Lysosomal damage sensing and lysophagy initiation by SPG20-ITCH.

Cells respond to lysosomal membrane permeabilization by membrane repair or selective macroautophagy of damaged lysosomes, termed lysophagy, but it is not fully understood how this decision is made. Here, we uncover a pathway in human cells that detects lipid bilayer perturbations in the limiting membrane of compromised lysosomes, which fail to be repaired, and then initiates ubiquitin-triggered lysophagy. We find that SPG20 binds the repair factor IST1 on damaged lysosomes and, importantly, integrates that with the detection of damage-associated lipid-packing defects of the lysosomal membrane. Detection occurs via sensory amphipathic helices in SPG20 before rupture of the membrane. If lipid-packing defects are extensive, such as during lipid peroxidation, SPG20 recruits and activates ITCH, which marks the damaged lysosome with lysine-63-linked ubiquitin chains to initiate lysophagy and thus triages the lysosome for destruction. With SPG20 being linked to neurodegeneration, these findings highlight the relevance of a coordinated lysosomal damage response for cellular homeostasis.

VCP/p97 Extracts Sterically Trapped Ku70/80 Rings from DNA in Double-Strand Break Repair.

During DNA double-strand break (DSB) repair, the ring-shaped Ku70/80 complex becomes trapped on DNA and needs to be actively extracted, but it has remained unclear what provides the required energy. By means of reconstitution of DSB repair on beads, we demonstrate here that DNA-locked Ku rings are released by the AAA-ATPase p97. To achieve this, p97 requires ATP hydrolysis, cooperates with the Ufd1-Npl4 ubiquitin-adaptor complex, and specifically targets Ku80 that is modified by K48-linked ubiquitin chains. In U2OS cells, chemical inhibition of p97 or siRNA-mediated depletion of p97 or its adapters impairs Ku80 removal after non-homologous end joining of DSBs. Moreover, this inhibition attenuates early steps in homologous recombination, consistent with p97-driven Ku release also affecting repair pathway choice. Thus, our data answer a central question regarding regulation of Ku in DSB repair and illustrate the ability of p97 to segregate even tightly bound protein complexes for release from DNA.

Do Corroles Stabilize Tetravalent Antimony?

Antimony corrole cations have been prepared by one-electron oxidation of antimony(III) congeners with silver(I) and copper(II) salts. Isolation and crystallization was successful for the first time, and the X-ray crystallographic investigation unraveled structural similarities with antimony(III)corroles. EPR experiments showed strong hyperfine interactions of the unpaired electron with the 121 Sb (I=5/2) and 123 Sb nuclei (I=7/2). A DFT analysis supports the description of the oxidized form as a SbIII corrole radical with less than 2 % SbIV character. In the presence of water or a fluoride source like PF6 - , the compounds undergo a redox disproportionation to yield known antimony(III)corroles and either difluorido-antimony(V)corroles, or bis-µ-oxido-di[antimony(V)corroles] via novel cationic hydroxo-antimony(V) derivatives.

VCP/p97 cooperates with YOD1, UBXD1 and PLAA to drive clearance of ruptured lysosomes by autophagy.

Rupture of endosomes and lysosomes is a major cellular stress condition leading to cell death and degeneration. Here, we identified an essential role for the ubiquitin-directed AAA-ATPase, p97, in the clearance of damaged lysosomes by autophagy. Upon damage, p97 translocates to lysosomes and there cooperates with a distinct set of cofactors including UBXD1, PLAA, and the deubiquitinating enzyme YOD1, which we term ELDR components for Endo-Lysosomal Damage Response. Together, they act downstream of K63-linked ubiquitination and p62 recruitment, and selectively remove K48-linked ubiquitin conjugates from a subpopulation of damaged lysosomes to promote autophagosome formation. Lysosomal clearance is also compromised in MEFs harboring a p97 mutation that causes inclusion body myopathy and neurodegeneration, and damaged lysosomes accumulate in affected patient tissue carrying the mutation. Moreover, we show that p97 helps clear late endosomes/lysosomes ruptured by endocytosed tau fibrils. Thus, our data reveal an important mechanism of how p97 maintains lysosomal homeostasis, and implicate the pathway as a modulator of degenerative diseases.

Targeting of parvulin interactors by diazirine mediated cross-linking discloses a cellular role of human Par14/17 in actin polymerization.

