A comparative analysis of the strengths and weaknesses of three different methods for radiologic evaluation of patellofemoral instability (PFI).Computed tomography (CT) and magnetic resonance imaging (MRI) were performed in 47 patients with or without PFI. The tibial tubercle-trochlear groove (TT-TG) distance was measured by two observers through conventional CT and three-dimensional CT reconstruction (TDR-TT-TG) respectively and the tibial tubercle-posterior cruciate ligament (TT-PCL) distance with MRI. The intraclass correlation coefficient (ICC) was used to evaluate the interobserver reliability. In addition, the differences of three measurements between different patients were compared. The consistency of TT-TG and TDR-TT-TG was analyzed by the Bland-Altman method.The ICCs of three measurements were high between two observers; the results were TT-TG (ICC = 0.852), TDR-TT-TG (ICC = 0.864), and TT-PCL (ICC = 0.758). The values of PFI patients were significantly higher than those of non-PFI patients, and the mean TT-TG, TDR-TT-TG, and TT-PCL distance in patients with PFI were 19.0 ± 3.8 mm, 19.0 ± 3.7 mm, and 25.1 ± 3.6 mm, respectively. There was no statistically significant difference between the TT-TG distance and the TDR-TT-TG distance, we found no significant difference. The Bland-Altman analysis showed that the TDR-TT-TG distance was in good agreement with the TT-TG distance.All three methods can be used to assess PFI; the TDR-TT-TG measurement method has superior operability and better interobserver consistency. It may be an alternative method to the conventional TT-TG distance measurement. Patellofemoral instability (PFI) refers to the fact that the patella cannot slide normally in the femoral trochlear groove, causing one or more subluxations or dislocations and abnormal patella slippage, resulting in a series of symptoms . PFI is After the failure of conservative treatment for PFI, surgery is frequently needed. To date, the operation for an unstable patella is mainly based on the principle of the balancing of soft tissue and bone structure , 11, 12.As we all know, the traditional measurement of TT-TG requires overlapping images of two planes, and the TT-PCL measurement requires three layers of superposition, identifying points containing the insertion point of PCL, trochlear groove, tibial tubercle, etc., whereas these markers are not in an axial plane , 26 on cForty-seven patients with PFI were admitted to the observation group from April 2017 to April 2019 in our hospital. Selection criteria were as follows: (1) clinical diagnosis of PFI and (2) no history of knee surgery. Exclusion criteria were as follows: (1) patients with MRI or CT contraindications; (2) multiple ligament injuries, especially in patients with posterior cruciate ligament injury; (3) patients with knee deformity or severe osteoarthritis; and (4) patients with periarticular fractures. Forty-seven non-PFI patients (non-acute stage gout patients) who underwent CT and MRI examinations on the ipsilateral knee in our hospital were selected as the control group.CT and MRI were performed in patients with PFI, and all gout patients underwent MRI and dual-source dual-energy CT examinations. Imaging operations were completed by unified trained technicians, and related parameters used in the scanning of each patient were uniformed. The specific procedures are as follows:Conventional CT scan was performed on the Siemens 64-slice CT . All patients were supine with leg and thigh in full extension. Straps were attached on the lower leg to avoid rotation. Images were taken with the following parameters: slice thickness 4 mm, scan time 9.28 s (nearly 5 to 10 s), rotation time 1 s, care dose , pitch 0.8 mm, and matrix 512 × 512.The dual-source dual-energy computed tomography (DSDECT) scan completed on the same instrument . The parameters were as follows: slice thickness 2 mm, scan time 17.14 s (nearly 10 to 20 s), rotation time 1.0 s, care dose , pitch 0.7 mm, and matrix 512 × 512.The conventional CT image was exported with Philips IntelliSpace Portal for transforming into TDR image, the bone window was selected for reconstruction image of the knee joint, and the patella was removed by excluding the Freehand function in clip&3D-segmentation, adjusted to the coronal position. Finally, a TDR image of the knee joint was obtained.All about MRI  scanner was routine: the patients were in a supine position, with a standard knee coil center level against the lower edge of the patella. The knee and hip joint naturally extended, and the feet were braced to prevent any movement. Four MRI sequences of knee were obtained. The scanning parameters of Andover are given below: (1) coronal proton density-weighted spectral attenuated inversion recovery (PDW-SPAIR) MR images ; (2) transverse PDW-SPAIR MR images ; (3) sagittal T1-weighted aTSE (turbo spin-echo) MR images ; and (4) sagittal proton density-weighted spectral inversion recovery (PDW-SPIR) MR images .As previously mentioned, the TT-TG distance was only measured on CT images according to the method described by Dejour et al. . Two speThe measurement was only performed on MRI images according to the TT-PCL distance described by Seitlinger et al. . Three sThe TDR-TT-TG distance was measured under TDR. First, the tangential line was taken from the two lowest points of the femoral condyle, and then draw two lines through the deepest point of the femoral trochlea (TG) and the most cephalic point of the tibial tuberosity (TT) respectively. Both TT line and TG line were perpendicular to the tangent of the distal femoral condyle. The horizontal distance between the two lines was measured, which was the TDR-TT-TG distance, with a measurement accuracy of 0.1 mm  was used to assess interobserver reliability to minimize the bias and confounding factors of measurement.T test and the Bland-Altman method for correlation analysis [Bland-Altman scatter plot was drawn by MedCalc v12.1.3 ]. P < 0.05 was considered statistically significant.All relevant data were analyzed by SPSS 22.0 . Continuous variables are expressed as the mean ± standard deviation. The comparison between two groups was conducted with an independent-samples The PFI group included 19 males and 28 females, aged 11–49 years old, with an average age of 20.7 ± 8.0 years; in the control group, 33 males and 14 females were included in the analysis, aged 23–56 years old, with a mean age of 31.8 ± 12.7 years old.The ICCs of the three different measurement methods between the two observers are shown in Table P = 0.000, P = 0.000, P = 0.000). There was no statistically significant difference between the TT-TG distance and the TDR-TT-TG distance, we found no significant difference. The TT-PCL distance was larger than both the TT-TG distance and the TDR-TT-TG distance; meanwhile, the difference was statistically significant  was − 2.71 to 2.78 mm. Figure Bland-Altman analysis of the two measurement methods showed that the mean difference between the TT-TG and TDR-TT-TG distances was In this study, we used a new method based on a TDR modeling to measure TT-TG distance. The TDR-TT-TG method is only required for the completion of measurement in one layer, and our results illustrated that it has a higher interobserver consistency (ICC = 0.852) than the other two methods in our study. Furthermore, the Bland-Altman analysis indicated that the data measured by the conventional CT and TDR had good consistency, and the bias was slight; thus, the two methods are interchangeable. These results demonstrated that the TDR measurement is a reliable and feasible method. Similarly, recent research by Nha et al.  also illThe TT-TG distance we measured was relatively close to that of Anley et al.  (TT-TG 1We found that the mean TT-PCL distance of PFI patients was 25.1 ± 3.6 mm, which was larger than the TT-PCL distance in the other studies , 30, 31.However, our study has some limitations. We ensured that the knee joint was in a 0° flexion position during CT examination, but knee coils for MRI testing we used could result in the knee being positioned in varus with slight knee flexion. Furthermore, although we tried to keep the knee as straight as possible during the examination, the angle of knee flexion may be different due to the different sizes of the limbs. Compared to TT-PCL, the measurement of TDR-TT-TG has a certain amount of radiation in the CT examination and a certain subjectivity in the selection of the lowest point of the femoral trochlea and the most cephalic point of the tibial tuberosity. While the consistency between observers was good, the clinical application of the TDR-TT-TG distance requires more studies.The TDR imaging technique for the measurement of the TT-TG distance has superior operability and better interobserver consistency. It may be an ideal alternative to the conventional TT-TG distance measurement.


