Premedication in upper gastrointestinal endoscopy for higher lesions detection rate has not been well studied so far. This study aimed to confirm whether premedication could improve the detection rate of early cancer or precancerous lesions and mucosal visibility.From July 2015 to December 2015, 7200 participants from 6 centers were screened by endoscopy with one of the 4 following premedications randomly: (1) water (group D); (2) pronase (group A); (3) simethicone (group B); (4) pronase and simethicone (group C). Early cancer and precancerous lesions detection rates were taken as the primary endpoints, and mucosal visibility was taken as the secondary endpoint. They were compared among four groups to determine different premedication effects in terms of different anatomical sites. Trial was registered at Chinese Clinical Trial Registry; the registration number is ChiCTR-IOR-17010985.p = 0.138 and 0.878). However, the visibility score distributions between control group (D) and premedication groups  were all statistically significant, with all anatomical sites p values < 0.001. Subgroup analyses, from 2 centers without screening before, also showed significant difference in esophageal  and overall  precancerous lesion detection rate.The upper gastrointestinal overall precancerous lesion detection rates among four groups were 8.7, 8.4, 10.0, and 10.3%, the overall early cancer detection rates were 1.3, 1.4%, 1.5, and 1.6%, both without significant difference (Premedication with pronase and simethicone may not increase lesion detection rates but could significantly increase the upper gastrointestinal mucosal visibility. Favorable esophageal and gastric mucosal visibility during upper gastrointestinal (UGI) endoscopy is essential for high-quality examination and minute malignant lesions identification. Mucus and foam can reduce the mucosal visibility and may increase the frequency of missing clinically relevant lesions. Therefore, premedication that reduces mucus and foam may be an effective strategy to improve the visibility of minimal lesions, such as early cancer or precancerous lesions, and could increase detection rates.Streptomyces griseus, which was already used as a mucolytic agent to improve visualization of the mucosa by its mucolytic effect [In many endoscopy centers, simethicone has been reported as an effective defoaming agent –3. Pronac effect –8. HowevFrom July 2015 to December 2015, a consecutive series of participants from the following six high UGI cancer incidence centers: Feicheng (Center I), Dongping (Center II), Linqu (Center III), Hebi (Center IV), Anqing (Center V), and Yanting (Center VI), who attended the Rural Cancer Early Detection & Treatment Program (RCEDTP) in China for UGI endoscopy screening, were enrolled in the randomized controlled trial. Inclusion and exclusion criteria were consistent with criteria from RCEDPT. Inclusion criteria were as follows: (1) aged between 40 and 69 years; (2) volunteered in the trial, and signed the informed consent. Exclusion criteria were as follows: (1) complete pyloric obstruction; (2) active gastrointestinal bleeding; (3) history of upper gastrointestinal surgery; (4) severe heart, liver, and kidney disease and unfit for endoscopy; (5) pregnant and lactating women; (6) has participated in other clinical studies within a month; (7) psychosis and severe neurosis; (8) allergic to drugs taken during the test.Informed consent was signed by all participants prior to enrollment. This clinical trial has been permitted by the ethics committee in Cancer Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College . The clinical trial is registered at Chinese Clinical Trial Registry, and the registration number is ChiCTR-IOR-17010985.We calculated different sample sizes according to the lesion detection rates of the esophagus, cardia, and stomach, respectively. Final sample size for the study was determined based on the gastric lesion detection rate, which is reported to be the lowest among the three anatomical sites. 0.85 and 0.05 were taken as the power of the test and α in our study, respectively. Since multiple comparisons among four groups were needed in our study, we adjusted α (0.05/6) when calculating the sample size. Because of low detection rate of gastric lesion in the population, we considered at least 1.5% increase of it as clinically relevant difference, between Group C and Group D. Based on these parameters, we got the total sample size of 7200 cases.3 at 40 °C (group A); (2) 100 mL of warm water containing 80 mg of simethicone at 40 °C (group B); (3) 100 mL of warm water containing 20,000 units of pronase, 80 mg of simethicone, and 1 g of NaHCO3 at 40 °C (group C); and (4) 100 mL of warm water (group D) at 40 °C. A total of 7200 random numbers were generated by SAS 9.2 statistical software for each participant. The participant ID number, the corresponding random number, and the corresponding group information were written on the same card, with the latter two in a sealed cover. After cards were manufactured, they were delivered to the study centers. All participants signed the informed consent of clinical trial before getting a card. The hidden information on the card was revealed by a designated person who did not enter the examination room and was not involved in the endoscopic procedures. Then, participants were administered drugs of the corresponding group. Both the study participants and the endoscopists remained blinded to the premedication drugs during the study.Based on the available participants of each center, 3200 samples were allocated to Center I, and 800 for each of the other centers Fig. . ParticiThe premedication was administered orally approximately 20 min before endoscopy. After premedication, the participants were given intravenous fluids, and guided to the examination room where the UGI endoscopy was performed. Participants underwent routine UGI endoscopic examinations using GIF-H260 video endoscope . All procedures were video recorded. After the removal of excess gastric solution, the visibility scores were evaluated separately at esophagus, cardia, fundus, gastric body, and antrum by the endoscopists. The visibility scores ranged from 1 to 3  Drug allergy was the major adverse events that happened in this study. Complications include bleeding, perforation, and aspiration pneumonia. Lesions include early cancer and precancerous lesions from esophageal, cardia, and stomach. Cardia area refers to the upper and lower range of 5 cm, respectively, of esophagogastric junction (EGJ) according to Siewert’s criteria. “Early cancer” means lesions with cancer cells detected pathologically, and endoscopic features meet the criterion of superficial lesions of digestive tract cancer according to Paris classification. “Precancerous lesions” were defined as lesions with low-grade intraepithelial neoplasia or high-grade intraepithelial neoplasia.UGI endoscopy was performed by four experienced endoscopists in Center I and two in each of the other centers. All endoscopic procedures were done under sedation, with intravenous anesthesia given by professional anesthetists.To minimize interobserver variation, frequent study meetings were held. Endoscopists and supervisors were provided with standardized instructions for standard operation and evaluation. All endoscopists and supervisors were required to have experience of performing more than 3000 and 5000 UGI endoscopic procedures, respectively. The supervisors reviewed 30% of the test video records randomly, and the data were considered reliable only when the error proportion fell within 5%.3) was added to neutralize the acidity of the gastric juice in Group A and Group C, the temperature of the premedication was maintained at 40 °C, and participants were instructed to took premedication 20 min before UGI endoscopy. Meanwhile, pure water was used to flush if clearing mucus and foam was needed during the examination. This could avoid the interference of the intraoperative flushing on the judgement of the visibility score.The maximal activity environment of pronase is pH 6–8 , temperaχ2 test. After confirming no significant difference in baseline information among different groups, χ2 tests were used to compare primary endpoints, and Kruskal–Wallis rank-sum tests were used for secondary endpoints comparison. For the potential reason of low baseline detection rates resulted from some centers’ secondary endoscopy surveillance leading to the negative results, the data from Center III and Center IV were merged for further subgroup analysis, and the aforementioned statistical analysis procedures were repeated.Per-protocol analysis was done with 7143 participants included in the analyzed dataset  and precancerous lesions  among the four groups also were not significantly different.The detection rate of early cancer and precancerous lesions of esophagus, cardia, and stomach had no significant difference among the four groups, respectively Table . CombiniThe visibility score distributions of esophagus, cardia, fundus, gastric body, and antrum were all significantly different among the four groups Table . To confp = 0.004) and overall  precancerous lesions detection rate.The data of Center III and Center IV, which participated RCEDTP for the first time, were analyzed together Table . ResultsPronase is a mucolytic agent used for digesting esophageal mucus, and simethicone is a silicone-based non-absorbable material that causes bursting of gas bubbles by reducing surface tension. Therefore, both the drugs can improve the mucosal visibility of UGI endoscopy , 10, 11.Our study validated the mucosal visibility effect of UGI endoscopy premedication again with a larger sample in multi-centers. The mucosal visibility of the pronase, simethicone, and combination group was better than that of the control group Table . In addiIt was expected that the lesion detection rate would increase after improvement in the UGI endoscopic visibility through premedication. However, for esophagus, cardia, stomach, and overall lesion detection rates, there was no significant difference among the four groups.This multicenter trial was based on the RCEDTP in China. The periodic interval of this program is 5 years, since the consensus is that 5-year periodic interval is sufficient for the occurrence and development of a disease. A previously screened area would undergo endoscopy surveillance every 5 years. Hence, the following endoscopy detections would cover many participants who previously had the primary screening. After the primary screening discovered early cancer or precancerous lesions of UGI, they were recommended to be treated timely. Although 5-year periodic interval was sufficiently long for the new lesions to occur, the baseline lesion detection rate in the secondary surveillance rural centers was significantly low compared to the primary screening. As the sample size for the study was determined based on the primary screening detection rate, inclusion of the participants who underwent secondary surveillance study could have resulted in lower baseline lesion detection rates seen in our study. Among all the centers, rural areas of Center I included in the study were secondary surveillance areas, so the baseline lesion detection rate of People’s Hospital in Center I was lower compared with the previous data. Meanwhile, the sample number People’s Hospital in Center I was 3200, accounting for nearly half of the entire sample. As a result, the relatively low lesion detection