Assessment of the efficacy of a multi-agent chemotherapy protocol in which cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) are administered in canine lymphoma is generally performed by physical measurement of lymph node diameter. However, no consistent correlation has been made with prognostic indicators and the length or absence of clinical remission based on lymph node size. RNA disruption measured mid-therapy has been correlated with increased disease-free survival in recent studies of human cancer and was assessed in this study of canine lymphoma patients. Fine needle aspirate samples were taken before treatment and at weeks 3, 6, and 11 of CHOP therapy. RNA was isolated from these samples and assessed using an Agilent Bioanalyzer. RNA disruption assay (RDA) analysis was performed on the data from the resulting electropherograms.An increased RNA disruption index (RDI) score was significantly associated with improved progression-free survival.Predicting the risk of early relapse during chemotherapy could benefit veterinary patients by reducing ineffective treatment and could allow veterinary oncologists to switch earlier to a more effective drug regimen. Canine high grade nodal lymphoma, the most common haematopoietic neoplasm in dogs , is routA number of studies have explored methods to predict outcome in canine lymphoma including refining diagnostic staging methods using cytology and sonography to evaluate the liver and spleen , measureThe desire to identify prior to or early during treatment those cancer patients that will derive a survival benefit from chemotherapy is common to both veterinary and human medicine. Approximately 20% of breast cancer patients treated with neoadjuvant chemotherapy (chemotherapy prior to surgery) achieve a complete response as assessed by histopathology and derive a survival benefit from chemotherapy ; yet, alIn this study, we investigated RNA disruption as a predictor of remission duration in canine lymphoma to understand if RNA disruption occurred and had clinical utility outside of breast cancer; tumour RNA disruption in human lymphoma patients has not yet been investigated. RNA disruption was measured in fine needle aspirate (FNA) samples of tumour tissue obtained at four time points  during CHOP therapy. This protocol permitted assessment of how early in treatment RNA disruption may be measured and how tumour RDI values change over time within a given patient. We then analysed RDI values in relation to clinical response and the progression free interval.Forty-one dogs fulfilled the inclusion criteria. Assessment of the clinical response during CHOP therapy found that 31 dogs achieved a CR for at least one timepoint, 4 dogs had only aPR, 1 dog had a PR which was followed by PD, 4 dogs had only PD, and 1 dog had only SD. Five dogs that initially had a CR relapsed during the course of CHOP therapy; the breakdown of all dogs by clinical response during treatment is shown in Table Summary of clinical response assessment of all dogs at weeks 2, 5, and 10 during CHOP therapy grouped by either consistent response throughout therapy or change in response during therapy.p-value = 0.04; likelihood ratio test).RNA concentrations and RDI values were determined for FNA samples taken pre-therapy and at approximate timepoints of 14, 35 and 70 days. A decrease in biopsy RNA concentration was found upon chemotherapy treatment which corresponded with clinical response measured at the same time point. A linear mixed effects model was fitted to the data using RNA concentration and clinical response as covariates. The median RNA concentration was about 10 fold higher from samples taken when the clinical response was determined to be progressive disease as opposed to those taken when there was a complete response )  were assessed and compared with the results of cytology measured in parallel samples acquired at identical timepoints.p-value = 0.008) and higher RNA concentration . Average RDI value and average RNA concentration for each neoplastic cell score (NCS) is shown in Table n = 5); however, the absence of neoplastic cells in these samples was noted as “No Diagnosis”. These samples also had overall cellularity scores of “0”, reflecting a complete absence of all cells. No conclusions can therefore be made on RDI values and a neoplastic score of “0” from this dataset.We then examined if there was a significant relationship between RDI value and the neoplastic cell score as determined by cytology. Two weighted linear regression models were fitted to the observed data (one for RDI and one for RNA concentration), and the hypothesis tests for the relationship between RDI/RNA concentration and cellularity were conducted. A higher proportion of neoplastic cells was significantly associated with lower RDI values , 1(1–24%), 2(25–49%), 3(> 50%). The RDI values and RNA concentrations were then grouped by neoplastic cell score and the average value, standard deviation and the range of values were generated.n = 22, T cell lymphoma n = 6) and the generated RDI values for all B cell lymphomas and all T cell lymphomas during CHOP therapy were graphed . As expected, the dogs with T cell lymphoma in this study were 5.7 times more likely  to have disease relapse compared to dogs within the B cell group .Immunophenotype was determined for 28 out of the 41 dogs in the study (B cell lymphoma n = 16), dogs that had a CR and subsequently relapsed during CHOP therapy (n = 9), and dogs that progressed during CHOP therapy (n = 6)  . The relative risk of relapse was lower for dogs with higher maximum tumour RDI compared to dogs with lower maximum tumour RDI. Dogs with a maximum RDI ≤0.7 had 4.1 times the risk of experiencing a relapse compared to dogs with maximum tumour RDI > 0.7 . These RDI cut points of 0.6 and 0.7 were then used to generate the Kaplan-Meier plots shown in Fig. p = 0.006). Similarly, as shown in Fig. The Kaplan-Meier plot in Fig. The utility of RDA in providing additional information to predict PFS was examined by comparing clinical response during CHOP therapy (standard of care) to the RDI values obtained during therapy. Clinical response was effective in predicting PFS for most dogs undergoing CHOP therapy, especially for those that responded well to treatment and those that showed no response. However, RDI values may be a useful discriminator in some cases. We identified three dogs that had complete clinical responses during treatment and yet relapsed before 6 months. The RDI values for these dogs fell below 1.4 at all time points and below 0.9 at time points up to 6 weeks, which included early-treatment and mid-treatment values  to evaluate how early in treatment RNA disruption may be measured and how tumour RDI values change over time within a given patient. RNA disruption, as measured by increases in tumour RDI value, occurred during CHOP therapy in FNA samples obtained from dogs with canine lymphoma. The increase in tumour RDI values from baseline can occur as early as two weeks after initiation of chemotherapy. It is often associated with ‘inadequate’ subsequent samples and is reflective of increased PFS. Although this pilot study was not powered to show statistically significant correlations at specific early time points, the clinical utility of RDA is in the assessment of RNA disruption early in therapy to allow veterinarians and owners to make decisions relating to the continuation or modification of chemotherapy.In this present study, adequacy rates for acquired samples were calculated at each time point, as RNA concentrations in a human breast cancer trial were found to be decreased at mid-therapy compared to pre-therapy samples . A subseIt is also likely that the extent of RNA disruption is relevant for outcome prediction. Narendrula et al. provided evidence that a threshold amount of RNA disruption was required for cell death . AlthougLymph nodes often have a number of non-neoplastic cells present which may not be susceptible to RNA disruption during chemotherapy. In this study samples with neoplastic cell scores of 2 and 1 had increased RDI values compared to samples with neoplastic cell scores of 3, suggesting that samples from tumours which are most actively responding to treatment have a larger amount of RNA disruption compared with samples from tumours that were not responding. The samples in this study with a complete absence of neoplastic cells were considered to be non-diagnostic and were devoid of all cellularity; no conclusions could be made on RDI values and neoplastic cell score of 0.While CHOP therapy is a standard protocol for canine B cell lymphoma, it has been reported to be less effective against T cell lymphoma . The ana. In vitro, RNA disruption was found to be initiated concurrently with apoptosis versus necrosis as indicated by Annexin-V and propidium iodide uptake measured by flow cytometry. Inhibition of caspase-3 activity significantly reduced RNA disruption in that study  and RNA concentration  in adequCytology slides were reviewed by a veterinary pathologist (DB) for the purpose of quantifying clinical response through estimation of tumour cellularity. Criteria evaluated by the pathologist for each sample included cellularity, cell size, cell integrity, neoplastic cells, anisocytosis, and the presence of red blood cells. Using these data, a morphologic diagnosis was made of either “Lymphoma” or “No Diagnosis” (due to insufficient nucleated cells). The proportion of neoplastic cells in cytosmears (‘neoplastic cell score’) was scored as 0 (0%), 1 (1–24%), 2 (25–50%), 3 (> 50%). All analyses were carried out in a blinded fashion; at the conclusion of the study RDI values and clinical data (including cytology and pathology results) were exchanged between OVC researchers and RnaDx personnel.Progression-free survival (PFS) time was calculated as the time from diagnosis to relapse. Statistical analyses included a linear mixed effects model and a likelihood test to assess RNA concentration and clinical response, weighted linear regression models and hypothesis tests for the relationships between RNA concentration/RDI value and neoplastic cell score. A Mann-Whitney test assessed the association of maximum RDI value with relapse timing (during or after CHOP therapy). A Cox proportional hazard model was used to assess the association of both maximum and average RDI values with PFS. Kaplan-Meier curves were generated for average and maximum RDI values and log-rank tests were used to compare median PFS.Additional file 1: Table A. 1. CHOP Therapy and FNA Schedule. Drugs and dosages of CHOP therapy by week with timing of FNA collection (taken prior to treatment).Additional file 2: Table A. 2. Dogs given Modified CHOP Regimen. Timing of the L-asparginase dose given as a modified CHOP regimen in four dogs and the resulting RDI values.


