Publications by authors named "Holger Scholz"

153 Publications

Kidney physiology and susceptibility to acute kidney injury: implications for renoprotection.

Nat Rev Nephrol 2021 Feb 5. Epub 2021 Feb 5.

Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.

Kidney damage varies according to the primary insult. Different aetiologies of acute kidney injury (AKI), including kidney ischaemia, exposure to nephrotoxins, dehydration or sepsis, are associated with characteristic patterns of damage and changes in gene expression, which can provide insight into the mechanisms that lead to persistent structural and functional damage. Early morphological alterations are driven by a delicate balance between energy demand and oxygen supply, which varies considerably in different regions of the kidney. The functional heterogeneity of the various nephron segments is reflected in their use of different metabolic pathways. AKI is often linked to defects in kidney oxygen supply, and some nephron segments might not be able to shift to anaerobic metabolism under low oxygen conditions or might have remarkably low basal oxygen levels, which enhances their vulnerability to damage. Here, we discuss why specific kidney regions are at particular risk of injury and how this information might help to delineate novel routes for mitigating injury and avoiding permanent damage. We suggest that the physiological heterogeneity of the kidney should be taken into account when exploring novel renoprotective strategies, such as improvement of kidney tissue oxygenation, stimulation of hypoxia signalling pathways and modulation of cellular energy metabolism.
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http://dx.doi.org/10.1038/s41581-021-00394-7DOI Listing
February 2021

Human plague: An old scourge that needs new answers.

PLoS Negl Trop Dis 2020 08 27;14(8):e0008251. Epub 2020 Aug 27.

Epidemiology and Clinical Research Unit, Institut Pasteur de Madagascar, Antananarivo, Madagascar.

Yersinia pestis, the bacterial causative agent of plague, remains an important threat to human health. Plague is a rodent-borne disease that has historically shown an outstanding ability to colonize and persist across different species, habitats, and environments while provoking sporadic cases, outbreaks, and deadly global epidemics among humans. Between September and November 2017, an outbreak of urban pneumonic plague was declared in Madagascar, which refocused the attention of the scientific community on this ancient human scourge. Given recent trends and plague's resilience to control in the wild, its high fatality rate in humans without early treatment, and its capacity to disrupt social and healthcare systems, human plague should be considered as a neglected threat. A workshop was held in Paris in July 2018 to review current knowledge about plague and to identify the scientific research priorities to eradicate plague as a human threat. It was concluded that an urgent commitment is needed to develop and fund a strong research agenda aiming to fill the current knowledge gaps structured around 4 main axes: (i) an improved understanding of the ecological interactions among the reservoir, vector, pathogen, and environment; (ii) human and societal responses; (iii) improved diagnostic tools and case management; and (iv) vaccine development. These axes should be cross-cutting, translational, and focused on delivering context-specific strategies. Results of this research should feed a global control and prevention strategy within a "One Health" approach.
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http://dx.doi.org/10.1371/journal.pntd.0008251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7451524PMC
August 2020

Application of Whole Genome Sequencing and Pan-Family Multi-Locus Sequence Analysis to Characterize Relationships Within the Family .

Front Microbiol 2020 14;11:1329. Epub 2020 Jul 14.

Department of Bacteriology, Animal and Plant Health Agency, Weybridge, United Kingdom.

The bacterial family is currently composed of seven genera, including species of the genus , a number of which are significant veterinary and zoonotic pathogens. The bacteriological identification of pathogenic spp. may be hindered by their close phenotypic similarity to other members of the , particularly of the genus . Additionally, a number of novel atypical taxa have recently been identified, which exhibit greater genetic diversity than observed within the previously described species, and which share genomic features with organisms outside of the genus. Furthermore, previous work has indicated that the genus is polyphyletic, raising further questions regarding the relationship between the genus and wider . We have applied whole genome sequencing (WGS) and pan-family multi-locus sequence analysis (MLSA) approaches to a comprehensive panel of type strains, in order to characterize relationships within the family. Phylogenies based on WGS core genome alignments were able to resolve phylogenetic relationships of 31 non- spp. type strains from within the family, alongside type strains of twelve species. A phylogeny based on concatenated pan-family MLSA data was largely consistent with WGS based analyses. Notably, recently described atypical isolates were consistently placed in a single clade with existing species, clearly distinct from all members of the genus and wider family. Both WGS and MLSA methods closely grouped spp. with a sub-set of species. However, results also confirmed that the genus is polyphyletic, with seven species forming a separate grouping. The pan-family MLSA scheme was subsequently applied to a panel of 50 field strains of the family , isolated from a wide variety of sources. This analysis confirmed the utility of the pan- MLSA scheme in placing field isolates in relation to recognized type strains. However, a significant number of these isolates did not cluster with currently identified type strains, suggesting the existence of additional taxonomic diversity within some members of the . The WGS and pan-family MLSA approaches applied here provide valuable tools for resolving the identity and phylogenetic relationships of isolates from an expanding bacterial family containing a number of important pathogens.
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http://dx.doi.org/10.3389/fmicb.2020.01329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372191PMC
July 2020

Specific Detection of Based on Receptor Binding Proteins of Phages.

Pathogens 2020 Jul 27;9(8). Epub 2020 Jul 27.

Deptartment of Bacteriology and Toxinology, Bundeswehr Institute of Microbiology (IMB), 80937 Munich, Germany.

