Rab11 regulates autophagy at dendritic spines in an mTOR- and NMDA-dependent manner.

Synaptic plasticity is a process that shapes neuronal connections during neurodevelopment and learning and memory. Autophagy is a mechanism that allows the cell to degrade its unnecessary or dysfunctional components. Autophagosomes appear at dendritic spines in response to plasticity-inducing stimuli. Autophagy defects contribute to altered dendritic spine development, autistic-like behavior in mice, and neurological disease. While several studies have explored the involvement of autophagy in synaptic plasticity, the initial steps of the emergence of autophagosomes at the postsynapse remain unknown. Here, we demonstrate a postsynaptic association of autophagy-related protein 9A (Atg9A), known to be involved in the early stages of autophagosome formation, with Rab11, a small GTPase that regulates endosomal trafficking. Rab11 activity was necessary to maintain Atg9A-positive structures at dendritic spines. Inhibition of mTOR increased Rab11 and Atg9A interaction and increased the emergence of LC3 positive vesicles, an autophagosome membrane-associated protein marker, in dendritic spines when coupled to NMDA receptor stimulation. Dendritic spines with newly formed LC3+ vesicles were more resistant to NMDA-induced morphologic change. Rab11 DN overexpression suppressed appearance of LC3+ vesicles. Collectively, these results suggest that initiation of autophagy in dendritic spines depends on neuronal activity and Rab11a-dependent Atg9A interaction that is regulated by mTOR activity.

Muscleblind-like proteins use modular domains to localize RNAs by riding kinesins and docking to membranes.

RNA binding proteins (RBPs) act as critical facilitators of spatially regulated gene expression. Muscleblind-like (MBNL) proteins, implicated in myotonic dystrophy and cancer, localize RNAs to myoblast membranes and neurites through unknown mechanisms. We find that MBNL forms motile and anchored granules in neurons and myoblasts, and selectively associates with kinesins Kif1bα and Kif1c through its zinc finger (ZnF) domains. Other RBPs with similar ZnFs associate with these kinesins, implicating a motor-RBP specificity code. MBNL and kinesin perturbation leads to widespread mRNA mis-localization, including depletion of Nucleolin transcripts from neurites. Live cell imaging and fractionation reveal that the unstructured carboxy-terminal tail of MBNL1 allows for anchoring at membranes. An approach, termed RBP Module Recruitment and Imaging (RBP-MRI), reconstitutes kinesin- and membrane-recruitment functions using MBNL-MS2 coat protein fusions. Our findings decouple kinesin association, RNA binding, and membrane anchoring functions of MBNL while establishing general strategies for studying multi-functional, modular domains of RBPs.

FMRP phosphorylation and interactions with Cdh1 regulate association with dendritic RNA granules and MEF2-triggered synapse elimination.

Fragile X Messenger Ribonucleoprotein (FMRP) is necessary for experience-dependent, developmental synapse elimination and the loss of this process may underlie the excess dendritic spines and hyperconnectivity of cortical neurons in Fragile X Syndrome, a common inherited form of intellectual disability and autism. Little is known of the signaling pathways that regulate synapse elimination and if or how FMRP is regulated during this process. We have characterized a model of synapse elimination in CA1 neurons of organotypic hippocampal slice cultures that is induced by expression of the active transcription factor Myocyte Enhancer Factor 2 (MEF2) and relies on postsynaptic FMRP. MEF2-induced synapse elimination is deficient in Fmr1 KO CA1 neurons, and is rescued by acute (24 h), postsynaptic and cell autonomous reexpression of FMRP in CA1 neurons. FMRP is an RNA binding protein that suppresses mRNA translation. Derepression is induced by posttranslational mechanisms downstream of metabotropic glutamate receptor signaling. Dephosphorylation of FMRP at S499 triggers ubiquitination and degradation of FMRP which then relieves translation suppression and promotes synthesis of proteins encoded by target mRNAs. Whether this mechanism functions in synapse elimination is not known. Here we demonstrate that phosphorylation and dephosphorylation of FMRP at S499 are both necessary for synapse elimination as well as interaction of FMRP with its E3 ligase for FMRP, APC/Cdh1. Using a bimolecular ubiquitin-mediated fluorescence complementation (UbFC) assay, we demonstrate that MEF2 promotes ubiquitination of FMRP in CA1 neurons that relies on activity and interaction with APC/Cdh1. Our results suggest a model where MEF2 regulates posttranslational modifications of FMRP via APC/Cdh1 to regulate translation of proteins necessary for synapse elimination.