The peptidyl-prolyl cis/trans isomerases (PPIases) Parvulin 14 (Par14) and Parvulin 17 (Par17) result from alternative transcription initiation of the PIN4 gene. Whereas Par14 is present in all metazoan, Par17 is only expressed in Hominidae. Par14 resides mainly within the cellular nucleus, while Par17 is translocated into mitochondria. Using photo-affinity labeling, cross-linking and mass spectrometry (MS) we identified binding partners for both enzymes from HeLa lysates and disentangled their cellular roles. Par14 is involved in biogenesis of ribonucleoprotein (RNP)-complexes, RNA processing and DNA repair. Its elongated isoform Par17 participates in protein transport/translocation and in cytoskeleton organization. Nuclear magnetic resonance (NMR) spectroscopy reveals that Par17 binds to ß-actin with its N-terminal region, while both parvulins initiate actin polymerization depending on their PPIase activity as monitored by fluorescence spectroscopy. The knockdown (KD) of Par17 in HCT116 cells results in a defect in cell motility and migration.

Differences in Attitudes Toward Online Interventions in Psychiatry and Psychotherapy Between Health Care Professionals and Nonprofessionals: A Survey.

Background:Although the use of e-mental health interventions and their evaluation is already well advanced in countries such as the United States and Australia, research in this area is still in the early stages in Germany. Moreover, existing programs are used only to a small extent by patients, although physicians and therapists generally have a positive attitude toward their use. To help promote the use of online interventions in the future, an analysis of the differences in opinions and attitudes toward e-mental health interventions between health care professionals and nonprofessionals is necessary.Objective:This study aimed to examine the differences in attitudes toward online interventions between health care professionals and nonprofessionals.Methods:This study examined 92 physicians, 36 psychotherapists, and 1,353 randomly recruited nonprofessionals with the eight-item questionnaire entitled "Attitudes on telemedicine in psychiatry and psychotherapy (ATiPP)."Results:The questionnaires of n = 62 physicians, n = 37 psychotherapists, and n = 1,353 nonprofessionals were included in the analysis. Overall, nonprofessionals rate the use of telemedicine more critically than professionals. The itemwise t tests show significant differences between health care professionals and nonprofessionals on six out of eight items. The analyses of variance with post hoc tests for each single item also found differences between the groups (physicians vs. therapists vs. telephone participants vs. practice sample).Conclusion:There are significant differences in attitudes toward online interventions between professionals and nonprofessionals.

FHOD1 regulates stress fiber organization by controlling the dynamics of transverse arcs and dorsal fibers.

The formin FHOD1 (formin homology 2 domain containing protein 1) can act as a capping and bundling protein in vitro. In cells, active FHOD1 stimulates the formation of ventral stress fibers. However, the cellular mechanisms by which this phenotype is produced and the physiological relevance of FHOD1 function are not currently understood. Here, we first show that FHOD1 controls the formation of two distinct stress fiber precursors differentially. On the one hand, it inhibits dorsal fiber growth, which requires the polymerization of parallel bundles of long actin filaments. On the other hand, it stimulates transverse arcs that are formed by the fusion of short antiparallel actin filaments. This combined action is crucial for the maturation of stress fibers and their spatio-temporal organization, and a lack of FHOD1 function perturbs dynamic cell behavior during cell migration. Furthermore, we show that the GTPase-binding and formin homology 3 domains (GBD and FH3) are responsible for stress fiber association and colocalization with myosin. Surprisingly, a version of FHOD1 that lacks these domains nevertheless retains its full capacity to stimulate arc and ventral stress fiber formation. Based on our findings, we propose a mechanism in which FHOD1 promotes the formation of short actin filaments and transiently associates with transverse arcs, thus providing tight temporal and spatial control of the formation and turnover of transverse arcs into mature ventral stress fibers during dynamic cell behavior.

The effect of myofascial release of the physiological chains on the pain and health status in patients with fibromyalgia, compared to passive muscle stretching and a control group: a randomized controlled clinical trial.

