Population Heterogeneity in Corynebacterium glutamicum ATCC 13032 Caused by Prophage CGP3

ABSTRACT The genome of Corynebacterium glutamicum type strain ATCC 13032 (accession number BX927147) contains three prophages, CGP1, CGP2, and CGP3. We recently observed that many genes within the CGP3 prophage region have increased mRNA levels in a dtxR deletion mutant that lacks the master regulator of iron homeostasis (J. Wennerhold and M. Bott, J. Bacteriol. 188:2907-2918, 2006). Here, we provide evidence that this effect is due to the increased induction of the prophage CGP3 in the dtxR mutant, possibly triggered by DNA damage caused by elevated intracellular iron concentrations. Upon induction, the CGP3 prophage region is excised from the genome and forms a circular double-stranded DNA molecule. Using quantitative real-time PCR, an average copy number of about 0.1 per chromosome was determined for circular CGP3 DNA in wild-type C. glutamicum. This copy number increased about 15-fold in the dtxR mutant. In order to visualize the CGP3 DNA within single cells, a derivative of the wild type was constructed that contained an array of tet operators integrated within the CGP3 region and a plasmid-encoded YFP-TetR fusion protein. As expected, one to two fluorescent foci that represented the chromosomally integrated CGP3 prophage were detected in the majority of cells. However, in a small fraction (2 to 4%) of the population, 4 to 10 CGP3 DNA molecules could be observed in a single cell. Interestingly, the presence of many CGP3 copies in a cell often was accompanied by an efflux of chromosomal DNA, indicating the lysis of the corresponding cell. However, evidence for the formation of functional infective CGP3 phage particles could not be obtained.

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A Transgenic Mouse Assay To Compare the Barrier Activities of the Chicken β-Globin 5′HS4, Human β-Globin 5′HS5 and Mouse 3′HS1.

Blood ◽

10.1182/blood.v108.11.362.362 ◽

2006 ◽

Vol 108 (11) ◽

pp. 362-362

Author(s):

Emily E. Devlin ◽

Cheryl Lynn Horton ◽

Lisa J. Garrett-Beal ◽

Patrick G. Gallagher ◽

David M. Bodine

Keyword(s):