Development of single-molecule ubiquitination mediated fluorescence complementation to visualize protein ubiquitination dynamics in dendrites.

The ubiquitination/proteasome system is important for the spatiotemporal control of protein synthesis and degradation at synapses, while dysregulation may underlie autism spectrum disorders (ASDs). However, methods allowing direct visualization of the subcellular localization and temporal dynamics of protein ubiquitination are lacking. Here we report the development of Single-Molecule Ubiquitin Mediated Fluorescence Complementation (SM-UbFC) as a method to visualize and quantify the dynamics of protein ubiquitination in dendrites of live neurons in culture. Using SM-UbFC, we demonstrate that the rate of PSD-95 ubiquitination is elevated in dendrites of FMR1 KO neurons compared with wild-type controls. We further demonstrate the rapid ubiquitination of the fragile X messenger ribonucleoprotein, FMRP, and the AMPA receptor subunit, GluA1, which are known to be key events in the regulation of synaptic protein synthesis and plasticity. SM-UbFC will be useful for future studies on the regulation of synaptic protein homeostasis.

Quality of Sexual Life after Native Tissue versus Polypropylene Mesh Augmented Pelvic Floor Reconstructive Surgery.

There are still controversies around reconstructive surgeries used in POP treatment. The aim of this study was to compare the QoSL after VNTR vs. TVM surgery due to POP via the use of PISQ-12 and FSFI questionnaires. The study included a group of 121 sexually active patients qualified for reconstructive surgery due to symptomatic POP, and 50 control. The average results of PISQ-12 before and after surgery were compared using the t-test. The significance of the mean differences in demographic groups was measured using the t-test for independent samples and one-way ANOVA. The results in the demographic groups were compared using the Mann-Whitney U test and the Kruskal-Wallis test. Fifty-eight women had VNTR, while 63 had TVM. Results of PISQ-12 revealed significant improvement in the sexual life after reconstructive surgery (27.24 vs. 32.43; p < 0.001, t = 8.48) both after VNTR and TVM. There were no significant differences in the assessment of the QoSL according to PISQ-12 and FSFI results between both analyzed groups of patients (PISQ-12: VNTR vs. TVM; t-test p = 0.19 and FSFI: VNTR vs. TVM; Mann-Whitney U test p = 0.54). VNTR is the treatment of choice in the case of uncomplicated primary POP.

The Optical Coherence Tomography and Raman Spectroscopy for Sensing of the Bone Demineralization Process.

The presented research was intended to seek new optical methods to investigate the demineralization process of bones. Optical examination of the bone condition could facilitate clinical trials and improve the safety of patients. The authors used a set of complementary methods: polarization-sensitive optical coherence tomography (PS-OCT) and Raman spectroscopy. Chicken bone samples were used in this research. To stimulate in laboratory conditions the process of demineralization and gradual removal of the hydroxyapatite, the test samples of bones were placed into 10% acetic acid. Measurements were carried out in two series. The first one took two weeks with data acquired every day. In the second series, the measurements were made during one day at an hourly interval (after 1, 2, 3, 5, 7, 10, and 24 h). The relation between the content of hydroxyapatite and images recorded using OCT was analyzed and discussed. Moreover, the polarization properties of the bones, including retardation angles of the bones, were evaluated. Raman measurement confirmed the disappearance of the hydroxyapatite and the speed of this process. This work presents the results of the preliminary study on the possibility of measuring changes in bone mineralization by means of the proposed methods and confirms their potential for practical use in the future.

Reliability of the Polish Pelvic Organ Prolapse/Urinary Incontinence Sexual Questionnaire (PISQ-12) and Assessment of Sexual Function before and after Pelvic Organ Prolapse Reconstructive Surgery-A Prospective Study.

It is estimated that 31-44% of all patients with symptomatic POP and/or UI suffer from sexual dysfunction. We aimed to validate the PISQ-12 in pre-and postmenopausal women and to assess the sexual function before and after POP reconstructive surgery. One hundred and forty sexually active patients were hospitalized due to symptomatic POP and 50 healthy controls were enrolled into the study. The patients were asked to complete PISQ-12, the FSFI and Beck's depression scale questionnaires twice. The Cronbach's alpha (α) was used to estimate the internal consistency. The scores were compared using the Intraclass Correlation Coefficient (ICC). Improvement in the QoSL (quality of sexual life) was observed in each age group of women. Pre-menopausal patients' QoSL was much better, both before and after surgery (29.62 and 34.64 points, respectively). The correlation between questionnaires before surgery was 0.63, and after was -0.76. The α value for the PISQ-12 was 0.83 before the procedure and 0.80 afterwards. In all the groups, the test-retest reliability was good-ICC = 0.72. Vaginal reconstructive surgeries improve the QoSL. The only demographic factor influencing the QoSL was the menopausal status. The Polish version of the PISQ-12 is a reliable and responsive instrument for assessing the sexual function in patients with diagnosed POP and/or UI.

