scholarly journals Structure and mechanism of a Hypr GGDEF enzyme that activates cGAMP signaling to control extracellular metal respiration

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Zachary F Hallberg ◽  
Chi Ho Chan ◽  
Todd A Wright ◽  
Philip J Kranzusch ◽  
Kevin W Doxzen ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Geobacter Sulfurreducens ◽  
Electricity Production ◽  
Signaling Proteins ◽  
Rna Seq ◽  
Model Combining ◽  
Ggdef Domain ◽  
Rare Opportunity

A newfound signaling pathway employs a GGDEF enzyme with unique activity compared to the majority of homologs associated with bacterial cyclic di-GMP signaling. This system provides a rare opportunity to study how signaling proteins natively gain distinct function. Using genetic knockouts, riboswitch reporters, and RNA-Seq, we show that GacA, the Hypr GGDEF in Geobacter sulfurreducens, specifically regulates cyclic GMP-AMP (3′,3′-cGAMP) levels in vivo to stimulate gene expression associated with metal reduction separate from electricity production. To reconcile these in vivo findings with prior in vitro results that showed GacA was promiscuous, we developed a full kinetic model combining experimental data and mathematical modeling to reveal mechanisms that contribute to in vivo specificity. A 1.4 Å-resolution crystal structure of the Geobacter Hypr GGDEF domain was determined to understand the molecular basis for those mechanisms, including key cross-dimer interactions. Together these results demonstrate that specific signaling can result from a promiscuous enzyme.

scholarly journals cGAMP signaling controls a transient surface-associated lifestyle

2018 ◽  
Author(s):  
Zachary F Hallberg ◽  
Chi Ho Chan ◽  
Todd A Wright ◽  
Philip J Kranzusch ◽  
Kevin W Doxzen ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Geobacter Sulfurreducens ◽  
Electricity Production ◽  
Signaling Proteins ◽  
Rna Seq ◽  
Model Combining ◽  
Ggdef Domain ◽  
Rare Opportunity

A newfound signaling pathway employs a GGDEF enzyme with unique activity compared to the majority of homologs associated with bacterial cyclic di-GMP signaling. This system provides a rare opportunity to study how signaling proteins natively gain distinct function. Using genetic knockouts, riboswitch reporters, and RNA-Seq, we show that GacA, the Hypr GGDEF in Geobacter sulfurreducens, specifically regulates cyclic GMP-AMP (3′,3′-cGAMP) levels in vivo to stimulate gene expression associated with metal reduction separate from electricity production. To reconcile these in vivo findings with prior in vitro results that showed GacA was promiscuous, we developed a full kinetic model combining experimental data and mathematical modeling to reveal mechanisms that contribute to in vivo specificity. A 1.4 Å-resolution crystal structure of the Geobacter Hypr GGDEF domain was determined to understand the molecular basis for those mechanisms, including key cross-dimer interactions. Together these results demonstrate that specific signaling can result from a promiscuous enzyme.

scholarly journals BRD4 PROTAC degrader ARV-825 inhibits T-cell acute lymphoblastic leukemia by targeting 'Undruggable' Myc-pathway genes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Shuiyan Wu ◽  
You Jiang ◽  
Yi Hong ◽  
Xinran Chu ◽  
Zimu Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Rna Seq ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Bet Protein

