RESUMO
Many microRNAs (miRNAs) are generated from primary transcripts containing multiple clustered stem-loop structures that are thought to be recognized and cleaved by the Microprocessor complex as independent units. Here, we uncover an unexpected mode of processing of the bicistronic miR-15a-16-1 cluster. We find that the primary miR-15a stem-loop is not processed on its own but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted pri-miR-15 cleavage and describe SAFB2 as an accessory protein of the Microprocessor. Notably, SAFB2-mediated cleavage expands to other clustered pri-miRNAs, indicating a general mechanism. Together, our study reveals an unrecognized function of SAFB2 in miRNA processing and suggests a scenario in which SAFB2 enables the binding and processing of suboptimal Microprocessor substrates in clustered primary miRNA transcripts.
Assuntos
Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , MicroRNAs/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Sequências Repetidas Invertidas/genética , Sequências Repetidas Invertidas/fisiologia , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , MicroRNAs/genética , Proteínas Associadas à Matriz Nuclear/genética , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores de Estrogênio/genéticaRESUMO
Members of the Old World Arenaviruses primarily utilize α-dystroglycan (α-DAG1) as a cellular receptor for infection. Mutations within the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) reduce or abrogate the binding affinity to α-DAG1 and thus influence viral persistence, kinetics, and cell tropism. The observation that α-DAG1 deficient cells are still highly susceptible to low affinity variants, suggests the use of an alternative receptor(s). In this study, we used a genome-wide CRISPR Cas9 knockout screen in DAG1 deficient 293T cells to identify host factors involved in α-DAG1-independent LCMV infection. By challenging cells with vesicular stomatitis virus (VSV), pseudotyped with the GP of LCMV WE HPI (VSV-GP), we identified the heparan sulfate (HS) biosynthesis pathway as an important host factor for low affinity LCMV infection. These results were confirmed by a genetic approach targeting EXTL3, a key factor in the HS biosynthesis pathway, as well as by enzymatic and chemical methods. Interestingly, a single point mutation within GP1 (S153F or Y155H) of WE HPI is sufficient for the switch from DAG1 to HS binding. Furthermore, we established a simple and reliable virus-binding assay, using directly labelled VSV-GP by intramolecular fusion of VSV-P and mWasabi, demonstrating the importance of HS for virus attachment but not entry in Burkitt lymphoma cells after reconstitution of HS expression. Collectively, our study highlights the essential role of HS for low affinity LCMV infection in contrast to their high affinity counterparts. Residual LCMV infection in double knockouts indicate the use of (a) still unknown entry receptor(s).
Assuntos
Proteoglicanas de Heparan Sulfato/metabolismo , Coriomeningite Linfocítica/metabolismo , Coriomeningite Linfocítica/transmissão , Vírus da Coriomeningite Linfocítica/metabolismo , Vírus da Coriomeningite Linfocítica/patogenicidade , Células HEK293 , Humanos , Receptores Virais/metabolismoRESUMO
Despite being mutated in cancer and RASopathies, the role of the activation segment (AS) has not been addressed for B-Raf signaling in vivo. Here, we generated a conditional knock-in mouse allowing the expression of the B-Raf(AVKA) mutant in which the AS phosphoacceptor sites T599 and S602 are replaced by alanine residues. Surprisingly, despite producing a kinase-impaired protein, the Braf(AVKA) allele does not phenocopy the lethality of Braf-knockout or paradoxically acting knock-in alleles. However, Braf(AVKA) mice display abnormalities in the hematopoietic system, a distinct facial morphology, reduced ERK pathway activity in the brain, and an abnormal gait. This phenotype suggests that maximum B-Raf activity is required for the proper development, function, and maintenance of certain cell populations. By establishing conditional murine embryonic fibroblast cultures, we further show that MEK/ERK phosphorylation and the immediate early gene response toward growth factors are impaired in the presence of B-Raf(AVKA). Importantly, alanine substitution of T599/S602 impairs the transformation potential of oncogenic non-V600E B-Raf mutants and a fusion protein, suggesting that blocking their phosphorylation could represent an alternative strategy to ATP-competitive inhibitors.
