Publications by authors named "Alexander Hahn"

49 Publications

Preoperative characteristics predictive of PROMIS Pain Interference two years after shoulder surgery.

J Orthop 2021 Sep-Oct;27:49-55. Epub 2021 Aug 16.

Department of Orthopaedic Surgery, University of Maryland School of Medicine, Baltimore, MD, USA.

Introduction: The objective of this study was to identify preoperative characteristics associated with worse Patient-Reported Outcomes Measurement Information System (PROMIS) Pain Interference (PI) two years after shoulder surgery.

Methods: This was a retrospective analysis of prospectively collected data on 293 patients who underwent elective shoulder surgery. Survey questionnaires were collected within one week of surgery and then two years postoperatively. Bivariate analysis was used to identify associations and multivariable analysis was used to control for confounding variables.

Results: Worse two-year PROMIS PI was significantly correlated with older age, higher BMI, greater comorbidities, more prior surgeries, and multiple socio-demographic factors. Less improvement in PROMIS PI was significantly correlated with greater comorbidities, more previous surgeries, unemployment, prior orthopaedic surgery on the operative joint, and a higher American Society of Anesthesiologists (ASA) score. Better scores on all preoperative patient-reported outcome measures correlated with better two-year PROMIS PI. Multivariable analysis demonstrated that worse two-year PROMIS PI was independently predicted by the following preoperative factors: Workers' Compensation claim, opioid use, worse whole body Numeric Pain Score, and worse PROMIS PI. Less improvement in two-year PROMIS PI was predicted by the same preoperative factors.

Conclusion: Worse PROMIS PI after shoulder surgery was associated with older age, greater comorbidities, mental health impairment, and lower socioeconomic status. Preoperative predictors of worse pain interference two years after shoulder surgery included Workers' Compensation, opioid use, worse whole body pain, and worse PROMIS PI.

Level Of Evidence: III.
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http://dx.doi.org/10.1016/j.jor.2021.08.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8405907PMC
August 2021

Inhibition of the NLRP3/IL-1β axis protects against sepsis-induced cardiomyopathy.

J Cachexia Sarcopenia Muscle 2021 Sep 2. Epub 2021 Sep 2.

Experimental and Clinical Research Center (ECRC), Charité-Universitätsmedizin Berlin, Max Delbrück Center (MDC) for Molecular Medicine in the Helmholtz Association, Berlin, Germany.

Background: Septic cardiomyopathy worsens the prognosis of critically ill patients. Clinical data suggest that interleukin-1β (IL-1β), activated by the NLRP3 inflammasome, compromises cardiac function. Whether or not deleting Nlrp3 would prevent cardiac atrophy and improve diastolic cardiac function in sepsis was unclear. Here, we investigated the role of NLRP3/IL-1β in sepsis-induced cardiomyopathy and cardiac atrophy.

Methods: Male Nlrp3 knockout (KO) and wild-type (WT) mice were exposed to polymicrobial sepsis by caecal ligation and puncture (CLP) surgery (KO, n = 27; WT, n = 33) to induce septic cardiomyopathy. Sham-treated mice served as controls (KO, n = 11; WT, n = 16). Heart weights and morphology, echocardiography and analyses of gene and protein expression were used to evaluate septic cardiomyopathy and cardiac atrophy. IL-1β effects on primary and immortalized cardiomyocytes were investigated by morphological and molecular analyses. IonOptix and real-time deformability cytometry (RT-DC) analysis were used to investigate functional and mechanical effects of IL-1β on cardiomyocytes.

Results: Heart morphology and echocardiography revealed preserved systolic (stroke volume: WT sham vs. WT CLP: 33.1 ± 7.2 μL vs. 24.6 ± 8.7 μL, P < 0.05; KO sham vs. KO CLP: 28.3 ± 8.1 μL vs. 29.9 ± 9.9 μL, n.s.; P < 0.05 vs. WT CLP) and diastolic (peak E wave velocity: WT sham vs. WT CLP: 750 ± 132 vs. 522 ± 200 mm/s, P < 0.001; KO sham vs. KO CLP: 709 ± 152 vs. 639 ± 165 mm/s, n.s.; P < 0.05 vs. WT CLP) cardiac function and attenuated cardiac (heart weight-tibia length ratio: WT CLP vs. WT sham: -26.6%, P < 0.05; KO CLP vs. KO sham: -3.3%, n.s.; P < 0.05 vs. WT CLP) and cardiomyocyte atrophy in KO mice during sepsis. IonOptix measurements showed that IL-1β decreased contractility (cell shortening: IL-1β: -15.4 ± 2.3%, P < 0.001 vs. vehicle, IL-1RA: -6.1 ± 3.3%, P < 0.05 vs. IL-1β) and relaxation of adult rat ventricular cardiomyocytes (time-to-50% relengthening: IL-1β: 2071 ± 225 ms, P < 0.001 vs. vehicle, IL-1RA: 564 ± 247 ms, P < 0.001 vs. IL-1β), which was attenuated by an IL-1 receptor antagonist (IL-1RA). RT-DC analysis indicated that IL-1β reduced cardiomyocyte size (P < 0.001) and deformation (P < 0.05). RNA sequencing showed that genes involved in NF-κB signalling, autophagy and lysosomal protein degradation were enriched in hearts of septic WT but not in septic KO mice. Western blotting and qPCR disclosed that IL-1β activated NF-κB and its target genes, caused atrophy and decreased myosin protein in myocytes, which was accompanied by an increased autophagy gene expression. These effects were attenuated by IL-1RA.

Conclusions: IL-1β causes atrophy, impairs contractility and relaxation and decreases deformation of cardiomyocytes. Because NLRP3/IL-1β pathway inhibition attenuates cardiac atrophy and cardiomyopathy in sepsis, it could be useful to prevent septic cardiomyopathy.
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http://dx.doi.org/10.1002/jcsm.12763DOI Listing
September 2021

SARS-CoV-2 variants B.1.351 and P.1 escape from neutralizing antibodies.

Cell 2021 04 20;184(9):2384-2393.e12. Epub 2021 Mar 20.

