Multiplexed CRISPR/CAS9‐mediated engineering of pre‐clinical mouse models bearing native human B cell receptors

B‐cell receptor (BCR) knock‐in (KI) mouse models play an important role in vaccine development and fundamental immunological studies. However, the time required to generate them poses a bottleneck. Here we report a one‐step CRISPR/Cas9 KI methodology to combine the insertion of human germline immunoglobulin heavy and light chains at their endogenous loci in mice. We validate this technology with the rapid generation of three BCR KI lines expressing native human precursors, instead of computationally inferred germline sequences, to HIV broadly neutralizing antibodies. We demonstrate that B cells from these mice are fully functional: upon transfer to congenic, wild type mice at controlled frequencies, such B cells can be primed by eOD‐GT8 60mer, a germline‐targeting immunogen currently in clinical trials, recruited to germinal centers, secrete class‐switched antibodies, undergo somatic hypermutation, and differentiate into memory B cells. KI mice expressing functional human BCRs promise to accelerate the development of vaccines for HIV and other infectious diseases.     

Intermittent hypoxia treatments cause cellular priming in human microglia

Obstructive sleep apnoea syndrome (OSAS) is a sleep-disordered breathing characterized by nocturnal collapses of the upper airway resulting in cycles of blood oxygen partial pressure oscillations, which lead to tissue and cell damage due to intermittent hypoxia (IH) episodes. Since OSAS-derived IH may lead to cognitive impairment through not fully cleared mechanisms, herein we developed a new in vitro model mimicking IH conditions to shed light on its molecular effects on microglial cells, with particular attention to the inflammatory response. The in vitro model was set-up and validated by measuring the hypoxic state, HIF-1α levels, oxidative stress by ROS production and mitochondrial activity by MTS assay. Then, the mRNA and protein levels of certain inflammatory markers (NF-κB and interleukin 6 (IL-6)) after different IH treatment protocols were investigated. The IH treatments followed by a normoxic period were not able to produce a high inflammatory state in human microglial cells. Nevertheless, microglia appeared to be in a state characterized by increased expression of NF-κB and markers related to a primed phenotype. The microglia exposed to IH cycles and stimulated with exogenous IL-1β resulted in an exaggerated inflammatory response with increased NF-κB and IL-6 expression, suggesting a role for primed microglia in OSAS-driven neuroinflammation.     

‘Cry-for-help’ in contaminated soil: a dialogue among plants and soil microbiome to survive in hostile conditions

An open question in environmental ecology regards the mechanisms triggered by root chemistry to drive the assembly and functionality of a beneficial microbiome to rapidly adapt to stress conditions. This phenomenon, originally described in plant defence against pathogens and predators, is encompassed in the ‘cry-for-help’ hypothesis. Evidence suggests that this mechanism may be part of the adaptation strategy to ensure the holobiont fitness in polluted environments. Polychlorinated biphenyls (PCBs) were considered as model pollutants due to their toxicity, recalcitrance and poor phyto-extraction potential, which lead to a plethora of phytotoxic effects and rise environmental safety concerns. Plants have inefficient detoxification processes to catabolize PCBs, even leading to by-products with a higher toxicity. We propose that the ‘cry-for-help’ mechanism could drive the exudation-mediated recruitment and sustainment of the microbial services for PCBs removal, exerted by an array of anaerobic and aerobic microbial degrading populations working in a complex metabolic network. Through this synergistic interaction, the holobiont copes with the soil contamination, releasing the plant from the pollutant stress by the ecological services provided by the boosted metabolism of PCBs microbial degraders. Improving knowledge of root chemistry under PCBs stress is, therefore, advocated to design rhizoremediation strategies based on plant microbiome engineering.     

Impact of diffused versus vasculature targeted DNA damage on the heart of mice depleted of telomeric factor Ft1

DNA damage is emerging as a driver of heart disease, although the cascade of events, its timing, and the cell types involved are yet to be fully clarified. In this context, the implication of cardiomyocytes has been highlighted, while that of vasculature smooth muscle cells has been implicated but not explored exhaustively. In our previous work we characterized a factor called Ft1 in mice and AKTIP in humans whose depletion generates telomere instability and DNA damage. Herein, we explored the effect of the reduction of Ft1 on the heart with the goal of comparatively defining the impact of DNA damage targeted to vasculature smooth muscle cells to that of diffuse damage. Using two newly generated mouse models, Ft1 constitutively knocked out (Ft1ko) mice, and mice in which we targeted the Ft1 depletion to the smooth muscle cells (Ft1sm22ko), it is shown that both genetic models display cardiac defects but with differences. Both Ft1ko and Ft1sm22ko mice display hypertrophy, fibrosis, and functional heart defects. Interestingly, Ft1sm22ko mice have early milder pathological traits that become manifest with age. Significantly, the defects of Ft1ko mice, including the alteration of the left ventricle and functional heart defects, are rescued by depletion of the DNA damage sensor p53. These results point to Ft1 deficiency as a driver of cardiac disease and show that Ft1 deficiency targeted to vasculature smooth muscle cells generates a pre-pathological profile exacerbated by age.     

Long non‐coding RNA TINCR suppresses metastatic melanoma dissemination by preventing ATF4 translation

Transition from proliferative‐to‐invasive phenotypes promotes metastasis and therapy resistance in melanoma. Reversion of the invasive phenotype, however, is challenged by the poor understanding of mechanisms underlying its maintenance. Here, we report that the lncRNA TINCR is down‐regulated in metastatic melanoma and its silencing increases the expression levels of invasive markers, in vitro migration, in vivo tumor growth, and resistance to BRAF and MEK inhibitors. The critical mediator is ATF4, a central player of the integrated stress response (ISR), which is activated in TINCR‐depleted cells in the absence of starvation and eIF2α phosphorylation. TINCR depletion increases global protein synthesis and induces translational reprogramming, leading to increased translation of mRNAs encoding ATF4 and other ISR proteins. Strikingly, re‐expression of TINCR in metastatic melanoma suppresses the invasive phenotype, reduces numbers of tumor‐initiating cells and metastasis formation, and increases drug sensitivity. Mechanistically, TINCR interacts with mRNAs associated with the invasive phenotype, including ATF4, preventing their binding to ribosomes. Thus, TINCR is a suppressor of the melanoma invasive phenotype, which functions in nutrient‐rich conditions by repressing translation of selected ISR RNAs.     

Melanoma and subcutaneous adipose tissue: Role of peritumoral adipokines in disease characterization and prognosis

In the last decades, the concept of adipose organ has emerged, giving adipose tissue an active endocrine and immunologic function through the secretion of multiple cytokines and chemokines that seem to be implicated in the development and progression of several cancer, including cutaneous melanoma. In this pilot experimental study, we analyzed the expression in the peritumor subcutaneous adipose tissue of the most significant adipokines involved in the processes of carcinogenesis and metastasis in a population of melanoma patients and in two control groups composed of melanocytic nevi and epidermoid cysts, respectively. We correlated the results obtained with the main disease prognostic factors observing a statistically significant increase in the expression of PAI1, LEP, CXCL1, NAMPT, and TNF-α at the level of the peritumor tissue of the melanoma samples compared to the control groups and a correlation of the same with the histopathological prognostic factor of melanoma. Our preliminary study shows that the overexpression of PAI1, LEP, CXCL1, NAMPT, and TNF-α may contribute to the growth and to the local aggressiveness of cutaneous melanoma. It opens the hypothesis of a direct oncogenic role of subcutaneous adipose tissue and adipokines in the tumorigenesis of melanoma.