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A new form of cell death has recently been proposed involving copper-induced cell death, termed cuproptosis. This new form of cell death has been widely studied in relation to a novel class of copper ionophores, including elesclomol and disulfiram. However, the exact mechanism leading to cell death remains contentious. The oldest and most widely accepted biological mechanism is that the accumulated intracellular copper leads to excessive build-up of reactive oxygen species and that this is what ultimately leads to cell death. Most of this evidence is largely based on studies using N-acetylcysteine (NAC), an antioxidant, to relieve the oxidative stress and prevent cell death. However, here we have demonstrated using inductively coupled mass-spectrometry, that NAC pretreatment significantly reduces intracellular copper uptake triggered by the ionophores, elesclomol and disulfiram, suggesting that reduction in copper uptake, rather than the antioxidant activity of NAC, is responsible for the diminished cell death. We present further data showing that key mediators of reactive oxygen species are not upregulated in response to elesclomol treatment, and further that sensitivity of cancer cell lines to reactive oxygen species does not correlate with sensitivity to these copper ionophores. Our findings are in line with several recent studies proposing the mechanism of cuproptosis is instead via copper mediated aggregation of proteins, resulting in proteotoxic stress leading to cell death. Overall, it is vital to disseminate this key piece of information regarding NAC's activity on copper uptake since new research attributing the effect of NAC on copper ionophore activity to quenching of reactive oxygen species is being published regularly and our studies suggest their conclusions may be misleading. [ABSTRACT FROM AUTHOR]

Background: Reliability of preclinical research is of critical concern. Prior studies have demonstrated the low reproducibility of research results and recommend implementing higher standards to improve overall quality and robustness of research. One understudied aspect of this quality issue is the harmony between the research hypotheses and the experimental design in published work., Methods and Findings: In this study we focused on highly cited cell culture studies and investigated whether commonly asserted cell culture claims such as viability, cytotoxicity, proliferation rate, cell death and apoptosis are backed with sufficient experimental evidence or not. We created an open access database containing 280 claims asserted by 103 different high-impact articles as well as the results of this study. Our findings revealed that only 64% of all claims were sufficiently supported by evidence and there were concerning misinterpretations such as considering the results of tetrazolium salt reduction assays as indicators of cell death or apoptosis., Conclusions: Our analysis revealed a discordance between experimental findings and the way they were presented and discussed in the manuscripts. To improve quality of pre-clinical research, we require clear nomenclature by which different cell culture claims are distinctively categorized; materials and methods sections to be written more meticulously; and cell culture methods to be selected and utilized more carefully. In this paper we recommend a nomenclature for selected cell culture claims as well as a methodology for collecting evidence to support those claims., Competing Interests: The authors have declared that no competing interests exist.

Adult T-cell leukemia/lymphoma (ATLL) originates from human T-cell leukemia virus type 1 (HTLV-1) infection due to the activation of the nuclear factor-κB (NF-κB) signaling pathway to maintain proliferation and survival. An important mechanism of the activated NF-κB signaling pathway in ATLL is the activation of the macroautophagy (herafter referred to as autophagy in the remainder of this manuscript)-lysosomal degradation of p47 (NSFL1C), a negative regulator of the NF-κB pathway. Therefore, we considered the use of chloroquine (CQ) or hydroxychloroquine (HCQ) (CQ/HCQ) as an autophagy inhibitor to treat ATLL; these drugs were originally approved by the FDA as antimalarial drugs and have recently been used to treat autoimmune diseases, such as systemic lupus erythematosus (SLE). In this paper, we determined the therapeutic efficacy of CQ/HCQ, as NF-κB inhibitors, in ATLL mediated by blockade of p47 degradation. Administration of CQ/HCQ to ATLL cell lines and primary ATLL cells induced cell growth inhibition in a dose-dependent manner, and the majority of cells underwent apoptosis after CQ administration. As to the molecular mechanism, autophagy was inhibited in CQ-treated ATLL cells, and activation of the NF-κB pathway was suppressed with the restoration of the p47 level. When the antitumor effect of CQ/HCQ was examined using immunodeficient mice transplanted with ATLL cell lines, CQ/HCQ significantly suppressed tumor growth and improved the survival rate in the ATLL xenograft mouse model. Importantly, HCQ selectively induced ATLL cell death in the ATLL xenograft mouse model at the dose used to treat SLE. Taken together, our results suggest that the inhibition of autophagy by CQ/HCQ may become a novel and effective strategy for the treatment of ATLL. [ABSTRACT FROM AUTHOR]

