The removal of functionally dispensable, infected or potentially neoplastic cells is driven by programmed cell death (PCD) pathways, highlighting their important roles in homeostasis, host defence ...against pathogens, cancer and a range of other pathologies. Several types of PCD pathways have been described, including apoptosis, necroptosis and pyroptosis; they employ distinct molecular and cellular processes and differ in their outcomes, such as the capacity to trigger inflammatory responses. Recent genetic and biochemical studies have revealed remarkable flexibility in the use of these PCD pathways and indicate a considerable degree of plasticity in their molecular regulation; for example, despite having a primary role in inducing pyroptosis, inflammatory caspases can also induce apoptosis, and conversely, apoptotic stimuli can trigger pyroptosis. Intriguingly, this flexibility is most pronounced in cellular responses to infection, while apoptosis is the dominant cell death process through which organisms prevent the development of cancer. In this Review, we summarize the mechanisms of the different types of PCD and describe the physiological and pathological processes that engage crosstalk between these pathways, focusing on infections and cancer. We discuss the intriguing notion that the different types of PCD could be seen as a single, coordinated cell death system, in which the individual pathways are highly interconnected and can flexibly compensate for one another.
The main protease, M
(or 3CL
) in SARS-CoV-2 is a viable drug target because of its essential role in the cleavage of the virus polypeptide. Feline infectious peritonitis, a fatal coronavirus ...infection in cats, was successfully treated previously with a prodrug GC376, a dipeptide-based protease inhibitor. Here, we show the prodrug and its parent GC373, are effective inhibitors of the M
from both SARS-CoV and SARS-CoV-2 with IC
values in the nanomolar range. Crystal structures of SARS-CoV-2 M
with these inhibitors have a covalent modification of the nucleophilic Cys145. NMR analysis reveals that inhibition proceeds via reversible formation of a hemithioacetal. GC373 and GC376 are potent inhibitors of SARS-CoV-2 replication in cell culture. They are strong drug candidates for the treatment of human coronavirus infections because they have already been successful in animals. The work here lays the framework for their use in human trials for the treatment of COVID-19.
The stromal vascular fraction (SVF) of adipose tissue contains an abundant population of multipotent adipose-tissue-derived stem cells (ASCs) that possess the capacity to differentiate into cells of ...the mesodermal lineage in vitro. For cell-based therapies, an advantageous approach would be to harvest these SVF cells and give them back to the patient within a single surgical procedure, thereby avoiding lengthy and costly in vitro culturing steps. However, this requires SVF-isolates to contain sufficient ASCs capable of differentiating into the desired cell lineage. We have investigated whether the yield and function of ASCs are affected by the anatomical sites most frequently used for harvesting adipose tissue: the abdomen and hip/thigh region. The frequency of ASCs in the SVF of adipose tissue from the abdomen and hip/thigh region was determined in limiting dilution and colony-forming unit (CFU) assays. The capacity of these ASCs to differentiate into the chondrogenic and osteogenic pathways was investigated by quantitative real-time polymerase chain reaction and (immuno)histochemistry. A significant difference (
P
= 0.0009) was seen in ASC frequency but not in the absolute number of nucleated cells between adipose tissue harvested from the abdomen (5.1 ± 1.1%, mean ± SEM) and hip/thigh region (1.2 ± 0.7%). However, within the CFUs derived from both tissues, the frequency of CFUs having osteogenic differentiation potential was the same. When cultured, homogeneous cell populations were obtained with similar growth kinetics and phenotype. No differences were detected in differentiation capacity between ASCs from both tissue-harvesting sites. We conclude that the yield of ASCs, but not the total amount of nucleated cells per volume or the ASC proliferation and differentiation capacities, are dependent on the tissue-harvesting site. The abdomen seems to be preferable to the hip/thigh region for harvesting adipose tissue, in particular when considering SVF cells for stem-cell-based therapies in one-step surgical procedures for skeletal tissue engineering.
D-dimer measurement is an important step in the diagnostic strategy of clinically suspected acute pulmonary embolism (PE), but its clinical usefulness is limited in elderly patients.
To prospectively ...validate whether an age-adjusted D-dimer cutoff, defined as age × 10 in patients 50 years or older, is associated with an increased diagnostic yield of D-dimer in elderly patients with suspected PE.
