Lipocalin 2 is a bacteriostatic protein that binds the siderophore enterobactin, an iron-chelating molecule produced by Escherichia coli (E. coli) that is required for bacterial growth. Infection of ...the lungs by E. coli is rare despite a frequent exposure to this commensal bacterium. Lipocalin 2 is an effector molecule of the innate immune system and could therefore play a role in hindering growth of E. coli in the lungs.
Lipocalin 2 knock-out and wild type mice were infected with two strains of E. coli. The lungs were removed 48 hours post-infection and examined for lipocalin 2 and MMP9 (a myeloid marker protein) by immunohistochemical staining and western blotting. Bacterial numbers were assessed in the lungs of the mice at 2 and 5 days after infection and mortality of the mice was monitored over a five-day period. The effect of administering ferrichrome (an iron source that cannot be bound by lipocalin 2) along with E.coli was also examined.
Intratracheal installation of E. coli in mice resulted in strong induction of lipocalin 2 expression in bronchial epithelium and alveolar type II pneumocytes. Migration of myeloid cells to the site of infection also contributed to an increased lipocalin 2 level in the lungs. Significant higher bacterial numbers were observed in the lungs of lipocalin 2 knock-out mice on days 2 and 5 after infection with E. coli (p < 0.05). In addition, a higher number of E. coli was found in the spleen of surviving lipocalin 2 knock-out mice on day 5 post-infection than in the corresponding wild-type mice (p < 0.05). The protective effect against E. coli infection in wild type mice could be counteracted by the siderophore ferrichrome, indicating that the protective effect of lipocalin 2 depends on its ability to sequester iron.
Lipocalin 2 is important for protection of airways against infection by E. coli.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
The myeloid transcription factor CEBPA is recurrently biallelically mutated (i.e., double mutated;
CEBPA
DM
) in acute myeloid leukemia (AML) with a combination of hypermorphic N-terminal ...mutations (
CEBPA
NT
), promoting expression of the leukemia-associated p30 isoform, and amorphic C-terminal mutations. The most frequently co-mutated genes in
CEBPA
DM
AML are
GATA2
and
TET2
, however the molecular mechanisms underlying this co-mutational spectrum are incomplete. By combining transcriptomic and epigenomic analyses of
CEBPA
-
TET2
co-mutated patients with models thereof, we identify
GATA2
as a conserved target of the
CEBPA
-
TET2
mutational axis, providing a rationale for the mutational spectra in
CEBPA
DM
AML. Elevated CEBPA levels, driven by
CEBPA
NT
, mediate recruitment of TET2 to the
Gata2
distal hematopoietic enhancer thereby increasing
Gata2
expression. Concurrent loss of TET2 in
CEBPA
DM
AML induces a competitive advantage by increasing
Gata2
promoter methylation, thereby rebalancing GATA2 levels. Of clinical relevance, demethylating treatment of
Cebpa-Tet2
co-mutated AML restores
Gata2
levels and prolongs disease latency.
Transcription factors are key regulators of hematopoietic stem cells (HSCs) and act through their ability to bind DNA and impact on gene transcription. Their functions are interpreted in the complex ...landscape of chromatin, but current knowledge on how this is achieved is very limited. C/EBPα is an important transcriptional regulator of hematopoiesis, but its potential functions in HSCs have remained elusive. Here we report that C/EBPα serves to protect adult HSCs from apoptosis and to maintain their quiescent state. Consequently, deletion of Cebpa is associated with loss of self-renewal and HSC exhaustion. By combining gene expression analysis with genome-wide assessment of C/EBPα binding and epigenetic configurations, we show that C/EBPα acts to modulate the epigenetic states of genes belonging to molecular pathways important for HSC function. Moreover, our data suggest that C/EBPα acts as a priming factor at the HSC level where it actively promotes myeloid differentiation and counteracts lymphoid lineage choice. Taken together, our results show that C/EBPα is a key regulator of HSC biology, which influences the epigenetic landscape of HSCs in order to balance different cell fate options.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
An RNA region associated with the donor substrate site, located at the base of the peptidyl transferase loop of 23 S rRNA, was subjected to a comprehensive single-site mutational study. Growth ...phenotypes of Escherichia coli cells were characterized on induction of synthesis of the mutated rRNAs and the mutated ribosomes were tested, selectively, for their capacity to generate peptide bonds under the conditions of the "fragment" assay. Most of the mutants exhibited dominant or recessive lethal growth phenotypes and, in general, defective growth correlated with low activities in peptide bond formation, although exceptions were observed with normal growth and low activities, and vice versa. All these phenotypes are consistent with defects occurring in the structure of the ribosomal donor site and/or the capacity of the donor substrate to enter or leave this site. A compensating base change approach was employed to test for Watson-Crick base-pairing interactions between the -CCA end of the P-site bound tRNA(Phe) and this region of the peptidyl-transferase loop. Single nucleotide substitutions were introduced into the -CCA end of tRNA(Phe) and the ability of the 3'-terminal pentanucleotide fragments to act as donor substrates was examined for ribosomes carrying the different mutated 23 S rRNAs. No evidence was found for the occurrence of Watson-Crick base-pairing interactions. However, the data are consistent with the formation of a Hoogsteen pair between the 3'-terminal adenosine base of the donor substrate and U2585 of the 23 S rRNA.