Infection Biology Unit, German Primate Center, Kellnerweg 4, 37077 Göttingen, Germany; Faculty of Biology and Psychology, Georg-August-University Göttingen, Wilhelmsplatz 1, 37073 Göttingen, Germany. Electronic address:

The global spread of SARS-CoV-2/COVID-19 is devastating health systems and economies worldwide. Recombinant or vaccine-induced neutralizing antibodies are used to combat the COVID-19 pandemic. However, the recently emerged SARS-CoV-2 variants B.1.1.7 (UK), B.1.351 (South Africa), and P.1 (Brazil) harbor mutations in the viral spike (S) protein that may alter virus-host cell interactions and confer resistance to inhibitors and antibodies. Here, using pseudoparticles, we show that entry of all variants into human cells is susceptible to blockade by the entry inhibitors soluble ACE2, Camostat, EK-1, and EK-1-C4. In contrast, entry of the B.1.351 and P.1 variant was partially (Casirivimab) or fully (Bamlanivimab) resistant to antibodies used for COVID-19 treatment. Moreover, entry of these variants was less efficiently inhibited by plasma from convalescent COVID-19 patients and sera from BNT162b2-vaccinated individuals. These results suggest that SARS-CoV-2 may escape neutralizing antibody responses, which has important implications for efforts to contain the pandemic.
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http://dx.doi.org/10.1016/j.cell.2021.03.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980144PMC
April 2021

Plxdc family members are novel receptors for the rhesus monkey rhadinovirus (RRV).

PLoS Pathog 2021 03 3;17(3):e1008979. Epub 2021 Mar 3.

German Primate Center - Leibniz Institute for Primate Research, Göttingen, Germany.

The rhesus monkey rhadinovirus (RRV), a γ2-herpesvirus of rhesus macaques, shares many biological features with the human pathogenic Kaposi's sarcoma-associated herpesvirus (KSHV). Both viruses, as well as the more distantly related Epstein-Barr virus, engage cellular receptors from the Eph family of receptor tyrosine kinases (Ephs). However, the importance of the Eph interaction for RRV entry varies between cell types suggesting the existence of Eph-independent entry pathways. We therefore aimed to identify additional cellular receptors for RRV by affinity enrichment and mass spectrometry. We identified an additional receptor family, the Plexin domain containing proteins 1 and 2 (Plxdc1/2) that bind the RRV gH/gL glycoprotein complex. Preincubation of RRV with soluble Plxdc2 decoy receptor reduced infection by ~60%, while overexpression of Plxdc1 and 2 dramatically enhanced RRV susceptibility and cell-cell fusion of otherwise marginally permissive Raji cells. While the Plxdc2 interaction is conserved between two RRV strains, 26-95 and 17577, Plxdc1 specifically interacts with RRV 26-95 gH. The Plxdc interaction is mediated by a short motif at the N-terminus of RRV gH that is partially conserved between isolate 26-95 and isolate 17577, but absent in KSHV gH. Mutation of this motif abrogated the interaction with Plxdc1/2 and reduced RRV infection in a cell type-specific manner. Taken together, our findings characterize Plxdc1/2 as novel interaction partners and entry receptors for RRV and support the concept of the N-terminal domain of the gammaherpesviral gH/gL complex as a multifunctional receptor-binding domain. Further, Plxdc1/2 usage defines an important biological difference between KSHV and RRV.
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http://dx.doi.org/10.1371/journal.ppat.1008979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7959344PMC
March 2021

SARS-CoV-2 and SARS-CoV Spike-Mediated Cell-Cell Fusion Differ in Their Requirements for Receptor Expression and Proteolytic Activation.

J Virol 2021 04 12;95(9). Epub 2021 Apr 12.

Nachwuchsgruppe Herpesviren, Abteilung Infektionsbiologie, Deutsches Primatenzentrum-Leibniz-Institut für Primatenforschung, Göttingen, Germany

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS2-S) with angiotensin-converting enzyme 2 (ACE2) and activation by proteases, in particular transmembrane protease serine 2 (TMPRSS2). Viruses can also spread through fusion of infected with uninfected cells. We compared the requirements of ACE2 expression, proteolytic activation, and sensitivity to inhibitors for SARS2-S-mediated and SARS-CoV-S (SARS1-S)-mediated cell-cell fusion. SARS2-S-driven fusion was moderately increased by TMPRSS2 and strongly by ACE2, while SARS1-S-driven fusion was strongly increased by TMPRSS2 and less so by ACE2 expression. In contrast to that of SARS1-S, SARS2-S-mediated cell-cell fusion was efficiently activated by batimastat-sensitive metalloproteases. Mutation of the S1/S2 proteolytic cleavage site reduced effector cell-target cell fusion when ACE2 or TMPRSS2 was limiting and rendered SARS2-S-driven cell-cell fusion more dependent on TMPRSS2. When both ACE2 and TMPRSS2 were abundant, initial target cell-effector cell fusion was unaltered compared to that of wild-type (wt) SARS2-S, but syncytia remained smaller. Mutation of the S2 cleavage (S2') site specifically abrogated activation by TMPRSS2 for both cell-cell fusion and SARS2-S-driven pseudoparticle entry but still allowed for activation by metalloproteases for cell-cell fusion and by cathepsins for particle entry. Finally, we found that the TMPRSS2 inhibitor bromhexine, unlike the inhibitor camostat, was unable to reduce TMPRSS2-activated cell-cell fusion by SARS1-S and SARS2-S. Paradoxically, bromhexine enhanced cell-cell fusion in the presence of TMPRSS2, while its metabolite ambroxol exhibited inhibitory activity under some conditions. On Calu-3 lung cells, ambroxol weakly inhibited SARS2-S-driven lentiviral pseudoparticle entry, and both substances exhibited a dose-dependent trend toward weak inhibition of authentic SARS-CoV-2. Cell-cell fusion allows viruses to infect neighboring cells without the need to produce free virus and contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2' cleavage site is essential for activation by TMPRSS2 and unravel important differences between SARS-CoV and SARS-CoV-2, among those, greater dependence of SARS-CoV-2 on ACE2 expression and activation by metalloproteases for cell-cell fusion. Bromhexine, reportedly an inhibitor of TMPRSS2, is currently being tested in clinical trials against coronavirus disease 2019. Our results indicate that bromhexine enhances fusion under some conditions. We therefore caution against the use of bromhexine in high dosages until its effects on SARS-CoV-2 spike activation are better understood. The related compound ambroxol, which similarly to bromhexine is clinically used as an expectorant, did not exhibit activating effects on cell-cell fusion. Both compounds exhibited weak inhibitory activity against SARS-CoV-2 infection at high concentrations, which might be clinically attainable for ambroxol.
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http://dx.doi.org/10.1128/JVI.00002-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8104116PMC
April 2021

Diagnostic Accuracy of Physical Examination Tests in Core Muscle Injury.

Am J Sports Med 2020 07 8;48(8):1983-1988. Epub 2020 Jun 8.

St Joseph's University Medical Center, Paterson, New Jersey, USA.

Background: Core muscle injury (CMI), often referred to as a sports hernia, is a common cause of groin pain in athletes characterized by concomitant injury to the insertion of the adductor longus and the rectus abdominis muscles. Currently, the literature on CMI is sparse with no standardized physical examination tests used in the diagnosis of this type of injury.

Purpose: To determine the diagnostic accuracy of various physical examination tests in the diagnosis of CMI.

Study Design: Cohort study (Diagnosis); Level of evidence, 3.

