Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases

In 2008 we published the fi rst set of guidelines for standardiz- ing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the fi eld. Our knowledge base and relevant new tech- nologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in differ- ent 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 meas urements that monitor the num- bers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the com- plete process including the amoun t and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accu mulation must be differentiated from stimuli that increas e autophagic activity, de fi ned as increased autophagy induction coupled with in creased delivery to, and degra- dation within, lysosomes (in most higher eukaryotes and some pro- tists such as Dictyostelium ) or the vacuole (in plants and fungi). In otherwords,itisespeciallyimportantthatinvestigatorsnewtothe fi eld understand that the appearance of more autophagosomes does not necessarily equate with mo re autophagy. In fact, in many cases, autophagosomes accumu late because of a block in traf fi cking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may re fl ect a reductionindegradativeactivity .Itisworthemphasizingherethat lysosomal digestion is a stage of au tophagy and evaluating its com- petence is a crucial part of the evaluation of autophagic fl ux, 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 appro- priate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no indi- vidual 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 speci fi c marker for an autophagic process. In these guidelines, we con- sider 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 fi eld

The cellular and molecular mechanisms underpinning tissue repair and its failure to heal are still poorly understood, and current therapies are limited. Poor wound healing after trauma, surgery, acute illness, or chronic disease conditions affects millions of people worldwide each year and is the consequence of poorly regulated elements of the healthy tissue repair response, including inflammation, angiogenesis, matrix deposition, and cell recruitment. Failure of one or several of these cellular processes is generally linked to an underlying clinical condition, such as vascular disease, diabetes, or aging, which are all frequently associated with healing pathologies. The search for clinical strategies that might improve the body's natural repair mechanisms will need to be based on a thorough understanding of the basic biology of repair and regeneration. In this review, we highlight emerging concepts in tissue regeneration and repair, and provide some perspectives on how to translate current knowledge into viable clinical approaches for treating patients with wound-healing pathologies.

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Characterization of the genetic landscape of Alzheimer's disease (AD) and related dementias (ADD) provides a unique opportunity for a better understanding of the associated pathophysiological processes. We performed a two-stage genome-wide association study totaling 111,326 clinically diagnosed/'proxy' AD cases and 677,663 controls. We found 75 risk loci, of which 42 were new at the time of analysis. Pathway enrichment analyses confirmed the involvement of amyloid/tau pathways and highlighted microglia implication. Gene prioritization in the new loci identified 31 genes that were suggestive of new genetically associated processes, including the tumor necrosis factor alpha pathway through the linear ubiquitin chain assembly complex. We also built a new genetic risk score associated with the risk of future AD/dementia or progression from mild cognitive impairment to AD/dementia. The improvement in prediction led to a 1.6- to 1.9-fold increase in AD risk from the lowest to the highest decile, in addition to effects of age and the APOE ε4 allele.

The previous edition of the consensus guidelines of the International Workshop on Chronic Lymphocytic Leukemia (iwCLL), published in 2008, has found broad acceptance by physicians and investigators caring for patients with CLL. Recent advances including the discovery of the genomic landscape of the disease, the development of genetic tests with prognostic relevance, and the detection of minimal residual disease (MRD), coupled with the increased availability of novel targeted agents with impressive efficacy, prompted an international panel to provide updated evidence- and expert opinion-based recommendations. These recommendations include a revised version of the iwCLL response criteria, an update on the use of MRD status for clinical evaluation, and recommendations regarding the assessment and prophylaxis of viral diseases during management of CLL.

A ciliopathy is a disease in which a hairlike cellular organelle called the cilium is dysfunctional. Most proteins altered in these single-gene disorders function at the level of the cilium–centrosome complex. This review considers the role of the cilium in disease.

Tissue repair after injury is a complex, metabolically demanding process. Depending on the tissue's regenerative capacity and the quality of the inflammatory response, the outcome is generally imperfect, with some degree of fibrosis, which is defined by aberrant accumulation of collagenous connective tissue. Inflammatory cells multitask at the wound site by facilitating wound debridement and producing chemokines, metabolites, and growth factors. If this well-orchestrated response becomes dysregulated, the wound can become chronic or progressively fibrotic, with both outcomes impairing tissue function, which can ultimately lead to organ failure and death. Here we review the current understanding of the role of inflammation and cell metabolism in tissue-regenerative responses, highlight emerging concepts that may expand therapeutic perspectives, and briefly discuss where important knowledge gaps remain.

