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Papers by Adam Grundhoff

(A) Northern blot analysis of total RNA from HEK293T cells transfecte... more (A) Northern blot analysis of total RNA from HEK293T cells transfected with indicated miRNA or putative miRNA expression vectors, with ethidium bromide stained low molecular weight RNA shown as a load control. This figure represents a single membrane that was probed first for control SV40 miRNA, then stripped and re-probed for each of the indicated miRNAs. Solid and outline arrows on left side correspond to pre-miRNAs and mature miRNAs, respectively, and approximate sizes are noted on right side. (B) Northern blot analysis of total RNA from HEK293T cells transfected with anti-Drosha siRNA or negative control (NC) siRNA, then re-transfected after 48 hours with respective siRNAs and indicated putative miRNA expression vectors. Total RNA was harvested after 48 hours. Ethidium bromide stained low molecular weight RNA shown as a load control. This figure represents a single membrane that was probed first for control SV40 miRNA, then stripped and re-probed for each of the indicated miRNAs. Membrane was additionally probed for HSUR (Herpesvirus saimiri U RNA 4) as a transfection control. Normalized to HSUR RNA, all indicated miRNAs are detected at higher intensity in negative-control-treated cells than Drosha knockdown cells, suggesting canonical dependence on Drosha processing. The numbers below each blot correspond to the ratio of mature miRNA signal in control cells compared to mature miRNA signal in Drosha-knockdown cells after each signal has been normalized to load control HSUR signal. Note: due to sequence similarity between putative HPV-derived miRNAs, cross-reactivity is seen in hpv17-miR-H1 lanes and hpv37-miR-H1 lanes. (C) Northern blot analysis of RNA from either HEK293T or Dicer KO (NoDice [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007156#ppat.1007156.ref068" target="_blank">68</a>]) cells transfected with the indicated miRNA expression vectors. Solid and outline arrows on left side correspond to pre-miRNAs and mature miRNAs, respectively. The numbers below each blot correspond to the ratio of mature miRNA signal to pre-miRNA signal, normalized to the same ratio in HEK293T cells. This figure represents a single membrane that was probed, then stripped and reprobed with the indicated oligonucleotide probes (SV40, FcPV1 F1 and F2, HPV41 miRNA probes) and a second membrane for HPV17 and HPV37 miRNAs. Ethidium bromide stained low molecular weight RNA is shown as a load control.</p

(A) Western blot analysis of HUVEC cells transduced with shRNA-expres... more (A) Western blot analysis of HUVEC cells transduced with shRNA-expressing lentiviruses directed against PML, Daxx, Sp100 or GFP. shDaxx transduced cells did not show a reduction of Daxx protein levels and were thus not included in further analyses. Although residual levels of PML and Sp100 are still visible in bulk protein extracts, immunofluorescence analysis suggests that a considerable number of cells is devoid of PML or Sp100 positive nuclear bodies (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004274#ppat.1004274.s009" target="_blank">Figure S9</a>). (B) FACS analysis of stably shRNA-expressing HUVEC cultures analysed 48 h ater infection with KSHV. The rightmost columns show cells which were treated with sodium butyrate (5 mM) immediately after infection. Mock infected cells were used to correct for background staining levels. Error bars represent SEM of three biological replicates. (C) Transcript levels of ORF50, ORF59, ORF64 and ORF73 in EA.hy cells at 48 hours post infection. Expression was calculated by normalization to GAPDH and is shown relative to shGFP controls (set to 1). Error bars represent SEM of at least three data sets.</p

(A) Western blot analysis and (B) confocal laser s... more (A) Western blot analysis and (B) confocal laser scanning immunofluorescence microscopy of PFSK-1 cells transfected with MCVSyn or MCVSyn-hpko and analyzed at the indicated time points. Material was derived from the same cultures shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004974#ppat.1004974.g008" target="_blank">Fig 8A to 8C</a>. The asterisk in (A) denotes the position of an unspecific background band. Antibody CM2B4 recognizes LT-Ag as well as the alternative splice product 57k T. The positions of both protein bands are indicated by arrowheads.</p