Methods: A consecutive series of patients evaluated by the senior author with symptoms consistent with CMI were included. Four physical examination tests were routinely performed in these patients by the senior author and were noted in each patient's chart as positive or negative: (1) pain with resisted cross-body sit-up in figure-of-4 position, (2) pain with straight-leg sit-up, (3) pain with resisted hip flexion in external rotation (external rotation Stinchfield test), and (4) the presence of an adductor contracture. CMI was independently diagnosed by a reference standard (magnetic resonance imaging [MRI]). All MRI scans were read by a musculoskeletal fellowship-trained radiologist. The sensitivity and specificity of each physical examination test alone and in combination were calculated based on this reference standard.

Results: A total of 81 patients were included in this study. MRI was positive for a CMI in 39 patients (48%) overall. Both the cross-body sit-up test and the presence of an adductor contracture were found to have a sensitivity of 100% (specificity, 3% for both). The external rotation Stinchfield test was found to have the highest specificity of 60% (sensitivity, 15%). The sensitivity of all 4 physical examination tests in combination was found to be 100% (specificity, 0%).

Conclusion: Certain physical examination maneuvers can be used to assist in the diagnosis of a CMI. The cross-body sit-up test and the presence of an adductor contracture are highly sensitive but nonspecific tests for CMI and therefore should be used in conjunction with diagnostic imaging before deciding on an appropriate treatment course.
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http://dx.doi.org/10.1177/0363546520926029DOI Listing
July 2020

Rhesus Monkey Rhadinovirus Isolated from Hemangioma Tissue.

Microbiol Resour Announc 2020 Mar 19;9(12). Epub 2020 Mar 19.

New England Primate Research Center, Southborough, Massachusetts, USA

We isolated a rhesus monkey rhadinovirus, isolate RRVmmu 209-07, from hemangioma tissue. The virion DNA was sequenced by Illumina-based sequencing. Isolate RRVmmu 209-07 is highly similar overall to RRV 26-95, but considerable differences exist in the 3' region of the genome.
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http://dx.doi.org/10.1128/MRA.01347-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082460PMC
March 2020

Is intramedullary screw fixation biomechanically superior to locking plate fixation and/or tension band wiring in transverse olecranon fractures? A cadaveric biomechanical comparison study.

Injury 2020 Apr 11;51(4):850-855. Epub 2020 Feb 11.

R Adams Cowley Shock Trauma Center, Department of Orthopaedics, University of Maryland School of Medicine, Baltimore, MD 21201, USA. Electronic address:

Objectives: To compare clinically important mechanical properties of three techniques used to fix transverse olecranon fractures (Arbeitsgemeinschaft fur Osteosynthesefragen and Orthopaedic Trauma Association class 2U1B1): (1) intramedullary (IM) screw, (2) locking plate, and (3) tension band wire in a realistic loading protocol using a cadaveric model.

Methods: Fresh frozen cadaveric transverse olecranon fracture models were fixed with an IM screw (n = 6), a locking plate (n = 6), or a tension band (n = 6). Compression after fixation was recorded using a pressure sensor in the fracture before samples were loaded through the triceps tendon for 500 cycles of 0-500 N, assessing implant survival. The primary outcome measure was compression force before loading. The secondary outcome was frequency of implant failure defined as breakage of the implant itself or fracture gapping >5 mm. Binary outcomes were compared with χ, and continuous variables were compared with unadjusted analysis of variance and a multivariable regression model adjusting for age, sex, dual-energy X-ray absorptiometry T-score, and testing order.

Results: No statistically significant difference was shown in fracture compression between IM screw (mean, 162 N; 95% confidence interval [CI], 27-297 N), locking plate (mean, 125 N; 95% CI, -9-260 N), and tension band (mean, 163 N; 95% CI, 29-298 N) in unadjusted (p = 0.89) and adjusted (p = 0.82) analyses. A 100% implant failure rate was observed with tension band compared with 0% implant failure with IM screw or locking plate (p < 0.01).

Conclusion: We found no statistically significant differences in compression across the fracture site among techniques. We did find a higher risk of implant failure with tension band compared with IM screw and locking plate during cyclic loading in cadaveric bone.
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http://dx.doi.org/10.1016/j.injury.2020.02.059DOI Listing
April 2020

Avoiding "conflicts of interest": a computational approach to scheduling parallel conference tracks and its human evaluation.

PeerJ Comput Sci 2019 11;5:e234. Epub 2019 Nov 11.

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States of America.

Conferences with contributed talks grouped into multiple concurrent sessions pose an interesting scheduling problem. From an attendee's perspective, choosing which talks to visit when there are many concurrent sessions is challenging since an individual may be interested in topics that are discussed in different sessions simultaneously. The frequency of topically similar talks in different concurrent sessions is, in fact, a common cause for complaint in post-conference surveys. Here, we introduce a practical solution to the conference scheduling problem by heuristic optimization of an objective function that weighs the occurrence of both topically similar talks in one session and topically different talks in concurrent sessions. Rather than clustering talks based on a limited number of preconceived topics, we employ a topic model to allow the topics to naturally emerge from the corpus of contributed talk titles and abstracts. We then measure the topical distance between all pairs of talks. Heuristic optimization of preliminary schedules seeks to balance the topical similarity of talks within a session and the dissimilarity between concurrent sessions. Using an ecology conference as a test case, we find that stochastic optimization dramatically improves the objective function relative to the schedule manually produced by the program committee. Approximate Integer Linear Programming can be used to provide a partially-optimized starting schedule, but the final value of the discrimination ratio (an objective function used to estimate coherence within a session and disparity between concurrent sessions) is surprisingly insensitive to the starting schedule. Furthermore, we show that, in contrast to the manual process, arbitrary scheduling constraints are straightforward to include. We applied our method to a second biology conference with over 1,000 contributed talks plus scheduling constraints. In a randomized experiment, biologists responded similarly to a machine-optimized schedule and a highly modified schedule produced by domain experts on the conference program committee.
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http://dx.doi.org/10.7717/peerj-cs.234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7924486PMC
November 2019

A Recombinant Rhesus Monkey Rhadinovirus Deleted of Glycoprotein L Establishes Persistent Infection of Rhesus Macaques and Elicits Conventional T Cell Responses.

J Virol 2020 01 6;94(2). Epub 2020 Jan 6.

