avoid selection bias instead of biopsy based on MRI risk assessment. Moreover, we not only examined mpMRI-TRUS fusion imaging but also combined it with mpUS. As a result, the imaging features of the fusion region were further characterized, which was deemed valuable for the accurate localization of PCa and the subsequent puncture process. By combining different techniques, the clinical detection limitations of each imaging modality can be balanced. In the future, we believe that this imaging modality can become a valuable tool for the clinical diagnosis of prostate cancer and further guide clinical decision-making.We found that TB can be used to improve the detection rate of csPCa, and thus, effectively used in the diagnosis and risk assessment of csPCa. The mpUS characteristics of the target areas can provide valuable diagnostic information for mpMRI-TRUS fusion imaging, which can be helpful for the further visualization of PCa foci. Eventually, we believe that the combination of mpUS with mpMRI-TRUS fusion imaging possesses a higher diagnostic value for csPCa and can provide valuable guidance for subsequent clinical treatment.


The melt-free radical grafting of glycidyl methacrylate (GMA) onto poly (lactic acid) (PLA) with styrene (St), α-methylstyrene (AMS), and epoxy resin (EP) as comonomers in a twin-screw extruder was used to prepare PLA-g-GMA graft copolymers. The prepared graft copolymers were then used as compatibilizers to prepare PLA/PPC/PLA-g-GMA blends by melt blending with PLA and polypropylene carbonate (PPC), respectively. The effects of different comonomers in the PLA-g-GMA graft copolymers on the thermal, rheological, optical, and mechanical properties and microstructure of the blends were studied. It was found that the grafting degree of PLA-g-GMA graft copolymers was increased to varying degrees after the introduction of comonomers in the PLA-g-GMA grafting reaction system. When St was used as the comonomer, the grafting degree of the PLA-g-GMA graft copolymer increased most significantly, from 0.8 to 1.6 phr. St as a comonomer also most improved the compatibility between PLA and PPC, and the haze of the blends was reduced while maintaining high transmittance. In addition, the PLA-g-GMA graft copolymer with the introduction of St as a comonomer significantly improved the impact toughness of the blends, while the thermal stability and tensile strength of the blends remained largely unchanged. Polylactic acid (PLA) is a biodegradable and biocompatible thermoplastic with a high modulus, high strength, high transparency, and excellent processing properties ,2,3, whig) , such asg) ,8,9, polg) ,11, polyg) ,13 and pg) . PPC is e effect ,16. PPC e effect ,18, so Pe effect . HoweverReactive compatibilization is a simple and effective method to improve the interfacial interaction between the two phases of blends. The reaction group contained in the reactive compatibilizer can generate block or graft copolymer between different polymer interfaces in the process of blending. This block or graft copolymer is similar to coupling agent or emulsifier, which can reduce the interfacial tension between the two phases of blending. Due to the decrease in interfacial tension, the molecular chains in the two phases can penetrate each other through the interface, which increased the thickness of the interface layer and the interfacial adhesion, thus greatly improving the physical properties of blend material. This technique has been shown to provide a better dispersion and more uniform phase morphology by chemical reaction during melt blending . Graft pBased on the above analysis, we selected a PLA-g-GMA graft copolymer as the reactive compatibilizer for the blending system of PLA and PPC in this work. On the one hand, PLA-g-GMA graft copolymer contained easily reactive functional groups that can react with PLA/PPC blends, which was particularly suitable for compatibilization between PLA and PPC with poor compatibility. Compared with non-reactive compatibilizers, PLA-g-GMA graft copolymer has the advantages of less dosage, low cost and good compatibilization effect. Therefore, it plays an important role in PLA blending research. We prepared PLA graft copolymers by the melt grafting of GMA onto PLA with the addition of St, AMS, and EP as comonomers. The prepared graft copolymers were then melt blended with PLA and PPC to prepare PLA/PPC/PLA-g-GMA blends. The effects of the different comonomers in the PLA-g-GMA graft copolymers on the thermal, rheological, optical, and mechanical properties of the blends were investigated, and the microscopic morphology of the blends was also studied.3 and a melt index of 7 g/min is supplied by Nature Works, USA, as 4032D. Polypropylene carbonate (PPC) is a solid particle, with a density of 1.28 g/cm3 and a melt index of 8 g/min, which is supplied by Boda Orient New Chemical Company, China, as BD-211. Glycidyl methacrylate (GMA)  is a liquid with content ≥ 99%, which is provided by Dow Chemicals, USA. Dicumyl peroxide (DCP)  is granular, with a purity of 99% and a melting point of 41.3 °C, which is provided by Chengdu Best Reagent Co., Ltd., Chengdu, China. Styrene (St) , a colorless and transparent liquid with 99% content and 0.909 g/mL density, is supplied by Tianjin Damao Chemical Reagent Plant, Tianjin, China. α- Methyl styrene (AMS) , a colorless transparent liquid, with a content of 99% and a density of 0.911 g/cm3, is provided by Sinopharm Chemical Reagent Company, Shanghai, China. Epoxy resin (EP) is a colorless viscous liquid with a viscosity of 14,000 and effective substance ≥ 99%, which is provided as E51 by the Shanghai Resin Factory Company, Shanghai, China.Polylactic acid (PLA) in pellet form with a density of 1.24 g/cmFor the functionalization of PLA, DCP was used as the initiator. The DCP was dissolved in a mixed solution of GMA and acetone, to which was added the appropriate amount of St, AMS, and EP, respectively. The mixtures were then mixed well with PLA at a certain mass ratio. After the acetone had completely volatilized, PLA-g-GMA and other graft copolymers were prepared using a twin-screw extruder. The specific composition of the graft copolymers is shown in About 2 g of each PLA-g-GMA graft copolymer was weighed, heated and refluxed in 25 mL of trichloromethane for 1 h, then precipitated and washed, and filtered in acetone three times to remove oligomers and unreacted monomers. The purified samples were dried under a vacuum at 70 °C for 12 h.d) of GMA onto PLA [d) of the graft copolymer was determined using Equation (1).0 is the volume of NaOH-ethanol standardized solution required to neutralize pure PLA (mL), V is the volume of NaOH-ethanol standardized solution required to neutralize the graft copolymer (mL), N is the molar concentration of the NaOH-ethanol standard solution, 142.15 is the molecular weight of GMA, and W is the weight of each graft copolymer sample (g). Grafting efficiency (Ge) was the ratio of the mass of GMA involved in grafting to the total mass of GMA added.Chemical titration was used to determine the grafting degree  analysis: The purified and dried PLA-g-GMA graft copolymers were molded into a film on a plate vulcanizer  and analyzed by FTIR. FTIR spectra were recorded on a NEXUS 470 PC. The scanning range of infrared test was 7400~350 cm1H-NMR) analysis: Weigh about 15 mg of PLA and PLA-g-GMA graft copolymer. Deuterated chloroform (CDCl3) and tetramethylsilane (TMS) were used as solvent and the internal standard, respectively. Bruker Ascend 500 ⅠⅠⅠ magnetic resonance analyzer was used for testing, scanning 128 times.Nuclear magnetic resonance analysis  analysis: Differential scanning calorimetry analysis  was performed by weighing approximately 5 mg of the PLA/PPC/PLA-g-GMA blends sample into a crucible. The temperature was raised from 0 to 200 °C at a rate of 10 °C/min and kept constant for 5 min to eliminate thermal history, which was further lowered to 20 °C at the same rate. The gas flow was 2.5 L/min.Thermogravimetric (TGA) analysis: Thermogravimetric analysis  was performed under flowing nitrogen (100 mL/min) at a heating rate of 10 °C/min. Approximately 15 mg of PLA/PPC/PLA-g-GMA blends were heated from room temperature to 500 °C.Rheological analysis: Rheological measurements of the blends were on a Dynamic mechanics analyzer . Frequency sweep for the PLA/PPC/PLA-g-GMA blends was under nitrogen using 25 mm plate geometry. The gap distance between the parallel plates was 0.8 mm. The sheet blends were about 1.0 mm in thickness. The angular frequency range used during testing was 0.1–100 rad/s with a shear strain of 1.0%. The temperature was plotted at 180 °C.Melt Flow Rate (MFR) analysis: The MFR was determined by a melt flow rate instrument  at 190 °C with a load of 2.16 kg (test standard was ASTM D1238-2013), MFR is calculated according to Equation (2):Optical property: The PLA/PPC/PLA-g-GMA blends were pressed into films of approximately 80 μm thickness using a flat vulcanizer and tested for haze and transmittance using a CS-700 haze meter .Mechanical property: According to GB/T1843/1-A, Izod notch impact strength was determined with a GT-7045-MD impact tester , the pendulum used was 2.750 J. The tensile properties were measured according to GB/T1040-1BA using a tensile testing machine  at a crosshead speed of 25 mm/min. The test was performed at room temperature, and the average values of at least five tests were reported.Scanning electron microscopy (SEM): The fracture surface of the PLA/PPC/PLA-g-GMA blends were sputter-coated with gold and then observed using a scanning electron microscope .