Bioorthogonal non-canonical amino acid tagging reveals translationally active subpopulations of the cystic fibrosis lung microbiota.
- 作者列表："Valentini TD","Lucas SK","Binder KA","Cameron LC","Motl JA","Dunitz JM","Hunter RC
:Culture-independent studies of cystic fibrosis lung microbiota have provided few mechanistic insights into the polymicrobial basis of disease. Deciphering the specific contributions of individual taxa to CF pathogenesis requires comprehensive understanding of their ecophysiology at the site of infection. We hypothesize that only a subset of CF microbiota are translationally active and that these activities vary between subjects. Here, we apply bioorthogonal non-canonical amino acid tagging (BONCAT) to visualize and quantify bacterial translational activity in expectorated sputum. We report that the percentage of BONCAT-labeled (i.e. active) bacterial cells varies substantially between subjects (6-56%). We use fluorescence-activated cell sorting (FACS) and genomic sequencing to assign taxonomy to BONCAT-labeled cells. While many abundant taxa are indeed active, most bacterial species detected by conventional molecular profiling show a mixed population of both BONCAT-labeled and unlabeled cells, suggesting heterogeneous growth rates in sputum. Differentiating translationally active subpopulations adds to our evolving understanding of CF lung disease and may help guide antibiotic therapies targeting bacteria most likely to be susceptible.
: 囊性纤维化肺部微生物群的独立培养研究对疾病的多微生物基础提供了很少的机制见解。破译个体分类群对CF发病机制的具体贡献需要全面了解其在感染部位的生态生理学。我们假设只有CF微生物群的一个子集是翻译活性的，并且这些活性在受试者之间不同。在这里，我们应用生物正交非经典氨基酸标签 (BONCAT) 来可视化和定量咳痰中的细菌翻译活性。我们报告了BONCAT标记的百分比 (即活性) 细菌细胞在受试者之间显著不同 (6-56%)。我们使用荧光激活细胞分选 (FACS) 和基因组测序将分类学分配给BONCAT标记的细胞。虽然许多丰富的分类群确实是有活性的，但通过常规分子谱检测到的大多数细菌种类显示出BONCAT标记和未标记细胞的混合群体，表明痰中的生长速率不均匀。区分翻译活性亚群增加了我们对CF肺病不断发展的理解，并可能有助于指导针对最可能易感细菌的抗生素疗法。
METHODS:OBJECTIVE:The aim of this study was to investigate the key molecular alterations in small primary pancreatic neuroendocrine tumors (PanNETs) associated with the development of liver metastases. BACKGROUND:Well-differentiated PanNETs with small size are typically indolent; however, a limited subset metastasize to the liver. METHODS:A total of 87 small primary PanNETs (<3 cm), including 32 metastatic cases and 55 nonmetastatic cases after a 5-year follow-up, were immunolabeled for DAXX/ATRX and analyzed for alternative lengthening of telomeres (ALT) by Fluorescence In Situ Hybridization. A subset of these cases, 24 that metastasized and 24 that did not metastasize, were assessed by targeted next-generation sequencing and whole-genome copy number variation. RESULTS:In the entire cohort, high Ki-67 (OR 1.369; 95% CI 1.121-1.673; P = 0.002), N-stage (OR 4.568; 95% CI 1.458-14.312; P = 0.009), and ALT-positivity (OR 3.486; 95% CI 1.093-11.115; P = 0.035) were independently associated with liver metastases. In the subset assessed by next-generation sequencing and copy number variation analysis, 3 molecular subtypes with differing risks of liver metastases were identified. Group 1 (n = 15; 73% metastasized) was characterized by recurrent chromosomal gains, CN-LOH, DAXX mutations, and ALT-positivity. Group 2 (n = 19; 42% metastasized, including 5 G1 tumors) was characterized by limited copy number alterations and mutations. Group 3 (n = 14; 35% metastasized) were defined by chromosome 11 loss. CONCLUSIONS:We identified genomic patterns of small PanNETs associated with a different risk for liver metastases. Molecular alterations, such as DAXX mutations, chromosomal gains, and ALT, are associated with an increased risk of metastasis in small PanNETs. Therefore, targeted sequencing and/or ALT analysis may help in the clinical decisions for these small PanNETs.
METHODS::Background: This retrospective comparative case series study aims to analyze the pancreatic fistula rates of internal and external stenting of the pancreatojejunostomy (PJ) anastomosis in patients who underwent pancreatoduodenectomy (PD) for periampullary tumors.Methods: Ninety-eight patients with periampullary tumors who were operated between 2010 and 2017 were enrolled in this study. A classic open PD with Roux-en-Y PJ reconstruction was performed in all cases.Results: The PJ anastomosis of 53 patients (54%) were stented internally whereas in 45 patients (46%) external stenting was preferred. Pancreatic fistula was observed in 29 patients (29.6%). Internal stenting and soft pancreatic tissue were found to be related to higher pancreatic fistula rates with odds ratios of 3.27 (p = .024) and 3.4 (p = .017), respectively. When only grade B and grade C fistulas were taken into account, the type of stenting has lost its significance but the texture of the remnant pancreas was still associated with 'clinically important' pancreatic fistula.Conclusions: We concluded that the external stenting of the PJ anastomosis may be considered as an effective approach for reducing postoperative pancreatic leaks in PD-planned patients for periampullary tumors. Although our study was retrospectively designed, we used standard charts to gather patient data and compared two stenting methods among homogeneous patient groups.
METHODS:BACKGROUND:Heavily pretreated pancreatic cancer patients have a grave prognosis. In this case series study, we evaluated the safety and efficacy of nab-paclitaxel-based chemotherapy for such patients. METHODS:The data of pancreatic adenocarcinoma patients (n = 40) treated with nab-paclitaxel after the failure of gemcitabine or fluoropyrimidines at our institution in 2013-2015 were reviewed. RESULTS:The median number of prior chemotherapy regimens was two (range, 1-6). Eighteen patients had an Eastern Cooperative Oncology Group performance status of ≥2. The regimens comprised nab-paclitaxel combined with the following drugs: gemcitabine (n = 28), gemcitabine and fluoropyrimidine (n = 3), platinum and fluoropyrimidine (n = 4), fluoropyrimidine (n = 4), and irinotecan and fluoropyrimidine (n = 1). The median dose of nab-paclitaxel was 63 (range, 51-72) mg/m2/dose, with the schedule of D1/15, D1/8, and D1/8/15 followed in 23, 14, and 3 patients, respectively. The median overall survival was 5.1 (95% CI, 4.6-5.7) months. Among 32 evaluable patients, two partial responses and six stable diseases were observed. The median progression-free survival was 2.6 (95% CI, 1.9-3.2) months. Grade 3/4 leucopenia or neutropenia was observed in three and two patients, respectively. Grade 3/4 anemia was observed in four patients. Other significant (grade 3 or more) nonhematological toxicities were not frequent, except for sepsis/infection (n = 7). However, more severe anemia or sepsis/infection was significantly associated with disease control. CONCLUSION:In heavily pretreated pancreatic adenocarcinoma patients, low-dose nab-paclitaxel-based chemotherapy was fairly tolerable with modest efficacy.