Prolonged exposure to simulated microgravity promotes stemness impairing morphological, metabolic and migratory profile of pancreatic cancer cells: a comprehensive proteomic, lipidomic and transcriptomic analysis.

BACKGROUND: The impact of the absence of gravity on cancer cells is of great interest, especially today that space is more accessible than ever. Despite advances, few and contradictory data are available mainly due to different setup, experimental design and time point analyzed. METHODS: Exploiting a Random Positioning Machine, we dissected the effects of long-term exposure to simulated microgravity (SMG) on pancreatic cancer cells performing proteomic, lipidomic and transcriptomic analysis at 1, 7 and 9 days. RESULTS: Our results indicated that SMG affects cellular morphology through a time-dependent activation of Actin-based motility via Rho and Cdc42 pathways leading to actin rearrangement, formation of 3D spheroids and enhancement of epithelial-to-mesenchymal transition. Bioinformatic analysis reveals that SMG may activates ERK5/NF-κB/IL-8 axis that triggers the expansion of cancer stem cells with an increased migratory capability. These cells, to remediate energy stress and apoptosis activation, undergo a metabolic reprogramming orchestrated by HIF-1α and PI3K/Akt pathways that upregulate glycolysis and impair ß-oxidation, suggesting a de novo synthesis of triglycerides for the membrane lipid bilayer formation. CONCLUSIONS: SMG revolutionizes tumor cell behavior and metabolism leading to the acquisition of an aggressive and metastatic stem cell-like phenotype. These results dissect the time-dependent cellular alterations induced by SMG and pave the base for altered gravity conditions as new anti-cancer technology.

Inhibitors of ADAM10 reduce Hodgkin lymphoma cell growth in 3D microenvironments and enhance brentuximab-vedotin effect.

Shedding of ADAM10 substrates, like TNFa or CD30, can affect both anti-tumor immune response and antibody-drug-conjugate (ADC)-based immunotherapy. We have published two new ADAM10 inhibitors, LT4 and MN8 able to prevent such shedding in Hodgkin lymphoma (HL). Since tumor tissue architecture deeply influences the outcome of anti-cancer treatments, we set up a new threedimensional (3D) culture systems to verify whether ADAM10 inhibitors can contribute to, or enhance, the anti-lymphoma effects of the ADC brentuximab-vedotin (BtxVed). In order to recapitulate some aspects of lymphoma structure and architecture, we assembled two 3D culture models: mixed spheroids made of HL lymph node (LN) mesenchymal stromal cells (MSC) and Reed Sternberg/Hodgkin lymphoma cells (HL cells) or collagen scaffolds repopulated with LN-MSC and HL cells. In these 3D systems we found that: i) the ADAM10 inhibitors LT4 and MN8 reduce ATP content or glucose consumption, related to cell proliferation, increasing lactate dehydrogenase release as a cell damage hallmark; ii) these events are paralleled by mixed spheroids size reduction and inhibition of CD30 and TNFa shedding; iii) the effects observed can be reproduced in repopulated HL LN-derived matrix or collagen scaffolds; iv) ADAM10 inhibitors enhance the anti-lymphoma effect of the anti-CD30 ADC BtxVed both in conventional cultures and in repopulated scaffolds. Thus, we provide evidence for a direct and combined antilymphoma effect of ADAM10 inhibitors with BtxVed, leading to the improvement of ADC effects; this is documented in 3D models recapitulating features of the LN microenvironment, that can be proposed as a reliable tool for anti-lymphoma drug testing.

Characterization of C2C12 cells in simulated microgravity: Possible use for myoblast regeneration.

Muscle loss is a major problem for many in lifetime. Muscle and bone degeneration has also been observed in individuals exposed to microgravity and in unloading conditions. C2C12 myoblst cells are able to form myotubes, and myofibers and these cells have been employed for muscle regeneration purposes and in myogenic regeneration and transplantation studies. We exposed C2C12 cells in an random position machine to simulate microgravity and study the energy and the biochemical challenges associated with this treatment. Simulated microgravity exposed C2C12 cells maintain positive proliferation indices and delay the differentiation process for several days. On the other hand this treatment significantly alters many of the biochemical and the metabolic characteristics of the cell cultures including calcium homeostasis. Recent data have shown that these perturbations are due to the inhibition of the ryanodine receptors on the membranes of intracellular calcium stores. We were able to reverse this perturbations treating cells with thapsigargin which prevents the segregation of intracellular calcium ions in the mitochondria and in the sarco/endoplasmic reticula. Calcium homeostasis appear a key target of microgravity exposure. In conclusion, in this study we reported some of the effects induced by the exposure of C2C12 cell cultures to simulated microgravity. The promising information obtained is of fundamental importance in the hope to employ this protocol in the field of regenerative medicine.

