The olfactory receptor Olfr78 promotes differentiation of enterochromaffin cells in the mouse colon.

The gastrointestinal epithelium constitutes a chemosensory system for microbiota-derived metabolites such as short-chain fatty acids (SCFA). Here, we investigate the spatial distribution of Olfr78, one of the SCFA receptors, in the mouse intestine and study the transcriptome of colon enteroendocrine cells expressing Olfr78. The receptor is predominantly detected in the enterochromaffin and L subtypes in the proximal and distal colon, respectively. Using the Olfr78-GFP and VilCre/Olfr78flox transgenic mouse lines, we show that loss of epithelial Olfr78 results in impaired enterochromaffin cell differentiation, blocking cells in an undefined secretory lineage state. This is accompanied by a reduced defense response to bacteria in colon crypts and slight dysbiosis. Using organoid cultures, we further show that maintenance of enterochromaffin cells involves activation of the Olfr78 receptor via the SCFA ligand acetate. Taken together, our work provides evidence that Olfr78 contributes to colon homeostasis by promoting enterochromaffin cell differentiation.

Comparative study of keratinocyte primary culture methods from paediatric skin biopsies for RNA-sequencing.

BACKGROUND: Keratinocyte culture is a standard method used to study gene expression, cell differentiation and proliferation. Numerous protocols exist, however their application is frequently unsuitable for small specimens, such as 4-mm punch skin biopsies. AIMS: This study compared 3 different methods of keratinocyte culture from paediatric skin biopsies to evaluate which one ensures adequate cell growth for RNA extraction and sequencing. MATERIALS AND METHODS: Thirty-six skin samples were obtained from 4-mm punch skin biopsies from residual human body material from healthy children. They were cultured in vitro according to 3 different methods: enzymatic method, epidermis explant and direct explant method. Keratinocytes were characterized by immunocytochemistry using pan-cytokeratin. RNA extraction was performed with RNeasy Mini kit. Quantity and quality of the extracted RNA was assessed to meet the requirements of library preparation for sequencing. RESULTS: The direct explant method had largely shown its superiority over the two other methods, with a 100% success rate and an average of 15 days of culture. RNA extraction yielded a mean of 8545.85 ng of RNA per sample with an RQN of 10. Cover-clip immunochemistry staining with pan-cytokeratin had confirmed the absence of fibroblast contamination. DISCUSSION: Although the enzymatic method is the most frequently used for keratinocyte culture, it is not suitable small samples required in dermatology. The direct explant method guarantees a high growth rate and the extraction of high quality RNA. Variation in the amount of RNA harvested are related to inter- and intra-individual variations and to the conditions of the experiment. CONCLUSION: This study allowed to conclude that the direct explant method is the most efficient and easy method to ensure cell growth when the samples are from 4-mm punch skin biopsies. This technique avoids fibroblasts contamination and obtains a sufficient quantity and quality of RNA to sequence it.

LGR5 controls extracellular matrix production by stem cells in the developing intestine.

The Lgr5 receptor is a marker of intestinal stem cells (ISCs) that regulates Wnt/b-catenin signaling. In this study, phenotype analysis of knockin/knockout Lgr5-eGFP-IRES-Cre and Lgr5-DTReGFP embryos reveals that Lgr5 deficiency during Wnt-mediated cytodifferentiation results in amplification of ISCs and early differentiation into Paneth cells, which can be counteracted by in utero treatment with the Wnt inhibitor LGK974. Conditional ablation of Lgr5 postnatally, but not in adults, alters stem cell fate toward the Paneth lineage. Together, these in vivo studies suggest that Lgr5 is part of a feedback loop to adjust the Wnt tone in ISCs. Moreover, transcriptome analyses reveal that Lgr5 controls fetal ISC maturation associated with acquisition of a definitive stable epithelial phenotype, as well as the capacity of ISCs to generate their own extracellular matrix. Finally, using the ex vivo culture system, evidences are provided that Lgr5 antagonizes the Rspondin 2-Wnt-mediated response in ISCs in organoids, revealing a sophisticated regulatory process for Wnt signaling in ISCs.

