NF-κB Signaling in Tumor Pathways Focusing on Breast and Ovarian Cancer.

Immune disorders and cancer share a common pathway involving NF-κb signaling. Through involvement with GM-CSF, NF-κB can contribute to proliferation and activation of T- and B- cells as well as immune cell migration to sites of inflammation. In breast cancer, this signaling pathway has been linked to resistance with endocrine and chemotherapies. Similarly, in ovarian cancer, NF-κB influences angiogenesis and inflammation pathways. Further, BRCA1 signaling common to both breast and ovarian cancer also has the capability to induce NF-κB activity. Immunotherapy involving NF-κB can also be implemented to combat chemoresistance. The complex signaling pathways of NF-κB can be harnessed for developing cancer therapeutics to promote immunotherapy for improving patient outcomes.

BRCA1/2 signaling and homologous recombination deficiency in breast and ovarian cancer.

Patients who have mutations of the genes BRCA1 or BRCA2 are at an increased risk for developing breast and ovarian cancer. BRCA1/2 function as tumor suppressor genes, responsible for regulating DNA repair, and play an essential role in homologous recombination. Mutation of BRCA1/2 results in homologous recombination deficiency and genomic instability which drives oncogenesis and cancer proliferation. Recently, BRCA1/2 gene expression has been implicated in regulating immune response. Here we discuss the signaling pathway of BRCA1/2 in relation to breast and ovarian cancer, with emphasis on how dysregulation facilitates the path to malignancy and current treatment options.

Targeted single-cell RNA sequencing of transcription factors enhances the identification of cell types and trajectories.

Single-cell RNA sequencing (scRNA-seq) is a widely used method for identifying cell types and trajectories in biologically heterogeneous samples, but it is limited in its detection and quantification of lowly expressed genes. This results in missing important biological signals, such as the expression of key transcription factors (TFs) driving cellular differentiation. We show that targeted sequencing of ∼1000 TFs (scCapture-seq) in iPSC-derived neuronal cultures greatly improves the biological information garnered from scRNA-seq. Increased TF resolution enhanced cell type identification, developmental trajectories, and gene regulatory networks. This allowed us to resolve differences among neuronal populations, which were generated in two different laboratories using the same differentiation protocol. ScCapture-seq improved TF-gene regulatory network inference and thus identified divergent patterns of neurogenesis into either excitatory cortical neurons or inhibitory interneurons. Furthermore, scCapture-seq revealed a role for of retinoic acid signaling in the developmental divergence between these different neuronal populations. Our results show that TF targeting improves the characterization of human cellular models and allows identification of the essential differences between cellular populations, which would otherwise be missed in traditional scRNA-seq. scCapture-seq TF targeting represents a cost-effective enhancement of scRNA-seq, which could be broadly applied to improve scRNA-seq resolution.

Pro-maturational Effects of Human iPSC-Derived Cortical Astrocytes upon iPSC-Derived Cortical Neurons.

Astrocytes influence neuronal maturation and function by providing trophic support, regulating the extracellular environment, and modulating signaling at synapses. The emergence of induced pluripotent stem cell (iPSC) technology offers a human system with which to validate and re-evaluate insights from animal studies. Here, we set out to examine interactions between human astrocytes and neurons derived from a common cortical progenitor pool, thereby recapitulating aspects of in vivo cortical development. We show that the cortical iPSC-derived astrocytes exhibit many of the molecular and functional hallmarks of astrocytes. Furthermore, optogenetic and electrophysiological co-culture experiments reveal that the iPSC-astrocytes can actively modulate ongoing synaptic transmission and exert pro-maturational effects upon developing networks of iPSC-derived cortical neurons. Finally, transcriptomic analyses implicate synapse-associated extracellular signaling in the astrocytes' pro-maturational effects upon the iPSC-derived neurons. This work helps lay the foundation for future investigations into astrocyte-to-neuron interactions in human health and disease.

Concentration effects of three common fish anesthetics on Pacific hagfish (Eptatretus stoutii).

The efficacy of three common fish anesthetics (clove oil, 2-phenoxyethanol, and tricaine methanesulfonate) was evaluated in the Pacific hagfish (Eptatretus stoutii). The overarching aim of our study was to identify the best anesthetic and concentration for the purposes of routine laboratory use of Pacific hagfish (i.e., short and consistent induction and recovery times and minimized stress and safety risk to hagfish). The objectives of our study were fourfold: (1) identify anesthetic stages of Pacific hagfish using clove oil anesthesia; (2) establish standardized anesthesia preparation procedures; (3) determine the optimal anesthetic and concentration for safely achieving stage V anesthesia; and (4) investigate the effects of repeatedly exposing Pacific hagfish to anesthesia. Experimental concentrations, ranging from 50 to 400 mg/L, of each anesthetic were tested on at least three Pacific hagfish individuals. We found the following: (1) Pacific hagfish exhibited similar stages of anesthesia to those described for bony fishes; (2) sufficient mixing of clove oil with seawater had a considerable effect on the consistency and timing of anesthetic induction; (3) concentration and anesthetic significantly impacted induction and recovery timing, whereas body mass had no impact on anesthetic trends; and (4) repeatedly exposing Pacific hagfish to optimal concentrations of clove oil or MS-222 had no effect on induction or recovery timing, whereas exposure number significantly impacted induction timing when using 2-PE. Due to consistent induction and recovery times, low risk of accidental overdose, and high safety margins for both handler and hagfish, we recommend 175 mg/L of clove oil as the ideal anesthetic and concentration for the routine laboratory use of Pacific hagfish.

