Reduced plate motion controlled timing of Early Jurassic Karoo-Ferrar large igneous province volcanism.

Past large igneous province (LIP) emplacement is commonly associated with mantle plume upwelling and led to major carbon emissions. One of Earth's largest past environmental perturbations, the Toarcian oceanic anoxic event (T-OAE; ~183 Ma), has been linked to Karoo-Ferrar LIP emplacement. However, the role of mantle plumes in controlling the onset and timing of LIP magmatism is poorly understood. Using global plate reconstruction models and Lower Toarcian sedimentary mercury (Hg) concentrations, we demonstrate (i) that the T-OAE occurred coevally with Karoo-Ferrar emplacement and (ii) that timing and duration of LIP emplacement was governed by reduced Pangean plate motion, associated with a reversal in plate movement direction. This new model mechanistically links Earth's interior and surficial processes, and the mechanism is consistent with the timing of several of the largest LIP volcanic events throughout Earth history and, thus, the timing of many of Earth's past global climate change and mass extinction events.

Allograft inflammatory factor-1 in myeloid cells drives autoimmunity in type 1 diabetes.

Allograft inflammatory factor-1 (AIF1) is a calcium-responsive cytoplasmic scaffold protein that directs hematopoiesis and immune responses within dendritic cells (DC) and macrophages. Although the role of AIF1 in transplant rejection and rheumatoid arthritis has been explored, little is known about its role in type 1 diabetes. Here, we show that in vivo silencing of AIF1 in NOD mice restrained infiltration of immune cells into the pancreas and inhibited diabetes incidence. Analyses of FACS-sorted CD45neg nonleukocyte populations from resected pancreatic islets showed markedly higher expression of insulin in the AIF1-silenced groups. Evaluation of CD45+ leukocytes revealed diminished infiltration of effector T cells and DC in the absence of AIF1. Transcriptional profiling further revealed a marked decrease in cDC1 DC-associated genes CD103, BATF3, and IRF8, which are required for orchestrating polarized type 1 immunity. Reduced T cell numbers within the islets were observed, with concomitant lower levels of IFN-γ and T-bet in AIF1-silenced cohorts. In turn, there was a reciprocal increase in functionally suppressive pancreas-resident CD25+Foxp3+CD4+ Tregs. Taken together, results show that AIF1 expression in myeloid cells plays a pivotal role in promoting type 1 diabetes and that its suppression restrains insulitis by shifting the immune microenvironment toward tolerance.

Pancreatic islets seeded in a novel bioscaffold forms an organoid to rescue insulin production and reverse hyperglycemia in models of type 1 diabetes.

Therapeutic approaches to combat type 1 diabetes (T1D) include donor pancreas transplantation, exogenous insulin administration and immunosuppressive therapies. However, these clinical applications are limited due to insufficient tissue compatible donors, side effects of exogenous insulin administration and/or increased onset of opportunistic infections attributable to induced global immunosuppression. An alternative approach to alleviate disease states is to utilize insulin-producing pancreatic islets seeded in a bioscaffold for implantation into diabetic recipients. The present studies now report that a newly developed cationic polymer biomaterial serves as an efficient bioscaffold for delivery of donor syngeneic pancreatic islet cells to reverse hyperglycemia in murine streptozotocin induced- or non-obese diabetic mouse models of T1D. Intraperitoneal implantation of pancreatic islets seeded within the copolymer bioscaffold supports long-term cell viability, response to extracellular signaling cues and ability to produce soluble factors into the microenvironment. Elevated insulin levels were measured in recipient diabetic mice upon implantation of the islet-seeded biomaterial coupled with reduced blood glucose levels, collectively resulting in increased survival and stabilization of metabolic indices. Importantly, the implanted islet-seeded biomaterial assembled into a solid organoid substructure that reorganized the extracellular matrix compartment and recruited endothelial progenitors for neovascularization. This allowed survival of the graft long-term in vivo and access to the blood for monitoring glucose levels. These results highlight the novelty, simplicity and effectiveness of this biomaterial for tissue regeneration and in vivo restoration of organ functions.