OBJECTIVE: To explore the potential effectiveness of myofascial release compared to passive muscle stretching and to a control group in modulating pain intensity and health status in adults diagnosed with fibromyalgia (FM). MATERIALS AND METHODS: A preliminary randomized controlled clinical trial was conducted, consisting of eight weekly sessions. The participants were divided into three groups: myofascial release group (RG = 13), a muscle stretching group (SG = 13), and a control group (CG = 12), which received advice from a rheumatologist. The outcomes measured were the visual analogue pain scale (VAS), the fibromyalgia impact questionnaire (FIQ) (representing health status), and the number of painful areas. Univariate analyzes of covariance (ANCOVA) were performed at baseline, after 4 weeks (during treatment), after 8 weeks (post-treatment), and after 12 weeks (follow-up). The International Physical Activity Questionnaire (IPAQ), the Beck Depression Inventory (BDI) and the Pain Catastrophizing Scale (PCS) were included as covariates. Clinical trial registration number: NCT: 03408496. RESULTS: After eight weeks, the RG showed lower VAS scores compared to the CG (mean difference 95% CI: -5.10 to -1.26) and the SG (mean difference 95% CI: -4.9 to -0.23) with no difference between the SG and the CG. The total FIQ score for the RG was lower than the CG after 4 weeks (95% CI: -49.92 to -5.61), and 8 weeks (mean difference 95% CI: -52.72 to -15.73), although there was no difference between the RG and SG, as well as between the SG and CG, at both time points. The number of painful body areas was similar in all groups at the four time points. CONCLUSION: Preliminary results suggest that the RG possibly showed greater improvements in pain intensity and health status compared to the CG, and possibly greater improvements in pain intensity compared to the SG.

Myofascial release in fibromyalgia may potentially have greater improvements on pain modulation and health status when compared to medical counseling.Myofascial release, when compared to stretching, may potentially offer greater improvement in pain modulation in fibromyalgia.Myofascial release was not effective in decreasing the number of painful body areas of patients with fibromyalgia.

Digital Content-Free Speech Analysis Tool to Measure Affective Distress in Mental Health: Evaluation Study.

BACKGROUND: Mood disorders and depression are pervasive and significant problems worldwide. These represent severe health and emotional impairments for individuals and a considerable economic and social burden. Therefore, fast and reliable diagnosis and appropriate treatment are of great importance. Verbal communication can clarify the speaker's mental state-regardless of the content, via speech melody, intonation, and so on. In both everyday life and clinical conditions, a listener with appropriate previous knowledge or a trained specialist can grasp helpful knowledge about the speaker's psychological state. Using automated speech analysis for the assessment and tracking of patients with mental health issues opens up the possibility of remote, automatic, and ongoing evaluation when used with patients' smartphones, as part of the current trends toward the increasing use of digital and mobile health tools. OBJECTIVE: The primary aim of this study is to evaluate the measurements of the presence or absence of depressive mood in participants by comparing the analysis of noncontentual speech parameters with the results of the Patient Health Questionnaire-9. METHODS: This proof-of-concept study included participants in different affective phases (with and without depression). The inclusion criteria included a neurological or psychiatric diagnosis made by a specialist and fluent use of the German language. The measuring instrument was the VoiceSense digital voice analysis tool, which enables the analysis of 200 specific speech parameters based on machine learning and the assessment of the findings using Patient Health Questionnaire-9. RESULTS: A total of 292 psychiatric and voice assessments were performed with 163 participants (males: n=47, 28.8%) aged 15 to 82 years. Of the 163 participants, 87 (53.3%) were not depressed at the time of assessment, and 88 (53.9%) participants had clinically mild to moderate depressive phases. Of the 163 participants, 98 (32.5%) showed subsyndromal symptoms, and 19 (11.7%) participants were severely depressed. In the speech analysis, a clear differentiation between the individual depressive levels, as seen in the Patient Health Questionnaire-9, was also shown, especially the clear differentiation between nondepressed and depressed participants. The study showed a Pearson correlation of 0.41 between clinical assessment and noncontentual speech analysis (P<.001). CONCLUSIONS: The use of speech analysis shows a high level of accuracy, not only in terms of the general recognition of a clinically relevant depressive state in the participants. Instead, there is a high degree of agreement regarding the extent of depressive impairment with the assessment of experienced clinical practitioners. From our point of view, the application of the noncontentual analysis system in everyday clinical practice makes sense, especially with the idea of a quick and unproblematic assessment of the state of mind, which can even be carried out without personal contact. TRIAL REGISTRATION: ClinicalTrials.gov NCT03700008; https://clinicaltrials.gov/ct2/show/NCT03700008.

The Taspase1/Myosin1f-axis regulates filopodia dynamics.

The unique threonine protease Tasp1 impacts not only ordered development and cell proliferation but also pathologies. However, its substrates and the underlying molecular mechanisms remain poorly understood. We demonstrate that the unconventional Myo1f is a Tasp1 substrate and unravel the physiological relevance of this proteolysis. We classify Myo1f as a nucleo-cytoplasmic shuttle protein, allowing its unhindered processing by nuclear Tasp1 and an association with chromatin. Moreover, we show that Myo1f induces filopodia resulting in increased cellular adhesion and migration. Importantly, filopodia formation was antagonized by Tasp1-mediated proteolysis, supported by an inverse correlation between Myo1f concentration and Tasp1 expression level. The Tasp1/Myo1f-axis might be relevant in human hematopoiesis as reduced Tasp1 expression coincided with increased Myo1f concentrations and filopodia in macrophages compared to monocytes and vice versa. In sum, we discovered Tasp1-mediated proteolysis of Myo1f as a mechanism to fine-tune filopodia formation, inter alia relevant for cells of the immune system.