Background and Purpose: Olfactory dysfunction (OD) is a common non-motor symptom of Parkinson disease (PD). However, the relationship between OD and neuropathologic proteins in cerebrospinal fluid (CSF) from PD patients remains unclear.Methods: 166 PD patients were included in the study. Overall olfactory function was assessed by summing up the scores of olfactory threshold, discrimination, and identification by a Sniffin' Sticks test, based on which, patients were divided into PD with OD (PD-OD) and PD with no OD (PD-NOD) groups. CSF samples were obtained from 76 PD patients. The levels of neuropathologic proteins, including α-Synuclein, Aβ1-42, total tau (T-tau), and multiple forms of phosphorylated tau (P-tau) in CSF were measured by an enzyme-linked immunosorbent assay.Results: out of the 166 PD patients, 103 cases (62.0%) had OD. The scores of overall olfactory functions, and olfactory threshold, discrimination, and identification in the PD-OD group were all significantly lower than that in the PD-NOD group (P < 0.001). α-Synuclein level in CSF was significantly higher in the PD-OD group than the PD-NOD group (P < 0.05), and was significantly and negatively correlated with the scores of overall olfactory function, and olfactory discrimination and identification (P < 0.05). Aβ1-42 level in CSF was higher in the PD-OD group than the PD-NOD group, and was significantly and negatively correlated with the olfactory identification score (P < 0.05). T-tau level in CSF was significantly lower in the PD-OD group than the PD-NOD group (P < 0.05), and was significantly and positively correlated with the olfactory discrimination score (P < 0.05). There was no significant difference in P-tau level in CSF between the PD-OD and PD-NOD groups and no correlation between OD score and P-tau level in CSF.Conclusions: PD-OD includes the impairments of olfactory threshold, discrimination, and identification, and is associated with the significant elevation of α-Synuclein and the decrease of the T-tau level in CSF. A pathological hallmark of Parkinson disease (PD) is Lewy bodies (LBs), which deposit in the substantia nigra (SN) and cause the progressive degeneration and death of dopaminergic neurons, leading to dopamine depletion in striatum and subsequent motor symptoms. Recent pathological investigations demonstrated that LBs beyond SN were associated with numerous non-motor symptoms   is the major component of neuritic plaque, a pathological biomarker of Alzheimer's disease (AD). In histologically diagnosed PD patients, Aβ was frequently observed in the brain, suggesting its relationship with PD11. Another investigation showed that Aβ1-42 level in CSF was significantly lowered in those with worse OD identified by the University of Pennsylvania Smell Identification Test (UPSIT)  is the major component of neurofibrillary tangle, another pathological hallmark of AD. When tau is over-phosphorylated, it leads to the degeneration and death of neurons  in 2015  family history of OD. Olfactory disorders can be inherited. For example, ciliary disease is a type of inherited pleiotropic genetic disease. The changes in the assembly, maintenance, and/or function of cilia are manifested in multiple organ systems. Anosmia is a clinical manifestation that has been confirmed in patients  were calculated as follows: LEDD = levodopa standard tablets × 1 + levodopa controlled-release tablet × 0.75 + (levodopa standard tablets × 1 + levodopa controlled-release tablet × 0.75) × 0.25 (if taking entacapone simultaneously) + piribedil sustained-release tablets × 1 + pramipexole hydrochloride tablet × 100 + selegiline hydrochloride tablets × 10.The olfactory function of PD patients was tested by Sniffin' Sticks from the Burghart Messtenik Company, German (product number: LA-13-00005). A total of 112 sticks were used for the test, in which 48, 48, and 16 were used for olfactory THR, DIS, and ID, respectively.Olfactory THR: participants were exposed to n-butyl alcohol from the lowest concentration to the highest one. The THR score was the number of Sniffin' Sticks that participants clearly identified with the minimal concentration of n-butyl alcohol. The lower the THR score, the worse the olfactory recognition.Olfactory DIS: participants were instructed to figure out the target odor that was different from the other two. The DIS score was the number of Sniffin' Sticks that participants correctly identified. The lower the DIS score, the worse the olfactory discrimination.Olfactory ID: participants were required to smell odor and choose the right answer from the given four. The ID score was the number of Sniffin' Sticks that participants correctly identified with the target odor. The lower the ID score, the worse the olfactory identification.Overall olfactory function was obtained by summing up the scores of THR, DIS, and ID (TDI).OD was identified by the following criteria from a cross-sectional study of 3,282 people by a Sniffin' Sticks test adjusted for sex and gender  group.CSF samples were obtained from 76 PD patients, including 35 males and 41 females, of which demographic variables were also collected.Anti-PD drugs were withheld for 12–14 h if the patient's condition allowed. In order to prevent the blood contamination of CSF, the lumber puncture for each patient was performed by a professionally trained neurologist, strictly following the standardized protocol. The first and second tubes may contain blood, as well as tissue fragments and contaminated skin microorganisms, thus, routinely, the third tube of CSF was retained to ensure that the CSF was not contaminated by blood to an acceptable degree. CSF samples from both the PD-OD and PD-NOD groups in this study all followed this standardized protocol, trying to avoid this potential bias. A total of 5 ml CSF was obtained through lumbar puncture in polypropylene tubes between 7-10 a.m. under fasting conditions. CSF samples were centrifuged immediately at 3,000 rpm at 4°C. Approximately 0.5 ml supernatant of CSF was aliquoted into separate Nunc cryotubes and kept frozen at −80°C until ready for assay. Each aliquot dedicated for each measure to avoid freeze-thawing and protein degradation.The levels of neuropathologic proteins in CSF from PD patients were measured using an enzyme-linked immunosorbent assay. We used the Human α-Synuclein (SNCA) ELISA Kit  produced by CUSABIO. According to the contents of the instructions and the company's response, this kit is designed for the full-length sequence of α-Synuclein. Therefore, it can both measure monomers and complexes, that is, total α-Synuclein was measured in this study. The CSB-E12011h Kit for T-tau was obtained from the Huamei Bioengineer Company . The Invitrogen-KHB8051 Kit for P-tau (T231), Invitrogen-KHB7041 Kit for P-tau (S199), Invitrogen-KHB7031 Kit for P-tau (S396), and Invitogen-KHO0631 Kit for P-tau (T181) were all obtained from the YuBo Bioscience Co. Ltd .P < 0.05 was statistically significant.Statistical analyses were performed with SPSS Statistics 22.0 . Demographic variables, the scores of olfactory THR, DIS, and ID, and the levels of neuropathologic proteins, including α-Synuclein, Aβ1-42, T-tau, and multiple forms of P-tau in CSF were compared between the PD-OD and PD-NOD groups.t-test. Bonferroni correction was performed in further comparisons between the PD-OD and PD-NOD groups. Continuous variables, if they were not normally distributed, were presented as median (quartile) and compared by a nonparametric test.Continuous variables, if they were normally distributed, were presented as means ± standard deviations and compared by a two-tailed Discrete variables were presented as percentages, and a Chi square test was used to make comparisons.Spearman correlation analyses were made between the scores of TDI, THR, DIS, and ID and the levels of neuropathologic proteins in CSF.Firstly, 166 PD patients were required to describe the status of their olfactory function, among which, 79 cases (47.6%) reported that they had OD.Secondly, the olfactory function of each PD patient was tested by a Sniffin' Sticks test. It was found that 103 out of the 166 cases (62.0%) actually had OD.Finally, in 103 PD patients with OD demonstrated by the Sniffin' Sticks test, 63 cases (61.2%) had OD before the occurrence of motor symptoms.Demographic variables of the PD-OD and PD-NOD groups were compared  and no sIn PD patients, the TDI score was 18.99 ± 5.87 points, in which the THR score was 3.50  points, the DIS score was 8.00  points, and the ID score was 7.00  points.Table 3).Further comparison analyses revealed that the TDI score as well as the scores of THR, DIS, and ID were all significantly lower than that in the PD-NOD group . It was found that the α-Synuclein level in CSF from the PD-OD group was significantly higher than that from the PD-NOD group, indicating that the elevation of α-Synuclein in CSF was related to PD-OD. Further analysis indicated that α-Synuclein level in CSF from PD patients was significantly and negatively correlated with TDI, DIS, and ID scores, but not with THR score (Table 4), indicating that OD, especially DIS and ID, deteriorated as the α-Synuclein level in CSF rose.Firstly, α-Synuclein levels of the PD-OD and PD-NOD groups were compared (Table 3). It was observed that the Aβ1-42 level in CSF from the two groups were not significantly different. Further analysis implied that the Aβ1-42 level in CSF had a significantly negative correlation with the ID score (Table 4), suggesting that ID worsened as Aβ1-42 level in CSF increased in PD patients.Secondly, the levels of Aβ1-42 in CSF in the PD-OD and PD-NOD groups were compared (Table 3). It was revealed that the T-tau level in CSF from the PD-OD group was significantly decreased when compared with that from the PD-NOD group, implying that the decrease of T-tau in CSF was associated with PD-OD. The levels of P-tau (S199), P-tau (S396), P-tau (T181), and P-tau (T231) between the two groups exhibited no significant differences. Thus, T-tau was associated with PD-OD. Further analysis revealed that the T-tau level in the CSF of PD patients had a significantly positive correlation with DIS score (Table 4), indicating that DIS progressed as T-tau level in CSF declined.Thirdly, the levels of T-tau and P-tau in CSF from the PD-OD and PD-NOD groups were compared . However, this percentage went up to 62.0% (103/166 cases) when olfactory function was objectively tested by Sniffin' Sticks and determined by the criteria adjusted for gender and sex  had significant olfactory function impairment before the occurrence of motor symptoms, illustrating that OD is a common non-motor symptom of PD prior to movement dysfunction  have different phagocytosis effects on different forms of α-Synuclein. TLR2 in microglia can bind with α-Synuclein oligomers and phagocytize them  , P-tau (S396), P-tau (T181), and P-tau (T231). Excessive P-tau results in the impairment of cell integrity, loss of physiological function, and the eventual degeneration and death of neurons, thus playing an important role in neurodegenerative diseases, such as PD. It was reported that tau was increased in PDD patients (Andreasson et al., A limitation of this study is the relatively insufficient CSF samples. It was difficult to obtain CSF from PD patients who were old, combined with low intracranial pressure, spinal deformity, and bone hyperplasia, etc. We will be collecting CSF from PD patients to confirm the conclusion of this study in the future.In summary, PD patients have a high frequency of OD, objective testing by Sniffin' Sticks is valuable for identifying more PD patients with OD, as most PD patients have significantly impaired olfactory function before the onset of motor symptoms. The OD of PD patients includes impairments of olfactory THR, DIS, and ID. PD-OD may be correlated with the elevation of α-Synuclein and the decrease of T-tau in CSF. Results from this investigation indicate the clinical features and potential neuropathologic