(A) SLK cells were transfected with KSHV BAC16 <a href="http:... more (A) SLK cells were transfected with KSHV BAC16 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004274#ppat.1004274-Brulois1" target="_blank">[143]</a> DNA and selected with hygromycin for 24 days to select for bacmid-carrying cells. Histone modifications were analyzed by high resolution ChIP on microarray analysis with antibodies directed against H3K4me3 (upper panel) or H3K27me3 (lower panel). (B) SLK cells were infected with KSHV and chromatin was prepared at indicated time points, and histone modification profiles were investigated by high resolution ChIP on microarray analysis with antibodies directed against H3K4me3 (upper panel) or H3K27me3 (lower panel). Arrows above H3K4me3 profiles denote peaks that are prominent at 24 h post infection, but were diminished upon acquisition of repressive H3K27me3 marks at later time points. Normalized signal intensity values from the profiles shown in A and B as well as from previously investigated SLK cells at 5 d post infection (5 dpi) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004274#ppat.1004274-Gunther1" target="_blank">[14]</a> were used to generate the heat maps shown in (C). The heat maps indicate the chromatin status as being either naïve (black), dominated by H3K4me3 (green) or H3K27me3 (red), or characterized by the presence of both modifications (yellow). The latter state is designated as ‘putative bivalent’ since co-occurrence of both modifications on the same nucleosome has formally been proven only for the ORF50/Rta promoter <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004274#ppat.1004274-Gunther1" target="_blank">[14]</a>.</p

(A+B) SLK-shSp100, SLK-shDaxx, SLK-shPML and SLK-shGFP cells were de novo</i... more (A+B) SLK-shSp100, SLK-shDaxx, SLK-shPML and SLK-shGFP cells were de novo infected with KSHV and chromatin was prepared at 36 h post infection. Deposition of activating H3K4me3 (A) or repressive H3K27me3 (B) marks was evaluated by ChIP-qPCR using specific primers as given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004274#ppat.1004274.s012" target="_blank">Table S1</a>. (C+D) ChIP-qPCR analysis of selected loci in de novo KSHV infected shSp100 (red squares) or shGFP (control; blue diamonds) SLK (C) or EA.hy (D) cells was performed to analyze deposition of H3K27me3 marks at the indicated time points.</p

(A) KSHV negative SLK cells or long-term infected SLKP<... more (A) KSHV negative SLK cells or long-term infected SLKP cells were treated with either the proteasome inhibitor MG-132 or DMSO for 8 h. Subsequently, soluble RIPA extracts were prepared and analyzed for Sp100 and LANA protein levels by immunoblotting. (B) Transcript levels of genes encoding the ND10 core components (PML, SP100, DAXX) and the three housekeeping genes GAPDH, ADH5 and VPS29. Transcript levels were analyzed by RNAseq (see complete dataset in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004274#ppat.1004274.s013" target="_blank">Dataset S1</a>) in mock infected (0 h value) or KSHV infected SLK cells after 2, 4, 8, 12, 16, 24, 48 or 96 hours of infection and are given as RPKM (reads per kilobase and million mapped reads) values. Baseline expression levels as observed in mock infected cells are marked across plots by a dashed gray line. The fold range (FR) of maximum up- or down-regulation across the entire time course is indicated in each panel.</p

