P < 0.0001, Mann–Whitney U-test). These measurements demonstrate a change and imbalance in the biochemical environment of the optic nerve. Its possible effect is discussed.The pathophysiology of vision loss and loss of visual field in patients with idiopathic intracranial hypertension with papilloedema is not fully understood. Although elevated CSF pressure induces damage to the optic nerve due to stasis of axoplasmic flow, there is no clear relationship between the severity of papilloedema and CSF pressure. Furthermore, there are cases of purely unilateral papilloedema and cases without papilloedema despite significantly elevated intracranial pressure as well as papilloedema that can persist despite a successfully lowered intracranial pressure. We hypothesize that at least in some of such cases, in addition to purely pressure-induced damage to the optic nerve, the biochemical composition of the CSF in the subarachnoid space surrounding the orbital optic nerve may play a role in the pathogenesis of vision loss. In this retrospective study, we report on lipocalin-type prostaglandin D synthase concentrations in the CSF within the perioptic and lumbar subarachnoid space in 14 patients with idiopathic intracranial hypertension  with chronic persistent papilloedema resistant to maximum-tolerated medical therapy and visual impairment. CSF was collected from the subarachnoid space of the optic nerve during optic nerve sheath fenestration and from the lumbar subarachnoid space at the time of lumbar puncture. CSF was analysed for lipocalin-type prostaglandin D synthase and the concentrations compared between the two sites using nephelometry. The mean lipocalin-type prostaglandin D synthase concentration in the perioptic subarachnoid space was significantly higher compared with the concentration in the lumbar subarachnoid space (69 ± 51 mg/l without correction of serum contamination and 89 ± 67 mg/l after correction of serum contamination versus 23 ± 8 mg/l;  et al. report significant higher concentrations of lipocalin-type prostaglandin D synthase in the perioptic subarachnoid space compared with the lumbar subarachnoid space. These findings indicate that next to cerebrospinal fluid pressure gradients, biochemical gradients might play a role in the pathophysiology of optic nerve damage in idiopathic intracranial hypertension.Pircher   This disorder affects predominantly women of childbearing age. In addition to headache, clinical symptoms are mainly visual disturbances  and pulsatile tinnitus.3Papilloedema is defined as swelling of the optic nerve (ON) head that results from stasis of axoplasmic flow leading to an accumulation of extracellular fluid due to elevated CSF pressure within the subarachnoid space (SAS) of the ON. The exact mechanism by which papilloedema causes visual disturbances is not fully understood. Although several studies have demonstrated CSF pressure–related damage to the ON due to stasis of axoplasmic flow, there is no clear relationship between the severity of papilloedema and CSF pressure.4 have been reported, such as a case of purely unilateral papilloedema verified by optical coherence tomography5 and even cases without papilloedema despite significantly elevated ICP.6 In addition, papilloedema sometimes persists despite a successfully lowered ICP.7 These phenomena suggest that ICP is not the only factor that plays a role in the pathophysiology of visual disturbances in the setting of papilloedema. The visual impairment that occurs in patients with IIH often is reversible if the papilloedema regresses but can become severe and irreversible, primarily if the papilloedema persists.8 It is not clear why optic disc oedema can persist despite normalized intracranial CSF pressure nor which mechanisms are causative for irreversible visual disturbances in patients with IIH and papilloedema.Papilloedema in patients with IIH is usually bilateral and symmetric, but some cases of bilateral markedly asymmetric papilloedema9 Specifically, CT cisternography in these patients demonstrated diminished contrast-loaded CSF within the SAS of the intraorbital ON with a minimum of contrast in the bulbar region of the ON just behind the lamina cribrosa. This finding is referred to as an ON sheath compartment syndrome. Due to compartmentation, the CSF pressure as well as the composition of CSF may differ from that in other CSF spaces within the compartmented ON sheath. The SAS compartmentation of the ON might not only disrupt the supply of newly produced CSF but also prevent the washout of potentially toxic substances. We performed an ON sheath fenestration (ONSF) in a series of patients with IIH and persistent papilloedema to allow outflow of accumulated CSF within the compartmented SAS in the retrobulbar ON. We also collected and examined the CSF released during this procedure for lipocalin-type prostaglandin D synthase (L-PGDS).A recently published study applying CT cisternography demonstrated disturbed CSF flow along the ON in patients with IIH who had persistent papilloedema.10 It consists of 168 amino acids and belongs to the lipocalin superfamily. In the brain, L-PGDS mainly is expressed in the arachnoid membrane of the cells of the leptomeninges and the choroid plexus from which it is secreted into the CSF, although it also is expressed in oligodendrocytes and astrocytes.12 L-PGDS is a multifunctional protein involved in the regulation of various neurological processes. For example, altered L-PGDS expression levels in the brain are associated with various neurological disorders, including Alzheimer’s disease related and other types of dementia, multiple sclerosis, spinal canal stenosis and normal-pressure hydrocephalus13-17 and may play a role also in ocular diseases like open-angle glaucoma18 and normal-tension glaucoma.19 Recently, it has been demonstrated that L-PGDS is a major neuroprotective Aβ chaperone, suggesting that it plays a role in neurodegenerative processes.20 In non-neuronal tissues, the L-PGDS gene is expressed in the heart and in male genitalia and is present in body fluids like plasma, seminal fluid and urine.21 The concentration of L-PGDS is low (0.6 mg/l) in serum, and it therefore is used clinically for the diagnosis of suspected CSF leakage into the nasal cavity.22L-PGDS, also known as beta-trace protein, is a brain-specific glycoprotein and the most abundant brain-derived protein and second-most  abundant protein in the CSF.24 The current study compares the CSF concentration of L-PGDS between the lumbar and the perioptic SAS in a homogeneous cohort of patients with IIH and persistent papilloedema and visual impairment, in order to evaluate the biochemical milieu within the CSF surrounding the ON that might play a role in the pathophysiology of the optic neuropathy that can develop in patients with IIH and persistent papilloedema.In previous studies in patients with different ON disorders and in a non-homogeneous cohort of patients with papilloedema from various causes, we found an uneven distribution of L-PGDS between the CSF in the lumbar SAS and the CSF in the SAS surrounding the ON just posterior to the ocular globe.This retrospective study was approved by the local ethical commission  and follows the tenets of the Declaration of Helsinki.From 2005 to 2020, 23 patients with the diagnosis of IIH were admitted to the department of ophthalmology because of therapy-resistant papilloedema and progressive visual impairment  despite weight-loss attempts and maximum-tolerated therapy with systemic acetazolamide. Of these, 21 patients showed ON sheath compartmentation (reduced contrast agent in the SAS of the ON on cisternography), and 18 of 21 patients (3 patients refused) subsequently underwent ONSF. In 14 of the 18 patients, L-PGDS could be measured in both the ON and the lumbar SAS and were included in this study.1 Finally, all patients underwent CT cisternography  to assess CSF dynamics along the ON and to look for evidence of ON compartmentation. In cases with compartmentation, ONSF was offered and performed.All patients initially underwent a full neuro-ophthalmological examination including best-corrected visual acuity, VF testing, testing of pupillary responses to light stimulation, slit-lamp biomicroscopy and ophthalmoscopic assessment. All patients also underwent MRI and lumbar puncture (LP) to establish the diagnosis of IIH. LP in all patients was performed in a standardized lateral decubitus position with a 20-gauge needle. The diagnosis was based on the updated modified Dandy criteria.CSF was sampled from the lumbar SAS during LP (L3–L4) and from the ON during ONSF. The LP was performed in a standard way in the lateral decubitus position using a 20-gauge spinal needle. The ONSF was performed via a medial transconjunctival orbitotomy under general anaesthesia. Special care was taken not to use fluids to moisten the cornea after the orbitotomy so as not to dilute the CSF. Multiple incisions were made in the dura of the ON with a 19-gauge blade, following which a piece of dura was excised. After the first incision, a gush of CSF was observed, followed by further slow CSF egress. CSF then was sampled with a syringe using a 37-gauge needle and immediately deep frozen.19 Both concentrations, L-PGDS with (native) and without contamination in the perioptic SAS, are shown in CSF from both sampling sites (lumbar and ON SAS) was examined for the content of L-PGDS and albumin. L-PGDS was chosen due to its abundance in the CSF and its exceedingly low serum concentration to avoid the possibility of false-positive results that often occur as a consequence of the presence of serum during CSF sampling. Albumin was measured to assess the amount of potential contamination of CSF with serum during the ONSF in the CSF samples from the ON SAS. Both L-PGDS and albumin levels also were measured in serum in order to estimate the amount of contamination with blood during ONSF. The concentration without blood contamination was calculated based on the albumin concentration measured in the lumbar SAS.P < 0.05.Statistical analysis was performed using SIGMAPLOT 10.0 , Microsoft Excel 2010 , and the SPSS 21.0  for Windows statistical package. Data were analysed using the Mann–Whitney U-test because normal distribution was not given. All data are expressed as mean and standard deviation or median and interquartile ranges, respectively. Differences were considered significant when the error probability was The authors confirm that the data supporting the findings of this study are available within the article.Fourteen patients (mean age 45 ± 13 years), 13 female (44 ± 13 years) and 1 male (57 years), with IIH and persistent papilloedema underwent CT-assisted cisternography that demonstrated a markedly reduced concentration of contrast-loaded CSF compatible with ON