mechanism of PD-OD.The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by ethics review board of Beijing Tiantan Hospital, Capital Medical University. The patients/participants provided their written informed consent to participate in this study.WZ: study concept and design, acquisition, analysis and interpretation of data, editing and critical revision of the manuscript, and study supervision. PG: acquisition and analysis of data, and study supervision. R-dW: acquisition and analysis of data, and manuscript editing. T-hL: acquisition and analysis of data. D-yD, W-JZ, J-hL, and H-yG: analysis of data and manuscript editing. D-nL, S-yY, LL, YH, L-jZ, and Q-jY: data acquisition. D-nL: data acquisition. L-xL: patient recruitment. X-mW: study design and supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Shape-memory composites have benefits for minimally invasive surgery, but their wider applications for bone repair are hindered by conflicts between the mechanical and memory performances, especially at load-bearing locations. In this study, we fabricated a graphene oxide shape-memory polyurethane composite through the chemical combination of graphene oxide and isocyanate, in order to realize satisfactory mechanical and shape-memory effects. As desired, a modulus of ~339 MPa and a shape recovery ratio of 98% were achieved, respectively, in the composite. In addition, finite element analysis demonstrated that, after being implanted in a defective bone through a minimally invasive treatment, where the highest stress was distributed at the implant–bone interface, this composite could offer a generated force during the recovery process. Furthermore, we also discuss the origins of the improved mechanical and memory properties of the composites, which arise from increased net-points and the stable molecular structure inside. Therefore, with its superior structure and properties, we envision that this shape-memory composite can provide new insights toward the practical application of shape-memory polymers and composites in the field of bone repair. T < Ttrans), respectively [There has been an interest in the study of smart materials, which are always closely related to human life, for more than fifty years. Since the term “shape-memory” was first reported as a smart material in 1941, shape-memory materials, especially shape-memory polymers (SMPs), have been developed quickly . SMPs arectively . Based oectively , heart sectively , and minectively . Our groectively , artificectively , and ortectively . Howeverectively . For insectively ,19.2-bonded carbon atoms, which has excellent mechanical, optical, and conductive properties, as well as good biocompatibility because of its special structure [One common approach to improving the mechanical performance of SMPs is to introduce nanofillers into the SMP matrix ,21. Graptructure ,23. Graptructure ,24,25,26tructure . Meng ettructure . Previoutructure , where pHerein, to address the limitations of the mechanical and memory performances in SMP composites, we developed an isocyanate modified GO/SMPU (SMPU/iGO) composite. GO was modified with MDI through covalent bonding in order to chemically blend it within the SMPU matrix. It is expected that the mechanical and memory properties of the composite could be significantly enhanced by the chemically bonded GO, by forming a stable molecular structure; thus, the composite could be used for minimally invasive treatments in bone repair. The physical blending of the SMPU/GO composite was regarded as the control group for comparison. As a proof of concept, Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, scanning electron microscopy (SEM), wide-angle X-ray diffraction (WAXD) and swelling tests were conducted to demonstrate the successful modifications of GO and the structures of the synthesized composites. In addition, differential scanning calorimetry (DSC), tensile tests, and bending tests were carried out to confirm the thermal and mechanical performances, as well as the shape-memory properties. Finally, finite element analysis (FEA) was performed to simulate the generated stress evolution that occurs during the recovery process on the interface between the SMP/iGO composite and cancellous bone in a minimally invasive bone-repairing model.PCL-diol  was obtained from Perstorp  Chemical Industry Co., LTD. 4,4-methylenebis (phenyl isocyanate) (MDI), 1,4-butanediol (BDO), and GO sheets were all obtained from Sigma-Aldrich . The PCL-diol and BDO were dried in advance under a vacuum at 100 °C for 24 h to remove the moisture. GO sheets were used as received.For the SMPU/GO composite, the SMPU was synthesized first through previously published procedures , and the-1 in order to investigate the structure of the SMP/GO composites. Raman spectra were collected on a Bayspec spectrometer  with a laser excitation wavelength of 785 nm, where the shift axis had been calibrated by silicon. SEM  at an accelerating voltage of 5 kV was utilized in order to recognize the surface morphologies of the composites. The wide-angle X-ray diffraction (WAXD) patterns of the samples were recorded using a Rigaku Smartlab XRD instrument , using a Cu Ka radiation source (1.54 Å). The composites were scanned from 2θ = 10° to 80°. The swelling degree was tested as follows: (1) samples were dipped in dimethylacetamide (DMAC) at 60 °C for 24 h to achieve the equilibrium state, and the weights of the samples before and after dipping were recorded; (2) the samples were dried at 40 °C for 24 h, and the weights of the samples before and after drying were recorded. Then, the swelling degree was calculated according to the literature [A 2000 FTIR spectrometer  with attenuated total reflectance accessories was used in the wavenumber range of 650–4000 cmterature .Tg) were measured by using differential scanning calorimetry . Samples were firstly heated to 120 °C and then cooled to 0 °C at a rate of 10 °C min−1. After remaining at this temperature for 5 min, a second scan was processed from 0 to 120 °C at the same rate, from which data were used for analyzing. Tensile tests of the SMP/GO composite were conducted at room temperature (RT) by using the INSTRON 5566 tester  with an extension rate of 5 mm min−1. Young’s modulus, strain at break and stress were derived from the stress-strain curves to compare the mechanical properties. Shape-memory performance was investigated through a bending test and by recording the changes of the angles [0, the maximum bending angle was θm at 80 °C, and the fixed angle of the sample at the equilibrium state was θf. After being recovered at 80 °C, the final angle was θr. Then, the shape fixity ratio (Rf) and shape recovery ratio (Rr) could be calculated according to the equations in the literature [Glass transition temperatures  to simulate the stress variety during the recovery process on the interface between the SMP/iGO composite and the bone tissues. We assumed that the device made of SMP/iGO was implanted into the defected bone at a compressed size, and we investigated the recovery stress at the interface when the temperature was 40 °C. The device–bone system was simplified to a cylinder-cube  model in this case, where all materials were regarded to be isotropic, homogeneous, and linearly elastic. According to the literature, the properties of the cylinder  and the cube (bone) were set respectively as follows–Young’s modulus: 339 MPa/1.5 GPa, Poisson’s ratio: 0.25/0.3, CTE: 0.13511/0 ,30.p < 0.05.All data were statistically analyzed using the Origin 8.5  and SPSS 22.0 software . Moreover, results were compared using one-way ANOVA, and significant difference was set to −1 represents the groups of –OH, and the strong bands at 1740 cm−1 and 1060 cm−1 are the stretching vibration of the C=O carbonyl groups and the C–O groups, respectively [−1 and the D bands at 1590cm−1 that arise from the sp2 and sp3 hybridizations of carbon, respectively, represent the existence of GO in the SMPU composites [ID/IG), indicating the disorder degree, were calculated, and the values in the spectra of the SMPU/iGO and SMPU/GO composites were 0.82 and 1.32, respectively [D/IG ratio of the SMPU/iGO suggested a highly ordered structure in the composite [Tg) of the SMPU composites were investigated by DSC, as shown in Tg of SMPU/GO and SMPU/iGO was around 46.3 and 58.7 °C, respectively. Tg could represent the mobility of the soft segment (polymer chains) in the matrix [Tg in the SMPU/iGO suggested that the chemically bonded GO enhanced the whole structure and hindered the movement of the polymer chains in the amorphous phase, which could achieve greater strength improvement in the SMPU/iGO than in the SMPU/GO.ectively . In the mposites ,33. The ectively . The lowomposite ,36, imple matrix , and theRf of ~80%, and the SMPU/GO had an Rf of ~95%, indicating that GO had more beneficial effects on the glassy state modulus of the physical blending composite rather than the chemically bonded one, since its shape was fixed in the glassy state. Nevertheless, the SMPU/iGO still had a satisfactory Rf to ensure good fixation ability for minimally invasive treatment. More importantly, the SMPU/iGO highly improved the Rr to ~98%. In general, the nanofillers could partly disturb the flexibility of the polymer in the elongated phase, meaning that the Rr would be lower, as with SMPU/GO (~80%), due to the aggregation of the nanofillers. However, in the structure of the SMPU/iGO, GO was regarded as the net-point in the new shape-memory system, resulting in a positive effect on the polymer’s elastic properties due to homogeneous dispersion. Rr is important in bone repair treatment, especially at load-bearing locations, because it corresponds to the recovery ability of the composites, the generated forces for providing the supporting function, and the mechanical stimulus to the surroundings in the defected site. The high Rr demonstrates that the SMPU/iGO has the potential for application in the field of bone repair.The bending test was conducted in order to investigate the shape-memory properties. The procedures and the corresponding angles can be found in According to previous studies, a mechanical stimulus might improve cell and tissue growth ,38,39. TRf than that of the SMPU/iGO. As for the SMPU/iGO, GO is combined with MDI through covalent bonds. Consequently, the net-points are constituted by both GO and short polymer chains, as shown in Here, we propose two models of modified GO SMP composites, based on the structural characterizations and the model we previously established for SMPs , in order of the SMPU/iGO was highly improved to ~98% compared with the physical blending composites. In addition to this, as expected, the SMPU/iGO presented a robust recovery process and generated force at the material–bone interface, as demonstrated by its behavior in the FEA. All in all, we believe that the developed SMPU/iGO with coalesced improved performances has the potential for practical application in the field of bone repair.This work proposes a chemically bonded SMPU composite using isocyanate modified GO for simultaneously improving the mechanical and memory properties. Physical blending SMPU/GO composites were used for comparison. The results demonstrated that the SMPU/iGO has a satisfactory modulus of 339 MPa  and a stress of ~21 MPa with a good stretchability (~92%). More importantly for bone repair, SMPU/iGO has a beneficial shape-memory effect. In particular, the R


