Pretreatment with GGD attenuated the levels of proinflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin- (IL-) 6, and IL-1β in serum by inhibiting Toll-like receptor 4 (TLR4)/NF-κB signaling pathway. These results indicated that GGD exhibits cardioprotective effects on myocardial I/R injury through inhibition of the TLR4/NF-κB signaling pathway, which led to reduced inflammatory response and the subsequent cardiomyocyte apoptosis.Guizhi Gancao Decoction (GGD) is a well-known traditional Chinese herbal medicine for the treatment of various cardiovascular diseases, such as myocardial ischemia-reperfusion (I/R) injury and arrhythmia. However, the mechanism by which GGD contributes to the amelioration of cardiac injury remains unclear. The aim of this study was to investigate the potential protective role of GGD against myocardial I/R injury and its possible mechanism. Consistent with the effect of the positive drug , we subsequently validated that GGD could ameliorate myocardial I/R injury as evidenced by histopathological examination and triphenyltetrazolium chloride (TTC) staining. Moreover, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay demonstrated that GGD suppressed myocardial apoptosis, which may be related to the upregulation of Bcl-2, PPAR Cardiovascular disease is one of the leading causes of human morbidity and mortality in developed countries . In the κB signaling pathway has been recently found to be a potential therapeutic target in the treatment of myocardial I/R injury [κB signaling pathway plays a substantial role in mediating inflammatory response and cardiomyocyte apoptosis following myocardial I/R injury [Inflammatory response and cardiomyocyte apoptosis have been recognized as hallmarks of myocardial I/R injury. Recent evidences suggest that cardiomyocyte apoptosis is primarily triggered or accelerated during I/R and partially contributes to overall cardiomyocyte death –8. InhibR injury , 16. AccR injury , 17, 18. Treatise on Febrile Diseases written by Zhongjing Zhang in Eastern Han Dynasty of ancient China, is a famous classic Traditional Chinese Medicine (TCM) formula consisting of two medicinal herbs, namely, Ramulus Cinnamomi (RC) and Radix Glycyrrhizae (RG) in the ratio of 2:1(w/w). RC and RG have been used extensively in China to treat inflammatory and ischemic diseases [2+ overload [κB signaling pathway and what is the underlying mechanism responsible for its actions.Guizhi Gancao Decoction (GGD), originally described indiseases –21. The diseases , 23. Cindiseases  and protdiseases –27, are overload , 29, andoverload , 31. Theα (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) ELISA kits were purchased from Nanjing KeyGEN Biotech . Antibodies against Bcl-2, Bax, caspase-3, caspase-9, PPARα, PPARγ, TLR4, NF-κB p65, and IκBα were purchased from Cell Signaling Technology .GGD extract, provided by the First Affiliated Hospital, Henan University of Traditional Chinese Medicine, consisted of RC and RG in a ratio of 2:1. Trimetazidine (TMZ) was produced by Servier Pharmaceutical Co., Ltd., . 2,3,5-triphenyltetrazolium chloride (TTC) was purchased from Sigma Chemical Co. . In situ cell death detection kit was purchased from Roche Molecular Biochemicals . Tumor necrosis factor- Treatise on Febrile Diseases). The experimental dose of rats was 6.3 times that of human beings, and from that the dose was calculated as RC:6.3∗(60∗1000/70) =5400mg =5.4 g/kg and RG:6.3∗(30∗1000/70) =2700mg =2.7 g/kg for rats. But this dose was actually too higher for rats. Referring to other classic prescriptions based on RC–RG herb pair, such as Guiganlongmu Decoction (RC:15g and RG:30g) and Guizhi Decoction (RC:45g and RG:24g), the final dose which was determined was 1.8~3.6g/kg, which was acceptable for gavage in rats.Male Sprague-Dawley rats (250-300g) were purchased from Animal Experiment Center of China Three Gorges University (Certificate no. SCXK 2017-0012). The animals were kept in rooms maintained at 23±2°C in a 12 h light/dark cycle and were fed a standard rodent diet with free access to water following international recommendations. All animal experiments in this study were performed in accordance with China Academy of Chinese Medical Sciences Guide for Laboratory Animals that conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . Rats were randomly divided into five groups (n = 18 per group) and treated as follows: (1) the control group; (2) the I/R group; (3) the I/R group treated with GGD solution at doses of 1.8g/kg and 3.6g/kg, respectively; (4) the I/R group treated with TMZ solution at the dose of 10mg/kg. GGD or TMZ was given intragastrically once a day for 14 consecutive days, while the control and I/R groups were given normal saline. The GGD decoction (90g) consisted of RC 60g and RG 30g . After tracheal intubation, the rats were ventilated with room air using an animal respirator. Left thoracotomy was performed at the fourth intercostals space and the heart was exposed. By a surgical needle, a 6-0 silk ligature was placed under the left anterior descending coronary artery (LAD) and encircled with a suture. The LAD was ligated to induce ischemia for 30 min. R wave amplification and ST segment depression were observed immediately in lead II of the attached electrode. Myocardial ischemia was indicated if the myocardia distal to the ligation line darkened. After 30 min of ischemia, the slipknot was released and the animal received 120 min of reperfusion. The control group (n=18) was sham-operated animals undergoing the same surgical procedure, except for the fact that the silk ligature placed around the left coronary artery was not tied. Except for accidental deaths due to anesthesia or failed surgery, the numbers of surviving rats in each group were as follows: I/R (n=15), I/R+GGD 1.8g/kg (n=14), I/R+GGD 3.6g/kg (n=12), and I/R+TMZ (n=14). Blood was collected after reperfusion by cardiac puncture and centrifuged at 2000The rats were sacrificed at 120min of reperfusion, and their hearts were immediately removed and frozen at -20°C for 15 min and then cut into five cross-section slices. Heart slices were incubated for 15 min in 1% TTC at 37°C. TTC-stained areas  and non-TTC-stained areas  were determined. The infarct size was analyzed by Image-Pro Plus image analysis software. Proportions of infarct myocardium to the whole myocardium tissues were then calculated using the following formula: Infarct area/whole heart area (INF/WH%).μm-thick sections, and evaluated for morphological changes with HE staining under a light microscope at a magnification of ×200. For immunohistochemical analyses of TLR4, tissue sections were deparaffinized and blocked with 10% rabbit serum to prevent nonspecific antibody binding. After washing three times with phosphate buffer saline (PBS), which contained (in g/L) NaCl 8, KCl 0.2, Na2HPO4 3.48, and KH2PO4 0.2, the sections were then incubated with TLR4 primary antibody at 4°C overnight. The sections were washed three times with PBS for 5min and incubated with secondary antibody at 37°C for 1 h. The sections were rinsed again with PBS for three times, incubated with diaminobenzidine for 10-15min, counterstained with hematoxylin, dehydrated, and mounted for microscopic observations with a magnification of ×400.Myocardial tissues were rapidly transferred to prepare 4% formaldehyde  and fixed overnight at 4°C before they were gradiently dehydrated with ethanol. The tissues were then embedded in paraffin, cut into 5Myocardial apoptosis was detected by TUNEL staining using in situ cell death detection kit. TUNEL assay was performed according to the manufacturer's protocol. Brown colored positive apoptotic cells were observed with TUNEL method and visualized at ×400 magnification.g for 15 min. The supernatants were collected for Western blotting and the protein concentrations were determined by Bradford assay . Equal amounts of proteins were separated by 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes . Membranes were blocked with 5 % nonfat dry milk and then washed. Primary antibodies, including antibodies against Bcl-2 (1:1000), Bax (1:1000), caspase-3 (1:1000), caspase-9 (1:1000), PPARα (1:1000), PPARγ (1:1000), TLR4 (1:1000), NF-κB p65 (1:1000), and IκBα (1:1000), were used to incubate the membranes overnight at 4°C. Horseradish peroxidase-conjugated secondary antibody  was used to incubate the membrane for 2h. Immunoreactivity was detected by ECL reagents  and a gel imaging system  was used to visualize the protein bands.Protein was extracted from myocardial tissues for Western blotting analysis. Briefly, myocardial tissues were homogenized with a glass dounce homogenizer in RIPA buffer  and then centrifuged at 20,000×α, IL-6, and IL-1β were analyzed spectrophotometrically according to the instruction of ELISA kits.The serum levels of TNF-P values less than 0.05 were considered statistically significant.All statistical analyses were performed using SPSS 16.0 software. Data were expressed as mean ± standard error (SEM) and analyzed using one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. Myocardial infarct size was assessed in the current study by TTC staining. As shown in In the present study, morphological changes of myocardial tissue were observed by HE staining. As shown in TUNEL assay was employed to ascertain the antiapoptotic effect of GGD. Apoptosis was determined by using a TUNEL detection kit according to the manufacturer's instructions. As shown in α and PPARγ were downregulated. To different extents, treatment with TMZ or GGD reversed the changes in apoptosis-related protein expressions induced by I/R. Among them, PPARγ expression was upregulated, but not significantly as compared with the IR group.To further demonstrate the antiapoptotic mechanism of GGD, we evaluated the expressions of apoptosis-related proteins by Western blotting analysis . ResultsProtein level of TLR4 was determined by immunohistochemistry and Western blotting in the current study. As shown in κB p65 and its inhibitory protein IKBɑ were determined using Western blotting. The results from Western blotting analyses showed that I/R injury induced an upregulation of NF-κB p65  and apoptotic regulatory proteins were dramatically reduced by pretreatment with GGD as compared with that of the I/R group. These findings were accompanied by decreased infarct size and improved morphological changes, indicating that attenuation of the subsequent myocardial necrosis could be attributed, at least partially, to antiapoptosis at the early stage of I/R.Cardiomyocyte apoptosis proves to be a major contributor to myocardial reperfusion injury, which is primarily triggered by ischemia and aggravated during the process of reperfusion and partially results in overall cardiomyocyte death . Inhibitand IL-6 . Cell apresponse –46. Subsresponse . Apoptosresponse . The casresponse . PPARs hresponse . Ligandsr organs –53. Acti effects , 55. TheκB signaling pathway and amelioration of subsequent inflammatory response and cardiomyocyte apoptosis.In conclusion, the present study demonstrated that pretreatment with GGD could protect the myocardia against I/R injury by downregulation of the TLR4/NF-