(A) Coverage plots of miDGE DNA-seq and small RNA-seq across the geno... more (A) Coverage plots of miDGE DNA-seq and small RNA-seq across the genomes of HPV18 and HPV17. Filled green plots at the top of each panel show DNA-seq coverage, the three plots underneath show mapped small RNA-seq from: PV: HEK293T-cells transfected with our papillomavirus miDGE library, PV filtered: same reads as in PV, but filtered to eliminate low-complexity reads JMRV: Serving as a negative control, derived from 293T-cells transfected with our JMRV miDGE library. JMRV read counts were normalized to correct for different sequencing depths between PV and JMRV miDGE experiments (see total read counts in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007156#ppat.1007156.t001" target="_blank">Table 1</a>). Asterisk indicates a previously purported miRNA candidate region suggested in the literature [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007156#ppat.1007156.ref039" target="_blank">39</a>], which is nonspecific (detected in the negative control JMRV miDGE analysis, lower plot) and can be eliminated by removing low complexity reads (center plot). (B) RNA-seq coverage for the most abundantly mapped HPV in 303 tumors in the TCGA CESC project [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007156#ppat.1007156.ref050" target="_blank">50</a>]. Each of the 303 libraries are represented on the X-axis (sorted based on Y-axis value). Y-axis indicates the percentage of the positions in the HPV genome with read mappings. Libraries with > = 50% coverage (213 libraries) were used for subsequent analysis. (C) Percentages of TCGA cervical squamous cell carcinoma (CESC) libraries with miRDeep2 miRNA identifications for each set of reference sequences. Number of libraries examined is 213. (D) Number of unique miRDeep2 miRNA identifications across TCGA CESC libraries for each set of reference sequences. Number of libraries examined is 213. (E) Percentages of Qian et al. [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007156#ppat.1007156.ref040" target="_blank">40</a>] libraries with miRDeep2 miRNA identifications for each set of reference sequences. Number of libraries examined is 12. (F) Number of unique miRDeep2 miRNA identifications across Qian et al. libraries for each set of reference sequences. Number of libraries examined is 12. (G) Raw read counts of all small RNAs mapping to the indicated reference sequences for each library from Qian et al.</p

Viruses, 2022

Background: The recently emerged SARS-CoV-2 B.1.1.529 lineage and its sublineages (Omicron varian... more Background: The recently emerged SARS-CoV-2 B.1.1.529 lineage and its sublineages (Omicron variant) pose a new challenge to healthcare systems worldwide due to its ability to efficiently spread in immunized populations and its resistance to currently available monoclonal antibody therapies. RT-PCR-based variant tests can be used to screen large sample-sets rapidly and accurately for relevant variants of concern (VOC). The aim of this study was to establish and validate a multiplex assay on the cobas 6800/8800 systems to allow discrimination between the two currently circulating VOCs, Omicron and Delta, in clinical samples. Methods: Primers and probes were evaluated for multiplex compatibility. Analytic performance was assessed using cell culture supernatant of an Omicron variant isolate and a clinical Delta variant sample, normalized to WHO-Standard. Clinical performance of the multiplex assay was benchmarked against NGS results. Results: In silico testing of all oligos showed no in...

Cells, 2022

The human skin and in particular its outermost layer, the epidermis, protects the body from poten... more The human skin and in particular its outermost layer, the epidermis, protects the body from potentially harmful substances, radiation as well as excessive water loss. However, the interference between the various stress responses of the epidermal keratinocytes, which often occur simultaneously, is largely unknown. The focus of this study was to investigate the interference between osmotic stress and DNA damage response. In addition to revealing the already well-described regulation of diverse gene sets, for example, cellular processes such as transcription, translation, and metabolic pathways (e.g., the KEGG citrate cycle and Reactome G2/M checkpoints), gene expression analysis of osmotically stressed keratinocytes revealed an influence on the transcription of genes also related to UV-induced DNA damage response. A gene network regulating the H2AX phosphorylation was identified to be regulated by osmotic stress. To analyze and test the interference between osmotic stress and DNA dam...

Additional file 2. Compressed rar file containing the bins generated from the biogas fermenter me... more Additional file 2. Compressed rar file containing the bins generated from the biogas fermenter metagenome, part 2 of 4.

