CyTRANSFINDER: a Cytoscape 3.3 plugin for three-component (TF, gene, miRNA) signal transduction pathway construction

Background: Biological research increasingly relies on network models to study complex phenomena. Signal Transduction Pathways are molecular circuits that model how cells receive, process, and respond to information from the environment providing snapshots of the overall cell dynamics. Most of the attempts to reconstruct signal transduction pathways are limited to single regulator networks including only genes/proteins. However, networks involving a single type of regulator and neglecting transcriptional and post-transcriptional regulations mediated by transcription factors and microRNAs, respectively, may not fully reveal the complex regulatory mechanisms of a cell. We observed a lack of computational instruments supporting explorative analysis on this type of three-component signal transduction pathways. Results: We have developed CyTRANSFINDER, a new Cytoscape plugin able to infer three-component signal transduction pathways based on user defined regulatory patterns and including miRNAs, TFs and genes. Since CyTRANSFINDER has been designed to support exploratory analysis, it does not rely on expression data. To show the potential of the plugin we have applied it in a study of two miRNAs that are particularly relevant in human melanoma progression, miR-146a and miR-214. Conclusions: CyTRANSFINDERsupportsthereconstructionofsmallsignaltransductionpathwaysamonggroupsof genes. Results obtained from its use in a real case study have been analyzed and validated through both literature data and preliminary wet-lab experiments, showing the potential of this tool when performing exploratory analysi

Lipid nanoparticle-mediated messenger RNA delivery for ex vivo engineering of natural killer cells

Natural killer (NK) cells participate in the immune system by eliminating cancer and virally infected cells through germline-encoded surface receptors. Their independence from prior activation as well as their significantly lower toxicity have placed them in the spotlight as an alternative to T cells for adoptive cell therapy (ACT). Engineering NK cells with mRNA has shown great potential in ACT by enhancing their tumor targeting and cytotoxicity. However, mRNA transfection of NK cells is challenging, as the most common delivery methods, such as electroporation, show limitations. Therefore, an alternative non-viral delivery system that enables high mRNA transfection efficiency with preservation of the cell viability would be beneficial for the development of NK cell therapies. In this study, we investigated both polymeric and lipid nanoparticle (LNP) formulations for eGFP-mRNA delivery to NK cells, based on a dimethylethanolamine and diethylethanolamine polymeric library and on different ionizable lipids, respectively. The mRNA nanoparticles based on cationic polymers showed limited internalization by NK cells and low transfection efficiency. On the other hand, mRNA-LNP formulations were optimized by tailoring the lipid composition and the microfluidic parameters, resulting in a high transfection efficiency (∼100%) and high protein expression in NK cells. In conclusion, compared to polyplexes and electroporation, the optimized LNPs show a greater transfection efficiency and higher overall eGFP expression, when tested in NK (KHYG-1) and T (Jurkat) cell lines, and cord blood-derived NK cells. Thus, LNP-based mRNA delivery represents a promising strategy to further develop novel NK cell therapies

Novel role of microRNA146b in promoting mammary alveolar progenitor cell maintenance

In this report, we have shown that miR146b promotes the maintenance of pregnancy-derived mammary luminal alveolar progenitors. MiR146b expression was significantly higher in the mammary glands of pregnant and lactating mice than in virgin mice. Furthermore, miR146b levels were significantly higher in mouse mammary glands exposed to the sex hormones, estrogen and progesterone, compared with those of untreated control animals. Pregnancy-derived primary mouse mammary epithelial cells in which miR146b was knocked down showed a significant reduction in the number of hollow acinar organoid structures formed on three-dimensional Matrigel and in β-casein expression. This demonstrates that miR146b promotes the maintenance of pregnancy-derived mammary luminal alveolar progenitors. It has been shown that mouse mammary luminal progenitors give rise to hollow organoid structures, whereas solid organoid structures are derived from stem cells. Among several miR146b targets, miR146b knockdown resulted in preferential STAT3β overexpression. In the primary mouse mammary epithelial cells, overexpression of STAT3β isoform caused mammary epithelial cell death and a significant reduction in β-casein mRNA expression. Therefore, we conclude that during pregnancy miR146b is involved in luminal alveolar progenitor cell maintenance, at least partially, by regulating STAT3β