Macrophages From Subjects With Isolated GH/IGF-I Deficiency Due to a GHRH Receptor Gene Mutation Are Less Prone to Infection by Leishmania amazonensis.

Isolated growth hormone (GH) deficiency (IGHD) affects approximately 1 in 4,000 to 1 in 10,000 individuals worldwide. We have previously described a large cohort of subjects with IGHD due to a homozygous mutation in the GH releasing hormone (GHRH) receptor gene. These subjects exhibit throughout the life very low levels of GH and its principal mediator, the Insulin Growth Factor-I (IGF-I). The facilitating role of IGF-I in the infection of mouse macrophages by different Leishmania strains is well-known. Nevertheless, the role of IGF-I in Leishmania infection of human macrophages has not been studied. This study aimed to evaluate the behavior of Leishmania infection in vitro in macrophages from untreated IGHD subjects. To this end, blood samples were collected from 14 IGHD individuals and 14 age and sex-matched healthy controls. Monocytes were isolated and derived into macrophages and infected with a strain of Leishmania amazonensis. In addition, IGF-I was added to culture medium to evaluate its effect on the infection. Cytokines were measured in the culture supernatants. We found that macrophages from IGHD subjects were less prone to Leishmania infection compared to GH sufficient controls. Both inflammatory and anti-inflammatory cytokines increase only in the supernatants of the control macrophages. Addition of IGF-I to the culture medium increased infection rates. In conclusion, we demonstrated that IGF-I is crucial for Leishmania infection of human macrophages.

Allograft Inflammatory Factor-1 Governs Hematopoietic Stem Cell Differentiation Into cDC1 and Monocyte-Derived Dendritic Cells Through IRF8 and RelB in vitro.

The multistep differentiation process from hematopoietic stem cells through common myeloid progenitors into committed dendritic cell (DC) subsets remains to be fully addressed. These studies now show that Allograft Inflammatory Factor-1 (AIF1) is required for differentiation of classical DC type 1 (cDC1) subsets and monocyte-derived DC (Mo-DC). Phenotypic studies found that AIF1 expression increased in committed subsets differentiating from common myeloid progenitors (CMP). However, silencing AIF1 expression in hematopoietic stem progenitors restrained the capacity to differentiate into Mo-DC and cDC1 cell subsets under GM-CSF or Flt3-L stimuli conditions, respectively. This was further marked by restrained expression of IRF8, which is critical for development of Mo-DC and cDC1 subsets. As a result, absence of AIF1 restrained the cells at the Lin-CD117+FcγR-CD34+ CMP stage. Further biochemical studies revealed that abrogating AIF1 resulted in inhibition of the NFκB family member RelB expression and p38 MAPK phosphorylation during differentiation of Mo-DC. Lastly, protein binding studies identified that AIF1 interacts with protein kinase C (PKC) to influence downstream signaling pathways. Taken together, this is the first report showing a novel role of AIF1 as a calcium-responsive scaffold protein that supports IRF8 expression and interacts with PKC to drive NFκB-related RelB for successfully differentiating hematopoietic progenitor cells into cDC and Mo-DC subsets under Flt3-L and GM-CSF stimuli, respectively.

IL-10 producing CD8+ CD122+ PD-1+ regulatory T cells are expanded by dendritic cells silenced for Allograft Inflammatory Factor-1.

Allograft Inflammatory Factor-1 (AIF1) is a cytoplasmic scaffold protein that contains Ca2+ binding EF-hand and PDZ interaction domains important for mediating intracellular signaling complexes in immune cells. The protein plays a dominant role in both macrophage- and dendritic cell (DC)-mediated inflammatory responses. This study now reports that AIF1 expression in DC is important in directing CD8+ T cell effector responses. Silencing AIF1 expression in murine CD11c+ DC suppressed antigen-specific CD8+ T cell activation, marked by reduced CXCR3, IFNγ and Granzyme B expression, and restrained proliferation. These primed CD8+ T cells had impaired cytotoxic killing of target cells in vitro. In turn, studies identified that AIF1 silencing in DC robustly expanded IL-10 producing CD8+ CD122+ PD-1+ regulatory T cells that suppressed neighboring immune effector responses through both IL-10 and PD-1-dependent mechanisms. In vivo studies recapitulated bystander suppression of antigen-responsive CD4+ T cells by the CD8+ Tregs expanded from the AIF1 silenced DC. These studies further demonstrate that AIF1 expression in DC serves as a potent governor of cognate T cell responses and present a novel target for engineering tolerogenic DC-based immunotherapies.