Anthropometric and glucometabolic changes in an aged mouse model of lipocalin-2 overexpression.

BACKGROUND: Lipocalin-2 (LCN2) is widely expressed in the organism with pleiotropic roles. In particular, its overexpression correlates with tissue stress conditions including inflammation, metabolic disorders, chronic diseases and cancer. OBJECTIVES: To assess the effects of systemic LCN2 overexpression on adipose tissue and glucose metabolism. SUBJECTS: Eighteen-month-old transgenic mice with systemic LCN2 overexpression (LCN2-Tg) and age/sex-matched wild-type mice. METHODS: Metabolic cages; histology and real-time PCR analysis; glucose and insulin tolerance tests; ELISA; flow cytometry; microPET and serum analysis. RESULTS: LCN2-Tg mice were smaller compared to controls but they ate (P = 0.0156) and drank (P = 0.0057) more and displayed a higher amount of visceral adipose tissue. Furthermore, LCN2-Tg mice with body weight ≥20 g showed adipocytes with a higher cell area (P < 0.0001) and altered expression of genes involved in adipocyte differentiation and inflammation. In particular, mRNA levels of adipocyte-derived Pparg (P ≤ 0.0001), Srebf1 (P < 0.0001), Fabp4 (P = 0.056), Tnfa (P = 0.0391), Il6 (P = 0.0198), and Lep (P = 0.0003) were all increased. Furthermore, LCN2-Tg mice displayed a decreased amount of basal serum insulin (P = 0.0122) and a statistically significant impaired glucose tolerance and insulin sensitivity consistent with Slc2a2 mRNA (P ≤ 0.0001) downregulated expression. On the other hand, Insr mRNA (P ≤ 0.0001) was upregulated and correlated with microPET analysis that demonstrated a trend in reduced whole-body glucose consumption and MRGlu in the muscles and a significantly reduced MRGlu in brown adipose tissue (P = 0.0247). Nevertheless, an almost nine-fold acceleration of hexokinase activity was observed in the LCN2-Tg mice liver compared to controls (P = 0.0027). Moreover, AST and ALT were increased (P = 0.0421 and P = 0.0403, respectively), which indicated liver involvement also demonstrated by histological staining. CONCLUSIONS: We show that LCN2 profoundly impacts adipose tissue size and function and glucose metabolism, suggesting that LCN2 should be considered as a risk factor in ageing for metabolic disorders leading to obesity.

LCN2 overexpression in bone enhances the hematopoietic compartment via modulation of the bone marrow microenvironment.

Lipocalin-2 (LCN2) is a member of the lipocalin family whose expression is modulated in several conditions, including cell differentiation, innate immunity, stress, and cancer. Although it is known that it is expressed in bone, its function in this tissue remains poorly studied. To this end, we took advantage of transgenic mice lines that expressed LCN2 driven by a bone specific type I collagen (LCN2-Tg). In the bone marrow (BM) of LCN2-Tg mice we observed an increased number of phenotypically long-term hematopoietic stem cells (LT-HSC) that also displayed a higher proliferation rate compared to wild-type controls (Wt). Furthermore, hematopoietic progenitor cells, obtained from LCN2-Tg BM showed an increased clonogenic capacity compared to those obtained from LCN2-Tg spleen, a higher concentration of serum erythropoietin and a higher number of mature erythrocytes in the peripheral blood of old LCN2-Tg animals compared to aged-matched wt. The findings of a combined increase in the BM of the LCN2-Tg mice of SDF-1, SCF, and TIMP-1 levels along with the reduction of both MMP-9 activity and cathepsin K concentration may explain the observed effects on the HSC compartment. This study shows that LCN2 overexpression in bones modifies the BM microenvironment via modulation of the expression of key secreted factors and cytokines, which in turn regulate the HSC niche behavior enhancing both HSC homing in young mice and erythrocytes production in older mice.

High ERp5/ADAM10 expression in lymph node microenvironment and impaired NKG2D ligands recognition in Hodgkin lymphomas.

