Deep sequencing deconvolution of multiple cDNA inserts.

<p><b>A.</b> Screening of the SK-MEL-23 melanoma cell line results in multiple clones with different patterns of cDNA inserts (upper) that all harbor NRAS based on an NRAS specific PCR (lower). <b>B.</b> The mutant NRAS allele harbors an A434T (G60G) and C435A (Q61K) dinucleotide substitution (underlined by thick black bar) that is present as 100% of sequence within a BaF3 transformant and as 50% or less in the SK-MEL-23 cDNA, plasmid acceptor library and retroviral library. <b>C.</b> Deep sequencing identified 12 distinct fusion sequences between the attB (black triangle) and 5′ UTR of NRAS from the SK-MEL-23 library. The number of individual reads for each of the sequences is provided. <b>D.</b> A subset of approximately 90,000 reads across the NRAS dinucleotide substitution are shown. The sequence is on the genomic negative strand and thus reversed from <b>C.</b> The C435A (G→T shown on the positive strand) substitution that results in Q61K is between the hashed marks. Gray reads have the paired-end on the same exon while brown reads have the paired-end on the next exon.</p

cDNA library screening identified an ALK translocation partner and resistance allele.

<p><b>A.</b> Immunohistochemistry using an antibody against ALK and karyotype of the anaplastic large cell lymphoma specimen 421. <b>B.</b> Multiple IL3-independent clones were recovered from screening of the cDNA library generated from 421, all of which contained ALK cDNA using primers within the flanking attB sites or within ALK itself. <b>C.</b> Immunoblot using antibody against ALK demonstrates ATIC-ALK expression in multiple clones but not in wild-type Ba/F3 cells. <b>D.</b> Effect on proliferation of 96 individual clones recovered from the 421 library with TAE684 300 nM, quantified using the ATP chemiluminescent agent CellTiterGlo. <b>E.</b> PCR amplification using the att-f and att-r primers with DNA from clone D3 demonstrates a full-length STAT5B allele that harbored a G210T (K70N) mutation.</p

Recovery of a transforming ERRB2 splice variant.

<p><b>A.</b> cDNA library screening of the melanoma cell lines 70 W (a sub-line of the MeWo) and HCC1569 resulted in IL3 independent Ba/F3 clones that contained ERBB2Δ16. <b>B.</b> Relative proliferation quantified using the ATP chemiluminescent agent CellTiterGlo. <b>C.</b> cDNA transcripts recovered from screening of MeWo, HCC1569 and 70 W cell lines. All of the transcripts lacked amino acids 2137–2184 that comprise exon 6. <b>D.</b> Expression of ERBB2 cDNA containing exon 16 using specific primers. Error bars indicate standard deviation. <b>E.</b> Expression of ERBB2Δ16 cDNA using specific primers, including a 5′ primer that overlaps exons 15 and 17 so does not amplify wild-type ERBB2.</p

cDNA library screening identifies EML4-ALK.

<p><b>A.</b> Screening of a library generated from H2228 cells generated 16 clones with distinct patterns of integrated cDNA. All clones contained one of two EML4-ALK transcripts that differed based on splicing of exon 6 and the length of the 5′ untranslated region, as indicated by the most 5′ nucleotide fused to the attB sequence. λ indicates size ladder. <b>B.</b> Screening of a library generated from H3122 cells generated Ba/F3 clones isolated at the indicated time point after withdrawal of IL3. PCR is shown using primers that amplify integrated cDNA. <b>C.</b> PCR amplification from eight clones generated from the H3122 library with different patterns of integrated cDNA. Five distinct EML4-ALK transcripts were identified based on differences in the length of the 5′ untranslated region, as in <b>A.</b></p

The mCME Project: A Randomized Controlled Trial of an SMS-Based Continuing Medical Education Intervention for Improving Medical Knowledge among Vietnamese Community Based Physicians’ Assistants

<div><p>Background</p><p>Community health workers (CHWs) provide critical services to underserved populations in low and middle-income countries, but maintaining CHW’s clinical knowledge through formal continuing medical education (CME) activities is challenging and rarely occurs. We tested whether a Short Message Service (SMS)-based mobile CME (mCME) intervention could improve medical knowledge among a cadre of Vietnamese CHWs (Community Based Physician’s Assistants–CBPAs) who are the leading providers of primary medical care for rural underserved populations.</p><p>Methods</p><p>The mCME Project was a three arm randomized controlled trial. Group 1 served as controls while Groups 2 and 3 experienced two models of the mCME intervention. Group 2 (passive model) participants received a daily SMS bullet point, and were required to reply to the text to acknowledge receipt; Group 3 (interactive model) participants received an SMS in multiple choice question format addressing the same thematic area as Group 2, entering an answer (A, B, C or D) in their response. The server provided feedback immediately informing the participant whether the answer was correct. Effectiveness was based on standardized examination scores measured at baseline and endline (six months later). Secondary outcomes included job satisfaction and self-efficacy.</p><p>Results</p><p>638 CBPAs were enrolled, randomized, and tested at baseline, with 592 returning at endline (93.7%). Baseline scores were similar across all three groups. Over the next six months, participation of Groups 2 and 3 remained high; they responded to >75% of messages. Group 3 participants answered 43% of the daily SMS questions correctly, but their performance did not improve over time. At endline, the CBPAs reported high satisfaction with the mCME intervention, and deemed the SMS messages highly relevant. However, endline exam scores did not increase over baseline, and did not differ between the three groups. Job satisfaction and self-efficacy scores also did not improve. Average times spent on self-study per week did not increase, and the kinds of knowledge resources used by the CBPAs did not differ between the three groups; textbooks, while widely available, were seldom used.</p><p>Conclusions</p><p>The SMS-based mCME intervention, while feasible and acceptable, did not result in increased medical knowledge. We hypothesize that this was because the intervention failed to stimulate lateral learning. For an intervention of this kind to be effective, it will be essential to find more effective ways to couple SMS as a stimulus to promote increased self-study behaviors.</p><p>Trial Registration</p><p>ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT02381743" target="_blank">NCT02381743</a></p></div

Weekly non-conforming answer rates (Group 3 only).

<p>This summarizes the week-by-week average rates of non-conforming answers sent back by Group 3 participants. Responses were interpretable only if in the formats ‘a’, ‘b’, ‘c’, ‘d’, or ‘A’, ‘B’, ‘C’, or ‘D’. All other responses were ‘non-conforming’. Non-conforming answers were most common in the initial few weeks of the study, and then fell to < 10%.</p

Impact of mCME intervention on key secondary endpoints: self-efficacy and job satisfaction.

<p>Impact of mCME intervention on key secondary endpoints: self-efficacy and job satisfaction.</p

Study flow diagram.

<p>Study flow diagram.</p

Weekly correct answer rates (Group 3 only).

<p>The figure summarizes the proportion of Group 3 participants who provided a correct response to the daily SMS question over the 6 months of the study. While there was considerable week-to-week variation, there was no trend to improvement over time.</p

Impact of mCME intervention on endline self-education behaviors.

<p>Impact of mCME intervention on endline self-education behaviors.</p