However, the differences in modulus of elasticity and compressive strength were not justified by the differences in wood density. Further infrared spectroscopy and X-ray diffraction analyses revealed additional differences in chemical composition and cellulose crystallinity between archaeological and contemporary wood. The results indicated the decrease in carbohydrate content and cellulose crystallinity in degraded wood, which, in addition to wood density, apparently contribute to the deterioration in mechanical strength of archaeological wood. The case study of the excavated archaeological wooden pipes shows that they have historical value but are not useful for practical purposes. It also revealed that not only wood density but also its chemical composition and cellulose crystallinity level has a substantial impact on the wood mechanical properties, particularly in compression.Large amounts of archaeological wood are often excavated during groundworks in cities and towns. Part of the unearthed artefacts is usually saved, conserved and then presented in museums. However, if the finding contains several similar objects, some of them could potentially be further employed for some other practical purposes. The research aimed to determine the mechanical performance of the remains of wooden water mains excavated at Bóżnicza street in Poznań, Poland and evaluate its potential usefulness for any practical purposes. First, wood density was determined along with its mechanical strength in compression. The density of archaeological wood identified as Scots pine was lower than contemporary pinewood (383 kg × m The history of wooden water pipes dates back to the 17–19th centuries. They replaced stone, clay and terracotta water mains and aqueducts invented by ancient Greeks and Romans and, together with complex networks of pumps, water towers and reservoirs, they enabled the development of modern cities worldwide ,3.Usually, the wooden water main consisted of several sections about 1.8- to 3.6-m long bored logs with an internal diameter at an opening of 80 to 150 mm . For earRecords about the existence of wooden water supply systems come from several different countries all around the world, including the United Kingdom  ,20,21, tSince the degradation processes change wood density and the composition of the main cell wall polymers, wood mechanical performance must also be affected ,39,40,41In 2018, during the groundwork for a new building at Bóżnicza street in Poznań, Poland, large amounts of archaeological wood were unearthed. It was recognised as remains of wooden water mains of Poznań city dated back to the turn of the 18th and 19th centuries. Some of the excavated water pipes looked relatively good B, while This research aimed to characterise the physico-mechanical properties of the excavated archaeological wooden pipes and evaluate their potential suitability for any other useful purposes. Therefore, except for identifying wood species and assessing its density, wood mechanical strength in compression was measured. Since the mechanical strength of archaeological wood turned out to be lower than what would result from its decreased density compared to contemporary sound wood of the same species, we decided to investigate what other factors affect the mechanical performance of the degraded wood. To achieve this, wood chemical composition was studied using Fourier-transform Infrared Spectroscopy along with the crystallinity of cellulose present in the cell wall using the X-ray diffraction method.Archaeological wood used in the study comes from excavations at Bóżnicza street in Poznań, Poland. It was unearthed at the turn of 2018 and 2019. Part of the excavated wooden logs that were visually relatively well-preserved B was usePinus sylvestris L.—see Wood was identified as Scots pine . Then, they were sectioned in each anatomical direction with a microtome. Microscope slides were observed under a light biological microscope Motic B3 Professional Series . Images of wood tissue were captured using a microscope-attached camera Moticam 2.0  coupled with a computer and analysed with a Motic Images Plus 2.0  ML image analysis software.Identification of the wood species of the excavated water pipes was made using light microscopy. First, small wood samples obtained from each wooden element were softened in a mixture of distilled water and glycerine  were cut out from the archaeological pine sapwood (a layer about 2–3 cm from the log perimeter). First, they were conditioned at room temperature (21 ± 1 °C) and ambient air relative humidity (40 ± 5%) until equilibrium moisture content was achieved. Then, the samples were weighed using an analytical balance accurate to 0.001 g . Next, their dimensions in the three principal anatomical directions were measured with a digital calliper accurate to 0.01 mm. Based on the results obtained, wood density was calculated as a ratio of the wood mass divided by its volume (according to ISO 13061-2:2014 ). After Similarly, contemporary pine sapwood samples with a macrostructure akin to archaeological wood were prepared.Since the correlation between wood density and mechanical strength is the most pronounced in compression ,48,49,50−1 resolution using a Bruker ALPHA FT-IR spectrometer . Infrared spectra in the 4000–400 cm−1 region were recorded in KBr pellets. Wood sample concentration was 2 mg/200 mg KBr. Five spectra for each sample were recorded, and the resulting average spectrum obtained was processed using Grams 9.1 software .Fourier-Transform Infrared Spectroscopy (FT-IR) method was used to characterise wood chemical composition. Small pieces of all 10 replicates of archaeological and contemporary pine wood specimens were air-dried, powdered together  and sieved. The wood powder fraction with an average particle diameter less than 0.2 mm was analysed with 4 cmCr.I. % is the crystallinity degree, crA is the crystalline area and tA is the total area under diffractogram.X-ray diffraction was applied to analyse cellulose crystallinity in archaeological and contemporary wood. Measurements were performed using a Diffractometer D8 ADVANCE  with Cu-K radiation (λ = 0.1542 nm), a parallel beam with Gobel mirror and a Dynamic Scintillation detector. The working conditions during the measurements were 40 kV and 30 mA, 2 s/step, 0.02 degree/step. The processing of the diffractograms, i.e., their deconvolution, was performed using Grams 9.1 software . For the amorphous background, a Voight profile was used, while mixed Gaussian-Lorenzian profiles were applied for the crystalline regions. The crystallinity degree was calculated according to the equation proposed by Hermans and Weidinger :(1)Cr.I.Pinus sylvestris L.) are presented in Wood identification was made based on its anatomy. The archaeological wood from water pipes was recognised as softwood; therefore, further species identification was based on radial sections. The most characteristic microscopic anatomical features that allowed recognising the wood as Scots pine , radial (R) and longitudinal (L) direction are 159, 189 and 4407 MPa, respectively  as the value of the elastic modulus measured divided by the wood density. The calculated sMOE values for both wood types are presented in c) was also measured to acquire a better picture of changes in the mechanical properties of the degraded wood . For contemporary pine, they are within the range of compressive strength specific for the species [c) values  for contemporary and archaeological samples, they are only 1.5, 2.3, and 2.0 times higher. Different Rc and sRc ratios for sound contemporary and degraded archaeological pine are in line with observed MOE and sMOE ratios. These indicate that not only wood density but also some other factors must have affected the mechanical properties of the degraded wood.Apart from MOE, the relative/compressive strength  1850–830 cm−1 assigned to different specific stretching and deformation vibrations from all wood components. Since the bands in the wood spectra are generally overlapped, the second derivative will usually give detailed information about the modifications in its structure during ageing. The infrared spectra of the archaeological and contemporary pine wood samples are presented in −1 assigned to C=O stretching vibration of carboxyl and acetyl groups decreases in intensity in the archaeological wood spectrum, compared to the contemporary one. The same behaviour was observed for the bands assigned to C–H deformation in cellulose and hemicellulose, C–O–C vibration in cellulose and hemicellulose, and C–O stretching mainly from C(3)–O(3)H in cellulose I at 1375, 1161, 1115 and 1065 cm−1. At the same time, aside from a decrease in intensity, shifting to lower wavenumber was observed for the band at 1228 cm−1 (assigned to C–O–C stretching mode of the pyranose ring). A slight increase in intensity can be seen for the bands from 1640, 1512, 1461, 1421 and 1268 cm−1, assigned to absorbed O–H, C=C of aromatic skeletal (lignin), C–H deformation in lignin and carbohydrates and C–H bending mode in cellulose and C–O stretch in lignin.As can be observed from both spectra and their derivatives, principal differences occur in the fingerprint region. The band from 1740/1741 cmThe decrease in intensity of the band assigned mainly to carbohydrates, combined with the increase in the intensity of the water band and of the bands assigned mainly to lignin, indicate the higher degradation of the carbohydrates with lower degradation of lignin. Moreover, higher intensity of the water band suggests that archaeological wood presents a more hydrophilic structure than contemporary wood.These observations—decreased concentrations of carbohydrates—indicate wood degradation. Moreover, they are well correlated with lower density usually observed for archaeological wood compared to the contemporary one since hemicelluloses and cellulose are the most available substrates for fungi and bacteria ,73,74.X-ray diffraction is mainly used to evaluate the degree of crystallinity in lignocellulosic materials. Among wood components, only cellulose shows a certain degree of crystallinity. Its regular structure and the presence of free hydroxyl groups that can be involved in different intra- and intermolecular hydrogen bonds may give rise to various ordered crystalline arrangements ,76.The natural form of cellulose in the wood cell wall (cellulose I) presents a typical diffractogram pattern with a high-intensity diffraction peak at 2theta = 22.3° , three other lower intensity diffraction bands at about 2theta = 14.8° , 2theta = 16.8°  and 2theta = 20.0° , and an amorphous background with its maximum at about 2theta = 18.5–19.2° ,77,78.The X-ray diffractograms of contemporary and archaeological pine wood are presented in The determined crystallinity degree for archaeological pine wood was 42.5%. It was about 25% lower than the value obtained for contemporary pine wood (56.9%). The structural modification (indicating the degradation of carbohydrates) and the reduced crystallinity degree contribute to the observed reduction in mechanical properties of archaeological wood and explain the differences for specific modulus of elasticity and specific compressive strength between sound contemporary and degraded archaeological wood.The mechanical analyses revealed that the elastic modulus in compression and compressive strength of archaeological wood is lower than expected from the decrease in its density compared to sound pine wood. Furthermore, FT-IR and XRD analyses showed changes in the chemical composition and structure of degraded wood, particularly the decrease in hemicelluloses and cellulose content and the reduced crystallinity degree, indicating the role of these components in the wood mechanical performance. Concluding, the case study shows that not only wood density but also its chemical composition and the crystallinity level has a substantial impact on the wood mechanical properties in compression. It also points out how both the physical and chemical properties of wood are crucial to understanding and predicting its performance. Moreover, it indicates how essential it is to characterise all the parameters to acquire a complete knowledge about the degraded wood artefact despite whether it is intended for conservation and exhibition in the museum or any other purposes.In the light of the results obtained in the study, it is clear that wooden water pipes unearthed at Bóżnicza street in Poznań can serve mainly as historical artefacts. The degree of wood degradation reflected in its deteriorated mechanical properties makes the archaeological pinewood unsuitable for practical uses requiring mechanical strength. However, it could be used for decorative purposes that do not need to carry mechanical loads.


