Background: RNA-Sequencing (RNA-seq) experiments have been popularly applied to transcriptome studies in recent years. Such experiments are still relatively costly. As a result, RNA-seq experiments often employ a small number of replicates. Power analysis and sample size calculation are challenging in the context of differential expression analysis with RNA-seq data. One challenge is that there are no closed-form formulae to calculate power for the popularly applied tests for differential expression analysis. In addition, false discovery rate (FDR), instead of family-wise type I error rate, is controlled for the multiple testing error in RNA-seq data analysis. So far, there are very few proposals on sample size calculation for RNA-seq experiments.Results: In this paper, we propose a procedure for sample size calculation while controlling FDR for RNA-seq experimental design. Our procedure is based on the weighted linear model analysis facilitated by thevoommethod which has been shown to have competitive performance in terms of power and FDR control for RNA-seq differential expression analysis. We derive a method that approximates the average power across the differentially expressed genes, and then calculate the sample size to achieve a desired average power while controlling FDR. Simulation results demonstrate that the actual power of several popularly applied tests for differential expression is achieved and is close to the desired power for RNA-seq data with sample size calculated based on our method.Conclusions: Our proposed method provides an efficient algorithm to calculate sample size while controlling FDR for RNA-seq experimental design. We also provide an R packagessizeRNAthat implements our proposed method and can be downloaded from the Comprehensive R Archive Network (http://cran.r-project.org).

The unfolded protein response (UPR) is a highly conserved response that protects plants from adverse environmental conditions. The UPR is elicited by endoplasmic reticulum (ER) stress, in which unfolded and misfolded proteins accumulate within the ER. Here, we induced the UPR in maize (Zea mays) seedlings to characterize the molecular events that occur over time during persistent ER stress. We found that a multiphasic program of gene expression was interwoven among other cellular events, including the induction of autophagy. One of the earliest phases involved the degradation by regulated IRE1-dependent RNA degradation (RIDD) of RNA transcripts derived from a family of peroxidase genes. RIDD resulted from the activation of the promiscuous ribonuclease activity of ZmIRE1 that attacks the mRNAs of secreted proteins. This was followed by an upsurge in expression of the canonical UPR genes indirectly driven by ZmIRE1 due to its splicing of Zmbzip60 mRNA to make an active transcription factor that directly upregulates many of the UPR genes. At the peak of UPR gene expression, a global wave of RNA processing led to the production of many aberrant UPR gene transcripts, likely tempering the ER stress response. During later stages of ER stress, ZmIRE1’s activity declined, as did the expression of survival modulating genes, Bax inhibitor1 and Bcl-2-associated athanogene7, amid a rising tide of cell death. Thus, in response to persistent ER stress, maize seedlings embark on a course of gene expression and cellular events progressing from adaptive responses to cell death.

BACKGROUND: Homozygous familial hypercholesterolemia is a genetic disease characterized by extremely high levels of low-density lipoprotein cholesterol (LDL-C) and a high risk of premature cardiovascular events. The proof-of-concept study ORION-2 (A Study of Inclisiran in Participants With Homozygous Familial Hypercholesterolemia) showed that inclisiran, a small interfering RNA that prevents production of the hepatic PCSK9 protein (proprotein convertase subtilisin/kexin type 9), could lead to durable reductions in LDL-C levels when added to statins and ezetimibe in patients with homozygous familial hypercholesterolemia. METHODS: ORION-5 was a phase 3, 2-part, multicenter study in 56 patients with homozygous familial hypercholesterolemia and elevated LDL-C levels despite maximum tolerated doses of LDL-C–lowering therapies with or without lipoprotein apheresis. Patients eligible for part 1 (double-blind, 6 months) were randomized 2:1 to receive either 300 mg of inclisiran sodium (equivalent to 284 mg of inclisiran) or placebo. Placebo-treated patients from part 1 were transitioned to inclisiran in part 2 (open-label, 18 months). The primary end point was the percentage change in LDL-C levels from baseline to day 150. RESULTS: The mean age of the patients was 42.7 years, and 60.7% were women. The mean baseline LDL-C levels were 294.0 mg/dL and 356.7 mg/dL in the inclisiran and placebo groups, respectively. The placebo-corrected percentage change in LDL-C level from baseline to day 150 was −1.68% (95% CI, −29.19% to 25.83%; P =0.90), and the difference was not statistically significant between the inclisiran and placebo groups. The placebo-corrected percentage change in PCSK9 levels from baseline to day 150 was −60.6% with inclisiran treatment ( P <0.0001); this was sustained throughout the study, confirming the effect of inclisiran on its biological target of PCSK9. No statistically significant differences between the inclisiran and placebo groups were observed in the levels of other lipids and lipoproteins (apolipoprotein B, total cholesterol, and non−high-density lipoprotein cholesterol). Adverse events and serious adverse events did not differ between the inclisiran and placebo groups throughout the study. CONCLUSIONS: Inclisiran treatment did not reduce LDL-C levels in patients with homozygous familial hypercholesterolemia despite substantial lowering of PCSK9 levels. Inclisiran was well-tolerated, and the safety findings were consistent with previously reported studies and the overall safety profile. REGISTRATION: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT03851705.