Conjunctival inflammation disturbs the blood–tear barrier and thus affects the tear film stability and composition. We aimed to develop a non-invasive and reliable method to induce conjunctivitis in dogs, a large animal model for translational work on ocular surface disease in humans. Six beagle dogs underwent a randomized, vehicle-controlled, balanced crossover trial—on six separate days, one eye received topical artificial tears (vehicle), while the other eye received one of six concentrations of histamine solution (0.005–500 mg/ml). At sequential times after eyedrop administration, a conjunctivitis score was given to each eye based on the degree of palpebral and bulbar conjunctival hyperemia and chemosis, ocular pruritus, and discharge. Total protein content (TPC) and serum albumin were quantified in tear fluid at baseline and 20 min. Additionally, 13 dogs presenting for various ophthalmic diseases with associated conjunctivitis were examined. Experimentally induced conjunctivitis developed rapidly (<1 min) following topical histamine administration and lasted for 1–3 h (four lowest doses) to 6–8 h (two highest doses). The severity of conjunctivitis was dose-dependent. Histamine was overall well tolerated, although transient blepharitis, aqueous flare, and ocular hypertension occurred in a few dogs receiving histamine ≥375 mg/ml. TPC and serum albumin levels increased in tears of eyes receiving histamine ≥1.0 mg/ml, being significantly higher than vehicle and baseline in eyes receiving histamine ≥375 mg/ml. Lacrimal albumin levels were also increased in 13 dogs with naturally acquired conjunctivitis, up 2.7–14.9 fold compared to contralateral healthy eyes. Histamine-induced conjunctivitis represents a robust model for translational work on the ocular surface given the low cost, non-invasiveness, self-resolving nature, ability to adjust the duration and severity of the disease, and shared features with naturally occurring ocular diseases. Histamine solutions of 1, 10, and 375 mg/ml induce mild, moderate, and severe conjunctivitis in dogs, respectively. Leakage of serum albumin in tear fluid of eyes with conjunctivitis suggests a breakdown of the blood–tear barrier. Conjunctivitis, or inflammation of the vascularized mucous membrane lining the inside of the eyelids, anterior sclera, and (when present) the nictitating membrane, is a common ocular surface disease in both humans and veterinary patients . In addiin vivo model of conjunctivitis will guide future studies to gain a deeper understanding of the disease, elucidating the impact of conjunctivitis on drug pharmacokinetics, tear film dynamics, metabolomics, and proteomics, among others.Despite the prevalence of conjunctivitis, there is limited knowledge about the disease impact on the ocular surface in clinical patients. Are tear film composition and quality affected by conjunctivitis-induced breakdown of the blood–tear barrier? Is there an impact on the pharmacokinetic profile of drugs on the ocular surface? Do medications administered systemically reach the tear film compartment at higher concentrations? Such knowledge can be gained from experimentally induced models of conjunctivitis in animals, such as intraperitoneal injection of ovalbumin in guinea pigs  and topiSix beagle dogs  were used in the study. The gender and neuter status was the same for all subjects , as sex hormones are known to be key regulators of vascular tone in various organs, including the eye . All dog® organics, Geel, Belgium) in 1.4% polyvinyl alcohol lubricating eye drops  using a sterile manner under a laminar flow hood. The pH of each solution was tested with a pH meter  and adjusted to 6.5 by adding 1% sodium hydroxide (prepared from granules mixed with sterile water), one drop at a time until the target pH was reached. The following 12 concentrations of histamine solution were compounded into 15-ml sterile eyedropper bottles  by the pharmacist at Iowa State University’s Lloyd Veterinary Medical Center: 0.001, 0.005, 0.01, 0.0375, 0.1, 0.5, 1.0, 10, 100, 375, 500, and 1,000 mg/ml. The vehicle solution consisted of artificial tears solution adjusted to pH of 6.5 with a minute amount of 1% sodium hydroxide (< 5 drops in 15-ml bottle). Histamine and vehicle solutions were used within 24 h of preparation and kept in the dark at room temperature (18–24°C) before use and in between experiments.Histamine ophthalmic solutions were formulated by mixing histamine powder  was randomly allocated to one of the 12 histamine concentrations . A single drop of histamine solution was instilled onto the ocular surface, followed by slit-lamp biomicroscopy and conjunctivitis scoring at 1, 3, 5, 7, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120, 180, 240, 300, 360, and 420 min. Additionally, the following ocular and physical parameters were recorded at 1, 10, 20, 30, 60, 120, and 240 min post-histamine administration: intraocular pressure, aqueous flare grading, body temperature, heart rate, respiratory rate, respiratory efforts, capillary refill time, and indirect blood pressure .A pilot study was conducted to assess the tolerability of histamine solutions in dogs  and the most appropriate concentrations to select for the crossover trial. Each of the 12 eyes  while the other eye received vehicle solution, and this order was kept consistent throughout the study. Each dog received all six histamine solutions over six consecutive days, using one solution per day (once in the morning) in a balanced crossover trial . The six histamine solutions are listed as follows: HTear collection: At baseline and at 20 min post-eyedrop administration, tear fluid was collected from both eyes and total protein content (TPC) was analyzed as previously described . After estimating the volume of extracted tear fluid with a pipette, each tear sample was diluted 1:3 with phosphate-buffered saline . TPC was calculated with Direct Detect™ infrared spectrometer and expressed in mg/ml after adjusting for the threefold dilution of each sample. Subsequently, the residual tear fluid was diluted fourfold with diluent provided with the albumin ELISA kit . Serum albumin and various cytokines/chemokines/growth factors  were quantified in each tear sample following the manufacturer’s protocol.escribed . BrieflyConjunctivitis score: At baseline and at selected times post-eyedrop administration (similar to pilot study), one examiner (LS) who was masked to which eye received histamine or vehicle solution performed slit-lamp biomicroscopy of both eyes to determine a conjunctivitis score at each time point. The conjunctivitis score was calculated as the sum of the following categories, each graded on a three- to four-point scale  for clinical ophthalmic disease with associated conjunctivitis were enrolled. A verbal informed consent was obtained from all owners, which was sufficient for the hospital’s ethics committee given that Schirmer tear test is part of routine clinical care. Dogs were examined by a board-certified veterinary ophthalmologist (LS or RA) for diagnosis and treatment of various ocular complaints including corneal ulceration, uveitis, and glaucoma. Details of the dogs  and their clinical diagnosis are presented in post hoc test was used to compare the six histamine doses for i) duration of conjunctivitis, determined as the time for conjunctivitis score to return to zero, and ii) severity of conjunctivitis, determined by the area under the curve of conjunctivitis score from 0 to 180 min. Within each histamine dose, the one-way repeated measures ANOVA and Bonferroni post hoc test was used to assess differences in conjunctivitis scores between time points. The Student t-test was used to evaluate differences between vehicle and histamine-treated eyes for TPC and albumin levels, both at baseline and at 20 min following eyedrop administration. Further, a ratio was calculated for TPC and albumin levels for each histamine dose as follows: (Concentration histamine 20 min/histamine baseline)/(Concentration vehicle 20 min/vehicle baseline). This ratio describes the percentage change in protein levels between baseline and 20 min post-induction of conjunctivitis, taking into account the variability inherent to the tear collection method itself by adding the vehicle-treated eyes as the denominator , and values P < 0.05 were considered statistically significant.Normality of the data was assessed with the Shapiro–Wilk test. The one-way ANOVA and Tukey ominator . The one4 administration (10 mg/ml histamine solution), while 5 (375 mg/ml histamine solution). Data were normally distributed such that results are presented as mean ± standard deviation (range).All eyes receiving the vehicle solution were scored at zero for all time points. In eyes receiving histamine, the ocular surface was examined from different angles to evaluate the canine conjunctiva in a comprehensive manner, including a view from the front (Conjunctivitis developed rapidly (<1 min) for all doses (P < 0.001), ranging from 61 ± 37 min (25–120 min) for H1, 110 ± 36 min (90–180 min) for H2, 115 ± 52 min (60–180 min) for H3, 190 ± 45 min (120–240 min) for H4, 390 ± 33 min (360–420 min) for H5, and 400 ± 31 min (360–420 min) for H6. All pairwise comparisons were statistically significant (P ≤ 0.029) except for H1–H3 (P = 0.201) and H5–H6 (P = 0.998).The duration of conjunctivitis was statistically different among histamine doses (P < 0.001), with an AUC0–180min (in score x min) ranging from 59.2 ± 36.8 (21.5–119) for H1, 200.2 ± 53.9 (107.5–254.5) for H2, 277.2 ± 128.3 (144.5–495.5) for H3, 522.3 ± 131.8 (358–735) for H4, 1,168.5 ± 266.4  for H5, and 1,186.5 ± 242.4  for H6. All pairwise comparisons were statistically significant (P ≤ 0.037) except for H5–H6 (P = 1.000) and H2–H3 , a standard deviation of 0.9, a power of 80%, and an α value of 0.05.A 5 and H6: i) Blepharitis, manifested by mild blepharedema and erythema  Aqueous flare, subtle in intensity (trace to 1+), was noted in 2/6 dogs receiving H5 and 6/6 dogs receiving H6, developing within 25–90 min and self-resolving within 1–3 h. Aqueous flare was commonly accompanied by miosis (5 at 30 min (IOP = 32 mmHg), although it was not statistically greater than baseline IOP (P = 0.529) and it self-resolved within 10 min. Ocular hypertension was also noted in 3/6 dogs receiving H6 in which IOP was significantly higher at 30 min  and 60 min  compared to baseline (16.8 ± 2.6 mmHg). Importantly, no fluorescein stain uptake or corneal changes were noted for any histamine dose.Locally, histamine administration was very well tolerated in dogs except for transient adverse effects noted with Hy miosis , was not1 to H6 developed systolic hypotension (<90 mmHg). However, the systolic blood pressure dropped from 130 to 88 mmHg in a single dog receiving 1,000 mg/ml histamine during the pilot phase, and the dog exhibited transient depression and weakness until blood pressure returned to baseline 10 min later.Systemically, all the vital parameters monitored were stable