Transgenic Mice ◽

Copy Number ◽

Globin Gene ◽

Red Cells ◽

Ctcf Binding ◽

Mrna Levels ◽

Wild Type ◽

Globin Mrna ◽

Enhancer Element ◽

Insulator Elements

Abstract Insulator elements are found at the boundary between euchromatin and heterochromatin, and are responsible for maintaining the correct chromatin configuration for a locus. The best characterized vertebrate insulator element, 5′ Hypersensitive Site (HS) 4 from the chicken β-globin locus (ch5′HS4), has two separable activities: enhancer blocking, which requires binding of the transcription factor CTCF, and barrier, which prevents transgene silencing. We have previously reported that transgenic mice carrying a wild-type erythrocyte ankyrin promoter (ANK-1E)/γ-globin gene showed uniform (γ-globin in 100% of red cells), position-independent (32/32 lines express), copy number-dependent (p=0.0005) expression of γ-globin mRNA and protein. Mutations in the ANK-1E promoter at positions −108 and −153 cause ankyrin-deficient Hereditary Spherocytosis. Transgenic mice with the −108/−153 ANK-1E/γ-globin transgene showed variegated (γ-globin in 0–80% of red cells), position-dependent (8/14 lines express), copy number-independent (p=0.27) expression of γ-globin. Flanking the −108/−153 ANK-1E/γ-globin transgene with the ch5′HS4 insulator restored uniform, position-independent (9/9 lines), copy number-dependent expression (p=0.003) at levels identical to the wild-type ANK-1E promoter. We hypothesized that we could test sequences for barrier activity by assaying their ability to restore normal expression to the −108/−153 ANK-1E/γ-globin gene in transgenic mice. In mammalian β-globin loci, human 5′HS5 and mouse 3′HS1 have been proposed to be insulator elements, similar to chicken 5′HS4, based on their ability to block enhancer element function. To test barrier function, we generated transgenic mice containing the −108/−153 ANK-1E/γ-globin transgene flanked by human 5′HS5, mouse wild-type 3′HS1, or mouse 3′HS1 with mutations that disrupt the binding of CTCF (×CTCF). A total of 5 lines of transgenic mice were generated containing the 5′HS5/−108/−153 ANK-1E/γ-globin transgene. γ-globin mRNA and protein were undetectable in 3/5 lines, indicating that expression was position-dependent, and in the two positive lines, mRNA levels did not correlate with copy number. A total of 9 lines of 3′HS1-flanked transgenic mice were generated, 3 of which did not express γ-globin mRNA and protein, demonstrating position-dependent expression. Among the 6 expressing lines, two lines showed variegated expression and the correlation between γ-globin mRNA level and copy number was significant (p=0.0117). In contrast, 3′HS1×CTCF transgenic mice expressed γ-globin in a uniform, position-independent (7/7 lines express), copy number-dependent (p=0.0005) manner. The levels of γ-globin mRNA in both the 3′HS1 and 3′HS1×CTCF transgenic mice were 2-fold greater than the levels measured in transgenic mice with the wild-type ANK-1E promoter (p=0.019; 0.0003 respectively), suggesting that 3′HS1 may contain an enhancer element. Our results indicate that while human 5′HS5 and mouse 3′HS1 block the effects of enhancer elements, neither are barrier elements as defined by the ability to prevent gene silencing. We hypothesize that the mutation of the CTCF binding sites allows the ANK-1E promoter to take greater advantage of the 3′HS1 enhancer, leading to a more uniform, position-independent, and copy number-dependent pattern of expression, as has been described for other enhancer elements in the β-globin Locus Control Region.

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Identification and Characterization of the Dicarboxylate Uptake System DccT in Corynebacterium glutamicum

Journal of Bacteriology ◽

10.1128/jb.00780-08 ◽

2008 ◽

Vol 190 (19) ◽

pp. 6458-6466 ◽

Cited By ~ 63

Author(s):

Jung-Won Youn ◽

Elena Jolkver ◽

Reinhard Krämer ◽

Kay Marin ◽

Volker F. Wendisch

Keyword(s):

Corynebacterium Glutamicum ◽

Tricarboxylic Acid Cycle ◽

Carbon Sources ◽

Mrna Levels ◽

Uptake System ◽

Dependent Manner ◽

Wild Type ◽

Prolonged Incubation ◽

In The Wild ◽

Biochemical Analyses

ABSTRACT Many bacteria can utilize C4-carboxylates as carbon and energy sources. However, Corynebacterium glutamicum ATCC 13032 is not able to use tricarboxylic acid cycle intermediates such as succinate, fumarate, and l-malate as sole carbon sources. Upon prolonged incubation, spontaneous mutants which had gained the ability to grow on succinate, fumarate, and l-malate could be isolated. DNA microarray analysis showed higher mRNA levels of cg0277, which subsequently was named dccT, in the mutants than in the wild type, and transcriptional fusion analysis revealed that a point mutation in the promoter region of dccT was responsible for increased expression. The overexpression of dccT was sufficient to enable the C. glutamicum wild type to grow on succinate, fumarate, and l-malate as the sole carbon sources. Biochemical analyses revealed that DccT, which is a member of the divalent anion/Na+ symporter family, catalyzes the effective uptake of dicarboxylates like succinate, fumarate, l-malate, and likely also oxaloacetate in a sodium-dependent manner.

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Transcriptional milestones in Dictyostelium development

10.1101/2021.05.27.445976 ◽

2021 ◽

Author(s):

Mariko Katoh-Kurasawa ◽

Karin Hrovatin ◽

Shigenori Hirose ◽

Amanda Webb ◽

Hsing-I Ho ◽

...

