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The texture of a yarn describes the orientation of individual fibers in a yarn group. This yarn structure is determined by the application. The OE-rotor spinning system is a very flexible solution among staple fiber spinning processes, since it allows yarn manipulation through the use of various spinning elements. For example, the design of the navel has a significant influence on the yarn structure. The opening roller and rotor are responsible for a large proportion of: the measured number of imperfections, the yarn uniformity, and damage to the fibers. The principal item however, the navel, is able to broadly manipulate yarn volume and hairiness, without necessarily having a negative effect on processing parameters, such as tenacity and elongation. The character of a textile product can be predominantly controlled by the navel.

Adjustment of the cellular metabolism of pro-inflammatory macrophages is essential for their bactericidal function; however, it underlies the development of many human diseases if induced chronically. Therefore, intervention of macrophage metabolic polarisation has been recognised as a potent strategy for their treatment. Although many small-molecule inhibitors affecting macrophage metabolism have been identified, their in vivo administration requires a tool for macrophage-specific delivery to limit their potential side effects. Here, we establish Drosophila melanogaster as a simple experimental model for in vivo testing of macrophage-specific delivery tools. We found that yeast-derived glucan particles (GPs) are suitable for macrophage-specific delivery of small-molecule inhibitors. Systemic administration of GPs loaded with atorvastatin, the inhibitor of hydroxy-methyl-glutaryl-CoA reductase (Hmgcr), leads to intervention of mevalonate pathway specifically in macrophages, without affecting HMGCR activity in other tissues. Using this tool, we demonstrate that mevalonate pathway is essential for macrophage pro-inflammatory polarisation and individual's survival of infection., (© 2024 The Authors. Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

Human cytomegalovirus (CMV) infection is the leading non-genetic cause of congenital malformation in developed countries, causing significant fetal injury, and in some cases fetal death. The pathogenetic mechanisms through which this host-specific virus infects then damages both the placenta and the fetal brain are currently ill-defined. We investigated the CMV modulation of key signaling pathway proteins for these organs including dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) and Sonic Hedgehog (SHH) pathway proteins using human first trimester placental trophoblast (TEV-1) cells, primary human astrocyte (NHA) brain cells, and CMV-infected human placental tissue. Immunofluorescence demonstrated the accumulation and re-localization of SHH proteins in CMV-infected TEV-1 cells with Gli2, Ulk3, and Shh re-localizing to the CMV cytoplasmic virion assembly complex (VAC). In CMV-infected NHA cells, DYRK1A re-localized to the VAC and DYRK1B re-localized to the CMV nuclear replication compartments, and the SHH proteins re-localized with a similar pattern as was observed in TEV-1 cells. Western blot analysis in CMV-infected TEV-1 cells showed the upregulated expression of Rb, Ulk3, and Shh, but not Gli2. In CMV-infected NHA cells, there was an upregulation of DYRK1A, DYRK1B, Gli2, Rb, Ulk3, and Shh. These in vitro monoculture findings are consistent with patterns of protein upregulation and re-localization observed in naturally infected placental tissue and CMV-infected ex vivo placental explant histocultures. This study reveals CMV-induced changes in proteins critical for fetal development, and identifies new potential targets for CMV therapeutic development.