Herein we describe that in classic Hodgkin lymphomas (cHL, n = 25) the lymph node (LN) stroma displayed in situ high levels of transcription and expression of the disulfide-isomerase ERp5 and of the disintegrin-metalloproteinase ADAM10, able to shed the ligands for NKG2D (NKG2D-L) from the cell membrane. These enzymes were detected both in LN mesenchymal stromal cells (MSCs) and in Reed-Sternberg (RS) cells; in addition, MIC-A and ULBP3 were present in culture supernatants of LN MSCs or RS cells. NKG2D-L-negative RS cells could not be killed by CD8(+)αßT or γδT cells; tumor cell killing was partially restored by treating RS cells with valproic acid, which enhanced NKG2D-L surface expression. Upon coculture with LN MSCs, CD8(+)αßT and γδT cells strongly reduced their cytolytic activity against NKG2D-L(+) targets; this seems to be the result of TGF-ß, present at the tumor site, produced in vitro by LN MSCs and able to down-regulate the expression of NKG2D on T lymphocytes. In addition, CD8(+)αßT and γδT cells from the lymph nodes of cHL patients, cocultured in vitro with LN MSCs, underwent TGF-ß-mediated down regulation of NKG2D. Thus, in cHL the tumor microenvironment is prone to inhibit the development of an efficient antitumor response.

Aminobisphosphonates prevent the inhibitory effects exerted by lymph node stromal cells on anti-tumor Vδ 2 T lymphocytes in non-Hodgkin lymphomas.

In this study, we analyzed the influence of mesenchymal stromal cells derived from lymph nodes of non-Hodgkin's lymphomas, on effector functions and differentiation of Vdelta (δ)2 T lymphocytes. We show that: i) lymph-node mesenchymal stromal cells of non-Hodgkin's lymphoma inhibit NKG2D-mediated lymphoid cell killing, but not rituximab-induced antibody-dependent cell-mediated cytotoxicity, exerted by Vδ2 T lymphocytes; ii) pre-treatment of mesenchymal stromal cells with the aminobisphosphonates pamidronate or zoledronate can rescue lymphoma cell killing via NKG2D; iii) this is due to inhibition of transforming growth factor-ß and increase in interleukin-15 production by mesenchymal stromal cells; iv) aminobisphosphonate-treated mesenchymal stromal cells drive Vδ2 T-lymphocyte differentiation into effector memory T cells, expressing the Thelper1 cytokines tumor necrosis factor-α and interferon-γ. In non-Hodgkin's lymphoma lymph nodes, Vδ2 T cells were mostly naïve; upon co-culture with autologous lymph-node mesenchymal stromal cells exposed to zoledronate, the percentage of terminal differentiated effector memory Vδ2 T lymphocytes increased. In all non-Hodgkin's lymphomas, low or undetectable transcription of Thelper1 cytokines was found. In diffused large B-cell lymphomas and in a group of follicular lymphoma, transcription of transforming growth factor ß and interleukin-10 was enhanced compared to non-neoplastic lymph nodes. Thus, in non-Hodgkin lymphomas mesenchymal stromal cells interfere with Vδ2 T-lymphocyte cytolytic function and differentiation to Thelper1 and/or effector memory cells, depending on the prominent in situ cytokine milieu. Aminobisphosphonates, acting on lymph-node mesenchymal stromal cells, can push the balance towards Thelper1/effector memory and rescue the recognition and killing of lymphoma cells through NKG2D, sparing rituximab-induced antibody-dependent cell-mediated cytotoxicity.

Altered bone development and turnover in transgenic mice over-expressing lipocalin-2 in bone.

Lipocalin-2 (LCN2) is a protein largely expressed in many tissues, associated with different biological phenomena such as cellular differentiation, inflammation and cancer acting as a survival/apoptotic signal. We found that LCN2 was expressed during osteoblast differentiation and we generated transgenic (Tg) mice over-expressing LCN2 in bone. Tg mice were smaller and presented bone microarchitectural changes in both endochondral and intramembranous bones. In particular, Tg bones displayed a thinner layer of cortical bone and a decreased trabecular number. Osteoblast bone matrix deposition was reduced and osteoblast differentiation was slowed-down. Differences were also observed in the growth plate of young transgenic mice where chondrocyte displayed a more immature phenotype and a lower proliferation rate. In bone marrow cell cultures from transgenic mice, the number of osteoclast progenitors was increased whereas in vivo it was increased the number of mature osteoclasts expressing tartrate-resistant acid phosphatase (TRAP). Finally, while osteoprotegerin (OPG) levels remained unchanged, the expression of the conventional receptor activator of nuclear factor-κB ligand (RANKL) and of the IL-6 was enhanced in Tg mice. In conclusion, we found that LCN2 plays a role in bone development and turnover having both a negative effect on bone formation, by affecting growth plate development and interfering with osteoblast differentiation, and a positive effect on bone resorption by enhancing osteoclast compartment.

