The process of programmed cell death, or apoptosis, is generally characterized by distinct morphological characteristics and energy-dependent biochemical mechanisms. Apoptosis is considered a vital component of various processes including normal cell turnover, proper development and functioning of the immune system, hormone-dependent atrophy, embryonic development and chemical-induced cell death. Inappropriate apoptosis (either too little or too much) is a factor in many human conditions including neurodegenerative diseases, ischemic damage, autoimmune disorders and many types of cancer. The ability to modulate the life or death of a cell is recognized for its immense therapeutic potential. Therefore, research continues to focus on the elucidation and analysis of the cell cycle machinery and signaling pathways that control cell cycle arrest and apoptosis. To that end, the field of apoptosis research has been moving forward at an alarmingly rapid rate. Although many of the key apoptotic proteins have been identified, the molecular mechanisms of action or inaction of these proteins remain to be elucidated. The goal of this review is to provide a general overview of current knowledge on the process of apoptosis including morphology, biochemistry, the role of apoptosis in health and disease, detection methods, as well as a discussion of potential alternative forms of apoptosis.

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Apoptosis: A Review of Programmed Cell Death

http://web.stanford.edu/group/brunet/Cell1999

Akt Promotes Cell Survival by Phosphorylating and Inhibiting a Forkhead Transcription Factor

Autophagy in the Pathogenesis of Disease

Autophagy, or cellular self-digestion, is a cellular pathway involved in protein and organelle degradation, with an astonishing number of connections to human disease and physiology. For example, autophagic dysfunction is associated with cancer, neurodegeneration, microbial infection and ageing. Paradoxically, although autophagy is primarily a protective process for the cell, it can also play a role in cell death. Understanding autophagy may ultimately allow scientists and clinicians to harness this process for the purpose of improving human health.

/pdf/autophagy-fights-disease-through-cellular-self-digestion-5i6zc9bv8a.pdf

Autophagy fights disease through cellular self-digestion

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Verfahren zur 3-D-Datenerfassung mittels einer Reihe von Projektionsbildern eines Untersuchungsobjektes aus unterschiedlichen Aufnahmewinkeln wahrend eines Rotationslaufs mit einem Biplan-C-Bogen-System mit zwei getrennten C-Bogen-Ebenen, die in einem ”Zwischenwinkel” zueinander angeordnet sind, wobei aus den Projektionsbildern dreidimensionale Bilddaten rekonstruierbar sind, gekennzeichnet durch folgende Schritte: a) Erfassung der Herztatigkeit mittels eines EKGs, b) Ermittlung der Herzfrequenz des Untersuchungsobjektes und Berechnung einer Start-Herzphase, c) Berechnung von Parametern der C-Bogen-Ebenen aus den gemas Schritt b) ermittelten Werten und Einstellung der Parameter, d) Start der Akquisition der Daten in der Start-Herzphase (τ Heart,Forward ) nach erfolgter Einstellung der Parameter, e) gleichmasige Rotation beider C-Bogen-Ebenen mit derselben Geschwindigkeit in einer ersten Drehrichtung als Vorwartslauf uber einen Winkelbereich und Aufnahme von Projektionsbildern in verschiedenen Winkelbereichen bei verschiedenen Herzphasen des Patienten, f) Start der Akquisition der Daten nach Beendigung des Vorwartslaufs in der Start-Herzphase (τ Heart,Backward ) des Ruckwartslaufes, g) gleichmasige Rotation beider C-Bogen-Ebenen mit derselben Geschwindigkeit in einer zweiten Drehrichtung als Ruckwartslauf uber einen Winkelbereich und Aufnahme verschiedener Winkelbereiche bei verschiedenen Herzphasen des Patienten, und h) Rekonstruktion der gewonnenen Daten nach Beendigung des Ruckwartslaufs.

Verfahren zur 3-D-Datenerfassung mit einem Biplan-C Bogen-System mit Biplan-Akquisitions-Multiplexing

Shailaja Kasibhatla, Gustavo P. Amarante-Mendes, Deborah Finucane, Thomas Brunner, Ella Bossy-Wetzel and Douglas R. Green This protocol was adapted from “Apoptosis Assays,” Chapter 15, in Cells (eds. Spector et al.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. This three-volume set is now out of print; however, some of the microscopy methods were republished in Basic Methods in Microscopy, by David L. Spector and Robert D. Goldman.

Analysis of DNA Fragmentation Using Propidium Iodide (PI) Fluorescence of Individual Nuclei.

Liver Receptor Homolog-1 (LRH-1/NR5A2) is a nuclear receptor that has been shown to promote apoptosis resistance in various tissues and disease contexts, however, its role in liver cell death remains unexplored. Deletion of LRH-1 in hepatocytes developed into a mild steatosis and inflammation already under steady-state conditions. Unexpectedly, hepatocyte-specific deletion of LRH-1 also resulted in a profound protection of mice from TNF-induced hepatocyte apoptosis and associated hepatitis. LRH-1-deficient hepatocytes showed elevated NF-ⲕB activity, while LRH-1 overexpression inhibited NF-ⲕB activity. This inhibition was based on direct physical interaction of the ligand-binding domain of LRH-1 and the Rel homology domain of NF-ⲕB subunit RelA. Mechanistically, we found that increased transcription of anti-apoptotic NF-ⲕB target genes, together with proteasomal degradation of pro-apoptotic BIM via regeneration-driven EGF receptor signaling, prevented mitochondrial apoptosis, ultimately protecting mice from TNF-induced liver damage. Collectively, our study demonstrates that LRH-1 is a critical modulator of cell death and inflammation in the healthy and diseased liver. Highlights Hepatic LRH-1 deletion causes mild liver steatosis, fibrosis, and inflammation. Female LRH-1-deficient mice are protected from TNF-induced liver damage. LRH-1 interacts with NF-ⲕB and inhibits its activity. LRH-1 deletion-provoked inflammation causes degradation of pro-apoptotic protein BIM. Graphical abstract