A multicenter, multinational, prospective management outcome study in 19 centers in Belgium, France, the Netherlands, and Switzerland between January 1, 2010, and February 28, 2013.
All consecutive outpatients who presented to the emergency department with clinically suspected PE were assessed by a sequential diagnostic strategy based on the clinical probability assessed using either the simplified, revised Geneva score or the 2-level Wells score for PE; highly sensitive D-dimer measurement; and computed tomography pulmonary angiography (CTPA). Patients with a D-dimer value between the conventional cutoff of 500 µg/L and their age-adjusted cutoff did not undergo CTPA and were left untreated and formally followed-up for a 3-month period.
The primary outcome was the failure rate of the diagnostic strategy, defined as adjudicated thromboembolic events during the 3-month follow-up period among patients not treated with anticoagulants on the basis of a negative age-adjusted D-dimer cutoff result.
Of the 3346 patients with suspected PE included, the prevalence of PE was 19%. Among the 2898 patients with a nonhigh or an unlikely clinical probability, 817 patients (28.2%) had a D-dimer level lower than 500 µg/L (95% CI, 26.6%-29.9%) and 337 patients (11.6%) had a D-dimer between 500 µg/L and their age-adjusted cutoff (95% CI, 10.5%-12.9%). The 3-month failure rate in patients with a D-dimer level higher than 500 µg/L but below the age-adjusted cutoff was 1 of 331 patients (0.3% 95% CI, 0.1%-1.7%). Among the 766 patients 75 years or older, of whom 673 had a nonhigh clinical probability, using the age-adjusted cutoff instead of the 500 µg/L cutoff increased the proportion of patients in whom PE could be excluded on the basis of D-dimer from 43 of 673 patients (6.4% 95% CI, 4.8%-8.5%) to 200 of 673 patients (29.7% 95% CI, 26.4%-33.3%), without any additional false-negative findings.
Compared with a fixed D-dimer cutoff of 500 µg/L, the combination of pretest clinical probability assessment with age-adjusted D-dimer cutoff was associated with a larger number of patients in whom PE could be considered ruled out with a low likelihood of subsequent clinical venous thromboembolism.
clinicaltrials.gov Identifier: NCT01134068.
Activated caspases are a hallmark of apoptosis induced by the intrinsic pathway, but they are dispensable for cell death and the apoptotic clearance of cells in vivo. This has led to the suggestion ...that caspases are activated not just to kill but to prevent dying cells from triggering a host immune response. Here, we show that the caspase cascade suppresses type I interferon (IFN) production by cells undergoing Bak/Bax-mediated apoptosis. Bak and Bax trigger the release of mitochondrial DNA. This is recognized by the cGAS/STING-dependent DNA sensing pathway, which initiates IFN production. Activated caspases attenuate this response. Pharmacological caspase inhibition or genetic deletion of caspase-9, Apaf-1, or caspase-3/7 causes dying cells to secrete IFN-β. In vivo, this precipitates an elevation in IFN-β levels and consequent hematopoietic stem cell dysfunction, which is corrected by loss of Bak and Bax. Thus, the apoptotic caspase cascade functions to render mitochondrial apoptosis immunologically silent.
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•Activation of Bak and Bax, essential mediators of apoptosis, triggers mtDNA release•mtDNA stimulates the cGAS/STING pathway to induce IFN-β production•Activation of the apoptotic caspase cascade blocks the cGAS/STING pathway•Inhibition or genetic deletion of caspases causes apoptotic cells to secrete IFN-β
In the absence of a functional caspases, dying cells behave as if virally infected, activating the cGAS/STING pathway to produce type I IFN, showing that the apoptotic caspase cascade functions to render apoptosis immunologically silent.
•This study relates to the social implications of (mobile) technology diffusion.•I hypothesise that phone diffusion undermines non-adopters’ healthcare access.•I use a panel of 12,003 sick households ...across rural India in 2005 and 2012.•Poor non-adopters’ access to private healthcare worsens during fast diffusion.•Wealthier households and public healthcare access are insulated from this trend.
The gains from digital technology diffusion are deemed essential for international development, but they are also distributed unevenly. Does the uneven distribution mean that not everyone benefits from new technologies to the same extent, or do some people experience an absolute disadvantage during this process? I explore this question through the case study of curative healthcare access in the context of rapid mobile phone uptake in rural India, contributing thus to an important yet surprisingly under-researched aspect of the social implications of (mobile) technology diffusion.
