Identification, isolation and characterization of human LGR5-positive colon adenoma cells.

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 (DKK4) and SPARC-related modular calcium binding 2 (SMOC2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.

Safety and activity of crizotinib for paediatric patients with refractory solid tumours or anaplastic large-cell lymphoma: a Children's Oncology Group phase 1 consortium study.

BACKGROUND: Various human cancers have ALK gene translocations, amplifications, or oncogenic mutations, such as anaplastic large-cell lymphoma, inflammatory myofibroblastic tumours, non-small-cell lung cancer (NSCLC), and neuroblastoma. Therefore, ALK inhibition could be a useful therapeutic strategy in children. We aimed to determine the safety, recommended phase 2 dose, and antitumour activity of crizotinib in children with refractory solid tumours and anaplastic large-cell lymphoma. METHODS: In this open-label, phase 1 dose-escalation trial, patients older than 12 months and younger than 22 years with measurable or evaluable solid or CNS tumours, or anaplastic large-cell lymphoma, refractory to therapy and for whom there was no known curative treatment were eligible. Crizotinib was given twice daily without interruption. Six dose levels (100, 130, 165, 215, 280, 365 mg/m(2) per dose) were assessed in the dose-finding phase of the study (part A1), which is now completed. The primary endpoint was to estimate the maximum tolerated dose, to define the toxic effects of crizotinib, and to characterise the pharmacokinetics of crizotinib in children with refractory cancer. Additionally, patients with confirmed ALK translocations, mutations, or amplification (part A2 of the study) or neuroblastoma (part A3) could enrol at one dose level lower than was currently given in part A1. We assessed ALK genomic status in tumour tissue and used quantitative RT-PCR to measure NPM-ALK fusion transcript in bone marrow and blood samples of patients with anaplastic large-cell lymphoma. All patients who received at least one dose of crizotinib were evaluable for response; patients completing at least one cycle of therapy or experiencing dose limiting toxicity before that were considered fully evaluable for toxicity. This study is registered with ClinicalTrials.gov, NCT00939770. FINDINGS: 79 patients were enrolled in the study from Oct 2, 2009, to May 31, 2012. The median age was 10.1 years (range 1.1-21.4); 43 patients were included in the dose escalation phase (A1), 25 patients in part A2, and 11 patients in part A3. Crizotinib was well tolerated with a recommended phase 2 dose of 280 mg/m(2) twice daily. Grade 4 adverse events in cycle 1 were neutropenia (two) and liver enzyme elevation (one). Grade 3 adverse events that occurred in more than one patient in cycle 1 were lymphopenia (two), and neutropenia (eight). The mean steady state peak concentration of crizotinib was 630 ng/mL and the time to reach this peak was 4 h (range 1-6). Objective tumour responses were documented in 14 of 79 patients (nine complete responses, five partial responses); and the anti-tumour activity was enriched in patients with known activating ALK aberrations (eight of nine with anaplastic large-cell lymphoma, one of 11 with neuroblastoma, three of seven with inflammatory myofibroblastic tumour, and one of two with NSCLC). INTERPRETATION: The findings suggest that a targeted inhibitor of ALK has antitumour activity in childhood malignancies harbouring ALK translocations, particularly anaplastic large-cell lymphoma and inflammatory myofibroblastic tumours, and that further investigation in the subset of neuroblastoma harbouring known ALK oncogenic mutations is warranted. FUNDING: Pfizer and National Cancer Institute grant to the Children's Oncology Group.

Benchmarking Single-Cell mRNA-Sequencing Technologies Uncovers Differences in Sensitivity and Reproducibility in Cell Types With Low RNA Content.

Single-cell RNA sequencing (scRNA-seq) has the ability to classify each cell and determine the transcriptomic profile of specific cell types and cells of a given disease state; however, sensitivity of the gene count for each cell can be a critical component to the success of a single-cell study. The recently introduced SMART-Seq Single Cell PLUS Kit (SSsc PLUS) claims to provide higher sensitivity and reproducibility versus popular methods for the sequencing analysis of single cells. Here, the cDNA-generation component of the kit, SMART-Seq Single Cell Kit (SSsc), was compared with the popular homebrew protocol, Smart-seq2, and its update, Smart-seq3. The SMART-Seq Library Prep Kit from SSsc PLUS was benchmarked against a commonly used scRNA-seq library preparation method, Illumina Nextera XT. Finally, the SSsc chemistry was tested in both full and fractional volumes on 2 popular liquid-handler devices to investigate whether the high sensitivity was maintained in miniaturization. We demonstrate that SSsc PLUS outperforms these other full-length methods in convenience, sensitivity, gene identification, and reproducibility while also offering full compatibility with automation platforms.