Single-cell transcriptome analysis reveals thyrocyte diversity in the zebrafish thyroid gland.

The thyroid gland regulates growth and metabolism via production of thyroid hormone in follicles composed of thyrocytes. So far, thyrocytes have been assumed to be a homogenous population. To uncover heterogeneity in the thyrocyte population and molecularly characterize the non-thyrocyte cells surrounding the follicle, we developed a single-cell transcriptome atlas of the region containing the zebrafish thyroid gland. The 6249-cell atlas includes profiles of thyrocytes, blood vessels, lymphatic vessels, immune cells, and fibroblasts. Further, the thyrocytes show expression heterogeneity, including bimodal expression of the transcription factor pax2a. To validate thyrocyte heterogeneity, we generated a CRISPR/Cas9-based pax2a knock-in line that monitors pax2a expression in the thyrocytes. A population of pax2a-low mature thyrocytes interspersed in individual follicles can be distinguished. We corroborate heterogeneity within the thyrocyte population using RNA sequencing of pax2a-high and pax2a-low thyrocytes, which demonstrates 20% differential expression in transcriptome between the two subpopulations. Our results identify and validate transcriptional differences within the presumed homogenous thyrocyte population.

The mononuclear phagocyte system contributes to fibrosis in post-transplant obliterans bronchiolitis.

Bronchiolitis obliterans syndrome (BOS) is a fibrotic disease that is heavily responsible for the high mortality rates after lung transplantation. Myofibroblasts are primary effectors of this fibrotic process, but their origin is still debated. The purpose of this work was to identify the precursors of mesenchymal cells responsible for post-transplant airway fibro-obliteration.Lineage-tracing tools were used to track or deplete potential sources of myofibroblasts in the heterotopic tracheal transplantation model. Allografts were analysed by histology, confocal microscopy, flow cytometry or single-cell transcriptomic analysis. BOS explants were evaluated by histology and confocal microscopy.Myofibroblasts in the allografts were recipient-derived. When recipient mice were treated with tacrolimus, we observed rare epithelial-to-mesenchymal transition phenomena and an overall increase in donor-derived myofibroblasts (p=0.0467), but the proportion of these cells remained low (7%). Haematopoietic cells, and specifically the mononuclear phagocyte system, gave rise to the majority of myofibroblasts found in occluded airways. Ablation of Cx3cR1+ cells decreased fibro-obliteration (p=0.0151) and myofibroblast accumulation (p=0.0020). Single-cell RNA sequencing revealed similarities between myeloid-derived cells from allografts and both murine and human samples of lung fibrosis. Finally, myofibroblasts expressing the macrophage marker CD68 were increased in BOS explants when compared to controls (14.4% versus 8.5%, p=0.0249).Recipient-derived myeloid progenitors represent a clinically relevant source of mesenchymal cells infiltrating the airways after allogeneic transplantation. Therapies targeting the mononuclear phagocyte system could improve long-term outcomes after lung transplantation.

Digenic inheritance of human primary microcephaly delineates centrosomal and non-centrosomal pathways.

Primary microcephaly (PM) is characterized by a small head since birth and is vastly heterogeneous both genetically and phenotypically. While most cases are monogenic, genetic interactions between Aspm and Wdr62 have recently been described in a mouse model of PM. Here, we used two complementary, holistic in vivo approaches: high throughput DNA sequencing of multiple PM genes in human patients with PM, and genome-edited zebrafish modeling for the digenic inheritance of PM. Exomes of patients with PM showed a significant burden of variants in 75 PM genes, that persisted after removing monogenic causes of PM (e.g., biallelic pathogenic variants in CEP152). This observation was replicated in an independent cohort of patients with PM, where a PM gene panel showed in addition that the burden was carried by six centrosomal genes. Allelic frequencies were consistent with digenic inheritance. In zebrafish, non-centrosomal gene casc5 -/- produced a severe PM phenotype, that was not modified by centrosomal genes aspm or wdr62 invalidation. A digenic, quadriallelic PM phenotype was produced by aspm and wdr62. Our observations provide strong evidence for digenic inheritance of human PM, involving centrosomal genes. Absence of genetic interaction between casc5 and aspm or wdr62 further delineates centrosomal and non-centrosomal pathways in PM.

