In 2006, the Bowel Cancer Screening Programme (BCSP) in England began offering biennial faecal occult blood testing (FOBt) at ages 60–69 years. Although FOBt is aimed at detecting colorectal neoplasms, other conditions can affect the result. In a large UK prospective study, we examined associations, both before and after screening, between FOBt positivity and 10 conditions that are often associated with gastrointestinal bleeding.By electronically linking BCSP and Million Women Study records, we identified 604 495 women without previous colorectal cancer who participated in their first routine FOBt screening between 2006 and 2012. Regression models, using linked national hospital admission records, yielded adjusted relative risks (RRs) in FOBt-positive versus FOBt-negative women for colorectal cancer, adenoma, diverticular disease, inflammatory bowel disease, haemorrhoids, upper gastrointestinal cancer, oesophagitis, peptic ulcer, anaemia and other haematological disorders.P < 0.001 for all RRs. In the 12 months after screening, the RR for inflammatory bowel disease was 26.3 (95% CI 19.9–34.7), and ranged between 2 and 5 for the upper gastrointestinal or haematological disorders. The RRs of being diagnosed with any of the eight conditions other than colorectal neoplasms before screening, and in the 12–24 months after screening, were 1.81 (95% CI 1.81–2.01) and 1.92 (95% CI 1.66–2.13), respectively.RRs in FOBt-positive versus FOBt-negative women were 201.3 (95% CI 173.8–233.2) for colorectal cancer and 197.9 (95% CI 180.6–216.8) for adenoma within 12 months after screening and 3.49 (95% CI 2.31–5.26) and 4.88 (95% CI 3.80–6.26), respectively, 12–24 months after screening; Whereas FOBt positivity is associated with a substantially increased risk of colorectal neoplasms after screening, eight other gastrointestinal and haematological conditions are also associated with FOBt positivity, both before and after screening. In the first 12 months after screening, women who are FOBt-positive have about a 200-fold increase in the risk of colorectal neoplasia. This excess risk remains slightly elevated in the 12–24 months after screening.FOBt positivity is associated with an increased risk of being diagnosed with non-neoplastic colorectal conditions that can also cause gastrointestinal bleeding. The greatest relative risk, both before and after FOBt positivity, is for inflammatory bowel disease.Bleeding from upper gastrointestinal conditions, especially peptic ulcers, makes a sizeable contribution to FOBt positivity. However, the proportion of FOBt-positive women subsequently diagnosed with upper gastrointestinal cancer is exceedingly low.About one in eight women who were FOBt-positive did not undertake follow-up investigations through services provided by the national Bowel Cancer Screening Programme, but many of them had pre-existing conditions associated with gastrointestinal bleeding and appear to have chosen to seek follow-up through other services where they were already under care. Colorectal cancer is the third most common cause of cancer deaths in the UK, and accounted for 12% of all new cancers in 2014.The National Health Service (NHS) Bowel Cancer Screening Programme (BCSP) in England was launched in 2006 to provide biennial colorectal cancer screening using guaiac-based FOBt. In this report, we link FOBt results in the BCSP with information on cause-specific hospital admissions in the large prospective study of women’s health, the Million Women Study. We investigated associations between FOBt positivity and 10 pre-specified conditions related to gastrointestinal bleeding.,The BCSP in England began offering biennial screening with FOBt to men and women aged 60–69 years from 2006 (extended to 74 years from 2010).,www.millionwomenstudy.org.The Million Women Study is a large nationwide study of women’s health. A total of 1.3 million women, representing one in four of all UK women born in 1935–50, joined the study in 1996–2001. Participants answered a questionnaire on sociodemographic, health and lifestyle characteristics, and consented to follow-up through medical records. Details of the study design have been published elsewhere.Using their unique NHS number and other identifying details, study participants are followed by linkage to electronic, routinely collected NHS records for deaths, cancer registrations and hospital admissions. Data for England are provided through NHS Digital. The Hospital Episode Statistics (HES) inpatient dataset in England contains details of all NHS inpatient (overnight and day case) admissions, including private patients treated in NHS hospitals. Diagnoses are recorded using the Tenth Revision of the International Classification of Diseases (ICD-10).With approval from the Cambridge South Research Ethics Committee and by the NHS Bowel Cancer Screening Programme Research Committee, the BCSP database was linked to the Million Women Study database in 2013 .Linked HES records were searched for hospital admission with diagnoses of the following 10 pre-specified gastrointestinal and haematological conditions: colorectal cancer, colorectal adenoma, inflammatory bowel disease, diverticular disease, haemorrhoids, peptic ulcer disease, oesophagitis, upper gastrointestinal cancer, anaemia and other bleeding disorders. Conditions were defined as being diagnosed ‘before screening’ if the first hospital record for the condition was before the first BCSP screening invitation. Conditions were defined as being diagnosed ‘after screening’ if the first hospital record of the condition was after the first screening invitation. First diagnoses after screening were further divided into those in the first 12 months after screening invitation (i.e. including results of diagnostic investigations following a positive screening test) and those first recorded in the 12–24 months after screening. Diagnoses after 24 months were not included, as the BCSP would have offered many women routine screening again 24 months after the first invitation. For FOBt-positive patients who underwent subsequent colonoscopy, the information was obtained from hospital diagnoses in the HES database, as colonoscopies are generally performed as inpatient ‘day cases’ in NHS hospitals.n = 278 705), had any solid or haematological malignancy registered before recruitment into Million Women Study (n = 15 144), were lost to follow-up before first BCSP invitation (n = 22) or had a hospital record of colorectal cancer before FOBt screening (n = 800). The remaining 604 495 participants, first screened for bowel cancer between 2006 and 2012, were included in the analyses.Record linkage identified 899 166 Million Women Study participants who received their first invitation for routine bowel cancer screening from the BCSP . For these analyses, we excluded women who did not accept the invitation for screening , for various diagnoses in FOBt-positive versus FOBt-negative women. For conditions first diagnosed after screening, Cox regression models, with attained age as the underlying time variable, estimated hazard ratios, henceforth also referred to as relative risks (RRs), for first hospital diagnoses in FOBt-positive versus FOBt-negative women in the first 12 months after screening, and 12–24 months after screening. For every RR estimate, 95% confidence intervals (CI) were calculated.2), as these have been associated increased risk of colorectal cancer. Information relating to these variables was obtained at recruitment; women with missing data on adjustment variables (<2% for each) were included in the analyses as a separate category. Information on family history of colorectal cancer and use of antiplatelet or anticoagulant medications were not collected at recruitment. Analyses of diagnoses other than colorectal neoplasms (cancer or adenoma) excluded women with colorectal cancer diagnosed in the 24 months after screening. All analyses were performed using Stata version 14.0.All analyses were adjusted for area deprivation  were FOBt-positive and referred for further investigations in the Bowel Cancer Screening Programme (BCSP). All FOBt screening was performed between 13 July 2006 and 13 March 2012.The baseline characteristics and the frequency of conditions recorded in hospital admission data before the FOBt screen are shown in P <0.0001) and anaemia .Before screening, FOBt-positive women were more likely than FOBt-negative women to have had previous diagnoses of colorectal adenoma or non-neoplastic colorectal conditions , as well as upper gastrointestinal conditions . FOBt-positive women were also more likely than FOBt-negative women to have had a previous admission for anaemia or other haematological disorders. The proportions of women admitted with at least one of the eight conditions  before FOB testing were 25% of FOBt-positive and 16% of FOBt-negative participants. The pre-screening diagnoses most strongly associated with FOBt positivity were inflammatory bowel disease  attended the BCSP for further investigations, which for almost all women was a colonoscopy. Among the 1174 (13%) FOBt-positive women who did not attend the BCSP for further investigation, hospital admission records indicate that they were significantly more likely than the attenders to have had previous hospital admissions for each of the nine pre-selected gastrointestinal and haematological conditions, particularly inflammatory bowel disease and anaemia .In all, 700 (7.9%) FOBt-positive women had a hospital diagnosis for colorectal cancer and 1704 (19%) for colorectal adenoma in the first 12 months after screening. Among the FOBt-negative women, the corresponding numbers were 247 and 649, yielding relative risks of 201.3 (173.8 – 233.2) and 197.9 (180.6 – 216.8) for colorectal cancer and for adenoma in FOBt-positive compared with FOBt-negative women. Results for colorectal cancer were similar, based on cancers ascertained from the cancer registry data, at 212.0 (184.0 – 246.5).P <0.0001 for all the RRs quoted , 612 with haemorrhoids  and 70 with inflammatory bowel disease ; s quoted . VirtualAfter excluding those diagnosed with colorectal cancer after screening, FOBt positivity was associated with a 5-fold risk of upper gastrointestinal cancer and 2-fold risk of peptic ulcer or oesophagitis in the first 12 months after screening . It was P <0.0001 for all the RRs quoted. In validation analysis, the RRs for colorectal cancer in attenders and non-attenders, using diagnoses ascertained from the cancer registry data, were similar to those based on HES data, at 232.0 (201.1 – 269.9) and 78.4 (55.8 – 110.1), respectively. The mean time between FOBt screening invitation and HES record of colorectal cancer diagnosis was also similar in the 662 attenders and 38 non-attenders, at 3.0 [standard deviation (SD) 1.6] and 4.9 (SD 2.9) months, respectively. For upper gastrointestinal cancer and anaemia, RRs in the first 12 months after screening were significantly greater in non-attenders than in attenders for BCSP investigations.Women who attended the BCSP for diagnostic colonoscopies had higher relative risks for colorectal cancer and adenoma, although those who did not attend also had considerably raised risks for both . The RRsP <0.0001) and for colorectal adenoma  remain elevated in in FOBt-positive versus FOBT-negative women, although much lower than in the preceding 12 months . There was also an increased risk of being diagnosed with other colorectal, upper gastrointestinal and haematological conditions  in the 12 months after FOBt. Virtually all the diagnoses were made at around the time of colonoscopy, which is generally recorded as day case hospital admission. Our findings are in line with results reported by other organised screening programmes, that 24–38% of FOBt-positive individuals have non-neoplastic colorectal conditions identified during diagnostic colonoscopies.About 15% of FOBt-positive women had a hospital admission with benign causes of upper gastrointestinal bleeding before screening and in 1% this was first diagnosed in the 12 months after screening, consistent with previous findings.The relative risks of being diagnosed with anaemia (>95% were iron deficiency anaemia) in FOBt-positive versus FOBt-negative women, before and after screening, range from 3 to 5. The anaemia itself may not necessarily be caused by gastrointestinal bleeding but could be a manifestation of some other underlying haematological condition. Approximately 6% of the FOBt-positive women had a previous hospital diagnosis of other specified haematological conditions which could predispose to gastrointestinal bleeding, and about 1.2% had a first diagnosis of one of these conditions in the 12 months after FOB testing. The associated relative risks are approximately 3–4, suggesting that the presence of these conditions influences FOBt positivity. A small part of the excess risks for non-colorectal conditions diagnosed after screening could be due to increased contact with health professionals.About one in eight who were FOBt-positive did not attend further investigations organised by the BCSP. Non-attenders were more likely than the attenders to have the pre-existing conditions associated here with gastrointestinal bleeding. Some have suggested that the most common reasons for non-attendance at BCSP after FOBt positivity include unwillingness to undergo a colonoscopy or inopportune timing,The findings here are for a population-based NHS screening programme using guaiac-based FOBt, and may not be directly translatable to other screening programmes that use immunochemical-based FOBt . The characteristics of screen-detected and interval cancers are being reported elsewhere.Some of the important strengths of this study include having a virtually complete dataset on cause-specific NHS hospital admissions, both before and after FOBt screening. By linking BCSP data to information collected in the Million Women Study, it was possible to obtain information on clinical diagnoses recorded before screening as well as after screening, not only for the 87% of the FOBt-positive women who attended the BCSP for further investigations, but also for the 13% who did not. The main limitation of this study is that diagnoses are restricted to those associated with hospital admissions (overnight or day case stays) and that it involved only women, who have lower rates of FOBt positivity and lower risks of colorectal cancer and adenoma compared with men. A systematic review has found the accuracy of primary diagnosis codes in these hospital data to be 96% after 2002.Our study confirms that FOBt positivity in routine bowel cancer screening is associated with a substantially increased risk of colorectal neoplasms diagnosed in the first 12 months after screening, and a small excess risk in the 12–24 months after screening. It also showed that FOBt positivity is associated with eight other gastrointestinal or haematological conditions related to gastrointestinal bleeding. These results emphasise the importance of reviewing relevant medical history and discussion about other conditions associated with gastrointestinal bleeding might be considered when counselling patients with positive FOBt results.This was work was supported by the UK Medical Research Council and Cancer Research UK, as well as the White-Walker Cancer Research Scholarship and an Australian National Health and Medical Research Council Postgraduate Scholarship (GNT1093776) for E.H. The study sponsors had no part in designing the study question nor in the analysis or interpretation of results. They also did not take part in the writing of the article nor in the decision to submit for publication.E.H. performed the literature search, participated in the study design and electronic data linkage and was responsible for statistical analysis, data interpretation and drafting of the manuscript. R.A., R.B., K.P. and G.R. participated in the study design, electronic data linkage and statistical analysis. R.W., R.S., J.P. and K.C. provided expert content advice and assisted in interpretation of study findings. J.G. and V.B. oversaw the study, including the study design, statistical analysis and drafting of the manuscript. All authors reviewed the study’s findings and read and approved the final manuscript.Conflict of interest: None declared.