Background: Immune checkpoint inhibitors (ICIs) have evolved for the treatment of solid tumors. In addition to the efficacy of ICIs for cancer, the adverse events (AEs) of ICIs are also noteworthy for gradually more extensive clinical use.Objective: To conduct a systematic review and network meta-analysis to evaluate the treatment-related AEs that occurred in clinical trials using different kinds of ICIs, to explore the differences in AEs among ICIs for treating non–small cell lung cancer (NSCLC) and melanoma, and to compare select immune-related AEs.Methods: PubMed, EMBASE, Cochrane Library, ClinicalTrials.gov, and other available sources were systematically searched for published reports up to January 1, 2019. Two reviewers independently selected reports about phase II/III randomized controlled trials to compare among ICIs and between ICIs and chemotherapy. After the bias assessment of all included trials, a Bayesian network meta-analysis was performed. The primary outcomes were any-grade and high-grade treatment-related AEs from all ICIs. The secondary outcomes were AEs in patients with NSCLC and melanoma and the presence of the select AEs pneumonitis/pneumonia and colitis.Results: Eighteen randomized controlled trials containing 11,223 patients with NSCLC or melanoma were included. A total network meta-analysis was conducted. The meta-analysis showed that atezolizumab 1,200 mg and pembrolizumab 2 mg/kg every 3 weeks were generally more tolerable than other ICIs. ICI combined with chemotherapy might suggest a higher risk of treatment-related AEs than monotherapy with a single ICI, except durvalumab and ipilimumab. In the NSCLC subgroup, pembrolizumab was associated with a higher risk of high-grade AEs than nivolumab. In addition, ICIs  led to a lower risk of any/high-grade treatment-related AEs than traditional chemotherapy and ICI combination chemotherapy. However, ICIs did not present preferable safety and tolerability compared to chemotherapy in treating melanoma. Compared with chemotherapy, nivolumab, durvalumab, two ICIs, and ICI combined chemotherapy led to more pneumonitis/pneumonia. However, when treating NSCLC, different types of ICIs did not differ significantly regarding the incidence of pneumonitis/pneumonia. A combination of nivolumab and ipilimumab had the highest risk for colitis, while pembrolizumab and atezolizumab had a lower possibility than the other ICIs.Conclusion: Atezolizumab 1,200 mg and pembrolizumab 2 mg/kg every 3 weeks were ordinarily safer than other ICIs. When treating NSCLC, nivolumab had the lowest risk; when treating melanoma, pembrolizumab had the lowest toxicity. Since ipilimumab, an anti–cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) therapy, was approved by the Food and Drug Administration (FDA) in 2011, remarkable progress has been made in immunotherapy. As the first approved checkpoint inhibitor, ipilimumab is indicated only for melanoma. Another checkpoint inhibitor against the programmed death 1(PD-1)/programmed death ligand 1 (PD-L1) has also shown prominent success for patients with advanced solid tumors. In the National Comprehensive Cancer Network guidelines for non–small cell lung cancer (NSCLC) , an updaCompared with standard chemotherapy, immune checkpoint inhibitors (ICIs) showed great clinical benefits in prolonging the overall survival and progression-free survival for patients with solid tumors . This reAcknowledging the AEs caused by ICIs is necessary for better clinical management. In a study by The purpose of this study was to systematically review and conduct a network meta-analysis on the safety and toxicity of different ICIs in treating NSCLC and melanoma. The risks for select specific treatment-related AEs (colitis and pneumonitis/pneumonia) were also compared among these different treatment patterns.The present report was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and the PRISMA extension statement for network meta-analysis . Two autOnly randomized controlled clinical trials were included. Articles that met the following criteria were included: (a) phase II or phase III clinical trials on patients with NSCLC or melanoma; (b) studies with outcomes reporting of the rates of any all-grade and high-grade (3–4) AEs or treatment-related AEs that led to discontinuation or treatment-related death; and (c) at least one ICI as the intervention. It has been proven that autoimmune AEs occur, such as colitis, pneumonitis, skin AEs, endocrine dysfunction, and hepatitis . It was Two researchers (Q-QC and J-YD) independently conducted the data extraction. The following data were summarized: first author, title, year of publication, study ID, tumor site, trial phase, treatments, median follow-up time, version of the Common Terminology Criteria for Adverse Events, any AEs, treatment-related AEs, specific AEs, specific treatment-related AEs, treatment-related AEs leading to discontinuation, and treatment-related deaths.The qualities of the trials were ranked by the Jadad scale based on the original article, updated references and P < 0.05).The primary objective of this article was to compare the toxicity and AEs among all ICIs and standard chemotherapy. Additionally, the differences in AEs between patients with NSCLC and melanoma were studied. Pairwise meta-analysis (PWMA) was applied for direct evidence that was pooled in random-effects models if heterogeneity existed (I2 index (P > 0.05), pooled odds ratios/RRs and their 95% confidence intervals were reported in a fixed-effects model; otherwise, a random-effects model was applied.A total network was built containing all the included trials, and both direct and indirect comparisons were conducted. The consistency between the direct and indirect evidence was statistically confirmed by node-splitting analyses. The incidence of specific treatment-related toxicity, relative risk (RR) for any AEs, and odds ratio for high-grade AEs were calculated with 95% confidence intervals. When treatment-related AEs were not observed in the original studies, a relative index of any AEs that occurred during treatment was taken as a replacement. Heterogeneity among the trials was verified by the Cochrane Q statistic and quantified with the 2 index . When emSubgroups were created based on the cancer site, specific treatment-related AEs, and different ICIs. All analyses involved the use of the packages “gemtc” and “pcnetmeta” in R v3.5.1, and PWMA was conducted in Review Manager v5.3.The selection and exclusion criteria of the study are presented in Detailed characteristics of the included trials are shown in All relative outcomes of any-grade or high-grade treatment-related AEs in the NMA are presented in The patients were divided into NSCLC and melanoma subgroups. Group NSCLC involved 11 original studies with 7,033 patients, while the melanoma group involved 4,190 patients from seven articles. In the selected AE analyses, indirect comparisons were conducted on pneumonitis/pneumonia and colitis. The results suggested that nivolumab, durvalumab, two ICIs, and ICI combination chemotherapy would remarkably increase the risk of any-grade pneumonitis/pneumonia compared with chemotherapy. Avelumab was the only ICI that might be ranked higher (lower risk) than chemotherapy. However, the risks did not vary in the NSCLC subgroup among different ICIs.In the colitis analysis, ipilimumab and two ICIs (nivolumab + ipilimumab) had the highest risk of occurrence. In a sensitive analysis that ignored durvalumab and did not report the risk of colitis, we found that nivolumab combined with ipilimumab could cause more colitis than other ICIs. In general, pembrolizumab and atezolizumab had a lower possibility of leading colitis than other ICIs. All the outcomes are shown in I2 > 50%, P < 0.05). For the one direct comparison, obvious inconsistency existed between the network meta-analysis and direct comparison for durvalumab, which presented a drastically higher risk than chemotherapy for any- and high-grade treatment-related AEs in the PWMA.The node-splitting analysis indicated no significant inconsistencies except for the comparison between nivolumab and two ICIs Table 2.As the number of FDA approvals for ICIs increases, the indications for different ICIs have also expanded. However, different ICIs have distinct immunologic mechanisms and should not be taken as a whole category; even ICIs that belong to the same mechanism might lead to unlikely treatment effects and tolerability in different diseases . This reSeveral meta-analyses and network meta-analyses concerning the safety and tolerability of ICIs have been reported . These pIn our analysis, most clinical trials used chemotherapy as a controlled arm, and we performed direct and indirect analyses to compare all types of ICIs, not only head-to-head trials. This process was different from that of a previous meta-analysis, which only contained direct comparisons. More importantly, pneumonia and colitis (two specific AEs related to ICI treatment) were analyzed among different ICIs. This study indicated that ICI leads to more pneumonitis/pneumonia and colitis than chemotherapy. Avelumab has the lowest risk for pneumonitis/pneumonia among all comparators, including chemotherapy. Compared with pembrolizumab and avelumab, the combination of two ICIs (nivolumab + ipilimumab) might lead to a higher risk of any-grade pneumonitis/pneumonia  were reported in this review to identify the different immune-related effects.Limitations also exist in this analysis. Due to the nature of network meta-analyses, missing values always exist in published articles. In the current analysis, we conducted a comprehensive assessment of the evidence we collected and excluded low-quality evidence to improve the quality of this review. Second, some treatments  were adopted in only one clinical trial, which might lead to a biased evaluation without enough head-to-head evidence. Third, as the reported AE types were different among the original trials, the specific treatment-related AEs could not be completely evaluated. Thus, we focused on any-grade and high-grade treatment-related AEs as the primary outcome, which could suggest the overall safety and tolerability. Additionally, specific AEs related to ICIs for NSCLC and melanoma were selected to distinguish the differences between tumor types. Third, the incidences of immune-related AEs (including pneumonitis/pneumonia and colitis) were not high, especially those of serious lung toxicities and colitis . The lowIn summary, atezolizumab 1,200 mg and pembrolizumab 2 mg/kg every 3 weeks were generally safer than other ICIs. Nivolumab and pembrolizumab were safer for NSCLC and melanoma than other ICIs, respectively.Q-QC is the first author of this NMA, and she is responsible for the modification of this paper. J-YD is the second author. JZ is the third author. BW is the corresponding author, and he takes responsibility for the authenticity of the paper and also the modification.This work was sponsored by unrestricted grants from the National Natural Science Foundation of China (NO. 7172810).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.