Department of Pathology, Miller School of Medicine, University of Miami, Miami, Florida, USA

A replication-competent, recombinant strain of rhesus monkey rhadinovirus (RRV) expressing the Gag protein of SIVmac239 was constructed in the context of a glycoprotein L (gL) deletion mutation. Deletion of gL detargets the virus from Eph family receptors. The ability of this gL-minus Gag recombinant RRV to infect, persist, and elicit immune responses was evaluated after intravenous inoculation of two RRV-naive rhesus monkeys. Both monkeys responded with an anti-RRV antibody response, and quantitation of RRV DNA in peripheral blood mononuclear cells (PBMC) by real-time PCR revealed levels similar to those in monkeys infected with recombinant gL RRV. Comparison of RRV DNA levels in sorted CD3 versus CD20 versus CD14 PBMC subpopulations indicated infection of the CD20 subpopulation by the gL-minus RRV. This contrasts with results obtained with transformed B cell lines , in which deletion of gL resulted in markedly reduced infectivity. Over a period of 20 weeks, Gag-specific CD8 T cell responses were documented by major histocompatibility complex class I (MHC-I) tetramer staining. Vaccine-induced CD8 T cell responses, which were predominantly directed against the Mamu-A*01-restricted GagCM9 epitope, could be inhibited by blockade of MHC-I presentation. Our results indicate that gL and the interaction with Eph family receptors are dispensable for the colonization of the B cell compartment following high-dose infection by the intravenous route, which suggests the existence of alternative receptors. Further, gL-minus RRV elicits cellular immune responses that are predominantly canonical in nature. Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with a substantial disease burden in sub-Saharan Africa, often in the context of human immunodeficiency virus (HIV) infection. The related rhesus monkey rhadinovirus (RRV) has shown potential as a vector to immunize monkeys with antigens from simian immunodeficiency virus (SIV), the macaque model for HIV. KSHV and RRV engage cellular receptors from the Eph family via the viral gH/gL glycoprotein complex. We have now generated a recombinant RRV that expresses the SIV Gag antigen and does not express gL. This recombinant RRV was infectious by the intravenous route, established persistent infection in the B cell compartment, and elicited strong immune responses to the SIV Gag antigen. These results argue against a role for gL and Eph family receptors in B cell infection by RRV and have implications for the development of a live-attenuated KSHV vaccine or vaccine vector.
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http://dx.doi.org/10.1128/JVI.01093-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6955260PMC
January 2020

Serum amyloid A1 mediates myotube atrophy via Toll-like receptors.

J Cachexia Sarcopenia Muscle 2020 02 23;11(1):103-119. Epub 2019 Aug 23.

Experimental and Clinical Research Center, Charité-Universitätsmedizin Berlin, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany.

Background: Critically ill patients frequently develop muscle atrophy and weakness in the intensive-care-unit setting [intensive care unit-acquired weakness (ICUAW)]. Sepsis, systemic inflammation, and acute-phase response are major risk factors. We reported earlier that the acute-phase protein serum amyloid A1 (SAA1) is increased and accumulates in muscle of ICUAW patients, but its relevance was unknown. Our objectives were to identify SAA1 receptors and their downstream signalling pathways in myocytes and skeletal muscle and to investigate the role of SAA1 in inflammation-induced muscle atrophy.

Methods: We performed cell-based in vitro and animal in vivo experiments. The atrophic effect of SAA1 on differentiated C2C12 myotubes was investigated by analysing gene expression, protein content, and the atrophy phenotype. We used the cecal ligation and puncture model to induce polymicrobial sepsis in wild type mice, which were treated with the IкB kinase inhibitor Bristol-Myers Squibb (BMS)-345541 or vehicle. Morphological and molecular analyses were used to investigate the phenotype of inflammation-induced muscle atrophy and the effects of BMS-345541 treatment.

Results: The SAA1 receptors Tlr2, Tlr4, Cd36, P2rx7, Vimp, and Scarb1 were all expressed in myocytes and skeletal muscle. Treatment of differentiated C2C12 myotubes with recombinant SAA1 caused myotube atrophy and increased interleukin 6 (Il6) gene expression. These effects were mediated by Toll-like receptors (TLR) 2 and 4. SAA1 increased the phosphorylation and activity of the transcription factor nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-κB) p65 via TLR2 and TLR4 leading to an increased binding of NF-κB to NF-κB response elements in the promoter region of its target genes resulting in an increased expression of NF-κB target genes. In polymicrobial sepsis, skeletal muscle mass, tissue morphology, gene expression, and protein content were associated with the atrophy response. Inhibition of NF-κB signalling by BMS-345541 increased survival (28.6% vs. 91.7%, P < 0.01). BMS-345541 diminished inflammation-induced atrophy as shown by a reduced weight loss of the gastrocnemius/plantaris (vehicle: -21.2% and BMS-345541: -10.4%; P < 0.05), tibialis anterior (vehicle: -22.7% and BMS-345541: -17.1%; P < 0.05) and soleus (vehicle: -21.1% and BMS-345541: -11.3%; P < 0.05) in septic mice. Analysis of the fiber type specific myocyte cross-sectional area showed that BMS-345541 reduced inflammation-induced atrophy of slow/type I and fast/type II myofibers compared with vehicle-treated septic mice. BMS-345541 reversed the inflammation-induced atrophy program as indicated by a reduced expression of the atrogenes Trim63/MuRF1, Fbxo32/Atrogin1, and Fbxo30/MuSA1.

Conclusions: SAA1 activates the TLR2/TLR4//NF-κB p65 signalling pathway to cause myocyte atrophy. Systemic inhibition of the NF-κB pathway reduced muscle atrophy and increased survival of septic mice. The SAA1/TLR2/TLR4//NF-κB p65 atrophy pathway could have utility in combatting ICUAW.
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http://dx.doi.org/10.1002/jcsm.12491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015249PMC
February 2020

Kaposi Sarcoma in Mantled Guereza.

Emerg Infect Dis 2019 08;25(8):1552-1555

We identified a novel Kaposi's sarcoma herpesvirus-related rhadinovirus (Colobine gammaherpesvirus 1) in a mantled guereza (Colobus guereza kikuyensis). The animal had multiple oral tumors characterized by proliferation of latent nuclear antigen 1-positive spindle cells and was not co-infected with immunosuppressive simian viruses, suggesting that it had Kaposi sarcoma caused by this novel rhadinovirus.
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http://dx.doi.org/10.3201/eid2508.181804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6649314PMC
August 2019

EphA7 Functions as Receptor on BJAB Cells for Cell-to-Cell Transmission of the Kaposi's Sarcoma-Associated Herpesvirus and for Cell-Free Infection by the Related Rhesus Monkey Rhadinovirus.

J Virol 2019 08 17;93(15). Epub 2019 Jul 17.

