Efficacy and safety of a SOD1-targeting artificial miRNA delivered by AAV9 in mice are impacted by miRNA scaffold selection.

Toxic gain-of-function mutations in superoxide dismutase 1 (SOD1) contribute to approximately 2%-3% of all amyotrophic lateral sclerosis (ALS) cases. Artificial microRNAs (amiRs) delivered by adeno-associated virus (AAV) have been proposed as a potential treatment option to silence SOD1 expression and mitigate disease progression. Primary microRNA (pri-miRNA) scaffolds are used in amiRs to shuttle a hairpin RNA into the endogenous miRNA pathway, but it is unclear whether different primary miRNA (pri-miRNA) scaffolds impact the potency and safety profile of the expressed amiR in vivo. In our process to develop an AAV amiR targeting SOD1, we performed a preclinical characterization of two pri-miRNA scaffolds, miR155 and miR30a, sharing the same guide strand sequence. We report that, while the miR155-based vector, compared with the miR30a-based vector, leads to a higher level of the amiR and more robust suppression of SOD1 in vitro and in vivo, it also presents significantly greater risks for CNS-related toxicities in vivo. Despite miR30a-based vector showing relatively lower potency, it can significantly delay the development of ALS-like phenotypes in SOD1-G93A mice and increase survival in a dose-dependent manner. These data highlight the importance of scaffold selection in the pursuit of highly efficacious and safe amiRs for RNA interference gene therapy.

Characterizing the composition of iPSC derived cells from bulk transcriptomics data with CellMap.

Induced pluripotent stem cell (iPSC) derived cell types are increasingly employed as in vitro model systems for drug discovery. For these studies to be meaningful, it is important to understand the reproducibility of the iPSC-derived cultures and their similarity to equivalent endogenous cell types. Single-cell and single-nucleus RNA sequencing (RNA-seq) are useful to gain such understanding, but they are expensive and time consuming, while bulk RNA-seq data can be generated quicker and at lower cost. In silico cell type decomposition is an efficient, inexpensive, and convenient alternative that can leverage bulk RNA-seq to derive more fine-grained information about these cultures. We developed CellMap, a computational tool that derives cell type profiles from publicly available single-cell and single-nucleus datasets to infer cell types in bulk RNA-seq data from iPSC-derived cell lines.

BioInfograph: An Online Tool to Design and Display Multi-Panel Scientific Figure Interactively.

Many fit-for-purpose bioinformatics tools generate plots to interpret complex biological data and illustrate findings. However, assembling individual plots in different formats from various sources into one high-resolution figure in the desired layout requires mastery of commercial tools or even programming skills. In addition, it is a time-consuming and sometimes frustrating process even for a computationally savvy scientist who frequently takes a trial-and-error iterative approach to get satisfactory results. To address the challenge, we developed bioInfograph, a web-based tool that allows users to interactively arrange high-resolution images in diversified formats, mainly Scalable Vector Graphics (SVG), to produce one multi-panel publication-quality composite figure in both PDF and HTML formats in a user-friendly manner, requiring no programming skills. It solves stylesheet conflicts of coexisting SVG plots, integrates a rich-text editor, and allows creative design by providing advanced functionalities like image transparency, controlled vertical stacking of plots, versatile image formats, and layout templates. To highlight, the sharable interactive HTML output with zoom-in function is a unique feature not seen in any other similar tools. In the end, we make the online tool publicly available at https://baohongz.github.io/bioInfograph while releasing the source code at https://github.com/baohongz/bioInfograph under MIT open-source license.

Organotypic Brain Slice Culture Microglia Exhibit Molecular Similarity to Acutely-Isolated Adult Microglia and Provide a Platform to Study Neuroinflammation.