Background: Once prostate cancer developed bone metastasis, the quality of life and prognosis of patients are seriously affected as no effective treatment is currently available. It is necessary to explore the mechanism of bone metastasis and new therapeutic targets.Purpose: To find out the differentially expressed serum proteins in prostate cancer patients with bone metastasis and analyze the expression of key proteins in prostate cancer tissues, serum and prostate cancer cell lines. So as to provide a basis for revealing the mechanism of bone metastasis and designing new therapeutic targets.Methods: iTRAQ-based proteomics method was used to compare serum differential proteins in prostate cancer patients with and without bone metastasis. Three key proteins  which had significant fold changes were selected to validate the results of mass spectrometry. Immunohistochemistry and ELISA were applied to tissues and serum samples from prostate cancer patients, respectively, for validation. Finally, western blot, flow cytometry, and immunocytochemistry were used to analyze the expression of the three differentially expressed proteins in the prostate cancer cell lines PC3, LNCap, and Du145.Results: Thirty-two differentially expressed proteins related to bone metastasis of prostate cancer were identified, of which 11 were up-regulated and 21 were down-regulated. CD59 and haptoglobin were up-regulated in prostate cancer with bone metastasis while tetranectin was down-regulated. Tetranectin showed differential expression in epithelial and stromal cells of prostate cancer and hyperplasia tissues.The expression of CD59 was highest in PC3 and lowest in LNCap, while the expression of haptoglobin and tetranectin was the highest in DU145 and lowest in PC3.Conclusion: Mass spectrometry analysis showed that there were more differentially expressed proteins in the serum of patients with bone metastasis than those without metastasis. It has been verified that CD59, haptoglobin and tetranectin are prostate cancer bone metastasis related proteins. Prostate cancer is one of the most common malignancies in Europe and the United States, with a mortality rate that is second only to lung cancer Bone metastasis of prostate cancer is a complex biological process affected by various factors. Bone is the third most common site of metastasis, second only to lung and liver, suggesting that bone/marrow has a favorable microenvironment for tumor growth In this study, isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic technology was used to analyze the differentially expressed proteins (DEPs) in the serum of prostate cancer patients with bone metastasis and non-metastasis. Three such DEPs with large fold changes were selected for further analysis and validation using immunohistochemistry and ELISA. We also analyzed the expression of the three DEPs in three commonly used prostate cancer cell lines. Our results provide new clues for further study of the role of these DEPs in the occurrence and development of bone metastasis of prostate cancer and for exploring possible therapeutic targets.Clinical samples and data collected comply with the regulations of China Medical Ethics Committee. All sample contributors had signed informed consent. The serum samples of 30 prostate cancer patients without metastasis and 30 prostate cancer patients with bone metastasis were collected from the urology department of Zhujiang Hospital of Southern Medical University. All of them were confirmed by prostate biopsy. No castration therapy was performed before blood collection in all patients. Bone metastasis were confirmed by single photon emission computed tomography and magnetic resonance imaging. The age of the patients ranged from 57-73 years, with the average age being 65. Five milliliters whole blood samples were collected without anticoagulant. Samples were stored in refrigerator at 4°C for 2 h and then centrifuged at 4°C for 10 min at 3000 rpm. The supernatant was collected and stored at -80°C.Prostate cancer with bone metastasis group and without metastasis group each consisted of 30 cases, giving 30 serum samples. Each group was divided into two subgroups, each subgroup consisting of 15 cases. The 15 serum samples of each subgroup were mixed together with equal volume to extract and purify the protein. The samples from cases with bone metastasis were labeled with iTRAQ114 and 121 and those without metastasis were labeled with iTRAQ116 and 118 respectively . Experiment was performed in two replicates, i.e. 4 repetitions in each group. Repeated manipulation begins at the stage of enzymatic hydrolysis and labeling of proteins.The serum samples were thawed and purified with ProteoPrep blue albumin & IgG depletion Kit . A 4-fold volume of acetone was added to the purified filtrate and then left for precipitation overnight at -20°C. The precipitated protein was centrifuged at 12000 rpm, for 20 minutes, at 4°C. The supernatant was removed and the pellet was dried. L3 lysis buffer (200 μL) was added to the dried protein lumps, and then dissolved by ultrasonic assistance, and centrifuged at 12000 rpm, for 20 min at 4°C. The supernatant was transferred to new microcentrifuge tubes and protein quantification was performed using BCA Protein Assay Kit .To 200 µg protein solution, 4 µL TCEP Reducing Reagent was added, and the reaction was incubated at 60°C for 1 h. Post-incubation, 2 µL MMTS Cysteine-Blocking Reagent was added, and the reaction was placed in 25°C for 30 min. The reduced alkylated protein solution was added into a 10K ultrafiltration tube  and centrifuged at 4°C for 20 min, after which the solution in the collection tube was discarded. Then, 100 µL 8 M urea was added and the bottom solution in the collection tube was discarded after centrifugation at 12 000 rpm for 20 min at 4°C; this step was repeated twice. Next, 100 µL of 0.25 M tetraethylammonium tetrahydroborate (TEAB) was added and samples were centrifuged at 12000 rpm for 20 min at 4°C, after which the solution in the collection tube was discarded; this step was repeated three times. The collection tube was then replaced with a new one, and 50 µL 0.5 M TEAB and trypsin (trypsin to protein mass ratio 1:50) was added into the ultrafiltration tube for overnight reaction at 37°C. Trypsin was added (trypsin to protein mass ratio 1:100) and reaction was kept at 37°C for 4 h and then centrifuged at 12000 rpm for 20 min at 4°C. After adding 50 µL 0.5 M TEAB into the ultrafiltration tube and centrifuging at 4°C 12000 rpm for 20 min, 100 µL samples were obtained at the bottom of the collection tube.Isopropanol (150 µL) was added to the iTRAQ reagents in a microcentrifuge tube, vortexed and centrifuged. Samples  were transferred to a new microcentrifuge tube and the iTRAQ reagent was added to the sample. After vortexing and a short spin, the samples were left at 25°C for 2 h, after which the reaction was terminated by adding 100 µL water. The labeled samples were then dried by vacuum freeze-centrifugation.2O, NH3 in H2O, pH = 10) and 5% buffer B  for 30 min. The dried iTRAQ-labeled peptide samples were then dissolved in 200 µL buffer A solution and loaded onto the Phenomenex column. Peptides were separated by a linear gradient formed from buffer A and buffer B, from 5% to 95% of buffer B at a flow rate of 0.2 mL/min. The whole elution process was monitored using 214 nm ultraviolet light and 15 fractions were collected from the linear gradient according to the peak shape. These fractions were analyzed by LC-MS/MS after vacuum drying.Before high-performance liquid chromatography , Phenomenex column  was equilibrated with 95% buffer A (HPeptide fractions collected through HPLC were dissolved in formic acid (FA)-acrylonitrile (ACN) solution , thoroughly vortexed, and centrifuged at 13,500 rpm for 20 min at 4°C. The supernatant was transferred to the loading tube, and 3 µg sample from every fraction was taken for further separation by two-dimensional liquid chromategraphy using a Dionex column. Peptides were separated by a linear gradient formed from mobile phase A  and mobile phase B , from 5% to 90% of mobile phase B for 65 min, at a flow rate of 300 nL/min. The separated peptides directly entered the mass spectrometer  for detection. MS spectra were acquired across the range of 350-1800 m/z in high resolution mode , using 100 ms accumulation time per spectrum. A maximum of 20 precursors per cycle were chosen for fragmentation from each MS spectrum with 100 ms minimum accumulation time for each precursor and dynamic exclusion for 20 s. Tandem mass spectra were recorded in high sensitivity mode (resolution : 17500), with turned on rolling collision and iTRAQ reagent collision energy adjustment. The protein quantification was based on unique peptides.TM 4.5  for protein retrieval. A strict cutoff for protein identification was applied with an unused ProtScore ≥ 1.3, which corresponded to a confidence limit of 95%, to minimize false positive results. At least two peptides with 95% confidence and coefficient variation (CV) < 0.5 were considered for protein quantification. False discovery rate (FDR) analysis was performed using the integrated tools in ProteinPilot (FDR < 0.01). For data analysis, protein quantification data with a fold change of > 1.3 and < 0.77 and p-value < 0.05 was selected as the significantly differently expressed protein (DEP). The interaction network of DEPs was analyzed by String online tool.Mass spectrometry data in RAW format were acquired by mass spectrometer. The RAW file was converted into mascot generic format (MGF) file by Thermo Proteome Discoverer. The MGF file and protein retrieval library were inputted into the ProteinPilot SoftwarePathological sections including 5 cases of bone metastasis tissue derived from prostate cancer, 30 cases of prostate cancer with bone metastasis, 30 cases of non-metastatic prostate cancer, and 30 cases of benign prostatic hyperplasia were collected. Three sections were prepared of every case and stained with antibodies for CD59, haptoglobin and tetranectin, respectively. Unstained sections were de-paraffinized and rehydrated using standard methods. Endogenous peroxidase activity was quenched by slide immersion in 3% hydrogen peroxide solution for 10 min followed by a PBS rinse. For antigen retrieval, slides were incubated in citric acid antigen repair solution in a