The highly pathogenic bacterium is the causative agent of plague, a notorious infectious zoonotic disease. When transmitted from person to person as pneumonic plague via droplets, is highly contagious and in most cases is fatal if left untreated. Thus, when plague is suspected, rapid diagnosis is crucial, as a serious course of the infection is only averted by early antibiotic therapy. The bacterium is easy to cultivate, accessible and has a high potential for nefarious use such as bioterrorism. Highly specific, rapid and easy-to-use confirmatory diagnostic methods are required to reliably identify the pathogen independently from PCR-based methods or F1 antigen-based immunological detection. specific phages such as L-413C and ΦA1122 are already used for detection of in bacterial plaque or biosensor assays. Here, we made use of the host specificities conferred by phage receptor binding (or tail fiber/spike) proteins (RBP) for developing a specific, fast and simple fluorescence-microscopy-based detection method for . Genes of putative RBP of phages L-413C () and ΦA1122 () were fused with those of fluorescent proteins and recombinant receptor-reporter fusion proteins were produced heterologously in . When first tested on attenuated strain EV76, RBP-reporters bound to the bacterial cell surface. This assay could be completed within a few minutes using live or formaldehyde-inactivated cells. Specificity tests using cultures of closely related species and several inactivated fully virulent strains exhibited high specificities of the RBP-reporters against . The L-413C RBP proved to be especially specific, as it only detected at all temperatures tested, whereas the RBP of ΦA1122 also bound to strains at 37 °C (but not at 28, 20 or 6 °C). Finally, the -specific capsule, produced when grown at 37 °C, significantly reduced binding of phage ΦA1122 RBP, whereas the capsule only slightly diminished binding of L-413C RBP.
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http://dx.doi.org/10.3390/pathogens9080611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460101PMC
July 2020

Expanding the host range: infection of a reptilian host (Furcifer pardalis) by an atypical Brucella strain.

Antonie Van Leeuwenhoek 2020 Oct 22;113(10):1531-1537. Epub 2020 Jul 22.

Department of Bacteriology and Toxinology, Bundeswehr Institute of Microbiology, Neuherbergstrasse 11, 80937, Munich, Germany.

Atypical brucellae show deviant phenotypes and/or genotypes. Besides Brucella inopinata, B. microti and B. vulpis, atypical strains have been described infecting humans, rodents, amphibians and fish. They represent potential zoonotic agents. Here, we provide evidence that reptiles as the remaining poikilothermic vertebrate class also represent susceptible hosts for atypical Brucella.
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http://dx.doi.org/10.1007/s10482-020-01448-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7481142PMC
October 2020

Autosomal dominant polycystic kidney disease in absence of renal cyst formation illustrates genetic interaction between and .

J Med Genet 2020 May 7. Epub 2020 May 7.

Medical Department III - Endocrinology, Nephrology, Rheumatology, University of Leipzig Medical Center, Leipzig, Saxony, Germany

Purpose: Autosomal dominant polycystic kidney disease (ADPKD), caused by pathogenic variants of either or , is characterised by wide interfamilial and intrafamilial phenotypic variability. This study aimed to determine the molecular basis of marked clinical variability in ADPKD family members and sought to analyse whether alterations of (Wilms tumour 1), encoding a regulator of gene expression, may have an impact on renal cyst formation.

Methods: ADPKD family members underwent clinical and molecular evaluation. Functionally, mRNA and protein expression upon knockdown was evaluated in mouse embryonic kidneys and mesonephric M15 cells.

Results: By renal gene panel analysis, we identified two pathogenic variants in an individual with maternal history of ADPKD, however, without cystic kidneys but polycystic liver disease: a known missense variant (c.8311G>A, p.Glu2771Lys) and a known de novo splice site variant (c.1432+4C>T). The latter was previously associated with imbalanced +/-KTS isoform ratio of . In ex vivo organ cultures from mouse embryonic kidneys, knockdown resulted in decreased expression on mRNA and protein level.

Conclusion: While the role of WT1 in glomerulopathies has been well established, this report by illustrating genetic interaction with proposes as potential modifier in ADPKD.
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http://dx.doi.org/10.1136/jmedgenet-2019-106633DOI Listing
May 2020

A headache with surprising outcome: first case of brucellosis caused by Brucella suis biovar 1 in Germany.

Infection 2019 Oct 9;47(5):863-868. Epub 2019 May 9.

Division of Infectious Diseases, Department of Medicine II, University of Freiburg, Medical Center and Faculty of Medicine, 79106, Freiburg, Germany.

In July 2018, brucellosis was diagnosed in a German patient without a travel history to regions endemic for Brucella. Microbiological analysis, including whole-genome sequencing, revealed Brucella suis biovar 1 as the etiologic agent. Core-genome-based multilocus sequence-typing analysis placed the isolate in close proximity to strains originating from Argentina. Notably, despite a strong IgM response, the patient did not develop Brucella-specific IgG antibodies during infection. Here, we describe the clinical course of infection, the extensive epidemiological investigations, and discuss possible routes of transmission.
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http://dx.doi.org/10.1007/s15010-019-01312-7DOI Listing
October 2019

Intermittent hypoxia: Friend and foe.

Acta Physiol (Oxf) 2019 06 11;226(2):e13276. Epub 2019 Apr 11.

Institut für Vegetative Physiologie, Charité-Universitätsmedizin Berlin, Berlin, Germany.

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http://dx.doi.org/10.1111/apha.13276DOI Listing
June 2019

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

Biochim Biophys Acta Gene Regul Mech 2019 01 20;1862(1):71-83. Epub 2018 Nov 20.

Institut für Vegetative Physiologie, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Charitéplatz 1, 10117 Berlin, Germany. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbagrm.2018.11.003DOI Listing
January 2019

Core Genome Multilocus Sequence Typing and Single Nucleotide Polymorphism Analysis in the Epidemiology of Brucella melitensis Infections.