CD8 T cells are the predominant effector cells of adaptive immunity in preventing cytomegalovirus (CMV) multiple-organ disease caused by cytopathogenic tissue infection. The mechanism by which CMV-specific, naïve CD8 T cells become primed and clonally expand is of fundamental importance for our understanding of CMV immune control. For CD8 T-cell priming, two pathways have been identified: direct antigen presentation by infected professional antigen-presenting cells (pAPCs) and antigen cross-presentation by uninfected pAPCs that take up antigenic material derived from infected tissue cells. Studies in mouse models using murine CMV (mCMV) and precluding either pathway genetically or experimentally have shown that, in principle, both pathways can congruently generate the mouse MHC/H-2 class-I-determined epitope-specificity repertoire of the CD8 T-cell response. Recent studies, however, have shown that direct antigen presentation is the canonical pathway when both are accessible. This raised the question of why antigen cross-presentation is ineffective even under conditions of high virus replication thought to provide high amounts of antigenic material for feeding cross-presenting pAPCs. As delivery of antigenic material for cross-presentation is associated with programmed cell death, and as CMVs encode inhibitors of different cell death pathways, we pursued the idea that these inhibitors restrict antigen delivery and thus CD8 T-cell priming by cross-presentation. To test this hypothesis, we compared the CD8 T-cell responses to recombinant mCMVs lacking expression of the apoptosis-inhibiting protein M36 or the necroptosis-inhibiting protein M45 with responses to wild-type mCMV and revertant viruses expressing the respective cell death inhibitors. The data reveal that increased programmed cell death improves CD8 T-cell priming in mice capable of antigen cross-presentation but not in a mutant mouse strain unable to cross-present. These findings strongly support the conclusion that CMV cell death inhibitors restrict the priming of CD8 T cells by antigen cross-presentation. Author summary: In patients as well as in experimental mouse models, CD8 T cells represent the most potent antiviral effector cells in preventing CMV disease in immunocompromised recipients of hematopoietic cell transplantation. Despite the clinical relevance of mounting a protective response, the mode of CMV antigen presentation to naïve CD8 T cells was long debated. In principle, naïve CD8 T cells can be sensitized through "direct antigen presentation" on the surface of infected professional antigen-presenting cells (pAPCs) or through "antigen cross-presentation" by uninfected pAPCs that take up antigenic material derived from infected cells following cell death. As CMVs are cytopathogenic, eventually killing host cells, and as they encode immune evasion proteins interfering with the MHC/HLA class-I pathway of direct antigen presentation, it seemed reasonable to propose a dominant role for antigen cross-presentation. Mouse models precluding either mode of antigen presentation, however, revealed that both can raise an equivalent CD8 T-cell response, and recent work has identified direct antigen presentation as the canonical pathway taken when both are accessible. Here we show that virus-encoded inhibitors of two programmed cell death modalities, apoptosis and necroptosis, reduce antigen release to below a level required for efficient cross-presentation. This solves an open question in CMV immunology. [ABSTRACT FROM AUTHOR]

Understanding the dynamics of acute HIV infection can offer valuable insights into the early stages of viral behavior, potentially helping uncover various aspects of HIV pathogenesis. The standard viral dynamics model explains HIV viral dynamics during acute infection reasonably well. However, the model makes simplifying assumptions, neglecting some aspects of HIV infection. For instance, in the standard model, target cells are infected by a single HIV virion. Yet, cellular multiplicity of infection (MOI) may have considerable effects in pathogenesis and viral evolution. Further, when using the standard model, we take constant infected cell death rates, simplifying the dynamic immune responses. Here, we use four models—1) the standard viral dynamics model, 2) an alternate model incorporating cellular MOI, 3) a model assuming density-dependent death rate of infected cells and 4) a model combining (2) and (3)—to investigate acute infection dynamics in 43 people living with HIV very early after HIV exposure. We find that all models qualitatively describe the data, but none of the tested models is by itself the best to capture different kinds of heterogeneity. Instead, different models describe differing features of the dynamics more accurately. For example, while the standard viral dynamics model may be the most parsimonious across study participants by the corrected Akaike Information Criterion (AICc), we find that viral peaks are better explained by a model allowing for cellular MOI, using a linear regression analysis as analyzed by R2. These results suggest that heterogeneity in within-host viral dynamics cannot be captured by a single model. Depending on the specific aspect of interest, a corresponding model should be employed. Author summary: We conducted a study to better understand the dynamics of early HIV-1 infection using four different mathematical models. These models tested various biological hypotheses, including the presence of cellular coinfection and phenomenological model capturing aspects of immune responses. We analyzed viral load data from 43 participants in a rich dataset of people recently infected. The models we used were the standard viral dynamics model, a model incorporating density-dependent cell death of infected cells, a model with coinfection of infected cells (an adaptation of a macroparasite model to HIV-1 dynamics), and a model that combined cellular coinfection and density-dependent cell death. Overall, the model incorporating density-dependent cell death of infected cells performed the best according to the Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC). We also assessed the models' performance by comparing them based on measures such as viral growth rate, peak magnitude and timing, decay rate, and setpoint. We find that different models describe differing features of the dynamics more accurately. For example, viral peaks are better explained by a model allowing for cellular multiplicity of infection (MOI). These results suggest that heterogeneity in within-host viral dynamics cannot be captured by a single model. Therefore, depending on the aspect of interest, a corresponding appropriate model should be employed. [ABSTRACT FROM AUTHOR]