steamer for 8 min at 100°C, followed by cooling down to 25°C naturally and a PBS rinse. Normal goat serum (10%) was placed on sections for 30 min for antigen blocking. Slides were incubated in primary antibody diluted in 10% goat serum overnight. The following primary antibodies and concentrations were used: anti-CD59 , anti-haptoglobin ; anti-tetranectin . Slides were rinsed in PBS followed by incubation in secondary antibody  for 30 min. Diaminobenzidine (DAB) detection, hematoxylin counterstaining, dehydration and sealing were performed according to conventional methods.Image-pro Plus 6.0 software was used to calculate the optical density of the area of interest in each slice, and the average optical density of each group was compared. Haptoglobin and CD59 were only expressed in glandular epithelial cells; thus, glandular epithelium was selected as the area of interest. Tetranectin was expressed in both glandular epithelium and stroma; thus, the glandular epithelium and stroma area were both analyzed for tetranectin.Serum samples were collected from 50 patients with prostate cancer and bone metastasis, 50 patients with prostate cancer without metastasis and 50 patients with benign prostatic hyperplasia. The Human CD 59 ELISA Kit , Human Haptoglobin Quantikine ELISA Kit , and Custom Human CLEC3B ELISA Kit  were used to detect the levels of CD59, haptoglobin and tetranectin, respectively. All operations were carried out in strict accordance with the manufacturer's operation manual.2. The cell lines were authenticated using short-tandem repeat profiling provided by the vendor and routinely monitored for mycoplasma contaminationPC3, LNCap, and Du145 human prostate cancer cell lines used in this study were purchased from American Type Culture Collection. LNCaP cell line is derived from lymph node metastasis of prostate cancer patients and has low metastasis potential, representing the growth characteristics of early androgen-dependent prostate cancer. PC3 cell line is derived from bone metastasis of prostate cancer and has moderate metastasis potential. DU145 cell line is derived from brain metastasis tissue of prostate cancer patients and has strong metastasis potential. Cells were cultured in RPMI-1640 medium , supplemented with 10% FBS , 1% antibiotic-antimycotic , and 1% glutaMAX  at 37°C and 5% CO6/100 μL. Primary antibody (1 μL) was added to the test tube and incubated at 4°C for 30 min. All tubes, including blank control tube, were washed with 500 μL PBS and then treated with anti-mouse IgG (whole molecule)-FITC antibody produced in goat  and incubated at 4°C for 30 min. The samples were then washed with 500 μL PBS and re-suspended in flow cytometry staining buffer . All samples were analyzed on a FACSCalibur flow cytometer .Cells were harvested by incubation with TrypLE for 3 min at 37°C. Each group of cells was divided into negative control and test group with each tube containing a total of 100 μL. The cell concentration was adjusted to 1×10The cell lysates were prepared by lysis buffer consisting of 50 mM Tris-HCl buffer (pH 7.4), supplemented with 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, and HALT™ protease inhibitor cocktail at 1× concentration. Protein concentrations were determined using BCA Protein Assay Kit . 40μg of lysate proteins were denatured with loading buffer by boiling for 5 min. The denatured lysates were then resolved by SDS-polyacrylamide gel electrophoresis. The separating gel concentration used for haptoglobin, tetracycline and CD59 were 8%, 12% and 15% respectively. The stacking gel concentration used for all three proteins was 5%.The resolved proteins were transferred to PVDF membranes . The membranes were blocked with 5% skimmed milk solution for 1 h at 25°C and washed three times in TBS-Tween. The blocked membranes were probed overnight at 4°C with the primary antibody solution (1:1000). The primary antibody used is the same as the immunohistochemical part. After washing three times in TBS-Tween, the blots were probed with the corresponding secondary antibody (1:3000) conjugated with horse radish peroxidase for 1 h at 4°C. After the final washes, the signal was developed with the Immobilon Western Chemilum HRP substrate , as recommended by the manufacturer, and the bands were captured on to Carestream autoradiography film .Coverslips were coated with 0.5% gelatin  and three different cell lines of prostate cancer were seeded onto different coverslips and cultured in RPMI-1640 medium supplemented with 10% FBS. When cells grew to 70% confluence on the coverslips, they were fixed with 4% paraformaldehyde for 30 min and incubated with 0.2% Triton X-100 for 5 min at 25°C. The cells were then treated with 10% normal goat serum , and incubated with primary mouse anti-human antibodies against CD59, Haptoglobin, and Tetranectin  at a dilution of 1:100 at 4°C overnight, followed by Alexa Fluor® 488-conjugated goat anti-mouse antibody at a dilution of 1:200  at 25°C for 1 h in the dark. After staining with DAPI  for 5 min to visualize the nuclei, cells were imaged by fluorescence microscopy .p-value < 0.05.The optical density of immunohistochemical sections and the concentration of each index measured by ELISA in each group are expressed as mean ± standard error. Statistical comparisons between groups were carried out using one-way ANOVA. Results were considered statistically significant at the Based on the screening conditions mentioned above (Section 2.2.6), 32 differentially expressed proteins (DEPs) were identified, of which 11 were up-regulated and 21 were down-regulated in samples obtained from the prostate cancer with bone metastasis group. All up and down-regulated DEPs are shown in Table The protein interaction network of the 32 prostate cancer bone metastasis-related proteins was analyzed by the STRING online tool. The results showed that F5, SEPP1, THBS1, HRG, SERPINA4, and CLEC3B interacted with each other in the whole network, but no other DEPs formed an interaction network .By immunohistochemical staining, we found that CD59 was expressed in cell membrane and cytoplasm of prostate cancer epithelial cells, and positive staining particles were found in the cytoplasm. CD59 was most strongly expressed in bone metastatic tumor of prostate cancer, but rarely expressed in glandular epithelial cells of prostate hyperplasia, which was nearly negative for CD59 Fig. . The meap =0.767), while significant differences were found between the other groups (p < 0.001).Haptoglobin was also expressed in prostate cancer epithelial cells, with positive expression in nucleus and cytoplasm. There were many positive staining granules around the cells. The intensity of haptoglobin expression in bone metastatic tumor was significantly higher than that in prostate cancer tissue with or without bone metastasis. The expression of haptoglobin in prostate hyperplasia was the weakest Fig. . The meap < 0.001). Since the bone metastasis tumor tissues were all dense prostate cancer epithelial cells, it was impossible to analyze the optical density of the mesenchyme, so only three groups of data were analyzed. Thus, the mean optical density in prostate cancer tissue with bone metastasis, prostate cancer tissue with no metastasis, and benign prostatic hyperplasia tissue was 0.112 ± 0.003, 0.175 ± 0.004, and 0.252 ± 0.006, respectively .Tetranectin was expressed in both cytoplasm of epithelial cells and mesenchyme. In epithelial cells, its expression in bone metastatic tumor, prostate cancer tissue with bone metastasis, prostate cancer tissue without metastasis, and benign prostatic hyperplasia tissue decreased in that order Fig. . The meap < 0.05)  in serum of prostate cancer patients with bone metastasis, prostate cancer without metastasis and benign prostatic hyperplasia were 57.94 (± 17.05), 28.92 (± 6.35), and 15.79 (± 5.29), respectively. The mean haptoglobin concentrations (in mg/mL) were 3.21 (± 0.93), 2.67 (± 0.92), and 1.87 (± 0.91), respectively. The mean concentrations of tetranectin (in μg/mL) were 7.33 (± 2.23), 9.47 (± 2.62), and 12.15 (± 4.59), respectively. The differences were statistically significant among the three groups for each protein  derived from different metastasis of prostate cancer indicated that PC3, which is derived from bone metastasis, had the lowest expression. This indicates that the expression of haptoglobin in prostate cancer cells is influenced by the microenvironment in different metastatic sites. The specific mechanism needs further study.Tetranectin, encoded by the clec3b gene and belonging to the C type lectin family, is a plasminogen kringle-4 binding protein that can be detected in the plasma and the extracellular matrix. In plasma or serum of healthy adults, the mean concentration of tetranectin is 10.8 mg/L (9-12.1 mg/L) Chen et al. reported that high intratumoral expression of tetranectin is associated with poor prognosis of patients with gastric cancer after gastrectomy Bone metastasis of prostate cancer is a complex biological process, involving a variety of gene and protein changes. In this study, 32 DEPs related to bone metastasis of prostate cancer were identified by proteomic analysis of serum samples from prostate cancer patients. Three such DEPs with significant fold changes were validated in prostate cancer tissues and serum samples, respectively. Moreover, we analyzed the expression of the three proteins in the three commonly used prostate cancer cell lines to provide a basis for the selection of cell lines for further in vitro study of bone metastasis mechanism. However, the proteins associated with bone metastasis of prostate cancer may not be all detectable in serum, and local pathological and physiological changes in prostate tissue may be important. Meanwhile, a large proportion of the identified DEPs could be secondary changes. Therefore, further screening is essential to identify key proteins and signal pathways, in order to design novel diagnostic and therapeutic approaches for the treatment of prostate cancer with bone metastasis.This work was mainly supported by the joint fund of Guizhou provincial Science & Technology Department and Guizhou provincial People's Hospital [LH(2016) 7164], and partially supported by the joint fund of Guizhou provincial Science & Technology Department and the Affiliated Hospital of Guizhou Medical University [LH(2015) 7392].


