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Neisseria meningitidis adhesin A (NadA) is a meningococcus surface protein thought to assist in the adhesion of the bacterium to host cells. We have previously shown that NadA also promotes bacterial internalization in a heterologous expression system. Here we have used the soluble recombinant NadA (rNadA) lacking the membrane anchor region to characterize its internalization route in Chang epithelial cells. Added to the culture medium, rNadA internalizes through a PI3K-dependent endocytosis process not mediated by the canonical clathrin or caveolin scaffolds, but instead follows an ARF6-regulated recycling pathway previously described for MHC-I. The intracellular pool of rNadA reaches a steady state level within one hour of incubation and colocalizes in endocytic vesicles with MHC-I and with the extracellularly labeled chaperone Hsp90. Treatment with membrane permeated and impermeable Hsp90 inhibitors 17-AAG and FITC-GA respectively, lead to intracellular accumulation of rNadA, strongly suggesting that the extracellular secreted pool of the chaperone is involved in rNadA intracellular trafficking. A significant number of intracellular vesicles containing rNadA recruit Rab11, a small GTPase associated to recycling endosomes, but do not contain transferrin receptor (TfR). Interestingly, cell treatment with Hsp90 inhibitors, including the membrane-impermeable FITC-GA, abolished Rab11-rNadA colocalization but do not interfere with Rab11-TfR colocalization. Collectively, these results are consistent with a model whereby rNadA internalizes into human epithelial cells hijacking the recycling endosome pathway and recycle back to the surface of the cell via an ARF6-dependent, Rab11 associated and Hsp90-regulated mechanism. The present study addresses for the first time a meningoccoccal adhesin mechanism of endocytosis and suggests a possible entry pathway engaged by N. meningitidis in primary infection of human epithelial cells.

While pulmonary adenocarcinoma (ADC) is morphologically heterogeneous, little is known about intra-tumor gene mutation heterogeneity (ITH). We therefore subjected 20 ADC nodules, 5 mutated for EGFR and 5 for KRAS , 5 with an ALK translocation, and 5 wild type (WT) for these alterations, to unsupervised next-generation sequencing of tumor regions from diverse architectural patterns. When 2 or more different gene mutations were found in a single tumor, this fulfilled the criteria for ITH. In the 84 studied tumor regions with diverse architecture, 71 gene mutations and 34 WT profiles were found. ITH was observed in 9/15 (60 %) ADC, 3 with an EGFR , 3 with a KRAS , and 3 with an ALK aberration, as reflected in 5, 6, and 9 additional mutations, respectively, detected in these tumors. EGFR mutations were observed in 21/22 and KRAS mutations in 18/22 tumor regions, suggesting that they appear early and have a driver role (dominant or trunk mutations). Branching mutations (in EZH2 , PIK3CA , TP53 , and EGFR exon 18) occurred in two or more regions, while private mutations (in ABL1 , ALK , BRAF , HER2 , KDR , LKB1 , PTEN , MET , SMAD4 , SMARCB1 , and SRC ) were confined to unique tumor samples of individual lesions, suggesting that they occurred later on during tumor progression. Patients with a tumor showing branching mutations ran a worse clinical course, independent of confounding factors. We conclude that in ADC, ITH exists in a pattern suggesting spatial and temporal hierarchy with dominant, branching, and private mutations. This is consistent with diverse intra-tumor clonal evolution, which has potential implications for patient prognosis or development of secondary therapy resistance.

Whether invasive mucinous adenocarcinoma (IMA) and colloid adenocarcinoma (ICA) of the lung represent separate tumour entities, or simply lie within a spectrum of phenotypic variability, is worth investigating. Fifteen ICA, 12 IMA, 9 ALK‐rearranged adenocarcinomas (ALKA), 8 non‐mucinous KRAS‐mutated adenocarcinomas (KRASA) and 9 mucinous breast adenocarcinomas (MBA) were assessed by immunohistochemistry for alveolar (TTF1, cytoplasmic MUC1), intestinal (CDX‐2, MUC2), gastric (membrane MUC1, MUC6), bronchial (MUC5AC), mesenchymal (vimentin), neuroendocrine (chromogranin A, synaptophysin), sex steroid hormone‐related (oestrogen and progesterone receptors), pan‐mucinous (HNF4A) and pan‐epithelial (keratin 7) lineage biomarkers and by targeted next generation sequencing (TNGS) for 50 recurrently altered cancer genes. Unsupervised clustering analysis using molecular features identified cluster 1 (IMA and ICA), cluster 2 (ALKA and KRASA) and cluster 3 (MBA) (p < 0.0001). Cluster 1 showed four histology‐independent sub‐clusters (S1 to S4) pooled by HFN4A and MUC5AC but diversely reacting for TTF1, MUC1, MUC2, MUC6 and CDX2. Sub‐cluster S1 predominantly featured intestinal‐alveolar, S2 gastrointestinal, S3 gastric and S4 alveolar differentiation. In turn, KRASA and ALKA shared alveolar lineage alongside residual MUC5AC expression, with additional focal CDX2 and diffuse vimentin, respectively. A proximal‐to‐distal scheme extending from terminal (TB) and respiratory (RB) bronchioles to alveolar cells was devised, where S3 originated from distal TB (cellular mucinous adenocarcinoma), S2 from proximal RB (secreting mucinous adenocarcinoma), S1 from intermediate RB (mucin lake‐forming colloid adenocarcinoma), S4 from distal RB (colloid alveolar adenocarcinoma), KRASA from juxta‐alveolar RB (KRAS‐mutated non‐mucinous adenocarcinoma) and ALKA from juxta‐bronchial alveolar cells (ALK‐translocated adenocarcinoma). TNGS analysis showed KRAS, LKB1, TP53, APC and CDKN2A mutation predominance. In conclusion, IMA and ICA are basket categories, which likely originate from distinct domains of stem/progenitor cells spatially distributed along bronchioles upon common molecular features and genetic alterations.

