Normalization of large-scale behavioural data collected from zebrafish

Many contemporary neuroscience experiments utilize high-throughput approaches to simultaneously collect behavioural data from many animals. The resulting data are often complex in structure and are subjected to systematic biases, which require new approaches for analysis and normalization. This study addressed the normalization need by establishing an approach based on linear-regression modeling. The model was established using a dataset of visual motor response (VMR) obtained from several strains of wild-type (WT) zebrafish collected at multiple stages of development. The VMR is a locomotor response triggered by drastic light change, and is commonly measured repeatedly from multiple larvae arrayed in 96-well plates. This assay is subjected to several systematic variations. For example, the light emitted by the machine varies slightly from well to well. In addition to the light-intensity variation, biological replication also created batch-batch variation. These systematic variations may result in differences in the VMR and must be normalized. Our normalization approach explicitly modeled the effect of these systematic variations on VMR. It also normalized the activity profiles of different conditions to a common baseline. Our approach is versatile, as it can incorporate different normalization needs as separate factors. The versatility was demonstrated by an integrated normalization of three factors: light-intensity variation, batch-batch variation and baseline. After normalization, new biological insights were revealed from the data. For example, we found larvae of TL strain at 6 days post-fertilization (dpf) responded to light onset much stronger than the 9-dpf larvae, whereas previous analysis without normalization shows that their responses were relatively comparable. By removing systematic variations, our model-based normalization can facilitate downstream statistical comparisons and aid detecting true biological differences in high-throughput studies of neurobehaviour

Expression profiling of the retina of pde6c, a zebrafish model of retinal degeneration

Retinal degeneration often affects the whole retina even though the disease-causing gene is specifically expressed in the light-sensitive photoreceptors. The molecular basis of the retinal defect can potentially be determined by gene-expression profiling of the whole retina. In this study, we measured the gene-expression profile of retinas microdissected from a zebrafish pde6cw59 (pde6c) mutant. This retinal-degeneration model not only displays cone degeneration caused by a cone-specific mutation, but also other secondary cellular changes starting from 4 days postfertilization (dpf). To capture the underlying molecular changes, we subjected pde6c and wild-type (WT) retinas at 5 dpf/ 120 h postfertilization (hpf) to RNA sequencing (RNA-Seq) on the Illumina HiSeq 2,000 platform. We also validated the RNA-Seq results by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) of seven phototransduction genes. Our analyses indicate that the RNA-Seq dataset was of high quality, and effectively captured the molecular changes in the whole pde6c retina. This dataset will facilitate the characterization of the molecular defects in the pde6c retina at the initial stage of retinal degeneration

TLS-bridged co-prediction of tree-level multifarious stem structure variables from worldview-2 panchromatic imagery: a case study of the boreal forest

In forest ecosystem studies, tree stem structure variables (SSVs) proved to be an essential kind of parameters, and now simultaneously deriving SSVs of as many kinds as possible at large scales is preferred for enhancing the frontier studies on marcoecosystem ecology and global carbon cycle. For this newly emerging task, satellite imagery such as WorldView-2 panchromatic images (WPIs) is used as a potential solution for co-prediction of tree-level multifarious SSVs, with static terrestrial laser scanning (TLS) assumed as a ‘bridge’. The specific operation is to pursue the allometric relationships between TLS-derived SSVs and WPI-derived feature parameters, and regression analyses with one or multiple explanatory variables are applied to deduce the prediction models (termed as Model1s and Model2s). In the case of Picea abies, Pinus sylvestris, Populus tremul and Quercus robur in a boreal forest, tests showed that Model1s and Model2s for different tree species can be derived (e.g. the maximum R2 = 0.574 for Q. robur). Overall, this study basically validated the algorithm proposed for co-prediction of multifarious SSVs, and the contribution is equivalent to developing a viable solution for SSV-estimation upscaling, which is useful for large-scale investigations of forest understory, macroecosystem ecology, global vegetation dynamics and global carbon cycle.This work was financially supported in part by the National Natural Science Foundation of China [grant numbers 41471281 and 31670718] and in part by the SRF for ROCS, SEM, China. (41471281 - National Natural Science Foundation of China; 31670718 - National Natural Science Foundation of China; SRF for ROCS, SEM, China)http://www-tandfonline-com.ezproxy.bu.edu/doi/abs/10.1080/17538947.2016.1247473?journalCode=tjde20Published versio

How to calculate sample size in animal and human studies

One of the most important statistical analyses when designing animal and human studies is the calculation of the required sample size. In this review, we define central terms in the context of sample size determination, including mean, standard deviation, statistical hypothesis testing, type I/II error, power, direction of effect, effect size, expected attrition, corrected sample size, and allocation ratio. We also provide practical examples of sample size calculations for animal and human studies based on pilot studies, larger studies similar to the proposed study—or if no previous studies are available—estimated magnitudes of the effect size per Cohen and Sawilowsky

Increased Expression of INHBA Is Correlated With Poor Prognosis and High Immune Infiltrating Level in Breast Cancer

