Automated microraft platform to identify and collect non-adherent cells successfully gene-edited with CRISPR-Cas9.

Microraft arrays have been used to screen and then isolate adherent and non-adherent cells with very high efficiency and excellent viability; however, manual screening and isolation limits the throughput and utility of the technology. In this work, novel hardware and software were developed to automate the microraft array platform. The developed analysis software identified microrafts on the array with greater than 99% sensitivity and cells on the microrafts with 100% sensitivity. The software enabled time-lapse imaging and the use of temporally varying characteristics as sort criteria. The automated hardware released microrafts with 98% efficiency and collected released microrafts with 100% efficiency. The automated system was used to examine the temporal variation in EGFP expression in cells transfected with CRISPR-Cas9 components for gene editing. Of 11,499 microrafts possessing a single cell, 220 microrafts were identified as possessing temporally varying EGFP-expression. Candidate cells (n=172) were released and collected from the microraft array and screened for the targeted gene mutation. Two cell colonies were successfully gene edited demonstrating the desired mutation.

Epidemiology and clinical features of toxicity following recreational use of synthetic cannabinoid receptor agonists: a report from the United Kingdom National Poisons Information Service.

CONTEXT: Toxicity from the use of synthetic cannabinoid receptor agonists (SCRAs) has been encountered increasingly frequent in many countries. OBJECTIVE: To characterise presentation rates, demographic profiles and reported clinical features for users of SCRAs referred by health professionals in the United Kingdom to the National Poisons Information Service (NPIS), to compare reported toxicity between commonly used branded products, and to examine the impact of legal control measures on enquiry numbers. METHODS: NPIS telephone enquiry records were searched for SCRA-related terms for the 8-year period 1st January 2007 to 31st December 2014, consolidating multiple enquiries about the same case into a single record. Demographic data, reported exposure details, clinical features and poisoning severity were analysed, excluding cases where SCRA exposure was unlikely. RESULTS: Enquiries to the NPIS were made concerning 510 individuals relating to probable SCRA use, with annual numbers increasing year on year. Most patients were male (80.8%) and <25 years old (65.1%). Common clinical features reported in the 433 (84.9%) patients reporting SCRA use without other substances included tachycardia (n = 73, 16.9%), reduced level of consciousness (n = 70, 16.2%), agitation or aggression (n = 45, 10.4%), vomiting (n = 30, 6.9%), dizziness (n = 26, 6.0%), confusion (n= 21, 4.8%), mydriasis (n = 20, 4.6%) and hallucinations (n = 20, 4.6%). The Maximum Poisoning Severity Score (PSS) indicated severe toxicity in 36 cases (8.3%). Legal control of "second generation" SCRAs did not affect the rate of growth in enquiry numbers or the proportion with severe toxicity. The three most commonly reported products were "Black Mamba" (n= 88, 20.3%), "Pandora's Box" (n= 65, 15.0%) and "Clockwork Orange" (n= 27, 6.2%). Neurological and general features were recorded more often with "Clockwork Orange" than for "Black Mamba" and "Pandora's Box", but moderate or severe toxicity was significantly less common after reported use of this product. CONCLUSIONS: Enquiries about SCRA-related toxicity have become increasingly frequent in the UK in spite of legal controls and commonly involve younger males. Differences in the patterns of toxicity associated with different branded preparations may occur, although further work with larger patient numbers is needed to confirm this.

Microfluidics for the detection of minimal residual disease in acute myeloid leukemia patients using circulating leukemic cells selected from blood.