Despite promising results with targeted drugs, chemoimmunotherapy with fludarabine, cyclophosphamide (FC), and rituximab (R) remains the standard therapy for fit patients with untreated chronic lymphocytic leukemia (CLL). Herein, we present the long-term follow-up of the randomized CLL8 trial reporting safety and efficacy of FC and FCR treatment of 817 treatment-naïve patients with CLL. The primary end point was progression-free survival (PFS). With a median follow-up of 5.9 years, median PFS were 56.8 and 32.9 months for the FCR and FC group (hazard ratio [HR], 0.59; 95% confidence interval [CI], 0.50-0.69, P < .001). Median overall survival (OS) was not reached for the FCR group and was 86.0 months for the FC group (HR, 0.68; 95% CI, 0.54-0.89, P = .001). In patients with mutated IGHV (IGHV MUT), FCR improved PFS and OS compared with FC (PFS: HR, 0.47; 95% CI, 0.33-0.68, P < .001; OS: HR, 0.62; 95% CI, 0.34-1.11, P = .1). This improvement remained applicable for all cytogenetic subgroups other than del(17p). Long-term safety analyses showed that FCR had a higher rate of prolonged neutropenia during the first year after treatment (16.6% vs 8.8%; P = .007). Secondary malignancies including Richter's transformation occurred in 13.1% in the FCR group and in 17.4% in the FC group (P = .1). First-line chemoimmunotherapy with FCR induces long-term remissions and highly relevant improvement in OS in specific genetic subgroups of fit patients with CLL, in particular those with IGHV MUT. This trial was registered at www.clinicaltrials.gov as #NCT00281918.

Influx of macrophages plays a crucial role in tissue repair. However, the precise function of macrophages during the healing response has remained a subject of debate due to their functional dichotomy as effectors of both tissue injury and repair. We tested the hypothesis that macrophages recruited during the diverse phases of skin repair after mechanical injury exert specific functions to restore tissue integrity. For this purpose, we developed a mouse model that allows conditional depletion of macrophages during the sequential stages of the repair response. Depletion of macrophages restricted to the early stage of the repair response (inflammatory phase) significantly reduced the formation of vascularized granulation tissue, impaired epithelialization, and resulted in minimized scar formation. In contrast, depletion of macrophages restricted to the consecutive mid-stage of the repair response (phase of tissue formation) resulted in severe hemorrhage in the wound tissue. Under these conditions, transition into the subsequent phase of tissue maturation and wound closure did not occur. Finally, macrophage depletion restricted to the late stage of repair (phase of tissue maturation) did not significantly impact the outcome of the repair response. These results demonstrate that macrophages exert distinct functions during the diverse phases of skin repair, which are crucial to control the natural sequence of repair events.

Abstract The genetic make-up of an individual contributes to the susceptibility and response to viral infection. Although environmental, clinical and social factors have a role in the chance of exposure to SARS-CoV-2 and the severity of COVID-19 1,2 , host genetics may also be important. Identifying host-specific genetic factors may reveal biological mechanisms of therapeutic relevance and clarify causal relationships of modifiable environmental risk factors for SARS-CoV-2 infection and outcomes. We formed a global network of researchers to investigate the role of human genetics in SARS-CoV-2 infection and COVID-19 severity. Here we describe the results of three genome-wide association meta-analyses that consist of up to 49,562 patients with COVID-19 from 46 studies across 19 countries. We report 13 genome-wide significant loci that are associated with SARS-CoV-2 infection or severe manifestations of COVID-19. Several of these loci correspond to previously documented associations to lung or autoimmune and inflammatory diseases 3–7 . They also represent potentially actionable mechanisms in response to infection. Mendelian randomization analyses support a causal role for smoking and body-mass index for severe COVID-19 although not for type II diabetes. The identification of novel host genetic factors associated with COVID-19 was made possible by the community of human genetics researchers coming together to prioritize the sharing of data, results, resources and analytical frameworks. This working model of international collaboration underscores what is possible for future genetic discoveries in emerging pandemics, or indeed for any complex human disease.

