Miracle-dz/UniFAR-FTUs huggingface dataset:
A quantitative TR-FRET plate reader immunoassay for measuring autophagy

Autophagy involves the isolation and targeting of unwanted cellular components to lysosomes for their digestion and reuse. Autophagic dysregulation has recently been implicated in a wide range of disease processes, yet facile methods for quantifying autophagy have been lacking in the field. Here we describe the generation of a quantitative plate reader assay for measuring the autophagic activity of cells. One of the best characterized autophagy markers is the protein LC3B, which normally resides in the cytosol (LC3B-I) but upon induction of autophagy becomes lipidated and embedded in autophagosomal membranes (LC3B-II). To quantify autophagy, we reasoned that GFP-tagged LC3B could serve as a time-resolved fluorescence resonance energy transfer (TR-FRET) acceptor upon cell lysis in the presence of terbium-labeled LC3B antibodies. Using this TR-FRET immunoassay approach, we screened a panel of LC3B antibodies and identified an antibody that exhibits strong preferential affinity toward autophagosome-associated LC3B-II and thereby facilitates specific detection of autophagic activity. The plate reader format provides both a quantitative and an objective result, thus overcoming some of the key limitations of the traditional immunoblotting and imaging approaches used to monitor autophagy. Moreover, since the assay step requires only a single addition of cell lysis buffer containing the detection antibody its simple workflow is both automation-friendly and scalable, which renders it suitable for high-throughput screening. We demonstrate how this TR-FRET immunoassay for GFP-tagged LC3B-II can be applied to quantitatively detect changes in the autophagy activity of cells, including estimating effects on autophagic flux.
Oxidative stress impairs autophagic flux in prion protein-deficient hippocampal cells

We previously reported that autophagy is upregulated in Prnp-deficient (Prnp0/0) hippocampal neuronal cells in comparison to cellular prion protein (PrPC)-expressing (Prnp+/+) control cells under conditions of serum deprivation. In this study, we determined whether a protective mechanism of PrPC is associated with autophagy using Prnp0/0 hippocampal neuronal cells under hydrogen peroxide (H2O2)-induced oxidative stress. We found that Prnp0/0 cells were more susceptible to oxidative stress than Prnp+/+ cells in a dose- and time-dependent manner. In addition, we observed enhanced autophagy by immunoblotting, which detected the conversion of microtubule-associated protein 1 light chain 3 β (LC3B)-I to LC3B-II, and we observed increased punctate LC3B immunostaining in H2O2-treated Prnp0/0 cells compared with H2O2-treated control cells. Interestingly, this enhanced autophagy was due to impaired autophagic flux in the H2O2-treated Prnp0/0 cells, while the H2O2-treated Prnp+/+ cells showed enhanced autophagic flux. Furthermore, caspase-dependent and independent apoptosis was observed when both cell lines were exposed to H2O2. Moreover, the inhibition of autophagosome formation by Atg7 siRNA revealed that increased autophagic flux in Prnp+/+ cells contributes to the prosurvival effect of autophagy against H2O2 cytotoxicity. Taken together, our results provide the first experimental evidence that the deficiency of PrPC may impair autophagic flux via H2O2-induced oxidative stress.
Stem Cell Therapy in Cartilage Repair—Culture-Free and Cell Culture–Based Methods

Abstract In order to overcome potential problems associated with autologous chondrocyte implantation, mesenchymal stem cell-based therapies could be potential alternatives. Conventional stem cell-based therapy accompanies the separation of cells from tissue followed by monolayer culture for the expansion of cell numbers. On the other hand, the cost of cell culture under quality control is high, which could be a potential barrier for industrialization. In order to reduce the cost associated cell culture, culture-free cell-based therapies have been investigated with the use of bone marrow aspirate. In this chapter, we will introduce the three stem cell-based therapies in cartilage repair. The first two procedures are using cell culture methods and the last one with cell-free method. All the three methods have been into the stage of clinical trials and their surgical procedures as well as their preliminary results will be reported.
Hypertonic stress promotes autophagy and microtubule-dependent autophagosomal clusters