Steroid-refractory (SR) acute graft-versus-host disease (aGvHD) remains a significant complication after allogeneic hematopoietic cell transplantation. Systemic corticosteroids are first-line therapy for aGvHD, but apart from ruxolitinib, there are no approved treatments for SR aGvHD. Vedolizumab is approved for treatment of ulcerative colitis and Crohn’s disease, and may be effective for treatment of SR intestinal aGvHD. We conducted a phase 2a trial (NCT02993783) to evaluate the clinical efficacy, tolerability, and safety of vedolizumab 300 and 600 mg for SR intestinal aGvHD. This study was terminated before full enrollment was completed because early results failed to demonstrate positive proof-of-concept in efficacy. Before termination, 17 participants had enrolled and an early response in intestinal aGvHD was observed in 11 and eight participants at days 15 and 28, respectively. All adverse events observed were consistent with those expected in a population with SR intestinal aGvHD. Overall, vedolizumab did not meet the primary efficacy endpoint , likely owing to premature study drug discontinuation, lack of efficacy, and the competing risks inherent with a population with advanced SR intestinal aGvHD. Nevertheless, this study provides valuable insights into the considerations needed when conducting studies in patients with SR intestinal aGvHD. Allogeneic hematopoietic cell transplantation is an important and potentially curative therapy for many hematologic malignancies, but carries the significant risk of graft-versus-host disease (GvHD) , 2. AcutAfter conventional allogeneic hematopoietic cell transplantation, the incidence of aGvHD ranges from 10% to 80%  in patients receiving T-cell-replete allogeneic grafts, despite standard measures of prophylaxis –7.First-line therapy for patients who develop aGvHD is systemic corticosteroids, but these are effective only in ~50% of patients, with durable responses observed in only one-third , 9. AparVedolizumab, a humanized immunoglobulin G1 monoclonal antibody directed against the α4β7 integrin expressed on lymphocytes and implicated in gastrointestinal (GI) trafficking, was developed for the treatment of moderate–severe ulcerative colitis and Crohn’s disease –16. VedoHere we present data from a phase 2a clinical trial (NCT02993783), which prospectively investigated the clinical efficacy, tolerability, and safety of vedolizumab for the treatment of SR intestinal aGvHD.This was a phase 2a, randomized, parallel, open-label study with two dose groups evaluating the clinical efficacy, tolerability, and safety of vedolizumab  IV for treatment of SR intestinal aGvHD.Participants were recruited and enrolled from the investigator’s practice or on referrals from other physicians. If alternative recruitment strategies were employed (such as advertisement) these were reviewed by the institutional review board (IRB)/independent ethics committee (IEC). All participants provided written informed consent before any study-required procedures were conducted, unless such procedure formed part of their standard of care.Participants were 18 years or older, had undergone a first allogeneic hematopoietic cell transplant, and had developed SR intestinal aGvHD  doses and schedule of vedolizumab were selected based on those approved for the treatment of ulcerative colitis and Crohn’s disease (300 mg IV every 8 weeks) . BecauseParticipants were randomized 1:1 to receive 300 or 600 mg doses of vedolizumab IV on days 1, 15, 43, 71, and 99 on the basis of evidence from a previous case series . InvestiIt was intended that after ten participants had enrolled in each dose group and had been followed through day 28, safety, tolerability, efficacy, and pharmacokinetic (PK) results would be assessed to determine an appropriate dose for subsequent participants. The group at the chosen dose would have been expanded by approximately 18 additional participants to assess the tolerability and efficacy of vedolizumab further . However, early results failed to demonstrate a positive proof-of-concept in efficacy (described below) and the trial was terminated early.The primary endpoints of the study were overall response (OR) . PK characterization of vedolizumab and the presence of anti-vedolizumab antibodies at baseline and at the end of the exposure period were also performed, and durable intestinal OR rate  was evaluated as an exploratory endpoint.All participants were planned to be followed for overall survival every 3 months until death, withdrawal of consent, termination of study by the sponsor, or for a maximum of 1 year after the last subject was enrolled in the study.Severity of aGvHD was assessed at each visit using the Blood and Marrow Transplant Clinical-Trials Network (BMT CTN)-modified International Blood and Marrow Transplant Research (IBMTR) database index . A positive proof-of-concept in efficacy was considered if the posterior probability of day 28 OR rate >54% was at least 85% and day 28 OR rate being at least 70% was at least 75%. Early review of patient-level data was performed ahead of the planned analysis to assess safety, PK, and efficacy data in order to facilitate decision-making in the study.Enrolled participants who received any dose of study drug comprised the safety analysis set. The efficacy analysis set included all participants from the safety analysis set who had a baseline efficacy assessment and at least one postbaseline efficacy assessment. The PK analysis set comprised all participants in the safety analysis set who had at least one post-dose PK sample collected.Approximately 38 patients were planned for enrollment. The sample size was based on clinical considerations and considered sufficient to determine a recommended dose and regimen and to describe the efficacy, tolerability, and safety of vedolizumab IV.On the basis of an early review of patient-level data and the totality of evidence available, it was concluded that the ability to evaluate overall efficacy was impaired owing to (a) the competing risks in the population, (b) premature discontinuation of study drug, and (c) observed mortality. It was determined that the proportion of participants achieving the primary efficacy outcome was not expected to reach 54% and the study was terminated early (9 May 2018) by the sponsor. Participants were recruited and followed from 28 April 2017 until study termination.Before termination, 17 participants were enrolled and treated at 11 sites in Belgium, France, Norway, and the USA  had isolated intestinal aGvHD, four (23.5%) had skin and intestinal involvement, and one (5.9%) had liver and intestinal involvement. Twelve participants (70.6%) had Grade C or D aGvHD . At baseline, 13 participants (76.5%) had Stage 2 or greater intestinal aGvHD .Baseline disease characteristics and outcomes for each participant are presented in Supplementary Table n = 1) or because of an unsatisfactory therapeutic response (n = 4). Four participants (30.8%) discontinued owing to ‘other’ reasons, including death (n = 3) and withdrawal (n = 1), three participants (23.1%) owing to a TEAE , and one participant (7.7%) owing to progression of an underlying malignancy (relapse of acute myeloid leukemia).Thirteen participants (76.5%) discontinued the study drug prematurely Fig. . Five ofn = 8), at the last study assessment only two were recorded as having no intestinal aGvHD involvement, suggesting that most had not responded to treatment at the time that they discontinued the study drug.Of those who discontinued for reasons other than unsatisfactory therapeutic response or progression of aGvHD  had an OR in all organs involved, and six of the eight participants with an OR in intestinal aGvHD at day 28 experienced a durable response (response maintained through day 43). An OR in intestinal aGvHD was recorded in 11 participants (64.7%) and eight participants (47.1%) at day 15 and day 28, respectively  were alive: four  in the vedolizumab 300 mg and one  in the vedolizumab 600 mg group, with a median overall survival of 161 days  in the 300 mg group and 58 days  in the 600 mg group  were considered unrelated to the study drug. Overall, five participants (29.4%) experienced a TEAE, and two participants (11.8%) experienced a serious adverse event considered to be related to the study drug.An overview of TEAEs is presented in Supplementary Table The most frequently reported adverse events were infections occurring in 15 participants (88.2%), including three participants 17.6%) with sepsis and four participants (23.5%) with cytomegalovirus (CMV) related infections, of which two (11.8%) were CMV colitis. The events of CMV colitis were both serious and considered related to the study drug for one participant (vedolizumab 600 mg). Commonly occurring infection-related TEAEs are presented in Supplementary Table .6% with No participants had detectable anti-vedolizumab antibodies at any scheduled time point.n = 5) or sepsis (n = 3). Other causes of death in the remaining participants (n = 5) who died during the TEAE period were due to the development of peritonitis (percutaneous endoscopic gastrostomy probe placement related), thrombotic microangiopathy, mediastinitis, or acute kidney injury; one cause of death was not reported. The death of the participant after the TEAE period was due to a fatal relapse of acute myeloid leukemia. Additional details at the time of death for each participant are provided in Table During the TEAE reporting period, 13 participants (76.5%) died  of participants dying from complications of aGvHD. An early review (prior to day 28) of patient-level data for the 17 enrolled participants concluded that the expected efficacy outcome for the study would not be achieved and the decision was made to terminate the study prior to completion of planned enrollment.The primary objective of this phase 2a study was to characterize the efficacy, tolerability, and safety of vedolizumab to identify a recommended dose for further evaluation of the treatment of SR intestinal aGvHD. While early clinical benefit (64.7% OR rate at day 15) was observed in approximately two-thirds of participants, less than half obtained an OR in intestinal aGvHD by day 28, with a significant number . Moreover, more than 70% of participants had SR Grade C or D aGvHD, and ~30% had multiorgan involvement at baseline. Among patients with aGvHD intestinal tract involvement, GI Stage 3–4 and SR aGvHD in