Validation of a New PCR-Based Screening Method for Prevention of Serratia marcescens Outbreaks in the Neonatal Intensive Care Unit.

BACKGROUND: Serratia marcescens may cause severe nosocomial infections, mostly in very low birth weight infants. Since S. marcescens exhibits by far the highest adjusted incidence rate for horizontal transmission, it can cause complex outbreak situations in neonatal intensive care units. OBJECTIVE: The aim of this study was to establish a fast and highly sensitive colonization screening for prompt cohorting and barrier nursing strategies. METHODS: A probe-based duplex PCR assay targeting the 16S rRNA gene of S. marcescens was developed and validated by using 36 reference strains, 14 S. marcescens outbreak- and nonoutbreak isolates, defined by epidemiological linkage and molecular typing, and applied in 1,347 clinical specimens from 505 patients. RESULTS AND CONCLUSIONS: The novel PCR assay proved to be highly specific and had an in vitro sensitivity of 100 gene copies per reaction (∼15 bacteria). It showed a similar (in laryngeal/tracheal specimens) or even higher (in rectal/stoma swabs) in vivo sensitivity in comparison to routine microbial culture and was much quicker (<24 h vs. 2 days). By combining different oligonucleotide primers, there was robust detection of genetic variants of S. marcescens strains. PCR inhibition was low (1.6%) and observed with rectal swabs only. Cohort analysis illustrated applicability of the PCR assay as a quick tool to prevent outbreak scenarios by allowing rapid decisions on cohorting and barrier nursing. In summary, this novel molecular screening for colonization by S. marcescens is specific, highly sensitive, and substantially accelerates detection.

Adaptation of the Oxygen Sensing System during Lung Development.

During gestation, the most drastic change in oxygen supply occurs with the onset of ventilation after birth. As the too early exposure of premature infants to high arterial oxygen pressure leads to characteristic diseases, we studied the adaptation of the oxygen sensing system and its targets, the hypoxia-inducible factor- (HIF-) regulated genes (HRGs) in the developing lung. We draw a detailed picture of the oxygen sensing system by integrating information from qPCR, immunoblotting, in situ hybridization, and single-cell RNA sequencing data in ex vivo and in vivo models. HIF1α protein was completely destabilized with the onset of pulmonary ventilation, but did not coincide with expression changes in bona fide HRGs. We observed a modified composition of the HIF-PHD system from intrauterine to neonatal phases: Phd3 was significantly decreased, while Hif2a showed a strong increase and the Hif3a isoform Ipas exclusively peaked at P0. Colocalization studies point to the Hif1a-Phd1 axis as the main regulator of the HIF-PHD system in mouse lung development, complemented by the Hif3a-Phd3 axis during gestation. Hif3a isoform expression showed a stepwise adaptation during the periods of saccular and alveolar differentiation. With a strong hypoxic stimulus, lung ex vivo organ cultures displayed a functioning HIF system at every developmental stage. Approaches with systemic hypoxia or roxadustat treatment revealed only a limited in vivo response of HRGs. Understanding the interplay of the oxygen sensing system components during the transition from saccular to alveolar phases of lung development might help to counteract prematurity-associated diseases like bronchopulmonary dysplasia.

The circadian clock regulates rhythmic erythropoietin expression in the murine kidney.

Generation of circadian rhythms is cell-autonomous and relies on a transcription/translation feedback loop controlled by a family of circadian clock transcription factor activators including CLOCK, BMAL1 and repressors such as CRY1 and CRY2. The aim of the present study was to examine both the molecular mechanism and the hemopoietic implication of circadian erythropoietin expression. Mutant mice with homozygous deletion of the core circadian clock genes cryptochromes 1 and 2 (Cry-null) were used to elucidate circadian erythropoietin regulation. Wild-type control mice exhibited a significant difference in kidney erythropoietin mRNA expression between circadian times 06 and 18. In parallel, a significantly higher number of erythropoietin-producing cells in the kidney (by RNAscope®) and significantly higher levels of circulating erythropoietin protein (by ELISA) were detected at circadian time 18. Such changes were abolished in Cry-null mice and were independent from oxygen tension, oxygen saturation, or expression of hypoxia-inducible factor 2 alpha, indicating that circadian erythropoietin expression is transcriptionally regulated by CRY1 and CRY2. Reporter gene assays showed that the CLOCK/BMAL1 heterodimer activated an E-box element in the 5' erythropoietin promoter. RNAscope® in situ hybridization confirmed the presence of Bmal1 in erythropoietin-producing cells of the kidney. In Cry-null mice, a significantly reduced number of reticulocytes was found while erythrocyte numbers and hematocrit were unchanged. Thus, circadian erythropoietin regulation in the normoxic adult murine kidney is transcriptionally controlled by master circadian activators CLOCK/BMAL1, and repressors CRY1/CRY2. These findings may have implications for kidney physiology and disease, laboratory diagnostics, and anemia therapy.