compartmentation in one or both ONs . These p2O. In the remaining three patients, the lumbar CSF pressures were only documented to be >25 cmH2O and, therefore, were not included in the calculations. The clinical findings in the 14 patients in this study are detailed in All 14 patients had visual symptoms, including blurred vision, transient visual obscurations, VF loss or a combination of these despite attempted weight loss and maximum-tolerated doses of acetazolamide. Twelve patients had headaches (more pronounced in recumbent position) and one patient reported diplopia that was due to a sixth nerve palsy. The time from diagnosis to CT cisternography varied from 5 to 45 months; the mean time was 17 ± 10 months. The time from CT cisternography to ONSF varied from 4 to 57 days; the mean time was 24 ± 17 days. At time of cisternography, all patients were being treated with oral acetazolamide . MRI and CT demonstrated flattening of the posterior globe and distention of the ON sheath in all patients. In addition, three patients showed evidence of an empty sella. The mean body mass index was 33 ± 3. The mean visual acuity in the 14 patients using logMAR charts was 0.2. The mean VF mean deviation (MD) in seven of the patients using automated perimetry  measured −5.3  dB; in the other seven patients, VFs were assessed using a Goldmann perimeter which showed enlarged blind spots, inferonasal VF cuts or general VF constrictions. The mean CSF opening pressure in 11 of the 14 patients measured by LP was 39 ± 8 cmHONSF was performed in 18 of 28 eyes in the cases in which cisternography demonstrated a distinct ONS compartmentation. Four patients underwent bilateral ONSFs, whereas 10 patients underwent unilateral ONSF. For the patients in whom bilateral ONSFs were performed, one randomly selected ON was used for statistical analysis. The randomize function in Microsoft Excel 2010  was used to randomly select the included side.P < 0.0001 without correction of serum contamination; P < 0.0001 with correction of serum contamination; Mann–Whitney U-test; P < 0.0001, Mann–Whitney U-test). The mean L-PGDS concentration in the serum measured 0.6 ± 0.1 mg/l. The mean albumin concentration in the serum measured 40 400 ± 2355 mg/l.The mean L-PGDS concentration measured 23 ± 8 mg/l  in the lumbar SAS, 69 ± 51 mg/l  in the perioptic SAS without correction of serum contamination and 89 ± 67 mg/l  in the perioptic SAS after correction of serum contamination . There wThe present study demonstrates a significantly higher concentration of L-PGDS in the CSF in the perioptic SAS compared with the concentration in the lumbar SAS and evidence of ON compartmentation in 14 IIH patients with persistent papilloedema. This finding indicates a biochemical alteration in the CSF surrounding the retrobulbar portion of the ON, with CSF flow along the ON being at least partially discontinuous from the intracranial CSF circulation. We consider that the significant difference in concentration of L-PGDS in the lumbar SAS and the SAS surrounding the ON results from disturbed CSF inflow and/or outflow, increased perioptic production of L-PGDS, or both. L-PGDS is a brain-derived protein that was chosen as a marker of altered CSF. Due to its abundance in the CSF, it might be representative for other brain-derived proteins in the CSF.Well-regulated CSF flow is crucial for maintaining a continuous CSF exchange that allows the transport of nutrients to neurones, axons and glial cells as well as providing steady removal of potentially toxic waste products. Reduced perioptic CSF flow in the ONs of the patients in this study will therefore not only cause impaired inflow of newly produced CSF but also is likely to result in impaired removal of L-PGDS from the perioptic SAS with subsequent accumulation of L-PGDS as measured in the retrobulbar ON. This of course might not only apply to L-PGDS but also to other proteins, peptides, neurotransmitters and minerals in the CSF.25Papilloedema is a sequela of elevated ICP that is propagated via the optic canal into the perioptic SAS surrounding the orbital segment of the ON where it causes damage to the axoplasmic transport, leading to swelling of the ON axons and accumulation of extracellular fluid. However, in the 14 patients included in this series, the transmission of CSF pressure from intracranially to the eye is interrupted, which raises the question of what other mechanisms might be involved in optic disc swelling in these patients. We hypothesize that two mechanisms may be possible: (i) increased ICP that is partially transmitted along the ON despite the SAS compartmentation and (ii) increased mechanical pressure within the SAS of the ON due to compartmentation. These mechanisms also could explain the persistence of papilloedema despite lowered ICP due to weight loss and treatment with acetazolamide. Similar processes have been postulated to be responsible for the optic disc swelling and other ocular manifestations that occur in astronauts with ‘Spaceflight-Associated Neuro-ocular Syndrome’.27 Indeed, three of the five astronauts in the original NASA 2011 study presented with optic disc swelling and ONS asymmetry suggestive of compartmentation.26 Cephalad fluid shifts and the fragile flow equilibrium in the densely septated cul-de-sac-like anatomic connection between the intracranial and orbital SAS, coupled with the pressure-induced growth of meningothelial cells within the lining the ONS, may lead to a one-way, ball-valve-like CSF flow and the gradual sequestration of CSF within the SAS surrounding the orbital portion of the ON in these astronauts similar to the patients in this study.28 This sequestration could result in locally elevated pressures within the ONS and the accumulation of toxic substances that could impact local structures with or without elevated brain CSF pressures.28 Asymmetric, chronic ONS compartmentation, possibly in conjunction with toxic metabolites, may have set the stage for tissue remodelling of the ON head and posterior sclera. Interestingly, persistent posterior globe flattening has been documented in several astronauts years following long-duration space flight, again suggesting that toxic chemicals may cause permanent sclera remodelling.25Astronauts exposed to long-duration space flight have been documented to have optic disc oedema, globe flattening, choroidal folds and hyperopic shifts in refraction. This combination of findings has been explained as being due to a relative rise in ICP due to microgravity-related reduced venous outflow during long-duration space flight and compartmentation of CSF within the orbital SAS both during and after the flight.29 A possible scenario therefore could be that the elevated lumbar CSF pressure measured in patients with IIH and transmitted to the SAS of the ON leads to proliferation of meningothelial cells and, thus, to a narrowing and compartmentation of the ON SAS. Unlike the ICP, the exact pressure in such an orbital ON compartment is not known. Due to the small and complex anatomy of the SAS of the ON, reliable CSF pressure measurements in the orbital ON SAS are not yet possible; however, the enlarged ONS diameters in the retrobulbar ON seen on CT scans in our patients  production.Next to toxic effects of L-PGDS, neuroprotective effects of L-PGDS are known as well. Recently, an 37 As the albumin concentration normally is much higher in serum than in CSF, the significant difference in albumin concentration between the lumbar SAS and the perioptic SAS in our patients is most likely due to contamination with blood during ONSF. The concentration of L-PGDS is, however, extremely low in serum. Thus, false-positive results caused by serum contamination are highly unlikely.The albumin concentrations in both the lumbar SAS and the serum of the patients in this study are similar to those reported in several other studies.3 Considering the large amount of CSF proteins that are recycled with a frequency of up to five times a day, we hypothesize that stagnant CSF with an accumulation of toxic components also might inflict damage to axons and glial cells of the ON, thus leading to visual disturbances. This hypothesis may be especially true for patients with persistent papilloedema, such as those in this study, as exposure time might play a crucial role.Clinical observations in patients with IIH suggest that in addition to purely pressure-induced damage to the ON, other pathophysiological mechanisms may be involved, among them ischaemic damage.Visual impairment in IIH patients often is reversible. The reversibility may be explained by the recovery of the obstructed axoplasmic flow when CSF pressure decreases. However, chronic papilloedema can also lead to irreversible VF constriction and, eventually, permanently reduced visual acuity. This is the case in pressure-induced ischaemia where the axons of the ON might be irreversible damaged, and recovery of visual symptoms is not possible. We hypothesize that a similar scenario may also occur as a consequence of neurotoxic substances that accumulate in the SAS surrounding the orbital portion of the ON. These substances could damage directly the integrity of ON axons, damage the vessels comprising the pial plexus of the ON leading to ON ischaemia or both. This, in turn, raises the question as to whether in IIH patients with persistent papilloedema, ONSF might be preferable to shunt procedures or stenting as it treats the problem more locally. All three procedures lower ICP, but only ONSF potentially could release stagnant CSF containing toxic substances from around the ON.This study has several weaknesses. Firstly, there is no control group for obvious ethical reasons. However, a homogeneous distribution of particles (molecules and proteins) in a closed fluid system is the normal state according to the second law of thermodynamics. Secondly, the number of subjects in this study is small. Thirdly, the mean time difference between LP and ONSF was 24 ± 17 days, and we do not know if the CSF concentration of L-PGDS changed during this time. Fourthly, all patients had therapy-resistant papilloedema and severe ON-related visual disturbances and, thus, represent a specific population of IIH patients. Finally, we assessed only the concentration of L-PGDS in the lumbar and perioptic SAS. Ideally, the concentrations of other potentially toxic substances should be studied. Nevertheless, this study demonstrates a difference in the biochemical milieu between the lumbar SAS and the SAS of the ON in IIH patients with persistent papilloedema. Alteration in the biochemical content of CSF, in addition to pressure-induced ON damage, may play a role in the pathophysiology of the optic neuropathy that can occur in these patients and raises a question regarding the optimal treatment of such patients.


