Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study

BACKGROUND: Several high throughput technologies have been employed to identify differentially regulated genes that may be molecular targets for drug discovery. Here we compared the sets of differentially regulated genes discovered using two experimental approaches: a subtracted suppressive hybridization (SSH) cDNA library methodology and Affymetrix GeneChip(® )technology. In this "case study" we explored the transcriptional pattern changes during the in vitro differentiation of human monocytes to myeloid dendritic cells (DC), and evaluated the potential for novel gene discovery using the SSH methodology. RESULTS: The same RNA samples isolated from peripheral blood monocyte precursors and immature DC (iDC) were used for GeneChip microarray probing and SSH cDNA library construction. 10,000 clones from each of the two-way SSH libraries (iDC-monocytes and monocytes-iDC) were picked for sequencing. About 2000 transcripts were identified for each library from 8000 successful sequences. Only 70% to 75% of these transcripts were represented on the U95 series GeneChip microarrays, implying that 25% to 30% of these transcripts might not have been identified in a study based only on GeneChip microarrays. In addition, about 10% of these transcripts appeared to be "novel", although these have not yet been closely examined. Among the transcripts that are also represented on the chips, about a third were concordantly discovered as differentially regulated between iDC and monocytes by GeneChip microarray transcript profiling. The remaining two thirds were either not inferred as differentially regulated from GeneChip microarray data, or were called differentially regulated but in the opposite direction. This underscores the importance both of generating reciprocal pairs of SSH libraries, and of real-time RT-PCR confirmation of the results. CONCLUSIONS: This study suggests that SSH could be used as an alternative and complementary transcript profiling tool to GeneChip microarrays, especially in identifying novel genes and transcripts of low abundance

Earliest Triassic microbialites in the South China Block and other areas; controls on their growth and distribution

Earliest Triassic microbialites (ETMs) and inorganic carbonate crystal fans formed after the end-Permian mass extinction (ca. 251.4 Ma) within the basal Triassic Hindeodus parvus conodont zone. ETMs are distinguished from rarer, and more regional, subsequent Triassic microbialites. Large differences in ETMs between northern and southern areas of the South China block suggest geographic provinces, and ETMs are most abundant throughout the equatorial Tethys Ocean with further geographic variation. ETMs occur in shallow-marine shelves in a superanoxic stratified ocean and form the only widespread Phanerozoic microbialites with structures similar to those of the Cambro-Ordovician, and briefly after the latest Ordovician, Late Silurian and Late Devonian extinctions. ETMs disappeared long before the mid-Triassic biotic recovery, but it is not clear why, if they are interpreted as disaster taxa. In general, ETM occurrence suggests that microbially mediated calcification occurred where upwelled carbonate-rich anoxic waters mixed with warm aerated surface waters, forming regional dysoxia, so that extreme carbonate supersaturation and dysoxic conditions were both required for their growth. Long-term oceanic and atmospheric changes may have contributed to a trigger for ETM formation. In equatorial western Pangea, the earliest microbialites are late Early Triassic, but it is possible that ETMs could exist in western Pangea, if well-preserved earliest Triassic facies are discovered in future work

Dose Comparison Study of Allogeneic Mesenchymal Stem Cells in Patients With Ischemic Cardiomyopathy (The TRIDENT Study)

RATIONALE: Cell dose and concentration play crucial roles in phenotypic responses to cell-based therapy for heart failure. OBJECTIVE: To compare the safety and efficacy of two doses of allogeneic bone marrow-derived human mesenchymal stem cells (hMSC) identically delivered in patients with ischemic cardiomyopathy (ICM). METHODS AND RESULTS: Thirty patients with ICM received in a blinded manner either 20 million (20M, n=15) or 100 million (100M, n=15) allogeneic hMSCs via transendocardial injection (10 0.5 cc injections/patient). Patients were followed for 12-months for safety and efficacy endpoints. There were no treatment-emergent serious adverse events (SAE) at 30 days or treatment related SAEs at 12 months. The Major Adverse Cardiac Event rate was 20.0% (95% CI, 6.9%, 50.0%) in 20M and 13.3% (95% CI, 3.5%, 43.6%) in 100M (p=0.58). Worsening heart failure re-hospitalization was 20.0% (95% CI, 6.9%, 50.0%) in 20M and 7.1% (95% CI, 1.0%, 40.9%) in 100M (p=0.27). Whereas scar size reduced to a similar degree in both groups: 20M by −6.4g (IQR, −13.5g, −3.4g, p=0.001) and 100M by −6.1g (IQR, −8.1g, −4.6g, p=0.0002), the ejection fraction (EF) improved only with 100M by 3.7 units (IQR, 1.1, 6.1, p=0.04). NYHA class improved at 12 months in 35.7% (95% CI, 12.7%, 64.9%) in 20M and 42.9% (95% CI, 17.7%, 71.1%) in 100M. Importantly, pro-BNP increased at 12 months in 20M by 0.32 log pg/mL (95% CI, 0.02, 0.62, p=0.039), but not in 100M (−0.07 log pg/mL; 95% CI, −0.36, 0.23, p=0.65; between group p=0.07). CONCLUSION: Although both cell doses reduced scar size, only the 100M dose increased EF. This study highlights the crucial role of cell dose in the responses to cell therapy. Determining optimal dose and delivery is essential to advance the field, decipher mechanism(s) of action, and enhance planning of pivotal Phase III trials. CLINICAL TRIAL REGISTRATION: NCTO2013674 [https://clinicaltrials.gov/ct2/show/NCT02013674