Priming of Colorectal Tumor-Associated Fibroblasts with Zoledronic Acid Conjugated to the Anti-Epidermal Growth Factor Receptor Antibody Cetuximab Elicits Anti-Tumor Vδ2 T Lymphocytes.

Tumor-associated fibroblasts (TAF) exert immunosuppressive effects in colorectal carcinoma (CRC), impairing the recognition of tumor cells by effector lymphocytes, including Vδ2 T cells. Herein, we show that CRC-derived TAF can be turned by zoledronic acid (ZA), in soluble form or as antibody-drug conjugate (ADC), into efficient stimulators of Vδ2 T cells. CRC-TAF, obtained from patients, express the epidermal growth factor receptor (EGFR) and the butyrophilin family members BTN3A1/BTN2A1. These butyrophilins mediate the presentation of the phosphoantigens, accumulated in the cells due to ZA effect, to Vδ2 T cells. CRC-TAF exposed to soluble ZA acquired the ability to trigger the proliferation of Vδ2 T cells, in part represented by effector memory cells lacking CD45RA and CD27. In turn, expanded Vδ2 T cells exerted relevant cytotoxic activity towards CRC cells and CRC-TAF when primed with soluble ZA. Of note, also the ADC made of the anti-EGFR cetuximab (Cet) and ZA (Cet-ZA), that we recently described, induced the proliferation of anti-tumor Vδ2 T lymphocytes and their activation against CRC-TAF. These findings indicate that ZA can educate TAF to stimulate effector memory Vδ2 T cells; the Cet-ZA ADC formulation can lead to the precise delivery of ZA to EGFR+ cells, with a double targeting of TAF and tumor cells.

Lipid Metabolism Reprogramming and Trastuzumab Resistance in Breast Cancer Cell Lines Overexpressing the ERBB2 Membrane Receptor.

Trastuzumab (Tz), an antibody targeting ERBB2, has significantly improved the prognosis for breast cancer (BCa) patients with overexpression of the ERBB2 receptor. However, Tz resistance poses a challenge to patient outcomes. Numerous mechanisms have been suggested to contribute to Tz resistance, and this study aimed to uncover shared mechanisms in in vitro models of acquired BCa Tz resistance. Three widely used ERBB2+ BCa cell lines, adapted to grow in Tz, were examined. Despite investigating potential changes in phenotype, proliferation, and ERBB2 membrane expression in these Tz-resistant (Tz-R) cell lines compared to wild-type (wt) cells, no common alterations were discovered. Instead, high-resolution mass spectrometry analysis revealed a shared set of differentially expressed proteins (DEPs) in Tz-R versus wt cells. Bioinformatic analysis demonstrated that all three Tz-R cell models exhibited modulation of proteins associated with lipid metabolism, organophosphate biosynthesis, and macromolecule methylation. Ultrastructural examination corroborated the presence of altered lipid droplets in resistant cells. These findings strongly support the notion that intricate metabolic adaptations, including lipid metabolism, protein phosphorylation, and potentially chromatin remodeling, may contribute to Tz resistance. The detection of 10 common DEPs across all three Tz-resistant cell lines offers promising avenues for future therapeutic interventions, providing potential targets to overcome Tz resistance and potentially improve patient outcomes in ERBB2+ breast cancer.

The importance of gravity vector on adult mammalian organisms: Effects of hypergravity on mouse testis.