Spectroscopic Optical Coherence Tomography for Thin Layer and Foil Measurements.

The main goal of this research was to assess if it is possible to evaluate the thickness of thin layers (both thin films on the surface and thin layers below the surface of the tested object) and foils using optical coherence tomography (OCT) for thickness assessment under the resolution of the standard commercially available OCT measurement system. In the proposed solution, light backscattered from the evaluated thin layer has been expressed as a multiple beam interference. Therefore, the OCT system was modeled as a two-beam interferometer (e.g., Michelson), in which one beam propagates from the reference arm and the other comes from a Fabry-Pérot interferometer. As a consequence, the mathematical model consists of the main Michelson interferometer, in which the measuring arm represents the Fabry-Pérot interferometer. The parameters of the layer (or foil) are evaluated by analyzing the minimum value of the interference contrast. The model developed predicts the behavior of the thin layers made from different materials (with different refractive indexes) with different thickness and located at different depths. To verify the correctness of the proposed model, an experiment with a wedge cell has been carried out. The wedge cell was shifted across the scanning beam using a linear translation stage with a micrometer screw under the scanning head. The relationship between the thickness of the gap of the wedge cell and the OCT output signal is presented. For the additional verification of the proposed model, the results of the measurements of the thickness of the thin foil were compared with the theoretical results of the simulations. The film thickness was evaluated based on the calculated positions of the minimum value of interference contrast. A combination of the standard potentialities of OCT with the proposed approach to analyzing the signal produces new metrological possibilities. The method developed allows us to evaluate thickness under the resolution of the system and the location of the layer as well. This produces the possibility of measuring a layer which is covered by another layer. Moreover, it is possible to create a thickness map with high sensitivity to thickness changes. These experiments and simulations are the culmination of preliminary research for evaluating the potential of the proposed measurement method.

Urine Proteomic Study in OAB Patients-Preliminary Report.

Overactive bladder (OAB) is defined by International Urogynecological Association (IUGA)/ International Continence Society (ICS) as urinary urgency, usually accompanied by frequency and nocturia, with or without urgency urinary incontinence, in the absence of urinary tract infection (UTI) or other obvious pathology. The pathophysiology of OAB is not well understood, however a number of different proteins and cytokines including vascular cell adhesion molecule-1 (VCAM-1) were found to be important in regulating structural integrity of the bladder wall. Proteome analysis may thus provide significant information with regard to OAB and may help in discovering novel diagnostic disease biomarkers. Sixteen Caucasian women aged 32-78 were included in the study. Patients were placed within 2 groups: OAB group (n = 8) and control group (n = 8). Urine samples were collected, immediately preserved in a protease inhibitor mixture, and frozen at -80 ℃. All samples were then further processed according to the isobaric tags for relative and absolute quantification (iTRAQ) manual. Proteins were labeled and analyzed in the mass spectrometer conjugated with liquid chromatograph (data are available via ProteomeXchange with the identifier PXD017799). There were no statistically significant differences in demographic data between control and OAB groups. VCAM-1 was the only protein that reached statistical significance as a differentiating protein in both of our experiments assessing the proteomic constitution in OAB patients. Studies involving a larger group of patients may provide further information on urinary bladder proteomics.

Sexual function specific questionnaires as a useful tool in management of urogynecological patients - Review.

Urinary incontinence, fecal incontinence and pelvic organ prolapse are one of the most prevalent gynaecological conditions and constitute a huge global problem affecting approximately 20% of women, increasing with age. Pelvic floor disorders can have negative influence on women's quality of life, decreasing social, psychological, occupational, physical and sexual well-being. Pelvic organ prolapse results in anatomical changes to the urogenital tract and it is perceived to be one of the main factor influencing sexual function. Because treatment of pelvic organ prolapse and complications related to it may cause discomfort, the most important outcome of the therapy, including anatomical restoration, is relief in symptoms and improvement in quality of life. Psychometrical instruments for measuring health-related quality of life are essential during evaluation of women with pelvic floor disorders. Assessing severity of pelvic organ prolapse, its' impact on quality of life, therapy planning and the inclusion of sexuality questionnaires as an outcome measure in urogynecological patients allows to analyze impact of surgical treatment on women's sexual life. For this purpose, condition - specific instruments were developed and published. The aim of this study is to present particular questionnaires and their proper practical application in clinical practice, especially before surgical treatment and follow-up. Furthermore, those questionnaires are essential in order to describe patients' expectations during tailored clinical management.