Abstract Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a high risk of induction failure and poor outcomes, with relapse due to drug resistance. Recent studies show that bromodomains and extra-terminal (BET) protein inhibitors are promising anti-cancer agents. ARV-825, comprising a BET inhibitor conjugated with cereblon ligand, was recently developed to attenuate the growth of multiple tumors in vitro and in vivo. However, the functional and molecular mechanisms of ARV-825 in T-ALL remain unclear. This study aimed to investigate the therapeutic efficacy and potential mechanism of ARV-825 in T-ALL. Methods Expression of the BRD4 were determined in pediatric T-ALL samples and differential gene expression after ARV-825 treatment was explored by RNA-seq and quantitative reverse transcription-polymerase chain reaction. T-ALL cell viability was measured by CCK8 assay after ARV-825 administration. Cell cycle was analyzed by propidium iodide (PI) staining and apoptosis was assessed by Annexin V/PI staining. BRD4, BRD3 and BRD2 proteins were detected by western blot in cells treated with ARV-825. The effect of ARV-825 on T-ALL cells was analyzed in vivo. The functional and molecular pathways involved in ARV-825 treatment of T-ALL were verified by western blot and chromatin immunoprecipitation (ChIP). Results BRD4 expression was higher in pediatric T-ALL samples compared with T-cells from healthy donors. High BRD4 expression indicated a poor outcome. ARV-825 suppressed cell proliferation in vitro by arresting the cell cycle and inducing apoptosis, with elevated poly-ADP ribose polymerase and cleaved caspase 3. BRD4, BRD3, and BRD2 were degraded in line with reduced cereblon expression in T-ALL cells. ARV-825 had a lower IC50 in T-ALL cells compared with JQ1, dBET1 and OTX015. ARV-825 perturbed the H3K27Ac-Myc pathway and reduced c-Myc protein levels in T-ALL cells according to RNA-seq and ChIP. In the T-ALL xenograft model, ARV-825 significantly reduced tumor growth and led to the dysregulation of Ki67 and cleaved caspase 3. Moreover, ARV-825 inhibited cell proliferation by depleting BET and c-Myc proteins in vitro and in vivo. Conclusions BRD4 indicates a poor prognosis in T-ALL. The BRD4 degrader ARV-825 can effectively suppress the proliferation and promote apoptosis of T-ALL cells via BET protein depletion and c-Myc inhibition, thus providing a new strategy for the treatment of T-ALL.

scholarly journals The BET Inhibitor JQ1 Augments the Antitumor Efficacy of Gemcitabine in Preclinical Models of Pancreatic Cancer

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3470
Author(s):  
Aubrey L. Miller ◽  
Patrick L. Garcia ◽  
Samuel C. Fehling ◽  
Tracy L. Gamblin ◽  
Rebecca B. Vance ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Dna Damage ◽  
Cholesterol Biosynthesis ◽  
Antitumor Efficacy ◽  
Rna Seq ◽  
Bet Inhibitor ◽  
Bet Inhibitors ◽  
Xenograft Models

Gemcitabine is used to treat pancreatic cancer (PC), but is not curative. We sought to determine whether gemcitabine + a BET bromodomain inhibitor was superior to gemcitabine, and identify proteins that may contribute to the efficacy of this combination. This study was based on observations that cell cycle dysregulation and DNA damage augment the efficacy of gemcitabine. BET inhibitors arrest cells in G1 and allow increases in DNA damage, likely due to inhibition of expression of DNA repair proteins Ku80 and RAD51. BET inhibitors (JQ1 or I-BET762) + gemcitabine were synergistic in vitro, in Panc1, MiaPaCa2 and Su86 PC cell lines. JQ1 + gemcitabine was more effective in vivo than either drug alone in patient-derived xenograft models (P < 0.01). Increases in the apoptosis marker cleaved caspase 3 and DNA damage marker γH2AX paralleled antitumor efficacy. Notably, RNA-seq data showed that JQ1 + gemcitabine selectively inhibited HMGCS2 and APOC1 ~6-fold, compared to controls. These proteins contribute to cholesterol biosynthesis and lipid metabolism, and their overexpression supports tumor cell proliferation. IPA data indicated that JQ1 + gemcitabine selectively inhibited the LXR/RXR activation pathway, suggesting the hypothesis that this inhibition may contribute to the observed in vivo efficacy of JQ1 + gemcitabine.

scholarly journals MODL-16. ABEMACICLIB, A SELECTIVE CDK4/6 INHIBITOR, RESTRICTS GROWTH OF PEDIATRIC GLIAL-LINEAGE TUMORS IN VITRO AND IN VIVO

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii414-iii414
Author(s):  
Muh-Lii Liang ◽  
Tsung-Han Hsieh ◽  
Tai-Tong Wong
Keyword(s):  
Dna Repair ◽  
Therapeutic Strategy ◽  
Target Genes ◽  
Rna Seq ◽  
Downstream Target ◽  
Rb Phosphorylation ◽  
Glial Lineage

