Massively parallel base editing to map variant effects in human hematopoiesis.

Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow for rich phenotyping through single-cell RNA sequencing readouts and separately for characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases.

SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract.

The mode of acquisition and causes for the variable clinical spectrum of coronavirus disease 2019 (COVID-19) remain unknown. We utilized a reverse genetics system to generate a GFP reporter virus to explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and a luciferase reporter virus to demonstrate sera collected from SARS and COVID-19 patients exhibited limited cross-CoV neutralization. High-sensitivity RNA in situ mapping revealed the highest angiotensin-converting enzyme 2 (ACE2) expression in the nose with decreasing expression throughout the lower respiratory tract, paralleled by a striking gradient of SARS-CoV-2 infection in proximal (high) versus distal (low) pulmonary epithelial cultures. COVID-19 autopsied lung studies identified focal disease and, congruent with culture data, SARS-CoV-2-infected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively. These findings highlight the nasal susceptibility to SARS-CoV-2 with likely subsequent aspiration-mediated virus seeding to the lung in SARS-CoV-2 pathogenesis. These reagents provide a foundation for investigations into virus-host interactions in protective immunity, host susceptibility, and virus pathogenesis.

A Method for the Acute and Rapid Degradation of Endogenous Proteins.

Methods for the targeted disruption of protein function have revolutionized science and greatly expedited the systematic characterization of genes. Two main approaches are currently used to disrupt protein function: DNA knockout and RNA interference, which act at the genome and mRNA level, respectively. A method that directly alters endogenous protein levels is currently not available. Here, we present Trim-Away, a technique to degrade endogenous proteins acutely in mammalian cells without prior modification of the genome or mRNA. Trim-Away harnesses the cellular protein degradation machinery to remove unmodified native proteins within minutes of application. This rapidity minimizes the risk that phenotypes are compensated and that secondary, non-specific defects accumulate over time. Because Trim-Away utilizes antibodies, it can be applied to a wide range of target proteins using off-the-shelf reagents. Trim-Away allows the study of protein function in diverse cell types, including non-dividing primary cells where genome- and RNA-targeting methods are limited.

Dynamic interactome of the MHC I peptide loading complex in human dendritic cells.

Dendritic cells (DCs) orchestrate immune responses by presenting antigenic peptides on major histocompatibility complex (MHC) molecules to T cells. Antigen processing and presentation via MHC I rely on the peptide-loading complex (PLC), a supramolecular machinery assembled around the transporter associated with antigen processing (TAP), which is the peptide transporter in the endoplasmic reticulum (ER) membrane. We studied antigen presentation in human DCs by isolating monocytes from blood and differentiating them into immature and mature DCs. We uncovered that during DC differentiation and maturation, additional proteins are recruited to the PLC, including B-cell receptor-associated protein 31 (BAP31), vesicle-associated membrane protein-associated protein A (VAPA), and extended synaptotagmin-1 (ESYT1). We demonstrated that these ER cargo export and contact site-tethering proteins colocalize with TAP and are within 40 nm proximity of the PLC, suggesting that the antigen processing machinery is located near ER exit- and membrane contact sites. While CRISPR/Cas9-mediated deletion of TAP and tapasin significantly reduced MHC I surface expression, single-gene deletions of the identified PLC interaction partners revealed a redundant role of BAP31, VAPA, and ESYT1 in MHC I antigen processing in DCs. These data highlight the dynamics and plasticity of PLC composition in DCs that previously was not recognized by the analysis of cell lines.

Virus neutralization assays for human respiratory syncytial virus using airway organoids.

