Idiopathic bilateral vestibulopathy (IBV) is an acquired bilateral peripheral vestibular dysfunction of unknown etiology, with persistent unsteadiness but without sensorineural hearing loss (SNHL) other than age-related hearing loss (ARHL).The prevalence of IBV is unknown. The most common symptom is persistent unsteadiness, particularly in darkness and/or on uneven ground. The other main symptom is oscillopsia during head and body movements. IBV is neither associated with SNHL, except for ARHL, nor any other neurological dysfunction that causes balance disorders. The clinical time course of IBV can generally be divided into two main types: progressive type and sequential type. The progressive type involves gradually progressive persistent unsteadiness without episodes of vertigo. The sequential type involves recurrent vertigo attacks accompanied by persistent unsteadiness. Originally, IBV was found to exhibit bilateral dysfunction in the lateral semicircular canals (LSCCs) and the superior vestibular nerve (SVN) system. However, recently, with the development of more sophisticated vestibular function tests of the otolith organs and vertical semicircular canals, it has been revealed that IBV involves peripheral vestibular lesions other than those already identified in the LSCC and the SVN system. Furthermore, novel subtypes of IBV that do not involve bilateral dysfunction of the LSCC and/or the SVN system have been proposed. Therapeutically, exercise-based vestibular rehabilitation in adult bilateral vestibulopathy (BVP) patients has resulted in improved gaze and postural stability moderately. There are several technical approaches for the treatment of BVP such as vestibular implants, sensory substitution devices and noisy galvanic vestibular stimulation.Combined use of various vestibular function tests, including recently developed tests, revealed the diversity of lesion sites in IBV. Further studies are required to determine the therapeutic effects of the technical approaches on IBV. Idiopathic bilateral vestibulopathy  , idiopatThe true prevalence of IBV is not known yet due to insufficient data. A previous study based on the 2008 United States National Health Interview Survey Balance and Dizziness Supplement reported that the prevalence of bilateral vestibulopathy (BVP) was 28/100,000 adults . HoweverIBV by definition does not have a clear etiology or pathogenesis. In a previous immunological study, sera from IBV patients for antibodies against the inner ear was screened . IgG antThe involvement of multiple factors in IBV is also indicated by the fact that some cases involve vertigo attacks, while others do not . VestibuThe most common symptom of IBV is persistent unsteadiness . In darkThe clinical time course of IBV is generally divided into two main types: progressive type and sequential type . The proWith regard to BVP, the diagnostic criteria were recently released by the Classification Committee of the Barany Society (see Appendix ). These As a baseline for diagnosis, IBV is characterized by acquired bilateral peripheral vestibular dysfunction with unknown cause, with persistent unsteadiness, without SNHL other than ARHL . OscilloRevealing bilateral dysfunction of the LSCC and/or the SVN system using the caloric test and the rotation test, both of which assess the vestibulo-ocular reflex (VOR) at LSCC plane, has historically been the most common method of diagnosing IBV , 38, 41.The development of VEMP testing made it possible to measure function in the otolith organs –9. The cSince a decrease in VOR gain demonstrated by vHIT or scleral-coil HIT at the LSCC plane is included in the diagnostic criteria for BVP , some ofThe main problem for the establishment of an understanding of otolithic involvement in BVP is the accuracy of assessment of bilateral losses of otolith function. While assessment of unilateral dysfunction of the otolith organs is quite easy, judgement of bilateral dysfunction of the otolith organs is not easy. In previous reports of IBV, bilaterally absent cVEMP responses was regarded as bilaterally abnormal cVEMPs –16. The A previous study assessed postural stability using foam posturography in IBV patients according to whether the SVN systems, the IVN systems or both of the vestibular nerve systems were affected . While fA previous study investigated vergence eye movements in IBV patients, by using a light emitting diode display with targets along the median plane . ConvergA previous study reported the abnormality of vestibular perceptual thresholds in IBV patients . SubjectIn BVP patients, gaze stabilization fails and can lead to decrease in visual acuity during head movements. There are various testing paradigms to evaluate dynamic visual acuity in BVP patients, such as reading an optotype chart, during fast head movements , passiveIt was reported that BVP led to a significant decrease in the gray matter mid-hippocampal volume and posterior parahippocampal volume . BVP alsRecently, a systematic review about the impact of BVP on spatial and non-spatial cognition was performed . In thisThe treatment of IBV has not been distinguished so far from that of standard BVP. Here, we describe the treatment of BVP.The effect of vestibular rehabilitation on BVP remains controversial due to conflicting results in previous reports. One systematic review of the effects of vestibular rehabilitation on adult BVP patients revealed moderate evidence for improved gaze and postural stability  following exercise-based vestibular rehabilitation .There are several technical approaches for the treatment of BVP. Vestibular implants stimulate the peripheral vestibular nerve through electrical pulses, and has been proposed as a candidate for the treatment of BVP –50. RestCombined use of various vestibular function tests, including recently developed tests, revealed the diversity of lesion sites in IBV. Further studies are required to determine the therapeutic effects of the technical approaches such as vestibular implants, sensory substitution devices and nGVS on IBV.


In life-threatening situations such as holding yourself from crossing the street when noticing a fast approaching car, efficient inhibitory control is even key to survival. Despite the fact that observing or measuring inhibition may be challenging as it implies absence of behavior, clearly, the role of inhibitory motor control should not be underestimated as it is of key importance for adaptability in many real-world situations. Clinical expressions of deficits in inhibitory function illustrate this such as for example Tourette Syndrome where inhibitory failure leads to production of awkward movements or sounds. Attention-Deficit/Hyperactivity Disorder also illustrates how lack of inhibition compromises everyday function and mental development. More recently, inhibition has also become a prominent focus of attention in aging research because there is mounting evidence that inhibitory motor function gradually declines with advancing age [The concept of inhibition is deeply rooted in our society. This is not only evident in our day-to-day activities but also in our scientific endeavors to reveal its underlying mechanisms. Indeed, inhibition is expressed in various functional domains such as cognition, action and emotion. Being able to suppress preferred, addictive or intended behaviors is at the heart of our well-being and how we succeed in society. There is no single construct that fully captures the different expressions of inhibition. Here we focus on motor inhibition, defined as the ability to successfully cancel a planned or already initiated action. This cing age . This doA and GABAB receptor activity. However, whereas TMS studies can only provide an indirect measure of inhibitory mechanisms and its implicated neurotransmitter, recent advances in medical imaging techniques allow scientists to accurately quantify the level of neurochemical compounds such as GABA in the brain by means of magnetic resonance spectroscopy (MRS) [Interestingly, recent developments in brain-behavioral approaches have provided a new window into the neural mechanisms mediating efficient inhibitory control in the context of healthy aging . This topy (MRS) . FurtherA and GABAB receptors at the postsynaptic level, (3) a reduction of GABA level assessed with MRS, and (4) altered intracortical inhibitory circuits as measured with TMS [To date, considerable research efforts have already been devoted to the study of the GABAergic system in the aging brain. In that regard, both animal and human research has shown age-related alterations in the GABA system, either related to (1) the synthesis of GABA at the presynaptic level, (2) altered subunit composition of both GABAMRS, and  altered with TMS . These fRecently, we used MRS to register baseline GABA levels within key regions of the motor inhibition network in order to identify its role in age-related deterioration of inhibitory control . We acquThere is mounting evidence that the integrity of the GABA system is critically important for efficient motor inhibition. More specifically, the registration of brain GABA levels within the motor inhibition network provides a window into the mechanisms mediating age-related declines in inhibitory motor control. But GABA is not only critical for inhibitory control. Modulation of GABA is important for inducing processes of neuroplasticity that enable the shaping of our actions and the generation of new behaviors. In view of the demographic evolution of society, the importance of research on age-related alterations in the GABA system cannot be underestimated. This work may contribute to a body of knowledge that may lead to the development of interventions targeting the GABA system with the ultimate goal to ameliorate age-related declines in motor inhibition.