Epigenetic basis for monocyte dysfunction in patients with severe alcoholic hepatitis.

BACKGROUND & AIMS: Severe forms of alcohol-related liver disease are associated with increased susceptibility to infections which are associated with poor prognosis. The cellular and molecular mechanisms responsible for this altered host defense are incompletely understood. METHODS: We performed whole blood phenotypic analysis and ex vivo stimulation with various pathogen-associated molecular patterns (PAMPs). We included 34 patients with alcohol-related cirrhosis (18 of whom had biopsy-proven severe alcoholic hepatitis [sAH]), 12 healthy controls and 11 patients with chronic alcohol consumption without significant liver disease. We also evaluated the transcriptomic (RNA-seq) and chromatin accessibility (ATAC-seq) profiles of CD14+ monocytes from a subset of patients. RESULTS: Circulating monocytes and conventional dendritic cells (DCs) from patients with sAH displayed complex alterations characterized by increased expression of both activating and inhibitory surface markers and an impaired pro-inflammatory response upon stimulation with PAMPs representative of gram-negative bacteria (lipopolysaccharide, Pam3CSK4) or fungal pathogens (Zymosan). Their decreased ability to produce more than 1 cytokine (polyfunctionality) upon PAMP stimulation correlated with the risk of developing infection at 28 days or mortality at 90 days. The presence of acute-on-chronic liver failure in patients with sAH did not significantly modify the immune profile of monocytes and DCs. Moreover, CD14+ monocytes of patients with sAH displayed altered transcriptional and epigenomic profiles characterized by downregulation of key innate immune and metabolic pathways and upregulation of important immunomodulatory factors. CONCLUSIONS: In patients with sAH, the altered transcriptional program and functional properties of monocytes that contribute to patients' susceptibility to infection have strong epigenetic determinants. LAY SUMMARY: Patients with severe alcoholic hepatitis are at increased risk of infections, which contribute to the poor prognosis associated with the disease. Herein, we show that epigenetic determinants underly the immune cell dysfunction and inappropriate responses to pathogens that are associated with severe alcoholic hepatitis.

Trop2 marks transient gastric fetal epithelium and adult regenerating cells after epithelial damage.

Mouse fetal intestinal progenitors lining the epithelium prior to villogenesis grow as spheroids when cultured ex vivo and express the transmembrane glycoprotein Trop2 as a marker. Here, we report the characterization of Trop2-expressing cells from fetal pre-glandular stomach, growing as immortal undifferentiated spheroids, and their relationship with gastric development and regeneration. Trop2(+) cells generating gastric spheroids differed from adult glandular Lgr5(+) stem cells, but appeared highly related to fetal intestinal spheroids. Although they shared a common spheroid signature, intestinal and gastric fetal spheroid-generating cells expressed organ-specific transcription factors and were committed to intestinal and glandular gastric differentiation, respectively. Trop2 expression was transient during glandular stomach development, being lost at the onset of gland formation, whereas it persisted in the squamous forestomach. Undetectable under homeostasis, Trop2 was strongly re-expressed in glands after acute Lgr5(+) stem cell ablation or following indomethacin-induced injury. These highly proliferative reactive adult Trop2(+) cells exhibited a transcriptome displaying similarity with that of gastric embryonic Trop2(+) cells, suggesting that epithelium regeneration in adult stomach glands involves the partial re-expression of a fetal genetic program.

Kinetochore KMN network gene CASC5 mutated in primary microcephaly.