Toxoplasma gondii Toxolysin 4 Contributes to Efficient Parasite Egress from Host Cells.

Egress from host cells is an essential step in the lytic cycle of T. gondii and other apicomplexan parasites; however, only a few parasite secretory proteins are known to affect this process. The putative metalloproteinase toxolysin 4 (TLN4) was previously shown to be an extensively processed microneme protein, but further characterization was impeded by the inability to genetically ablate TLN4. Here, we show that TLN4 has the structural properties of an M16 family metalloproteinase, that it possesses proteolytic activity on a model substrate, and that genetic disruption of TLN4 reduces the efficiency of egress from host cells. Complementation of the knockout strain with the TLN4 coding sequence significantly restored egress competency, affirming that the phenotype of the Δtln4 parasite was due to the absence of TLN4. This work identifies TLN4 as the first metalloproteinase and the second microneme protein to function in T. gondii egress. The study also lays a foundation for future mechanistic studies defining the precise role of TLN4 in parasite exit from host cells. IMPORTANCE After replicating within infected host cells, the single-celled parasite Toxoplasma gondii must rupture out of such cells in a process termed egress. Although it is known that T. gondii egress is an active event that involves disruption of host-derived membranes surrounding the parasite, very few proteins that are released by the parasite are known to facilitate egress. In this study, we identify a parasite secretory protease that is necessary for efficient and timely egress, laying the foundation for understanding precisely how this protease facilitates T. gondii exit from host cells.

Copper distributed by Atx1 is available to copper amine oxidase 1 in Schizosaccharomyces pombe.

Copper amine oxidases (CAOs) have been proposed to be involved in the metabolism of xenobiotic and biogenic amines. The requirement for copper is absolute for their activity. In the fission yeast Schizosaccharomyces pombe, cao1(+) and cao2(+) genes are predicted to encode members of the CAO family. While both genes are expressed in wild-type cells, we determined that the expression of only cao1(+) but not cao2(+) results in the production of an active enzyme. Site-directed mutagenesis identified three histidine residues within the C-terminal region of Cao1 that are necessary for amine oxidase activity. By use of a cao1(+)-GFP allele that retained wild-type function, Cao1-GFP was localized in the cytosol (GFP is green fluorescent protein). Under copper-limiting conditions, disruption of ctr4(+), ctr5(+), and cuf1(+) produced a defect in amine oxidase activity, indicating that a functionally active Cao1 requires Ctr4/5-mediated copper transport and the transcription factor Cuf1. Likewise, atx1 null cells exhibited substantially decreased levels of amine oxidase activity. In contrast, deletion of ccc2, cox17, and pccs had no significant effect on Cao1 activity. Residual amine oxidase activity in cells lacking atx1(+) can be restored to normal levels by returning an atx1(+) allele, underscoring the critical importance of the presence of Atx1 in cells. Using two-hybrid analysis, we demonstrated that Cao1 physically interacts with Atx1 and that this association is comparable to that of Atx1 with the N-terminal region of Ccc2. Collectively, these results describe the first example of the ability of Atx1 to act as a copper carrier for a molecule other than Ccc2 and its critical role in delivering copper to Cao1.

[The molecular bases for copper uptake and distribution: lessons from yeast]. / Les bases moléculaires de l'approvisionnement en cuivre: leçons tirées de la levure.

Copper exists in two oxidation states, cuprous (Cu1+) and cupric (Cu2+), which, respectively, can donate or accept electrons. The fact that copper has two readily interconvertible redox states makes it a catalytic co-factor for many important enzymes. Over the past years, work in a number of laboratories has clearly demonstrated that studies in yeast have served as a springboard for identifying cellular components and processes involved in copper uptake and distribution. In several cases, it has been shown that mammalian proteins are capable of functionally replacing yeast proteins, thereby revealing their remarkable functional conservation. For high-affinity copper transport into cells, it has been shown that copper transporters of the Ctr family are required. Upon entering the cell, copper is partitioned to different proteins and into different compartments within the cell. Given the potential toxicity of copper, specialized proteins bind copper after it enters the cell and subsequently donate the bound copper to their corresponding recipient proteins. Three copper-binding proteins, Ccs1, Cox17, and Atx1, have been identified that serve as "copper chaperones" to deliver copper. double dagger.