Keyword(s):

Gene Expression ◽

Genetic Background ◽

Web Application ◽

Morphological Changes ◽

Single Cells ◽

Sequencing Analysis ◽

Wild Type ◽

Chromosomal Dna ◽

Gene Annotations ◽

Developmental Conditions

Development of the social amoeba Dictyostelium discoideum begins by starvation of single cells and ends in multicellular fruiting bodies 24 hours later. These major morphological changes are accompanied by sweeping gene expression changes, encompassing nearly half of the 13,000 genes in the genome. To explore the relationships between the transcriptome and developmental morphogenesis, we performed time-series RNA-sequencing analysis of the wild type and 20 mutant strains with altered morphogenesis. These strains exhibit arrest at different developmental stages, accelerated development, or terminal morphologies that are not typically seen in the wild type. Considering eight major morphological transitions, we identified 1,371 milestone genes whose expression changes sharply between two consecutive transitions. We also identified 1,099 genes as members of 21 regulons, which are groups of genes that remain coordinately regulated despite the genetic, temporal, and developmental perturbations in the dataset. The gene annotations in these milestones and regulons validate known transitions and reveal several new physiological and functional transitions during development. For example, we found that DNA replication genes are co-regulated with cell division genes, so they are co-expressed in mid-development even though chromosomal DNA is not replicated at that time. Altogether, the dataset includes 486 transcriptional profiles, across developmental and genetic conditions, that can be used to identify new relationships between gene expression and developmental processes and to improve gene annotations. We demonstrate the utility of this resource by showing that the cycles of aggregation and disaggregation observed in allorecognition-defective mutants involve a dedifferentiation process. We also show unexpected variability and sensitivity to genetic background and developmental conditions in two commonly used genes, act6 and act15, and robustness of the coaA gene. Finally, we propose that gpdA should be used as a standard for mRNA quantitation because it is less sensitive to genetic background and developmental conditions than commonly used standards. The dataset is available for democratized exploration without the need for programming skills through the web application dictyExpress and the data mining environment Orange.

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Generation of Iron-Independent Siderophore-Producing Agaricus bisporus through the Constitutive Expression of hapX

Genes ◽

10.3390/genes12050724 ◽

2021 ◽

Vol 12 (5) ◽

pp. 724

Author(s):

Min-Seek Kim ◽

Hyeon-Su Ro

Keyword(s):

Agaricus Bisporus ◽

Recombinant Strain ◽

Minimal Medium ◽

Binary Vector ◽

Constitutive Expression ◽

Pcr Analysis ◽

Mrna Levels ◽

Wild Type ◽

Chromosomal Dna ◽

In The Wild

Agaricus bisporus secretes siderophore to uptake environmental iron. Siderophore secretion in A. bisporus was enabled only in the iron-free minimal medium due to iron repression of hapX, a transcriptional activator of siderophore biosynthetic genes. Aiming to produce siderophore using conventional iron-containing complex media, we constructed a recombinant strain of A. bisporus that escapes hapX gene repression. For this, the A. bisporushapX gene was inserted next to the glyceraldehyde 3-phosphate dehydrogenase promoter (pGPD) in a binary vector, pBGgHg, for the constitutive expression of hapX. Transformants of A. bisporus were generated using the binary vector through Agrobacterium tumefaciens-mediated transformation. PCR and Northern blot analyses of the chromosomal DNA of the transformants confirmed the successful integration of pGPD-hapX at different locations with different copy numbers. The stable integration of pGPD-hapX was supported by PCR analysis of chromosomal DNA obtained from the 20 passages of the transformant. The transformants constitutively over-expressed hapX by 3- to 5-fold and sidD, a key gene in the siderophore biosynthetic pathway, by 1.5- to 4-fold in mRNA levels compared to the wild-type strain (without Fe3+), regardless of the presence of iron. Lastly, HPLC analysis of the culture supernatants grown in minimal medium with or without Fe3+ ions presented a peak corresponding to iron-chelating siderophore at a retention time of 5.12 min. The siderophore concentrations of the transformant T2 in the culture supernatant were 9.3-fold (−Fe3+) and 8-fold (+Fe3+) higher than that of the wild-type A. bisporus grown without Fe3+ ions, while no siderophore was detected in the wild-type supernatant grown with Fe3+. The results described here demonstrate the iron-independent production of siderophore by a recombinant strain of A. bisporus, suggesting a new application for mushrooms through molecular biological manipulation.