Assuntos
Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Animais , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células Cultivadas , Ativação Enzimática/genética , Ativação Enzimática/fisiologia , Feminino , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Masculino , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Mutação , Fosforilação , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Although the essential role of the adaptor protein SLP-65 in pre-B cell differentiation is established, the molecular mechanism underlying its function is poorly understood. In this study, we uncover a link between SLP-65-dependent signaling and the phosphoinositide-3-OH kinase (PI(3)K)-protein kinase B (PKB)-Foxo pathway. We show that the forkhead box transcription factor Foxo3a promotes light chain rearrangement in pre-B cells. Our data suggest that PKB suppresses light chain recombination by phosphorylating Foxo proteins, whereas reconstitution of SLP-65 function counteracts PKB activation and promotes Foxo3a and Foxo1 activity in pre-B cells. Together, these data illuminate a molecular function of SLP-65 and identify a key role for Foxo proteins in the regulation of light chain recombination, receptor editing and B cell selection.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Linfócitos B/imunologia , Fatores de Transcrição Forkhead/metabolismo , Genes de Cadeia Leve de Imunoglobulina/genética , Proteínas Tirosina Quinases/metabolismo , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/genética , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Recombinação GenéticaRESUMO
BACKGROUND: Elevated water temperature, as is expected through climate change, leads to masculinization in fish species with sexual plasticity, resulting in changes in population dynamics. These changes are one important ecological consequence, contributing to the risk of extinction in small and inbred fish populations under natural conditions, due to male-biased sex ratio. Here we investigated the effect of elevated water temperature during embryogenesis on sex ratio and sex-biased gene expression profiles between two different tissues, namely gonad and caudal fin of adult zebrafish males and females, to gain new insights into the molecular mechanisms underlying sex determination (SD) and colour patterning related to sexual attractiveness. RESULTS: Our study demonstrated sex ratio imbalances with 25.5% more males under high-temperature condition, resulting from gonadal masculinization. The result of transcriptome analysis showed a significantly upregulated expression of male SD genes (e.g. dmrt1, amh, cyp11c1 and sept8b) and downregulation of female SD genes (e.g. zp2.1, vtg1, cyp19a1a and bmp15) in male gonads compared to female gonads. Contrary to expectations, we found highly differential expression of colour pattern (CP) genes in the gonads, suggesting the 'neofunctionalisation' of those genes in the zebrafish reproduction system. However, in the caudal fin, no differential expression of CP genes was identified, suggesting the observed differences in colouration between males and females in adult fish may be due to post-transcriptional regulation of key enzymes involved in pigment synthesis and distribution. CONCLUSIONS: Our study demonstrates male-biased sex ratio under high temperature condition and support a polygenic SD (PSD) system in laboratory zebrafish. We identify a subset of pathways (tight junction, gap junction and apoptosis), enriched for SD and CP genes, which appear to be co-regulated in the same pathway, providing evidence for involvement of those genes in the regulation of phenotypic sexual dimorphism in zebrafish.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Gônadas/metabolismo , Diferenciação Sexual , Razão de Masculinidade , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Animais , Cor , Feminino , Temperatura Alta , Masculino , Maturidade Sexual , Transcriptoma , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genéticaRESUMO
Precursor B lymphocytes expand upon expression of a pre-B cell receptor (pre-BCR), but then transit into a resting state in which immunoglobulin light chain gene recombination is initiated. This bi-phasic sequence is orchestrated by the IL-7 receptor (IL-7R) and pre-BCR signaling, respectively, but little is known about microRNAs fine-tuning these events. Here, we show that pre-B cells lacking miR-15 family functions exhibit prolonged proliferation due to aberrant expression of the target genes cyclin E1 and D3. As a consequence, they fail to trigger the transcriptional reprogramming normally accompanying their differentiation, resulting in a developmental block at the pre-B cell stage. Intriguingly, our data indicate that the miR-15 family is suppressed by both IL-7R and pre-BCR signaling, suggesting it is actively integrated into the regulatory circuits of developing B cells. These findings identify the miR-15 family as a novel element required to promote the switch from pre-B cell proliferation to differentiation.