ZBP1 phosphorylation at serine 181 regulates its dendritic transport and the development of dendritic trees of hippocampal neurons.

Local protein synthesis occurs in axons and dendrites of neurons, enabling fast and spatially restricted responses to a dynamically changing extracellular environment. Prior to local translation, mRNA that is to be translated is packed into ribonucleoprotein particles (RNPs) where RNA binding proteins ensure mRNA silencing and provide a link to molecular motors. ZBP1 is a component of RNP transport particles and is known for its role in the local translation of ß-actin mRNA. Its binding to mRNA is regulated by tyrosine 396 phosphorylation, and this particular modification was shown to be vital for axonal growth and dendritic branching. Recently, additional phosphorylation of ZBP1 at serine 181 (Ser181) was described in non-neuronal cells. In the present study, we found that ZBP1 is also phosphorylated at Ser181 in neurons in a mammalian/mechanistic target of rapamycin complex 2-, Src kinase-, and mRNA binding-dependent manner. Furthermore, Ser181 ZBP1 phosphorylation was essential for the proper dendritic branching of hippocampal neurons that were cultured in vitro and for the proper ZBP1 dendritic distribution and motility.

Evaluation of exhaled breath temperature (EBT) as a marker and predictor of asthma exacerbation in children and adolescents.

INTRODUCTION: Noninvasive and easy-to-use tools to monitor airway inflammation in asthma are needed to maintain disease control, particularly in pediatric population. The aim of the study was to evaluate exhaled breath temperature (EBT) in pediatric respiratory clinic setting. METHODS: We evaluated 37 children and adolescents with asthma (5-17 years; median: 11 years). The patients were followed up in stable condition and during exacerbations (paired observations in n = 19 subjects). We evaluated medication use, EBT, fractional exhaled nitric oxide (FeNO), spirometry and atopic status of patients. RESULTS: EBT was significantly higher in children with asthma exacerbation {entire group: median [interquartile range (IQR)]: 32.3 [1.1]°C vs. 33.8 [1.7]°C; p < 0.001 and mean ± SD: 33.1 ± 1.0°C vs. 33.6 ± 1.1°C; p = 0.038 for paired observations}. Significant correlation was observed between EBT and FeNO in the entire group (r = 0.22; p = 0.03). No difference was observed in EBT median values in atopic and non-atopic subjects in the entire group (median [IQR]: 32.6 [1.6] vs. 32.7 [2.0]; p = 0.88) and in subgroups. There was no difference in EBT values in patients receiving systemic or inhaled glucocorticosteroids (p = 0.45 and 0.83). There was no significant correlation between EBT and body or room temperature. The only significant predictor of exacerbation in logistic regression model was EBT {aOR = 2.4; 95% [confidence interval (CI)]: 1.4-4.1}. ROC analysis demonstrated applicability of EBT as a marker of asthma exacerbation in children (AUC = 0.748; p < 0.001; cut-off = 33.3°C; sensitivity: 64.3%; specificity: 82.1%). CONCLUSIONS: We suggest that EBT may serve as marker and predictor of asthma exacerbation in children. EBT follow-up may be useful in asthma monitoring in children and adolescents.

Molecular neurobiology of mTOR.

Mammalian/mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that controls several important aspects of mammalian cell function. mTOR activity is modulated by various intra- and extracellular factors; in turn, mTOR changes rates of translation, transcription, protein degradation, cell signaling, metabolism, and cytoskeleton dynamics. mTOR has been repeatedly shown to participate in neuronal development and the proper functioning of mature neurons. Changes in mTOR activity are often observed in nervous system diseases, including genetic diseases (e.g., tuberous sclerosis complex, Pten-related syndromes, neurofibromatosis, and Fragile X syndrome), epilepsy, brain tumors, and neurodegenerative disorders (Alzheimer's disease, Parkinson's disease, and Huntington's disease). Neuroscientists only recently began deciphering the molecular processes that are downstream of mTOR that participate in proper function of the nervous system. As a result, we are gaining knowledge about the ways in which aberrant changes in mTOR activity lead to various nervous system diseases. In this review, we provide a comprehensive view of mTOR in the nervous system, with a special focus on the neuronal functions of mTOR (e.g., control of translation, transcription, and autophagy) that likely underlie the contribution of mTOR to nervous system diseases.

Limited Clinical Significance of Dimeric Form of Pyruvate Kinase as a Diagnostic and Prognostic Biomarker in Non-small Cell Lung Cancer.