and not a single dog receiving HP ≥ 0.365). Twenty minutes following eyedrop administration, lacrimal TPC varied from 2.3 to 36.3 mg/ml (9.9 ± 5.6 mg/ml) and was statistically greater in histamine vs. vehicle-treated eyes for H5 (P = 0.009) and H6 (P = 0.021) but no other doses  and no differences were noted between vehicle and histamine-treated eyes for any dose (P ≥ 0.394). Twenty minutes following eyedrop administration, albumin levels varied from 0.019 to 9.595 mg/ml (1.158 ± 2.098 mg/ml) and were statistically greater in histamine vs. vehicle-treated eyes for H5 (P = 0.021) and H6 (P = 0.029) but no other doses  and no differences were noted between vehicle and histamine eyes for any dose (The levels of interferon-gamma (IFNγ), interleukin-2 (IL-2), and beta nerve growth factor (NGF-β) were below limits of quantification in all tear samples. Tear concentrations of other chemokines/cytokines/growth factors, described as mean ± standard deviation (range), were as follows Figure 7–P < 0.001) compared to lacrimal concentrations noted in contralateral unaffected eyes (0.13–3.06 mg/ml). Re-examination of two dogs following therapy of the underlying ocular disease  and sustained (120–420 min) development of conjunctivitis. Second, the changes noted in tear fluid levels of TPC and albumin strongly suggest a breakdown of the blood–tear barrier. In particular, albumin represents a marker of vascular permeability and plasma leakage given it is not produced by the lacrimal gland or corneo-conjunctival tissue . Our finIn addition to protein quantification, the present study investigated a panel of cytokines/chemokines/growth factors in tear samples of dogs. Similar to human subjects with ocular surface disease such as dry eye or vernal keratoconjunctivitis , an incrpost hoc power analysis showed that n = 5 dogs were sufficient to detect significant differences between histamine doses for the main study outcome . Furthermore, although the subjectivity of our clinical scoring may be perceived as a drawback, given that photograph-based methods are mainstream in human studies  studies , while o studies . Future In summary, histamine-induced conjunctivitis in dogs represents a robust and reliable model for translational research on the ocular surface. The model is particularly appealing given the low cost, non-invasiveness, self-resolving nature, ability to adjust the duration and severity of the disease, and shared features with naturally occurring diseases in human and veterinary medicine. The model could be used to assess new therapeutics and to better understand the impact of conjunctivitis on drug pharmacokinetics–pharmacodynamics, tear film dynamics, and tear film composition, among many other applications.The datasets generated for this study are available on request to the corresponding author.The study was approved by the Institutional Animal Care and Use Committee of Iowa State University (log # 2-18-8704-K) and adhered to the Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision Research.LS conceptualized and designed the study in consultation with RA and JM. LS, RA, and AW performed the experiments. LS, Y-JS, and JM analyzed the data. All authors wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Introduction: Protein supplementation is proposed to promote recovery and adaptation following endurance exercise. While prior literature demonstrates improved performance when supplementing protein during or following endurance exercise, chronic supplementation research is limited.Methods: Runners (VO2peak = 53.6 ± 8.9 ml/kg/min) were counter-balanced into a placebo group  or protein group  based on sex and VO2peak, and underwent 10 weeks of progressive endurance training. Prior to training, body composition, blood cell differentials, non-invasive mitochondrial capacity using near-infrared spectroscopy, and a 5 km treadmill time trial (TT) were evaluated. Progressive training then commenced (5–10% increase in weekly volume with a recovery week following 3 weeks of training) whereby PRO supplemented with 25 g of whey protein following workouts and prior to sleep . PLA supplemented similarly with a < 1 g sugar pill per day. Following training, participants were reanalyzed for the aforementioned tests.Results: VO2peak and initial 5 km TT were not significantly different between groups. PRO consumed significantly more dietary protein throughout the training period . Running volume increased significantly over time, but was not significantly different between groups throughout training. Blood measures were unaltered with training or supplementation. Mitochondrial capacity trended toward improving over time (time p = 0.063) with no difference between groups. PLA increased lean mass 0.7 kg (p < 0.05) while PRO experienced infinitesimal change . PLA improved 5 km TT performance 6.4% (1 min 31 s), while PRO improved only 2.7% (40 s) (interaction p = 0.080).Conclusion: This is the first evidence to suggest long-term protein supplementation during progressive run training is not beneficial for runners. Protein supplementation has been used to assist in recovery from exercise as well as increase caloric and protein intake. Current dietary protein recommendations for endurance athletes range from 1.2 to 2.0 g/kg/day  has suggProtein ingestion during endurance exercise has been shown to improve performance –14. FollGiven the improvements from protein supplementation on muscle quality measures, the eccentric contractions and weight-bearing nature of running compared to cycling could better unveil the ergogenic potential of protein supplementation. In this regard, Luden et al.  utilizedThe discrepant findings on protein supplementation in endurance athletes in tandem with the lack of long-term training studies led us to examine if protein supplementation could affect physiological and performance variables in runners during 10 weeks of progressive run training. We hypothesized protein supplementation would improve body composition and 5 km TT performance compared to a non-isocaloric placebo based on the anabolic and recovery properties associated with protein supplementation and/or the addition of energy .This study was approved by the Auburn University Institutional Review Board and was in compliance with the Helsinki Declaration (IRB protocol: #17-231 MR 1706). Participants read and signed an informed consent form prior engaging in the study. Inclusion criteria were as follows: (a) participants could be males or females between the ages of 18–45 years old, (b) participants had to partake in at least 32 km (~20 mi) per week of run training for at least one month prior to the study, (c) participants had to be healthy and free of any known disease determined by medical history questionnaire, and (d) participants had to abstain from supplemental protein or amino acids for 3 months prior to participating. A physical activity questionnaire and medical history form were filled out prior to participation to establish physical activity requirements were met and to identify potential risk factors that could be aggravated by training.2peak test and 5 km time trial (TT) familiarization. Participants were then counter-balanced based on sex and VO2peak into a placebo group (PLA) or protein group (PRO). Pretesting was performed 48 h later and consisted of: (a) dual-energy x-ray absorptiometry (DEXA), (b) venipuncture from an antecubital vein for blood cell measurements, (c) mitochondrial capacity measurement using near-infrared spectroscopy (NIRS), and (d) 5 km treadmill TT performance. At the conclusion of pretesting, participants were given canisters of protein powder or canisters of placebo sugar pills (described under “Supplementation and Food Logs”). Notably, this study was single-blinded and participants were informed that either supplement may be more beneficial based on the sparse chronic protein supplementation literature in endurance athletes.Participants completed an initial session consisting of a VO2peak testing was performed to determine maximal aerobic capacity. Participants reported to all testing sessions not having consumed alcohol in 24 h, chewed/smoked tobacco in 24 h, consumed caffeine in 8 h, consumed calories in 4 h, or engaged in strenuous exercise for 36 h. Participants were then fitted with a Polar heart rate monitor  and mounted a Woodway Treadmill . Following mouthpiece and headgear fitting, expired gases were continuously analyzed using a ParvoMedics TrueMax 2400 metabolic measurement system . Participants walked at 1.34 m/s (3.0 mi/h) for 3 min and then lightly jogged at 2.46 m/s (5.5 mi/h) for 2 min for a warm-up. Participants' speed was then increased to a pace which he/she deemed suitable for a 30-min run, and grade was increased to 1% to start the test. Grade was increased 1% every 1 min until volitional fatigue. Heart rate (HR), rating of perceived exertion (RPE), and respiratory exchange ratio (RER) were recorded every minute until cessation of the test. A test was accepted if heart rate was within 5% of age-predicted max HR, RPE ≥ 17, and RER ≥ 1.05. Average age for females and males was 30.9 ± 10.3 and 31.0 ± 7.2 years old, respectively. Average VO2peak for females and males was 47.0 ± 4.2 and 58.1 ± 8.5, respectively. Based on average age and values put forth by the American College of Sports Medicine .VOMedicine , both th2peak testing, participants were familiarized for the 5 km TT. Participants mounted the treadmill and were instructed to run 5 km (3.11 mi) as fast as possible. Participants were blinded to time and speed in order to avoid motivation from these variables and practiced starting the treadmill under these conditions. Once comfortable, grade was placed at 1%, and the TT began once the participant increased the treadmill speed. Upon starting, participants raised the speed until a self-selected pace was achieved which could then be manipulated throughout the TT based on desire to run faster or slower. Participants were monitored throughout the TT, but no verbal encouragement was given from the research staff. Once participants reached 5 km, time to completion was recorded, and participants walked for 5 min at a self-selected pace to cool-down.Following VO2peak testing and familiarization, testing began with assessment of hydration status measured through urine testing by an ATAGO 2392 handheld refractometer . Participants were considered hydrated if urine specific gravity level was < 1.020 g/ml which all participants met. Following hydration testing, participants underwent a dual-energy X-ray absorptiometry (DEXA) scan on a GE Corporation Lunar Prodigy  to determine body composition. Reliability for this test has been previously reported by our laboratory (intraclass correlation coefficient = 0.999) (−1). Detailed set-up, mitochondrial capacity assessment using NIRS procedures, analysis, and reliability of the device have been detailed elsewhere  where at least one training session each week was a high intensity workout . Each participant was assigned an alias to use the training mobile application “Strava” . Participants