rate strongly affected the overall baseline detection rate of the trial, and finally leading to non-significant difference. Actually, except Center III and Center IV, which involved RCEDTP for the first time, the other centers more or less contained secondary surveillance participants.When designing the trail, 1.5% increase of gastric lesions detection rate was selected as the meaningful clinical difference. The determination of this parameter was based on the primary screening detection rate. However, in this study neither the detection rates of early cancer nor the precancerous lesions were significantly improved. In addition to the inclusion of secondary surveillance participants in those centers, which lead to lower incidence of the UGI lesions, the overestimate clinical meaningful difference cut-off of 1.5% may be another potential reason. In other words, baseline detection rate determines improvement potential. Translating this into clinical practice, for experienced endoscopists, lesions could be found through proper water flushing despite no premedication. In fact, their baseline detection rate may be very close to peak, and the premedication effect may have limited effect for them, so this could be another potential reason for the negative results in our study.To exclude the impact of secondary endoscopy surveillance, the data of each center were analyzed separately. By analyzing the data of Center III and Center IV together, it was found that the esophagus and overall detection rate had statistical difference in precancerous lesions. Since cardia and stomach did not have detection rate difference among four groups, we think that the overall detection rate difference mainly resulted from esophagus. Even though it was just a subgroup analysis, we want to show its significance here. Firstly, comparing esophagus precancerous lesion detection rate in group D Tables , 4, the The European Society of Gastrointestinal Endoscopy (ESGE) guidelines have recommended procedure time for standard endoscopy,  and our Our study has some limitations. First, because of the unbalanced sample distribution among six centers and unexpected low baseline detection rates, the negative result of Center I highly affected and even determined the total result. Second, endoscopies were performed by selected highly skilled endoscopists, the results may not be generalizable to community endoscopists. For experienced endoscopists, the UGI lesions detection rates did not increase obviously with premedication of pronase and simethicone. It needs further research whether this conclusion could be expanded to all endoscopists. Third, despite having specialized staff supervising the study, it was hard to ensure that the premedication was given at 20 min before the study, which might have affected their activity and effect. Fourth, according to the CONSORT guidelines, a written card with a covered part is not considered a correct allocation concealment, so our allocation concealment may have had some limitations.In conclusion, this was the first large sample multicenter randomized controlled double-blind study to show the influence of different premedication methods before UGI endoscopy on the UGI lesion detection rate and visibility. The combination agents group had a better mucosal visibility compared with single agent or control group. Besides, premedication with simethicone can better improve the visibility than pronase. However, improved mucosal visibility may not obviously increase lesion detection rates for experienced endoscopists. Whether it makes sense or not for inexperienced endoscopists in higher detection rate areas needs further study.


All dogs had cutaneous lesions and were azotemic with oliguria or anuria. All dogs underwent at least one PEX cycle; one dog had a single cycle PEX, three dogs two cycles PEX, and two dogs had one cycle PEX and one cycle of prolonged intermittent renal replacement treatment. Complications seen during PEX therapy included hypothermia (n = 4), tachycardia (n = 2), hypotension (n = 2), and hypocalcemia (n = 6). Two dogs survived to discharge, the remaining four dogs were euthanized. The positive outcome in two dogs treated with PEX despite the reported high mortality rate once acute kidney injury with oliguria/anuria occurs does not confirm success of this treatment. However, survival in two dogs that were initially oligoanuric highlights that further consideration and evaluation of PEX for this patient group is warranted for this specific disease. Additional studies are urgently needed to identify the underlying etiology of CRGV before more targeted therapies can be developed. Based on our findings, further evaluation of the role of PEX in this specific disease are warranted.Cutaneous and renal glomerular vasculopathy (CRGV) is a rare disease affecting dogs, with a recent apparent increase in prevalence since 2012 in the UK. This disease is characterized by a vasculopathy affecting small vessels of the kidney and skin, leading to thrombotic microangiopathy. The underlying etiology remains unknown although clinicopathological and histological findings resemble features of certain forms of thrombotic microangiopathy in people, for which plasma exchange (PEX) is considered an important component of therapy. The objective of the present study is to describe the use of PEX as adjunctive treatment in dogs diagnosed with CRGV. A retrospective review of dogs diagnosed with CRGV between 2014 and 2016 treated with PEX was performed. Clinical records were reviewed and data relating to signalment, diagnostic tests and management strategies were summarized. Information and complications relating to PEX were recorded. Six dogs were diagnosed with CRGV ( Thrombotic microangiopathy (TMA) syndromes have been described in people and in dogs with the hallmark of these conditions being endothelial damage and subsequent vascular occlusion leading to anemia, thrombocytopenia and organ dysfunction . ThromboA recent retrospective study in dogs in the UK described both the clinical and histopathological findings associated with this condition in detail; patients typically presented with ulcerative skin lesions affecting the distal limbs, variable degrees of anemia, thrombocytopenia, and developed acute kidney injury (AKI) and kidney failure within 7–10 days .E. coli Shiga toxin), atypical HUS , secondary HUS  and idiopathic HUS  and Hemolytic Uremic Syndrome (HUS). Both TTP and HUS can manifest in acquired and congenital forms . Hemolytthic HUS , 6, 7 althic HUS .In the pathogenesis of TMA syndromes, the presence of specific circulating blood constituents is common, offering potential targets for therapy. Therapeutic plasma exchange (PEX), also known as plasmapheresis or total plasma exchange, consists of the removal of the patient's plasma (and its constituent components) via centrifugation or filtration through an extracorporeal circuit and its replacement with allogenic plasma transfusion, human serum albumin, isotonic crystalloids or synthetic colloids .Therefore, the goal of PEX in this context is to remove molecules responsible for the pathogenesis of the microangiopathy, such as autoantibody-inhibited ADAMTS13 in TTP, or complement regulators , in aHUS . Plasma People with HUS often have a more severe degree of kidney injury compared to TTP, therefore PEX is recommended alongside early treatment with renal replacement therapy in the attempt to halt progression to irreversible kidney injury .The clinical use of PEX in dogs has been previously described in several conditions such as systemic lupus erythematosus , hyperviDogs presented to the Queen Mother Hospital for Small Animals (QMHA) between January 2014 and December 2016 and diagnosed with CRGV were included in the study. Eligible dogs were identified by searching the hospital medical database using the terms “CRGV” and “plasmapheresis.” Inclusion criteria were dogs clinically diagnosed with CRGV receiving at least one cycle of PEX. A diagnosis of CRGV was made on the basis of renal histopathology if post-mortem examination had been performed or on the basis of compatible historical data, clinical signs including ulcerative skin lesions, hematological and biochemical findings and dermal histopathology compatible with the description provided by Holm et al. 2015 , serum biochemistry using iLab 600® , activated partial thromboplastin time, prothrombin time using coagulation profile, ST Hemostasis Analyzer  or IDEXX Coag Dx™ , urinalysis , sediment analysis and culture), imaging , infectious disease including detection for Leptospirosis antibodies by using microscopic agglutination test, , Leptospirosis polymerase chain reaction test to detect Leptospira spp.  and test for tick-borne diseases by using SNAP® 4Dx® . Histopathological data from biopsies of skin lesions or post-mortem examination were reviewed. Clinical records were evaluated for information relating to the PEX therapy including the specific dialysis catheter used, type of anticoagulant provided, replacement fluids, each patient's PEX prescription, number of cycles, duration of cycles, requirements of additional transfusion of blood products, complications and outcome.Descriptive statistics are provided and where indicated, results are reported as median (range).Six dogs met the inclusion criteria during the study period. There was one dog diagnosed with CRGV during the study period which did not undergo PEX and was thus excluded. Two dogs were female entire and 4 dogs were female neutered. The median age was 4 years (10 months to 8 years) and median body weight was 21.8 kg (range 11.2–27). Breeds represented included 2 Labradors Retrievers, 2 mixed breed dogs, 1 Cocker Spaniel and 1 Dalmatian.n = 3, case 2, 4, 6), inappetence , hemorrhagic diarrhea  and vomiting . All dogs had been hospitalized for a median of 3 days (range 1–7) at the referring practice prior to admission to our center.All dogs were reported to have developed cutaneous lesions. The length between observation of the cutaneous lesions and admission to the primary veterinary surgeon was 6 days (range 1–9); 5/6 dogs were reported to have progressive lameness and a stiff gait. Other presenting complaints included lethargy  and thoracic limbs ], ventrum , and on the head with both cutaneous and oral lesions . All dogs were normothermic at time of admission .At presentation, the abnormalities on physical examination and initial assessment included cutaneous lesions, which were mainly identified at the extremities  at time of presentation. These dogs were normotensive with evidence of volume overload. In total 5/6 dogs had evidence of volume overload. Volume overload was identified as weight gain in comparison to the weight reported in the history when healthy (n = 4), Grade IV (n = 1), Grade V (n = 1) during the first 24–48 h of hospitalization. During the time of hospitalization, the median peak concentration of creatinine was 693.5 μmol/L (range 509–1199), which corresponded to progression to IRIS AKI grade IV (n = 5) and V (n = 1). The lowest concentration of albumin measured during hospitalization was 19.85 g/L (range 10–22.7).Clinicopathological findings for all dogs are summarized in Tables 9/L (range 15–80 × 109/L) (RI 150–190 × 109/L); see Table Hematology revealed a non-regenerative anemia in 4 dogs  and thrombocytopenia in all dogs [median estimated manual platelet count was 75 × 10n = 6) showed a median urine specific gravity of 1.015 [range 1.009–1.021 (RI 1.018–1.050)], with