Metabolism of tumor tissue differs from the normal one by the intensity of protein synthesis and glycolysis. The dimeric pyruvate kinase (PKM2) is a specific enzyme for tumor glycolysis. The aim of this study was to determine the relationship between the activity of PKM2 and the type and stage of non-small cell lung cancer (NSCLC). A second objective was to compare the expression of PKM2 with disease progression and prognosis. We studied 65 patients divided into two groups: 45 patients with lung cancer and 20 non-cancer healthy subjects taken as control. The serum activity of PKM2 was assessed spectrophotometrically. We found that PKM2 activity was greater, on average, by 136 % for adenocarcinoma and for 126 % for squamous cell carcinoma compared with that present in control subjects. The higher PKM2 activity was associated only with Stage III of cancer (p < 0.001). Sensitivity of PKM2 as a cancer marker was 79 % for adenocarcinoma and 81 % for squamous cell carcinoma and specificity was 50 % for both cancer types. We conclude that PKM2 activity is higher in patients with NSCLC than in healthy subjects. The level of PKM2 activity is associated with advanced stage of cancer. Nonetheless, low specificity of PKM2 assessment makes it of limited utility in NSCLC diagnosis or evaluation of cancer progression.

Tuberous sclerosis complex: From molecular biology to novel therapeutic approaches.

Tuberous sclerosis complex (TSC) is a rare multi-system disorder, primary manifestations of which are benign tumors and lesions in various organs of the body, including the brain. TSC patients often suffer from epilepsy, mental retardation, and autism spectrum disorder (ASD). Therefore, TSC serves as a model of epilepsy, ASD, and tumorigenesis. TSC is caused by the lack of functional Tsc1-Tsc2 complex, which serves as a major cellular inhibitor of mammalian Target of Rapamycin Complex 1 (mTORC1). mTORC1 is a kinase controlling most of anabolic processes in eukaryotic cells. Consequently, mTORC1 inhibitors, such as rapamycin, serve as experimental or already approved drugs for several TSC symptoms. However, rapalogs, although quite effective, need to be administered chronically and likely for a lifetime, since therapy discontinuation results in tumor regrowth and epilepsy recurrence. Recent studies revealed that metabolism and excitability (in the case of neurons) of cells lacking Tsc1-Tsc2 complex are changed, and these features may potentially be used to treat some of TSC symptoms. In this review, we first provide basic facts about TSC and its molecular background, to next discuss the newest findings in TSC cell biology that can be used to improve existing therapies of TSC and other diseases linked to mTORC1 hyperactivation. © 2016 IUBMB Life, 68(12):955-962, 2016.

[The assessment of nutritional knowledge of persons with eating disorders]. / Ocena poziomu wiedzy zywieniowej u osób z zaburzeniami odzywiania.

The main purpose of this work was to examine the level of nutritional knowledge of persons with eating disorders. The study was performed in the group of 60 persons (30 persons diagnosed with anorexia nervosa and 30 persons with diagnosis bulimia nervosa) and 60 controls. We found that ill persons possess the higher level of nutritional knowledge than person from the control group, yet the average of correct answers amounted to 51%. Our results point to the necessity of nutritional education in persons with eating disorders.

Alpha-1-antitrypsin (AAT) anomalies are associated with lung disease due to rapidly growing mycobacteria and AAT inhibits Mycobacterium abscessus infection of macrophages.

Rapidly growing mycobacteria (RGM) are ubiquitous in the environment but cause lung disease in only a fraction of exposed individuals. This variable susceptibility to disease implies vulnerability to RGM infection due to weakness in host defense. Since most persons who contract RGM lung disease have no known host defense defect, it is likely that uncharacterized host deficiencies exist that predispose to RGM infection. Alpha-1-antitrypsin (AAT) is a host factor that may protect individuals from respiratory infections. Therefore, we assessed AAT protein anomalies as a risk factor for RGM lung disease. In a cohort of 100 patients with RGM lung disease, Mycobacterium (M.) abscessus was the most prevalent organism, isolated in 64 (64%) subjects. Anomalous AAT proteins were present in 27% of the cohort, which is 1.6 times the estimated prevalence of anomalous AAT proteins in the United States population (p=0.008). In in vitro studies, both AAT and a synthetic inhibitor of serine proteases suppressed M. abscessus infection of monocyte-derived macrophages by up to 65% (p<0.01). AAT may be an anti-RGM host-defense factor, and anomalous AAT phenotypes or AAT deficiency may constitute risk factors for pulmonary disease due to RGM.