In the age of space exploration, the effect of hypergravity on human physiology is a relatively neglected topic. However, astronauts have several experiences of hypergravity during their missions. The main disturbance of altered gravity can be imputed to cell cytoskeleton alteration and physiologic homeostasis of the body. Testis has proved to be a particularly sensible organ, subject to environmental alteration and physiological disturbance. This makes testis an organ eligible for investigating the alteration following exposure to altered gravity. In our study, mice were exposed to hypergravity (3g for 14 days) in the Large Diameter Centrifuge machine (ESA, Netherland). We have observed a morphological alteration of the regular architecture of the seminiferous tubules of testis as well as an altered expression of factors involved in the junctional complexes of Sertoli cells, responsible for ensuring the morpho-functional integrity of the organ. The expression of key receptors in physiological performance, such as Androgen Receptors and Interstitial Cells Stimulating Hormone receptors, was found lower expressed. All these findings indicate the occurrence of altered physiological organ performance such as the reduction of the spermatozoa number and altered endocrine parameters following hypergravity exposure.

SARS-CoV-2 infection induces DNA damage, through CHK1 degradation and impaired 53BP1 recruitment, and cellular senescence.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the RNA virus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Although SARS-CoV-2 was reported to alter several cellular pathways, its impact on DNA integrity and the mechanisms involved remain unknown. Here we show that SARS-CoV-2 causes DNA damage and elicits an altered DNA damage response. Mechanistically, SARS-CoV-2 proteins ORF6 and NSP13 cause degradation of the DNA damage response kinase CHK1 through proteasome and autophagy, respectively. CHK1 loss leads to deoxynucleoside triphosphate (dNTP) shortage, causing impaired S-phase progression, DNA damage, pro-inflammatory pathways activation and cellular senescence. Supplementation of deoxynucleosides reduces that. Furthermore, SARS-CoV-2 N-protein impairs 53BP1 focal recruitment by interfering with damage-induced long non-coding RNAs, thus reducing DNA repair. Key observations are recapitulated in SARS-CoV-2-infected mice and patients with COVID-19. We propose that SARS-CoV-2, by boosting ribonucleoside triphosphate levels to promote its replication at the expense of dNTPs and by hijacking damage-induced long non-coding RNAs' biology, threatens genome integrity and causes altered DNA damage response activation, induction of inflammation and cellular senescence.

Lysyl-Oxidase Dependent Extracellular Matrix Stiffness in Hodgkin Lymphomas: Mechanical and Topographical Evidence.

PURPOSE: The biochemical composition and architecture of the extracellular matrix (ECM) is known to condition development and invasiveness of neoplasms. To clarify this point, we analyzed ECM stiffness, collagen cross-linking and anisotropy in lymph nodes (LN) of Hodgkin lymphomas (HL), follicular lymphomas (FL) and diffuse large B-cell lymphomas (DLBCL), compared with non-neoplastic LN (LDN). METHODS AND RESULTS: We found increased elastic (Young's) modulus in HL and advanced FL (grade 3A) over LDN, FL grade 1-2 and DLBCL. Digital imaging evidenced larger stromal areas in HL, where increased collagen cross-linking was found; in turn, architectural modifications were documented in FL3A by scanning electron microscopy and enhanced anisotropy by polarized light microscopy. Interestingly, HL expressed high levels of lysyl oxidase (LOX), an enzyme responsible for collagen cross-linking. Using gelatin scaffolds fabricated with a low elastic modulus, comparable to that of non-neoplastic tissues, we demonstrated that HL LN-derived mesenchymal stromal cells and HL cells increased the Young's modulus of the extracellular microenvironment through the expression of LOX. Indeed, LOX inhibition by ß-aminopropionitrile prevented the gelatin stiffness increase. CONCLUSIONS: These data indicate that different mechanical, topographical and/or architectural modifications of ECM are detectable in human lymphomas and are related to their histotype and grading.

Simulated Microgravity Modulates Focal Adhesion Gene Expression in Human Neural Stem Progenitor Cells.

We analyzed the morphology and the transcriptomic changes of human neural stem progenitor cells (hNSPCs) grown on laminin in adherent culture conditions and subjected to simulated microgravity for different times in a random positioning machine apparatus. Low-cell-density cultures exposed to simulated microgravity for 24 h showed cell aggregate formation and significant modulation of several genes involved in focal adhesion, cytoskeleton regulation, and cell cycle control. These effects were much more limited in hNSPCs cultured at high density in the same conditions. We also found that some of the genes modulated upon exposure to simulated microgravity showed similar changes in hNSPCs grown without laminin in non-adherent culture conditions under normal gravity. These results suggest that reduced gravity counteracts the interactions of cells with the extracellular matrix, inducing morphological and transcriptional changes that can be observed in low-density cultures.