–1 in all spectra was the characteristic absorption peak of carbonyl C=O in the PLA backbone. Comparing the FTIR spectra of PLA and the PLA-g-GMA graft copolymers, it can be seen that the spectra of all samples were approximately the same  were found in the spectra of any of the graft copolymers, indicating that the unreacted monomers were completely removed during the purification process.the same a, differtructure b. In theture . The new multipeak confirmed that GMA was successfully grafted onto the PLA  and grafting efficiency (Ge) of PLA-g-GMA graft copolymers containing different comonomers. As can be seen, the grafting degree of the PLA-g-GMA graft copolymers all increased to different degrees after the introduction of the different comonomers in the grafting reaction system, increasing 2 times, 1.6 times, and 1.1 times when St, AMS, and EP, respectively, were used as the copolymers. This was mainly due to the competition between grafting and chain scission (β-scission) of the PLA macro radicals in a single monomer grafting system. The PLA macro radicals underwent more extensive β-scission due to the ‘‘bite’’ reaction with the terminal hydroxyl (–OH), or carboxyl, group (–COOH), resulting in a low grafting degree of PLA-g-GMA graft copolymers. However, in the dual-monomer grafting system, the carbon–carbon double bonds (C=C) in St were more reactive than those in GMA. When St was added as a comonomer in the melt grafting system, the conjugated double bond of St enabled it to react rapidly with PLA macromolecular radicals to form stable styryl macromolecular radicals. GMA copolymerizes easily with styryl macromolecular radicals, and the reaction rate of this reaction was greater than that of the reaction with GMA with PLA macromolecular radicals. Similarly, AMS can form AMS-co-GMA (PAG) oligomers with GMA, which then react with PLA macromolecular free radicals to graft GMA onto the main chain of PLA through PAG, which improves the grafting degree of the PLA-g-GMA graft copolymer. It is well known that the copolymerization of two monomers is determined by the reactivity ratio of the monomers. The Q-e scheme has been developed to determine the ratios for each pair of monomers in a particular copolymerization [1 and Q2 are measures of the reactivity of monomers and related to the resonance stabilization of the monomer, and the constants e1 and e2 are measures of the polarity of the monomers. According to the Q-e scheme, copolymerization will proceed more suitably between monomers with similar Q values. The Q values of GMA, St, and AMS are 0.98, 1.00, and 0.53, respectively. Therefore, GMA was more likely to copolymerize with styrene macromolecular radicals, which was the reason for the significantly higher GMA grafting degree when St was used as a comonomer [rization :(3)r1=Q1omonomer ,40. Whenomonomer . The rea2, respectively. After PPC and PLA are blended, the elongation at break and impact strength of PLA/PPC blends are improved to a certain extent. In order to further improve the performance of the blends, PLA-g-GMA graft copolymer is introduced as a reactive compatibilizer in the blending process. From 2, respectively. Previous studies have found that the mechanical properties of such materials are influenced by interfacial strength [It can be seen from strength . When PLstrength , which istrength . In addiIn conclusion, the mechanical properties of the PLA/PPC/PLA-g-GMA blends were best when the PLA-g-GMA graft copolymer was 10 phr of the blends. Therefore, in subsequent experiments, the content of graft copolymer was kept as 10 phr.g) of the PLA and PPC phases, respectively. As g decreased from 22.98 to 22.05 °C, which indicated that the compatibility between PLA and PPC was improved, but the ΔTg only decreased by 0.93 °C. The ΔTg of the blends was further reduced when the copolymer monomer was introduced into the PLA-g-GMA graft copolymer. When St was used as the copolymer monomer, ΔTg decreased from 22.98 °C to 20.42 °C; when AMS was used as the copolymer monomer, ΔTg decreased from 22.98 °C to 20.96 °C, a decrease of 2.56 and 2.02 °C, respectively. When EP was used as the comonomer, the ΔTg decreased to 21.05 °C, a decrease of only 1.93 °C. This phenomenon is due to the grafting degree of the PLA-g-GMA graft copolymer, as shown in m) and crystallinity (Xc) of the blends gradually decreased after the graft copolymer-containing comonomers were introduced into the blends. This was because the reaction point worked as a physical cross-linking point, and the entangled structures in the amorphous area impeded the transportation of macromolecular chains between polymer chains, restricting the ability of PLA to crystallize and form a more amorphous phase, so the crystallinity of the blends was reduced [m of the blends [ reduced . In addie blends .5% of the blends decreased by 6.75 °C, and the thermal stability also decreased. This was probably because the preparation of the PLA-g-GMA graft copolymers was accompanied by degradation, cross-linking, and other side reactions, so the thermal stability of the blends was reduced to some extent after the graft copolymers were added to the blends. After the introduction of PLA-g-GMA graft copolymers containing different comonomer into the blends, the thermal stability of the blends did not further decrease but instead increased slightly. When St was used as a comonomer, the T5% of the blends increased by 5.68 to 326.53 °C. This was because the conjugated double bond of St can rapidly react with PLA macromolecular free radicals, and the St radicals were more stable and had a longer half-life, which can better subsequent reactions with GMA, thus inhibiting PLA chain degradation, cross-linking, and other side reactions during the grafting reaction. When AMS was used as a comonomer, the T5% of the blends increased by just 2.17 to 323.02 °C. Although AMS improved the grafting degree of PLA-g-GMA to some extent and increased the intermolecular forces between PLA and PPC, there are weak bonds in the AMS sequence, and the copolymers containing the AMS structure were prone to chain breakage, and under heating the blends depolymerized at their weak bonds and formed chain radicals, which could further initiate other chemical reactions [5%. When EP was used as a comonomer, the T5% of the blends was 320.38 °C, which was close to that when no comonomer was introduced, which may be caused by the small effect of EP as a comonomer on the grafting degree of PLA-g-GMA graft copolymer. It can also be seen from max of the blends almost does not change significantly, which indicated that the PLA-g-GMA graft copolymer did not cause the blends to degrade significantly at a lower temperature.eactions ,45, resu–1), and the higher the grafting degree of the PLA-g-GMA graft copolymer, the larger the G′ and G″ of the blends. The significant increase in G′ indicated that the elasticity of the blend melt was improved, resulting in enhanced interaction between PLA and PPC, and, thus, the compatibility of the blend was improved. In addition, after the introduction of the graft copolymers into the blends, the molecular weight of the blends became higher, and the friction resistance between the molecular chain segments gradually increased. Since the internal friction hinders the movement of the molecular chain, G″ increased.As As Han plots, which consist of the energy storage modulus as the ordinate and the loss modulus as the abscissa, can be used to compare the differences between elastic and viscous properties, with linear viscoelastic data being the most sensitive to structural differences. The diagonal line (G′ = G″) divides the graph into two parts and defines the transition from viscous behavior (G′ < G″) to elastic behavior (G′ > G″). The Han plots of all blends are shown in MFR analysis was used to determine the melt flow rate of the PLA/PPC/PLA-g-GMA blends. The effect of different comonomers in the PLA-g-GMA graft copolymer on the MFR of the blends is shown in As seen in 2, respectively, while the elongation at break of PPC was as high as 761.32%. It can be seen from 2. The PLA-g-GMA graft copolymer improved the toughness of the blends to a degree, which was due to a small rise in the compatibility between PLA and PPC. After the introduction of the comonomers in the PLA-g-GMA graft copolymers, the elongation at break and impact strength of the blends first increased and then decreased. When St was used as the comonomer, the elongation at break and impact strength of the blends increased the most significantly , followed by AMS , while the mechanical properties of the blends when EP was used as the comonomer were close to those when no comonomer was introduced . This was because St can best enhance the grafting degree of PLA-g-GMA graft copolymer, and the higher the grafting degree, the more epoxy groups that can react with PLA or PPC end groups, and the better the interfacial compatibility between PLA and PPC. With the improvement of the compatibility of the blends, the physical entanglement of the molecular chains of the blends increased, the intermolecular forces were enhanced, and the interfacial tension was reduced. At the same time, the stress can be effectively transferred from the PLA phase to the PPC phase, and the toughness of the blends was improved. When AMS was used in place of St as the comonomer, its larger substituent size led to a larger spatial site resistance effect, so the reactivity with PLA macromolecular radicals was reduced, and the degree of improvement of the grafting degree of the PLA-g-GMA graft copolymer was reduced, resulting in the decrease in the degree of improvement of the elongation at break and impact strength of the blends. When EP was the comonomer, it did not promote the reaction between GMA and PLA but instead reacted directly with PLA to form PLA-g-EP graft copolymer, and the PLA-g-EP graft copolymer did not improve the compatibility between PLA and PPC, so it had little effect on the impact toughness of the blends. In addition, it can also be seen from To investigate the effect of different comonomers in PLA-g-GMA graft copolymers on the fracture morphology of the PLA/PPC/PLA-g-GMA blends, the impact cross-section of the blends was observed by SEM, as shown in The addition of St, AMS, and EP as comonomer to the melt-grafting system of GMA onto PLA was to prepare PLA-g-GMA graft copolymers containing different comonomers. The prepared graft copolymers were melt blended with PLA and PPC to prepare PLA/PPC/PLA-g-GMA blends. The thermal, optical, and mechanical properties and micromorphology of these blends were extensively studied. The main conclusions can be summarized as follows:1H-NMR analysis showed that GMA, St, AMS, and EP had been successfully grafted onto the PLA molecular chains to form PLA-g-GMA, PLA-g-GMA/St, PLA-g-GMA/AMS, and PLA-g-GMA/EP graft copolymers. After the introduction of the comonomer in the grafting reaction, the grafting degree of PLA-g-GMA graft copolymer was improved to varying degrees, and when St was used as the comonomer, the grafting degree was increased most significantly.FTIR and The introduction of PLA-g-GMA graft copolymers improved the toughness of PLA/PPC blends to the degree that depended on the graft copolymer content, with the elongation at break and impact strength of the blends reaching a maximum when the graft copolymer was present at 10 phr.The PLA-g-GMA graft copolymers containing comonomers can further improve the compatibility between PLA and PPC; when St was used as the comonomer, the improvement effect was most obvious. At this time, the storage modulus, loss modulus, and complex viscosity of the blends were maximized without any negative effect on the thermal stability of the blends.2, respectively) when St was used as a comonomer. At this time, the impact fracture mode of the blends showed obvious ductile fracture characteristics.When the PLA-g-GMA graft copolymer contained comonomers, the blends film reduced the haze while maintaining high transmittance, reflecting good optical properties. Meanwhile, the elongation at break and impact strength of the blends were further improved, while the tensile strength remained almost unchanged. The elongation at break and impact strength of the blends reached the peak value (286.35% and 6858.6 J/m