Assuntos
Diferenciação Celular , Proliferação de Células , MicroRNAs/imunologia , MicroRNAs/metabolismo , Células Precursoras de Linfócitos B/fisiologia , Animais , Linfócitos B/imunologia , Ciclina D3/genética , Ciclina E/genética , Ativação Linfocitária , Linfopoese , Camundongos , MicroRNAs/genética , Proteínas Oncogênicas/genética , Receptores de Antígenos de Linfócitos B/genética , Receptores de Interleucina-7/genética , Transdução de SinaisRESUMO
Although canonical NF-κB signaling is crucial to generate a normal mature B-cell compartment, its role in the persistence of resting mature B cells is controversial. To resolve this conflict, we ablated NF-κB essential modulator (NEMO) and IκB kinase 2 (IKK2), two essential mediators of the canonical pathway, either early on in B-cell development or specifically in mature B cells. Early ablation severely inhibited the generation of all mature B-cell subsets, but follicular B-cell numbers could be largely rescued by ectopic expression of B-cell lymphoma 2 (Bcl2), despite a persisting block at the transitional stage. Marginal zone (MZ) B and B1 cells were not rescued, indicating a possible role of canonical NF-κB signals beyond the control of cell survival in these subsets. When canonical NF-κB signaling was ablated specifically in mature B cells, the differentiation and/or persistence of MZ B cells was still abrogated, but follicular B-cell numbers were only mildly affected. However, the mutant cells exhibited increased turnover as well as functional deficiencies upon activation, suggesting that canonical NF-κB signals contribute to their long-term persistence and functional fitness.
Assuntos
Linfócitos B/citologia , Linfócitos B/imunologia , Sobrevivência Celular/imunologia , NF-kappa B/imunologia , Transdução de Sinais/imunologia , Animais , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BLRESUMO
In T lymphocytes, expression of miR-148a is induced by T-bet and Twist1, and is specific for pro-inflammatory Th1 cells. In these cells, miR-148a inhibits the expression of the pro-apoptotic protein Bim and promotes their survival. Here we use sequence-specific cholesterol-modified oligonucleotides against miR-148a (antagomir-148a) for the selective elimination of pro-inflammatory Th1 cells in vivo. In the murine model of transfer colitis, antagomir-148a treatment reduced the number of pro-inflammatory Th1 cells in the colon of colitic mice by 50% and inhibited miR-148a expression by 71% in the remaining Th1 cells. Expression of Bim protein in colonic Th1 cells was increased. Antagomir-148a-mediated reduction of Th1 cells resulted in a significant amelioration of colitis. The effect of antagomir-148a was selective for chronic inflammation. Antigen-specific memory Th cells that were generated by an acute immune reaction to nitrophenylacetyl-coupled chicken gamma globulin (NP-CGG) were not affected by treatment with antagomir-148a, both during the effector and the memory phase. In addition, antibody titers to NP-CGG were not altered. Thus, antagomir-148a might qualify as an effective drug to selectively deplete pro-inflammatory Th1 cells of chronic inflammation without affecting the protective immunological memory.
Assuntos
Antagomirs/genética , Colite/imunologia , Colo/imunologia , Inflamação/imunologia , MicroRNAs/genética , Células Th1/fisiologia , Animais , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismoRESUMO
Tyrosine kinase inhibitors (TKIs) are widely used to treat patients with leukaemia driven by BCR-ABL1 (ref. 1) and other oncogenic tyrosine kinases. Recent efforts have focused on developing more potent TKIs that also inhibit mutant tyrosine kinases. However, even effective TKIs typically fail to eradicate leukaemia-initiating cells (LICs), which often cause recurrence of leukaemia after initially successful treatment. Here we report the discovery of a novel mechanism of drug resistance, which is based on protective feedback signalling of leukaemia cells in response to treatment with TKI. We identify BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukaemia-initiating subclones.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Inibidores de Proteínas Quinases/farmacologia , Fator 1 de Ribosilação do ADP/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-6 , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Metastatic disease is largely resistant to therapy and accounts for almost all cancer deaths. Myeloid cell leukemia-1 (MCL-1) is an important regulator of cell survival and chemo-resistance in a wide range of malignancies, and thus its inhibition may prove to be therapeutically useful. METHODS: To examine whether targeting MCL-1 may provide an effective treatment for breast cancer, we constructed inducible models of BIMs2A expression (a specific MCL-1 inhibitor) in MDA-MB-468 (MDA-MB-468-2A) and MDA-MB-231 (MDA-MB-231-2A) cells. RESULTS: MCL-1 inhibition caused apoptosis of basal-like MDA-MB-468-2A cells grown as monolayers, and sensitized them to the BCL-2/BCL-XL inhibitor ABT-263, demonstrating that MCL-1 regulated cell survival. In MDA-MB-231-2A cells, grown in an organotypic model, induction of BIMs2A produced an almost complete suppression of invasion. Apoptosis was induced in such a small proportion of these cells that it could not account for the large decrease in invasion, suggesting that MCL-1 was operating via a previously undetected mechanism. MCL-1 antagonism also suppressed local invasion and distant metastasis to the lung in mouse mammary intraductal xenografts. Kinomic profiling revealed that MCL-1 antagonism modulated Src family kinases and their targets, which suggested that MCL-1 might act as an upstream modulator of invasion via this pathway. Inhibition of MCL-1 in combination with dasatinib suppressed invasion in 3D models of invasion and inhibited the establishment of tumors in vivo. CONCLUSION: These data provide the first evidence that MCL-1 drives breast cancer cell invasion and suggests that MCL-1 antagonists could be used alone or in combination with drugs targeting Src kinases such as dasatinib to suppress metastasis.