varying degrees of proteinuria . Granular casts were documented in 3/6 dogs . Four of six dogs  had an evaluation of UPC ratios performed, with a median of 2.95 [range 0.49–11.16 (RI < 0.5)]; of these only one dog had an inactive urine sediment analysis with low cellularity. Urine culture was performed in 4/6 dogs. The collection of urine via cystocentesis for bacterial culture was deemed contraindicated in two dogs due to severe thrombocytopenia. A urinary tract infection was diagnosed in 1 dog (case 1); this was suspected to be hospital-acquired following urinary catheterization and the following organisms were cultured: Escherichia coli, Enterococcus spp., and Staphylococcus aureus. This dog, at time of presentation, had a negative urine culture prior to urinary catheterization. When the diagnosis of a urinary tract infection was made following repeat urine analysis the antibiotic therapy was selected based on culture and sensitivity test results.Urine analysis  tested by using SNAP 4DX. Leptospirosis testing was performed in all six dogs via MAT. Two dogs were also tested using urine PCR (case 2 and 4). Four dogs  had Leptospirosis titers between 1:100 and 1:200, which are not suggestive of active infection and in the remaining two dogs all titers were negative (case 4 and 6) (Testing for 4 and 6) ; rising Escherichia coli, Staphylococcus pseudointermedius, Streptococcus canis, and Pseudomonas aeruginosa.Histopathological evaluation of skin biopsies in all dogs revealed necroulcerative dermatitis with vasculitis and thrombosis. The main findings included extensive dermal ulceration with necrotizing folliculitis and perifolliculitis, and adnexal necrosis with occasional vasculitis. The lesions were largely centered to the hair follicles and adnexa. Two dogs (case 1 and 2) had a skin culture taken which revealed a growth of Assessment for the presence of peritoneal effusion was performed by point-of-care ultrasound and peritoneal effusion was present in 4/6 dogs  which included presence of fluids in 1–2 out of 4 quadrants of the abdomen. A complete abdominal ultrasound was performed in all dogs. In 4/6 dogs  an enlarged and edematous pancreas with mixed echogenicity was detected. One of these dogs (case 5) also had gallbladder wall edema reported. One dog (case 6) had a thickened gastric wall with loss of layering, likely due to inflammation. One dog (case 6) had changes to the descending colon, consistent with ongoing diarrhea and possible colonic ulceration, with a maintained intestinal wall layering and normal thickness. One dog (case 5) had a 3.5 mm cortical nephrolith detected in the right kidney, otherwise no ultrasonographic changes were reported in the kidneys of other dogs.Thoracic radiography was performed in all dogs following placement of the dialysis catheter, with no abnormalities reported on intrathoracic structures. There was only 1 dog (case 5) that had a scant pleural effusion present at time of arrival on point-of-care ultrasound of thorax. Three dogs had echocardiographic assessment by a board-certified cardiologist : one dog (case 5) had signs of aortic insufficiency and one dog (case 2) had a mild depression of systolic function and dilatation of the center ventricle.n = 5 case 1, 2, 3, 4, 6), doxycycline (Ronaxan®) , marbofloxacin (Marbocare®) , generic ampicillin (Ampicillin®) , generic cefuroxime  and generic cephalexin (Rilexine™) . All dogs also received antiemetic therapy: maropitant (Cerenia®) (n = 6), metoclopramide (Emeprid®)  and generic ondansetron (Ondansetron®) . All six dogs received a proton pump inhibitor (Omeprazole®). Further medical management consisted of amlodipine (Amodip®)  and pentoxifylline  (n = 4). Pentoxifylline was administered as a potential adjunctive treatment for the presumed presence of vasculitis. One dog (case 4) required anti-seizure medication with diazepam (Diazemuls®) and levetiracetam (Keppra®). Analgesia was required in 5/6 dogs: methadone (Synthadon®)  and buprenorphine (Buprecare®) .Medical management from time of admission to the hospital prior to PEX therapy varied. Only one of the six dogs (case 6) received intravenous fluid therapy (Aquapharm 11®), at time of admission due the presence of clinical dehydration. All dogs received antimicrobials in the event the pathogenesis was related to an infectious cause. The following antimicrobials were administered: potentiated amoxicillin (Augmentin®) (Within the first 24 h of monitoring urine output (UOP), four dogs  received furosemide (Dimazon®) either as a bolus or constant rate infusion in the attempt to improve urine production. Urinary output was quantified using an indwelling urinary catheter and a closed collection system in 4/6 dogs  and estimated through monitoring of body weight and assessment of fluid balance by assessing presence of cutaneous edema and cavitary effusions in all dogs. Prior to initiation of diuretic therapy, UOP measured in 4 dogs was 0, 0.25, 0.3, and 0.5 mL/kg/h. After initiation of diuretics the UOP increased to 1.55, 2.81, 2.87, and 3.64 mL/kg/h. This was the UOP measured during hospitalization at the time of PEX therapy. The two dogs without initial urinary catheterization were oliguric with minimal urination prior to first PEX cycle, and both dogs had a urinary catheter placed at the time of PEX. One of these dogs (case 6) was clinically dehydrated and received fluid therapy which resulted in polyuria with a median UOP 7.4 mL/kg/h (range 5.5–17.9 mL/kg/h) on day 2 of hospitalization. Another dog (case 2) became polyuric over a short period of time after initiation of diuretics.The decision to perform plasma exchange was made within 24–48 h of hospitalization due to lack of clinical improvement, worsening azotemia and persistent oliguria or anuria despite advanced medical management as described.Three dogs case 1, 3, 4) underwent one PEX cycle and two of these dogs (case 3 and 4) also required a cycle of prolonged intermittent renal replacement treatment (PIRRT) with veno-venous hemodiafiltration to treat the severe azotemia and hyperkalemia the following day. The duration of PIRRT treatment was respectively 5 and 6.5 h, which resulted in a urea reduction ratio (URR) of 45.6 and 56%, respectively. The three other dogs case 2, 5, 6) underwent two cycles of PEX within 48–72 h. Only case 1 received a single cycle of PEX, the remaining 4 dogs received two extracorporeal therapy cycles each  in the jugular vein. The chosen size of the jugular catheter was based on the size of each dog, ranging from 11.5–13.5 Fr to 19–24 cm. Four dogs had the catheter placed in the right jugular vein and two dogs in the left jugular vein. The procedure was performed under general anesthesia using surgical aseptic conditions with modified Seldinger technique used. Anticoagulation for the PEX cycles was achieved with regional anticoagulation, infusing Citrate ACD-A (Haemonetics®) through the pre-blood pump in all 6 dogs.Our citrate protocol consisted of administering Citrate ACD-A at a rate of 5.65 mmol citrate per liter of circulating blood for 30 min. Following this we aimed to reduce the citrate infusion to 1.69–2.26 mmol/L of blood. The citrate infusion was titrated to achieve an extracorporeal plasma ionized calcium concentration of < 0.35 mmol/L. Calcium gluconate  was administered diluted to 2.4% with NaCl 0.9% (Aquapharm 1®) at a starting rate (ml/h) of 0.4 times the citrate infusion (ml/h) and subsequently titrated to achieve a target plasma ionized calcium in the patient between 1.1 and 1.3 mmol/L .The median Plasma Volume (PV) exchanged with PEX was 1.8 range 1–3.3) with a median volume of 1426 mL . The PV was calculated by the following equation; PV = (BW(kg) × 0.09) × (1-hematocrit) . PEX was performed over a median time of 3 h (range 2.5–4) ). However, only one patient (case 6) developed clinical signs attributable to hypocalcemia, with facial twitching which resolved with an additional 1 mL/kg IV bolus of 10 % calcium gluconate. Hypothermia (<37°C) was identified in 4 dogs despite the use of active heating with forced air heating blankets from the outset , and the use of a blood warmer on the extracorporeal circuit return line immediately after connection. Sinus tachycardia  was observed in 2 patients (case 3 and 6).Two dogs (case 3 and 6) developed severe hypotension (Doppler systolic pressure 60 mmHg), one during a cycle of PEX and the other during a cycle of PIRRT. Case 6 developed hypotension during her first PEX cycle due to hypovolemia from bleeding from venipuncture and the catheter insertion sites. This patient was severely thrombocytopenic. The hypotension did not respond to fluid resuscitation [45 mL/kg bolus of Hartmann's Solution (Aquapharm 11®)] and required vasopressor therapy with a noradrenaline (Norepinephrine®) constant rate infusion. Later in the same PEX cycle, this patient developed neurological signs (obtundation and nystagmus) at the end of treatment cycle requiring interruption of PEX and the return of the extracorporeal blood to the patient. Due to ongoing bleeding this patient was further stabilized with a transfusion of packed red blood cells (pRBC).The second dog (case 3) developed hypotension during her PIRRT cycle which did not respond to fluid resuscitation, also requiring noradrenaline constant rate infusion. This patient later developed neurological signs (mitotic pupils and loss of muscle tone) which was stabilized with a bolus of hypertonic saline (Braun Vetcare hypertonic NaCl solution 7.5%®) and interruption of PIRRT. Following the return of extracorporeal blood to the patient and hypertonic saline administration, this patient regained consciousness and became normotensive. Median blood pressure in all dogs during PEX was 140 mmHg (range 60–220). Two dogs (case 4 and 6) required additional blood transfusions with pRBC. The PEX therapy was performed the day following admission at our center in 4/6 dogs  and on day 2 of hospitalization in 2/6 dogs.Median length of hospitalization until either discharge from hospital or time of death was 5.5 days (range 4–23 days). Four dogs  were euthanized at the owners' request due to lack of clinical improvement, ongoing costs and grave prognosis. A post-mortem examination was performed in all non-surviving dogs and the results were characteristic of CRGV as described by Holm et al. , median time to euthanasia was 5 days (4–6 days). Case 3 continued with severe clinical deterioration despite repeat cycles with both PEX and PIRRT. Her clinical deterioration consisted of obtundation, worsening azotemia, marked pelvic limb vasculitis with the concern that she would not tolerate the required PIRRT cycles for management of her AKI. Therefore, the owner elected euthanasia. Case 4 was euthanized due to neurological deterioration with ongoing seizure activity; post-mortem examination showed multifocal neuronal degeneration and necrosis with hemorrhage and edema. Case 5 developed septic shock and hypoxemia; post-mortem examination revealed the presence of severe pneumonia and bacterial endocarditis. This patient had a sudden clinical deterioration following the second PEX cycle, which was performed 48 h after placement of the dialysis catheter. Echocardiography in this patient was performed prior to catheter placement and did not reveal any signs of endocarditis or systolic dysfunction. Case 6 was euthanized due to sudden development of respiratory failure requiring further life-support with mechanical ventilation; heart failure and volume overload were excluded