Inactivation of Six2 in mouse identifies a novel genetic mechanism controlling development and growth of the cranial base.

The cranial base is essential for integrated craniofacial development and growth. It develops as a cartilaginous template that is replaced by bone through the process of endochondral ossification. Here, we describe a novel and specific role for the homeoprotein Six2 in the growth and elongation of the cranial base. Six2-null newborn mice display premature fusion of the bones in the cranial base. Chondrocyte differentiation is abnormal in the Six2-null cranial base, with reduced proliferation and increased terminal differentiation. Gain-of-function experiments indicate that Six2 promotes cartilage development and growth in other body areas and appears therefore to control general regulators of chondrocyte differentiation. Our data indicate that the main factors restricting Six2 function to the cranial base are tissue-specific transcription of the gene and compensatory effects of other Six family members. The comparable expression during human embryogenesis and the high protein conservation from mouse to human implicate SIX2 loss-of-function as a potential congenital cause of anterior cranial base defects in humans.

Expressing Hoxa2 across the entire endochondral skeleton alters the shape of the skeletal template in a spatially restricted fashion.

Hox genes control morphogenesis along the antero-posterior axis. The skeleton of vertebrates offers an exemplar readout of their activity: Hox genes control the morphology of the skeleton by defining type of vertebrae, and structure of the limbs. The head skeleton of vertebrates is formed by cranial neural crest (CNC), and mainly by a Hox-free domain of the CNC. Ectopic expression of anterior Hox genes in the CNC prevents the formation of the facial skeleton. These inhibitory effects on skeletogenesis are at odds with the recognized function of Hox genes in patterning the developing skeleton. To clarify these controversial effects, we overexpressed Hoxa2 across the entire developing endochondral skeleton in mouse. This gave rise to strong and spatially restricted effects: the most noticeable abnormalities were detected in the cranial base and consisted in a failure of bones to form or in a transformed morphology of bones. The rest of the skeleton exhibited milder defects, which never consisted in the absence or the transformation of any skeletal components. Analyses at early stages of endochondral bone development showed disorganized cell condensations in the cranial base of Col2a1-Hoxa2 transgenic embryos. We show that the distribution of Hoxa2-positive cells in Col2a1-Hoxa2 embryos does not match the wild-type developing cartilages. The Hoxa2-positive cells detected in atypical, non-chondrogenic location in the cranial base, remain as chondrocytes and lay down cartilage, indicating that Hoxa2 does not alter the fate of chondrocytes, but interferes with their spatial distribution. We propose that the ability of Hoxa2 to change the spatial distribution of cells accounts for the different phenotypes observed in Col2a1-Hoxa2 embryos; it also provides an explanation for the apparent inconsistency between the inhibitory effects of Hoxa2 on skeletal development, and the ability of Hox genes to establish the morphology of the vertebrate skeleton.

Lipocalin-2 controls the expression of SDF-1 and the number of responsive cells in bone.

Lipocalin-2 (LCN2) is a member of the lipocalin family, small secreted proteins functioning as modulators of many different physiological processes including cell differentiation, proliferation and apoptosis. LCN2 expression is also up-regulated in several pathological conditions, including inflammation and cancer. LCN2 synthesis has been described in epithelia, bone and cells of the immune system. Despite its wide expression the role of LCN2 remains to be fully elucidated. To better understand the role of this lipocalin in the bone/bone marrow system we generated transgenic mice over-expressing LCN2 specifically in bone under the control of a type I collagen promoter. In the bone marrow of these transgenic mice we observed an increased expression of SDF-1 that correlated with an increased number of CD34+/CXCR4+ (SDF-1 receptor) cells. To some extent, this appeared due to an enhanced cell proliferation rate. The higher level of the factor synthesis and the increased number of cells expressing its receptor was maintained during animal aging. Our results show that LCN2 could play a role in determining the number of CD34+/CXCR4+ precursor cells in the bone marrow thus contributing to the control of the bone marrow microenvironment.

The novel diterpene 7ß-acetoxy-20-hydroxy-19,20-epoxyroyleanone from Salvia corrugata shows complex cytotoxic activities against human breast epithelial cells.

