Role of TGF-β/SMAD4/c-MYC-regulated NLE1 in tumor growth and liver metastasis of colorectal cancer

TGF-β signaling plays an essential role in colorectal cancer (CRC) progression. Patients frequently suffer from mutations in the TGFBR2 gene or deletions of SMAD4. These mutations correlate with a poorer survival rate in CRC patients. How these genetic changes contribute to tumor growth and metastatic spread is not fully understood. Thus, an in-depth analysis of the gene expression profiles of CRC patients acquiring a SMAD4 deletion could help to identify new therapeutic options. Here, we used the CRISPR/Cas9 approach to introduce a deletion of SMAD4 in patient-derived tumor organoids (PDTOs) and investigate their effect on the gene expression profile when exposed to a TGF-β-rich environment. Thereby, we could observe that SMAD4-deficient PDTOs acquire an enrichment in the expression of c-MYC target genes and human colonic stem cell gene sets. Only 11 of the 284 deregulated genes (FBL, GINS2, IFITM3, LSM6, MYC, NAT10, NLE1, POLR3K, PRMT1, PSMG4, RRP1) confer an advantage for tumor cell fitness according to DepMap. Four of these genes (FBL, NAT10, NLE1, RRP1) are part of ribosome biogenesis. They might contribute to the increased protein biosynthesis levels observed in advanced CRC. Since the role of NLE1 in the progression of CRC is not fully understood, we decided to focus our studies on the regulation and function of NLE1 in CRC. We found that NLE1 expression was upregulated upon SMAD4 loss in TGF-β-exposed PDTOs and that c-MYC can bind to the promoter of NLE1. Thereby, c-MYC prevents TGF-β-mediated downregulation of NLE1. Furthermore, NLE1 levels were higher in different CRC cohorts than in normal tissues and significantly enriched in Wnt/MYC CRC molecular subtypes. After the deletion of NLE1 in different CRC cell lines and PDTOs, we could observe an apparent reduction in de novo protein biosynthesis rates. In accordance, the deletion of NLE1 also resulted in slower proliferation kinetics, reduced colony formation, and less anchorage-independent growth in CRC cell lines. Moreover, NLE1 knockout cells showed higher levels of apoptosis, a reduced migratory/invasive capacity, and underwent cell cycle arrest and apoptosis. In the case of PDTOs, smaller organoid sizes and reduced clonogenicity during the loss of NLE1 were observed. This phenotype was also reflected in an endoscopy-guided orthotopic mouse transplantation model. Primary tumors lacking NLE1 expression were smaller than NLE1 wild-type derived tumors, and the affected animals showed less metastatic burden in the liver. As a downstream effect, NLE1-deficient cells showed activation of p38/MAPK, accumulation of p62- and LC3-positive structures, which defines impaired autophagy, and higher ROS levels. Furthermore, deletion of NLE1 in TP53 proficient cancer and normal human colonic epithelial cells led to cell cycle arrest rather than apoptosis, and NLE1 mRNA levels predicted relapse-free survival in CRC patients. In summary, we could show that a TGF-β/SMAD4/c-MYC axis regulates NLE1 and represents a limiting factor for de novo protein biosynthesis and the tumorigenic potential of advanced CRC. To which extent therapeutic targeting of NLE1 can be used as a treatment of CRC in a clinical setting warrants further investigation. TGF-β signaling plays an essential role in colorectal cancer (CRC) progression. Patients