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Dasatinibe/farmacologia , Resistencia a Medicamentos Antineoplásicos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Metastatic ovarian cancer has a dismal prognosis and current chemotherapeutic approaches have very limited success. Metadherin (MTDH) is expressed in human ovarian cancer tissue and its expression inversely correlates with patients overall survival. Consistent with these studies, we observed MTDH expression in tissue specimens of FIGO Stage III ovarian carcinomas (72/83 cases). However, we also observed this in normal human ovarian epithelial (OE) cells, which raised the question of whether MTDH-variants with functional differences exist. We identified a novel MTDH exon 11 skipping variant (MTDHdel) which was seen at higher levels in ovarian cancer compared to benign OE cells. We analyzed MTDH-binding partner interactions and found that 12 members of the small ribosomal subunit and several mRNA binding proteins bound stronger to MTDHdel than to wildtype MTDH which indicates differential effects on gene translation. Knockdown of MTDH in ovarian cancer cells reduced the amount of distant metastases and improved the survival of ovarian cancer-bearing mice. Selective overexpression of the MTDHdel enhanced murine and human ovarian cancer progression and caused a malignant phenotype in originally benign human OE cells. MTDHdel was detectable in microdissected ovarian cancer cells of some human tissue specimens of ovarian carcinomas. In summary, we have identified a novel MTDH exon 11 skipping variant that shows enhanced binding to small ribosomal subunit members and that caused reduced overall survival of ovarian cancer bearing mice. Based on the findings in the murine system and in human tissues, MTDHdel must be considered a major promalignant factor for ovarian cancer.
Assuntos
Moléculas de Adesão Celular/genética , Proteínas de Membrana/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Deleção de Sequência , Animais , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Éxons , Feminino , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Transplante de Neoplasias , Proteínas de Ligação a RNARESUMO
BACKGROUND: The identification of functionally or structurally important non-conserved residue sites in protein MSAs is an important challenge for understanding the structural basis and molecular mechanism of protein functions. Despite the rich literature on compensatory mutations as well as sequence conservation analysis for the detection of those important residues, previous methods often rely on classical information-theoretic measures. However, these measures usually do not take into account dis/similarities of amino acids which are likely to be crucial for those residues. In this study, we present a new method, the Quantum Coupled Mutation Finder (QCMF) that incorporates significant dis/similar amino acid pair signals in the prediction of functionally or structurally important sites. RESULTS: The result of this study is twofold. First, using the essential sites of two human proteins, namely epidermal growth factor receptor (EGFR) and glucokinase (GCK), we tested the QCMF-method. The QCMF includes two metrics based on quantum Jensen-Shannon divergence to measure both sequence conservation and compensatory mutations. We found that the QCMF reaches an improved performance in identifying essential sites from MSAs of both proteins with a significantly higher Matthews correlation coefficient (MCC) value in comparison to previous methods. Second, using a data set of 153 proteins, we made a pairwise comparison between QCMF and three conventional methods. This comparison study strongly suggests that QCMF complements the conventional methods for the identification of correlated mutations in MSAs. CONCLUSIONS: QCMF utilizes the notion of entanglement, which is a major resource of quantum information, to model significant dissimilar and similar amino acid pair signals in the detection of functionally or structurally important sites. Our results suggest that on the one hand QCMF significantly outperforms the previous method, which mainly focuses on dissimilar amino acid signals, to detect essential sites in proteins. On the other hand, it is complementary to the existing methods for the identification of correlated mutations. The method of QCMF is computationally intensive. To ensure a feasible computation time of the QCMF's algorithm, we leveraged Compute Unified Device Architecture (CUDA).The QCMF server is freely accessible at http://qcmf.informatik.uni-goettingen.de/.