Early diagnosing clinically significant prostate cancer (csPCa) through Magnetic Resonance Imaging (MRI) is very challenging and, nowadays, csPCa confirmation comes exclusively from prostate biopsy. However, biopsy is an invasive procedure and it also frequently causes csPCa misclassification. This study develops a non-invasive machine learning method for early predicting csPCa based on radiomic MRI image analysis. The main novelty of this study is investigating the radiomic differences between progressive risk groups of PCa, attributed according to the biopsy outcome and Gleason Grade group stratification. Besides predicting csPCa with very good performance (sensitivity = specificity = 84% in the test phase), this study highlights patients with GG = 2 as non-csPCa , so that GG = 2 can be counselled for follow up, whilst GG ≥ 3 admitted to radical treatments. Not least, this study provides a plausible clinical interpretation of radiomic features, by discussing their values with respect to the histological meaning.p < 0.001), to achieve only four features. After data augmentation, the features were exploited to train a support vector machine classifier, subsequently validated on a test set. To assess the results, Kruskal–Wallis and Wilcoxon rank-sum tests (p < 0.001) and receiver operating characteristic (ROC)-related metrics were used. GG1 and GG2 were equivalent (p = 0.26), whilst clear separations between either GG and GG ≥ 3 exist  by multiparametric magnetic resonance imaging is consistent, also using the updated PIRADS score and although different definitions of csPCa, patients with Gleason Grade group (GG) ≥ 3 have a significantly worse prognosis. This study aims to develop a machine learning model predicting csPCa  by mpMRI radiomic features and analyzing similarities between GG groups. One hundred and two patients with 117 PIRADS ≥ 3 lesions at mpMRI underwent target+systematic biopsy, providing histologic diagnosis of PCa, 61 GG < 3 and 56 GG ≥ 3. Features were generated locally from an apparent diffusion coefficient and selected, using the LASSO method and Wilcoxon rank-sum test ( In the last decade, the strategy used to diagnose prostate cancer (PCa) in patients with a clinical suspicion of PCa has radically changed from systematic biopsy to the routine use of multiparametric magnetic resonance imaging (mpMRI) in the first instance. The so-called “MRI-pathway”  has dramTo note, discerning csPCa from benign processes or non-csPCa is crucial since patients’ management may vary dramatically. Different definitions are reported to identify csPCa . HoweverRecently, radiomics has emerged as the application of well-established machine learning (ML) and artificial intelligence techniques to medical image analysis ,10,11. TAccordingly, the main goal of our research is to develop a ML model to predict csPCa, namely, GG ≥ 3. In particular, this study sets up an image processing procedure based on extracting local radiomic features, enabling the visual assessment of colorimetric maps on the whole prostate gland, through which similarities and differences between GG1, GG2 and GG ≥ 3 are analyzed. This allows the prediction of high-risk diseases even before any biopsy examination.This study was observational, retrospective and single-centered. It was approved by our local Institutional Review Board (IRB), which waived the need for informed consent, and it was conducted in accordance with institutional guidelines, including the Declaration of Helsinki (Ethics Committee code: 784/2021/Oss/AOUBo). Two hundred and twenty patients with at least one PIRADS ≥ 3 lesion at mpMRI, according to the European Society of Urogenital Radiology (ESUR) guidelines v2.1, performed at our Radiology Unit from September 2020 to December 2021, were enrolled. The inclusion criteria were the following: (1) having undergone fusion-TB of the index lesion at our Radiology Unit and (2) having a diagnosis of PCa at a histopathological report of TB from a dedicated pathologist of the Pathology Unit of our Institution. The exclusion criteria were the following: (1) mpMRI protocol not completely adhering to the suggested imaging protocols described in the ESUR guidelines; (2) previous prostatic surgery or hormone therapy; (3) the presence of severe artifacts not allowing the evaluation of one or more sequences of the mpMRI due to uni/bilateral hip prostheses or other causes. Finally, 102 patients are analyzed, and the flowchart of patient enrolment is reported in The mpMRI examinations were carried out using a 1.5 T scanner  and a pelvic phased-array surface coil combined with a disposable endorectal coil. Although the use of the 3 T scanner is recommended by many authors, the ESUR guidelines also specify that an optimization of the acquisition parameters and adequate technical requirements provide adequate and reliable diagnostic examinations even with the 1.5 T scanner. Therefore, the mpMRI study of the prostate gland and seminal vesicles is optimized according to the ESUR guidelines and includs Fast Relaxation Fast Spin Echo (FR-FSE) T2w, DWI and Dynamic Contrast-Enhanced (DCE) sequences; the scan parameters were described in a previous study . SupplemAll PIRADS ≥ 3 lesions underwent target biopsy performed transrectally by two experienced radiologists using the mpMRI-TRUS fusion image guide after antibiotic prophylaxis and a cleansing rectal enema using a non-disposable biopsy gun  with a disposable 18-gauge needle and a US platform  with an end-fire TRUS probe as previously described . The biohttps://imagej.nih.gov/ij, accessed in 13 August 2020), a Java-based public-domain software  mmp < 10s (d) and Starting from 132 features, LASSO detected a preliminary subset of 17 features. Finally, 88 4-dim feature vectors’ results were significant  is reported, whilst clear separations between either GG = 1 or GG = 2 and GG = 3 exist , with No significant difference between GG = 1 and GG = 2  , whilst Our model shows very high performance in predicting GG ≥ 3, employing only four features. Furthermore, the signature highlights a strong similarity between GG1 and GG2 and significant differences between GG =  and GG ≥ 3. These findings seem to not support the current tendency to assume intermediate/high-risk PCa as belonging to the same class as csPCa . Indeed,From a methodological point of view, our radiomic features yield parametric colormaps corresponding to ADC slices. This helps clinicians, in a multidisciplinary team, in feature interpretation and broader comprehension of tissue properties. In this regard, the radiomic signature highlights different properties of GG ≥ 3 compared to GG1 and GG2 lesions. First of all, Furthermore, the capability to implement an integration of conventional mpMRI evaluation with radiomic features by machine learning models could improve the discrimination between csPCa and non-csPCa, besides the re-assessment of mpMRI imaging by experienced radiologists. Moreover, the proposed parametric maps from ADC provide clinicians (primarily urologists and radiologists) with a useful tool that allows PCa lesions to be characterized even beyond the visible lesion boundaries. Besides that, additional advantages can be derived from multivariate hybrid models, including clinical parameters too, for csPCa patient stratification. The pre-biopsy evaluation of radiomic features by machine learning methods can help clinicians spare patients unnecessary biopsies, especially in borderline cases. For instance, patients with PIRADS 3 lesions can be jointly submitted to clinical and machine learning-based evaluation so that those with a high risk of csPCa by machine learning should be referred to target biopsy, whilst those with a low risk of csPCa could be counseled for follow up. At the same time, for those patients for which biopsy is required; this approach could even pave the way to radiomic-driven biopsy. Besides that, investigating the spatial correlation between the parametric feature maps and the digital scans of prostate histological samples can unveil cluster properties on the samples. In conclusion, with a wider population, mpMRI radiomic features can be integrated with clinic parameters and multi-modality features, including Prostate-Specific Membrane Antigen Positron Emission Tomography (PSMA-PET), to increase the precision of diagnosis of aggressive PCa needing to be treated.Despite several strengths, our study is not devoid of limitations. First, the number of patients included in the study is limited; this possibly bounds the model’s generalizability, although that number allowed us to perform out-of-sample model validation. In addition, patients undergo mpMRI and MRI-target fusion biopsy in a single experienced center, thus increasing the homogeneity of the study population. Second, a ML model relying on bioptic samples may affect the accuracy in discriminating pathologic GG from GG3 due to possible significant discrepancies between the clinic and pathologic GG. A further step is validating the model relying on pathologic specimens of patients who underwent radical prostatectomy.