We report a highly sensitive microfluidic assay to detect minimal residual disease (MRD) in patients with acute myeloid leukemia (AML) that samples peripheral blood to search for circulating leukemic cells (CLCs). Antibodies immobilized within three separate microfluidic devices affinity-selected CLC subpopulations directly from peripheral blood without requiring pre-processing. The microfluidic devices targeted CD33, CD34, and CD117 cell surface antigens commonly expressed by AML leukemic cells so that each subpopulation's CLC numbers could be tracked to determine the onset of relapse. Staining against aberrant markers (e.g. CD7, CD56) identified low levels (11-2684 mL(-1)) of CLCs. The commonly used platforms for the detection of MRD for AML patients are multi-parameter flow cytometry (MFC), typically from highly invasive bone marrow biopsies, or PCR from blood samples, which is limited to <50% of AML patients. In contrast, the microfluidic assay is a highly sensitive blood test that permits frequent sampling for >90% of all AML patients using the markers selected for this study (selection markers CD33, CD34, CD117 and aberrant markers such as CD7 and CD56). We present data from AML patients after stem cell transplant (SCT) therapy using our assay. We observed high agreement of the microfluidic assay with therapeutic treatment and overall outcome. We could detect MRD at an earlier stage compared to both MFC and PCR directly from peripheral blood, obviating the need for a painful bone marrow biopsy. Using the microfluidic assay, we detected MRD 28 days following one patient's SCT and the onset of relapse at day 57, while PCR from a bone marrow biopsy did not detect MRD until day 85 for the same patient. Earlier detection of MRD in AML post-SCT enabled by peripheral blood sampling using the microfluidic assay we report herein can influence curative clinical decisions for AML patients.

Peptide/MHC tetramer-based sorting of CD8⁺ T cells to a leukemia antigen yields clonotypes drawn nonspecifically from an underlying restricted repertoire.

Testing of T cell-based cancer therapeutics often involves measuring cancer antigen-specific T-cell populations with the assumption that they arise from in vivo clonal expansion. This analysis, using peptide/MHC tetramers, is often ambiguous. From a leukemia cell line, we identified a CDK4-derived peptide epitope, UNC-CDK4-1 (ALTPVVVTL), that bound HLA-A*02:01 with high affinity and could induce CD8⁺ T-cell responses in vitro. We identified UNC-CDK4-1/HLA-A*02:01 tetramer⁺ populations in 3 of 6 patients with acute myeloid leukemia who had undergone allogeneic stem cell transplantation. Using tetramer-based, single-cell sorting and T-cell receptor ß (TCRß) sequencing, we identified recurrent UNC-CDK4-1 tetramer-associated TCRß clonotypes in a patient with a UNC-CDK4-1 tetramer⁺ population, suggesting in vivo T-cell expansion to UNC-CDK4-1. In parallel, we measured the patient's TCRß repertoire and found it to be highly restricted/oligoclonal. The UNC-CDK4-1 tetramer-associated TCRß clonotypes represented >17% of the entire TCRß repertoire-far in excess of the UNC-CDK4-1 tetramer⁺ frequency-indicating that the recurrent TCRß clonotypes identified from UNC-CDK-4-1 tetramer⁺ cells were likely a consequence of the extremely constrained T-cell repertoire in the patient and not in vivo UNC-CDK4-1-driven clonal T-cell expansion. Mapping recurrent TCRß clonotype sequences onto TCRß repertoires can help confirm or refute antigen-specific T-cell expansion in vivo.

Size distributions and dispersions along a 485-year chronosequence for sand dune vegetation.

Using a sand dune chronosequence that spans 485 years of primary succession, we collected nearest-neighbor vegetation data to test two predictions associated with the traditional "size-advantage" hypothesis for plant competitive ability: (1) the relative representation of larger species should increase in later stages of succession; and (2) resident species that are near neighbors should, over successional time, become more similar in plant body size and/or seed size than expected by random assembly. The first prediction was supported over the time period between mid to later succession, but the second prediction was not; that is, there was no temporal pattern across the chronosequence indicating that either larger resident species, or larger seeded resident species, increasingly exclude smaller ones from local neighborhoods over time. Rather, neighboring species were generally more different from each other in seed sizes than expected by random assembly. As larger species accumulate over time, some relatively small species are lost from later stages of succession, but species size distributions nevertheless remain strongly right-skewed-even in late succession-and species of disparate sizes are just as likely as in early succession to coexist as immediate neighbors. This local-scale coexistence of disparate sized neighbors might be accounted for-as in traditional interpretations-in terms of species differences in "physical-space-niches" (e.g., involving different rooting depths), combined with possible facilitation effects. We propose, however, that this coexistence may also occur because competitive ability involves more than just a size advantage, with traits associated with survival (tolerance of intense competition) and fecundity (offspring production despite intense competition) being at least equally important.