Inspired by a previous analysis of cross-sectional data from rural India, I hypothesise that health systems increasingly adapt to mobile phone users where phones have diffused widely. This adaptation will leave poor non-adopters worse off than before and increases healthcare inequities. I use a panel of 12,003 rural households with an illness in 2005 and 2012 from the Indian Human Development Survey to test this hypothesis. Based on village-cluster robust fixed-effects linear probability models, I find that (a) mobile phone diffusion is significantly and negatively linked to various forms of rural healthcare access, suggesting that health systems increasingly adapt to phone use and discriminate against non-users; that (b) poor rural households without mobile phones experience more adverse effects compared to more affluent households, which indicates a struggle and competition for healthcare access among marginalised groups; and that (c) no effects emerge for access to public doctors, which implies that some healthcare providers are less responsive to mobile phone use than others.
Overall, my findings indicate that the rural Indian healthcare system gradually adapts to increasing mobile phone use at the expense of non-users. I conclude that rapid mobile phone diffusion creates an opportunity to improve people’s access to healthcare in rural India, but it also creates new forms of marginalisation among poor rural households.
The CRISPR/Cas9 technology enables the introduction of genomic alterations into almost any organism; however, systems for efficient and inducible gene modification have been lacking, especially for ...deletion of essential genes. Here, we describe a drug-inducible small guide RNA (sgRNA) vector system allowing for ubiquitous and efficient gene deletion in murine and human cells. This system mediates the efficient, temporally controlled deletion of MCL-1, both in vitro and in vivo, in human Burkitt lymphoma cell lines that require this anti-apoptotic BCL-2 protein for sustained survival and growth. Unexpectedly, repeated induction of the same sgRNA generated similar inactivating mutations in the human Mcl-1 gene due to low mutation variability exerted by the accompanying non-homologous end-joining (NHEJ) process. Finally, we were able to generate hematopoietic cell compartment-restricted Trp53-knockout mice, leading to the identification of cancer-promoting mutants of this critical tumor suppressor.
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•A lentiviral CRISPR/Cas9 platform for the conditional targeting of essential genes•Efficient mutation of genes in mouse and human cell lines and primary mouse cells•Identification of cancer-promoting mutations in the tumor suppressor p53
The CRISPR/Cas9 system is an exciting methodology for genetic modification. Aubrey et al. have advanced this technology by developing an inducible lentiviral system. This platform facilitates efficient gene targeting that permits phenotypic assessment following deletion of essential genes and identification of tumor-promoting mutations in vivo.
A huge number of bacterial species are motile by flagella, which allow them to actively move toward favorable environments and away from hazardous areas and to conquer new habitats. The general ...perception of flagellum-mediated movement and chemotaxis is dominated by the
Escherichia coli
paradigm, with its peritrichous flagellation and its famous run-and-tumble navigation pattern, which has shaped the view on how bacteria swim and navigate in chemical gradients. However, a significant amount-more likely the majority-of bacterial species exhibit a (bi)polar flagellar localization pattern instead of lateral flagella. Accordingly, these species have evolved very different mechanisms for navigation and chemotaxis. Here, we review the earlier and recent findings on the various modes of motility mediated by polar flagella.
The tumour suppressor gene TP53 is mutated in ~50% of human cancers. In addition to its function in tumour suppression, p53 also plays a major role in the response of malignant as well as ...nontransformed cells to many anticancer therapeutics, particularly those that cause DNA damage. P53 forms a homotetrameric transcription factor that is reported to directly regulate ~500 target genes, thereby controlling a broad range of cellular processes, including cell cycle arrest, cell senescence, DNA repair, metabolic adaptation and cell death. For a long time, induction of apoptotic death in nascent neoplastic cells was regarded as the principal mechanism by which p53 prevents tumour development. This concept has, however, recently been challenged by the findings that in striking contrast to Trp53-deficient mice, gene-targeted mice that lack the critical effectors of p53-induced apoptosis do not develop tumours spontaneously. Remarkably, even mice lacking all mediators critical for p53-induced apoptosis, G1/S boundary cell cycle arrest and cell senescence do not develop any tumours spontaneously. In this review we discuss current understanding of the mechanisms by which p53 induces cell death and how this affects p53-mediated tumour suppression and the response of malignant cells to anticancer therapy.
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