Alexander disease (AxD) is a fatal neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP), which supports the structural integrity of astrocytes. Over 70 GFAP missense mutations cause AxD, but the mechanism linking different mutations to disease-relevant phenotypes remains unknown. We used AxD patient brain tissue and induced pluripotent stem cell (iPSC)-derived astrocytes to investigate the hypothesis that AxD-causing mutations perturb key post-translational modifications (PTMs) on GFAP. Our findings reveal selective phosphorylation of GFAP-Ser13 in patients who died young, independently of the mutation they carried. AxD iPSC-astrocytes accumulated pSer13-GFAP in cytoplasmic aggregates within deep nuclear invaginations, resembling the hallmark Rosenthal fibers observed in vivo. Ser13 phosphorylation facilitated GFAP aggregation and was associated with increased GFAP proteolysis by caspase-6. Furthermore, caspase-6 was selectively expressed in young AxD patients, and correlated with the presence of cleaved GFAP. We reveal a novel PTM signature linking different GFAP mutations in infantile AxD. Alexander disease is a rare and fatal neurodegenerative condition. It is caused by mutations in a gene that is crucial for the structure of astrocytes, a type of brain cell whose role is to support neurons. The gene codes for a protein called GFAP, which is made almost exclusively in astrocytes. In Alexander disease, mutated versions of the gene make GFAP collect in disordered clumps or aggregates, which interfere with the astrocytes’ normal activities.All Alexander disease patients develop GFAP aggregates, but the type of mutation they have in the gene for GFAP does not predict how their illness will progress. The age of onset of disease, for example, can vary between less than one year old to more than 70 years old. Battaglia et al. sought to understand how GFAP aggregates form in the cells of Alexander disease patients. One way that GFAP can be altered in the cell is by a process called phosphorylation. Enzymes called kinases add phosphate groups to GFAP, which can regulate the protein’s activity, stability and interactions with other proteins.Battaglia et al. found high levels of phosphorylation at one specific site in the GFAP protein in people who had very early onset of Alexander disease. This phosphorylation was not related to any particular mutation in the gene for GFAP. An added phosphate group at this location in the protein made GFAP more likely to be broken into two pieces by an enzyme called caspase-6. One of the breakdown products is already known to play a role in aggregation. Young patients with Alexander disease had high levels of GFAP breakdown products and caspase-6. The phosphorylated protein and this enzyme were found to accumulate in astrocyte aggregates.The findings provide a basis for investigating new strategies to treat Alexander disease that target phosphorylation – as removing or preventing the addition of phosphate groups can be done with drugs. But before exploring how to do this, it will be necessary to find out which enzyme is responsible for phosphorylating GFAP at this particular position. These studies may also give a broader understanding of other GFAP-like proteins , which are involved in over 70 human diseases. GFAP, which encodes glial fibrillary acidic protein (GFAP), cause AxD  is a rare and invariably fatal neurological disorder that affects primarily infants and small children, but can also manifest later in life . Autosomause AxD . GFAP isause AxD . The accause AxD . Silencil of AxD . While twww.interfil.org). Disease-associated IF proteins share two key molecular features: abnormal post-translational modifications (PTMs)   and paths (PTMs) , simple s (PTMs) , desmin s (PTMs) , vimentis (PTMs) , neurofis (PTMs)  and the s (PTMs) . There aLike all IF proteins, GFAP contains three functional domains: amino-terminal ‘head’ domain, central α-helical ‘rod’ domain and carboxy-terminal ‘tail’ domain . The gloHere, we identified a critical phosphorylation site in the GFAP head domain that is selectively and strongly upregulated in the brain tissues of AxD patients who died very young, independently of the position of the disease mutation that they carried. Further, we show that this site-specific phosphorylation promotes GFAP aggregation and is a marker of perinuclear GFAP aggregates associated with deep nuclear invaginations in AxD patient astrocytes, but not in isogenic control astrocytes. Finally, we demonstrate a correlation between site-specific GFAP phosphorylation and caspase cleavage in cells and in post-mortem brain tissue from AxD patients. Although our study does not establish a causal relationship between GFAP phosphorylation and caspase cleavage, we show that caspase-6 is a new marker for the most severe form of human AxD.Collectively, our results reveal a new PTM signature that is associated with defective GFAP proteostasis in the most severe form of AxD. Future interventional studies targeting these PTMs will determine whether they contribute to, or are the consequence of, disease severity.IFs undergo protein synthesis-independent turnover and re-organization to meet cellular demands . PTMs arStrikingly, we found that the pSer13-GFAP peptide was selectively elevated in the cortex tissue from AxD patients who died very young  . OverallTo determine the functional significance of pSer13 on GFAP filament organization, we analyzed the filament properties of non-phosphorylatable (S13A) and phospho-mimic (S13D and S13E) GFAP mutants. We optimized a transient over-expression system in the SW13 vimentin-negative adrenocarcinoma cells (SW13vim-) for this assay, which resulted in primarily filamentous WT GFAP and insoluble aggregated forms of common AxD mutants of GFAP . CompareGFAP  using CRISPR/Cas9 mediated gene editing  relative to the isogenic controls  via neural progenitor cells (NPCs), as described in the Materials and methods and shown schematically in controls . This iscontrols  and mouscontrols . In addicontrols . Finallycontrols , whereascontrols . In vivocontrols . The in controls .Next, we determined if pSer13-GFAP was present in the AxD iPSC-astrocytes, similar to what we observed in the human brain tissues. As shown in To understand the mechanism for how GFAP phosphorylation may promote GFAP aggregation, we conducted a biochemical analysis of the S13A, S13D and S13E GFAP mutants. In line with our immunofluorescence result , we obseTo determine how blocking GFAP cleavage affects aggregation, we performed site-directed mutagenesis to block cleavage of GFAP at Asp225. As shown in Caspase-6 is not expressed highly in the normal human brain, especially after birth . TherefoNext we asked whether AxD patients, particularly young AxD patients that exhibit more pSer13-GFAP and caspase-6 expression, also displayed increased GFAP cleavage. To determine the extent of caspase-6-cleaved GFAP in AxD patient brains, we utilized an antibody that specifically recognizes N-terminally caspase-6-cleaved GFAP (D225) . We deteOur study reveals that missense mutations, affecting discrete domains on the GFAP molecule, share a common PTM signature that is associated with compromised GFAP proteostasis in the severe form of AxD. Using patient brain tissue and human iPSC-derived AxD astrocytes, we show that head domain phosphorylation promotes defective filament assembly and perinuclear accumulation and incorporation of mutant GFAP within nuclear invaginations. By taking an unbiased mass spectrometry proteomic approach, we were able to identify GFAP phospho-peptides that were selectively elevated in human AxD brain tissue, and subsequently validated these results using a phospho-specific antibody against the most abundant epitope (pSer13-GFAP). We demonstrate the importance of the Ser13 site for GFAP assembly in vitro and in cells. Phospho-mimetic mutation S13D completely abolished the ability of GFAP to form filaments in vitro, without leading to aggregation. In transfected SW13vim- cells, phospho mimic S13D-and S13E-GFAP mutants formed highly abnormal perinuclear aggregates that correlated with increased cleavage of GFAP by caspase-6. We detected a dramatic increase in caspase-6 expression, in association with Ser13 phosphorylation and cleavage of GFAP, in the brain tissue of AxD patients who succumbed to the disease very early in life. While the N-terminal caspase-6 fragment of GFAP promotes filament aggregation in vitro , presentOur study does not address whether Ser13 phosphorylation directly promotes caspase cleavage of GFAP, or if these two PTMs are independent markers of an increased cellular stress response in AxD. One possibility is that Ser13 phosphorylation destabilizes the filament structure, thereby promoting access of caspase-6 to the rod domain Asp225 residue, where the cleavage occurs. Another likely possibility is that the increased cleavage of GFAP is an indirect result of stress-dependent caspase-6 activation in the more severe form of AxD. This is supported by previous studies showing that AxD mutations promote activation and nuclear accumulation of p53 , which cGiven our findings that pSer13-GFAP is enriched in the most aggressive form of AxD, monitoring the levels of this phospho-epitope  in AxD patient cerebrospinal fluid or blood may provide added sensitivity for disease activity . Phospho222VELD225 in vitro generates an N-terminal 26 kDa fragment and a C-terminal 24 kDa fragment. The N-terminal fragment directly impairs assembly of full-length GFAP and promotes aggregation in vitro  and Alzheimer’s Disease (AD) . SimilarRecently, iPSC-derived patient astrocyte models have emerged as an important system for dissecting the cellular mechanisms in AxD. For example, these novel tools have revealed that AxD astrocytes have defects in the secretory pathway, impaired ATP release, and attenuated calcium waves ; that thInvaginations of the nucleus, such as those we observe here, have been described in physiological and pathological states . ControlThe following antibodies were used: rabbit anti-GFAP , rabbit anti-caspase-6 , rabbit anti-Caspase-6 , rabbit anti-D225 , mouse aSW13vim- cells were provided by Dr Bishr Omary and cultured in DMEM with 10% fetal bovine serum and 1% penicillin-streptomycin. Authentication of the cell line was done by short tandem repeat (STR) profiling by ATCC. Fibroblasts from a male 6 year old type I AxD patient were obtained from the Coriell institute . Sanger sequencing was performed to confirm the AxD mutation was present in the cells . The cell lines used tested negative for mycoplasma contamination, as assayed using the Universal Mycoplasma Detection Kit (ATCC 30–1012K).De-identified post-mortem fresh-frozen and fixed AxD patient and control brain tissues were provided by the NIH NeuroBioBank and are described in Sample Preparation: HSEs from AxD patient post-mortem brain cortex tissue were prepared as described previously . Samples were injected onto an Easy Spray PepMap C18 column  (Thermo Fisher Scientific) and separated over a 1 hr method. The gradient for separation consisted of 5–40% mobile phase B at a 250 nl/min flow rate, where mobile phase A was 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in 80% ACN. The QExactive HF was operated in data-dependent mode where the 15 most intense precursors were selected for subsequent fragmentation. Resolution for the precursor scan (m/z 300–1600) was set to 120,000 with a target value of 3 × 106 ions. MS/MS scans resolution was set to 15,000 with a target value of 1 × 105 ions. The normalized collision energy was set to 27% for HCD. Dynamic exclusion was set to 30 s, peptide