based on physical examination, patient-side echocardiography and thoracic radiographs. The owners elected euthanasia and a cause for the respiratory distress was not identified despite post-mortem examination. There were no significant pathological changes on histological examination of the heart and lungs of this patient.Two dogs (case 1 and 2) survived to discharge from hospital. The survivors were hospitalized for 10 and 23 days, respectively. During hospitalization both dogs were hypertensive and anuric. Case 1 received 1 PEX cycle, and case 2 received 2 PEX cycles without major complications. One of the survivors (case 2) became polyuric after starting diuretic therapy. The patient remained polyuric throughout its single PEX cycle and was weaned off diuretics 19 h post PEX. At the time of discharge, case 1 had mild azotemia  and hyperphosphatemia . This was an improvement compared with pre-treatment values . The second survivor (case 2) also had mild azotemia at time of discharge  compared to a moderate azotemia while hospitalized . These dogs were still alive at the time of last follow up 3 years and 2 months (case 1) and 1 year and 9 months (case 2), respectively after discharge from hospital. By that time, the dogs were doing well clinically with no requirements for further medical management. The azotemia had completely resolved for case 1, whilst case 2 had a slightly increased urea (9.2 mmol/L) but a normal creatinine concentration (101 μmol/L). Both cases were normotensive and were considered as IRIS Chronic Kidney Disease stage 1 . During This study describes the use of PEX in 6 dogs clinically diagnosed with CRGV. Unfortunately, only 2/6 dogs survived to discharge. Overall, PEX was tolerated by dogs; however, commonly reported complications of apheresis therapy were identified. Current human recommendations for the treatment of TMA would be to perform daily cycles for three days, though this approach does not have strong evidence base . The decThrombotic microangiopathy in people can occur secondary to several causes, including TTP and HUS. A detailed review of the causes of TMA in people is beyond the scope of this study and readers are referred elsewhere , 6, 7. IE. Coli or drugs   . Managemibitors) . Atypicaibitors) , 26; theibitors) , 8. Antiibitors) , 27. Foribitors) .Therefore, the major goals of PEX in TTP are the removal of the antibodies against ADAMTS-13 and the supplementation of ADAMTS-13 via FFP, preventing further thrombi formation. Similarly, in aHUS PEX can remove the aberrant complement and provide replacement with normal complement factors . In peopE. coli or Shigella dysenteriae Shiga toxin or by Salmonella or Campylobacter infections, which can be identified by fecal culture   , 3, 5 alAll patients described in this study developed cutaneous lesions, with evidence of microangiopathic damage, thrombocytopenia and AKI with 4/6 dogs also developing anemia. Renal biopsy was available for confirmation of CRGV in 4/6 dogs. In the remaining 2 dogs clinicopathological features, combined with compatible dermal histopathology, strongly indicated that CRGV was the underlying disease process despite the lack of renal biopsies. Definitive ante-mortem confirmation of TMA in the 2 surviving dogs would have required renal biopsy, but the risk of potential bleeding in the face of thrombocytopenia was considered to outweigh the benefit of confirming the diagnosis.At the time of manuscript preparation, no treatment has been demonstrated to reduce the high mortality associated with this disease in dogs developing oliguric or anuric AKI . The casThe decision to perform PEX in the dogs included in this study was made when there was an apparent progression of their CRGV syndrome, with the aim of intervening early to achieve removal of suspected immune complexes that could be involved in the pathophysiological mechanism of CRGV. Nevertheless, the patients included in this study were already at an advanced stage in the disease process on the basis that they presented with azotemia and oligoanuria. The optimal time to intervene with therapies such as PEX has not been established. However, from clinical experience, intervention after the development of severe AKI appears to be associated with a guarded prognosis. This raises questions as to whether the most appropriate time to intervene with therapies such as PEX is at the time point when non-azotemic AKI is identified or UOP first begins to fall. However, lack of a definitive diagnostic test, other than renal biopsy, the intense and invasive nature of PEX therapy that is not without the potential for complications, makes this a challenging judgment call to make, particularly considering that based on clinical experience some of the less severely affected but clinically suspected cases may show improvement with standard management for AKI. In light of the poor prognosis associated with this condition, once AKI and oliguria develop, we considered PEX to be the best alternative to manage this disease as long-term dialytic therapy was not a clinically or financially viable option for these dogs currently in the UK. These cases were particularly severely affected. It is possible that patients with CRGV can present with a spectrum of clinical signs, some of which may be azotemic and managed in primary care practice recovering with supportive treatment and not reaching the level of severity of the disease of those cases that make it to referral practice.The role that PEX played in the positive outcome of 2 of the 6 cases is difficult to ascertain and represents a major limitation of this report. It is possible that these patients would have improved in terms of their clinical status with continued supportive therapy, particularly given that there was indication of improved UOP after furosemide administration in one of the surviving cases. As previously reported, a positive response in UOP following furosemide administration in patients with AKI may reflect less severe renal injury . NeverthAll of the 4 non-survivors  were euthanized at their owner's request within the first week of hospitalization and often requested the day following a PEX cycle. There were various reasons for the decision for euthanasia including clinical deterioration, grave prognosis and costs. All owners were aware of the timeframes expected for renal recovery, the need of repeat cycles as well as the financial investment these advanced therapeutic options require from the outset. Given the complexity of these cases, various factors may have influenced some of the clients to request humane euthanasia instead of continuing with management early in the course of the disease.Extracorporeal therapy requires specially trained staff, clinical expertise and special equipment. PEX is not a risk-free therapy, but studies in people have shown that there can be improved survival rates with the use of PEX in TMA patients  and thatThe complications associated with PEX observed in the current study are similar to those previously reported, such as hypotension and citrate-induced hypocalcemia –18. OthePrevious studies have reported that glomerular disease often leads to a UPC ratio >2.0 in dogs . Renal lTwo dogs (case 3 and 6) developed tachycardia during PEX therapy. Case 6, as described above, became hypotensive possibly secondary to hypovolemia which may explain the compensatory tachycardia. However, case 3, was normotensive throughout the PEX cycle and as such it seems unlikely that volume changes contributed to tachycardia. Case 3 did, however, become agitated and required additional sedation with opioid analgesia and benzodiazepine constant rate infusion which improved the tachycardia. As PEX therapy is an invasive procedure, this requires patients to be calm during the cycle to allow good blood flow and avoid catheter occlusion related issues.In total, three dogs  developed neurological abnormalities. Case number 3 and 6 developed neurological signs with abnormal mentation during PIRRT and PEX therapy respectively due to hypovolemia. Case 4 already had abnormal neurological signs prior to PEX with seizure activity at the time of presentation. It is difficult to ascertain if these were a consequence of the clinical progression of the disease; neurological post-mortem examination was performed in case 4 and documented evidence of CNS necrosis with hemorrhages.The current recommendation in human medicine regarding PEX therapy is to perform 1–1.5 PV exchange during a cycle . The extThe non-survivors that did not benefit from PEX may have presented at later stages of the disease where renal function was irreversibly compromised. In such patients, repeated cycles of PEX combined with hemodialysis or PIRRT may have been necessary in order to achieve a successful outcome. In people with TMA, and specifically TTP, repeated cycles are often necessary and recovery times often extend to weeks , 8, 33.However, 3 out of 4 non-survivors  were ultimately euthanized due to co-morbidities that occurred in addition to their renal injury; namely neurological deterioration, pneumonia/endocarditis and respiratory failure. It can be argued that the number of cycles of PEX performed in the non-surviving dogs was insufficient to allow appropriate renal recovery and/or that these patients should have been supported with renal replacement therapies e.g., hemodialysis in order to reduce the morbidity. However, this was not possible in the patients described in this study. In addition, in the cases reported, the decision to perform euthanasia was not limited to renal recovery.n = 30) received PEX as a therapeutic option but three dogs received PIRRT. Sadly, all 30 dogs died or were euthanized. However, Holm et al. (This study has some inherent limitations, such as the small number of dogs included and the lack of a control group. Holm et al.  retrospem et al.  also repFuture prospective randomized studies comparing dogs with CRGV treated with and without PEX and stratified by illness severity score would be needed to assess therapeutic efficacy. Additional studies evaluating for the presence of autoantibodies to ADAMTS13 in dogs with CRGV, as observed in people with TTP, would also be recommended to clarify the pathogenesis of this disease.Another limitation regarding PEX therapy in this study included the available fluids for replacement and the lack of guidelines indicating the optimal timing of plasma infusion initiation. Alternative fluids, such as synthetic colloids might have been beneficial for these cases although concerns regarding their safety is still unknown, especially in patients with AKI . NeverthIn this retrospective study, we have described the novel application of PEX as an adjunctive treatment in dogs with severe CRGV. Plasma exchange is an invasive procedure, requiring special equipment and specially-trained staff. However, the good outcome in the two survivors, in light of the high mortality reported with CRGV, warrants further consideration whether PEX could prove helpful in selected cases. Moreover, our finding should encourage further studies aimed at better understanding the pathogenesis of this condition, and further evaluation of the potential role of PEX in the treatment of affected dogs.This study was approved by the Clinical Research Ethical Review Board. URN: M2016 0086.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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. The reviewer AS and handling Editor declared their shared affiliation.