frequently suffer from mutations in the TGFBR2 gene or deletions of SMAD4. These mutations correlate with a poorer survival rate in CRC patients. How these genetic changes contribute to tumor growth and metastatic spread is not fully understood. Thus, an in-depth analysis of the gene expression profiles of CRC patients acquiring a SMAD4 deletion could help to identify new therapeutic options. Here, we used the CRISPR/Cas9 approach to introduce a deletion of SMAD4 in patient-derived tumor organoids (PDTOs) and investigate their effect on the gene expression profile when exposed to a TGF-β-rich environment. Thereby, we could observe that SMAD4-deficient PDTOs acquire an enrichment in the expression of c-MYC target genes and human colonic stem cell gene sets. Only 11 of the 284 deregulated genes (FBL, GINS2, IFITM3, LSM6, MYC, NAT10, NLE1, POLR3K, PRMT1, PSMG4, RRP1) confer an advantage for tumor cell fitness according to DepMap. Four of these genes (FBL, NAT10, NLE1, RRP1) are part of ribosome biogenesis. They might contribute to the increased protein biosynthesis levels observed in advanced CRC. Since the role of NLE1 in the progression of CRC is not fully understood, we decided to focus our studies on the regulation and function of NLE1 in CRC. We found that NLE1 expression was upregulated upon SMAD4 loss in TGF-β-exposed PDTOs and that c-MYC can bind to the promoter of NLE1. Thereby, c-MYC prevents TGF-β-mediated downregulation of NLE1. Furthermore, NLE1 levels were higher in different CRC cohorts than in normal tissues and significantly enriched in Wnt/MYC CRC molecular subtypes. After the deletion of NLE1 in different CRC cell lines and PDTOs, we could observe an apparent reduction in de novo protein biosynthesis rates. In accordance, the deletion of NLE1 also resulted in slower proliferation kinetics, reduced colony formation, and less anchorage-independent growth in CRC cell lines. Moreover, NLE1 knockout cells showed higher levels of apoptosis, a reduced migratory/invasive capacity, and underwent cell cycle arrest and apoptosis. In the case of PDTOs, smaller organoid sizes and reduced clonogenicity during the loss of NLE1 were observed. This phenotype was also reflected in an endoscopy-guided orthotopic mouse transplantation model. Primary tumors lacking NLE1 expression were smaller than NLE1 wild-type derived tumors, and the affected animals showed less metastatic burden in the liver. As a downstream effect, NLE1-deficient cells showed activation of p38/MAPK, accumulation of p62- and LC3-positive structures, which defines impaired autophagy, and higher ROS levels. Furthermore, deletion of NLE1 in TP53 proficient cancer and normal human colonic epithelial cells led to cell cycle arrest rather than apoptosis, and NLE1 mRNA levels predicted relapse-free survival in CRC patients. In summary, we could show that a TGF-β/SMAD4/c-MYC axis regulates NLE1 and represents a limiting factor for de novo protein biosynthesis and the tumorigenic potential of advanced CRC. To which extent therapeutic targeting of NLE1 can be used as a treatment of CRC in a clinical setting warrants further investigation.Der TGF-β Signalweg spielt eine zentrale Rolle bei der Entstehung von Darmkrebs. Häufig zeigen Patienten Mutationen im TGFBR2 Gen oder Deletionen von SMAD4. Diese Mutationen korrelieren mit einer schlechten Überlebensrate von Darmkrebspatienten. Wie diese genetischen Veränderungen