match was set to preferred, and precursors with unknown charge or a charge state of 1 and ≥7 were excluded. Data Analysis: Raw data files were processed using Proteome Discoverer version 2.1 (Thermo Fisher Scientific). Peak lists were searched against a reviewed Uniprot human database, appended with a common contaminants database, using Sequest. The following parameters were used to identify tryptic peptides for protein identification: 10 ppm precursor ion mass tolerance; 0.02 Da product ion mass tolerance; up to two missed trypsin cleavage sites; phosphorylation of Ser, Thr and Tyr were set as variable modifications. The ptmRS node was used to localize the sites of phosphorylation. Peptide false discovery rates (FDR) were calculated by the Percolator node using a decoy database search and data were filtered using a 5% FDR cutoff. The peak areas for the identified peptides were extracted and used for relative quantitation across samples.eviously  and in FMutagenesis of GFAP  was performed using the QuikChange II mutagenesis kit (Agilent) to generate the designated point mutants. Sanger sequencing of the entire coding sequence of GFAP was performed to confirm the wild-type and mutant sequences. We used established procedures for the purification and in vitro assembly of GFAP . For traHigh salt extracts (HSEs) and triton-X (TX) lysates were prepared as previously described . Total lSkin fibroblasts were reprogrammed under feeder free conditions using Cytotune –iPS 2.0 Sendai Reprogramming kit and individual iPSC clones were picked for propagation in culture for 10 passages. To confirm stemness and differentiation capabilities of reprogrammed and edited iPSCs, we used the qPCR based TaqMan human Pluripotent Stem Cell Scorecard Panel (Thermo Fisher Scientific). iPSCs were differentiated into all three germ layers using STEMdiff Trilineage Differentiation Kit , and a monolayer-based protocol was used to directly differentiate hES cells in parallel into the three germ layers (~1 week). Non-differentiated and differentiated cells were lysed and total RNA purified using the RNeasy kit (QIAGEN). RNA reverse transcription was performed following the Taqman Scorecard’s manufacture guidelines and the qRT-PCR was carried out using the QuantStudio 7 Flex Real-Time PCR system. The TaqMan PCR assay combines DNA methylation mapping, gene expression profiling, and transcript counting of lineage marker genes . Reprogr5 iPSCs were electroporated with the ribonucleoprotein mix. Seventy-two hours after electroporation, cells were dissociated into single cells, diluted, and seeded on Matrigel-coated 96-well plates. Single-cell colonies were selected after two weeks and tested for gene correction. Genomic DNA of single clones was extracted and the gene of interest amplified by PCR using allele specific primers. Sanger sequencing of positive clones demonstrated single or double allele gene correction. Off-target sites within the exons of genes were predicted via selection of the top candidates using the MIT software (CRISPR.mit.edu). The analysis was performed via PCR of 400 bp fragments, which flanked the predicted off-target cut site followed by Sanger sequencing. The chromatograms for edited clones were compared to sequences from the original AxD patient cells.We used the TrueCut Cas9 Protein V2, sgRNAs and the Neon Transfection system (Thermo Fisher Scientific) to edit iPSCs. The recombinant TrueCut Cas9 V2 was diluted in resuspension buffer R provided in the kit and mixed with 900 ng of sgRNA and 2700 ng of single-stranded donor oligonucleotide, incubated 15 min at room temperature and then a total of 3 × 106 cells per well. At day five, EBs were seeded on poly-ornithine and laminin (PLO/LAM)-coated dishes in NIM. Rosette selection was performed after 12 days using Rosette Selection Reagent (StemCell Technologies). NPCs were expanded for 7 days in Neural Progenitor Medium (StemCell Technologies). NPCs were then differentiated into astrocyte precursors by seeding dissociated single cells at 1 × 105 cells/cm2 density on PLO/LAM dishes in STEMdiff astrocyte differentiation medium (StemCell Technologies). Astrocyte precursors were maintained for 20 days with medium changes every 48 hr and splitting every week with Accutase . Astrocytes were expanded for up to 120 days in STEMdiff astrocyte maturation medium (StemCell Technologies).iPSCs were maintained on Matrigel in StemFlex medium (Thermo Fisher Scientific) and passaged every 3–4 days with 0.5 mM EDTA dissociation solution. iPSCs were differentiated into neural progenitor cells (NPC) using an embryoid body (EB) protocol. Briefly, iPSCs at 80% confluence were collected, resuspended in Neural Induction Medium  and seeded on one well of an Aggrewell 800 plate (StemCell Technologies) at 3 × 10en face to the substrate with a diamond knife and Leica UCT Ultramicrotome . Ultrathin sections (70 nm) were mounted on 200 mesh copper grids and stained with 4% uranyl acetate and lead citrate. The sections were observed and digital images were taken using a JEOL JEM-1230 transmission electron microscope operating at 80kV  equipped with a Gatan Orius SC1000 CCD Digital Camera .AxD iPSC-astrocytes grown on a polystyrene dish were fixed in 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer, pH 7.4, for one hour at room temperature and stored at 4°C. The cells were washed 3 times in 0.1M sodium cacodylate buffer followed by post-fixation in 1% buffered osmium tetroxide for 1 hr. After three washes in deionized water, the cells were dehydrated in ethanol, infiltrated and embedded in situ in PolyBed 812 epoxy resin . The cell monolayer was sectioned  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:We are particularly excited about the findings that Alexander Disease-causing mutations perturb key post-translational modifications (PTMs) on GFAP. The selective phosphorylation of GFAP-Ser13 in patients who died young raises the hypothesis that Ser13 phosphorylation and increased GFAP proteolysis by caspase-6 facilitated GFAP aggregation, which leads to disease state.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Huda Zoghbi as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: W Christopher Risher (Reviewer #2).Thank you for submitting your article "Phosphorylation-dependent proteolysis perturbs GFAP proteostasis, implicating caspase-6 in early onset Alexander Disease" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Battaglia and co-authors have identified a posttranslational modification of the GFAP protein and potential mechanism involving caspase-6 that appear to correlate with disease severity in the rare but lethal disorder Alexander disease (AxD). In this report post-mortem samples from individuals with known GFAP mutations were analyzed for GFAP-PTM by mass spectrometry. Phospho-Ser13 was identified primarily in samples from AxD patients with early onset, regardless of the mutational variant. Subsequent transfection experiments with Ser13 phospho- and de-phospho mimics further suggest that Ser13 phosphorylation promotes protein aggregation. In addition, the authors show that pSer13-GFAP along with caspase-6 localize to aggregates found in astrocytes derived from AxD iPSCs, and that the phospho-mimic S13D leads to caspase-6 cleavage of GFAP, similar to AxD mutations. Finally, they show that pSer13, caspase-6 protein, and caspase-6 cleaved GFAP are present in brains from early onset AxD patients.Essential revisions:All three reviewers found the question interesting and the results potentially significant.1) The major critique from all three reviewers is that the causality between phosphorylation of GFAP and cleavage of caspase-6 in relation to the pathology of AxD are not explored. For example, does blocking phosphorylation of this site on GFAP in the patient iPSC astrocytes prevent/reverse the GFAP aggregation and perinuclear accumulation of GFAP? Are there known effects on astrocyte health/reactivity in addition to GFAP aggregation that can be monitored, to see if they are also rescued? Experiments along these lines would greatly strengthen the study.Specific comments along this line include "The key pieces of evidence suggesting cause and effect are the transfection experiments with phopho-mimetic constructs. Although N-terminal substitutions are rare in AxD, how do we know that the S13D mutation does not cause GFAP aggregation in itself ? Did the authors try other substitutions, e.g. S13V, S13E (or D225E to prevent caspase-6 cleavage)? Since it is known that over-expression of wild-type GFAP causes aggregation, how are GFAP expression levels controlled between transfections? If phosphorylation of GFAP-Ser13 promotes caspase-6 cleavage and GFAP aggregation, shouldn't elimination of pSer13 alleviate aggregate formation in the R79H/S13A transfection? Would caspase-6 inhibitors (Ac-VEID-CHO) prevent aggregation in either the SW13vim-cells or iPSC astrocytes? The authors claim in the Discussion that impairment of GFAP assembly by the S13D phospho-mimic is due to caspase-6 cleavage, but this link is not demonstrated by the data shown. "2) The Western blots in the figures  used Coomassie staining as the loading control, but no details were given. Given that antibody labeling of Coomassie-stained gels is notoriously difficult, did the authors use different gels for determining protein amounts vs. blotting? This would not be an appropriate normalization method for the actual blotted membranes, and would therefore need to be replaced. If these Coomassie controls were done on the same gels that went on to be blotted for antibodies, please provide technical details in the Materials and methods, as this is a non-standard approach.eLife. Your revised article has been favorably evaluated by Huda Zoghbi (Senior Editor), a Reviewing Editor, and three reviewers.Thank you for resubmitting your work entitled "Phosphorylation-dependent proteolysis perturbs GFAP proteostasis, implicating caspase-6 in early onset Alexander Disease" for further consideration at eLife, but there are some remaining issues that need to be addressed before acceptance, as outlined below:The reviewers agreed that the manuscript has been improved and is a strong candidate for The main remaining issue is on how strong the conclusions can be drawn. Please make modifications to the text and potentially to the title to reflect the concerns of reviewer 3.Reviewer #1:eLife.The authors have added a number of new experiments to the paper that strengthen the conclusion linking phosphorylation of GFAP to caspase-6 cleavage of GFAP and to the pathology of Alexander disease. While major point 1 is not fully addressed (determining whether these changes are a cause or a consequence of more severe disease mutations), the paper does make significant new findings in the area of Alexander disease that warrant publication in Reviewer #2:The authors have satisfactorily addressed all of my major and minor concerns; I have no further issues. Additionally, the mechanistic insight provided by the new experiments and analyses has significantly elevated the impact of the study.Reviewer #3:The authors have made a significant effort in adding important data to address the reviewers' concerns and questions. These include in vitro assembly analysis for the GFAP S13D phospho-mimic, the addition of the S13E phospho-mimic and analysis of GFAP-R79H phosphorylation sites in transfection experiments (SW13vim-), and both genetic and pharmacological inhibition of caspase-6 to assess the effects of GFAP cleavage on protein aggregation in SW13vim- cells. Important controls were also added for the iPS cell and AxD patient experiments. Most of these