Different antiproliferative effects of matuzumab and cetuximab in A431 cells are associated with persistent activity of the MAPK pathway.

Preclinical studies have shown the potential antitumour efficacy of monoclonal antibodies (MAbs) directed to the epidermal growth factor receptor (EGFR). In this report, we investigated the cytotoxic effects of the MAb matuzumab (EMD 72000) towards A431 cells and compared it to cetuximab. While cetuximab induced cell cycle arrest and inhibited A431 cell proliferation, matuzumab did not. Both MAbs inhibited growth factor induced EGFR, HER2 and AKT phosphorylation; however, only cetuximab inhibited ERK 1/2 phosphorylation. Taken together, the data indicate that each antibody may elicit different responses on EGFR downstream signalling pathways with a distinct impact on A431 cell line survival. When combined, MAbs synergistically inhibited cell proliferation and induced EGFR down-regulation with a strong inhibition of ERK1/2 and AKT phosphorylation. In addition, both MAbs efficiently inhibited VEGF expression and induced ADCC, highlighting their therapeutic potential in vivo when used either as a single agent or in combination.

Tumorigenic activity and therapeutic inhibition of Rheb GTPase.

The AKT-mTOR pathway harbors several known and putative oncogenes and tumor suppressors. In a phenotypic screen for lymphomagenesis, we tested candidate genes acting upstream of and downstream from mTOR in vivo. We find that Rheb, a proximal activator of mTORC1, can produce rapid development of aggressive and drug-resistant lymphomas. Rheb causes mTORC1-dependent effects on apoptosis, senescence, and treatment responses that resemble those of Akt. Moreover, Rheb activity toward mTORC1 requires farnesylation and is readily blocked by a pharmacological inhibitor of farnesyltransferase (FTI). In Pten-deficient tumor cells, inhibition of Rheb by FTI is responsible for the drug's anti-tumor effects, such that a farnesylation-independent mutant of Rheb renders these tumors resistant to FTI therapy. Notably, RHEB is highly expressed in some human lymphomas, resulting in mTORC1 activation and increased sensitivity to rapamycin and FTI. Downstream from mTOR, we examined translation initiation factors that have been implicated in transformation in vitro. Of these, only eIF4E was able to enhance lymphomagenesis in vivo. In summary, the Rheb GTPase is an oncogenic activity upstream of mTORC1 and eIF4E and a direct therapeutic target of farnesyltransferase inhibitors in cancer.

Genetic analysis of Dengue 3 virus subtype III 5' and 3' non-coding regions.

The emergence of dengue hemorrhagic fever in Sri Lanka in 1989 correlated with the appearance of a genetic variant of Dengue 3 virus (DENV-3) subtype III (group B), closely related to the endemic group A variant. We studied the 5' and 3' non-coding regions (NCRs) of 15 DENV-3 subtype III isolates from Sri Lanka, Nicaragua and Martinique and found variability in the 3' NCRs. This included an 11-nucleotide insertion common to all isolates examined and two nucleotide differences that segregated viruses geographically into an American and a Sri Lankan group. Comparisons also identified three nucleotide differences shared by all group A Sri Lankan DENV-3 III isolates linked to mild disease epidemics but not group B Sri Lankan and group B-associated American isolates linked to severe disease epidemics. Clustering of the Latin American/Caribbean isolates with Sri Lankan group B DENV-3 in phylogenetic analyses supports the proposed common East African origin for all these strains and confirms the use of the 3' NCR for molecular epidemiologic studies of DENV-3. The differences in the 3' NCRs reported here, as well as potential alterations in the structural and non-structural coding genes, may contribute to the increased pathogenicity of group B DENV-3.

MNK, EIF4E and targeting translation for therapy.