In this study, the effects of programming and the addition of SiO2 particles to the thermodynamic properties of the specimens were detected using differential scanning calorimetry (DSC), the optimal shape memory programming temperature of which is 72.9 °C. Combined with scanning electron microscopy (SEM) and shape memory effect test, the particles are evenly distributed between the two phases, and the shape fixation rate (Rf) of 98.15% and the shape recovery rate (Rr) of 97.31% show that the composite has a good shape memory effect. Fourier transform infrared spectroscopy (FTIR) and dynamic infrared dichroism illustrate the change of the hydrogen bond of soft and hard segments with the SiO2 particles in the shape memory cycle, revealing the optimal shape memory programming process. This study provides an insight into the reinforcement mechanism of SiO2 nanoparticles in SMPU matrix and verify whether it can meet the engineering requirements of expansion joints when used as a sealant of concrete pavement.Expansion joint failure is one of the main causes that lead to the damages of concrete pavement. The silicon dioxide/shape memory polyurethane (SiO Cement concrete pavement has become one of the main paving types of high-grade pavement due to its high strength, good stability and long service . Most ofNowadays, many kinds of sealants have been used to seal expansion joints on concrete pavement, such as hot-poured, cold-poured or preformed. Due to the damages of the existing sealants (cohesive and adhesive failure), more and more new materials are being researched . Among tHowever, there is a thermodynamic incompatibility between soft and hard segments in SMPU due to their different chemical constituents. This incompatibility causes the segregation between the soft and hard segments in SMPU, and hard segments are mutually aggregated. As a result, two phase structure is formed in SMPU, including a soft segment region (soft segment phase) and a hard segment region (hard segment phase), which is the microphase separation in SMPU .At the same time, the chemical connection between soft and hard segments reduces macro phase separation in SMPU. The sizes of hard segments in SMPU are usually from 5 nm to 100 nm with fibrous, spherical, cylindrical and plate shapes. In addition to covalent interactions, there are non-covalent interactions between the hard segment chain and/or the hard segment region in SMPU, such as hydrogen bonding, dipole interaction, etc. . Under tIt was known from the shape memory mechanism of SMPU that the phase separation microstructure is constantly changed during the shape memory effect cycle . Zhang eAdditionally, Priyanka et al.  investig2 nanoparticles on thermal behaviors, shape memory effect, interaction among microphase structure and molecular orientation of porous SMPU matrix were not discussed.In our previous study , we foun2 nanoparticle is a three-dimensional chain structure, which interacts with the electron cloud of SMPU matrix. A network structure is formed in SMPU by the effective entanglement based on physical reaction, which is conductive to improve the mechanical properties, aging resistance and thermal stability of SMPU. Therefore, properties of SiO2/SMPU nanocomposite were paid more and more attention [It was found that the addition of particle reinforcement could enhance mechanical performance and shape recovery force of SMPU . Among tttention .2/SMPU composite and found that it had great shape memory effect and mechanical properties when SiO2 particle content was 15%. Zhang et al. [2 particles on the surface could effectively enhance UV resistance, thermal stability and interfacial shear strength. Yousefi et al. [2/SMPU nanocomposite. Huang et al. [Shi et al.  preparedg et al.  pointed i et al.  optimizeg et al.  used the2 nanoparticle on thermal stability, shape memory performance, interfacial microphase structure and molecular orientation of SMPU were rarely investigated. Further, the filling effects of SiO2 nanoparticles on molecular orientation of porous SMPU matrix as well as the interaction between SiO2 nanoparticles and SMPU were seldom reported. As a result, it is difficult to reveal the reinforcement mechanism of SiO2 nanoparticles and shape memory mechanism of SiO2/SMPU nanocomposite. The objective of this study is to better understand thermal behavior and shape memory effect of SiO2/SMPU nanocomposite used as a sealant for expansion joint on concrete pavement, as well as characterize changes in interfacial microstructure and molecular orientation of SiO2/SMPU during a shape memory cycle. This provides an insight into the reinforcement mechanism of SiO2 nanoparticles on SMPU matrix and the shape memory mechanism of SiO2/SMPU nanocomposite, which further explain that programmed sealant can better adapt to the engineering application requirements of expansion joints in a working environment that meets the road temperature.Currently, it is noted that influences of SiO2/SMPU nanocomposite was synthesized by the in-situ polymerization method. The glass transition temperature (Tg) and the phase transition temperature (Ttrans) of SiO2/SMPU nanocomposite was determined by DSC tests, further confirming whether the Ttrans meets the working requirements. Rf and Rr were calculated to evaluate shape memory effect of SiO2/SMPU nanocomposite. Then, FESEM was used to observe the microscopic morphology changes of original, programmed and recovered SiO2/SMPU nanocomposites, respectively. After that, FTIR and dynamic infrared dichroism tests were conducted to discuss microstructure changes at the interface between SiO2 nanoparticles and SMPU matrix, as well as molecular orientation changes of original, programmed and recovered SiO2/SMPU nanocomposite, revealing the reinforcement mechanism of SiO2 nanoparticles on SMPU matrix and the shape memory mechanism of SiO2/SMPU nanocomposite. Generally, the highest temperature of pavement in summer as the response temperature of SiO2/SMPU nanocomposite sealant, it realizes the self-healing of the sealant by recovering to the original shape in presence of an external stimuli, which greatly prolongs the service life and reduces the maintenance cost.In this study, SiO2 powder with the particle size of 16 nm .Main materials used in this study include polyadipate-1,4-butanediol diol , toluene diisocyanate ), 1,4-butanediol , and SiO2/SMPU nanocomposite was prepared through uniformly dispersing 15% by volume of SiO2 nanoparticles in SMPU matrix. SiO2/SMPU nanocomposite was synthesized by in-situ polymerization. The calculated amount of PBAG was placed in a 250 mL 4-neck round-bottom flask, which was equipped with a thermometer, a mechanical stirrer, and nitrogen inlet and outlet tubes. PBAG was dehydrated at 120 °C for 1.5 h under vacuum environments (vacuum degree > 0.095 MPa). When the temperature was lowered to 80 °C, a calculated amount of TDI was added. Under the protection of nitrogen, the temperature was maintained at 80 °C for 2 h to obtain SMPU prepolymer.SiO2 nanoparticles were added into the prepolymer. The mixture was stirred using a mechanical stirrer for about 10 min for uniform dispersion of SiO2 particles. After that, when the temperature was lowered to 70 °C, the required amount of chain extender of BDO was added dropwise into the mixture which was rapidly stirred for 30 min. The synthesized SiO2/SMPU nanocomposite was immediately injected into polytetrafluoroethylene molds where the homogeneous mixture was cooled to room temperature and cured. Thus, a SiO2/SMPU nanocomposite sample was obtained after demolding. Finally, this sample was subjected to various property characterizations. The schematic diagram of chemical structures of molecular segments in SiO2/SMPU nanocomposite was shown in Then SiO2/SMPU composite. About 15 mg SiO2/SMPU sample was heated from −40 °C to 200 °C at a heating rate of 10 °C/min in the nitrogen environments. After that, SiO2/SMPU sample was cooled from 200 °C to −40 °C at a cooling rate of 20 °C/min, and then was heated to 200 °C at the heating rate of 20 °C/min. DSC test results in the second heating process were obtained to analyze thermal properties of SiO2/SMPU composite through eliminating the thermal history. This experiment was repeated three times.DSC  was utilized to discuss the thermal behaviors of soft and hard segments and determined the transition temperature (Ttrans) of SiO2/SMPU nanocomposite with shape memory effect, it was usually subjected to a typical five-step thermodynamic cycle for programming and free recovery as shown in To endow SiO2/SMPU composite with shape memory effects, it was usually subjected to a typical shape memory cycle called programming and recovery.To endow SMPU and SiO2 contents on shape memory effects of SMPU, the prepared pure SMPU and SiO2/SMPU specimens which were first machined into dog bone fixed on the fixture separately, and then placed in a heating chamber without any loading. Meanwhile, the chamber was heated to Ttrans and held for 20 min to make the temperature distribute uniformly in the sample.To evaluate influences of SiOOn this basis, the sample was taken out after programming for 2 h, and cooled to room temperature. A continuous load is required during the entire process of programming and cooling, then the load was removed and put the sample at room temperature for 24 h.trans to recovery for two hours. The pre-strain of programmed specimen reached 25%, and the shape fixity ratio (Rf) and shape recovery ratio (Rr) were calculated to evaluate shape memory effects, which were expressed as follows.l0, l1, l2 and l3 are the tagged middle lengths of original, programmed with loading at room temperature, programmed without loading at room temperature and recovered specimens, respectively.Finally, the sample stood for 24 h was put back without any loading in the chamber from the room temperature to T2/SMPU samples were characterized using FESEM , respectively. Samples were first fixed on an aluminum stub and sputtered with gold under vacuum conditions. The 10 mm × 10 mm × 10 mm cross-sections samples cut from the pure SMPU and SiO2/SMPU specimens in different states were first fixed on an aluminum stub and further sputtered with gold under vacuum conditions. Then, the sample chamber was opened to place samples. Finally, the morphologies of the samples were observed using FESEM.Morphology changes of original, programmed and recovered SiO2/SMPU samples, respectively. The sample was prepared by KBr tableting method, and a small amount of KBr crystal was thoroughly ground using an agate mortar. Then, the tested solid sample was added by 5%, and mixed until the mixture was uniform. Finally, the powder was transferred to a metal mold, and placed in a hydraulic press for tableting, and then FTIR tests were performed.To study the changes of molecular segments during a five-step thermodynamic cycle, FTIR tests were conducted on the original, programmed and recovered SiO2/SMPU nanocomposite in the original, programmed and recovered states, respectively, Fourier transform infrared spectrometer  was used to perform dynamic infrared dichroism tests. The spectral resolution was 4 cm–1, and each spectral line was the average of 32 scanning values. SiO2/SMPU samples were ground to light permeation, respectively. The orientation function value (f) of the particular represented bond was calculated according to the following Equation (3) to further quantitatively analyze shape memory property of SiO2/SMPU nanocomposite [f was orientation function, α was the angle between the direction in oscillating transition moment and molecular chain, and R was the dichroic ratio of measured particular absorption band, and its value was calculated based on Equation (4).