Osmotic homeostasis is fundamental for most cells, which face recurrent alterations of environmental osmolality that challenge cell viability. Protein damage is a consequence of hypertonic stress, but whether autophagy contributes to the osmoprotective response is unknown. Here, we investigated the possible implications of autophagy and microtubule organization on the response to hypertonic stress. We show that hypertonicity rapidly induced long-lived protein degradation, LC3-II generation and Ptdlns3K-dependent formation of LC3- and ATG12-positive puncta. Lysosomotropic agents chloroquine and bafilomycin A1, but not nutrient deprivation or rapamycin treatment, further increased LC3-II generation, as well as ATG12-positive puncta, indicating that hypertonic stress increases autophagic flux. Autophagy induction upon hypertonic stress enhanced cell survival since cell death was increased by ATG12 siRNA-mediated knockdown and reduced by rapamycin. We additionally showed that hypertonicity induces fast reorganization of microtubule networks, which is associated with strong reorganization of microtubules at centrosomes and fragmentation of Golgi ribbons. Microtubule remodeling was associated with pericentrosomal clustering of ATG12-positive autolysosomes that colocalized with SQSTM1/p62 and ubiquitin, indicating that autophagy induced by hypertonic stress is at least partly selective. Efficient autophagy by hypertonic stress required microtubule remodeling and was DYNC/dynein-dependent as autophagosome clustering was enhanced by paclitaxel-induced microtubule stabilization and was reduced by nocodazole-induced tubulin depolymerization as well as chemical (EHNA) or genetic [DCTN2/dynactin 2 (p50) overexpression] interference of DYNC activity. The data document a general and hitherto overlooked mechanism, where autophagy and microtubule remodeling play prominent roles in the osmoprotective response.
Risk and Economic Consequences of Contagious Animal Disease Introduction

Risk and economic consequences of contagious animal disease introduction Risico op en economische gevolgen van insleep van besmettelijke dierziekten Horst,H.S. , 1997 The research described in this thesis focused on risks and economic consequences of the introduction of contagious animal diseases into the Netherlands, in particular of Classical Swine Fever (CSF) and Foot-and-Mouth Disease (FMD). Special emphasis was placed on the development of a virus introduction model. Using an integrated approach, this model was then combined with an existing model describing virus spread and an economic model focusing on quantification of losses due to epidemics. As objective quantitative information on underlying aspects of virus introduction was only scarcely available, additional information to base the proposed simulation model on was required and therefore obtained from experts. In order to elicit this information in an objective and quantitative way, various questionnaire techniques were evaluated and combined within a computerized design. In particular the questionnaire techniques Conjoint Analysis and ELI, which had not or only scarcely been used in the area of animal health economics thus far, proved to be useful methods to elicit expert knowledge. These techniques were used to elicit the relative importance of risk factors related to virus introduction (Conjoint Analysis) and probability density functions on the duration of the interval first infectionfirst detection (ELI). The expert knowledge thus obtained formed, combined with quantitative data obtained from databases and experiments described in the literature, the input on which the virus introduction model was based. This Monte Carlo simulation model, called VIRiS (Virus Introduction Risk Simulation), simulates the introduction of virus into the Netherlands. The results show that important causes of outbreaks are the import of live animals and trucks used for export and re-entering the country. These two factors together are expected to account for more than 70% of the primary outbreaks of CSF and FMD in the Netherlands. Insights and results obtained by VIRiS were combined with outcomes of models describing spread and economic consequences of epidemics. In this way alternative prevention strategies were evaluated as to their ability to reduce annual losses due to outbreaks. For example, the complete elimination of risks related to the risk factor 'returning trucks' reduces the expected annual losses due to FMD and CSF epidemics together by approximately US$ 9 million. The approach presented in this thesis is general and may be used for other diseases and countries as well. PhD-thesis, Department of Economics and Management, Wageningen Agricultural University, Hollandseweg 1, 6706 KN Wageningen, the Netherlands.
Bone marrow stromal cells protect myeloma cells from bortezomib induced apoptosis by suppressing microRNA-15a expression