Junior Research Group Herpesviruses, German Primate Center-Leibniz Institute for Primate Research, Göttingen, Germany

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma and is associated with two B cell malignancies, primary effusion lymphoma (PEL) and the plasmablastic variant of multicentric Castleman's disease. On several adherent cell types, EphA2 functions as a cellular receptor for the gH/gL glycoprotein complex of KSHV. KSHV gH/gL also has previously been found to interact weakly with other members of the Eph family of receptor tyrosine kinases (Ephs), and other A-type Ephs have been shown to be able to compensate for the absence of EphA2 using overexpression systems. However, whether these interactions are of functional consequence at endogenous protein levels has remained unclear so far. Here, we demonstrate for the first time that endogenously expressed EphA7 in BJAB B cells is critical for the cell-to-cell transmission of KSHV from producer iSLK cells to BJAB target cells. The BJAB lymphoblastoid cell line often serves as a model for B cell infection and expresses only low levels of all Eph family receptors other than EphA7. Endogenous EphA7 could be precipitated from the cellular lysate of BJAB cells using recombinant gH/gL, and knockout of EphA7 significantly reduced transmission of KSHV into BJAB target cells. Knockout of EphA5, the second most expressed A-type Eph in BJAB cells, had a similar, although less pronounced, effect on KSHV infection. Receptor function of EphA7 was conserved for cell-free infection by the related rhesus monkey rhadinovirus (RRV), which is relatively even more dependent on EphA7 for infection of BJAB cells. Infection of B cells is relevant for two KSHV-associated malignancies, the plasmablastic variant of multicentric Castleman's disease and PEL. Therefore, elucidating the process of B cell infection is important for the understanding of KSHV pathogenesis. While the high-affinity receptor for the gH/gL glycoprotein complex, EphA2, has been shown to function as an entry receptor for various types of adherent cells, the gH/gL complex can also interact with other Eph receptor tyrosine kinases with lower avidity. We analyzed the Eph interactions required for infection of BJAB cells, a model for B cell infection by KSHV. We identified EphA7 as the principal Eph receptor for infection of BJAB cells by KSHV and the related rhesus monkey rhadinovirus. While two analyzed PEL cell lines exhibited high EphA2 and low EphA7 expression, a third PEL cell line, BCBL-1, showed high EphA7 and low EphA2 expression, indicating a possible relevance for KSHV pathology.
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http://dx.doi.org/10.1128/JVI.00064-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6639272PMC
August 2019

Dimers of mitochondrial ATP synthase induce membrane curvature and self-assemble into rows.

Proc Natl Acad Sci U S A 2019 03 13;116(10):4250-4255. Epub 2019 Feb 13.

Department of Structural Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany

Mitochondrial ATP synthases form dimers, which assemble into long ribbons at the rims of the inner membrane cristae. We reconstituted detergent-purified mitochondrial ATP synthase dimers from the green algae sp. and the yeast into liposomes and examined them by electron cryotomography. Tomographic volumes revealed that ATP synthase dimers from both species self-assemble into rows and bend the lipid bilayer locally. The dimer rows and the induced degree of membrane curvature closely resemble those in the inner membrane cristae. Monomers of mitochondrial ATP synthase reconstituted into liposomes do not bend membrane visibly and do not form rows. No specific lipids or proteins other than ATP synthase dimers are required for row formation and membrane remodelling. Long rows of ATP synthase dimers are a conserved feature of mitochondrial inner membranes. They are required for cristae formation and a main factor in mitochondrial morphogenesis.
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http://dx.doi.org/10.1073/pnas.1816556116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6410833PMC
March 2019

Isolation and sequence analysis of a novel rhesus macaque foamy virus isolate with a serotype-1-like env.

Arch Virol 2018 Sep 2;163(9):2507-2512. Epub 2018 Jun 2.

Deutsches Primatenzentrum - Leibniz-Institut für Primatenforschung, Kellnerweg 4, 37077, Göttingen, Germany.

SFVmmu-DPZ9524 represents the third completely sequenced rhesus macaque simian foamy virus (SFV) isolate, alongside SFVmmu_K3T with a similar SFV-1-type env, and R289HybAGM with a SFV-2-like env. Sequence analysis demonstrates that, in gag and pol, SFVmmu-DPZ9524 is more closely related to R289HybAGM than to SFVmmu_K3T, which, outside of env, is more similar to a Japanese macaque isolate than to the other two rhesus macaque isolates SFVmmu-DPZ9524 and R289HybAGM. Further, we identify bel as another recombinant locus in R289HybAGM, confirming that recombination contributes to sequence diversity in SFV.
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http://dx.doi.org/10.1007/s00705-018-3892-9DOI Listing
September 2018

Structure, mechanism, and regulation of the chloroplast ATP synthase.

Science 2018 05;360(6389)

Department of Structural Biology, Max Planck Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt am Main, Germany.

The chloroplast adenosine triphosphate (ATP) synthase uses the electrochemical proton gradient generated by photosynthesis to produce ATP, the energy currency of all cells. Protons conducted through the membrane-embedded F motor drive ATP synthesis in the F head by rotary catalysis. We determined the high-resolution structure of the complete cFF complex by cryo-electron microscopy, resolving side chains of all 26 protein subunits, the five nucleotides in the F head, and the proton pathway to and from the rotor ring. The flexible peripheral stalk redistributes differences in torsional energy across three unequal steps in the rotation cycle. Plant ATP synthase is autoinhibited by a β-hairpin redox switch in subunit γ that blocks rotation in the dark.
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http://dx.doi.org/10.1126/science.aat4318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116070PMC
May 2018

ATP Synthase Diseases of Mitochondrial Genetic Origin.

Front Physiol 2018 4;9:329. Epub 2018 Apr 4.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.

Devastating human neuromuscular disorders have been associated to defects in the ATP synthase. This enzyme is found in the inner mitochondrial membrane and catalyzes the last step in oxidative phosphorylation, which provides aerobic eukaryotes with ATP. With the advent of structures of complete ATP synthases, and the availability of genetically approachable systems such as the yeast , we can begin to understand these molecular machines and their associated defects at the molecular level. In this review, we describe what is known about the clinical syndromes induced by 58 different mutations found in the mitochondrial genes encoding membrane subunits and of ATP synthase, and evaluate their functional consequences with respect to recently described cryo-EM structures.
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http://dx.doi.org/10.3389/fphys.2018.00329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5893901PMC
April 2018

A conserved Eph family receptor-binding motif on the gH/gL complex of Kaposi's sarcoma-associated herpesvirus and rhesus monkey rhadinovirus.

PLoS Pathog 2018 02 12;14(2):e1006912. Epub 2018 Feb 12.