Several needleless techniques have been developed to outcome the inherent disadvantages of the traditional needle stitching technique for graft preparation, such as tendon damage through the needle, time consumption, and the potential risk of needlestick injury. The purpose of the present study is to compare the graft preparation time and the biomechanical performance between an efficient needleless technique and the traditional needle stitching technique for graft preparation in anterior cruciate ligament reconstruction (ACLR).t-test was used to compare the means of the two groups.The time required to perform a complete suture on 20 hamstring tendons during ACLRs was measured. The grafts from one side were prepared using the needle stitching technique. The grafts from the other side used the needleless grasping suture technique. For the second part of the study, 12 fresh-frozen porcine flexor tendons were divided into two groups using two techniques and were mounted in an electric tensile test system. Each group was pretensioned to 100 N to simulate the maximum initial graft tension. The suturing state of sutures and graft (intact and damaged) and the load-elongation curve were recorded for each group. A Student’s p < 0.05) than the needle stitching technique group . The state of sutures in each group was intact. The mean elongation was 11.75 ± 1.38 (range: 9.47–12.99) mm and 10.59 ± 1.02 (range: 9.12–11.76) mm in the needleless stitching technique group and the needle grasping suture technique group, respectively. There was no statistically significant difference in the elongation between the two groups (p > 0.05).In operation, the needleless grasping suture technique group  was significantly faster (The needleless grasping suture technique was a convenient and efficient method for graft preparation in ACLR. Anterior cruciate ligament (ACL) rupture is one of the most common injuries in sports medicine, especially in young and athletic populations. The estimated number of ACL injuries per year in the United States has increased to over 200,000 annually, and the number of ACL reconstruction (ACLR) performed has reached 120,000 per year . Among tTraditionally, several suturing techniques , 6 have The previous study  showed tThe clinical study was approved by the Ethics Committee (Approve number: L2021100). The patients were treated with arthroscopic ACLR. The inclusion criteria of the recruited patients were (i) they should have suffered from acute trauma within the previous 6 weeks, (ii) should have had symptoms of knee instability and other clinical evidence of ACL insufficiency verified by positive Lachman tests, (iii) MRI scans should reveal no other ligament injury except for ACL rupture, with or without accompanied meniscus injury, (iv) patients should be in good health, (v) patients should not have undergone previous ipsilateral knee joint surgery, and (vi) patients should not have been treated with medications known to affect bone metabolism. During ACLR, the time for the graft preparation of 20 HTs using the needleless grasping and the needle stitching technique (Whipstitch)  was recopost hoc power analysis with 83% power , 12 fresh-frozen porcine flexor tendons were collected from a local abattoir when the pigs were slaughtered (Approve number: 202101260). The porcine flexor tendons divided into two groups were cut to the same length (10 cm) for mechanical testing . The significance level (α) was set at 0.05. A post hoc power analysis  was conducted with G*power software 3.1.9  according to the results of the graft preparation time and the elongation of grafts.All data were normally distributed and expressed as mean ± standard deviation. A Student’s post hoc power analyses indicated a calculated 83% power to detect the effects on the graft preparation time and the elongation of grafts.The p < 0.05) (The mean graft preparation time was 19.8 ± 4.4 s (range: 13.5–32.9 s) and 52.7 ± 12.7 s (range: 36.0–87.5 s) in the needleless grasping suture technique group and the needle stitching technique group, respectively. Therefore, the needleless grasping suture technique groups were faster than the needle stitching technique groups ( < 0.05) .p > 0.05) (The suturing state of the sutures and grafts in each group was intact. The mean elongation was 11.8 ± 1.4 mm (range: 9.5–13.0mm) and 10.6 ± 1.0 mm (range: 9.1–11.8 mm) in the needleless stitching and the needle grasping suture technique, respectively. There was no statistically significant difference in the elongation between the two groups under the loading ( > 0.05) .The principal finding of the present study was that the time consumed for the graft preparation of the newly introduced needleless technique was significantly shorter than that of Whipstitch, but with similar elongation within 100 N. The results of the present study might suggest that the presented needleless technique might be a reliable alternative without the inherent disadvantages of the traditional needle stitching technique for graft preparation, such as tendon damage through the needle, time consumption, and the potential risk of needlestick injury.in vitro biomechanical test compared several methods  and lack of financial support, the SpeedTrapTM technique was not included in this study for difference comparison.Our study has several limitations. First, only one kind of needle stitching technique and suture material was used in our study, including real operation and mechanical tests. Second, we adopted the whipstitch and chose the braided absorbable suture with the needle  according to our routine clinical practice. Third, due to the expensive equipment (SpeedTrapIn conclusion, the needleless grasping suture technique was a convenient and efficient method for graft preparation following ACLR without expensive equipment and complex teaching operations. The newly introduced needleless grasping suture technique demonstrated a shorter graft preparation time but pretensioning of the ligament compared with the needle whipstitch technique, suggesting that the presented needleless technique might be a reliable alternative.


In March 2019, we launched Neurological Research and Practice (NRP), the official English language, open access, online journal of the German Society of Neurology (Deutsche Gesellschaft für Neurologie) . In our I'm proud to say that we have achieved several significant milestones. First, NRP has now published more than 150 peer-reviewed articles. As submissions have increased, our acceptance rate has dropped from around 60% to below 40%. While most articles originate from Germany, about 30% have first authors and affiliations from outside the country. This number has also increased over the years.Second, last year NRP was listed in PubMed®, a major accomplishment for a journal less than three years after its first publication. PubMed® was largely responsible for the remarkable increase in full article downloads Fig.  and inteThird, the citation of articles is still one of the most prominent aspects of the impact and visibility of a journal. For NRP, the citation rates are quite good, especially for a new journal. Table One of the next milestones we are looking forward to is NRP becoming indexed in the Emerging Sources Citation Index produced by Clarivate Analytics. Becoming indexed would further improve the journal's visibility and contribute to its Impact Factor. After just three years in operation, this would be a major achievement. Let's keep our fingers crossed that I will be able to report on that next year.https://neurolrespract.biomedcentral.com). The section editors, international editorial board members, and many other colleagues have supported NRP as authors and reviewers. Our single, blinded peer review protocol requires at least two and sometimes more reviews. As we all know, it's getting increasingly difficult to find qualified reviewers who commit to the swift delivery of high-quality reviews, especially for a new journal. Unfortunately, Data Protection Regulations prevent us from listing the reviewers who supported the Journal and express our gratefulness for their work. Some of the quick and reliable reviewers have been invited frequently and contributed a lot. The list is led by 5 colleagues, who contributed more than 15 Reviews. These are Christos Krogias [At the beginning of 2022, after 3 years of me being the sole editor of the Journal, we appointed section editors for different scientific areas (you can find a list of section editors on the website starting page.  Krogias . Uta Mey Krogias , and JulAll in all, we have had a very rewarding first 3 years of the journal. Once we receive an Impact Factor, we can declare that NRP is out of "early childhood" and about to enter the next phase of its development.Thank you to all who supported this endeavor and helped with a successful takeoff.