Additional file 9. High-resolution version of Fig.Â 5a with continuous labeling of all bacterial ... more Additional file 9. High-resolution version of Fig.Â 5a with continuous labeling of all bacterial bins. Bin-IDs are color coded according to assigned phylum.

West Nile virus (WNV) belongs to a group of medically important single-stranded, positive-sense R... more West Nile virus (WNV) belongs to a group of medically important single-stranded, positive-sense RNA viruses causing deadly disease outbreaks around the world. The 3 0 untranslated region (3 0-UTR) of the flavivirus genome, in particular the terminal 3 0 stem-loop (3 0 SL) fulfils multiple functions in virus replication and virus-host interactions. Using the Kunjin strain of WNV (WNV KUN), we detected a virally encoded small RNA, named KUN-miR-1, derived from 3 0 SL. Transcription of WNV KUN pre-miRNA (3 0 SL) in mosquito cells either from plasmid or Semliki Forest virus (SFV) RNA replicon resulted in the production of mature KUN-miR-1. Silencing of Dicer-1 but not Dicer-2 led to a reduction in the miRNA levels. Further, when a synthetic inhibitor of KUN-miR-1 was transfected into mosquito cells, replication of viral RNA was significantly reduced. Using cloning and bioinformatics approaches, we identified the cellular GATA4 mRNA as a target for KUN-miR-1. KUN-miR-1 produced in mosquito cells during virus infection or from plasmid DNA, SFV RNA replicon or mature miRNA duplex increased accumulation of GATA4 mRNA. Depletion of GATA4 mRNA by RNA silencing led to a significant reduction in virus RNA replication while a KUN-miR-1 RNA mimic enhanced replication of a mutant WNV KUN virus producing reduced amounts of KUN-miR-1, suggesting that GATA4-induction via KUN-miR-1 plays an important role in virus replication.

Kidney International, 2021

BK polyomavirus-associated nephropathy is a common complication after kidney transplantation lead... more BK polyomavirus-associated nephropathy is a common complication after kidney transplantation leading to reduced graft function or loss. The molecular pathogenesis of BK polyomavirus-induced nephropathy is not well understood. A recent study had described a protective effect of the activating natural killer cell receptor KIR3DS1 in BK polyomavirus-associated nephropathy, suggesting a role of NK cells in modulating disease progression. Using an in vitro cell culture model of human BK polyomavirus infection and kidney biopsy samples from patients with BK polyomavirus-associated nephropathy, we observed significantly increased surface expression of the ligand for KIR3DS1, HLA-F, on BK polyomavirus-infected kidney tubular cells. Upregulation of HLA-F expression resulted in significantly increased binding of KIR3DS1 to BK polyomavirus-infected cells and activation of primary KIR3DS-positive natural killer cells. Thus, our data provide a mechanism by which KIR3DS-positive natural killer cells can control BK polyomavirus infection of the kidney, and rationale for exploring HLA-F/KIR3DS1 interactions for immunotherapeutic approaches in BK polyomavirus-associated nephropathy.

Highlights d Importin-a3 is the most abundantly expressed isoform in the mammalian lung d Importi... more Highlights d Importin-a3 is the most abundantly expressed isoform in the mammalian lung d Importin-a3 is highly conserved across species d Importin-a3 is one of the major nuclear transporters of NF-kB d Importin-a3 acts as an immune sensor of influenza A virus infections

mBio, 2020

Kaposi’s sarcoma-associated herpesvirus (KSHV) causes the aggressive disease primary effusion lym... more Kaposi’s sarcoma-associated herpesvirus (KSHV) causes the aggressive disease primary effusion lymphoma (PEL). Here, we show that a viral transcription factor (vIRF3) cooperates with the cellular transcription factor IRF4 to control an oncogenic gene expression program in PEL cells. These proteins promote KSHV-mediated B cell transformation by activating the expression of prosurvival genes through super-enhancers. Our report thus demonstrates that this DNA tumor virus encodes a transcription factor that functions with cellular IRF4 to drive oncogenic transcriptional reprogramming.