zum Tumorwachstum und zur Ausbreitung von Metastasen beitragen, ist bisher nicht vollständig verstanden. Daher könnte eine umfassende Analyse der Genexpressionsprofile von Darmkrebspatienten, die eine SMAD4-Deletion erwerben, helfen, neue therapeutische Ansätze zu identifizieren. Zunächst wurde mittels CRISPR/Cas9 eine Deletion von SMAD4 in Patienten-abgeleiteten Tumororganoiden (PDTOs) eingeführt, um dann ihre Wirkung auf das Genexpressionsprofil zu untersuchen, wenn sie einer TGF-β reichen Umgebung ausgesetzt sind. Dabei konnten wir beobachten, dass SMAD4-defiziente PDTOs eine Anreicherung für c-MYC-Zielgene und humane Kolonstammzellgene erwerben. Interessanterweise zeigten nur 11 der 284 deregulierten Gene (FBL, GINS2, IFITM3, LSM6, MYC, NAT10, NLE1, POLR3K, PRMT1, PSMG4, RRP1) einen Vorteil für die Fitness von Tumorzellen. 4 dieser Gene (FBL, NAT10, NLE1, RRP1) sind Teil der Ribosomenbiogenese und könnten zu einer erhöhten Proteinbiosynthese beitragen, die bei fortgeschrittenem Darmkrebs beobachtet wird. Da die Rolle von NLE1 beim Fortschreiten von Darmkrebs noch nicht vollständig geklärt ist, hatten wir uns entschlossen, unseren Fokus daraufzulegen. Dabei fanden wir heraus, dass die Expression von NLE1 nach SMAD4 Verlust in TGF-β-exponierten PDTOs hochreguliert wurde und dass c-MYC in der Lage ist, an den Promotor von NLE1 zu binden. Dadurch verhindert c-MYC das TGF-β-vermittelte Herunterregeln von NLE1. Darüber hinaus war die Expression von NLE1 in verschiedenen Kohorten von Darmkrebspatienten höher im Vergleich zu Normalgewebe und insbesondere in Wnt/MYC Subgruppen von Darmkrebspatienten angereichert. Nach Deletion von NLE1 in verschiedenen kolorektalen Krebszelllinien und PDTOs konnten wir eine deutliche Reduktion der de-novo-Proteinbiosyntheseraten beobachten. Dementsprechend führte die Deletion von NLE1 auch zu einer langsameren Proliferationskinetik, weniger Koloniebildung und weniger verankerungsunabhängigem Wachstum in kolorektalen Krebszelllinien. Darüber hinaus zeigten NLE1-defiziente Zellen verstärkt Apoptose, eine geringere Migrations-/Invasionskapazität und einen Stillstand des Zellzyklus. Bei den PDTOs wurde eine kleinere Organoidgröße und eine verringerte Klonogenität nach Verlusts von NLE1 beobachtet. Dieser Phänotyp spiegelte sich auch in einem Endoskopie-gestützten orthotopen Maustransplantationsmodell wieder. Primärtumore von NLE1-defizienten Zellen waren im Vergleich zu Tumoren, die vom NLE1 Wildtyp Zellen stammten, kleiner und zeigten auch eine geringere Fähigkeit zur Ausbildung von Metastasen in der Leber. Des Weiteren zeigten NLE1-defiziente Zellen eine Aktivierung von p38/MAPK, eine Akkumulation von p62- und LC3-positiven Strukturen, was eine beeinträchtigte Autophagie definiert, und vermehrt oxidativen Stress. Darüber hinaus führte die Deletion von NLE1 in TP53 Wildtyp Krebs- und normalen menschlichen Dickdarmepithelzellen eher zu einem Zellzyklusstillstand als zu Apoptose, und die NLE1 mRNA Expression prognostizierte ein Rezidiv-freies Überleben bei Darmkrebspatienten. Zusammenfassend konnte gezeigt werden, dass NLE1 durch eine TGF-β/SMAD4/c-MYC-Achse reguliert wird und ein limitierender Faktor für die de-novo-Proteinbiosynthese und das tumorogene Potenzial von fortgeschrittenem Darmkrebs darstellt. Inwieweit eine Blockierung von NLE1 als therapeutischer Ansatzpunkt im Darmkrebs dienen kann, müssen weitere Studien erst noch zeigen