MSC-Regulated MicroRNAs Converge on the Transcription Factor FOXP2 and Promote Breast Cancer Metastasis

SummaryMesenchymal stem/stromal cells (MSCs) are progenitor cells shown to participate in breast tumor stroma formation and to promote metastasis. Despite expanding knowledge of their contributions to breast malignancy, the underlying molecular responses of breast cancer cells (BCCs) to MSC influences remain incompletely understood. Here, we show that MSCs cause aberrant expression of microRNAs, which, led by microRNA-199a, provide BCCs with enhanced cancer stem cell (CSC) properties. We demonstrate that such MSC-deregulated microRNAs constitute a network that converges on and represses the expression of FOXP2, a forkhead transcription factor tightly associated with speech and language development. FOXP2 knockdown in BCCs was sufficient in promoting CSC propagation, tumor initiation, and metastasis. Importantly, elevated microRNA-199a and depressed FOXP2 expression levels are prominent features of malignant clinical breast cancer and are associated significantly with poor survival. Our results identify molecular determinants of cancer progression of potential utility in the prognosis and therapy of breast cancer

The effect of autistic traits on disembedding and mental rotation in neurotypical women and men

Funder: Autism Research TrustFunder: Templeton World Charitable FundFunder: NIHR Cambridge Biomedical Research Centre; doi: http://dx.doi.org/10.13039/501100018956Funder: National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health ResearchFunder: Care East of England at Cambridgeshire and Peterborough NHS Foundation TrustAbstractRecent data has revealed dissociations between social and non-social skills in both autistic and neurotypical populations. In the present study, we investigated whether specific visuospatial abilities, such as figure disembedding and mental rotation, are differently related to social and non-social autistic traits, in neurotypical women and men. University students (N = 426) completed the Autism Spectrum Quotient (AQ), figure disembedding and mental rotation of two-dimensional figures tasks. AQ social skills (AQ-social) and attention-to-details (AQ-attention) subscales were used as measures of social and non-social autistic traits, respectively. Mental rotation was affected by a significant interaction between sex, social and non-social traits. When non-social traits were above the mean (+ 1 SD), no sex differences in mental rotation were found. Instead, below this value, sex differences depended on the social traits, with men on average outperforming women at middle-to-high social traits, and with a comparable performance, and with women on average outperforming men, at lower social traits. A small positive correlation between figure disembedding and social traits was observed in the overall sample. These results are interpreted in terms of the hyper-systemizing theory of autism and contribute to the evidence of individual differences in the cognitive style of autistic people and neurotypical people with autistic traits.</jats:p

The effect of autistic traits on disembedding and mental rotation in neurotypical women and men

Funder: Autism Research TrustFunder: Templeton World Charitable FundFunder: NIHR Cambridge Biomedical Research Centre; doi: http://dx.doi.org/10.13039/501100018956Funder: National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health ResearchFunder: Care East of England at Cambridgeshire and Peterborough NHS Foundation TrustAbstractRecent data has revealed dissociations between social and non-social skills in both autistic and neurotypical populations. In the present study, we investigated whether specific visuospatial abilities, such as figure disembedding and mental rotation, are differently related to social and non-social autistic traits, in neurotypical women and men. University students (N = 426) completed the Autism Spectrum Quotient (AQ), figure disembedding and mental rotation of two-dimensional figures tasks. AQ social skills (AQ-social) and attention-to-details (AQ-attention) subscales were used as measures of social and non-social autistic traits, respectively. Mental rotation was affected by a significant interaction between sex, social and non-social traits. When non-social traits were above the mean (+ 1 SD), no sex differences in mental rotation were found. Instead, below this value, sex differences depended on the social traits, with men on average outperforming women at middle-to-high social traits, and with a comparable performance, and with women on average outperforming men, at lower social traits. A small positive correlation between figure disembedding and social traits was observed in the overall sample. These results are interpreted in terms of the hyper-systemizing theory of autism and contribute to the evidence of individual differences in the cognitive style of autistic people and neurotypical people with autistic traits.</jats:p