Neutralizing antibodies are considered a correlate of protection against severe human respiratory syncytial virus (HRSV) disease. Currently, HRSV neutralization assays are performed on immortalized cell lines like Vero or A549 cells. It is known that assays on these cell lines exclusively detect neutralizing antibodies (nAbs) directed to the fusion (F) protein. For the detection of nAbs directed to the glycoprotein (G), ciliated epithelial cells expressing the cellular receptor CX3CR1 are required, but generation of primary cell cultures is expensive and labor-intensive. Here, we developed a high-throughput neutralization assay based on the interaction between clinically relevant HRSV grown on primary cells with ciliated epithelial cells, and validated this assay using a panel of infant sera. To develop the high-throughput neutralization assay, we established a culture of differentiated apical-out airway organoids (Ap-O AO). CX3CR1 expression was confirmed, and both F- and G-specific monoclonal antibodies neutralized HRSV in the Ap-O AO. In a side-by-side neutralization assay on Vero cells and Ap-O AO, neutralizing antibody levels in sera from 125 infants correlated well, although titers on Ap-O AO were consistently lower. We speculate that these lower titers might be an actual reflection of the neutralizing antibody capacity in vivo. The organoid-based neutralization assay described here holds promise for further characterization of correlates of protection against HRSV disease.

Characterization of primary models of human trophoblast.

Two recently developed models, trophoblast organoids and trophoblast stem cells (TSCs), are useful tools to further the understanding of human placental development. Both differentiate from villous cytotrophoblast (VCT) to either extravillous trophoblast (EVT) or syncytiotrophoblast (SCT). Here, we compare the transcriptomes and miRNA profiles of these models to identify which trophoblast they resemble in vivo. Our findings indicate that TSCs do not readily undergo SCT differentiation and closely resemble cells at the base of the cell columns from where EVT derives. In contrast, organoids are similar to VCT and undergo spontaneous SCT differentiation. A defining feature of human trophoblast is that VCT and SCT are human leukocyte antigen (HLA) null, whereas EVT expresses HLA-C, -G and -E molecules. We find that trophoblast organoids retain these in vivo characteristics. In contrast, TSCs express classical HLA-A and HLA-B molecules, and maintain their expression after EVT differentiation, with upregulation of HLA-G. Furthermore, HLA expression in TSCs differs when grown in 3D rather than in 2D, suggesting that mechanical cues are important. Our results can be used to select the most suitable model for the study of trophoblast development, function and pathology.

Short-term response of primary human meniscus cells to simulated microgravity.

BACKGROUND: Mechanical unloading of the knee articular cartilage results in cartilage matrix atrophy, signifying the osteoarthritic-inductive potential of mechanical unloading. In contrast, mechanical loading stimulates cartilage matrix production. However, little is known about the response of meniscal fibrocartilage, a major mechanical load-bearing tissue of the knee joint, and its functional matrix-forming fibrochondrocytes to mechanical unloading events. METHODS: In this study, primary meniscus fibrochondrocytes isolated from the inner avascular region of human menisci from both male and female donors were seeded into porous collagen scaffolds to generate 3D meniscus models. These models were subjected to both normal gravity and mechanical unloading via simulated microgravity (SMG) for 7 days, with samples collected at various time points during the culture. RESULTS: RNA sequencing unveiled significant transcriptome changes during the 7-day SMG culture, including the notable upregulation of key osteoarthritis markers such as COL10A1, MMP13, and SPP1, along with pathways related to inflammation and calcification. Crucially, sex-specific variations in transcriptional responses were observed. Meniscus models derived from female donors exhibited heightened cell proliferation activities, with the JUN protein involved in several potentially osteoarthritis-related signaling pathways. In contrast, meniscus models from male donors primarily regulated extracellular matrix components and matrix remodeling enzymes. CONCLUSION: These findings advance our understanding of sex disparities in knee osteoarthritis by developing a novel in vitro model using cell-seeded meniscus constructs and simulated microgravity, revealing significant sex-specific molecular mechanisms and therapeutic targets.

Comparative Analysis of Primary Ovarian Cancer Cells and Established Cell Lines as a New Tool for Studies on Ovarian Cancer Cell Complexity.