German Primate Center - Leibniz Institute for Primate Research, Göttingen, Germany.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus associated with Kaposi's sarcoma and two B-cell malignancies. The rhesus monkey rhadinovirus (RRV) is a virus of nonhuman primates that is closely related to KSHV. Eph family receptor tyrosine kinases (Ephs) are cellular receptors for the gH/gL glycoprotein complexes of both KSHV and RRV. Through sequence analysis and mutational screens, we identified conserved residues in the N-terminal domain of KSHV and RRV glycoprotein H that are critical for Eph-binding in vitro. Homology-based structural predictions of the KSHV and RRV gH/gL complexes based on the Epstein-Barr-Virus gH/gL crystal structure located these amino acids in a beta-hairpin on gH, which is likely stabilized by gL and is optimally positioned for protein-protein interactions. Guided by these predictions, we generated recombinant RRV and KSHV strains mutated in the conserved motif as well as an RRV gL null mutant. Inhibition experiments using these mutants confirmed that disruption of the identified Eph-interaction motif or of gL expression resulted in complete detargeting from Ephs. However, all mutants were infectious on all cell types tested, exhibiting normal attachment but a reduction in infectivity of up to one log order of magnitude. While Eph-binding-negative RRV mutants were replication-competent on fibroblasts, their infectivity was comparatively more reduced on endothelial cells with a substantial subpopulation of endothelial cells remaining resistant to infection. Together, this provides evidence for a cell type-specific use of Ephs by RRV. Furthermore, our results demonstrate that gL is dispensable for infection by RRV. Its deletion caused a reduction in infectivity similar to that observed after mutation of Eph-binding residues in gH. Our findings would be compatible with an ability of KSHV and RRV to use other, less efficient entry mediators in lieu of Ephs, although these host factors may not be uniformly expressed by all cells.
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http://dx.doi.org/10.1371/journal.ppat.1006912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5825162PMC
February 2018

Gammaherpesviral Tegument Proteins, PML-Nuclear Bodies and the Ubiquitin-Proteasome System.

Viruses 2017 10 21;9(10). Epub 2017 Oct 21.

Institute for Clinical and Molecular Virology, University Hospital Erlangen, Friedrich Alexander University Erlangen-Nuremberg, 91054 Erlangen, Germany.

Gammaherpesviruses like Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) subvert the ubiquitin proteasome system for their own benefit in order to facilitate viral gene expression and replication. In particular, viral tegument proteins that share sequence homology to the formylglycineamide ribonucleotide amidotransferase (FGARAT, or PFAS), an enzyme in the cellular purine biosynthesis, are important for disrupting the intrinsic antiviral response associated with Promyelocytic Leukemia (PML) protein-associated nuclear bodies (PML-NBs) by proteasome-dependent and independent mechanisms. In addition, all herpesviruses encode for a potent ubiquitin protease that can efficiently remove ubiquitin chains from proteins and thereby interfere with several different cellular pathways. In this review, we discuss mechanisms and functional consequences of virus-induced ubiquitination and deubiquitination for early events in gammaherpesviral infection.
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http://dx.doi.org/10.3390/v9100308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5691659PMC
October 2017

Secreted Frizzled-Related Protein 2 and Inflammation-Induced Skeletal Muscle Atrophy.

Crit Care Med 2017 Feb;45(2):e169-e183

1Department of Molecular Cardiology, Experimental and Clinical Research Center (ECRC), Charité-Universitätsmedizin Berlin, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany. 2Department of Anesthesiology and Operative Intensive Care Medicine, Campus Virchow and Campus Mitte, Charité-Universitätsmedizin Berlin, Berlin, Germany. 3Berlin Institute of Health (BIH), Kapelle-Ufer 2, Berlin, Germany. 4Center for Musculoskeletal Surgery, Campus Virchow, Charité-Universitätsmedizin Berlin, Berlin, Germany. 5Department of Cardiology, Heart Center Brandenburg and Medical University Brandenburg (MHB), Bernau, Germany.

Objective: In sepsis, the disease course of critically ill patients is often complicated by muscle failure leading to ICU-acquired weakness. The myokine transforming growth factor-β1 increases during inflammation and mediates muscle atrophy in vivo. We observed that the transforming growth factor-β1 inhibitor, secreted frizzled-related protein 2, was down-regulated in skeletal muscle of ICU-acquired weakness patients. We hypothesized that secreted frizzled-related protein 2 reduction enhances transforming growth factor-β1-mediated effects and investigated the interrelationship between transforming growth factor-β1 and secreted frizzled-related protein 2 in inflammation-induced atrophy.

Design: Observational study and prospective animal trial.

Setting: Two ICUs and research laboratory.

Patients/subjects: Twenty-six critically ill patients with Sequential Organ Failure Assessment scores greater than or equal to 8 underwent a skeletal muscle biopsy from the vastus lateralis at median day 5 in ICU. Four patients undergoing elective orthopedic surgery served as controls. To search for signaling pathways enriched in muscle of ICU-acquired weakness patients, a gene set enrichment analysis of our recently published gene expression profiles was performed. Quantitative reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemistry were used to analyze secreted frizzled-related protein 2 expression and protein content. A mouse model of inflammation-induced skeletal muscle atrophy due to polymicrobial sepsis and cultured myocytes were used for mechanistic analyses.

Interventions: None.

Measurements And Main Results: Gene set enrichment analysis uncovered transforming growth factor-β1 signaling activation in vastus lateralis from ICU-acquired weakness patients. Muscular secreted frizzled-related protein 2 expression was reduced after 5 days in ICU. Likewise, muscular secreted frizzled-related protein 2 expression was decreased early and continuously in mice with inflammation-induced atrophy. In muscle, secreted frizzled-related protein 2 was predominantly contained in fast twitch/type II myofibers. Secreted frizzled-related protein 2 physically interacted and colocalized with transforming growth factor-β1 through its cysteine-rich domain. Finally, secreted frizzled-related protein 2 prevented transforming growth factor-β1-induced atrophy in C2C12 myotubes.

Conclusions: Muscular secreted frizzled-related protein 2 is down-regulated in ICU-acquired weakness patients and mice with inflammation-induced muscle atrophy. Decreased secreted frizzled-related protein 2 possibly establishes a positive feedback loop enhancing transforming growth factor-β1-mediated atrophic effects in inflammation-induced atrophy.
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http://dx.doi.org/10.1097/CCM.0000000000002056DOI Listing
February 2017

Structure of a Complete ATP Synthase Dimer Reveals the Molecular Basis of Inner Mitochondrial Membrane Morphology.

Mol Cell 2016 08 30;63(3):445-56. Epub 2016 Jun 30.

Department of Structural Biology, Max Planck Institute of Biophysics, Max-von-Laue-Str. 3, 60438 Frankfurt am Main, Germany. Electronic address:

We determined the structure of a complete, dimeric F1Fo-ATP synthase from yeast Yarrowia lipolytica mitochondria by a combination of cryo-EM and X-ray crystallography. The final structure resolves 58 of the 60 dimer subunits. Horizontal helices of subunit a in Fo wrap around the c-ring rotor, and a total of six vertical helices assigned to subunits a, b, f, i, and 8 span the membrane. Subunit 8 (A6L in human) is an evolutionary derivative of the bacterial b subunit. On the lumenal membrane surface, subunit f establishes direct contact between the two monomers. Comparison with a cryo-EM map of the F1Fo monomer identifies subunits e and g at the lateral dimer interface. They do not form dimer contacts but enable dimer formation by inducing a strong membrane curvature of ∼100°. Our structure explains the structural basis of cristae formation in mitochondria, a landmark signature of eukaryotic cell morphology.
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http://dx.doi.org/10.1016/j.molcel.2016.05.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4980432PMC
August 2016

Viral FGARAT Homolog ORF75 of Rhesus Monkey Rhadinovirus Effects Proteasomal Degradation of the ND10 Components SP100 and PML.