Several genes expressed at the centrosome or spindle pole have been reported to underlie autosomal recessive primary microcephaly (MCPH), a neurodevelopmental disorder consisting of an important brain size reduction present since birth, associated with mild-to-moderate mental handicap and no other neurological feature nor associated malformation. Here, we report a mutation of CASC5 (aka Blinkin, or KNL1, or hSPC105) in MCPH patients from three consanguineous families, in one of which we initially reported the MCPH4 locus. The combined logarithm of odds score of the three families was >6. All patients shared a very rare homozygous mutation of CASC5. The mutation induced skipping of exon 18 with subsequent frameshift and truncation of the predicted protein. CASC5 is part of the KMN network of the kinetochore and is required for proper microtubule attachment to the chromosome centromere and for spindle-assembly checkpoint (SAC) activation during mitosis. Like MCPH gene ASPM, CASC5 is upregulated in the ventricular zone (VZ) of the human fetal brain. CASC5 binds BUB1, BUBR1, ZWINT-1 and interestingly it binds to MIS12 through a protein domain which is truncated by the mutation. CASC5 localized at the equatorial plate like ZWINT-1 and BUBR1, while ASPM, CEP152 and PCTN localized at the spindle poles in our patients and in controls. Comparison of primate and rodent lineages indicates accelerated evolution of CASC5 in the human lineage. Our data provide strong evidence for CASC5 as a novel MCPH gene, and underscore the role of kinetochore integrity in proper volumetric development of the human brain.

Starvation-resistant cavefish reveal conserved mechanisms of starvation-induced hepatic lipotoxicity.

Starvation causes the accumulation of lipid droplets in the liver, a somewhat counterintuitive phenomenon that is nevertheless conserved from flies to humans. Much like fatty liver resulting from overfeeding, hepatic lipid accumulation (steatosis) during undernourishment can lead to lipotoxicity and atrophy of the liver. Here, we found that although surface populations of Astyanax mexicanus undergo this evolutionarily conserved response to starvation, the starvation-resistant cavefish larvae of the same species do not display an accumulation of lipid droplets upon starvation. Moreover, cavefish are resistant to liver atrophy during starvation, providing a unique system to explore strategies for liver protection. Using comparative transcriptomics between zebrafish, surface fish, and cavefish, we identified the fatty acid transporter slc27a2a/fatp2 to be correlated with the development of fatty liver. Pharmacological inhibition of slc27a2a in zebrafish rescues steatosis and atrophy of the liver upon starvation. Furthermore, down-regulation of FATP2 in Drosophila larvae inhibits the development of starvation-induced steatosis, suggesting the evolutionarily conserved importance of the gene in regulating fatty liver upon nutrition deprivation. Overall, our study identifies a conserved, druggable target to protect the liver from atrophy during starvation.

Starvation resistant cavefish reveal conserved mechanisms of starvation-induced hepatic lipotoxicity.

Starvation causes the accumulation of lipid droplets in the liver, a somewhat counterintuitive phenomenon that is nevertheless conserved from flies to humans. Much like fatty liver resulting from overfeeding, hepatic lipid accumulation (steatosis) during undernourishment can lead to lipotoxicity and atrophy of the liver. Here, we found that while surface populations of Astyanax mexicanus undergo this evolutionarily conserved response to starvation, the starvation-resistant cavefish larvae of the same species do not display an accumulation of lipid droplets upon starvation. Moreover, cavefish are resistant to liver atrophy during starvation, providing a unique system to explore strategies for liver protection. Using comparative transcriptomics between zebrafish, surface fish, and cavefish, we identified the fatty acid transporter slc27a2a/fatp2 to be correlated with the development of fatty liver. Pharmacological inhibition of slc27a2a in zebrafish rescues steatosis and atrophy of the liver upon starvation. Further, down-regulation of FATP2 in drosophila larvae inhibits the development of starvation-induced steatosis, suggesting the evolutionary conserved importance of the gene in regulating fatty liver upon nutrition deprivation. Overall, our study identifies a conserved, druggable target to protect the liver from atrophy during starvation.

Lgr4 is required for Paneth cell differentiation and maintenance of intestinal stem cells ex vivo.