Primary cancer cells reflect the genetic background and phenotype of a tumor. Immortalized cells with higher proliferation activity have an advantage over primary cells. The aim of the study was to immortalize the primary ovarian cancer (OvCa) cells using the plasmid-carrying human telomerase reverse transcriptase (hTERT) gene and compare their phenotype and biological activity with the primary cells. The primary OvCa3 A and OvCa7 A cells were isolated from the ascitic fluid of two high-grade serous ovarian cancer patients and were characterized using immunocytochemical methods, flow cytometry, real-time RT-PCR, Western blot, metabolic activity, and migratory potential. Both immortalized ovarian cancer cell lines mirrored the phenotype of primary cancer cells, albeit with modifications. The OvCa3 A hTERT cells kept the mesenchymal stem cell phenotype of CD73/CD90/CD105-positivity and were CD133-negative, whereas the cell population of OvCa7 A hTERT lost CD73 expression, but almost 90% of cells expressed the CD133 characteristic for the CSCs phenotype. Immortalized OvCa cells differed in gene expression level with respect to Sox2 and Oct4, which was associated with stemness properties. The OvCa7 A hTERT cells showed higher metabolic and migratory activity and ALDH1 expression than the corresponding primary OvCa cells. Both primary and immortalized cell lines were able to form spheroids. The newly established unique immortalized cell line OvCa7 A hTERT, with the characteristic of a serous ovarian cancer malignancy feature, and with the accumulation of the p53, Pax8, and overexpression of the CD133 and CD44 molecules, may be a useful tool for research on therapeutic approaches, especially those targeting CSCs in ovarian cancer and in preclinical 2D and 3D models.

Effects of selective cyclooxygenase-2 inhibitor robenacoxib on primary cells derived from feline injection-site sarcoma.

Feline injection-site sarcomas (FISSs) are highly invasive malignant mesenchymal neoplasms that arise from injection sites in cats. Although the tumorigenesis of FISSs is still uncertain, there is a consensus that FISS is associated with chronic inflammation caused by irritation of injection-related trauma and foreign chemical substances. Chronic inflammation can provide a proper microenvironment for tumour development, which has been known as one of the risk factors of tumorigenesis in many tumours. To investigate the tumorigenesis of FISS and screen for its potential therapeutic targets, cyclooxygenase-2 (COX-2), an inflammation-enhancing enzyme, was selected as a target for this study. In vitro experiments using FISS- and normal tissue-derived primary cells and robenacoxib, a highly selective COX-2 inhibitor, were performed. The results demonstrated that expression of COX-2 could be detected in formalin-fixed and paraffin-embedded FISS tissues and FISS-derived primary cells. Cell viability, migration and colony formation of FISS-derived primary cells were inhibited, and cell apoptosis was enhanced by robenacoxib in a dose-dependent manner. However, susceptibility to robenacoxib varied in different lines of FISS primary cells and was not completely correlated with COX-2 expression. Our results suggest that COX-2 inhibitors could be potential adjuvant therapeutics against FISSs.

Integrated analysis of the tumor microenvironment using a reconfigurable microfluidic cell culture platform.

The tumor microenvironment (TME) is a complex network of non-malignant cells and stroma that perform a wide array of vital roles in tumor growth, immune evasion, metastasis, and therapeutic resistance. These highly diverse roles have been shown to be critically important to the progression of cancers and have already shown potential as therapeutic targets. Therefore, there has been a tremendous push to elucidate the pathways that underlie these roles and to develop new TME-directed therapies for cancer treatment. Unfortunately, TME-focused research has been limited by a lack of translational in vitro culture platforms that can model this highly complex niche and can support the integrated analysis of cell biology and function. In the current study, we investigate whether an independently developed reconfigurable microfluidic platform, known as Stacks, can address the critical need for translational multi-cellular tumor models and integrated analytics in TME research. We present data on multi-cellular culture of primary human cells in Stacks as well as the orthogonal analysis of cellular polarization, differentiation, migration, and cytotoxicity in this reconfigurable system. These expanded capabilities of Stacks are highly relevant to the cancer research community with the potential to enhance clinical translation of pre-clinical TME studies and to yield novel biological insight into TME crosstalk, metastasis, and responses to novel drug combinations or immune therapies.