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Deletion of a fur-Like Gene Affects Iron Homeostasis and Magnetosome Formation in Magnetospirillum gryphiswaldense

Journal of Bacteriology ◽

10.1128/jb.00319-10 ◽

2010 ◽

Vol 192 (16) ◽

pp. 4192-4204 ◽

Cited By ~ 47

Author(s):

René Uebe ◽

Birgit Voigt ◽

Thomas Schweder ◽

Dirk Albrecht ◽

Emanuel Katzmann ◽

...

Keyword(s):

Iron Uptake ◽

Iron Homeostasis ◽

Wild Type ◽

Intracellular Iron ◽

Magnetospirillum Gryphiswaldense ◽

Cellular Iron ◽

Iron Supply ◽

Iron Requirement ◽

Magnetite Biomineralization ◽

Titration Assay

ABSTRACT Magnetotactic bacteria synthesize specific organelles, the magnetosomes, which are membrane-enveloped crystals of the magnetic mineral magnetite (Fe3O4). The biomineralization of magnetite involves the uptake and intracellular accumulation of large amounts of iron. However, it is not clear how iron uptake and biomineralization are regulated and balanced with the biochemical iron requirement and intracellular homeostasis. In this study, we identified and analyzed a homologue of the ferric uptake regulator Fur in Magnetospirillum gryphiswaldense, which was able to complement a fur mutant of Escherichia coli. A fur deletion mutant of M. gryphiswaldense biomineralized fewer and slightly smaller magnetite crystals than did the wild type. Although the total cellular iron accumulation of the mutant was decreased due to reduced magnetite biomineralization, it exhibited an increased level of free intracellular iron, which was bound mostly to a ferritin-like metabolite that was found significantly increased in Mössbauer spectra of the mutant. Compared to that of the wild type, growth of the fur mutant was impaired in the presence of paraquat and under aerobic conditions. Using a Fur titration assay and proteomic analysis, we identified constituents of the Fur regulon. Whereas the expression of most known magnetosome genes was unaffected in the fur mutant, we identified 14 proteins whose expression was altered between the mutant and the wild type, including five proteins whose genes constitute putative iron uptake systems. Our data demonstrate that Fur is a regulator involved in global iron homeostasis, which also affects magnetite biomineralization, probably by balancing the competing demands for biochemical iron supply and magnetite biomineralization.

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The hypoferremic response to acute inflammation is maintained in thalassemia mice even under parenteral iron loading

PeerJ ◽

10.7717/peerj.11367 ◽

2021 ◽

Vol 9 ◽

pp. e11367

Author(s):

Chanita Sanyear ◽

Buraporn Chiawtada ◽

Punnee Butthep ◽

Saovaros Svasti ◽

Suthat Fucharoen ◽

...

Keyword(s):

Mrna Expression ◽

Serum Iron ◽

Acute Inflammation ◽

Iron Homeostasis ◽

Mrna Levels ◽

Iron Dextran ◽

Iron Loading ◽

Wild Type ◽

Altered Expression ◽

Parenteral Iron

Background Hepcidin controls iron homeostasis by inducing the degradation of the iron efflux protein, ferroportin (FPN1), and subsequently reducing serum iron levels. Hepcidin expression is influenced by multiple factors, including iron stores, ineffective erythropoiesis, and inflammation. However, the interactions between these factors under thalassemic condition remain unclear. This study aimed to determine the hypoferremic and transcriptional responses of iron homeostasis to acute inflammatory induction by lipopolysaccharide (LPS) in thalassemic (Hbbth3/+) mice with/without parenteral iron loading with iron dextran. Methods Wild type and Hbbth3/+ mice were intramuscularly injected with 5 mg of iron dextran once daily for two consecutive days. After a 2-week equilibration, acute inflammation was induced by an intraperitoneal injection of a single dose of 1 µg/g body weight of LPS. Control groups for both iron loading and acute inflammation received equal volume(s) of saline solution. Blood and tissue samples were collected at 6 hours after LPS (or saline) injection. Iron parameters and mRNA expression of hepcidin as well as genes involved in iron transport and metabolism in wild type and Hbbth3/+ mice were analyzed and compared by Kruskal–Wallis test with pairwise Mann–Whitney U test. Results We found the inductive effects of LPS on liver IL-6 mRNA expression to be more pronounced under parenteral iron loading. Upon LPS administration, splenic erythroferrone (ERFE) mRNA levels were reduced only in iron-treated mice, whereas, liver bone morphogenetic protein 6 (BMP6) mRNA levels were decreased under both control and parenteral iron loading conditions. Despite the altered expression of the aforementioned hepcidin regulators, the stimulatory effect of LPS on hepcidin mRNA expression was blunt in iron-treated Hbbth3/+ mice. Contrary to the blunted hepcidin response, LPS treatment suppressed FPN1 mRNA expression in the liver, spleen, and duodenum, as well as reduced serum iron levels of Hbbth3/+ mice with parenteral iron loading. Conclusion Our study suggests that a hypoferremic response to LPS-induced acute inflammation is maintained in thalassemic mice with parenteral iron loading in a hepcidin-independent manner.