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IJS, IMTLJ, KILJ, KISLJ, NUK, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Lipocalin‐2 (LCN2) was originally isolated from human neutrophils and termed neutrophil gelatinase‐associated lipocalin (NGAL). However, the functions of LCN2 and the cell types that are primarily ...responsible for LCN2 production remain unclear. To address these issues, hepatocyte‐specific Lcn2 knockout (Lcn2Hep–/–) mice were generated and subjected to bacterial infection (with Klesbsiella pneumoniae or Escherichia coli) or partial hepatectomy (PHx). Studies of Lcn2Hep–/– mice revealed that hepatocytes contributed to 25% of the low basal serum level of LCN2 protein (∼62 ng/mL) but were responsible for more than 90% of the highly elevated serum LCN2 protein level (∼6,000 ng/mL) postinfection and more than 60% post‐PHx (∼700 ng/mL). Interestingly, both Lcn2Hep–/– and global Lcn2 knockout (Lcn2–/–) mice demonstrated comparable increases in susceptibility to infection with K. pneumoniae or E. coli. These mice also had increased enteric bacterial translocation from the gut to the mesenteric lymph nodes and exhibited reduced liver regeneration after PHx. Treatment with interleukin (IL)‐6 stimulated hepatocytes to produce LCN2 in vitro and in vivo. Hepatocyte‐specific ablation of the IL‐6 receptor or Stat3, a major downstream effector of IL‐6, markedly abrogated LCN2 elevation in vivo. Furthermore, chromatin immunoprecipitation (ChIP) assay revealed that STAT3 was recruited to the promoter region of the Lcn2 gene upon STAT3 activation by IL‐6. Conclusion: Hepatocytes are the major cell type responsible for LCN2 production after bacterial infection or PHx, and this response is dependent on IL‐6 activation of the STAT3 signaling pathway. Thus, hepatocyte‐derived LCN2 plays an important role in inhibiting bacterial infection and promoting liver regeneration. (Hepatology 2015;61:692‐702)
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The naturally occurring streptogramin B antibiotic,
pristinamycin IA, which inhibits peptide elongation, can
produce two modifications in 23S rRNA when bound to the
Escherichia coli 70S ribosome and ...irradiated at
365 nm. Both drug-induced effects map to highly conserved
nucleotides within the functionally important peptidyl
transferase loop of 23S rRNA at positions m2A2503/Ψ2504
and G2061/A2062. The modification yields are influenced
strongly, and differentially, by P-site-bound tRNA and
strongly by some of the peptidyl transferase antibiotics
tested, with chloramphenicol producing a shift in the latter
modification to A2062/C2063. Pristinamycin IA can also
produce a modification on binding to deproteinized, mature
23S rRNA, at position U2500/C2501. The same modification
occurs on an ∼37-nt fragment, encompassing positions
∼2496–2532 of the peptidyl transferase loop that
was excised from the mature rRNA using RNAse H. In contrast,
no antibiotic-induced effects were observed on in vitro
T7 transcripts of full-length 23S rRNA, domain V, or on
a fragment extending from positions ∼2496–2566,
which indicates that one or more posttranscriptional modifications
within the sequence Cm-C-U-C-G-m2A-Ψ-G2505
are important for pristinamycin IA binding and/or the antibiotic-dependent
modification of 23S rRNA.