Gene inactivation of the orphan G protein-coupled receptor LGR4, a paralogue of the epithelial-stem-cell marker LGR5, results in a 50% decrease in epithelial cell proliferation and an 80% reduction in terminal differentiation of Paneth cells in postnatal mouse intestinal crypts. When cultured ex vivo, LGR4-deficient crypts or progenitors, but not LGR5-deficient progenitors, die rapidly with marked downregulation of stem-cell markers and Wnt target genes, including Lgr5. Partial rescue of this phenotype is achieved by addition of LiCl to the culture medium, but not Wnt agonists. Our results identify LGR4 as a permissive factor in the Wnt pathway in the intestine and, as such, as a potential target for intestinal cancer therapy.

Role of Epac and protein kinase A in thyrotropin-induced gene expression in primary thyrocytes.

cAMP pathway activation by thyrotropin (TSH) induces differentiation and gene expression in thyrocytes. We investigated which partners of the cAMP cascade regulate gene expression modulations: protein kinase A and/or the exchange proteins directly activated by cAMP (Epac). Human primary cultured thyrocytes were analysed by microarrays after treatment with the adenylate cyclase activator forskolin, the protein kinase A (PKA) activator 6-MB-cAMP and the Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP (007) alone or combined with 6-MB-cAMP. Profiles were compared to those of TSH. Cultures treated with the adenylate cyclase- or the PKA activator alone or the latter combined with 007 had profiles similar to those induced by TSH. mRNA profiles of 007-treated cultures were highly distinct from TSH-treated cells, suggesting that TSH-modulated gene expressions are mainly modulated by cAMP and PKA and not through Epac in cultured human thyroid cells. To investigate whether the Epac-Rap-RapGAP pathway could play a potential role in thyroid tumorigenesis, the mRNA expressions of its constituent proteins were investigated in two malignant thyroid tumor types. Modulations of this pathway suggest an increased Rap pathway activity in these cancers independent from cAMP activation.

Homeostatic PD-1 signaling restrains EOMES-dependent oligoclonal expansion of liver-resident CD8 T cells.

The co-inhibitory programmed death (PD)-1 signaling pathway plays a major role in the context of tumor-specific T cell responses. Conversely, it also contributes to the maintenance of peripheral tolerance, as patients receiving anti-PD-1 treatment are prone to developing immune-related adverse events. Yet, the physiological role of the PD-1/PDL-1 axis in T cell homeostasis is still poorly understood. Herein, we show that under steady-state conditions, the absence of PD-1 signaling led to a preferential expansion of CD8+ T cells in the liver. These cells exhibit an oligoclonal T cell receptor (TCR) repertoire and a terminally differentiated exhaustion profile. The transcription factor EOMES is required for the clonal expansion and acquisition of this differentiation program. Finally, single-cell transcriptomics coupled with TCR repertoire analysis support the notion that these cells arise locally from liver-resident memory CD8+ T cells. Overall, we show a role for PD-1 signaling in liver memory T cell homeostasis.

CDK4 phosphorylation status and rational use for combining CDK4/6 and BRAF/MEK inhibition in advanced thyroid carcinomas.