∥ and A⊥ represented the infrared spectrum absorption intensities which were parallel and perpendicular to the stretching direction of SiO2/SMPU specimen during the programming, respectively.To further verify the changes in molecular orientation degree of prepared SiOomposite .f = . Because the SMPU is a kind of typical microphase separation structure polymer [Ms, the molecular kinergety of soft phase in SMPU is too low to overcome the rotation barriers within the main chains [Dm (around 72.9 °C), which can be seen as a phase separation temperature which short hard segments segregate from the mixed phase and aggregate into the micro-hard phase [Mh), which shown obvious endothermic peaks near 164.5 °C, the specimens gradually fail to maintain its morphology, it is indicated that the soft segments provide elastomeric features, whereas the hard segments act as reinforcing components and provide dimensional stability [It can be deduced from  polymer , when thn chains ; thereford phase . As the tability .2/SMPU composites, the Tms shift to the left with the SiO2 particles adding, this is due to the fact that the addition of SiO2 affects the symmetry of the soft segment molecular chain, and the hydrogen bonds forming with the soft segment increase the binding force among the soft segments [ms both decreases, Simultaneously, the exothermic peak area of the SiO2/SMPU composite is slightly larger than pure SMPU, which is due to the added SiO2 particles form hydrogen bonding with the soft segment, and with some hard segments, in order for some short hard segment molecular chains to be pulled by hydrogen bonds and separated from the mixed phase [2, so that the peak end of the melting endotherm of the hard segment does not move.Compared to the pure SMPU and SiOsegments . As crysed phase . Howevertrans.When the programming is performed at a temperature higher than that which corresponds with the exothermic peak of 72.9 °C, the hydrogen bonds between the soft and hard segments are reduced during the heating process as a result. Without the strong restriction of the hard segments, the soft segments can possess greater agility and lower stiffness , and thaMh are almost constant, the TMs shifts slightly to the left, and at the same time, the exothermic peak which represents the phase separation movement gradually disappears. Compared with the pure SMPU and the composite material after programming, it can be observed that due to the addition of SiO2 particles, the microphase separation of the material is hindered, which means that the soft segment cannot be completely distorted and requires further verification by the shape memory effect.Based on the premise that the T2/SMPU specimens, respectively. Test results of specimen lengths, calculated Rf and Rr of pure SMPU and SiO2/SMPU nanocomposite are summarized in 2/SMPU specimens are elongated in the stretching direction. Then, when the temperature is raised to Ttrans °C and maintained for 30 min, the two specimens are almost restored to their original lengths after the free recovery process. This is because the elastic strain energy is prestored in the programmed SMPU and SiO2/SMPU specimens during the programming, thus providing a driving force for the thermally induced SMPU and SiO2/SMPU specimens to restore to their original shapes.It is seen from f and Rr of SMPU are 100%; however, the average values of Rf and Rr of SiO2/SMPU nanocomposite are 98.15% and 97.31%, respectively, which are slightly less than 100%. The reason for this is that the mixture and microphase separation are directly affected by the crystallization of pure SMPU matrix. However, the addition of SiO2 nanoparticles lowers the orderliness of microcrystals and paracrystals in SMPU. Crystal imperfections are increased to slightly lower the shape memory effect of SiO2/SMPU nanocomposite [2 nanoparticles cause the damages and deformation of formed stable network in SMPU during the programming and recovery. However, the prepared SiO2/SMPU nanocomposite still shows an excellent shape memory performance.As shown in omposite . Another2 nanoparticles and the microstructure differences among original, programmed and recovered SiO2/SMPU nanocomposites, the 2/SMPU nanocomposites.In order to discuss the distribution of SiO2/SMPU nano composite can be found in 2 nanoparticles is more evenly distributed in the two-phase structure of SMPU. Further compared between 2 nanoparticles are distributed at the specimen surface where some of the protrusions are present. These apparent protrusions are due to the aggregation of soft segments and hard segments to form respectively their own microdomains, indicating that SiO2/SMPU shows crystallization [2 nanoparticles with different sizes also indicate that the appropriate filling amount can be formed by electrostatic or van der Waals force to form a moderately sized particle size, thereby effectively filling part of the pores formed during the SMPU reaction. [The microstructure of the pure SMPU and SiOlization . The SiOeaction. 2/SMPU sample has been subjected to unidirectional horizontal stretching at Ttrans °C, and molecular segments are oriented along the stretching direction. The molecular chain length is increased under the influence of micro-Brown motion. Some step-like pleats are seen on the surface of programmed SiO2/SMPU. This is because there are obvious differences in the modulus between soft and hard segments, and their borne loads are different. Additionally, it is worth noting that some segments are damaged, suggesting that there are microscopic losses during the five-step thermodynamic cycle.As shown in 2/SMPU sample. This is because molecular chains in soft segments are restored to original curled states after experiencing a free recovery process at Ttrans °C. As a result, the step-like pleats on SiO2/SMPU sample disappear.From 2/SMPU nanocomposite during a five-step thermodynamic cycle  on FTIR spectra are discussed as shown in To further characterize orientation changes of soft and hard segments in original, programmed and recovered SiO2/SMPU samples are basically similar, the intensities of stretching vibration peaks at 1645 and 1638 cm−1 become weaker, and two new stretching vibration peaks at 1636 and 1629 cm−1 appear on FTIR spectra of programmed SiO2/SMPU sample.It is observed from −1 are assigned to the short-range disordered and long-range disordered hydrogen bonds between soft and hard segments, respectively [−1 are attributed to the orderly hydrogen bonds in hard segments of SMPU [2/SMPU specimen, thereby increasing the number of hydrogen bonds, and changing synchronously the hydrogen bond type [Among them, the absorption peaks at 1645 and 1636 cmectively . However of SMPU . The posond type .−1 almost disappear on FTIR spectra of recovered SiO2/SMPU, while the intensities of stretching vibration peaks at 1645 and 1638 cm−1 are higher than that of programmed SiO2/SMPU, but lower than that of original SiO2/SMPU. This is due to the fact that orientated molecular segments are restored to freely curled states during the recovery of SiO2/SMPU nanocomposite. Thus C=O groups are restored to free state because of some hydrogen bond cleavages, leading to the increase in stretching vibration peak intensity of C=O in the freely vibrating amide at 1680 cm−1 [As shown in 680 cm−1 .2/SMPU samples. 2/SMPU nanocomposite [To further study the relationship among intermolecular or intramolecular hydrogen bonds of characteristic functional groups, the hydrogen bond changes of characteristic functional groups in hard segments are discussed according to FTIR spectra of SiOomposite .2/SMPU nanocomposite system, including N–H in urethane, –OH in SiO2 and –OH in PBAG, respectively, as well as two proton acceptors, namely C=O in urethane and C=O in 1,4-butanediol ester [It is seen from ol ester . The ureol ester .N–H) and C=O (RC =O) groups in the SMPU are calculated using Equations (5) and (6). 2/SMPU [The stretching vibration regions of urethane N–H and C=O groups are commonly used to analyze the nature of the formed hydrogen bonds . The hyd. 2/SMPU .RN–H = N-H of programmed SiO2/SMPU is decreased, while RC=O is increased when compared with that of original SiO2/SMPU. The reason for this is, on the one hand, some new hydrogen bonds are formed between SiO2 nanoparticle and molecular chain segments as soft segments are stretched during the programming of SiO2/SMPU.As shown in 2/SMPU and don’t participate in the formation of hydrogen bonds, are orderly rearranged as the stretching strain is increased during the programming of SiO2/SMPU. Therefore, these urethane segments are combined with hard segments to form hydrogen bonds, entering into hard segments and bringing ordered structures in hard segments [On the other hand, some urethane segments, which are dispersed in the curled soft segments of original SiOsegments .At the same time, some hydrogen bonds formed by –NH groups in urea and C=O groups in PBAG are reduced. This is because some original hydrogen bonds are broken as molecular chains move during the orderly rearrangement of soft segments from naturally curled state to ordered structure along the stretching direction. However, the hydrogen bonds between –NH and C=O groups of amides in hard segments are increased. This is due to the fact that –NH groups move toward hard segments and combine with free-vibrating C=O groups of amides in hard segments to form hydrogen bonds as the stretching strain is increased .2/SMPU nanocomposite, while the stretching vibration peaks of hydrogen-bonded C=O groups in the amide move towards to lower wavenumber at 1645 and 1638 cm−1 as the hydrogen bond number is changed. These are consistent with the test results of FTIR.Thus, the total number change of hydrogen bond in –NH group is not obvious, but the total number of hydrogen bonds of C=O groups in hard segments is increased. As the hydrogen bond interaction among hard segments is enhanced, it is more conducive to the separation of microphase structure, thus effectively optimizing its mechanical properties . The cha2/SMPU nanocomposite during a five-step thermodynamic cycle , f (1645), f (3338) and f (2943) at above characteristic absorption peaks are calculated as shown in To further quantitatively discuss the orientation of main molecular chains, such characteristic absorption peaks at 1645, 1680, 2943 and 3338 cmectively . The orif (1680), f (1645), f (3338) and f (2943) at above characteristic absorption peaks of original SiO2/SMPU