Abstract BACKGROUND Glial-lineage tumors constitute a heterogeneous group of neoplasms, comprising gliomas, oligodendrogliomas, and ependymomas, which account for 40%–50% of all pediatric central nervous system tumors. Advances in modern neuro-oncological therapeutics are aimed at improving neoadjuvant chemotherapy and deferring radiotherapy because radiation exposure may cause long-term side effects on the developing brain in young children. Despite aggressive treatment, more than half the high-grade gliomas (pHGGs) and one-third of ependymomas exhibit recurrence within 2 years of initial treatment. METHODS By using integrated bioinformatics and through experimental validation, we found that at least one gene among CCND1, CDK4, and CDK6 was overexpressed in pHGGs and ependymomas. RESULTS The use of abemaciclib, a highly selective CDK4/6 inhibitor, effectively inhibited cell proliferation and reduced the expression of cell cycle–related and DNA repair–related gene expression, which was determined through RNA-seq analysis. The efficiency of abemaciclib was validated in vitro in pHGGs and ependymoma cells and in vivo by using subcutaneously implanted ependymoma cells from patient-derived xenograft (PDX) in mouse models. Abemaciclib demonstrated the suppression of RB phosphorylation, downstream target genes of E2F, G2M checkpoint, and DNA repair, resulting in tumor suppression. CONCLUSION Abemaciclib showed encouraging results in preclinical pediatric glial-lineage tumors models and represented a potential therapeutic strategy for treating challenging tumors in children.

scholarly journals lncRNA CASC19 Contributes to Radioresistance of Nasopharyngeal Carcinoma by Promoting Autophagy via AMPK-mTOR Pathway

2021 ◽  
Vol 22 (3) ◽  
pp. 1407
Author(s):  
Hongxia Liu ◽  
Wang Zheng ◽  
Qianping Chen ◽  
Yuchuan Zhou ◽  
Yan Pan ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Cell Line ◽  
Malignant Tumors ◽  
Underlying Mechanism ◽  
Mtor Pathway ◽  
Rna Seq ◽  
Cellular Autophagy ◽  
First Time

Nasopharyngeal carcinoma (NPC) is one of the most frequent head and neck malignant tumors and is majorly treated by radiotherapy. However, radiation resistance remains a serious obstacle to the successful treatment of NPC. The aim of this study was to discover the underlying mechanism of radioresistance and to elucidate novel genes that may play important roles in the regulation of NPC radiosensitivity. By using RNA-seq analysis of NPC cell line CNE2 and its radioresistant cell line CNE2R, lncRNA CASC19 was screened out as a candidate radioresistance marker. Both in vitro and in vivo data demonstrated that a high expression level of CASC19 was positively correlated with the radioresistance of NPC, and the radiosensitivity of NPC cells was considerably enhanced by knockdown of CASC19. The incidence of autophagy was enhanced in CNE2R in comparison with CNE2 and another NPC cell line HONE1, and silencing autophagy with LC3 siRNA (siLC3) sensitized NPC cells to irradiation. Furthermore, CASC19 siRNA (siCASC19) suppressed cellular autophagy by inhibiting the AMPK/mTOR pathway and promoted apoptosis through the PARP1 pathway. Our results revealed for the first time that lncRNA CASC19 contributed to the radioresistance of NPC by regulating autophagy. In significance, CASC19 might be a potential molecular biomarker and a new therapeutic target in NPC.

scholarly journals xMD-miRNA-seq to generate near in vivo miRNA expression estimates in colon epithelial cells

2018 ◽  
Author(s):  
Avi Z. Rosenberg ◽  
Carrie Wright ◽  
Karen Fox-Talbot ◽  
Anandita Rajpurohit ◽  
Courtney Williams ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Small Rna ◽  
Mirna Expression ◽  
Low Cost ◽  
Expression Patterns ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
Cell Type