particular are associated with substantial morbidity and mortality, with a significantly increased risk of infections, and a limited response to salvage therapies –38. The Given the data from this study, it is also certainly possible that treatment with vedolizumab was unable to exert an effect rapid enough and/or of significant magnitude to allow the injured GI epithelium to recover sufficiently and prevent disease-related complications. It has been proposed that if enough tissue injury has occurred, T-cell migration to the GI endothelium is of limited importance, and α4β7 integrin is no longer needed for the propagation of aGvHD . From thA number of case series on the use of vedolizumab in SR intestinal aGvHD have reported variable outcomes , 41, 42.There have been two recent large retrospective multicenter case series published. Danylesko et al. described 29 patients with SR intestinal aGvHD treated with vedolizumab IV 300 mg and demonstrated OR rates of 79%  after 7–10 days. Twelve patients (41%) were able to discontinue immunosuppressive therapy. Overall survival rates at 6 and 12 months were 41.4% and 27.5%, respectively, with the majority of successful outcomes observed in patients who were treated early in their disease course . FløisanTaken together, the early OR results observed in this study, along with the anecdotal evidence from retrospective case studies, justify pursuing the evaluation of vedolizumab for lower intestinal SR GvHD. However, considering the mechanism of action and kinetics of GI epithelial regeneration in the context of aGvHD, vedolizumab may be more effective if administered earlier in the disease course, such as for prevention, pre-emptive use, or initial therapy.With regard to safety, observed adverse events were comparable with prior reports in a patient population with SR GvHD . The morOverall, this study was unable to demonstrate a positive proof-of-concept in efficacy in participants with SR GvHD treated with vedolizumab. While early and durable responses to intestinal aGvHD following treatment with vedolizumab were observed in some participants, vedolizumab was unable to generate a satisfactory response in this study. Evaluating the true efficacy of vedolizumab in patients with advanced severe intestinal aGvHD and associated complications is clearly challenging and perhaps not realistically possible. It may be that vedolizumab may be more suited to use for intestinal GvHD prevention, and this is currently being evaluated in a phase 3 clinical trial (NCT03657160).The observations made in this study are nevertheless important, as they provide vital information on the severity of SR intestinal aGvHD and management of complications that need to be considered when designing and conducting future studies in such patients if positive efficacy outcomes are to be demonstrated.Supplementary material


Anoplopoma fimbria) are becoming aquaculturally and economically more important, this manuscript provides essential reference intervals (RI) for their plasma biochemistry and hematology along with reference photomicrographs of blood cells in healthy, fasted sablefish. Blood cell morphology can differ between fish species. In addition, blood cell counts and blood chemistry can vary between fish species, demographics, water conditions, seasons, diets, and culture systems, which precludes the use of RI’s from other fish species. For this study, blood was collected for plasma biochemistry and hematology analysis between June 20 and July 18, 2019, from healthy, yearling sablefish, hatched and reared in captivity on a commercial diet. Overnight fast of 16–18 hours did not sufficiently reduce lipids in the blood, which led to visible lipemia and frequent rupture of blood cells during analysis. Therefore, sablefish should be fasted for 24 to 36 hours before blood is collected to reduce hematology artifacts or possible reagent interference in plasma biochemistry analysis. Lymphocytes were the most dominant leukocytes (98%), while eosinophils were rare, and basophils were not detected in sablefish. Neutrophils were very large cells with Döhle bodies. In mammals and avian species, Döhle bodies are usually signs of toxic change from inflammation, but no such association was found in these fish. In conclusion, lipemia can interfere with sablefish blood analysis, and available removal methods should be evaluated as fasting for up to 36 h might not always be feasible. Also, more studies are required to establish RI for different developmental stages and rearing conditions.Plasma biochemistry and hematology reference intervals are integral health assessment tools in all medical fields, including aquatic animal health. As sablefish ( Anoplopoma fimbria), colloquially known as butterfish or black cod, are a highly valuable and fast-growing demersal species native to the continental slope of the North Pacific Ocean. Between 2009 and 2019, U.S. landing values of wild sablefish have ranged between $89.1 and $184.6 million [Sablefish  –12. InveHigh stocking density within any culture system increases disease pressure and is a ubiquitous challenge for producers and aquatic veterinarians in charge of maintaining the health status of valuable finfish . ResearcBlood parameters are essential indicators of fish health and normal physiology. However, the blood is a complex physiological system, and parameters are sensitive to intrinsic and extrinsic factors, including, in part: fish species, developmental and maturation stage, sex, water chemistry, temperature, nutrition, and management systems , 19. ForThis study’s objective was to develop diagnostic references for normal ranges  of plasma biochemistry and hematology values and to characterize blood cell morphology of healthy cultured sablefish. Producers and aquaculture healthcare professionals will be able to use these RI as a guideline and a diagnostic tool.Mixed-sex sablefish were obtained from a captive brood-stock and rearing program conducted by the National Oceanic and Atmospheric Administration’s (NOAA) Northwest Fisheries Science Center (NWFSC) at the Manchester Research Station . The fish were reared for one year in land-based tanks with artificial recirculating seawater at the NWFSC  and transferred to NOAA’s Newport Research Station  on April 18, 2019. At the start of holding, the fish had an average weight of 186 g and grew to an average weight of 440 g on the final day of sampling. In Newport, the sablefish were reared in a 2-m diameter fiberglass tank in ca. 1500 L, with flow-through seawater at ca. 10–14.5°C. The salinity ranged from 29 to 33.9 ppt and dissolved oxygen ranged from 6.9 to 9.6 mg/L. The indoor tank was lit from above with a combination of natural (skylights) and artificial lighting. The artificial lighting was on a daily timer, providing 9 hours of light, turning on after sunrise, and turning off before sunset. Internal air temperatures ranged from 17.9 to 23.9°C.Fish were fed BioOregon’s Biobrood (4 mm) diet at least twice per day five days per week, at approximately 1% of body weight. This diet consists of marine fish oils and fishmeal protein. The feed’s composition, per manufacturer, includes 48% minimum protein; 20% minimum oil; 8.5% maximum moisture; 1.0% maximum fiber; 11% maximum ash; 18.2 MJ/kg digestible energy; and 60 ppm astaxanthin (a carotenoid). The sablefish were visually inspected daily Monday through Friday. There were no mortalities or signs of illness observed in the sablefish throughout the duration of the study.Pre-analytical procedures were consistent with methodologies applicable to health assessments in aquatic medicine . In brieAn aliquot of heparinized blood (1.5 mL) was centrifuged at 1200 x g at 14°C for 10 minutes. Plasma was harvested and transferred to a sterile 2 mL empty tube. Heparinized whole blood and plasma samples from each fish were stored in a chilled container and transported within 2–4 hours to Oregon State University Veterinary Diagnostic Laboratory in Corvallis, Oregon.Blood was collected from a total of 87 sablefish. However, data from 6 fish  were excluded from analysis due to grossly high lipid content in the blood that could potentially cause aberrant biochemical and hematological analyses and skewing statistical results. Therefore, reference intervals were ultimately determined from a total of 81 sablefish of the same lineage, age, and environmental conditions. Fish were collected on June 24 (2 fish), 25 (3 fish), 26 (3 fish), and July 5 (1 fish), 10 (32 fish), and 17 (40 fish), 2019. This study was reviewed and permitted by the Oregon State University Animal Care and Use Committee (ACUP #5154). Tricaine methanesulfonate (MS-222) was used for euthanasia.Heparinized whole blood and plasma samples were immediately processed upon receipt by the Veterinary Diagnostic Laboratory at the Oregon State University Carlson College of Veterinary Medicine in Corvallis, Oregon. The lab evaluated the biochemical and hematologic parameters.Plasma biochemical profiles were selected based upon prior studies in other fish species –24. BiocMeasurement of all hematologic parameters was performed manually within 24 hours of receipt. Heparinized whole blood was utilized to assess packed cell volume (PCV), total protein (TP), and manual cell counting. PCV was obtained by the collection of whole blood into hematocrit tubes and measurement post-5-minute centrifugation. Total plasma protein was evaluated by a refractometer .Manual cell counts were performed by two board-certified clinical pathologists (EG and JJ). Leukocyte and thrombocytes were stained by transferring 20 μL whole blood into 1.98 mL Natt-Herrick Solution  for a 1:100 dilution. The mixture was allowed to incubate for 20–30 minutes at room temperature then counted via Neubauer hemocytometer  using a standard protocol , 26, 27.Blood smears were submitted on glass slides and stained with Modified Wright’s stain (Thermo Fisher Scientific) via an automated method . Leukocytes were identified based upon characteristics described for other related marine species , 29, andBlood values were collected into Excel spreadsheets and reference intervals calculated with the macro add-in Reference Value Advisor V 2.1 software . The sofThe reference intervals for measured plasma biochemistry parameters in the 81 sufficiently fasted sablefish is presented in Hematologic parameters, including packed cell volume (PCV), plasma protein (PP), and cell counts, were collected manually from the 72 fish sampled on July 10 and 17, 2019. Thrombocyte counts were only available from 60 animals due to early technical difficulties with staining. All data are presented in Morphology of blood cells was similar to those noted in other fish species , 29, 32.In sablefish, lymphocytes predominated and comprised up to 98% of the total leukocytes present in healthy individuals. Lymphocyte morphology was similar to that of other species, and lymphocytes were identified as round cells with high nucleus to cytoplasm ratios (N:C ratios), round nuclei and condensed chromatin . Cell boMonocytes were larger than lymphocytes and similar in appearance to monocytes in other non-mammalian species Figs , 3 and 4This study addresses an urgent need associated with the development of a sustainable and highly profitable emerging aquaculture species. Establishing a sablefish aquaculture industry would contribute to the livelihoods of coastal (and inland) communities along the northern Pacific Ocean. Species-specific biochemistry and hematologic reference intervals (RI) derived from a healthy reference population are crucial for adequate health monitoring of any species, particularly species new to culturing. Management and feeding practices, and disease treatment regimens can be appropriately adjusted in correlation to physiological parameters of the fish determined from health monitoring. Blood cell counts are valuable tools in assessing the general health status of the fish and can alert a health care provider to infections and environmental or management issues, e.g., problems with water quality or nutrition.An important observation was that fasting sablefish for 24–36 hours was necessary to obtain blood samples with lipid content low enough to not interfere with plasma biochemistry or hematology analysis. High amounts of triglycerides in human blood has been found to significantly interfere with measurements of phosphorus, calcium, creatinine, and total protein due to an interaction with the analyzer and reagents . Plasma The fish used in this study received a commercial diet at 1% of their body weight distributed over at least two feeding events per day. Recent studies recommend feeding sablefish to satiation once every other day to allow for adequate feed conversion and consistent intake of the feed , 43. RedThe possibility exists that the observed affinity for high blood lipid concentration reflects the general tender consistency of the meat in this species, which gave rise to the colloquial name “butterfish”. However, the energy density of sablefish is relatively low compared with other pelagic fish from the North Pacific, and only low to moderate amounts of the wet mass are attributed to lipids . A musclAnimal husbandry and management can have a substantial impact on blood reference ranges. In a study of blood RI in sunshine bass, the only biochemical reference intervals that were similar in three different holding systems were glucose and cortisol . SimilarSex can also affect blood reference intervals . All-femS1 FigPhotograph of lipemic blood sample from fish #34 on June 20th, 2019, after centrifugation. A packed cell fraction (bottom of tube) is overlaid by a turbid, pale-beige plasma. The turbidity indicates a significant amount of blood lipids.(DOCX)Click here for additional data file.S1 AppendixData set includes lipemic samples, RI calculation reports for each biochemistry parameter, and a comparison of the RI of 87 fish (with lipemic samples) versus 81 fish (without lipemic samples), plus all conversions from the conventional units to the SI units.(XLSX)Click here for additional data file.


Actinobacteria and Firmicutes, while the second cluster contained a high prevalence of Fusobacteria.The aim of this study was to characterize the bacterial profile of semen collected from Holstein Friesian breeding bulls via a high-throughput sequencing approach for a 16S rRNA gene variability analysis. A total of 55 fresh semen samples of sexually mature breeding bulls were used in the study. They were gathered from Holstein Friesian breeding bulls at Slovak Biological Services in Nitra, Slovak Republic. To amplify the V4 region of the 16S rRNA bacterial gene, universal primers 515F and 806R enhanced by a 6 bp barcode identification sequence were used. The 16S rRNA high-throughput sequencing strategy was used. Two microbial clusters were identified among the analyzed samples—the first cluster was based on n = 55) were examined for the natural variability in the present bacteria. Semen doses were selected randomly from Slovak Biological Services in Nitra, Slovak Republic. The most predominant phyla within the whole dataset were Firmicutes (31%), Proteobacteria (22%), Fusobacteria (18%), Actinobacteria (13%) and Bacteroidetes (12%). Samples of semen were divided into two separate clusters according to their microbiome compositions using a cording partition around a medoids analysis. Microbiomes of the first cluster (CL1) of samples (n = 20) were based on Actinobacteria  and Firmicutes , while the second cluster  contained samples characterized by a high prevalence of Fusobacteria . Some important indicator microbial groups were differentially distributed between the clusters.Bacterial contamination of semen is an important factor connected to the health status of bulls that may significantly affect semen quality for artificial insemination. Moreover, some important bovine diseases may be transmitted through semen. Up to now, only a very limited number of complex studies describing the semen microbiome of bulls have been published, as many bacteria are hard to cultivate using traditional techniques. The 16S rRNA high-throughput sequencing strategy allows for the reliable identification of bacterial profiles of bovine semen together with the detection of noncultivable bacterial species. Fresh samples from Holstein Friesian breeding bulls ( Fertilization in the cattle industry is a complex of different multicomponent cascades and processes that are associated with various factors based on genetic, health and environment circumstances ,2,3. WitBacterioidetes, Actinobacteria, Proteobacteria, Firmicutes, Fusobacteria or Cyanobacteria by classical methods [Staphylococcus, Streptococcus, Mycoplasma, Pseudomonas, Corynebacterium or Bacillus may even be present in ejaculates of clinically healthy bulls [Bacillus cereus, Staphylococcus cohnii, Staphylococcus klosii, Micrococcus luteus, Bacillus licheniformis, Staphylococcus xylosus, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus lentus, Staphylococcus epidermidis, Bacillus mycoides and Staphylococcus haemolyticus [The microbiological checkup of ejaculates as well as doses used for artificial insemination (AI) have become an inevitable part of strategies to eliminate the prevalence of bacteriospermia, which is reported to oscillate in a quite wide range from 7% up to 99% of ejaculates ,11,12,13 methods . Some ophy bulls . Naturalhy bulls ,17 as wehy bulls ,19. An ahy bulls  as the bhy bulls ,22,23,24hy bulls . A previolyticus .Nevertheless, no core microbiome has been defined for bull semen. A common core microbiome characterizes the most widespread microbial taxa within a host population . HoweverThe aim of this study was to characterize the bacterial profile of semen collected from Holstein Friesian breeding bulls via a high-throughput sequencing approach for a 16S rRNA gene variability analysis.9 sperm/mL were processed further (n = 55).A total of 55 fresh semen samples of sexually mature breeding bulls were used in the study. Each sample was obtained from an individual bull, and the bulls were not mating with a cow. The samples were gathered from Holstein Friesian breeding bulls at Slovak Biological Services in Nitra, Slovak Republic, during winter and early spring of 2019 and 2020, respectively. The animals were 4–6 years old and were fed a standard diet based on green and cereal fodder, berseem, straw and concentrated mixtures. Water was supplied constantly. The animals were kept loose in individual enclosures with access to outdoor exercise. The animals were regularly examined by a veterinarian to ensure proper health conditions. To maintain the external sterility, the artificial vagina was sterilized before sample acquisition and high standards of hygiene were followed during the whole process of sample collection. Prior to semen collection, the animals were allowed to urinate and their external genitalia were properly washed to avoid contamination of the ejaculate. Single-use gloves were changed between each collection. The obtained semen samples were transported into the laboratory immediately in a thermos to maintain a constant temperature of 10 °C in the vials that were disinfected with absolute ethanol . For subsequent experiments, the vials were kept in a sterile Class II laminar flow hood. Only samples with at least a 70% motility and 1 × 10−1 of each primer and KAPA HiFi HotStart ReadyMix (1×)  in a SureCycler 8800 thermal cycler . The following thermal profile was used: 90 s of denaturation at 98 °C, 35 cycles  with a final elongation step of 120 s at 72 °C. The PCR products were checked on 2% agarose gels in a TBE buffer containing ethidium bromide and were purified with a PCR purification kit . Concentrations of the obtained PCR products were measured using a Qubit 2.0 Fluorometer . The DNA of the samples were adjusted to an equal concentration and pooled together. The adapters were attached via a Truseq LT PCR-free kit . A MiSeq Reagent Kit v3 (600-cycle)  was used for sequencing.Genomic DNA was extracted from the semen samples via a DNeasy UltraClean microbial DNA kit . The quantitative and qualitative parameters (A260 and A280) of the extracted DNA were analyzed via the Nanodrop NanoPhotometer . To amplify the V4 region of the 16S rRNA bacterial gene, universal primers 515F and 806R enhanced by a 6 bp barcode identification sequence were used ,37. PCR The obtained basic raw sequencing data were processed via the Seed 2 software  to identThe sequencing of the bacterial metagenomic amplicon libraries prepared for bovine semen samples resulted in 565, 035 reads after Q30 quality filtering, i.e., 10,273 reads per sample . After subsequent processing including denoising, chimera discarding and mitochondria removal, high-quality reads were assigned to 1206 ASVs. According to a rarefaction