sample are all slightly less than zero. This indicates that some molecular chains of SMPU are perpendicular to the stretching direction at a micro level [2 nanoparticles in SMPU matrix, forming an interfacial region together with soft and hard segments. The –OH groups of SiO2 nanoparticles are combined with C=O groups in soft and hard segments to form hydrogen bonds. Therefore, this causes the ordered arrangement of some molecular chains in the original SiO2/SMPU sample.As shown in ro level . It is df values at all selected characteristic absorption peaks of original, programmed and recovered SiO2/SMPU samples are different. Among them, the f (2943) value is increased obviously to 0.11 when the stretching strain reaches 25% which is higher than those at other characteristic absorption peaks. This suggests that more obvious orientation occurs in soft segments.From 2 nanoparticles. This hinders the entanglement between some soft and hard segments, and inhibits the movements of hard segments along the stretching direction during the programming of SiO2/SMPU specimen [The reasons for this are that the rigidity of hard segment is increased due to the addition of SiOspecimen . Thus, t2 nanoparticles. The f (1645) and f (1680) values are increased with the increase in stretching strain during the programming. This suggests that molecular chains of C=O groups in hard segments are oriented along the stretching direction. This is due to the fact that hydrogen bonds are formed among C=O groups in hard segments, molecular chains in soft segments and SiO2 nanoparticles. Thus, molecular chains in hard segments tend to rearrangement as molecular chains of soft segments and SiO2 nanoparticles are moved along the stretching direction.The orientation behavior of hard segments is relatively complicated due to the addition of SiOf (3338) value is a negative and its absolute value becomes larger as the stretching strain is increased from 0% to 10%. The possible reason is that molecular chains in soft segments which wrap around N–H molecular chains are orderly rearranged along the stretching direction so that partial hydrogen bonds between molecular chains in soft segments and N–H molecular chains in hard segments are broken [f (3338) absolute value.It is also seen from e broken . Howeverf values are consistent with the changes of hydrogen bond indexes of RN–H and RC=O in Further, some new hydrogen bonds are formed between molecular chains of N–H and C=O groups in hard segments when the stretching strain is increased from 10% to 25%. As a result, N–H molecular chains are also oriented along the stretching direction as C=O molecular chains in hard segments are orderly rearranged . These rf (2943) value of soft segment in recovered SiO2/SMPU nanocomposite is almost the same with those of original sample. However, f (1645), f (1680) and f (3338) values of hard segments in recovered SiO2/SMPU nanocomposite are slightly larger than those of original SiO2/SMPU sample.As shown in 2/SMPU specimen is stretched to the strain of 25% during the programming, SiO2 nanoparticles are filled in the pores of porous SMPU along with the movement of hard segment to simultaneously extrude some lamella crystals. This facilitates lamella crystals to be oriented along the stretching direction and partial lamella crystals are damaged at the same time so that programmed SiO2/SMPU specimen is not completely restored to its original shape. Therefore, this also explains the reason why Rf and Rr cannot reach 100% at a microscopic level as shown in This is due to the fact that hard segment is regularly arranged to form microcrystalline or para crystalline structures . When Si2/SMPU nanocomposite is prepared, and its thermal behaviors and shape memory performance are characterized. Changes in microstructure, interaction among microphase structures, and molecular orientation of SiO2/SMPU are discussed during a five-step thermodynamic cycle. Main conclusions are obtained as follows.2/SMPU nanocomposite, which matches the highest working temperature of expansion joints on concrete pavement in China.The phase separation temperature (72.9 °C) is regarded as the best shape memory switching temperature of prepared SiO2 nano particles with an average size of about 70–180 nm could be well dispersed in SMPU matrix and the addition of SiO2 nano particles has quite small influence on the thermal behavior and microstructure of SMPU.The SiO2 nano particles, in the one shape memory cycle, the average values of Rf and Rr of SiO2/SMPU are 98.2% and 97.3%, respectively, showing excellent shape memory effect although crystal imperfections are increased due to the addition of SiO2 nanoparticles.The shape memory performance of SMPU is affected by SiO2/SMPU nanocomposite in shape memory process could be ascribed to the interactions between SiO2 nano particles and segments of SMPU. In programming process, the hard and soft segments at the surface have a perpendicular direction at the small strain, and then possess a parallel orientation at higher deformation.The peculiar orientation behaviors of SiO2/SMPU nanocomposite, showing obvious anisotropy. The programmed specimen is not completely recovered to the original shape because partial hydrogen bonds between hard and soft segments are broken, and some crystal structures are also damaged.Calculation results of orientation functions suggest that the tensile programming leads to the molecular orientation in SiOIn this study, SiO2 nano particles on shape memory performance, further study of mechanical properties such as durability will be considered, and multiple shape memory programming of SiO2/SMPU nanocomposite will be carried out in combination with actual application scenarios and the optimization of materials to have better self-healing properties.For the next phase, based on the influence of SiO


GDAP1 encodes for a mitochondrial outer membrane protein. Mouse and insect models with mutations in Gdap1 have reproduced several traits of the human disease. However, the precise function in the cell types affected by the disease remains unknown. Here, we use induced-pluripotent stem cells derived from a Gdap1 knockout mouse model to better understand the molecular and cellular phenotypes of the disease caused by the loss-of-function of this gene. Gdap1-null motor neurons display a fragile cell phenotype prone to early degeneration showing (1) altered mitochondrial morphology, with an increase in the fragmentation of these organelles, (2) activation of autophagy and mitophagy, (3) abnormal metabolism, characterized by a downregulation of Hexokinase 2 and ATP5b proteins, (4) increased reactive oxygen species and elevated mitochondrial membrane potential, and (5) increased innate immune response and p38 MAP kinase activation. Our data reveals the existence of an underlying Redox-inflammatory axis fueled by altered mitochondrial metabolism in the absence of Gdap1. As this biochemical axis encompasses a wide variety of druggable targets, our results may have implications for developing therapies using combinatorial pharmacological approaches and improving therefore human welfare.Charcot-Marie-Tooth disease is a chronic hereditary motor and sensory polyneuropathy targeting Schwann cells and/or motor neurons. Its multifactorial and polygenic origin portrays a complex clinical phenotype of the disease with a wide range of genetic inheritance patterns. The disease-associated gene  Charcot-Marie-Tooth (CMT) disease is the most prevalent hereditary sensory and motor neuropathy 1 in 2500). CMT disease is a clinically and genetically heterogeneous group of disorders sharing common phenotypical features, including weakness, and wasting of distal limb muscles, waned deep tendon reflexes, distal sensory loss, and skeletal deformities. Overall, the clinical traits of the disease result from progressive loss of sensory and motor nerves; affecting either the myelin shaft  or the axon  of motor neurons (MNs). Together, these cellular and structural defects lead to a progressive decrease in patient welfare [ in 2500.Several pathological mechanisms underlying CMT2 subtypes have been described, and include defects in mitochondrial dynamics, organelle-organelle contacts, and axonal transport . This paGanglioside-induced Differentiation-Associated Protein 1 , encodes an integral mitochondrial outer membrane protein expressed predominantly in MNs and Schwann cells [Gdap1 knockout cellular model, which renders no functional protein, previous research has defined that disease-associated missense mutations in GDAP1 result in a premature stop codon and a shortened protein [Gdap1 have reproduced several traits found in human CMT disease, the precise function of GDAP1 in healthy settings and its mechanistic link to CMT disease remains uncertain [Gdap1–/– mice into MNs and found that absence of this protein results in a fragile phenotype characterized by mitochondrial dysfunction and activation of the innate immune response.The CMT-associated gene nn cells , 8. Simi protein . GDAP1 p protein , 15, reg protein , 17, oxi protein , mitocho protein –21. Alth protein , 23 and  protein  models wncertain –28. To aMnx1  promoter [Gdap1WT and Gdap1–/– iPSCs [To facilitate the identification of MNs in our studies we generated reporter iPSC lines by introducing EGFP under the control of the promoter  in Gdap1/– iPSCs . MN diff/– iPSCs , 31. On Mnx1 gene expression , flow cytometry and immunofluorescence. On d7, EGFP fluorescent signal  in EGFP-positive MN somas was assessed by microscopy using TOM20 as a marker for these organelles  and mitochondrial superoxide anion, using TMRM and MitoSOX fluorescent probes, respectively. opy Fig. .Gdap1–/– cells compared to controls  and extracellular acidification (ECAR) rates were measured as proxies of oxidative phosphorylation (OXPHOS) and glycolysis, respectively. Data analysis showed higher OCR in ols Fig. , while gres Fig. .Fig. 4InGdap1–/– cultures , Hk2 and Ldh enzyme expression decreased in res Fig. . Interesing Fig. . While klls Fig. , when GDlls Fig. .Fig. 5GdGdap1–/– MEFs displayed marked upregulation of genes associated with innate immune response  and after seeding, at d8  and d10 .Next, gene expression of these markers was analyzed during MN differentiation Fig. . Gdap1-nA relationship between ROS, innate immune response and MAPK activation does exist –39. ActiGdap1 induces a fragile phenotype in MNs characterized by mitochondrial dysfunction and activation of the innate immune response. Our results provide insight into Gdap1 function in health and disease.In this study, we found that lack of GDAP1 mRNA in SH-SY5Y neuroblastoma cells can induce mitochondrial fragmentation [Gdap1 deficiency in murine models has mild [GDAP1 is a protein of the outer mitochondrial membrane described as an accessory protein promoting fission of these organelles , 40, 41.entation  and Gdaphas mild , 23 or ahas mild  on mitocMitochondria are main sources of cellular ROS production , and uncGdap1–/– cell cultures showed a decrease in glycolysis associated with HK2 and LDH downregulation, and a rise in oxidative