This study assessed the influence of donor, environmental, and logistical factors on the contamination rates of the conjunctival swabs and organ culture media of human donor eyes.In total, 1008 conjunctival swabs and 418 organ culture media samples from 504 consecutive human donor eyes were analyzed. Cross-tabulation, chi-squared tests, and Fisher's exact tests were used to assess the influence of the different factors on the contamination rates of the conjunctival swabs and organ culture media.P = .007]. The highest conjunctival swab contamination rate was found in the corneas procured in external institutions  . Hospitalization times of 2 to 7 days prior to death were associated with an increased conjunctival swab contamination risk . However, the sex, age, cause of donor death, differentiation between septic and aseptic donors, differentiation between heart-beating brain-dead multiorgan donors and cadaveric donors, a warmer mean monthly temperature, and death to corneoscleral disc excision time did not significantly increase the conjunctival swab contamination risk. In addition, none of these factors affected the organ culture media contamination risk. Moreover, a positive conjunctival swab did not significantly increase the media contamination risk (P = .08). Surprisingly, the microorganisms causing media contamination were present at 50% of the amount detected on the conjunctival surface of the respective donor eye.The overall contamination rates were 28.4% for the conjunctival swabs and 1.0% for the organ culture media. A prolonged time between death and the conjunctival swab collection was associated with an increased conjunctival swab contamination rate [odds ratio (OR) = 1.9, 95% confidence interval (CI) = 1.2–3.0, A prolonged time between death and the conjunctival swab collection, a hospitalization time of 2 to 7 days prior to death, and corneal collection outside the university hospital seemed to be the main factors responsible for an increased conjunctival swab contamination risk. In addition, our investigation illustrated that a positive conjunctival swab is not a strong indicator for organ culture media contamination. Critical discussion is necessary regarding the validity of conjunctival swabs as prognostic parameters for organ culture media contamination. Until January 2018, the German guidelines on the collection and processing of donor corneas have required 2 microbiological examinations of the donor's cornea using conjunctival swabs after disinfection and before the corneal excision or enucleation.,23 In spite of donor eye decontamination with povidone-iodine, persistent microorganismal colonization has been demonstrated.–27The transplantation of contaminated corneal tissue may lead to postoperative endophthalmitis, which represents one of the most serious post-keratoplasty complications, and it may have devastating consequences for a patient's vision.multiorgan donor versus cadaveric donor, septic donor versus aseptic donor, hospitalization time prior to death, procurement site, death to conjunctival swab collection time, enucleation to processing time, and mean monthly temperature.In this study, we investigated the influence and correlations of several factors on and between the contamination of conjunctival swabs and organ culture media, like sex, donor age, cause of death, heart-beating brain-dead To the best of our knowledge, the evaluation of so many factors on the contamination of conjunctival swabs and organ culture media has never been undertaken before. Nor have the correlations been determined between the microbial growth in the conjunctival swabs of corneal donors and organ culture media contamination in an eye bank, during a study period without changing standards, while simultaneously considering the microbiological species.22.1 We came to the conclusion that older donors cannot generally be excluded from corneal donation due to the scarcity of grafts available for keratoplasties in Germany.In the period from July 2015 to September 2017, 504 corneas from 252 consecutive donors were stored at the Tübingen Eye Bank in Tübingen, Germany. No maximum donor age limit was set, and the minimum donor age was 14 years old. In 2016, our study group showed that our collective would have lost nearly 14% of the grafts collected for transplantation by using a maximum donor age (>79 years old).A detailed medical history of each donor was obtained from interviews with the family, the last attending doctor, the donor's family doctor, and a review of any hospital medical records. The consent and medical history of each donor were recorded. The donor serology was analyzed from a blood sample taken up to 24 hours postmortem. A culture period beginning up to 72 hours postmortem was accepted. The beginning of the culture period was defined as the time when the prepared and excised corneoscleral discs were first placed in the tissue culture flasks  filled with culture medium  with 2.5% fetal bovine serum . Any potential donors with high-risk sexual behaviors or intravenous drug use, and consequently, at high risk for any of the infectious pathogens  were not eligible to donate.Blood samples had been drawn for the mandatory infectious disease tests, including human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), and syphilis. The infectious disease testing was done as per previously published methods.This study was approved by the institutional review board of the University of Tübingen, and it adhered to the tenets of the Declaration of Helsinki.2.2The periocular region  and the ocular surface  were cleaned using a 0.75% povidone-iodine solution [1 mL of 7.5% Braunol  diluted with 10 mL of sterile 0.9% NaCl ] for at least 3 minutes. The face and the head of the donor were covered with sterile surgical drapes, leaving the eye region open. The medical doctor performing the procedure wore a sterile gown, sterile gloves, a surgical cap, and a mask. Before enucleation, 2 conjunctival swabs  were taken from each eye . All corneas were procured by members of the Tübingen eye bank which means that the swabs and samples taken in external institutions were taken by the same doctors taking the samples in the principal institutions. After a limbal-based conjunctival incision, a 360° peritomy was performed. Following the blunt dissection of the muscles from the conjunctiva and Tenon's capsule, the muscles were transected using a muscle hook and scissors. The optic nerve was cut with curved blunt scissors to allow a complete enucleation.2, and 95% humidity. Sterile instruments were used throughout the whole process, including the enucleation, corneoscleral disc excision, and endothelial examination. The processing of each whole globe was performed in a class II biological safety cabinet by one experienced technician.The 2 globes were stored separately in sterile urine cups . We placed each eye in gauze at the bottom of the sterile cup, which was filled with 10 mL of 0.9% physiological NaCl (B. Braun Melsungen AG) and 5 mL of gentamicin eye drops . The eyes were transported to our eye bank on a cool pack in a cool box (temperature between 1°C and 10°C). Here, the donor globes were stored in a refrigerator at 4°C until the preparation of the corneoscleral discs in a class II biological safety cabinet. Before preparing the corneoscleral discs, all excess conjunctiva was removed from the donor globes, and they were immersed separately in a 0.375% povidone-iodine solution [2.5 mL of 7.5% Braunol diluted with 50 mL of sterile 0.9% NaCl (B. Braun Melsungen AG)] for 5 min, followed by rinsing with 50 mL of sterile 0.9% NaCl (B. Braun Melsungen AG). Then, a 15-mm trephine was used to cut the sclera around the cornea, and the cut was completed using scissors. The corneoscleral disc was carefully separated from the iris and the uveal tissue using scissors and tweezers. After that, the corneoscleral disc was placed on a corneal holder  and put into a tissue culture flask (Corning Incorporated) filled with Culture Medium I (Biochrom AG) with 2.5% FBS (Merck Millipore). Culture Medium I is supplemented with 60 μg/mL of penicillin G sodium, 130 μg/mL of streptomycin sulfate, and 2.5 μg/mL of amphotericin B. The corneoscleral discs were stored in an incubator at 37°C, 5% CO2.32.3.1ead after 24 and 48 hours and after 10 days.The conjunctival swabs were transported and stored at room temperature. All the samples were tested immediately at the Institute of Medical Microbiology and Hygiene . Supplemented Columbia sheep blood agar plates , Endo agar plates, and supplemented brain heart infusion (BHI) agar plates  were incubated at 37°C to test for bacterial contamination. A liver broth was also used. Additionally, yeast-gentamicin plates  were used to detect fungal contamination, and they were incubated at 30°C. The plates were incubated for 10 days. They were rAny proof of bacterial or fungal microorganisms was documented as contamination.If there was cultural growth, the microorganisms were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry . 2.3.2ead after 24 and 48 hours and after 7 days. The contaminated corneas were removed from the eye bank. Two days before transplantation, the culture medium was exchanged and retested for contamination. Any proof of bacterial or fungal microorganisms was documented as contamination.The culture flasks underwent daily visual inspections for turbidity. After 3 days of incubation in the Tübingen Eye Bank the culture media were routinely tested for contamination. For this, each corneal medium was exchanged in a class II biological safety cabinet, and it was tested for bacterial and fungal contamination at the Institute of Medical Microbiology and Hygiene. The supplemented Columbia sheep blood agar plates (Oxoid GmbH), Endo agar plates, and supplemented BHI agar plates  were incubated at 37°C to test for bacterial contamination. A liver broth was also used. Yeast-gentamicin plates  were used to detect fungal contamination, and they were incubated at 30°C. The plates were incubated for 7 days, and they were r2.4multiorgan donor versus cadaveric donor, septic donor versus aseptic donor, hospitalization time prior to death, procurement site, death to conjunctival swab collection time, enucleation to processing time, and the mean monthly temperature. To explore whether the seasonal temperature changes corresponded to the contamination rates of the conjunctival swabs and organ culture media, the donation months were divided into 2 separate groups: May through September, which included the warmer months, and October through April, which included the cooler months.We collected the relevant donor and storage data, including the sex, donor age, cause of death, heart-beating brain-dead  There is no weather station in Tübingen; therefore, we used the data from the next closest weather station (Stuttgart-Echterdingen). The distance as the crow flies is around 20 kilometers.The mean monthly temperature data of Stuttgart-Echterdingen, which is close to Tübingen, was obtained from the German meteorological service homepage.2.5P < .05 was considered to be statistically significant.The statistical analysis of the data was conducted using IBM SPSS Statistics for Windows, version 24.0 . The categorical data were analyzed using cross-tabulations and Pearson's chi-squared tests. Fisher's