additions have strengthened the manuscript and support a connection between GFAP phosphorylation and aggregation. However, the new data showing caspase-6 inhibition in itself disrupts GFAP filament organization and fails to rescue S13D aggregation complicate interpretation of the results, and evidence for direct cause and effect between caspase-6 GFAP cleavage and protein aggregation is still lacking. The findings in this report are important in identifying PTMs as new markers of disease severity and should be published, but as noted in the original review, the authors should be more conservative with their conclusions.The authors indicate a direct link between GFAP-pSer13, caspase-6 cleavage, and protein aggregation in the title, Introduction and Discussion. A major question is still whether the observed phenomena contribute to disease severity or are downstream in the stress response and simply reflect the more severe pathology in early onset AxD. The following 3 areas contribute to this concern:1) In agreement with a more severe phenotype and astrocyte response, GFAP levels are higher in early onset AxD cases compared to those with late onset . GFAP has been shown to be phosphorylated in reactive gliosis associated with certain injury models , and the overall damage response could have a broader effect in activating caspase-6; p53 for example is activated in AxD  and is known to transactivate caspase-6 . The authors hypothesize that GFAP phosphorylation directly affects caspase-6 accessibility, but aggregation of GFAP-S13D and S13E in SW13vim- cells likely causes a cellular stress response that could also indirectly activate caspase-6. While I agree with the authors that investigating these mechanisms is beyond the scope of the current manuscript, they should address this possibility in their discussion and conclusions.2) New experiments to inhibit caspase-6 cleavage of GFAP produce ambiguous results. Inhibition of caspase-6 alone leads to GFAP bundling in SW13vim- cells and in combination with the S13D phospho-mimic, aggregates are not resolved. Western analysis shows clearance of a 100 kD (hmm) band, but a ~200 kD band remains and no western data is shown to demonstrate the level of WT-GFAP oligomerization induced by caspase-6 inhibition. As the authors suggest in the discussion, the role of caspase-6 activation is complex and may also be involved in cytoskeletal reorganization.3) The supplemental data added to Figure 7 introduce important evidence for a link between GFAP phosphorylation, aggregation and caspase-6 cleavage. Immunoblotting shows de-phospho-mimics of serine residues (S to A) within the S13-S17 motif of mutant R79H GFAP eliminate high molecular weight oligomerization and significantly reduce caspase-6 cleavage fragments. However, these data are complicated by the limited contribution of pSer13 in GFAP-R79H aggregation in SW13vim- cells and the more dominant effects of pS16 and pS17, which have not been otherwise explored in this report. Although the SW13vim- cell line is commonly used to test the effects of GFAP mutation, these results demonstrate the limitations of the system for these experiments. Future work should include similar experiments in the iPSC derived astrocytes. Essential revisions:All three reviewers found the question interesting and the results potentially significant.1) The major critique from all three reviewers is that the causality between phosphorylation of GFAP and cleavage of caspase-6 in relation to the pathology of AxD are not explored. For example, does blocking phosphorylation of this site on GFAP in the patient iPSC astrocytes prevent/reverse the GFAP aggregation and perinuclear accumulation of GFAP? Are there known effects on astrocyte health/reactivity in addition to GFAP aggregation that can be monitored, to see if they are also rescued? Experiments along these lines would greatly strengthen the study.We present new experimental evidence  and kinase motif-based predictions (Supplementary file 4) revealing the complexity of this PTM mechanism on GFAP . Although we are actively working to identify the critical kinase(s) involved in the phosphorylation of S13 (through candidate and unbiased approaches), we currently do not have strong and conclusive evidence for the specific involvement of a major kinase that we can selectively inhibit to measure the effects. However, we fully agree with the reviewers that this is of key importance and it is an avenue that we are actively exploring, using a combination of in vitro and in vivo approaches to find the most promising target for intervention.Specific comments along this line include "The key pieces of evidence suggesting cause and effect are the transfection experiments with phopho-mimetic constructs. Although N-terminal substitutions are rare in AxD, how do we know that the S13D mutation does not cause GFAP aggregation in itself ?To address this question, we purified WT-, S13A-, and S13D-GFAP and performedin vitro filament assembly assays. As shown in new Figure 3C, the S13D mutant is unable to assemble into filaments, and it does not aggregate on its own. This is in contrast to the phenotype of the N-terminal caspase-cleaved fragment, which does aggregate on its own .Did the authors try other substitutions, e.g. S13V, S13E (or D225E to prevent caspase-6 cleavage)?We now include new data on the effects of an S13E substitution, which phenocopied S13D with respect to aggregation  and caspase cleavage . We also generated D225E mutant alone, and in combination with S13D. We present new data to show that D225E mutation essentially eliminates S13D-GFAP cleavage, and that this correlates with a decrease in aggregation . We note that D225E on its own interfered with GFAP filament formation, which is not surprising as caspase-cleavage is an important mechanism used to re-organize IFs.Since it is known that over-expression of wild-type GFAP causes aggregation, how are GFAP expression levels controlled between transfections?For all transfection experiments, we initially carried out titration of WT-GFAP cDNA amounts (250ng-1µg) in a 24-well format using three different transfection reagents . We combined this with a time-course experiments . Based on these initial optimization experiments, we selected to use Lipofectamine 2000/500ng cDNA/24hr conditions. Under these conditions, there is minimal aggregation of WT-GFAP (based on protein solubility in TritonX-100 buffer and filament organization). In contrast, under these same conditions, AxD-associated GFAP mutants are significantly less soluble and form the expected aggregates. We now show this as a supplement .If phosphorylation of GFAP-Ser13 promotes caspase-6 cleavage and GFAP aggregation, shouldn't elimination of pSer13 alleviate aggregate formation in the R79H/S13A transfection?We present new data that R79H in the over-expression system is highly phosphorylated at nearby serines 16/17 . Mutagenesis of these residues to non-phosphorylatable alanines decreases GFAP cleavage and oligomerization of the R79H mutant. Therefore, our current hypothesis is that phosphorylation of S13 is a coordinated event that works in tandem with nearby phosphorylation sites. This is also predicted by the kinase motif analysis (Supplementary file 4). Another contributing factor may be that the S13A substitution leads to the formation of abnormal filaments, based on the in vitro assembly assay . Given these results, it is not surprising that S13A does not alleviate R79H aggregation.Would caspase-6 inhibitors (Ac-VEID-CHO) prevent aggregation in either the SW13vim-cells or iPSC astrocytes?These data are shown in new Figure 8E-G. In the presence of the caspase-6 inhibitor, S13D aggregates in the SW13vim- cells were reduced in size, but we observed more filament bundles (consistent with previous studies on other IFs showing that caspase cleavage is required for filament re-organization). Additionally, the presence of a high-molecular-mass (100kDa) S13D-GFAP oligomer was significantly reduced by caspase-6 inhibition.The authors claim in the Discussion that impairment of GFAP assembly by the S13D phospho-mimic is due to caspase-6 cleavage, but this link is not demonstrated by the data shown.We modified this section of the Discussion to include the new results with the recombinant protein and propose a potential mechanism regarding the aggregation mechanism of the S13D mutant.2) The Western blots in the figures  used Coomassie staining as the loading control, but no details were given. Given that antibody labeling of Coomassie-stained gels is notoriously difficult, did the authors use different gels for determining protein amounts vs. blotting? This would not be an appropriate normalization method for the actual blotted membranes, and would therefore need to be replaced. If these Coomassie controls were done on the same gels that went on to be blotted for antibodies, please provide technical details in the Materials and methods, as this is a non-standard approach.The stained gels are the same gels that were transferred onto the PVDF membranes. We routinely stain all gels after wet transfer overnight at 40V. Regardless of transfer technique, there is typically protein remaining on the gel after the transfer of total cell and total tissue lysates. We find that in most cases this is a more reliable loading control than a ‘housekeeping’ protein, which can change with conditions. However, to address the concern regarding equal protein loading, we re-ran the gels in question and included pan-actin blots for the membranes in new Figure 4E  and new Figure 9B  to show that our original conclusions remain. We also modified the Materials and methods section to include information about post-transfer gel staining.The main remaining issue is on how strong the conclusions can be drawn. Please make modifications to the text and potentially to the title to reflect the concerns of reviewer 3.Reviewer #1:The authors have added a number of new experiments to the paper that strengthen the conclusion linking phosphorylation of GFAP to caspase-6 cleavage of GFAP and to the pathology of Alexander disease. While major point 1 is not fully addressed (determining whether these changes are a cause or a consequence of more severe disease mutations), the paper does make significant new findings in the area of Alexander disease that warrant publication in eLife.We thank reviewer #1 for their careful and positive evaluation of our revised manuscript. We have made changes to the title, Introduction, and Discussion to clarify that causality was not established with regards to the PTMs and AxD disease severity, and also in the answers to reviewer 3 comments below.Reviewer #2:The authors have satisfactorily addressed all of my major and minor concerns; I have no further issues. Additionally, the mechanistic insight provided by the new experiments and analyses has significantly elevated the impact of the study.We thank reviewer #2 for their careful and positive evaluation of our revised manuscript and the new data.Reviewer #3:The authors have made a significant effort in adding important data to address the reviewers' concerns and questions. These include in vitro assembly analysis for the GFAP S13D phospho-mimic, the addition of the S13E phospho-mimic and analysis of GFAP-R79H phosphorylation sites in transfection experiments (SW13vim-), and both genetic and pharmacological inhibition of caspase-6 to assess the effects of GFAP cleavage on protein aggregation in SW13vim- cells. Important controls were also added for the iPS cell and AxD patient experiments. Most of these additions have strengthened the manuscript and support