Innate immune receptor signaling induces transient melanoma dedifferentiation while preserving immunogenicity.

BACKGROUND: Immune-stimulatory agents, like agonists of the innate immune receptor RIG-I, are currently tested in clinical trials as an intratumoral treatment option for patients with unresectable melanoma, aiming to enhance anti-tumor T cell responses. Switching of melanoma toward a dedifferentiated cell state has recently been linked to T cell and therapy resistance. It remains to be determined whether RIG-I agonists affect melanoma differentiation, potentially leading to T cell resistance. METHODS: Patient metastases-derived melanoma cell lines were treated with the synthetic RIG-I agonist 3pRNA, and effects on tumor cell survival, phenotype and differentiation were determined. Transcriptomic data sets from cell lines and metastases were analyzed for associations between RIG-I (DDX58) and melanoma differentiation markers and used to define signaling pathways involved in RIG-I-driven dedifferentiation. The impact of 3pRNA-induced melanoma dedifferentiation on CD8 T cell activation was studied in autologous tumor T cell models. RESULTS: RIG-I activation by 3pRNA induced apoptosis in a subpopulation of melanoma cells, while the majority of tumor cells switched into a non-proliferative cell state. Those persisters displayed a dedifferentiated cell phenotype, marked by downregulation of the melanocytic lineage transcription factor MITF and its target genes, including melanoma differentiation antigens (MDA). Transition into the MITFlow/MDAlow cell state was JAK-dependent, with some cells acquiring nerve growth factor receptor expression. MITFlow/MDAlow persisters switched back to the proliferative differentiated cell state when RIG-I signaling declined. Consistent with our in vitro findings, an association between melanoma dedifferentiation and high RIG-I (DDX58) levels was detected in transcriptomic data from patient metastases. Notably, despite their dedifferentiated cell phenotype, 3pRNA-induced MITFlow/MDAlow persisters were still efficiently targeted by autologous CD8 tumor-infiltrating T lymphocytes (TILs). CONCLUSIONS: Our results demonstrate that RIG-I signaling in melanoma cells drives a transient phenotypic switch toward a non-proliferative dedifferentiated persister cell state. Despite their dedifferentiation, those persisters are highly immunogenic and sensitive toward autologous TILs, challenging the concept of melanoma dedifferentiation as a general indicator of T cell resistance. In sum, our findings support the application of RIG-I agonists as a therapeutic tool for the generation of long-term clinical benefit in non-resectable melanoma.

MTBP phosphorylation controls DNA replication origin firing.

Faithful genome duplication requires regulation of origin firing to determine loci, timing and efficiency of replisome generation. Established kinase targets for eukaryotic origin firing regulation are the Mcm2-7 helicase, Sld3/Treslin/TICRR and Sld2/RecQL4. We report that metazoan Sld7, MTBP (Mdm2 binding protein), is targeted by at least three kinase pathways. MTBP was phosphorylated at CDK consensus sites by cell cycle cyclin-dependent kinases (CDK) and Cdk8/19-cyclin C. Phospho-mimetic MTBP CDK site mutants, but not non-phosphorylatable mutants, promoted origin firing in human cells. MTBP was also phosphorylated at DNA damage checkpoint kinase consensus sites. Phospho-mimetic mutations at these sites inhibited MTBP's origin firing capability. Whilst expressing a non-phospho MTBP mutant was insufficient to relieve the suppression of origin firing upon DNA damage, the mutant induced a genome-wide increase of origin firing in unperturbed cells. Our work establishes MTBP as a regulation platform of metazoan origin firing.

Development of a Digital Content-Free Speech Analysis Tool for the Measurement of Mental Health and Follow-Up for Mental Disorders: Protocol for a Case-Control Study.