Transcriptome analysis is emerging as emerging as a promising tool to enhance precision of diagnosis and monitoring in solid organ transplantation. Clinical progress has however been hampered by the current reliance on samples from core needle biopsies. This proof-of-principle study examined whether fine needle aspirates, being less invasive, permit the ascertainment of the identical molecular information as core biopsies.n = 11). RNA expression patterns of two gene sets (156 genes) were executed using targeted RNA sequencing in samples from fine needle vs. core needle samples. A subgroup of kidneys (n = 6) also underwent whole transcriptome RNA sequencing from core biopsies of tumor and peri-tumoral normal tissue .We collected fine needles aspirates from various needle sizes  and the corresponding core biopsies (G16 needle) of non-tumor tissue of full nephrectomy specimens from patients suffering from clear cell renal cell carcinoma . This close correlation was further documented by heat map, Principal Component Analyses (PCA) and whole transcription RNA sequencing. The similarity between fine neddle aspirates and core needle biopsies was additionally confirmed in the subgroup with complete RNA sequencing.Samples from all needle sizes except two G25 aspirates yielded RNA potentially suitable for sequencing of both gene sets. The mRNA expression patterns of the two gene sets were highly correlated between fine needle aspirates (G23) and corresponding (G16) core biopsies (Fine needle biopsies yield similar genomic information to core needle biopsies. The less invasive nature of fine needle biopsies may therefore permit more frequent molecular monitoring and a more targeted use of core needle biopsies in native and especially in transplanted kidneys. The diagnosis of rejection in renal transplantation is currently reliant on core needle biopsies given the lack of specificity of renal function measurements in blood or urine. Core biopsies are generally accepted to be safe but are expensive, labor-intensive, and can be accompanied by serious complications that may require hospitalization, such as gross hematuria requiring blood transfusions as well as even surgical interventions or angiographic embolizations (1–2.5% of kidney biopsies) [A less-invasive method of monitoring allograft status would facilitate understanding and management of allograft dysfunction. Such a tool would be especially relevant where protocol biopsies are recommended to detect subclinical T cell or antibody-mediated rejection, prior to overt functional allograft deterioration, or where response to therapy may have been suboptimal in routine practice .Molecular analyses are emerging as important techniques to complement or in certain cases to even replace tissue histology in diagnosis of transplant dysfunction. Robust transcript sets have been identified which predict future tissue alterations and/or reflect early acute or chronic allograft rejection in renal allografts , 5. A seGiven the inherent risk of repeated core needle biopsies, but the high potential clinical value of obtaining transcriptome information, we have investigated whether FNA, being less invasive, yields equivalent molecular information to regular core needle biopsies. In this proof-of-principle study we compared RNA sequencing results from two well defined gene sets (cell cycle and Wnt panels) in aspirates and biopsies from nephrectomy specimens.n = 11) with suspected renal cell carcinoma at the time of radical nephrectomy. All patients had normal kidney function (eGFR > 60 ml/min/1.73m2) and had undergone total nephrectomy. Key characteristics of the patients are outlined in Table Kidney tissue samples were obtained from patients n = 1 with sun = 6) of the total group of subjects (n = 11), stored as formalin-fixed, paraffin-embedded (FFPE) tissues, had previously been analyzed by whole transcriptome RNA sequencing  (Table n = 6) were included to investigate potential differences between whole transcriptome and targeted sequencing.Concurrently harvested kidney biopsies G16) from the tumor and adjacent normal tissue from a nested subset  were immediately placed in RNA later® . FNA samples (G19–25) were placed directly into 700 μl of Qiazol lysis buffer . Samples were stored at − 80 °C.Core needle kidney biopsy samples were weighed and cut into pieces of approximately 5 mg. Homogenization of biopsy cores and FNA samples was performed in 700 μl Qiazol lysis buffer with ceramic beads. All samples were homogenized for 3 × 10 seconds at 6800 rpm on a Precellys homogenizer . Total RNA was extracted using the miRNeasy Micro kit . RNA was stored at -80C until further use for RNA sequencing.RNA concentration was measured using a NanoDrop 1000 spectrophotometer . RNA integrity was assessed using the Agilent RNA 6000 Nano kit on a 2100 Bioanalyzer . The DV200 value (the percentage of RNA fragments longer than 200 nucleotides) was used to reflect RNA quality for subsequent sequencing from FFPE tissues . A minimRNA sequencing and analyses were conducted using targeted RNA expression, heatmaps and PCA. RNA sequencing was performed using two different predesigned TruSeq Targeted RNA panels from Illumina©. Panel sequencing was selected because, if successful, sequencing of defined gene panels (e.g. related to rejection processes) is much more likely to be integrated into routine clinical practice than full transcriptome sequencing.http://www.support.illumina.com/content/illumina-marketing/en/products/truseq-targeted-rna-expressioncell-cycle-panel.html. This panel was selected because cell cycle genes would reflect injury-repair mechanisms in the renal allograft [http://www.support.illumina.com/content/illumina-marketing/en/products/truseq-targeted-rna-expressionwnt-panel.html. This panel was selected given its general importance in biological processes, and because the wnt pathway has been implicated in chronic renal allograft injury [To test the potential utility of FNA-derived RNA sequencing, gene sets with relevance in kidney allografts were selected. The TruSeq Targeted RNA Expression “Cell Cycle Panel” includes 63 genes/transcripts participating in the cell cycle and DNA replication. The complete gene set of this expression panel is available at llograft . The Trut injury .Sequencing libraries were generated according to manufacturer’s instructions  with RNA normalized to inputs of 200 ng total RNA from each sample. Equal amounts of libraries were pooled  to a final concentration of 18 pM and subjected to cluster and single end read sequencing.http://www.ensembl.org/).Sequencing was performed for 50 cycles using MiSeq Reagent Kit v3 on a MiSeq® instrument . Base calling was done on the instrument and Fastq files were generated using bcl2fastq v.2.18 with default settings for the RNA-seq data. Transcript expression values were determined using the Salmon software  with thehttps://bioconductor.org/packages/release/bioc/html/tximport.html). Genes with less than two counts total were filtered out prior to analysis.Gene counts from the 2 gene sets were combined in the tximport package (https://bioconductor.org/packages/release/bioc/html/DESeq2.html) using the rlog transform on count data. Heatmaps for expression data were generated using the pheatmap package (https://cran.r-project.org/web/packages/pheatmap) and hierarchical clustering of genes and samples in the heatmap was generated using the Ward’s clustering method and correlation distances. Principal Component Analysis (PCA) was performed on variance transformed data (rlog) and visualized using the ggfortify package (https://cran.r-project.org/web/packages/ggfortify). The average expression values for the G16 biopsy (n = 11) and G23 FNA (n = 11) gene panel data, as well as for the prior G16 biopsy (n = 6) whole transcriptome data, were entered into a generalized pair plot using the GGally package .Variance transformed expression data was generated with the DESeq2 package  underwent both, prior sequencing of whole transcriptome RNA (as recently published [n = 6) served as an internal control and comparator (n = 6) to detect differences between whole transcriptome and targeted panel sequencing.A subset of nephrectomy samples (ublished ) and seqRNA quality for sequencing was expressed by the RIN and the DV200 values. The quantity Fig.  and qualp ≥ 0.13 for all comparisons.The mean DV200 values (95% CI) were 56% (43–69) for G23 FNA samples and 68% (62–74) for G16 core biopsy samples. The DV200 percentages well exceed the 30% minimum required for RNA sequencing . The RINhttps://www.illumina.com/documents/products/technotes/technote-trusec-rna-access.pdf and recently described [These RIN values reflected the expected RNA degradation from our FFPE tissues. However, RIN values derived from FFPE samples are not a reliable predictor of RNA quality or of successful library preparation using the Illumina TruSeq RNA Access Kit®, as investigated by the vendor, escribed . Mean RNescribed , 16, 17.p ≥ 0.11 for all comparisons.As expected, one-way ANOVA with Tukey’s multiple comparisons test revealed a significantly lower RNA yield from all FNA compared to G16 core biopsy samples . RNA sequencing analyses were therefore performed using the G23 FNA aspirates, as the yield was reliable and this needle size is routine used for FNA in clinical practice.FNA from needles of all tested sizes consistently yielded sufficient quantities of RNA for sequencing with the exception of two G25 aspirates, which would have permitted sequencing of only one gene panel. Also, the total RNA yield from some of these G25 size aspirations was, however, at the threshold of 50 ng/sample required for RNA input for both sequencing panels according to Illumina  and the corresponding 11 core needle biopsy samples (G16); samples of some patients (e.g. 50 N) clustered