Разработка технологии получения твердой дозированной лекарственной формы Грамицидина С методом влажной грануляции во взвешенном слое

Работа посвящена исследованию процесса грануляции пептидного антибиотика с бета-циклодекстрином во взвешенном слое. Получен и охарактеризован комплекс включения антибиотика с бета-циклодекстрином. Изучены параметры, влияющие на эффективность процесса инкапсуляции. Анализ высвобождения показал растворимость грамицидина С в водном растворе. В работе были исследованы технологические свойства смеси и свойства таблетированной формы. Разработана технологическая схема производства.In this study granulation of a peptide antibiotic with beta-cyclodextrin in bed fluidization was investigated. The inclusion complex of antibiotic with beta-cyclodextrin was obtained and characterized. The parameters of granulation affecting on encapsulation process were studied. A release analysis showed the solubility of gramicidin S in aqueous solution. The processability of the mixture was investigated and the property of the bulk product was analyzed. The technological scheme of production is developed

Pelagic microbial heterotrophy in response to a highly productive bloom of <i>Phaeocystis antarctica</i> in the Amundsen Sea Polynya, Antarctica

Abstract Heterotrophic bacteria play a key role in marine carbon cycling, and understanding their activities in polar systems is important for considering climate change impacts there. One goal of the ASPIRE project was to examine the relationship between the phytoplankton bloom and bacterial heterotrophy in the Amundsen Sea Polynya (ASP). Bacterial abundance, production (BP), respiration, growth efficiency, and extracellular enzyme activity (EEA) were compared to nutrient and organic matter inventories, chlorophyll a (Chl a), viral and microzooplankton abundance, and net primary production (NPP). Bacterial production and respiration clearly responded (0.04–4.0 and 10–53 µg C L−1 d−1, respectively) to the buildup of a massive Phaeocystis antarctica bloom (Chl a: 0.2–22 µg L−1), with highest rates observed in the central polynya where Chl a and particulate organic carbon (POC) were greatest. The highest BP rates exceeded those reported for the Ross Sea or any other Antarctic coastal system, yet the BP:NPP ratio (2.1–9.4%) was relatively low. Bacterial respiration was also high, and growth efficiency (2–27%; median = 10%) was similar to oligotrophic systems. Thus, the integrated bacterial carbon demand (0.8–2.8 g C m−2 d−1) was a high fraction (25–128%; median = 43%) of NPP during bloom development. During peak bloom, activity was particle-associated: BP and EEA correlated well with POC, and size fractionation experiments showed that the larger size fraction (> 3 µm) accounted for a majority (∼ 75%) of the BP. The community was psychrophilic, with a 5x reduction in BP when warmed to 20°C. In deeper waters, respiration remained relatively high, likely fueled by the significant downward particle flux in the region. A highly active, particle-associated, heterotrophic microbial community clearly responded to the extraordinary phytoplankton bloom in the ASP, likely limiting biological pump efficiency during the early season

Human metapneumovirus driven IFN-β production antagonizes macrophage transcriptional induction of IL1-β in response to bacterial pathogens

Human metapneumovirus (HMPV) is a pneumovirus that may cause severe respiratory disease in humans. HMPV infection has been found to increase susceptibility to bacterial superinfections leading to increased morbidity and mortality. The molecular mechanisms underlying HMPV-mediated increase in bacterial susceptibility are poorly understood and largely understudied. Type I interferons (IFNs), while critical for antiviral defenses, may often have detrimental effects by skewing the host immune response and cytokine output of immune cells. It is currently unknown if HMPV skews the inflammatory response in human macrophages triggered by bacterial stimuli. Here we report that HMPV pre-infection impacts production of specific cytokines. HMPV strongly suppresses IL-1β transcription in response to LPS or heat-killed Pseudomonas aeruginosa and Streptococcus pneumonia, while enhancing mRNA levels of IL-6, TNF-α and IFN-β. We demonstrate that in human macrophages the HMPV-mediated suppression of IL-1β transcription requires TANK-binding kinase 1 (TBK1) and signaling via the IFN-β-IFNAR axis. Interestingly, our results show that HMPV pre-infection did not impair the LPS-stimulated activation of NF-κB and HIF-1α, transcription factors that stimulate IL-1β mRNA synthesis in human cells. Furthermore, we determined that sequential HMPV-LPS treatment resulted in accumulation of the repressive epigenetic mark H3K27me3 at the IL1B promoter. Thus, for the first time we present data revealing the molecular mechanisms by which HMPV shapes the cytokine output of human macrophages exposed to bacterial pathogens/LPS, which appears to be dependent on epigenetic reprogramming at the IL1B promoter leading to reduced synthesis of IL-1β. These results may improve current understanding of the role of type I IFNs in respiratory disease mediated not only by HMPV, but also by other respiratory viruses that are associated with superinfections.</p

Human metapneumovirus driven IFN-β production antagonizes macrophage transcriptional induction of IL1-β in response to bacterial pathogens

