Functional Characterization of the HuR:CD83 mRNA Interaction

Maturation of dendritic cells (DC) is characterized by expression of CD83, a surface protein that appears to be necessary for the effective activation of naïve T-cells and T-helper cells by DC. Lately it was shown that CD83 expression is regulated on the posttranscriptional level by interaction of the shuttle protein HuR with a novel posttranscriptional regulatory RNA element (PRE), which is located in the coding region of the CD83 transcript. Interestingly, this interaction commits the CD83 mRNA to efficient nuclear export via the CRM1 pathway. To date, however, the structural basis of this interaction, which potentially involves three distinct RNA recognition motifs (RRM1–3) in HuR and a complex three-pronged RNA stem-loop element in CD83 mRNA, has not been investigated in detail. In the present work we analyzed this interaction in vitro and in vivo using various HuR- and CD83 mRNA mutants. We are able to demonstrate that both, RRM1 and RRM2 are crucial for binding, whereas RRM3 as well as the HuR hinge region contributed only marginally to this protein∶RNA interaction. Furthermore, mutation of uridine rich patches within the PRE did not disturb HuR:CD83 mRNA complex formation while, in contrast, the deletion of specific PRE subfragments from the CD83 mRNA prevented HuR binding in vitro and in vivo. Interestingly, the observed inhibition of HuR binding to CD83 mRNA does not lead to a nuclear trapping of the transcript but rather redirected this transcript from the CRM1- towards the NXF1/TAP-specific nuclear export pathway. Thus, the presence of a functional PRE permits nucleocytoplasmic trafficking of the CD83 transcript via the CRM1 pathway

Highly significant antiviral activity of HIV-1 LTR-specific tre-recombinase in humanized mice

Stable integration of HIV proviral DNA into host cell chromosomes, a hallmark and essential feature of the retroviral life cycle, establishes the infection permanently. Current antiretroviral combination drug therapy cannot cure HIV infection. However, expressing an engineered HIV-1 long terminal repeat (LTR) site-specific recombinase (Tre), shown to excise integrated proviral DNA in vitro, may provide a novel and highly promising antiviral strategy. We report here the conditional expression of Tre-recombinase from an advanced lentiviral self-inactivation (SIN) vector in HIV-infected cells. We demonstrate faithful transgene expression, resulting in accurate provirus excision in the absence of cytopathic effects. Moreover, pronounced Tre-mediated antiviral effects are demonstrated in vivo, particularly in humanized Rag2−/−γc−/− mice engrafted with either Tre-transduced primary CD4+ T cells, or Tre-transduced CD34+ hematopoietic stem and progenitor cells (HSC). Taken together, our data support the use of Tre-recombinase in novel therapy strategies aiming to provide a cure for HIV

The acidic protein rich in leucines Anp32b is an immunomodulator of inflammation in mice

ANP32B belongs to a family of evolutionary conserved acidic nuclear phosphoproteins (ANP32A-H). Family members have been described as multifunctional regulatory proteins and proto-oncogenic factors affecting embryonic development, cell proliferation, apoptosis, and gene expression at various levels. Involvement of ANP32B in multiple processes of cellular life is reflected by the previous finding that systemic gene knockout (KO) of Anp32b leads to embryonic lethality in mice. Here, we demonstrate that a conditional KO of Anp32b is well tolerated in adult animals. However, after immune activation splenocytes isolated from Anp32b KO mice showed a strong commitment towards Th17 immune responses. Therefore, we further analyzed the respective animals in vivo using an experimental autoimmune encephalomyelitis (EAE) model. Interestingly, an exacerbated clinical score was observed in the Anp32b KO mice. This was accompanied by the finding that animal-derived T lymphocytes were in a more activated state, and RNA sequencing analyses revealed hyperactivation of several T lymphocyte-associated immune modulatory pathways, attended by significant upregulation of Tfh cell numbers that altogether might explain the observed strong autoreactive processes. Therefore, Anp32b appears to fulfill a role in regulating adequate adaptive immune responses and, hence, may be involved in dysregulation of pathways leading to autoimmune disorders and/or immune deficiencies

Evaluation of Processed Nerve Allograft in Peripheral Nerve Surgery : A Systematic Review and Critical Appraisal

Background: Peripheral nerve injuries cause substantial problems when not treated properly. A specific problem is reconstruction of nerve defects, which can be treated in different ways. This study aimed to systematically review whether processed nerve allograft (PNA) is justified in reconstruction of a nerve defect in patients after posttraumatic or iatrogenic peripheral nerve injury and to compare PNA with other established methods. Methods: A systematic review with a focused question, PICO (patient, intervention, comparison, outcome) and constraints, was performed. A structured literature search, including several databases, was done to evaluate the existing evidence for outcomes and postoperative complications related to PNA. The certainty of evidence was classified according to Grading of Recommendations, Assessment, Development and Evaluations. Results: No conclusions, concerning differences in outcome of nerve reconstruction using PNA compared with the use of nerve autograft or conduits, could be drawn. The level of certainty for all evaluated outcomes was very low. Most published studies lack a control group to patients treated with PNA; being only descriptive, making it difficult to compare PNA with established methods without substantial risk of bias. For studies including a control group, the scientific evidence was of very low certainty, due to a low number of included patients, and large, undefined loss of patients during follow-up, rendering a high risk of bias. Finally, the authors often had financial disclosures. Conclusion: Properly conducted randomized controlled trial studies on the use of PNA in reconstruction of peripheral nerve injuries are needed to establish recommendations in clinical practice

African tick bite fever - papulovesicular exanthem with fever after staying in South Africa