exact tests were used as tests of association, as appropriate. The quantitative data were reported as the mean with the standard deviation. The odds ratios (ORs) were quoted with 95% confidence intervals (CIs), and 32. The mean time between death and the conjunctival swab collection was 12.4 ± 15.0 hours, and the mean time between death and the corneoscleral disc excision was 27.3 ± 12.3 hours. The time period between death and the conjunctival swab collection was divided into 2 groups: group 1 was < 6 hours and group 2 was 6 to 72 hours. The contamination rates of the conjunctival swabs were 20.9% in group one and 33.1% in group 2 (Table P = .007). The organ culture media contamination rates were 1.6% in group 1 and 0.7% in group 2. The time interval between death and the conjunctival swab collection had no statistically significant influence on the contamination rate (P = .6).This retrospective study included the microbiological testing of 1008 conjunctival swabs and 418 organ culture media samples from 504 consecutive donor eyes. Around 86 corneas and their culture media samples were discarded before the 3 days of incubation were over. The main reasons therefore were serology-reactive or nonevaluable blood samples of the donors. The male-to-female ratio was 62% to 38%. The overall contamination rate of the conjunctival swabs was 28.4%, and that of the organ culture media was 1.0%. The mean donor age was 68 ± 15 years old (range 17–96 years). The most common causes of death were cardiovascular disease (34.8%), infection (14.4%), cancer (12.4%), and multiple organ dysfunction syndrome (12.4%). The mean endothelial cell density at beginning of the storage time was 2261 ± 490 cells/mmP = .003). The second highest conjunctival swab contamination rate was found in the corneas procured in the pathology department of the University Hospital Tübingen    , cause of donor death , donor age , heart-beating brain-dead multiorgan donor versus cadaveric donor , septic donor versus aseptic donor , and mean monthly temperature of the warmer months .Hospitalization times of 2 to 7 days were associated with an increased risk of conjunctival swab contamination , Enterococcus spp. (3.8%), and Escherichia coli (3.3%). Around 5% of the conjunctival swab contamination organisms were of fungal origin, exclusively Candida spp. Around 75% of the organ culture media contamination organisms were of fungal origin, exclusively Candida spp., and 25% were of bacterial origin, exclusively gram-positive organisms. There is no apparent common factor with the patients that gave positive fungal results for the culture media contamination.A total of 95% of the conjunctival swab contamination organisms were of bacterial origin. The most common bacteria belonging to the normal skin bacterial flora (70.7%), including coagulase negative staphylococci, The microorganisms causing the medium contamination were a mere 50% equivalent to those detected on the conjunctival surface of the respective donor eye.4Our investigation illustrates that a prolonged time between death and the conjunctival swab collection, hospitalization times of 2 to 7 days prior to death, and corneal collection outside the university hospital seemed to be mainly responsible for the increased conjunctival swab contamination risk in 1008 conjunctival swabs from 504 consecutive donor eyes at the Tübingen Eye Bank. The different factors evaluated did not have an effect on the 8 organ culture media samples tested, and a positive conjunctival swab did not significantly increase the medium contamination risk.–32 This wide range may be due to the varying decontamination protocols and different conjunctival swab origins. While Reddy and Paul found a 7.0% positive rate in patients admitted for cataract surgery (eyes in a living state-control group) after inserting one drop of proparacaine into the conjunctival sack, Mindrup et al, Matsumoto et al, and Fuest et al–32 obtained rates in line with our results of 28.9%, 36.7%, and 22.8%, respectively, after disinfection with povidone-iodine in corneal donor cadavers. Without decontamination before the swab collection, Capriotti et al obtained a rate of 86.2%.The positive conjunctival swab rate of 28.4% found in our investigation can be placed in the range of the 7.0% to 86.2% positive swabs found in previous studies.Staphylococcus spp., Corynebacterium spp., and Streptococcus spp. (excluding Streptococcus pneumonia) were the predominant microorganisms found on the conjunctival swabs in our study, which fits the bacterial spectrum of the human conjunctiva described by Matsumoto et al, Reddy and Paul, Fuest et al, and Wilhelm et al.,31,32,34 They demonstrated that low concentrations  were more rapidly bactericidal than a full strength treatment .Donor eye decontamination before graft processing plays a significant role in preventing tissue contamination. Many years ago, povidone-iodine was known to be one of the best antiseptic solutions as a skin preparation for the reduction of microorganisms. In 1982, the bactericidal activity of various concentrations of povidone-iodine was tested by Berkelman et al. recommended the use of a 0.75% povidone-iodine solution for at least 3 minutes to decontaminate the donor eyes. However, in total, we decontaminated the donor eyes for at least 8 minutes in low concentrations of povidone-iodine and transported the eyes separately between the 2 decontaminations in sterile cups filled with 5 mL of gentamicin eye drops.The main reasons for the very low organ culture media contamination rate in our investigation in contrast to other studies could be the different graft preparations and processing. Decontamination protocols and decontamination procedures vary in many ways. In our study, the periocular region, the fornices, and the ocular surface were cleaned using a 0.75% povidone-iodine solution for at least 3 minutes. After sterile enucleation, the donor globes were also immersed separately in a 0.375% povidone-iodine solution for 5 min. In accordance with our study, Laubichler et al who cleaned the periocular region with a 7.5% povidone-iodine solution spray and the ocular surface with a 1.25% povidone-iodine solution for 3 and 5 minutes, respectively. Furthermore Li et al abstained from the use of gentamicin, and they prepared the corneoscleral disc directly at the donor site instead of in a class II biological safety cabinet, without decontaminating twice, as in our study. Despite the slightly similar steps, no complete execution of the entire detailed decontamination protocol described in our investigation could be found in any of the other studies considered.Our extensive decontamination protocol, as described above, could explain why our organ culture media contamination rate of 1.0% was lower than the rates of 4.5% to 10.8% found by Li et al,With regard to the hospitalization times of more than 7 days prior to death, no significantly increased conjunctival swab contamination risk was seen. The reason for this might be that these patients were typically submitted to extensive systemic antibiotic and antimycotic treatments before death. For this reason, the microbiological flora in the conjunctival compartment may also have been reduced. determined that cancer, infections, and respiratory diseases as causes of death significantly increased the culture media contamination risk. The fact that there was no significant factor influencing the culture media contamination rate in this study was probably due to the low contamination rate of 1%.Contrary to our investigation not discovering any factors influencing the culture media contamination rate, Armitage et al,39 However, this should be tested in vitro on human corneas over a longer culture period of time before routine use.Because the microorganisms causing the culture media contamination were tested as sensitive to several antibiotic and antifungal substances in our study, the addition of different antibiotics and antifungals to the culture medium can be discussed in the future. The use of voriconazole, as a triazole reacting differently than the currently used polyene amphotericin, is conceivable. The aminoglycoside streptomycin and beta-lactam penicillin could be supplemented by the fluoroquinolone levofloxacin in the future. Both levofloxacin and voriconazole have been described as nontoxic to corneal endothelial cells.Nevertheless, some points should be considered before drawing hasty conclusions. The main limitation of our evaluation was the pilot nature of the observations. Studies in the future will require a larger sample size, which would increase the power of the analysis and the validity of its findings. Another limiting factor of this study was the limited comparability of these data with other publications on the results of the microbiological testing of conjunctival swabs and organ culture media. The other studies used different methods of excising the corneoscleral disc only versus enucleating the entire globe, different decontamination protocols at different concentrations and repetitions, and different methods of microbiological testing. In future studies the investigation on antibiotic and antimycotic treatments before donor death and its influence on the contamination rates of the conjunctival swabs could be considered as well as the addition of different antibiotics and antifungals to the culture medium. To increase the comparability with other publications on this topic the microbiological testing could distinguish between different germs instead of summarizing several bacteria as normal skin bacterial flora.In conclusion, this investigation illustrates that a prolonged time between death and the conjunctival swab collection, hospitalization times of 2 to 7 days prior to death, and corneal collection outside the university hospital seemed to be the main factors responsible for an increased conjunctival swab contamination risk. Surprisingly, the microorganisms causing the medium contamination were only 50% equivalent to the organisms detected on the conjunctival surface of the respective donor eye. Moreover, a positive conjunctival swab was not a strong indicator for corneal culture medium contamination. Therefore, a critical discussion is necessary about the validity of conjunctival swabs as prognostic parameters for organ culture media contamination.Conceptualization: Tobias Röck, Gunnar Blumenstock.Data curation: Tobias Röck, Johanna Landenberger, Daniel Röck.Formal analysis: Tobias Röck, Johanna Landenberger.Investigation: Tobias Röck, Johanna Landenberger, Michael Buhl, Efdal Yoeruek, Daniel Röck.Methodology: Tobias Röck, Johanna Landenberger, Efdal Yoeruek, Karl Ulrich Bartz-Schmidt, Matthias Bramkamp, Gunnar Blumenstock, Daniel Röck.Project administration: Tobias Röck, Daniel Röck.Resources: Michael Buhl, Efdal Yoeruek.Software: Johanna Landenberger, Gunnar Blumenstock.Supervision: Tobias Röck, Karl Ulrich Bartz-Schmidt, Matthias Bramkamp, Gunnar Blumenstock, Daniel Röck.Validation: Tobias Röck, Johanna Landenberger, Michael Buhl, Matthias Bramkamp, Gunnar Blumenstock, Daniel Röck.Visualization: Tobias Röck, Gunnar Blumenstock.Writing – original draft: Tobias Röck, Daniel Röck.Writing – review & editing: Tobias Röck, Michael Buhl, Efdal Yoeruek, Karl Ulrich Bartz-Schmidt, Matthias Bramkamp, Gunnar Blumenstock.