Deregulation of protein translation is a common event in cancer and occurs frequently as a result of mutational activation of the AKT signaling pathway. We had previously reported the in vivo oncogenic activity of the translation initiation factor eIF4E, which acts downstream AKT and mTOR. We now identified an absolute requirement for Ser209 phosphorylation by the MNK1/2 kinases for eIF4E's oncogenic action. MNK1/2 kinases are dispensable for normal development in mammals. This potential difference between normal and cancer cells may provide a therapeutic avenue for targeting translational requirements in cancer.

Dissecting eIF4E action in tumorigenesis.

Genetically engineered mouse models are powerful tools for studying cancer genes and validating targets for cancer therapy. We previously used a mouse lymphoma model to demonstrate that the translation initiation factor eIF4E is a potent oncogene in vivo. Using the same model, we now show that the oncogenic activity of eIF4E correlates with its ability to activate translation and become phosphorylated on Ser 209. Furthermore, constitutively activated MNK1, an eIF4E Ser 209 kinase, promotes tumorigenesis in a manner similar to eIF4E, and a dominant-negative MNK mutant inhibits the in vivo proliferation of tumor cells driven by mutations that deregulate translation. Phosphorylated eIF4E promotes tumorigenesis primarily by suppressing apoptosis and, accordingly, the anti-apoptotic protein Mcl-1 is one target of both phospho-eIF4E and MNK1 that contributes to tumor formation. Our results provide insight into how eIF4E contributes to tumorigenesis and pinpoint a level of translational control that may be suitable for therapeutic intervention.

The Arf tumor suppressor regulates platelet-derived growth factor receptor beta signaling: a new view through the eyes of Arf(-/-) mice.

Arf is a key mammalian tumor suppressor gene known to be activated in response to aberrant mitogenic signals leading to both p53-dependent and -independent effects. We recently uncovered a new and somewhat unexpected function for mouse Arf as a regulator of mural cell accumulation within an ocular vascular bed destined to regress in the postnatal period. We found that the Arf gene product, p19(Arf), blocks mural cell proliferation driven by Platelet-derived growth factor receptor beta (Pdgfrbeta) in the developing vitreous. In vivo studies and analyses of cultured cells indicate that p19(Arf) dampens the expression of Pdgfrbeta. In cultured mouse embryo fibroblasts, p19(Arf) accomplishes this independently of two established effectors - Mdm2 and p53. Our findings indicating that p19(Arf) responds to specific developmental cues to disrupt Pdgfrbeta signaling in the developing eye extend existing paradigms for Arf tumor suppressor gene biology.

Arf-dependent regulation of Pdgf signaling in perivascular cells in the developing mouse eye.

We have established that the Arf tumor suppressor gene regulates mural cell biology in the hyaloid vascular system (HVS) of the developing eye. In the absence of Arf, perivascular cells accumulate within the HVS and prevent its involution. We now demonstrate that mural cell accumulation evident at embryonic day (E) 13.5 in Arf(-/-) mice was driven by excess proliferation at E12.5, when Arf expression was detectable in vitreous pericyte-like cells. Their expression of Arf overlapped with Pdgf receptor beta (Pdgfrbeta), which is essential for pericyte accumulation in the mouse. In cultured cells, p19Arf decreased Pdgfrbeta and blocked Pdgf-B-driven proliferation independently of Mdm2 and p53. The presence of a normal Arf allele correlated with decreased Pdgfrbeta in the embryonic vitreous. Pdgfrbeta was required for vitreous cell accumulation in the absence of Arf. Our findings demonstrate a novel, p53- and Mdm2-independent function for p19Arf. Instead of solely sensing excessive mitogenic stimuli, developmental cues induce Arf to block Pdgfrbeta-dependent signals and prevent the accumulation of perivascular cells selectively in a vascular bed destined to regress.

Comparative analysis of amplified and nonamplified RNA for hybridization in cDNA microarray.