curve analysis and Good’s coverage, all samples contained a sufficient number of sequences and curves became saturated before 2000 reads. According to a prior diversity analysis, all samples were rarified to 2710 sequences, which was a minimal count per sample.p = 0.001). The differences between samples in these clusters are visible on all taxonomic levels; thus, we considered them as samples with truly separated types of semen microbiomes.A partition around medoids based on weighted unifrac distancies found two clusters to be the most suitable to describe the distribution of samples according to their ASVs . The strp = 0.003). Similar differences were observed in the Shannon’s index where the mean value of cluster 1 was 5.52 (SD = 0.74) while the value of cluster 2 was 4.60 (SD = 0.45). The samples in cluster 2 also showed a significantly lower Pielou’s evenness than cluster 1 (0.77 (SD = 0.06) and 0.86 (SD = 0.04), respectively).An analysis of the diversity indexes  confirmeA total of 16 phyla were detected in the semen samples  while onThe same five dominant phyla were found as a part of microbiota in human seminal plasma: Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Fusobacteria . This isActinobacillus and Bacteroides were detected in cluster 2 samples while Cutibacterium, Staphylococcus or Prevotella were more abundant in cluster 1  that are not usually used in animal breeding. As such, semen is often contaminated by microorganisms, and it is difficult to distinguish the source of certain members of the microbiome .The microbiome composition obtained in this study is similar to the preputial microbiome of bulls recorded by . The micBesides commensal bacteria present in bovine ejaculates, pathogens could potentially be transmitted via bovine semen while being an inevitable part of the whole microbiome in different portions. The most refereed potential bacterial pathogens transmitted via semen belong to the genera Campylobacter, Brucella, Leptospira, Coxiella, Histophilus, Ureaplasma, Mycobacterium, Chlamydia and Mycoplasma .Some members of the genus Campylobacter were detected in 5 of 20 samples from cluster 1 and in 9 of 35 samples in the cluster 2. However, their share of the total microbiome remains low, with a maximum frequency of 1% in sample HF144. Campylobacteriosis is reported as a venereal disease with a worldwide distribution . ReportiHaemophilus was detected in seven samples from cluster 1 and in 21 samples from cluster 2. A total of four ASVs were identified as Haemophilus, and ASV45 (99.5% similarity to Haemophilus paracuniculus according to BLAST) was found in 16 samples that belonged to cluster 2. Haemophilus was reported to be one of the most abundant bacterial genera in human semen samples along with Proteobacteria members . With reThe semen microbiome is reported to have an important impact on the qualitative parameters of sperm . DiffereIn the case of specific specimens, such as semen samples, it is still difficult to identify all bacteria using conventional microbiological techniques, particularly because, if the samples are cultured first, the colonies that are detected fully depend on the culture conditions. Furthermore, some bacteria are either slow or not growing at all, or may be in a competitive regime with other species ,57. MoreThe identification of noncultivable members of microbiomes relies on shotgun metagenomics or targeted loci (amplicon sequencing). The shotgun metagemic approach generates data that also describe the distribution of microbial functional genes, but it is significantly more expensive and requires an extensive bioinformatic analysis in comparison to amplicon sequencing. As 16S rRNA gene sequences constitute a base of the phylogenetic taxonomic system, practically all prokaryotic microorganisms, including the noncultivable ones, may be classified by an analysis of this gene. In the case of some species complexes, identification and classification should involve additional genes  to achieve a species level of resolution . On the Here, a typical bacterial community was described in the semen of healthy Holstein Friesian breeding bulls. Only limited information exists currently for this type of specimen. The analysis of 55 samples revealed the microbiome members typical of the urogenital tracts of bulls. Two different clusters were found among the samples. The first cluster is characterized by the presence of Firmicutes and Actinobacteria. The second cluster exhibits a high portion of Fusobacteria. More knowledge in this field may reinforce our understanding of the microbial communities typical of the bovine reproductive system and may help to manage assisted reproductive technologies in a more precise manner. 16S high-throughput sequencing seems to be a viable alternative to classical microbiological methods in the study of the bacterial composition of seminal fluid, not only in bulls but also in other species of domestic animals.


To examine the value of energetic-spectrum computed tomography  quantitative parameters in renal cell carcinoma (RCC) microvascular angiogenesis.The authors evaluated 32 patients with pathologically confirmed RCC who underwent triple-phase contrast-enhanced CT with spectral CT imaging mode from January 2017 to December 2019. Quantitative parameters include parameters derived from iodine concentration (IC) and water concentration (WC) of 120 keV monochromatic images. All specimens were evaluated including the microvascular density (MVD), microvascular area (MVA) and so on. The correlation between IC and WC  with microvascular parameters were analyzed with Pearson or Spearman rank correlation coefficients.1, WCD2, NWCD2 and ICD1 . The MVA of all tumors was (16.16 ± 8.98) % per field at × 400 magnification. The MVA of RCC correlated positively with the mean IC, mean WC, mean NWC, mean NIC, random IC, random NIC in renal cortical, mean WC and mean NWC in renal parenchymal phase, WCD1, WCD2, WCD3, NWCD2, and NWCD3 . Microvascular grading correlated positively with the mean NWC, mean NIC and random NIC in renal cortical phase, mean NWC in renal parenchymal phase, NWCD2, WCD3, NWCD3, NICD2 and NICD3 . As for tumor diameter (55.19 ± 19.15), μm, only NWCD3 was associated with it .The MVD of all tumors was 26.00 (15.00–43.75) vessels per field at × 400 magnification. The MVD of RCC correlated positively with the mean IC, mean WC, mean NWC, mean NIC, random IC, random NIC in renal cortical phase, WCDICD and WCD of spectral CT have a potential for evaluating RCC microvascular angiogenesis. MVD, MVA and microvascular grade showed moderate positive correlation with ICD and WCD. ICD displayed more relevant than that of WCD. The parameters of renal cortical phase were the best in three phases. NICD and NWCD manifested stronger correlation with microvascular parameters than that of ICD and WCD. Renal cell carcinoma (RCC) is a kind of tumor originated from renal epithelial cells, accounting for more than 90% of renal cell carcinoma, consisting of 2% of adult malignancies , 2. The Due to the formation of new microvessels, the structure of microvessels in malignant tumors is different from that in normal tumors. In malignant tumors, microvessels are curved and organized more irregularly. In addition, their walls are more fragile due to the weak connection among endothelial cells, other pericyte layers and basement membrane . These cIn recent years, spectral imaging has been introduced as a new application of dual energy CT and an advanced CT scanning technology . EnergetThe study was approved by the Institutional Ethics Committee of the First Hospital of Jilin University, Changchun, China. Written informed consent was obtained from related patients by email or letter. We retrospectively reviewed the Picture Archiving and Communication System for renal cell carcinoma patients who had undergone renal CT plain scan plus three-phase enhancement before surgery from January 2017 to December 2019. The inclusion criteria were as follows: 1) patients aged between 18 and 80 years old; 2) renal CT plain scan plus three-phase enhancement before surgery; 3) patients who had undergone CT performed according to our standard protocol; 4) patients who had received standard nephrectomy; 5) The interval between CT scan and surgery was no more than 2 weeks. When evaluating tumor angiogenesis index, in order to ensure the consistency of baseline level, the following inclusion criteria should also be met: 1) RCCs were all single without distant metastasis; 2) The maximum diameter of RCC was between 3 and 5 cm.The exclusion criteria were as follows: 1) Imaging data with poor quality due to body movements or other artifacts were excluded; 2) Patients who were found to be allergic to iodine contrast agent before enhanced CT examination were excluded; 3) Patients who are RCC complicated with hemorrhage were excluded; 4) Cystic RCC and RCC with large cystic lesions were excluded to ensure the same baseline level of tumor angiogenesis index.Renal three-phase enhancement CT was performed on a Revolution GSI CT scanner . Scanning range was conducted from “diaphragmatic apex to lower pole of kidney” with supine position. The CT scan was conducted with the following parameters: tube voltage, automatically selected by machine according to the patient’s weight; tube current, 485 mA; pitch, 0.984; field of view (FOV), medium; image matrix, 512 × 512; rotation speed, 0.6; slice thickness/gap, 5/5 mm. Renal three-phase enhancement scan: high pressure syringe was utilized to inject non-ionic contrast agent iohexol or iopatol through elbow vein, with the dosage of 1.0–1.5 ml/kg and the speed of 3.5 ml/s. The cortical phase scanning was performed at 25 s after injection, renal parenchymal phase scanning was performed at (60 ± 5) s, and excretion phase scanning was performed at (180 ± 30) s after injection. During the scanning, the patient was required to hold his breath after deep inspiration to avoid motion artifacts affecting the image quality.ao) at the same slice of the lesion was obtained. Normalized iodine concentration (NIC) was normalized to the ICao using the formula NIC = IC/ICao. Likewise, the normalized WC (NWC) was obtained with the formula NWC = WC/WCao. (N)WCD1/ICD1 = The difference of (N)WC/IC between renal cortical phase and parenchymal phase. (N)WCD2/ICD2 = The difference of (N)WC/IC between renal parenchymal phase and excretory phase. (N)WCD3/ICD3 = The difference of (N) WC /IC between renal cortical phase and excretory phase. The above measurements were conducted independently by three radiologists with more than 10 years working experience. All the values were taken as the average of the three radiologists.The iodine and water concentration of renal three-phase enhancement CT were measured in Picture Archiving and Communication Systems . The IC of each tumor demands to take the average of six values on the decomposition images for iodine (120kev). These six values include four values in the axial maximum plane and two values in the coronal maximum plane. The four measurement values of the maximum axial plane include the measurement values of the directions in front, back, left and right. The region of interest (ROI) of these four directions should not cross each other and keep as far as possible to avoid obvious necrosis. It’s required to keep the area of ROI consistent. The WC of each tumor was obtained on the water equivalent images in the same way as above. The iodine concentration in the aorta  for 3 times. After DAB staining, the nuclei were stained with hematoxylin. Finally,the slide was sealed.Five sections were offered for per patient, the average value of microvessel density, area and grading was taken from the five sections. As long as no less than one slice has characteristic blood vessels, the characteristic blood vessels can be defined as positive.1) Mean microvessel density (MVD): After staining, the high vascular density area was found under low power microscope. The top five regions with the higher vascular density in each slice were selected. The number of microvessels in each field of vision was counted. The average value of the five regions is calculated as mean MVD. When counting, only a single endothelial cell or cell cluster is counted, otherwise the vascular lumen is not counted as microvessel.2) Microvascular area (MVA): Percentage of microvessel area in tumor tissue area.3) Microvascular classification: Punctiform microvessels displayed that the microvessels were divided by tumor cells and distributed in punctiform shape, with no communication between the microvessels. The linear microvessel manifest that there were punctate microvessels on both sides of the microvessel, with the middle long and narrow, which was linear. The annular microvessels are characterized by thick microvessels, which encircle the tumor cells in an island shape and communicate with each other. Strip type microvessels showed thick microvessels and paralleled to tumor trabeculae, communicating with each other as well as showing a thick network. The tumor microvascular classification was semi quantified: the punctiform type was grade I, the linear type was grade II, and the annular or strip type was grade III Fig. . The mic4) Analysis of special vascular structure: Record whether there are vascular lakes, vascular trees (One main vessel accompanied by branch vessels), thick muscular vessels or vessels with diameter more than 50 μm appearing.P < 0.05 was considered statistically significant.Pearson’s or Spearman’s correlation test was applied to test the correlation between WC and IC values  with microvessel parameters . All statistical analyses were performed using SPSS 21.0 (SPSS). p < 0.001). Table 1, ICD2, NICD2, NICD3, NWCD2 and NWCD3) are skew distribution.A total of 32 patients with RCC were included in the study. The nuclear grade of RCC was positively correlated with MVD , as shown in Table p < 0.05), as displayed in Table p < 0.05), and the correlation between the mean NIC and microvascular grading was stronger than that between the mean NWC with microvascular grading.The correlations between the WC/IC in renal cortical phase and microvessel parameters were analyzed. The mean WC, mean IC, mean NWC, mean NIC, random IC and random NIC from renal cortical phase scanning correlated positively with the MVD of RCC . Besides, the correlation between the mean NWC and MVA was slightly stronger than that between the mean WC with MVA. At the same time, the mean NWC was also discovered to be positively correlated with microvascular grading .The relationships between microvascular parameters and WC/IC of renal parenchymal phase among the 32 patients enrolled in the study were presented in Table p > 0.05) were found.The correlations between the WC/IC indexes in renal excretory phase and microvascular parameters of RCC were demonstrated in Table 1, WCD2, and NWCD2 correlated positively with the MVD of RCC . And the correlation between WCD1 and MVD was higher than that between WCD2 and MVD. Moreover, the correlation between the NWCD2 was lower than that between WCD2 and MVD.The correlations between WCD indicators and microvessel parameters were exhibited in Table 1, WCD2, NWCD2, WCD3, and NWCD3 correlated positively with the MVA of RCC . According to the strength of association, the order is as follows: WCD2,>WCD3 > WCD1. The correlations between the WCD2 and WCD3 with MVA were stronger than those between the NWCD2 and NWCD3 with MVA.For MVA, the WCD3, NWCD2, and NWCD3 correlated positively with the microvascular grading of RCC . The correlation between the WCD3 and microvascular grading was weaker than that between the NWCD3 and microvascular grading. What’s more, DNWC3 was moderately correlated with the vessel diameter .The WCD1 correlated positively with the MVD of RCC . The NICD2 and NICD3 correlated positively with the MVD of RCC . The correlation between the NICD2 and microvascular grading (r = 0.397) was stronger than that between the NICD3 and microvascular grading (r = 0.383).The correlations between DIC indicators and microvessel parameters were displayed in Table The incidence rate of RCC has raised in recent years, ranking the seventh in the most frequently diagnosed malignancies . Renal cCT and MR perfusion are utilized to assess tumor angiogenesis. Nevertheless, perfusion parameters are not correlated to microvascular angiogenesis. Perfusion index (PI) reflects the ability of blood perfusion. The high PI of tumor tissue is not only related to the number of microvessels, but also related to that of large vessels. It may be the reason that microvascular parameters such as MVD sometimes don’t have obvious correlation with CTP and MTP parameters. Nevertheless, due to the limitation of exam methods, the above opinions need to be supplemented by new means. The minimum voltage (80kvp) and the maximum voltage (140kvp) are usually used to achieve the maximum energy separation to distinguish different substances. Spectral CT scan quantitatively analyze the iodine and water density of RCC by generating decomposition image Spectral CT can be combined with iodine contrast to calculate iodine concentration in lesions. It can be used to evaluate the microvascular parameters of various solid tumors including MVD, MVA, etc.In our study, iodine and water were utilized as the bascs material pair in material decomposition and were also used to measure IC and WC in kidney. The density of iodine and water in tumor can be quantitatively analyzed by spectral CT. the MVD, MVA and other microvascular parameters were evaluated on the basis of macroscopic imaging . Only thIC mainly reflects the blood supply of microvascular RCC in the renal cortical phase, while IC may indicate the flow and retention of blood supply in the renal medullary phase. It is worth noting that cortical phase enhancement is more prominent in RCC, suggesting that cortical phase IC is more useful for the detection of angiogenesis in RCC. The IC of renal cortical phase image reflects the density of tumor capillaries, and the difference of renal cortical phase and medullary phase image reflects the clearance of tumor capillaries and tumor matrix . In renaNIC and NWC were calculated in order to normalize the differences in individual circulation between patients. We discovered that the correlation between NIC and NWC with microvascular parameters was not significantly better than that of IC and WC. NIC and NWC did not show more stable as an index, which was slightly inconsistent with previous studies . Some st3. That is to say, in this study, the larger the tumor, the faster the water excretion from the cortical phase to the renal pelvis secretory phase. The present study showed that among the difference of cortical phase,medullary phase as well as medullary phase, the correlation between WC derived indexes and angiogenesis seems to be more significant than that of IC derived indexes. It is becuase the water outflow from tumor tissue is more easily affected by the changes of microvascular structure with the increase of microvascular area and the complexity of microvascular structure [It has been shown that the average iodine density of larger tumors is significantly lower than that of the smaller ones , which itructure . In addiSeveral limiting factors must be considered in this study. Firstly, the study included a relatively small number of RCC patients. We believe that our research may encourage future research, and a large multicenter study will be desirable. Secondly, the relationship between the prognosis of these RCC patients and IC and WC is worth exploring. Moreover, although pathologists and radiologists are particularly careful in sampling, it is difficult to match histopathological sections with imaging sections accurately. RCCs may show different characteristics in different perspects of view. In clinical practice, the ROI is delineated at the maximum level of axial and coronal tumor, also the average value of each measurement is calculated as the final result to minimize the systemic errors. Although strict inclusion and exclusion criteria were established, selection bias still existed due to the retrospective study. Further studies will be carried out with expanded samples. Finally, the correlation between WC and IC parameters on microvascular status is still in research stage, and it needs to be further verified in the follow-up study before applied in clinical practice.ICD and WCD of spectral CT have potential for evaluating RCC microvascular angiogenesis. MVD, MVA and microvascular grade showed moderate positive correlation with ICD and WCD. ICD displayed more relevant than that of WCD. The parameters of renal cortical phase were the best in three phases. NICD and NWCD demonstrated stronger correlation with microvascular parameters than that of ICD and WCD.