J Clin Microbiol 2018 09 27;56(9). Epub 2018 Aug 27.

National and OIE Reference Laboratory for Brucellosis, Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise G. Caporale, Teramo, Italy

The use of whole-genome sequencing (WGS) using next-generation sequencing (NGS) technology has become a widely accepted method for microbiology laboratories in the application of molecular typing for outbreak tracing and genomic epidemiology. Several studies demonstrated the usefulness of WGS data analysis through single-nucleotide polymorphism (SNP) calling from a reference sequence analysis for , whereas gene-by-gene comparison through core-genome multilocus sequence typing (cgMLST) has not been explored so far. The current study developed an allele-based cgMLST method and compared its performance to that of the genome-wide SNP approach and the traditional multilocus variable-number tandem repeat analysis (MLVA) on a defined sample collection. The data set was comprised of 37 epidemiologically linked animal cases of brucellosis as well as 71 isolates with unknown epidemiological status, composed of human and animal samples collected in Italy. The cgMLST scheme generated in this study contained 2,704 targets of the 16M reference genome. We established the potential criteria necessary for inclusion of an isolate into a brucellosis outbreak cluster to be ≤6 loci in the cgMLST and ≤7 in WGS SNP analysis. Higher phylogenetic distance resolution was achieved with cgMLST and SNP analysis than with MLVA, particularly for strains belonging to the same lineage, thereby allowing diverse and unrelated genotypes to be identified with greater confidence. The application of a cgMLST scheme to the characterization of strains provided insights into the epidemiology of this pathogen, and it is a candidate to be a benchmark tool for outbreak investigations in human and animal brucellosis.
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http://dx.doi.org/10.1128/JCM.00517-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113479PMC
September 2018

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

J Biol Chem 2017 12 17;292(49):20281-20291. Epub 2017 Oct 17.

Institut für Vegetative Physiologie, 10117 Berlin, Germany. Electronic address:

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 , 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 transcripts in cultured embryonic gonads at multiple developmental stages. Furthermore, WT1 bound to the promoter in the chromatin of embryonic testes and ovaries. Forced expression of the WT1(-KTS) isoform, which functions as a transcription factor, increased 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 promoter. Treatment with the KDR tyrosine kinase inhibitor SU1498 or the KDR ligand VEGFA revealed that KDR signaling represses the testis-promoting gene in embryonic XX gonads. WT1 knockdown abrogated the stimulatory effect of SU1498-mediated KDR inhibition on expression. Exposure to 1% O to mimic the low-oxygen conditions in the embryo increased expression but did not affect mRNA levels in gonadal explants. However, incubation in 1% O in the presence of SU1498 significantly reduced transcripts in cultured testes and increased levels in ovaries. These findings demonstrate that both the local oxygen environment and WT1, which enhances expression, contribute to sex-specific expression in developing murine gonads.
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http://dx.doi.org/10.1074/jbc.M117.816751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5724013PMC
December 2017

Lysophosphatidylcholine activates caspase-1 in microglia via a novel pathway involving two inflammasomes.

J Neuroimmunol 2017 09 10;310:107-110. Epub 2017 Jul 10.

Charité - Universitätsmedizin Berlin, Institute of Physiology, 10117 Berlin, Germany. Electronic address:

Inflammasomes regulate microglial caspase-1 activation and subsequent neuroinflammatory processes in brain pathology. In the present study, we have identified inflammasomes causing caspase-1 activation following stimulation of microglia with lysophosphatidylcholine (LPC), a proinflammatory lipid generated under pathological conditions in the brain. LPC-induced caspase-1 activation in microglia was found to depend on LPS prestimulation, inflammasome NLRP3 and adaptor molecule ASC. Furthermore, knockdown of inflammasome NLRC4 inhibited LPC-stimulated caspase-1 activity in microglia, suggesting the requirement of two inflammasomes for optimal caspase-1 activity.
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http://dx.doi.org/10.1016/j.jneuroim.2017.07.004DOI Listing
September 2017

Prevalence, Host Range, and Comparative Genomic Analysis of Temperate Phages.

Front Microbiol 2017 30;8:1207. Epub 2017 Jun 30.

Department of Biological Safety, German Federal Institute for Risk AssessmentBerlin, Germany.

and are closely related bacteria that populate different habitats and differ in their pathogenic properties. Only little is known about mobile genetic elements in these genera which might be important for survival and virulence. Previous studies on lysogeny indicated that active phages are rare in this genus. To gain insight into the presence and nature of prophages in , temperate phages were isolated from various species and characterized in detail. analyses disclosed numerous prophages in published genomes. Induction experiments showed that prophages can be induced by various stress factors and that some strains released phage particles even under non-induced conditions. Sixty percent of lysates prepared from 125 strains revealed lytic activity. The host range and DNA similarities of 19 phages belonging to the families , or were determined suggesting that they are highly diverse. Some phages showed relationship to the temperate phage BiPB01. The genomic sequences of the myovirus POA1180 (41,655 bp) and podovirus POI1126 (60,065 bp) were analyzed. Phage POA1180 is very similar to a prophage recently identified in a strain isolated from an exotic frog. The POA1180 genome contains genes which may confer resistance to chromate and the ability to take up sulfate. Phage POI1126 is related to podoviruses of (PCB5), (Pep14), and (BcepIL02) and almost identical to an unnamed plasmid of the strain LMG 3301. Further experiments revealed that the POI1126 prophage indeed replicates as an extrachromosomal element. The data demonstrate for the first time that active prophages are common in and suggest that atypical brucellae also may be a reservoir for temperate phages.
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http://dx.doi.org/10.3389/fmicb.2017.01207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5492332PMC
June 2017

Ex vivo cultures combined with vivo-morpholino induced gene knockdown provide a system to assess the role of WT1 and GATA4 during gonad differentiation.