Despite unsurpassed anti-tumor activity of bortezomib for multiple myeloma (MM), drug resistance has emerged as a challenge, especially when MM cells adhere to the stroma. This study aimed to determine whether bone marrow stromal cells (BMSCs) have a role in the development of chemoresistance in MM. Our data demonstrate that the secretion of interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), and cell-to-cell contact with microenvironment-derived stromal cells from patients with multiple myeloma (MM-BMSCs) significantly decreased the sensitivity of myeloma cells to bortezomib treatment. Mechanistically, we found that microRNA (miRNA)- 15a expression was up-regulated in U266 and NCI-H929 cells treated by bortezomib, which was inhibited by MM-BMSCs. miRNA-15a transfected myeloma cells were arrested in G1/S checkpoint and secreted less VEGF compared to control transfected cells, although no significant difference was found in VEGF mRNA levels. In conclusion, our data suggest that via suppressing miRNA-15a expression, BMSCs provide survival support and protect myeloma cells from bortezomib induced apoptosis.
Tumor suppressor gene PDCD4 negatively regulates autophagy by inhibiting the expression of autophagy-related gene ATG5

PDCD4 (programmed cell death protein 4), a suppressor of gene transcription and translation, plays a crucial inhibitory role in several types of human tumors. However, its underlying mechanisms remain unclear. Autophagy, an evolutionarily conserved catabolic process, maintains cellular homeostasis under stress conditions such as starvation and plays a crucial role in tumor initiation and progression. We report here that PDCD4 inhibits autophagy in multiple cell types both in vitro and in vivo, which in turn contributes to its tumor suppressor activity. Importantly, PDCD4 inhibits the expression of an essential autophagy related gene, ATG5 and the formation of an ATG12–ATG5 complex, and its ma3 domains are required for PDCD4-mediated inhibition of autophagy. Unlike most tumor suppressors that act as positive or dual regulators of autophagy, our findings indicate that PDCD4 negatively regulates autophagy by targeting ATG5, which provides a novel mechanism of tumor suppression.
Prognostic significance of neuron-specific enolase in patients with diffuse large B-cell lymphoma treated with rituximab-based immunochemotherapy

Abstract The present study examined the clinical significance of serum neuron-specific enolase (NSE) in patients with diffuse large B-cell lymphoma (DLBCL). Serum NSE values were measured using a electrochemiluminescence assay in 106 patients and tissue NSE expression was examined using immunohistochemistry in 65 patients. Fifty-four percent of patients had a positive expression of serum NSE (>15.20 ng/mL), and cytoplasmic NSE was pathologically demonstrated in 26/65 cases, which showed a positive correlation with that of serum NSE expression. The serum NSE value was closely correlated with performance status, serum lactate dehydrogenase (LDH) level, International Prognostic Index (IPI) score, and Ann Arbor stage, and declined significantly in patients who responded to treatment. In the rituximab-immunochemotherapy group, there was a significant difference in the 5-year overall survival (OS) rate between the NSE-positive and -negative groups (93% vs. 44%, p = 0.001), and the serum NSE level was found to be an independent prognostic factor. Serum NSE may be a novel marker of disease aggressiveness as well as a prognostic factor for DLBCL in the era of rituximab.
PINK1 is degraded through the N-end rule pathway

PINK1, a mitochondrial serine/threonine kinase, is the product of a gene mutated in an autosomal recessive form of Parkinson disease. PINK1 is constitutively degraded by an unknown mechanism and stabilized selectively on damaged mitochondria where it can recruit the E3 ligase PARK2/PARKIN to induce mitophagy. Here, we show that, under steady-state conditions, endogenous PINK1 is constitutively and rapidly degraded by E3 ubiquitin ligases UBR1, UBR2 and UBR4 through the N-end rule pathway. Following precursor import into mitochondria, PINK1 is cleaved in the transmembrane segment by a mitochondrial intramembrane protease PARL generating an N-terminal destabilizing amino acid and then retrotranslocates from mitochondria to the cytosol for N-end recognition and proteasomal degradation. Thus, sequential actions of mitochondrial import, PARL-processing, retrotranslocation and recognition by N-end rule E3 enzymes for the ubiquitin proteosomal degradation defines the rapid PINK1 turnover. PINK1 steady-state elimination by the N-end rule identifies a novel organelle to cytoplasm turnover pathway that yields a mechanism to flag damaged mitochondria for autophagic elimination.
Stroke subtypes in southern Iran

