Eye Lens Organoids Made Simple: Characterization of a New Three-Dimensional Organoid Model for Lens Development and Pathology.

Cataract, the opacification of the lens, is the leading cause of blindness worldwide. Although effective, cataract surgery is costly and can lead to complications. Toward identifying alternate treatments, it is imperative to develop organoid models relevant for lens studies and drug screening. Here, we demonstrate that by culturing mouse lens epithelial cells under defined three-dimensional (3D) culture conditions, it is possible to generate organoids that display optical properties and recapitulate many aspects of lens organization and biology. These organoids can be rapidly produced in large amounts. High-throughput RNA sequencing (RNA-seq) on specific organoid regions isolated via laser capture microdissection (LCM) and immunofluorescence assays demonstrate that these lens organoids display a spatiotemporal expression of key lens genes, e.g., Jag1, Pax6, Prox1, Hsf4 and Cryab. Further, these lens organoids are amenable to the induction of opacities. Finally, the knockdown of a cataract-linked RNA-binding protein encoding gene, Celf1, induces opacities in these organoids, indicating their use in rapidly screening for genes that are functionally relevant to lens biology and cataract. In sum, this lens organoid model represents a compelling new tool to advance the understanding of lens biology and pathology and can find future use in the rapid screening of compounds aimed at preventing and/or treating cataracts.

Eye lens organoids going simple: characterization of a new 3-dimensional organoid model for lens development and pathology.

The ocular lens, along with the cornea, focuses light on the retina to generate sharp images. Opacification of the lens, or cataract, is the leading cause of blindness worldwide. Presently, the best approach for cataract treatment is to surgically remove the diseased lens and replace it with an artificial implant. Although effective, this is costly and can have post-surgical complications. Toward identifying alternate treatments, it is imperative to develop organoid models relevant for lens studies and anti-cataract drug screening. Here, we demonstrate that by culturing mouse lens epithelial cells under defined 3-dimensional (3D) culture conditions, it is possible to generate organoids that display optical properties and recapitulate many aspects of lens organization at the tissue, cellular and transcriptomic levels. These 3D cultured lens organoids can be rapidly produced in large amounts. High-throughput RNA-sequencing (RNA-seq) on specific organoid regions isolated by laser capture microdissection (LCM) and immunofluorescence assays demonstrate that these lens organoids display spatiotemporal expression of key lens genes, e.g. , Jag1 , Pax6 , Prox1 , Hsf4 and Cryab . Further, these lens organoids are amenable to induction of opacities. Finally, knockdown of a cataract-linked RNA-binding protein encoding gene, Celf1 , induces opacities in these organoids, indicating their use in rapidly screening for genes functionally relevant to lens biology and cataract. In sum, this lens organoid model represents a compelling new tool to advance the understanding of lens biology and pathology, and can find future use in the rapid screening of compounds aimed at preventing and/or treating cataract.

Mitochondrial remodeling and energy metabolism adaptations in colonic crypts during spontaneous epithelial repair after colitis induction in mice.

Mucosal healing has emerged as a therapeutic goal to achieve lasting clinical remission in ulcerative colitis. Intestinal repair in response to inflammation presumably requires higher energy supplies for the restoration of intestinal barrier and physiological functions. However, epithelial energy metabolism during intestinal mucosal healing has been little studied, whereas inflammation-induced alterations have been reported in the main energy production site, the mitochondria. The aim of the present work was to assess the involvement of mitochondrial activity and the events influencing their function during spontaneous epithelial repair after colitis induction in mouse colonic crypts. The results obtained show adaptations of colonocyte metabolism during colitis to ensure maximal ATP production for supporting energetic demand by both oxidative phosphorylation and glycolysis in a context of decreased mitochondrial biogenesis and through mitochondrial function restoration during colon epithelial repair. In parallel, colitis-induced mitochondrial ROS production in colonic epithelial cells was rapidly associated with transient expression of GSH-related enzymes. Mitochondrial respiration in colonic crypts was markedly increased during both inflammatory and recovery phases despite decreased expression of several mitochondrial respiratory chain complex subunits after colitis induction. Rapid induction of mitochondrial fusion was associated with mitochondrial function restoration. Finally, in contrast with the kinetics expression of genes involved in mitochondrial oxidative metabolism and in glycolysis, the expression of glutaminase was markedly reduced in the colonic crypts both during colitis and repair phases. Overall, our data suggest that the epithelial repair after colitis induction is characterized by a rapid and transient increased capacity for mitochondrial ATP production in a context of apparent restoration of mitochondrial biogenesis and metabolic reorientation of energy production. The potential implication of energy production adaptations within colonic crypts to sustain mucosal healing in a context of altered fuel supply is discussed.