Little is known about molecular testing on tumor tissue retrieved from stained sections, for which there may be a clinical need. We retrospectively analyzed 112 sections from 56 tumor patients using either fluorescence in situ hybridization (FISH) with different probes (19 sections from 17 patients) or Sanger or targeted next generation sequencing for detection of BRAF , EGFR , KRAS , C-KIT , and TP53 mutations (93 sections from 39 patients). Tumor tissue sections had been stained by hematoxylin and eosin (H&E) (42 sections) or by immunohistochemistry for cytoplasmic or nuclear/nuclear-cytoplasmic markers (70 sections) with a peroxidase (P-IHC, with 3,3′-diaminobenzidine as chromogen) or alkaline phosphatase label (AP-IHC, with Warp Red™ as chromogen). For FISH analysis, the concordance rate between the original diagnosis and that obtained on H&E- or P-IHC-stained tissue sections (AP-IHC was not on record for this set of patients) was 95 % (18 out of 19 tumor sections). Only one tumor sample, diffusely positive for MLH1, did not yield any nuclear hybridization signal. For sequencing analysis, the concordance rate was 100 % on negative P-IHC and positive AP-IHC-stained sections, regardless of the subcellular localization of the reaction product. Mutations were detected in only 52 % of cases expressing nuclear/nuclear-cytoplasmic markers, regardless of the sequencing technology used ( p  = 0.0002). In conclusion, stained sections may be a valuable resource for FISH or sequencing analysis, but on cases expressing nuclear markers sequencing results need to be interpreted cautiously.

Neisseria meningitidis adhesin A (NadA) is a meningococcus surface protein thought to assist in the adhesion of the bacterium to host cells. We have previously shown that NadA also promotes bacterial internalization in a heterologous expression system. Here we have used the soluble recombinant NadA (rNadA) lacking the membrane anchor region to characterize its internalization route in Chang epithelial cells. Added to the culture medium, rNadA internalizes through a PI3K-dependent endocytosis process not mediated by the canonical clathrin or caveolin scaffolds, but instead follows an ARF6-regulated recycling pathway previously described for MHC-I. The intracellular pool of rNadA reaches a steady state level within one hour of incubation and colocalizes in endocytic vesicles with MHC-I and with the extracellularly labeled chaperone Hsp90. Treatment with membrane permeated and impermeable Hsp90 inhibitors 17-AAG and FITC-GA respectively, lead to intracellular accumulation of rNadA, strongly suggesting that the extracellular secreted pool of the chaperone is involved in rNadA intracellular trafficking. A significant number of intracellular vesicles containing rNadA recruit Rab11, a small GTPase associated to recycling endosomes, but do not contain transferrin receptor (TfR). Interestingly, cell treatment with Hsp90 inhibitors, including the membrane-impermeable FITC-GA, abolished Rab11-rNadA colocalization but do not interfere with Rab11-TfR colocalization. Collectively, these results are consistent with a model whereby rNadA internalizes into human epithelial cells hijacking the recycling endosome pathway and recycle back to the surface of the cell via an ARF6-dependent, Rab11 associated and Hsp90-regulated mechanism. The present study addresses for the first time a meningoccoccal adhesin mechanism of endocytosis and suggests a possible entry pathway engaged by N. meningitidis in primary infection of human epithelial cells.

Neisseria meningitidis adhesin A (NadA) is a meningococcus surface protein thought to assist in the adhesion of the bacterium to host cells. We have previously shown that NadA also promotes bacterial internalization in a heterologous expression system. Here we have used the soluble recombinant NadA (rNadA) lacking the membrane anchor region to characterize its internalization route in Chang epithelial cells. Added to the culture medium, rNadA internalizes through a PI3K-dependent endocytosis process not mediated by the canonical clathrin or caveolin scaffolds, but instead follows an ARF6-regulated recycling pathway previously described for MHC-I. The intracellular pool of rNadA reaches a steady state level within one hour of incubation and colocalizes in endocytic vesicles with MHC-I and with the extracellularly labeled chaperone Hsp90. Treatment with membrane permeated and impermeable Hsp90 inhibitors 17-AAG and FITC-GA respectively, lead to intracellular accumulation of rNadA, strongly suggesting that the extracellular secreted pool of the chaperone is involved in rNadA intracellular trafficking. A significant number of intracellular vesicles containing rNadA recruit Rab11, a small GTPase associated to recycling endosomes, but do not contain transferrin receptor (TfR). Interestingly, cell treatment with Hsp90 inhibitors, including the membrane-impermeable FITC-GA, abolished Rab11-rNadA colocalization but do not interfere with Rab11-TfR colocalization. Collectively, these results are consistent with a model whereby rNadA internalizes into human epithelial cells hijacking the recycling endosome pathway and recycle back to the surface of the cell via an ARF6-dependent, Rab11 associated and Hsp90-regulated mechanism. The present study addresses for the first time a meningoccoccal adhesin mechanism of endocytosis and suggests a possible entry pathway engaged by N. meningitidis in primary infection of human epithelial cells.