Assuntos
Algoritmos , Mutação , Proteínas/química , Proteínas/genética , Sequência de Aminoácidos , Aminoácidos/química , Sequência Conservada , Receptores ErbB/química , Receptores ErbB/genética , Glucoquinase/química , Glucoquinase/genética , Humanos , Conformação Proteica , Teoria Quântica , Alinhamento de SequênciaRESUMO
Epithelial polarization and polarized cargo transport are highly coordinated and interdependent processes. In our search for novel regulators of epithelial polarization and protein secretion, we used a genome-wide CRISPR/Cas9 screen and combined it with an assay based on fluorescence-activated cell sorting (FACS) to measure the secretion of the apical brush-border hydrolase dipeptidyl peptidase 4 (DPP4). In this way, we performed the first CRISPR screen to date in human polarized epithelial cells. Using high-resolution microscopy, we detected polarization defects and mislocalization of DPP4 to late endosomes/lysosomes after knockout of TM9SF4, anoctamin 8, and ARHGAP33, confirming the identification of novel factors for epithelial polarization and apical cargo secretion. Thus, we provide a powerful tool suitable for studying polarization and cargo secretion in epithelial cells. In addition, we provide a dataset that serves as a resource for the study of novel mechanisms for epithelial polarization and polarized transport and facilitates the investigation of novel congenital diseases associated with these processes.
Assuntos
Dipeptidil Peptidase 4 , Células Epiteliais , Humanos , Dipeptidil Peptidase 4/metabolismo , Células Epiteliais/metabolismo , Intestinos , Microvilosidades/metabolismo , Transporte Proteico , Polaridade Celular , Proteínas de Membrana/metabolismoRESUMO
The nonreceptor protein spleen tyrosine kinase (Syk) is a key mediator of signal transduction in a variety of cell types, including B lymphocytes. We show that deregulated Syk activity allows growth factor-independent proliferation and transforms bone marrow-derived pre-B cells that are then able to induce leukemia in mice. Syk-transformed pre-B cells show a characteristic pattern of tyrosine phosphorylation, increased c-Myc expression, and defective differentiation. Treatment of Syk-transformed pre-B cells with a novel Syk-specific inhibitor (R406) reduces tyrosine phosphorylation and c-Myc expression. In addition, R406 treatment removes the developmental block and allows the differentiation of the Syk-transformed pre-B cells into immature B cells. Because R406 treatment also prevents the proliferation of c-Myc-transformed pre-B cells, our data indicate that endogenous Syk kinase activity may be required for the survival of pre-B cells transformed by other oncogenes. Collectively, our data suggest that Syk is a protooncogene involved in the transformation of lymphocytes, thus making Syk a potential target for the treatment of leukemia.