Long-acting injectable formulations represent a rapidly emerging category of drug delivery systems that offer several advantages compared to orally administered medicines. Rather than having to frequently swallow tablets, the medication is administered to the patient by intramuscular or subcutaneous injection of a nanoparticle suspension that forms a local depot from which the drug is steadily released over a period of several weeks or months. The benefits of this approach include improved medication compliance, reduced fluctuations of drug plasma level, or the suppression of gastrointestinal tract irritation. The mechanism of drug release from injectable depot systems is complex, and there is a lack of models that would enable quantitative parametrisation of the process. In this work, an experimental and computational study of drug release from a long-acting injectable depot system is reported. A population balance model of prodrug dissolution from asuspension with specific particle size distribution has been coupled with the kinetics of prodrug hydrolysis to its parent drug and validated using in vitro experimental data obtained from an accelerated reactive dissolution test. Using the developed model, it is possible to predict the sensitivity of drug release profiles to the initial concentration and particle size distribution of the prodrug suspension, and subsequently simulate various drug dosing scenarios. Parametric analysis of the system has identified the boundaries of reaction- and dissolution-limited drug release regimes, and the conditions for the existence of a quasi-steady state. This knowledge is crucial for the rational design of drug formulations in terms of particle size distribution, concentration and intended duration of drug release., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Spoken language is constantly undergoing change: Speakers within and across social and regional groups influence each other's speech, leading to the emergence and drifts of accents in a language. These processes are driven by mutual unintentional imitation of the phonetic details of others' speech in conversational interactions, suggesting that continuous auditory-motor adaptation takes place in interactive language use and plasticity of auditory-motor representations of speech persists across the lifespan. The brain mechanisms underlying this large-scale social-linguistic behavior are still poorly understood., Research Aim: To investigate the role of cerebellar and basal ganglia dysfunctions in unintended adaptation to the speech rhythm and articulation rate of a second speaker., Methods: Twelve patients with spinocerebellar ataxia type 6 (SCA6), 15 patients with Parkinson's disease (PD), and 27 neurologically healthy controls (CTRL) participated in two interactive speech tasks, i.e., sentence repetition and "turn-taking" (i.e., dyadic interaction with sentences produced by a model speaker). Production of scripted sentences was used as a control task. Two types of sentence rhythm were distinguished, i.e., regular and irregular, and model speech rate was manipulated in 12 steps between 2.9 and 4.0 syllables per second. Acoustic analyses of the participants' utterances were performed to determine the extent to which participants adapted their speech rate and rhythm to the model., Results: Neurologically healthy speakers showed significant adaptation of rate in all conditions, and of rhythm in the repetition task and partly also the turn-taking task. Patients with PD showed a stronger propensity to adapt than the controls. In contrast, the patients with cerebellar degeneration were largely insensitive to the model speaker's rate and rhythm. Contrary to expectations, sentences with an irregular speech rhythm exerted a stronger adaptive attraction than regular sentences in the two patient groups., Conclusions: Cerebellar degeneration inhibits the propensity to covertly adapt to others' speech. Striatal dysfunction in Parkinson's disease spares or even promotes the tendency to accommodate to other speakers' speech rate and rhythm., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Human cytomegalovirus congenital infection is the leading non-genetic cause of fetal malformation in developed countries. There are currently no safe antivirals for use during pregnancy. Placental trophoblast cells specifically secrete exosomes containing miRNA from the chromosome 19 miRNA cluster (C19MC) which confer viral resistance to recipient cells. We show the highly expressed C19MC miRNA miR-517a-3p inhibits HCMV replication and viral protein expression in both fibroblast and trophoblast cell cultures (71.6% and 50.4% inhibition of HCMV DNA at 7 days post infection respectively; p < 0.05). This naturally occurring molecule has potential for opening-up antiviral therapeutic strategies for pregnancy., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objective: To investigate how surgeons approach ethically challenging scenarios that arise in penile prosthesis surgery and identify patient-related factors that impact their approach., Methods: A survey was distributed to the Society for Urologic Prosthetic Surgeons membership consisting of 6 ethically challenging scenarios: an HIV+ patient, a patient with cognitive disability, a registered sex offender, a nonverbal patient, a litigious patient, and an uncontrolled diabetic patient whose insurance will lapse soon. Additional clinical information was provided to assess how the likelihood to offer surgery might change. The primary outcome was the likelihood of offering surgery in each scenario., Results: The response rate was 15.6% (n = 29). When compared to the baseline patient, respondents had a lower likelihood of offering surgery in all scenarios except the HIV+ patient, with the lowest likelihood of offering surgery to a sex offender (P < .01). Within each scenario, factors associated with an increased odds of offering surgery included knowledge that a patient with Down Syndrome is high functioning (odds ratio [OR] 5.0, confidence interval [CI]: 1.4-17.8), that a prior sex offender is currently married (OR 16.5, CI:3.5-99.8), that a litigious patient sued a surgeon for a retained sponge (OR 6.3, CI:1.7-24.3), and that a nonverbal patient had expressed prior interest in penile prosthesis surgery (OR 4.5, CI: 1.3-16.2)., Conclusion: Ethical principles, including respect for autonomy, nonmaleficence, beneficence, and justice, are appropriately applied by urological prosthetic surgeons when ethical challenges arise. While the likelihood of offering penile prosthesis surgery is decreased with most ethical dilemmas, specific clinical factors often augment decision-making., (Copyright © 2020. Published by Elsevier Inc.)

