UNC-45A breaks the microtubule lattice independently of its effects on non-muscle myosin II.

In invertebrates, UNC-45 regulates myosin stability and functions. Vertebrates have two distinct isoforms of the protein: UNC-45B, expressed in muscle cells only, and UNC-45A, expressed in all cells and implicated in regulating both non-muscle myosin II (NMII)- and microtubule (MT)-associated functions. Here, we show that, in vitro and in human and rat cells, UNC-45A binds to the MT lattice, leading to MT bending, breakage and depolymerization. Furthermore, we show that UNC-45A destabilizes MTs independent of its C-terminal NMII-binding domain and even in the presence of the NMII inhibitor blebbistatin. These findings identified UNC-45A as a novel type of MT-severing protein with a dual non-mutually exclusive role in regulating NMII activity and MT stability. Because many human diseases, from cancer to neurodegenerative diseases, are caused by or associated with deregulation of MT stability, our findings have profound implications in the biology of MTs, as well as the biology of human diseases and possible therapeutic implications for their treatment.This article has an associated First Person interview with the joint first authors of the paper.

LFA-1 cluster formation in T-cells depends on L-plastin phosphorylation regulated by P90RSK and PP2A.

The integrin LFA-1 is crucial for T-cell/ APC interactions and sensitive recognition of antigens. Precise nanoscale organization and valency regulation of LFA-1 are mandatory for an appropriate function of the immune system. While the inside-out signals regulating the LFA-1 affinity are well described, the molecular mechanisms controlling LFA-1 avidity are still not fully understood. Here, we show that activation of the actin-bundling protein L-plastin (LPL) through phosphorylation at serine-5 enables the formation of clusters containing LFA-1 in high-affinity conformation. Phosphorylation of LPL is induced by an nPKC-MEK-p90RSK pathway and counter-regulated by the serine-threonine phosphatase PP2A. Interestingly, recruitment of LFA-1 into the T-cell/APC contact zone is not affected by LPL phosphorylation. Instead, for this process, activation of the actin-remodeling protein cofilin through dephosphorylation is essential. Together, this study reveals a dichotomic spatial regulation of LFA-1 clustering and microscale movement in T-cells by two different actin-binding proteins, LPL and cofilin.

How to measure staff continuity in intensive psychiatric home treatment: a routine data and single case analysis.

Background: Intensive forms of outreach mental health care (IOC) such as crisis resolution or home treatment teams are increasingly implemented as alternatives to inpatient admission, providing recovery-oriented treatment at home at comparable costs and outcomes. However, one issue with IOC is the lack of continuity regarding staff members who provide home visits, complicating relationship building and meaningful therapeutic exchange. The aim of this study is to validate existing primarily qualitative findings using performance data and to explore a possible correlation between the number of staff involved within IOC treatment and the service users' length of stay (LOS). Methods: Routine data from an IOC team in a catchment area in Eastern Germany were analyzed. Basic parameters of service delivery were calculated and an in-depth descriptive analysis regarding staff continuity was performed. Further, an exploratory single case analysis was conducted, presenting the exact sequence of all treatment contacts for one case with low and one with high staff continuity. Results: We analyzed 10.598 face-to-face treatment contacts based on 178 IOC users. The mean LOS was 30.99 days. About 75% of all home visits were conducted by two or more staff members simultaneously. Service users saw an average of 10.24 different staff per treatment episode. On 11% of the care days, only unknown staff, and on 34% of the care days at least one unknown staff member conducted the home visit. 83% of the contacts were performed by the same three staff members and 51% were made by one and the same staff member. A significant positive correlation (p = 0.0007) was found between the number of different practitioners seen by a service user in the first seven days of care and the LOS. Conclusion: Our results suggest that a high number of different staff in the early period of IOC episodes correlates with an extended LOS. Future research must clarify the exact mechanisms of this correlation. Furthermore, it should be investigated how the multiple professions within IOC teams influence the LOS and the quality of treatment and what quality indicators may be suitable to ensure treatment processes.

Bioactive reagents used in mesotherapy for skin rejuvenation in vivo induce diverse physiological processes in human skin fibroblasts in vitro- a pilot study.

The promise of mesotherapy is maintenance and/or recovery of a youthful skin with a firm, bright and moisturized texture. Currently applied medications employ microinjections of hyaluronic acid, vitamins, minerals and amino acids into the superficial layer of the skin. However, the molecular and cellular processes underlying mesotherapy are still elusive. Here we analysed the effect of five distinct medication formulas on pivotal parameters involved in skin ageing, that is collagen expression, cell proliferation and morphological changes using normal human skin fibroblast cultures in vitro. Whereas in the presence of hyaluronic acid, NCTF135(®) and NCTF135HA(®) , cell proliferation was comparable to control cultures; however, with higher expression of collagen type-1, matrix metalloproteinase-1 and tissue inhibitor of matrix metalloproteinase-1, addition of Soluvit(®) N and Meso-BK led to apoptosis and/or necrosis of human fibroblasts. The data indicate that bioactive reagents currently applied for skin rejuvenation elicit strikingly divergent physiological processes in human skin fibroblast in vitro.