Counseling students' interest in gerocounseling: a survey study.

Recent trends in counselor education are moving away from a standardized academic track in gerocounseling. In response to this, we surveyed 385 counseling students regarding issues related to gerocounseling, such as their interest in gerocounseling topics, willingness to participate in gerocounseling training, how prepared they feel to work with older adults, and if they would engage in specialized training. Results showed counseling students have a substantial interest in gerocounseling, and many are willing to seek specialized training. Strategies are provided for counselor education faculty who wish to develop a gerocounseling specialization within their program.

Medical students' perceptions of an undergraduate research elective.

Recent years have seen a steady decline in the number of new physician-investigators (Association of American Medical Colleges, 2000). To encourage medical students to select research careers, the Queen's University Faculty of Health Sciences curriculum includes a mandatory Critical Enquiry elective in the 2nd year. An anonymous written survey was administered to medical students before and after the elective to determine their perceptions of the value of the elective and its impact on their decision to pursue a career in medical research. There was a significant increase in the number of students expressing an interest in pursuing a research career following the elective (35-42%, p = 0.029). Students recognized other benefits including the development of critical appraisal, information literacy, and critical thinking skills; and the opportunity to select an area of and form contacts for postgraduate training. Even students who choose not to pursue careers in medical research perceive benefits to a mandatory undergraduate research elective.

Proficiency testing performance: a case study with modeling.

OBJECTIVES: Previous literature has approached proficiency testing (PT) performance by defining the minimum levels, and combinations of imprecision and bias, necessary to meet PT requirements. In this case report, current PT performance was assessed and modeling performed to prioritize our quality improvement efforts. METHODS: A total of 1,006 chemistry challenge results from Ontario's Laboratory Proficiency Testing Program (LPTP, now QMPLS) performed on 69 tests during 1999 and 2000 were used for this retrospective analysis. Peer group means, all method means and results from reference labs were used for comparison. QMPLS flagging and recommended performance criteria were compiled, and modeling performed to predict different levels of performance. RESULTS: Our internal imprecision is <5% for 72% of our 69 tests; however, only 20% of our tests had a CV/PT <25%. Of the 1,006 challenges performed, 136 (13.5%) results were outside PT limits, 55 (5.5%) results were flagged, and 12 requests were received from QMPLS seeking clarification on 24 (2.4%) results. Follow-up identified 9 (38%) nonanalytical errors, 8 (33%) method bias errors, 4 (17%) random errors, 2 poor methods, and one with no error identified. Modeling predicted flagging rates of 2.4% using QMPLS recommended precision performance, 1.6% using our current internal imprecision, 2.2% or 7.0% if we included an overall 20% or 50% relative bias rate with our current imprecision levels, or 15.0% when an estimate of our actual bias for each analyte was considered along with our current imprecision levels. CONCLUSIONS: If imprecision were the only cause of PT errors, our flagging rate for this study period would be 1.6%, and we would need to formally investigate 8 results a year. In practice, strict application of the QMPLS PT criteria would result in 68 investigations annually; however, judicial review of the results before request for clarification significantly reduced this number to 12 investigations (of which 38% were nonanalytical errors). At the present time bias is a significant cause of poor PT performance in a variety of assays. Individual laboratories need to address the problem of bias, and ultimately so do manufacturers. It would be helpful if PT programs also acknowledged this necessary evolution in both their criteria and processes.