/pdf/liver-receptor-homolog-1-lrh-1-nr5a2-orchestrates-hepatic-2m7wp9u3.pdf

Liver Receptor Homolog-1 (LRH-1/NR5A2) orchestrates hepatic inflammation and TNF-induced cell death

Abstract The need for new options in lung cancer treatment inevitably leads back to basic research. The tumor itself and the tumor environment especially the interaction with the immune system need to be better understood to develop targeted therapies. In the context of lung cancer glucocorticoids (GC) are mainly known as a combination drug to attenuate side-effects of chemotherapies. However, endogenous extra-adrenal GC have been shown to substantially regulate local immune responses within various tissues, including the lung. In this study we investigated whether primary lung tumors have maintained the capacity to synthesize GC and may thereby regulate anti-tumor immune responses. We show that several non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC) cell lines express key steroidogenic enzymes and synthesize bioactive GC under steady state conditions. We also show that tumor-derived GC can inhibit splenic T cell activation, thus demonstrating their immunoregulatory potential. Moreover, steroidogenic enzymes were detected by quantitative RT-PCR and immunohistochemistry in tissue sections of different human lung tumors, further strengthening the idea that human lung carcinomas regulate their microenvironment by releasing immunoregulatory GC, which potentially contributes to immune evasion and treatment resistance. 

https://www.nature.com/articles/s41435-023-00194-y.pdf

Human lung carcinomas synthesize immunoregulatory glucocorticoids

Verfahren (1) zur Darstellung eines Zielbereiches (62) eines Hohlorgans (20) eines Untersuchungsobjektes (64), in den eine Katheterspitze (14) eines Katheters (12) eingefuhrt ist, umfassend folgende Verfahrensschritte: S1) Entgegennahme eines, wenigstens den Zielbereich (62) des Hohlorgans (20) umfassenden, 3D-Bilddatensatzes (30); S2) Entgegennahme eines Kennwertes zur Bestimmung des Hohlorgans (20) in dem 3D-Bilddatensatz (30), Bestimmung des Hohlorgans (20) in dem 3D-Bilddatensatz (30) und Bestimmung einer Zentrallinie (44) des Hohlorgans (20) des 3D-Bilddatensatzes (30), wobei die Zentrallinie (44) des Hohlorgans (20) bei Gefaszu- bzw. Gefasabgangen stetig interpoliert wird; S3) Entgegennahme eines 2D-Durchleuchtungsbildes (10), das wenigstens die Katheterspitze (14) des eingefuhrten Katheters umfasst; S4) Registrierung des 2D-Durchleuchtungsbildes (10) mit dem 3D-Bilddatensatz (30) und Bestimmung der Position der Katheterspitze (14) des eingefuhrten Katheters innerhalb des 3D-Bilddatensatzes (30), wobei die Hohe bzw. Tiefe der Katheterspitze (14) auf die Hohe bzw. Tiefe der Zentrallinie (44) gesetzt wird; S5) Bestimmen einer ersten und einer zweiten Projektion (26, 28) des 3D-Bilddatensatzes (30), wobei die Projektionen (26, 28) ihren Ursprung (42) in der registrierten Position der Katheterspitze (14) des eingefuhrten Katheters (12) innerhalb des 3D-Bilddatensatzes (30) haben, und wobei die Projektionsrichtungen (36, 38) in einer Bildebene (40) des 2D-Durchleuchtungsbildes (10) sind, die die registrierte Position der Katheterspitze (14) des eingefuhrten Katheters (12) innerhalb des 3D-Bilddatensatzes (30) umfasst, und wobei die Projektionsrichtungen (36, 38) senkrecht zur Zentrallinie (44) des Hohlorgans (20) an der registrierten Position der Katheterspitze (14) des eingefuhrten Katheters (12) innerhalb des 3D-Bilddatensatzes (30) sind und wobei die Projektionsrichtungen (36, 38) antiparallel zueinander verlaufen; S6) Darstellung der ersten und/oder der zweiten Projektion (26, 28) des 3D-Bilddatensatzes (30).

Thomas Brunner

Papers

Verfahren zur 3-D-Datenerfassung mit einem Biplan-C Bogen-System mit Biplan-Akquisitions-Multiplexing

Analysis of DNA Fragmentation Using Propidium Iodide (PI) Fluorescence of Individual Nuclei.

Liver Receptor Homolog-1 (LRH-1/NR5A2) orchestrates hepatic inflammation and TNF-induced cell death

Human lung carcinomas synthesize immunoregulatory glucocorticoids

Verfahren und Vorrichtung zur Darstellung eines Zielbereiches eines Hohlorgans eines Untersuchungsobjektes