[Biometric properties of QUALIFY: a tool for assessing quality indicators]. / Biometrische Eigenschaften des QUALIFY-Instruments zur Bewertung von Qualitätsindikatoren.

INTRODUCTION: Accurate health care evaluation using quality indicators (QIs) is of vital clinical importance for a quality-oriented health care system. The QUALIFY tool is the current research standard for assessing QIs of health care; however, its biometric properties in psychiatry have not yet been evaluated empirically. Our aim was to evaluate the internal consistency and structure of QUALIFY. METHODS: This study applies a literature-based post-hoc analytical design to a sample of 289 QIs of mental health care. First, the indicators were assessed on the basis of nineteen ordinal QUALIFY criteria as a single measuring tool. Second, using Cronbach's alpha the internal consistency of the measuring tool was evaluated and the structure of QUALIFY using an explorative principal component analysis was tested. RESULTS AND DISCUSSION: The tool showed an acceptable internal consistency (Cronbach's α=0.75), with three criteria (consideration of potential risks/side effects when using the indicator, implementation barriers taken into account, and the ability to influence the indicator) being inconsistent with the full scale. If these three criteria were not taken into account, the tool had a good internal consistency (Cronbach's α=0.81). The QUALIFY structural matrix comprises three components, one of which reflected six from eight original quality criteria of the scientific category. The other two components represent the semiotic structure of the QIs. CONCLUSION: QUALIFY is an internally inconsistent instrument, which may be useful to assess mental health care QIs. The information about the structure of QUALIFY can be applied for the purposes of research planning as well as the interpretation and development of QIs.

Inflammation induces pro-NETotic neutrophils via TNFR2 signaling.

Cytokines released during chronic inflammatory diseases induce pro-inflammatory properties in polymorphonuclear neutrophils (PMNs). Here, we describe the development of a subgroup of human PMNs expressing CCR5, termed CCR5+ PMNs. Auto- and paracrine tumor necrosis factor (TNF) signaling increases intracellular neutrophil elastase (ELANE) abundance and induces neutrophil extracellular traps formation (NETosis) in CCR5+ PMNs, and triggering of CCR5 amplifies NETosis. Membranous TNF (mTNF) outside-in signaling induces the formation of reactive oxygen species, known activators of NETosis. In vivo, we find an increased number of CCR5+ PMNs in the peripheral blood and inflamed lamina propria of patients with ulcerative colitis (UC). Notably, failure of anti-TNF therapy is associated with higher frequencies of CCR5+ PMNs. In conclusion, we identify a phenotype of pro-NETotic, CCR5+ PMNs present in inflamed tissue in vivo and inducible in vitro. These cells may reflect an important component of tissue damage during chronic inflammation and could be of diagnostic value.

Supported Employment, Participation at Work, and Peer Support: A Qualitative, Participatory Case Study Report of the Geesthacht Model.

Background: For people who have experienced mental health crises or psychosocial disabilities, it is considerably more difficult to receive support to participate in work on an equal basis with others. In the town of Geesthacht, in Northern Germany, an integrative care network was implemented that allows for acute psychiatric treatment as well as participation in work and activities. This paper aims to explore the principles, advantages, and challenges of this innovative project. Methodology: Within the context of a participatory and collaborative process evaluation of a prospective controlled cohort study (PsychCare), researchers with and without experiential expertise conducted expert interviews and focus groups to evaluate the experiences of 37 employees, with and without lived experience, from various institutions associated with this care network. The data was analyzed using qualitative content analysis. Results: It was the change from financial compensation paid on a daily basis to a global treatment budget that allowed for a significant reduction of hospital beds in Geesthacht and freed up resources to implement a complex care network. Since then, various possibilities for participation, work, and activities for former service users, some of which are compensated financially, have been made available. These developments now allow for a less bureaucratic and often smooth transition from being a service user to involvement in participatory activities in the role of a peer, which is frequently perceived to be empowering and beneficial by participants with lived experience. At the same time, this care model has led to multiple role conflicts and different challenges for all parties involved. Conclusion: This innovative project in Geesthacht demonstrates the multifaceted potential of a global treatment budget system in the field of mental health care. To address certain downsides of the Geesthacht model, further development is necessary.

Keratinocytes costimulate naive human T cells via CD2: a potential target to prevent the development of proinflammatory Th1 cells in the skin.