would upload global positioning system (GPS) tracked run data or manually upload data  to “Strava.” Participants were instructed to upload training data to “Strava” on a daily basis so researchers could ensure compliance and health. The research staff used a shared account to track and log every participant's training sessions. Care was taken by the research staff to contact participants if several days lapsed with no training to check for soreness or injury. Participants had the freedom to dictate how weekly volume was divided for each session. If participants underwent a training session that seemed excessive in volume based on training status then the research team contacted the participant to advise him or her about accumulating fatigue and increasing injury risk potential.Initially, participants informed the research staff of baseline training volume. Progressive training was induced by increasing weekly volume 5–10% each week whereby participants with lower weekly volume at baseline increased ~10% , and participants with higher weekly volume at baseline increased ~5% . Mesocyles lasted 4 weeks where training volume increased across weeks 1–3 while week 4 was a recovery week where participants would repeat week 1 volume within that mesocycle. Therefore, volume was increased weeks 1–3, 5–7, and 9, and weeks 4 and 8 were recovery weeks. Week 10 was considered a taper leading into post testing where participants reduced volume to week 1 values (~50% reduction).Supplementation included either placebo pills or whey protein powder. Participants in the PLA were instructed to consume two pills following a training session and ingest two pills before bed on workout days. On non-workout days, participants in PLA were instructed to consume two pills between meals and two pills before bed to emulate PRO. Participants in the whey protein group (PRO) were instructed to consume protein by mixing one scoop of protein powder with 350–500 ml of water. Similarly, on workout days PRO was instructed to consume one scoop immediately following a workout and another scoop before bed. On non-workout days, PRO was instructed to consume one scoop breakfast and lunch or lunch and dinner and one scoop before bed to mitigate catabolic intervals throughout the day based on muscle protein synthesis following protein ingestion , 31. AdhFood logs were filled out by participants during weeks 1, 5, and 10 to gain perspective on eating habits. Participants were given detailed instructions on how to determine food proportions and thoroughness of meal description needed for accurate assessment. Each participant entered their daily intake via “MyFitnessPal” mobile application  and submitted spreadsheets constructed by “MyFitnessPal” detailing daily consumption. Four food logs (2 weekdays [Mon-Fri] and 2 weekend days [Sat and Sun]) were submitted by each subject at each time point for a total of 12 food logs. Participants were instructed not to add supplementation to their food logs. Once the study was completed, protein supplementation macronutrients were added manually to the existing totals for PRO (nothing was added to PLA), and then averaged to represent participants' eating habits.p < 0.05.Data are reported throughout as mean ± standard deviation (SD) values. Prior to statistical analyses, all variables were tested for normality using Shapiro-Wilk tests with an alpha level set at t-tests for absolute and relative macronutrient intake at each time point were purposefully performed on these measures to fully elucidate any possible differences between groups.Self-reported macronutrient intake was not normally distributed and certain time points did not pass Levene's test for homogeneity of variances. Forced independent samples t-tests were also purposefully performed on these measures to fully elucidate any possible differences between groups.Training data was not normally distributed and certain time points did not pass Levene's test for homogeneity of variances. A (2 × 10) repeated measures ANOVA was utilized where group  and time (week 1–week 10) were analyzed. A Huynh-Feldt correction was used if sphericity was violated. Forced independent samples 2peak to determine which variable had a greater relationship with 5 km TT performance. ANCOVAs were also performed on all measures to control for baseline and were found to be inconsequential to the results and therefore are not presented. All of the aforementioned statistical analyses were performed using SPSS version 24.0 , and alpha level was set at p < 0.05. Effect sizes and 95% confidence intervals were calculated for body composition, mitochondrial assessment, and 5 km TT in Microsoft Excel  using the formula for Cohen's d: [(PLA delta mean–PRO delta mean)/pooled SD] where delta refers to post–pre value for each participant. Effect sizes were noted if “large” (d ≥ 0.80).Monocyte levels were not normally distributed, and white blood cell levels did not pass Levene's test for homogeneity of variances; however, these variables were analyzed using parametric statistics given the majority of blood data was normally distributed and contained homogenous variances. Body composition, mitochondrial assessment, and 5 km TT were normally distributed and passed the assumption for homogeneity of variances using Levene's tests. A 2 × 2 repeated measures ANOVA was utilized for blood measures, body composition, mitochondrial assessment, and 5 km TT where group  and time  were analyzed. If a significant interaction was identified then pairwise comparisons were utilized for within and between group differences. Pearson correlation coefficients were calculated for mitochondrial capacity and VOd = 0.914 (extrapolated from provided means and standard error) for improvements in 4-km run time following 7 days of whey protein and carbohydrate supplementation. Using this effect size with a power of 80% indicated that 40 total participants, 20 in each group, would be needed for appropriate statistical power. We sought to achieve this number, however had difficulty in recruiting due to rigor of training program, individual training season and plans, and initial running experience.5 km TT performance was the variable we were most interested in. Thus, power was calculated based on this variable. Hansen et al.  reported2peak (p = 0.698) or 5 km TT performance (p = 0.166). One female participant in PRO did not complete the post 5 km TT yielding a sample size of eight for this variable. Sex comparisons were considered, but ultimately removed due to small sample size. Of note, independent t-tests conducted between sexes on the delta scores for body composition metrics, mitochondrial assessment, or time trial performance did not reveal significant differences between sexes (p > 0.05). VO2peak showed a significant, strong correlation with Pre 5 km TT  and Post 5 km TT  (data not shown).Participant and baseline characteristics can be found in Table t-tests were utilized to detect any possible differences between groups. Relative (g/kg) and absolute (g) carbohydrate (CHO) intake was not different between groups at weeks 1, 5, 10, or overall . Relative fat intake was significantly different between groups during week 1 and overall (p < 0.05).Self-reported macronutrient data for participants is presented in Table p < 0.05), and nearly double the relative amount of the PLA group (p < 0.05).Relative and absolute protein consumption was significantly different during weeks 1, 5, 10, and overall. The PRO group consumed ~50 more grams of protein compared to the PLA group (p < 0.10). The protein group consumed nearly 400 more kilocalories overall compared to the placebo group which came primarily from the increased protein intake.Relative kilocalorie intake was significantly different between groups at weeks 1, 5, 10, and overall. Absolute kilocalorie intake approached significance weeks 1, 5, 10, and overall . Pairwise comparisons, independent of group, revealed differences between week 1 (46.2 ± 28.7 km) and week 6 , week 7 , week 8 , and week 9  demonstrating an increase in training volume  to peak training volume–week 7 . Likewise, PRO significantly increased training volume from week 1 (50.5 ± 37.1 km) to peak training volume–week 8 . There were no between-group differences based on the ANOVA, and forced independent samples t-tests demonstrated no significant differences between groups for distance ran during each week (p > 0.05).Forced dependent samples p = 0.003, group p = 0.471, and group × time p = 0.240). Pairwise comparisons, independent of group, demonstrated training time differences between week 1 (4.01 ± 2.08 h) and week 6 , week 7 , week 8 , and week 9  demonstrating an increase in time spent training .Similar differences were seen for average time spent training. An increase in weekly time spent training was observed  where PLA values were higher than PRO values independent of time. Data are not shown—see Supplementary Table No significant interactions or main effects were found between groups for white blood cells, neutrophils, monocytes, red blood cells, or hemoglobin. There was not a significant group × time interaction or time effect; however, there was a significant group effect for lymphocytes (p = 0.052), however there was not a significant group × time interaction, group effect, or meaningful effect size. Average value and standard deviation at pre for PLA (72.7 ± 8.0 kg) and PRO (65.9 ± 11.8 kg), and at post for PLA (71.7 ± 8.8 kg) and PRO (64.6 ± 10.8 kg). Ninety five percent confidence interval for the mean at pre for PLA (66.0–79.4 kg) and PRO (56.8–74.9 kg), and at post for PLA (64.4–79.1 kg) and PRO (56.3–72.9 kg).Body mass Figure  approachp = 0.049) and large effect size (d = 1.032). Pairwise comparisons showed a significant increase from Pre to Post for PLA (p = 0.024), but not for PRO. Pairwise comparisons did not demonstrate significant group differences at Pre or Post. Average value and standard deviation at pre for PLA (52.4 ± 7.3 kg) and PRO (50.0 ± 10.6 kg), and at post for PLA (53.1 ± 6.7 kg) and PRO (49.9 ± 10.3 kg). Ninety five percent confidence interval for the mean at pre for PLA (46.3–58.5 kg) and PRO (41.9–58.1 kg), and at post for PLA (47.6–58.7 kg) and PRO (41.9–57.8 kg).Total lean mass Figure  demonstrp = 0.016) where PLA possessed more fat mass than PRO. A time effect was also observed (p = 0.006) whereby both groups decreased fat mass from Pre to Post. Average value and standard deviation at pre for PLA (17.5 ± 2.9 kg) and PRO (13.1 ± 3.6 kg), and at post for PLA (15.5 ± 3.1 kg) and PRO (12.0 ± 3.1 kg). Ninety five percent confidence interval for the mean at Pre for PLA (15.1–19.9 kg) and PRO (10.3–15.8 kg), and at Post for PLA (12.9–18.1 kg) and PRO (9.6–14.3 kg).Fat mass Figure  did not p = 0.063) where both groups improved mitochondrial capacity from Pre to Post. There was not a significant group effect, group × time interaction, or meaningful effect size  and PRO (1.67 ± 0.53 min−1), and at post for PLA (1.51 ± 0.37 min−1) and PRO (1.80 ± 0.67 min−1). Ninety five percent confidence interval for the mean at Pre for PLA (1.13–1.69 min−1) and PRO (1.26–2.08 min−1), and at Post for PLA (1.20–1.82 min−1) and PRO (1.29–2.32 min−1).Mitochondrial capacity assessment using Near-Infrared Spectrometry (NIRS) approached a significant time effect p = 0.06 where bor = −0.390; p = 0.136), Post mitochondrial capacity and Post 5 km TT , Pre mitochondrial capacity and change in 5 km TT , or change in mitochondrial capacity and change in 5 km TT  (data not shown).Independent of supplementation, there was not a significant correlation between Pre mitochondrial capacity and Pre 5 km TT (p = 0.080), and demonstrated a large effect size between groups (d = 0.945). There was a significant time effect (p < 0.001) showing both groups improved 5 km TT performance from Pre to Post; however, there was no significant group effect. PLA improved 5 km TT performance 1min and 31 s, where PRO improved by 40 s . 