As a promising alternative to traditional prepreg, carbon fiber/poly(ether ether ketone) (CF/PEEK) hybrid composites have attracted wide public interest for their flexibility and conformability. However, modification methods focused on the hybrid premix have not been previously studied. In the present work, the interfacial strength of the hybrid composite was improved by treating the carbon and PEEK fibers together in a radiofrequency (RF) plasma containing one of the following gases to achieve surface activation: air, Ar, or Ar–air. After plasma treatment, the increased roughness of CF and the grafted chemical groups of CFs and PEEK fibers were propitious to the mechanical interlocking and interfacial strength. Significant interfacial shear strength (IFSS) enhancement was achieved after Ar 1 min, air 1 min plasma treatment. This study offers an alternative method for improving the interfacial properties of CF/PEEK composites by focusing on the boundary layer and modifying and controlling the fiber–matrix interface. With the increasing demand for high-performance composites for aerospace, automobile, and other structural applications in engineering, great efforts have been devoted to the study and development of fiber-reinforced thermoplastic composites ,2,3. AmoIn recent years, an increasing number of researchers have focused on the plasma modification technology, mainly for its advantages compared with other methods, such as high efficiency and surface limitation ,16. PlasPlasma treatment has been widely applied for CF modifications using high-energy particles, such as electrons and ions, to cause oxidation and candles on the surface of fibers. Hence, this method can achieve the following: remove the weak interfacial layer on CF surfaces; increase the polarity and roughness of the surface; enhance the chemical bonding and physical chimerism between fibers and resin matrices; and improve the interfacial strength of composites . HoweverIn our study, plasma treatment by air, Ar, or Ar–air was applied. The physicochemical properties of CFs and PEEK fibers before and after plasma treatment were investigated. Surface morphology changes were characterized by scanning electron microscopy (SEM). The chemical structures and specific properties of CFs were studied by Raman spectra and single-fiber tensile tests. Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry/thermogravimetric analysis (DSC/TGA) were carried out to study PEEK fibers and compare their structure and properties before and after plasma treatment. Element content and surface group composition were studied by X-ray photoelectron spectroscopy (XPS). The microdroplet test was used to assess the interfacial shear strength (IFSS). The results were then analyzed.2) with a mean diameter of 7.5 μm were supplied by Toray Industries Inc. . PEEK fibers (850D/144F), with an average diameter of 27 μm, was purchased from Changzhou Co–Win Novel Materials Co, Ltd. . The fibers were all cleaned using ethyl alcohol and acetone. They were then dried in a vacuum oven at 80 °C for 1 h, and the dust, grease, and chemical residues were removed without altering the sizing on fibers.The commercial polyacrylonitrile (PAN)-based carbon fiber tows  power supply , which is usually used to clean nanoscale organic pollutants on substrates or crystals. This equipment is composed of a vacuum chamber, a needle valve, a resistance vacuum gauge, a vacuum pump, and a radio source a. The plBy definition, plasma is a neutrally ionized gas on a spatial scale, containing an equal number of negative and positive ions, a small percentage of free radicals, a few parts of atoms, excited molecules and free electrons, and a large amount of extremely energetic vacuum ultraviolet light b. The baScanning electron microscope  measurements were used to observe surface topography and provide a general analysis for the material before and after modification.−1 to obtain the first- and second-order Raman bands for CF. The band intensity, band width, and band position were obtained from the first-order Raman spectra using a Gauss curve-fitting procedure.Raman spectroscopy is an effective experimental technique used to determine information on carbon fiber microstructure. Raman spectroscopy data were collected using a INVIA010410 Laser micro confocal Raman spectrometer  with a wavelength and laser beam size of 514.5 nm of argon laser (green) and ~2 µm diameter. A 500× magnification microscope was used to select a point of focus for the laser beam spot on the fiber’s surface. The scanning range was set at 500–4000 cmSingle-fiber tensile tests were performed on CFs treated with different plasma conditions using a Sintech universal testing machine following the ASTM D3379 standard test method. The test sample was obtained by cutting a section of 12 cm fiber, pulling out to get the monofilament, and then fixing it on the paper frame using glue and tensile force to keep it straight, as shown in −1 wavelength.The chemical structure of PEEK before and after plasma treatment was examined based on the Nicolet iS50 Fourier transform infrared spectra , using the KBr pellet method at 500–4000 cm−1 in a nitrogen atmosphere. The thermogravimetric analysis tests for PEEK fibers (~10–15 mg) were conducted in a nitrogen atmosphere from 25 °C to 800 °C at a heating rate of 10 °C min−1 to study changes in the PEEK fiber’s thermal stability induced by plasma treatment.Differential scanning calorimetry measurements were performed in order to study the effect of plasma treatment on the melting point of PEEK fibers using STA449F3 synchronous thermal analyzer . The fibrous PEEK, cut into small length sizes with ~10 mg weight, were thermally cycled between 25 °C and 400 °C twice at a constant heating/cooling rate of 10 °C minhv = 1486.6 eV) monochromated X-ray source at 15 kV and 150 W. The pass energy and energy step were 20.0 eV and 0.05 eV, respectively. A nonlinear least-square curve-fitting program  was used to deconvolve the XPS data. To compensate for the charging effects, all spectra were calibrated with graphitic carbon as the reference at a binding energy (BE) of 284.6 eV. The spectra were deconvolved by subtracting a Shirley + linear background, and a Lorentzian–Gaussian (GL = 20%) mixed function. The surface chemical composition was calculated from the peak areas of relevant spectra.XPS analyses were conducted using the ESCALAB 250Xi  to study the surface elements and groups of CFs and PEEK fibers after plasma treatment. XPS spectra were obtained using an Al Ka —to directly test the interfacial sheer strength (IFSS) between CF and PEEK. The process is illustrated in The surface morphology of CFs at different plasma treatment times are shown in Typical SEM images of the PEEK fibers before and after plasma treatment are shown in These data show that plasma treatment etches the surface of CFs and PEEK fibers and significantly changes the surface morphology. Although the etching depth is determined by time and instrument, the etching effect closely corresponds with the type of plasma gas. Prolonging plasma treatment time causes outstanding etching, resulting in a more complex surface morphology with increased surface roughness for both CFs and PEEK fibers . Certain2-bonded amorphous carbons possess strong Raman scatter despite their intense optical absorption. A Raman peak at ~1585 cm−1 is typical of bulk crystalline graphite, called the G band (ordered or graphitic). This peak represents the basic vibration mode of graphite crystal, and its intensity is closely related to the crystal size. The Raman peak at 1360 cm−1 originates from vibrations of the edge of graphite carbon and is called the D band, which represents disordered carbon. Thus, R-the ratio of D and G bands (D/IGI) is usually calculated and regarded as a measure of the size of graphite crystallites and the amount of amorphous carbon phase [R values were obtained from the ratio of curve areas at ~1360 cm−1 and 1585 cm−1, and the crystalline size aL was calculated using the following relationship:C = 44 Å.Graphitic carbon and other spon phase ,31,32. IR and aL of untreated and treated carbon fibers obtained from R and aL did not significantly change with different plasma treatments and times. Thus, it can be concluded that plasma treatment might not change the crystalline or graphitic structure of carbon fibers because plasma treatments can only influence surfaces on a nanometer range.The Raman spectra and its cumulative fit peak for CF before and after different plasma treatments are shown in 0), Gamma function (Γ(σ0)), slope B, and intercept A of the straight line, are listed in As the etching effect depends on the plasma treatment time and equipment, we focused on the tensile strength of CFs treated for different times. The Weibull plots and fitted straight lines for single-fiber tensile tests are presented in −1 for PEEK and different plasma-treated PEEK are shown in We used FTIR spectroscopy to investigate PEEK fibers before and after plasma treatment. The wide spectra from 4000 to 500 cmTg) and thermal stability. DSC and TG curves of PEEK fibers treated with plasma for different lengths of time are shown in Tg of PEEK at 343 °C did not change after different durations of plasma treatment. To study the effect of plasma treatments on the thermal properties of PEEK fibers, DSC/TGA was applied to test the samples’ glass transition temperature , sp3-hybridized carbon atoms (C–C), C–O, –C=O, and O–C=O, respectively [2 and sp3 carbon atoms were activated, damaged, and replaced with new oxygen on the surface during plasma treatment. Therefore, various oxidative reactions induced by plasma were predicted to occur. As reported, oxygen and argon plasma were able to create free radicals that coupled with active species in the plasma environment [The bonding energies corresponding to the groups that deconvolve from C1s of CFs and PEEK fibers and their concentrations are listed in ectively ,37. The ironment . Due to IFSS before and after plasma treatment was determined using the microdroplet test, and the destruction are shown in In this work, the physical and chemical structures of CFs and PEEK fibers before and after plasma treatments were investigated by both surface morphology and surface chemical structure analyses using SEM, AFM, Raman spectra, FTIR, and XPS. In addition, IFSS was tested using the microdroplet test.SEM results demonstrated the surface morphology of fibers, thus providing a brief explanation of the physical combination and interlock between CFs and PEEK fibers. Improving CF roughness is propitious to the enhancement of its IFSS. The significant properties of CFs and PEEK fibers were studied to examine the effect of plasma over different testing times. In conclusion, plasma treatments were unable to affect the tensile strength of carbon fibers and the thermal properties of PEEK fibers within 10 min in our study. XPS analyses indicated the chemical state of the surface. Plasma treatment was shown to be an effective method for increasing oxygen content and active groups on CFs and PEEK fibers.Active groups partly revealed the interfacial interaction and adhesion between CF and PEEK during the molding process. To make the work more satisfactory, IFSS values after different plasma treatments were tested, and composites treated with Ar for 1 min and air for 1 min in sequence had the highest IFSS of 59.73 ± 3.88 MPa, with an increment of 41.01%, indicating the effectiveness of plasma modification.This study offers an in-depth understanding of the intrinsic properties of CFs and PEEK fibers after plasma modification and demonstrates the interfacial strength between them. Further investigations will focus on performing experimental studies on the fabrication of CF/PEEK composites using CF/PEEK hybrid fabrics and comparing properties of CF/PEEK hybrid composites before and after plasma treatment.