Idiopathic pulmonary fibrosis (IPF) is a severe interstitial lung disease for which two effective antifibrotics, nintedanib and pirfenidone, are available. However, many patients receive a reduced ...dosage or pause treatment due to side effects although the impact of antifibrotic treatment reduction is uncertain.
We retrospectively investigated the impact of antifibrotic treatment reduction on death in a large real-life IPF cohort. The primary endpoint of the analyses was time until death by any cause. Five patient groups were defined based on treatment intensity (full, reduced or no treatment) and the antifibrotic drug type (pirfenidone or nintedanib). Between group survival was compared using Cox proportional hazards analysis adjusted for age, sex, smoking status, and lung function at baseline.
375 patients from the Danish PFBIO-cohort were followed from April 2016 until November 2021 with a median follow-up time of 1.84 years. Of patients receiving nintedanib and pirfenidone, 80.19% and 67.42% had reduced treatment, respectively, when considering the entire follow-up period.
Treatment with nintedanib and pirfenidone was associated with improved survival compared to no antifibrotic treatment independent of treatment intensity (nintedanib: HR: 0.31, 95%-CI: 0.19–0.53, p < 0.001 & pirfenidone: HR: 0.26, 95%-CI: 0.16–0.42, p < 0.001). Nintedanib and pirfenidone in lower intensities were not associated with worse survival outcomes.
A substantial proportion of patients with IPF receive reduced antifibrotic treatment to ameliorate the side effects associated with a full dosage regime. Treatment with nintedanib and pirfenidone, independent of treatment intensity, was preferable over no antifibrotic treatment in improving survival and reduced dose appears to be a good alternative if full dose is not tolerated.
•A remarkably high number of patients with IPF receive reduced antifibrotic treatment.•Low antifibrotic treatment adherence in IPF despite best-case scenario conditions.•Antifibrotics seem superior in improving survival in IPF.•Survival benefits of antifibrotics remain even after treatment reduction in IPF.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Chromatin-Immunoprecipitation coupled with deep sequencing (ChIP-seq) is used to map transcription factor occupancy and generate epigenetic profiles genome-wide. The requirement of nano-scale ChIP ...DNA for generation of sequencing libraries has impeded ChIP-seq on in vivo tissues of low cell numbers.
We describe a robust, simple and scalable methodology for ChIP-seq of low-abundant cell populations, verified down to 10,000 cells. By employing non-mammalian genome mapping bacterial carrier DNA during amplification, we reliably amplify down to 50 pg of ChIP DNA from transcription factor (CEBPA) and histone mark (H3K4me3) ChIP. We further demonstrate that genomic profiles are highly resilient to changes in carrier DNA to ChIP DNA ratios.
This represents a significant advance compared to existing technologies, which involve either complex steps of pre-selection for nucleosome-containing chromatin or pre-amplification of precipitated DNA, making them prone to introduce experimental biases.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Objectives
Familial cases of hematological malignancies are associated with germline mutations. In particular, heterozygous mutations of SRP72 correlate with the development of myelodysplasia and ...bone marrow aplasia in two families. The signal recognition particle 72 kDa protein (SRP72) is part of the SRP complex, responsible for targeting of proteins to the endoplasmic reticulum. The main objective of this study is to investigate the role of SRP72 in the hematopoietic system, thus explaining why a reduced dose could increase susceptibility to hematological malignancies.
Methods
We developed an Srp72 null mouse model and characterized its hematopoietic system using flow cytometry, bone marrow transplantations, and gene expression analysis.
Results
Heterozygous loss of Srp72 in mice is not associated with major changes in hematopoiesis, although causes mild reductions in blood and BM cellularity and minor changes within the stem/progenitor compartment. We did not observe any hematological disorder. Interestingly, gene expression analysis demonstrated that genes encoding secreted factors, including cytokines and receptors, were transcriptionally down‐regulated in Srp72+/− animals.
Conclusions
The Srp72+/− mouse model only partially recapitulates the phenotype observed in families with inherited SRP72 lesions. Nonetheless, these results can provide mechanistic insights into why SRP72 mutations are associated with aplasia and myelodysplasia in humans.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
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