Reproducibility of Molecular Phenotypes after Long-Term Differentiation to Human iPSC-Derived Neurons: A Multi-Site Omics Study.

Reproducibility in molecular and cellular studies is fundamental to scientific discovery. To establish the reproducibility of a well-defined long-term neuronal differentiation protocol, we repeated the cellular and molecular comparison of the same two iPSC lines across five distinct laboratories. Despite uncovering acceptable variability within individual laboratories, we detect poor cross-site reproducibility of the differential gene expression signature between these two lines. Factor analysis identifies the laboratory as the largest source of variation along with several variation-inflating confounders such as passaging effects and progenitor storage. Single-cell transcriptomics shows substantial cellular heterogeneity underlying inter-laboratory variability and being responsible for biases in differential gene expression inference. Factor analysis-based normalization of the combined dataset can remove the nuisance technical effects, enabling the execution of robust hypothesis-generating studies. Our study shows that multi-center collaborations can expose systematic biases and identify critical factors to be standardized when publishing novel protocols, contributing to increased cross-site reproducibility.

Investigating the Experiences of Childhood Cancer Patients and Parents Participating in Optional Nontherapeutic Clinical Research Studies in the UK.

BACKGROUND: While the majority of childhood cancer clinical trials are treatment related, additional optional research investigations may be carried out that do not directly impact on treatment. It is essential that these studies are conducted ethically and that the experiences of families participating in these studies are as positive as possible. METHODS: A questionnaire study was carried out to investigate the key factors that influence why families choose to participate in optional nontherapeutic research studies, the level of understanding of the trials involved, and the experiences of participation. RESULTS: A total of 100 participants from six UK centers were studied; 77 parents, 10 patients >16 years, and 13 patients aged 8-15 years. Ninety-seven percent of parents and 90% of patients felt that information provided prior to study consent was of the right length, with 52% of parents and 65% of patients fully understanding the information provided. Seventy-four percent of parents participated in research studies in order to "do something important", while 74% of patients participated "to help medical staff". Encouragingly, <5% of participants felt that their clinical care would be negatively affected if they did not participate. Positive aspects of participation included a perception of increased attention from medical staff. Negative aspects included spending longer periods in hospital and the requirement for additional blood samples. Ninety-six percent of parents and 87% of patients would participate in future studies. CONCLUSIONS: The study provides an insight into the views of childhood cancer patients and their parents participating in nontherapeutic clinical research studies. Overwhelmingly, the findings suggest that participation is seen as a positive experience.

Assessing similarity to primary tissue and cortical layer identity in induced pluripotent stem cell-derived cortical neurons through single-cell transcriptomics.

Induced pluripotent stem cell (iPSC)-derived cortical neurons potentially present a powerful new model to understand corticogenesis and neurological disease. Previous work has established that differentiation protocols can produce cortical neurons, but little has been done to characterize these at cellular resolution. In particular, it is unclear to what extent in vitro two-dimensional, relatively disordered culture conditions recapitulate the development of in vivo cortical layer identity. Single-cell multiplex reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to interrogate the expression of genes previously implicated in cortical layer or phenotypic identity in individual cells. Totally, 93.6% of single cells derived from iPSCs expressed genes indicative of neuronal identity. High proportions of single neurons derived from iPSCs expressed glutamatergic receptors and synaptic genes. And, 68.4% of iPSC-derived neurons expressing at least one layer marker could be assigned to a laminar identity using canonical cortical layer marker genes. We compared single-cell RNA-seq of our iPSC-derived neurons to available single-cell RNA-seq data from human fetal and adult brain and found that iPSC-derived cortical neurons closely resembled primary fetal brain cells. Unexpectedly, a subpopulation of iPSC-derived neurons co-expressed canonical fetal deep and upper cortical layer markers. However, this appeared to be concordant with data from primary cells. Our results therefore provide reassurance that iPSC-derived cortical neurons are highly similar to primary cortical neurons at the level of single cells but suggest that current layer markers, although effective, may not be able to disambiguate cortical layer identity in all cells.