Nuclear egress is a common herpesviral process regulating nucleocytoplasmic capsid release. For human cytomegalovirus (HCMV), the nuclear egress complex (NEC) is determined by the pUL50-pUL53 core that regulates multicomponent assembly with NEC-associated proteins and capsids. Recently, NEC crystal structures were resolved for α-, β- and γ-herpesviruses, revealing profound structural conservation, which was not mirrored, however, by primary sequence and binding properties. The NEC binding principle is based on hook-into-groove interaction through an N-terminal hook-like pUL53 protrusion that embraces an α-helical pUL50 binding groove. So far, pUL50 has been considered as the major kinase-interacting determinant and massive phosphorylation of pUL50-pUL53 was assigned to NEC formation and functionality. Here, we addressed the question of phenotypical changes of ORF-UL50-mutated HCMVs. Surprisingly, our analyses did not detect a predominant replication defect for most of these viral mutants, concerning parameters of replication kinetics (qPCR), viral protein production (Western blot/CoIP) and capsid egress (confocal imaging/EM). Specifically, only the ORF-UL50 deletion rescue virus showed a block of genome synthesis during late stages of infection, whereas all phosphosite mutants exhibited marginal differences compared to wild-type or revertants. These results (i) emphasize a rate-limiting function of pUL50 for nuclear egress, and (ii) demonstrate that mutations in all mapped pUL50 phosphosites may be largely compensated. A refined mechanistic concept points to a multifaceted nuclear egress regulation, for which the dependence on the expression and phosphorylation of pUL50 is discussed., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Nuclear egress is a rate-limiting step of herpesviral replication, restricting the nucleocytoplasmic transport of viral capsids. The process is regulated by two viral nuclear egress proteins (core NEC pUL50-pUL53), which recruit additional cellular and viral proteins. The multicomponent NEC mediates disassembly of the nuclear lamina barrier and the docking of nuclear capsids. The quantitation of nuclear egress has been accomplished by electron microscopic analysis, but is generally hampered by the low number of detectable cytoplasmic capsids. A newly established method for the quantitation of viral nuclear egress improves the characterization of viral mutants, host cell permissiveness and antiviral drug efficacy. In this study, various strains of human cytomegalovirus (HCMV) were used to measure the replication efficiencies in primary human fibroblasts, applying methods of cell fractionation, DNase digestion, sucrose cushions and quantitative PCR. Several stages of optimization led to a reliable quantitative assay that allowed the characterization of viral nuclear egress efficacy. Using this assay, recovery of the nuclear egress of a NEC-defective HCMV mutant was quantitatively assessed by applying an inducible NEC-expressing fibroblast culture for trans-complementation. This novel assay system can be further used to accurately quantitate and characterize the functionality of nuclear egress of HCMV or other herpesviruses., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Crown Copyright © 2020. Published by Elsevier B.V. All rights reserved.)

Nuclear egress is a regulated process shared by α-, β- and γ-herpesviruses. The core nuclear egress complex (NEC) is composed of the membrane-anchored protein homologs of human cytomegalovirus (HCMV) pUL50, murine cytomegalovirus (MCMV) pM50, Epstein-Barr virus (EBV) BFRF1 or varicella zoster virus (VZV) Orf24, which interact with the autologous NEC partners pUL53, pM53, BFLF2 or Orf27, respectively. Their recruitment of additional proteins leads to the assembly of a multicomponent NEC, coordinately regulating viral nucleocytoplasmic capsid egress. Here, the functionality of VZV, HCMV, MCMV and EBV core NECs was investigated by coimmunoprecipitation and confocal imaging analyses. Furthermore, a recombinant MCMV, harboring a replacement of ORF M50 by UL50, was analyzed both in vitro and in vivo. In essence, core NEC interactions were strictly limited to autologous NEC pairs and only included one measurable nonautologous interaction between the homologs of HCMV and MCMV. A comparative analysis of MCMV-WT versus MCMV-UL50-infected murine fibroblasts revealed almost identical phenotypes on the levels of protein and genomic replication kinetics. In infected BALB/c mice, virus spread to lung and other organs was found comparable between these viruses, thus stating functional complementarity. In conclusion, our study underlines that herpesviral core NEC proteins are functionally conserved regarding complementarity of core NEC interactions, which were found either virus-specific or restricted within subfamilies.