metabolism. While overexpression of GDAP1 induced increased MMP [LDH silencing can activate OXPHOS in several tumor cell lines [Gdap1–/– MNs suggests that mitochondria may be unable to consume MMP, possibly due to ATP5B protein downregulation. This altered metabolic profile of Gdap1–/– cells correlates with a dysfunctional mitochondrial state that is likely a key factor in GDAP1 deficiency-associated cellular defects in CMT disease.Defective OXPHOS can generate ROS . Gdap1–/ased MMP , LDH silll lines . Our resGdap1 increased oxidative stress in mice [GDAP1 (associated with CMT2K) usually fall on the GST domains of the protein [Oxidative stress has been proposed as a crucial element in various neurological disorders . GDAP1 c in mice . In this protein  and it h protein .Gdap1–/– MNs have a fragile phenotype prone to degeneration associated with activation of the p38α MAPK, a key player in apoptotic cell death [Neuronal cell death is a hallmark of neurodegenerative diseases . Here well death . In factll death –58, inclll death .Gdap1–/– mice presented chronic neuroinflammation in the spinal cord and sciatic nerve [GDAP1-linked CMT variants.Also, it has been observed that chronic neuroinflammation is present in several neurodegenerative diseases –63. Inteic nerve . The chrGdap1 leads to downregulation of Hk2 in MNs, the possibility that GDAP1 deficiency could lead to the opening of the mitochondrial permeability transition pore thus mounting the innate immune response is a possibility that deserves further investigation, as it may provide the molecular link to MN degeneration in CMT2, opening therefore new avenues for its therapeutical intervention.HK1 and HK2 proteins localize to the mitochondrial surface by direct interaction with the voltage-dependent anion channel (VDAC). It is known that the interaction of VDAC with these hexokinases protects cells from mitochondrial ROS production  and supppMnx1-EFGP reporter [iPSC lines were cultured in the presence of hLIF as previously described . Motor nreporter . Differereporter , 31. Difreporter . Furtherhttp://rsb.info.nih.gov/ij/plugins/track/jacop.html) for ImageJ analysis software, as previously described [Cells were fixed in 4% PFA (in PBS), permeabilized with 0.5% Triton X-100 in PBS, blocked with 3% BSA in PBS, and incubated overnight at 4 °C with indicated primary antibodies. Next day, samples were washed with PBS and incubated with secondary antibodies in the dark. After washing with PBS, cells were incubated with Hoechst 33342  as nuclear counterstaining, washed with PBS, rinsed with water, and mounted. For analysis of mitochondrial membrane potential, cells were incubated with 10 nm TMRM  for 20 min at 37 °C in the dark. For the analysis of mitochondrial superoxide, cells were incubated with 5 µM MitoSOX  for 10 min at 37 °C in the dark. Colocalization of Tom20 and Lc3b staining was evaluated by calculating the Pearson Correlation Coefficient (PCC) using the freely available JACoP plug-in  or 5 μM MitoSOX  for 5 min at 37 °C in the dark. Measurements were taken with the BD FACSCanto II cytometer (BD Biosciencies) and results analyzed with FlowJo . Data from at least 10,000 cell events per sample were acquired.2, 37 °C). Cells were imaged every hour . Images were analyzed using ImageJ software  and the number of neurites per soma was quantified by direct observation of the images.Images of MNs in culture were captured using an IN-Cell Analyzer 2000 microscope  with a 40X objective under controlled atmosphere conditions  supplemented with phosphatase and protease inhibitors. Cellular lysates were used for immunoblotting with indicated antibodies. Signals in membranes were detected using ECL prime (Amersham) and images automatically captured in an Alliance Mini HD9  system. Acquired images were analyzed with ImageJ. Antibodies used are listed in supplementary information. For LC3B immunoblots, cells were incubated with 50 µM Chloroquine  for 4 h prior lysis with ice-cold RIPA buffer. Uncropped blots can be found in Supplementary materials.Total RNA was extracted using TRI reagent  and cDNA synthesized using SuperScript III reverse transcriptase . 2–3 µl of cDNA were amplified in the StepOnePlus Real-Time PCR system (Applied Biosystems) using SYBR Premix ExTaq . Each RT-qPCR assay was performed in duplicate and repeated at least 3 times. Primers used are listed in supplementary information.Microarray data has been described . In-siliBasal and uncoupled oxygen consumption rates (OCR), or extracellular acidification rates (ECAR), were measured using a Seahorse bioanalyzer (XF96) and the Mito or Glycolysis stress test kits , respectively, following the manufacturer recommendations. Measurements were normalized to total number of cells per well by fluorescence microscopy on a replica-seeded plate stained with Hoechst 33342. Each experiment was conducted in quadruplicate and repeated at least 3 times. Further details can be found in supplementary information.t-test was used to estimate statistical significance between two categories. Relative values (percentages) were normalized using arcsine transformation before carrying out their statistical comparison. In all assays at least 3 different iPS cell clones (n ≥ 3) per genotype were tested. Each data point was independently repeated at least three times. These independently repeated experiments were considered as replicates for statistical analysis. In all figures, panels display representative images from 1–2 different iPS cell clones. Bar diagrams displaying numerical results were obtained from at least 3 independent iPS cell clones (n) from each genotype.Where indicated, Student’s Supplemental dataFigure S1Figure S2Figure S3Original data WB


TM (PL), and sEMG-TL during a running maximum oxygen uptake (2max) test. The study investigated ten healthy participants. On day one, participants performed a three-speed treadmill test at 8, 10, and 12 km·h−1 for 2 min at each speed. On day two, participants performed a 2max test. Analysis of variance found significant differences in sEMG-TL at all three speeds (p < 0.05). A significantly weak positive relationship between sEMG-TL and %2max  was established, while significantly strong relationships for 8 out of 10 participants at the individual level  were found. Meanwhile, the accelerometry PL was not significantly related to %2max (p > 0.05) and only demonstrated significant correlations in 3 out of 10 participants at the individual level. Therefore, the sEMG shorts-derived training load was sensitive in detecting a work rate difference of at least 2 km·h−1. sEMG-TL may be an acceptable metric for the measurement of internal loads and could potentially be used as a surrogate for oxygen consumption.The purpose of this study was two-fold: (1) to determine the sensitivity of the sEMG shorts-derived training load (sEMG-TL) during different running speeds; and (2) to investigate the relationship between the oxygen consumption, heart rate (HR), rating of perceived exertion (RPE), accelerometry-based PlayerLoad Monitoring of the training load (TL) is an integral part of the training process in numerous sports and exercise regimes. The TL informs coaches of the physical and psychological stressors imposed on athletes during training and is categorised into two theoretical constructs: internal and external . The intr = 0.66), but no other GPS metrics, such as high-intensity running, sprinting, and maximum speed [It is recommended that a variety of internal and external load metrics are captured simultaneously as they are usually not interchangeable . For exaum speed . Contextum speed . Thus, oSurface electromyography (sEMG) is a useful method for the evaluation of muscle activity, particularly in dynamic exercise. It has gained extensive usage in studying the electrophysiological aspects of muscle contractions and force generation within the realms of sports and exercise medicine . NormallPrevious research has shown the strong validity and reliability of textile sEMG sensors embedded into compression shorts compared to a gold-standard BIOPAC system during knee flexion and extension, performed at different intensities . Current2max test. To date, one study has demonstrated how sEMG shorts may be used as a viable predictor of lactate threshold work rates during an incremental cycling protocol [The association between the integrated EMG (iEMG) of the quadriceps, hamstrings, and calf muscles and oxygen consumption suggests that muscle activation may be a representation of individuals’ physical exertion ,20,21. Tprotocol . This deTM utilises a triaxial accelerometer to calculate the PlayerLoadTM (PL), an external load variable expressed as the square root of the sum of squared instantaneous acceleration changes in X, Y, and Z vectors, which has been validated in various sports and exercise research protocols [Catapultrotocols ,23.External loads, such as PL, are often combined with internal loads such as HR measures to understand how prescribed training loads relate to individual athlete responses ,25. HoweTherefore, this study aims to (1) investigate the sensitivity of sEMG-TL across different running speeds; (2) explore and compare the relationships of sEMG-TL, HR, RPE, and PL with oxygen consumption during a maximal running test; and (3) examine the associations of sEMG-TL with HR, RPE, and PL. Understanding these relationships will contribute to advancing future research in the field of sports and exercise TL monitoring. Moreover, the findings will have practical implications, guiding the potential application of sEMG shorts as a tool for the monitoring of training loads in sports and exercise settings.Six healthy, recreationally active males and four healthy, recreationally active females volunteered to participate in this study . Written®, COSMED, Rome, Italy). Recording of the body fat percentage was deemed necessary as excess subcutaneous fat has been shown to weaken sEMG signals and dampen the peak amplitude [TM, Redwood City, CA, USA). A neoprene vest that involved holding a triaxial accelerometer unit  between the scapulae was also fitted to each participant. Participants were familiarised with the wearable technology and a stationary sEMG calibration protocol, which was undertaken to configure the sEMG thresholds for each muscle group. The sEMG sensors were positioned to measure four muscle groups on each leg . Following the calibration protocol, participants completed a 3-speed treadmill running test. This test was conducted to assess whether the sEMG-TL could detect small changes in workload, as, to date, no research has explored the sEMG-TL. Participants visited the laboratory on two consecutive days. On the first day, they undertook preliminary anthropometric tests, including body fat percentage recording using an air displacement plethysmograph (BodPodmplitude . The par2max) test, during which various internal loads  and external loads (accelerometery) were continuously monitored. On the second day, participants completed a treadmill running maximum oxygen uptake  to measure 2 (mL·kg−1·min−1), which was averaged over consecutive 60 s