Correction: Impaired cellular bioenergetics caused by GBA1 depletion sensitizes neurons to calcium overload

n/

Activation of the Integrated Stress Response and ER Stress Protect from Fluorizoline-Induced Apoptosis in HEK293T and U2OS Cell Lines

The prohibitin (PHB)-binding compound fluorizoline as well as PHB-downregulation activate the integrated stress response (ISR) in HEK293T and U2OS human cell lines. This activation is denoted by phosphorylation of eIF2 alpha and increases in ATF4, ATF3, and CHOP protein levels. The blockage of the activation of the ISR by overexpression of GRP78, as well as an increase in IRE1 activity, indicate the presence of ER stress after fluorizoline treatment. The inhibition of the ER stress response in HEK293T and U2OS led to increased sensitivity to fluorizoline-induced apoptosis, indicating a pro-survival role of this pathway after fluorizoline treatment in these cell lines. Fluorizoline induced an increase in calcium concentration in the cytosol and the mitochondria. Finally, two different calcium chelators reduced fluorizoline-induced apoptosis in U2OS cells. Thus, we have found that fluorizoline causes increased ER stress and activation of the integrated stress response, which in HEK293T and U2OS cells are protective against fluorizoline-induced apoptosis

Mitochondrial Signature in Human Monocytes and Resistance to Infection in C. elegans During Fumarate-Induced Innate Immune Training.

Monocytes can develop immunological memory, a functional characteristic widely recognized as innate immune training, to distinguish it from memory in adaptive immune cells. Upon a secondary immune challenge, either homologous or heterologous, trained monocytes/macrophages exhibit a more robust production of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, than untrained monocytes. Candida albicans, β-glucan, and BCG are all inducers of monocyte training and recent metabolic profiling analyses have revealed that training induction is dependent on glycolysis, glutaminolysis, and the cholesterol synthesis pathway, along with fumarate accumulation; interestingly, fumarate itself can induce training. Since fumarate is produced by the tricarboxylic acid (TCA) cycle within mitochondria, we asked whether extra-mitochondrial fumarate has an effect on mitochondrial function. Results showed that the addition of fumarate to monocytes induces mitochondrial Ca2+ uptake, fusion, and increased membrane potential (Δψm), while mitochondrial cristae became closer to each other, suggesting that immediate (from minutes to hours) mitochondrial activation plays a role in the induction phase of innate immune training of monocytes. To establish whether fumarate induces similar mitochondrial changes in vivo in a multicellular organism, effects of fumarate supplementation were tested in the nematode worm Caenorhabditis elegans. This induced mitochondrial fusion in both muscle and intestinal cells and also increased resistance to infection of the pharynx with E. coli. Together, these findings contribute to defining a mitochondrial signature associated with the induction of innate immune training by fumarate treatment, and to the understanding of whole organism infection resistance

Investigating Mitochondrial Ca²⁺ Dynamics in Isolated Mitochondria and Intact Cells: Application of Fluorescent Dyes and Genetic Reporters

Mitochondrial Ca2+ buffering is a hallmark of eukaryotic cellular physiology, contributing to the spatiotemporal shaping of the cytosolic Ca2+ signals and regulation of mitochondrial bioenergetics. Often, this process is altered in a pathological context; therefore, it can be scrutinized experimentally for therapeutic intervention. In this chapter, we describe fluorescence and bioluminescence measurement of mitochondrial Ca2+ in both isolated mitochondria and intact cells

Assessing the Redox Status of Mitochondria Through the NADH/FAD²⁺ Ratio in Intact Cells

This section aims to describe the measurement of NADH and FAD2+ levels in intact cells using fluorescence microscopy. Both NADH and FADH2 are major electron donors for the electron transport chain through shifting of their redox status. Furthermore, within their redox couples, only NADH and FAD2+ are fluorescent. Therefore, calibration of the NADH and FAD2+ fluorescence signal is a crucial factor in accurately assessing mitochondrial function and redox status

Attitude, perception and feedback of second year medical students on teaching-learning methodology and evaluation methods in pharmacology: A questionnaire-based study

Background: To assess the student′s attitude, perception and feedback on teaching-learning methodology and evaluation methods in pharmacology. Materials and Methods: One hundred and forty second year medical students studying at Smt. Kashibai Navale Medical College, Pune, were selected. They were administered a pre-validated questionnaire containing 22 questions. Suggestions were also asked regarding the qualities of good pharmacology teachers and modification in pharmacology teaching methods. Descriptive statistics were used and results were expressed as percentage. Results: Majority of the students found cardiovascular system (49.25%) as the most interesting topic in pharmacology, whereas most of the students opined that cardiovascular system (60.10%), chemotherapy (54.06%) and central nervous system (44.15%) are going to be the most useful topics in internship. 48.53% students preferred clinical/patient-related pharmacology and 39.13% suggested use of audiovisual-aided lectures. Prescription writing and criticism of prescription were amongst the most useful and interesting in practical pharmacology. Students expressed interest in microteaching and problem-based learning, whereas seminars, demonstrations on manikin and museum studies were mentioned as good adjuvants to routine teaching. Multiple Choice Question (MCQ) practice tests and theory viva at the end of a particular system and periodical written tests were mentioned as effective evaluation methods. Students were found to have lot of interest in gathering information on recent advances in pharmacology and suggested to include new drug information along with prototype drugs in a comparative manner. Conclusion: There is a need of conducting few microteaching sessions and more of clinical-oriented problem-based learning with MCQ-based revisions at the end of each class in the pharmacology teaching at undergraduate level

Pathological consequences of MICU1 mutations on mitochondrial calcium signalling and bioenergetics