together closely, others did not.The heatmap Fig.  depicts n = 11; and G16, n = 11), and ii) the two sample sets from the subset of patients with additional complete RNA sequencing of renal cell carcinoma tissues  and their adjacent renal tissue . The two components (PC1 and PC2) of the PCA indicate that the expression patterns of the whole-genome based tumor tissues differs from non-tumor tissues, but taken together these two whole genome groups are much more dissimilar to the targeted sequencing samples. Therefore, the internal comparator group with complete RNA sequencing underscores the similarity between fine (G23) and core needle biopsies (G16) with targeted sequencing. The FNA (G23) and core biopsy (G16) groups show a high degree of overlap, indicating similar expression patterns in the sequencing results of the two gene panels.The Principal Component Analysis (PCA) describes the variance across the four groups Table . Figure The validity of RNA sequencing obtained from FNA samples is further underlined by the PCA in Fig. To test the true utility of FNA against current standard core biopsy samples, we analyzed the correlation of gene expression between the two gene panels measured by sequencing in each of the three different sample groups: 11 FNA samples (G23), 11 core needle biopsies (G16) and 6 non-tumor whole genome tissue samples.n = 63) and “Wnt” panel (n = 93) genes between the FNA and the respective core biopsies . A minority of genes (n = 28) from the total of 156 genes were differentially expressed between the G23 and G16 groups, including genes such as MMP-9, MMP-2, IL-6, and FOXN1 which play a role in inflammatory or immunologic processes (data not shown); this variability could be explained by the respective G23 and G16 needle location. The correlation of mRNA expression patterns between G23 and G16 samples and the respective results of the fully sequenced group  was lower .Figure This study demonstrates that RNA sequencing of FNA samples provides equivalent molecular information to that of core needle biopsy samples. These proof of principle findings may justify a less invasive approach to immune monitoring in organ transplantation, which thus far has heavily relied on full core biopsies.Currently percutaneous FNA is often performed for cancer diagnosis in various solid organs, such as breast, liver, and thyroid gland as well as in accessible lymph nodes . FNAs arThe present study demonstrates that sufficient quantities of RNA suitable for RNA sequencing can be obtained with a wide variety of fine needle sizes. Moreover, the comparative analyses indicate excellent correlation of mRNA expression patterns abundances obtained by FNA and full core biopsies. The internal comparator group with complete RNA sequencing further highlighted the similarity between fine and core needle biopsies with targeted sequencing.This study has several important strengths. Current FNA and core biopsy samples were obtained under direct vision after nephrectomy and permitting stringent comparability of RNA expression from the various procedures taken from the same tissue area. The quality and quantity of RNA obtained by FNA was robust and therefore strongly suggest that FNA may be a viable diagnostic tool in selected patients. Despite these encouraging findings however, our study has several limitations that must be acknowledged. Samples were obtained from explanted kidneys which were no longer perfused, therefore potential confounding of FNA reproducibility by circulating blood cells as compared with core biopsy samples cannot be addressed. Comparative studies in perfused organs are required to assess the influence of peripheral blood and biopsy/aspiration site on the results. Clinical complications could not be assessed in this study, however a proof-of-principal study such as ours is a necessary ethical pre-requisite prior to consideration of clinical studies. Further limitations to our study include the facts that we provide panel sequencing and not complete RNA sequencing of the whole genome and that we did not determine the variability of RNA expression between multiple FNA aspirates and the same biopsy. Moreover, FNA is not able to deliver the transcriptome of individual cell types and a detailed pathology report cannot be assigned to an FNA. In addition, as with all transcriptome studies, it remains to be shown if mRNA results of our selected panel genes can be extrapolated to the respective gene product on the protein level and to other genes and pathways.Transcriptome analysis has not yet reached the clinical mainstream and therefore the true clinical impact of facilitated transcriptome/RNA monitoring also requires prospective studies. It could be argued that the current hesitation in performing multiple biopsies in the same patient may have hindered progress in understanding the clinical value of the transcriptome. The use of FNA may therefore accelerate understanding of its clinical relevance. The close correlation of gene expression from RNA obtained with FNA as compared with core biopsies, as well as the relative safety, justifies further investigation and confirmation of the value of FNA in prospective clinical studies.It is important to acknowledge however that FNA is not an adequate source for histological investigations. Therefore, we do not suggest that FNA can replace all histologic analysis, which remains important for diagnosis of disease recurrence, borderline rejection changes, viral infections etc. We continue to support the need for histology especially for the first biopsy to ensure that unexpected and transplant-unrelated diagnoses are not missed. It is conceivable however that FNA could replace protocol biopsies, especially where allograft function appears normal. More frequent monitoring with serial protocol FNA in immunologically high-risk kidney allografts might detect rejection processes earlier than histological changes and/or an overt decline in kidney function. Candidate gene panels for transplant rejection have already been defined , 23. FNAIn conclusion, fine needles aspirates reproducibly reflect molecular information in the kidney. FNA may therefore provide a simple and safe tool to monitor native and especially transplanted kidneys by supplementing the more invasive core needle biopsies and could provide accessible information to guide clinical decision making.


Xiphinema index. Grapevines in diseased vineyards are often infected by multiple genetic variants of GFLV but no information is available on the molecular composition of virus variants retained in X. index following nematodes feeding on roots. In this work, aviruliferous X. index were fed on three naturally GFLV-infected grapevines for which the virome was characterized by RNAseq. Six RNA-1 and four RNA-2 molecules were assembled segregating into four and three distinct phylogenetic clades of RNA-1 and RNA-2, respectively. After 19 months of rearing, single and pools of 30 X. index tested positive for GFLV. Additionally, either pooled or single X. index carried multiple variants of the two GFLV genomic RNAs. However, the full viral genetic diversity found in the leaves of infected grapevines was not detected in viruliferous nematodes, indicating a genetic bottleneck. Our results provide new insights into the complexity of GFLV populations and the putative role of X. index as reservoirs of virus diversity.Grapevine fanleaf virus (GFLV) is responsible for a widespread disease in vineyards worldwide. Its genome is composed of two single-stranded positive-sense RNAs, which both show a high genetic diversity. The virus is transmitted from grapevine to grapevine by the ectoparasitic nematode  One of the main characteristics of RNA viruses is their high mutation rates. This is due to their highly error-prone RNA polymerase that produces mistakes during replication , stimulaBottleneck contributes also to the evolution of plant virus populations. A bottleneck consists of a sharp reduction at the population (quantitative) or at the genetic  levels. Bottlenecks can arise within a host-cell during various steps of the infectious cycle and during colonization of host organs by the virus or by grafting in the case of grapevine . BottlenParatrichodorus pachydermus [Many studies have been performed to predict the effect of quantitative and genetic bottlenecks on the overall fitness of plant virus quasispecies ,8,9,10. hydermus .Vitis spp.), one of the oldest domesticated crops [in planta. Both RNAs can be found in some particles as it was observed in purified preparations of GFLV [Xiphinema index [Longidoridae family and is one of the 13 nematode species described as vectors of grapevine soil-borne viruses [Xiphinema index acquires and releases GFLV particles during the feeding process when its stylet is deeply inserted in the parenchyma tissues of growing root tips. GFLV can be retained several years by X. index, even after extended periods of fallow beyond 4 years [X. index occurs in a non-circulative and non-propagative manner [Grapevine fanleaf virus (GFLV) is one of the most-studied nematode transmitted viruses due to its negative economic impact on grapevine  named VA6, VA7 and VA8 derived from cuttings of about 20 years old vines  in a fanleaf diseased vineyard in Chablis, France . Cuttings were collected in July 2016 and propagated under greenhouse conditions. Plants were grown in small pots before their transplantation in a 10 L container in the presence of GFLV-free nematodes. Aviruliferous X. index were reared on fig plants (Ficus carica) as previously described [X. index were allowed to feed on the three aforementioned grapevines starting in September 2016. Prior to setting up the feeding process, 5–6 leaves from each GFLV-infected grapevine were sampled, flash frozen in liquid nitrogen and stored at −80 °C for