Hepatocellular carcinomas, exhibiting intratumor fibrosis, express cancer-specific extracellular matrix remodeling and WNT/TGFB signatures, associated with poor outcome.

BACKGROUND AND AIMS: HCC, the third leading cause of cancer-related death, arises in the context of liver fibrosis. Although HCC is generally poorly fibrogenic, some tumors harbor focal intratumor extracellular matrix (ECM) deposits called "fibrous nests." To date, the molecular composition and clinical relevance of these ECM deposits have not been fully defined. APPROACH AND RESULTS: We performed quantitative matrisome analysis by tandem mass tags mass spectrometry in 20 human cancer specific matrisome (HCCs) with high or low-grade intratumor fibrosis and matched nontumor tissues, as well as in 12 livers from mice treated with vehicle, carbon tetrachloride, or diethylnitrosamine. We found 94 ECM proteins differentially abundant between high and low-grade fibrous nests, including interstitial and basement membrane components, such as several collagens, glycoproteins, proteoglycans, enzymes involved in ECM stabilization and degradation, and growth factors. Pathway analysis revealed a metabolic switch in high-grade fibrosis, with enhanced glycolysis and decreased oxidative phosphorylation. Integrating the quantitative proteomics with transcriptomics from HCCs and nontumor livers (n = 2,285 samples), we identified a subgroup of fibrous nest HCCs, characterized by cancer-specific ECM remodeling, expression of the WNT/TGFB (S1) subclass signature, and poor patient outcome. Fibrous nest HCCs abundantly expressed an 11-fibrous-nest - protein signature, associated with poor patient outcome, by multivariate Cox analysis, and validated by multiplex immunohistochemistry. CONCLUSIONS: Matrisome analysis highlighted cancer-specific ECM deposits, typical of the WNT/TGFB HCC subclass, associated with poor patient outcomes. Hence, histologic reporting of intratumor fibrosis in HCC is of clinical relevance.

The mechanosensitive TRPV2 calcium channel promotes human melanoma invasiveness and metastatic potential.

Melanoma is a highly aggressive cancer endowed with a unique capacity of rapidly metastasizing, which is fundamentally driven by aberrant cell motility behaviors. Discovering "migrastatics" targets, specifically controlling invasion and dissemination of melanoma cells during metastasis, is therefore of primary importance. Here, we uncover the prominent expression of the plasma membrane TRPV2 calcium channel as a distinctive feature of melanoma tumors, directly related to melanoma metastatic dissemination. In vitro as well as in vivo, TRPV2 activity is sufficient to confer both migratory and invasive potentials, while conversely TRPV2 silencing in highly metastatic melanoma cells prevents aggressive behavior. In invasive melanoma cells, TRPV2 channel localizes at the leading edge, in dynamic nascent adhesions, and regulates calcium-mediated activation of calpain and the ensuing cleavage of the adhesive protein talin, along with F-actin organization. In human melanoma tissues, TRPV2 overexpression correlates with advanced malignancy and poor prognosis, evoking a biomarker potential. Hence, by regulating adhesion and motility, the mechanosensitive TRPV2 channel controls melanoma cell invasiveness, highlighting a new therapeutic option for migrastatics in the treatment of metastatic melanoma.

PIM2 kinase has a pivotal role in plasmablast generation and plasma cell survival, opening up novel treatment options in myeloma.