The impact of konjac glucomannan (KGM) with different molecular weight (Mw) on the retrogradation properties of pea starch, such as color, viscoelasticity, gel strength, water holding capacity (WHC), moisture distribution and crystallinity, was investigated. At the same time as the Mw of KGM decreased, the lightness, elastic modulus, gel strength, water freedom and crystallinity of pea starch showed an increasing trend, whereas the viscosity modulus and WHC showed a decreasing trend. At one day of storage, compared with single pea starch, KGM with low Mw made gel strength increase from 40 g to 45 g, WHC decrease from 82% to 65% and crystallinity increase from 21.3% to 24.0%. Therefore, KGM with low Mw could promote retrogradation of pea starch in the short-term. At 7 days or even 14 days of storage, KGM with medium-high Mw had smaller indices than those of pure pea starch, including the lightness, storage modulus, gel strength, water freedom and crystallinity. This indicated that KGM with medium-high Mw could inhibit the long-term retrogradation of starch. The larger the Mw of KGM, the more noticeable the inhibition effect. Pea starch (PS) is widely available and inexpensive. Compared with cereal starch and potato starch, its amylose content is higher, accounting for about 30–65% of the total starch . Pea staThe retrogradation of starch is defined as the recombination of starch chains (amylose and amylopectin) in the gelatinized paste, giving rise to the formation of a more ordered structure . RegulatKonjac glucomannan (KGM) is a kind of neutral polysaccharide extracted from the tubers of konjac. It is mainly composed of D-glucose and D-mannose, which are linked by β- glycosidic bonds in a molar ratio of 1:1.6 ,8,9,10. Moreover, the molecular weight (Mw) of the polysaccharide also has an influence on the aging properties of starch. For the same polysaccharide, the larger the Mw, the more effective it was at restraining starch aging, such as wheat bran arabinoxylan , water-eHowever, there are few reports concerning the influence of KGM with various Mw on the retrogradation properties of pea starch. Therefore, the aim of this work was to explore the impact of KGM with different Mw at the fixed mixing ratio on the retrogradation properties of pea starch using a colorimeter, a dynamic rheometer, a textural analyzer, water holding capacity (WHC), low-field nuclear magnetic resonance (LF-NMR) and an X-ray diffractometer (XRD).6 Da, 1.0 × 106 Da and 3.9 × 105 Da, which were labeled as HKGM, MKGM and LKGM, respectively. In order to explore the effects of the Mw of a polysaccharide on the retrogradation properties of starch, three different Mw of KGM were prepared using hydrothermal degradation. Their Mw were 1.5 × 10At one day of storage, the L value of pea starch blended with LKGM was significantly higher than the others. However, as the storage time was extended to 7 days or even 14 days, the L values of PS-LKGM were almost equal to those of pure pea starch. It was speculated that LKGM could promote regeneration of pea starch in the short-term but had no effect on starch regeneration in the long-term.For the other two Mw of KGM, the L values of HKGM and MKGM were lower than that of single pea starch throughout the storage period. Especially in the late stages, it was evident that the larger the Mw of KGM, the smaller the L value. Accordingly, it was supposed that KGM with medium and high Mw could inhibit the long-term retrogradation of starch.The influence of frequency on the storage modulus G′ and the loss modulus G″ of PS- KGM mixtures at different times is depicted in At the same time, the order of G′ from all samples was PS-LKGM > PS > PS-MKGM > PS-HKGM. Similar phenomena were reported that at the same angular frequency starch gels with HWBAX (wheat bran arabinoxylan with a larger Mw and branching degree) revealed the smaller G′ compared to starch paste containing the same amount of LWBAX (wheat bran arabinoxylan lower Mw and branching degree) . MoreoveThere is a good correlation between the hardness of food and the aging of starch. As time persists, the hardness of starch-based food becomes greater, and the sensory quality of food decreases, which is attributed to the aging of starch. Therefore, the gel strength was measured to reflect the hardness of gel and starch aging . The effStarch retrogradation is accompanied by dehydration, resulting in the degradation of food sensory quality. Therefore, WHC can be adopted to reflect the regeneration degree of starch indirectly. If the WHC of the sample increased, the retrogradation degree of starch declined. The effect of various Mw of KGM on the WHC of pea starch with time is presented in In addition, it was worth noting that the WHC of PS-LKGM was lower than that of pure pea starch during the entire storage period. At one day of storage, LKGM caused the WHC of pea starch decline to 65%. KGM with small Mw not only was not conducive to the water retention of the samples but also accelerated water loss and promoted regeneration to a certain extent.2) can represent the different molecular mobility as well as the combination degree between the sample and moisture. A lower T2 indicates the closer binding of water molecules in the samples [2 values of PS-KGM mixtures at different storage times. The T2 value of each sample decreased over time. It was known that the retrogradation of starch required the participation of water. During the rearrangement of starch, a part of the water became increasingly stable in the system, which caused the decline of T2 values [Low-field nuclear magnetic resonance (LF-NMR) is a fast and non-destructive detection method, which is used to analyze the motion of water molecule in food systems. The spin–spin relaxation time  was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. . Food-grade konjac glucomannan (KGM) was obtained from Hubei Konson Konjac Gum Co., Ltd. .6 Da, 1.0 × 106 Da and 3.9 × 105 Da, marked as HKGM, MKGM and LKGM, respectively.KGM with different molecular weight (Mw) were obtained according to the method of Chen, with some modification . Native The mixtures of PS-KGM were prepared as follows: The above konjac flour was dispersed in distilled water and mechanically stirred to form a sol with a mass fraction of 1%. Afterwards, pea starch, KGM sol and water were blended evenly. Within the mixture, the mass fractions of pea starch and konjac flour were fixed at 5% and 0.5%, respectively ,33. AfteThe lightness (L) value of the sample was measured using a colorimeter . Prior to determination, the colorimeter was calibrated by a white plate.Dynamic rheological experiments were performed using a controlled-stress Kinexus 2500 rheometer . The experimental parameters were set as follows: the geometry was a cone-plate (CP4/40), the frequency range was 0.1–20 Hz, the strain value was 1% and the temperature was 25 °C. Indicators, such as storage modulus G′ and loss modulus G″, were recorded in real time.The gel strength was measured using a TA-XT plus Texture Analyzer . The measurements were performed with a cylindrical probe (P/0.5) at pre-test speed of 1 mm/s, test speed of 1 mm/s, post-test speed of 1 mm/s, compression distance of 4 mm and trigger force of 5 g .1 represents the weight of sample, and W2 represents the weight of supernatant.The sample was transferred to a centrifuge tube (diameter 30 mm) and subsequently centrifuged at 4800 rpm for 30 min at 25 °C. The supernatant was removed, and the mass of the precipitate was weighed. Water holding capacity (WHC) was calculated as follows:2) was recorded using the sequence of Carr–Purcell–Meiboom–Gill (CPMG). Other parameters were set as SW = 100 kHz, SF = 21 MHz, RG = 20 dB, TW = 4000 ms, NS = 4, TE = 0.2 ms and NECH = 18,000.LF-NMR tests were carried out using an instrument . About 2 g of the sample was packed into a glass tube with a diameter of 15 mm and inserted into the instrument. The tests were operated at 25 °C. The spin-spin relaxation time . The tests were conducted under the conditions of 40 kV, 20 mA and the Cu Kα radiation wavelength of 0.1542 nm. The range of the diffraction angle was from 5° to 40° with a step-scan of 0.01°. The relative crystallinity (RC) was calculated using the following equation:p < 0.05. Each experiment was carried out at least three times.These data were calculated using an analysis of variance (ANOVA) with the software SPSS 19.0  and were presented as the formation of mean plus standard deviations. Statistical differences were defined as


The novel coronavirus (SARS-CoV-2) caused lethal infections worldwide during an unprecedented pandemic. Identification of the candidate viral epitopes is the first step in the design of vaccines against the viral infection. Several immunoinformatic approaches were employed to identify the SARS-CoV-2 epitopes that bind specifically with the major histocompatibility molecules class I (MHC-I). We utilized immunoinformatic tools to analyze the whole viral protein sequences, to identify the SARS-CoV-2 epitopes responsible for binding to the most frequent human leukocyte antigen (HLA) alleles in the Egyptian population. These alleles were also found with high frequency in other populations worldwide.Molecular docking approach showed that using the co-crystallized MHC-I and T cell receptor (TCR) instead of using MHC-I structure only, significantly enhanced docking scores and stabilized the conformation, as well as the binding affinity of the identified SARS-CoV-2 epitopes. Our approach directly predicts 7 potential vaccine subunits from the available SARS-CoV-2 spike and ORF1ab protein sequence. This prediction has been confirmed by published experimentally validated and in silico predicted spike epitope. On the other hand, we predicted novel epitopes (RDLPQGFSA and FCLEASFNY) showing high docking scores and antigenicity response with both MHC-I and TCR. Moreover, antigenicity, allergenicity, toxicity, and physicochemical properties of the predicted SARS-CoV-2 epitopes were evaluated via state-of-the-art bioinformatic approaches, showing high efficacy of the proposed epitopes as a vaccine candidate.Our predicted SARS-CoV-2 epitopes can facilitate vaccine development to enhance the immunogenicity against SARS-CoV-2 and provide supportive data for further experimental validation. Our proposed molecular docking approach of exploiting both MHC and TCR structures can be used to identify potential epitopes for most microbial pathogens, provided the crystal structure of MHC co-crystallized with TCR.The online version contains supplementary material available at 10.1186/s43141-022-00344-1. A virus that causes infectious pneumonia broke out at the end of 2019 and rapidly spread worldwide , and theFurthermore, T cells detect viral antigens presented by MHC class I (the immunogenic peptide–MHC class I complexes), which will enhance CD8+ T cell cytokine production and cytotoxic activity (active effector CTLs) . The alpThe presentation of a stable immunogenic peptide–MHC class I (MHC) complex is dependent on the fitting between the peptide and the MHC groove, but it is not the only factor. The other factors affecting the formation of MHC complex include protease activity, the accessibility of chaperones, or the antigen. The binding groove of MHC class I is closed on both ends by conserved tyrosine residues, limiting the size of peptides that bind to MHC molecules to roughly 8–10 residues at their C-terminal end docking into the F-pocket , 20.The main objective of our study is to predict the most antigenic SARS-CoV-2 epitopes that are compatible with HLA haplotypes of the Egyptian population. We chose Spike and ORF1ab proteins, as they have a robust scores in several prediction tools including binding prediction with MHC, antigenicity