Human metapneumovirus (HMPV) is a pneumovirus that may cause severe respiratory disease in humans. HMPV infection has been found to increase susceptibility to bacterial superinfections leading to increased morbidity and mortality. The molecular mechanisms underlying HMPV-mediated increase in bacterial susceptibility are poorly understood and largely understudied. Type I interferons (IFNs), while critical for antiviral defenses, may often have detrimental effects by skewing the host immune response and cytokine output of immune cells. It is currently unknown if HMPV skews the inflammatory response in human macrophages triggered by bacterial stimuli. Here we report that HMPV pre-infection impacts production of specific cytokines. HMPV strongly suppresses IL-1β transcription in response to LPS or heat-killed Pseudomonas aeruginosa and Streptococcus pneumonia, while enhancing mRNA levels of IL-6, TNF-α and IFN-β. We demonstrate that in human macrophages the HMPV-mediated suppression of IL-1β transcription requires TANK-binding kinase 1 (TBK1) and signaling via the IFN-β-IFNAR axis. Interestingly, our results show that HMPV pre-infection did not impair the LPS-stimulated activation of NF-κB and HIF-1α, transcription factors that stimulate IL-1β mRNA synthesis in human cells. Furthermore, we determined that sequential HMPV-LPS treatment resulted in accumulation of the repressive epigenetic mark H3K27me3 at the IL1B promoter. Thus, for the first time we present data revealing the molecular mechanisms by which HMPV shapes the cytokine output of human macrophages exposed to bacterial pathogens/LPS, which appears to be dependent on epigenetic reprogramming at the IL1B promoter leading to reduced synthesis of IL-1β. These results may improve current understanding of the role of type I IFNs in respiratory disease mediated not only by HMPV, but also by other respiratory viruses that are associated with superinfections

The PeptideAtlas project

The completion of the sequencing of the human genome and the concurrent, rapid development of high-throughput proteomic methods have resulted in an increasing need for automated approaches to archive proteomic data in a repository that enables the exchange of data among researchers and also accurate integration with genomic data. PeptideAtlas (http://www.peptideatlas.org/) addresses these needs by identifying peptides by tandem mass spectrometry (MS/MS), statistically validating those identifications and then mapping identified sequences to the genomes of eukaryotic organisms. A meaningful comparison of data across different experiments generated by different groups using different types of instruments is enabled by the implementation of a uniform analytic process. This uniform statistical validation ensures a consistent and high-quality set of peptide and protein identifications. The raw data from many diverse proteomic experiments are made available in the associated PeptideAtlas repository in several formats. Here we present a summary of our process and details about the Human, Drosophila and Yeast PeptideAtlas build

The PeptideAtlas project

The completion of the sequencing of the human genome and the concurrent, rapid development of high-throughput proteomic methods have resulted in an increasing need for automated approaches to archive proteomic data in a repository that enables the exchange of data among researchers and also accurate integration with genomic data. PeptideAtlas () addresses these needs by identifying peptides by tandem mass spectrometry (MS/MS), statistically validating those identifications and then mapping identified sequences to the genomes of eukaryotic organisms. A meaningful comparison of data across different experiments generated by different groups using different types of instruments is enabled by the implementation of a uniform analytic process. This uniform statistical validation ensures a consistent and high-quality set of peptide and protein identifications. The raw data from many diverse proteomic experiments are made available in the associated PeptideAtlas repository in several formats. Here we present a summary of our process and details about the Human, Drosophila and Yeast PeptideAtlas builds

Stopping Speed in the Stop-Change Task: Experimental Design Matters!

Previous research comparing the speed of inhibiting a motor response in no-foreknowledge vs. foreknowledge conditions revealed inconsistent findings. While some studies found stopping to be faster in the no-foreknowledge condition, others reported that it was faster in the foreknowledge condition. One possible explanation for the heterogeneous results might be differences in experimental design between those studies. Given this, we wanted to scrutinize whether it makes any difference if foreknowledge and no-foreknowledge are investigated in a context in which both conditions are presented separated from each other (block design) vs. in a context in which both conditions occur intermingled (event-related design). To address this question a modified stop-change task was used. In Experiment 1 no-foreknowledge and foreknowledge trials were imbedded in a block design, while Experiment 2 made use of an event-related design. We found that inhibition speed as measured with the stop signal reaction time (SSRT) was faster in the foreknowledge as compared to the no-foreknowledge condition of the event-related study, whereas no differences in SSRT between both conditions were revealed in the block design study. Analyses of reaction times to the go stimulus reflect that participants tended to slow down their go responses in both experimental contexts. However, in the foreknowledge condition of the event-related study, this strategic slowing was especially pronounced, a finding we refer to as strategic delay effect (SDE), and significantly correlated with SSRT. In sum our results suggest that inhibition speed is susceptible to strategic bias resulting from differences in experimental setup