Recent research has debated the effects of milk and forage feeding regimes in the first weeks of life on the future performance of dairy calves. However, little is known about how feeding regime can affect behavioural and physiological responses, which have the potential to impact on calf health and well-being. Traditional methods of assessing calf health and welfare such as behavioural observations and blood sampling can be time consuming and impractical for producers and invasive for animals involved. Developments in technology have increased the availability of on-farm non-invasive devices which allow automatic and remote collection of behavioural and physiological data linked to animal health and welfare. This study aimed to use devices to measure lying behaviour, heart rate, heart rate variability and infrared temperature of calves offered high or low levels of milk replacer and different types of forage throughout the first ten weeks of life. Calves displayed changes in lying behaviour and heart rate variability as a result of changes in milk replacer feeding frequency. Additionally, infrared temperature changes were detected during periods of vaccination which corresponded with a rise in core body temperature. Results have highlighted that these sensors can provide important and useable data regarding overall calf well-being on commercial farms.This study aimed to examine the use of non-invasive monitoring technologies as a means of capturing behavioural, physiological and health responses of calves allocated to different nutritional regimes. Seventy-four Holstein Friesian calves were individually penned and allocated to receive either high (HML) or conventional (CML) milk replacer (MR) levels between 5–70 days of age. Additionally calves were allocated to one of four forage treatments: (i) chopped straw offered between 14–70 days of age (CS14), (ii) chopped straw offered between 56–70 days of age (CS56), (iii) grass silage offered between 56–70 days of age (GS56), and (iv) no forage in the pre-wean period (NF). A representative sample of calves from each treatment were fitted with activity sensors and heart rate monitors throughout the experimental period to examine lying behaviour and heart rate variability, respectively. Thermal images of the eye and rectal area of each calf were taken 5 days/week between 5–77 days of age. Faecal and respiratory scoring of each individual calf was carried out on a daily basis throughout the experimental period. Milk replacer feeding level had limited effects on measures of calf health, although HML calves tended to have an increased likelihood for receiving treatment for scour than CML calves. Daily lying time (min/d) was lower in HML calves following reduction in MR feeding frequency at 43 days of age and weaning at 71 days of age when compared with CML calves. Additionally, HML calves displayed a lower heart rate variability following weaning, this suggestive of increased stress load. There were limited effects of forage treatment, however, CS14 calves displayed a greater daily lying time following MR step-down at 68 days of age, this potentially indicating increased rumination. Results of the present study highlight the benefits of using remote monitoring technologies as a means of detecting behavioural and physiological changes as a result of nutritional management strategy in individually housed dairy calves. Traditional calf feeding programs have involved providing calves with restricted levels of milk or milk replacer (MR) as a means of encouraging starter intake and thus facilitating earlier weaning and rumen development . HoweverInsufficient nutrition in the pre-wean period can impact negatively on immune function and increase the potential for susceptibility to infectious disease , it coulRecently, Peli et al  reportedThis study was conducted at the Agri-Food and Bioscience Institute (AFBI) research farm in Hillsborough . The study commenced on 16th February 2016 and ended on 10th July 2016. The study described in this manuscript was approved by the Agri-Food and Biosciences Institute Animal Welfare and Ethical Review Body and all procedures and treatments were conducted under a license from the Department of Health, Social Services and Public Safety for Northern Ireland in accordance with the Animals (Scientific Procedures) Act 1986.Seventy-four Holstein Friesian calves  born between 16th February and 11th April 2016 were allocated to the study following weighing at ≤12 h of age which was used as the birth weight (40.8 ± 5.9 kg). All calves received 3 L colostrum via oesophageal feeding tube or teated bottle before transfer to the calf-rearing unit. Single point blood samples were taken via jugular venipuncture using 10 mL clot activated serum separation vacutainer tubes  at 24–36 h old to allow determination of calf immune status using the zinc sulphate turbidity (ZST) technique, as described by McEwan et al. . All calCalves were individually penned in order of birth and allocated to treatment in a 2 (milk replacer feeding level) × 4 (forage provision) factorial design . Milk reIndividual pens (1m × 1.8m) were constructed using hurdles within two open ended concrete walled and floored housing blocks. A base layer of woodchip was added to the floor of each pen with sawdust bedding material added on top and replenished on a daily basis. Within these pens, calves had visual contact with all other calves within the housing block and nose-to-nose contact with calves from neighboring individual pens. Upon arrival to the calf rearing accommodation at ≤12 h of age all calves were fitted with jackets which remained on until calves reached 29 days of age. Calves remained in individual pens until the end of study at day 84.Liquid diets were offered via a teated bucket twice a day on days 1 to 4. On days 1 to 3 each feed consisted of 2 L colostrum/transition milk, and on day 4 each 2L feed consisted of a mixture of half transition milk/half whey-based milk replacer. From 5 to 70 days of age, calves were offered milk replacer at a rate of 150g/L. Further details of milk replacer feeding regimes are provided in Individual daily milk replacer and concentrate intakes were recorded on a daily basis from 5 days of age. Individual water intake was recorded 5 days/week from 5 days of age. Forage intake was recorded on a daily basis where applicable. Live weight was recorded at birth as well as weekly according to individual calf age between days 7 and 84 . These results will be reported elsewhere.Faecal consistency was qualitatively scored on a daily basis following morning feeding time using the scale of 1 = normal Consistency, 2 = slightly liquid consistency, 3 = moderately liquid and 4 = primarily liquid consistency . A calf ® software  with maximum, average and minimum temperature of images recorded. Ambient temperature and relative humidity (RH) within the rearing accommodation were recorded using a calibrated EBI 20-TH data logger  throughout the experimental period. Average temperature and RH values obtained during the time period when images were taken were entered into the software program during image processing to allow for atmospheric changes during the sampling period. Analysis of eye images focused on the medial, posterior, palpebral border of the lower eyelid and the lacrimal caruncle as these have been found to be the area of most consistent temperature [Thermal images of each calf were taken 2 h post AM feed 5 days/week between days 7 and 77. Images were taken by the same trained operator using a calibrated FLIR E8 camera  and were of the right eye (plus a 1 cm area surrounding the eye) and the anus (plus a 1cm area surrounding the anus). Images were taken at a consistent distance (~0.5 m) and angle (~90°) whilst the calf was standing. For purposes of standardisation, images were taken within the individual pens prior to introduction of any potential stressors such as weighing or blood sampling. As calves quickly became habituated to the process, very limited handling from the camera operator was required. Images were processed using FLIRperature .