PLoS One 2017 20;12(4):e0176296. Epub 2017 Apr 20.

Institut für Vegetative Physiologie, Charité-Universitätsmedizin Berlin, Campus Charité Mitte, Berlin, Germany.

Gonad morphogenesis relies on the correct spatiotemporal expression of a number of genes that together fulfill the differentiation of the bipotential gonad into testes or ovaries. As such, the transcription factors WT1 and GATA4 are pivotal for proper gonadal development. Here we address the contributions of GATA4 and WT1 to the sex differentiation phase in testes and ovaries. We applied an ex vivo technique for cultivating gonads in hanging droplets of media that were supplemented with vivo-morpholinos to knockdown WT1 and GATA4 either alone or in combination at the same developmental stage. We show that WT1 is equally important for both, the initial establishment and the maintenance of the sex-specific gene expression signature in testes and ovaries. We further identified Foxl2 as a novel putative downstream target gene of WT1. Moreover, knockdown of WT1 reduced mRNA levels of several molecular components of the hedgehog signaling pathway in XY gonads, whereas Gata4 vivo-morpholino treatment increased transcripts of Dhh and Ptch1 in embryonic testes. The data suggest that for its proper function, WT1 relies on the correct expression of the GATA4 protein. Furthermore, GATA4 down-regulates several ovarian promoting genes in testes, such as Ctnnb1, Fst, and Bmp2, suggesting that this repression is required for maintaining the male phenotype. In conclusion, this study provides novel insights into the role of WT1 and GATA4 during the sex differentiation phase and represents an approach that can be applied to assess other proteins with as yet unknown functions during gonadal development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0176296PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5398674PMC
September 2017

Whole genome sequencing of Brucella melitensis isolated from 57 patients in Germany reveals high diversity in strains from Middle East.

PLoS One 2017 7;12(4):e0175425. Epub 2017 Apr 7.

Bundeswehr Institute of Microbiology, Munich, Germany.

Brucellosis, a worldwide common bacterial zoonotic disease, has become quite rare in Northern and Western Europe. However, since 2014 a significant increase of imported infections caused by Brucella (B.) melitensis has been noticed in Germany. Patients predominantly originated from Middle East including Turkey and Syria. These circumstances afforded an opportunity to gain insights into the population structure of Brucella strains. Brucella-isolates from 57 patients were recovered between January 2014 and June 2016 with culture confirmed brucellosis by the National Consultant Laboratory for Brucella. Their whole genome sequences were generated using the Illumina MiSeq platform. A whole genome-based SNP typing assay was developed in order to resolve geographically attributed genetic clusters. Results were compared to MLVA typing results, the current gold-standard of Brucella typing. In addition, sequences were examined for possible genetic variation within target regions of molecular diagnostic assays. Phylogenetic analyses revealed spatial clustering and distinguished strains from different patients in either case, whereas multiple isolates from a single patient or technical replicates showed identical SNP and MLVA profiles. By including WGS data from the NCBI database, five major genotypes were identified. Notably, strains originating from Turkey showed a high diversity and grouped into seven subclusters of genotype II. MLVA analysis congruently clustered all isolates and predominantly matched the East Mediterranean genetic clade. This study confirms whole-genome based SNP-analysis as a powerful tool for accurate typing of B. melitensis. Furthermore it allows special allocation and therefore provides useful information on the geographic origin for trace-back analysis. However, the lack of reliable metadata in public databases often prevents a resolution below geographic regions or country levels and corresponding precise trace-back analysis. Once this obstacle is resolved, WGS-derived bacterial typing adds an important method to complement epidemiological surveys during outbreak investigations. This is the first report of a detailed genetic investigation of an extensive collection of B. melitensis strains isolated from human cases in Germany.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0175425PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384748PMC
September 2017

The Wilms tumor protein WT1 stimulates transcription of the gene encoding insulin-like growth factor binding protein 5 (IGFBP5).

Gene 2017 Jul 30;619:21-29. Epub 2017 Mar 30.

Institut für Vegetative Physiologie, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany. Electronic address:

Insulin-like growth factor (IGF) binding proteins (IGFBPs) constitute a family of six secreted proteins that regulate the signaling of insulin-like growth factors (IGFs). IGFBP5 is the most conserved family member in vertebrates and the major IGF binding protein in bone. IGFBP5 is required for normal development of the musculoskeletal system, and various types of cancer frequently express high levels of IGFP5. Here we identify the gene encoding IGFBP5 as a novel downstream target of the Wilms tumor protein WT1. IGFBP5 and WT1 are expressed in an overlapping pattern in the condensing metanephric mesenchyme of embryonic murine kidneys. Down-regulation of WT1 by transfection with antisense vivo-morpholino significantly decreased Igfbp5 transcripts in murine embryonic kidney explants. Likewise, silencing of Wt1 in a mouse mesonephros-derived cell line reduced Igfbp5 mRNA levels by approximately 80%. Conversely, induction of the WT1(-KTS) isoform, whose role as transcriptional regulator has been firmly established, significantly increased IGFBP5 mRNA and protein levels in osteosarcoma cells. IGFBP5 expression was not significantly changed by WT1(+KTS) protein, which exhibits lower DNA binding affinity than the WT1(-KTS) isoform and has a presumed role in post-transcriptional gene regulation. Luciferase reporter constructs harboring 0.8 and 1.6 kilobases of the murine Igfbp5 promoter, respectively, were stimulated approximately 5-fold by co-transfection of WT1(-KTS). The WT1(+KTS) variant had no significant effect on IGFBP5 promoter activity. Binding of WT1(-KTS), but not of WT1(+KTS) protein, to the IGFBP5 promoter in human osteosarcoma cells was proven by chromatin immunoprecipitation (ChIP) and confirmed by electrophoretic mobility shift assay. These findings demonstrate that WT1 activates transcription of the IGFBP5 gene with possible implications for kidney development and bone (patho)physiology.
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http://dx.doi.org/10.1016/j.gene.2017.03.037DOI Listing
July 2017

Genetic Diversity of Reference and Non-reference Phages and Its Impact on -Typing.