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Tmprss6, An Inhibitor of Hepatic Bmp/Smad Signaling, Is Required for Hepcidin Suppression and Iron Loading In a Mouse Model of β-Thalassemia

Blood ◽

10.1182/blood.v116.21.164.164 ◽

2010 ◽

Vol 116 (21) ◽

pp. 164-164

Author(s):

Karin E. Finberg ◽

Rebecca L. Whittlesey ◽

Stefano Rivella ◽

Nancy C. Andrews

Keyword(s):

Iron Deficiency ◽

Iron Homeostasis ◽

Heme Iron ◽

Mrna Levels ◽

Iron Loading ◽

Wild Type ◽

Smad Signaling ◽

Hepcidin Expression ◽

Non Heme Iron ◽

Hepcidin Mrna

Abstract Abstract 164 TMPRSS6, a transmembrane protease produced by the liver, is an essential regulator of mammalian iron homeostasis. TMPRSS6 inhibits the expression of hepcidin, a circulating peptide that decreases intestinal iron absorption and macrophage iron release, by down-regulating hepatic BMP/SMAD signaling for hepcidin production. Accordingly, TMPRSS6 mutations result in elevated hepcidin levels, impaired absorption of dietary iron, and systemic iron deficiency. Interestingly, in congenital iron loading anemias such as β-thalassemia, hepcidin levels are inappropriately low relative to body iron stores, a finding that has been postulated to result from the production of a hepcidin-repressing factor in the setting of ineffective erythropoiesis. Here we asked if Tmprss6 is required to achieve the hepcidin suppression present in Hbbth3/+ mice, a model of β-thalassemia intermedia. To test this, we bred Hbbth3/+ mice to mice harboring a targeted disruption of the Tmprss6 serine protease domain. We generated mice of various Hbb-Tmprss6 genotype combinations and compared parameters of systemic iron homeostasis at 8 weeks of age. Consistent with prior studies of Hbbth3/+ mice, Hbbth3/+ mice harboring 2 wild-type Tmprss6 alleles (Hbbth3/+Tmprss6+/+ mice) showed non-heme iron concentrations of liver, spleen, and kidney that were significantly elevated compared to wild-type controls. Homozygosity for Tmprss6 mutation, however, ameliorated the iron overload phenotype of Hbbth3/+ mice and led to systemic iron deficiency. Tissue non-heme iron concentrations were markedly reduced in Hbbth3/+Tmprss6−/− mice as compared to Hbbth3/+Tmprss6+/+ mice and were similar to levels observed in Tmprss6−/− mice harboring 2 wild-type Hbb alleles. Hbbth3/+Tmprss6−/− mice had hemoglobin levels similar to the thalassemic levels present in Hbbth3/+Tmprss6+/+ mice. However, compared to Hbbth3/+Tmprss6+/+ mice, Hbbth3/+Tmprss6−/− mice showed markedly reduced erythrocyte mean corpuscular volume and serum transferrin saturation, as well as increased red blood cell count. Interestingly, homozygous loss of Tmprss6 in Hbbth3/+ mice also led to marked reduction in splenomegaly and improvement in peripheral red blood cell morphology. Consistent with prior studies of Hbbth3/+ mice, Hbbth3/+Tmprss6+/+ mice displayed hepatic hepcidin mRNA levels that were similar to wild-type and were, therefore, inappropriately decreased relative to their increased hepatic iron stores. Hepatic mRNA levels of Bmp6, encoding a Bmp ligand that is transcriptionally regulated by iron and acts as a key regulator of hepcidin expression in vivo, were significantly elevated in Hbbth3/+Tmprss6+/+ mice, suggesting that their relative hepcidin deficiency does not result from impaired Bmp6 transcription. While Hbbth3/+Tmprss6+/+ mice showed suppressed hepcidin levels, homozygous loss of Tmprss6 alleviated their hepcidin suppression and led to elevated hepcidin mRNA levels similar to Tmprss6−/− mice harboring 2 wild-type Hbb alleles. Hbbth3/+Tmprss6−/− mice also showed elevated hepatic mRNA encoding Id1, another transcriptional target of Bmp/Smad signaling. These findings indicate that Tmprss6 is required to achieve the suppression of hepatic hepcidin expression that underlies systemic iron overload in Hbbth3/+ mice. Furthermore, our results demonstrate that, by up-regulating hepatic Bmp/Smad signaling for hepcidin production, genetic loss of Tmprss6 induces profound changes in systemic iron homeostasis in this thalassemia model. Disclosures: No relevant conflicts of interest to declare.