IGF1) gene has been reported to affect cancer susceptibly in several studies. However, the results of the relevant studies are inconsistent. We conduct a current meta-analysis to investigate the association between rs1520220 and cancer susceptibly.The rs1520220 polymorphism in the insulin-like growth factor 1  were searched for studies regarding the relationship between rs1520220 and cancer susceptibly. Odds ratios (ORs) and the related 95% confidence intervals (CIs) were employed to assess the strength of the associations. A stratified analysis was performed according to cancer type, ethnicity, and quality score, and when results were obtained from no fewer than two studies, these results were pooled.p = 0.040; homozygote model OR = 1.22, 95%Cl = 1.01–1.47, p = 0.040; dominant model OR = 1.19, 95%Cl = 1.01–1.39, p = 0.033). No significantly association was detected in Caucasian populations. The analysis stratified by cancer type suggested that rs1520220 was not associated with susceptibility to breast cancer.There was no positive association between rs1520220 and overall cancer risk. However, the analysis stratified by ethnicity revealed that rs1520220 significantly increased cancer susceptibility in Asian populations  contains supplementary material, which is available to authorized users. The occurrence of cancer depends on both genetic and environmental factors , 2. ReleIGF1 SNPs affect plasma IGF1 levels and thus influence the risk of cancer [IGF1 is a growth factor that involves in many important biological and pathological processes , 9. The f cancer , 15.IGF1 gene which might lead to alternative splicing and a subsequent change in protein function [rs1520220 is located in intron 3 of function . It has function , 17. Howfunction –25. For function , but Li We searched for relevant studies in three databases: PubMed, Embase, and Web of Science. The search conditions limited the language to English and the data of publication prior to February 28, 2018. The following keywords were used: “IGF1 or IGF-1 or insulin-like growth factor 1 or rs1520220,” “cancer or tumor or carcinoma,” and “SNP or polymorphism or variant or mutation.” We also checked the references of the identified articles to ensure that we obtained all potentially relevant studies.The inclusion criteria of this meta-analysis are as follows: studies must (1) concern the relationship between rs1520220 and cancer susceptibility, (2) be case-control or cohort study, and (3) contain sufficient genotyping data to allow for the pooling of the results . The exclusion criteria are as follows: (1) when subjects of two studies overlap, the one containing fewer subjects was excluded, and (2) reviews and meta-analyses are excluded.The following information was extracted from the included studies by two authors independently: first author’s name, year of publication, country, cancer type, ethnicity, genotyping methods, control source, genotype distributions of cases and controls, and Hardy-Weinberg equilibrium (HWE) for controls. Disagreements were resolved via discussion.We assessed the quality of the included studies based on the following five factors : case soQ test and quantified by I2 [P > 0.1), the fixed-effects model was used [P values less than 0.05 were considered to indicate significant disequilibrium. Stratified analyses were conducted by ethnicity, cancer type, and quality score. Only results synthesized from no fewer than two studies are shown. Sensitivity analyses were performed via metainf command which investigates the influence of each individual study on the overall meta-analysis summary estimate by omitting each study in turn [We estimated the strengths of the associations using pooled ORs with corresponding 95% CIs. Five genetic models are employed: the allele model (C vs. G), the homozygote model (CC vs. GG), the heterozygote model (GC vs. GG), the dominant model (CC + GC vs. GG), and the recessive model (CC vs. GC + GG). The heterogeneity was evaluated using a ed by I2 . When hewas used . Otherwiwas used . Hardy-W in turn . Publica in turn , 32. AllWe obtained 2086 relevant articles through database searching after removing duplicates. Then, by screening the titles and abstracts, we excluded 1953 articles, and 133 articles remained. We read the full texts of these 133 articles and ultimately identified eight articles that meet the inclusion criteria . Thus, no significant association was detected in Caucasian populations  and head and neck cancer (HNC) susceptibility in women [IGF1 gene. It has been reported that rs35767 is significantly associated with increased susceptibility of childhood acute lymphoblastic leukemia (ALL) [Several tibility –39. Thesin women . The rs3ia (ALL)  and thatia (ALL) .IGF1 gene, and it has a minor allele frequency (MAF) about 10~ 40% in the populations included in the human 1000 Genomic Project phase 3 . After removing duplicate documents, 2086 related articles were initially obtained, which ensured the maximum possible recall rate. Through meta-analysis, we found that rs1520220 was not related to cancer susceptibility in the overall analysis based on the present epidemiology studies. Thus, in the analysis stratified by ethnicity, we revealed that rs1520220 increased cancer susceptibility in Asian populations.In this meta-analysis, we systematically searched for literature on IGF1 rs1520220 polymorphism on serum IGF1 are inconsistent [The present studies regarding the effect of nsistent , 18. In nsistent , and thinsistent . TherefoOur meta-analysis has several limitations. Firstly, we found that rs1520220 increased cancer susceptibility in Asians. The molecular mechanism via which the rs1520220 C allele increases plasma IGF1 levels and thus cancer risk remains unclear. Secondly, we did not consider potential external factors, such as gender, age, diet, and tobacco and alcohol intake habits or gene-gene interactions. Thirdly, the number of studies included in the meta-analysis is limited. We only included studies written in English, and important-related studies in other languages may have been overlooked.IGF1 rs1520220 is not significantly associated with overall cancer susceptibility. However, we did find that rs1520220 significantly increased cancer susceptibility in Asian populations. We also suggest that rs1520220 was not associated with susceptibility to breast cancer. There is a need for additional well-designed epidemiology and molecular biology studies to verify these conclusions and provide new insights into the role of SNPs in the etiology of cancer.The present meta-analysis showed that Additional file 1:Table S1. Quality score assessment. Table S2. Sensitivity analyses for IGF1 rs1520220 polymorphism and cancer susceptibility. Table S3. MAFs of IGF1 rs1520220 polymorphism in the populations from the 1000 Genomes Project Phase 3. (DOCX 30 kb)


An urgent hemodialysis was initiated, and he was dependent on regular hemodialysis subsequently. Computed tomography renal scan showed diffuse nonenhancing hypodense lesion in both renal parenchyma. Diagnosis of diffuse large B cell lymphoma with germinal center type, CD20 positive, and proliferative index 95% was confirmed via renal biopsy, and there was no bone marrow infiltrates. Unfortunately, the patient succumbs prior to initiation of chemotherapy.We reported a case of primary renal lymphoma (PRL) presented with non-oliguric acute kidney injury and bilateral kidney infiltrates in an individual with human immunodeficiency virus (HIV) disease. Acute kidney injury secondary to lymphoma infiltrates is very rare . A 37-year-old gentleman with underlying human immunodeficiency virus (HIV) disease was on combined antiretroviral therapy since diagnosis. He presented to our center with uremic symptoms and gross hematuria. Clinically, bilateral kidneys massively enlarged and were ballotable. Blood investigations showed hemoglobin of 3.7 g/L, urea of 65.6 mmol/L, and serum creatinine of 1630  Primary renal lymphoma (PRL) is one of the rare extranodal non-Hodgkin lymphoma. It is defined as non-Hodgkin lymphoma arising in renal parenchyma and not invasion from neighboring lymphomatous lesion. The first case was reported by Coggins in 1980 . There iIn adult age from 18 to 50 years old, PRL usually presented with abdominal and flank pain while weight loss and gross hematuria are seen more commonly in adults more than 50 years old . Kidney µmol/L. After 12 months of treatment, his HIV viral load was less than 20 cells/L, and CD4 increased to 178 cells/microliter and serum creatinine of 193 µmol/L. During his follow-up in February 2017, serum creatinine rose markedly to 1051 µmol/L but remained asymptomatic; he was advised for admission and further investigation, but the patient refused due to some family issues.A 37-year-old gentleman with underlying human immunodeficiency virus (HIV) diagnosed in June 2015 was under infectious disease clinic follow-up from another center. He was started on combined antiretroviral therapy (tenofovir-emtricitabine and efavirenz) since diagnosis but defaulted treatment a month prior to his admission to our center. His CD4 count upon diagnosis was 20 cells/microliter and serum creatinine of 108 He presented to our center in April 2017 with vomiting and gross hematuria for one week associated with abdominal pain and distention. He was also symptomatic of anemia. He had loss of weight and loss of appetite. He had good urine output (more than one liter in a day) and no frothy urine. He did not take any nephrotoxic drugs, that is, herbal supplementation or nonsteroidal anti-inflammatory drugs. Clinically, he was pale, and bilateral kidneys were ballotable and massively enlarged with an irregular surface and hard in consistency. There were no hepatomegaly and splenomegaly. Peripheral lymph nodes were not palpable as well.9/L, lymphocyte count of 1.7 × 109/L, and neutrophil count of 5.4 × 109/L and urea of 65.6 mmol/L, serum creatinine of 1630 µmol/L, potassium of 5.4 mmol/L, sodium of 132 mmol/L, hyperphosphatemia of 3.70 mmol/L, and corrected serum calcium of 2.48 mmol/L. Venous blood gas showed metabolic acidosis with pH of 7.18 and bicarbonate of 11 mmol/L. Urinalysis showed urine protein 2 + (0.75 g/L) and blood 3 + (250 μ/L). An internal jugular double-lumen catheter was inserted, and hemodialysis was initiated. Ultrasound abdomen (Blood investigations showed hemoglobin of 3.7 g/L (normochromic and normocytic), total white count of 7.6 × 10 abdomen  and 4-ph abdomen  were donThe ultrasound showed enlarged bilateral kidneys with calculus, and his 4-phase renal CT showed diffuse nonenhancing hypodense lesion in both renal parenchyma. The lesion at the right kidney extends medially to the paraaortic region, forming another hypodense mass component, measuring 6.4 cm × 8.1 cm × 11 cm. The mass is displacing the branches of the aorta, duodenum, and pancreas anteriorly. Subcentimetre nodes were seen in the mesenteric