Herpesviruses uniquely express two essential nuclear egress-regulating proteins forming a heterodimeric basic structure of the nuclear egress complex (core NEC). These core NECs serve as a hexameric lattice-structured platform for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina- and membrane-rearranging functions (multicomponent NEC). Here, we report the X-ray structures of β- and γ-herpesvirus core NECs obtained through an innovative recombinant expression strategy based on NEC-hook::NEC-groove protein fusion constructs. This approach yielded the first structure of γ-herpesviral core NEC, namely the 1.56 Å structure of Epstein-Barr virus (EBV) BFRF1-BFLF2, as well as an increased resolution 1.48 Å structure of human cytomegalovirus (HCMV) pUL50-pUL53. Detailed analysis of these structures revealed that the prominent hook segment is absolutely required for core NEC formation and contributes approximately 80% of the interaction surface of the globular domains of NEC proteins. Moreover, using HCMV::EBV hook domain swap constructs, computational prediction of the roles of individual hook residues for binding, and quantitative binding assays with synthetic peptides presenting the HCMV- and EBV-specific NEC hook sequences, we characterized the unique hook-into-groove NEC interaction at various levels. Although the overall physicochemical characteristics of the protein interfaces differ considerably in these β- and γ-herpesvirus NECs, the binding free energy contributions of residues displayed from identical positions are similar. In summary, the results of our study reveal critical details of the molecular mechanism of herpesviral NEC interactions and highlight their potential as an antiviral drug target., (© 2020 Muller et al.)

Infections with human herpesviruses share several molecular characteristics, but the diversified medical outcomes are distinct to viral subfamilies and species. Notably, both clinical and molecular correlates of infection are a challenging field and distinct patterns of virus-host interaction have rarely been defined; this study therefore focuses on the search for virus-specific molecular indicators. As previous studies have demonstrated the impact of herpesvirus infections on changes in host signalling pathways, we illustrate virus-modulated expression levels of individual cellular protein kinases. Current data reveal (i) α-, β- and γ-herpesvirus-specific patterns of kinase modulation as well as (ii) differential levels of up-/downregulated kinase expression and phosphorylation, which collectively suggest (iii) defined signalling patterns specific for the various viruses (VSS) that may prove useful for defining molecular indicators. Combined, the study confirms the correlation between herpesviral replication and modulation of signalling kinases, possibly exploitable for the in vitro characterization of viral infections.

Mutations in the cytomegalovirus UL97 kinase gene contribute to antiviral resistance. Mutations A594S and G598D from two clinical isolates were analyzed, and bacterial artificial chromosome (BAC)-engineered A594S recombinant cytomegalovirus exhibited a ganciclovir-resistant phenotype on plaque reduction. Viral replication was comparable to that of the wild type. Cell-based kinase activity and autophosphorylation of ectopically expressed proteins showed that mutants retained some kinase activity. This study showed that patient-derived cytomegalovirus with different ganciclovir sensitivities retained replication efficiency and exhibited some kinase activity in vitro ., (Copyright © 2019 American Society for Microbiology.)

Infections with the human cytomegalovirus (HCMV) cause serious medical problems including organ rejection and congenital infection. Treatment of HCMV infections with currently available medication targeting viral enzymes is often accompanied with severe side effects and the occurrence of drug-resistant viruses. This demands novel therapeutical approaches like targeting genetically stable host cell proteins that are crucial for virus replication. Although numerous experimental drugs with promising in vitro efficacy have been identified, the lack of available data in animal models limits their potential for further clinical development. Recently, we described the very strong in vitro antiherpesviral activity of the NF-κB inhibitor TF27 and the CDK7 inhibitor LDC4297 at low nanomolar concentrations. In the present study, we present first data for the in vivo efficacy of both experimental drugs using an established cytomegalovirus animal model (murine CMV replication in immunodefective Rag -/- mice). The main findings of this study are (i) a strong inhibitory potency against beta- and gamma-herpesviruses of both compounds in vitro, (ii) even more important, a pronounced anticytomegaloviral activity also exerted in vivo, that resulted from (iii) a restriction of viral replication to the site of infection, thus preventing organ dissemination, (iv) in the absence of major compound-associated adverse events. Thus, we provide evidence for a strong antiviral potency in vivo and proof-of-concept for both drugs, which may encourage their further drug development, possibly including pharmacologically optimized derivatives, for a potential use in future antiherpesviral treatment., (Copyright © 2018. Published by Elsevier B.V.)

The nuclear phase of herpesvirus replication is regulated through the formation of regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely pointing to the formation of a viral core nuclear egress complex (NEC) that recruits a multimeric assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the mechanism of the association of human cytomegalovirus (HCMV) capsids with the NEC, which in turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide electron microscopy-based data demonstrating the association of both nuclear capsids and NEC proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells, nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking and NEC-capsid interaction., Competing Interests: The authors declare no conflict of interest.