The differentiation of B cells into plasmablasts (PBs) and then plasma cells (PCs) is associated with extensive cell reprogramming and new cell functions. By using specific inhibition strategies (including a novel morpholino RNA antisense approach), we found that early, sustained upregulation of the proviral integrations of Moloney virus 2 (PIM2) kinase is a pivotal event during human B-cell in vitro differentiation and then continues in mature normal and malignant PCs in the bone marrow. In particular, PIM2 sustained the G1/S transition by acting on CDC25A and p27Kip1 and hindering caspase 3-driven apoptosis through BAD phosphorylation and cytoplasmic stabilization of p21Cip1. In PCs, interleukin-6 triggered PIM2 expression, resulting in antiapoptotic effects on which malignant PCs were particularly dependent. In multiple myeloma, pan-PIM and myeloid cell leukemia-1 (MCL1) inhibitors displayed synergistic activity. Our results highlight a cell-autonomous function that links kinase activity to the newly acquired secretion ability of the PBs and the adaptability observed in both normal and malignant PCs. These findings should finally prompt the reconsideration of PIM2 as a therapeutic target in multiple myeloma.

An EMT-primary cilium-GLIS2 signaling axis regulates mammogenesis and claudin-low breast tumorigenesis.

The epithelial-mesenchymal transition (EMT) and primary ciliogenesis induce stem cell properties in basal mammary stem cells (MaSCs) to promote mammogenesis, but the underlying mechanisms remain incompletely understood. Here, we show that EMT transcription factors promote ciliogenesis upon entry into intermediate EMT states by activating ciliogenesis inducers, including FGFR1. The resulting primary cilia promote ubiquitination and inactivation of a transcriptional repressor, GLIS2, which localizes to the ciliary base. We show that GLIS2 inactivation promotes MaSC stemness, and GLIS2 is required for normal mammary gland development. Moreover, GLIS2 inactivation is required to induce the proliferative and tumorigenic capacities of the mammary tumor­initiating cells (MaTICs) of claudin-low breast cancers. Claudin-low breast tumors can be segregated from other breast tumor subtypes based on a GLIS2-dependent gene expression signature. Collectively, our findings establish molecular mechanisms by which EMT programs induce ciliogenesis to control MaSC and MaTIC stemness, mammary gland development, and claudin-low breast cancer formation.

Exposure of human fetal kidneys to mild analgesics interferes with early nephrogenesis.

Acetaminophen, aspirin, and ibuprofen are mild analgesics commonly used by pregnant women, the sole current recommendation being to avoid ibuprofen from the fifth month of gestation. The nephrotoxicity of these three analgesics is well documented in adults, as is their interference with prostaglandins biosynthesis. Here we investigated the effect of these analgesics on human first trimester kidneys ex vivo. We first evaluated prostaglandins biosynthesis functionality by performing a wide screening of prostaglandin expression patterns in first trimester human kidneys. We demonstrated that prostaglandins biosynthesis machinery is functional during early nephrogenesis. Human fetal kidney explants aged 7-12 developmental weeks were exposed ex vivo to ibuprofen, aspirin or acetaminophen for 7 days, and analyzed by histology, immunohistochemistry, and flow cytometry. This study has revealed that these analgesics induced a spectrum of abnormalities within early developing structures, ranging from cell death to a decline in differentiating glomeruli density. These results warrant caution for the use of these medicines during the first trimester of pregnancy.

Sciadonic acid derived from pine nuts as a food component to reduce plasma triglycerides by inhibiting the rat hepatic Δ9-desaturase.

Sciadonic acid (Scia) is a Δ5-olefinic fatty acid that is particularly abundant in edible pine seeds and that exhibits an unusual polymethylene-interrupted structure. Earlier studies suggested that Scia inhibited the in vitro expression and activity of the Stearoyl-CoA Desaturase 1 (SCD1), the hepatic Δ9-desaturase involved in the formation of mono-unsaturated fatty acids. To confirm this hypothesis, rats were given 10% Scia in diets balanced out with n-6 and n-3 fatty acids. In those animals receiving the Scia supplement, monoene synthesis in the liver was reduced, which was partly attributed to the inhibition of SCD1 expression. As a consequence, the presence of Scia induced a 50% decrease in triglycerides in blood plasma due to a reduced level of VLDL-secreted triglycerides from the liver. In non-fasting conditions, results showed that Scia-induced inhibition of SCD1 led to a decrease in the proportions of 16:1n-7 and 18:1n-7 in the liver without impacting on the level of 18:1n-9, suggesting that only triglycerides with neosynthesized monoenes are marked out for release. In conclusion, this in vivo study confirms that Scia highly inhibits SCD1 expression and activity. The work was performed on normo-triglyceride rats over six weeks, suggesting promising effects on hyper-triglyceridemic models.