Pooling, room temperature, and extended storage time increase the release of adult-specific biologic response modifiers in platelet concentrates: a hidden transfusion risk for neonates?

BACKGROUND: Adult donor platelets (PLTs) are frequently transfused to prevent or stop bleeding in neonates with thrombocytopenia. There is evidence for PLT transfusion-related morbidity and mortality, leading to the hypothesis on immunomodulatory effects of transfusing adult PLTs into neonates. Candidate factors are biologic response modifiers (BRMs) that are expressed at higher rates in adult than in neonatal PLTs. This study investigated whether storage conditions or preparation methods impact on the release of those differentially expressed BRMs. STUDY DESIGN AND METHODS: Pooled PLT concentrates (PCs) and apheresis PCs (APCs) were stored under agitation for up to 7 days at room temperature (RT) or at 2 to 8°C. The BRMs CCL5/RANTES, TGFß1, TSP1, and DKK1 were measured in PCs' supernatant, lysate, and corresponding plasma. PLT function was assessed by light transmission aggregometry. RESULTS: Concerning the preparation method, higher concentrations of DKK1 were found in pooled PCs compared to APCs. In supernatants, the concentrations of CCL5, TGFß1, TSP1, and DKK1 significantly increased, both over standard (≤4 days) and over extended storage times (7 days). Each of the four BRMs showed an up to twofold increase in concentration after storage at RT compared to cold storage (CS). There was no difference in the aggregation capacity. CONCLUSION: This analysis shows that the release of adult-specific BRMs during storage is lowest in short- and CS APCs. Our study points to strategies for reducing the exposure of sick neonates to BRMs that can be specifically associated to PLT transfusion-related morbidity.

Deletion of an intronic HIF-2α binding site suppresses hypoxia-induced WT1 expression.

Hypoxia-inducible factors (HIFs) play a key role in the adaptation to low oxygen by interacting with hypoxia response elements (HREs) in the genome. Cellular levels of the HIF-2α transcription factor subunit influence the histopathology and clinical outcome of neuroblastoma, a malignant childhood tumor of the sympathetic ganglia. Expression of the Wilms tumor gene, WT1, marks a group of high-risk neuroblastoma. Here, we identify WT1 as a downstream target of HIF-2α in Kelly neuroblastoma cells. In chromatin immunoprecipitation assays, HIF-2α bound to a HRE in intron 3 of the WT1 gene, but not to another predicted HIF binding site (HBS) in the first intron. The identified element conferred oxygen sensitivity to otherwise hypoxia-resistant WT1 and SV40 promoter constructs. Deletion of the HBS in the intronic HRE by genome editing abolished WT1 expression in hypoxic neuroblastoma cells. Physical interaction between the HRE and the WT1 promoter in normoxic and hypoxic Kelly cells was shown by chromosome conformation capture assays. These findings demonstrate that binding of HIF-2α to an oxygen-sensitive enhancer in intron 3 stimulates transcription of the WT1 gene in neuroblastoma cells by hypoxia-independent chromatin looping. This novel regulatory mechanism may have implications for the biology and prognosis of neuroblastoma.

The Erythropoietin Promoter Variant rs1617640 Is Not Associated with Severe Retinopathy of Prematurity, Independent of Treatment with Erythropoietin.

In this case-control study, the erythropoietin (EPO) promoter variant s1617640, linked to high intravitreal EPO concentrations and increased risk of diabetic retinopathy, was not associated with severe retinopathy of prematurity. This finding was observed both in infants with and without recombinant EPO administration.

Wilms tumor protein-dependent transcription of VEGF receptor 2 and hypoxia regulate expression of the testis-promoting gene Sox9 in murine embryonic gonads.