Nuclear egress of herpesvirus capsids is mediated by a multi-component nuclear egress complex (NEC) assembled by a heterodimer of two essential viral core egress proteins. In the case of human cytomegalovirus (HCMV), this core NEC is defined by the interaction between the membrane-anchored pUL50 and its nuclear cofactor, pUL53. NEC protein phosphorylation is considered to be an important regulatory step, so this study focused on the respective role of viral and cellular protein kinases. Multiply phosphorylated pUL50 varieties were detected by Western blot and Phos-tag analyses as resulting from both viral and cellular kinase activities. In vitro kinase analyses demonstrated that pUL50 is a substrate of both PKCα and CDK1, while pUL53 can also be moderately phosphorylated by CDK1. The use of kinase inhibitors further illustrated the importance of distinct kinases for core NEC phosphorylation. Importantly, mass spectrometry-based proteomic analyses identified five major and nine minor sites of pUL50 phosphorylation. The functional relevance of core NEC phosphorylation was confirmed by various experimental settings, including kinase knock-down/knock-out and confocal imaging, in which it was found that (i) HCMV core NEC proteins are not phosphorylated solely by viral pUL97, but also by cellular kinases; (ii) both PKC and CDK1 phosphorylation are detectable for pUL50; (iii) no impact of PKC phosphorylation on NEC functionality has been identified so far; (iv) nonetheless, CDK1-specific phosphorylation appears to be required for functional core NEC interaction. In summary, our findings provide the first evidence that the HCMV core NEC is phosphorylated by cellular kinases, and that the complex pattern of NEC phosphorylation has functional relevance.

Infection with human cytomegalovirus (HCMV) is a serious medical problem, particularly in immunocompromised individuals and neonates. The success of (val)ganciclovir therapy is hampered by low drug compatibility and induction of viral resistance. A novel strategy of antiviral treatment is based on the exploitation of cell-directed signaling, e. g. pathways with a known relevance for carcinogenesis and tumor drug development. Here we describe a principle for putative antiviral drugs based on targeting dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs). DYRKs constitute an evolutionarily conserved family of protein kinases with key roles in the control of cell proliferation and differentiation. Members of the DYRK family are capable of phosphorylating a number of substrate proteins, including regulators of the cell cycle, e.g. DYRK1B can induce cell cycle arrest, a critical step for the regulation of HCMV replication. Here we provide first evidence for a critical role of DYRKs during viral replication and the high antiviral potential of DYRK inhibitors (SC84227, SC97202 and SC97208, Harmine and AZ-191). Using established replication assays for laboratory and clinically relevant strains of HCMV, concentration-dependent profiles of inhibition were obtained. Mean inhibitory concentrations (EC50) of 0.98 ± 0.08 μM/SC84227, 0.60 ± 0.02 μM/SC97202, 6.26 ± 1.64 μM/SC97208, 0.71 ± 0.019 μM/Harmine and 0.63 ± 0.23 μM/AZ-191 were determined with HCMV strain AD169-GFP for the infection of primary human fibroblasts. A first analysis of the mode of antiviral action suggested a block of viral replication at the early-late stage of HCMV gene expression. Moreover, rhesus macaque cytomegalovirus (RhCMV), varicella-zoster virus (VZV) and herpes simplex virus (HSV-1) showed a similarly high sensitivity to these compounds. Thus, we conclude that DYRK signaling represents a promising target pathway for the development of novel anti-herpesviral strategies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The nuclear lamina lines the inner nuclear membrane providing a structural framework for the nucleus. Cellular processes, such as nuclear envelope breakdown during mitosis or nuclear export of large ribonucleoprotein complexes, are functionally linked to the disassembly of the nuclear lamina. In general, lamina disassembly is mediated by phosphorylation, but the precise molecular mechanism is still not completely understood. Recently, we suggested a novel mechanism for lamina disassembly during the nuclear egress of herpesviral capsids which involves the cellular isomerase Pin1. In this study, we focused on mechanistic details of herpesviral nuclear replication to demonstrate the general importance of Pin1 for lamina disassembly. In particular, Ser22-specific lamin phosphorylation consistently generates a Pin1-binding motif in cells infected with human and animal alpha-, beta-, and gammaherpesviruses. Using nuclear magnetic resonance spectroscopy, we showed that binding of Pin1 to a synthetic lamin peptide induces its cis/trans isomerization in vitro. A detailed bioinformatic evaluation strongly suggests that this structural conversion induces large-scale secondary structural changes in the lamin N-terminus. Thus, we concluded that a Pin1-induced conformational change of lamins may represent the molecular trigger responsible for lamina disassembly. Consistent with this concept, pharmacological inhibition of Pin1 activity blocked lamina disassembly in herpesvirus-infected fibroblasts and consequently impaired virus replication. In addition, a phospho-mimetic Ser22Glu lamin mutant was still able to form a regular lamina structure and overexpression of a Ser22-phosphorylating kinase did not induce lamina disassembly in Pin1 knockout cells. Intriguingly, this was observed in absence of herpesvirus infection proposing a broader importance of Pin1 for lamina constitution. Thus, our results suggest a functional model of similar events leading to disassembly of the nuclear lamina in response to herpesviral or inherent cellular stimuli. In essence, Pin1 represents a regulatory effector of lamina disassembly that promotes the nuclear pore-independent egress of herpesviral capsids., Competing Interests: The authors have declared that no competing interests exist.