TNF-α and IL-10 Control CXCL13 Expression in Human Macrophages.

The chemokine CXCL13 controls the normal organization of secondary lymphoid tissues and the neogenesis of ectopic lymphoid structures in nonlymphoid organs, particularly the lungs. The progression and severity of idiopathic pulmonary fibrosis (IPF), a fatal and irreversible interstitial lung disease, is predicted by the circulating blood concentrations of CXCL13. Although CXCL13 is produced by pulmonary tissues, it has not been determined which cells are involved. This study examines CXCL13 production by lung tissue macrophages from patients with IPF and the signaling pathways controlling CXCL13 gene expression in human alveolar macrophages (AM) and monocyte-derived macrophages (MoDM). CXCL13 is found in CD68- and CD206-positive AM from patients with IPF, and the CXCL13 gene is induced in these macrophages and MoDM when they are stimulated with LPS. We found that TNF-α and IL-10 control optimal CXCL13 gene expression in MoDM and possibly in AM by activating the NF-κB and JAK/STAT pathways, respectively. We also found that blood TNF-α and CXCL13 concentrations are significantly correlated in patients with IPF, suggesting that TNF-α contributes to CXCL13 production in humans. In conclusion, the results of this study demonstrate that AM from patients with IPF produces CXCL13 and that the NF-κB and JAK/STAT pathways are required to induce the expression of this major chemokine.

Dietary Protein Intake Level Modulates Mucosal Healing and Mucosa-Adherent Microbiota in Mouse Model of Colitis.

Mucosal healing after an inflammatory flare is associated with lasting clinical remission. The aim of the present work was to evaluate the impact of the amount of dietary protein on epithelial repair after an acute inflammatory episode. C57BL/6 DSS-treated mice received isocaloric diets with different levels of dietary protein: 14% (P14), 30% (P30) and 53% (P53) for 3 (day 10), 6 (day 13) and 21 (day 28) days after the time of colitis maximal intensity. While the P53 diet worsened the DSS- induced inflammation both in intensity and duration, the P30 diet, when compared to the P14 diet, showed a beneficial effect during the epithelial repair process by accelerating inflammation resolution, reducing colonic permeability and increasing epithelial repair together with epithelial hyperproliferation. Dietary protein intake also impacted mucosa-adherent microbiota composition after inflammation since P30 fed mice showed increased colonization of butyrate-producing genera throughout the resolution phase. This study revealed that in our colitis model, the amount of protein in the diet modulated mucosal healing, with beneficial effects of a moderately high-protein diet, while very high-protein diet displayed deleterious effects on this process.

Implication of nigral dopaminergic lesion and repeated L-dopa exposure in neuropsychiatric symptoms of Parkinson's disease.

This study aims to investigate the contribution of nigral dopaminergic (DA) cell loss, repeated exposure to DA medication and the combination of both to the development of neuropsychiatric symptoms observed in Parkinson's disease (PD). A bilateral 6-OHDA lesion of the substantia nigra pars compacta (SNc) was performed in rats. A set of animals was repeatedly administered with L-dopa (20 mg/kg/day) and benserazide (5 mg/kg/day) over 10 days starting from day 11 post-lesion. Behavioural testing was performed in week 3 post-lesion: novel object recognition (NOR), elevated plus maze (EPM) social interaction (SI) tests, and amphetamine-induced hyperlocomotion (AIH). Immunohistochemical analysis revealed a significant partial lesion (48%) in 6-OHDA versus sham rats. This lesion was not associated with motor impairment. However, lesioned rats displayed a significant deficit in the NOR, which was reversed by acute treatment with l-dopa/benserazide (12.5 mg/kg and 15 mg/kg respectively). Lesioned rats also displayed a deficit in the EPM which was not reversed by acute treatment with l-dopa. No difference was observed in the SI test or in the AIH assay. In all assays, no effect of chronic l-dopa exposure was observed. This study provides new insights into the neuropathophysiology associated with neuropsychiatric symptoms of PD. Our data strongly emphasises a not previously clearly identified critical role in cognition for the SNc. The results suggest that DA pathways were less directly involved in lesion-induced anxiety-like behaviour. We did not report any effect of chronic l-dopa exposure in the context of partial nigral cell loss.