response, and high docking scores with both MHC and TCR. These scores offer significant stability of the provided epitopes, whereas epitope prediction scores measure the affinity between the proposed epitopes and MHC molecules, while antigenicity response measures the ability of the proposed epitopes to elicit an immune response. The scores thus express the stimulation of the immune response against the proposed epitopes. Moreover, molecular docking scores evaluate the most conformational stability of our proposed epitopes with both MHC molecules and T cell receptors. The methods have been selected for their high accuracy in predicting binding conformation and are more fitting with our approach for protein-protein interaction. For example, HDock provides a robust homology modeling strategy for molecular docking via exploiting the FASTA format of the input data instead of the 3D structure prediction molecules. This improves the molecular docking results compared to feeding the 3D structures directly to the docking software. In this case, the software implements different conformation of the predicted epitopes according to their fitting in the binding pocket of both MHC and TCR. Additionally, NetMHCpan4.1 server  has a hiFor a more reliable characterization of the epitopes, we used additional tools. Vaxijen –24 is a In this study, several SARS-CoV-2 epitopes have been identified using a whole viral protein sequence analysis, exploiting the most updated version of tools for epitope prediction, antigenicity response, and molecular docking. These tools represented the most common and accurate platforms for epitope prediction analysis –24. Theshttps://www.ebi.ac.uk/Tools/st/emboss_transeq), and multiple sequence alignment of amino acid sequences was implemented via ClustalW using Molecular Evolutionary Genetics Analysis software “MegaX” [Genomic sequences of SARS-CoV-2 isolates Egyptian strains [GISAID database   [https://ddg-pharmfac.net/AllergenFP/index.html) respectively. Physicochemical properties, including hydropathicity, charge, half-life, instability index, pI , and molecule weight, were predicted by ExPASy ProtParam Tool [The toxicity and allergenic response of the proposed epitopes were predicted by ToxinPred server (inpred/)  and Alleram Tool .http://hdock.phys.hust.edu.cn/), which is currently exploited for protein docking based on two methods; template-based and template-free methods, both methods have been exploited to determine the most accurate one in providing high docking scores with both MHC and TCR. We found that template-free method provides more robust docking scores than template-based method. We provide both the crystal structures of MHC-I and TCR chains in PDB format, while the ligands are in their FASTA format. In the molecular docking, we substituted the crystallized epitopes bound between the groove of the MHC and TCR of 5YXN and 4PRP  to investigate the number of interacting bonds between the structures, as depicted in  residue at position 7713 to glycine (G) residue in S protein. The second, presented in only one Egyptian strain SARS-CoV-2 isolate was a mutation of lysine (K) residue at position 2798, to arginine (R) in ORF1ab protein  and template-based (PDB format) approaches of HDock. We tested both approaches by using the homology modeling structures of the candidate epitopes in their PDB format, which were obtained from the PEP-FOLD3 server, and the epitope protein sequences in FASTA format. We found that the template-free-based model provides higher docking scores than the template-based method. Moreover, by applying our docking approach in providing the alpha and beta chains of TCR, which were co-crystallized with MHC-I molecules, the docking scores and the number of hydrogen bonds increased significantly. This enhanced our analysis and presented a new docking approach by binding the query ligand to both TCR and HLA molecules that stabilize the binding and show a more confident docking conformation.We located the most favorable vaccine candidates in the Spike and ORF1ab proteins. Similar to other coronaviruses, the Spike protein is a trimeric class I transmembrane glycoprotein located on the surface of SARS-CoV-2 . SARS-Co+ epitope (YLQPRTFLL) has been validated experimentally, which also shows similarity with MERS-CoV epitope for the same HLA haplotype [Our study proposed seven immunogenic epitopes, with no toxicity, and with a high antigenicity response that is compatible with their physiochemical properties. Some epitopes are novel and others were predicted in-silico or by experimental techniques –70. The aplotype , 68. Anoaplotype , 70. We aplotype –73. AddiThe exploited HLA haplotypes represented the highest frequencies in the Egyptian population and also in worldwide population  . The preWe identified seven SARS-CoV-2 epitopes from Spike and ORF1ab proteins, according to the most common HLA allele frequencies of the Egyptian population. Some of these epitopes were previously validated in vitro and in silico and others are novel SARS-CoV-2 epitopes, characterized by a high probability of eliciting an immune response and stable molecular interaction. This was indicated by the high antigenicity, highest docking scores, and docking stability of these epitopes with both MHC class I and TCR chains that were stabilized by several hydrogen bonds. Importantly, our molecular docking approach is more feasible and useful when using the structure of MHC molecules co-crystallized with their TCR chains, and not only using the crystal structure of MHC molecules as followed in many recent studies. This molecular docking approach of utilizing both MHC and TCR structures for epitope prediction can be extended to most microbial infections. Experimental validation of these proposed epitopes should ultimately confirm their binding and interaction with specific TCRs, immunogenic response, and therapeutic potential against SARS-CoV-2.Additional file 1: Figure S1. Molecular docking of  ORF1ab and (c – e) Spike epitopes  ORF1ab and (c – e) Spike epitopes (No. 24, 79, 12, & 29 in Additional file 2: Supplementary Table 1.


Quadruped robots are widely applied in real-world environments where they have to face the challenges of walking on unknown rough terrains. This paper presents a control pipeline that generates robust and compliant legged locomotion for torque-controlled quadruped robots on uneven terrains. The Cartesian motion planner is designed to be reactive to unexpected early and late contacts using the estimated contact forces. Moreover, we present a novel scheme of optimal stiffness modulation that aims to coordinate desired compliance and tracking performance. It optimizes joint stiffness and contact forces coordinately in a quadratic programming (QP) formulation, where the constraints of non-slipping contacts and torque limits are imposed as well. In addition, the issue of stability under variable stiffness control is solved by imposing a tank-based passivity constraint explicitly. We finally validate the proposed control pipeline on our quadruped robot CENTAURO in experiments on uneven terrains and, through comparative tests, demonstrate the improvements of the variable stiffness locomotion. Quadruped robots have found extensive applications in real-world scenarios such as field exploration, industrial inspection, logistics and delivery, and disaster rescue  due to tIn case of unexpected interactions, impedance control  is able Different approaches have been studied in the literature to implement variable stiffness control. An exponential-function-based modulation of joint stiffness is implemented in , where tFrom the perspective of energy, modulating the stiffness of a robot may inject too much energy into it, which will lead the robot to instability. The works mentioned above, however, do not take into account stability issues in variable stiffness control. To this end, several approaches have been studied in the literature. An energy tank-based method is introduced in  to analy• A novel scheme for optimal stiffness modulation is proposed, where the desired stiffness gains and the desired contact forces are generated coordinately within a single optimization problem.• The stability issue of stiffness modulation is resolved theoretically by introducing an energy tank-based constraint to the coordinated optimization. To the best of the authors’ knowledge, this is the first time that such a scheme with guaranteed stability is proposed and applied to quadruped robot locomotion.• A control pipeline for torque-controlled quadruped robots, integrating the aforementioned scheme, is developed, enabling locomotion on uneven terrains with optimal modulation of joint stiffness. It is validated extensively on the quadruped robot CENTAURO seen in  through In this paper, we present a control pipeline for torque-controlled quadruped robots. It features optimal modulation of joint stiffness with guaranteed stability and touchdown adaptation that allows overcoming uncertainties in terrain geometries using only proprioceptive information. To be specific, the motion planner relies on estimated contact forces to transit between gait phases, which endows the robot with adaptations to early or late contacts in unknown environments. Moreover, we propose a QP-based optimization over both contact forces and joint stiffness based on desired motions and desired Cartesian compliance. An energy tank-based approach is exploited as a passivity constraint to restrict the variation rate of the joint stiffness, thus ensuring stable behaviors of the robot. Other types of constraints such as unilaterality of contact forces and torque limits are also considered in the optimization to ensure practically feasible results. The contributions of this paper are summarized as follows.The remainder of this paper is organized as follows. The proposed control pipeline for legged locomotion mainly consists of three modules: 1) a position control module that generates Cartesian reference trajectories and the corresponding joint motions; 2) a torque/impedance control module that computes feedforward and feedback torque commands; and 3) a contact estimation module that estimates ground contact forces and determines the actual contact states. An overview diagram of the proposed scheme is shown in xcom and xzmp denote the positions of the CoM and the ZMP, respectively, in the world frame, and then the reference motions of the CoM are generated by optimizing the jerks The generation of the reference motions for the center of mass (CoM) majorly follows our previous work  and it wxb in the last objective denotes the geometric center of the robot base and the reference pc,i denotes the positions of the feet and There are three objectives in the optimization . The firTaking into account that CENTAURO weighs about 110 kg and is driven by actuators with high gear ratios, the robot adopts a quasi-static gait pattern in the locomotion tasks. The gait schedule of the swing leg is periodic, which follows the sequence left-front leg (LF) → right-back leg (RB) → right-front leg (RF) → left-back leg (LB). The feet in contact are required to keep stationary, while the reference motions of the swing foot are formulated by two segments of quintic polynomials, one for the lifting phase and the other for the landing phase.a priori in blind locomotion, contact-triggered touchdown adaptation is implemented.In this work, it is assumed that visual perception is not integrated to the locomotion controller. In order to adapt to uneven terrains that are unknown ta and ts denote the periods of the All-Stance Phase and the Swing Phase, respectively. The red dashed arrows represent transitions that can only be triggered by detected contact events. The motion of the swing foot is adjusted