RACK1 is an interaction partner of ATG5 and a novel regulator of autophagy

Autophagy is biological mechanism allowing recycling of long-lived proteins, abnormal protein aggregates, and damaged organelles under cellular stress conditions. Following sequestration in double- or multimembrane autophagic vesicles, the cargo is delivered to lysosomes for degradation. ATG5 is a key component of an E3-like ATG12-ATG5-ATG16 protein complex that catalyzes conjugation of the MAP1LC3 protein to lipids, thus controlling autophagic vesicle formation and expansion. Accumulating data indicate that ATG5 is a convergence point for autophagy regulation. Here, we describe the scaffold protein RACK1 (receptor activated C-kinase 1, GNB2L1) as a novel ATG5 interactor and an autophagy protein. Using several independent techniques, we showed that RACK1 interacted with ATG5. Importantly, classical autophagy inducers (starvation or mammalian target of rapamycin blockage) stimulated RACK1-ATG5 interaction. Knockdown of RACK1 or prevention of its binding to ATG5 using mutagenesis blocked autophagy activation. Therefore, the scaffold protein RACK1 is a new ATG5-interacting protein and an important and novel component of the autophagy pathways

Study of protein-protein interactions in autophagy research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper

Prolonged breast-feeding is an independent risk factor for postmenopausal osteoporosis.

Objectives: This study investigated the effects of parity and age at first pregnancy and breast-feeding, as well as duration of BF for total and per child on postmenopausal osteoporosis

The accuracy of three-dimensional ultrasonography in the diagnosis of Müllerian duct anomalies and its concordance with magnetic resonance imaging

This retrospective study was performed to comparatively evaluate the diagnostic accuracies of three-dimensional ultrasonography (3D-US) and magnetic resonance imaging (MRI) for identification of Müllerian duct anomalies (MDAs). A total of 27 women with suspected MDAs underwent gynaecological examination, 2D-US, 3D-US and MRI, respectively. The MDAs were classified with respect to the European Society of Human Reproduction and Embryology–European Society for Gynaecological Endoscopy (ESHRE/ESGE) and American Society of Reproductive Medicine (ASRM) systems. Based on the ESHRE/ESGE classification, there was a discrepancy for only one patient between US and MRI. Thus, the concordance between US and MRI was 26/27 (96.3%). With respect to ASRM classification, there was a disagreement between MRI and 3D-US in three patients, thus the concordance between MRI and 3D-US was 24/27 (88.9%). To conclude, the 3D-US has a good level of agreement with MRI for recognition of MDAs.Impact Statement What is already known on this subject? Müllerian duct anomalies (MDAs) are relatively common malformations of the female genital tract and they may adversely affect the reproductive potential. The establishment of accurate and timely diagnosis of these malformations is critical to overcome clinical consequences of MDAs. What the results of this study add? The concordance between US and MRI for diagnosis of MDAs based on ESHRE-ESGE classification and ASRM were 96.3% and 88.9%, respectively. These results indicate that 3D US has a satisfactory level of diagnostic accuracy for MDAs and it can be used in conjunction with MRI. Minimisation of diagnostic errors is important to improve reproductive outcome and to avoid unnecessary surgical interventions. What the implications are of these findings for clinical practice and/or further research? Efforts must be spent to eliminate the discrepancies between the clinical and radiological diagnosis of MDAs. Further trials should be implemented for establishment and standardisation of radiological images for identification and classification of MDAs

A novel ATG5 interaction with Ku70 potentiates DNA repair upon genotoxic stress

The maintenance of cellular homeostasis in living organisms requires a balance between anabolic and catabolic reactions. Macroautophagy (autophagy herein) is determined as one of the major catabolic reactions. Autophagy is an evolutionarily conserved stress response pathway that is activated by various insults including DNA damage. All sorts of damage to DNA potentially cause loss of genetic information and trigger genomic instability. Most of these lesions are repaired by the activation of DNA damage response following DNA repair mechanisms. Here we describe, a novel protein complex containing the autophagy protein ATG5 and the non-homologous end-joining repair system proteins. We discovered for the first time that ATG5 interacted with both Ku80 (XRCC5) and Ku70 (XRCC6). This novel interaction is facilitated mainly via Ku70. Our results suggest that this interaction is dynamic and enhanced upon genotoxic stresses. Strikingly, we identified that ATG5-Ku70 interaction is necessary for DNA repair and effective recovery from genotoxic stress. Therefore, our results are demonstrating a novel, direct, dynamic, and functional interaction between ATG5 and Ku70 proteins that plays a crucial role in DNA repair under genotoxic stress conditions

Performance of tuberculin skin test and interferon gamma assay for the diagnosis of latent tuberculosis infection in juvenile idiopathic arthritis