Acacetin, an important ingredient of acacia honey and a component of several medicinal plants, exhibits therapeutic effects such as antioxidative, anticancer, anti-inflammatory, and anti-plasmodial activities. However, to date, studies reporting a systematic investigation of the in vivo fate of orally administered acacetin are limited. Moreover, the in vitro physicochemical and biopharmaceutical properties of acacetin in the gastrointestinal (GI) tract and their pharmacokinetic impacts remain unclear. Therefore, in this study, we aimed to systematically investigate the oral absorption and disposition of acacetin using relevant rat models. Acacetin exhibited poor solubility (≤119 ng/mL) and relatively low stability (27.5–62.0% remaining after 24 h) in pH 7 phosphate buffer and simulated GI fluids. A major portion (97.1%) of the initially injected acacetin dose remained unabsorbed in the jejunal segments, and the oral bioavailability of acacetin was very low at 2.34%. The systemic metabolism of acacetin occurred ubiquitously in various tissues , resulting in very high total plasma clearance of 199 ± 36 mL/min/kg. Collectively, the poor oral bioavailability of acacetin could be attributed mainly to its poor solubility and low GI luminal stability. Betula pendula (silver birch), Carthamus tinctorius (safflower), Robinia pseudoacacia (black locust), and Turnera diffusa (damiana) [R. pseudoacacia flowers by bees [Acacetin, an O-methylated flavone, is present in several medicinal plants, such as damiana) ,2,3. Predamiana) ,5,6,7,8. by bees . This ho by bees ,11.Despite their numerous health benefits, many flavonoids commonly exhibit very low oral bioavailability and insufficient levels in blood to produce therapeutic efficacy, which limits their successful clinical application . Regardi1/2), peak plasma concentration (Cmax), and area under plasma concentration versus time curve from time zero to time infinity (AUCinf) following the intravenous dose of acacetin in rats [According to a recent systematic study on the in vitro and in vivo metabolism of acacetin in rats , the domTherefore, we aimed to elucidate the factors that determine the oral bioavailability of acacetin, such as its physicochemical properties, GI stability, GI absorption, hepatic first-pass extraction, and systemic clearance, based on the concepts of mass balance and a well-stirred hepatic clearance model. This study provides quantitative biopharmaceutical and pharmacokinetic insights to facilitate the design of efficient oral formulations and the precise prediction of herb–drug interactions for acacetin.Acacetin (purity > 98%) and ketoconazole (KCZ) were purchased from Tokyo Chemical Industry Co. . Chlorpropamide (purity > 97%), quinidine (QND), sulfaphenazole (SPZ), α-naphthoflavone (NF), phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO), and polyethylene glycol 400 (PEG 400) were purchased from Sigma-Aldrich Co. . Nicotinamide adenine dinucleotide phosphate (NADPH), uridine diphosphate glucuronic acid (UDPGA), and rat liver microsomes  were purchased from Corning, Inc. . Pooled plasma  was purchased from Innovative Research, Inc. . The protocol for the animal studies conducted in this report was approved by the Institutional Animal Care and Use Committee at Pusan National University . Eight-week-old male Sprague-Dawley rats weighing approximately 250 g were purchased from DBL Co. . The rats were housed and acclimatized before use, as reported previously [v/v): started at 80:20 at 0 min, ramped from 80:20 to 10:90 for 0.5 min, maintained at 10:90 for 0.5 min, back to 80:20 for 0.5 min, and maintained for 2 min . The parameters related to the ion source were as follows: collision gas pressure, 1.5 mTorr; sheath gas pressure, 30 Arb; capillary temperature, 300 °C; vaporizer temperature, 300 °C; and auxiliary valve flow rate, 10 Arb. The method was validated according to the FDA guidelines [The concentration of acacetin in the samples obtained in this study was determined via a validated LC-MS/MS method using chlorpropamide as an internal standard (IS). The TSQ Vantage triple quadrupole mass spectrometer  was coupled to an HPLC  with the TurboIon interface operated in positive ion mode using selected reaction monitoring. Chromatographic separation was performed using a reversed phase column . Gradient elution of the mobile phase (consisting of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B)) was performed at a flow rate of 0.3 mL/min, and the procedure was as follows  sodium chloride, 0.32% (w/v) pepsin, and 0.7% (v/v) HCl . Simulated intestinal fluid (SIF) was prepared by dissolving 3 mM sodium taurocholate and 0.1% (w/v) pancreatin in phosphate buffer . The solubility of acacetin was measured by adding excessive amounts of acacetin into glass vials containing 1.5 mL of water, pH 7.4 phosphate buffer, SGF, and SIF. The mixtures were allowed to reach equilibrium in a shaking incubator  at 500 rpm at 37 °C for 48 h. The samples were centrifuged at 16,000× g for 5 min, and the supernatant was passed through a 0.20-μm syringe filter to remove excess acacetin. Further, the concentration of acacetin in the filtrate was determined using the LC-MS/MS method. The distribution coefficients of acacetin in water (log P) and pH 7.4 phosphate buffer (log D7.4) were determined using an octanol–water system at 25 ± 0.5 °C. The two phases were pre-saturated with each other by vigorous vortex-mixing for 15 min and further stabilized overnight for phase separation. A certain amount of acacetin (less than its solubility) was dissolved in the pre-saturated octanol, vortex mixed with the same volume of pre-saturated water for 1 h, shaken for 24 h, and further centrifuged at 16,000× g for 30 min. Finally, the acacetin concentrations in the octanol and water layers were determined using the LC-MS/MS method. The membrane permeability of acacetin and reference compounds (verapamil as a reference for high permeability and ranitidine as a reference for low permeability) was assessed by parallel artificial membrane permeability assay (PAMPA). The PAMPA was conducted using a commercially available PAMPA kit  according to the manufacturer’s protocol. The permeability was calculated using pION PAMPA Explorer ver. 3.8 software .Simulated gastric fluid (SGF) consisted of 0.2% (P), microsomes (fuMIC), SGF (fuSGF), and SIF(fuSIF) were measured using a rapid equilibrium dialysis device  as previously described with slight modifications [B) of acacetin was determined as described previously [g for 5 min to prepare the plasma sample. The concentrations of acacetin in the aforementioned plasma and buffer samples were measured using the LC-MS/MS method.The fractions of unbound acacetin in plasma , plasma, RLM, SGF, and SIF. The samples spiked with acacetin (10 μM) were incubated at 37 °C, and 120 μL aliquots of the samples were collected at 0, 15, 30, 60, 120, 180, 240, 360, 420, 600, and 1440 min. The collected samples were diluted with acetonitrile to obtain a proper concentration and analyzed by the LC-MS/MS method.v/v/v)) was administered intravenously (10 mg/kg) or orally (100 mg/kg) to rats. Approximately 250 μL of blood was collected via the femoral artery at 0, 2, 5, 15, 30, 45, 60, 120, and 180 min after intravenous administration and at 0, 2, 5, 15, 30, 45, 60, 90, 120, and 180 min after oral administration. The cannula was flushed with heparin  at each time point to prevent blood clotting. Following centrifugation of the blood sample at 8000× g and 4 °C for 2 min, 120 μL of plasma was obtained. Urine was collected at 1-h intervals in an ice-cold container over 24 h. After urine collection at 24 h, each rat was sacrificed by cervical dislocation; the entire GI tract (including its contents and feces) was excised, transferred into a beaker containing 100 mL methanol, and cut into small pieces using scissors. After manual stirring with a glass rod for 1 min, a 50-μL aliquot of the supernatant was collected. The plasma, urine (Ae24h), and GI tract (GI24h) samples were stored at −80 °C until further LC-MS/MS analysis.The rats were fasted overnight before the experiment and anesthetized using an intramuscular injection of zoletil at 20 mg/kg . The femThe in situ closed-loop study was performed as previously described with slight modification . Brieflyg for 30 min, and the supernatant fraction was stored at −80 °C until further use. The reaction mixture consisted of substrate, plasma, or tissue S9 fractions (protein concentration: 0.5 mg/mL) and NADPH (1 mM) or UDPGA (2 mM) in Tris buffer . After preincubation at 37 °C for 5 min, acacetin  was added to initiate the enzyme reaction. After 5 min of incubation with shaking at 37 °C, the reaction was terminated by mixing the reaction mixture with acetonitrile containing IS (volume four times that of the reaction mixture). After vortex-mixing and centrifugation at 16,000× g for 10 min, 100 μL of the supernatant was obtained and stored at −80 °C until further LC-MS/MS assay.The rat gut, spleen, kidney, liver, heart, brain, and lung were excised after cervical dislocation, rinsed with cold saline, blotted dry with tissue paper, and weighed. Each tissue sample was homogenized using a homogenizer  in cold 0.25 M sucrose solution. The homogenate was centrifuged at 9000× m and Vmax are the Michaelis–Menten constant and maximal metabolic rate, respectively. The unbound intrinsic metabolic clearance (CLint) was calculated as Vmax/(Km × fuMIC). Furthermore, the disappearance of acacetin (20 μM) in the absence or presence of specific CYP isoform-selective inhibitors in RLM was determined to study the hepatic CYP-mediated metabolism of acacetin. Microsomal incubation mixtures  were prepared as follows: RLM , 1 mM NADPH, 10 mM MgCl2, 50 mM potassium phosphate buffer, acacetin, with or without CYP isoform-selective inhibitors . At 0 and 5 min after initiating the metabolic reaction, a 100-μL aliquot of the incubation mixture was taken, transferred into a clean 1.5 mL microcentrifuge tube containing 100 μL cold acetonitrile to terminate the metabolic reaction, and finally treated in the same manner as described for the tissue S9 fraction samples.To investigate hepatic phase I enzymes- and UGT-dependent metabolism of acacetin, the disappearance rate of acacetin at concentrations of 1–200 µM in RLM was measured in the presence of both NADPH and UDPGA. The substrate (acacetin) concentration ((S); µM) versus the initial metabolic rate  profiles were interpreted based on Michaelis–Menten kinetics, and relevant parameters were estimated via nonlinear regression analysis  using the following Michaelis–Menten equation:inf, area under plasma concentration versus time curve from time zero to the last sampling time (AUClast), t1/2, total body clearance (CL), apparent volume of distribution at steady state (Vss), and absolute oral bioavailability (F) [max and time to reach Cmax (Tmax) were directly obtained from the plasma concentration versus time data.A noncompartmental analysis  was performed to determine the following pharmacokinetic parameters: the AUClity (F) . The Cmap-value < 0.05 was considered statistically significant, calculated using a t-test to compare the means of two groups or Tukey’s honestly significant difference test with a posteriori analysis of variance (ANOVA) to compare the means of more than three groups. Unless indicated otherwise, all data are expressed as the mean ± standard deviation, except for median (ranges) for Tmax, and they are rounded to three significant digits.A m/z 285.22 and 277.59, respectively. The prominent product ions were detected at m/z 242.17 and 175.04, respectively. The selectivity of the LC-MS/MS method was assessed by comparing chromatograms from blank rat plasma. Isocratic elution using the reversed-phase column resulted in relatively short retention times of 2.2 min for acacetin and 2.1 min for the IS. No interference peak was detected around the retention times of the analyte in the blank plasma traces, indicating the good selectivity of the present method. For the evaluation of linearity, the calibration curves for acacetin were plotted with eight concentration levels, which were linear within the concentration range of 0.5–2000 ng/mL. The representative calibration curve for acacetin was y = 0.00314x + 0.253. The correlation coefficients of the four calibration curves were ≥0.996, indicating decent linearity. The lower limit of quantitation (LLOQ) value for acacetin was 0.5 ng/mL, which could offer sufficient sensitivity