accordingly in the cases of early touchdown and late touchdown. When contact is detected in advance of the nominal moment, the swing foot is switched to contact state and the nominal reference motion is truncated immediately. On the other hand, if expected touchdown is not established within the duration ts, the swing foot will move downwards at a constant speed so as to search for contact. When contact is detected, in both of the two cases, the motion planner will then switch to the All-Stance Phase, update the internal model with the actual configurations and start a new planning loop. The adaptation to early and late touchdown implemented in this chapter will enable perception-less locomotion over moderately uneven grounds.The motion planner adopts a finite state machine with time/event-driven transitions as illustrated in fup on the z-axis, the contact state of that leg will switch from swing to contact and a contact signal will be sent simultaneously to trigger the state machine shown in The contact event is detected by a threshold trigger of the estimated ground reaction forces, the calculation of which will be introduced later in CartesI/O (The Cartesian reference motions are then transformed into joint space by the whole-body controller which solves an inverse kinematics (IK) problem at 200 Hz. The IK problem is formulated as a hierarchical QP-based optimization inside artesI/O , a ROS-bi = 1, 2) in the following formulation.i = 1 is assumed to be superior to the problem with i = 2. The first objective in i-th problem and n DoFs of the actuated joints λ is selected to be λ = 1 × 10–4 in our implementation. The inequality constraint To be specific, the whole-body IK controller used in our control pipeline solves a cascade of two constrained optimization problems . m is the mass of the robot. gc = T m/s2 is the gravity acceleration vector. i-th foot. The equations above can be rewritten in matrix form asTo start with, the dynamics  is adopt seen in , it is rα1, α2 are two positive weight factors. The inequality n DoFs and The following optimization is solved at every control loop to compute the desired contact forces.τff is finally obtained by mapping the optimal contact forces fd into joint space throughτfb is calculated as followsThe feedforward torque fest are estimated from the measurements of joint torques and the dynamics of the robot throughi-th leg and (⋅)+ denotes the pseudo-inverse operation. S and the vector h are the same as defined in CENTAURO is not equipped with foot force/torque sensors and, therefore, the ground reaction forces Due to noisy measurements from the sensors and inaccurate parameters of the model, the contact forces estimated by The proposed scheme of optimal stiffness modulation will be introduced in detail in this section. First of all, it is worth clarifying that the stability of variable stiffness control is different from the locomotion stability for quadruped robots  . Looselye,q~˙l) . Althougtank-based passivity constraint. That is, we may store the dissipated energy into a virtual tank and withdraw the energy to cover some non-passive actions without loss of passivity. From the perspective of control theories, the tank energy depicts a sort of passivity margin which tells the controller how much variation of stiffness can be afforded now.Following the analysis above, imposing such a conditionSome observations and considerations on the computation of joint stiffness gains are first provided before presenting our scheme of the stiffness optimization with guaranteed stability.τ = τff + τfb is subject to boundary constraints imposed by the actuators, i.e., τ ∈ . It is worth noting that the values of the desired contact forces fd and the joint stiffness matrix KP affect each other. This is because the feedforward torques τff, which are mapped from fd by τfb and thus constrain the feasible range of the stiffness gains, i.e., there areFirst of all, the torque command fd are actually consistent to achieve the Cartesian reference motions. Therefore, it is the authors’ belief that the computation of the desired contact forces and the joint stiffness is better to be considered together, which should permit more possibilities to achieve desired motions and desired compliance in a coordinated manner.Another observation is that the position-based commands calculated by the whole-body IK controller and the desired contact forces Additionally, it is worth reminding that the sign of the tk, there is approximatelyT being the servo time of the controller, which is typically a small value (in our case it is 5 ms). In this paper, we assume that the stiffness and damping are decoupled among joints. That is, the stiffness matrix Motivated by these thoughts, we propose an optimization over both contact forces and joint stiffness by extending the previous optimization α3-related term in kp. ‖ ⋅‖F denotes the Frobenius norm of matrices. As known in the literature  that allows for long-term operation without triggering the overheat protection. However, the continuous torque limit is a rather conservative constraint because it is usually less than half of the peak torque limit, and exceeding this limit slightly and occasionally does not prevent continuous operation in practice. Consequently, the inequality i-th element being 1 + ϵi (ϵi is the i-th element of ϵ).Besides the peak torque limit, there also exists a continuous torque limit i-leg and (⋅)i denotes the subvector or the submatrix corresponding to the joints on the i-leg. In addition, as pointed out in  safe robot control framework developed in our lab, which also provides interfaces to non-RT processes  at IIT . The robrocesses .z-axis of the world frame, the magnitude of which may exceed 600 N sometimes. Aiming to mainly reduce the contact impulse in locomotion, we apply the proposed optimal stiffness modulation only to the 12 pitch joints of the legs  Nms/rad. As mentioned in Emax. It is set to be 5 J in our cases based on the observation that 5 J is a relatively conservative bound but allows to keep the tank energy positive, i.e., enabling continuous modulation of the joint stiffness gains, in most experimental trials. The design of the desired Cartesian stiffness will be introduced later in For selection of parameters, the torque limit orted in , is set Unless otherwise stated, other parameters used in the following experiments are listed in The experimental results of applying the proposed control pipeline on our robot CENTAURO are displayed in this part. The workspace consists of two wooden steps as shown in The results of using the proposed control pipeline are displayed in this section.fd and the feedforward reference torques τff. The dashed boxes indicate the periods when the corresponding leg is in swing, within which the expected contact forces should be 0. However, the corresponding feedforward reference torques are not 0 within the swing phases since the compensation for gravity and Coriolis effects has already been considered by the mapping 4 N/m) at the second half of swing phases while remaining at a higher value (3 × 104 N/m) at the other times, which aims to balance the requirements of supporting the body steadily and touching down compliantly.The design of the desired Cartesian stiffness introduced in The optimized joint stiffness gains for each leg are shown in ϵ in ϵ to 0, the better the reference joint torques comply with the constraints. As explained in α6, which controls the computation of ϵ, is selected to be a relatively huge value in order to obey the torque constraints as much as possible. The values of ϵ shown in The slack factor We also compare the proposed control pipeline with other strategies on the robot and the results will be provided in the following part.Compared with locomotion with fixed joint stiffness, the proposed variable stiffness locomotion may achieve a better balance between tracking performance and contact compliance. To this end, we repeat the task above with different configurations of joint stiffness.In this test, the stiffness for the hip pitch, knee pitch and ankle pitch joints on each leg are fixed to be  Nm/rad. In this test, the stiffness for the hip pitch, knee pitch and ankle pitch joints on each leg are fixed to be  Nm/rad. The impulse with the ground has even reduced in this case as shown in contact signal and transit from the Swing Phase directly to the All-Stance Phase after a period of ts (seen in In order to validate the effectiveness of the implemented touchdown adaptation, we disable this function and then repeat the variable stiffness locomotion in the same task. To be specific, the Cartesian motion planner will ignore any  seen in . The exeAs mentioned in A variable stiffness controller with guaranteed stability is developed in  for the The tank-based approach is introduced by  to studymatrix nearness technique. However, that matrix-optimization approach seems impossible to account for constraints such as torque limits and the energy tank-based passivity constraint, which may produce infeasible or unstable behaviors for the real robots. An alternative method to mitigate the aforementioned restriction is proposed in (In this paper, we assume the stiffness and damping matrices, i.e. d out in . A possid out in  to optimposed in , where tposed in , it is aWe present a control pipeline in this paper that enables our quadruped robot to walk on moderately uneven terrains robustly and compliantly without perception aided. One of our ongoing topics is to combine the proposed locomotion controller with perception in order to traverse more challenging environments. Due to some properties of the objects, the light conditions or the noise from the sensors, the perception may introduce uncertainties of about several centimeters in the terrain information, which, as demonstrated in the experiments, can be well solved by the proposed control pipeline. That work will eventually allow our quadruped robot CENTAURO overcoming more challenging terrains with various obstacles.


The role of culturing the graft preservation fluid (PF) is controversial and its impact on graft arteritis development remains unclear.Systematic literature search retrieving all-languages observational studies comparing SOT recipients with culture positive PF versus culture negative PF. Graft site arteritis within 180 days from transplant was selected as primary outcome. Data were independently extracted using a pre-specified form, and quality of included studies was independently assessed according to ROBINS-I tool for observational studies. Meta-analysis was performed using Mantel-Haenszel random-effect models.Enterococcus spp., beta-hemolytic streptococci and Staphylococcus aureus) and 1,351 (61.2%) as low-risk (mostly coagulase negative Staphylococci) pathogens. A significant higher risk of graft arteritis was found in SOT recipients with a PF yielding a high-risk pathogen , with low heterogeneity (I²=2.24%). Similar results were found considering separately high-risk bacteria  and fungi , with no heterogeneity (I²=0%), and in the subgroup analyses of liver  and kidney  recipients. However, data about diagnostic features of graft arteritis were very limited, indeed for only 11 of the 93 events histological or microbiological results were reported.2,492 articles were screened, and twenty-one observational studies  were included in the meta-analyses. Among positive PF, 857 (38.8%) were classified as high-risk group pathogens (fungi, Gram negative, Our results may support the performance of PF culturing and a preemptive diagnostic or therapeutic management upon isolation of high-risk pathogen. Further studies based on reliable diagnosis of graft arteritis are needed to confirm our findings.All Authors: No reported disclosures.