Tools for drug discovery and disease modeling- the future is upon us.

Although technical prowess in screening for drugs has increased dramatically with the development of high content imaging, resonance energy transfer- and intensiometric biosensors, translation into the clinic has stagnated and not all drugs work in all patients. This is likely due to 1) our rudimentary understanding of disease mechanisms, and 2) our increasing use of generic, cell-based screens which have moved us away from biologically relevant tissues, organs, and patients. Here, we focus on emerging tools to undertake screening and evaluate drug actions in models ranging from heterologous expression systems, primary cells, patient-derived induced pluripotent stem cells and organoids to in vivo models.

Long-term osteogenic differentiation of human bone marrow stromal cells in simulated microgravity: novel proteins sighted.

Microgravity-induced bone loss is a major concern for space travelers. Ground-based microgravity simulators are crucial to study the effect of microgravity exposure on biological systems and to address the limitations posed by restricted access to real space. In this work, for the first time, we adopt a multidisciplinary approach to characterize the morphological, biochemical, and molecular changes underlying the response of human bone marrow stromal cells to long-term simulated microgravity exposure during osteogenic differentiation. Our results show that osteogenic differentiation is reduced while energy metabolism is promoted. We found novel proteins were dysregulated under simulated microgravity, including CSC1-like protein, involved in the mechanotransduction of pressure signals, and PTPN11, SLC44A1 and MME which are involved in osteoblast differentiation pathways and which may become the focus of future translational projects. The investigation of cell proteome highlighted how simulated microgravity affects a relatively low number of proteins compared to time and/or osteogenic factors and has allowed us to reconstruct a hypothetical pipeline for cell response to simulated microgravity. Further investigation focused on the application of nanomaterials may help to increase understanding of how to treat or minimize the effects of microgravity.

Clinical Consequences and Functional Impact of the Rare S737F CFTR Variant and Its Responsiveness to CFTR Modulators.

S737F is a Cystic Fibrosis (CF) transmembrane conductance regulator (CFTR) missense variant. The aim of our study was to describe the clinical features of a cohort of individuals carrying this variant. In parallel, by exploiting ex vivo functional and molecular analyses on nasal epithelia derived from a subset of S737F carriers, we evaluated its functional impact on CFTR protein as well as its responsiveness to CFTR modulators. We retrospectively collected clinical data of all individuals bearing at least one S737F CFTR variant and followed at the CF Centre of Tuscany region (Italy). Nasal brushing was performed in cooperating individuals. At study end clinical data were available for 10 subjects (mean age: 14 years; range 1-44 years; 3 adult individuals). Five asymptomatic subjects had CF, 2 were CRMS/CFSPID and 3 had an inconclusive diagnosis. Ex vivo analysis on nasal epithelia demonstrated different levels of CF activity. In particular, epithelia derived from asymptomatic CF subjects and from one of the subjects with inconclusive diagnosis showed reduced CFTR activity that could be rescued by treatment with CFTR modulators. On the contrary, in the epithelia derived from the other two individuals with an inconclusive diagnosis, the CFTR-mediated current was similar to that observed in epithelia derived from healthy donors. In vitro functional and biochemical analysis on S737F-CFTR expressed in immortalized bronchial cells highlighted a modest impairment of the channel activity, that was improved by treatment with ivacaftor alone or in combination with tezacaftor/elexacaftor. Our study provide evidence towards the evaluation of CFTR function on ex vivo nasal epithelial cell models as a new assay to help clinicians to classify individuals, in presence of discordance between clinical picture, sweat test and genetic profile.

SERINC5 Can Enhance Proinflammatory Cytokine Production by Primary Human Myeloid Cells in Response to Challenge with HIV-1 Particles.