Assuntos
Linfócitos B/metabolismo , Diferenciação Celular/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transferência Adotiva , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/transplante , Benzamidas , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia/genética , Leucemia/patologia , Leucemia/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Oxazinas/farmacologia , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Fosforilação/efeitos dos fármacos , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Receptores de Antígenos de Linfócitos B/genética , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/patologia , Quinase Syk , TransfecçãoRESUMO
BACKGROUND: The Andes-Amazon basin of Peru and Bolivia is one of the most data-poor, biologically rich, and rapidly changing areas of the world. Conservation scientists agree that this area hosts extremely high endemism, perhaps the highest in the world, yet we know little about the geographic distributions of these species and ecosystems within country boundaries. To address this need, we have developed conservation data on endemic biodiversity (~800 species of birds, mammals, amphibians, and plants) and terrestrial ecological systems (~90; groups of vegetation communities resulting from the action of ecological processes, substrates, and/or environmental gradients) with which we conduct a fine scale conservation prioritization across the Amazon watershed of Peru and Bolivia. We modelled the geographic distributions of 435 endemic plants and all 347 endemic vertebrate species, from existing museum and herbaria specimens at a regional conservation practitioner's scale (1:250,000-1:1,000,000), based on the best available tools and geographic data. We mapped ecological systems, endemic species concentrations, and irreplaceable areas with respect to national level protected areas. RESULTS: We found that sizes of endemic species distributions ranged widely (< 20 km2 to > 200,000 km2) across the study area. Bird and mammal endemic species richness was greatest within a narrow 2500-3000 m elevation band along the length of the Andes Mountains. Endemic amphibian richness was highest at 1000-1500 m elevation and concentrated in the southern half of the study area. Geographical distribution of plant endemism was highly taxon-dependent. Irreplaceable areas, defined as locations with the highest number of species with narrow ranges, overlapped slightly with areas of high endemism, yet generally exhibited unique patterns across the study area by species group. We found that many endemic species and ecological systems are lacking national-level protection; a third of endemic species have distributions completely outside of national protected areas. Protected areas cover only 20% of areas of high endemism and 20% of irreplaceable areas. Almost 40% of the 91 ecological systems are in serious need of protection (= < 2% of their ranges protected). CONCLUSIONS: We identify for the first time, areas of high endemic species concentrations and high irreplaceability that have only been roughly indicated in the past at the continental scale. We conclude that new complementary protected areas are needed to safeguard these endemics and ecosystems. An expansion in protected areas will be challenged by geographically isolated micro-endemics, varied endemic patterns among taxa, increasing deforestation, resource extraction, and changes in climate. Relying on pre-existing collections, publically accessible datasets and tools, this working framework is exportable to other regions plagued by incomplete conservation data.
Assuntos
Biodiversidade , Conservação dos Recursos Naturais/métodos , Demografia , Ecossistema , Modelos Teóricos , Animais , Bolívia , Geografia , Mapas como Assunto , Peru , Especificidade da EspécieRESUMO
Trajectory or path planning is a fundamental issue in a wide variety of applications. In this article, we show that it is possible to solve path planning on a maze for multiple start point and endpoint highly efficiently with a novel configuration of multilayer networks that use only weighted pooling operations, for which no network training is needed. These networks create solutions, which are identical to those from classical algorithms such as breadth-first search (BFS), Dijkstra's algorithm, or TD(0). Different from competing approaches, very large mazes containing almost one billion nodes with dense obstacle configuration and several thousand importance-weighted path endpoints can this way be solved quickly in a single pass on parallel hardware.
RESUMO
Active networks of biopolymers and motor proteins in vitro self-organize and exhibit dynamic structures on length scales much larger than the interacting individual components of which they consist. How the dynamics is related across the range of length scales is still an open question. Here, we experimentally characterize and quantify the dynamic behavior of isolated microtubule bundles that bend due to the activity of motor proteins. At the motor level, we track and describe the motion features of kinesin-1 clusters stepping within the bending bundles. We find that there is a separation of length scales by at least 1 order of magnitude. At a run length of <1 µm, kinesin-1 activity leads to a bundle curvature in the range of tens of micrometers. We propose that the distribution of microtubule polarity plays a crucial role in the bending dynamics that we observe at both the bundle and motor levels. Our results contribute to the understanding of fundamental principles of vital intracellular processes by disentangling the multiscale dynamics in out-of-equilibrium active networks composed of cytoskeletal elements.
