BackgroundCancer immunotherapy has demonstrated significant clinical activity in different cancers. T cells represent a crucial component of the adaptive immune system and are thought to mediate anti-tumoral immunity. Antigen-specific recognition by T cells is via the T cell receptor (TCR) which is unique for each T cell. Next generation sequencing (NGS) of the TCRs can be used as a platform to profile the T cell repertoire. Though there are a number of software tools available for processing repertoire data by mapping antigen receptor segments to sequencing reads and assembling the clonotypes, most of them are not designed to track and examine the dynamic nature of the TCR repertoire across multiple time points or between different biologic compartments (e.g., blood and tissue samples) in a clinical context.ResultsWe integrated different diversity measures to assess the T cell repertoire diversity and examined the robustness of the diversity indices. Among those tested, Clonality was identified for its robustness as a key metric for study design and the first choice to measure TCR repertoire diversity. To evaluate the dynamic nature of T cell clonotypes across time, we utilized several binary similarity measures (such as Baroni-Urbani and Buser overlap index), relative clonality and Morisita’s overlap index, as well as the intraclass correlation coefficient, and performed fold change analysis, which was further extended to investigate the transition of clonotypes among different biological compartments. Furthermore, the application of differential testing enabled the detection of clonotypes which were significantly changed across time. By applying the proposed “3D” analysis pipeline to the real example of prostate cancer subjects who received sipuleucel-T, an FDA-approved immunotherapy, we were able to detect changes in TCR sequence frequency and diversity thus demonstrating that sipuleucel-T treatment affected TCR repertoire in blood and in prostate tissue. We also found that the increase in common TCR sequences between tissue and blood after sipuleucel-T treatment supported the hypothesis that treatment-induced T cell migrated into the prostate tissue. In addition, a second example of prostate cancer subjects treated with Ipilimumab and granulocyte macrophage colony stimulating factor (GM-CSF) was presented in the supplementary documents to further illustrate assessing the treatment-associated change in a clinical context by the proposed workflow.ConclusionsOur paper provides guidance to study the diversity and dynamics of NGS-based TCR repertoire profiling in a clinical context to ensure consistency and reproducibility of post-analysis. This analysis pipeline will provide an initial workflow for TCR sequencing data with serial time points and for comparing T cells in multiple compartments for a clinical study.

BackgroundThe roles of carcinoembryonic antigen (CEA), cytokeratin 19 fragments (CYFRA21-1) and neuron-specific enolase (NSE) in metastases occurrence and poor diagnosis in specific histological classifications of lung cancer need further exploring. In this study, we investigated relationship between elevated levels of three biomarkers of CEA, CYFRA21-1 and NSE (individually and in combination) and metastasis, survival status and prognosis in lung cancer patients.MethodsEight hundred and sixty eight lung cancer patients including adenocarcinoma (ADC, N = 445), squamous cell carcinoma (SCC, N = 215), small cell lung cancer (SCLC, N = 159) and other types (N = 49) were categorized into negative, moderate and high groups according to serum levels of biomarkers, and were then categorized into negative, single, double and triple groups according to any positive combination of three biomarkers. The cutoff values of three biomarkers for groupings were developed on the training group (N = 432) and verified in a validation group (N = 436). Clinical and laboratory characteristics were then assessed for correlation with occurrence of metastasis, survival status and prognosis between the two groups. Further correlation analyses were also conducted by different subtypes (ADC, SCC and SCLC) and tumor stages (I + II, III and IV) of lung cancers.ResultsThe consistent results between training and validation group confirmed the rationality of grouping methods. CYFRA21-1 levels had stronger association with metastases and survival status than CEA and NSE in all lung cancer patients. When stratified by subtypes, these significances only existed in ADC patients for CYFRA21-1. Cox regression analyses showed that CYFRA21-1 and NSE were independent prognostic factors for lung cancer patients. However, only CYFRA21-1 was an independent prognostic factor in ADC and SCLC patients subtypes. Cox-regression results also indicated that CYFRA21-1 could act as independent prognostic factor in different stages (I + II, III and IV) of lung cancer.ConclusionCYFRA21-1 was more important in metastasis occurrence and in predicting poor prognosis in lung cancer patients than CEA, NSE and positive numbers of biomarkers.

BackgroundSterile and fertile flowers are important evolutionary developmental phenotypes in angiosperm flowers. The development of floral organs, critical in angiosperm reproduction, is regulated by microRNAs (miRNAs). However, the mechanisms underpinning the miRNA regulation of the differentiation and development of sterile and fertile flowers remain unclear.ResultsHere, based on investigations of the morphological differences between fertile and sterile flowers, we used high-throughput sequencing to characterize the miRNAs in the differentiated floral organs of Viburnum macrocephalum f. keteleeri. We identified 49 known miRNAs and 67 novel miRNAs by small RNA (sRNA) sequencing and bioinformatics analysis, and 17 of these known and novel miRNA precursors were validated by polymerase chain reaction (PCR) and Sanger sequencing. Furthermore, by comparing the sequencing results of two sRNA libraries, we found that 30 known and 39 novel miRNA sequences were differentially expressed, and 35 were upregulated and 34 downregulated in sterile compared with fertile flowers. Combined with their predicted targets, the potential roles of miRNAs in V. macrocephalum f. keteleeri flowers include involvement in floral organogenesis, cell proliferation, hormonal pathways, and stress responses. miRNA precursors and targets were further validated by quantitative real-time PCR (qRT-PCR). Specifically, miR156a-5p, miR156g, and miR156j expression levels were significantly higher in fertile flowers than in sterile flowers, while SPL genes displayed the opposite expression pattern. Considering that the targets of miR156 are predicted to be SPL genes, we propose that miR156 may be involved in the regulation of stamen development in V. macrocephalum f. keteleeri.ConclusionsWe identified miRNAs differentially expressed between fertile and sterile flowers in V. macrocephalum f. keteleeri and provided new insights into the important regulatory roles of miRNAs in the differentiation and development of fertile and sterile flowers.