Objective: To investigate about frequency of different subtypes of stroke in south of IranMethods: This is a retrospective, single-center study conducted at Namazi Hospital, Shiraz, south of Iran. Age, sex, length of hospitalization and mortality of stroke patients were recorded by reviewing hospital medical records.Results: 16 351 patients (53.6% male, 46.4%female) were recruited. Ischemic stroke (10750 patients, 65.7%), intracerebral hemorrhage(3282 patients,20.1%) and subarachnoid hemorrhage(1057 patients,6.5%) were the most common subtypes of stroke, respectively. In 1262 patients (7.7%) the stroke subtype could not been specified. Ischemic stroke and intracerebral hemorrhage were more common in men but subarachnoid hemorrhage was more common in women. Subarachnoid hemorrhage occurred significantly in younger patients. Mortality was significantly higher in intracerebral hemorrhage. Hospital stay was significantly longer in subarachnoid hemorrhage group.Conclusion: the distribution of the different subtypes of stroke in Iran is similar to Caucasians.
The accumulation of misfolded proteins in the mitochondrial matrix is sensed by PINK1 to induce PARK2/Parkin-mediated mitophagy of polarized mitochondria

Defective mitochondria exert deleterious effects on host cells. To manage this risk, mitochondria display several lines of quality control mechanisms: mitochondria-specific chaperones and proteases protect against misfolded proteins at the molecular level, and fission/fusion and mitophagy segregate and eliminate damage at the organelle level. An increase in unfolded proteins in mitochondria activates a mitochondrial unfolded protein response (UPRmt) to increase chaperone production, while the mitochondrial kinase PINK1 and the E3 ubiquitin ligase PARK2/Parkin, whose mutations cause familial Parkinson disease, remove depolarized mitochondria through mitophagy. It is unclear, however, if there is a connection between those different levels of quality control (QC). Here, we show that the expression of unfolded proteins in the matrix causes the accumulation of PINK1 on energetically healthy mitochondria, resulting in mitochondrial translocation of PARK2, mitophagy and subsequent reduction of unfolded protein load. Also, PINK1 accumulation is greatly enhanced by the knockdown of the LONP1 protease. We suggest that the accumulation of unfolded proteins in mitochondria is a physiological trigger of mitophagy.
Renal impairment is not an independent adverse prognostic factor in patients with multiple myeloma treated upfront with novel agent-based regimens

Abstract Renal impairment (RI) is a common presenting complication of multiple myeloma associated with significant morbidity and early mortality, while it has been associated with inferior survival in patients treated with conventional regimens. We assessed the impact of RI in 203 unselected consecutive patients treated upfront with novel agents (thalidomide, lenalidomide, bortezomib). RI was assessed by the estimated glomerular filtration rate (eGFR). RI (eGFR <60 mL/min) was present in 93 (45.8%) of patients at diagnosis and was associated with advanced age, advanced International Staging System (ISS) stage, poorer performance status, hypercalcemia, urine Bence-Jones proteinuria, anemia and thrombocytopenia. Myeloma response rates were similar for patients with or without RI. In univariate analysis RI was associated with shorter survival and a higher rate of early death (7% vs. 3.5%); however, when adjusted for other prognostic factors, RI was not independently associated with survival. In conclusion, in unselected newly diagnosed patients treated with novel agent-based therapies, RI is not independently associated with inferior survival, probably due to the significant activity of novel agents even in the context of RI.
Job satisfaction traits of Nigerian physical therapists

In recent years emigration of highly trained health personnel has caused manpower shortages that were of real concern to the developing countries. This study was designed to (1) determine whether physical therapists (PTs) in Nigeria were satisfied with their jobs, and (2) assess whether Nigerian PTs practicing in the United States (U.S.) were more satisfied with their jobs than the PTs in Nigeria. A questionnaire was administered to PTs in Nigeria ( N = 110) and Nigerian PTs based in the US ( N = 31). The questionnaire consisted of two parts: Part 1 sought demographic information and Part 2 consisted of ten statements relating to paperwork, job challenge, physical demand, job autonomy, fulfillment, and stress. Overall, these cohorts of PTs were satisfied with the clinical practice aspects of their jobs. The two groups were comparable except in areas relating to stress, paperwork, and physical demand. Nigerian PTs practicing in the U.S. were not more satisfied than their counterparts in Nigeria. The study suggests that factors other than those related to clinical practice aspects of physical therapy were responsible for the wave of emigration of PTs from Nigeria in the past decade.
Why do psychiatrists have difficulty disengaging with the out-patient clinic?