Background: CDK4/6 inhibitors (CDK4/6i) have been established as standard treatment against advanced Estrogen Receptor-positive breast cancers. These drugs are being tested against several cancers, including in combinations with other therapies. We identified the T172-phosphorylation of CDK4 as the step determining its activity, retinoblastoma protein (RB) inactivation, cell cycle commitment and sensitivity to CDK4/6i. Poorly differentiated (PDTC) and anaplastic (ATC) thyroid carcinomas, the latter considered one of the most lethal human malignancies, represent major clinical challenges. Several molecular evidence suggest that CDK4/6i could be considered for treating these advanced thyroid cancers. Methods: We analyzed by two-dimensional gel electrophoresis the CDK4 modification profile and the presence of T172-phosphorylated CDK4 in a collection of 98 fresh-frozen tissues and in 21 cell lines. A sub-cohort of samples was characterized by RNA sequencing and immunohistochemistry. Sensitivity to CDK4/6i (palbociclib and abemaciclib) was assessed by BrdU incorporation/viability assays. Treatment of cell lines with CDK4/6i and combination with BRAF/MEK inhibitors (dabrafenib/trametinib) was comprehensively evaluated by western blot, characterization of immunoprecipitated CDK4 and CDK2 complexes and clonogenic assays. Results: CDK4 phosphorylation was detected in all well-differentiated thyroid carcinomas (n=29), 19/20 PDTC, 16/23 ATC and 18/21 thyroid cancer cell lines, including 11 ATC-derived ones. Tumors and cell lines without phosphorylated CDK4 presented very high p16CDKN2A levels, which were associated with proliferative activity. Absence of CDK4 phosphorylation in cell lines was associated with CDK4/6i insensitivity. RB1 defects (the primary cause of intrinsic CDK4/6i resistance) were not found in 5/7 tumors without detectable phosphorylated CDK4. A previously developed 11-gene expression signature identified the likely unresponsive tumors, lacking CDK4 phosphorylation. In cell lines, palbociclib synergized with dabrafenib/trametinib by completely and permanently arresting proliferation. These combinations prevented resistance mechanisms induced by palbociclib, most notably Cyclin E1-CDK2 activation and a paradoxical stabilization of phosphorylated CDK4 complexes. Conclusion: Our study supports further clinical evaluation of CDK4/6i and their combination with anti-BRAF/MEK therapies as a novel effective treatment against advanced thyroid tumors. Moreover, the complementary use of our 11 genes predictor with p16/KI67 evaluation could represent a prompt tool for recognizing the intrinsically CDK4/6i insensitive patients, who are potentially better candidates to immediate chemotherapy.

Cold atmospheric plasma differentially affects cell renewal and differentiation of stem cells and APC-deficient-derived tumor cells in intestinal organoids.

Cold atmospheric plasma (CAP) treatment has been proposed as a potentially innovative therapeutic tool in the biomedical field, notably for cancer due to its proposed toxic selectivity on cancer cells versus healthy cells. In the present study, we addressed the relevance of three-dimensional organoid technology to investigate the biological effects of CAP on normal epithelial stem cells and tumor cells isolated from mouse small intestine. CAP treatment exerted dose-dependent cytotoxicity on normal organoids and induced major transcriptomic changes associated with the global response to oxidative stress, fetal-like regeneration reprogramming, and apoptosis-mediated cell death. Moreover, we explored the potential selectivity of CAP on tumor-like Apc-deficient versus normal organoids in the same genetic background. Unexpectedly, tumor organoids exhibited higher resistance to CAP treatment, correlating with higher antioxidant activity at baseline as compared to normal organoids. This pilot study suggests that the ex vivo culture system could be a relevant alternative model to further investigate translational medical applications of CAP technology.

Identification of distinct functional thymic programming of fetal and pediatric human γδ thymocytes via single-cell analysis.

Developmental thymic waves of innate-like and adaptive-like γδ T cells have been described, but the current understanding of γδ T cell development is mainly limited to mouse models. Here, we combine single cell (sc) RNA gene expression and sc γδ T cell receptor (TCR) sequencing on fetal and pediatric γδ thymocytes in order to understand the ontogeny of human γδ T cells. Mature fetal γδ thymocytes (both the Vγ9Vδ2 and nonVγ9Vδ2 subsets) are committed to either a type 1, a type 3 or a type 2-like effector fate displaying a wave-like pattern depending on gestation age, and are enriched for public CDR3 features upon maturation. Strikingly, these effector modules express different CDR3 sequences and follow distinct developmental trajectories. In contrast, the pediatric thymus generates only a small effector subset that is highly biased towards Vγ9Vδ2 TCR usage and shows a mixed type 1/type 3 effector profile. Thus, our combined dataset of gene expression and detailed TCR information at the single-cell level identifies distinct functional thymic programming of γδ T cell immunity in human.

Functional reprogramming of monocytes in patients with acute and convalescent severe COVID-19.