a connection between GFAP phosphorylation and aggregation.We thank reviewer #3 for their careful evaluation of our revised manuscript and our new data further linking GFAP phosphorylation and aggregation.However, the new data showing caspase-6 inhibition in itself disrupts GFAP filament organization and fails to rescue S13D aggregation complicate interpretation of the results, and evidence for direct cause and effect between caspase-6 GFAP cleavage and protein aggregation is still lacking. The findings in this report are important in identifying PTMs as new markers of disease severity and should be published, but as noted in the original review, the authors should be more conservative with their conclusions.We agree with the reviewer that additional experiments would be required to conclusively establish if there is a direct cause and effect between GFAP caspase-6 cleavage and aggregation in AxD patient cells. We modified the Introduction and Discussion to reflect that point, as noted in the responses to the specific comments below. We also agree that the PTMs highlighted in our report represent new markers of AxD severity, which is now reflected in the revised title of the manuscript.The authors indicate a direct link between GFAP-pSer13, caspase-6 cleavage, and protein aggregation in the title, Introduction and Discussion. A major question is still whether the observed phenomena contribute to disease severity or are downstream in the stress response and simply reflect the more severe pathology in early onset AxD.We agree with the reviewer that more work is needed to establish where in the disease process these PTMs occur. To address this concern, we revised the statements indicating a direct link and made the following additional changes:“Site-specific phosphorylation and caspase cleavage of GFAP are new markers of Alexander Disease severity”1) Title was changed to: 2) We added the following statement to the Introduction: “Collectively, our results reveal a new PTM signature that is associated with defective GFAP proteostasis in the most severe form of AxD. Future interventional studies targeting these PTMs will determine whether they contribute to, or are the consequence of, disease severity.”3) We added the following statement to the Discussion: “While the N-terminal caspase-6 fragment of GFAP promotes filament aggregation in vitro , presently we do not have direct evidence of cause and effect between caspase-6 cleavage and GFAP aggregation in AxD patient cells.”The following 3 areas contribute to this concern:1) In agreement with a more severe phenotype and astrocyte response, GFAP levels are higher in early onset AxD cases compared to those with late onset . GFAP has been shown to be phosphorylated in reactive gliosis associated with certain injury models , and the overall damage response could have a broader effect in activating caspase-6; p53 for example is activated in AxD  and is known to transactivate caspase-6 . The authors hypothesize that GFAP phosphorylation directly affects caspase-6 accessibility, but aggregation of GFAP-S13D and S13E in SW13vim- cells likely causes a cellular stress response that could also indirectly activate caspase-6. While I agree with the authors that investigating these mechanisms is beyond the scope of the current manuscript, they should address this possibility in their discussion and conclusions.We agree that the PTMs could be upstream or downstream of the stress response, and have revised the text to reflect this point as follows:1) We added the following statement to the Introduction: “Finally, we demonstrate a correlation between site-specific GFAP phosphorylation and caspase cleavage in cells and in post-mortem brain tissue from AxD patients. Although our study does not establish a causal relationship between GFAP phosphorylation and caspase cleavage, we show that caspase-6 is a new marker for the most severe form of human AxD.”2) We added the following paragraph to the Discussion: “Our study does not address whether Ser13 phosphorylation directly promotes caspase cleavage of GFAP, or if these two PTMs are independent markers of an increased cellular stress response in AxD. One possibility is that Ser13 phosphorylation destabilizes the filament structure, thereby promoting access of caspase-6 to the rod domain Asp225 residue, where the cleavage occurs. Another likely possibility is that the increased cleavage of GFAP is an indirect result of stress-dependent caspase-6 activation in the more severe form of AxD. This is supported by previous studies showing that AxD mutations promote activation and nuclear accumulation of p53 , which can directly induce caspase-6 expression . Future studies in AxD iPSC-astrocytes and animal models will be required to determine the timing of GFAP phosphorylation and caspase-6 activation in relationship to GFAP cleavage and aggregation.”2) New experiments to inhibit caspase-6 cleavage of GFAP produce ambiguous results. Inhibition of caspase-6 alone leads to GFAP bundling in SW13vim- cells and in combination with the S13D phospho-mimic, aggregates are not resolved. Western analysis shows clearance of a 100 kD (hmm) band, but a ~200 kD band remains and no western data is shown to demonstrate the level of WT-GFAP oligomerization induced by caspase-6 inhibition. As the authors suggest in the discussion, the role of caspase-6 activation is complex and may also be involved in cytoskeletal reorganization.We agree that the role of caspase-6 is complex and requires thorough investigation. With respect to the hmm GFAP bands, we focused primarily on the 100kDa band in the S13D mutant because of the significant difference in the intensity of this band compared to WT GFAP , and after caspase-6 inhibition . We hypothesize that the 200kDa band likely reflects presence of normal GFAP tetramer, but we have not tested that specifically.We also added the following statement to the Discussion:“However, we were not able to demonstrate in cells that inhibition of caspase-6, or mutagenesis of the cleavage site on GFAP, can resolve aggregate formation. These results point to a more complex function for caspase-6, likely involving cytoskeletal remodeling in response to stress.”3) The supplemental data added to Figure 7 introduce important evidence for a link between GFAP phosphorylation, aggregation and caspase-6 cleavage. Immunoblotting shows de-phospho-mimics of serine residues (S to A) within the S13-S17 motif of mutant R79H GFAP eliminate high molecular weight oligomerization and significantly reduce caspase-6 cleavage fragments. However, these data are complicated by the limited contribution of pSer13 in GFAP-R79H aggregation in SW13vim- cells and the more dominant effects of pS16 and pS17, which have not been otherwise explored in this report. Although the SW13vim- cell line is commonly used to test the effects of GFAP mutation, these results demonstrate the limitations of the system for these experiments. Future work should include similar experiments in the iPSC derived astrocytes.We agree that the limitations of the various systems need to be taken into account, and have emphasized this point in the Discussion: “This remains to be addressed in the future using the appropriate model systems, as over-expression studies in cancer cell lines (such as the SW13vim- cells we used here) may not be truly reflective of the signaling that occurs in astrocytes. In particular, it remains to be resolved whether phosphorylation of GFAP on Ser13 is part of a sequentially priming phosphorylation cascade involving nearby Ser16/17  or if Ser16/17 phosphorylation is unique to the SW13 over-expression system.”


To The EditorCystic fibrosis (CF) and asthma are not always easily distinguished and both can coexistin the same patient.Specific IgE for at least one aeroallergen, present in 67% by skin tests and 80% byradioallergosorbent test (RAST), indicates that subjects with CF should be evaluated andtreated for coexisting allergy.®).D.pteronyssinus. However, a positive response to AF was observed in 9/40(23%) and specific IgE ≥0.35 kU/L in 10 (25%) patients. There was a correlation betweenSPT and specific IgE (p<0.0001). Allergic rhinitis was observed in 22 (55%) patientsand 2 had sputum culture positive for Aspergillus sp., despite negativeSPT/specific IgE being for its antigens. Three patients had a diagnosis of ABPA with radiological changes (infiltrates and/orbronchiectasis), and of which two of them presented serological ABPA.Patients with CF  were investigated for allergicbronchopulmonary aspergillosis (ABPA) by thoracic computed tomography, skinprick/puncture testing (SPT) for aeroallergens, blood eosinophil counts, sputum culture,total serum IgE levels and serum specific IgE to Aspergillus fumigatus (AF) (ImmunoCAPAspergillus in18.6% of their patients (n=86) with CF, as well as positive allergic cutaneous tests(ACT) in 29.5% of them (n=78), results which are similar to those in the presentstudy.Aguiar, Damaceno and Forte found serum specific IgE for Aspergillus, allergicsensitization and clinically proven ABPA.Although AF sensitization is crucial for the diagnosis of ABPA in cystic fibrosis, it isnecessary to differentiate lung colonization by Aspergillus-specific IgE are the minimal criteria for ABPA.Patients with CF should be periodically examined for ABPA, and those sensitized should bemonitored more often. Clinical deterioration, high total IgE, andMonitoring by SPT and serology, as suggested


Pinctada fucata martensii) meat protein hydrolysate with alkaline protease by ultrafiltration, polyethylene glycol methyl ether modified immobilized metal ion affinity medium, and reverse-phase high performance liquid chromatography. Both peptides exhibited high ACE inhibitory activity with IC50 values of 458.06 ± 3.24 μM and 109.25 ± 1.45 μM, respectively. Based on the results of a Lineweaver-Burk plot, HLHT and GWA were found to be non-competitive inhibitor and competitive inhibitor respectively, which were confirmed by molecular docking. Furthermore, the pearl oyster meat protein hydrolysate exhibited an effective antihypertensive effect on SD rats. These results conclude that pearl oyster meat protein is a potential resource of ACE inhibitory peptides and the purified peptides, HLHT and GWA, can be exploited as functional food ingredients against hypertension.Angiotensin-I-converting enzyme (ACE) inhibitory peptides derived from natural products have shown a blood pressure lowering effect with no side effects. In this study, two novel ACE inhibitory peptides  were purified from pearl oyster ( Hypertension is a well-established risk factor for many problems like cardiovascular diseases, chronic kidney diseases, arteriosclerosis, and stroke . The numPinctada fucata martensii), a kind of marine pearl shellfish, is widely cultured for pearl production in Southern China with a large number of oyster meat (more than 2000 metric tons) [Pearl oyster (ic tons) . As a riic tons) . ACE inhic tons) . Howeveric tons) ,18. Howeic tons) ,20. BasiCredited to these reports on the application of modified IMAM, in this study, ACE inhibitory peptides in pearl oyster meat protein hydrolysate (POMPH) were purified through ultrafiltration, mPEG modified IMAM (IMAM@mPEG) and reverse-phase HPLC (RP-HPLC). The sequences of purified peptides were identified by matrix assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) while their inhibition patterns were illustrated by a Lineweaver-Burk plot and molecular docking. Moreover, the antihypertensive potency of POMPH in vivo was evaluated in SD rats.4Cl (containing 0.5 M NaCl). The ACE inhibitory activity of the eluate was