and inguinal region. In view of definite diagnosis cannot be made via imaging, a renal biopsy was arranged. Histopathology and immunochemistry result of left renal tissue showed diffuse large B cell lymphoma with the germinal center type Figures –5.A bone marrow aspirate and trephine (BMAT) was done and showed no bone marrow infiltration. Positron emission tomography CT scan (PET-CT) concluded extensive malignant lesion in the upper abdomen involving the kidneys, stomach, and pancreas. The liver, spleen, lungs, and brain have normal uptake. There were malignant lesions also found in the right cervical node, left tonsils, and posterior chest wall. Some small bone metastasis was also detected.His repeat CD4 count was 84 cells/microliter, and the viral load was 6974 copies per ml. He was restarted on antiretroviral treatment: oral Kivexa (abacavir/lamivudine) 1 tab daily and oral efavirenz 600 mg daily, added oral trimethoprim and sulfamethoxazole (Bactrim) 2 tablets daily as prophylaxis prior to starting chemotherapy. He underwent regular hemodialysis and was transfused with packed red blood cell. He was planned for standard treatment of rituximab-cyclophosphamide, hydroxydaunorubicin, Oncovin, and prednisone (R-CHOP), but the patient developed upper gastrointestinal bleed and succumbed to death before starting the chemotherapy.The clinical presentation of PRL can be similar to other renal pathologies such as renal cell carcinoma, renal abscess, renal tuberculosis, or secondary metastasis. The symptoms range from hematuria, acute or chronic kidney injury, and loin pain or weight loss. Imaging findings of poorly enhancing or hypoechoic masses, retroperitoneal tumors directly invading the kidneys, bilateral renal enlargement, and perirenal soft-tissue masses are suggestive of PRL . HoweverTo our knowledge, the reported cases of primary renal lymphoma in the HIV individual are scarce, and it is one of the AIDS-defining malignancies . Even thIn the HIV patient, lymphomagenesis can happen due to chronic B-cell activation secondary to the immunodeficient state and proliferation of oncogenic viruses (Epstein–Barr virus and Kaposi's sarcoma herpesvirus) and impaired immune control of tumor suppression gene and host genetic alterations. However, despite the early commencement of combined antiretroviral therapy with immune reconstitution, there is no decrease in the incidence of lymphoma among HIV individuals. There are some recent data to suggest that there is a human immunodeficiency virus- (HIV-) encoded protein p17 gene sequences that directly contribute to lymphomagenesis, and strategies to target p17 proteins in the treatment of lymphoma can be beneficial if this association is established .In lymphoma patients with initial presentation of acute kidney injury (AKI), the assessment has to be based on prerenal, renal, and postrenal causes. Volume depletion secondary to poor oral intake and gastrointestinal loss is the commonest cause of prerenal AKI. Intrarenal cause of acute kidney injury in lymphoma can be due to ischemic or nonischemic acute tubular necrosis, direct tumor infiltrates, and renovascular disorder due to the prothrombotic state associated with malignancy or glomerulopathy. However, postrenal causes can occur from direct tumor or lymph nodes compression, and the patient usually presents with oliguric AKI.AKI primarily due to lymphomatous infiltration is rare and is seen in less than 1% of hematological malignancies . The assHowever, in the HIV patients treated with tenofovir, nephrotoxicity secondary to tenofovir needs to be considered when the patient presented with acute kidney injury. A review article reported that tenofovir cytotoxicity happens at the mitochondria of the proximal tubule and causes Fanconi syndrome and acute-on-chronic tubulointerstitial nephritis . Thus, cµmol/L to 193 µmol/L after 12 months of starting tenofovir. The limitation is no sufficient urinalysis and imaging to look for any early lymphomatous infiltrates at that point of time.As described, the likelihood of AKI in this case is due to direct lymphomatous infiltration to the kidney parenchyma as the patient presented with gross hematuria and bilateral massive kidney enlargement which was confirmed by renal biopsy and also PET scan. However, tenofovir toxicity and concomitant renal calculi cause need to be taken into consideration as well because there was an increment of serum creatinine from 108 There is still no data for prognosis of PRL in HIV individuals. In a study that analyzes the efficacy of R-CHOP in HIV-positive diffuse large B cell lymphoma (DLBCL) and HIV-negative DLBCL, there is no difference in the outcome of both groups of patients . Thus, tIn conclusion, PRL is rare, and it is still one of the differential diagnoses for consideration when a patient presented with renal mass and AKI. The gold standard to confirm the diagnosis is by doing renal biopsy in order to get histopathological sample for examination together with BMAT and PET-CT for staging of the disease. Currently, the standard treatment of PRL is still R-CHOP. Generally, the prognosis of PRL is based on the stage of disease upon diagnosis.


Patients with end-stage renal disease (ESRD) undergoing haemodialysis (HD) experience oxidative/carbonyl stress, which is postulated to increase after the HD session. The influence of diabetes mellitus and sex on oxidation of plasma proteins in ESRD has not yet been clarified despite that diabetic nephropathy is the most common cause of ESRD in developed and developing countries and despite the increasingly emerging differences between males and females in epidemiology, pathophysiology, clinical manifestations, and outcomes for several diseases. Therefore, this study aimed to evaluate the possible effect of type 2 diabetes mellitus, gender, and dialysis filter on plasma level of protein carbonyls (PCO) in ESRD patients at the beginning and at the end of a single HD session. Results show that mean post-HD plasma PCO levels are significantly higher than mean pre-HD plasma PCO levels and that the type of dialysis filter and dialysis technique are unrelated to plasma PCO levels. The mean level of plasma PCO after a HD session increases slightly but significantly in nondiabetic ESRD patients compared to diabetic ones, whereas it increases more markedly in women than in men. These novel findings suggest that women with ESRD are more susceptible than men to oxidative/carbonyl stress induced by HD. Compared to the general population, patients with chronic kidney disease (CKD) are at higher risk for cardiovascular disease (CVD) because of higher prevalence of traditional  and nontraditional cardiovascular risk factors. The latter include uraemia, anaemia, inflammation, and oxidative stress, which all together form part of the malnutrition-inflammation complex (or cachexia) syndrome, which is a strong predictor of morbidity and mortality in these patients. –7. The sS-thiolation). S-thiolated plasma proteins, measured as protein thiolation index (PTI) [In haemodialysed patients, oxidative stress, which may act synergistically with inflammation, is involved in the development of long-term complications such as amyloidosis, atherosclerosis, and CVD , 3, 11. ex (PTI) , are index (PTI) , 13. Othex (PTI) , 16 as wex (PTI) –19. Simiex (PTI) –22.While it appears that oxidative stress in haemodialysed patients may result from uraemia per se , 23, theOnly a few studies have focused specifically on the impact of a single HD session on plasma PCO levels in ESRD patients , 24, 25.The study was approved by the institutional review board before initiation and carried out according to the Code of Ethics of the World Medical Association (Declaration of Helsinki). All the 69 Caucasian patients enrolled in the study belong to stage 5 of CKD and are referred to as ESRD patients on maintenance HD. In addition to HD, patients are treated with a pharmacological treatment that varies upon the clinical necessities and consists mainly on the treatment of ESRD complications. Most of the patients assume drugs for anaemia  and bone mineral disorder . In addition, patients may also take specific drugs for other comorbidities, for example, hypertension, diabetes mellitus, ischemic cardiopathy, and other vasculopathies. Blood samples were collected, after informed written consent, from ESRD patients undergoing maintenance HD at the Nephrology and Dialysis Unit of the Humanitas Clinical and Research Center . The samples were collected at the arterial line at the beginning and at the end of the HD session. The presence of a clinically overt infectious process was the only exclusion criteria. For every patient, an anamnestic record was collected. A deidentification of the samples was performed before any additional data processing. The baseline clinical characteristics of recruited patients are shown in β2-microglobulin clearance of over 20 ml/min) synthetic biocompatible membranes. In particular, the filters used are the following, classified by the dialysis technique:n = 41)2, thickness: 35 μm), for 23 patientsRevaclear™ 300, whose membrane is made of polyarylethersulphone (PAES) + polyvinylpyrrolidone (PVP) , for 14 patientsRevaclear™ 400, whose membrane is made of PAES + PVP , for 4 patientsFiltryzer® 1.6, whose membrane is made of polymethyl metacrylate (PMMA)  (n = 27)2, thickness: 50 μm), for 14 patientsPolyflux™ 170 H, whose membrane is made of PAES + PVP + polyamide (PA) , for 11 patientsPolyflux™ 210 H, whose membrane is made of PAES + PVP + PA , for one patientNephral™ ST 400, whose membrane is made of acrylonitrile/methallyl sulphonate copolymer, coated with high-molecular-weight polyethyleneimine (PEI) , for one patientNephral™ ST 500, whose membrane is made of acrylonitrile/methallyl sulphonate copolymer, coated with PEI (surface: 2.15 mDiffusive plus convective techniques (online haemodiafiltration (online HDF) and acetate-free biofiltration (AFB))  reducing SDS-PAGE gels and electroblotted onto a polyvinylidene difluoride (PVDF) membrane. Protein carbonylation was detected, after derivatization with DNPH, with anti-DNP antibodies specific for the 2,4-dinitrophenyl hydrazone-carbonyl adduct by Western blot immunoassay as previously reported [Plasma proteins were fractionated on 12.5%  in each ELISA plate well. We incubated ELISA plate overnight at 4°C to allow protein binding. ELISA assay was performed according to the manufacturer's instructions. Absorbance of plate wells was read at 450 nm using the Plate Reader TECAN Infinite® 200 PRO. In all the performed assays, calibration line showed an R2 close to 0.99. We then calculated carbonyl content of samples by using the regression factors (intercept with the y-axis and line slope) obtained from standard curve.Plasma PCO were measured using the ELISA kit manufactured by Enzo Life Sciences (ALX-850-312-KI01). Carbonylated protein standard (40 mg/ml containing 0-0.12-0.22-0.42-0.7-0.9 