J Virol 2016 09 12;90(17):8013-28. Epub 2016 Aug 12.

Virologisches Institut, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany

Unlabelled: Nuclear domain 10 (ND10) components restrict herpesviral infection, and herpesviruses antagonize this restriction by a variety of strategies, including degradation or relocalization of ND10 proteins. The rhesus monkey rhadinovirus (RRV) shares many key biological features with the closely related Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) and readily infects cells of both human and rhesus monkey origin. We used the clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) technique to generate knockout (ko) cells for each of the four ND10 components, PML, SP100, DAXX, and ATRX. These ko cells were analyzed with regard to permissiveness for RRV infection. In addition, we analyzed the fate of the individual ND10 components in infected cells by immunofluorescence and Western blotting. Knockout of the ND10 component DAXX markedly increased RRV infection, while knockout of PML or SP100 had a less pronounced effect. In line with these observations, RRV infection resulted in rapid degradation of SP100, followed by degradation of PML and the loss of ND10 structures, whereas the protein levels of ATRX and DAXX remained constant. Notably, inhibition of the proteasome but not inhibition of de novo gene expression prevented the loss of SP100 and PML in cells that did not support lytic replication, compatible with proteasomal degradation of these ND10 components through the action of a viral tegument protein. Expression of the RRV FGARAT homolog ORF75 was sufficient to effect the loss of SP100 and PML in transfected or transduced cells, implicating ORF75 as the viral effector protein.

Importance: Our findings highlight the antiviral role of ND10 and its individual components and further establish the viral FGARAT homologs of the gammaherpesviruses to be important viral effectors that counteract ND10-instituted intrinsic immunity. Surprisingly, even closely related viruses like KSHV and RRV evolved to use different strategies to evade ND10-mediated restriction. RRV first targets SP100 for degradation and then targets PML with a delayed kinetic, a strategy which clearly differs from that of other gammaherpesviruses. Despite efficient degradation of these two major ND10 components, RRV is still restricted by DAXX, another abundant ND10 component, as evidenced by a marked increase in RRV infection and replication upon knockout of DAXX. Taken together, our findings substantiate PML, SP100, and DAXX as key antiviral proteins, in that the first two are targeted for degradation by RRV and the last one still potently restricts replication of RRV.
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http://dx.doi.org/10.1128/JVI.01181-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988134PMC
September 2016

Management of Adult Elbow Fracture Dislocations.

Orthop Clin North Am 2016 Jan;47(1):97-113

Department of Orthopedic Surgery and Sports Medicine, Temple University Hospital, 3501 North Broad Street, Philadelphia, PA 19140, USA.

Elbow fracture dislocations are complicated injuries that are difficult to manage and fraught with complications. A complete series of radiographs is typically complemented with CT scan to evaluate the elbow and assist preoperative planning. Typically, operative intervention is necessary and a systematic approach to the elbow injuries should be chosen. This article addresses the coronoid and proceeds to the radial head, lateral soft tissues, and finally the medial ligaments if elbow instability persists. With a focused, systematic surgical approach, improved outcomes have been demonstrated and patients may recover full function and range of motion in the affected elbow.
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http://dx.doi.org/10.1016/j.ocl.2015.08.001DOI Listing
January 2016

Glioma-derived versican promotes tumor expansion via glioma-associated microglial/macrophages Toll-like receptor 2 signaling.

Neuro Oncol 2015 02 1;17(2):200-10. Epub 2014 Dec 1.

Cellular Neurosciences, Max Delbrück Center for Molecular Medicine, Berlin, Germany (F.H., O.D.a D., A.H., S.A.W., H.K.); Cancer Genetics and Cellular Stress Responses, Max Delbrück Center for Molecular Medicine, Berlin, Germany (Y.Y.); Mass Spectrometry, Max Delbrück Center for Molecular Medicine, Berlin, Germany (R.J.S., G.D.); Robinson Institute, University of Adelaide, Adelaide, Australia (A.K.K., K.R.D., C.R.); Department of Neuropathology, Charité Medical University, Berlin, Germany (J.L.R., F.L.H.); Department of Neurology and Center for Anatomy, Charité Medical University, Berlin, Germany (S.L.); Department of Neurosurgery, Charité Medical University, 13353 Berlin, Germany (M.S.)Present affiliation: Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 430030 Wuhan, People's Republic of China (F.H.).

Background: Accumulation and infiltration of microglia/brain macrophages around and into glioma tissue promote tumor invasion and expansion. One tumor-promoting mechanism of microglia/brain macrophages is upregulation of membrane type 1 matrix metalloprotease (MT1-MMP), which promotes the degradation of extracellular matrix. MT1-MMP upregulation is induced by soluble factors released by glioma cells activating microglial Toll-like receptor 2 (TLR2).

Methods: Versican identified by proteomics was silenced in glioma cells by short interference RNA and short hairpin RNA approaches and studied in vitro and after injection into mouse brains or organotypic brain slices.

Results: The splice variants V0/V1 of the endogenous TLR2 ligand versican are highly expressed in mouse and human glioma tissue. Versican-silenced gliomas induced less MT1-MMP expression in microglia both in vitro and in vivo, which resulted in smaller tumors and longer survival rates as compared with controls. Recombinant versican V1 induced significantly higher levels of MT1-MMP in wild-type microglia compared with untreated and treated TLR2 knockout microglial cells. Using glioma-injected organotypic brain slices, we found that the impact of versican signaling on glioma growth depended on the presence of microglia. Moreover, we found that TLR2 expression is upregulated in glioma-associated microglia but not in astrocytes. Additionally, an established TLR2 neutralizing antibody reduced glioma-induced microglial MT1-MMP expression as well as glioma growth ex vivo.

Conclusions: Our results show that versican released from glioma promotes tumor expansion through glioma-associated microglial/macrophage TLR2 signaling and subsequent expression of MT1-MMP. This signaling cascade might be a novel target for glioma therapies.
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http://dx.doi.org/10.1093/neuonc/nou324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288527PMC
February 2015

Whole genome and transcriptome analyses of environmental antibiotic sensitive and multi-resistant Pseudomonas aeruginosa isolates exposed to waste water and tap water.

Microb Biotechnol 2015 Jan 3;8(1):116-30. Epub 2014 Sep 3.

Institute of Functional Interfaces (IFG), Campus North, Karlsruhe Institute of Technology (KIT), Hermann von Helmholtz Platz 1, Eggenstein-Leopoldshafen, D-76344, Germany.