Thymoquinone protects rat liver after partial hepatectomy under ischaemia/reperfusion through oxidative stress and endoplasmic reticulum stress prevention.

Ischaemia reperfusion (I/R) is associated with liver injury and impaired regeneration during partial hepatectomy (PH). The aim of this study was to investigate the effect of thymoquinone (TQ), the active compound of essential oil obtained from Nigella sativa seeds, on rat liver after PH. Male Wistar rats were divided equally into four groups (n = 6) receiving an oral administration of either vehicle solution (sham and PH groups) or TQ at 30 mg/kg (TQ and TQ + PH groups) for 10 consecutive days. Then, rats underwent PH (70%) with 60 minutes of ischaemia followed by 24 hours of reperfusion (PH and TQ + PH groups). Alanine aminotransferase (ALT) activity and histopathological damage were determined. Also, antioxidant parameters, liver regeneration index, hepatic adenosine triphosphate (ATP) content, endoplasmic reticulum (ER) stress and apoptosis were assessed. In response to PH under I/R, liver damage was significantly alleviated by TQ treatment as evidenced by the decrease in ALT activity (P < .01) and histological findings (P < .001). In parallel, TQ preconditioning increased hepatic antioxidant capacities. Moreover, TQ improved mitochondrial function (ATP, P < .05), attenuated ER stress parameters and repressed the expression of apoptotic effectors. Taken together, our results suggest that TQ preconditioning could be an effective strategy to reduce liver injury after PH under I/R. The protective effects were mediated by the increase of antioxidant capacities and the decrease of ER stress and apoptosis.

Thymoquinone prevents endoplasmic reticulum stress and mitochondria-induced apoptosis in a rat model of partial hepatic warm ischemia reperfusion.

This study was undertaken to evaluate the protective effect of thymoquinone (TQ), the bioactive compound of Nigella sativa seeds, against warm ischemia-reperfusion (I/R) injury in liver. Rats were given an oral administration of a vehicle solution (sham group) or TQ at the appropriate dose (10, 20, 30 and 40mg/kg) for ten days consecutively. Following, they were subjected to 60min of partial hepatic ischemia followed by 24h of reperfusion. .Transaminase activities, histopathological changes, TNFα and antioxidant parameters were evaluated. Also, endoplasmic reticulum (ER) stress, mitochondrial damage and apoptosis were studied. In addition, ERK and P38 phosphorylation was determined by Western blot technique. We found that TQ at 30mg/kg is the effective dose to protect rat liver against I/R injury. Moreover, 30mg/kg of TQ prevented histological damages, inflammation and oxidative stress. Interestingly, it decreased the expression of ER stress parameters including GRP78, CHOP and caspase-12. In parallel, it improved mitochondrial function and attenuated the expression of apoptotic parameters. Furthermore, TQ significantly enhanced ERK and P38 phosphorylation. In conclusion, we demonstrated the potential of TQ to protect the rat liver against I/R injury through the prevention of ER stress and mitochondrial dysfunction. These effects implicate the prevention of oxidative stress.

CD95-Mediated Calcium Signaling Promotes T Helper 17 Trafficking to Inflamed Organs in Lupus-Prone Mice.

CD95 ligand (CD95L) is expressed by immune cells and triggers apoptotic death. Metalloprotease-cleaved CD95L (cl-CD95L) is released into the bloodstream but does not trigger apoptotic signaling. Hence, the pathophysiological role of cl-CD95L remains unclear. We observed that skin-derived endothelial cells from systemic lupus erythematosus (SLE) patients expressed CD95L and that after cleavage, cl-CD95L promoted T helper 17 (Th17) lymphocyte transmigration across the endothelial barrier at the expense of T regulatory cells. T cell migration relied on a direct interaction between the CD95 domain called calcium-inducing domain (CID) and the Src homology 3 domain of phospholipase Cγ1. Th17 cells stimulated with cl-CD95L produced sphingosine-1-phosphate (S1P), which promoted endothelial transmigration by activating the S1P receptor 3. We generated a cell-penetrating CID peptide that prevented Th17 cell transmigration and alleviated clinical symptoms in lupus mice. Therefore, neutralizing the CD95 non-apoptotic signaling pathway could be an attractive therapeutic approach for SLE treatment.