Loss of function mutations of the protein MICU1, a regulator of mitochondrial Ca(2+) uptake, cause a neuronal and muscular disorder characterised by impaired cognition, muscle weakness and an extrapyramidal motor disorder. We have shown previously that MICU1 mutations cause increased resting mitochondrial Ca(2+) concentration ([Ca(2+)]m). We now explore the functional consequences of MICU1 mutations in patient derived fibroblasts in order to clarify the underlying pathophysiology of this disorder. We propose that deregulation of mitochondrial Ca(2+) uptake through loss of MICU1 raises resting [Ca(2+)]m, initiating a futile Ca(2+) cycle, whereby continuous mitochondrial Ca(2+) influx is balanced by Ca(2+) efflux through the sodium calcium exchanger (NLCXm). Thus, inhibition of NCLXm by CGP-37157 caused rapid mitochondrial Ca(2+) accumulation in patient but not control cells. We suggest that increased NCLX activity will increase sodium/proton exchange, potentially undermining oxidative phosphorylation, although this is balanced by dephosphorylation and activation of pyruvate dehydrogenase (PDH) in response to the increased [Ca(2+)]m. Consistent with this model, while ATP content in patient derived or control fibroblasts was not different, ATP increased significantly in response to CGP-37157 in the patient but not the control cells. In addition, EMRE expression levels were altered in MICU1 patient cells compared to the controls. The MICU1 mutations were associated with mitochondrial fragmentation which we show is related to altered DRP1 phosphorylation. Thus, MICU1 serves as a signal-noise discriminator in mitochondrial calcium signalling, limiting the energetic costs of mitochondrial Ca(2+) signalling which may undermine oxidative phosphorylation, especially in tissues with highly dynamic energetic demands. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech

Legislative Documents

Also, variously referred to as: House bills; House documents; House legislative documents; legislative documents; General Court documents

Clinicopathological correlation of radiologic measurement of post-therapy tumor size and tumor volume for pancreatic ductal adenocarcinoma

ObjectivesTumor size measurement is critical for accurate tumor staging in patients with pancreatic ductal adenocarcinoma (PDAC). However, accurate tumor size measurement is challenging in patients who received neoadjuvant therapy before resection, due to treatment-induced fibrosis and tumor invasion beyond the grossly identified tumor area. In this study, we evaluated the correlation between the tumor size and tumor volume measured on post-therapy computed tomography (CT) scans and the pathological measurement. Also, we investigated the correlation between these measurements and clinicopathological parameters and survival.Materials and methodsRetrospectively, we evaluated 343 patients with PDAC who received neoadjuvant therapy, followed by pancreaticoduodenectomy and had pre-operative pancreatic protocol CT imaging. We measured the longest tumor diameter (RadL) and the radiological tumor volume (RadV) on the post-therapy CT scan, then we categorized RadL into four radiologic tumor stages (RTS) based on the current AJCC staging (8th edition) protocol and RadV based on the median. Pearson correlation or Spearman's coefficient (δ), T-test and ANOVA was used to test the correlation between the radiological and pathological measurement. Chi-square analysis was used to test the correlation with the tumor pathological response, lymph-node metastasis and margin status and Kaplan-Meier and Cox-proportional hazard for survival analysis. P-value < 0.05 was considered significant.ResultsAs a continuous variable, RadL showed a positive linear correlation with the post-therapy pathologic tumor size in the overall patient population (Pearson correlation coefficient: 0.72, P < 0.001) and RadV (δ: 0.63, p < 0.0001). However, there was no correlation between RadL and pathologic tumor size in patients with ypT0 and those with pathologic tumor size of ≤1.0 cm. Post-therapy RTS and RadV group correlated with ypT stage, tumor response grades using either CAP or MDA grading system, distance of superior mesenteric artery margin and tumor recurrence/metastasis.ConclusionAlthough RadL tends to understage ypT in PDAC patients who had no radiologically detectable tumor or small tumors (RTS0 or RTS1), radiologic measurement of post-therapy tumor size may be used as a marker for the pathologic tumor staging and tumor response to neoadjuvant therapy

Predictive Modeling for Voxel-Based Quantification of Imaging-Based Subtypes of Pancreatic Ductal Adenocarcinoma (PDAC): A Multi-Institutional Study

Previously, we characterized qualitative imaging-based subtypes of pancreatic ductal adenocarcinoma (PDAC) on computed tomography (CT) scans. Conspicuous (high delta) PDAC tumors are more likely to have aggressive biology and poorer clinical outcomes compared to inconspicuous (low delta) tumors. Here, we developed a quantitative classification of this imaging-based subtype (quantitative delta; q-delta). Retrospectively, baseline pancreatic protocol CT scans of three cohorts (cohort#1 = 101, cohort#2 = 90 and cohort#3 = 16 [external validation]) of patients with PDAC were qualitatively classified into high and low delta. We used a voxel-based method to volumetrically quantify tumor enhancement while referencing normal-pancreatic-parenchyma and used machine learning-based analysis to build a predictive model. In addition, we quantified the stromal content using hematoxylin- and eosin-stained treatment-naïve PDAC sections. Analyses revealed that PDAC quantitative enhancement values are predictive of the qualitative delta scoring and were used to build a classification model (q-delta). Compared to high q-delta, low q-delta tumors were associated with improved outcomes, and the q-delta class was an independent prognostic factor for survival. In addition, low q-delta tumors had higher stromal content and lower cellularity compared to high q-delta tumors. Our results suggest that q-delta classification provides a clinically and biologically relevant tool that may be integrated into ongoing and future clinical trials