In the wake of expanding international tourism, rickettsioses are increasingly observed also in central Europe. African tick bite fever is a recently described, acute febrile illness with characteristic skin lesions. It is caused by Rickettsia africae, which is transmitted to humans by ticks of the Amblyomma genus. A 60-year-old woman presented with a papulovesic-ular exanthem, fever, and headache after returning from South Africa. A purple nodule with central necrosis ("tache noire"or "inoculation eschar") was noticed on the lower leg. Antibodies against rickettsia of the spotted fever group were detected serologically. Oral doxycycline led to clearance of the disease after few days of treatment

Posttranslational hypusination of the eukaryotic translation initiation factor-5A regulates Fusarium graminearum virulence

Activation of eukaryotic translation initiation factor eIF5A requires a posttranslational modification, forming the unique amino acid hypusine. This activation is mediated by two enzymes, deoxyhypusine synthase, DHS, and deoxyhypusine hydroxylase, DOHH. The impact of this enzymatic complex on the life cycle of a fungal pathogen is unknown. Plant pathogenic ascomycetes possess a single copy of the eIF5A activated by hypusination. We evaluated the importance of imbalances in eIF5A hypusination in Fusarium graminearum, a devastating fungal pathogen of cereals. Overexpression of DHS leads to increased virulence in wheat, elevated production of the mycotoxin deoxynivalenol, more infection structures, faster wheat tissue invasion in plants and increases vegetatively produced conidia. In contrast, overexpression of DOHH completely prevents infection structure formation, pathogenicity in wheat and maize, leads to overproduction of ROS, reduced DON production and increased sexual reproduction. Simultaneous overexpression of both genes restores wild type-like phenotypes. Analysis of eIF5A posttranslational modification displayed strongly increased hypusinated eIF5A in DOHH overexpression mutant in comparison to wild type, and the DHS overexpression mutants. These are the first results pointing to different functions of differently modified eIF5A

Hybrid-Mode-Assisted Long-Distance Excitation of Short-Range Surface Plasmons in a Nanotip-Enhanced Step-Index Fiber

We propose and experimentally demonstrate a monolithic nanowire-enhanced fiber-based nanoprobe for the broadband delivery of light (550–730 nm) to a deep subwavelength scale using short-range surface plasmons. The geometry is formed by a step index fiber with an integrated gold nanowire in its core and a protruding gold nanotip with sub-10 nm apex radius. We present a novel coupling scheme to excite short-range surface plasmons, whereby the radially polarized hybrid mode propagating inside the nanowire section excites the plasmonic mode close to the fiber endface, which is in turn superfocused down to nanoscale dimensions at the tip apex. We show that in this all-integrated fiber-plasmonic coupling scheme the wire length can be orders of magnitude longer than the attenuation length of short-range plasmon polaritons, yielding a broadband plasmon excitation and reducing demands in fabrication. We observe that the scattered light in the far-field from the nanotip is axially polarized and preferentially excited by a radially polarized input, unambiguously revealing that it originates from a short-range plasmon propagating on the nanotip, in agreement with simulations. This novel excitation scheme will have important applications in near-field microscopy and nanophotonics and potentially offers significantly improved resolution compared to current delivery near-field probes

RRM1 and RRM2 of HuR are necessary for efficient CD83 mRNA recognition.

<p><b>A.</b> Schematic diagram of the domain structure of HuR. The positions of the three RNA recognition motifs (RRM1–3) and the hinge region (H) in the human 326 amino acid (aa) HuR protein are indicated. Recombinant proteins (indicated by probe #) which were subsequently analyzed for CD83 PRE binding are depicted. Probe numbers correspond to gel lanes in panel B and C. <b>B.</b> Coomassie-stained SDS-PAGE of the recombinantly expressed and purified proteins (indicated by arrowheads). M, marker proteins. <b>C.</b> Radiolabelled CD83wt PRE coding sequence RNA was incubated either with GST (negative control) or with various HuR-derived GST-fusion proteins. Lane 1: GST; lane 2: full-length HuR; lane 3: HuR aa 1–244 (RRM1-RRM2-H); lane 4: HuR aa 27–93 (RRM1); lane 5: HuR aa 108–174 (RRM2); lane 6: HuR aa 246–317 (RRM3); lane 7: HuR aa 103–244 (RRM2-H); lane 8: HuR aa 190–328 (H-RRM3); HuR aa 175–245 (H). CD83 PRE RNA:protein interaction was analyzed by gel retardation assay as before.</p

CRM1-mediated nuclear export of CD83 mRNA PRE.

<p><b>A.</b> COS7 cells were transiently transfected either with an expression vector encoding for human CD83 cDNA flanked by the homologous 5′- and 3′-UTR (wt; lane 1–6) or with a related vector in which essential PRE sequences were deleted (PREΔSubL1–3; lane 7–12). At day two posttransfection cultures were exposed to 10 nM of the CRM1 inhibitor LMB or DMSO (solvent control) for 8 hours. Total, cytoplasmic and nuclear RNA was isolated, subjected to CD83- and GAPDH-specific (negative control) PCR and analyzed by gel electrophoresis. <b>B.</b> Quantitative real-time PCR of the RNA probes shown in panel A. RNA ratios +LMB/−LMB are depicted. <b>C.</b> Total, cytoplasmic and nuclear RNA was isolated from cell cultures which were cotransfected with the CD83 PREΔSubL1–3 expression vector and either a construct expressing four tandem repeats of the MPMV CTE (4×CTE) or the respective parental vector (negative control). For NXF1/TAP-independent control, mitochondrial cytochrome C oxidase mRNA was detected in total and cytoplasmic RNA, while GAPDH-specific transcripts were detected in nuclear RNA. <b>D.</b> Quantitative real-time PCR of the RNA probes shown in panel C. RNA ratios +/− NXF1/TAP inactivation (+CTE/−CTE) are depicted.</p