Wilms tumor protein 1 (WT1) has been implicated in the control of several genes in sexual development, but its function in gonad formation is still unclear. Here, we report that WT1 stimulates expression of Kdr, the gene encoding VEGF receptor 2, in murine embryonic gonads. We found that WT1 and KDR are co-expressed in Sertoli cells of the testes and somatic cells of embryonic ovaries. Vivo-morpholino-mediated WT1 knockdown decreased Kdr transcripts in cultured embryonic gonads at multiple developmental stages. Furthermore, WT1 bound to the Kdr promoter in the chromatin of embryonic testes and ovaries. Forced expression of the WT1(-KTS) isoform, which functions as a transcription factor, increased KDR mRNA levels, whereas the WT1(+KTS) isoform, which acts presumably on the post-transcriptional level, did not. ChIP indicated that WT1(-KTS), but not WT1(+KTS), binds to the KDR promoter. Treatment with the KDR tyrosine kinase inhibitor SU1498 or the KDR ligand VEGFA revealed that KDR signaling represses the testis-promoting gene Sox9 in embryonic XX gonads. WT1 knockdown abrogated the stimulatory effect of SU1498-mediated KDR inhibition on Sox9 expression. Exposure to 1% O2 to mimic the low-oxygen conditions in the embryo increased Vegfa expression but did not affect Sox9 mRNA levels in gonadal explants. However, incubation in 1% O2 in the presence of SU1498 significantly reduced Sox9 transcripts in cultured testes and increased Sox9 levels in ovaries. These findings demonstrate that both the local oxygen environment and WT1, which enhances KDR expression, contribute to sex-specific Sox9 expression in developing murine gonads.

Common genetic variants at the 11q13.3 renal cancer susceptibility locus influence binding of HIF to an enhancer of cyclin D1 expression.

Although genome-wide association studies (GWAS) have identified the existence of numerous population-based cancer susceptibility loci, mechanistic insights remain limited, particularly for intergenic polymorphisms. Here, we show that polymorphism at a remote intergenic region on chromosome 11q13.3, recently identified as a susceptibility locus for renal cell carcinoma, modulates the binding and function of hypoxia-inducible factor (HIF) at a previously unrecognized transcriptional enhancer of CCND1 (encoding cyclin D1) that is specific for renal cancers characterized by inactivation of the von Hippel-Lindau tumor suppressor (pVHL). The protective haplotype impairs binding of HIF-2, resulting in an allelic imbalance in cyclin D1 expression, thus affecting a link between hypoxia pathways and cell cycle control.

Wilms' tumor protein Wt1 regulates the Interleukin-10 (IL-10) gene.

We identified the Wilms' tumor protein, Wt1, as a novel transcriptional activator of the immunosuppressant cytokine interleukin-10 (IL-10). Silencing of Wt1 by RNA interference reduced IL-10 mRNA levels by approximately 90%. IL-10 transcripts were increased more than 15-fold upon forced expression of Wt1. Electrophoretic mobility shift assay and chromatin immunoprecipitation revealed a cis-element that was responsible for activation of the IL-10 promoter by Wt1 in murine macrophages. Mutation of the Wt1 binding motif abrogated stimulation of the IL-10 promoter by tumor necrosis factor-α (TNFα). These results suggest a novel immune regulatory function of Wt1 in controlling IL-10 gene expression.

Wilms' tumour protein Wt1 stimulates transcription of the gene encoding vascular endothelial cadherin.

The Wilms' tumour gene, Wt1, encodes a zinc finger protein, which is mutated in a subset of paediatric renal carcinomas known as Wilms' tumours (nephroblastomas). Recent findings indicate that Wt1, beside its role in genitourinary development, is also necessary for normal vascularisation of the embryonic heart, and may even be involved in tumour angiogenesis. The original purpose of this study was to decipher potential downstream signalling pathways of Wt1 for blood vessel formation. We found that the Wt1(-KTS) protein, which functions as a transcription factor, stimulated the expression of cadherin 5 (CDH5, vascular endothelial (VE) cadherin) and other vascular genes, i.e. those encoding vascular endothelial growth factor receptors 1 and 2, and angiopoietin-2. Furthermore, an enhancer element was identified in the first intron of the CDH5 gene, which bound to the Wt1(-KTS) protein and was necessary for reporter gene activation by Wt1(-KTS) in transiently transfected cell lines. Wt1 and VE-cadherin proteins could be co-localised by double immunofluorescence staining in maturating glomeruli of embryonic murine kidneys. VE-cadherin transcripts were reduced in some but not all tissues of Wt1-deficient mouse embryos. These results indicate that Wt1 can stimulate vascular gene transcription. By demonstrating that Wt1(-KTS) protein trans-activates an enhancer element in the first intron we identified CDH5 as a novel target gene of Wt1. It is suggested that transcriptional activation of CDH5 by Wt1 fulfils regulatory functions during vascular development and kidney formation.