® IceQube® automatic activity sensors  at 5 days of age and remained on calves throughout the experimental period. Sensors were affixed to the lateral side of the right rear leg in accordance with current best practice [A total of 28 calves, balanced across milk replacer and forage treatments were fitted with IceRoboticspractice ,30 with A total of 36 calves, balanced across milk replacer and forage treatments, were fitted with a Polar Equine RS800CX Science  heart monitor on days −2, −1, 0, +1 and +2 where day 0 = day of 1st vaccination, day of 2nd vaccination and days 29, 43, 57, 68 and 71 of age. These ages represented calf jacket removal (d29), changes in milk feeding frequency and quantity  and weaning (d71). Monitors were fitted 1–2 h post AM feed and remained on calves for 1–2 h to allow data capture during the period when calves were undisturbed and most likely to be resting. Sampling was conducted at the same time each day to help decrease any effects of circadian rhythms. Electrode gel  was applied to ensure contact between the calf and monitor . The tim® . All statistical models included sex as a fixed effect and birth weight as a covariate unless otherwise stated. A probability of p < 0.05 was selected as the level of significance, and where data was significant it was subjected to Fisher’s protected least significant difference (PLSD) test. All data were analysed using GenStatAs variation in daily faecal and total respiratory scores was low, they were averaged on a weekly basis for the first four and complete ten weeks of the experimental period, respectively. Mean weekly scores were then fitted to a repeated measures residual maximum likelihood estimation (REML) mixed effects model with fixed effects and associated interactions of milk replacer level and forage treatment. Likelihood of an animal receiving treatment for scour, pneumonia or a high temperature with signs of respiratory illness was analyzed by modelling of binomial proportions using logistic regression. Fixed effects of sex, milk level, forage treatment and interactions of milk level and forage treatment were fitted individually to the model and back transformed means are reported.Thermal imaging and rectal temperature data were assigned to four age bands; d7–21 to represent the period over which first vaccination occurred, d22–42 to represent the period following first vaccination and prior to first milk step-down of accelerated calves, d43–56 to represent the period following first milk step-down and the time during which second vaccination occurred and d57–77 to represent the period over which milk step downs and weaning occurred. Data were fitted to a repeated measures REML model with fixed effects and associated interactions of milk replacer level, forage treatment and time. Ambient temperature was included as a covariate. These data were also fitted to a repeated measures REML model with fixed effects and associated interactions of milk replacer level, forage treatment, time and method of temperature assessment. Correlation coefficients between each of the three temperature methods were determined by linear regression using the predicted means obtained through the repeated measures analysis.Daily and hourly lying time along with number and duration of lying bouts and heart rate and heart rate variability data were fitted to the same repeated measures REML model as described above. Results pertaining to hourly lying behaviour are presented in p = 0.010). There was no effect of milk replacer level or forage treatment on average faecal score between days 5–13, 21–27 or 28–34, or of forage treatment between d14–20 (p > 0.1). No faecal scores above 1 were recorded after 34 days of age. CML calves had an increased average respiratory score between days 49–55 when compared to HML calves (p = 0.022), however, despite the observed increase, average respiratory scores across both treatments remained below the threshold deemed indicative of bovine respiratory disease. There was no effect of forage treatment on respiratory score (p > 0.1). There was no effect of milk level offered on the probability of receiving treatment for pneumonia (p = 0.189) or pyrexia plus respiratory type symptoms (p = 0.599). Calves offered chopped straw at 56 days of age (CS56) had an increased likelihood of receiving treatment for pneumonia (p = 0.008), however, no interactive effects of MR level and forage treatment were observed (p = 0.831). An increased likelihood of receiving treatment for scour was observed in HML calves (p = 0.059) with values of 0.47 and 0.26 for HML and CML calves respectively. There was no significant effect of forage treatment (p = 0.130) or MR level × forage treatment (p = 0.196) on likelihood of receiving treatment for scour.Calves offered high (HML) levels of milk replacer had an increased average faecal score between days 14–20 when compared to calves offered conventional (CML) levels of milk replacer . Average core body, IR eye and IR rectal temperatures recorded between d7–77 are presented in p = 0.057), however, there were no differences in IR eye or IR rectal temperature. There were no differences between MR or forage treatments in core body, IR eye or IR rectal temperature when data were analysed over the 7 days prior to and following calf jacket removal, step down in MR feeding frequency or quantity or weaning. Although unaffected by MR feeding level, there was an effect of day with an increase in all temperature parameters in the 2 days following 1st and 2nd vaccination (p < 0.001).There were no effects of MR feeding level or forage treatment on core body, infrared (IR) eye or IR rectal temperature between days 7–21, 22–42, or 43–56 ; p > 0.1Calf temperature was affected by method of temperature assessment across all time periods. Positive correlations were found between core body and IR eye temperature when data were analysed between 7–21, 22–42, 43–56 and 57–77 days of age . Positivp = 0.017), and was characterized by an increased number (p < 0.001) of shorter (p = 0.002) lying bouts when compared with CML calves. During this period, calves offered chopped straw from 14 days of age (CS14) displayed an increased number (20.0 vs 15.4) of shorter (56.2 vs 71.6 min) lying bouts than those offered chopped straw at 56 days of age (CS56). However, despite this variation, there no was no significant impact of forage treatment on total daily lying time (p = 0.306). Daily lying time was 40 min/d lower in HML calves in the period following their step down from three to two MR feeds at 43 days of age when compared with CML calves (p = 0.021). There was no significant effect of MR feeding level on daily lying behaviour between d57–62 (p > 0.05), however, there was an effect of forage treatment whereby CS56 calves had fewer lying bouts (p = 0.028) than CS14 and GS56 calves (19.3 vs 22.0 vs 22.7). Lying behaviour was not significantly impacted by MR feeding level in the period following reduction of MR to 2L/day at 68 days of age (p > 0.1), however, there was an effect of forage treatment whereby total daily lying time was higher (p = 0.022) in CS14 calves when compared with GS56 and NF calves (1112 vs 976 vs 986 min/d). Daily lying time was higher in CML calves in the period following weaning at 71 days of age (p = 0.026), lying bouts also tended to be in longer in these calves when compared to HML calves (p = 0.098). There was no effect of forage treatment on lying behaviour during the post weaning period, however, there was an effect of MR level × forage treatment on lying bout duration (p = 0.014), with bouts approximately 10.8 min shorter in HML calves receiving chopped straw at 56 days of age. Diurnal lying behaviour over periods of milk step-down and weaning is presented in Daily lying time was increased in HML calves between days 36–42  and lower RMSSD (p = 0.058) at 72 days of age. There was no significant effect of forage treatment on HR or RMSSD throughout the experimental period (There was no effect of MR feeding level on heart rate (HR) or heart rate variability (HRV) as measured by root mean square of successive differences (RMSSD) during the period following calf jacket removal at 29 days of age. This was also true in the periods following step down of HML calves from three to two and two to one MR meals/d at 43 and 57 days of age, respectively, and following reduction of MR level to 2L/day across treatments at 68 days of age. Both heart rate and RMSSD values were unaffected by MR treatment in the period following weaning at 71 days of age, however, there was an interactive effect of MR treatment and day, with HML calves displaying increased heart rate (l period .p < 0.001). As such it is likely that CML calves in the present study were spending more time standing to consume concentrate that HML calves. Calves offered chopped straw at 14 days of age (CS14) also displayed an increased number of shorter lying bouts during this period. Castells, et al. [Responses to common management practices such as restricted feeding and weaning have previously been assessed using measures of calf activity, with technological developments meaning that standing and lying behaviour can now be recorded automatically . De Paul, et al.  reported, et al.  and like, et al. , a decre, et al. , as suchHeart rate monitors have previously been shown to provide increased opportunities for non-invasive monitoring of physiological responses to stress . An incrIll-health in early calfhood can have a significant impact on animal behaviour, welfare and productivity, all of which can affect the economic efficiency of dairy operations ,41. EnteIn the present study, it could have been considered that calves offered high (HML) levels of MR would have been less likely to suffer from ill-health when compared to those offered conventional (CML) levels of MR. However, comparable to that reported by Quigley, et al. , HML calOne of the primary detectable physiological responses to infection and inflammation is a change in core body temperature, and as such it is often used as an indicator of ill-health . In addiThere is limited published research addressing the correlation between infrared (IR) temperature and core body temperature in very young calves. Although variable in the present study, correlation between IR eye temperature and core body temperature was within the range to that found in Jersey heifers by Salles, et al.  and compUnder the management conditions described in the present study, MR feeding level had limited effects on calf health and physiological responses to common management practices. However, changes in activity which could be linked with hunger as a result of conventional feeding levels and following reduction in MR volume in calves offered high levels of MR in early life were detectable using automatic activity sensors. Additionally, the higher lying time observed in CS14 calves between 68–70 days of age could point to increased rumination as a result of earlier forage introduction. This suggests that providing calves with a forage source in addition to concentrate feed could be beneficial to rumen development. In the present study, calves were individually housed which perhaps contributed to the low level of ill-health amongst calves. Calves were also monitored on a daily basis with a combination of traditional and novel indicators, this potentially resulting in earlier detection and treatment of ill-health, as such, future research should incorporate techniques described here into group housing systems. Results of the present study also provide evidence for the role of remote-sensing technologies within calf rearing systems. Using a range of traditional and novel methods increases the possibilities for collection of behavioural and physiological data. This will allow producers to obtain an overall indicator of calf well-being and responses to common management practices encountered in early life. Further research which aims to compare and correlate these technologies with hormonal, biochemical and immunological parameters could help in the development of early warning systems for calf health and welfare issues.