downstream high throughput sequencing (HTS) analyses. After 19 months (April 2018), nematodes were extracted from soil samples using the sieving method as previously described [X. index were individually handpicked under a binocular. Single nematodes (annotated i1 to i20) and pools of 30 individuals (annotated P1 to P5) were stored at −20 °C in sterile water prior to molecular analyses.All experiments were conducted in greenhouse located at the INRA-Grand Est Experimental Unit in Colmar, France . Three GFLV-infected grapevines , as previously described . Purity X. index, total RNA extractions were performed as described previously [For eviously  from nemcDNA libraries were prepared from total RNA grape leaf extracts after a poly-A selection at the GeT-PlaGe Genotoul platform facility . The HTS approach was a paired-end 2 × 150 pb RNAseq completed on a Hiseq 3000  following the manufacturer’s instructions. A double indexing strategy was used for all samples. After the demultiplexing steps being performed by GeT-PlaGe Genotoul, all dataset analyses were finalized using the CLC Genomics Workbench v11.0 software .de novo assembly followed by BLAST [The virome of each grapevine sample was obtained by (i) mapping reads onto a curated list of grapevine-infecting virus references, and (ii) performing by BLAST  analysesby BLAST . CompletAll GFLV nucleotide sequences (GenBank numbers MN496417 to MN496426) were aligned manually using CLC workbench v11.0 with basic parameters . Phylogenetic trees were reconstructed using the Neighbor Joining method with nucleotide distance measures following Jukes-Cantor model. Bootstrapping analysis was performed with 1000 replicates. Phylogenetic trees were visualized using ITOL .® DNA polymerase PCR kit , in a final volume of 50 µL as per the manufacturer’s recommendations. Degenerated primers targeting specifically GFLV RNA-1 were designed  with a mix of oligo(dT) in a final volume of 40 µL. Then, PCR was performed using 2 µL of the cDNA template using GoTaqdesigned  to ampliX. index actin (ACT) mRNA and Vitis vinifera glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA  mRNA . The lisDH) mRNA .TM real-time system). Briefly, PCR was performed in 96-well optical reaction plates for 30 s at 95 °C followed by 40 cycles of denaturation for 5 s at 95 °C, annealing and elongation for 5 s at 65 °C. After each run, a melting curve analysis was carried out to ascertain that a single amplicon was produced in each well. Each sample/primer combination was carried out in triplicate from which average threshold cycle (Ct) values were obtained and used for calculation. Samples were considered positive only when all three biological replicates exhibited (i) a Ct value, (ii) a Standard Error (SE) of the triplicates below 0.5 and (iii) a Ct value + SE below Ct – SE of the negative control. Absolute quantification of GFLV RNA-1 was expressed as molecule of viral RNA per ng of total RNA and deduced from a linear regression of Ct values obtained from a serial dilution of plasmid containing cDNA from GFLV RNA-1 [To further investigate the accumulation of viruses in nematodes, RT-qPCR was performed using the same aforementioned cDNA. Eva Green qPCR mix  was used with specific primers  targetinLV RNA-1 . RT-qPCRAvaI/AvaII or EcoRI (amplicon 1) or StyI (amplicon 2) with 8 µL of PCR reaction from grapevine and pools of nematodes samples and 12 µL of PCR reaction from single nematodes samples in a final volume of 25 µL. Restriction enzymes were from New England Biolabs, Ipswich, MA, USA. Restriction digests were resolved by electrophoresis on 11% polyacrylamide (37.5:1) gels at 120 V for 90 min.RT-PCR amplicons 1 and 2 corresponding to fragments of cDNA of GFLV RNA-1 and RNA-2 respectively, were separately digested overnight at 37 °C. Digestions were performed with ® gel and PCR cleanup kit . Thirteen to forty-two clones per PCR products were Sanger sequenced  using M13 universal primers.The RT-PCR products of GFLV RNA-1 and RNA-2 from selected pools of nematodes and single nematodes were cloned into pGEM-T Easy Vector (Promega), as per the manufacturer’s recommendation, after a PCR cleanup step using the Nucleospinde novo assembly analyses. From the three datasets, six GFLV-RNA1 and four GFLV RNA-2 consensus molecules were assembled. Each RNA molecule was identified in the text with the following code: the name of the sample, the number of the GFLV genomic RNA and the number of the molecular variant  were obtained in all three plants following  variant . These s variant . The cla variant . The ran variant . The num variant . ConsequTymoviridae family were also identified with grapevine redglobe virus (GRGV) in VA7 and with three molecular variants of grapevine rupestris vein feathering virus (GRVFV) in VA6  in VA6 . Arabis Amplicons of the expected length for RNA-1 and RNA-2 were obtained by RT-PCR for all grapevine samples using the same total RNA extracts as for HTS  but only 11 out of 20 single nematodes tested positive for ACT  primers  where RNA-1 molecular variants from only two clades (Ia and Ib) were detected. Regarding RNA-2, results were similar to those of pooled nematodes with variants from clades IIa and IIb being detected in single nematode i11. As molecular variants from these clades were present in different plants, these results revealed a complex GFLV RNA-2 diversity within a single nematode deriving from at least two different grapevines, VA6 and VA7 and/or VA8.Tymoviridae and Betaflexiviridae families, respectively. ArMV is specifically transmitted by the dagger nematode X. diversicaudatum while mealybugs and soft scale insects transmit GVB from the Vitivirus genus [X. index and single X. index were examined by RT-PCR  and more than 80% when focusing on RNA-1 . This reX. index could enhance the number of possible combinations between RNA-1 and RNA-2 molecules that it might deliver during the inoculation phase of the transmission process. This could increase potential reassortments, one of the main driving forces of virus evolution, among mutation and recombination events.In addition, we demonstrated that a single nematode could acquire and retain different variants of GFLV after feeding not only on one, but possibly on two different plants. For example, it is clear that single nematode #11 carried RNA-2 variants derived from at least 2 vines, VA6, VA7 and/or VA8 b. This cDespite the limited number of nematodes being tested, the genetic diversity of GFLV from the three grapevines was almost but not completely recovered in nematodes, with RNA molecules VA6-1-2 and VA6-2-1 not detected in nematodes. This partial genetic bottleneck could be explained by: (i) a non-uniform distribution of populations of GFLV within the plant VA6 (molecules VA6-1-2 and VA6-2-1 were hardly detected by RFLP in some samples from roots of grapevine cuttings VA6-A and VA6-B), and (ii) as previously mentioned , cloningX. index are transferred into plants during inoculation and can be detected in bait grapevines. Yet, if our results can be transferred to the field, the spread of GFLV within a vineyard will tend to be maintained in the long term, due to the capacity of a single nematode to retain multiple molecular variants. These findings are fundamental concerning GFLV evolution and for the successful establishment of a robust systemic infection in grapevine regardless of challenges encountered in distinct organs and under different environmental pressure.To date, we do not know whether all the GFLV variants carried by viruliferous X. index, ultimately improving their efficiency to survive and disseminate the virus. Second, concerning the mode of transmission, and as previously stated [X. index. Third, unlike insect vectors, nematodes behave more like colonizers than migrators, implying a slow but inexorable spread of an infection within a vineyard, and that the viral genetic diversity, if needed to be conserved, must be assured by transmission with a limited number of vectors. In addition, it is interesting to notice that within vineyards, grapevine is often mixed-infected with multiple GFLV variants [In this work, we report that the large diversity and genetic composition of GFLV populations found in grapevines was maintained quite well in a population of nematodes as well as in single nematodes. This might reflect the putative long co-evolution between GFLV and its nematode vector , for whiy stated , it is pvariants ,32, this