Cerebrospinal fluid (CSF) drainage is a technique that has significantlyreduced the incidence of spinal cord ischaemia (SCI). We present results ofa systematic review to assess the literature on this topic in relation tothoracoabdominal aortic aneurysm repair (TAAR).Major medical databases were searched to identify papers related to CSFbiomarkers measured during TAAAR.Fifteen papers reported measurements of CSF biomarkers with 265 patients intotal. CSF biomarkers measured included S-100ß, neuron-specificendolase (NSE), lactate, glial fibrillary acidic protein A (GFPa), Tau, heatshock protein 70 and 27 , and proinflammatory cytokines.Lactate and S-100ß were reported the most, but did not correlate withSCI, which was also the case with NSE and TAU. GFPa showed significant CSFlevel rises, both intra and postoperative in patients who suffered SCI andwarrants further investigation, similar results were seen with HSP70, HSP27and IL-8.Although there is significant interest in this topic, there still remains asignificant lack of high-quality studies investigating CSF biomarkers duringTAAR to detect SCI. A large and multicentre study is required to identifythe significant role of each biomarker. Surgical repair can offer a good prognosis comparedto conservative management, however, such type of surgery is associated with seriousmorbidities perioperatively and mortality.Spinal cord ischaemia (SCI) is a major complication post open thoracoabdominal aorticaneurysm repair (TAAR). The presence of TAAR is a major risk factor for suddendeath, it carries a mortality rate of 90% at 5 years if remainedunoperated. To protect against such complication, variousadjunctive techniques have been developed to maintain appropriate spinal cordcirculation and reduce the incidence of spinal cord ischaemia, among such techniquesare: cerebrospinal fluid (CSF) drainage to reduce CSF pressure and increase spinalcord perfusion, deep hypothermia which decreases the body’smetabolic activity and oxygen demand, sequential clamping and reattachment ofintercostal arteries, and distal aortic perfusion.One of the major perioperative complications is the development of paraplegia, whichis not only physically decapitating but also can lead to serious psychologicalissues. Such complication is consequence of spinal cord ischaemia, which comes fromlimited spinal cord blood flow during surgery, for which reports as high as 30% havebeen described,10. This method is used to alert surgeons toidentify impending spinal cord ischaemia and therefore have a chance to change theirintraoperative strategy to prevent postoperative paraplegia. However, in currentliterature, there is much debate and uncertainties as to the usefulness andapplication of MEPs and hence some centres do not routinely use it and yet, theyreport low rates of paraplegia. There does not currently exist any high-levelevidence to show that the use of MEPs significantly decreases the incidence ofpostoperative spinal cord ischaemia.A leading technique these days to assess spinal cord function is measuring motorevoked potentials (MEPs) intraoperatively, which potentially gives surgeons areal-time assessment of spinal cord function-16. This has been extrapolated into thoracoabdominalaneurysm repair because CSF drainage catheter is routinely applied in allthoracoabdominal aneurysm elective repairs and there are certain research outputssuggesting that there is an association of their appearance concurrently with spinalcord ischaemia. Our current research is in the development of a novel microwavesensor that can detect specific biomarkers in the CSF in real time, that we believecould offer a better real-time assessment of the spinal cord intra and postoperativethoracoabdominal aneurysm repair. In this review, we have systematically collatedall published information on the measured biomarkers in the CSF duringthoracoabdominal aneurysm repair and their relationship to SCI.Current evidence from the literature suggest that there is release of specificbiomarkers into the CSF when there an ischaemic insult to brain or spinalcordElectronic searches were performed in PubMed, MEDLINE, EMBASE, Google Scholar,SCOPUS, and Cochrane. No limits were placed on dates. Limits were placed onarticles published in the English language only. Search terms used includedthoracic aortic aneurysm OR thoracic aneurysm OR TAAR OR thoracic dissection ORthoracic aorta OR thoracic aortic dissection AND intrathecal OR cerebrospinalfluid OR CSF OR biomarker AND spinal cord ischaemia OR paraplegia OR paralysisOR neurological deficit. To achieve maximum sensitivity, all search terms werecombined with Boolean operators and searched as both keywords and MeSH  terms. Following exclusion of articles based on title orabstract, full text articles selected had reference lists searched for anypotential further articles to be included in this review.-30.In total, we found 15 papers that reported measurements of CSF biomarkers followingthoracoabdominal aneurysm repair. Those papers are summarized in -23. The results areambiguous as to whether high level of S-100β was associated with SCI orcan it be used as a marker to predict SCI intraoperatively as levels ofS-100β were not significantly higher in two studies for patients whosuffered from SCI compared to control group. In most studies,the rise seen in S-100β as a late change in the CSF and hence not usefulas early predictive factor for SCI.Seven studies reported levels of S-100β in the CSF as well as in patientswho suffered from SCI and compared to controls . In thisstudy, S-100β levels in the CSF could not be associated with silentneurological damage or adverse neurological complications postoperatively.However, CSF S-100β was higher in 75% of patients who suffered adverseneurological complications postoperatively than the 90th percentileof patients without neurological complications. The median concentration of CSFS-100β remained virtually unchanged and within reference range withoutadverse neurological complications.Lases et al. in their study described the measurements ofCSF S-100β in 8 patients undergoing open thoracoabdominal aneurysmrepair. CSF samples in those patients were collected at time of induction,application of cross-clamping to aorta, 5 minutes following reperfusion, at skinclosure, and 24 hours postoperatively. Among those 8 patients operated on, onesuffered from delayed-onset postoperative paraplegia, and it was noted that CSFS-100β continued to rise from skin closure and 24 hours postoperatively,unlike the other 7 patients in whom S-100β decreased following skinclosure. In the patient with postoperative paraplegia, the MEP and SSEPmeasurements intraoperatively did not show any significant abnormalities toindicate presence of SCI.Van Dongen et al. prospectively studied 23 patients undergoingTAAR or descending thoracic aortic aneurysm repair (DTAAR) who all had spinalcord drainage and CSF measurement of S-100β, the samples were taken attime of induction, 0, 6, 12, 18, 24, 48, and 72 hours postoperatively, amongthose 23 patients, four of them suffered from SCI . Inthis study, S-100β levels were statistically increased 6 hourspostoperatively in those with SCI compared to those without evidence of SCI(P<0.0010). These levels peaked at 48 hours (289± 454 µg/L). In this study, there was a clear correlation betweenS-100β CSF concentrations and SCI and the authors concluded thatS-100β seems to be a sensitive reliable biomarker to predict SCI.Kunihara et al. investigated 11 patients undergoing electiveDTAAR or TAAR and measured CSF S-100β level and furthermore measured theconcentrations of its isoforms (S-100A1B and S-100BB). Two out of elevenpatients suffered from postoperative neurological damage (1 stroke and 1paraplegia). The patient who suffered from paraplegia developed thiscomplication 3 days postoperatively. In this patient, the S-100βpreoperative levels rose 20 hours after surgery and peaked  on the 3rd postoperative day.Anderson et al. studied 13 patients undergoing TAAR andmeasured their CSF S-100β level. CSF samples were taken for analysis attime of induction, before and after application of aortic cross-clamping,following this every 10 minutes intraoperatively and then every 30 minutes, onceon intensive care, samples were taken 3 times, then twice daily until removal ofthe CSF drain. Among the 13 patients, two suffered from spinal cord, one of themsuffered severe cerebral ischaemia, as well as signs of SCI, peak CSFS-100β was 400.8 µg/L on the first postoperative day, while CSFS-100β levels continued to rise from the beginning of the operation inthose with SCI, but with highest levels seen at 6 hours.Khaladj et al. studied 8 patients who underwent thoracicendovascular stent grafting of the descending aorta. The patients were deemed athigh risk of suffering postoperative paraplegia as the aneurysm involvedcritical intercostal arteries. CSF measurements were taken at induction, duringstenting, 6 hours following completion and 20 hours after repair. In this study,no patient developed neurological deficit following graft stenting, theirbaseline CSF S-100β concentrations were 0.9 to 1.7 µg/L.Brunnekreef et al. describes 28 patients undergoing DTAAR or TAARand measurements of CSF S-100β. In this study, 3 patients developed SCIand a further patient developed both SCI and brain infarction. In the latter,CSF S-100β peaked at 965 µg/ml 48 hours postoperatively. Asubsequent patient with brain infarction without SCI showed a CSF S-100βpeak of 16.8 µg/ml. The other 3 patients who suffered SCI showedstatisically significant higher levels of CSF S-100β from 12 to 24 hourspostoperatively . Interestingly, Shiiya et al. describes 3 patients who sufferedfrom temporary neurological deficit, their CSF S-100β rose following thecompletion of surgery similar to the SCI group and above those withoutneurological injury, however, levels of CSF S-100β decreased 24 hourspostoperatively to near baseline.Shiiya et al.17) found that CSF S-100β did rise in 75% of those withSCI, yet remained unchanged in the other 25% of the patients. However, in therest of studies, CSF S-100β rose in all patients who suffered from SCI,interestingly the peak level of CSF S-100β was extremely variable betweenstudies,18,20. For the most of the studies, increase in CSFS-100β was a late marker of SCI and was noted following completion of theoperation, although a large majority of patients in these studies suffered fromlate-onset postoperative SCI.The current literature investigating the association of CSF S-100β to SCIis limited. Furthermore, the results displayed are equivocal as to whether thisis a potential useful biomarker. The largest of the studies ], however, only three studies comparedlactate CSF levels in patients with neurological deficit compared to controls and did not add value to the data obtained fromMEPs. The second study was conducted by Anderson et al.. In this study,the NSE level in patient with paraplegia was at the upper limit throughout theprocedure and this did not change significantly during the entire procedure,however this was non-specific as the same increase was noted in patients who didnot develop any evidence of SCI, on the contrary, it was noted that one of thepatients who developed stroke postoperatively had a high level of serum NSEwhich peaked at first postoperative day. Ultimately, their study shows poorcorrelation between CSF and serum NSE value and, thus, prediction of SCI.Neuron-specific enolase is a 78 kD gamma-homodimer and represents the dominantenolase-isoenzyme found in neuronal and neuroendocrine tissues. Its levels inother tissues, except erythrocytes, are negligible. The biological half-life ofNSE in body fluids is approximately 24 hours. A comparative NSE level in CSF wasmeasured in two studies (summarized in Tau is the major microtubule-associated protein (MAP) of a normal mature neuron,it has two major functions, its ability to promote assembly and to maintainstructure of microtubules of the neurons, and therefore derangements in tau cancause serious neurological issues., they reported the levels of Tau protein inCSF for 28 patients that underwent DTAAR or TAAR in the CSF of 37 patients that underwent TAAR. CSF samples were taken atbaseline, at aortic