The limit of detection and limit of quantification were 0.78 and 2.36 ng mL−1, respectively. The inter-day and intra-day accuracy and precision were −6.36 to 1.88 and 0.99 to 2.58%, respectively. The metabolic stability of NQT in the HLM matrix was calculated using the in vitro half-life  and intrinsic clearance . NQT is considered to be a moderate extraction ratio drug that is moderately excreted from the human body compared with other related TKIs. This proposed methodology is thought to be the first method for assessing NQT concentration and its metabolic stability.Naquotinib  is a novel third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKIs). NQT was found to be more effective than osimertinib against the EGFR L858R plus T790M mutation (L858R+T790M). A rapid resolution liquid chromatography (RRLC)-tandem mass spectrometry (MS/MS) method was developed and validated for NQT quantification and its metabolic stability was investigated. NQT and foretinib (FTB) as an internal standard (IS) were separated using a mobile phase under isocratic conditions with a C18 column (reversed phase system). The linearity of the analytical method ranged from 5 to 500 ng mL The first established LC-MS/MS method for NQT analysis. NQT was shown to be moderately excreted from the human body. The epidermal growth factor receptor (EGFR) signaling pathway attained considerable importance a few years ago as a therapeutic target in NSCLC.6 Tyrosine kinase inhibitors (TKIs) that control the EGFR are very efficacious in EGFR mutation treatment with a characteristic therapeutic window. First-line TKIs controlling EGFR (e.g. erlotinib and gefitinib) are characterized by an initially good response against these mutations.7,8 Unfortunately, acquired resistance in approximately 60% of patients and the toxicities associated with treatment9,10 have decreased their therapeutic efficacies.11,12 This encouraged scientists to develop second-generation irreversible EGFR TKIs .13,14 However, NSCLC was shown to acquire resistance to first- and second-generation EGFR-TKIs in approximately 1 year.15 The EGFR T790M mutation is found in approximately 50% of NSCLCs resistant to first- and second-generation EGFR-TKIs. The second-generation EGFR TKIs (e.g. dacomitinib and avitinib) address the drawbacks of first-line TKIs in the first year from the beginning of treatment.8,13Non-small cell lung cancer (NSCLC) is a common and widespread subgroup of lung cancers13,14 Naquotinib  is considered a novel third-generation EGFR-TKI and was found to be more effective than osimertinib against the L858R plus T790M mutation (L858R+T790M). Additionally, naquotinib and osimertinib showed a wide therapeutic window and similar efficacy for EGFR exon 20 insertions cells.16The third-generation TKIs maintain the second-generation drugs' benefits by preventing EGFR mutations and surmounting the T790M resistance mutation.in vivo bioavailability value.17 NQT could be considered a moderate extraction ratio drug and, hence, is moderately excreted from the human body compared to other TKIs.18–21 This implies a lower risk of drug accumulation inside the body in comparison to other TKIs (e.g. dacomitinib). Therefore, the metabolic stability of NQT was determined by computing two important parameters  that could be utilized to further calculate other physiological parameters .A review of the literature proved there were no published chromatographic methods for NQT assay; therefore, we were prompted to establish an LC-MS/MS methodology for this target. The estimation of bioavailability provides an idea of the compound metabolism. If the test drug is quickly metabolized, it will show a low 2.2.1.4COOH) and human liver microsomes, pooled  were procured from Sigma-Aldrich (USA). HPLC grade water (H2O) was prepared by a filtration system .Naquotinib (99.12%) and foretinib (99.81%) were procured from MedChemExpress (USA). Acetonitrile , formic acid (HCOOH), ammonium formate (NH2.2.All LC-MS/MS parameters were optimized to attain the best chromatographic separation of NQT and FTB (IS) with high resolution . FTB was2, 12 L min−1) was utilized as the drying gas in the ESI source while high-purity N2 (55 psi) was used as the collision gas. The ESI temperature (T) and capillary voltage (V) were set at 350 °C and 3500 V, respectively. The instruments and data acquisition were controlled using Mass Hunter software. NQT was quantified using the analyzer mode of multiple reaction monitoring (MRM) for the mass reactions (parent to fragment ions) from 563 → 463 and 563 → 323 for NQT and 633 → 128 for FTB (A triple quadrupole (QqQ) mass spectrometer with an electrospray ionization source interface  was utilized for detection. Low-purity nitrogen (N for FTB . The fra for FTB  and 2.2.3.−1 in DMSO) was diluted 10-fold in the mobile phase to make working NQT solution 1 , which was further diluted to prepare WK2 (20 μg mL−1). FTB (100 μg mL−1 in DMSO) was diluted 50-fold in the mobile phase to make WK3 (1 μg mL−1).Both NQT and FTB are soluble in dimethyl sulfoxide (DMSO). NQT , low quality control (LQC), medium quality control (MQC), and high quality control (HQC), respectively. NQT and FTB were extracted using an acetonitrile (ACN) protein precipitation method for the metabolic stability experiments.22 ACN (2 mL) was added to each milliliter of the standard solution, followed by centrifugation (14 000 rpm for 12 min) in a thermostatic centrifuge at 4 °C to precipitate proteins and refine the sample to remove unwanted materials. Then, 1 mL of each supernatant was collected and filtered using a syringe filter (0.22 μm pore size). WK3 (50 μL) was added, followed by transference of the mixtures to 1.5 mL vials. The injection volume was 1 μL to facilitate the adjustment of the peak shape by enhancing the sharpness. Control samples were similarly prepared as described above, using the stated phosphate buffer without the HLM matrix to verify that the components of HLM did not interfere at NQT and FTB retention times. A standard calibration curve was constructed by plotting the peak area ratio of NQT to FTB (y-axis) against NQT nominal values (x-axis). A linear regression equation was utilized to verify the linearity of the supposed method. The slope, intercept, and coefficient of correlation (r2) values were computed.WK2 was mixed with a specific HLM matrix (40.00 μL in 1 mL of phosphate buffer) to establish a calibration curve of 12 standards: 5.00, 10.00, 15.00, 30.00, 50.00, 80.00, 100.00, 150.00, 200.00, 300.00, 400.00, and 500.00 ng mL2.5.21–23 The following validation parameters, linearity, assay recovery, sensitivity, reproducibility, specificity, limit of quantification (LOQ), and limit of detection (LOD), were utilized for the computation. All validation parameters were computed for NQT. The least squares statistical method was utilized for statistical calculation of the calibration curve equations (y = ax + b). A linear fit was verified by the r2, which showed linearity in the range of 5–500 ng mL−1. The recovery of NQT in the spiked HLM matrix was 98.61 ± 2.42% (relative standard deviation [RSD] < 2.68%).Parameters for validation of the supposed LC-MS/MS method for NQT assay were described in detail in our previous articles.2.6.2, 3.3 mM). The mixture was pre-incubated for 10 min in a thermostated water bath (37 °C) and then the metabolic reactions were initiated and subsequently stopped by adding NADPH (1 mM) and 2 mL ACN, respectively, at specific time intervals: 0, 2.5, 5, 7.5, 10, 15, 20, 40, 50, and 70 min. The metabolic stability curve for NQT was subsequently constructed.The NQT metabolic stability was determined by assessing the NQT concentration remaining after incubation with HLMs. Briefly, 1 μM of NQT was incubated with HLMs  and the experiment was repeated three times for confirmation of the results. The metabolic reaction was performed in phosphate buffer (pH 7.4) containing magnesium chloride  was adjusted to 4.2 using formic acid because at a higher pH, peak tailing and an excessive increase in retention time were observed. The ratio between the aqueous and organic parts (ACN) was set to 55 : 45%, because an increase in the ACN ratio resulted in overlapping chromatographic peaks with bad peak resolution, whereas a decrease in the ratio increased the elution time. Various types of stationary phases such as HILIC columns were tested, but NQT and FTB were not retained and good results were finally attained using a C18 column. MRM was used for NQT quantification to remove any HLM matrix interference and enhance the sensitivity of the proposed LC-MS/MS method .The run time for complete elution of NQT and FTB was 4 min with good resolution. There was no carryover in the blank HLM matrix sample. 3.2.3.2.1.3.2.2.r2 for the supposed method were 5–500 ng mL−1 and ≥0.9999, respectively, in the HLM matrix. The regression equation of the NQT calibration curve was y = 1.5581x − 3.395. The LOD and LOQ were 0.78 and 2.36 ng mL−1, respectively, while the LLQC peak showed a high signal to noise (S/N) ratio and a good peak shape, suggesting the sensitivity of the developed LC-MS/MS method . These batches were marked as set 1. Set 2 was prepared using the mobile phase in place of the HLM matrix and, thus, the matrix effect was calculated using the following equation:The studied HLMs containing NQT and FTB showed matrix effects of 98.61 ± 2.42 and 98.7 ± 0.7%, respectively. The internal standard normalized matrix effect  was computed using the following equation:25 Accordingly, these results showed that the HLM matrix had no obvious influence on the ionization of NQT or FTB.The IS normalized MF was 1.01 and it was within the range of adequacy.3.3.y-axis against the time of incubation on the x-axis  and very long in vitro t1/2 value (67.96 min) that revealed the moderate clearance of NQT from the blood by the liver and possible well-controlled in vivo bioavailability. This drug could be utilized without accumulation inside the body or fast elimination from the blood.27These results revealed that the metabolic stability of NQT was characterized by a very low Cl4.in vitro t1/2 and Clint. These two outcomes indicate that NQT showed moderate clearance from the blood by the liver and possible well-controlled in vivo bioavailability.28–30 Therefore, this drug could be utilized without concerns about accumulation inside the body or fast elimination from the blood.An LC-MS/MS method was established and validated for quantifying NQT. The established method exhibited good sensitivity, was eco-friendly with the small volume of ACN used, fast, accurate, and showed high recovery. The LC-MS/MS method was utilized for NQT metabolic stability assessment in an HLM matrix using two parameters: The authors declare no conflict of interest.CECollision energyEGFREpidermal growth factor receptorFVFragmentor voltageHLMsHuman liver microsomesHQCHigh quality controlISInternal standardIS normalized MFInternal standard normalized matrix effectLC-MS/MSLiquid chromatography tandem mass spectrometryLODLimit of detectionLQCLower quality controlLOQLimit of quantificationMQCMedium quality controlNSCLCNon-small cell lung cancerNQTNaquotinibFTBForetinibQCQuality controlTKITyrosine kinase inhibitorRRLCRapid resolution liquid chromatography