The interplay between keratinocytes and immune cells, especially T cells, plays an important role in the pathogenesis of chronic inflammatory skin diseases. During psoriasis, keratinocytes attract T cells by releasing chemokines, while skin-infiltrating self-reactive T cells secrete proinflammatory cytokines, e.g., IFNγ and IL-17A, that cause epidermal hyperplasia. Similarly, in chronic graft-versus-host disease, allogenic IFNγ-producing Th1/Tc1 and IL-17-producing Th17/Tc17 cells are recruited by keratinocyte-derived chemokines and accumulate in the skin. However, whether keratinocytes act as nonprofessional antigen-presenting cells to directly activate naive human T cells in the epidermis remains unknown. Here, we demonstrate that under proinflammatory conditions, primary human keratinocytes indeed activate naive human T cells. This activation required cell contact and costimulatory signaling via CD58/CD2 and CD54/LFA-1. Naive T cells costimulated by keratinocytes selectively differentiated into Th1 and Th17 cells. In particular, keratinocyte-initiated Th1 differentiation was dependent on costimulation through CD58/CD2. The latter molecule initiated STAT1 signaling and IFNγ production in T cells. Costimulation of T cells by keratinocytes resulting in Th1 and Th17 differentiation represents a new explanation for the local enrichment of Th1 and Th17 cells in the skin of patients with a chronic inflammatory skin disease. Consequently, local interference with T cell-keratinocyte interactions may represent a novel strategy for the treatment of Th1 and Th17 cell-driven skin diseases.

UNC-45A is preferentially expressed in epithelial cells and binds to and co-localizes with interphase MTs.

UNC-45A is an ubiquitously expressed protein highly conserved throughout evolution. Most of what we currently know about UNC-45A pertains to its role as a regulator of the actomyosin system. However, emerging studies from both our and other laboratories support a role of UNC-45A outside of actomyosin regulation. This includes studies showing that UNC-45A: regulates gene transcription, co-localizes and biochemically co-fractionates with gamma tubulin and regulates centrosomal positioning, is found in the same subcellular fractions where MT-associated proteins are, and is a mitotic spindle-associated protein with MT-destabilizing activity in absence of the actomyosin system. Here, we extended our previous findings and show that UNC45A is variably expressed across a spectrum of cell lines with the highest level being found in HeLa cells and in ovarian cancer cells inherently paclitaxel-resistant. Furthermore, we show that UNC-45A is preferentially expressed in epithelial cells, localizes to mitotic spindles in clinical tumor specimens of cancer and co-localizes and co-fractionates with MTs in interphase cells independent of actin or myosin. In sum, we report alteration of UNC45A localization in the setting of chemotherapeutic treatment of cells with paclitaxel, and localization of UNC45A to MTs both in vitro and in vivo. These findings will be important to ongoing and future studies in the field that further identify the important role of UNC45A in cancer and other cellular processes.

Bodies for Anatomy Education in Medical Schools: An Overview of the Sources of Cadavers Worldwide.

The International Federation of Associations of Anatomists (IFAA) recommended in 2012 that only donated bodies be used for anatomy teaching and research. However, in many countries around the world, anatomists still depend on bodies that do not stem from voluntary donations by the deceased but, rather, are "unclaimed." A broad search of the literature was conducted to produce a baseline overview of the sources of cadavers used for anatomy teaching in undergraduate medical curricula on a global scale. Information from the literature search was supplemented with data from a 2016-2017 survey of selected senior local anatomists. Of 165 countries with medical schools, information was gathered for 71. In 22 (32%) of the 68 countries that use cadavers for anatomy teaching, body donation is the exclusive source of bodies. However, in most other countries, unclaimed bodies remain the main (n = 18; 26%) or exclusive (n = 21; 31%) source. Some countries import cadavers from abroad, mainly from the United States or India. In one country, bodies of executed persons are given to anatomy departments. The heterogeneous geographical distribution of body sources cannot easily be accounted for, but religion, culture, and folk beliefs about what should happen to bodies after death seem to play a role. Implementation of the IFAA recommendations still has a long way to go, but it is encouraging that functioning body donation programs exist on all continents and that there are examples of recent rises in donations and of anatomists initiating new donation programs.

Transposon mutagenesis screen in mice identifies TM9SF2 as a novel colorectal cancer oncogene.