cross-clamping, 1, 2, 12, 24 hours after cross-clamping, atthe completion of surgery, and if there were any signs of paraplegia. Fifteenpatients were reported as having postoperative paraplegia. In these patients thecumulative averages and mean values of both HSP70 and HSP27 were larger thanpatients who did not develop paraplegia. These values became statisticallysignificant at 12 and 24 hours postoperatively. Furthermore, they showed thatpatients with non-linear HSP measurements overtime or had larger positiveaverage changes from pre- to post-cross-clamp time were more likely toexperience paraplegia.Heat shock proteins are a group of proteins that are produced in response tostressful conditions; initially they were described in response to heat shock,however later they were noted in other stressful tissue situations. A study byHecker et al., GFAp is thought to help in maintainingastrocyte mechanical strength, however, it was first named, isolated andcharacterized by Eng et al. in 1969.Glial fibrillary acidic protein is an intermediate filament protein that isexpressed by many cell types of the central nervous system. First described in1971 reported the only study in which GFAp wasmeasured in 11 patients that underwent DTAAR or TAAR , midreperfusion (from 7 to 15 hours) and late reperfusion (over 15 hours).Measurements from the SCI group showed significant differences in glutamate butdid not show differences in glycine level at all measurement’s times, exceptbaseline. Aspartate showed significant increases in the SCI group during lateperfusion measurements only. Glutamate levels increased significantly across allmeasurements; nearly threefold at time of aortic cross-clamp application (375%),remained high at early reperfusion, but lowering to around 250%, before risingat late reperfusion to approximately 400% of baseline.The study by Brock et al.. Those monitoring tools have a key role as anadjunct-monitoring method while repairing the diseased aorta to predict the risk ofneurological injuries and provide rescue when it occurs and hence the outcomes couldbe better when using such methods. In a large study by Estrera et al., of 105complex aortic repairs CSF drainage, MEPs and SSEPs played a key role in earlydetection of SCIs, such injuries were noted through alterations in the signalsconduction and therefore immediate actions were taken to revertthis. Similar outcomes were also reported by Dong et al.of the fact the both MEPs and SSEPs can predict spinal ischaemia and theirintraoperative use can prevent the development of paraplegia. Finally, in asystematic review and meta-analysis by Fok et al., they have reported the beneficialuse of IONM including both MEPs and SSEPs, however there is lack of generalconsensus about the superiority or full recommendation of eithermethod.Surgical intervention remains the recommended treatment in patients withthoracoabdominal aortic aneurysms that cannot benefit from endovascular management,this option is particularly important to prevent dissection or rupture and,ultimately, death. Paraplegia remains a devastating complication of TAAR,particularly when considering that many of such patients are being operated on atelective basis. Several methods developed in light of the risk of paraplegia, suchas adjuncts to surgery like deep hypothermic circulatory arrest, circulatorysupport, CSF drainage, and the use of different forms of intraoperativeneurophysiological monitoring (IONM) in the form of MEPs andSSEP-37. This research has begun to trickle into theaortic world through small observational studies. Specific attention has focused onthe biomarkers lactate, S-100β, NSE and GFAp, which have all shown to be goodindicators correlating to injury magnitude in traumatic brain injury, as well asoutcomes and survival. The most promising potential biomarkers are S-100β,GFAp and HSP, according to our analysis and published literature.CSF biomarkers represent an area that has previously undergone much research withinthe neurosurgical field, with particular respects to traumatic brain injury wherethere are several ongoing clinical trials. S-100β is aneurotropic dimeric acidic Ca2+ -binding protein with a molecular weightof 21 kDa, existing in 2 β subunits (S-100αβ heterodimer andS-100ββ homodimer). One study in this review differentiated betweenthe two subunits but found no relevance between the two, the majority ofcommercially available assays do not differentiate between S-100αβheterodimer and S-100ββ homodimer,39.S-100β is part of the S100 protein family and the most widely studiedbiomarker and is highly conserved in the mammalian brain, with the majority found inlarge concentrations within the cytoplasm of astrocytes. Despite this, other smaller studies have foundstatistically significant associations of S-100β concentrations in CSF forpatients with neurological damage,20,21. One study described concentrations ofS-100β and patients with temporary neurological damage and S-100βwould rise in a fashion similar to those who suffered permanent neurological damage,but rather than plateau or continue to rise, would decrease back towardbaseline. This finding suggests that changes inS-100β concentration may reflect temporary or permanent neurological damage.However, the rise of S-100β was always a late sign of neurological damage,whether this is a reflection of sample timing could be argued.The results for S-100β as a potential biomarker in TAAR to detect SCI havebeen ambiguous. The largest of the studies involving 69 patients did not findsignificant changes in CSF S-100β in those who suffered neurologicaldamage, the data provided are suggestiveevidence that GFAp may serve as a biomarker for SCI in TAAR, or at least warrantsfurther investigation. GFAp is the principal 8-9 nm intermediate filament found inmature fibrillary astrocytes. Astrogliosis is the morphological and functionalchanges seen in astrocytes and occurs in response to a range of neurologicaldiseases or CNS injury, including ischaemia. This process has also been shown tocause the increased expression of GFAp. Anderson et al. noted, combined withthe relative insolubilty of GFAp, increased concentrations could reflect structuralCNS damage and release into CSF would imply disintegration of cells containingGFAp.Although only investigated in one study. They play crucial roles in folding/unfolding ofproteins, assembly of multiprotein complexes, transport/sorting of proteins intocorrect subcellular compartments, cell-cycle control and signalling, and cellprotection against stress/apoptosis. Furthermore, following their discovery they arereleased in a variety of other stresses, including ischaemia. Hecker et al. chooseto study HSP27 and HSP70 families for their association with cellular protection andrecovery after a near-lethal stress.The highly conserved large family of heat shock proteins often coined as molecularchaperones, found in all cells of all organisms, so called because of their initialdiscovery when their dramatic up-regulation was detected following subjection ofcells to heat shockOne of the main limiting factors in looking at the association between CSF biomarkersand SCI is that detection of these markers requires extensive and immediatelaboratory testing. Consequently, this requires time, which is not ideal forintraoperative use, specific equipment, which is expensive and furthermore needstechnical expertise. The ideal biomarker is one that is both sensitive and specific,that can be rapidly, inexpensively and easily detected. Furthermore, there must begood evidence to show that timely intervention in the presence of this biomarkeractually changes surgical outcomes. Although the quest for a biomarker continues, itwill require real-time detection to make intraoperative decisions to prevent SCI.These requisites make the search for the biomarker to detect SCI in TAAR verychallenging.CSF may potentially hold clinically relevant information regarding SCI, however, thisis an under-researched area in the thoracic aortic surgery world. So far, to theauthors’ knowledge, the most promising candidates are heat shock proteins, GFAp andS-100β. Detection of these biomarkers requires both time and financialsupport as they have very dynamic changes in their concentrations and are hard tomeasure experimentally. Our future research will be in the further development of areal-time sensor that can detect small changes in the concentration of theseproteins.


Ranae Oviductus. The fingerprint chromatograms of the Ranae Oviductus protein were established through an Agilent Zorbax 300SB-C8 column and diode array detection at 215 nm, using 0.085% TFA (v/v) in acetonitrile (A) and 0.1% TFA in ultrapure water (B) as mobile phase. The similarity was in the range of 0.779–0.980. The fingerprint chromatogram of Ranae Oviductus showed a significant difference with counterfeit products. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) successfully identified Ranae Oviductus from the samples. These results indicated that the method established in this work was reliable.This work demonstrated a method combining reversed-phase high-performance liquid chromatography (RP-HPLC) with chemometrics analysis to identify the authenticity of  Rana chensinensis is mainly distributed in the Changbai Mountain area, China. Ranae Oviductus is the dried oviduct of female Rana temporaria chensinensis David. The Ranae Oviductus is a potent traditional Chinese medicine that has been used in clinical studies for thousands of years. Today it is widely used as a nutrient food. It has been reported that Ranae Oviductus has significant effects in enhancing immunity, anti-fatigue, anti-aging, and lowering blood fat [Ranae Oviductus has been in short supply because of its limited production [Ranae Oviductus in the market [Ranae Oviductus, its authenticity identification has attracted more and more attention from the pharmacists, doctors, and medicinal scientists. The identification method of Ranae Oviductus is still under development. In the 2005 China Pharmacopoeia, the appearance and expansion degree were employed as discriminating items of Ranae Oviductus [Ranae Oviductus [Ranae Oviductus and counterfeit products using traditional methods [Ranae Oviductus.lood fat ,2,3,4. Aoduction . Its hige market ,7. Thoseviductus . Our groviductus . Accordi methods . TherefoRanae Oviductus are proteins and the proteins are the major bioactive components of Ranae Oviductus [Ranae Oviductus and counterfeit products using HPLC based on protein has not been studied yet. In addition, reversed-phase high-performance liquid chromatography (RP-HPLC) is a simple, fast, and effective technique for protein separation and characterization, as used for protein in milk, wheat gliadin, and transgenic zein [Angelica from related umbellifer herbs. Sun et al. analyzed polysaccharides from different Ganoderma. Li et al. established the fingerprint analysis of polyphenols, which were extracted from pomegranate peel, with reliable results [More than 40% of the components in viductus ,12. Howenic zein ,14,15. Onic zein . The Wornic zein . Many re results ,19,20.Ranae Oviductus were used as the study objects. We used RP-HPLC to establish a fingerprint method for the identification of Ranae Oviductus. Ten batches of Ranae Oviductus were collected from different main producing areas of the Changbai Mountains. A protein reference chromatogram was established using those Ranae Oviductus, based on protein composition similarity analysis. Furthermore, the difference between the authentic Ranae Oviductus and counterfeit products were investigated. The results were verified via a chemometric approach, utilizing principal component analysis and hierarchical clustering analysis. Both showed that the newly established Ranae Oviductus identification method was reliable.In this work, the main proteins components of The petroleum ether, guanidine hydrochloride, and ammonium sulfate analytical grade were purchased from Beijing Chemical Factory . The dithiothreitol (DTT) and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich . The HPLC-grade acetonitrile (MeCN) and HPLC-grade methanol were purchased from Fisher . The ultrapure water was obtained from a gradient water purification system .Ranae