Front Microbiol 2017 15;8:408. Epub 2017 Mar 15.

Department of Biological Safety, German Federal Institute for Risk Assessment Berlin, Germany.

Virulent phages have been used for many years to type isolates, but until recently knowledge about the genetic makeup of these phages remains limited. In this work the host specificity and genomic sequences of the original set (deposited in 1960) of VLA reference phages Tb, Fi, Wb, Bk2, R/C, and Iz were analyzed and compared with hitherto described brucellaphages. VLA phages turned out to be different from homonymous phages in other laboratories. The host range of the phages was defined by performing plaque assays with a wide selection of strains. Propagation of the phages on different strains did not alter host specificity. Sequencing of the phages Tb, Fi, Wb, and R/C revealed nucleotide variations when compared to same-named phages previously described by other laboratories. The phages Bk2 and Iz were sequenced for the first time. While Bk2 exhibited the same deletions as Wb, Iz possesses the largest genome of all reference phages. The duplication of a 301 bp sequence in this phage and the large deletion in Bk2, Wb, and R/C may be a result of recombination caused by repetitive sequences located in this DNA region. To identify new phages as potential candidates for lysotyping, the host range and Single Nucleotide Polymorphisms (SNPs) of 22 non-reference phages were determined. The phages showed lysis patterns different from those of the reference phages and thus represent novel valuable candidates in the typing set.
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http://dx.doi.org/10.3389/fmicb.2017.00408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5350156PMC
March 2017

Long-range dispersal moved into Western Europe from the East.

Microb Genom 2016 12 12;2(12):e000100. Epub 2016 Dec 12.

1​Department of Clinical Microbiology and the Laboratory for Molecular Infection Medicine Sweden, Umeå University, Umeå, Sweden.

For many infections transmitting to humans from reservoirs in nature, disease dispersal patterns over space and time are largely unknown. Here, a reversed genomics approach helped us understand disease dispersal and yielded insight into evolution and biological properties of , the bacterium causing tularemia. We whole-genome sequenced 67 strains and characterized by single-nucleotide polymorphism assays 138 strains, collected from individuals infected 1947-2012 across Western Europe. We used the data for phylogenetic, population genetic and geographical network analyses. All strains (=205) belonged to a monophyletic population of recent ancestry not found outside Western Europe. Most strains (=195) throughout the study area were assigned to a star-like phylogenetic pattern indicating that colonization of Western Europe occurred via clonal expansion. In the East of the study area, strains were more diverse, consistent with a founder population spreading from east to west. The relationship of genetic and geographic distance within the population was complex and indicated multiple long-distance dispersal events. Mutation rate estimates based on year of isolation indicated null rates; in outbreak hotspots only, there was a rate of 0.4 mutations/genome/year. Patterns of nucleotide substitution showed marked AT mutational bias suggestive of genetic drift. These results demonstrate that tularemia has moved from east to west in Europe and that has a biology characterized by long-range geographical dispersal events and mostly slow, but variable, replication rates. The results indicate that mutation-driven evolution, a resting survival phase, genetic drift and long-distance geographical dispersal events have interacted to generate genetic diversity within this species.
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http://dx.doi.org/10.1099/mgen.0.000100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5359409PMC
December 2016

Brucella spp. of amphibians comprise genomically diverse motile strains competent for replication in macrophages and survival in mammalian hosts.

Sci Rep 2017 03 16;7:44420. Epub 2017 Mar 16.

Bundeswehr Institute of Microbiology and German Center for Infection Research (DZIF), Munich, Germany.

Twenty-one small Gram-negative motile coccobacilli were isolated from 15 systemically diseased African bullfrogs (Pyxicephalus edulis), and were initially identified as Ochrobactrum anthropi by standard microbiological identification systems. Phylogenetic reconstructions using combined molecular analyses and comparative whole genome analysis of the most diverse of the bullfrog strains verified affiliation with the genus Brucella and placed the isolates in a cluster containing B. inopinata and the other non-classical Brucella species but also revealed significant genetic differences within the group. Four representative but molecularly and phenotypically diverse strains were used for in vitro and in vivo infection experiments. All readily multiplied in macrophage-like murine J774-cells, and their overall intramacrophagic growth rate was comparable to that of B. inopinata BO1 and slightly higher than that of B. microti CCM 4915. In the BALB/c murine model of infection these strains replicated in both spleen and liver, but were less efficient than B. suis 1330. Some strains survived in the mammalian host for up to 12 weeks. The heterogeneity of these novel strains hampers a single species description but their phenotypic and genetic features suggest that they represent an evolutionary link between a soil-associated ancestor and the mammalian host-adapted pathogenic Brucella species.
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http://dx.doi.org/10.1038/srep44420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5353553PMC
March 2017

Achaete-Scute Homolog 1 Expression Controls Cellular Differentiation of Neuroblastoma.