for the present study on rat pharmacokinetics. The within- and between-run precision was evaluated using triplicates of quality control samples at four concentration levels  on three different analytical runs  > acacetin (8.06 × 10−6 cm/s) > ranitidine (0.411 × 10−6 cm/s). The protein binding of acacetin was relatively extensive in plasma, moderate in RLM, and low in SGF/SIF. An RB of approximately 1 indicated an equal distribution of acacetin between red blood cells and plasma.The physicochemical properties of acacetin, such as lipophilicity, solubility, permeability, protein binding, and blood distribution were experimentally determined; the relevant parameters are listed in compound ,29. Its The stability profiles of acacetin in phosphate buffers of pH 1–13, plasma, RLM, SGF, and SIF are shown in 1/2. The urinary and GI (including biliary) excretion of unchanged acacetin contributed to minor fractions (1.5–12.8% of dose) of the CL of acacetin in rats. This indicates that a major fraction of acacetin administered intravenously is eliminated by metabolic processes. Following oral administration, considerable fluctuations and variations in plasma concentrations of acacetin were observed during the whole period of blood collection. Thus, the AUCinf, t1/2, and F of acacetin could not be estimated from the plasma concentration versus time curves, due to the absence of a discernible linear terminal phase. Assuming that acacetin follows linear pharmacokinetics with a constant renal clearance, the urinary excretion of unchanged drug is directly proportional to its plasma AUCinf [The time profiles of plasma acacetin concentration following intravenous (10 mg/kg) and oral (100 mg/kg) administration in rats are shown in a AUCinf . Thus, tp = 0.806).The in situ absorption of acacetin in the GI tract was evaluated using a closed-loop assay. p < 0.026). Similarly, in all tissues except the plasma, the disappearance rates were significantly higher in the presence of UDPGA/alamethicin than in the control (p < 0.034). Thus, the phase I enzymes- and UGT-mediated systemic metabolism of acacetin occurs significantly in most tissues. Notably, the liver exhibited significantly higher metabolizing activity than all the tested tissues; thus, the metabolic kinetics of acacetin was further investigated in RLM. The concentration dependency for the disappearance of acacetin in RLM in the presence of NADPH and UDPGA/alamethicin is shown in m, Vmax, and CLint were estimated to be 187 ± 14 μM, 95.0 ± 5.6 nmol/min/mg protein, and 508 ± 9 μL/min/mg protein, respectively. Although the phase I metabolism of acacetin is known to be mainly catalyzed by CYPs [p = 0.021 and 0.001, respectively). This suggests that the CYP2C, 2D, and 3A subfamilies were involved in the metabolism of acacetin in RLM.Tissue-dependent differences in the systemic metabolism of acacetin were assessed by measuring the disappearance rate of acacetin for 5 min in plasma and various tissue S9 fractions in the absence  and presence of NADPH or UDPGA/alamethicin . In all  by CYPs , the typThe aim of this study was to investigate biopharmaceutical and pharmacokinetic factors responsible for the low oral bioavailability and high systemic clearance of acacetin. The pH-dependent stability test A showed max of 52.1 ng/mL. A previous study reported that acacetin exhibited considerably high bioavailability of 84% and Cmax of 1668 ng/mL following oral administration at 10 mg/kg [max of 17 ng/mL at 5 mg/kg in mice [max of 19.02 ng/mL at 1.4–1.676 mg/kg in rats [In the present rat pharmacokinetic study, the systemic exposure of acacetin following oral administration was observed to be very low and variable, resulting in a relatively low F of 2.34% and C10 mg/kg . However10 mg/kg , this re in mice  and Cmax in rats . Thus, t–6 cm/s) [The PAMPA permeability values of acacetin seem to lie approximately in the middle of those of verapamil and ranitidine. This suggests that acacetin may have moderate membrane permeability via a passive diffusion pathway. Rats have been recognized as a good animal model for predicting the GI absorption of drugs, particularly via passive diffusion, in humans ,34. As a–6 cm/s) , the MAD24h values between intravenous and oral pharmacokinetic studies. However, as this method is valid only when the drug is stable in the GI lumen for 24 h, it may be inadequate to use it for acacetin that has poor stability. Thus, it is difficult for us to estimate a reliable and accurate MAD or unabsorbed fraction of acacetin from the current data. In our present study, the GI absorption of acacetin was indirectly assessed by the in situ closed-loop study. It is important to note that both GI luminal degradation and GI absorption of drug occur simultaneously in the in situ loop state, whereas GI luminal degradation only occurs in the in vitro isolated loop state (control group). Thus, the real amounts of drug absorbed from the GI loop can be indirectly estimated by comparison of the remaining fraction observed in the in situ closed loop condition with that observed in the in vitro isolated loop condition. As shown in p = 0.806) between the in vitro isolated loop and in situ loop groups (78.9% vs. 76.0%), suggesting that only a small portion (2.9%) of orally administered acacetin was absorbed from the loop. Assuming that these data hold true for the whole GI tract, the fraction of acacetin unabsorbed from the GI tract following oral administration can be estimated to be 97.1% of the dose (=100 − 2.9). Consequently, the fraction of acacetin dose eliminated by GI and hepatic first-pass metabolism before reaching the general circulation can be determined to be 0.56% of the dose (= 2.9 − 2.34). Thus, it can be speculated that the relative contribution of GI absorption and first-pass metabolism to the low oral bioavailability of acacetin is 97.1% and 0.56% of the dose, respectively. This suggests that the limited GI absorption caused by poor stability and solubility is mainly responsible for the low oral bioavailability of acacetin at the dose of 100 mg/kg. However, it appears that this result can be changed when either other dose levels vehicle systems, or both, are used, which warrants further investigation. Additionally, the total blood clearance  of acacetin is 191 ± 34 mL/min/kg, which far exceeds the hepatic blood flow rate of 50–80 mL/min/kg in rats [The fraction of a drug unabsorbed following oral administration can be estimated by comparing the GI in rats ,22. As tCollectively, the present results imply that the poor oral bioavailability of acacetin could be a consequence of the interplay among its poor solubility, low GI luminal stability, and intestinal permeability. Thus, formulation strategies to improve these unfavorable physicochemical properties could be effective for enhancing the oral bioavailability of acacetin ,16,37. IThe physicochemical compatibility, GI absorption, systemic metabolism, and pharmacokinetics of acacetin were comprehensively evaluated using relevant rat models. Acacetin exhibited poor solubility and relatively low stability in pH 7.4 phosphate buffer, SGF, and SIF. Moreover, the absorption of acacetin in rat jejunal segment was limited, and its F was very low at 2.34%. The systemic metabolism of acacetin occurred ubiquitously in various tissues , resulting in very high CL. Thus, the poor oral bioavailability of acacetin could be attributed mainly to its poor solubility and low GI luminal stability. To our knowledge, this is the first systematic study on the biopharmaceutical and pharmacokinetic factors responsible for the oral absorption and systemic disposition of acacetin.


Australovenator wintonensis, from the Upper Cretaceous Winton Formation  of central Queensland, is the most complete non-avian theropod found in Australia to date. In fact, the holotype of A. wintonensis and isolated megaraptorid teeth  constitute the only theropod body fossils reported from the Winton Formation. Herein, we describe a new fragmentary megaraptorid specimen from the Winton Formation, found near the type locality of A. wintonensis. The new specimen comprises parts of two vertebrae, two metatarsals, a pedal phalanx and multiple unidentifiable bone fragments. Although the new megaraptorid specimen is poorly preserved, it includes the only megaraptorid vertebrae known from Queensland. The presence of pleurocoels and highly pneumatic caudal centra with camerate and camellate internal structures permit the assignment of these remains to Megaraptora gen. et sp. indet. A morphological comparison revealed that the distal end of metatarsal II and the partial pedal phalanx II-1 of the new specimen are morphologically divergent from Australovenator. This might indicate the presence of a second megaraptorid taxon in the Winton Formation, or possibly intraspecific variation.The holotype specimen of the megaraptorid  These are: Rapator ornitholestoides, known only from a metacarpal I P186153) . Our pre8.Australovenator (AODF 604). Additionally, morphological discrepancies between Australovenator and the new specimens maybe representative of either ontogenetic/intraspecific variation or indicative of the presence of a second megaraptorid from the Winton Formation. If correct, the latter interpretation adds further support to previous claims [This paper describes the fragmentry remains of only the second non-avian theropod skeleton recovered from the Winton Formation in Central Queensland, Australia. The remains, presumed to have come from a single individual, are assigned to Megaraptoridae indet. based on the presence of camerate and camellate internal structures and the presence of pleurocoels in caudal vertebrae. Given the size of the distal ends of metatarsal II (AODF 972) and IV (AODF 979), this individual would have been larger than the holotype of s claims ,34 that


Neurological disorders, including minimally conscious state (MCS), may be associated with the presence of high concentrations of reactive oxygen species within the central nervous system. Regarding the documented role of mesenchymal stem cells (MSCs) in oxidative stress neutralization, the aim of this study is to evaluate the effect of bone marrow-derived MSC (BM-MSC) transplantation on selected markers of oxidative stress in MCS patients. Antioxidant capacity was measured in cerebrospinal fluid (CSF) and plasma collected from nine patients aged between 19 and 45 years, remaining in MCS for 3 to 14 months. Total antioxidant capacity, ascorbic acid and ascorbate concentrations, superoxide dismutase, catalase, and peroxidase activity were analyzed and the presence of tested antioxidants in the CSF and plasma was confirmed. Higher ascorbic acid (AA) content and catalase (CAT) activity were noted in CSF relative to plasma, whereas superoxide dismutase (SOD) activity and total antioxidant capacity were higher in plasma relative to CSF. Total antioxidant capacity measured in CSF was greater after BM-MSC transplantations. The content of ascorbates was lower and CAT activity was higher both in CSF and plasma after the administration of BM-MSC. The above results suggest that MSCs modulate oxidative stress intensity in MCS patients, mainly via ascorbates and CAT activity. Traumatic brain injury (TBI), structural brain lesions, acute endocrine-metabolic disorders, neuronal dysfunctions, and psychogenic unresponsiveness, among many others, provoke consciousness disturbances ,2,3 whicConsciousness disturbances are accompanied by an elevated level of oxidative stress, manifesting a high concentration of reactive oxygen species (ROS). Excessive ROS production and losing the scavenging capacity of the antioxidant response system, lead to extensive protein oxidation and lipid peroxidation, which seem to be particularly dangerous in a lipid-rich content nervous system ,6,7. The2O2), which is removed by other enzymes: CAT and GPx [The natural defense mechanisms against oxidative stress described in the nervous system can be divided