Requisite analytic and diagnostic performance characteristics for the clinical detection of BRAF V600E in hairy cell leukemia: a comparison of 2 allele-specific PCR assays.

Detection of high-frequency BRAF V600E mutations in hairy cell leukemia (HCL) has important diagnostic utility. However, the requisite analytic performance for a clinical assay to routinely detect BRAF V600E mutations in HCL has not been clearly defined. In this study, we sought to determine the level of analytic sensitivity needed for formalin-fixed, paraffin-embedded (FFPE) and frozen samples and to compare the performance of 2 allele-specific polymerase chain reaction (PCR) assays. Twenty-nine cases of classic HCL, including 22 FFPE bone marrow aspirates and 7 frozen specimens from blood or bone marrow were evaluated using a laboratory-developed allele-specific PCR assay and a commercially available allele-specific quantitative PCR assay-myT BRAF Ultra. Also included were 6 HCL variant and 40 non-HCL B-cell lymphomas. Two cases of classic HCL, 1 showing CD5 expression, were truly BRAF V600E-negative based on negative results by PCR and sequencing despite high-level leukemic involvement. Among the remaining 27 specimens, V600E mutations were detected in 88.9% (17/20 FFPE; 7/7 frozen) and 81.5% (15/20 FFPE; 7/7 frozen), for the laboratory-developed and commercial assays, respectively. No mutations were detected among the 46 non-HCL lymphomas. Both assays showed an analytic sensitivity of 0.3% involvement in frozen specimens and 5% in FFPE tissue. On the basis of these results, an assay with high analytic sensitivity is required for the clinical detection of V600E mutations in HCL specimens. Two allele-specific PCR assays performed well in both frozen and FFPE bone marrow aspirates, although detection in FFPE tissue required 5% or more involvement.

Toxoplasma gondii toxolysin 4 is an extensively processed putative metalloproteinase secreted from micronemes.

Proteases play central roles in cell invasion by Toxoplasma gondii and other apicomplexan parasites. Herein we report the cloning and characterization of a novel secretory putative metalloproteinase, Toxolysin 4 (TLN4). T. gondii tachyzoites store TLN4 in the micronemes and secrete it in response to elevated calcium, suggesting a possible role in cell invasion. TLN4 is initially synthesized as a large (∼260 kDa) precursor, which is extensively processed into multiple proteolytic fragments within the parasite secretory system. At least some of these proteolytic fragments remain associated in a large molecular complex. Whereas precomplementation with the TLN4 cDNA allowed disruption of the TLN4 gene, multiple attempts to directly knockout TLN4 without precomplementation failed. TLN4 knockout parasites were detected by PCR in transfected populations but were lost from the cultures during drug selection and growth suggesting that TLN4 contributes to parasite fitness.

Mechanisms of copper loading on the Schizosaccharomyces pombe copper amine oxidase 1 expressed in Saccharomyces cerevisiae.