Gastro-esophageal (GE) cancers are one of the major causes of cancer-related death in the world. There is a need for novel biomarkers in the management of GE cancers, to yield predictive response to the available therapies. Our study aims to identify leading genes that are differentially regulated in patients with these cancers. We explored the expression data for those genes whose protein products can be detected in the plasma using the Cancer Genome Atlas to identify leading genes that are differentially regulated in patients with GE cancers. Our work predicted several candidates as potential biomarkers for distinct stages of GE cancers, including previously identified CST1, INHBA, STMN1, whose expression correlated with cancer recurrence, or resistance to adjuvant therapies or surgery. To define the predictive accuracy of these genes as possible biomarkers, we constructed a co-expression network and performed complex network analysis to measure the importance of the genes in terms of a ratio of closeness centrality (RCC). Furthermore, to measure the significance of these differentially regulated genes, we constructed an SVM classifier using machine learning approach and verified these genes by using receiver operator characteristic (ROC) curve as an evaluation metric. The area under the curve measure was > 0.9 for both the overexpressed and downregulated genes suggesting the potential use and reliability of these candidates as biomarkers. In summary, we identified leading differentially expressed genes in GE cancers that can be detected in the plasma proteome. These genes have potential to become diagnostic and therapeutic biomarkers for early detection of cancer, recurrence following surgery and for development of targeted treatment. Likewise, esophageal cancer is the seventh most common cancer and the sixth leading cause of cancer-related death. Each year more than 500,000 new cases of esophageal cancer are diagnosed and about 509,000 people die from it1. Despite improvements in surgical and radiation treatments and the availability of newer agents, the prognosis of patients with recurrent GE cancers remains very poor4. The need for novel strategies to improve current therapy is therefore vital in the management of GE cancers.Cancers of the stomach and esophagus or gastro-esophageal (GE) cancers represent a highly aggressive disease and are one of the major causes of cancer-related death in the world. Stomach cancer is the fifth most common cancer and the third leading cause of cancer-related death worldwide. For example, in 2018, more than 1 million new cases of stomach cancer were diagnosed and about 783,000 people die from it5. Comparative assessment of genetic aberrations between cancerous and matched normal tissues as control, has facilitated identification of new biomarkers that may also serve as new therapeutics targets or predict various cancer-related outcomes. There are several known biomarkers that are associated with tumorigenesis or have prognostic and predictive values in patients with stomach and esophageal cancers10. For example, approximately 20% of stomach and gastroesophageal junction cancers are associated with the amplification of the HER2 gene that is an important therapeutic target and predicts response to trastuzumab8. Most biomarkers such as TP53 or CDH1, however, have limited therapeutic and predictive values and presence or absence of them does not alter treatment strategies9. There is a strong unmet need for novel biomarkers in the management of GE cancers to identify new therapeutic targets and to yield predictive response to the available therapies.It is well known that cancer development and progression are triggered by altered activities and dysregulated expression of genes that control cell proliferation and differentiation12. We also applied machine-learning approaches to test our predictive accuracy. Overall, the purpose of these analyses is to identify differentially expressed novel tumor-specific genes that code for the plasma proteins and use this information to develop blood-based prognostic biomarker studies in the near future.We conducted this study by intersecting the gene expression profiles from The Cancer Genome Atlas (TCGA) with the plasma proteome databases to identify leading genes that are differentially expressed (upregulated or downregulated) in patients with esophageal and stomach cancers11. Overall, gene expression data for 415 independent tumor samples and 35 matching normal tissue samples for STAD and for 185 ESCA cases with 11 matching normal tissue samples were available. We also downloaded the plasma proteome database from http://www.plasmaproteomedatabase.org. The database contained information on 1241 protein-coding genes, while gene expression profiles from TCGA mapped to 1232 protein-coding genes. To analyse plasma proteome genes, we used non-parametric Mann–Whitney-U test to identify genes that are expressed at significantly different levels (p < 0.05) in cancerous and normal tissues. The deregulated genes were grouped according to tumor stages based on the available patient data. This allowed identification of genes with expression significantly increased or decreased at multiple stages of cancer.We obtained the level-3 HiSeq RSEM gene-normalized RNA-seq gene expression data for stomach adenocarcinoma (STAD) and esophageal carcinoma (ESCA) from the TCGA databaseGene co-expression networks were used for analyzing the importance of genes and their relationships with other genes. In a weighted gene co-expression networks (WGCN), nodes represent the gene expression profiles, edges represent the pairwise correlation between gene expressions while the edge weights represented the correlation strengths. For our study, the correlation strength of a pair of genes was measured by their similarity, which was calculated by the Pearson correlation coefficient (PCC) between their expression profiles. Specifically, for each pair of genes 13, that is commonly used in recent studies15, to construct the weighted network for the stomach and esophageal cancer datasets. To filter out the noisy edges in WGCNs, we applied the soft thresholding scheme13 by raising the co-expression similarity to a soft power 13. The scale-free networks were constructed because they have strong ability to tolerate against errors16. We then removed the self-loop edges of nodes by setting the diagonal elements of the adjacency matrix as 0. Also, we applied a hard threshold to remove weak edges between nodes by setting the threshold as 0.01.We used WGCNA R package17. This closeness centrality is the inverse of the average shortest-path distance from one node to other nodes in the network. It also indicates the efficiency of one node to spread information through a network. Finally, based on the closeness centrality score of genes in networks, we defined a novel metric of gene importance in networks as the ratio of the closeness centrality (RCC) of a gene in its corresponding similarity network and its dissimilarity network, i.e.Next, we calculated the closeness centrality measures to identify the closeness of a particular node to all other nodes in a network18. The ROC curve is an evaluation metric for binary classification problems, which visualizes the trade-off between true positive rate (TPR) and false positive rate (FPR). We then measured the area under the curve (AUC), as higher the AUC, the better the performance of the model in distinguishing between normal and tumor samples.To test the significance of the differentially regulated genes, we constructed a support vector machine (SVM) classifier with linear kernel. The features were based on the leading upregulated genes, the leading downregulated genes and a set of randomly selected genes for each cancer type. Accordingly, we used Receiver Operator Characteristic (ROC) curve as the evaluation metric for the classification of cancer patients and normal samples as in previous studiesWe examined gene expression of 1232 protein-coding genes that were detected in plasma proteome in tumors of 185 patients with esophageal cancer and in the matching tissue of 11 subjects with no cancer. Among cancer patients, 18 (9.7%) had stage I tumors, 78 (42.2%) had stage II disease, 56 (30.3%) had stage III disease, and 9 (4.9%) had stage IV cancer. In 24 (13%) patients, cancer stage was not known. The comparison between esophageal tumors and healthy tissue showed BIRC5 (p = 2.61E−08), APOC2 (p = 3.23E−08), CENPF (p = 4.38E−08), STMN1 (p = 5.74E−08), and HNRPC (p = 8.21E−08) to be five leading genes overexpressed in esophageal cancer  had stage I cancer, 123 (29.6%) had stage II disease, 169 (40.7%) had stage III disease, and 41 (9.9%) had stage IV cancer. In 25 patients (6%) with stomach cancer, the disease stage was not known. Comparison between normal stomach tissue and stomach tumors showed that CST1 (p = 3.97E−21), INHBA (p = 9.22E−20), ACAN (p = 1.08E−19), HSP90AB1 (p = 2.62E−19), and HSPD1 (p = 3.91E−19) were the leading five genes that were overexpressed in stomach cancer , PNP (p = 0.007), SERPINB8 p = 0.042) and EHD1 (p = 0.046). In patients with stage II disease, MSN (p = 0.003), KRT5 (p = 0.004), TNC (p = 0.007), and NAP1L4 (p = 0.018) were overexpressed compared with other stages of the disease. In patients with stage IV esophageal cancer CYCS (p = 0.014), PON3 (p = 0.14), ACPP (p = 0.047), and RPL22 (p = 0.047) were significantly upregulated compared with patients with early-stage cancer. We did not notice a stage-specific upregulated gene in stage III esophageal cancer , AR (1.01E−07), CKB (1.17E−07), ADH1B (1.79E−07), and NCAM1 (2.15E−07) Fig. A. We didIn patients with stomach cancer following five genes were most significantly downregulated: GPX3 (1.65E−19), CLEC3B (5.70E−19), CFD (5.68E−18), GSN 4.5 IE−17), and CCL14 (1.12E−16)  of the identified biomarkers between normal and tumor samples for both cancer types and found them to be significant (p < 0.05). Based on these results, we are confident on our predictive accuracy of these genes as potential biomarkers.We next sought to evaluate the predictive accuracy of the leading candidates as potential biomarkers in GE cancers. Towards this, we calculated the similarity and dissimilarity matrices. These matrices were used to construct the co-expression network with weighted co-expression network analysis (WGCNA)Since the sample sizes of the esophageal and stomach cancer datasets were small, a fivefold cross validation was adopted to evaluate the performances of SVM models on each dataset. The corresponding mean ROC curves of 50 executions of fivefold cross validation are illustrated in Fig. 5. We noted that compared to normal tissue, BIRC5, CENPF, STMN1, APOC2, and HNRPC were the five most significantly upregulated genes in esophageal cancer. Furthermore, these genes were also overexpressed in stomach cancer.Our investigation identified leading genes that are upregulated or downregulated in patients with GE cancers. We specifically focussed on those genes whose protein products can be detected in the plasma, as measured in the plasma proteome database. Thus, our investigation has a direct translational impact. The abnormal gene expression plays a pivotal role in tumor development and progression21. CENPF gene encodes centromere protein F that associates with the centromere–kinetochore complex. CENP-F protein is thought to be a cell cycle regulated protein that may play a role in chromosome segregation during mitosis. Interestingly, there is evidence that CENPF expression is associated with inferior outcomes in patients with esophageal cancer and patients with lower CENPF expression had a better survival rate compared with those with higher CENPF expression22. The CENPF gene is also amplified in other solid tumors including hepatocellular