into two groups: low molecular weight antioxidants action and the antioxidant enzyme system ,16. The  and GPx .Although the prevalence of MCS has been estimated to be 112,000 to 280,000 adult and pediatric cases ,21 and aConsidering that recent investigations documented the ability of MSCs in oxidative stress neutralization and the wide range of MSC applications in regenerative medicine, it is hypothesized that autologous transplantation of bone marrow-derived MSCs (BM-MSCs) decreases the range of oxidative stress in patients in MCS. As far as it is known, this is the first study investigating the influence of BM-MSC therapy on oxidative stress in MCS patients.The study was conducted by the Department of Neurosurgery and Laboratory of Regenerative Medicine Stem Cells Bank, School of Medicine, Collegium Medicum, University of Warmia and Mazury in Olsztyn, Poland and the University Clinical Hospital in Olsztyn, Poland. The study was approved by the Bioethical Committee of the School of Medicine, University of Warmia and Mazury in Olsztyn, Poland . This study was performed in accordance with the Declaration of Helsinki. Nine patients aged between 19 and 45 years remaining in MCS for 3 to 14 months qualified for BM-MSC therapy. The characteristics of these patients are presented in n = 9) in local anesthesia from the posterior iliac crest.Approximately 200 mL of bone marrow were obtained from each patient . Cells were plated and expanded in a T-150 flask  and grown at 37 °C and 5% CO2 [The culture of purified BM-MSCs was prepared under aseptic conditions according to Good Manufacturing Practice (GMP) procedures . Briefly, a phosphate-buffered saline (PBS)-diluted  cell fraction of bone marrow was layered over a Ficoll density gradient , followed by centrifugation at 400 G at room temperature for 40 min. Nucleated cells were collected, diluted with two volumes of PBS, centrifuged twice at 200 G for 10 min, and finally resuspended in culture medium  was used to determine the MSC phenotype, according to MSC features established by the International Society for Cellular Therapy guidelines . Flow cy6 cells were injected intrathecally during the neurosurgical procedure by the neurosurgeon. The MSCs were administered to the patients three times every two months.Before transplantation, the cells were thawed in a basal culture medium without serum, detached, and washed three times with PBS 1× containing 1% human albumin . The number of cells was determined by analysis in a Bürker chamber  with Trypan blue  staining. A mean of 20 × 10The culture of purified MSCs was prepared under aseptic Good Manufacturing Practice conditions by the European Medicines Agency in 1999, where manufacturing facilities maintain a clean and hygienic manufacturing area in controlled environmental conditions. All manufacturing processes were clearly defined, controlled, and validated to ensure consistency and compliance with specifications. The laboratory has all the approvals and certificates required by Polish and European law.Immediately before MSC transplantation, 8 mL of cerebrospinal fluid (CSF) and up to 2 h before MSC transplantation, 8 mL of plasma sample were collected. CSF was collected from patients by lumbar puncture made by a neurosurgeon. Plasma and CSF collected before the first administration of MSCs provided control values for the analyzed parameters. The collected CSF and plasma samples were aliquoted and frozen at −80 °C for further analysis.Antioxidant capacity was measured in CSF and plasma collected from nine patients by examination of total antioxidant capacity , ascorbic acid and ascorbate concentrations , superoxide dismutase, and catalase activity , according to the manufacturer’s instructions. Peroxidase activity was measured by Peroxidase Activity Assay Kit  and methods described by Shannon et al.  and Maehp < 0.05). The analyses were performed using one-way ANOVA followed by Tukey’s multiple comparison test . All experimental data are shown as the mean ± SEM, and the differences were considered significantly different at a 95% confidence level (son test  or Stude (t-test .p < 0.05). In plasma, statistical differences in antioxidant capacity between examined groups were not found (p > 0.05). Antioxidant capacity was four-fold greater in plasma compared to CSF (p < 0.0001).Total antioxidant capacity, determined by evaluation of the ABTS  oxidation ability by active antioxidants in CSF a and plap < 0.05). The AA content was equalized to the control value after the second application of MSC (p > 0.05). In the plasma, the AA concentration was greater before MSC treatment compared to the content after the application of BM-MSC (p < 0.05). The AA concentration was three-fold greater in CSF towards plasma (p < 0.0001).The concentration of AA in CSF a and plap < 0.05). In plasma, the total concentration of ascorbates was lower after the first and second application of BM-MSC than before therapy (p < 0.05). The total content of ascorbates was two-fold greater in plasma compared to CSF (p < 0.0001).The concentration of ascorbates in CSF a and plap > 0.05) and plasma (p > 0.05). The SOD activity was greater in plasma in comparison to CSF (p < 0.0001).The activity of SOD in CSF a and plap < 0.05), whereas in plasma it was lower after the second application of BM-MSC in comparison to the first application (p < 0.05). The CAT activity was 200-fold greater in CSF in comparison to plasma (p < 0.0001).The CAT activity in CSF a and plaThe activity of peroxidase in CSF and plasma was measured for patients in MCS undergoing BM-MSC therapy. The results gained by using different protocols did not confirm the presence of active peroxidases in the examined material.Oxidative stress accompanies neurodegenerative disorders, causing neuronal dysfunctions and vascular damage ,44. The To select the potential mechanism of BM-MSC action involved in oxidative stress reduction in MCS, the concentration or activity of molecules regulating this process in CNS was examined. Among them was ascorbic acid (AA), which participates in neuromodulation ,50, exteThe analysis of AA concentrations in CSF from controls and patients with neurological disorders did not show significant differences ,65, whicThe current study found that AA concentration in CSF was significantly lower after the first autologous transplantation of BM-MSCs in comparison with the concentration before cell administration, which indicates that AA was intensively consumed in the CNS tissues in response to a BM-MSC application, and the level of oxidative stress accompanying MCS may be decreased in response to BM-MSC transplantation. Surprisingly, the effect of BM-MSC administration on AA concentration in CSF was compensated for after the second transplantation, which may be a result of a mechanism maintaining a high and relatively constant level of AA in the brain ,58. MoreAlthough the concentration of AA in CSF is relatively constant in patients with neurodegenerative disorders ,65, the The current study also comprises an analysis of the total reduction ability in CSF and plasma, involving measurements of the concentrations of AA with other molecules as electron donors neutralizing ROS. Although similar trends in total ascorbate content as in the case of AA concentration were observed, the influence of BM-MSC application on anti-oxidation capacity was weaker in CSF and stronger in plasma of patients in MCS. Furthermore, the current study found a three times greater content of AA and two times lower concentration of all electron donors in CSF in comparison to plasma, which indicates the importance of AA in CNS homeostasis. Moreover, among electron donors there are also anions, being transitional forms of AA, leading to DHA formation, which is a transport type of AA ,79. Incr2•- to H2O2. Catalase and GPx catalyze the conversion of H2O2 into oxygen and water. It is well known that three isoforms of SOD participate in the formation of the primary defense against ROS-mediated damage [The appropriate level of ROS in the CNS is also maintained by the enzyme system, including SOD, CAT, and GPx. The only known function of SOD is the dismutation of Od damage ,87,88. Id damage ,90. Althd damage ,91,92,93The analysis of oxidative stress in neurodegenerative disorders has shown a short-term effect of enzymatic antioxidant defense. Higher CAT activity and lower total SOD activity was detected in the plasma of patients within the first 24–72 h of acute ischemic stroke onset compared to healthy controls and returned to control values within five days ,95. Like2O2 in CSF of patients in MCS. These results are consistent with studies showing that CAT is more efficient in removing higher intracellular H2O2 concentrations, whereas at low concentrations this radical is disposed mainly by GPx [2O2 into harmless forms by GPx occurs with the concomitant oxidation of reduced glutathione (GSH) into the oxidized form (GSSG), and glutathione reductase recycles GSSG to GSH. Thus, it is supposed that the main limiting factor for GPx in scavenging higher amounts of H2O2 is the rate of recycling of GSH/thioredoxin, which is necessary to maintain the catalytic cycle [The results of the current study revealed increased CAT activity in CSF after the first and second BM-MSC transplantation compared to the control value. Surprisingly, GPx activity, known as a crucial antioxidant enzyme, oscillated at the limit of detection and did not allow reliable analysis to be performed or conclusions to be drawn. The observed changes may imply that CAT was the key enzyme responsible for catalyzing the decomposition of Hy by GPx ,100. Moric cycle ,102. On ic cycle . However2O2 degradation to rescue neurons from hypoxic conditions and revealed the highest turnover rates among known enzymes [2O2-mediated cytotoxicity by reducing cellular protein oxidation, DNA damage, loss of neuronal cytoskeleton structure, and plasma membrane integrity [2O2 produced not only by SOD but also by other cellular mechanisms [2O2 by CAT and GPx [It was documented that CAT may be an alternate source for oxygen upon H enzymes . Moreoventegrity . The incchanisms . Further and GPx , so we s and GPx . Moreove and GPx  and redu and GPx ; howeverThe qualification process of patients presenting disorders of consciousness (DOC) is still under development, and therefore, a number of diagnostic methods analyzing pathomechanism, clinical evaluation, and outcome predictions is required for accurate diagnosis . DespiteSummarizing, the presence of all examining antioxidants in CSF and plasma of patients undergoing therapy was confirmed, except GPx, both before and after BM-MSC transplantation. A greater content of AA and CAT activity was detected in CSF in comparison to plasma, suggesting the major role of these factors in maintaining the proper level of ROS in the CNS of MCS patients. Moreover, higher total antioxidant ability and lower AA concentration detected after BM-MSC transplantation than before therapy both in CSF and plasma, indicate that BM-MSC administration increased the capacity of CNS cells to ROS utilization and that the important tool of this action is intensively consumed AA. Furthermore, change in CSF:plasma ratio of AA concentration after BM-MSC transplantation indicates that during therapy AA sources were completed by the transport of AA from plasma to CSF. Moreover, CAT activity was changed after BM-MSC transplantation, which suggests that BM-MSC therapy for MCS patients also influenced the enzymatic antioxidant mechanism. The above study indicates the influence of BM-MSC therapy on the oxidative stress intensity in patients in MCS regardless of pathomechanism and clinical evaluation. These results confirm the hypothesis that BM-MSC transplantation is a promising therapeutic tool for neurodegenerative disorders. However, the results of ongoing clinical evaluations are needed to assess the effectiveness of BM-MSC application on the course of recovery in MCS patients.