We present a human miRNA tissue atlas by determining the abundance of 1997 miRNAs in 61 tissue biopsies of different organs from two individuals collected post-mortem. One thousand three hundred sixty-four miRNAs were discovered in at least one tissue, 143 were present in each tissue. To define the distribution of miRNAs, we utilized a tissue specificity index (TSI). The majority of miRNAs (82.9%) fell in a middle TSI range i.e. were neither specific for single tissues (TSI > 0.85) nor housekeeping miRNAs (TSI < 0.5). Nonetheless, we observed many different miRNAs and miRNA families that were predominantly expressed in certain tissues. Clustering of miRNA abundances revealed that tissues like several areas of the brain clustered together. Considering -3p and -5p mature forms we observed miR-150 with different tissue specificity. Analysis of additional lung and prostate biopsies indicated that inter-organism variability was significantly lower than inter-organ variability. Tissue-specific differences between the miRNA patterns appeared not to be significantly altered by storage as shown for heart and lung tissue. MiRNAs TSI values of human tissues were significantly (P = 10(-8)) correlated with those of rats; miRNAs that were highly abundant in certain human tissues were likewise abundant in according rat tissues. We implemented a web-based repository enabling scientists to access and browse the data (https://ccb-web.cs.uni-saarland.de/tissueatlas).

BACKGROUND: The BCL2 inhibitor venetoclax has shown activity in patients with chronic lymphocytic leukemia (CLL), but its efficacy in combination with other agents in patients with CLL and coexisting conditions is not known. METHODS: In this open-label, phase 3 trial, we investigated fixed-duration treatment with venetoclax and obinutuzumab in patients with previously untreated CLL and coexisting conditions. Patients with a score of greater than 6 on the Cumulative Illness Rating Scale (scores range from 0 to 56, with higher scores indicating more impaired function of organ systems) or a calculated creatinine clearance of less than 70 ml per minute were randomly assigned to receive venetoclax-obinutuzumab or chlorambucil-obinutuzumab. The primary end point was investigator-assessed progression-free survival. The safety of each regimen was also evaluated. RESULTS: deletion, mutation, or both and in patients with unmutated immunoglobulin heavy-chain genes. Grade 3 or 4 neutropenia occurred in 52.8% of patients in the venetoclax-obinutuzumab group and in 48.1% of patients in the chlorambucil-obinutuzumab group, and grade 3 or 4 infections occurred in 17.5% and 15.0%, respectively. All-cause mortality was 9.3% in the venetoclax-obinutuzumab group and 7.9% in the chlorambucil-obinutuzumab group. These differences were not significant. CONCLUSIONS: Among patients with untreated CLL and coexisting conditions, venetoclax-obinutuzumab was associated with longer progression-free survival than chlorambucil-obinutuzumab. (Funded by F. Hoffmann-La Roche and AbbVie; ClinicalTrials.gov number, NCT02242942.).

The cells in the human body are continuously challenged by a variety of genotoxic attacks. Erroneous repair of the DNA can lead to mutations and chromosomal aberrations that can alter the functions of tumor suppressor genes or oncogenes, thus causing cancer development. As a central tumor suppressor, p53 guards the genome by orchestrating a variety of DNA-damage-response (DDR) mechanisms. Already early in metazoan evolution, p53 started controlling the apoptotic demise of genomically compromised cells. p53 plays a prominent role as a facilitator of DNA repair by halting the cell cycle to allow time for the repair machineries to restore genome stability. In addition, p53 took on diverse roles to also directly impact the activity of various DNA-repair systems. It thus appears as if p53 is multitasking in providing protection from cancer development by maintaining genome stability.

Lung cancer remains one of the leading causes of cancer-related death in developed countries. Although lung adenocarcinomas with EGFR mutations or EML4-ALK fusions respond to treatment by epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) inhibition, respectively, squamous cell lung cancer currently lacks therapeutically exploitable genetic alterations. We conducted a systematic search in a set of 232 lung cancer specimens for genetic alterations that were therapeutically amenable and then performed high-resolution gene copy number analyses. We identified frequent and focal fibroblast growth factor receptor 1 (FGFR1) amplification in squamous cell lung cancer (n = 155), but not in other lung cancer subtypes, and, by fluorescence in situ hybridization, confirmed the presence of FGFR1 amplifications in an independent cohort of squamous cell lung cancer samples (22% of cases). Using cell-based screening with the FGFR inhibitor PD173074 in a large (n = 83) panel of lung cancer cell lines, we demonstrated that this compound inhibited growth and induced apoptosis specifically in those lung cancer cells carrying amplified FGFR1. We validated the FGFR1 dependence of FGFR1-amplified cell lines by FGFR1 knockdown and by ectopic expression of an FGFR1-resistant allele (FGFR1(V561M)), which rescued FGFR1-amplified cells from PD173074-mediated cytotoxicity. Finally, we showed that inhibition of FGFR1 with a small molecule led to significant tumor shrinkage in vivo. Thus, focal FGFR1 amplification is common in squamous cell lung cancer and associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in this cohort of patients.