NCOR1, GATA3, CDH1, ATM, AKT1, and PTEN) were significantly associated with the corresponding expression levels of the proteins. The proteins were involved in multiple pathways related to the development of cancer, including the PI3K‐Akt signaling pathway, pertinent microRNAs, and the MAPK signaling pathway. In addition, overall survival and recurrence‐free survival analysis revealed the close associations of the expression of GATA3, NCOR1, CDH1, and ATM with survival of BC patients. Therefore, detecting these gene mutations and exploring their corresponding expression could be valuable in predicting the prognosis of patients. The results of the high‐throughput data mining provide important fundamental bioinformatics information and a relevant theoretical basis for further exploring the molecular pathogenesis of BC and assessing the prognosis of patients.Advances in cancer biology have allowed early diagnosis and more comprehensive treatment of breast cancer (BC). However, it remains the most common cause of cancer death in women worldwide because of its strong invasiveness and metastasis. In‐depth study of the molecular pathogenesis of BC and of relevant prognostic markers would improve the quality of life and prognosis of patients. In this study, bioinformatics analysis of SNP‐related data from BC patients provided in the TCGA database revealed that six mutant genes ( Single nucleotide polymorphisms (SNPs) are DNA sequence polymorphisms caused by a single nucleotide variation. SNPs are the most common type of human heritable variation and are common in the human genome. Gene SNPs can cause changes in gene expression by affecting the binding, cleavage, methylation, and mRNA degradation of gene transcription factors, causing genetic differences among individuals.As a relatively small allelic variable, an SNP is an important genetic marker to study the characteristics of different cancers or cancers. As genome‐wide association studies have progressed, there is an increasing evidence that BC susceptibility is associated with genetic SNPs. For example, SNPs of ERCC5 have been associated with the development of certain cancers, including BC.The Cancer Genome Atlas (TCGA) database can be applied to high‐throughput genomic analyses to better demonstrate the genetic basis of disease by using genome sequencing and bioinformatics analysis of gene mutations responsible for cancer. The findings could improve our ability to diagnose, treat, and prevent cancer. To further explore the biological significance of DNA sequence polymorphisms in the diagnosis and prognosis of BC, we downloaded data of BC‐related SNPs from the TCGA database and used bioinformatics analysis methods, including mutation data, protein‐protein interaction (PPI) network, and correlation analyses, to mine mutation genes related to BC diagnosis and prognosis. The goal was to provide a scientific theoretical basis for personalized precision medical treatment for BC.22.1https://gdc.cancer.gov/).TCGA Data Portal was terminated, and all TCGA data were transferred to the newly established Genomic Data Commons (2.2https://david.ncifcrf.gov/)P < 0.05 as the cutoff condition, screening of molecular functions (MFs), biological processes, cellular components (CCs), and KEGG pathways for mutated gene enrichment can be accomplished.To better understand the dysfunction caused by these mutant genes, we used the DAVID  and overall survival (OS) data were downloaded from GEO (Affymetrix microarrays only), EGA, and TCGA. The primary purpose of the tool is a meta‐analysis‐based biomarker assessment.33.1P < 0.01 as the cutoff condition ‐Akt, calcium, and mitogen‐activated protein kinase (MAPK) signaling pathways, among others . Among them, the expression levels of CDH1, NCOR1, ATM, and PTEN in the mutant samples were decreased, while the expression of GATA3 and AKT1 increased  was found to be closely related to patients' OS and RFS. The OS and RFS curves revealed that high expression of NCOR1, ATM, and GATA3 was associated with higher OS and RFS, while high expression of CHD1 was associated with poor prognosis 8 rs11225394 and MMP9 rs3787268 are closely related to BC risk in the Chinese Han population.In the present study, our aim was to screen and identify prognostic biomarkers associated with SNP‐mediated expression through a series of bioinformatics analyses of BC‐related data in TCGA database. To further study the related molecular mechanisms involved in the direct involvement of these mutant genes, functional enrichment and pathway analysis were performed. The genes were enriched in the cytoplasm and nucleoplasm, and were mainly involved in certain transcriptional regulation, such as negative or positive regulation of transcription from RNA polymerase II promoter, positive regulation of transcription from DNA templates, and various binding pathways, such as protein and DNA binding. Pathway analysis indicated that BC mutated genes were mainly involved in the PI3K‐Akt and, calcium signaling pathways, and many other pathways related to cancer development. The functional enrichment and pathway analyses demonstrate the molecular mechanisms of SNP mutations in disease progression, and the functional level interaction of these genes.NCOR1, GATA3, CDH1, ATM, AKT1, and PTEN) were significantly correlated with the corresponding expression levels, and were enriched and involved in multiple cancer‐related pathways. GATA binding protein 3 (GATA3) is a transcription factor crucial for mammary gland morphology and cell differentiation and acts as a tumor suppressor.GATA3 could inhibit tumor growth and lung metastasis.GATA3 SNP mutations were identified in BC samples, and were positively correlated with expression levels, that is, the expression level of GATA3 was also increased in mutant samples. In addition, studies by Atlas et al have confirmed that the GATA3 gene is identified with mutations in >10% of all BC samples,Mutations in six genes  is a transcriptional co‐regulator that binds chromatin‐modifying enzymes to gene‐specific transcription factors and interacts with members of the BTB‐ZF transcription factor family to play important roles in T cell development and function. Low expression of NCOR1 is associated with acquired tamoxifen resistance in a mouse model of BC.NCOR1 expression is significantly associated with shorter RFS in BC patients, suggesting a poor prognosisNCOR1 gene was significantly reduced in the mutated samples, and the results of the correlation analysis showed that the SNP mutation of NCOR1 was negatively correlated with the expression level. Moreover, the OS and RFS analysis data supported the poor prognosis associated with low expression of NCOR1, which is consistent with previous studies and further confirms the validity of the present findings.Nuclear receptor corepressor 1 (CDH1) and Ataxia telangiectasia mutated (ATM) are tumor suppressor genes, which are enriched in multiple signaling pathways, including cancer pathways, miRNAs in cancer, apoptosis, and the p53 signaling pathway. CDH1 is frequently mutated in diffuse gastric cancer and lobular BC. Diffuse gastric cancer patients with CDH1 mutation have shorter survival time than those without the CDH1 mutation.ATM are closely associated with BC, ovarian cancer, and other cancers.ATM expression is down‐regulated in BC and suggests poor prognosis.ATM gene promoter might affect the DNA repair mechanism by causing the dysregulation of the ATM/p53 signaling pathway, thereby affecting tumor progression in BC.CDH1 and ATM were negatively correlated with the expression levels, with SNPs resulting in reduced expression. Among them, the main reason for the correlation between CDH1 SNP mutation and expression is the mutation of CC in CDH1 rs587783047 site. In addition, the OS and RFS analysis revealed that decreased expression of ATM detrimentally affects the prognosis of patients. Conversely, the increased expression of CDH1 detrimentally affects the prognosis of patients, which required further clinical investigation.E‐cadherin  were significantly associated with the corresponding expression levels and were involved in multiple pathways involved in cancer development. In addition, further analysis indicated that the SNP mutation at the AKT1 rs121434592, CDH1 rs587783047, and GATA3 rs763236375 sites were the important reasons for affecting gene expression. In addition, OS and RFS analysis found that the expression of NCOR1, GATA3, CDH1, and ATM were closely related to the survival of BC patients. Therefore, detecting gene mutations and exploring their corresponding expression can be used to predict the prognosis of patients. The findings will require validation in large‐scale clinical studies to determine their accuracy and sensitivity in tumorigenesis and predicting patient outcomes. However, the focus of this study is to provide new ideas for clinical diagnosis and evaluation of prognosis through bioinformatics analysis. Our results provide an important bioinformatics basis and relevant theoretical basis for guiding follow‐up studies on BC.Bioinformatics analysis revealed that SNPs in six genes (The authors declare that they have no competing interests.