Front Mol Neurosci 2016 21;9:156. Epub 2016 Dec 21.

Institut für Vegetative Physiologie, Charité-Universitätsmedizin Berlin Berlin, Germany.

Neuroblastoma, the major cause of infant cancer deaths, results from fast proliferation of undifferentiated neuroblasts. Treatment of high-risk neuroblastoma includes differentiation with retinoic acid (RA); however, the resistance of many of these tumors to RA-induced differentiation poses a considerable challenge. Human achaete-scute homolog 1 (hASH1) is a proneural basic helix-loop-helix transcription factor essential for neurogenesis and is often upregulated in neuroblastoma. Here, we identified a novel function for hASH1 in regulating the differentiation phenotype of neuroblastoma cells. Global analysis of 986 human neuroblastoma datasets revealed a negative correlation between hASH1 and neuron differentiation that was independent of the N-myc (MYCN) oncogene. Using RA to induce neuron differentiation in two neuroblastoma cell lines displaying high and low levels of hASH1 expression, we confirmed the link between hASH1 expression and the differentiation defective phenotype, which was reversed by silencing hASH1 or by hypoxic preconditioning. We further show that hASH1 suppresses neuronal differentiation by inhibiting transcription at the RA receptor element. Collectively, our data indicate hASH1 to be key for understanding neuroblastoma resistance to differentiation therapy and pave the way for hASH1-targeted therapies for augmenting the response of neuroblastoma to differentiation therapy.
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http://dx.doi.org/10.3389/fnmol.2016.00156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5174122PMC
December 2016

The Change of a Medically Important Genus: Worldwide Occurrence of Genetically Diverse Novel Brucella Species in Exotic Frogs.

PLoS One 2016 30;11(12):e0168872. Epub 2016 Dec 30.

Hessian State Laboratory (LHL), Schubertstrasse 60, Giessen, Germany.

The genus Brucella comprises various species of both veterinary and human medical importance. All species are genetically highly related to each other, sharing intra-species average nucleotide identities (ANI) of > 99%. Infections occur among various warm-blooded animal species, marine mammals, and humans. Until recently, amphibians had not been recognized as a host for Brucella. In this study, however, we show that novel Brucella species are distributed among exotic frogs worldwide. Comparative recA gene analysis of 36 frog isolates from various continents and different frog species revealed an unexpected high genetic diversity, not observed among classical Brucella species. In phylogenetic reconstructions the isolates consequently formed various clusters and grouped together with atypical more distantly related brucellae, like B. inopinata, strain BO2, and Australian isolates from rodents, some of which were isolated as human pathogens. Of one frog isolate (10RB9215) the genome sequence was determined. Comparative genome analysis of this isolate and the classical Brucella species revealed additional genetic material, absent from classical Brucella species but present in Ochrobactrum, the closest genetic neighbor of Brucella, and in other soil associated genera of the Alphaproteobacteria. The presence of gene clusters encoding for additional metabolic functions, flanked by tRNAs and mobile genetic elements, as well as by bacteriophages is suggestive for a different ecology compared to classical Brucella species. Furthermore it suggests that amphibian isolates may represent a link between free living soil saprophytes and the pathogenic Brucella with a preferred intracellular habitat. We therefore assume that brucellae from frogs have a reservoir in soil and, in contrast to classical brucellae, undergo extensive horizontal gene transfer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0168872PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5201264PMC
June 2017

Burkholderia humptydooensis sp. nov., a New Species Related to Burkholderia thailandensis and the Fifth Member of the Burkholderia pseudomallei Complex.

Appl Environ Microbiol 2017 03 15;83(5). Epub 2017 Feb 15.

Department of Biological Sciences and The Pathogen and Microbiome Institute, Northern Arizona University, Flagstaff, Arizona, USA

During routine screening for from water wells in northern Australia in areas where it is endemic, Gram-negative bacteria (strains MSMB43, MSMB121, and MSMB122) with a similar morphology and biochemical pattern to and were coisolated with on Ashdown's selective agar. To determine the exact taxonomic position of these strains and to distinguish them from and , they were subjected to a series of phenotypic and molecular analyses. Biochemical and fatty acid methyl ester analysis was unable to distinguish sp. nov. from closely related species. With matrix-assisted laser desorption ionization-time of flight analysis, all isolates grouped together in a cluster separate from other spp. 16S rRNA and sequence analyses demonstrated phylogenetic placement for sp. nov. in a novel clade within the group. Multilocus sequence typing (MLST) analysis of the three isolates in comparison with MLST data from 3,340 strains and related taxa revealed a new sequence type (ST318). Genome-to-genome distance calculations and the average nucleotide identity of all isolates to both and , based on whole-genome sequences, also confirmed sp. nov. as a novel species within the complex. Molecular analyses clearly demonstrated that strains MSMB43, MSMB121, and MSMB122 belong to a novel species for which the name sp. nov. is proposed, with the type strain MSMB43 (American Type Culture Collection BAA-2767; Belgian Co-ordinated Collections of Microorganisms LMG 29471; DDBJ accession numbers CP013380 to CP013382). is a soil-dwelling bacterium and the causative agent of melioidosis. The genus consists of a diverse group of species, with the closest relatives of referred to as the complex. A proposed novel species, sp. nov., was isolated from a bore water sample from the Northern Territory in Australia. sp. nov. is phylogenetically distinct from and other members of the complex, making it the fifth member of this important group of bacteria.
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http://dx.doi.org/10.1128/AEM.02802-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5311406PMC
March 2017

Isolation of a novel 'atypical' Brucella strain from a bluespotted ribbontail ray (Taeniura lymma).