The 'obesity epidemic' represents a major global socioeconomic burden that urgently calls for a better understanding of the underlying causes of increased weight gain and its associated metabolic comorbidities, such as type 2 diabetes mellitus and cardiovascular diseases. Improving our understanding of the cellular basis of obesity could set the stage for the development of new therapeutic strategies. The CNS plays a pivotal role in the regulation of energy and glucose homeostasis. Distinct neuronal cell populations, particularly within the arcuate nucleus of the hypothalamus, sense the nutrient status of the organism and integrate signals from peripheral hormones including pancreas-derived insulin and adipocyte-derived leptin to regulate calorie intake, glucose metabolism and energy expenditure. The arcuate neurons are tightly connected to other specialized neuronal subpopulations within the hypothalamus, but also to various extrahypothalamic brain regions, allowing a coordinated behavioral response. This At a Glance article gives an overview of the recent knowledge, mainly derived from rodent models, regarding the CNS-dependent regulation of energy and glucose homeostasis, and illustrates how dysregulation of the neuronal networks involved can lead to overnutrition and obesity. The potential impact of recent research findings in the field on therapeutic treatment strategies for human obesity is also discussed.

Mitochondrial fusion and structure depend on the dynamin-like GTPase OPA1, whose activity is regulated by proteolytic processing. Constitutive OPA1 cleavage by YME1L and OMA1 at two distinct sites leads to the accumulation of both long and short forms of OPA1 and maintains mitochondrial fusion. Stress-induced OPA1 processing by OMA1 converts OPA1 completely into short isoforms, inhibits fusion, and triggers mitochondrial fragmentation. Here, we have analyzed the function of different OPA1 forms in cells lacking YME1L, OMA1, or both. Unexpectedly, deletion of Oma1 restored mitochondrial tubulation, cristae morphogenesis, and apoptotic resistance in cells lacking YME1L. Long OPA1 forms were sufficient to mediate mitochondrial fusion in these cells. Expression of short OPA1 forms promoted mitochondrial fragmentation, which indicates that they are associated with fission. Consistently, GTPase-inactive, short OPA1 forms partially colocalize with ER-mitochondria contact sites and the mitochondrial fission machinery. Thus, OPA1 processing is dispensable for fusion but coordinates the dynamic behavior of mitochondria and is crucial for mitochondrial integrity and quality control.

BACKGROUND: While the leading symptoms during coronavirus disease 2019 (COVID-19) are acute and the majority of patients fully recover, a significant fraction of patients now increasingly experience long-term health consequences. However, most data available focus on health-related events after severe infection and hospitalisation. We present a longitudinal, prospective analysis of health consequences in patients who initially presented with no or minor symptoms of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection. Hence, we focus on mild COVID-19 in non-hospitalised patients. METHODS: 958 Patients with confirmed SARS-CoV-2 infection were observed from April 6th to December 2nd 2020 for long-term symptoms and SARS-CoV-2 antibodies. We identified anosmia, ageusia, fatigue or shortness of breath as most common, persisting symptoms at month 4 and 7 and summarised presence of such long-term health consequences as post-COVID syndrome (PCS). Predictors of long-term symptoms were assessed using an uni- and multivariable logistic regression model. FINDINGS: We observed 442 and 353 patients over four and seven months after symptom onset, respectively. Four months post SARS-CoV-2 infection, 8•6% (38/442) of patients presented with shortness of breath, 12•4% (55/442) with anosmia, 11•1% (49/442) with ageusia and 9•7% (43/442) with fatigue. At least one of these characteristic symptoms was present in 27•8% (123/442) and 34•8% (123/353) at month 4 and 7 post-infection, respectively. A lower baseline level of SARS-CoV-2 IgG, anosmia and diarrhoea during acute COVID-19 were associated with higher risk to develop long-term symptoms. INTERPRETATION: The on-going presence of either shortness of breath, anosmia, ageusia or fatigue as long-lasting symptoms even in non-hospitalised patients was observed at four and seven months post-infection and summarised as post-COVID syndrome (PCS). The continued assessment of patients with PCS will become a major task to define and mitigate the socioeconomic and medical long-term effects of COVID-19. FUNDING: COVIM:"NaFoUniMedCovid19"(FKZ: 01KX2021).