BACKGROUND: The prevalence of mental disorders worldwide is very high. The guideline-oriented care of patients depends on early diagnosis and regular and valid evaluation of their treatment to be able to quickly intervene should the patient's mental health deteriorate. To ensure effective treatment, the level of experience of the physician or therapist is of importance, both in the initial diagnosis and in the treatment of mental illnesses. Nevertheless, experienced physicians and psychotherapists are not available in enough numbers everywhere, especially in rural areas or in less developed countries. Human speech can reveal a speaker's mental state by altering its noncontent aspects (speech melody, intonations, speech rate, etc). This is noticeable in both the clinic and everyday life by having prior knowledge of the normal speech patterns of the affected person, and with enough time spent listening to the patient. However, this time and experience are often unavailable, leaving unused opportunities to capture linguistic, noncontent information. To improve the care of patients with mental disorders, we have developed a concept for assessing their most important mental parameters through a noncontent analysis of their active speech. Using speech analysis for the assessment and tracking of mental health patients opens up the possibility of remote, automatic, and ongoing evaluation when used with patients' smartphones, as part of the current trends toward the increasing use of digital and mobile health tools. OBJECTIVE: The primary objective of this study is to evaluate measurements of participants' mental state by comparing the analysis of noncontent speech parameters to the results of several psychological questionnaires (Symptom Checklist-90 [SCL-90], the Patient Health Questionnaire [PHQ], and the Big 5 Test). METHODS: In this paper, we described a case-controlled study (with a case group and one control group). The participants will be recruited in an outpatient neuropsychiatric treatment center. Inclusion criteria are a neurological or psychiatric diagnosis made by a specialist, no terminal or life-threatening illnesses, and fluent use of the German language. Exclusion criteria include psychosis, dementia, speech or language disorders in neurological diseases, addiction history, a suicide attempt recently or in the last 12 months, or insufficient language skills. The measuring instrument will be the VoiceSense digital voice analysis tool, which enables the analysis of 200 specific speech parameters, and the assessment of findings using psychometric instruments and questionnaires (SCL-90, PHQ, Big 5 Test). RESULTS: The study is ongoing as of September 2019, but we have enrolled 254 participants. There have been 161 measurements completed at timepoint 1, and a total of 62 participants have completed every psychological and speech analysis measurement. CONCLUSIONS: It appears that the tone and modulation of speech are as important, if not more so, than the content, and should not be underestimated. This is particularly evident in the interpretation of the psychological findings thus far acquired. Therefore, the application of a software analysis tool could increase the accuracy of finding assessments and improve patient care. TRIAL REGISTRATION: ClinicalTrials.gov NCT03700008; https://clinicaltrials.gov/ct2/show/NCT03700008. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/13852.

Efficacy of Manual Therapy on Pain, Impact of Disease, and Quality of Life in the Treatment of Fibromyalgia: A Systematic Review.

BACKGROUND: Myofascial mobilization has been used as an intervention for patients with fibromyalgia (FM) for acting on ascending nociceptive pathways possibly involved in the central sensitization process, modulating the pain experience. However, there is still a gap in its efficacy compared with another hands-on approach because manual therapy has nonspecific effects, such as placebo. OBJECTIVES: This systematic review aims to review the scientific literature for an overview of the efficacy of manual therapy in pain, disease impact, and quality of life in patients with FM compared with control or other treatments through randomized clinical trials. STUDY DESIGN: This study involved systematic review of published randomized controlled trials (RCTs). SETTING: This study examined all RCTs evaluating the effect of manual therapy on pain, impact of disease, and quality of life for patients with FM. METHODS: Systematic review. The research was performed in 9 databases: MEDLINE/PubMed, CINAHL, Web of Science, Scopus, ScienceDirect, Lilacs, SciELO, PEDro, and Cochrane. Searches were carried out from the end of the project until September 2019, with no language and year restrictions. Randomized controlled clinical trials that used the following outcome measures were included: Visual Analog Scale, Fibromyalgia Impact Questionnaire, and SF-36 Quality of Life Questionnaire. The risk of bias and quality of studies was assessed using the PEDro scale; the Cochrane risk-of-bias tool; and Grading of Recommendations Assessment, Development, and Evaluation System. RESULTS: Seven studies were included (368 patients). The quantitative analysis was performed on 4 studies because of the lack of data in the others. Myofascial release was the most used modality. The level of evidence ranged from very low to moderate, mainly because of the inconsistency and inaccuracy of results. LIMITATIONS: The present systematic review presented limitations because of the heterogeneity of the included studies and only a short-term analysis of the intervention results. It was observed that other information, such as pressure, repetition, and/or sustaining manual therapy techniques, could be better described in future protocols, aiming at a better comparison between the techniques and their subsequent reproducibility. CONCLUSIONS: Current evidence of manual therapy in patients with FM, based on a very low to moderate quality of evidence, was inconclusive and insufficient to support and recommend the use of manual therapy in this population. To date, only general osteopathic treatment has achieved clinically relevant pain improvement when compared with control.