Relationship of suicide rates with climate and economic variables in Europe during 2000-2012

The derived models explained 62.4 % of the variability of male suicidal rates. Economic variables alone explained 26.9 % and climate variables 37.6 %. For females, the respective figures were 41.7, 11.5 and 28.1 %. Male suicides correlated with high unemployment rate in the frame of high growth rate and high inflation and low GDP per capita, while female suicides correlated negatively with inflation. Both male and female suicides correlated with low temperature. Data from 29 European countries covering the years 2000-2012 and concerning male and female standardized suicidal rates (according to WHO), economic variables (according World Bank) and climate variables were gathered. The statistical analysis included cluster and principal component analysis and categorical regression. It is well known that suicidal rates vary considerably among European countries and the reasons for this are unknown, although several theories have been proposed. The effect of economic variables has been extensively studied but not that of climate. The current study reports that the climatic effect (cold climate) is stronger than the economic one, but both are present. It seems that in Europe suicidality follows the climate/temperature cline which interestingly is not from south to north but from south to north-east. This raises concerns that climate change could lead to an increase in suicide rates. The current study is essentially the first successful attempt to explain the differences across countries in Europe; however, it is an observational analysis based on aggregate data and thus there is a lack of control for confounders. RESULTS METHODS BACKGROUND DISCUSSIO

The first nucleotide sequence of an archaeal elongation factor 1β gene

An archaeal elongation factor 1β gene has been isolated for the first time from a Sulfolobus solfataricus genomic library. The sequenced clone (869 bp) contained two open reading frames, one coding for a protein made of 91 amino acid residues (SsEF-1β), the other one encoding a nonidentified product (ORF 115). The amino acid sequences of segments at the N-and C-terminal of the translated SsEF-1β were identical to those determined for the native protein. Northern and Southern analyses showed that the SsEF-1β gene is represented in S. solfataricus by a unique sequence. Compared to eubacterial or eukaryal corresponding genes the SsEF-1β is much shorter

Qualification of a flow cytometry-based method for the evaluation of in vitro cytotoxicity of GTA002 natural killer cell therapy

Background: Natural Killer (NK) cell-based therapies represent a ground-breaking opportunity for the treatment of solid tumors and hematological malignancies. NK cell manufacturing under good manufacturing practice (GMP) is complex and requires attentive assessment the product's safety and efficacy through quality control (QC). Release testing includes monitoring of in vitro cell expansion, differentiation, purity, phenotype, and cytotoxicity. As NK cells are biologically active products, the establishment of potency methods is particularly relevant; surrogate or improper assays can lead to rejection of qualifiable batches or to release of products that falsely meet potency specifications, potentially causing low efficacy during clinical trials. As cell-based therapeutics are highly heterogeneous, no universal guidelines for product characterization are available, and developers must invest significant effort in establishing and validating robust and fit-to-purpose assays. In this study, we describe the qualification procedure of a flow cytometry-based analytical method to assess in vitro potency of GTA002 NK cells, to be applied to oNKord®/inaleucel allogeneic off-the-shelf NK cell product from Glycostem Therapeutics, undergoing a Phase I/IIa clinical trial in acute myeloid leukemia (AML) patients (NCT04632316). Methods: First, we established multi-color flow cytometry panels to quantitatively determine the count of effector (E) GTA002 cells and leukemia target (T) K562 cells alone and in co-culture at different E:T ratios (10:1, 3:1, 1:1). Effector potency was then qualitatively expressed as percentage of cytotoxicity. Next, we defined protocols for method qualification to assess the pivotal features of the assays, including accuracy, precision, linearity, range, specificity, robustness, and carryover; quantitative acceptance criteria were determined for all parameters. Results of the qualification procedure are reported and discussed against pre-defined acceptance criteria. Results: Overall, our methods show robust performance across all parameters, ensuring QC-compliant assessment of NK cell potency as part of the release test panel for clinical batches. Notably, we identified relevant aspects to address when progressing towards method validation to support pivotal clinical studies. Conclusions: This article provides a “case-study” of how analytical method development for cell therapeutics is planned and executed from early clinical stages, anticipating the need to establish robust procedures to overcome scientific and regulatory challenges during method validation