In this issue of APT Mitchell & Selmes present an article detailing why patients miss appointments and how clinicians should respond. Many of the papers quoted relate to the psychiatric out-patient clinic. In this commentary, as well as picking up on some of the themes that emerge from their article, I explore the attachment that psychiatrists seem to have to this particular model of patient contact.
Restarting stalled autophagy a potential therapeutic approach for the lipid storage disorder, Niemann-Pick type C1 disease

Autophagy is essential for cellular homeostasis and its dysfunction in human diseases has been implicated in the accumulation of misfolded protein and in cellular toxicity. We have recently shown impairment in autophagic flux in the lipid storage disorder, Niemann-Pick type C1 (NPC1) disease associated with abnormal cholesterol sequestration, where maturation of autophagosomes is impaired due to defective amphisome formation caused by failure in SNARE machinery. Abrogation of autophagy also causes cholesterol accumulation, suggesting that defective autophagic flux in NPC1 disease may act as a primary causative factor not only by imparting its deleterious effects, but also by increasing cholesterol load. However, cholesterol depletion treatment with HP-β-cyclodextrin impedes autophagy, whereas pharmacologically stimulating autophagy restores its function independent of amphisome formation. Of potential therapeutic relevance is that a low dose of HP-β-cyclodextrin that does not perturb autophagy, coupled with an autophagy inducer, may rescue both the cholesterol and autophagy defects in NPC1 disease.
Glucocerebrosidase deficits in sporadic Parkinson disease

Parkinson disease (PD) is a progressive neurodegenerative movement disorder characterized pathologically by abnormal SNCA/α-synuclein protein inclusions in neurons. Impaired lysosomal autophagic degradation of cellular proteins is implicated in PD pathogenesis and progression. Heterozygous GBA mutations, encoding lysosomal GBA/glucocerebrosidase (glucosidase, β, acid), are the greatest genetic risk factor for PD, and reduced GBA and SNCA accumulation are related in PD models. Here we review our recent human brain tissue study demonstrating that GBA deficits in sporadic PD are related to the early accumulation of SNCA, and dysregulation of chaperone-mediated autophagy (CMA) pathways and lipid metabolism.
GABARAPL1 (GEC1) associates with autophagic vesicles

Gabarapl1 (gec1) was first described as an estrogen regulated gene which shares a high sequence homology with the gabarap gene. We previously demonstrated that GABARAPL1, like GABARAP, interacts with the GABAA receptor and tubulin and promotes tubulin polymerization. Previous work has demonstrated that the GABARAP family members (GABARAP, LC3, GATE-16 and Atg8) are not only involved in the transport of proteins or vesicles but are also implicated in various mechanisms such as autophagy, cell death, cell proliferation and tumor progression. We therefore asked whether GABARAPL1 might also play a role in autophagy. First, we showed that GABARAPL1 is cleaved at glycine 116, a residue which is conserved in other members of the family. We also demonstrated that GABARAPL1 is linked to phospholipids, delipidated by Atg4B, associated with intracellular membranes and accumulated in intracellular vesicles after inhibition of lysosomal activity. Finally, we showed that GABARAPL1 partially colocalizes with LC3 or Lysotracker green in intracellular vesicles. Taken together, our results demonstrate that GABARAPL1 associates with autophagic vesicles.
Proline-Glycine-Proline (PGP) and High Mobility Group Box Protein-1 (HMGB1): Potential Mediators of Cystic Fibrosis Airway Inflammation

Cystic fibrosis (CF) is chronic lung disease characterized by an unrelenting neutrophil-predominant airway inflammatory response. This inflammation leads to extracellular matrix (ECM) remodeling and eventually to the development of bronchiectasis. While many components of the immune response in CF have been well-characterized, recent data suggests that small molecules may play an important and underappreciated role in this inflammation. This review will examine two novel molecules: proline-glycine-proline (PGP) and high mobility group box protein-1 (HMGB1), and their potential impact in CF lung disease. This review will provide a brief overview of CF lung disease and background on both HMGB1 and PGP. It will then focus on these molecules in a murine model of CF-like airway disease and in human biological specimens from CF individuals. Finally, this manuscript will address possible mechanisms for therapeutic targeting of these bioactive mediators.