Microglia are central nervous system (CNS) resident immune cells that have been implicated in neuroinflammatory pathogenesis of a variety of neurological conditions. Their manifold context-dependent contributions to neuroinflammation are only beginning to be elucidated, which can be attributed in part to the challenges of studying microglia in vivo and the lack of tractable in vitro systems to study microglia function. Organotypic brain slice cultures offer a tissue-relevant context that enables the study of CNS resident cells and the analysis of brain slice microglial phenotypes has provided important insights, in particular into neuroprotective functions. Here we use RNA sequencing, direct digital quantification of gene expression with nCounter® technology and targeted analysis of individual microglial signature genes, to characterize brain slice microglia relative to acutely-isolated counterparts and 2-dimensional (2D) primary microglia cultures, a widely used in vitro surrogate. Analysis using single cell and population-based methods found brain slice microglia exhibited better preservation of canonical microglia markers and overall gene expression with stronger fidelity to acutely-isolated adult microglia, relative to in vitro cells. We characterized the dynamic phenotypic changes of brain slice microglia over time, after plating in culture. Mechanical damage associated with slice preparation prompted an initial period of inflammation, which resolved over time. Based on flow cytometry and gene expression profiling we identified the 2-week timepoint as optimal for investigation of microglia responses to exogenously-applied stimuli as exemplified by treatment-induced neuroinflammatory changes observed in microglia following LPS, TNF and GM-CSF addition to the culture medium. Altogether these findings indicate that brain slice cultures provide an experimental system superior to in vitro culture of microglia as a surrogate to investigate microglia functions, and the impact of soluble factors and cellular context on their physiology.

Cx3cr1-deficient microglia exhibit a premature aging transcriptome.

CX3CR1, one of the highest expressed genes in microglia in mice and humans, is implicated in numerous microglial functions. However, the molecular mechanisms underlying Cx3cr1 signaling are not well understood. Here, we analyzed transcriptomes of Cx3cr1-deficient microglia under varying conditions by RNA-sequencing (RNA-seq). In 2-mo-old mice, Cx3cr1 deletion resulted in the down-regulation of a subset of immune-related genes, without substantial epigenetic changes in markers of active chromatin. Surprisingly, Cx3cr1-deficient microglia from young mice exhibited a transcriptome consistent with that of aged Cx3cr1-sufficient animals, suggesting a premature aging transcriptomic signature. Immunohistochemical analysis of microglia in young and aged mice revealed that loss of Cx3cr1 modulates microglial morphology in a comparable fashion. Our results suggest that CX3CR1 may regulate microglial function in part by modulating the expression levels of a subset of inflammatory genes during chronological aging, making Cx3cr1-deficient mice useful for studying aged microglia.

Influence of cultivar and maturity at harvest on the essential oil composition, oleoresin and [6]-gingerol contents in fresh ginger from northeast India.

Severe flooding of the Brahmaputra River during the monsoon season and continuous rainfall in the northeast region (NER) of India cause an enormous loss of ginger crop every year. In this context, the present study investigates the variation in the essential oil composition and oleoresin and [6]-gingerol contents in 10 different fresh ginger cultivars harvested at 6- and 9-month maturity from five different states of NER. Monoterpenes, sesquiterpenes, and citral composition in the essential oil were evaluated to ascertain their dependence upon the maturity of ginger. Except Mizoram Thinglaidum, Mizoram Thingria, Nagaland Nadia, and Tripura I ginger cultivars, all other cultivars showed an increase in the citral content during the maturity that was observed for the first time. At 6-month maturity, a higher undecanone level was found in Nagaland Nadia (7.36 ± 0.61%), Tripura I (6.23 ± 0.61%), and Tripura III (9.17 ± 0.76%) cultivars, and these data can be used as a benchmark to identify those immature varieties. Interestingly, the Nagaland Nadia cultivar showed higher ar-curcumene (9.57 ± 0.58%) content than zingiberene (5.84 ± 0.24%), which was unique among all cultivars. Ginger harvested at 9-month maturity from the Tripura II cultivar had the highest citral content (22.03 ± 0.49%), and the Meghalaya Mahima cultivar had the highest zingiberene content (29.89 ± 2.92%). The oleoresin content was found to decrease with maturity in all cultivars, except Assam Fibreless and Manipur I. Moreover, the highest oleoresin (11.43 ± 0.58 and 9.42 ± 0.63%) and [6]-gingerol (1.67 ± 0.03 and 1.67 ± 0.05 g) contents were observed for Tripura II and Nagaland Nadia, respectively. This study suggests that Tripura and Nagaland are the most ideal locations in NER for ginger cultivation to obtain high yields of oleoresin and [6]-gingerol contents and harvesting at the 6-month maturation will compensate for the loss of ginger crop caused by the Brahmaputra River flooding in NER every year.