Oviductus, bullfrog oviduct, toad oviduct and frog oviduct were provided by Jilin Province Rana Industry Association which were collected from the Changbai Mountain area in the Jilin province of China. The specific location is shown on the map in Ranae Oviductus samples were collected from different regions from the main producing area of the Changbai Mountain range. The specific collection information is shown in Ranae Oviductus was pulverized into a powder (passing through a 20-mesh sieve) and degreased with petroleum ether at room temperature. After filtration, the powder was placed in an oven at 55 °C for 1 h. Afterward, 0.50 g of the sample was added to PBS buffer . After continuously stirring for 8 h, the mixture was centrifuged at 5000 r/min for 15 min. The supernatant was collected and the precipitate was extracted again. The two centrifugal supernatants were combined. To the supernatant, an ammonium sulfate solid was slowly added until to 60% saturation [Ranae Oviductus.The dried turation ,22. The turation ,24, and turation . The samturation . The prev/v with acetonitrile) and mobile phase B (0.1% TFA in v/v with ultrapure water) [The samples were separated using an Agilent Technologies 1200 Series liquid chromatograph  equipped with a quaternary pump, autosampler, thermostatted column compartment, diode array detector (DAD), and UV detector. The columns used were the Agilent Zorbax 300SB-C8 column  and Agilent Zorbax SB-C18 column  with mobile phase A (0.085% TFA in e water) ,28. Grade water) . The datRanae Oviductus sample (S1) was used to verify the RP-HPLC method. A precision analysis was carried out by repeatedly injecting the same solution 5 times on the same day. The repeatability was assessed by injecting 5 separate solutions obtained from the same Ranae Oviductus sample. The stability was evaluated by analyzing the same sample solution at different time periods of 0, 2, 4, 8, 16 and 24 h at room temperature.Ranae Oviductus were investigated using the professional software Similarity Evaluation System for the Chromatographic Fingerprint, according to the recommendations of the State Food and Drug Administration (SFDA). The HPLC fingerprint data of the samples were imported to the evaluation system (the solvent peaks in the first 4 min were removed and the time window was set at 0.2 s). The calibration method was multi-point calibration. The significant common peaks were labeled as mark peaks and the reference chromatogram fingerprint was generated with a mean value method. The similarity of the fingerprint data was represented by a correlation coefficient (similarity) and the higher similarity between the two samples resulted in a correlation coefficient value close to 1. The correlation coefficients of all chromatograms of 10 batches of Ranae Oviductus samples were calculated throughout the study and a correlation analysis was performed.The common characteristic peaks and similarities of fingerprint data of 10 batches of Hierarchical clustering analysis (HCA) is a cluster analysis technique that reflects the similarities and differences between samples in the form of a hierarchical tree diagram ,31. ThisPrincipal component analysis (PCA) is a classification method that uses dimensionality reduction techniques to simplify numerous original variables into several representative composite indicators ,33. AccoRanae Oviductus, the Ranae Oviductus (S1) collected from the China Changbai mountain area were systematically investigated. The RP-HPLC chromatography method was optimized through the detection wavelength, separation column, flow rate and temperature. Three classical UV detection conditions were previously reported: 215 nm corresponding to the maximum absorption of peptide bonds; 254 nm corresponding to the maximum absorption of phenylalanine residues; and 280 nm corresponding to tyrosine and maximum absorption of tyrosine residues and tryptophan residues [Ranae Oviductus using a diode array detector (DAD) with a wavelength range of 195–300 nm. The red region in the diagram indicated a larger absorption value. Although obvious solvent peaks around 215 nm were observed, the analysis of the core substance was not affected. The UV absorption diagram suggested that the separation effect at 215 nm was better than 254 nm and 280 nm.In order to improve the separation rate of the proteins in residues . Figure Ranae Oviductus. The results showed that the C8 column had a higher separation rate than the C18 column, which could be attributed to the large molecular weight of the proteins  were used to examine the column effect on the protein separation of proteins b. TherefSince the flow rate of the mobile phase can affect the isolation efficiency, three flow rates  were tested in this study. High flow rates showed that peaks overlapped c. The flOn the other hand, the temperature played an important role in the RP-HPLC separation. Theoretically, high temperatures can increase the motion rate of proteins. In this study, three different temperatures  were investigated d. From tRanae Oviductus sample S1) within one day. The relative standard deviations (RSD) of the retention times and peak areas of the 12 common peaks were smaller than 2.02% and 4.23%, respectively. The repeatability was determined by injecting five separate sample solutions of the Ranae Oviductus sample. The results showed that the RSD of the retention time and peak area of the 12 common peaks were smaller than 2.96% and 5.62%, which suggested that the RP-HPLC method had good repeatability. The stability test was carried out at room temperature for 0, 2, 4, 8, 16 and 24 h. The RSD of the retention times and peak area were smaller than 2.62% and 5.22%. All tests indicated that the RP-HPLC method established in this work satisfied the requirements of protein fingerprinting analysis of Ranae Oviductus.The accuracy of the RP-HPLC method was investigated through consecutive tests five times, using the same sample solution  . A reference fingerprint chromatographic spectrum of 10 batches of Ranae Oviductus was created  were performed depending on the aforementioned optimized RP-HPLC method. The results showed a significant difference. By comparing Ranae Oviductus. In contrary, the counterfeit products, including the bullfrog oviduct, showed four common-peaks  in 0–30 min and the toad oviduct, showed three common-peaks  in the same time period. Ranae Oviductus showed 12 common-peaks (peak1-peak12) in 30–80 min, whereas, the bullfrog oviduct and toad oviduct only showed five common-peaks. The frog oviduct only showed four tiny common-peaks , the bullfrog oviduct was classified in the b region, the toad oviduct was classified in the c region, and the frog oviduct was classified in the d region. The results were consistent with the HCA analysis, that both Ranae Oviductus and the counterfeit products were correctly classified. Comparing the similarity analysis with the HCA, PCA can provide a more visual comparison of the chromatograms.As an effective data analysis technique, PCA has been used to study the classification of samples . To direRanae Oviductus. Ten batches of Ranae Oviductus collected from the Changbai mountain area were used to analyze the protein components. The results showed 12 common-peaks in the reference fingerprint chromatographic spectrum. In combination with stoichiometry HCA and PCA, the results suggested that the method established in this work can satisfy the identification of Ranae Oviductus and counterfeit products. The method established in this work provides a promising approach for the identification of Ranae Oviductus and counterfeit products.This study used the RP-HPLC method and fingerprint technique to establish a chromatographic fingerprint of the proteins from


P<0.0001). Receiver operating characteristics (ROCs) to predict DR and VDTR demonstrated areas under the curve for RBP4 of 0.79 : 0.73–0.85) and 0.90 (95% CI: 0.85–0.94), respectively, which were superior to other factors. For each 1 μg/ml increase in plasma level of RBP4, the unadjusted and adjusted risk of DR would be increased by 8% (with the odds ratio (OR) of 1.08 (95% CI: 1.05–1.13), P<0.001) and 5% (1.05 (1.02–1.11), P=0.001), respectively. It was 12% (with the OR of 1.12 (95% CI: 1.07–1.18), P<0.001) and 9% (1.09 (1.05–1.15), P<0.001) for VTDR. The present study shows that elevated plasma levels of RBP4 were associated with DR and VDTR in Chinese patients with type 2 diabetes, suggesting a possible role of RBP4 in the pathogenesis of DR complications. Lowering RBP4 could be a new strategy for treating type 2 diabetes with DR.The retinol-binding protein 4 (RBP4) has been postulated to play a role in glucose homeostasis, insulin resistance, and diabetes mellitus in human and animal studies. The aim of the present study was to evaluate the role of RBP4 in Chinese patients with type 2 diabetes mellitus with and without diabetic retinopathy (DR). Plasma RBP4 concentrations were tested in 287 patients with type 2 diabetes. At baseline, demographic and clinical information including presence of DR and vision-threatening DR (VTDR) was collected. The relationship between RBP4 and DR (VTDR) was investigated using logistic regression. Patients with DR or VTDR had significantly higher plasma levels of RBP4 on admission ( The world’s largest diabetes epidemic is in China . The preRetinol-binding protein 4 (RBP4) protein belongs to the lipocalin family and is the specific carrier for retinol (vitamin A) in the blood . It was Intestinally, Huang et al.  reportedFrom January 2016 to December 2017, all patients with type 2 diabetes from Department of Endocrinology of The First Affiliated Hospital of Jinan University  were screened for the study. Type 2 diabetes was defined according to the criteria of the American Diabetes Association (a glycated hemoglobin (HbA1c) level ≥ 6.5% and/or a fasting plasma glucose (FPG) ≥ 7.0 mmol/l) . PatientIn addition, 150 normal people without type 2 diabetes from the Physical Examination Center were designated as the control group. Those people matched with the patients for sex, age, and body mass index (BMI). The study followed the principles of the Declaration of Helsinki and was approved by the Institute Ethics Committee of The First Affiliated Hospital of Jinan University. All participants were informed of the study protocol and their written informed consents were obtained.The following demographic and clinical data including age, sex, race, BMI, systolic and diastolic blood pressure, diabetes duration, presence and severity of DR, diabetic macular edema (DME) status, hypertension, hyperlipoproteinemia, smoking habits, alcohol abuse, and treatments  were recorded at admission.The Canon CR6-45NM ophthalmic digital imaging system and a Canon EOS 10D digital camera were used to take two digital images per eye through a non-pharmacologically dilated pupil (X.-X.C. and X.