HIV-1 has to overcome physical barriers posed by host cell restriction factors (RFs) for efficient replication. Some RFs, including Trim5α and tetherin, trigger antiviral signaling in addition to directly impairing HIV replication. SERINC5 (S5) is an RF that is incorporated into HIV-1 particles to potently impair their infectivity and is efficiently antagonized by the viral pathogenesis factor Nef. Since effects of S5 on HIV-1 infectivity were mostly studied in reporter cell lines, we analyzed the effects of S5 during infection of primary HIV-1 target cells. In activated CD4+ T lymphocytes, virion incorporation of S5 only moderately impaired virion infectivity and was not associated with altered innate immune recognition. In contrast, in monocyte-derived macrophages, S5 virion incorporation potentiated the production of proinflammatory cytokines with very potent but donor-dependent effects on virion infectivity. Nef counteracted effects of S5 on both cytokine production and virion infectivity. Similar S5-induced cytokine production was observed in immature monocyte-derived dendritic cells. Notably, S5-mediated enhancement of cytokine production was not linked to the efficacy of productive infection and could be overcome by using vesicular stomatitis virus glycoprotein (VSV-G) but not infectivity restriction-insensitive HIV-1 Env for cell entry. Moreover, inhibiting entry of S5-negative HIV-1 ΔNef particles increased proinflammatory cytokine production comparably to virion incorporation of S5. Together, these results describe the sensitization of noninfectious HIV-1 particles to proinflammatory cytokine production by myeloid target cells as an additional and Nef-sensitive activity of S5. Moreover, the study reveals important cell-type and donor-dependent differences in the sensitivity of HIV target cells for antiviral effects of S5.IMPORTANCE SERINC5 (S5) is a host cell restriction factor (RF) that impairs the infectivity of HIV-1 particles in target cell lines. To assess the potential physiological relevance of this restriction, we assessed the effects of S5 on HIV-1 infection of relevant primary human target cells. We found that effects of S5 on infection of CD4+ T lymphocytes were negligible. In myeloid target cells, however, virion incorporation of S5 potently suppressed infectivity and promoted innate immune recognition of HIV-1 particles characterized by proinflammatory cytokine production. Both effects were not observed in cells of all donors analyzed, were exerted independently of one another, and were counteracted by the HIV-1 pathogenesis factor Nef. These results identify the sensitization of HIV-1 particles for innate immune recognition by myeloid target cells as a novel activity of S5 and emphasize the need to study RF function in the context of primary target cells and taking donor variabilities into account.

Galectin network in osteoarthritis: galectin-4 programs a pathogenic signature of gene and effector expression in human chondrocytes in vitro.

Galectin-4 (Gal-4) is a member of the galectin family, which have been identified as galactose-binding proteins. Gal-4 possesses two tandem repeat carbohydrate recognition domains and acts as a cross-linking bridge in sulfatide-dependent glycoprotein routing. We herein document its upregulation in osteoarthritis (OA) in correlation with the extent of cartilage degradation in vivo. Primary human OA chondrocytes in vitro respond to carbohydrate-inhibitable Gal-4 binding with the upregulation of pro-degradative/-inflammatory proteins such as interleukin-1ß (IL-1ß) and matrix metalloproteinase-13 (MMP-13), as documented by RT-qPCR-based mRNA profiling and transcriptome data processing. Activation of p65 by phosphorylation of Ser536 within the NF-κB pathway and the effect of three p65 inhibitors on Gal-4 activity support downstream involvement of such signaling. In 3D (pellet) cultures, Gal-4 presence causes morphological and biochemical signs of degradation. Taken together, our findings strongly support the concept of galectins acting as a network in OA pathogenesis and suggest that blocking their activity in disease progression may become clinically relevant in the future.

Cell culture models to study retinal pigment epithelium-related pathogenesis in age-related macular degeneration.