nmol carbonyls/mg protein) and human plasma samples (60–75 mg/ml) were diluted 1 : 40 in DNPH solution and incubated 45 min to allow PCO derivatization. A 1 : 200 dilution in ELISA buffer was then performed before adding 200 by standardized methods at the clinical laboratory of the Humanitas Clinical and Research Center [Creatinine, C-reactive protein, white blood cell count, albumin, fibrinogen, haemoglobin, ferritin, total iron-binding capacity, urea, sodium, potassium, calcium, and phosphorus were measured h Center , 16.t-test was used to test whether differences in plasma PCO level in ESRD patients before (pre-HD) and after (post-HD) a single HD session were significant. The paired Student's t-test was also used to test for differences in plasma PCO levels before and after a single HD session by dividing the haemodialysed patients depending on the dialysis filter used, the cooccurrence of type 2 diabetes mellitus, and the gender. All the values are expressed as mean and standard errors (SE). A p value <0.05 was considered to be significant. The statistical significances are marked as ∗ = p < 0.05 and ∗∗ = p < 0.01. The relationship between pre-HD and post-HD PCO levels was investigated by simple linear regression analysis.The paired Student's PCO are considered the most general and the most commonly used biomarkers of severe oxidative protein damage. The results of protein carbonylation assessed by Western blotting using anti-DNP antibodies from five ESRD patients and five age-matched voluntary healthy donors are presented in t-test applied to the mean value of plasma PCO level measured in ESRD patients pre-HD (mean 0.1239 ± 0.0140 nmol/mg protein) and post-HD (mean 0.1332 ± 0.0140 nmol/mg protein) proved that the means are significantly different (p < 0.05)  . There wIn HD, patient's blood is allowed to flow through a filter , whereby waste products and excess water are removed across a semipermeable membrane separating flowing blood from the dialysate stream. The clean blood is then returned to the haemodialysed patient's body, while wastes are discharged. The main determinant of the quality of HD therapy is represented by the artificial membrane packed into the haemodialyser. The HD therapy per se, in particular the type of dialysis membrane, contributes to the increased production of ROS in ESRD patients . Indeed,n = 22; mean age: 71.6 ± 2.1 yrs; HD vintage: 5.1 ± 0.7 yrs) and nondiabetics  , whereas they are not significantly different in diabetics  (t-test for independent samples). Pre-HD plasma PCO levels were significantly positively correlated with post-HD plasma PCO concentrations both in nondiabetic   (Considering that diabetes mellitus occurs as an important comorbidity in the ESRD population (often composed predominantly of subjects in advanced age) and that some studies suggest that oxidative stress in diabetic patients leads to increased plasma PCO levels , 41, we 6 years) . Data we, resp.) ; in addi 0.0001)  and diab 0.0001)  ESRD patn = 45, mean age 70.1 ± 2.4 yrs, HD vintage 5.6 ± 0.6 yrs; females: n = 24, mean age 66.9 ± 3.1 yrs; HD vintage 6.1 ± 0.8 yrs) , whereas they are significantly different in females  (t-test). Pre-HD plasma PCO levels were significantly positively correlated with post-HD plasma PCO concentrations both in male    . Data we < 0.01) ; in addi 0.0001)  and fema 0.0001)  ESRD patProtein carbonylation, which may result from direct oxidation of lysine, arginine, proline, and threonine residues and interaction with reactive carbonyl species produced from carbohydrate and lipid oxidation or non-oxidative reactions with dicarbonyl compounds, is an indicator of oxidative protein damage . Its useThe increased levels of PCO measured in haemodialysed patients suggest that protein carbonylation may be an important biomarker of oxidative stress in ESRD patients as well. A number of studies compared biomarkers of oxidative stress between ESRD patients and age-matched healthy subjects , 28, 44 About the possible influence of the dialysis membrane on PCO levels, we have considered that during the HD session, the contact of blood with the dialysis membrane and the loss of antioxidants may promote oxidative stress. In a study conducted on 15 nondiabetic ESRD patients , PCO levels were found to be significantly increased after a HD session with a cuprophane membrane  and incrIt is well known that oxidative stress is increased in diabetes mellitus. In particular, some studies have shown that plasma PCO levels are increased in diabetics , 41. UnfIn contrast to what is observed in the general population, where females have a longer life expectancy than males , female In summary, all these results suggest that (i) the HD session increases plasma protein carbonylation; therefore, although important advances have been done in the field of dialysis biocompatibility, the HD session probably still represents a source of oxidative stress; (ii) plasma PCO level measurement may become an indicator of oxidative/carbonyl stress in ESRD and could be included in the routine monitoring of haemodialysed patients, since standard uraemia and inflammation biomarkers may not be sufficient on their own to describe the inflammatory/oxidative state of ESRD patients on HD; and (iii) haemodialysed women seem to be more susceptible to oxidative/carbonyl stress induced by the HD session than men. On the basis of these findings, it seems appropriate to suggest that the female sex could be considered a fundamental biologic variable (or a “risk factor”) associated with HD-induced plasma protein carbonylation in ESRD patients on maintenance HD. Therefore, this study shows that gender differences exist in plasma PCO levels of haemodialysed patients before and after a single HD session and highlights the critical importance of reporting of sex information in study description, data analyses, results, and their interpretation in basic science and medical/clinical research studies concerning ESRD. As a matter of fact, the prevalence of CKD stages 1–5 among US adults aged 18 years or older is higher in women than men (16% versus 13%); however, men are 64% more likely than women to develop ESRD , yet mosFinally, our study has some limitations. Firstly, it includes a relatively small number of ESRD male and female patients and has been performed in only one single HD center. Secondly, the majority of the patients studied in this cohort are from Italy and of Caucasian race, and the applicability of the study findings across nationalities and races remains unclear. However, we hope that these results stimulate further research with a larger number of men and women on HD recruited from different dialysis centers, possibly of different nationalities and races, to advance our understanding of the pathophysiology of sex  differences in CKD and improve clinical care of women with CKD. While some advances have been made in both clinical and basic research, much remains poorly understood, both at the molecular and clinical levels.Post-HD plasma PCO level increases in nondiabetic but not in diabetic ESRD patients, more markedly in women than in men. Women with ESRD are more susceptible than men to HD-induced oxidative/carbonyl stress.


RESULTS/ANTICIPATED RESULTS: Of the 500 hospitalizations reviewed, 430 patients had confirmed diabetes diagnosis, adequate data to calculate FI scores, and were included in this analysis. Patients were on average 66.9 (10.9) years old; 99% male and 75% were White. Overall, 268 patients (62.3%) were categorized as frail or very frail. The mean FI score was 0.23 (SD 0.07). FI scores were highest in patients receiving insulin alone (mean 0.26) compared with patients receiving metformin alone (mean 0.22), sulfonylurea alone (mean 0.23), or no medication (mean 0.22). The lowest mean frailty score was seen in patients taking all other drug combinations, 0.19. The differences across these patient groups were statistically significant with p<0.01. Further, 75% of patients on insulin alone were frail or very frail compared with 68% on sulfonylurea alone, 58% on metformin alone, and 58% on no medication. Framingham criteria for acute HF were present for 318 of 430 patients (74.0%). FI scores were higher in patients hospitalized for HF compared with non-HF hospitalizations . A higher proportion of patients hospitalized for HF were classified as frail or very frail compared with those hospitalized for non-HF diagnosis . DISCUSSION/SIGNIFICANCE OF IMPACT: This study demonstrates that certain antidiabetic medications are associated with patient frailty. In addition, those patients admitted for HF have higher FI scores than those admitted for non-HF diagnoses. Further investigation is planned to assess the degree to which frailty is captured by traditional covariates used in observational studies.OBJECTIVES/SPECIFIC AIMS: This study is part of a parent grant evaluating antidiabetic medications and risk for heart failure in an observational cohort of Veterans with type 2 diabetes (T2DM). Confounding by indication remains a concern in many observational studies of medications because difficult to measure confounders such as frailty may influence prescribing of different medications based on patient characteristics. Frailty is a multidimensional syndrome of loss of reserves  that gives rise to vulnerability to adverse outcomes. The objective of this study is to determine if frailty is a potential confounder in Veterans with T2DM, that is, independently associated with exposure to a specific antidiabetic medications and hospitalization for decompensated heart failure. METHODS/STUDY POPULATION: We conducted a cross-sectional study of patients with diabetes who were hospitalized within the Veterans Health Administration (VHA) Tennessee Valley Healthcare System from 2002 to 2012. Inclusion criteria were: age 18 years or older, receive regular VHA care (prescription fill or visit at least once every 180 d), a diagnosis of T2DM. A probability sample of HF and non-HF hospitalizations was collected. HF hospitalizations were selected on the basis of meeting either a primary diagnosis code (ICD-9) and/or disease related group (DRG) code for HF. For each hospitalization using a standardized chart abstraction tool, data was abstracted on: antidiabetic medication(s), patient frailty status, and reason for hospitalization (HF or non-HF). Antidiabetic medication regimens were categorized as follows: no medication treatment, metformin alone, sulfonylurea alone, insulin alone, insulin and one oral agent, and all other regimens. Patient frailty status was measured using a modified version of the Canadian Health and Aging frailty index (FI), which generates a score (range 0–1) by dividing the number of deficits present by the number of deficits measured. Established categories for FI scores are: non frail ≤0.10, vulnerable 0.10–0.21, frail 0.22–0.45, and very frail >0.45. Patient frailty status at the time of hospitalization was used as a surrogate for patient frailty at the time of prescription of antidiabetic medication; this is a limitation of this approach. Hospitalizations were classified as HF hospitalizations if 2 major or 1 major and 2 minor Framingham criteria were present. FI was compared across antidiabetic medication regimen categories and hospitalization type using analysis of variance (ANOVA) and Student