Antonie Van Leeuwenhoek 2017 Feb 26;110(2):221-234. Epub 2016 Oct 26.

Bundeswehr Institute of Microbiology, Neuherbergstrasse 11, 80937, Munich, Germany.

A pleomorphic Gram-negative, motile coccobacillus was isolated from the gills of a wild-caught bluespotted ribbontail ray after its sudden death during quarantine. Strain 141012304 was observed to grow aerobically, to be clearly positive for cytochrome oxidase, catalase, urease and was initially identified as "Brucella melitensis" or "Ochrobactrum anthropi" by Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and VITEK2-compact, respectively. Affiliation to the genus Brucella was confirmed by bcsp31 and IS711 PCR as well as by Brucella species-specific multiplex PCR, therein displaying a characteristic banding pattern recently described for Brucella strains obtained from amphibian hosts. Likewise, based on recA sequencing, strain 141012304 was found to form a separate lineage, within the so called 'atypical' Brucella, consisting of genetically more distantly related strains. The closest similarity was detected to brucellae, which have recently been isolated from edible bull frogs. Subsequent next generation genome sequencing and phylogenetic analysis confirmed that the ray strain represents a novel Brucella lineage within the atypical group of Brucella and in vicinity to Brucella inopinata and Brucella strain BO2, both isolated from human patients. This is the first report of a natural Brucella infection in a saltwater fish extending the host range of this medically important genus.
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http://dx.doi.org/10.1007/s10482-016-0792-4DOI Listing
February 2017

A High-Coverage Yersinia pestis Genome from a Sixth-Century Justinianic Plague Victim.

Mol Biol Evol 2016 11 30;33(11):2911-2923. Epub 2016 Aug 30.

Max Planck Institute for the Science of Human History, Jena, Germany.

The Justinianic Plague, which started in the sixth century and lasted to the mid eighth century, is thought to be the first of three historically documented plague pandemics causing massive casualties. Historical accounts and molecular data suggest the bacterium Yersinia pestis as its etiological agent. Here we present a new high-coverage (17.9-fold) Y. pestis genome obtained from a sixth-century skeleton recovered from a southern German burial site close to Munich. The reconstructed genome enabled the detection of 30 unique substitutions as well as structural differences that have not been previously described. We report indels affecting a lacl family transcription regulator gene as well as nonsynonymous substitutions in the nrdE, fadJ, and pcp genes, that have been suggested as plague virulence determinants or have been shown to be upregulated in different models of plague infection. In addition, we identify 19 false positive substitutions in a previously published lower-coverage Y. pestis genome from another archaeological site of the same time period and geographical region that is otherwise genetically identical to the high-coverage genome sequence reported here, suggesting low-genetic diversity of the plague during the sixth century in rural southern Germany.
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http://dx.doi.org/10.1093/molbev/msw170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5062324PMC
November 2016

Brucella vulpis sp. nov., isolated from mandibular lymph nodes of red foxes (Vulpes vulpes).

Int J Syst Evol Microbiol 2016 May 29;66(5):2090-2098. Epub 2016 Feb 29.

AGES, Institute for Veterinary Disease Control,Robert Koch-Gasse 17, A-2340 Mödling,Austria.

Two slow-growing, Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains F60T and F965), isolated in Austria from mandibular lymph nodes of two red foxes (Vulpes vulpes), were subjected to a polyphasic taxonomic analysis. In a recent study, both isolates were assigned to the genus Brucella but could not be attributed to any of the existing species. Hence, we have analysed both strains in further detail to determine their exact taxonomic position and genetic relatedness to other members of the genus Brucella. The genome sizes of F60T and F965 were 3 236 779 and 3 237 765 bp, respectively. Each genome consisted of two chromosomes, with a DNA G+C content of 57.2 %. A genome-to-genome distance of >80 %, an average nucleotide identity (ANI) of 97 % and an average amino acid identity (AAI) of 98 % compared with the type species Brucella melitensis confirmed affiliation to the genus. Remarkably, 5 % of the entire genetic information of both strains was of non-Brucella origin, including as-yet uncharacterized bacteriophages and insertion sequences as well as ABC transporters and other genes of metabolic function from various soil-living bacteria. Core-genome-based phylogenetic reconstructions placed the novel species well separated from all hitherto-described species of the genus Brucella, forming a long-branched sister clade to the classical species of Brucella. In summary, based on phenotypic and molecular data, we conclude that strains F60T and F965 are members of a novel species of the genus Brucella, for which the name Brucella vulpis sp. nov. is proposed, with the type strain F60T ( = BCCN 09-2T = DSM 101715T).
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http://dx.doi.org/10.1099/ijsem.0.000998DOI Listing
May 2016

Genotyping Yersinia pestis in Historical Plague: Evidence for Long-Term Persistence of Y. pestis in Europe from the 14th to the 17th Century.

PLoS One 2016 13;11(1):e0145194. Epub 2016 Jan 13.

Bundeswehr Institute of Microbiology, Munich, Germany.

Ancient DNA (aDNA) recovered from plague victims of the second plague pandemic (14th to 17th century), excavated from two different burial sites in Germany, and spanning a time period of more than 300 years, was characterized using single nucleotide polymorphism (SNP) analysis. Of 30 tested skeletons 8 were positive for Yersinia pestis-specific nucleic acid, as determined by qPCR targeting the pla gene. In one individual (MP-19-II), the pla copy number in DNA extracted from tooth pulp was as high as 700 gene copies/μl, indicating severe generalized infection. All positive individuals were identical in all 16 SNP positions, separating phylogenetic branches within nodes N07_N10 (14 SNPs), N07_N08 (SNP s19) and N06_N07 (s545), and were highly similar to previously investigated plague victims from other European countries. Thus, beside the assumed continuous reintroduction of Y. pestis from central Asia in multiple waves during the second pandemic, long-term persistence of Y. pestis in Europe in a yet unknown reservoir host has also to be considered.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0145194PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4712009PMC
July 2016

Detection of Babesia venatorum, Anaplasma phagocytophilum and Candidatus Neoehrlichia mikurensis in Ixodes persulcatus ticks from Mongolia.