Nuclear egress of herpesvirus capsids through the nuclear envelope is mediated by the multimeric nuclear egress complex (NEC). The human cytomegalovirus (HCMV) core NEC is defined by an interaction between the membrane-anchored pUL50 and its nuclear co-factor pUL53, tightly associated through heterodimeric corecruitment to the nuclear envelope. Cellular proteins, such as p32/gC1qR, emerin and protein kinase C (PKC), are recruited by direct interaction with pUL50 for the multimeric extension of the NEC. As a functionally important event, the recruitment of both viral and cellular protein kinases leads to site-specific lamin phosphorylation and nuclear lamina disassembly. In this study, interaction domains within pUL50 for its binding partners were defined by co-immunoprecipitation. The interaction domain for pUL53 is located within the pUL50 N-terminus (residues 10-169), interaction domains for p32/gC1qR (100-358) and PKC (100-280) overlap in the central part of pUL50, and the interaction domain for emerin is located in the C-terminus (265-397). Moreover, expression and formation of core NEC proteins at the nuclear rim were consistently detected in cells permissive for productive HCMV replication, including two trophoblast-cell lines. Importantly, regular nuclear-rim formation of the core NEC was blocked by inhibition of cyclin-dependent kinase (CDK) activity. In relation to the recently published crystal structure of the HCMV core NEC, our findings result in a refined view of NEC assembly. In particular, we suggest that CDKs may play an important regulatory role in NEC formation during HCMV replication.

This study examines entrainment of speech timing and rhythm with a model speaker in healthy persons and individuals with Parkinson's. We asked whether participants coordinate their speech initiation and rhythm with the model speaker, and whether the regularity of metrical structure of sentences influences this behaviour. Ten native German speakers with hypokinetic dysarthria following Parkinson's and 10 healthy controls heard a sentence ('prime') and subsequently read aloud another sentence ('target'). Speech material comprised 32 metrically regular and irregular sentences, respectively. Turn-taking delays and alignment of speech rhythm were measured using speech wave analyses. Results showed that healthy participants initiated speech more closely in rhythm with the model speaker than patients. Metrically regular prime sentences induced anticipatory responses relative to metrically irregular primes. Entrainment of speech rhythm was greater in metrically regular targets, especially in individuals with Parkinson's. We conclude that individuals with Parkinson's may exploit metrically regular cues in speech.