We compared HIV-1 env characteristics and the gag cytotoxic T-lymphocyte (CTL) escape mutations from CSF and plasma samples. Employing population-based Sanger sequencing, we sequenced HIV-1 env from CSF of 25 patients and plasma of 26 patients. For gag, 15 CSF and 21 plasma samples were successfully sequenced. Of these, 18 and 9 were paired env and gag CSF/plasma samples, respectively. There was no statistically significant difference in the proportion of CCR5-using strains in the CSF and plasma, (p = 0.50). Discordant CSF/plasma virus co-receptor use was found in 2/18 pairs (11.1%). The polymorphisms in the HIV-1 V3 loop were concordant between the two compartments. From the HIV-1 gag sequences, three pairs had discordant CTL escape mutations in three different epitopes of the nine analyzed. These findings suggest little variation in the HIV-1 env between plasma and CSF and that the CCR5-using strains predominate in both compartments. HIV-1 gag CTL escape mutations also displayed little variation in CSF and plasma suggesting similar CTL selective pressure.HIV-1 compartmentalization in reservoir sites remains a barrier to complete HIV eradication. It is unclear whether there is variation in HIV-1  Early in the course of infection, the human immunodeficiency virus (HIV) can cross the blood-brain-barrier and infect the central nervous system (CNS) ,2. Once env , where X is any amino acid except proline.The V2, V3, and V4 loop sequences of HIV-1 gp120 were used to identify PNGSs using the N-Glycosite tool in the Los Alamos HIV sequence database . In this study, we could not do any human leukocyte antigen (HLA) typing; however, relevant epitopes with HLA restrictions were selected from the epitope database of Los Alamos HIV Immunology Database and used to identify variations within or flanking known CTL epitopes as well as compensatory mutations in the HIV-1 gag region. The epitopes analyzed were TL9, GL9, RM9, KK10, NY10, ISW9, KF11, TW10, and QW9. These epitopes are restricted by HLAs that have a combined prevalence of 26.2% in Botswana [HIV-1 Botswana .p-values < 0.05 were considered to be statistically significant. All analysis was performed using R version 3.6.0 [A McNemar test was used to calculate the difference in the proportion of CCR5-using strains in CSF and plasma. The difference in amino acid sequence length and PNGS between CSF and plasma was calculated using a Wilcoxon signed-rank test. The on 3.6.0 . 10 CFU/mL : 31–44) years. The majority (78.5%) of the individuals were ART-naïve with a median CD4+ T-cell count of 28 (IQR: 10–44) cells/uL. The participants had median plasma and CSF HIV-1 VL of 5.1 (IQR: 4.8–5.7) and 4.6 (IQR: 3.7–4.9) copies/mL respectively, median CSF white blood cell count of 10 (3.0–65.8) cells/μL and median fungal burden of 5.0 (IQR: 4.1–5.7) log0 CFU/mL .env sequences from plasma (red) and CSF (black), of which 18 were paired sequences and the rest were unpaired  and plasma (red) pairs  pairs . The CSFenv sequences from plasma and CSF respectively, and these sequences were analyzed for co-receptor usage. Of these, 21 (80.8%) plasma and 21 (84%) CSF sequences had CCR5-using strains and the remaining were CXCR4-using strains.We assessed the co-receptor usage in plasma and CSF using three different algorithms namely Geno2pheno, PSSM and 11/25 rule. We then concluded on the final tropism based on the three methods. From the 41 participants, we successfully sequenced 26 and 25 HIV-1 p = 0.5). CSF/plasma viral tropism discordance was found in 11.1% (95% CI: 0.1–3.4). The individuals who showed viral tropism discordance were both ART-naïve.The sequences obtained formed 18 pairs from plasma and CSF and the proportion of CCR5 using strains obtained using the three algorithms are reported in p = 0.32), p-value could not be calculated. We also compared the amino acid sequence length in the three variable loops and there was also no significant difference between the CSF and plasma .We compared the proportion of PNGSs in the three variable loops  in paired CSF and plasma samples. There was no significant difference in the number of V2 PNGSs between CSF and plasma  were conserved in all paired samples except for one sequence (ID: 1452) from CSF that had lost its glycan site. The PNGS for the V2 loop are provided in The PNGS at position 301 in the HIV-1 We compared polymorphisms in the significant positions of the HIV-1 V3 loop from CSF and plasma. We had an overall of 51 sequences with 26 from plasma and 25 from CSF. We evaluated the prevalence of mutations A316T . This is consistent with other studies showing that the majority of individuals with CM or advanced HIV disease harboured CCR5-using strains in the CSF and plasma despite the chronicity of the HIV infection or presence of CM [HIV compartmentalization in the CNS has been intensively described in ART experienced individuals due to a report of poor penetration of some ARVs through the BBB into the CNS leading to the emergence of discordant drug resistance mutations in the CNS and peripheral blood ,32,33,34ce of CM ,10 and wce of CM .The prevalence of CSF/plasma viral tropism discordance in our study was 11.1%. These individuals were ART-naïve; however, due to the small sample size, we could not establish implications of ART status on HIV discordance between the CSF and plasma compartment. Previous studies have also shown relatively little discordance in the two compartments and a slightly higher prevalence of CCR5-using strains in the CSF than in plasma ,36,37. Tenv gp120 V4 loop that is associated with increased sensitivity to neutralizing antibodies if the glycan at this position is removed [env V4 region was removed in CSF and plasma sequence in only one participant. There are conflicting data on whether the sequon for epitope 2G12 in the V4 region including the N392 is highly conserved or not due to one study showing high conservation while another study reported this site to be poorly conserved [Prior studies have shown a difference in the average number of glycosylation sites between plasma and CSF as a result of potential selection pressure from neutralizing antibodies . However removed . This lo removed . The D38 removed . Due to onserved ,42. In oenv V3 loop were conserved and were concordant in paired CSF/plasma samples. This is not surprising because as previously reported, glycosylation at this position is highly conserved rendering the virus resistant to broadly neutralizing antibodies by blocking epitopes that can be recognized by these antibodies [env V2 loop (N160) were conserved in all sequences except sequence 1452 from CSF. The removal of this glycan may disrupt the PG16 conformational epitope in the variable loop leading to a decreased neutralization activity of the PG16 antibody [All PNGSs at position 301 in the HIV-1 tibodies ,44. All env V3 loop in the CSF and plasma.Analysis of the HIV-1 V3 loop is important in identifying mutations that may contribute to resistance to entry inhibitors. Mutations A316T and I323V have been reported to contribute to Maraviroc resistance . The selgag CTL escape mutations can compartmentalize as a result of positive selection from the host CTL response [gag CTL escape mutations between the CSF and plasma suggesting similar CTL selective pressure. Mutation T242N in the TW10 epitope that is commonly known for reducing viral replication capacity was observed in only two sequences. However, compensatory mutations H219Q, I223V and/or M228L, which are associated with T242N mutation were observed in most of our sequences. The occurrence of these compensatory mutations in the absence of T242N may indicate the reversion of T242N to wildtype following transmission to an HLA-mismatched host [HIV-1 response ,21. Howehed host ,51. Our study was limited by a relatively small number of participants. A lack of a control  has also been a limitation of this study because we could not confidently conclude on the effects of CM on compartmentalization. Using Sanger-based population sequencing limited our ability to assess minority variants in the two compartments that might also contribute to viral compartmentalization. Therefore, we recommend the use of robust methods such as single genome amplification or next generation sequencing to look in-depth at these genetic variations.env derived from CSF and plasma, furthermore, the CCR5-tropic strains predominate in these two compartments even in participants with advanced HIV disease and HIV-associated CM. However, a relatively high proportion of strains harbouring Maraviroc resistance-associated mutations were observed in both compartments leaving a question of whether they will be susceptible to this drug or not. Our study, which was one of the few studies that investigated discordance in the HIV-1 gag CTL escape mutations between the CSF and plasma showed little variation in these mutations between the two compartments. Although our results were not significant, they show the importance of carrying out studies that may help to understand the implications of these CTL escape mutations on potential novel treatments, therapeutic vaccines, and HIV cure strategies.Our results suggest that there is little variation in HIV-1C


Surface electromyography (sEMG) is a widely used technology in rehabilitation research and provides quantifiable information on the myoelectric output of a muscle. In this perspective, we discuss the barriers which have restricted the wide-spread use of sEMG in clinical rehabilitation of individuals with spinal cord injury (SCI). One of the major obstacles is integrating the time-consuming aspects of sEMG in the already demanding schedule of physical therapists, occupational therapists, and other clinicians. From the clinicians' perspective, the lack of confidence to use sEMG technology is also apparent due to their limited exposure to the sEMG technology and possibly limited mathematical foundation through educational and professional curricula. Several technical challenges include the limited technology-transfer of ever-evolving knowledge from sEMG research into the off-the-shelf EMG systems, lack of demand from the clinicians for systems with advanced features, lack of user-friendly intuitive interfaces, and the need for a multidisciplinary approach for accurate handling and interpretation of data. We also discuss the challenges in the application and interpretation of sEMG that are specific to SCI, which are characterized by non-standardized approaches in recording and interpretation of EMGs due to the physiological and structural state of the spinal cord. Addressing the current barriers will require a collaborative, interdisciplinary, and unified approach. The most relevant steps could include enhancing user-experience for students pursuing clinical education through revised curricula through sEMG-based case studies/projects, hands-on involvement in the research, and formation of a common platform for clinicians and technicians for self-education and knowledge share. The current state-of-the-art rehabilitation for individuals with spinal cord injuries (SCI) utilizes technologies such as neuromodulation using exoskeleton robotics, functional electrical stimulation (FES), treadmill training with and without body-weight support (BWS) in addition to the traditional exercise-based rehabilitation. The recent developments in neurorehabilitation research and technologies have resulted in a shift in focus toward the recovery of function through high intensity repetitive training after SCI . Some ofSurface electromyography (sEMG), a non-invasive technique for assessing the myoelectric output of a muscle, can provide objective answers to these significant questions. sEMG has shown great promise in neurorehabilitation research and has been a widely-utilized tool to assess neuromuscular outcomes in research . HoweverSeveral barriers can be identified that restrict the adoption of sEMG technology in a clinical environment.Needle EMG (nEMG) and fine wire EMG (fwEMG) are the invasive forms of EMG for accessing neurophysiological attributes of neuromuscular diseases. However, the invasiveness, discomfort, and limited applicability of these techniques on multiple muscles during dynamic tasks limit their use in the clinic. Nonetheless, nEMG still is gold standard for clinical diagnosis of nerve and muscle pathologies and preferred over non-invasive sEMG  for neurA study collected perspectives of 22 clinicians  and reported limited clinician time as one of the barriers to the uptake of sEMG technology in clinics . The timMost of the PT and OT programs offer wide-ranging coursework in the rehabilitation domain including human anatomy, neuroscience, biomechanics, kinesiology, movement analysis, evidence-based practice, pharmacological interventions, etc. Irrespective of the breadth of topics covered, there is minimal focus on the technological aspects of rehabilitation. As a result, rehabilitation tools such as sEMG are theoretically taught, but practical knowledge imparted is limited. Further, the educational content may not cover ever-evolving aspects of sEMG technology and its applications. Feldner et al. reported that many clinicians felt less confident to use sEMG in clinics due to their limited experience . AccordiThe field of sEMG is always evolving and new algorithms for sEMG processing, analysis, and classifications are continuously being developed. However, the rate at which these technological advances are frequently integrated into the existing sEMG systems is limited. For instance, many of the existing off-the-shelf sEMG systems have not gone beyond implementing the basic sEMG features such as mean and root-mean-square (RMS) amplitudes, moving average or RMS envelopes, basic filtering and rectifications, and basic Fourier-based analysis. Automatic burst or ON-OFF detections, activation timing analyses, signal decomposition, and time-frequency analyses are widely published –25 and aIn addition to the sEMG setup time, other challenges hinder the adoption of sEMG technology in the clinic. Such barriers include the functionality in multiple environments, portability, the facility layout, purchasing cost and maintenance, providing evidence to support returns on such investments, and staff training.is the muscle active?” “what is the strength of the activation?” or more complicated ones such as, “what is the volitional contribution and how it relates to the applied stimuli during