AbstractAccurate, RNA-seq based, microRNA (miRNA) expression estimates from primary cells have recently been described. However, this in vitro data is mainly obtained from cell culture, which is known to alter cell maturity/differentiation status, significantly changing miRNA levels. What is needed is a robust method to obtain in vivo miRNA expression values directly from cells. We introduce expression microdissection miRNA small RNA sequencing (xMD-miRNA-seq), a method to isolate cells directly from formalin fixed paraffin-embedded (FFPE) tissues. xMD-miRNA-seq is a low-cost, high-throughput, immunohistochemistry-based method to capture any cell type of interest. As a proof-of-concept, we isolated colon epithelial cells from two specimens and performed low-input small RNA-seq. We generated up to 600,000 miRNA reads from the samples. Isolated epithelial cells, had abundant epithelial-enriched miRNA expression (miR-192; miR-194; miR-200b; miR-200c; miR-215; miR-375) and overall similar miRNA expression patterns to other epithelial cell populations (colonic enteroids and flow-isolated colon epithelium). xMD-derived epithelial cells were generally not contaminated by other adjacent cells of the colon as noted by t-SNE analysis. xMD-miRNA-seq allows for simple, economical, and efficient identification of cell-specific miRNA expression estimates. Further development will enhance rapid identification of cell-specific miRNA expression estimates in health and disease for nearly any cell type using archival FFPE material.

scholarly journals Single cell RNA sequencing of calvarial and long bone endocortical cells

2019 ◽  
Author(s):  
Ugur M. Ayturk ◽  
Joseph P. Scollan ◽  
Alexander Vesprey ◽  
Christina M. Jacobsen ◽  
Paola Divieti Pajevic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cultured Cells ◽  
Neutralizing Antibody ◽  
Long Bone ◽  
Appendicular Skeleton ◽  
Rna Seq ◽  
Isolated Cells

ABSTRACTSingle cell RNA-seq (scRNA-seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether scRNA-seq could be used to detect the effect of environmental and pharmacologic perturbations on osteoblasts. We began with a commonly used in vitro system in which freshly isolated neonatal mouse calvarial cells are expanded and induced to produce a mineralized matrix. We used scRNA-seq to compare the relative cell type abundances and the transcriptomes of freshly isolated cells to those that had been cultured for 12 days in vitro. We observed that the percentage of macrophage-like cells increased from 6% in freshly isolated calvarial cells to 34% in cultured cells. We also found that Bglap transcripts were abundant in freshly isolated osteoblasts but nearly undetectable in the cultured calvarial cells. Thus, scRNA-seq revealed significant differences between heterogeneity of cells in vivo and in vitro. We next performed scRNA-seq on freshly recovered long bone endocortical cells from mice that received either vehicle or Sclerostin-neutralizing antibody for 1 week. Bone anabolism-associated transcripts were also not significantly increased in immature and mature osteoblasts recovered from Sclerostin-neutralizing antibody treated mice; this is likely a consequence of being underpowered to detect modest changes in gene expression, since only 7% of the sequenced endocortical cells were osteoblasts, and a limited portion of their transcriptomes were sampled. We conclude that scRNA-seq can detect changes in cell abundance, identity, and gene expression in skeletally derived cells. In order to detect modest changes in osteoblast gene expression at the single cell level in the appendicular skeleton, larger numbers of osteoblasts from endocortical bone are required.

scholarly journals Quercetin inhibits intrahepatic cholangiocarcinoma by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation via the NF-κB pathway

2021 ◽  
Author(s):  
Yinghui Song ◽  
Zhihua Zhang ◽  
Qin Chai ◽  
GuoYi Xia ◽  
Zhangtao Yu ◽  
...  
Keyword(s):  
Intrahepatic Cholangiocarcinoma ◽  
Clonogenic Assay ◽  
Direct Interaction ◽  
Rna Seq ◽  
Wound Healing Assay ◽  
Tumor Bearing ◽  
Mesenchymal Transformation ◽  
First Time

Abstract Intrahepatic cholangiocarcinoma (ICC) is a rare high-fatal hepatobiliary malignancy, the treatment option of ICC is very limited, and the prognosis is also poor. Recently, emerging evidence has shown the potential of quercetin (QE) for cancer therapy. We explored the effect and mechanism of QE on ICC in vitro and in vivo. CCK-8 assay and Clonogenic assay showed that QE could inhibit ICC cells proliferation and survival. PI staining suggested QE could induce ICC cells arrest in G1 phase. AV/PI staining suggested QE could promote ICC cells apoptosis. Wound Healing Assay and Transwell chamber experiment suggested QE could inhibit ICC cells EMT. RNA-seq, the changes in the structure of mitochondria by electron microscopy and the key markers of ferroptosis (free iron ions, MDA, SOD, GPX4) were supported QE could promote ferroptosis in ICC cells. Molecular docking showed that QE had direct interaction with NF-κB and GPX4. In vivo, treatment with QE inhibited tumor growth and prolonged survival time of tumor-bearing nude mice. Our data for the first time suggest that QE is a new ferroptosis inducer and combinative treatment of inhibiting NF-κB in ICC cells by inducing ferroptosis and inhibiting EMT, which will hopefully provide a prospective strategy for ICC patients.