Age-related macular degeneration (AMD) is a disease that affects the macula - the central part of the retina. It is a leading cause of irreversible vision loss in the elderly. AMD onset is marked by the presence of lipid- and protein-rich extracellular deposits beneath the retinal pigment epithelium (RPE), a monolayer of polarized, pigmented epithelial cells located between the photoreceptors and the choroidal blood supply. Progression of AMD to the late nonexudative "dry" stage of AMD, also called geographic atrophy, is linked to progressive loss of areas of the RPE, photoreceptors, and underlying choriocapillaris leading to a severe decline in patients' vision. Differential susceptibility of macular RPE in AMD and the lack of an anatomical macula in most lab animal models has promoted the use of in vitro models of the RPE. In addition, the need for high throughput platforms to test potential therapies has driven the creation and characterization of in vitro model systems that recapitulate morphologic and functional abnormalities associated with human AMD. These models range from spontaneously formed cell line ARPE19, immortalized cell lines such as hTERT-RPE1, RPE-J, and D407, to primary human (fetal or adult) or animal (mouse and pig) RPE cells, and embryonic and induced pluripotent stem cell (iPSC) derived RPE. Hallmark RPE phenotypes, such as cobblestone morphology, pigmentation, and polarization, vary significantly betweendifferent models and culture conditions used in different labs, which would directly impact their usability for investigating different aspects of AMD biology. Here the AMD Disease Models task group of the Ryan Initiative for Macular Research (RIMR) provides a summary of several currently used in vitro RPE models, historical aspects of their development, RPE phenotypes that are attainable in these models, their ability to model different aspects of AMD pathophysiology, and pros/cons for their use in the RPE and AMD fields. In addition, due to the burgeoning use of iPSC derived RPE cells, the critical need for developing standards for differentiating and rigorously characterizing RPE cell appearance, morphology, and function are discussed.

Receptor-proximal effectors mediating GnRH actions in the goldfish pituitary: Involvement of G protein subunits and GRKs.

In goldfish (Carassius auratus), two endogenous isoforms of gonadotropin-releasing hormone (GnRH) stimulate luteinizing hormone (LH) and growth hormone (GH) secretion. These isoforms, GnRH2 and GnRH3, act on a shared population of cell-surface GnRH receptors (GnRHRs) expressed on both gonadotrophs and somatotrophs, and can signal through unique, yet partially overlapping, suites of intracellular effectors, in a phenomenon known as functional selectivity or biased signalling. In this study, G-protein alpha (Gα) subunits were targeted with two inhibitors, YM-254890 and BIM-46187, to ascertain the contribution of specific G-protein subunits in GnRH signalling. Results with the Gαq/11-specific inhibitor YM-254890 on primary cultures of goldfish pituitary cells revealed the use of these subunits in GnRH control of both LH and GH release, as well as GnRH-induced elevations in phospho-ERK levels. Results with the pan-Gα inhibitor BIM-46187 matched those using YM-254890 in LH release but GH responses differed, indicating additional, non-Gαq/11 subunits may be involved in somatotrophs. BIM-46187 also elevated unstimulated LH and GH release suggesting that Gα subunits regulate basal hormone secretion. Furthermore, G-protein-coupled receptor kinase (GRK2/3) inhibition reduced LH responses to GnRH2 and GnRH3, and selectively enhanced GnRH2-stimulated GH release, indicating differential use of GRK2/3 in GnRH actions on gonadotrophs and somatotrophs. These findings in a primary untransformed system provide the first direct evidence to establish Gαq/11 as an obligate driver of GnRH signalling in goldfish pituitary cells, and additionally describe the differential agonist- and cell type-selective involvement of GRK2/3 in this system.

Oxidative potential and in vitro toxicity of particles generated by pyrotechnic smokes in human small airway epithelial cells.