Cell Reports, 2020

Importin-a adaptor proteins orchestrate dynamic nuclear transport processes involved in cellular ... more Importin-a adaptor proteins orchestrate dynamic nuclear transport processes involved in cellular homeostasis. Here, we show that importin-a3, one of the main NF-kB transporters, is the most abundantly expressed classical nuclear transport factor in the mammalian respiratory tract. Importin-a3 promoter activity is regulated by TNF-a-induced NF-kB in a concentration-dependent manner. High-level TNFa-inducing highly pathogenic avian influenza A viruses (HPAIVs) isolated from fatal human cases harboring human-type polymerase signatures (PB2 627K, 701N) significantly downregulate importin-a3 mRNA expression in primary lung cells. Importin-a3 depletion is restored upon back-mutating the HPAIV polymerase into an avian-type signature (PB2 627E, 701D) that can no longer induce high TNF-a levels. Importin-a3-deficient mice show reduced NF-kBactivated antiviral gene expression and increased influenza lethality. Thus, importin-a3 plays a key role in antiviral immunity against influenza. Lifting the bottleneck in importin-a3 availability in the lung might provide a new strategy to combat respiratory virus infections.

Life Science Alliance, 2020

HIV and EBV are human pathogens that cause a considerable burden to worldwide health. In combinat... more HIV and EBV are human pathogens that cause a considerable burden to worldwide health. In combination, these viruses are linked to AIDS-associated lymphomas. We found that EBV, which transforms B cells, renders them susceptible to HIV-1 infection in a CXCR4 and CD4-dependent manner in vitro and that CXCR4-tropic HIV-1 integrates into the genome of these B cells with the same molecular profile as in autologous CD4+ T cells. In addition, we established a humanized mouse model to investigate the in vivo interactions of EBV and HIV-1 upon coinfection. The respective mice that reconstitute human immune system components upon transplantation with CD34+ human hematopoietic progenitor cells could recapitulate aspects of EBV and HIV immunobiology observed in dual-infected patients. Upon coinfection of humanized mice, EBV/HIV dual-infected B cells could be detected, but were susceptible to CD8+ T-cell–mediated immune control.

Diagnostics, 2021

Background: The recent emergence of distinct and highly successful SARS-CoV-2 lineages has substa... more Background: The recent emergence of distinct and highly successful SARS-CoV-2 lineages has substantial implications for individual patients and public health measures. While next-generation-sequencing is routinely performed for surveillance purposes, RT-qPCR can be used to rapidly rule-in or rule-out relevant variants, e.g., in outbreak scenarios. The objective of this study was to create an adaptable and comprehensive toolset for multiplexed Spike-gene SNP detection, which was applied to screen for SARS-CoV-2 B.1.617 lineage variants. Methods: We created a broad set of single nucleotide polymorphism (SNP)-assays including del-Y144/145, E484K, E484Q, P681H, P681R, L452R, and V1176F based on a highly specific multi-LNA (locked nucleic acid)-probe design to maximize mismatch discrimination. As proof-of-concept, a multiplex-test was compiled and validated (SCOV2-617VOC-UCT) including SNP-detection for L452R, P681R, E484K, and E484Q to provide rapid screening capabilities for the novel ...

Viruses, 2021

So far, only a few reports about reinfections with SARS-CoV-2 have been published, and they often... more So far, only a few reports about reinfections with SARS-CoV-2 have been published, and they often lack detailed immunological and virological data. We report about a SARS-CoV-2 reinfection with a genetically distinct SARS-CoV-2 variant in an immunocompetent female healthcare worker that has led to a mild disease course. No obvious viral escape mutations were observed in the second virus variant. The infectious virus was shed from the patient during the second infection episode despite the presence of neutralizing antibodies in her blood. Our data indicate that a moderate immune response after the first infection, but not a viral escape, did allow for reinfection and live virus shedding.