-H.L.). DR was diagnosed according to the presence of one or more retinal microaneurysms or retinal blot hemorrhages by the Early Treatment Diabetic Retinopathy Study (ETDRS) grading standards . The DR 2) = 175 × Cr-1.234 age-0.179 sex .The fasting blood samples were collected on the first day of admission before any treatment had been implemented. After centrifugation, the plasma samples were immediately stored at −80°C before assay. Plasma RBP4 levels were batch analyzed using a commercially available ELISA assay . The detection range for RBP4 was 1.6–100 ng/ml. Inter-assay and intra-assay coefficients of variation were 4.5–9.0% and 4.0–7.0%, respectively. In the present study, a 1:100 dilution was used, due to the high levels of RBP4 (μg/ml) in the plasma sample. The median plasma level of RBP4 in the 150 normal individuals was 15.5 μg/ml and the 97.5th percentile was 28.5 μg/ml. Blood levels of high-sensitivity C-reactive protein (Hs-CRP), HbA1c, insulin, FPG, and Cr were tested using routine laboratory methods by ROCHE COBASC311 . The urinary albumin excretion (UAE) rate was measured by enzyme immunoassay using immunoturbidimetry with a 24-h urine collection . The homeostasis model assessment of insulin resistance (HOMA-IR) index was calculated from the fasting blood glucose and fasting plasma insulin concentrations by the formula: HOMA-IR = fasting plasma insulin (μU/ml) × fasting blood glucose (mmol/l)/22.5 . EstimatCategorical variables were expressed as percentages and continuous variables were expressed as medians . Differences between the two groups were compared by Chi-Square or Mann–Whitney U-test as appropriate. Correlations amongst continuous variables were tested by the Spearman’s rank correlation coefficient.P<0.05.To investigate whether RBP4 allows prediction of both DR and VTDR in diabetes, different statistical methods were used. First, the relation of RBP4 with the two points was investigated with the use of logistic regression models. We used crude models and multivariate models adjusted for all significant predictors and report odds ratios (ORs). For multivariate analysis, we included confounders, known risk factors, and other predictors as assessed in univariate analysis. For a more detailed exploration of the RBP4 and DR (VTDR), we also used multivariate analysis models to estimate adjusted OR and 95% confidence intervals (CIs) of DR (VTDR) for RBP4 quartiles (with quartiles 1–3 as reference). Second, receiver operating characteristic (ROC) curves were used to test the overall prediction accuracy of RBP4 and other markers, and results were reported as area under the curve (AUC). In addition, the predicted accuracy of combined model I (RBP4/HOMA-IR/diabetes duration) and combined model II (RBP4/HOMA-IR/diabetes duration/Hs-CRP/HbA1c) were calculated. All statistical analysis were performed with SPSS for Windows, version 22.0  and the ROCR package (version 1.0-2). Statistical significance was defined as P=0.33). Amongst those patients, 83 were defined as DR . As shown in the P<0.05). In addition, intensive glucose-lowering therapy was more common in patients with DR than in those without DR (P<0.05). Basal characteristic of diabetes patients with DR or without DR were listed in the In the present study, 287 patients with type 2 diabetes were included. The median age was 62 (IQR: 55–71) years and 155 (54.0%) were men. The diabetes duration of those patients was 12 (IQR: 6–15) years. One hundred and twenty-one (42.2%) out of 287 were taking insulin and 159 were taking oral hypoglycemic agents. The plasma levels of insulin in patients, who were taking insulin, were not different when compared with those who were not taking insulin (9.68 (IQR: 6.45–12.15) compared with 9.49 (6.38–12.04); P<0.001). In those patients with diabetes, plasma levels of RBP4 were positively correlated with HbA1c , BMI , Hs-CRP , and duration of illness . Furthermore, RBP4 was significantly correlated with the HOMA-IR .The data suggested that plasma levels of RBP4 in patients with diabetes were significantly higher than in normal controls (28.6 (IQR: 18.1–38.0) μg/ml compared with 15.5 (IQR: 11.0–22.8) μg/ml; P<0.001; P<0.001) and 5% (1.05 (1.02–1.11), P=0.001), respectively (P<0.001) after adjusting for possible confounders. In addition, unlike other factors, HbA1c, Hs-CRP, and HOMA-IR were also identified as DR indicators in multivariate analysis (Plasma levels of RBP4 were significantly higher in patients with DR than those without DR (39.7 (IQR: 29.8–47.7) μg/ml compared with 25.0 (IQR: 15.5–32.6) μg/ml; ectively . Furtheranalysis .P<0.001). With this AUC, RBP4 presented a greater discriminatory ability to diagnose DR compared with HbA1c , Hs-CRP , diabetes duration , and HOMA-IR . Interestingly, the combined model I (RBP4/HOMA-IR/diabetes duration) improved the ability of every single marker to diagnose DR . Furthermore, the combined model II (RBP4/HOMA-IR/diabetes duration/Hs-CRP/HbA1c) improved the ability of every single marker and model I to diagnose DR .Based on the ROC curve, the optimal cut-off value of plasma RBP4 as an indicator for DR diagnosis was projected to be 36.4 μg/ml, which yielded a sensitivity of 61.5% and a specificity of 83.3%, with an AUC of 0.79  and 9% (1.09 (1.05–1.15), P<0.001), respectively (P<0.001) after adjusting for possible confounders. In addition, unlike other factors, HbA1c, Hs-CRP, HOMA-IR, and diabetes duration were also identified as VTDR indictors in multivariate analysis . Plasma RBP4 levels were significantly higher in patients with VTDR than those without VTDR (47.1 (IQR: 41.1–49.8) μg/ml compared with 26.9 (IQR: 17.2–35.2) μg/ml; ectively . Furtheranalysis .P<0.001). With this AUC, RBP4 presented a greater discriminatory ability to diagnose VTDR compared with HbA1c , Hs-CRP , diabetes duration , and HOMA-IR . Interestingly, the combined model (RBP4/HOMA-IR/diabetes duration) improved the ability of every single marker to diagnose VTDR . Furthermore, the combined model II (RBP4/HOMA-IR/diabetes duration/Hs-CRP/HbA1c) did not improved the ability of model I to diagnose VTDR .Based on the ROC curve, the optimal cut-off value of plasma RBP4 as an indicator for VTDR diagnosis was projected to be 38.4 ug/ml, which yielded a sensitivity of 87.2% and a specificity of 82.6%, with an area AUC of 0.90  in the 10-year follow-up phase in Beijing Child and Adolescent Metabolic Syndrome study [Diabetes duration, glycemic control , and infme study . In factme study . Lastly,me study  revealedSome limitations should be considered when interpreting the result. First, without serial measurement of circulating RBP4, when and how long RBP4 affected our participants were not tested. In the present study, 121 patients were taking insulin and their serum insulin levels were measured for the calculation of HOMA-IR. Their insulin levels might be not properly accurate because they were taking insulin. However, the fasting blood samples were collected before insulin treatment. In addition, plasma levels of insulin in patients taking insulin were not different when compared with those patients not taking insulin. Second, the cross-sectional design does not allow for cause or effect relationships. Third, some patients with severe visual impairment (such as advanced DR) had been excluded. Fourth, genetic variants in the RBP4 gene may be associated with circulating RBP4 concentration and phenotypes related to glucose metabolism . HoweverThe present study shows that elevated plasma levels of RBP4 were associated with DR and VDTR in Chinese patients with type 2 diabetes, suggesting a possible role of RBP4 in the pathogenesis of DR complications. Further studies are required to validate our findings and elucidate the causal relationship of RBP4 in the pathology of the DR. Lowering RBP4 could be a new strategy for treating type 2 diabetes with DR.


Millions of drivers could experience shoulder muscle overload when rapidly rotating steering wheels and reduced steering ability at increased steering wheel angles. In order to address these issues for drivers with disability, surface electromyography (sEMG) sensors measuring biceps brachii muscle activity were incorporated into a steering assistance system for remote steering wheel rotation. The path-following accuracy of the sEMG interface with respect to a game steering wheel was evaluated through driving simulator trials. Human participants executed U-turns with differing radii of curvature. For a radius of curvature equal to the minimum vehicle turning radius of 3.6 m, the sEMG interface had significantly greater accuracy than the game steering wheel, with intertrial median lateral errors of 0.5 m and 1.2 m, respectively. For a U-turn with a radius of 7.2 m, the sEMG interface and game steering wheel were comparable in accuracy, with respective intertrial median lateral errors of 1.6 m and 1.4 m. The findings of this study could be utilized to realize accurate sEMG-controlled automobile steering for persons with disability. On confined residential roads or parking areas where sharp turns are rapidly executed at or below the speed limit of 30 km/h, a primary task for automobile drivers is to be safe by keeping their “eyes on the road and hands on the steering wheel” ,2,3. HowSome steering wheel users who may experience shoulder muscle overload or reduced steering portability are restricted to one-handed steering wheel operation due to hemiplegia, i.e., paralysis on one side of the body, or an amputated upper limb ,10,11,12In an actual automobile, an onboard vehicle computer would rely on the sEMG electrodes to detect electrical muscle activity, i.e., myoelectric activity, resulting from isometric contractions of the biceps brachii  13]. Th. Th13]. Although sEMG electrodes have been used to reliably control prosthetic limbs and to objectively evaluate the muscle burden, steering comfort, and the time for drivers to apply force on steering wheels during vehicle turning maneuvers, few studies have investigated sEMG controlled steering as a means of assisting drivers with disability ,22,23,24Since the safety of the test subjects was of the highest priority during the study, the proposed steering assistance interface was tested with a driving simulator as a safer alternative to an actual automobile. A further advantage over an actual automobile was the ability to use the driving simulator to execute turning maneuvers more consistently for each test subject. Myoelectric activity was measured by individual self-adhesive electrodes to provide steering control signals. If the mounting configuration of the electrodes was at least experimentally comparable to a game steering wheel, with respect to path-following accuracy, the configuration would be adapted to an automobile as an electrode armband for more efficient mounting .Since path following is the primary task of conventional automobile steering, the sEMG interface was subjected to driving simulator trials to evaluate path-following accuracy in comparison to a game steering wheel . DriversOne driving scenario involved a right U-turn with a radius of 3.6 m, which was equal to the minimum turning radius of the simulated automobile. The trajectory of this U-turn required the SWA to change from 0° to 65° to evaluate the accuracy of the sEMG interface in a driving scenario associated with high shoulder joint forces in steering wheel users . Since tThe rest of this paper is structured as follows: As described in The steering assistance interface  enables In order to prevent steering caused by inadvertent myoelectric activity or sEMG signal noise, the handle could be released to cause a photoelectric sensor on the handle to deactivate sEMG signal reception . AlternaHset, (2) leftward SWA, −δHset, and (3) the neutral position, where δH is 0°. “Set” in this case refers to the rightward and leftward SWAs being programmed into the vehicle computer according to the health conditions of drivers  control scheme that divides the SWA of the steering wheel into three states  18]:  . The groRelocating the ground electrode from the wrist to a location above the elbow, such as the lateral side of the upper arm, could accommodate some amputees . WhereasSimilar to the majority of studies on sEMG-controlled prosthetics, nondisabled drivers in the current study performed isometric contractions that could represent sEMG signals from the affected limbs of transhumeral amputees ,75,76,77The equipment for controlling the driving simulator with the steering assistance interface is diagramed in The sEMG data acquisition device (DAQ) consisted of a custom circuit that filtered signals below 2 Hz and above 530 Hz and amplified the remaining signals . The ampThe experimental setup for the force feedback game steering wheel (Driving Force™ GT) consists of: a laptop that runs the driving simulator, the steering wheel assembly, and the brake and accelerator pedal assembly a. In theThe game steering wheel and steering assistance interface were used to perform two types of U-turns. U-turn 1 had a radius of curvature of 3.6 m, which is equal to the minimum turning radius of the simulated automobile a, whereaThe change in SWA from 0° to a maximum of 65° throughout U-turn 1 a is assoTurning the game steering wheel to the maximum SWA to execute U-turn 1 a precludOversteer and understeer are vehicle characteristics that occur during steady-state cornering along a circular path ,34. Afte“Ideal trajectory” refers to the path that is followed by an ideal vehicle that can instantaneously transition from a longitudinal trajectory to a steady-state circular trajectory. However, since automobiles always require time to transition, a transient phase necessarily occurs before reaching steady-state. A longer transient phase extends the time to reach steady-state, resulting in higher lateral error. In order to minimize the transient phase, the steering wheel could be rotated by the driver as quickly as possible. In the case of the steering assistance interface, the shortest possible transient phase available in the driving simulator is 0.1 s. Since the maximum SWA of the simulated vehicle is 65° and the SWR of 720 deg/s is attainable through commercially available steering actuators, dividing the maximum SWA by the SWR of the actuator yields an approximate transient phase of 0.1 s . Thus, tF, of the front wheels. Based on the radius of curvature, R, of U-turn 2 and the wheelbase length, l, the front steer angle could be approximated as follows . . 