The fitness of sensitive and resistant Pseudomonas aeruginosa in different aquatic environments depends on genetic capacities and transcriptional regulation. Therefore, an antibiotic-sensitive isolate PA30 and a multi-resistant isolate PA49 originating from waste waters were compared via whole genome and transcriptome Illumina sequencing after exposure to municipal waste water and tap water. A number of different genomic islands (e.g. PAGIs, PAPIs) were identified in the two environmental isolates beside the highly conserved core genome. Exposure to tap water and waste water exhibited similar transcriptional impacts on several gene clusters (antibiotic and metal resistance, genetic mobile elements, efflux pumps) in both environmental P. aeruginosa isolates. The MexCD-OprJ efflux pump was overexpressed in PA49 in response to waste water. The expression of resistance genes, genetic mobile elements in PA49 was independent from the water matrix. Consistently, the antibiotic sensitive strain PA30 did not show any difference in expression of the intrinsic resistance determinants and genetic mobile elements. Thus, the exposure of both isolates to polluted waste water and oligotrophic tap water resulted in similar expression profiles of mentioned genes. However, changes in environmental milieus resulted in rather unspecific transcriptional responses than selected and stimuli-specific gene regulation.
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http://dx.doi.org/10.1111/1751-7915.12156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4321378PMC
January 2015

Binding of the Kaposi's sarcoma-associated herpesvirus to the ephrin binding surface of the EphA2 receptor and its inhibition by a small molecule.

J Virol 2014 Aug 4;88(16):8724-34. Epub 2014 Jun 4.

Department of Pathology, Miller School of Medicine, University of Miami, Miami, Florida, USA

Unlabelled: The ephrin receptor tyrosine kinase A2 (EphA2) is an entry receptor for Kaposi's sarcoma-associated herpesvirus (KSHV) that is engaged by the virus through its gH/gL glycoprotein complex. We describe here that natural ephrin ligands inhibit the gH/gL-EphA2 interaction. The effects of point mutations within EphA2 demonstrated that KSHV gH/gL interacts with EphA2 through a restricted set of the same residues that mediate binding of A-type ephrins. Two previously described inhibitors of the EphA2 interaction with ephrin A5 also inhibited binding of KSHV gH/gL to EphA2. The more potent of the two compounds inhibited KSHV infection of blood vessel and lymphatic endothelial cells in the micromolar concentration range. Our results demonstrate that interaction of KSHV with EphA2 occurs in a fashion similar to that of the natural ephrin ligands. Our data further indicate a new avenue for drug development against KSHV.

Importance: Our study reports two important findings. First, we show that KSHV engages its receptor, the receptor tyrosine kinase EphA2, at a site that overlaps the binding site of the natural ephrin ligands. Second, we demonstrate that KSHV infection of target cells can be blocked by a small-molecule inhibitor of the viral glycoprotein-EphA2 interaction. These findings represent a novel avenue for the development of strategies to treat KSHV-associated diseases.
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http://dx.doi.org/10.1128/JVI.01392-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4136269PMC
August 2014

Secretome analysis of Anabaena sp. PCC 7120 and the involvement of the TolC-homologue HgdD in protein secretion.

Environ Microbiol 2015 Mar 15;17(3):767-80. Epub 2014 Jul 15.

Institute of Molecular Biosciences, Cell Biology of Plants, Goethe University, Max-von-Laue Str. 9, Frankfurt/am Main, 60438, Germany.

Secretion of proteins is a central strategy of bacteria to influence and respond to their environment. Until now, there has been very few discoveries regarding the cyanobacterial secrotome or the secretion machineries involved. For a mutant of the outer membrane channel TolC-homologue HgdD of Anabaena sp. PCC 7120, a filamentous and heterocyst-forming cyanobacterium, an altered secretome profile was reported. To define the role of HgdD in protein secretion, we have developed a method to isolate extracellular proteins of Anabaena sp. PCC 7120 wild type and an hgdD loss-of-function mutant. We identified 51 proteins of which the majority is predicted to have an extracellular secretion signal, while few seem to be localized in the periplasmic space. Eight proteins were exclusively identified in the secretome of wild-type cells, which coincides with the distribution of type I secretion signal. We selected three candidates and generated hemagglutinin-tagged fusion proteins which could be exclusively detected in the extracellular protein fraction. However, these proteins are not secreted in the hgdD-mutant background, where they are rapidly degraded. This confirms a direct function of HgdD in protein secretion and points to the existence of a quality control mechanism at least for proteins secreted in an HgdD-dependent pathway.
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http://dx.doi.org/10.1111/1462-2920.12516DOI Listing
March 2015

An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge.

Genome Biol 2014 Mar 25;15(3):R53. Epub 2014 Mar 25.

Background: There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance.

Results: A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization.

Conclusions: The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups.
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http://dx.doi.org/10.1186/gb-2014-15-3-r53DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4073084PMC
March 2014

Short-term, daily intake of yogurt containing Bifidobacterium animalis ssp. lactis Bf-6 (LMG 24384) does not affect colonic transit time in women.

Br J Nutr 2014 Jan 8;111(2):279-86. Epub 2013 Oct 8.

Department of Food Science, The Pennsylvania State University, University Park, PA, USA.

The present study investigated the effect of Bifidobacterium animalis ssp. lactis Bf-6 (LMG 24 384) (Bf-6)-supplemented yogurt on colonic transit time (CTT). A triple-blinded, randomised, placebo-controlled, two-period cross-over trial was conducted with sixty-eight women with a self-reported history of straining during bowel movements or hard or lumpy stools in the past 2 years. As per regulatory requirements for probiotic studies, eligible women were generally healthy and not actively constipated at the time of enrolment. Participants consumed both Bf-6 and placebo yogurts for 14 d each in a randomised order, with a 6-week washout period between the treatments. The primary outcome, CTT, was assessed via Sitz marker X-rays. The average CTT was 42·1 h for the active period and 43·3 h for the control period (mean difference 1·2 h, 95 % CI - 4·9, 7·4). Since the statistical tests for the cross-over study implied that the mean CTT for the active and control periods in period 2 were biased, the standard protocol suggests examining the results of only period 1 as a traditional randomised controlled trial. This showed that the mean CTT was 35·2 h for the active period v. 52·9 h for the control period (P= 0·015). Bootstrapping demonstrated that both the mean and median differences remained significant (P= 0·016 and P= 0·045, respectively). Few adverse events were noted, with no differences among the active and control periods. The paired analysis showed no differences between the active and control periods during the cross-over trial. Further trials should be conducted in populations with underlying problems associated with disordered transit to determine the potential value of probiotic supplementation more accurately.
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http://dx.doi.org/10.1017/S0007114513002237DOI Listing
January 2014
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