5 km TT performance indicated a large effect and approached significance shown through a 6.4% improvement in PLA and only a 2.7% improvement in PRO. These results corroborate and expand on work by Macdermid et al.  who deteThe training program utilized was designed to maximize outcomes and minimize injury. Positive outcomes have been shown using a linear model of training by strictly increasing volume , 35, howp = 0.063). With an adequate sample size we speculate this finding would be significantly different with training based on prior literature where mitochondrial volume and function improve . Past literature does suggest, however, acute protein supplementation seems beneficial in altering biomarkers related to improved recovery  or an alPR and MDR conceived the research question. MDR procured materials. All authors recruited and collected data. PR, MDR, and HK analyzed the data. PR and PM conducted statistical analyses. PR wrote the manuscript. MDR provided revisions following initial manuscript draft. All authors revised, edited, and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Thus far, the preparation of the recombinant enzyme for enzymatic and structural in vitro studies has been performed mainly using a baculovirus expression system. More recently, it was reported that the enzyme could be exogenously expressed and isolated from E. coli, although limited amounts of active protein were obtained. We herein describe two novel methods to yield pure recombinant enzyme. The first describes the expression and purification of the full-length enzyme from eukaryotic cells HEK293-F, whilst the second concerns the C-terminal domain of hsEH obtained from the cost-effective and rapid E. coli prokaryotic system. The two methods successfully generated satisfactory amounts of functional enzyme, with virtually identical enzymatic activity. Overall, the protocols described in this paper can be employed for the recombinant expression and purification of active hsEH, to be used in future biomedical investigations and for high-throughput screening of inhibitors for potential use in the treatment of cardiovascular disease.The human soluble Epoxide Hydrolase (hsEH) is an enzyme involved in the hydrolysis of endogenous anti-inflammatory and cardio-protective signalling mediators known as epoxyeicosatrienoic acids (EETs). EETs’ conversion into the corresponding diols by hsEH generates non-bioactive molecules, thereby the enzyme inhibition would be expected to enhance the EETs bioavailability, and their beneficial properties. Numerous inhibitors have been developed to target the enzyme, some of which are showing promising antihypertensive and anti-inflammatory properties  •hsEH is a key regulator of cardiovascular homeostasis.•A HEK293-F mammalian expression system for hsEH full-length (FL) was developed.•E. coli expression system for the hsEH C-terminal Domain (CTD) was established.An •in vitro.Both proteins exhibited the same enzymatic specific activity •The CTD preparation provides benefits of easy operation, and high yield and purity. The purified enzyme showed virtually identical specific activity and inhibition properties of the recombinant enzyme prepared from insect cells. However, the yield of purified hsEH full-length (hsEH FL) obtained from prokaryotic cells was reported to be rather low (around 2 mg/L). Herein, we present a novel protocol of expression and purification of the recombinant hsEH FL, that yields milligrams of pure and active recombinant enzyme. This alternative method utilises the mammalian cells FreeStyle™ HEK293 (HEK293-F) [E. coli, very little detail on the protein expression and purification system was provided in the previous report. In this paper, we describe a thorough protocol of prokaryotic expression of the isolated CTD, along with an optimised purification method to yield milligram amounts per litre of pure protein for biological investigations and drug discovery studies.The human soluble Epoxide Hydrolase (hsEH – EC 3.3.2.10) is composed by two independently folded domains , an N-teisorders ,12. Of tisorders  and AUDAisorders  failed iisorders ,16. Two isorders  have beeisorders ,19. As npression . Howeverpression  reportedEK293-F) . Beyond EK293-F)  reportedIn addition to protein cloning, expression, and purification of the hsEH FL and CTD, the enzymatic specific activity of the recombinant enzymes was also compared and will be herein described.22.1® vector (Invitrogen) using the pcDNA3.1 Directional TOPO® Expression Kit (Invitrogen). The hsEH FL cDNA was PCR-amplified using primers designed to facilitate directional cloning . The mammalian expression vector cloned with the hsEH FL cDNA was finally amplified in E. coli DH5α C2987 competent cells (NEB) and purified using the Pure Yield™ Plasmid Maxiprep system (Promega), out of a 0.5 L bacterial culture.The cDNA of hsEH FL was kindly provided by Dr C Morisseau (UC Davis) and was cloned into the pcDNA3.1D/V5-His-TOPO cloning , as deta2.27 viable FreeStyle™ 293-F cells (HEK293-F – ThermoFisher Scientific) was transferred into a disposable Erlenmeyer flask (Sigma) previously filled with 30 mL of pre-warmed FreeStyle 293 Expression Serum-Free Medium™ (SFM – ThermoFisher Scientific). The cells were cultured for two days at 37 °C, 8% CO2, and 100 rpm, in a shacking incubator, up to a density of 1–3 × 106 cells/mL and 90% viability. Cells were sub-cultured four times, every two days of incubation, in 30 mL of fresh SFM to 0.2–0.5 × 106 cells/mL. During the fifth and last passage, the cells were seeded in 125 mL of fresh medium to 1 × 106 cells/mL. After 48 h, the cells were prepared for transfection, being resuspended at a cell density of 2 × 106 cells/mL in 250 mL of fresh pre-warmed SFM. The vector and the transfection agent polyethylenimine (Sigma) were added to the cells to a final concentration of respectively 3 μg/mL and 9 μg/mL. After 24 h, the culture was diluted 1:1 in pre-warmed SFM supplemented with 2.2 mM Valproic Acid (Sigma). Three days later the cells were collected by centrifugation at 1200 rpm for 20 min at room temperature and stored at −20 °C.A vial containing 1 × 102.3® cloning ligation-independent system (Clontech Laboratories Inc.) [® primer design web tool, characterised by a sequence that overlaps with the gene of interest, and a region complementary to the vector (. coli competent cells. Successful cloning was confirmed by sequencing (Source Bioscience).A modified pET3a plasmid, containing a hexahistidine tag (His-tag) and a TEV (tobacco etch virus) cleavage site (MHHHHHHSTENLYFQGSS) in the multiple cloning site, was provided by the Protein Production Facility of King's College London. The cDNA of the hsEH CTD (amino acids 230–555) was cloned in the plasmid using the IN-Fusiones Inc.) . The cDNe vector . The tem2.4The pET3a containing hsEH CTD cDNA was transformed in chemically competent Ros2™(DE3) (EMD Millipore). A single colony of transformed cells was inoculated in 50 mL of ZYP5052 auto-induction medium  and cult2.52, 0.2 mM EDTA, 15 μg/mL benzamidine, a tablet of cOmplete™ protease inhibitor cocktail (Roche) per 50 mL of buffer), and the cells broken with three thaw-and-freeze cycles in liquid nitrogen. Pellets from bacterial cultures were resuspended in 2 mL/g of wet cells of lysis buffer , to be sonicated on ice with a Vibra-Cell™ sonicator (Sonics) for 10 min (3 s pulse ON and 10 s pulse OFF). Both mammalian and bacterial lysates were then centrifuged at 38800 × g for 1 h at 4 °C, and the supernatants passed through a 0.45 μm filter (Millipore). The soluble fractions were loaded onto a 5 mL HisTrap™ FF Nickel Sepharose Column , previously equilibrated with binding buffer . The bound species were eluted in 1.5 mL fractions, applying a 0–60% gradient in 30 column volumes (CVs) of elution buffer . The fractions containing hsEH were pooled and dialysed at 4 °C overnight, in 20 mM MOPS pH 7.4, 75 mM NaCl, 1 mM EDTA, 2 mM DTT.The pellets obtained from mammalian cells growths were resuspended in 5 mL/g of wet cells of lysis buffer  were poured onto a 15 mL polypropylene gravity column (Pierce), and washed with 10 CVs of water, 10 CVs of 50% MeOH, and 10 CVs of 0.1 M NaOH. The resin was then transferred in a 100 mL frosted neck round-bottom flask, containing 20 mL of 0.3 M NaOH, and 2 mL of 1,4-butanediol diglycidyl ether (activation solution), then cooled down for 30 min on ice while shaking. 60 mg of NaBH4 were added, and the epoxy-activation of the Sepharose was carried out at room temperature for 5 h on an orbital shaker in a fume hood . The activated resin was transferred in a sintered-glass funnel and washed with 20 CVs of water pH 6, 20 CVs of 50% MeOH, 20 CVs of MeOH, 20 CVs of 50% MeOH, and 20 CVs of water pH 6. The resin was resuspended in 20 mL of activation solution, added with 50 mL of benzyl mercaptan, and left to swirl for 24 h at room temperature in a fume hood. The derived resin was transferred on a sintered glass funnel, and washed with 40 CVs of 50% MeOH, 40 CVs of MeOH, 40 CVs of 50% MeOH, 40 CVs of water, 40 CVs of 0.5 M NaCl, 40 CVs of water, 40 CVs of 0.001 M HCl to hydrolyse the unreacted epoxy-groups, 40 CVs of water, and 40 CVs of 50% EtOH. The resin was then split in aliquots of 2.5 mL in gravity columns and washed with 10 CVs of EtOH. The final BTS resin was stored at 4 °C in 0.1% of butylated hydroxyanisole.The Benzylthio-Sepharose (BTS) is an improved affinity chromatography resin for the purification of Epoxide Hydrolases. The resin was synthesised in-house using a protocol adapted from Winxtrom et al. . 