Ticks Tick Borne Dis 2016 Mar 11;7(2):357-60. Epub 2015 Dec 11.

Institute of Animal Hygiene and Veterinary Public Health, University of Leipzig, Leipzig, Germany. Electronic address:

Information about the prevalence and geographical distribution of tick-borne pathogens Anaplasma phagocytophilum, Candidatus Neoehrlichia mikurensis, and Babesia spp. is still rare in Mongolia. We tested 275 Ixodes persulcatus ticks for A. phagocytophilum, Cand. N. mikurensis and Babesia spp. and 125 Dermacentor nuttalli ticks especially for Babesia spp. using different PCR methods. Ticks were collected from three provinces (Selenge, Arkhangai, Khentii) in Mongolia. DNA of A. phagocytophilum, Cand. N. mikurensis and Babesia spp. were found with a prevalence of 6.2%, 1.5% and 3.3% in each case in I. persulcatus ticks. This is the first time Cand. N. mikurensis was found in ticks from Mongolia. Sequence analysis of Babesia spp.-positive amplicons showed exclusively B. venatorum, which had also not been mentioned in Mongolia before. On the contrary, all D. nuttalli ticks tested negatively for Babesia spp. This study demonstrates that all three zoonotic pathogens are present in I. persulcatus ticks in Mongolia, and justify the need for further investigations of a more detailed genetic characterization of these pathogens.
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http://dx.doi.org/10.1016/j.ttbdis.2015.12.007DOI Listing
March 2016

Hypoxia inhibits nephrogenesis through paracrine Vegfa despite the ability to enhance tubulogenesis.

Kidney Int 2015 Dec 22;88(6):1283-1292. Epub 2015 Jul 22.

Department of Nephrology and Hypertension, Friedrich-Alexander University Erlangen-Nürnberg (FAU), Erlangen, Germany.

Reduced nephron number predisposes to hypertension and kidney disease. Interaction of the branching ureteric bud and surrounding mesenchymal cells determines nephron number. Since oxygen supply may be critical for intrauterine development, we tested whether hypoxia and hypoxia-inducible factor-1α (HIF-1α) influence nephrogenesis. We found that HIF-1α is required for branching of MDCK cells. In addition, culture of metanephric mouse kidneys with ureteric bud cell-specific stabilization or knockout of HIF-1α revealed a positive impact of HIF-1α on nephrogenesis. In contrast, widespread stabilization of HIF-1α in metanephric kidneys through hypoxia or HIF stabilizers impaired nephrogenesis, and pharmacological HIF inhibition enhanced nephrogenesis. Several lines of evidence suggest an inhibitory effect through the hypoxia response of mesenchymal cells. HIF-1α was expressed in mesenchymal cells during nephrogenesis. Expression of the anti-branching factors Bmp4 and Vegfa, secreted by mesenchymal cells, was increased upon HIF stabilization. The conditioned medium from hypoxic metanephric kidneys inhibited MDCK branching, which was partially rescued by Vegfa antibodies. Thus, the effect of HIF-1α on nephrogenesis appears context dependent. While HIF-1α in the ureteric bud is of importance for proper branching morphogenesis, the net effect of hypoxia-induced HIF activation in the embryonic kidney appears to be mesenchymal cell-dependent inhibition of ureter branching.
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http://dx.doi.org/10.1038/ki.2015.214DOI Listing
December 2015

The GYF domain protein CD2BP2 is critical for embryogenesis and podocyte function.

J Mol Cell Biol 2015 Oct 16;7(5):402-14. Epub 2015 Jun 16.

Institute for Chemistry and Biochemistry, Freie Universität Berlin, 14195 Berlin, Germany Leibniz-Institut fuer Molekulare Pharmakologie, 13125 Berlin, Germany

Scaffolding proteins play pivotal roles in the assembly of macromolecular machines such as the spliceosome. The adaptor protein CD2BP2, originally identified as a binding partner of the adhesion molecule CD2, is a pre-spliceosomal assembly factor that utilizes its glycine-tyrosine-phenylalanine (GYF) domain to co-localize with spliceosomal proteins. So far, its function in vertebrates is unknown. Using conditional gene targeting in mice, we show that CD2BP2 is crucial for embryogenesis, leading to growth retardation, defects in vascularization, and premature death at embryonic day 10.5 when absent. Ablation of the protein in bone marrow-derived macrophages indicates that CD2BP2 is involved in the alternative splicing of mRNA transcripts from diverse origins. At the molecular level, we identified the phosphatase PP1 to be recruited to the spliceosome via the N-terminus of CD2BP2. Given the strong expression of CD2BP2 in podocytes of the kidney, we use selective depletion of CD2BP2, in combination with next-generation sequencing, to monitor changes in exon usage of genes critical for podocyte functions, including VEGF and actin regulators. CD2BP2-depleted podocytes display foot process effacement, and cause proteinuria and ultimately lethal kidney failure in mice. Collectively, our study defines CD2BP2 as a non-redundant splicing factor essential for embryonic development and podocyte integrity.
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http://dx.doi.org/10.1093/jmcb/mjv039DOI Listing
October 2015