Left atrial appendage morphology has been proved to be an important predictor of left atrial thrombus (LAT) and left atrial spontaneous echo contrast (LASEC) and stroke in patients with non-valvular atrial fibrillation (NVAF). However, the relation between left atrial appendage (LAA) lobes and LAT or LASEC is still unknown. The aim of this study is to investigate the correlation between the number of left atrial appendage lobes and LAT/LASEC in patients with NVAF.This monocentric cross-sectional study enrolled 472 consecutive patients with non-valvular atrial fibrillation, who had transthoracic echocardiography (TTE) and transesophageal echocardiography (TEE) prior to cardioversion or left atrial appendage closure (LAAC) from July 2009 to August 2015 in department of cardiology of Shanghai Tenth People’s Hospital. Patients who had significant mitral or aortic valve disease, previous cardiac valvular surgery and other complicated cardiac diseases were excluded. Individuals were divided into two groups:the LAT/LASEC group (16.95%), which comprised patients with LAT or LASEC, as confirmed by TEE; and a negative control group (83.05%).Baseline overall group characterization with demographic, clinical, laboratory data and echocardiographic parameters, alongside with information on medication was obtained for all patients. Subgroup analysis with line chart was applied for exploring the association between LAA lobes and LAT/LAESC. Receptor-operating curves (ROC) were used to test the value of LA anteroposterior diameter detected by different echocardiography methods predicting LAT or LASEC. Multivariable logistic regression analysis was used to investigate independent predictors of LAT/LASEC.2DS2-VASc score , larger LAD , lower left upper pulmonary venous flow velocity (LUPVFV) , more left atrial appendage lobes . There was a good discriminative capacity for LAD detected by TTE (area under the curve (AUC), 0.67, CI 95% 0.61–0.73, p < 0.001) and LAD detected by TEE . The subgroup analysis based on gender and different LAA lobes yielded similar results  that the number of LAA lobes were significantly associated with LA thrombus or SEC. In multivariable logistic regression analysis, both the number of LAA lobes  and the persistent AF  provided independent and incremental predictive value beyond CHA2DS2-VASc score.Among 472 patients, 23 (4.87%) had LA/LAA thrombus and 57 (12.1%) had LA spontaneous echo contrast. Compared to the negative group, patients in LAT/LASEC group had higher CHAThe number of LAA lobes is an independent risk factor and has a moderate predictive value for LAT/LASEC among NVAF patients in China. Atrial fibrillation (AF) is one of the most commonly observed arrhythmias in clinical practice with an incidence of 0.77% in China and approximately 1.5–2.0% in the developed world. This arrhythmia is associated with a five-fold risk of stroke, and 20–30% of all strokes are due to AF, thus a higher mortality compared with those without AF , 5. LAA Remarkably, it is observed that even in patients with AF, the incidence of AF associated stroke varied widely, ranging from 1 to 20% annually . One posWe studied 472 patients with all types of non-vavular AF. Left atrial thrombus and spontaneous echo contrast were analyzed by 2D-TEE and classified into two groups (LAT or LASEC positive group and negative group). Simultaneously, LAA lobes were counted during TEE procedure. Then, univariate analysis was performed using the Student’s t-test and chi-square test. Predictors from univariate analysis were used for obtaining logistic regression models that could determine the relative importance of independent predictors of LAAT and LASEC .This single-center cross-sectional study enrolled patients undergoing TTE and TEE prior to catheter ablation or left atrial appendage closure (LAAC) during a non-valvular AF episode. A total of 472 consecutive participants  who were hospitalized at Department of Cardiology of Shanghai Tenth People’s Hospital of Tongji University from July 2009 to August 2015 were referred to our center. AF was identified by an electrocardiogram and met the diagnostic criteria used in 2011 ACCF/AHA/HRS Guidelines for the management of patients with AF  a visible outpouching part demarcated by an external crease from the body of LAA; [ii] the inner diameter was at least 2-mm; [iii] the direction of lobe could be opposite with the main tubular body of LAA; [iv] the anatomic plane was occasionally but not necessarily lain in a same anatomic plane than the main tubular body; [v] the LAA had at least one lobe , 30. FigP value < 0.05 was considered statistically significant.All statistical analyses were performed by SPSS for Windows version 22 . All continuous data are presented as the mean ± SD and were compared using Student’s t-test and one-way analysis of variance (ANOVA) test for two-level and multiple level grouping variables, respectively. Categorical variables are described as count and percent and were compared using Pearson’s chi-square test (or Fisher’s exact test whenever needed). Then sub-analysis based on gender and different numbers of LAA lobes was used to explore the correlation between LAA lobes and LAT/LASEC, the same as LUPVFV. The receiver operating characteristic (ROC) curve was used to discriminate the power of the LA anteroposterior diameter measured by TTE and TEE in identifying TEE surrogate markers of stroke  . Comparin = 23; LASEC: n = 57) by TEE examination. As shown in the table, patients with LAT or LASEC were significantly older but with nearly equal heart rate, systolic blood pressure (SBP), diastolic blood pressure (DBP) and without significant predilection for gender. The percentage distribution of prior congestive heart failure, diabetes, hypertension and previous vascular disease were significantly different between 2 patient groups, more in LAT/LASEC group. Simultaneously, patients with thrombosis had a longer duration of AF and more previous stroke events compared to patients without thrombosis; but these differences were statistically insignificant. In addition, persistent AF during hospitalization was more frequent and CHA2DS2-VASc score as expected, significantly higher in patients with LAT/LASEC.Baseline characteristics of 472 patients were shown in Table P = .049), larger LA anteroposterior diameter measured by TTE  or TEE , slightly thicker ventricular septal , lower LVEF , higher pulmonary artery pressure , more LAA lobes  and lower LUPVFV . Importantly, both T-test and Chi-Square test showed that LAA lobes of patients with LAT/LASEC were significantly more than other patients (Table These patients’ baseline laboratory examinations and echocardiographic characteristics were shown in Table p < 0.001). The value of LAD by TEE with a sensitivity of 72.2% and a specificity of 63.6% was 48.5 mm, AUC = 0.73  .ROC analysis demonstrated that a cut-off value of anteroposterior LAD with 44.5 mmby TTEcould predict the presence of LAT/LASEC. At this level, sensitivity was 76.3% and specificity was 51.2%, Area under the curve (AUC) = 0.67  (Table P < 0.001) and female (P = 0.004) subgroup  Table . MoreoveP = 0.032) (Table p = 0.78) and female (p = 0.12) subgroup  had been detected LUPVFV by TEE. The average LUPVFV was 0.63 ± 0.24 m/s. According to analysis of variance, LUPVFV decreased in patients with LAT/LASEC, compared with non-thrombosis patients . Multivariate linear regression analysis was performed to determine the relative importance of independent predictors of LAT/LASEC, the number of LAA lobes , AF types  and antiplatelet or anticoagulation therapy  were independent predictors of LAT/LASEC , and double lobes 17.9%. These variations may be attributable to the race discrepancy, sample sizes of population and subjective judgment of LAA morphology.Previous studies indicated that the number of lobes was variable, with the prevalence of single lobe ranging from 20 to 42.7%, double lobes from 25.2 to 64.3%, multiple lobes from 26.0 to 35.7% , 30. How.Thus, our results support the findings of previous studies: patient with complicated morphology (like non-chicken LAA) had more commonly multilobed LAA, lower LAAFV and was more likely to develop thrombus and SEC than patient with a single lobe LAA. Further research should explore the correlation between LAA lobes and LAAFV.LAA morphology was often classified into four types: (i) Chicken Wing LAA, a main lobe (> 4 cm) with a folded angle under 100°; (ii) Windsock LAA, a main lobe (> 4 cm) with a folded angle over 100°; (iii) Cactus LAA, a main lobe (< 4 cm) with more than two lobes over 1 cm; and (iv) Cauliflower LAA, a main lobe (< 4 cm) with no forked lobes. Such a division of LAA morphology which was originally designed to help practical planning for a transcatheter LAA closure device placement is now widely recognized .AccordinAdditionally, it is generally believed that Chicken Wing LAA is similar to Windsock LAA, and it is also difficult to differentiate Cactus LAA from Cauliflower LAA by morphology. Therefore, such category method is subjective and conflicting. However, the number of LAA lobes by TEE is objective and easy to test, while cardiac CT and MRI are expensive and deleterious, thus we can apply TEE to acquire the number of LAA lobes and examine LAT and LASEC simultaneously.Additionally, our study documented that left upper pulmonary venous flow velocity (LUPVFV) decreased in LAT/LASEC group. LUPVFV can reflect LA pressure and LV diastolic filling pressure (E/e’), which are important influential factors for LAAFV , 42. Con2DS2-VASc score of 2.86. Additionally, the rate of anticoagulation therapy among these patients was high (80.1%), which may affect the rate of LAT and LASEC and thromboembolic events. Thus, our findings could not be adapted to a high-risk AF population.This study has several limitations. First, this study is a single-center study with a relatively small sample. Second, the retrospective design of the study is an additional limitation. Large-scale studies with long-term follow-up are warranted to evaluate the predictive value of LAA lobes for stroke. Third, we investigated a relatively low risk population reflected by a mean CHAMore left atrial appendage lobes are associated with significantly higher risk of left atrial thrombus or left atrial spontaneous echo contrast in patients with non-valvular atrial fibrillation. Therefore, the number of LAA lobes is an independent risk factor and has a moderate predictive value for LAT/LASEC among NVAF patients in China.