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Loss of ferroportin induces memory impairment by promoting ferroptosis in Alzheimer’s disease

Cell Death and Differentiation ◽

10.1038/s41418-020-00685-9 ◽

2021 ◽

Author(s):

Wen-Dai Bao ◽

Pei Pang ◽

Xiao-Ting Zhou ◽

Fan Hu ◽

Wan Xiong ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Memory Impairment ◽

Iron Homeostasis ◽

Critical Role ◽

Gene Set Enrichment Analysis ◽

Molecular Characteristics ◽

Intracellular Iron

AbstractIron homeostasis disturbance has been implicated in Alzheimer’s disease (AD), and excess iron exacerbates oxidative damage and cognitive defects. Ferroptosis is a nonapoptotic form of cell death dependent upon intracellular iron. However, the involvement of ferroptosis in the pathogenesis of AD remains elusive. Here, we report that ferroportin1 (Fpn), the only identified mammalian nonheme iron exporter, was downregulated in the brains of APPswe/PS1dE9 mice as an Alzheimer’s mouse model and Alzheimer’s patients. Genetic deletion of Fpn in principal neurons of the neocortex and hippocampus by breeding Fpnfl/fl mice with NEX-Cre mice led to AD-like hippocampal atrophy and memory deficits. Interestingly, the canonical morphological and molecular characteristics of ferroptosis were observed in both Fpnfl/fl/NEXcre and AD mice. Gene set enrichment analysis (GSEA) of ferroptosis-related RNA-seq data showed that the differentially expressed genes were highly enriched in gene sets associated with AD. Furthermore, administration of specific inhibitors of ferroptosis effectively reduced the neuronal death and memory impairments induced by Aβ aggregation in vitro and in vivo. In addition, restoring Fpn ameliorated ferroptosis and memory impairment in APPswe/PS1dE9 mice. Our study demonstrates the critical role of Fpn and ferroptosis in the progression of AD, thus provides promising therapeutic approaches for this disease.

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Surfactant protein A reduces TLR4 and inflammatory cytokine mRNA levels in neonatal mouse ileum

Scientific Reports ◽

10.1038/s41598-021-82219-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Lidan Liu ◽

Chaim Z. Aron ◽

Cullen M. Grable ◽

Adrian Robles ◽

Xiangli Liu ◽

...