A pH-sensitive fluorescent protein sensor to follow the pathway of calcium phosphate nanoparticles into cells.

Calcium phosphate nanoparticles (100 nm) were fluorescently labelled with poly(ethyleneimine) (PEIATTO490LS; red fluorescence). They were loaded with a Tandem fusion protein consisting of mRFP1-eGFP (red and green fluorescence in the same molecule)that acts as smart biological pH sensor to trace nanoparticles inside cells. Its fluorescence is also coupled to the structural integrity of the protein, i.e. it is also a label for a successful delivery of a functional protein into the cell. At pH 7.4, the fluorescence of both proteins (red and green) is detectable. At a pH of 4.5-5 inside the lysosomes, the green fluorescence is quenched due to the protonation of the eGFP chromophore, but the pH-independent red fluorescence of mRFP1 remains. The nanoparticles were taken up by cells (cell lines: HeLa, Caco-2 and A549) via endocytic pathways and then directed to lysosomes. Time-resolved confocal laser scanning microscopy confirmed mRFP1 and nanoparticles co-localizing with lysosomes. The fluorescence of eGFP was only detectable outside lysosomes, i.e. most likely inside early endosomes or at the cell membrane during the uptake, indicating the neutral pH at these locations. The Tandem fusion protein provides a versatile platform to follow the intracellular pathway of bioactive nanocarriers, e.g. therapeutic proteins. The transfection with a Tandem-encoding plasmid by calcium phosphate nanoparticles led to an even intracellular protein distribution in cytosol and nucleoplasm, i.e. very different from direct protein uptake. Neither dissolved protein nor dissolved plasmid DNA were taken up by the cells, underscoring the necessity for a suitable carrier like a nanoparticle. STATEMENT OF SIGNIFICANCE: A pH-sensitive protein ("tandem") was used to follow the pathway of calcium phosphate nanoparticles. This protein consists of a pH-sensitive fluorophore (eGFP; green) and a pH-independent fluorophore (mRFP1; red). This permits to follow the pathway of a nanoparticle inside a cell. At a low pH inside an endolysosome, the green fluorescence vanishes but the red fluorescence persists. This is also a very useful model for the delivery of therapeutic proteins into cells. The delivery by nanoparticles was compared with the protein expression after cell transfection with plasmid DNA encoding for the tandem protein. High-resolution image analysis gave quantitative data on the intracellular protein distribution.

VCP maintains lysosomal homeostasis and TFEB activity in differentiated skeletal muscle.

Differentiated tissue is particularly vulnerable to alterations in protein and organelle homeostasis. The essential protein VCP, mutated in hereditary inclusion body myopathy, amyotrophic lateral sclerosis and frontotemporal dementia, is critical for efficient clearance of misfolded proteins and damaged organelles in dividing cells, but its role in terminally differentiated tissue affected by disease mutations is less clear. To understand the relevance of VCP in differentiated tissue, we inactivated it in skeletal muscle of adult mice. Surprisingly, knockout muscle demonstrated a necrotic myopathy with increased macroautophagic/autophagic proteins and damaged lysosomes. This was not solely due to a defect in autophagic degradation because age-matched mice with muscle inactivation of the autophagy essential protein, ATG5, did not demonstrate a myopathy. Notably, myofiber necrosis was preceded by upregulation of LGALS3/Galectin-3, a marker of damaged lysosomes, and TFEB activation, suggesting early defects in the lysosomal system. Consistent with that, myofiber necrosis was recapitulated by chemical induction of lysosomal membrane permeabilization (LMP) in skeletal muscle. Moreover, TFEB was activated after LMP in cells, but activation and nuclear localization of TFEB persisted upon VCP inactivation or disease mutant expression. Our data identifies VCP as central mediator of both lysosomal clearance and biogenesis in skeletal muscle. Abbreviations: AAA: ATPases Associated with diverse cellular Activities; TUBA1A/α-tubulin: tubulin alpha 1a; ATG5: autophagy related 5; ATG7: autophagy related 7; ACTA1: actin alpha 1, skeletal muscle; CLEAR: coordinated lysosomal expression and regulation; CTSB/D: cathepsin B/D; Ctrl: control; DAPI: diamidino-2-phenylindole; EBSS: Earle's balanced salt solution; ELDR: endolysosomal damage response; ESCRT: endosomal sorting complexes required for transport; Gastroc/G: gastrocnemius; H&E: hematoxylin and eosin; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; IBMPFD/ALS: inclusion body myopathy associated with Paget disease of the bone, frontotemporal dementia and amyotrophic lateral sclerosis; i.p.: intraperitoneal; LAMP1/2: lysosomal-associated membrane protein 1/2; LLOMe: Leu-Leu methyl ester hydrobromide; LGALS3/Gal3: galectin 3; LMP: lysosomal membrane permeabilization; MTOR: mechanistic target of rapamycin kinase; MYL1: myosin light chain 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MSP: multisystem proteinopathy; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; Quad/Q: quadriceps; RHEB: Ras homolog, mTORC1 binding; SQSTM1: sequestosome 1; TFEB: transcription factor EB; TA: tibialis anterior; siRNA: small interfering RNA; SQSTM1/p62, sequestosome 1; TARDBP/TDP-43: TAR DNA binding protein; TBS: Tris-buffered saline; TXFN, tamoxifen; UBXN6/UBXD1: UBX domain protein 6; VCP: valosin containing protein; WT: wild-type.