For this aim, we analyzed the variations of the cis-acting elements in the 5′ and 3′ untranslated regions (UTRs) between the PEDV TW, CV777, and other reference strains. We also determined the previously unidentified transcription regulatory sequence (TRS), a sequence motif required for coronavirus transcription, and found that a nucleotide deletion in the TW strain, in comparison with CV777 strain, immediately downstream of the leader core sequence alters the identity between the leader TRS and the body TRS. Functional analyses using coronavirus defective interfering (DI) RNA revealed that such variations in cis-acting elements for the TW strain compared with the CV777 strain have an influence on the efficiency of gene expression. The current data show for the first time the evolution of PEDV in terms of cis-acting elements and their effects on gene expression, and thus may contribute to our understanding of recent PED outbreaks worldwide.In 2013, the outbreak of porcine epidemic diarrhea (PED) in Taiwan caused serious economic losses. In this study, we examined whether the variations of the  Alphacoronavirus of subfamily Coronavirinae, which is in the family Coronaviridae, order Nidovirales [cis-acting RNA elements [cis-acting RNA element required for subgenomic mRNA (sgmRNA) synthesis that is located downstream of the leader sequence and upstream of each ORF in the 3′-proximal genome region [Porcine epidemic diarrhea virus (PEDV) belongs to the genus ovirales ,2. The covirales . RNA eleelements . For thee region . An upste region .Alphacoronavirus 5′ UTRs have revealed four conserved RNA secondary structures, including stem-loops (SLs) 1, 2, 4, and 5, which are also conserved for Betacoronavirus [Betacoronavirus have determined a requirement of these 5′ secondary structures for coronaviral RNA synthesis [Betacoronavirus 3′ UTRs, including the 5′-most bulged stem-loop (BSL) [Alphacoronavirus [Alphacoronavirus and Betacoronavirus contain the conserved octanucleotide sequence 5′-GGAAGAGC-3′ within the HVR [Analyses of onavirus . Moreoveynthesis . RNA strop (BSL) ,10, hairop (BSL) , and 3′-op (BSL) . HoweverSynthesis of sgmRNA in coronaviruses requires a conserved TRS sequence motif, which is located at the 3′ end of the leader sequence (TRS-L) and precedes each 3′ proximal gene (TRS-B). The TRS contains a conserved sequence (CS) flanked by variable sequences at its 5′ (5′ TRS) and 3′ (3′ TRS) ends . During The PEDV prototype strain CV777 was first identified in 1978, and suckling piglets infected with this strain exhibited only mild diarrhea ,21,22. Icis-acting elements in PEDV has not been previously analyzed. In this study, we determined whether the cis-acting elements that are required for gene expression are altered in the PEDV Taiwan (TW) strain in comparison with those of the PEDV prototype strain CV777. Functional analyses were also performed to determine whether the identified variations in cis-acting elements between the two strains alter the efficiency of translation, replication (genomic RNA synthesis), and transcription (sgmRNA synthesis) of the virus. The results may extend our understanding for the recent PED outbreaks in Taiwan and other countries.The evolution of Fecal and intestinal samples were collected from the Animal Disease Diagnostic Center of National Chung Hsing University and National Pingtung University of Science and Technology between January 2014 and December 2016. The total sample size was 38. Samples were homogenized, and RNA was extracted using TRIzol  according to the manufacturer’s instructions.Random hexamer primers were used for reverse transcription (RT) with SuperScript III reverse transcriptase , and the resulting cDNA was used for PCR with PfuUltra II high-fidelity DNA polymerase  and oligonucleotides PEDV 25(−) and PEDV 450(+) for the 5′ UTR and PEDV 27488 (−) and PEDV 3′end (+) for the 3′ UTR . The resTo construct the PEDV TW defective interfering (DI) RNA pTWDI, an overlap PCR mutagenesis procedure was performed, as previously described , but wit5 TCID50/mL. Vero cells in 35-mm dishes were infected with 200 µL of 105 TCID50/mL of PEDVPT-P5. The DNA constructs pTWDI, pCV777DI, pΔA S-TRS, and pS-TRS were linearized with MluI, transcribed in vitro to synthesize RNA transcripts with the mMessage mMachine T7 transcription kit (Thermo Fisher Scientific) according to the manufacturer’s instructions and passed through a Biospin 6 column . After 2 h of infection with the helper virus, 3 µg of RNA transcript was transfected into the cells. To detect replication of PEDV DI RNA and DI RNA-derived sgmRNAs (sgm DI RNA), the supernatant was collected at 48 hour posttransfection (hpt) and then used to infect fresh Vero cells . Ten micrograms of TRIzol-extracted total cellular RNA at 48 hpi of VP1 was used for the RT reaction with the reporter oligonucleotide TGEV(+) and SuperScript III reverse transcriptase (Thermo Fisher Scientific). To examine synthesis of DI RNA, SYBR® green amplification mix  and primers PEDV476(−) and TGEV(+) were used for qPCR according to the manufacturer’s protocol. To analyze the synthesis of sgm DI RNA, primers PEDVL20(−), which binds to the leader sequence, and 5′GD(+), which binds to the reporter gene, were employed to differentiate the DI RNA from the helper virus PEDV. In these experiments, dilutions of plasmids containing the same gene as the detected DI RNA or sgm DI RNA were always run in parallel with the quantitated cDNA for use in standard curves (dilutions ranged from 108 to 10 copies of each plasmid). The amount of synthesized RNA was normalized to the levels of internal controls, including helper virus genomic RNA, 18S rRNA, and DI RNA. The reactions were performed with an initial pre-incubation at 95 °C for 5 min, followed by 35 amplification cycles of 95 °C for 15 s and 60 °C for 60 s.The PEDV TW strain (GenBank Accession No. KP276252) was obtained from Dr. Hui-Wen Chang   and usedMluI, transcribed in vitro with the mMessage mMachine T7 transcription kit (Thermo Fisher Scientific) according to the manufacturer’s instructions, and passed through a Biospin 6 column (Bio-Rad). Vero cells were then transfected with 3 µg of transcript RNA, and proteins in cell lysates were collected at 16 h posttransfection. The protein samples were separated using 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and electrotransferred onto nitrocellulose membranes . An antibody against enhanced green fluorescent protein (EGFP) was used as the primary antibody; goat anti-mouse IgG conjugated to horseradish peroxidase (HRPO) was the secondary antibody . Detected EGFP was visualized using Western Lightning™ Chemiluminescence Reagent  and X-ray film .The DNA constructs pTWDI-EP and pCV777DI-EP were linearized with t-test using Prism 6.0 software . Values are presented as the mean ± standard deviation (SD) (n = 3); * p < 0.05, ** p < 0.01, and *** p < 0.001. The MegAlign program  was used for the alignment of nucleotide and deduced amino acid sequences.Statistical analysis of the data was performed with Student’s unpaired Alphacoronavirus form four secondary structures: SL1, SL2, SL4, and SL5 [Alphacoronaviruses [Madhugiria et al. showed that the ≈310 nts of the 5′ terminal genome region of  and SL5 , four SLUsing a sequencing analysis of 3′ UTR, either U or C at nt −49 was found from PEDV-positive samples (TW strain). The PEDV TW strain was therefore divided into two groups: Taiwan group A (with U at nt −49) and Taiwan group B (with C at nt −49) A. RegardAlphacoronaviruses, which may explain why the conserved counterpart of the Betacoronavirus BSL structure [Alphacoronaviruses [While analyzing the 3′ UTR sequences of TW strain, with the primers annealing the 5′ leader sequence and 3′ UTR, the reverse transcription polymerase chain reaction (RT-PCR) products with a length of ≈1700 base pairs (bp) and ≈250 bp were observed A, lane 2tructure ,10 couldaviruses . For PEDaviruses , as showEGFP gene  E after 4cis-acting elements required for coronavirus sgmRNA synthesis that are located at the 3′ end of the leader sequence (TRS-L) and preceded by each gene (TRS-B). The TRS contains a CS flanked by variable sequences at its 5′ (5′ TRS) and 3′ (3′ TRS) ends of the CS. The CS of TRS-L (CS-L) in PEDV CV777 is CUAAAC. The CS of TRS-B (CS-B) for CV777 strain E, M, and N genes has been experimentally determined to be CUAGAC, AUAAAC, and CUAAAC, respectively [E, M, and N genes). Although the CS-B for the S and ORF3 genes for CV777 strain was previously assumed to be GUAAAC and CUAGAC, respectively [S, ORF3, E, M, and N genes was determined  and between the 3′ TRS-B and 3′ TRS-L (1 for CV777 and 3 for TW) is observed. Based on the argument above, it is currently assumed that the CS of TRS-B (CS-B) for CV777 strain S and ORF3 genes is CGUAAA  genes in TW may alter the relative efficiency of sgmRNA synthesis.According to the current study shown in ynthesis ,17, the S gene was inserted into TW DI  in comparison with those of the PEDV prototype strain CV777. Functional analyses suggest that such variations in cis-acting elements for the TW strain compared with the CV777 strain have an influence on the efficiency of gene expression. Therefore, it is speculated that variations in cis-acting elements may also be one of the factors potentially contributing to the pathogenesis. Although the cis-acting elements of the Thailand reference strain are the same as those of the CV777 strain, we still cannot exclude the importance of the cis-acting elements because it is not clear whether the Thailand reference strain is virulent or not based on the insufficient information from GenBank. Accordingly, we also cannot rule out other factors that also play roles in the PED outbreak. Further study is required to determine to what extent the variations in cis-acting elements affect the virulence and pathogenesis using a reverse genetic system.Overall, we have determined that the cis-acting elements in the 5′ and 3′ UTRs between the PEDV TW and prototype CV777 strains. We also determined the previously unidentified cis-acting element TRS and found that (i) a nucleotide deletion in the TW strain, in comparison with CV777 strain, immediately downstream of the leader core sequence alters the identity between the leader TRS and the body TRS, and (ii) the altered sequence identity has influence on coronavirus subgenomic mRNA synthesis. Functional analyses revealed that such variations in the 5′ and 3′ UTRs of the TW strain also alter the efficiency of gene expression in comparison with that of the CV777 strain. Because the analyzed cis-acting elements for the TW strain are the same as those of the reference strains for USA and Korea, our results may extend our understanding of PEDV evolution in terms of cis-acting elements and the recent outbreaks of PED worldwide. Further study using reverse genetic system is required to determine whether the altered gene expression is associated with the increased severity of the recent PED outbreaks. In addition, a putative ORF within the 3′ UTR and a BSL structure within the putative ORF were also found. The biological significance of the ORF and BSL structure remains to be experimentally demonstrated.In this study, we found variations in