10 mL o2HPO4, 3 mM KH2PO4, pH 7.4, 0.2 mM EDTA, 1 mM DTT.Proteins subjected to dialysis after IMAC purification were loaded onto the BTS resin previously equilibrated with binding buffer . After washing the column with 2.5 CVs of binding buffer, hsEH proteins were eluted with 10 CVs of elution buffer . Purity and state of the protein were verified by SDS-PAGE, and the fractions containing protein were dialysed at 4 °C overnight in 137 mM NaCl, 2.7 mM KCl, 10 mM Na2.72HPO4, 3 mM KH2PO4, pH 7.4, 0.2 mM EDTA, 1 mM DTT, 5% glycerol. Fractions containing hsEH proteins were pooled together and dialysed overnight at 4 °C in 50 mM TRIS-HCl pH 7.4, 300 mM NaCl, 20% glycerol. After dialysis, samples were concentrated to 1 mg/mL  and loaded onto a HiLoad 16/600 Superdex 75pg SEC , previously equilibrated with 137 mM NaCl, 2.7 mM KCl, 10 mM NaASY tool ), flash 2.8®-20), the membranes were exposed to the primary antibody in PBS-T added with 2% milk , shaken for 1 h at room temperature. After being washed three times for 10 min with 5 mL PBS-T, the membranes were exposed to HRP-conjugated secondary antibody in PBS-T added with 2% milk . After 1 h in agitation at room temperature, the membranes were washed three times for 10 min, two times with 5 mL PBS-T, and a last time with PBS. The detection was carried out using the Enhanced Chemiluminescent Luminol (Promega).For immunoblotting, the samples were initially run on SDS-PAGE (using Laemmli protocol ) at 170 2.9The intact protein MS experiments were performed using a maXis (Bruker) calibrated using the tuning mix ES-TOF (ESI) positive (Agilent). The proteins were dialysed overnight at 4 °C in 25 mM HEPES pH 7.4, 300 mM NaCl, 10% glycerol, 1 mM DTT, to be then concentrated to 5–10 mg/mL. The samples for ESI-MS were prepared diluting the proteins at a final concentration of 60 μg/mL in 0.1% formic acid, to be then loaded onto a Hamilton syringe and directly injected into the spectrometer using a motorised pump with a flow rate of 10–50 μL/min. The spectra were recorded for 3 min and a spectral rate of 1 Hz, with an end plate offset of −500 V, a capillary voltage of −4500 V, and a nebuliser pressure of 3.0 Bar. The detection of the spectra was performed setting the funnel and multipole RF at 400 Vpp, tuning the ISCID energy between 80 and 110 eV to optimise the signal-to-noise ratio, and selecting an ion energy of 3 eV.2.10t-test and reported in the plots as average ± SEM. The statistical significance was set up at minimum p 0.05.The enzyme specific activity was evaluated using a rapid continuous spectrofluorometric method developed by Wolf et al. . The ass33.1® Expression Kit, a set of primers were designed  using the auto-induction media ZYP5052, given that induction with isopropyl-β-D-thiogalactopyranoside (IPTG) led to recombinant protein accumulation in inclusion bodies. We tested whether IPTG induction of Ros2™(DE3) cultured in Luria Broth or Terrific Broth produced insoluble hsEH CTD: in agreement with what was observed for the full-length recombinant enzyme [data not shown). However, using the autoinduction ZYP5052 media, the amount of hsEH CTD present in the soluble fraction increased noticeably. Transformed Ros2™(DE3) cells were cultured at 37 °C, up to three different levels of optical density (OD600nm) , then the temperature was lowered to 18 °C and the cells cultured for either 16 or 24 h. Although most of the protein was still found in the insoluble fractions, detectable levels of soluble protein were observed when the cells were cultured at 37 °C up to an OD600 of 0.5, followed by 24 h incubation at 18 °C  cells, pellets of 10–15 g/L of cells were harvested. As per the full-length protein, the first step of purification was a Niwo peaks A, of whiwo peaks B. The prwo peaks C. A finawo peaks E, indicawo peaks E. Approx3.6m/z intensity of the species in solution. The spectra showed a Gaussian-type distribution of peaks ranging from m/z 1275.2 to 2475.7 for the CTD, and 1196.3–2289.7 m/z for the FL (Electrospray ionisation mass spectrometry (ESI-MS) and Western blotting confirmed the identity of the purified proteins. The ESI-MS experiments were performed spraying hsEH CTD and FL in positive ionisation conditions, detecting the r the FL A and C. r the FL C. In addWestern blotting analyses were performed on purified hsEH CTD and FL, stained with two different primary antibodies: a mouse anti-His-tag monoclonal antibody (taking advantage of the His-tag engineered in the recombinant enzymes) A and C aE. coli and HEK293-F cells respectively.Western Blot and ESI-MS experiments confirmed the identity of the recombinant hsEH CTD and FL expressed and purified from 3.7via a spectrofluorometric assay, using the synthetic substrate PHOME [The specific activity of hsEH CTD and FL was determined te PHOME . Protein2 ≥ 0.9)  B and plo(2 ≥ 0.9 C. The exc t-test . The hsE4S. frugiperda [E. coli BL21(DE3) cells, obtaining around 2 mg/L of pure and active protein: nonetheless, in our hands, this method yielded even lower amounts of pure hsEH. We have therefore developed alternative expression and purification methods of both full-length protein and its functional C-terminal domain, which is necessary and sufficient for the EETs epoxy-hydrolytic activity of the enzyme. hsEH FL was obtained from mammalian HEK293-F cells, obtaining around 2 mg of protein from only 250 mL of culture. The optimised expression and purification of hsEH CTD using bacterial expression, the fastest and cheapest method of protein production available, yielded approximately 6 mg of pure protein per L of culture. Moreover, we herein demonstrated that the FL and CTD exhibited virtually identical turnover frequencies, suggesting that the CTD in isolation can be utilised in future studies of hsEH epoxy hydrolytic enzymatic activity and inhibition.hsEH is considered a key regulator of the cardiovascular homeostasis, a role exerted through the hydrolysis of endothelial signalling modulators termed epoxyeicosatrienoic acids (EETs) . Accumulugiperda  that theugiperda . Since tugiperda  reportedIn conclusion, we have provided new tools to prepare active hsEH full-length or CTD, which will assist future drug discovery studies and investigations of its biological function and mechanisms.All the authors contributed to the planning of the study. GA performed cloning and optimisation of bacterial expression of hsEH CTD, development and optimisation of purification of hsEH FL and CTD, characterisation of hsEH FL and CTD, and their specific activity measurements. RLC carried out cloning and optimisation of mammalian expression of the hsEH FL. GA, RLC, and MRC contributed to the writing of the manuscript. All the authors contributed to the results discussion. MRC and PE obtained funding for this work.Conflicts of interest: none.


The repurposed sensors have enhanced aqueous solubility and the ability to quantitatively measure biologically relevant concentrations of Ca2+ (0.25 mM–1 mM), Zn2+ (6.25 µM–50 µM), and GSH (0.25 mM–1 mM) by fluorescence in aqueous media. In addition, the liposomal sensors are nontoxic to HEK293 cells and have the ability to detect exogenously added Zn2+ (1 mM), Ca2+ (1 mM), or GSH (1 mM) near cells without internalisation. This new sensing platform provides a means to repurpose a range of intracellular fluorescent sensors to specifically detect extracellular analytes, while also improving biocompatibility for overall enhanced use in a wide range of biomedical applications.Cell-permeable fluorescent chemosensors  have been incorporated into yeast total lipid extract-based liposomes to suppress inherent cell permeability to allow the detection of extracellular Ca The extracellular environment proximal to a cell membrane is critical to cellular function, directly influencing cell growth , differe2+, Ca2+, and GSH were chosen as analytes for the study, as they are known to play a critical role in early embryonic development. For example, changes in concentrations of extracellular Ca2+, Zn2+, and GSH are associated with the fertilization of an embryo [2+, Zn2+, and GSH levels then provides an opportunity to identify the success, or otherwise, of fertilisation in clinical applications such as IVF. To address this need, three distinct fluorescent sensors  unless otherwise stated, and used without further purification. Calcein was purchased from TCI  and mCB from Sigma-Aldrich. SP2 was synthesised as previously described . All lipw/w to lipid) and mixed with a solution containing 25 mg of lipid , 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), L-α-phosphatidylcholine from chicken eggs (egg PC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or total lipids extracts from Escherichia coli or Saccharomyces cerevisiae (yeast) in 1 mL buffer (20 mM 3-(N-morpholino)propanesulfonic acid (MOPS), 5% maltose, pH 7.2)). Large multilamellar liposomes were formed by sonicating and vortexing the lipid-sensor mixture for 1 h. The mixture was clarified to remove unincorporated insoluble sensor by low speed centrifugation at 14,000 rpm for 20 min at room temperature, followed by ultracentrifugation at 50,000 rpm for 1 h at 25 °C to isolate the liposome fraction. The pellet containing liposomes was washed twice with a buffer (1 mL) to remove any unincorporated sensor and then resuspended in a buffer (1 mL). Unilamellar liposomes were obtained by extruding this mixture through a 0.4 µm membrane. After 11 passages, liposomes with a median size distribution of ~200 nm in diameter were generated for analysis. The fluorescence of all liposomal complexes (100 µL) in a buffer was then measured by a Synergy H4 microplate reader with λex/em = 480/518 nm for all calcein complexes, λex/em = 380/480 nm for all mCB complexes and λex/em = 532/620 nm for all SP2 complexes. The mCB, calcein, and SP2 liposomal complexes were then separately mixed with GSH (1 mM), Ca2+ (1 mM) and Zn2+ (50 µM), respectively, and incubated in the dark for 10 min at room temperature. All concentrations of analytes reported are final concentrations of the solutions after mixing. The resultant fluorescence emission was similarly recorded. The experiments were carried out in duplicates. The fluorescence intensity of each sample was normalized to the fluorescence of the corresponding sample without analyte and plotted in GraphPad Prism 7.0.The fluorescent sensor  was dissolved in DMSO . The fluorescence of each sample was measured on the microplate reader and the intensity was normalised to the sample without analyte. This was then plotted using GraphPad Prism 7 as 2+ (0–1 mM), Zn2+ (0–50 µM), and GSH (0–1 mM), respectively, for 10 min in the dark at room temperature. All analytes were first dissolved in water to make a concentrated stock solution. All concentrations of analytes reported are final concentrations of the solution after mixing. The resultant fluorescence spectra of LP–Cal (λex = 480 nm), LP–mCB (λex = 380 nm), and LP–SP2 (λex = 532 nm) of each concentration of analyte were recorded on the plate reader. The experiments were carried out in duplicate in the dark. The fluorescence intensities at 518 nm for LP–Cal, 480 nm for LP–mCB, and 620 nm for LP–SP2 were plotted against analyte concentrations in µM, respectively, to produce a standard curve of calibration for each sensor. A linear trendline was fitted to the plots by GraphPad Prism 7.0. Blank liposomes without sensors were also prepared with yeast total lipid extract and their fluorescence in the presence and absence of analytes  was similarly measured as a negative control. An averaged excitation spectrum  in an MOPS buffer was incubated for 10 min with or without analyte , respectively, overnight. A LIVE/DEAD® Viability/Cytotoxicity Kit  was prepared