Copper amine oxidases (CAOs) are found in almost every living kingdom. Although Saccharomyces cerevisiae is one of the few yeast species that lacks an endogenous CAO, heterologous gene expression of CAOs from other organisms produces a functional enzyme. To begin to characterize their function and mechanisms of copper acquisition, two putative cao(+) genes from Schizosaccharomyces pombe were expressed in S. cerevisiae. Expression of spao1(+) resulted in the production of an active enzyme capable of catalysing the oxidative deamination of primary amines. On the other hand, expression of spao2(+) failed to produce an active CAO. Using a functional spao1(+)-GFP fusion allele, the SPAO1 protein was localized in the cytosol. Under copper-limiting conditions, yeast cells harbouring deletions of the MAC1, CTR1 and CTR3 genes were defective in amine oxidase activity. Likewise, atx1Delta null cells exhibited no CAO activity, while ccc2Delta mutant cells exhibited decreased levels of amine oxidase activity, and mutations in cox17Delta and ccs1Delta did not cause any defects in this activity. Copper-deprived S. cerevisiae cells expressing spao1(+) required a functional atx1(+) gene for growth on minimal medium containing ethylamine as the sole nitrogen source. Under these conditions, the inability of the atx1Delta cells to utilize ethylamine correlated with the lack of SPAO1 activity, in spite of the efficient expression of the protein. Cells carrying a disrupted ccc2Delta allele exhibited only weak growth on ethylamine medium containing a copper chelator. The results of these studies reveal that expression of the heterologous spao1(+) gene in S. cerevisiae is required for its growth in medium containing ethylamine as the sole nitrogen source, and that expression of an active Schiz. pombe SPAO1 protein in S. cerevisiae depends on the acquisition of copper through the high-affinity copper transporters Ctr1 and Ctr3, and the copper chaperone Atx1.

Functional dissection of Ctr4 and Ctr5 amino-terminal regions reveals motifs with redundant roles in copper transport.

Copper uptake in the fission yeast Schizosaccharomyces pombe is carried out by a heteromeric complex formed by two proteins, Ctr4 and Ctr5. In this study, a stable expression system using integrative plasmids was developed to investigate the respective roles of Ctr4 and Ctr5 in copper transport. It was shown that expression of full-length Ctr4 or truncated Ctr4 containing residues 106-289 was required for localization of Ctr5 to the plasma membrane. Likewise, when the full-length Ctr5 or truncated Ctr5 from residues 44-173 was co-expressed with Ctr4, this protein was visualized at the periphery of the cell. To determine the importance of the Mets motifs (consisting of five methionines arranged as Met-X2-Met-X-Met, where X is any amino acid) of Ctr4 and Ctr5 in the heteroprotein complex, we co-expressed Ctr5 lacking the Mets motif and Cys-X-Met-X-Met sequence with wild-type Ctr4 or its mutant derivatives. Conversely, Ctr4 lacking the Mets motif and Met(122) was expressed with wild-type Ctr5 or its mutant derivatives. These experiments revealed that the five Mets motifs of Ctr4 and the Ctr4 residue Met(122) have equally important roles in copper assimilation. Furthermore, the two partially overlapping Mets motifs and the Cys-X-Met-X-Met sequence in Ctr5 have redundant functions in copper transport, with the latter sequence making a greater contribution than the former. Together, the data reveal that co-expression of both Ctr4 and Ctr5 is necessary for the proper function and localization of the heteroprotein complex to the plasma membrane. Once on the cell surface, the N-terminal regions of Ctr4 and Ctr5 can function independently to transport copper; however, the greatest efficiency is achieved when both N termini are present.

The Schizosaccharomyces pombe Pccs protein functions in both copper trafficking and metal detoxification pathways.

Because copper is both an essential cofactor and a toxic metal, different strategies have evolved to appropriately regulate its homeostasis as a function of changing environmental copper levels. In this report, we describe a metallochaperone-like protein from Schizosaccharomyces pombe that maintains the delicate balance between essentiality and toxicity. This protein, designated Pccs, has four distinct domains. SOD activity assays reveal that the first three domains of Pccs are necessary and sufficient to deliver copper to its target, copper-zinc superoxide dismutase (SOD1). Pccs domain IV, which is absent in Saccharomyces cerevisiae CCS1, contains seventeen cysteine residues, eight pairs of which are in a potential metal coordination arrangement, Cys-Cys. We show that S. cerevisiae ace1Delta mutant cells expressing the full-length Pccs molecule are resistant to copper toxicity. Furthermore, we demonstrate that the Pccs domain IV enhances copper resistance of the ace1Delta cells by an order of magnitude compared with that observed in the same strain expressing a pccs+ I-II-III allele encoding Pccs domains I-III. We consistently found that S. pombe cells disrupted in the pccs+ gene exhibit an increased sensitivity to copper and cadmium. Furthermore, we demonstrate that overexpression of pccs+ is associated with increased copper resistance in fission yeast cells. Taken together, our findings suggest that Pccs activates apo-SOD1 under copper-limiting conditions through the use of its first three domains and protects cells against metal ion toxicity via its fourth domain.