Severe COVID-19 disease is associated with dysregulation of the myeloid compartment during acute infection. Survivors frequently experience long-lasting sequelae, but little is known about the eventual persistence of this immune alteration. Herein, we evaluated TLR-induced cytokine responses in a cohort of mild to critical patients during acute or convalescent phases (n = 97). In the acute phase, we observed impaired cytokine production by monocytes in the patients with the most severe COVID-19. This capacity was globally restored in convalescent patients. However, we observed increased responsiveness to TLR1/2 ligation in patients who recovered from severe disease, indicating that these cells display distinct functional properties at the different stages of the disease. In patients with acute severe COVID-19, we identified a specific transcriptomic and epigenomic state in monocytes that can account for their functional refractoriness. The molecular profile of monocytes from recovering patients was distinct and characterized by increased chromatin accessibility at activating protein 1 (AP1) and MAF loci. These results demonstrate that severe COVID-19 infection has a profound impact on the differentiation status and function of circulating monocytes, during both the acute and the convalescent phases, in a completely distinct manner. This could have important implications for our understanding of short- and long-term COVID-19-related morbidity.

Preclinical evaluation of CDK4 phosphorylation predicts high sensitivity of pleural mesotheliomas to CDK4/6 inhibition.

Malignant pleural mesothelioma (MPM) is an aggressive cancer with limited therapeutic options. We evaluated the impact of CDK4/6 inhibition by palbociclib in 28 MPM cell lines including 19 patient-derived ones, using various approaches including RNA-sequencing. Palbociclib strongly and durably inhibited the proliferation of 23 cell lines, indicating a unique sensitivity of MPM to CDK4/6 inhibition. When observed, insensitivity to palbociclib was mostly explained by the lack of active T172-phosphorylated CDK4. This was associated with high p16INK4A (CDKN2A) levels that accompany RB1 defects or inactivation, or (unexpectedly) CCNE1 overexpression in the presence of wild-type RB1. Prolonged palbociclib treatment irreversibly inhibited proliferation despite re-induction of cell cycle genes upon drug washout. A senescence-associated secretory phenotype including various potentially immunogenic components was irreversibly induced. Phosphorylated CDK4 was detected in 80% of 47 MPMs indicating their sensitivity to CDK4/6 inhibitors. Its absence in some highly proliferative MPMs was linked to very high p16 (CDKN2A) expression, which was also observed in public datasets in tumours from short-survival patients. Our study supports the evaluation of CDK4/6 inhibitors for MPM treatment, in monotherapy or combination therapy.

Vitamin D deficiency-induced hypertension is associated with vascular oxidative stress and altered heart gene expression.

Vitamin D deficiency (VDD) is associated with an increased cardiovascular risk. We investigated the effect of VDD on the cardiovascular system of growing male rats fed with a vitamin D-deficient diet. Using isolated rat aorta, we assessed both superoxide anion and endothelial-dependent relaxations. Microarray technology was used to identify changes induced by VDD in cardiac gene expression. Compared with control, VDD increased systolic blood pressure (P < 0.05) and superoxide anion production in the aortic wall (P < 0.05) and tended to increase serum levels of angiotensin II and atrial natriuretic peptide (P < 0.15). However, VDD slightly improved maximal relaxation to acetylcholine from 75 % ± 3% to 83% ± 2% (P < 0.05). Incubation of aortic rings either with nitro-l-arginine methyl ester (l-NAME) or catalase did not eliminate the enhancement of endothelial-mediated relaxation observed in vitamin D-deficient rats. Only incubation with indometacin or calcium-activated potassium channels blockers suppressed this difference. Compared with control, the expression of 51 genes showed different expression, including several genes involved in the regulation of oxidative stress and myocardial hypertrophy. In conclusion, VDD in early life increases arterial blood pressure, promotes vascular oxidative stress, and induces changes in cardiac gene expression. However, the endothelial-mediated regulation of vasomotor tone is maintained throughout the enhancement of an NO-independent compensatory pathway.