determined as 84.14 ± 2.63%, which was much higher than that of D-POMPH at the same protein concentration. This result indicated that IMAM@mPEG can effectively enrich ACE inhibitory peptides in D-POMPH. The D-POMPH and eluate of IMAM@mPEG were analyzed by GPC  and greater than 5 kDa (T-POMPH) were obtained by ultrafiltration and their ACE inhibitory activity was determined as shown in d by GPC  using thp < 0.05) was observed between these fractions. Among these fractions, F5 and F6 showed higher ACE inhibitory activity and were thus further separated using a second RP-HPLC run as shown in 5 and F6 each was fractionated into three fractions. Their ACE inhibitory activity assayed at a protein concentration of 0.54 mg/mL revealed that fractions F51 and F62 exhibited higher ACE inhibitory activity (64.73 ± 1.32% and 84.71 ± 1.27% respectively). All fractions showed a significant difference (p < 0.05) in ACE Inhibitory activity.The eluate of IMAM@mPEG with higher ACE inhibitory activity was further separated through RP-HPLC as shown in 51 and F62 were determined by MALDI-TOF-TOF-MS  and were found to exhibit ACE inhibitory activity in terms of IC50 values of 452.23 ± 2.36 μM and 98.92 ± 5.73 μM, respectively, which were greatly similar to those purified from POMPH. The purified peptides HLHT and GWA are novel ACE inhibitory peptides and have not been reported previously.Molecular masses and amino acid sequences of peptides FF-TOF-MS  revealinAccording to previous reports, ACE inhibitory peptides are usually composed of 2–12 amino acids  and thosStichopus horren [Lineweaver-Burk plots were applied to analyze the inhibition patterns of the purified peptides. As shown in s horren .2+ [2+. These findings were in accordance with their ACE inhibition patterns determined by Lineweaver-Burk plots, and hence it was further concluded that the variation in bioactivities of the two peptides could be due to their different natures of interaction with residues of ACE.The intermolecular interaction and potential binding sites of HLHT and GWA with ACE were further elaborated by molecular docking. The main interaction residues at the active site of ACE have been divided into three active pockets, including S1 , S2 , and S1′ (Glu162 residue) . As seenThe antihypertensive efficacy of captopril and POMPH in vivo was investigated in terms of changes in physiological hypertensive parameters including systolic blood pressure (SBP) and diastolic blood pressure (DBP) after intravenous administration to SD rats and the results are compiled in Pinctada fucata martensii) meat was obtained from pearl culture base in Beihai, Guangxi Province, China. Alkaline protease (≥250 units/mg) was purchased from Pangbo biological engineering Co., Ltd. . Toluene and 3-aminopropyl triethoxysilane (APTES) were purchased from Kelon . NaBH4 was purchased from Sinopharm reagent . mPEG (Mn 5000), His-His-Leu (HHL) and ACE (≥2.0 units/mg) were purchased from Sigma-Aldrich . Peptides HLHT and GWA were obtained from GL Biochem . Trifluoroacetic acid (TFA), and HPLC grade methanol and acetonitrile were purchased from Fluka . Fresh pearl oyster (Pearl oyster meat protein was hydrolyzed according to reported method . Briefly2) was isolated with a magnet and successively washed with ethanol and deionized water, respectively. Then, mPEG was subjected to aldehyde modification according to a previous report [2 (0.2 g) was dissolved in 80 mL mixed solution of ethanol/water (v/v = 1:1) containing aldehyde modified mPEG . This mixture was stirred at room temperature for 24 h and was mixed with NaBH4 (0.076 g). The reaction was continued for 24 h after which the product was dialyzed in distilled water for 48 h. The obtained IMAM@mPEG was magnetically separated and dried in a vacuum freeze drier.Magnetic IMAM was prepared according to a previously reported procedure  and modis report . IMAM-NHw/v) for 40 min at 35 °C followed by separating IMAM@mPEG through a magnet, which was washed several times with the same buffer until absorbance of the rinsed buffer at 280 nm reached to a baseline. The adsorbed peptides were eluted with 1.5 M NH4Cl (containing 0.5 M NaCl) and isolated with the help of a magnet as mentioned above. The molecular weight distribution of D-POMPH and eluate of IMAM@mPEG were analyzed by GPC at a constant mobile phase of PBS  using Shodex Protein KW-802.5 column with a flow rate of 0.5 mL/min for 50 min at 280 nm. The eluate of IMAM@mPEG was further applied to an RP-HPLC column  for separation using two solvents (solvent A as 0.1% (v/v) TFA in water, and solvent B as 0.1% (v/v) TFA in acetonitrile). The gradient  at a flow rate of 0.5 mL/min at 30 °C was used and monitored at 280 nm. The fractions with high ACE-inhibitory activity were collected for the second step RP-HPLC with a linear gradient of solvent B from 1–35% in 4.5 min at a flow rate of 0.5 mL/min at 30 °C. These purification procedures were repeated until sufficient samples were collected to perform an assay of the ACE inhibitory activity and sequencing. The prepared D-POMPH (MW < 5 kDa) was dissolved in 0.1 M PBS with a concentration of 1 mg/mL. The IMAM@mPEG was mixed with D-POMPH at a ratio of 5:1  was determined by logarithmic regression analysis.ACE inhibitory activity in vitro was measured according to the reported method . The actTM Analyzer . During analysis, each sample was desorbed and ionized at 337 nm and operated in positive ion delayed extraction reflector mode. Spectra were recorded over mass/charge (m/z) range of 100–1500. Mass spectrometry/mass spectrometry (MS-MS) experiments were achieved through collision-induced dissociation while peptide sequencing was performed via manual calculation.Molecular mass and amino acid sequence of the purified peptides were determined using a 4800 Plus MALDI TOF/TOFThe inhibitory patterns of purified peptides HLHT and GWA on ACE were evaluated by incubating various concentrations  of HHL with ACE in the absence and presence of inhibitory peptides. The concentrations of HLHT were set as 0, 295.86, and 591.72 μM while those of GWA were set as 0, 98.62, and 197.24 μM. The purified ACE inhibitory peptide (30 µL) at various concentrations and ACE solution (30 µL) were mixed and pre-incubated at 37 °C for 10 min. Subsequently, HHL (40 µL) was added and incubated for 5 min at 37 °C. The reaction was terminated by adding 1 M HCl (150 µL). The released hippuric acid (HA) was quantified using HPLC at 228 nm. The ACE inhibitory pattern in the presence of the inhibitor was determined using the Lineweaver–Burk plot, where the reciprocal of HHL concentration was used as the independent variable (X-axis) and the reciprocal of production rate of HA as the dependent variable (Y-axis).The mechanism of interaction of the purified ACE inhibitory peptides bonding with ACE and legitimacy of their activity were elaborated via molecular docking using Autodock software package . The three-dimensional crystal structure of human testicular ACE was obtained from the Protein Data Bank (PDB: 1O86), and the peptides structure were drawn and energy-minimized using Chem Office 2015 software . AutoDocktools was employed to prepare both ACE and peptides for docking. Through Autodock Vina, binding free energy was calculated and peptide displaying the lowest binding affinity to protein was chosen as the best conformation. The visualizations of the protein-ligand structure were shown using PyMol molecular graphics system.The antihypertensive efficacy of POMPH was evaluated in male SD rats . All rats were cared and fed following the standards for laboratory animals established by People’s Republic of China (GB14925-2001)  and animp < 0.05 was considered statistically significant. All the experiments were performed in triplicate and mean values with ± SD (standard deviation) were reported. All data were analyzed by one-way ANOVA, using SPSS 17.0 software . A value of 50 values of 458.06 ± 3.24 μM and 109.25 ± 1.45 μM were purified from POMPH using multi-step purification including ultrafiltration, IMAM@mPEG, and RP-HPLC. HLHT and GWA exhibited non-competitive and competitive inhibition routes respectively, which were further confirmed via molecular docking simulations. The antihypertensive effect of POMPH was practically confirmed by lowering blood pressure in SD rats. This study concludes POMPH as a promising source of ACE inhibitory peptides and HLHT and GWA purified from POMPH could be deemed as potential anti-hypertensive ingredients for functional food.In summary, two novel ACE inhibitory peptides (HLHT and GWA) with respective IC


Malignant pleural mesothelioma (MPM) is a rare, predominantly asbestos-related and biologically highly aggressive tumor associated with a dismal prognosis. Multimodal therapy consisting of platinum-based chemotherapy is the treatment of choice. The reasons underlying the rather poor efficacy of platinum compounds remain largely unknown. Kinase activity might influence cellular response to these regimens.  For this exploratory study, we screened MPM cell lines  differing in response to cisplatin and benign control fibroblasts (MRC-5) for overall phosphorylation patterns as well as kinase activity with respect to cellular response to cisplatin-based therapeutics. We analysed the cell lines for cellular kinases in a high-throughput manner using the highly innovative technique PamGene. Cell state analysis including apoptosis, necrosis, and cell viability was performed by using enzyme activity and fluorescent-based assays.  Cisplatin alters cellular phosphorylation patterns affecting cell cycle, migration, adhesion, signal transduction, immune modulation, and apoptosis. In cisplatin-responsive cell lines, phosphorylation of AKT1 and GSK3B was decreased but could not be influenced in cisplatin-resistant NCI-H2452 cells. Cisplatin-responsive cell lines showed increased phosphorylation levels of JNK1/2/3 but decreased phosphorylation in cisplatin-resistant NCI-H2452 cells.  Kinase phosphorylation and activity might play a crucial role in cellular response to cytostatic agents. Cisplatin influences phosphorylation patterns with distinct features in cisplatin-resistant cells. These alterations exert a significant impact on cell cycle, migration, adhesion, signal transduction, immune modulation, and apoptosis of the respective tumor cells. Based on our results, the induction of p38 or JNK1/3, or inhibition of AKT1 by, for example, BIA-6, might offer a positive synergistic effect by induction of an apoptotic response to cisplatin-based treatment, thus potentially enhancing the clinical outcome of MPM patients. Malignant pleural mesothelioma (MPM) is a rare, predominantly asbestos-related tumor and associated with a dismal prognosis , 2. In tBesides pemetrexed, platinum compounds are standard chemotherapeutic agents and still a hallmark of chemotherapy for MPM . In clinPlatinum cytotoxicity is based on forming bulky DNA adducts by chemically altering DNA bases by covalent binding of platinum , leadingResistance to antitumor agents such as platin compounds has been correlated to a broad spectrum of mechanisms. It is known since the early 1990s that the activity of several proteins involved in the development of antitumor drug resistance is regulated by protein phosphorylation . EspeciaAgainst this background, we aimed to investigate the impact of overall phosphorylation patterns as well as kinase activity in cellular response to cisplatin-based therapeutics. Therefore, we analysed different MPM cell lines for cellular kinases in a high-throughput manner using the highly innovative technique PamGene.MPM cell lines MSTO-211H (biphasic subtype and moderately cisplatin sensitive) and NCI-H2052 (epithelioid subtype and cisplatin sensitive) as well as the cell line NCI-H2452 (sarcomatoid subtype and cisplatin-resistant) were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium . The human lung-fibroblast cell line MRC-5 was used as a control cell line. MRC-5 cells were cultured in Minimal Essential Medium (Thermo Fisher Scientific). All culture media were supplemented with 10% fetal calf serum and 1% penicillin and streptomycin (Thermo Fisher Scientific).5 cells/well were seeded in a 24-well plate. After 12 h of incubation at 37°C and 5% CO2, 10 μM of cisplatin  was added to cells. After 48 h of incubation with cisplatin, protein isolation was performed according to the protocol 1160 from the PamGene platform . Therefore, cells were lysed by using M-PER Mammalian Protein Extraction Reagent containing HALT phosphatase inhibitor cocktail and HALT protease inhibitor cocktail EDTA-free (Thermo Fisher Scientific). Lysed cells were harvested by using a cell scraper. Lysates were stored in 5–20 μl aliquots at −80°C.The effect of cisplatin on kinase activity was analysed in each cell line. Therefore, 1.6 × 10The protein concentration was determined via fluorometric quantification  using the protein assay kit according to the manufacturers' instructions.