and breast cancers and correlates with patients’ outcomes25. As cancer cells undergo active division, perhaps the up regulation of genes like BIRC5, CENPF could be a direct consequence of active mitosis.The baculoviral IAP repeat containing 5 (BIRC5) gene, also known as survivin, is a member of the inhibitor of apoptosis (IAP) family, where it encodes regulatory proteins that prevent apoptotic cell death. Survivin localizes to the mitotic spindle and participates in regulating mitosis. In addition to GE cancers, it is highly expressed in various other malignancies and is associated with poor outcomes including a shorter survival period27. There is some evidence that it may have a prognostic significance in the early-stage gastric cancer. For example, a study that evaluated STMN1 role in both operable and advanced gastric cancers showed that in the operable cohort, STMN1 expression correlated with cancer recurrence, and resistance to adjuvant therapies28.STMN1 belongs to the stathmin family of genes and encodes a cytosoplasmic phosphoprotein stathmin 1. The encoded protein belongs to the family of microtubule-destabilizing proteins that control the assembly and disassembly of the mitotic spindle and thereby, regulate mitosis. Similar to esophageal cancer, STMN1 is highly expressed in various cancers, including leukemia, breast, prostate and lung cancer, and is a promising target for cancer therapy30. HNRNPC gene encodes a protein that belongs to the subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs). The hnRNPs are RNA binding proteins and are associated with pre-mRNAs in the nucleus. These proteins are involved in pre-mRNA processing and other aspects of mRNA metabolism and transport along with cell proliferation and differentiation31. However, functions of hnRNPs in tumorigenesis and cancer progression in solid and hematological malignancies are not well understood32.In contrast to the relatively known roles of BIRC5, CENPF, and STMMN1 in malignancies, functions of APOC2 and HNRNPC genes in cancer cell are less well defined. The APOC2 gene encodes a lipid-binding protein that belongs to the apolipoprotein family and is a component of the very low-density lipoprotein. This protein activates the enzyme lipoprotein lipase, which hydrolyzes triglycerides. APOC2 mutations could cause hyperlipoproteinemia type IB, characterized by hypertriglyceridemia, xanthomas, and early atherosclerosis33. CST1 has also been considered as a potential tumor marker in various epithelial malignancies34. Of note, a study involving patients with esophageal squamous cell carcinoma whose tumors express high levels of Cystatin SN showed favorable survival compared with those patients with low Cystatin SN expression35. Inhibin-βA (INHBA), a ligand belonging to the transforming growth factor-β superfamily, is associated with cell proliferation in cancer. INHBA is overexpressed in various types of cancers including esophageal and stomach tumors37. Overexpression of the INHBA gene is considered a useful independent predictor of outcomes in patients with gastric cancer after the curative surgery. High INHBA gene expression has shown to be associated with significantly poorer 5-year overall survival compared with low expression cases in patients with stomach cancer37. HSPD1 and HSP90AB1 belong to heath shock protein (HSP) group and encodechaperonin family proteins38. HSPD1 encodes a mitochondrial protein, which is important for assembly of imported proteins in the mitochondria and may function as a signaling molecule in the immune system. HSP90AB1 is thought to play a role in gastric apoptosis and inflammation. HSPs control a wide variety of signaling and cellular responses and have been classified into several subfamilies such as the HSP60s, HSP70s, HSP90s, and HSP100s39. HSP expression often correlates with patient prognosis in various malignancies42 For example, HSP60 has been identified as an independent prognostic factor for both overall survival and recurrence-free survival in patients with early-stage stomach cancer42. The ACAN gene is a member of the aggrecan/versican proteoglycan family. The encoded protein is an integral part of the extracellular matrix in cartilaginous tissue, and it withstands compression in cartilage. Mutations in this gene may be involved in skeletal dysplasia and spinal degeneration, however, its role in cancer is not well understood.Our analysis showed that compared with normal stomach tissue, CST1, INHBA, ACAN, HSP90AB1, and HSPD1 were the leading five genes that were overexpressed in stomach cancer. These genes were also upregulated in patients with esophageal cancer. The CST1 gene encodes a secretory peptide called Cystatin SN, which is a cysteine proteinase inhibitor. Cysteine proteases are involved in tissue remodeling during development, and they support the migration of cancer cells. CST1 itself is known to promote proliferation, clone formation, and metastasis in breast cancer cells and high CST1 expression is negatively correlated with breast cancer survival43. Its role in tumorigenesis and progression has not been elucidated yet. The androgen-receptor (AR) gene encodes AR. Once AR binds its hormone ligand testosterone, it translocates into the nucleus, and stimulates transcription of androgen responsive genes44. In vitro evidence suggests a significant influence of sex hormones upon cancer growth45. For example, AR pathway plays an important role in the development of prostate cancer and various other epithelial malignancies including bladder, kidney, lung, breast, liver and ovary45. However, AR role in GE cancers development and progression is not known46. CKB or creatinine kinase B gene encodes a cytoplasmic enzyme that is involved in energy homeostasis. Its dysregulation could promote cancer invasiveness and progression47. Similar to AR, its disease modulating effect in GE cancers is unknown. ADHIB encodes alcohol dehydrogenase 1B enzyme. Evidence suggests that genetic polymorphisms of this enzyme has been associated with the increased risk of the aerodigestive cancer triggered by alcohol consumption48. The NCAM1 gene encodes a cell adhesion protein, a member of the immunoglobulin superfamily that is involved in both cell to cell and cell to matrix interactions. Its downregulation has been linked to cancer progression and development of metastases in gastrointestinal and other malignancies49.With respect to downregulated genes, C16orf89, AR, CKB, ADH1B, and NCAM1 were the leading downregulated genes in patients with esophageal cancer. C16orf89 is predominantly expressed in the thyroid gland and is involved in the development and function of the thyroid50. Consistent with our observation of its downregulation, promoter hypermethylation and downregulation of GPX3 in melanoma, stomach, head and neck, cervical and lung cancers suggest that GPX3 serves as a tumor suppressor in these cancers51. C-Type Lectin Domain Family 3 Member B (CLEC3B) is a member of the C-type lectin superfamily that encodes tetranectin. Dysregulation of CLEC3B has been reported in various epithelial cancers including stomach cancer53. Chen and others using TCGA database also noted downregulation of CLEC3B in stomach cancer. However, when they evaluated 328 patients with early-stage stomach cancer, high intratumoral tetranectin level was significantly associated with tumor invasion, lymph node metastasis, advanced TNM stage, and a shorter overall survival53. CFD or complement factor D encodes a serine protease that catalyze breakdown of factor B a rate limiting step of alternative pathway of complement activation. Impaired balance of complement activation could promote inflammation and tumorigenesis resulting in malignant cells proliferation, migration, invasiveness and metastasis55. GSN or Gelsolin gene encodes a protein that is involved in assembly and disassembly of actin filaments. Gelsolin has been attributed in prostate tumorigenesis and malignant transformation57. The C–C type chemokine 14 gene is known to induce targeted cell migration and is thought to play a role in carcinogenesis and metastasis of certain malignancies including breast cancer59.In patients with stomach cancers, GPX3, CLEC3B, CFD, GSN, and CCL14 were the leading five genes that were most significantly downregulated. GPX3 encodes glutathione peroxidase that belongs to a family of selenocysteine-containing redox enzymes that play important roles in cell signaling and immune modulation60. Conversely, AHNAK can act as a tumour suppressor gene and mediates the negative regulation of cell growth61.Aside from the leading upregulated and downregulated genes in patients with GE cancers, we also noted a stage-wise upregulation of several genes, such as APOC2, IL8, RNASE2, SERPINE1, and CETP, and stage-wise downregulation of other genes that play important role in and survival, including AHNAK, MEGF8, MMRN1, PROC, REG1A, SECTM1, TNFRSF10C, and TPPP3. The stage-related expression of these genes suggests their potential role in the disease progression and utility as monitoring markers or therapeutic targets. The cholesteryl ester transfer protein (CETP) for example maintains cholesterol homeostasis and has been identified as a potential target for estrogen positive breast cancer64. However, deep learning networks was not adopted in this study. This is primarily because, deep learning algorithms are non-linear and normally has millions of parameters65. Since the aim of our study is to identify key biomarkers for GE cancers, the explanation of model is crucial to evaluate the significance of genes. Contrarily, the classical machine learning models, such as linear models, provide a direct relationship between features and their prediction which makes it relatively straightforward to reason the decision mechanism of the model. Also, to avoid overfitting, more data are needed for the training of deep learning models. Moreover, the variations of the training data are necessary to construct a robust model. As the dataset sizes are not large in this study, the deep learning model will result in overfitting, if we apply deep learning to these datasets.Furthermore, we also evaluated the predictive accuracy and the significance of the genes we identified. In recent years, deep neural networks have achieved enormous successes for such applications7. Furthermore, we did not have information on histopathology of these cancer types and therefore, were not able to segregate the data based on histopathology. Finally, while we examine the up/down regulated genes, solely from the perspective of their differential expression, it will be interesting to investigate these candidates in cohorts of immunodeficient patients as it will provide additional knowledge on how these candidates may promote adaptive alterations of host gut- and tissue-based microbiome66. In summary, the present study identified leading upregulated and downregulated genes in GE cancers. Since expression of the upregulated genes was minimal in both stomach and esophageal normal tissues, these genes have a strong potential to become diagnostic and therapeutic biomarkers for screening and early detection of cancer, recurrence following surgery and for anti-cancer therapies. Future studies will be required for validating diagnostic, therapeutic and prognostic importance of these genes. Our group plans to prospectively evaluate prognostic and predictive values of selected genes in a cohort of patients with metastatic gastroesophageal cancer who are treated with combination chemotherapy.While our work provides a significant amount of novel information regarding the behavior of cancer-related molecules in GE cancers, it does not assess the level of gene expression based on the molecular classification of stomach and esophageal cancersSupplementary Information 1.