87]. For the U-turn with a radius of curvature equal to 3.6 m, the steering assistance interface is comparable or greater in path-following accuracy to the game steering wheel.For the U-turn with a radius of curvature equal to 7.2 m, the steering assistance interface is comparable or greater in path following accuracy to the game steering wheel, even if the sEMG interface is used without steering wheel correction.U-turns illustrated in As indicated in Trajectories from the first three successful attempts of each condition were used for data analysis to ensure that enough data would be available for processing. Median trajectories across trials were plotted on two-dimensional coordinate planes to compare the trajectories of the interfaces with respect to the ideal trajectories. Note that 1.1 m is the shortest distance between the edge of any road cone and the ideal trajectories . The latp < 0.05, i.e., the hypothesis that the population of the data is normally distributed is rejected, if p is less than 0.05. Since p < 0.05 for the data from conditions 1 to 4 in p < 0.05, i.e., if p < 0.05 for one of the tested U-turn types, there is a significant difference between the two interfaces, and one rejects the hypothesis that the median intertrial difference in lateral error is zero between the interfaces [Comparisons between interfaces were made with statistical analyses conducted in MATLAB. The Shapiro–Wilk test was performed, where terfaces . For hypHset, set to 65° was more accurate than the game steering wheel. The difference between the sEMG interface and the game steering wheel, with respect to the intertrial median lateral error, was statistically significant with p < 0.01. In the case of U-turn 2 (Hset equal to 33°, was comparable in accuracy to the game steering wheel, since there was no statistically significant difference in path following accuracy. Based on intertrial lateral error data across all participants, the sEMG interface was compared to the game steering wheel with respect to path following accuracy . For U-tU-turn 2 b, with aMedian trajectories for each interface are shown in Based on calculations from experimental data, intertrial median errors and IQRs are summarized in According to the intertrial median lateral errors in The transient phase of the sEMG interface in the case of U-turn 2 b was setUnlike U-turn 1 a, undersThe trajectory data from the driving simulator trials suggest that steering assistance could provide path-following accuracy that is comparable to a steering wheel interface, even in scenarios where steering wheel correction may be performed. Accuracy in the context of the tested scenarios  is depenSince data were collected from 16 participants with Japanese driver’s licenses, future studies could recruit a larger sample of test subjects to more accurately represent the population of drivers in Japan. Participants in this study were all persons without disabilities, and thus the results are at least relevant to drivers with one healthy arm. It is expected that a driver who has hemiplegia or upper limb amputation affecting only the left side of the body could use an unaffected right upper limb to operate the sEMG interface with accuracy that is comparable to current experimental results. As for sEMG input from upper limbs with unilateral amputations above the right wrist or right elbow, comparable operation is possible, if the residual biceps brachii repeatedly provides sEMG signals that are similar in amplitude and activation period to the sEMG signals of intact limbs. Otherwise, future studies including drivers with amputations could be compared to this study to quantify any statistically significant difference in path-following accuracy. As the interface continues to be implemented for drivers with disability, the suitability of the interface in relation to specific drivers and automobiles would be assessed by specialists of disability, such as occupational therapists and physiatrists . Based on present and future evaluations of the sEMG interface with respect to path following accuracy, further interface modifications could be realized. One planned modification is the replacement of the disposable wet electrodes with reusable dry electrodes for more efficient mounting and to eliminate the need for drivers to remove conductive gel and electrode adhesive from the skin after using the interface. Additions to the interface handle would include a photoelectric motion sensor to detect the hand of the driver, as explained in A steering assistance interface with sEMG electrodes underwent driving simulator trials to evaluate path-following accuracy relative to a game steering wheel. Although further testing was required to confirm the accuracy of the sEMG interface in most cases, trajectory data from the trials indicated that the steering assistance interface was at least comparable to the game steering wheel, with respect to the tested U-turns . For a U


CSF levels of LC3B represented a potential diagnostic and prognostic biomarker of early-stage PD in patients. Based on our findings, molecular biological changes in PD are associated with dysregulation of the lysosomal autophagy pathway.The roles of autophagy-related proteins as diagnostic or monitoring biomarkers in Parkinson’s disease (PD) have not been clearly elucidated. We recruited 32 patients with early-stage PD and 28 control subjects, and evaluated parkinsonian motor symptoms and dopamine transporter imaging data. Cerebrospinal fluid (CSF) levels of LC3B, Beclin1, and LAMP-2 were estimated using ELISAs, and CSF levels of ATG5, ATG7, and p62 were examined by immunoblotting. Additionally, we also assessed the levels of α-synuclein, total tau, and phosphorylated tau in CSF using ELISAs. Significant differences in the levels of LC3B, LAMP-2, and Beclin1 were observed between the PD and control groups. Using 29.8 pg/mL as the cut-off value for a diagnostic biomarker of PD, CSF LC3B levels exhibited high sensitivity (96.9%) and specificity (89.3%) with an area under the curve of 0.982. Furthermore, LC3B was significantly correlated with the asymmetry index in the caudate and putamen, as estimated by a semi-quantitative analysis of [ However, all current treatment modalities are only designed to manage symptoms by stimulating the dopaminergic system, and not modify the underlying disease in patients with PD2. The first step in the development of disease-modifying strategies is to identify ideal biomarkers for early diagnosis and monitoring disease progression3; thus, suitable biomarkers of PD in patients are urgently needed4.Parkinson’s disease (PD) is a common and disabling neurodegenerative disease characterized by various motor and non-motor symptoms4. In this respect, the cerebrospinal fluid (CSF) is a potentially reliable source of biomarkers of PD, because peptides or proteins in the CSF might directly reflect the pathological changes or brain-specific activities in neurodegenerative process5. Although biomarkers derived from diverse mechanisms, including free radicals, mitochondrial dysfunction, excitotoxicity, and inflammation, have been proposed for PD, many studies have reported the critical role of abnormal misfolded proteins, including α-synuclein, tau, and amyloid-β, in PD7. However, a consensus regarding the use of the aforementioned proteins as biomarkers in patients with PD is still lacking10. In terms of α-synuclein levels in CSF, some studies, but not others, have reported decreased levels of α-synuclein in patients with PD11. Furthermore, a large overlap in the CSF α-synuclein level was observed between patients with PD and patients with other neurodegenerative diseases12. Similarly, conflicting results for other misfolded proteins have been reported in previous studies14.The effective biomarkers of PD should be based on the pathogenic processes observed in patients with PD15. Moreover, lysosomal activity is required for α-synuclein degradation, and the activation of autophagy reduces toxic aggregates in PD models17. Recently, several studies have examined the GBA gene encoding β-glucocerebrosidase (GCase), which is related to autophagy function18. Reduced GCase activity has been observed in post-mortem brain tissues and CSF from patients with PD, and GCase activity was evaluated as a biomarker for differentiating PD from neurological controls18. Therefore, CSF biomarkers reflecting the lysosomal autophagy pathway could be useful for patients with PD.Among the various suggested mechanisms of PD, dysregulation of the lysosomal autophagy pathway has been observed in patients with PD and animal models19. However, in that study, the sample size was very small and only limited numbers of ATG proteins were evaluated. Moreover, the authors enrolled patients with PD, regardless of disease stage, and focused on the roles of biomarkers in differentiating between PD and atypical parkinsonism, not on the use of biomarker for early diagnosis or monitoring disease severity.However, few investigations have directly measured the levels of autophagy-related (ATG) proteins as biomarkers of PD, and only one study reported the differential expression of specific ATG proteins in patients with PDTherefore, we compared Beclin1, ATG5, ATG9, L3CB, p62, and LAMP-2, levels between patients with PD and normal controls, and investigated their correlations with clinical severity, quantitative dopamine transporter imaging data, and conventional CSF biomarkers in patients with PD to investigate the roles of various ATG proteins as biomarkers of the pathogenic process in patients with PD.18F] N-(3-fluoropropyl)-2β-carbon ethoxy-3β-(4-iodophenyl) nortropane (FP-CIT) positron emission tomography (PET), significantly lower uptake of the radiolabel was observed in both the putamen and caudate of patients with PD than in control group. The asymmetry index (AI) of the caudate and putamen in the PD group was also significantly increased compared with the control group , of other specific anatomical structures in our study. Thus, an association between dysregulated autophagy and nigrostrital degeneration was observed at least in patients with early-stage PD, strengthening the usefulness of ATG proteins as potential biomarkers of early-stage PD.Interestingly, in our study, the LC3B level was significantly correlated with the severity of motor symptoms, dopamine transporter imaging data, and conventional CSF biomarkers, including α-synuclein and total tau. In previous studies, α-synuclein was correlated with Unified Parkinson’s Disease Rating Scale (UPDRS) part 3 score or Hoehn and Yahr (HY) stage, but these correlations were not found in many other studies34. Considering the significant correlation between LC3B and α-synuclein in our study, ATG proteins and α-synuclein may interact. The activation of macroautophagy reduces the overexpression of the mutant α-synuclein protein, while inhibition of macroautophagy leads to a remarkable increase in the levels of endogenous or overexpressed α-synuclein in dopaminergic neurons. Conversely, α-synuclein itself may modulate the macroautophagy process, and transgenic mice overexpressing α-synuclein exhibited increased autophagosomes in previous studies35. However, α-synuclein might also affect chaperone-mediated autophagy, and the blockade autophagy flux and various molecular pathways