intervals. At the end of each 60 s interval, the participants’ heart rate (bpm) and RPE  were also measured.On the second day, participants completed an incremental treadmill exercise test to determine The Athos™ unit electronics subsystem is responsible for signal reception, transmission, and conditioning. Within the electronics subsystem is a signal conditioning module that filters the sEMG data. Sampled sEMG signals were captured at 1 kHz. The anti-aliasing filter applied prior to sampling prevented high-frequency noise greater than 500 Hz from aliasing into the sEMG spectrum. Filtering included a linear band-pass with a cut-off frequency from 10 Hz to 500 Hz (23 dB frequencies with centre frequency at 120 Hz); a notch  filter, rectification, and linear envelope were applied. The linear envelope was down-sampled by a factor of 25 and smoothed using a 16-sample root-mean-square (RMS) transformation along with signal conversion from analogue to digital. sEMG signals were averaged at a 10 s window. sEMG recordings were considered viable when impedance was below 5 kΩ. Poor contact quality was deemed if loss of the contact signal occurred over 10% of the time. These filtering and signal processes were predetermined by the Athos™.The sEMG peak amplitude, which was captured through the calibration protocol, provided individualised reference points for each muscle group, which the raw integrated sEMG output  could be normalised against. The integrated sEMG for each muscle group was measured as a percentage of the MVC. The accumulation of the normalised integrated sEMG values across all muscle groups relative to the MVC peak sEMG amplitude represented the sEMG-TL reported in arbitrary units (a.u.). A single arbitrary unit was equivalent to one muscle activating at 100% of the MVC for 1-s.The triaxial accelerometer sampled at 100 Hz. The data were accumulated to generate a measure of the external load (PL). The equation for PL can be seen below:2max) using a Shapiro–Wilk test was performed. Results showed that the distributions for all variables  departed significantly from normality , as well as the 3-speed test data . Descriptive statistics were determined for the participants’ sEMG-TL data from the 3-speed test and for all variables during each minute in the 2max test. The sensitivity of the sEMG-TL in detecting 2 km⋅h−1 changes in running intensity during the 3-speed treadmill test was assessed by a Kruskal–Wallis one-way analysis of variance by ranks test, and pairwise comparisons were performed using a Dunn–Bonferroni post hoc test. Statistical significance was set at p < 0.05. Raw accelerometry data and 2 (mL·kg−1·min−1) were averaged at every 60 s interval during the 2max test. 2 was normalised to 2max (%2max). Thus, a non-parametric Spearman’s correlation test was applied to determine the correlations between all variables in the composite data set, while separate correlations were applied on the individual data sets for sEMG-TL, HR, RPE, PL, and %2max. The significance level was set at p < 0.05. Correlations were classified as previously suggested: 0 = no correlation, (0 < rs < 0.2) = very weak correlation, (0.2 ≤ rs < 0.4) = weak correlation, (0.4 ≤ rs < 0.6) = moderate correlation, (0.6 ≤ rs < 0.8) = strong correlation, (0.8 ≤ rs < 1.0) = very strong correlation, and 1 = perfect correlation [The data were analysed using the statistical software package SPSS (version 26.0). Since we had a small sample size, determination of the distribution of the data for each variable  = 11.50, p < 0.05. Post hoc pairwise comparisons using the Dunn–Bonferroni test demonstrated that the sEMG-TL at a low running speed was significantly different from both the moderate and high running speeds (adjusted p < 0.05), but no statistical difference was established between moderate and high running speeds (adjusted p > 0.05).sEMG-TL is presented as a median (interquartile range (IQR)) for each running speed. 2max test. Generally, the median load for each variable increased at every 1 min interval during the 2max test. There were 74 individual recordings included in the analysis for each dependent variable. Median (IQR) loads are presented in s = 0.70, p = 0.002) and rs = 0.91, p = 0.001, respectively) with %2max, sEMG-TL revealed a very weak correlation . In contrast, no correlation was established between PL and %2max . sEMG-TL showed weak significant correlations between RPE , HR  and PL .2max in nine out of ten participants, while only three participants showed very strong non-significant positive correlations between PL and %2max. In addition, Correlation coefficients at the individual level are presented in −1 in running velocity. As expected, higher running velocities were associated with an increased sEMG-TL. The purpose of this study was to (1) investigate the sensitivity of sEMG-TL across different running speeds; (2) explore and compare the relationships of sEMG-TL, HR, RPE, and PL with oxygen consumption during a maximal running test; and (3) examine the associations of sEMG-TL with HR, RPE, and PL. An important feature of this study is the novelty of the textile sEMG sensors embedded in compression shorts to provide an sEMG-based internal TL metric as a method for the objective monitoring of the muscular load during dynamic exercise. The results show that sEMG-TL exhibited significant differences among three different running intensities, distinguished by increments of 2 km⋅h2max was established  for energy. This was reflected through the 2 measured from the participant. The higher force output requirements and associated fatigue as the test progresses will stimulate the muscles to increase the electrical discharge to compensate for the decreasing muscle strength, resulting in an increased sEMG amplitude [2max [Considering the second aim of the study, a significant yet somewhat modest association between sEMG-TL and %ablished . Howevericipants . The intmax test . Howevermax test ,32. Durimplitude . During [V˙O2max . 2max both in the composite data set and at the individual level for all participants. These findings align with the previous literature, suggesting that both HR and RPE are reliable indicators of intensity and load in the context of load monitoring [2max. HR, being a measure of the heart’s response to exercise, appears to be particularly effective in representing the oxygen output during physical activity, as anticipated, given its direct reflection of the cardiovascular system’s response to exertion. These results support the use of HR and RPE for load monitoring, as they highlight the utility of HR and RPE as practical measures to assess 2max during exercise. By capturing complementary aspects of the participants’ physiological responses, HR and RPE provide valuable insights into the intensity of the exercise and are more strongly related to 2max than sEMG-TL and PL.HR and RPE demonstrated very strong significant correlations with nitoring ,35. It i2max for the grouped data. This highlights the different parameters that internal and external player monitoring systems capture during exercise [2max. It has been previously shown that sEMG from the quadriceps and hamstrings may better reflect energy expenditure during uphill and downhill locomotion compared to accelerometry [PL demonstrated no correlation with exercise ,36. The erometry . The meterometry ,39. Thus2max test, which in part may explain this relationship. At the individual level, sEMG-TL demonstrated very strong significant correlations with RPE in seventy percent of participants. Establishing a relationship between RPE and sEMG-TL is an important finding as this could help with exercise prescription and in designing routines for specific muscle loads.In regard to the third aim of the study, the current findings established a significant weak association between sEMG-TL and RPE in the composite data set . PreviouIn addition, a significant, very weak correlation was found between HR and sEMG-TL in the composite data, yet very strong significant correlations were established in eighty percent of participants when the HR and sEMG-TL correlations were analysed at the individual level. While there is no direct causal relationship between sEMG and HR, they are often positively associated during exercise ,43. As m2max at the individual level; thus, the shorts could serve as a potential surrogate for the measurement of 2 during incremental running. Most of the previous research conducted on textile sEMG sensors embedded in shorts for the monitoring of muscle activity involves isokinetic exercises, general locomotion, or low-level functional exercise ,16,17,18Individual biomechanical and physiological characteristics likely impact the relationship between sEMG-TL and the other load variables ,31,32. AAnother limitation of the current study is that, while HR and RPE are global measurements of internal load, sEMG represents a portion of superficial muscle , yet the participants likely experienced physiological stress elsewhere in the body. Therefore, caution should be taken when using the data to inform exercise loading parameters where exercise includes more muscles other than the upper leg. In the future, research might consider using a full-body sEMG compression garment that encompasses many more sEMG sensors to better reflect the whole-body muscle load.This study aimed to investigate the sensitivity of sEMG-TL across different running speeds, explore its relationship with oxygen consumption, and examine its associations with HR, RPE, and PL. The novelty of using textile sEMG sensors embedded in compression shorts provided an objective method for the monitoring of the muscular load during dynamic exercise. 2max at the group level, but strong individual correlations were found. HR and RPE showed very strong significant correlations with 2max, supporting their reliability for the assessment of exercise loads. However, PL demonstrated no correlation with 2max, indicating the limitations of accelerometry in capturing loads during treadmill running with gradient increases in the uphill slope. Additionally, a significant weak association was found between sEMG-TL and RPE at the composite level, while showing more substantial individual-level correlations. The study’s strengths include the use of sEMG shorts to derive a TL metric and its potential application for individual exercise prescription. However, factors such as the small sample size and the need to control contextual factors were identified as study limitations. The results revealed significant differences in sEMG-TL among different running intensities, with higher velocities associated with increased sEMG-TL. The study also established a significant yet weak association between sEMG-TL and In conclusion, this research emphasises the sensitivity of sEMG-TL in detecting small changes in exercise intensity and highlights the potential for future research to be conducted with training load monitoring using sEMG. Future research should consider larger sample sizes and use full-body sEMG compression garments to better reflect the whole-body muscle load during exercise.