Keyword(s):

Proinflammatory Cytokine ◽

Inflammatory Cytokine ◽

Protein A ◽

Surfactant Protein ◽

Surfactant Protein A ◽

Mrna Levels ◽

Wild Type ◽

Cytokine Mrna ◽

Protein Levels ◽

Cytokine Levels

AbstractLevels of intestinal toll-like receptor 4 (TLR4) impact inflammation in the neonatal gastrointestinal tract. While surfactant protein A (SP-A) is known to regulate TLR4 in the lung, it also reduces intestinal damage, TLR4 and inflammation in an experimental model of necrotizing enterocolitis (NEC) in neonatal rats. We hypothesized that SP-A-deficient (SP-A−/−) mice have increased ileal TLR4 and inflammatory cytokine levels compared to wild type mice, impacting intestinal physiology. We found that ileal TLR4 and proinflammatory cytokine levels were significantly higher in infant SP-A−/− mice compared to wild type mice. Gavage of neonatal SP-A−/− mice with purified SP-A reduced ileal TLR4 protein levels. SP-A reduced expression of TLR4 and proinflammatory cytokines in normal human intestinal epithelial cells (FHs74int), suggesting a direct effect. However, incubation of gastrointestinal cell lines with proteasome inhibitors did not abrogate the effect of SP-A on TLR4 protein levels, suggesting that proteasomal degradation is not involved. In a mouse model of experimental NEC, SP-A−/− mice were more susceptible to intestinal stress resembling NEC, while gavage with SP-A significantly decreased ileal damage, TLR4 and proinflammatory cytokine mRNA levels. Our data suggests that SP-A has an extrapulmonary role in the intestinal health of neonatal mice by modulating TLR4 and proinflammatory cytokines mRNA expression in intestinal epithelium.

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Single Cell Genetic Profiling of Tumors of Breast Cancer Patients Aged 50 Years and Older Reveals Enormous Intratumor Heterogeneity Independent of Individual Prognosis

Cancers ◽

10.3390/cancers13133366 ◽

2021 ◽

Vol 13 (13) ◽

pp. 3366

Author(s):

Anna-Sophie Liegmann ◽

Kerstin Heselmeyer-Haddad ◽

Annette Lischka ◽

Daniela Hirsch ◽

Wei-Dong Chen ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Patients ◽

Copy Number ◽

Genome Instability ◽

Single Cells ◽

Gene Mutations ◽

Intratumor Heterogeneity ◽

Breast Cancer Patients ◽

Luminal A ◽

Pik3ca Mutations

Purpose: Older breast cancer patients are underrepresented in cancer research even though the majority (81.4%) of women dying of breast cancer are 55 years and older. Here we study a common phenomenon observed in breast cancer which is a large inter- and intratumor heterogeneity; this poses a tremendous clinical challenge, for example with respect to treatment stratification. To further elucidate genomic instability and tumor heterogeneity in older patients, we analyzed the genetic aberration profiles of 39 breast cancer patients aged 50 years and older (median 67 years) with either short (median 2.4 years) or long survival (median 19 years). The analysis was based on copy number enumeration of eight breast cancer-associated genes using multiplex interphase fluorescence in situ hybridization (miFISH) of single cells, and by targeted next-generation sequencing of 563 cancer-related genes. Results: We detected enormous inter- and intratumor heterogeneity, yet maintenance of common cancer gene mutations and breast cancer specific chromosomal gains and losses. The gain of COX2 was most common (72%), followed by MYC (69%); losses were most prevalent for CDH1 (74%) and TP53 (69%). The degree of intratumor heterogeneity did not correlate with disease outcome. Comparing the miFISH results of diploid with aneuploid tumor samples significant differences were found: aneuploid tumors showed significantly higher average signal numbers, copy number alterations (CNAs) and instability indices. Mutations in PIKC3A were mostly restricted to luminal A tumors. Furthermore, a significant co-occurrence of CNAs of DBC2/MYC, HER2/DBC2 and HER2/TP53 and mutual exclusivity of CNAs of HER2 and PIK3CA mutations and CNAs of CCND1 and PIK3CA mutations were revealed. Conclusion: Our results provide a comprehensive picture of genome instability profiles with a large variety of inter- and intratumor heterogeneity in breast cancer patients aged 50 years and older. In most cases, the distribution of chromosomal aneuploidies was consistent with previous results; however, striking exceptions, such as tumors driven by exclusive loss of chromosomes, were identified.

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