Unlabelled: The regulatory protein pUL69 of human cytomegalovirus acts as a viral mRNA export factor, facilitating the cytoplasmic accumulation of unspliced RNA via interaction with the cellular mRNA export factor UAP56. Here we provide evidence for a posttranslational modification of pUL69 via arginine methylation within the functionally important N terminus. First, we demonstrated a specific immunoprecipitation of full-length pUL69 as well as pUL69aa1-146 by a mono/dimethylarginine-specific antibody. Second, we observed a specific electrophoretic mobility shift upon overexpression of the catalytically active protein arginine methyltransferase 6 (PRMT6). Third, a direct interaction of pUL69 and PRMT6 was confirmed by yeast two-hybrid and coimmunoprecipitation analyses. We mapped the PRMT6 interaction motif to the pUL69 N terminus and identified critical amino acids within the arginine-rich R1 box of pUL69 that were crucial for PRMT6 and/or UAP56 recruitment. In order to test the impact of putative methylation substrates on the functions of pUL69, we constructed various pUL69 derivatives harboring arginine-to-alanine substitutions and tested them for RNA export activity. Thus, we were able to discriminate between arginines within the R1 box of pUL69 that were crucial for UAP56/PRMT6-interaction and/or mRNA export activity. Remarkably, nuclear magnetic resonance (NMR) analyses revealed the same α-helical structures for pUL69 sequences encoding either the wild type R1/R2 boxes or a UAP56/PRMT6 binding-deficient derivative, thereby excluding the possibility that R/A amino acid substitutions within R1 affected the secondary structure of pUL69. We therefore conclude that the pUL69 N terminus is methylated by PRMT6 and that this critically affects the functions of pUL69 for efficient mRNA export and replication of human cytomegalovirus., Importance: The UL69 protein of human cytomegalovirus is a multifunctional regulatory protein that acts as a viral RNA export factor with a critical role for efficient replication. Here, we demonstrate that pUL69 is posttranslationally modified via arginine methylation and that the protein methyltransferase PRMT6 mediates this modification. Furthermore, arginine residues with a crucial function for RNA export and for binding of the cellular RNA export factor UAP56 as well as PRMT6 were mapped within the arginine-rich R1 motif of pUL69. Importantly, we demonstrated that mutation of those arginines did not alter the secondary structure of R1, suggesting that they may serve as critical methylation substrates. In summary, our study reveals a novel posttranslational modification of pUL69 which has a significant impact on the function of this important viral regulatory protein. Since PRMTs appear to be amenable to selective inhibition by small molecules, this may constitute a novel target for antiviral therapy., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Herpesviral capsids are assembled in the host cell nucleus before being translocated into the cytoplasm for further maturation. The crossing of the nuclear envelope represents a major event that requires the formation of the nuclear egress complex (NEC). Previous studies demonstrated that human cytomegalovirus (HCMV) proteins pUL50 and pUL53, as well as their homologs in all members of Herpesviridae, interact with each other at the nuclear envelope and form the heterodimeric core of the NEC. In order to characterize further the viral and cellular protein content of the multimeric NEC, the native complex was isolated from HCMV-infected human primary fibroblasts at various time points and analyzed using quantitative proteomics. Previously postulated components of the HCMV-specific NEC, as well as novel potential NEC-associated proteins such as emerin, were identified. In this regard, interaction and colocalization between emerin and pUL50 were confirmed by coimmunoprecipitation and confocal microscopy analyses, respectively. A functional validation of viral and cellular NEC constituents was achieved through siRNA-mediated knockdown experiments. The important role of emerin in NEC functionality was demonstrated by a reduction of viral replication when emerin expression was down-regulated. Moreover, under such conditions, reduced production of viral proteins and deregulation of viral late cytoplasmic maturation were observed. Combined, these data prove the functional importance of emerin as an NEC component, associated with pUL50, pUL53, pUL97, p32/gC1qR, and further regulatory proteins. Summarized, our findings provide the first proteomics-based characterization and functional validation of the HCMV-specific multimeric NEC., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Our understanding of brainstem swallowing centers is mainly based on experimental animals. In order to solve this problem also in humans, a clinical-anatomical study on dysphagic patients with different lesion patterns was performed., Patients and Methods: We studied 43 consecutively admitted dysphagic patients with unilateral paresis of the vagal nerve (PVN), Avellis' syndrome (AS), Wallenberg's syndrome (WS), posterior fossa tumour (PFT) or cerebellar hemorrhage (CH) with regard to clinical and anatomical aspects., Findings: There was a continuum with regard to functional outcome from neurogenic dysphagia (ND): Patients with PFT or CH had a significantly worse outcome than patients with WS; the outcome of WS patients was significantly worse than that of patients with PVN or AS. In AS only the Nucleus ambiguus (NA) and its surrounding reticular formation (RF) were affected. In all patients with WS, the infarctions of the dorsolateral medulla were situated in the rostral third of the medulla and affected the NA and the Nucleus tractus solitarii (NTS) with their surrounding RF. In patients with PFT and CH, the NTS and its surrounding RF were affected on both sides. The overlap area of WS and PFT lesions is situated in the NTS and the surrounding RF, especially in its Nucleus parvocellularis., Interpretation: Our results point to the fact, that in humans the dorsomedial central pattern generators (CPGs) for swallowing are situated in the rostral part of the dorsal medulla oblongata near the NTS/surrounding RF (especially Nucleus parvocellularis) and that the dorsomedial CPGs are superior to the ventrolateral CPGs (near the NA/surrounding RF) with regard to their swallowing-relevance. Furthermore, we hypothesize that due to the individual asymmetry of the swallowing-dominant forebrain hemisphere - the outcome from ND in WS depends on the side of the medullary infarction.