The objective of this prospective cross-sectional study was to compare a Mycobacterium tuberculosis-specific interferon gamma (IFN-gamma) enzyme linked immunosorbent assay [QuantiFERON-TB Gold In-Tube (QFT-GIT)] test with tuberculin skin test (TST) for detection of latent tuberculosis infection (LTBI) in patients with juvenile idiopathic arthritis (JIA). To our knowledge, this is the first study evaluating the performance of QFT-GIT in comparison with TST in JIA. A cross-sectional study of 39 children with JIA and 40 healthy controls was conducted in A degrees zmir, Turkey. Blood was for drawn for the QFT-GIT assay prior to administration of the TST using 5 tubercullin units (TU) of purified protein derivative (PPD-S). A positive TST was defined as a parts per thousand yen10 mm for JIA and a parts per thousand yen15 mm for controls. Statistical analysis was performed using SPSS version 16.0 for Windows. There were no significant differences between JIA patients and controls for age, sex, and Bacillus Calmette-Gu,rin (BCG) vaccination. Of patients, 70% had active JIA disease. The median TST induration was 5.8 mm (+/- 5.7 mm) for JIA and 10.7 mm (+/- 4.5 mm) for the control group, which was statistically significant (p = 0.000). The rate of patients who showed no reaction to TST was 38%, of which 93% had active disease. There were two patients who had positive IFN-gamma results but negative TST, who had systemic and polyarticular type JIA, respectively. Overall agreement between TST and QFT-GIT was low both in JIA and control group (kappa value =0.06 and 0.10, respectively). TST may be inadequate to diagnose LTBI in JIA patients. The IFN-gamma assay may be useful to identify false negative TST response in cases with latent M. tuberculosis infection. The combination of IF QFT-GIT method with TST would provide successful diagnostic screening for LTBI in JIA, particularly prior to anti-tumor necrosis factor treatment. Long-term prospective studies are still necessary to appreciate the advantages and the applicability of these tests in pediatrics

Hydrodynamic cavitation kills prostate cells and ablates benign prostatic hyperplasia tissue

Hydrodynamic cavitation is a physical phenomenon characterized by vaporization and bubble formation in liquids under low local pressures, and their implosion following release to a higher pressure environment. Collapse of the bubbles releases high energy and may cause damage to exposed surfaces. We recently designed a set-up to exploit the destructive nature of hydrodynamic cavitation for biomedical purposes. We have previously shown that hydrodynamic cavitation could kill leukemia cells and erode kidney stones. In this study, we analyzed the effects of cavitation on prostate cells and benign prostatic hyperplasia (BPH) tissue. We showed that hydrodynamic cavitation could kill prostate cells in a pressure and time dependent manner. Cavitation did not lead to programmed cell death, i.e. classical apoptosis or autophagy activation. Following the application of cavitation, we observed no prominent DNA damage and cells did not arrest in the cell cycle. Hence, we concluded that cavitation forces directly damaged the cells, leading to their pulverization. Upon application to BPH tissues from patients, cavitation could lead to a significant level of tissue destruction. Therefore similar to ultrasonic cavitation, we propose that hydrodynamic cavitation has the potential to be exploited and developed as an approach for the ablation of aberrant pathological tissues, including BPH

RACK1 is an interaction partner of ATG5 and a novel regulator of autophagy

Autophagy is biological mechanism allowing recycling of long-lived proteins, abnormal protein aggregates and damaged organelles under cellular stress conditions. Following sequestration in double or multimembrane autophagic vesicles, the cargo is delivered to lysosomes for degradation. ATG5 is a key component of an E3-like ATG12-ATG5-ATG16 protein complex that catalyzes conjugation of the MAP1LC3 protein to lipids, thus controlling autophagic vesicle formation and expansion. Accumulating data indicate that ATG5 is a convergence point for autophagy regulation. Here, we describe the scaffold protein RACK1 (Receptor Activated C-Kinase 1, GNB2L1), as a novel ATG5 interactor and an autophagy protein. Using several independent techniques, we showed that RACK1 interacted with ATG5. Importantly, classical autophagy inducers (starvation or mTOR blockage) stimulated RACK1-ATG5 interaction. Knockdown of RACK1 or prevention of its binding to ATG5 using mutagenesis blocked autophagy activation. Therefore, the scaffold protein RACK1 is a new ATG5-interacting protein and an important and novel component of the autophagy pathways

Increased Risk of Unfavorable Metabolic Outcome during Short-Term Follow-Up in Subjects with Nonfunctioning Adrenal Adenomas

Objective: To demonstrate long-term changes in the prevalence of several types of metabolic derangements in subjects with nonfunctioning adrenal adenomas. Subjects and Methods: 273 subjects with adrenal adenomas, including 231 with nonfunctioning adenoma and 42 with subclinical Cushing's syndrome (sCS), were evaluated with respect to anthropometric and laboratory characteristics and prevalence of type 2 diabetes mellitus (T2DM), hypertension, dyslipidemia, metabolic syndrome (MS), prediabetes and cardiovascular disease (CVD). Median duration was 24 months. Follow-up data of 114 participants with nonfunctioning adrenal adenomas are also presented while those of 117 were missing. Follow-up data regarding changes in anthropometric and laboratory parameters and prevalence rates of metabolic disturbances were obtained from the medical records. Results: The prevalence rates for both patients with nonfunctioning adenoma and sCS were: dyslipidemia: 161 (59%), hypertension: 147 (54%), MS: 128 (47%), prediabetes: 62 (23%), T2DM: 49 (18%), and CVD: 21 (8%). Hypertension and CVD were prevalent in subjects with sCS compared to participants with nonfunctioning adenoma. In follow-up, body mass index (p = 0.005), systolic blood pressure (p < 0.001), waist circumference (p = 0.005), homeostasis model assessment (p = 0.046), high-sensitivity C-reactive protein (p = 0.023), total cholesterol (p < 0.001) and low-density lipoprotein cholesterol (p < 0.001) and prevalence of hypertension (p < 0.001), dyslipidemia (p < 0.001), prediabetes (p < 0.001) and MS (p < 0.01) significantly increased in subjects with nonfunctioning adenoma. Conclusion: The data showed that nonfunctioning adrenal adenomas were associated with the development or deterioration of atherosclerotic risk factors. Therefore, follow-up and management strategies should be developed to decrease atherosclerotic morbidity in those individuals. Copyright (c) 2012 S. Karger AG, Base