Retinal ganglion cells can be classified into more than 40 distinct subtypes, whether by functional classification or transcriptomics. The examination of these subtypes in relation to their physiology, projection patterns, and circuitry would be greatly facilitated through the identification of specific molecular identifiers for the generation of transgenic mice. Advances in single cell transcriptomic profiling have enabled the identification of molecular signatures for cellular subtypes that are only rarely found. Therefore, we used single cell profiling combined with hierarchical clustering and correlate analyses to identify genes expressed in distinct populations of Parvalbumin-expressing cells and functionally classified RGCs. RGCs were manually isolated based either upon fluorescence or physiological distinction through cell-attached recordings. Microarray hybridization and RNA-Sequencing were employed for the characterization of transcriptomes and in situ hybridization was utilized to further characterize gene candidate expression. Gene candidates were identified based upon cluster correlation, as well as expression specificity within physiologically distinct classes of RGCs. Further, we identified Prph, Ctxn3, and Prkcq as potential candidates for ipRGC classification in the murine retina. The use of these genes, or one of the other newly identified subset markers, for the generation of a transgenic mouse would enable future studies of RGC-subtype specific function, wiring, and projection.

The morphological development of the leaf greatly influences plant architecture and crop yields. The maize leaf is composed of a leaf blade, ligule and sheath. Although extensive transcriptional profiling of the tissues along the longitudinal axis of the developing maize leaf blade has been conducted, little is known about the transcriptional dynamics in sheath tissues, which play important roles in supporting the leaf blade. Using a comprehensive transcriptome dataset, we demonstrated that the leaf sheath transcriptome dynamically changes during maturation, with the construction of basic cellular structures at the earliest stages of sheath maturation with a transition to cell wall biosynthesis and modifications. The transcriptome again changes with photosynthesis and lignin biosynthesis at the last stage of sheath tissue maturation. The different tissues of the maize leaf are highly specialized in their biological functions and we identified 15 genes expressed at significantly higher levels in the leaf sheath compared with their expression in the leaf blade, including the BOP2 homologs GRMZM2G026556 and GRMZM2G022606, DOGT1 (GRMZM2G403740) and transcription factors from the B3 domain, C2H2 zinc finger and homeobox gene families, implicating these genes in sheath maturation and organ specialization.

Avian pathogenic Escherichia coli (APEC), an extraintestinal pathogenic E. coli (ExPEC), constitutes an animal health and a potential zoonotic risk. Most studies focus on the response of a single tissue to APEC infection. Understanding interactions among lymphoid tissues is of importance in controlling APEC infection. Therefore, we studied bone marrow, bursa, and thymus transcriptomes because of these tissues' crucial roles in development of pre-lymphocytes, B cells, and T cells, respectively. Using lesion scores of liver, pericardium, and air sacs, infected birds were classified as either resistant or susceptible. Little difference in gene expression was detected in resistant birds in bone marrow versus bursa or thymus, while there were large differences between tissues in susceptible birds. Phagosome, lysosome and cytokine interactions were strongly enhanced in thymus versus bone marrow in susceptible birds, and T cell receptor (TCR), cell cycle, and p53 signaling were significantly decreased. B cell receptor (BCR) was also significantly suppressed in bursa versus bone marrow in susceptible birds. This research provides novel insights into the complex developmental changes in gene expression occurring across the primary lymphoid organs and, therefore, serves as a foundation to understanding the cellular and molecular basis of host resistance to APEC infection.