BackgroundHigh on-treatment platelet reactivity (HPR) represents a strong risk factor for thrombotic events after PCI. We aim to evaluate the efficacy and safety of individualizing intensified dual antiplatelet therapy (DAPT) in PCI-treated patients with HPR based on platelet function testing (PFT).MethodsElectronic databases were searched for randomized control trials that reported the clinical outcomes of using an intensified antiplatelet protocol with P2Y12 receptor inhibitor comparing with standard maintenance dose of clopidogrel on the basis of platelet function testing. Clinical endpoints were assessed.ResultsFrom 2005 to 2016, thirteen clinical studies comprising 7290 patients were included for analysis. Compared with standard antiplatelet therapy with clopidogrel, the intensified protocol based on platelet function testing was associated with a significant reduction in major adverse cardiovascular events (RR:0.55, 95% CI: 0.36–0.84, p = 0.005), cardiovascular death (RR:0.60, 95% CI: 0.38–0.96, p = 0.03), stent thrombosis (RR:0.58, 95% CI: 0.36–0.93, p = 0.02) and target vessel revascularization (RR:0.33, 95% CI: 0.14–0.76, p = 0.009). No significant difference was found in the rate of bleeding events between intensified and standard protocol.ConclusionsCompared with standard clopidogrel therapy, individualized intensified antiplatelet therapy on the basis of platelet reactivity testing reduces the incidence of cardiovascular events in patient undergoing PCI, without increasing the risk of bleeding.

BackgroundThe roles of carcinoembryonic antigen (CEA), cytokeratin 19 fragments (CYFRA21-1) and neuron-specific enolase (NSE) in metastases occurrence and poor diagnosis in specific histological classifications of lung cancer need further exploring. In this study, we investigated relationship between elevated levels of three biomarkers of CEA, CYFRA21-1 and NSE (individually and in combination) and metastasis, survival status and prognosis in lung cancer patients.MethodsEight hundred and sixty eight lung cancer patients including adenocarcinoma (ADC, N = 445), squamous cell carcinoma (SCC, N = 215), small cell lung cancer (SCLC, N = 159) and other types (N = 49) were categorized into negative, moderate and high groups according to serum levels of biomarkers, and were then categorized into negative, single, double and triple groups according to any positive combination of three biomarkers. The cutoff values of three biomarkers for groupings were developed on the training group (N = 432) and verified in a validation group (N = 436). Clinical and laboratory characteristics were then assessed for correlation with occurrence of metastasis, survival status and prognosis between the two groups. Further correlation analyses were also conducted by different subtypes (ADC, SCC and SCLC) and tumor stages (I + II, III and IV) of lung cancers.ResultsThe consistent results between training and validation group confirmed the rationality of grouping methods. CYFRA21-1 levels had stronger association with metastases and survival status than CEA and NSE in all lung cancer patients. When stratified by subtypes, these significances only existed in ADC patients for CYFRA21-1. Cox regression analyses showed that CYFRA21-1 and NSE were independent prognostic factors for lung cancer patients. However, only CYFRA21-1 was an independent prognostic factor in ADC and SCLC patients subtypes. Cox-regression results also indicated that CYFRA21-1 could act as independent prognostic factor in different stages (I + II, III and IV) of lung cancer.ConclusionCYFRA21-1 was more important in metastasis occurrence and in predicting poor prognosis in lung cancer patients than CEA, NSE and positive numbers of biomarkers.

BackgroundNitrite inhalants have become popular as recreational drugs among the homosexual population in some developed countries since the 1980s. These drugs, also called RUSH in China, have become attractive among Chinese men who have sex with men (MSM) in the past few years. The aim of this cross-sectional study was to understand the knowledge, attitude, and status of nitrite inhalant use among Chinese MSM.MethodsThe study participants were recruited from Tianjin, China between April and August 2012. Information, including demographics, sexual behavior, and RUSH use, was obtained through structured interviewer questionnaires. Blood samples were also collected to identify the status of HIV, HSV, and syphilis infections.ResultsA total of 500 participants were interviewed. Of the participants, 64.0% knew that RUSH could increase sexual pleasure and 38.6% of the participants had used RUSH at least once. The mean duration of RUSH use was 1.5 years. Among the participants who were familiar with RUSH, 60.0% had heard of RUSH for the first time after 2011, 55% received information about RUSH via the internet, and only 42.2% knew the side effects of RUSH. RUSH users were more likely to work in companies (Odds ratio [OR]: 2.61; 95% CI: 1.65–4.12), live with homosexual partners (OR: 1.88; 95% CI: 1.19–2.92), not live alone (OR: 2.26; 95% CI: 1.29–3.96), smoke cigarettes (OR: 1.49; 95% CI: 1.02–2.17), use alcohol (OR: 1.63; 95% CI: 1.12–2.39), and seek sexual partners on the internet (OR: 2.59; 95% CI: 1.50–4.50).ConclusionsThe impact of RUSH abuse on the expanding HIV epidemic among MSM has been demonstrated in China. Our findings suggest that the communication and awareness of health hazard of recreational drugs should be reinforced in HIV prevention education, especially through new media. Future research is needed to further explore how integrative strategies should be used to reduce the substance abuse and risky sexual behaviors.