μl of 2% BSA (PamGene). Master mix was prepared by using the reagent kit for PTK PamChip arrays (PamGene). 5 μg of sample protein lysate was applied. As required for the mastermix, ATP (4 mM) was diluted 1 : 25.The Protein Tyrosine Kinase Assay  was performed according to the manufacturers' instructions. The PamChip®-4 was prepared by a blocking step with 30 μl of 2% BSA (PamGene). Master mix was prepared by using the reagent kit for STK PamChip arrays (PamGene). 0.5 μg of sample protein lysate was applied. As required for the mastermix, ATP (4 mM) was diluted 1 : 25.The Serine/Threonine Kinase Assay  was performed according to the manufacturers' instructions. The PamChip®-4 was prepared by a blocking step with 30 Analysis of the results of PTK and STK assay was performed by using the BioNavigator software (PamGene).http://www.kinhub.org/kinmap/).Image analysis and log2 transformation of the results were performed by using the BioNavigator software (PamGene). Based on substrate phosphorylation pattern, kinase activities of each specific kinase were estimated using the kinase upstream analysis algorithm (BioNavigator). Each corresponding kinase was classified by specificity of each kinase, and dependency power levels were calculated. To visualize kinase activity changes before and after cisplatin treatment, kinase trees were generated by using the KinHub platform .Statistical and graphical analyses of specific phosphosite phosphorylation levels were performed with the t-test (parametric) was applied. For ordinal variables with more than two groups, either the Kruskal–Wallis test (nonparametric) or ANOVA (parametric) was used to detect group differences.Before starting the explorative data analysis, the Shapiro–Wilk test was applied to test for normal distribution of the data. Based on the results, either parametric or nonparametric test was used. For dichotomous variables, either the Wilcoxon Mann–Whitney rank sum test (nonparametric) or two-sided student's Double dichotomous contingency tables were analysed using Fisher's exact test. To test dependency of ranked parameters with more than two groups, Pearson's chi-squared test was used. Correlations between metric variables were tested by using Spearman's rank correlation test as well as Pearson's product moment correlation coefficient for linear modelling.p values were adjusted by using the false discovery rate (FDR). The level of statistical significance was defined as p ≤ 0.05 after adjustment.Due to the multiple statistical testing, all MRC-5 cells showed minor changes in phosphorylation of phosphosites when comparing cisplatin treatment (highlighted by black bars) and medium (highlighted by grey bars)  showed an induction in their phosphorylation. In The comparison of significantly altered phosphorylation levels between the different cell lines revealed 62 alterations. The majority of differences  could beOverall, high phosphorylation of phosphosites Suppl.  depicts Analysis of the phosphorylation pattern with respect to MAPK signaling pathway (KEGG hsa04010), cell cycle (KEGG hsa04110), and pathways in cancer (KEGG hsa05200) was performed for each cell line. Induction of phosphorylation by cisplatin is indicated by green labels, and reduction of phosphorylation is indicated in red labels . In MRC-5 cells, the phosphosite was not anymore phosphorylated during cisplatin treatment. The result for EFS is shown in Suppl. Proteins contributing to cell adhesion/migration and membrane properties showed a cluster with remarkable response to cisplatin treatment in NCI-H2052 and MSTO-211H. EPHA1  showed similar results as depicted in Suppl. Figures BTLA, CD3E, CD247, CD79A, PTK2B, HAVCR2, PECAM1, and ZAP70 modulate immune response and showed differential phosphorylation during therapy. NCI-H2052 showed the strongest decrease in phosphorylation of these proteins during cisplatin therapy. In addition, phosphorylation was much higher before therapy compared to the other cell lines investigated. Similar high phosphorylation was also found in NCI-H2452 before therapy, but phosphorylation reduced less during therapy compared to NCI-H2052. Phosphorylation in MSTO-211H was much lower compared to the other two MPM cell lines. During therapy, only a minor reduction of phosphorylation was monitored for proteins modulating immune response. MRC-5 phosphorylation was low in general but decreased during therapy to a nonmeasurable extent.Phosphorylation of proteins driving cell cycle control showed major changes during therapy . The changes were similar to the above presented results. Again, NCI-H2052 showed the strongest changes, followed by NCI-H2452. Similar, but with general lower phosphorylation, MSTO-211H was comparable with the other two MPM cell lines. Again, MRC-5 presented with generally low phosphorylation of the reported proteins and reduced during therapy—in most cases, phosphorylation was absent due to cisplatin treatment.Kinase trees were created for each cell line  and observed this effect by activation of caspases . This coZhao et al. observed associations between increased apoptosis by high expression of phosphorylated c-Jun N-terminal kinase (JNK) and subsequently elevated expression levels of p53 in ovarian cancer cells during treatment of platinum containing drugs . This suKinase phosphorylation and activity might play a crucial role in cellular response to cytostatic agents. Cisplatin treatment results in altered phosphorylation patterns in both the MPM cell lines and the lung fibroblast cell line. These alterations have consequences for cell cycle, migration, adhesion, signal transduction, immune modulation, and apoptosis of the cell. Cisplatin-resistant MPM cells showed a clearly distinct phosphorylation pattern compared to cells showing response to cisplatin, indicating a specific sensitivity-profile. Our results indicate that inhibition of AKT1 by, e.g., BIA-6, or, in another approach, induction of p38 or JNK1/3 of the MAPK pathway, might offer positive synergistic effects through induction of an apoptotic response to cisplatin-based treatment and thus potentially enhance patients' clinical outcome.


To the Editor:NCT02546921, www.clinicaltrials.gov). A major impediment in the field relates to lack of efficient cloning and production strategies for recombinant IgE at high enough yields for preclinical and clinical studies.AllergoOncology, the emerging discipline of cancer immunology aiming to exploit features of allergy-related immunity to treat tumors,A). To prevent promoter silencing, we developed a novel dual-plasmid system containing Ubiquitous Chromatin Opening Element (UCOE) sequences located upstream of the transgene promoter.B)C) by using Polymerase Incomplete Primer Extension (PIPE) cloning. UCOE enables higher transfection efficiency and higher proportions of medium- and high-expressing transfectomas than pVITRO1 (see www.jacionline.org). Vectors were linearized before transfection to allow correct integration into the host genome, and transgene-expressing cells were selected. The choice of Expi293F cells as hosts was based on human-like glycosylation profiles, ability to grow in suspension, high-density and serum-free conditions, and characteristics crucial for expediting production, scaling up, and adaptability to good manufacturing practice conditions. We adapted Expi293F cells from suspension to adherent growth conditions and vice versa. Adherent cells were transfected and seeded in selection medium to promote host genome integration of exogenous DNA. Resistant cells were cloned by limiting dilution. We designed a cell-based flow cytometric method to detect functional IgE recognizing natively expressed antigens to screen antibody-secreting clones . Our strategy incorporates seamless cloning, selection, and fast antibody production at high yields , A. To pones see . Clones www.jacionline.org).After selecting the highest-expressing clone, we optimized culture conditions to maximize IgE production and minimize time and resources. We observed a slow decrease in specific daily antibody productivity consistent with cell growth rate and consumption of culture medium nutrients. This productivity decrease was due to nutrient depletion in the medium rather than cell density (see 6 cells/mL generating 2 mg/d (D).To maximize yields, we tested different seeding Expi-CSPG4 IgE cell concentrations in fresh medium, measuring secreted antibody daily for 5 days. As expected, higher starting cell concentrations yielded faster and greater antibody production, with cells seeded at 11 × 10g 2 mg/d , D.6 and 11 × 106 cells/mL), which place cells under stress, we analyzed production consistency over time by passaging every 2 days at 5 × 106 cells/mL (5M/mL 2D) or every day at 5 × 106 cells/mL (5M/mL 1D) or 11 × 106 cells/mL (11M/mL 1D), replacing medium at every passage. After 4 days, all conditions yielded consistent antibody production (E). The 11M/mL 1D and 5M/mL 2D conditions yielded similar production per passage. However, 11M/mL 1D resulted in the highest production per day (see www.jacionline.org), suggesting this is optimal for reducing resources and time.Using high cell concentrations , with the ability to repeat the process with the same cells at least 3 times without losing production efficiency. Yields in 4 days in small shaking flask cultures were approximately 33-fold greater than the most optimal 14-day stable IgE production recorded in shaking flask conditions and 13-fold higher than 14-day IgE production reported by using bioreactors.Different culture conditions can affect antibody quality and structural and functional properties, including posttranslational modifications, such as glycosylation.A). Lectin blot and liquid chromatography–mass spectrometry (LC-MS) glycosylation analyses revealed no significant differences among IgE produced with our method (B and C), particularly with regard to oligomannose structures, removal of which is reported to abrogate anaphylaxis.D). All preparations triggered significant and comparable levels of mast cell degranulation when cross-linked by polyclonal anti-IgE or target antigen on CSPG4high melanoma cells (E). The hapten-specific NIP-IgE cross-linked by polyclonal anti-IgE, but not by CSPG4high cells, triggered significant degranulation. These suggest that different preparations and density conditions preserve receptor recognition and antibody potency. Importantly, reduced MAN5 in the new IgE is insufficient to affect IgE binding or functionality.Size-exclusion HPLC showed very similar antibody main peak profiles , A. Lectr method , B and Cma cells , E. The www.jacionline.org). This offers new opportunities to expedite the design of novel therapeutic antibodies with enhanced effector functions suitable for basic and translational research, scaling up, process development, and manufacturing for clinical trials of IgE therapy in patients with cancer. Rapid generation of IgE antibodies recognizing allergens, cancer antigens, or parasitic targets can find direct applications for exploring multifaceted functions of IgE in allergy, oncology, and AllergoOncology.Therefore we present a novel process for serum-free production of IgE with comparable structural and functional characteristics to the previous pVITRO method but at higher yield and in less time than any documented stable platforms and with less resources than transient systems (see