Besides discovering new applications of the cell type-specific secretome, the proposed strategy could allow the recognition of the cocktail of bioactive factors which might be responsible for the effects of conditioned media, thus providing a solid rationale to the implementation of a cell-free approach in several clinical scenarios involving tissue regeneration.The adult stem cell secretome is currently under investigation as an alternative to cell-based therapy in regenerative medicine, thanks to the remarkable translational opportunity and the advantages in terms of handling and safety. In this perspective, we recently demonstrated the efficient performance of the adipose-derived mesenchymal stem/stromal cell (ASC) secretome in contrasting neuroinflammation in a murine model of diabetic neuropathy, where the administration of factors released by dermal fibroblasts (DFs) did not exert any effect. Up to now, the complex mixture of the constituents of the conditioned medium from ASCs has not been fully deepened, although its appropriate characterization is required in the perspective of a clinical use. Herein, we propose the differential proteomic approach for the identification of the players accounting for the functional effects of the cell secretome with the aim to unravel its appropriate applications. Out of 967 quantified proteins, 34 and 62 factors were found preponderantly or exclusively secreted by ASCs and DFs, respectively. This approach led to the recognition of distinct functions related to the conditioned medium of ASCs and DFs, with the former being involved in the regulation of neuronal death and apoptosis and the latter in bone metabolism and ossification. The proosteogenic effect of DF secretome was validated  Currently, the therapeutic effect of MSC administration has been tested for the treatment of numerous acute and chronic pathologies, spanning from cardiovascular disorders to musculoskeletal and immune diseases [Adult stem cell-based therapies have been proven effective in resolving a wide array of clinical questions, opening the way to their translation from preclinical models to medical practice. Up to date, 301 clinical trials explored or are currently investigating the safety and performance of mesenchymal stem/stromal cells (MSCs), a class of adult stem cells that can be conveniently harvested from several tissue sources  and DFs possess peculiar features that allow their discrimination through Raman spectroscopy with an accuracy of 93.7% . In our ne field , 23. HerUnless otherwise stated, reagents and chemicals were purchased from Sigma-Aldrich .2 in complete culture medium  added with 10% FBS . The medium was replaced every other day and, at 70–80% confluence, cells were detached with 0.5% trypsin/0.2% EDTA, plated at a density of 10,000 cells/cm2 for ASCs and OBs, 5000 cells/cm2 for DFs, and expanded.All the cell types used in this study were isolated from waste tissues of healthy donors undergoing plastic (abdominoplasty and liposuction) or orthopaedic surgery, after written consent and following the procedure PQ 7.5.125, version 4, dated 2015-01-22, approved by the IRCCS Galeazzi Orthopaedic Institute. ASCs were isolated from the subcutaneous adipose tissue of 3 female donors (age range: 26–65 y/o) while DFs were isolated from the deepidermised dermis of 3 female patients (age range: 26–46 y/o). Cells were isolated following previously described protocols . Brieflyg for 15 minutes to remove cell debris and concentrated using Amicon® Ultra-15 centrifugal filter columns with a 3 kDa molecular weight cutoff . Protein concentration was measured by a Bradford assay .Conditioned medium was prepared as previously described . Once atμg of total proteins from each sample were in-solution digested using the Filter Aided Sample Preparation (FASP) protocol as reported in literature [μm i.d., 12 cm long) reverse phase silica capillary column, packed with 1.9 μm ReproSil-Pur 120 C18-AQ . A gradient of eluents A (distilled water with 0.1% v/v formic acid) and B (acetonitrile with 0.1% v/v formic acid) was used to achieve separation (300 nl/min flow rate), from 2% B to 40% B in 88 minutes. Full-scan spectra were acquired with the lock-mass option, resolution set to 70,000 and mass range from m/z 300 to 2000 Da. The ten most intense doubly- and triply-charged ions were selected and fragmented. All MS/MS samples were analysed using the Mascot  search engine to search the human_proteome 20171122 . Searches were performed with the following settings: trypsin as proteolytic enzyme; 2 missed cleavages allowed; carbamidomethylation on cysteine as fixed modification; protein N-terminus-acetylation, methionine oxidation as variable modifications; and mass tolerance was set to 5 ppm and to 0.02 Da for precursor and fragment ions, respectively. To quantify proteins, the raw data were loaded into the MaxQuant software version 1.5.2.8 [t-test in order to define significantly differently expressed proteins with a p value < 0.05, followed by hierarchical clustering analysis, using MeV software v. 4_9_0 [Conditioned media samples were delivered to ProMiFa  to perform nLC-MS/MS analysis. 20 terature . Aliquot 1.5.2.8 . Label-fv. 4_9_0 .3 osteoblasts/cm2 were plated in triplicate on 96-well plates and maintained in culture for 16 days in complete culture medium. At days 2, 4, 7, 9, 11, and 14, cells were treated with CM deriving from 10,000 ASCs or DFs (recipient to donor cell ratio 1 : 10) and viability/proliferation was monitored through time as previously described [μl of supernatant was then transferred to black-bottom 96-well plates and fluorescence  was read with a Wallac Victor 2 plate reader . At the end of the experiment, cells were fixed and stained by Diff-Quik, following standard protocol . Statistical analysis was performed by two-way ANOVA and Bonferroni's multiple comparison test using GraphPad Prism 5 Software .3 × 10escribed . Briefly2 and cultured in standard conditions until confluence. Then, cells were treated with conditioned medium deriving from 120,000 ASCs or DFs (recipient to donor cell ratio 1 : 5). After 24 hours, the expression of osteogenic marker genes  and of VEGF  . Data were normalized on ACTB (Hs01060665_g1) and the relative quantification was determined using the delta delta CT (ΔΔCT) method.OBs were plated at a density of 6000 cells/cmneration ) was assneration . Briefly2 and cultured in standard conditions until confluence. Cells were treated with conditioned medium deriving from 400,000 ASCs or DFs (recipient to donor cell ratio 1 : 5) for 72 hours. Cells were then lysed in 65 mM Tris-HCl, pH 6.8, with 2% sodium dodecyl sulfate (SDS) supplemented with protease-inhibitor cocktail. 15 μg of whole cell lysates, quantified by BCA Protein Assay (Thermo Fisher Scientific), were resolved into 8% SDS-PAGE and transferred to nitrocellulose membranes . Membranes were probed overnight with either rabbit polyclonal antibody raised against osteopontin  or mouse monoclonal antibody raised against SPARC . Goat polyclonal anti-GAPDH antibody  was employed as a housekeeping protein. Proteins of interest were detected after 45 minutes of incubation with appropriate HRP-conjugated secondary antibodies  using LiteAblot® Turbo Extra-Sensitive Chemiluminescent Substrate . Images were acquired through ChemiDoc Imaging System™ and analysed through Image Lab™ software .OBs were seeded at a density of 8000 cells/cme − 07) being associated to extracellular exosomes. No relevant functional pathway was associated to these proteins, even though DF-specific factors appeared to be involved in sugar metabolism .We performed a nLC-MS/MS analysis to identify differentially secreted proteins between ASCs and DFs. Three conditioned media of each cell type were analysed. 1208 proteins were identified, 976 of which were quantified. Following a hierarchical clustering approach, we identified two groups of factors that were differently secreted among ASCs and DFs . The rest test with no further correction, we identified additional factors differentially secreted from ASCs (n = 18) and DFs (n = 35). Finally, we compared the CM of same-donor ASCs and DFs and the factors released uniquely by one population were manually selected. These data confirmed that the two cell types peculiarly release a multitude of different factors (31 ASC-specific proteins and 91 DF-specific proteins), regardless of the donor features. However, since many proteins were solely selected with this criterion  and/or are involved in neuron death and apoptosis . To widen our analysis and deepen our understanding of the differentially secreted proteins, we applied different criteria. At first, we manually checked the data to identify factors exclusively secreted by either ASCs or DFs. Among the proteins released exclusively by one cell type , some factors were not identified through the previous clustering (1 for ASCs and 6 for DFs) . Moreoveriterion , they weg for 34  and 62 (g for 34  factors e − 05 and 34 proteins for DF—FDR: 7.84e − 13). Moreover, it reinforced the previous observation that CM-DFs contain proteins involved in metabolic processes , thus supporting our hypothesis of a proosteoblastic action of CM-DFs. By contrast, CM-ASCs reduced the expression of these genes of about 4 times while COLL I expression was slightly diminished by both treatments. Finally, we investigated the effect of a longer CM treatment (72 hours) on the intracellular protein levels of SPARC and OPN  ASCs and DFs, on cultured human primary osteoblasts. At first, we investigated the influence of CM treatments on osteoblast viability over a period of two weeks . While t and OPN , two impIn light of the fact that conditioned media could be used in the future as biotechnological products for regenerative medicine, it is essential to carry out their characterization before proceeding to clinics. Here, we have implemented differential proteomics to investigate the secretome of ASCs and DFs, focusing on the potential applications in the regenerative medicine field. We applied different criteria to select proteins that were differentially or exclusively released by one cell type and we came up with 34 and 62 factors uniquely or prevalently secreted by ASCs and DFs, respectively. The STRING analysis of these factors led to the recognition of distinct functions related to the CM of ASCs and DFs which are consistent with previous findings on ASC-CM neuroprotective effects , 32–34 aβ-induced new bone formation [in vitro test which provided evidence of the proosteogenic function predicted from the proteomic analysis. In this study, CM-DFs switched from a term of comparison, convenient to point out the specific effectors contained in CM-ASCs, to an interesting source of bioactive factors. With our results, we do not want to minimize the proosteogenic potential of the MSC secretome, which has been already documented especially for BMSCs [On the other hand, the analysis of the factors preponderantly or specifically released by DFs produced unexpected outcomes. CM-DFs resulted particularly enriched in proteins involved in sugar metabolism and further investigations should aim at deciphering whether these factors might be involved in the production of glycosaminoglycans and/or in other metabolic processes. Of note, several proteins, which are involved in bone metabolism and ossification , were also highlighted. Among them, Tenascin C (TNC) is an extracellular matrix glycoprotein implicated in osteoblastic differentiation and mineralization within the bone, probably acting as a mediator of TGF-ormation . C-type ormation . Anotherormation , suggestormation , 56. Intormation , supportor BMSCs , but to or BMSCs . FurtherInterestingly, most of the factors differentially released by ASCs and DFs are contained in extracellular vesicles, but only half of the proteins involved in the regenerative functions are released as vesicular cargos (STRING). This remark is consistent with recent observations of a major effect exerted by the whole conditioned medium compared to the exosomes only   to be used in different regenerative medicine applications.