Pyrotechnic smokes are widely used in civilian and military applications. The major issue arise from the release of particles after smoke combustion but the health risks related to their exposure are poorly documented whereas toxicity of airborne particles on the respiratory target are very well known. Therefore, this study aimed to explore the in vitro toxicity of the particle fraction of different pyrotechnic smokes. Particles from a red signalling smoke (RSS), an hexachloroethane-based obscuring smoke (HC-OS) and an anti-intrusion smoke (AIS) were collected from the cloud. RSS particles displayed the highest organic fraction (quinones and polycyclic aromatic hydrocarbons) of the three samples characterized. AIS particles contained K and cholesterol derivatives. HC-OS particles were mainly metallic with very high concentrations of Al, Fe and Ca. Intrinsic oxidative potential of smoke particles was measured with two assays. Depletions of DTT by RSS particles was greater than depletion obtained with AIS and HC-OS particles but depletion of acid ascorbic (AA) was only observed with HC-OS particles. In vitro toxicity was assessed by exposing human small airway epithelial cells (SAEC) to various concentrations of particles. After 24 h of exposure, cell viability was not affected but significant modifications of mRNA expression of antioxidant (SOD-1 and -2, catalase, HO-1, NQO-1) and inflammatory markers (IL-6, IL-8, TNF-α) were observed and were dependent on smoke type. Particles rich in metal, such as HC-OS, induced a greatest depletion of AA and a greatest inflammatory response, whereas particles rich in organic compounds, such as RSS, induced a greatest DTT depletion and a greatest antioxidant response. In conclusion, the three smoke particles have an intrinsic oxidative potential and triggered a cell adaptive response. Our study improved the knowledge of particle toxicity of pyrotechnic smokes and scientific approach developed here could be used to study other type of particles.

In vitro cultivation of primary intestinal cells from Eisenia fetida as basis for ecotoxicological studies.

The earthworm Eisenia fetida is a commonly used model organism for unspecific soil feeders in ecotoxicological studies. Its intestinal cells are the first to encounter possible pollutants co-ingested by the earthworm, which makes them prime candidates for studies of toxic effects of environmental pollutants on the cellular as compared to the organismic level. In this context, the aim of this study was to demonstrate the suitability of preparations of primary intestinal E. fetida cells for in vitro ecotoxicological studies. For this purpose, a suitable isolation and cultivation protocol was established. Cells were isolated directly from the intestine, maintaining >85% viability during subsequent cultivations (up to 144 h). Exposure to established pollutants and soil elutriates comprising silver nanoparticles and metal ions (Cu2+, Cd2+) induced a significant decrease in the metabolic activity of the cells. In case of microplastic particles (MP particles), namely 0.2, 0.5, 2.0, and 3.0 µm diameter polystyrene (PS) beads as well as 0.5 and 2.0 µm diameter polylactic acid (PLA) beads, no active uptake was observed. Slight positive as well as negative dose and size dependent effects on the metabolism were seen, which to some extent might correlate with effects on the organismic level.

Primary TSC2-/meth Cells Induce Follicular Neogenesis in an Innovative TSC Mouse Model.

Cutaneous lesions are one of the hallmarks of tuberous sclerosis complex (TSC), a genetic disease in which mTOR is hyperactivated due to the lack of hamartin or tuberin. To date, novel pharmacological treatments for TSC cutaneous lesions that are benign but still have an impact on a patient's life are needed, because neither surgery nor rapamycin administration prevents their recurrence. Here, we demonstrated that primary TSC2-/meth cells that do not express tuberin for an epigenetic event caused cutaneous lesions and follicular neogenesis when they were subcutaneously injected in nude mice. Tuberin-null cells localized in the hair bulbs and alongside mature hairs, where high phosphorylation of S6 and Erk indicated mTOR hyperactivation. Interestingly, 5-azacytidine treatment reduced hair follicles, indicating that chromatin remodeling agents might be effective on TSC lesions in which cells lack tuberin for an epigenetic event. Moreover, we demonstrated that the primary TSC2-/meth cells had metastatic capability: when subcutaneously injected, they reached the bloodstream and lymphatics and invaded the lungs, causing the enlargement of the alveolar walls. The capability of TSC2-/meth cells to survive and migrate in vivo makes our mouse model ideal to follow the progression of the disease and test potential pharmacological treatments in a time-dependent manner.