Calcium phosphate nanoparticle-mediated transfection in 2D and 3D mono- and co-culture cell models.

The transfer of nucleic acids into living cells, i.e. transfection, is a major technique in current molecular biology and medicine. As nucleic acids alone are not able to penetrate the cell membrane, an efficient carrier is needed. Calcium phosphate nanoparticles can serve as carrier due to their biocompatibility, biodegradability and high affinity to nucleic acids like DNA or RNA. Their application was extended here from two-dimensional (2D) to three-dimensional (3D) cell culture models, including co-cultures. Compared to 2D monolayer cell cultures, a 3D culture system represents a more realistic spatial, biochemical and cellular environment. The uptake of fluorescent calcium phosphate nanoparticles (diameter 40-70 nm; cationic) was studied in 2D and 3D cell culture models by confocal laser scanning microscopy. The transfection of eGFP by calcium phosphate nanoparticles was compared in 2D and 3D cell culture, including co-cultures of green fluorescing HeLa-eGFP cells and MG-63 cells in 2D and in 3D models with the red fluorescent protein mCherry. This permitted a cell-specific assessment of the local transfection efficiency. In general, the penetration of nanoparticles into the spheroids was significantly higher than that of a model oligonucleotide carried by Lipofectamine. The transfection efficiency was comparable in 3D cell cultures with 2D cell cultures, but it occurred preferentially at the surface of the spheroids, following the uptake pathway of the nanoparticles. STATEMENT OF SIGNIFICANCE: Three-dimensional cell culture models can serve as a bridge between the in-vitro cell cultures and the in-vivo situation, especially when mass transfer effects have to be considered. This is the case for nanoparticles where the incubation effect in a two-dimensional cell culture strongly differs from a three-dimensional cell culture or a living tissue. We have compared the uptake of nanoparticles and a subsequent transfection of fluorescent proteins in two-dimensional and three-dimensional cell culture models. An elegant model to investigate the transfection in co-cultures was developed using HeLa-eGFP cells (green fluorescent) together with MG-63 cells (non-fluorescent) that were transfected with the red-fluorescing protein mCherry. Thereby, the transfection of both cell types in the co-culture was easily distinguished.

Delivery of the autofluorescent protein R-phycoerythrin by calcium phosphate nanoparticles into four different eukaryotic cell lines (HeLa, HEK293T, MG-63, MC3T3): Highly efficient, but leading to endolysosomal proteolysis in HeLa and MC3T3 cells.

Nanoparticles can be used as carriers to transport biomolecules like proteins and synthetic molecules across the cell membrane because many molecules are not able to cross the cell membrane on their own. The uptake of nanoparticles together with their cargo typically occurs via endocytosis, raising concerns about the possible degradation of the cargo in the endolysosomal system. As the tracking of a dye-labelled protein during cellular uptake and processing is not indicative of the presence of the protein itself but only for the fluorescent label, a label-free tracking was performed with the red-fluorescing model protein R-phycoerythrin (R-PE). Four different eukaryotic cell lines were investigated: HeLa, HEK293T, MG-63, and MC3T3. Alone, the protein was not taken up by any cell line; only with the help of calcium phosphate nanoparticles, an efficient uptake occurred. After the uptake into HeLa cells, the protein was found in early endosomes (shown by the marker EEA1) and lysosomes (shown by the marker Lamp1). There, it was still intact and functional (i.e. properly folded) as its red fluorescence was detected. However, a few hours after the uptake, proteolysis started as indicated by the decreasing red fluorescence intensity in the case of HeLa and MC3T3 cells. 12 h after the uptake, the protein was almost completely degraded in HeLa cells and MC3T3 cells. In HEK293T cells and MG-63 cells, no degradation of the protein was observed. In the presence of Bafilomycin A1, an inhibitor of acidification and protein degradation in lysosomes, the fluorescence of R-PE remained intact over the whole observation period in the four cell lines. These results indicate that despite an efficient nanoparticle-mediated uptake of proteins by cells, a rapid endolysosomal degradation may prevent the desired (e.g. therapeutic) effect of a protein inside a cell.