The purpose of this investigation is to determine the effects of exercise induced muscle damage (EIMD) on balance and knee joint torque. Thirteen males and females volunteered to participate in the study. Following a familiarization session, baseline measures were obtained for isometric torque measured during a maximal voluntary isometric contraction (MVIC) for knee flexors and extensors, and ankle dorsi-flexors and plantar-flexors. Additionally, balance performance was tested in double leg (DL), and right single leg (RSL) static and dynamic unstable stability was measured. Participants then performed the muscle damage protocol of front loaded Bulgarian split squats. All measurements were re-assessed for torque and balance immediately and up to 72 h afterwards. A one-way repeated-measures analysis of variance (ANOVA) was used to analyze differences between baseline and all time-points for torque and balance measures. There was a significant time effect for knee extensors MVIC torque, where baseline measures are greater than post EIMD, 24 h and 48 h post EIMD. There was no significant time effect for all balance conditions. These results provide evidence of EIMD following high intensity eccentric exercises with significant reductions in knee extensor torque up to at least 48 h and show that balance was not compromised following EIMD. Exercise induced muscle damage (EIMD) results from unaccustomed exercise or by performing an exercise that is of high intensity and/or duration ,2. It isThe maintenance of upright balance is a complex task governed by the postural control system, which involves intact sensory and motor neuromuscular systems  that worPrevious studies have also well documented the impact of EIMD on neuromuscular function. Exercise induced muscle damage results in an immediate and prolonged reduction in force and power generation capacity from the muscle as well as an increased physiological demand . Addition = 5) and females (n = 8)  volunteered to participate in a five-session protocol that was approved by the university Institutional Review Board and meets the ethical standards in sport and exercise science research [Thirteen recreationally trained males  by performing 3 sets to volitional failure on each leg. The Jones Machine® was front loaded with 40% of their body weight. During the split squat, the back leg was placed on a padded bench for support and the knee was placed into 90° of flexion, allowing focus on single leg performance of the front leg. We provided assistance on the concentric phase after the participants reached 90° of knee flexion of the front knee on the exercising leg, allowing greater focus on the eccentric phase [A repeated measures design was implemented with one familiarization day that included informed consent, anthropometrics, and familiarization of all experimental protocols. Following the familiarization session, participants visited the laboratory for testing on 4 consecutive days. All baseline measures were assessed for balance and right knee and ankle joint torque (explained under measures) on day 1 following a dynamic warm-up. The right leg only was tested for torques and single leg balance measures, to keep consistency between subjects. The dynamic warm-up consisted of 2 sets of 15 m jogging, leg swings, high knees, exaggerated lunges, and walking planks. After baseline measures were taken, participants completed the exercise induced muscle damage protocol, which consisted of split squats using a Jones Machineic phase ,27. ImmeIsometric torque values were assessed using the BioDex Isokinetic dynamometer . Maximal voluntary isometric contractions were performed for right knee extensors (quadriceps), knee flexors (hamstrings), ankle dorsiflexors; tibialis anterior (TA) and ankle plantar flexors; gastrocnemius (GM) with 3 trials of 3 s each. Participants were seated with the hip at 90° of right leg flexion and knee fixed in a flexed position at an angle of 60° below horizontal for quadriceps (QUAD) and hamstring (HAM) measurement. For right ankle joint torque, participants were seated with the hip at 90° of flexion and right ankle in a fixed position at an angle of 45°. Participants were instructed to produce maximal amount of force for each trial and were verbally encouraged during contractions. Maximum torque from the 3 trials was used for analysis for each muscle. Static and dynamic balance was assessed using Biodex Balance System SD . Each participant performed bilateral double leg (DL) static stability, DL dynamic unstable surface stability (level 4), unilateral right single leg (RSL) static stability, and RSL dynamic unstable surface stability (level 4) balance test. Anterior/Posterior (AP) sway, Medial/Lateral (ML) sway, and an overall sway scores were used for analysis for each balance test. Participants performed all balance tests without shoes on and hands next to their side. Each test consisted of three 20 s trials, and those trials were averaged for analysis. The Biodex Balance System SD ranges from levels 12 to 1 for dynamic balance (12 = most stable and 1 = least stable); level 4 is commonly used in balance assessment with active individuals. Smaller sway values represent more balance and postural stability, and larger sway values represent less balance and postural stability.p < 0.05.A one-way repeated-measures analysis of variance (ANOVA) test was used to determine changes from baseline in balance and muscle pain over time. If main effects for time occurred, they were followed up with Bonferroni post-hoc analyses for pairwise differences with an adjusted alpha for comparisons from baseline. A paired-sample t-test was used to determine differences in total volume-load between left and right leg during EIMD protocol. All analyses were conducted using IBM SPSS™© software . Statistical significance was determined as p = 0.60) differences between left leg (1470.06 ± 503.07 kg) and right leg (1393.46 ± 340.53 kg) total volume-load during the EIMD protocol.There were no significant  time effect for QUAD MVIC torque, where baseline measures were greater than post EIMD (p = 0.001), 24 h post (p = 0.003), and 48 h post (p = 0.02)  time effect for HAM MVIC torque from pre  to immediate post (IM post) , 24 h post , 48 h post , and 72 h post . There was no significant  time effect for GM MVIC torque from pre  to IM-post , 24 h post , 48 h post , and 72 h post . There was no significant  time effect for TA MVIC torque from pre  to IM-post , 24 h post , 48 h post , and 72 h post .There was a significant ( = 0.02) . There wp = 0.17; effect size = 0.12), AP sway , and ML sway . There were no significant time effects for RSL dynamic unstable balance in overall sway  and ML sway . There was a significant  time effect for RSL dynamic unstable balance in AP sway; however, post-hoc analysis revealed no significant differences from baseline to IM post (p = 0.16), 24 h (p = 0.84), 48 h (p = 0.15), and 72 h (p = 0.05) post EIMD. There was no significant time effect for DL static balance in overall sway , AP sway , and ML sway  following EIMD. There was no significant time effect for DL dynamic unstable balance in overall sway (p = 0.51), AP sway (p = 0.22), and ML sway (p = 0.72) following EIMD , which has the capability to test the three afferent systems  individually , can helThe impact of EIMD on torque generation capability from the lower extremity muscles and on balance performance over the course of 72 h from the induction of muscle damage was analyzed in this study. Significant reductions in torque from the knee extensor muscle was evident immediately following the muscle damaging protocol and was significantly reduced until 48 h, providing evidence of muscle damage. Balance performance was not affected after muscle damage, suggesting that the visual and vestibular system might have provided the necessary sensory feedback to compensate for the altered feedback from the proprioceptive system due to muscle damage. Findings from the study help us understand the impact of EIMD on athletic performance and provide the time-course of changes in torque production and balance variables until 72 h after muscle damage. This information could be helpful in planning a return to exercise or rehabilitation for individuals who are recreationally trained and exposed to EIMD.


Cytoplasmic male sterility (CMS) plays a crucial role in the utilization of heterosis and various types of CMS often have different abortion mechanisms. Therefore, it is important to understand the molecular mechanisms related to anther abortion in wheat, which remain unclear at present.In this study, five isonuclear alloplasmic male sterile lines (IAMSLs) and their maintainer were investigated. Cytological analysis indicated that the abortion type was identical in IAMSLs, typical and stainable abortion, and the key abortive period was in the binucleate stage. Most of the 1,281 core shared differentially expressed genes identified by transcriptome sequencing compared with the maintainer in the vital abortive stage were involved in the metabolism of sugars, oxidative phosphorylation, phenylpropane biosynthesis, and phosphatidylinositol signaling, and they were downregulated in the IAMSLs. Key candidate genes encoding chalcone--flavonone isomerase, pectinesterase, and UDP-glucose pyrophosphorylase were screened and identified. Moreover, further verification elucidated that due to the impact of downregulated genes in these pathways, the male sterile anthers were deficient in sugar and energy, with excessive accumulations of ROS, blocked sporopollenin synthesis, and abnormal tapetum degradation.Through comparative transcriptome analysis, an intriguing core transcriptome-mediated male-sterility network was proposed and constructed for wheat and inferred that the downregulation of genes in important pathways may ultimately stunt the formation of the pollen outer wall in IAMSLs. These findings provide insights for predicting the functions of the candidate genes, and the comprehensive analysis of our results was helpful for studying the abortive interaction mechanism in CMS wheat. Triticum aestivum L.) is one of the most important grain and forage crops throughout the world, where it accounts for 20% of the total energy consumed by humans. However, wheat breeding is still underdeveloped and only a small amount of the available diversity is exploited at present [Aegilops kotschyi cytoplasm, Va706A with Ae. vavilovii cytoplasm, Ju706A with Ae. juvenalis cytoplasm, C706A with Ae. crassa cytoplasm, and U706A with Ae. uniaristata cytoplasm, have many superior advantages that can be utilized in hybrid wheat production, where they can improve the wheat quality, are easy to restore, enhance powdery mildew resistance, and increase the growth potential [Increasing crop yields in a sustainable and efficient manner is essential to meet the growing global demand for food . Wheat , many key genes are also involved in pollen and anther development, such as MYB39, A6, AMS, MS1, and TSM1, which are associated with tapetum development and pollen wall formation and the changes of these genes may be important cause of pollen abortion and male sterility [Arabidopsis, UDP-glucose pyrophosphorylase1 (UGP1) and UDP-glucose pyrophosphorylase2 (UGP2) involving in sugar metabolic pathway are essential for pollen starch accumulation and callose deposition, and their suppression results in pollen abortion and male sterility [orf) of mitochondrial F0 ATP synthase complex gene, such as atp6, have been identified as one of the candidates for CMS pepper by RNA editing and northern blot analysis, and further indicating the CMS trait is related to mitochondrial dysfunction [In recent years, transcriptome sequencing has been employed as a powerful tool for studying global transcription networks to provide high resolution data and it has been applied widely in many crops, such as soybean , cotton terility . In planterility . In riceterility , 19. Mitterility . In eukaterility . In peppfunction . Previoufunction , 24. Durfunction . TherefoIn the present study, we used five types of IAMSLs and their maintainer line to obtained a comprehensive overview of the cellular and metabolic changes related to pollen abortion, and a core transcriptional regulatory network in male sterile wheat was further constructed by bioinformatics analyses and various experimental verification. Meanwhile, these findings provide novel insights into the core mechanism of CMS in wheat and the pollen development process.2– KI staining to observe the pollen grains of the trinucleate stage, in contrast to B706, the pollen was 100% sterile in all of the IAMSLs and the pollen abortion type was typical and stainable abortion  and B706 in various developmental stages were observed by stereomicroscopy and scanning electron microscopy. Compared with the IAMSLs, at the trinucleate stage, the anthers from B706 were normally dehiscent with the shedding of mature pollen grains, whereas the anthers did not crack in the IAMSLs. Unlike the mature fertile plants, the epidermis cell and inner epidermal Ubisch bodies in the IAMSLs had irregular shapes with a disordered arrangement  were generated by assembling these clean reads , ABC transporter , and cytochrome P450   were screened based on set criteria (FDR < 0.05 and |Log2FC| > 1) in each sterile line and the maintainer line . Compared with B706, 3,091 DEGs were