Background: Inhibin, beta A (INHBA) is a member of the transforming growth factor-β superfamily and is associated with carcinogenesis and cancer progression in several types of human cancers. However, its significance in breast cancer has not been evaluated. Here, we investigated the prognostic value of INHBA and its correlation with tumor-infiltration immune cells in the microenvironment of breast cancer.Methods: In this study, we analyzed the INHBA expression profile in the Oncomine database and Tumor Immune Estimation Resource 2.0 (TIMER2.0) site. Using Breast Cancer Gene-Expression Miner (bc-GenExMiner v4.7) tool and the UALCAN cancer database, we further evaluated the correlation of INHBA expression with clinicopathological factors in breast cancer. Then, we assessed the clinical prognostic value of INHBA using Kaplan–Meier Plotter and the PrognoScan databases. The correlations between INHBA and tumor-infiltrating immune cells were investigated via TIMER2.0. In addition, correlations between INHBA expression and gene markers of immune infiltrates were analyzed by TIMER2.0 and Gene Expression Profiling Interactive Analysis 2.Results: Compared with the level in normal tissues, the INHBA mRNA expression was upregulated in different subtypes of breast cancer, and its expression was positively correlated with progesterone receptor, human epidermal growth factor receptor-2 status, and PAM50 subtypes but negatively related to age and basal-like status. The INHBA protein was also highly expressed in primary breast cancer and closely related to the pathological stage. Patients with high INHBA expression levels showed worse overall survival, relapse-free survival, and distant metastasis-free survival. Also, high INHBA expression was significantly associated with worse overall survival and relapse-free survival in positive lymph nodes. Of interest, INHBA expression was negatively correlated with infiltrating levels of activated NK cells, NKT, and CD4+ T cells but was positively correlated with tumor infiltration of CD8+ T cells, neutrophils, especially macrophages and cancer-associated fibroblasts. Moreover, INHBA expression showed strong correlations with various markers of monocytes/macrophages and cancer-associated fibroblasts.Conclusion: High INHBA expression is correlated with poor prognosis and the infiltration of immune cells in the tumor microenvironment. These findings suggest that INHBA may be involved in immune escape and can serve as a potential biomarker of prognosis and tumor-infiltrating immune cells

Characterization of the complete chloroplast genomes of five Populus species from the western Sichuan plateau, southwest China: comparative and phylogenetic analyses

Species of the genus Populus, which is widely distributed in the northern hemisphere from subtropical to boreal forests, are among the most commercially exploited groups of forest trees. In this study, the complete chloroplast genomes of five Populus species (Populus cathayana, P. kangdingensis, P. pseudoglauca, P. schneideri, and P. xiangchengensis) were compared. The chloroplast genomes of the five Populus species are very similar. The total chloroplast genome sequence lengths for the five plastomes were 156,789, 156,523, 156,512, 156,513, and 156,465 bp, respectively. A total of 130 genes were identified in each genome, including 85 protein-coding genes, 37 tRNA genes and eight rRNA genes. Seven genes were duplicated in the protein-coding genes, whereas 11 genes were duplicated in the RNA genes. The GC content was 36.7% for all plastomes. We analyzed nucleotide substitutions, small inversions, simple sequence repeats and long repeats in the chloroplast genomes and found nine divergence hotspots (ccsA+ccsA-ndhD, ndhC-trnV, psbZ-trnfM, trnG-atpA, trnL-ndhJ, trnR-trnN, ycf4-cemA, ycf1, and trnR-trnN), which could be useful molecular genetic markers for future population genetic and phylogenetic studies. We also observed that two genes (rpoC2 and rbcL) were subject to positive selection. Phylogenetic analysis based on whole cp genomes showed that P. schneideri had a close relationship with P. kangdingensis and P. pseudoglauca, while P. xiangchengensis was a sister to P. cathayana

Plastome Sequences Help to Resolve Deep-Level Relationships of Populus in the Family Salicaceae

Populus, a core genus of Salicaceae, plays a significant ecological role as a source of pioneer species in boreal forests. However, interspecific hybridization and high levels of morphological variation among poplars have resulted in great difficulty in classifying species for systematic and comparative evolutionary studies. Here, we present phylogenetic analyses of 24 newly sequenced Populus plastomes and 36 plastomes from GenBank, which represent seven genera of Salicaceae, in combination with a matrix of eighteen morphological characters of 40 Populus taxa to reconstruct highly supported relationships of genus Populus. Relationships among the 60 taxa of Salicaceae strongly supported two monophyletic genera: Populus and Salix. Chosenia was nested within the genus Salix, and five clades within Populus were divided. Clade I included the three taxa P. euphratica, P. pruinosa, and P. ilicifolia. Clade II contained thirteen taxa [P. adenopoda, P. alba, P. bolleana, P. davidiana, P. hopeiensis, P. nigra, P. qiongdaoensis, P. rotundifolia, P. rotundifolia var. duclouxiana, P. tremula, P. tremula × alba, P. tomentosa, and P. tomentosa (NC)]. Clade III included the ten taxa P. haoana, P. kangdingensis, P. lasiocarpa, P. pseudoglauca, P. qamdoensis, P. schneideri, P. simonii, P. szechuanica, P. szechuanica var. tibetica, and P. yunnanensis. Clade IV included P. cathayana, P. gonggaensis, P. koreana, P. laurifolia, P. trinervis, P. wilsonii, and P. xiangchengensis. The last clade comprised P. angustifolia, P. balsamifera, P. deltoides, P. deltoides × nigra, P. fremontii, P. mexicana, and P. trichocarpa. This phylogeny is also supported by morphological traits, including bark smoothness, bud size, petiole shape, leaf inflorescence, male anther length and male anther tip