electrically induced activations?” Answers to such questions remain unclear as there is no standardized approach to first process and then interpret such data. The existing off-the-shelf systems are not specifically tuned to address these SCI-specific challenges. For example, the most significant barrier in using sEMG during FES is interpreting the recorded sEMG signals due to the overpowering presence of stimulation artifact. The stimulation artifact is a broadband signal with widespread stimulation frequency harmonics at high amplitudes that engulf the myoelectric responses in sEMG. Particularly when a train of ES pulses is applied, the sEMG recordings are accompanied by ES artifact spikes with magnitudes that are manifold compared to the actual MU outputs. Moreover, the presence of stimulation artifact is not confined in the time-domain; it is also observed in the frequency domain. The harmonics of stimulation frequency overlap with the majority of the energy bands in a typical sEMG frequency spectrum (20–350 Hz). As a result, traditional selected-filtering of frequency bands, to remove ES artifacts, is ineffective and results in significant data loss . Studies have demonstrated the presence of intact neuronal axons across the lesion, even after cSCI . For insata loss . The ES ata loss . In the Rehabilitation professionals' acceptance and adoption of technologies rely on conditions that facilitate their use such as scheduling, support and a conductive environment . The folThe Consensus for Experimental Design in Electromyography (CEDE) project, an international initiative which aims to guide decision-making in recording, analysis, and interpretation of sEMG have published the guidelines on the sEMG electrode selection and amplitude normalization . The interfaces running the EMG data collection and processing algorithms with minimal user inputs could be beneficial for their widespread implementations. Another goal could be set to successfully transfer EMG-related research products  into a clinical environment. Irrespective of the programming platforms  on which these algorithms are built upon, simple user-interfaces, application programming interfaces (APIs) and/or open-source executables can be created for their unobstructive and intuitive use by the clinicians with non-technical backgrounds. A centralized knowledge-base can be used to create and disseminate the sEMG tutorials on topics ranging from the basics of sEMG technology to step-by-step guidelines for data processing. Such a centralized open-source platform can also facilitate the collaborations among investigators and sEMG users with overlapping interests. With the help of well-established societies such as International Society of Electromyography and Kinesiology (ISEK), IEEE Engineering in Medicine and Biology Society (EMBS), Society for Neuroscience (SFN), and several societies of clinical motion analysis , the long-term goal can be set to developing international scientific meetings or chapters specific to sEMG applications in specific rehabilitation domain  where the specific pool of researchers can meet, share knowledge and collaborate. In recent years, efforts have been made to provide open-access tutorials and consensus articles on sEMG-related best practices, such as the consensus standards and guidelines on the sEMG detection , sEMG silization , 32. Desad hoc. This could result in more effective interventions for better long-term benefits.The instantaneous quantification of muscle response can serve as an important marker to track the impairment as well as recovery during rehabilitation. With access to the EMG in real-time, the clinicians or researchers can quantify, track, and manipulate levels of voluntary efforts by modulating intervention parameters. For example, if a clinician observes that the FES frequency of 100 Hz is causing a muscle to fatigue faster with less voluntary participation (shown by EMG features such as amplitude), s(he) could change to a lower stimulation frequency, which could potentially increase voluntary contribution and reduce fatigue due to stimulation, thus making the session still productive. Such modulations could happen simply by patient's own feedback on fatigue but the data-driven nature of this decision making could make the training more objective, patient-specific, safe and less Rank 1 – sEMG signal can be classified as no activity, baseline noise; Rank 2 – Sparse MU action potentials; Rank 3 – Burst of activity but no clear correlation to stimulation profile ; Rank 4 – Burst of activity with partial correlation to stimulation; Rank 5 – Repeated burst of visible activity that is significantly correlated with applied stimulation. Ranking procedures can be validated by visual inspection as well as automated, software-driven inspections. Such a standardized approach can track progress during or after different interventions. Once accepted and implemented, common standardized outcomes would enable comparing different interventions for efficacy.Motivated by the ranking system provided by Heald et al. , a standFor many of the SCI patients, functional or motor changes may not be present but electrophysiological changes or residual voluntary muscle activations may still be present , 33, 34.In summary, addressing the current barriers in widespread use of sEMG in SCI rehabilitation will require a collaborative, interdisciplinary, and unified approach. Nonetheless, sEMG technology has the potential to present significant opportunities that can allow clinicians and researchers to transform future interventions into effective and impactful rehabilitation modalities for individuals with SCI.rpilkar@kesslerfoundation.org.The data sharing will be contingent upon the regulations applied by the funding agency. Requests to access the datasets should be directed to The studies involving human participants were reviewed and approved by Kessler Foundation Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.RP and KM drafted the manuscript. All authors contributed to the revisions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rapid lethal exacerbation and recurrence featuring acute leukemoid reaction (ALR) after retrolaparoscopic radical nephrectomy (RN) is a relatively rare clinical incident. Performing the reoperation for the patient and analyzing the tissue-based genetic mutation information postoperatively are a skill-demanding and meaningful task, which have been even more rarely reported.We present a case with a large right renal mass (13.0 × 10.0 × 8.0 cm). This 71-year-old male patient underwent the retrolaparoscopic RN in our department. The operation was technically precise and successful with final pathological diagnosis of hybrid (clear cell and papillary type) renal cell carcinoma (RCC). However, 10 days after the patient was discharged, he was readmitted with the chief complaint of high fever with severe right flank pain. CT scanning revealed that right retroperitoneal hematoma and the blood routine showed the dramatic elevation of white blood cell count (WBC). Even though the immediate broad-spectrum antibiotics were administered without delay and subsequent percutaneous puncturing and drainage was performed, the patient’s condition still exacerbated rapidly. In spite of the reoperation of hematoma evacuation, the patient died of multiple organ failure 10 days after the reoperation. The pathological result of reoperation showed the necrotic and hematoma tissue blended with RCC tumor cells (nuclear grading III), and both of the postoperative tissue-originated comprehensive genomic profiling by using the specimens from the RN and reoperation respectively indicated significant mutations of some oncogenes which might have potential relevance with ALR. Besides, both of the immunohistochemical (IHC) staining results from primary surgical renal mass and reoperative resected tissue revealed the positive expressions of granulocyte colony-stimulating factor (G-CSF).ALR may be a predictor of poor prognosis in patients with RCC, and comprehensive genomic profiling as well as the alterative expression of G-CSF can help to provide potential valuable genetic etiological information and evidence for guiding the potential effective molecular-targeting therapy. The copied information was analyzed and reconstructed into the 3D-DM by using a postprocessing software named three-dimensional medical image reconstructing and guiding system . The retroperitoneal space along with critical anatomic structures including renal tumor, the relevant vasculature, the kidney, and the renal collecting system on the affected side were reconstructed and marked by using different colors simultaneously Fig. .The retrolaparoscopic RN was performed under the assisted navigation of 3D-DM at the Department of Urology. The right kidney along with the right adrenal gland was dissected and excised completely. The hemostasis was achieved carefully.9/L at the first day postoperatively, which further dropped down to 7.3 × 109/L 2 days later. The recovery course was uneventful, and the patient was discharged after 7 days postoperatively.The operative time was 1 h and 30 min, with no intraoperative complications happened. The estimated blood loss was about 20 mL. The size of resected tumor was 10.0 × 8.0 × 6.0 cm. The final pathological diagnosis was hybrid (clear cell and papillary type) renal cell carcinoma (nuclear grading III) Fig. . WBC cou9/L). At the same time, the body temperature was high (37.9 to 40.5 °C). ALR was established. He was treated with systemic antibiotics and the temperature decreased, but the WBC count still remained on high level. After the consultation of multi-disciplinary team and department discussion, we decided to perform the percutaneous puncturing and drainage for him after a 5-day systemic antibiotic therapy. Even though there was bloody drainage fluid with the amount of about 300 mL each day, the patient’s condition still exacerbated rapidly and the CT scanning result showed no significant shrinkage for the retroperitoneal hematoma  Fig. . Furtherphosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and epidermal growth factor receptor (EGFR)  which provides the service of genetic testing. Both of the postoperative tissue-originated comprehensive genomic profiling by using the specimens from the RN and reoperation respectively indicated significant mutations of several genes Fig. . There aGiven some cases of G-CSF-producing RCC with leukemoid reactions have been reported previously , 15, theRenal tumor is rarely associated with ALR; only few papers reported several sporadic cases before –14. ALR EGFR/PIK3CA pathway has been reported as an important regulating role in the progression of RCC [EGFR signaling pathway can retard the tumor progression [PIK3CA pathway is highly activated in RCC progression [Ataxia telangiectasia and Rad3 Serine/Threonine Kinase (ATR) responds to a broader spectrum of DNA damage and replication interference, including single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) adjacent to ssDNA, adducts, cross-links, and inhibition of DNA polymerase [protein kinase, DNA-activated, catalytic subunit (PRKDC), which is an important DNA-dependent protein kinase catalytic subunit, has been reported the over-expression in multiple human RCC tissues, and its inhibition via pharmacological inhibitors or siRNA/shRNA knockdown can significantly reduce the RCC cell proliferation in vitro and in vivo [Given the fact that RCC progression is usually accompanied with uncontrollable proliferation, distant metastasis, and recurrence, and the exact molecular mechanism of RCC is still unclear and needs to be further investigated, the genetic mutation analysis is becoming more and more important in the course of diagnosis and treatment for RCC. As for the outcome of postoperative tissue-originated comprehensive genomic profiling, the mutations of several vital oncogenes were indicated in this case. The n of RCC . The expn of RCC , and supgression . Moreovegression . Ataxia lymerase . This silymerase , 23. Bes in vivo .EGFR is due to polyploidy of chromosome 7. The EGFR copy number gain in tumor cells is associated with increased mutant allele transcription and gene activity. EGFR can activate the RAS/RAF/MEK pathway, which has a central role in the development and progression of cancer. A previous study reported that several growth factors including EGFR can induce G-CSF expression by a RAS/RAF/MEK-dependent mechanism, and the activation of the RAS/RAF/MEK signaling pathway triggered by EGFR can result in enhanced G-CSF expression and induce neutrophil recruitment significantly [EGFR, but the authors did not further explore the potential relevance between EGFR and leukemoid reaction in this case [EGFR induced the activation of RAS/RAF/MEK pathway and resulted in the upregulation of G-CSF, which caused ALR eventually. Besides, the PI3K/AKT/mTOR signaling pathway plays a central role in a wide spectrum of cellular activities, including cell proliferation, survival, and differentiation. Missense mutation of PIK3CA pathway components are involved in tumor development and occur in many cancer types. A previous study reported that inhibition of PI3K signaling decreases the production of proinflammatory cytokines including G-CSF [PIK3CA and G-CSF, inducing a leukocytosis. In this case, the high intratumoral G-CSF expression and secretion, interacting with many oncogenes including EGFR and PIK3CA, might play a central role in the induction of ALR and could be viewed as an adverse prognosticator.As for the relevance between ALR and our genetic findings, we found that the increased copy number of ficantly . Anotherhis case . In thisng G-CSF . Anotherng G-CSF . We specOn the other hand, we speculate that the carbon dioxide pneumoperitoneum may be a potential risk factor for the tumor recurrence in this case. It has been a controversial issue whether the laparoscopic pneumoperitoneum enhances the tumor metastasis. Many reports addressed different conclusions based on their own outcomes –32, and ALR associated with overexpression of G-CSF may be a predictor of poor prognosis in patients with RCC, and comprehensive genomic profiling can help to provide potential valuable genetic etiological information and evidence for guiding the potential effective molecular-targeting therapy.Additional file 2: Supplemental 2 Surgical videoAdditional file 1: Supplemental 1