Limiting amounts of RNA is a major issue in cDNA microarray, especially when one is dealing with fresh tissue samples. Here we describe a protocol based on template switch and T7 amplification that led to efficient and linear amplification of 1300x. Using a glass-array containing 368 genes printed in three or six replicas covering a wide range of expression levels and ratios, we determined quality and reproducibility of the data obtained from one nonamplified and two independently amplified RNAs (aRNA) derived from normal and tumor samples using replicas with dye exchange (dye-swap measurements). Overall, signal-to-noise ratio improved when we used aRNA (1.45-fold for channel 1 and 2.02-fold for channel 2), increasing by 6% the number of spots with meaningful data. Measurements arising from independent aRNA samples showed strong correlation among themselves (r(2)=0.962) and with those from the nonamplified sample (r(2)=0.975), indicating the reproducibility and fidelity of the amplification procedure. Measurement differences, i.e, spots with poor correlation between amplified and nonamplified measurements, did not show association with gene sequence, expression intensity, or expression ratio and can, therefore, be compensated with replication. In conclusion, aRNA can be used routinely in cDNA microarray analysis, leading to improved quality of data with high fidelity and reproducibility.

cDNA microarray analysis of genes associated with ERBB2 (HER2/neu) overexpression in human mammary luminal epithelial cells.

To investigate changes in gene expression associated with ERBB2, expression profiling of immortalized human mammary luminal epithelial cells and variants expressing a moderate and high level of ERBB2 has been carried out using cDNA microarrays corresponding to approximately 6000 unique genes/ESTs. A total of 61 significantly up- or downregulated (2.0-fold) genes were identified and further validated by RT-PCR analysis as well as microarray comparisons with a spontaneously ERBB2- overexpressing breast cancer cell line and ERBB2-positive primary breast tumors. The expression and clinical relevance of proteins predicted to be associated with ERBB2 overexpression in breast cancers were analysed together with their clinical relevance by antibody screening using a tissue array. Differentially regulated genes include those involved in cell-matrix interactions including proline 4-hydroxylase (P4HA2), galectin 1 (LGALS1) and galectin 3 (LGALS3), fibronectin 1 (FN1) and p-cadherin (CDH3), and cell proliferation (CRIP1, IGFBP3) and transformation (S100P, S100A4). A number of genes associated with MYC signalling were also differentially expressed, including NDRG1, USF2 and the epithelial membrane proteins 1 and 3 (EMP1, EMP3). These data represent profiles of the transcriptional changes associated with ERBB2-related pathways in the breast, and identify novel and potentially useful targets for prognosis and therapy.

Differentially expressed genes in gastric tumors identified by cDNA array.

Using cDNA fragments from the FAPESP/lICR Cancer Genome Project, we constructed a cDNA array having 4512 elements and determined gene expression in six normal and six tumor gastric tissues. Using t-statistics, we identified 80 cDNAs whose expression in normal and tumor samples differed more than 3.5 sample standard deviations. Using Self-Organizing Map, the expression profile of these cDNAs allowed perfect separation of malignant and non-malignant samples. Using the supervised learning procedure Support Vector Machine, we identified trios of cDNAs that could be used to classify samples as normal or tumor, based on single-array analysis. Finally, we identified genes with altered linear correlation when their expression in normal and tumor samples were compared. Further investigation concerning the function of these genes could contribute to the understanding of gastric carcinogenesis and may prove useful in molecular diagnostics.

Characterization of a vaccinia-like virus isolated in a Brazilian forest.

The SPAn232 virus (SPAnv) was isolated from sentinel mice in the forest of Cotia, São Paulo, Brazil. It was grouped originally as a Cotia virus (CV) sample due to serological cross-reaction with the latter. However, SPAnv presented genetic characteristics that differed from CV and indicated that SPAnv is a member of the vaccinia virus (VV) subgroup. SPAnv showed a HindIII-digested DNA pattern similar to those of the WR and Lister strains of VV. Also, SPAnv presented genes homologous to the vaccinia growth factor, thymidine kinase and A-type inclusion (ATI) genes from VV. RFLP analysis of the SPAnv ATI homologous gene indicated that the virus belongs to the VV group. Nucleotide sequences from SPAnv genes showed up to 99% similarity with the same genes from VV. Such a relationship was confirmed visually through the drawing of phylogenetic trees. The results point out the occurrence of a VV strain that is possibly in active circulation in the forests of Southeast Brazil.