Avidin-conjugated calcium phosphate nanoparticles as a modular targeting system for the attachment of biotinylated molecules in vitro and in vivo.

Avidin was covalently conjugated to the surface of calcium phosphate nanoparticles, coated with a thin silica shell and terminated by sulfhydryl groups (diameter of the solid core about 50nm), with a bifunctional crosslinker connecting the amino groups of avidin to the sulfhydryl group on the nanoparticle surface. This led to a versatile nanoparticle system where all kinds of biotinylated (bio-)molecules can be easily attached to the surface by the non-covalent avidin-biotin-complex formation. It also permits the attachment of different biomolecules on the same nanoparticle (heteroavidity), creating a modular system for specific applications in medicine and biology. The variability of the binding to the nanoparticle surface of the was demonstrated with various biotinylated molecules, i.e. fluorescent dyes and antibodies. The accessibility of the conjugated avidin was demonstrated by a fluorescence-quenching assay. About 2.6 binding sites for biotin were accessible on each avidin tetramer. Together with a number of about 240 avidin tetramer units per nanoparticle, this offers about 600 binding sites for biotin on each nanoparticle. The uptake of fluorescently labelled avidin-conjugated calcium phosphate nanoparticles by HeLa cells showed the co-localization of fluorescent avidin and fluorescent biotin, indicating the stability of the complex under cell culture conditions. CD11c-antibody functionalized nanoparticles specifically targeted antigen-presenting immune cells (dendritic cells; DCs) in vitro and in vivo (mice) with high efficiency. STATEMENT OF SIGNIFICANCE: Calcium phosphate nanoparticles have turned out to be very useful transporters for biomolecules into cells, both in vitro and in vivo. However, their covalent surface functionalization with antibodies, fluorescent dyes, or proteins requires a separate chemical synthesis for each kind of surface molecule. We have therefore developed avidin-terminated calcium phosphate nanoparticles to which all kinds of biotinylated molecules can be easily attached, also as a mixture of two or more molecules. This non-covalent bond is stable both in cell culture and after injection into mice in vivo. Thus, we have created a highly versatile system for many applications, from the delivery of biomolecules over the targeting of cells and tissue to in vivo imaging.

Development of a Questionnaire to Measure the Attitudes of Laypeople, Physicians, and Psychotherapists Toward Telemedicine in Mental Health.

BACKGROUND: In the field of psychiatry and psychotherapy, there are now a growing number of Web-based interventions, mobile phone apps, or treatments that are available via remote transmission screen worldwide. Many of these interventions have been shown to be effective in studies but still find little use in everyday therapeutic work. However, it is important that attitude and expectation toward this treatment are generally examined, because these factors have an important effect on the efficacy of the treatment. To measure the general attitude of the users and prescribers toward telemedicine, which may include, for instance, Web-based interventions or interventions through mobile phone apps, there are a small number of extensive tests. The results of studies based on small groups of patients have been published too, but there is no useful short screening tool to give an insight into the general population's attitude. We have developed a screening instrument that examines such attitude through a few graded questions. OBJECTIVE: This study aimed to explore the Attitude toward Telemedicine in Psychiatry and Psychotherapy (ATiPP) and to evaluate the results of general population and some subgroups. METHODS: In a three-step process, the questionnaire, which is available in three versions (laypeople, physicians, and psychologists), was developed. Afterwards, it was evaluated by four groups: population-representative laypeople, outpatients in different faculties, physicians, and psychotherapists. RESULTS: The results were evaluated from a total of 1554 questionnaires. The sample population included 1000 laypeople, 455 outpatients, 62 physicians, and 37 psychotherapists. The reliability of all three versions of the questionnaire seemed good, as indicated by the Cronbach alpha values of .849 (the laypeople group), .80 (the outpatients' group), .827 (the physicians' group), and .855 (the psychotherapists' group). CONCLUSIONS: The ATiPP was found to be useful and reliable for measuring the attitudes toward the Web-based interventions in psychiatry and psychotherapy and should be used in different studies in this field in the future to evaluate and reflect the attitude of the participants.