with 1 µM calcein-AM and 10 µM ethidium homodimer-1. Cells were washed twice with warm PBS, then lifted and resuspended in an appropriate LIVE/DEAD® Viability/Cytotoxicity assay buffer. In each well, 8 × 105 cells were plated using a black walled 96-well plate. The plate was measured using a microplate reader.HEK293T cells were cultured in DMEM (Dulbecco’s Modified Eagle Medium) medium  containing 10% heat inactivated fetal bovine serum (FBS) (Gibco)), 1% penicillin-streptomycin, 1% L-glutamine , and 2 ‰ fungizone . Cells were plated at 2.7 × 105 cells/well in 96-well plates, and the fluorescence signal levels were measured with a FLUOstar Optima microplate reader  after 2 h or 24 h of incubation with LP–Cal, LP–mCB, or LP–SP2 in PBS to obtain quantitative measures of cell viability. Cells incubated in mercuric chloride (25 μM) were used as a negative control for cell viability and untreated cells were used as a positive control for cell viability.Cell viability was quantified using the AlamarBlue assay , as previously described . The HEK5 cells/mL density in an 8-well ibidi μ-slide . Cells were incubated for 2 h in a PBS buffer containing 50 µL of LP–Cal, LP–SP2, or LP–mCB solution. For cell samples containing LP–Cal, LP–SP2, or LP–mCB, Ca2+ (1 mM), Zn2+ (1 mM), or GSH (1 mM) was added, respectively, and the samples were allowed to incubate for 30 min in the dark. The plate was then imaged on an Olympus FluroView V10i confocal microscope  with excitation and emission wavelengths of 473 nm, 490–590 nm for LP–Cal; 405 nm, 420–520 nm for LP–mCB; and 559 nm, 570–670 nm for LP–SP2. Images were processed using ImageJ. Under “Brightness/Contrast” setting, the minimum level was set to 0 and the maximum level to 80 for all images. The fluorescence intensities reported in HEK293T cells were cultured in DMEM medium  containing 10% heat inactivated fetal bovine serum , 1% penicillin-streptomycin , 1% L-glutamine , and 0.2% fungizone . Cells were plated at 2.7 × 102+, mCB, and calcein [2+ was also used in order to expand the scope of the study. Calcein was selected due to its wide availability and common use with cells. Moreover, calcein is a member of the well-characterised fluorescein-based chemosensor family. Though the sensitivity of calcein to Ca2+ is pH dependent, it is capable to detect Ca2+ at a concentration used in all subsequent experiments at physiological pH (em = 480 nm), calcein in the green range (λem = 518 nm), and SP2 in the red range (λem = 620 nm). All three sensors have been reported to function in biological environments to detect Ca2+, GSH, or Zn2+ by fluorescence [E. coli and yeast. These liposome–sensor complexes were assembled by mixing a DMSO solution of the sensor (8 mg/mL) with the lipid solution in an MOPS buffer  by sonication for 1 h at room temperature. The unincorporated sensors were subsequently removed by low-speed centrifugation  for 20 min. The resulting liposomes were isolated by high-speed ultracentrifugation  for 1 h. Unilamellar liposomes were then obtained by extrusion of this mixture through a 0.4 µm membrane. The size of the liposomes was measured by DLS (dynamic light scattering) with the thusly obtained Z-Ave and polydispersity index (PDI) reported in Well-documented sensors for the detection of GSH and Ca calcein ,28,29,30 calcein  red-emitgical pH . Importarescence ,31,32,332+ for calcein, Zn2+ for SP2 and GSH for mCB complexes), was measured in an MOPS buffer and the results are shown in 2+ (1 mM) and Zn2+ (50 µM), respectively. A 1.6-fold increase was observed for the liposome–mCB complex (LP–mCB) with added GSH (1 mM). All liposomes derived from yeast total lipid extract, LP–Cal, LP–mCB, and LP–SP2, present as large unilamellar vesicles (LUV), with consistent sizes between 165–175 nm as determined by dynamic light scattering analysis  were diluted with an MOPS buffer (70 µL) and 10 µL of a range of concentrations of Ca2+ (0–1 mM), GSH (0–1 mM), or Zn2+ (0–50 µM) were added. All concentrations reported are final concentrations. The mixtures were incubated for 10 min at room temperature in the dark. The resultant fluorescence was then measured using excitation wavelengths of 480 nm for calcein [2+, GSH, and Zn2+ to LP–Cal, LP–mCB, and LP–SP2, respectively (2+), 0.25 mM (GSH), and 6.25 µM (Zn2+), respectively. By contrast, fluorescence was not observed for yeast-derived liposomes without an embedded sensor, with and without added analyte . The cells were then imaged on an Olympus FluroView V10i confocal microscope with excitation and emission wavelengths of λex/λem = 473/490–590 nm, λex/λem = 405/420–520 nm and λex/λem = 559/570–670 nm for LP–Cal, LP–mCB, and LP–SP2, respectively, in order to measure the fluorescence response of these complexes upon the addition of each analyte. Weak fluorescence was observed for any of the yeast-derived liposomal complexes in the absence of analyte, see 2+, Zn2+, and GSH by fluorescence in a cell-based environment.Importantly, the incubation of each of the yeast-derived liposomal complexes with HEK293 cells over 24 h did not induce cytotoxicity relative to untreated cells, as evaluated by two separate viability assays, see 2+, Zn2+, and GSH, respectively. These sensors are chemically distinctive and encompass emission wavelength ranges commonly used in sensing, with mCB, calcein, and SP2 representing the blue region (400 nm–480 nm), the green region (500 nm–550 nm), and the orange/red region (600 nm–700 nm), respectively. The nature of lipid used for the construction of the liposome is critical. Here, we established that yeast total lipid extract provided an optimal scaffold for all three sensors, as it generated liposomes of consistent size and retained the greatest level of fluorescence response upon exposure to analytes. Quantitative sensing of Ca2+ (0–1 mM), Zn2+ (0–50 µM), and GSH (0–1 mM) in aqueous solution was demonstrated using yeast liposome complexes, LP–Cal, LP–SP2, and LP–mCB, respectively. Further, we show the use of the yeast-derived liposomal sensors near HEK293 cells, without internalisation. Collectively, this work shows the repurposing of cell permeable sensors for extracellular sensing applications, negating the need for the synthesis and development of new sensor analogues.In summary, small molecule fluorescent sensors, calcein, mCB, and SP2, can be embedded into a liposomal membrane to improve biocompatibility, while retaining their ability to detect Ca


The RNA genome of Cam13 possesses 8,155 nucleotides and a poly(A) tail, and it has a typical sapelovirus genome organization consisting of a 5’ terminal untranslated region, a large open reading frame (ORF), and a 3’ terminal untranslated region. The ORF encodes a single polyprotein that is subsequently processed into a leader protein (L), four structural proteins  and seven functional proteins . We confirmed that 293 T, HepG2/C3A, Hep2C, Huh7 and primary cynomolgus monkey kidney cells were susceptible to SSV infection. In contrast, PK-15, Vero, Vero E6, RD-A, A549, and primary green monkey kidney cells were not susceptible to SSV infection. We established an ELISA for the detection of IgG antibodies against SSV by using the virus particles as the antigen. A total of 327 serum samples from cynomolgus monkeys and 61 serum samples from Japanese monkeys were examined, and the positive rates were 88.4% and 18%, respectively. These results demonstrated that SSV infection occurred frequently in the monkeys. Since Cam13 shared 76.54%–79.52% nucleotide sequence identities with other known SSVs, and constellated in a separate lineage in the phylogeny based on the entire genome sequence, we propose that Cam13 is a new genotype of the simian sapelovirus species.We isolated a novel simian sapelovirus (SSV), Cam13, from fecal specimen of a cynomolgus monkey by using PLC/PRF/5 cells. The SSV infection of the cells induced an extensive cytopathic effect. Two types of virus particles with identical diameter (~32 nm) but different densities (1.348 g/cm Most of these viruses have been classified into the genus Enterovirus of the family Picornaviridae9. It has been demonstrated that some SV strains  are distinct from other simian enteroviruses, and rather related to porcine enterovirus 8 (PEV8)11. Based on the entire genome sequences, SV2 and PEV8 were classified into a new genus Sapelovirus11, which currently consists of two species: Sapelovirus A (formerly known as porcine sapelovirus [PSV]), and Sapelovirus B (formerly named as simian sapelovirus [SSV])12. A third species (Avian sapelovirus) including duck picornavirus (DPV) TW90A has been renamed Anativirus A and moved to a new genus, Anativirus (http://www.picornaviridae.com/sapelovirus/sapelovirus.htm)13.Many simian viruses (SV) were isolated from various primate tissues during the development of tissue culture methods as well as from specimens derived from primates used in biomedical researchhttp://www.picornaviridae.com/). In addition, sapeloviruses possess a leader protein (L) at the N-terminus of the polyprotein14.Sapelovirus is a nonenveloped positive-sense single-stranded RNA viruses. It contains approximately 7.5~ 8.2 kb genomic RNA. The genome organization is similar to that of other picornaviruses: a 5′ untranslated region (UTR), a large open reading frame (ORF), a 3′ UTR, and poly(A) tail. The large ORF encodes a single polyprotein that is subsequently processed into four structural proteins  and seven non-structural proteins   sequences the SSVs can be separated into three groups 11, but no clear genotyping for these viruses has been reported, and the epidemiology, antigenicity, and pathogenicity of the SSVs remain unclear. In the present study, we used a human hepatocarcinoma cell line, PLC/PRF/5, to isolate an SSV from fecal specimens of cynomolgus monkeys. The next-generation sequence analysis (NGS) of the entire genome of our isolate (Cam13) suggested that this virus is a novel member of SSV. We also proposed the introduction of a genotyping system to classify SSV isolates among Sepelovirus B.PSV has been detected in pigs worldwide, and the entire genome sequences of several PSV strains have been analyzed20. The monkeys were 3–4 years old and imported from a monkey farm in Cambodia in 2017. We also used a total of 297 serum samples including 36 sera collected from cynomolgus monkeys bred and grown at the Tsukuba Primate Research Center, Japan in 1995; 123 serum samples from cynomolgus monkeys imported from China in 2017; 58 serum samples from wild cynomolgus monkeys imported from the Philippines in 1985; and 80 sera from wild cynomolgus monkeys imported from Malaysia in 1969.A set of serum and fecal samples was collected from 30 cynomolgus monkeys (C1 to C30) originally to detect hepatitis E virus (HEV)All of the serum samples from the imported monkeys were collected right after the monkeys were imported into Japan. In addition to cynomolgus monkeys, 61 serum samples collected from Japanese monkey from 1995 to 2004 in Japan were used. The serum samples and stool suspensions were stored at −80 °C until use. The imported monkeys used in these