Background: No studies exist dealing with the outcome of dysphagic patients with posterior fossa (IV. ventricle) tumours (PFT) or cerebellar hemorrhage (CH), and the outcome of patients with Wallenberg's syndrome (WS) after functional swallowing therapy (FST) has so far not been studied in detail. Patients and methods. 208 patients with neurogenic dysphagia (ND) who were consecutively admitted for functional swallowing therapy (FST) over a 3 year period to our hospital were examined clinically, by use of a videofluoroscopic swallowing study (VFSS) and/or fibreoptic evaluation of swallowing (FEES). The most frequent etiology was stroke (48%), followed by CNS tumours (13%). In the present study we defined three groups. Group 1 comprised 8 patients with PFT or CH. Group 2 consisted of 27 patients with WS, which was the leading cause among patients with non-hemispheric stroke. Since in WS a vagal nerve paresis due to affection of the Nucleus ambiguus occurs, 8 patients with Avellis' syndrome or unilateral paresis of the vagal nerve served as controls and were defined as group 3. Findings. In the three groups, functional feeding status showed significant improvement after FST comprising methods of restitution, compensation and adaptation, each of which were applied in more than 80% of patients. Outcome was, however, significantly worse in group 1 as compared to group 2 and in group 2 as compared to group 3. Dysfunction of the upper esophageal sphincter and reflex triggering were significantly more severely disturbed in groups 1 and 2 as compared to group 3. Group 1 showed significantly more severe disturbances of the oral phase as compared to groups 2 and 3. After FST, more than 50% (5/8) of group 1 and 30% (8/27) of WS patients (group 2) were dependent on tube feeding, whereas all patients of group 3 were full-oral feeders. Interpretation. This is the first study dealing with the outcome of dysphagic patients with PFT or CH. Based on our results it can be assumed that in these patients pressure is exerted on both dorsomedial central pattern generators (DMCPGs) for swallowing in a posterior-anterior direction. Due to the importance of the DMCPGs for swallowing, bilateral (and often MRI-invisible) lesions seem to be very harmful. For a better understanding of the pathomechanism responsible for ND in patients with PFT or CH, modern imaging methods such as proton magnetic resonance spectroscopy should be used for studying metabolic changes in the dorsal medulla in the future. Since the outcome of patients with WS with regard to dependence of tube feeding was not associated with the site or size of the lesion, it may due to the individual asymmetry of the swallowing-dominant forebrain hemisphere - depend on the side of the medullary infarction.

Over 30% of persons with multiple sclerosis (pwMS) suffer from swallowing symptoms, a higher rate than previously assumed. Neurogenic dysphagia (ND) may cause many different kinds of oropharyngeal sensorimotor dysfunctions in pwMS, and is associated with both the amount of disability and brainstem signs. About 15% of pwMS with mild disability may also suffer from ND. Diagnostic tools comprise history taking, bedside screening examination (50 ml water test combined with assessment of pharyngeal sensation or with pulse oximetry) and sometimes a videofluoroscopic swallowing study (VFSS) and fibreoptic endoscopic evaluation of swallowing (FEES). VFSS and FEES are complementary methods and both have advantages and disadvantages. Interventions for ND in pwMS are mainly based on functional swallowing therapy, including methods of restitution, compensation and adaptation. The aim of intervention is to prevent aspiration and aspiration pneumonia. Outcome assessment should focus on clinically relevant parameters, such as activity limitation, participation restriction and health-related quality of life.

To examine whether kinematic analysis of laryngeal movements (which are closely linked to pharyngeal swallowing) can differentiate between normal and disturbed swallowing, we used a three-dimensional ultrasound movement recording system to measure the movements of the larynx during swallowing in 32 patients with neurogenic dysphagia caused by central nervous system lesions and in 32 age- and sex-matched healthy individuals. At the beginning of an inpatient rehabilitation swallowing program, laryngeal movements in 24 patients were highly disturbed in terms of velocity curve irregularities. After rehabilitation, the majority of patients with hitherto irregular velocity profiles exhibited laryngeal kinematics that were indistinguishable from those of 32 healthy subjects. Kinematic analysis of laryngeal movements, therefore, is suitable for monitoring motor recovery of swallowing disturbances in patients with neurogenic dysphagia while undergoing swallowing rehabilitation.