affecting the lysosomal autophagy pathway are potential confounders in the interpretation of our results37. Furthermore, the significance of the α-synuclein level as a biomarker of PD remains controversial39. Therefore, the significance of the simple correlation between α-synuclein and LC3B levels in CSF remains unclear and further studies aiming to elucidate the precise significance of the interaction between these two proteins in the CSF are necessary.Tau and α-synuclein exhibit close interactions. The aggregation of α-synuclein is accompanied by hyperphosphorylated tau, and α-synuclein facilitates the aggregation of the tau protein8. In addition, total and p-tau levels in the CSF correlated with dopamine transporter imaging data in only limited ROIs in our study, unlike a previous study27. Nevertheless, conflicting results have been reported for conventional CSF biomarkers, and the discrepancies among these studies might be due to differences in the sample size, diagnostic inaccuracy, the contributions of comorbid conditions, and a lack of standardization in the sampling protocol24.Although a significant reduction in the total α-synuclein and total tau levels was observed in patients with PD, considerable overlap between the PD and control groups was also observed in our study. These findings are consistent with several previous studies, but one study examining drug-naïve patients with early-stage PD reported a significant reduction in the p-tau, total α-synuclein and total tau levels in patients with PDTherefore, our results should be interpreted with caution, and further studies with strict protocols for data acquisition, CSF handling, and delicate adjustments of confounding factors are needed, although we confirmed that LC3B displayed a higher sensitivity and specificity for the diagnosis of PD compared to α-synuclein and total tau levels.41. Although LC3B levels were markedly reduced in the PD group and strongly correlated with imaging data, clinical parameters and other CSF biomarkers in our study, the alteration in the LC3B level in the CSF was also observed in the two studies mentioned above. We adopted very strict inclusion criteria for patients with PD to minimize the possible confounding effects of comorbidities. Additionally, the PD-specific lysosomal autophagy pathway should be studied in the future. Second, ATG proteins, including LC3B, do not precisely reflect autophagy flux. A decreased LC3B level might not only be due to reduced autophagosome formation but also to increased autophagosome degradation. Furthermore, ATG proteins might be entrapped in autophagosomes due to the lack of lysosomal activity. CSF levels of autophagy marker proteins in patients with PD are likely affected by the dynamics of ATG proteins and may not reflect an actual change in the autophagy process. Therefore, the meaning of the decreased levels of ATG proteins in patients with PD remains unclear and an investigation of autophagy markers in brain specimens should be simultaneously conducted along with the monitoring of autophagy flux in vivo instead of simply measuring ATG protein levels to answer this question. In addition, the stability of these proteins in the CSF could be another concern. We tried to collect CSF samples under the same conditions, such as daytime collection from drug-naïve patients, as much as possible to minimize this limitation. Third, our results showed some discrepancies from previous studies, potentially due to the use of strict inclusion and exclusion criteria for enrolment to exclude possible confounding factors. Additionally, we enrolled drug-naïve patients with early-stage PD in the present study. Consequently, the sample size was relatively small and ATG proteins were not evaluated in patients with advanced PD. Finally, our study was designed as a cross-sectional study, and thus a longitudinal study with a larger population is required to determine the suitability of using CSF ATG proteins as biomarkers of PD progression.Our study has some important limitations. First, impairments in the lysosomal autophagy pathway are not a PD-specific mechanism and could be related to other neurodegenerative diseases. LAMP-1, LAMP-2, and LC3 levels are significantly increased in the CSF from patients with Alzheimer’s disease, and the autophagy biomarkers LC3B and Beclin1 correlate with clinical outcomes and disease severity in patients who experienced an acute strokeDespite these limitations, our study examined a relatively homogenous PD group through the use of very strict inclusion and exclusion criteria to minimize possible confounding effects. We excluded subjects with white matter changes, comorbidities or medications that potentially affected levels of ATG proteins in the CSF. Furthermore, we also excluded subjects with any RBCs in their CSF to minimize contamination from RBCs, which can influence α-synuclein levels.In conclusion, autophagy and lysosomes are involved in the pathogenesis of early-stage PD. Additionally, CSF ATG proteins, particularly LC3B, represents a potential biomarker for the diagnosis of early-stage PD. Moreover, LC3B levels in the CSF might also indicate disease severity in patients with early-stage PD.42. Non-motor symptoms were investigated in patients with PD using the Non-motor Symptoms Scale (NMSS)43. Additionally, Beck’s depression scale and the Korean version of Montreal Cognitive Assessment (MoCA-K) were performed to assess mood and cognitive symptoms in all enrolled subjects45. All clinical assessments were performed before patients were treated with dopaminergic medications. Basic demographic and laboratory data, including age, gender, haemoglobin level, haematocrit, and blood urea nitrogen, creatinine, aspartate aminotransferase, alanine aminotransferase levels were obtained in this study.This study was approved by the Institutional Review Board of Gangneung Asan Hospital, and performed according to the principles of the Declaration of Helsinki. All enrolled subjects provided written informed consent. We prospectively recruited 32 drug-naïve patients with early-stage PD and 28 healthy controls at the Movement Disorders Clinic of Gangneung Asan Hospital from January to December, 2016. PD was diagnosed using the United Kingdom Parkinson’s Disease Society Brain Bank criteria, and FP-CIT PET. Parkinsonian motor symptoms were evaluated with the UPDRS part 3 and the HY stage, early-stage PD was defined as HY stage less than 3We excluded subjects 1) with abnormalities on the magnetic resonance imaging; 2) with a cognitive deficit (MoCA-K score less than 23); 3) who were taking medications known to regulate the autophagy pathway, including statins, carbamazepine, and vitamin D supplements; 4) who were diagnosed with other diseases, including cerebrovascular disease, diabetes, nervous or systemic infection, malignant diseases such as renal or hepatic failure, recent head trauma or surgery; and 5) whose CSF sample contained any number of red blood cell (RBCs) or white blood cell (WBCs) in a routine analysis.46. All CSF samples were collected between 9 and 12 a.m. after an overnight fasting from subjects with a drug-naïve status at admission to minimise interference from medications and lifestyle. CSF samples were collected in sterile, siliconized polypropylene tubes, and the first 2 mL of the CSF sample were sent to the laboratory for routine testing for cell counts, total protein levels, and glucose levels. An additional 15 mL of CSF was transferred to a 5-mL conical polypropylene tube at room temperature, mixed gently, centrifuged at 1500 g for 10 minutes at room temperature, and transferred to precooled 1.0-mL siliconized polypropylene aliquot tubes followed by immediate freezing on dry ice and storage in a −80 °C freezer. All CSF analyses were performed by a single laboratory technician who was blinded to the clinical data. All standards, aqueous controls, and CSF samples were analysed in duplicate in each run. The value of each result was defined as the arithmetic mean of the calculated concentration of duplicates.CSF sampling and collection were performed according to a published protocol and guidelineConcentrations of three PD biomarkers, total tau, tau phosphorylated at threonine 181 (p-tau) and α-synuclein, in the CSF were measured with commercially available Human Total tau, p-tau and α-synuclein ELISA Kits  according to the manufacturer’s protocol. Briefly, 100 μL/well of diluted standards from reconstituted stocks and duplicate CSF samples (100 μL/well) were added to the capture antibody-coated plate and incubated at room temperature for 3 hours. 100 μL/well of biotinylated detection antibody were added, and the plate was incubated at room temperature for 2 hours. After the incubation, 100 μL/well of streptavidin horseradish peroxidase were added and incubated for 30 minutes at room temperature. After washing the plate four times, a mixture of 2 different chemiluminescent substrates was added and the optical density was measured using a Microplate reader  at a wavelength of 450 nm. The proteins were quantified from standard curves using a linear regression analysis. Similarly, Beclin1, LAMP-2 and LC3B ELISA Kits  were used with a similar method to detect autophagy biomarkers. The coefficients of variation for all CSF markers in the laboratory are: α-synuclein ; total tau ; p-tau ; LC3B ; LAMP-2 .CSF samples were added to equal volumes of loading buffer , and heated at 99 °C for 7 mins. Equal amounts of protein (15 μg) were loaded on an SDS-PAGE gel, electrophoretically separated, and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h and then incubated with primary antibodies  in TBST at 4 °C overnight. After three washes with TBST, membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies  for 1 h at room temperature. Antibodies were detected using enhanced ECL Advance Western Blotting Detection Reagents  and LAS-4000 film . Albumin  served as a loading control.http://www.fil.ion.ucl.ac.uk/spm) software implemented in MATLAB 12 for Windows  as previously described47. Regional mean standardized uptake values (SUVmean) were extracted from the left and right caudate, the left and right putamen and the occipital cortex using volume-of-interest templates drawn in the MNI space47. The semi-quantitative analyses were performed using the SNBR and the AI.FP-CIT PET scans were obtained 2 hours after the intravenous injection of an average of 185 MBq (5 mCi) of FP-CIT. All enrolled subjects underwent PET imaging in a Discovery ST8 scanner . The emission PET data were acquired for 15 minutes, and the computed tomography data were used for attenuation correction. The dopamine transporter binding state was evaluated through a semi-quantitative analysis of FP-CIT PET. The FP-CIT PET images were spatially normalized to the Montreal Neurological Institute (MNI) space using a standard FP-CIT PET template and the Statistical Parametric Mapping 8 /occipital SUVmeanThe AIs of the putamen and caudate were calculated as follows: / higher striatal SNBR.p-value < 0.05 was considered statistically significant.All data are presented as means ± standard deviations. All statistical analyses were performed with a commercial statistical software program . Categorical variables were compared using the chi-square test, and continuous variables with normally distributed data were compared using a standard t-test. The Mann-Whitney U test was used to compare variables that did not meet the assumption for a normal distribution. A receiver operating characteristic (ROC) curve analysis was performed using the ‘pROC’ package to determine whether specific proteins served as diagnostic biomarkers, and we used the ‘OptimalCutpoints’ package to calculate the optimal cut-off value for the difference between the control and PD groups by measuring Youden’s Index (R version 3.4.1). The relationships between the FP-CIT PET values and levels of ATG proteins, including LC3B, Beclin1, ATG5, and LAMP-2, in patients with PD were explored using partial Spearman’s correlation analyses adjusted for age. Correlations between autophagy biomarkers and conventional CSF markers, including total tau, p-tau and α-synuclein, were also evaluated using Pearson’s correlation test. A Dataset 1