scholarly journals Plasmodium vivax and Drug Resistance

2021 ◽  
Author(s):  
Puji Budi Setia Asih ◽  
Din Syafruddin
Keyword(s):  
Drug Resistance ◽  
Molecular Basis ◽  
Indirect Evidence ◽  
Antimalarial Drugs ◽  
In Vivo Studies ◽  
In Vitro Cultivation ◽  
Radical Cure ◽  
The Status

Resistance to antimalarial drugs is a threat to global efforts to eliminate malaria by 2030. Currently, treatment for vivax malaria uses chloroquine or ACT for uncomplicated P. vivax whereas primaquine is given to eliminate latent liver stage infections (a method known as radical cure). Studies on P. vivax resistance to antimalarials and the molecular basis of resistance lags far behind the P. falciparum as in vitro cultivation of the P. vivax has not yet been established. Therefore, data on the P. vivax resistance to any antimalarial drugs are generated through in vivo studies or through monitoring of antimalarial treatments in mixed species infection. Indirect evidence through drug selective pressure on the parasites genome, as evidenced by the presence of the molecular marker(s) for drug resistance in areas where P. falciparum and P. vivax are distributed in sympatry may reflect, although require validation, the status of P. vivax resistance. This review focuses on the currently available data that may represent the state-of-the art of the P. vivax resistance status to antimalarial to anticipate the challenge for malaria elimination by 2030.

scholarly journals A Comparative Transcriptome Analysis of Human and Porcine Choroid Plexus Cells in Response to Streptococcus suis Serotype 2 Infection Points to a Role of Hypoxia

2021 ◽  
Vol 11 ◽  
Author(s):  
Alexa N. Lauer ◽  
Rene Scholtysik ◽  
Andreas Beineke ◽  
Christoph Georg Baums ◽  
Kristin Klose ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Inflammatory Response ◽  
Choroid Plexus ◽  
Cellular Proliferation ◽  
Inflammatory Responses ◽  
Streptococcus Suis ◽  
Gene Set Enrichment Analysis ◽  
Rna Seq

Streptococcus suis (S. suis) is an important opportunistic pathogen, which can cause septicemia and meningitis in pigs and humans. Previous in vivo observations in S. suis-infected pigs revealed lesions at the choroid plexus (CP). In vitro experiments with primary porcine CP epithelial cells (PCPEC) and human CP epithelial papilloma (HIBCPP) cells demonstrated that S. suis can invade and traverse the CP epithelium, and that the CP contributes to the inflammatory response via cytokine expression. Here, next generation sequencing (RNA-seq) was used to compare global transcriptome profiles of PCPEC and HIBCPP cells challenged with S. suis serotype (ST) 2 infected in vitro, and of pigs infected in vivo. Identified differentially expressed genes (DEGs) were, amongst others, involved in inflammatory responses and hypoxia. The RNA-seq data were validated via quantitative PCR of selected DEGs. Employing Gene Set Enrichment Analysis (GSEA), 18, 28, and 21 enriched hallmark gene sets (GSs) were identified for infected HIBCPP cells, PCPEC, and in the CP of pigs suffering from S. suis ST2 meningitis, respectively, of which eight GSs overlapped between the three different sample sets. The majority of these GSs are involved in cellular signaling and pathways, immune response, and development, including inflammatory response and hypoxia. In contrast, suppressed GSs observed during in vitro and in vivo S. suis ST2 infections included those, which were involved in cellular proliferation and metabolic processes. This study suggests that similar cellular processes occur in infected human and porcine CP epithelial cells, especially in terms of inflammatory response.

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