AIMS: The aim of this study was the characterization of the in vitro cytotoxic properties of a recently isolated diterpene compound, 7ß-acetoxy-20-hydroxy-19,20-epoxyroyleanone (compound 1), extracted from Salvia corrugata, versus human cell lines. MAIN METHODS: We used as model study immortalized breast epithelial cells MCF10A and two ERBB2+ breast cancer (BCa) cell lines, SKBR-3 and BT474. Compound 1 was isolated by methanolic extraction from regenerated shoots of Salvia corrugata Vahl, and purified by high pressure liquid chromatography (HPLC). Flow cytometry (FCM) was employed for cell cycle, apoptosis and reactive oxygen species (ROS) analysis. Cell morphology was assessed by immunofluorescence and transmission electron microscopy (TEM). KEY FINDINGS: Compound 1 inhibited cell survival of all breast cell lines. In particular, compound 1 promoted cell cycle arrest in the G0/G1 phase and apoptosis along with impairment of the mitochondrial function, which was reflected in a gross alteration of the mitochondrial network structure. Furthermore, we also detected a potent activation of the ERK1/2 kinase, which suggested the induction of reactive oxygen species (ROS). Partial rescue of survival obtained with n-acetylcysteine (NAC) when coadminstered with compound 1 further supported a contribution of ROS mediated mechanisms to the growth-arrest and proapoptotic activity of compound 1 in both BCa cell lines. ROS production was indeed confirmed in SKBR-3. SIGNIFICANCE: Our findings show that compound 1 has a cytotoxic activity against both human normal and cancer cell lines derived from breast epithelia, which is mediated by ROS generation and mitochondrial damage.

High-Resolution X-Ray Tomography: A 3D Exploration Into the Skeletal Architecture in Mouse Models Submitted to Microgravity Constraints.

Bone remodeling process consists in a slow building phase and in faster resorption with the objective to maintain a functional skeleton locomotion to counteract the Earth gravity. Thus, during spaceflights, the skeleton does not act against gravity, with a rapid decrease of bone mass and density, favoring bone fracture. Several studies approached the problem by imaging the bone architecture and density of cosmonauts returned by the different spaceflights. However, the weaknesses of the previously reported studies was two-fold: on the one hand the research suffered the small statistical sample size of almost all human spaceflight studies, on the other the results were not fully reliable, mainly due to the fact that the observed bone structures were small compared with the spatial resolution of the available imaging devices. The recent advances in high-resolution X-ray tomography have stimulated the study of weight-bearing skeletal sites by novel approaches, mainly based on the use of the mouse and its various strains as an animal model, and sometimes taking advantage of the synchrotron radiation support to approach studies of 3D bone architecture and mineralization degree mapping at different hierarchical levels. Here we report the first, to our knowledge, systematic review of the recent advances in studying the skeletal bone architecture by high-resolution X-ray tomography after submission of mice models to microgravity constrains.

Overexpression of the C-type natriuretic peptide (CNP) is associated with overgrowth and bone anomalies in an individual with balanced t(2;7) translocation.

Longitudinal bone growth is determined by the process of endochondral ossification in the cartilaginous growth plate, which is located at both ends of vertebrae and long bones and involves many systemic hormones and local regulators. We report the molecular characterization of a de novo balanced t(2;7)(q37.1;q21.3) translocation in a young female with Marfanoid habitus and skeletal anomalies. The translocation was characterized by fluorescence in situ hybridization (FISH), checked for other abnormalities by array-comparative genomic hybridization (CGH), and finally, the breakpoints were cloned, sequenced, and compared. Biochemical dosage was applied to study the possible mechanisms that may cause the proposita's phenotype. The breakpoint on chromosome 2 disrupts the hypothetical gene MGC42174 (HUGO-approved symbol DIS3L2) and is located in the proximity of the NPPC gene coding for C-type natriuretic peptide (CNP), a molecule that regulates endochondral bone growth. CNP plasma concentration was doubled in the proband compared to five normal controls, while NPPC was substantially overexpressed in her fibroblasts. A transgenic mouse generated to target NPPC overexpression in bone showed a phenotype highly reminiscent of the patient's phenotype. The breakpoint on chromosome 7 is localized proximally at about 75 kb from the COL1A2 gene. The COL1A2 allele on the derivative chromosome was strongly underexpressed in fibroblasts, but total collagen was not significantly different from controls. Several evidences support the conclusion that the proband's abnormal phenotype is associated with C-type natriuretic peptide overexpression.