Alzheimer´s disease is considered a neurodegenerative disease and is diagnosed by exclusion, while the detection of specific cerebrospinal fluid (CSF) biomarkers, namely amyloid-beta (Aβ) peptides Aβ1–42 (Aß42), phospho-tau , and total-tau (T-tau), has been shown to improve diagnostic accuracy. Recently, a new generation of sample tubes  for the Elecsys CSF immunoassay for the determination of Alzheimer´s disease biomarkers in CSF was introduced, promising better measurability. However, the pre-analytic influencing factors have not yet been sufficiently investigated.In 29 patients without Alzheimer’s disease diagnosis, CSF concentrations of Aß42, P-tau and T-tau were examined in native CSF and after different influencing interventions using the Elecsys immunoassay test method. The following influencing factors were analyzed: contamination with blood , 14-day storage at 4 °C, blood contamination of CSF and 14-day storage at 4 °C, 14-day freezing at -80 °C in Sarstedt tubes or glass vials, 3-month intermediate storage at -80 °C in glass vials.Both storage at -80 °C for 14 days in Sarstedt false-bottom tubes and in glass vials and storage at -80 °C for 3 months in glass vials resulted in significant decreases in Aß42 , P-tau  and T-tau  concentrations in CSF. No significant differences were found for the other pre-analytical influencing factors.Measurements of the concentrations of Aß42, P-tau, and T-tau in CSF with use of the Elecsys immunoassay are robust to the pre-analytical influencing factors of blood contamination and duration of storage. Freezing at -80 °C results in significant reduction of biomarker concentrations regardless of the storage tube and must be considered in retrospective analysis.The online version contains supplementary material available at 10.1186/s42466-023-00257-5. Alzheimer´s disease is considered as a neurodegenerative disorder, which is mainly diagnosed by clinical assessment, neuropsychological testing, and brain imaging as well as exclusion of differential diagnoses . The accWhen determining CSF biomarkers for Alzheimer´s disease, pre-analytical influencing factors must be considered, since sample handling and storage as well as inter-laboratory variances have been reported to influence the measurement results –8. In paDifferent studies have already investigated different influencing factors for the determination of CSF biomarkers of Alzheimer´s disease with the Elecsys immunoassay. However, some influencing factors, which are frequently observed in routine laboratory work-up, and thus represent the “real world”, have not been investigated so far. Therefore, the aim of the present study was to investigate the influence of pre-analytical interventions in daily clinical routine possibly determining more accurate threshold values.A total of 29 patients presenting to Hannover Medical School (MHH) in 2022 and 2023 were included in this prospective study . Some of these patients suffered from severe, long-lasting or therapy-refractory disease courses of underlying psychiatric disorders, thus comprehensive diagnostics with cerebral magnetic resonance imaging and CSF analysis were carried out to exclude possible underlying neurological diseases.Lumbar puncture was performed in seated patients with an atraumatic needle (21 G) and introducer (19 G) after extensive dermal disinfection by the same investigator (FFK). Lumbar puncture was performed after breakfast between 10 and 12 a.m., while CSF analyses were performed within 30 minutes after sampling. CSF for the determination of Aß42, P-Tau and T-Tau was collected in false-bottom polypropylene tubes  designed for this test. The first two ml of the CSF were used for different other analyses. Four samples (2.5 ml) were obtained for the investigation of blood contamination and storage duration from the first patient subset. One sample was used for immediate measurement (“native”), one for measurement after storage (14 days), one for blood contamination and immediate measurement and the last one for blood contamination and measurement after storage (14 days). Three additional samples (2.5 ml) were obtained for the study of freezing as a possible influencing factor from a different subgroup of patients. Two samples were used for the comparison of freezing at -80 °C for 14 days in glass vials vs. Sarstedt false-bottom tubes and one sample for the study of freezing at -80 °C for 3 months. The tube was kept upright during entire course of the study.Samples were analyzed without prior centrifugation. Immediately after sample collection, Alzheimer´s disease CSF biomarkers were measured using Elecsys β-Amyloid (1–42) CSF II, Elecsys Phospho-Tau (181P) CSF, and Elecsys Total-Tau CSF immunoassays on the cobas e 801 analyzer (Roche Diagnostics). We also investigated whether pre-analytical interventions, which are frequently observed in laboratory routine diagnostics, have an influence on Alzheimer´s disease biomarker concentrations. The influence of contamination with 10,000 erythrocytes/µL CSF (n = 5) or 20,000 erythrocytes/µL CSF was investigated (n = 12). In addition, CSF samples with and without blood contamination were stored at 4 °C for 14 days and then compared with baseline values (n = 17). Furthermore, baseline concentrations of Alzheimer´s disease biomarkers were compared with concentrations after intermediate storage in glass vials and freezing at -80 °C (for 3 months) and subsequent thawing (n = 17). Samples intermediately stored in glass vials were transferred to Sarstedt false-bottom tubes to enable the peptide and protein measurement.Finally, the baseline concentrations of Alzheimer´s disease biomarkers were compared with concentrations after 14-day storage at -80 °C in glass vials or Sarstedt false-bottom tubes (n = 12). Samples stored in glass vials were transferred to Sarstedt false-bottom tubes to enable the peptide and protein measurement.Statistical analysis was performed using GraphPad Prism . The statistical significance level was set at 5%. The Shapiro-Wilk normality test was used to assess the normal distribution of values. Data were expressed as minimum, maximum (min-max), and mean unless otherwise stated. Fisher´s exact test was used for group comparison of binary variables, and either the Mann-Whitney U-Test or paired t-test was used for metric variables. Longitudinal data were analyzed with the Friedman test with Dunn posthoc test for multiple comparisons.Since several authors have already investigated some pre-analytical factors of CSF concentrations of Alzheimer´s disease biomarkers using the Elecsys immunoassay, their findings have been summarized in supplemental Table Some of the included patients suffered from psychiatric disorders, as shown in Table Comparison of the baseline concentrations of Aß42, P-tau, and T-tau and the concentration quotients of P-tau/Aß42 and T-tau/Aß42 with the concentrations and quotients after blood contamination with 10,000 erythrocytes/µl CSF  revealed no significant differences. Similarly, blood contamination with 20,000 erythrocytes/µl CSF  did not result in significantly different concentrations and quotients of Aß42, P-tau, T-tau, P-tau/Aß42 and T-tau/Aß42  led to significant concentration differences compared to baseline values.The concentrations of Aß42, P-tau, and T-tau decreased by an average of 13% ±7.5 (p = 0.0005), 9% ±6 (p = 0.0091), and 12% ±7.9 (p = 0.0059), respectively, after storage in Sarstedt false-bottom tubes at -80 °C and thawing , 13% ±6 (p = 0.0010), and 32% ±19.1 (p = 0.0038), respectively, after storage in glass vials at -80 °C, thawing, and transfer into Sarstedt false-bottom tubes for measurement . P-tau and the quotients P-tau/Aß42 and T-tau/Aß42 were not significantly different.Intermediate storage in glass vials and freezing at -80 °C for 3 months, followed by thawing and sample transfer into Sarstedt false-bottom tubes for the determination of Alzheimer´s disease biomarkers resulted in significant concentration differences compared to baseline values. Concentrations of Aß42, P-tau, and T-tau decreased by an average of 42% ±13.4 (p < 0.0001), 12% ±11.5 (p = 0.0017), and 20% ±14.2 (p = 0.0011), respectively, after intermediate storage and thawing Fig. . In contA new generation of sample tubes  for the Elecsys CSF immunoassay of Alzheimer´s disease biomarkers has been introduced recently. The promise of a better measurability, especially of Aß42, with less influence by pre-analytical factors was made, thus relatively high cut-off values for pathological concentrations have been proposed , 11, 17.Hansson et al. reported stability of Aß42 CSF concentrations up to a blood contamination of 1% of the sample volume and storage duration of 8 days at room temperature and up to 15 days at 2-8 °C . In the The determination of specific thresholds for blood contamination is important for routine diagnostics since blood contamination of 2,500 erythrocytes/µl CSF is known to refine CSF total protein measurements, whereas 5,000 erythrocytes/µl CSF leads to sophistication of CSF IgM determinations . HoweverIn addition, Alzheimer´s disease biomarker measurement using the Elecsys immunoassay was described as exceptionally robust and accurate, thus Aß42 determination in plasma was proposed as possible rapid and easily available prescreening tool for Alzheimer´s disease , 20–22. A very high susceptibility of Alzheimer´s disease biomarkers in CSF  to non-specific adsorption to non-polypropylene materials was already described –25. In lThe use of the Elecsys automated assay for CSF Aß42, P-tau, and T-tau shows robust results on the concentrations of Aß42, P-tau, and T-tau, as well as the concentration quotient of P-tau/Aß42 and T-tau/Aß42, which are not affected by the pre-analytical influencing factors blood contamination of the CSF and storage at 4 °C. However, freezing at -80 °C for 14 days has a significant effect on biomarker concentrations and must be considered in retrospective analysis.Below is the link to the electronic supplementary material.Supplemental table 1. Investigated pre-analytical influencing factors and different analytics involving the Elecsys immunoassay and determination of Alzheimer’s disease biomarkers in cerebrospinal fluid (CSF)Supplemental table 2. Investigated comparisons of different analytics involving the Elecsys immunoassay for the determination of Alzheimer’s disease biomarkers in cerebrospinal fluid (CSF)