RESUMO
MicroRNAs are small non-coding RNAs that have emerged as post-transcriptional regulators involved in development and function of different types of immune cells, and aberrant miRNA expression has often been linked to cancer. One prominent miRNA family in the latter setting is the miR-15 family, consisting of the three clusters miR-15a/16-1, miR-15b/16-2 and miR-497/195, which is best known for its prominent tumor suppressive role in chronic lymphocytic leukemia (CLL). However, little is known about the physiological role of the miR-15 family. In this study, we provide a comprehensive in vivo analysis of the physiological functions of miR-15a/16-1 and miR-15b/16-2, both of which are highly expressed in immune cells, in early B cell development. In particular, we report a previously unrecognized physiological function of the miR-15 family in restraining progenitor B cell expansion, as loss of both clusters induces an increase of the pro-B as well as pre-B cell compartments. Mechanistically, we find that the miR-15 family mediates its function through repression of at least two different types of target genes: First, we confirm that the miR-15 family suppresses several prominent cell cycle regulators such as Ccne1, Ccnd3 and Cdc25a also in vivo, thereby limiting the proliferation of progenitor B cells. Second, this is complemented by direct repression of the Il7r gene, which encodes the alpha chain of the IL-7 receptor (IL7R), one of the most critical growth factor receptors for early B cell development. In consequence, deletion of the miR-15a/16-1 and miR-15b/16-2 clusters stabilizes Il7r transcripts, resulting in enhanced IL7R surface expression. Consistently, our data show an increased activation of PI3K/AKT, a key signaling pathway downstream of the IL7R, which likely drives the progenitor B cell expansion we describe here. Thus, by deregulating a target gene network of cell cycle and signaling mediators, loss of the miR-15 family establishes a pro-proliferative milieu that manifests in an enlarged progenitor B cell pool.
Assuntos
MicroRNAs , Receptores de Interleucina-7 , Proliferação de Células/genética , MicroRNAs/metabolismo , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Receptores de Interleucina-7/genéticaRESUMO
MiRNAs are small noncoding RNAs that promote the sequence-specific repression of their respective target genes, thereby regulating diverse physiological as well as pathological processes. Here, we identify a novel role of the miR-26 family in early B cell development. We show that enhanced expression of miR-26 family members potently blocks the pre-B to immature B cell transition, promotes pre-B cell expansion and eventually enables growth factor independency. Mechanistically, this is at least partially mediated by direct repression of the tumor-suppressor Pten, which consequently enhances PI3K-AKT signaling. Conversely, limiting miR-26 activity in a more physiological loss-of-function approach counteracts proliferation and enhances pre-B cell differentiation in vitro as well as in vivo. We therefore postulate a rheostat-like role for the miR-26 family in progenitor B cells, with an increase in mature miR-26 levels signaling cell expansion, and facilitating pre-B to the immature B cell progression when reduced.
Assuntos
MicroRNAs , Fosfatidilinositol 3-Quinases , Proliferação de Células/genética , MicroRNAs/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genéticaRESUMO
Checkpoint kinase 1 (CHK1; encoded by CHEK1) is an essential gene that monitors DNA replication fidelity and prevents mitotic entry in the presence of under-replicated DNA or exogenous DNA damage. Cancer cells deficient in p53 tumor suppressor function reportedly develop a strong dependency on CHK1 for proper cell cycle progression and maintenance of genome integrity, sparking interest in developing kinase inhibitors. Pharmacological inhibition of CHK1 triggers B-Cell CLL/Lymphoma 2 (BCL2)-regulated cell death in malignant cells largely independently of p53, and has been suggested to kill p53-deficient cancer cells even more effectively. Next to p53 status, our knowledge about factors predicting cancer cell responsiveness to CHK1 inhibitors is limited. Here, we conducted a genome-wide CRISPR/Cas9-based loss-of-function screen to identify genes defining sensitivity to chemical CHK1 inhibitors. Next to the proapoptotic BCL2 family member, BCL2 Binding Component 3 (BBC3; also known as PUMA), the F-box protein S-phase Kinase-Associated Protein 2 (SKP2) was validated to tune the cellular response to CHK1 inhibition. SKP2 is best known for degradation of the Cyclin-dependent Kinase Inhibitor 1B (CDKN1B; also known as p27), thereby promoting G1-S transition and cell cycle progression in response to mitogens. Loss of SKP2 resulted in the predicted increase in p27 protein levels, coinciding with reduced DNA damage upon CHK1-inhibitor treatment and reduced cell death in S-phase. Conversely, overexpression of SKP2, which consequently results in reduced p27 protein levels, enhanced cell death susceptibility to CHK1 inhibition. We propose that assessing SKP2 and p27 expression levels in human malignancies will help to predict the responsiveness to CHK1-inhibitor treatment.