New therapeutic targets for advanced colorectal cancer (CRC) are critically needed. Our laboratory recently performed an insertional mutagenesis screen in mice to identify novel CRC driver genes and, thus, potential drug targets. Here, we define Transmembrane 9 Superfamily 2 (TM9SF2) as a novel CRC oncogene. TM9SF2 is an understudied protein, belonging to a well conserved protein family characterized by their nine putative transmembrane domains. Based on our transposon screen we found that TM9SF2 is a candidate progression driver in digestive tract tumors. Analysis of The Cancer Genome Atlas (TCGA) data revealed that approximately 35% of CRC patients have elevated levels of TM9SF2 mRNA, data we validated using an independent set of CRC samples. RNAi silencing of TM9SF2 reduced CRC cell growth in an anchorage-independent manner, a hallmark of cancer. Furthermore, CRISPR/Cas9 knockout of TM9SF2 substantially diminished CRC tumor fitness in vitro and in vivo. Transcriptome analysis of TM9SF2 knockout cells revealed a potential role for TM9SF2 in cell cycle progression, oxidative phosphorylation, and ceramide signaling. Lastly, we report that increased TM9SF2 expression correlates with disease stage and low TM9SF2 expression correlate with a more favorable relapse-free survival. Taken together, this study provides evidence that TM9SF2 is a novel CRC oncogene.

Identification of a novel anoikis signalling pathway using the fungal virulence factor gliotoxin.

Anoikis is a form of apoptosis induced by cell detachment. Integrin inactivation plays a major role in the process but the exact signalling pathway is ill-defined. Here we identify an anoikis pathway using gliotoxin (GT), a virulence factor of the fungus Aspergillus fumigatus, which causes invasive aspergillosis in humans. GT prevents integrin binding to RGD-containing extracellular matrix components by covalently modifying cysteines in the binding pocket. As a consequence, focal adhesion kinase (FAK) is inhibited resulting in dephosphorylation of p190RhoGAP, allowing activation of RhoA. Sequential activation of ROCK, MKK4/MKK7 and JNK then triggers pro-apoptotic phosphorylation of Bim. Cells in suspension or lacking integrin surface expression are insensitive to GT but are sensitised to ROCK-MKK4/MKK7-JNK-dependent anoikis upon attachment to fibronectin or integrin upregulation. The same signalling pathway is triggered by FAK inhibition or inhibiting integrin αV/ß3 with Cilengitide. Thus, GT can target integrins to induce anoikis on lung epithelial cells.

Soluble cysteine-rich tick saliva proteins Salp15 and Iric-1 from E. coli.

Ticks transmit numerous pathogens, including borreliae, which cause Lyme disease. Tick saliva contains a complex mix of anti-host defense factors, including the immunosuppressive cysteine-rich secretory glycoprotein Salp15 from Ixodes scapularis ticks and orthologs like Iric-1 from Ixodes ricinus. All tick-borne microbes benefit from the immunosuppression at the tick bite site; in addition, borreliae exploit the binding of Salp15 to their outer surface protein C (OspC) for enhanced transmission. Hence, Salp15 proteins are attractive targets for anti-tick vaccines that also target borreliae. However, recombinant Salp proteins are not accessible in sufficient quantity for either vaccine manufacturing or for structural characterization. As an alternative to low-yield eukaryotic systems, we investigated cytoplasmic expression in Escherichia coli, even though this would not result in glycosylation. His-tagged Salp15 was efficiently expressed but insoluble. Among the various solubility-enhancing protein tags tested, DsbA was superior, yielding milligram amounts of soluble, monomeric Salp15 and Iric-1 fusions. Easily accessible mutants enabled epitope mapping of two monoclonal antibodies that, importantly, cross-react with glycosylated Salp15, and revealed interaction sites with OspC. Free Salp15 and Iric-1 from protease-cleavable fusions, despite limited solubility, allowed the recording of (1)H-(15)N 2D NMR spectra, suggesting partial folding of the wild-type proteins but not of Cys-free variants. Fusion to the NMR-compatible GB1 domain sufficiently enhanced solubility to reveal first secondary structure elements in (13)C/(15)N double-labeled Iric-1. Together, E. coli expression of appropriately fused Salp15 proteins may be highly valuable for the molecular characterization of the function and eventually the 3D structure of these medically relevant tick proteins.

Mapping the ligand-binding region of Borrelia hermsii fibronectin-binding protein.

Many pathogenic microorganisms express fibronectin-binding molecules that facilitate their adherence to the extracellular matrix and/or entry into mammalian cells. We have previously described a Borrelia recurrentis gene, cihC that encodes a 40-kDa surface receptor for both, fibronectin and the complement inhibitors C4bp and C1-Inh. We now provide evidence for the expression of a group of highly homologues surface proteins, termed FbpA, in three B. hermsii isolates and two tick-borne relapsing fever spirochetes, B. parkeri and B. turicatae. When expressed in Escherichia coli or B. burgdorferi, four out of five proteins were shown to selectively bind fibronectin, whereas none of five proteins were able to bind the human complement regulators, C4bp and C1-Inh. By applying deletion mutants of the B. hermsii fibronectin-binding proteins a putative high-affinity binding site for fibronectin was mapped to its central region. In addition, the fibronectin-binding proteins of B. hermsii were found to share sequence homology with BBK32 of the Lyme disease spirochete B. burgdorferi with similar function suggesting its involvement in persistence and/or virulence of relapsing fever spirochetes.