BACKGROUND: Significant evidence indicates that the failing heart is energy starved. During the development of heart failure, the capacity of the heart to utilize fatty acids, the chief fuel, is diminished. Identification of alternate pathways for myocardial fuel oxidation could unveil novel strategies to treat heart failure. METHODS AND RESULTS: Quantitative mitochondrial proteomics was used to identify energy metabolic derangements that occur during the development of cardiac hypertrophy and heart failure in well-defined mouse models. As expected, the amounts of proteins involved in fatty acid utilization were downregulated in myocardial samples from the failing heart. Conversely, expression of β-hydroxybutyrate dehydrogenase 1, a key enzyme in the ketone oxidation pathway, was increased in the heart failure samples. Studies of relative oxidation in an isolated heart preparation using ex vivo nuclear magnetic resonance combined with targeted quantitative myocardial metabolomic profiling using mass spectrometry revealed that the hypertrophied and failing heart shifts to oxidizing ketone bodies as a fuel source in the context of reduced capacity to oxidize fatty acids. Distinct myocardial metabolomic signatures of ketone oxidation were identified. CONCLUSIONS: These results indicate that the hypertrophied and failing heart shifts to ketone bodies as a significant fuel source for oxidative ATP production. Specific metabolite biosignatures of in vivo cardiac ketone utilization were identified. Future studies aimed at determining whether this fuel shift is adaptive or maladaptive could unveil new therapeutic strategies for heart failure.

Tissue homeostasis requires maintenance of functional integrity under stress. A central source of stress is mechanical force that acts on cells, their nuclei, and chromatin, but how the genome is protected against mechanical stress is unclear. We show that mechanical stretch deforms the nucleus, which cells initially counteract via a calcium-dependent nuclear softening driven by loss of H3K9me3-marked heterochromatin. The resulting changes in chromatin rheology and architecture are required to insulate genetic material from mechanical force. Failure to mount this nuclear mechanoresponse results in DNA damage. Persistent, high-amplitude stretch induces supracellular alignment of tissue to redistribute mechanical energy before it reaches the nucleus. This tissue-scale mechanoadaptation functions through a separate pathway mediated by cell-cell contacts and allows cells/tissues to switch off nuclear mechanotransduction to restore initial chromatin state. Our work identifies an unconventional role of chromatin in altering its own mechanical state to maintain genome integrity in response to deformation.

ABSTRACT The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods to characterize single EVs. Nanoparticle Tracking Analysis (NTA) is commonly used to determine EV concentration and diameter. As the EV field is lacking methods to easily confirm and validate NTA data, questioning the reliability of measurements remains highly important. In this regard, a comparison addressing measurement quality between different NTA devices such as Malvern's NanoSight NS300 or Particle Metrix' ZetaView has not yet been conducted. To evaluate the accuracy and repeatability of size and concentration determinations of both devices, we employed comparative methods including transmission electron microscopy (TEM) and single particle interferometric reflectance imaging sensing (SP‐IRIS) by ExoView. Multiple test measurements with nanospheres, liposomes and ultracentrifuged EVs from human serum and cell culture supernatant were performed. Additionally, serial dilutions and freeze‐thaw cycle‐dependent EV decrease were measured to determine the robustness of each system. Strikingly, NanoSight NS300 exhibited a 2.0–2.1‐fold overestimation of polystyrene and silica nanosphere concentration. By measuring serial dilutions of EV samples, we demonstrated higher accuracy in concentration determination by ZetaView (% BIAS range: 2.7–8.5) in comparison with NanoSight NS300 (% BIAS range: 32.9–36.8). The concentration measurements by ZetaView were also more precise (% CV range: 0.0–4.7) than measurements by NanoSight NS300 (% CV range: 5.4–10.7). On the contrary, quantitative TEM imaging indicated more accurate EV sizing by NanoSight NS300 (% D TEM range: 79.5–134.3) compared to ZetaView (% D TEM range: 111.8–205.7), while being equally repeatable (NanoSight NS300% CV range: 0.8–6.7; ZetaView: 1.4–7.8). However, both devices failed to report a peak EV diameter below 60 nm compared to TEM and SP‐IRIS. Taken together, NTA devices differ strongly in their hardware and software affecting measuring results. ZetaView provided a more accurate and repeatable depiction of EV concentration, whereas NanoSight NS300 supplied size measurements of higher resolution.