To assess brake reaction time  in patients receiving transfemoral coronary angiography (CAG). We assumed that patients would have a significantly impaired BRT after the procedure.A prospective, observational study design was applied. Consecutive patients undergoing right-sided transfemoral CAG as part of the clinical routine were included. An experimental driving simulator was used to determine BRT after receiving a visual stimulus. The subjects applied the brake with their right foot as quickly as possible when a red-light signal appeared. The time interval between stimulus and brake application was taken as BRT. In addition to the total BRT, also its components were determined: neurologic reaction time, foot transfer time and brake travel time. BRT was determined before and 1 day after CAG (pre-post comparison).p = 0.270). Similarly, also the BRT components ‘foot transfer time’ and ‘brake travel time’ did not show significant differences between the two test occasions. However, neurologic reaction time decreased from 269 ± 67 to 255 ± 64 ms (p = 0.036).71 patients were included in the analysis . Total BRT was 594 ± 188 and 591 ± 198 ms before and after the CAG procedure, respectively (We found no impairment of BRT on the first day after puncture of the right-sided femoral artery in patients undergoing CAG. Therefore, with regard to BRT, it is regarded safe to resume driving from day 1 after CAG. Other factors of driving safety beyond BRT must also be considered. In the context of standard procedures of the lower limbs, physicians are often confronted with questions regarding driving ability after invasive procedures. Such questions are of obvious importance for patient safety as well as for the safety of other traffic participants. Driving ability is crucial for an individual’s participation in contemporary social life and an important task in activities of daily living. Therefore, an excessive driving prohibition following interventional or surgical treatment is not useful. When weighing the interests of safety vs. independence, it would be beneficial to have specific scientific knowledge about postoperative driving impairments following invasive medical procedures.Among the various factors contributing to driving ability , previous studies reported “brake reaction time” (BRT) to be a key parameter  –3. BRT wPrevious research provided good evidence on driving ability in the context of elective orthopaedic surgery such as total joint arthroplasty –8, knee Therefore, the purpose of this study was to assess brake reaction time as a key factor in driving ability in patients undergoing puncture and temporary catheter sheath implantation in the right-sided femoral artery for invasive coronary angiography (CAG). We assumed that BRT might be significantly increased after the procedure in our patients .We designed a prospective, observational study that was approved by the Ethics Committee of our medical university (No. AN2016-0050 360/4.2 367/5.4 (3922a)) and was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. Consecutive patients undergoing CAG as part of the clinical routine of our university hospital were considered for participation and included after granting written informed consent. The only exclusion criteria were: (a) acute coronary syndrome, (b) no driver’s licence, (c) known neurologic diseases that would impair BRT.Patients with stable angina pectoris scheduled for elective CAG via a transfemoral route were included. All patients were pretreated with 100 mg Aspirin at least the day before and on the day of the procedure. For CAG, we gained vascular access to the right common femoral artery after local anaesthesia with 10 cc 1% lidocaine. Using the Seldinger technique we inserted a vascular access sheath  in the femoral artery. Patients undergoing PCI of a coronary artery received a 600 mg loading dose of Clopidogrel and 60–70 IE/kg of unfractionated Heparin immediately prior to guide wire placement. After PCI, administration of Aspirin and Clopidogrel was continued once daily in all patients until the end of the study. After CAG completion, we removed the sheath and closed the vascular access site using the FemoSeal™ vascular closure device . All patients additionally received an inguinal compression bandage and rested in bed for the following six hours before mobilization. This procedure was identical in all patients regardless if they were treated with PCI or not. Patients with inguinal hematoma (> 5 cm diameter), clinically apparent pseudoaneurysm (vascular murmur) or immobilizing pain at the vascular access site were excluded from the follow-up measurement. For study exclusion due to access site complications, we only used clinical assessments. If clinically indicated, color-coded Doppler sonography was used to guide the further treatment strategies.Based on apparatuses described and validated in the published literature , 2, we dThe procedure was commenced by the patient fully depressing the accelerator, which was confirmed when a green lamp lit up. This prevented the patient from driving in a ‘ready-to-brake fashion’. After an interval of 5–10 s, the investigator pressed a switch concealed from the patient, which activated the red lamp  and the electronic clock. Before starting the procedure, the subjects were instructed in a standardized manner to apply the brake with their right foot as quickly as possible when the red-light signal appeared. The time interval until the subject operated the brake pedal with 160 N was measured and taken as the BRT.In addition to the total BRT, the integrated stopwatch also automatically determined individual components of BRT: neurologic reaction time , foot transfer time  and brake travel time .Patients exclusively used their right leg to operate the pedals of the apparatus, while the left foot rested on the clutch pedal. After the subject had familiarized himself with the apparatus in three training runs, BRT was measured consecutively 10 times as described. An interval of 5 s was maintained between measurements. Total BRT and its components were calculated as the arithmetic average of the 10 measurements. All participants were given the same standardized instructions. BRT was determined before CAG to obtain a reference value. Another BRT measurement was performed in all patients on day 1 after CAG. On day 1 after CAG patients also rated their level of inguinal pain on a 10-point numeric rating scale (NRS).Data analysis was performed with SPSS . Data were not normally distributed as indicated by the Kolmogorov–Smirnov test. As descriptive values medians and interquartile ranges were determined. Wilcoxon tests were applied to test for significant differences in BRT between measurements 1 and 2. Alpha was defined as 0.05 (two-tailed).Eighty-four patients were included and underwent BRT measurement at the first test. Of these patients, 13 did not participate in the second BRT measurement for the following reasons: organizational reasons: four, cancelled CAG: one, haematoma > 5 cm: three, immobilizing pain: three, refused: two. The remaining 71 patients were included in the analysis . Patient characteristics are provided in Table p = 0.270). Similarly, also the BRT components ‘foot transfer time’ and ‘brake travel time’ did not show significant differences between the two test occasions. However, neurologic reaction time decreased from 269 ± 67 to 255 ± 64 ms (p = 0.036) (Table Total BRT was 594 ± 188 and 591 ± 198 ms before and after the CAG procedure, respectively p = 0.70. SimilThe most important finding of the study was that BRT was not impaired on day 1 after puncture of the right-sided femoral artery. Therefore, it is deemed safe to resume driving from day 1 after CAG.When trying to compare our findings with previous research, it appears that no such studies have been conducted to date. This is surprising because so many other standard medical procedures involving the lower limb have been investigated with regard to BRT. Those studies dealt with inguinal hernia repair –17, totaAs mentioned above, on the basis of our findings on BRT we do not recommend a ban on driving following right-sided puncture of the femoral artery with temporary catheter sheath implantation. Our findings might also be applicable to patients who undergo puncture of the right-sided femoral artery for endovascular therapy of peripheral arterial occlusive disease. Although we believe that also such patients should be allowed to resume driving, this must be viewed with more caution as we tested only patients who underwent CAG.We did not analyze left-sided puncture of the femoral artery although invasive procedures of the left leg can potentially affect BRT, as was shown for total knee arthroplasty . The exaWe detected a slight decrease in neurologic reaction time in the post-CAG measurements compared to the baseline measurements. This difference was presumably based on a certain training effect as the assessments were conducted on two consecutive days. A difference of 14 ms was found, probably without actual clinical relevance. Additionally, experienced drivers would not experience the described training effect in their own car in real circumstances, i.e. no 14 ms delay due to the initial use of an artificial car simulator.The following limitations must be acknowledged. Thirteen patients could not be included in the second BRT measurement, thus giving a drop-out rate of 15%. Therefore, our results must be interpreted with caution. Moreover, the above-mentioned recommendations for patients with endovascular therapy of peripheral arterial occlusive disease must be viewed with even more caution as we tested individuals only following CAG. We studied driving safety only in terms of BRT. Although BRT was described as the most important factor . As an eThe study findings are regarded as clinically relevant. No previous studies have investigated driving ability after the standard medical procedure of transfemoral coronary angiography. When weighing the interests of safety vs. independence, it is mandatory to have specific scientific knowledge about postoperative driving impairments following invasive medical procedures. Even if the transradial approach is increasingly used in CAG and BRT is very likely not affected by it, the transfemoral access in CAG is still widely used . We, theWe found no BRT impairment on day 1 after puncture of the right-sided femoral artery in patients undergoing CAG. Therefore, with regard to BRT, it is deemed safe for patients to resume driving from day 1 following CAG. Other driving safety factors beyond BRT must also be considered.