experiments were reviewed and approved by the institutional ethics committee of the National Institute of Infectious Diseases (NIID), and all of the animal experiments were carried out according to the “Guides for Animal Experiments Performed at NIID”.2 atmosphere. For virus inoculation, the confluent cells were trypsinized, diluted 1:3 and cultured in a 25-cm2 tissue culture flask. After 24 h of incubation, the medium was removed and the cells were washed two times with phosphate-buffered saline (PBS)21.The cell line PLC/PRF/5 (JCRB0406) was obtained from the Health Science Research Resources Bank . The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with L-glutamine and high glucose supplemented with 10% (v/v) heat-inactivated fetal bovine serum , 100 U penicillin  and 100 mg streptomycin (Gibco) at 37 °C in a humidified 5% CO20. The fecal specimens were diluted with 10 mM PBS to prepare a 10% (w/v) stool suspension. The suspension was shaken at 4 °C for 1 h, clarified by centrifugation at 10,000 g for 30 min, and passed through a 0.45 µm membrane filter 22.Ten fecal specimens from monkeys that were positive for anti-HEV IgM antibody by an enzyme-linked immunosorbent assay (ELISA) were selected to inoculate PLC/PRF/52. Further incubation was done at 36 °C. The culture medium was collected and supplemented with new medium every 4 days22.One ml of the 10% stool suspension was inoculated onto PLC/PRF/5 cells, and the adsorption was performed at 37 °C for 1 h. The cells were washed two times with PBS and then supplemented with 10 ml of maintenance medium consisting of medium 199  containing 2% (v/v) heat-inactivated FBS and 10 mM MgCl2 atmosphere.Two green monkey kidney cell lines (Vero and Vero E6), green monkey primary kidney cells (PGMKC), cynomolgus monkey primary kidney cells (PCMKC), a human embryonic kidney cell line (293 T), a porcine kidney cell line (PK-15), human hepatocellular carcinoma cell lines , a human lung carcinoma cell line (A549), a human cervix adenocarcinoma cell line (Hep2c), and an embryonal rhabdomyosarcoma cell line (RD-A) were used to examine the susceptibility to SSV. These cells were inoculated with the supernatant of SSV-infected PLC/PRF/5 cells. All of the cells were cultured with DMEM supplemented with 10% FBS at 37 °C in a humidified 5% COg for 60 min to remove the cells and the debris. The supernatant was then centrifuged at 32,000 rpm for 3 h in a Beckman SW32Ti rotor. The resulting pellet was re-suspended in PBS buffer at 4 °C overnight, and purified by an equilibrium CsCl gradient centrifugation at 35,000 rpm for 24 h at 10 °C in a Beckman SW55Ti rotor. The gradient was fractionated into 250 μl aliquots, and each fraction was weighed to estimate the buoyant density and isopycnic point. Finally, each fraction was diluted with PBS and centrifuged for 2 h at 50,000 rpm in a Beckman TLA55 rotor to remove CsCl16.The virus–infected PLC/PRF/5 cells were harvested when CPE completely appeared and then centrifuged at 10,000 16.The viral proteins in each fraction were separated by SDS-PAGE using 5%–20% e-Pagel  and then stained with Coomassie blue. For Western blotting, the separated proteins were electrophoretically transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skim milk in 50 mM Tris-HCl (pH 7.4) containing 150 mM NaCl, and then incubated with 1:500 diluted monkey serum. Detection of the monkey IgG antibody was achieved using phosphatase-labeled goat anti-monkey IgG (H + L) (1:1000 dilution) . Nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate P-toluidine were used as coloring agents 23.The purified virus particles were placed on a carbon-coated grid for 45 sec, rinsed with distilled water, and stained with a 2% uranyl acetate solution. The grids were observed under a transmission electron microscope  at 80 kV24. The 3′-terminus nucleotide sequence was further confirmed by RT-PCR. The reverse transcription was performed by using Superscript™ II RNase H− reverse transcriptase  and oligonucleotide (dT) primer TX30SXN (5′-GACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3′)25. The 3′-terminal sequence was amplified by PCR using the primers, camF7879 (5′-ATGGACACGTAGCGCTGCTA-3′) and TX30SXN. The nucleotide sequencing was carried out with the primer camF7879 using an ABI 3130 Genetic Analyzer automated sequencer .The RNA was extracted from the purified virus particles by using a MagNA Pure LC system with a MagNA Pure LC Total Nucleic Acid isolation kit  according to the manufacturer’s recommendations. The nucleotide sequences of the entire genomes were determined by NGS as described previously26. Briefly, 96-well polystyrene microplates  were coated with the purified empty particles of Cam13  and incubated overnight at 4 °C. The plates were washed twice with 10 mM PBS containing 0.05% Tween 20 (PBS-T), blocked with 200 µl of 5% skimmed milk  dissolved in PBS-T for 1 h at 37 °C, and washed three times as described above. Next, 100 µl of the diluted serum samples (1:200 dilutions) was added and incubated at 37 °C for 1 h.In reference to the detection of the anti-HEV IgG antibodies, we established an ELISA for the detection of anti-SSV IgG antibodies by using SSV particles as the antigen2O2 was added to each well. The plates were incubated in a dark room at room temperature for 30 min, and then 50 µl of 2 M H2SO4 was added to each well. Optical density (OD) values were measured at 492 nm.After three consecutive washes, each well was supplemented with 100 µl of horseradish peroxidase (HRP)-conjugated goat anti-monkey IgG-heavy and light chain antibody  . The plates were incubated at 37 °C for 1 h and then were washed four times with PBS-T. The substrate orthophenylenediamine (100 µl)  supplemented with HThe nucleotide (nt) and amino acid (aa) sequences were aligned in the ClustalW software followed by phylogenetic analysis via the maximum-likelihood method in the MEGA 6 software. The phylogenetic trees were evaluated by bootstrap analysis with 1000 replicates.20. However, when we used the sterile-filtered 10% stool suspensions to inoculate PLC/PRF/5 cells, an unexpected CPE was observed on day 7 post-inoculation (p.i.) in the cells that received the suspension from 1 of 10 monkeys imported from Cambodia (C13), although no HEV RNA was detected in the supernatant. We thus collected the cell culture supernatant and used it again to inoculate PLC/PRF/5 cells. The CPE was clearly observed at day 2 p.i., and again no HEV RNA was detected in the supernatant, indicating that the CPE was unlikely to be caused by HEV infection.Monkey fecal specimens were originally used to isolate HEV, as described previously3, respectively , we harvested the infected PLC/PRF/5 cells at day 4 p.i.; the supernatant was concentrated, and the pelletized agent(s) was subjected to equilibrium CsCl density gradient ultracentrifugation. The gradient was separated into 20 fractions and each fraction was examined by SDS-PAGE. The protein bands were primarily distributed in fractions 6 and 11–14 with densities of 1.348 and 1.295 g/cmely Fig. .Figure 1In fraction 6, at least three protein bands were observed with molecular masses of ~34 kDa, ~26 kDa and ~7 kDa, whereas two protein bands with molecular masses of ~34 kDa, and ~26 kDa were observed in fractions 11–14 Fig. . By westFor our analysis of the entire genome sequences of the CPE agent (s), the RNA was extracted from the purified particles in fraction 6 and subjected to NGS analyses. The sequence analyses revealed that the genomes consisted of 8,155 nucleotides (nt) and a poly (A) tail (GenBank accession no. LC503602). The 5′-terminal untranslated region (5′UTR) and the 3′UTR contained 773 and 95 nucleotides, respectively, and the strains contained a single large ORF with 7,287 nt encoding 2,428 aa. The predicted protease cleavage sites Q/G were found in the aa positions 393/394, 630/631, 927/928, 1,218/1,219, 1,330/1,331, 1,663/1,664, 1,757/1,758, 1,779/1,780 and 1,962/1,963 of the polyprotein 28 and SV2 strain (AY064708)11, respectively. In contrast, Cam13 shared 76.58%, 78.12%, 79.21% and 79.52 of the nucleotide sequence identity with VRDL1, SV2, JX627573 and JX627574, respectively. In addition, the VP1 of Cam13 shared 60.00%, 70.25%, 76.11% and 76.83% of the nucleotide sequence identity with that of VRDL1, SV2, JX627573 and JX627574, respectively . As shown in Table In our previous research we confirmed that PSV is capable of infecting not only PLC/PRF/5 cells but also PK-15, Vero E6, and PGMKC cellsWe used purified Cam13 particles from fraction 6 Fig. . When 50Since the OD values of the serum samples of the cynomolgus monkeys from Tsukuba, Cambodia, and China ranged from 1.098 to 3.134, 2.610 to 3.537, and 1.504 to 3.564, respectively Fig. , all of We examined the antigenic cross-reactivity between SSV and PSV by performing the ELISA. We used the purified virus particles of SSV (Cam13) and PSV Jpsv1315) to detect the IgG antibodies. Six anti-SSV IgG-positive serum samples from cynomolgus monkeys and six anti-PSV IgG-positive serum samples from swine were used. As shown in Fig.  to detecTo data, several nucleotide sequences including a partial or entire SSV genome have been analyzed, but the epidemiology, biology, and pathology of this virus has remained unclear. In this study we isolated a novel SSV strain, Cam13, from fecal specimens of a cynomolgus monkey by cell culture with PLC/PRF/5 cells and we analyzed physicochemical and immunological characteristics of this virus.Based on the results of sequence analyses, four capsid proteins  were assumed, but only three protein bands were observed by SDS-PAGE from purified particles Fig. . This isThe preparation of the purified Cam13 allowed us to establish an ELISA for the detection of anti-SSV IgG antibody in Japanese monkeys and cynomolgus monkeys from China, Cambodia, Malaysia and the Philippines. The sero-prevalence of SSV was as high as 88.4% (289/327) in the cynomolgus monkeys and 18% (11/61) in Japanese monkeys. These results suggested that (1) all of the monkeys from monkey farms or monkey facilities were exposed to SSV, and (2) SSV infection is common in cynomolgus and Japanese monkeys. In addition, the positive rates of anti-SSV IgG in the wild monkeys were 79.3% (Philippines) and 67.5% , which is significant lower than the rate in the monkeys from monkey farm or facility in Cambodia, China and Tsukuba, Japan. These results indicated that SSV infection in monkeys seems to spread more easily among farms than in wild environment.Cam13 was isolated from a healthy cynomolgus monkey, and although most of the monkeys described herein have been exposed to SSV, none of the monkeys showed significant clinical signs, suggesting that SSV infection does not cause any serious diseases in monkeys.As the receptor molecule(s) of SSV is unknown, we compared the susceptibilities of several cell lines to sapeloviruses to obtain clues. We observed that PCMKC, Hep2C, Huh-7.5.1, PK-15, Vero E6, PGMKC, and RD-A cells showed different susceptibilities to PSV and SSV, suggesting that these viruses use different receptors for infection.