identified in K706A, as well as 11,606 DEGs in Va706A, 3,951 DEGs in Ju706A, 18,246 DEGs in C706A, and 7,156 DEGs in U706A   was associated with both “cell wall” and “oxidation-reduction process”. Indeed, a previous study demonstrated that pectinesterase is associated with pollen fertility, pollen germination, and anther dehiscence in Arabidopsis thaliana [TraesCS1B02G046200) annotated as the chalcone--flavonone isomerase was included in two GO terms , thereby indicating that this gene probably plays a vital role in CMS wheat. These results indicate that pollen abortion in CMS wheat might be related to changes in genes associated with the cell wall, thereby affecting pollen wall formation and anther dehiscence to ultimately lead to sterility.The core DEGs annotated using the GO database were classified into three principal categories from three ontologies: “biological process”, “cellular component”, and “molecular function”. According to the GO classifications at four levels, enrichment of the core DEGs demonstrated that 44 GO terms were significant p < 0.0  and putative pectinesterase 53  in “pentose and glucuronate interconversions” and “starch and sucrose metabolism”, pyruvate decarboxylase (TraesCS2B02G104000) in “glycolysis/gluconeogenesis” and (TraesCS2D02G086000) in “pentose and glucuronate interconversions”, peroxidase  in “phenylpropanoid biosynthesis”, and atp6–1 (EPlTAEG00000010144 and EPlTAEG00000010116) in “oxidative phosphorylation”, which are associated with metabolic regulation, pollen development and energy synthesis. These genes were severely suppressed in the IAMSLs, which indicates that they may be related to male sterility, thereby providing a valuable resource for studying male sterility in CMS wheat.KEGG enrichment analysis was performed to further identify the functions of the core DEGs in specific biological pathways. The annotated DEGs were assigned to 81 KEGG pathways, where “pentose and glucuronate interconversions”, “starch and sucrose metabolism”, and “glycolysis/gluconeogenesis” were the extremely representative based on the bubble diagram. Interestingly, similar to the KOG and GO results where the “signal transduction” category occupied the main position, the “phosphatidylinositol (PI) signaling system” pathway was also significant according to KEGG enrichment analysis Fig. a-c. Manyr = 0.87, p = 3e-06) was highly correlated with male sterility in the 18 samples , UTP--glucose-1-phosphate uridylyltransferase (TraesCS6A02G050100), and pectinesterase 53   was performed using the non-redundant core DEGs to further elucidate the genes associated with male sterility in the five IAMSLs Fig. a. AnalysWe analyzed the total soluble sugar contents in the anthers from the IAMSLs and maintainer in the binucleate stage to verify whether the downregulated expression of these genes in sugar metabolic pathways may affect the maintenance of pollen growth and development. Compared with B706, the soluble starch contents were significantly lower in the anthers from the five IAMSLs Fig. a. Thus, In order to clarify the effect of down-regulated genes of oxidative phosphorylation pathway on pollen development, we measured the ATP and ROS contents in the anthers using a spectrophotometric method. We found that the ATP contents were significantly lower in the IAMSLs than B706, but excess ROS accumulated in the five IAMSLs Fig. b-d. ThusBased on the above KEGG and hierarchical clustering analysis, our results suggest that the downregulation of these DEGs might limit the phenylpropanoid biosynthesis process. Therefore, TUNEL and transmission electron microscopy assays were conducted using anther sections from the critical stages of abortion. As shown in Fig. We validated the accuracy of the RNA-seq data by conducting quantitative real time polymerase chain reaction (RT-qPCR) analysis, where 10 DEGs that had probable relationships with CMS were selected based on their functional classifications, WGCNA analysis, and the results of previous studies. The expression patterns of these candidate genes according to RT-qPCR were consistent with those determined by RNA-seq Fig. . FurtherIn plants, sucrose and starch are accumulated as energy reserves and carbon skeleton sources, and thus they are necessary for pollen and anther development in the later mature developmental stages by providing basic materials for constructing plant cell walls and fibers. Pentose and glucuronate interconversions, starch and sucrose metabolism, and glycolysis/gluconeogenesis are important sugar metabolism pathways during anther development, and the disruption of sugar metabolism can severely damage pollen development to eventually cause male sterility. In general, the pollen will mature early if the starch level is kept below a certain threshold . For exa2+ and the activity of protein kinase C in the cells [The plasma membrane is an important structure for maintaining cell viability. PI is one of the major phospholipids in the cell membrane and a major phospholipid signaling molecule because of its special molecular structure, where the PI signal transduction process has many important roles . PI-speche cells . The PI he cells . Vesiclehe cells . When thMitochondria are important sites of cellular metabolism and energy metabolism . VariousAccording to previously published results and based on the putative functions and changes in the expression patterns of 1,281 DEGs and their experimental verification in the present study, we propose an intriguing transcriptome-mediated male sterility network for wheat, as shown in Fig. Based on our results, during the vital abortive stage, we found most of the 1281 core shared DEGs were downregulated in the IAMSLs. Furthermore, the key candidate genes related to male sterility were screened and identified, which encoding glycolipid transfer protein, pectinesterase, and UDP-glucose pyrophosphorylase. Moreover, according to our bioinformatics analysis and further verification, we proposed an intriguing core transcriptome-mediated male-sterility network for wheat, and inferred that due to the downregulation of genes in sugars, oxidative phosphorylation, phenylpropane biosynthesis, and phosphatidylinositol signaling pathways, the sugar and energy of anthers were deficient, with excessive accumulations of ROS, thereby triggering the abnormal tapetum degradation and blocked sporopollenin synthesis. These results provide new insights into the metabolic pathways involved with anther abortion in CMS.Aegilops kotschyi cytoplasm, Va706A with Ae. vavilovii cytoplasm, Ju706A with Ae. juvenalis cytoplasm, C706A with Ae. crassa cytoplasm, and U706A with Ae. uniaristata cytoplasm, and their maintainer line B706 as a control, where they all had the same nuclear background . The sterile lines were obtained from stable sterile lines by backcrossing over 20 times with B706. To confirm the stability of sterility, the five IAMSLs were planted during October 2015 in wheat experimental fields at Northwest A&F University, Yangling, China . During April 2016, each IAMSL was checked by bagging and the self-setting rates of the lines were all zero, thereby indicating that male sterility was stable and confirming previous findings [Five types of IAMSLs were used in this study, i.e., K706A with findings . In orde2-KI to evaluate the viability of mature pollen grains [The stages of microspore development were identified by staining the nuclei with acetic acid magenta and microscopy, as described by Ba et al . Anthersn grains . Anthersn grains , and imaTotal RNA from each of 18 samples  was isolated using an RNAiso for Polysaccharide-rich Plant Tissue kit  Co. Ltd., China) according to the instructions provided by the manufacturer. For each RNA sample, the concentration was measured with a NanoDrop 2000 spectrophotometer . The integrity of RNA was confirmed using RNase-free agarose gel electrophoresis, before measuring the concentration with a 2100 Bioanalyzer . mRNA enrichment and purification were conducted using 5 μg of total RNA with oligo (dT)-attached magnetic beads, before breaking into short fragments. The cleaved mRNA fragments were used as templates for first-strand cDNA synthesis with random hexamers and SuperScript II. Second-strand cDNA was synthesized with DNA polymerase I, dNTPs, and RNaseH. The cDNA fragments were then subjected to end-repair, before adding poly-(A) tails together with a ligation sequencing adapter, and purifying the fragments, which were then selected by size with AMPure XP beads. Subsequently, PCR amplification was employed for enrichment of the purified cDNA templates. Finally, Illumina sequencing was performed with the Illumina HiSeq PE150 platform by Guangzhou Sagene Biotechnology Co. Ltd. .ftp://ftp.ncbi.nlm.nih.gov/genbank) and the mapped reads were removed. Next, based on the wheat reference genome , all of the high-quality clean reads excluding rRNA were mapped using HISAT2 (https://ccb.jhu.edu/software/hisat2/index.shtml). The mapped clean reads were assembled with StringTie (https://ccb.jhu.edu/software/stringtie/index.shtml) [p-adjust < 0.05 [In order to obtain high-quality clean reads (clean data) for sequence analysis, adaptor sequences, low-quality reads (base number SQ ≤ 20 in more than 50% of the whole reads), and reads with over 10% unknown bases (N bases) were removed . The higx.shtml) . The expt < 0.05 .http://string-db.org/) based on the KOGs database (ftp://ftp.ncbi.nih.gov/pub/COG/KOG/). GO (http://www.geneontology.org/) and KEGG (http://www.genome.jp/kegg/) functional annotations for the shared DEGs were obtained using BLAST2GO (http://www.blast2go.com/) and BLASTx/BLASTp 2.2.24+, respectively. GO term and KEGG pathway enrichment analysis were performed using the shared DEGs with Goatools and KOBAS software, where the threshold was a corrected P-value ≤0.05 [The shared DEGs in the IAMSLs were functionally classified using BLASTx 2.2.24+ software with the STRING9.0 database (ue ≤0.05 . The relue ≤0.05 . The heaWGCNA was performed for the shared DEGs by using WGCNA in the R package to infer the co-expressed gene modules that had strong associations with sterility . The adjhttp://www.philes.cn/) with a Soluble Sugars Assay kit and an ATP Detection Kit , respectively, according to the instructions supplied with the kits. The total sugar contents (mg g− 1) and ATP contents (μmol g− 1) were calculated based on the formulae provided with the instructions. The rate of superoxide anion (O2−) production and the H2O2 contents were measured using 0.5 g of anthers from the IAMSLs and B706 as described previously [Anthers (0.2 g) were collected during the binucleate stage from each material to determine the soluble sugar  and ATP contents. The total sugar contents and ATP contents were measured at 620 nm and 700 nm using an ultraviolet spectrophotometer  for 10 min, before washing with phosphate-buffered saline for 15 min. TUNEL assays were conducted with an In Situ Cell Death Detection Kit  according to the manufacturer’s instructions. The sections were then observed with a fluorescence confocal scanning microscope . The green TUNEL fluorescent signal and blue DAPI fluorescence were analyzed at excitation and emission wavelengths of 450 nm and 515 nm and 358 nm and 461 nm, respectively. In addition, we counted the TUNEL-positive nuclei/total nuclei ratio in the key stages using IPP 6.0 software.Paraffin sections of anthers (6 μm) were dehydrated using a graded ethanol series and incubated in 20 μg L− 1, pH 7.2), the anthers were fixed in 1% osmium tetroxide and dehydrated with an ethanol series . The anthers were prepared with a UC6 ultramicrotome (Leica) and stained using 0.2% lead citrate [To obtain ultrathin sections, anthers from the late uninucleate and binucleate stages were fixed in 2.5% glutaraldehyde for > 24 h at 4 °C. After washing with phosphate buffer  according to the manufacturer’s instructions. The  RT-qPCR . The prit method .p < 0.05; ** p < 0.01) using SPSS statistical software and Excel Office.Statistical analyses were performed for the measurements of physiological indexes results using one-way analysis of variance. Significant differences in the expression levels between the IAMSLs and B706 were evaluated with the Duncan test  classification. Abscissa for the number of genes, Ordinate for each KOG classified content. Figure S6. GO annotation classification of the core differentially expressed genes. Figure S7. GO enrichment analysis of the core differentially expressed genes. (A) cellular component GO categories enrichment analysis; (B) molecular function GO categories enrichment analysis; (C) biological process GO categories enrichment analysis. Figure S8. Bubble diagram for KEGG enrichment in the DEGs of magenta module. Figure S9. Cytoscape representation of co-expressed genes in the magenta module. Figure S10. Sectional area of the tapetum cell (A) and TUNEL positive nuclei/total nuclei proportion for key stages (B). Figure S11. Coefficient analysis of fold change data between RT-qPCR and RNA-seq. Data indicating relative transcript level from RT-qPCR are means of three replicates. Scatterplots were generated by log2 expression ration from RNA-seq (x-axis) and RT-qPCR (y-axis).Additional file 2: Table S1. Sequence-specific primers used for RT-qPCR.Additional file 3: Table S2. Summary and evaluation of sequencing results.Additional file 4: Table S3. Annotation and FPKM of 1281 core genes.