This study aims to investigate if high-concentration HOCl fogging disinfection causes cytotoxicity and genotoxicity to cultured primary human skin fibroblasts. The cells were exposed to a dry fog of HOCl produced from solutions with a concentration of 300 ppm (5.72 mM) or 500 ppm (9.53 mM). After four times when fibroblasts were exposed to aerosolized HOCl at a concentration of 500 ppm for 9 minutes, significant cytotoxicity and genotoxicity effects were observed. Significant changes in the morphology of fibroblasts and cell death due to membrane disruption were observed, independent of the number of exposures. Flow cytometry analyses performed under these experimental conditions indicated a decrease in the number of cells with an intact cell membrane in the exposed samples compared to the sham samples, dropping to 49.1% of the total cells. Additionally, under the same conditions, the neutral comet assay results demonstrated significant DNA damage in the exposed cells. However, no analogous damages were found when the cells were exposed to aerosolized HOCl generated from a 300-ppm solution for 3 minutes, whether once or four times. Therefore, we have concluded that aerosolized HOCl in dry fog, with a concentration exceeding 300 ppm, can cause cytotoxic and genotoxic effects on human skin fibroblasts. [ABSTRACT FROM AUTHOR]

Legionella pneumophila strains harboring wild-type rpsL such as Lp02rpsLWT cannot replicate in mouse bone marrow-derived macrophages (BMDMs) due to induction of extensive lysosome damage and apoptosis. The bacterial factor directly responsible for inducing such cell death and the host factor involved in initiating the signaling cascade that leads to lysosome damage remain unknown. Similarly, host factors that may alleviate cell death induced by these bacterial strains have not yet been investigated. Using a genome-wide CRISPR/Cas9 screening, we identified Hmg20a and Nol9 as host factors important for restricting strain Lp02rpsLWT in BMDMs. Depletion of Hmg20a protects macrophages from infection-induced lysosomal damage and apoptosis, allowing productive bacterial replication. The restriction imposed by Hmg20a was mediated by repressing the expression of several endo-lysosomal proteins, including the small GTPase Rab7. We found that SUMOylated Rab7 is recruited to the bacterial phagosome via SulF, a Dot/Icm effector that harbors a SUMO-interacting motif (SIM). Moreover, overexpression of Rab7 rescues intracellular growth of strain Lp02rpsLWT in BMDMs. Our results establish that L. pneumophila exploits the lysosomal network for the biogenesis of its phagosome in BMDMs. Author summary: The bacterial pathogen Legionella pneumophila extensively modulates vesicle trafficking pathways to create a membrane-bound vauole to support its intracellular growth. Although the role of the early phase of secretory pathway between the endoplasmic reticulum and the Golgi apparatus in the biogenesis of the Legionella-containing vacule (LCV) has been well-established, the role of the endosomal network in this process is poorly defined. Earlier studies using a bona fide wild-type L. pneumophila strain showed that Rab7, the small GTPase critical for the late endocytic pathway is enriched on the LCV but the mechanism of such recruitment is not understood. In this study, we used a genome-wide CRISPR/Cas9 screen to identify factors in mouse bone marrow-derived macrophages (BMDMs) that restrict intracellular growth of L. pneumophila strains such as Lp02RpsLWT harboring chromosomally encoded wild-type rpsL gene. We found that inactivation of Hmg20a, a gene involved in epigenetic regulation, allowed strain Lp02RpsLWT to grow in BMDMs. Further analysis revealed that Hmg20a indirectly governs this process by negatively controlling the expression of an array of genes involved in lysosomal biogenesis, including rab7. We found that overexpression of Rab7 alleviates the restriction of strain Lp02RpsLWT by BMDMs and only SUMOylated Rab7 can be recruited to the LCV. Finally, we identified the Dot/Icm substrate SulF as the bacterial factor responsible for Rab7 recruitment, which functions by binding to the SUMO moiety. Our results have established a molecular link between the LCV and the late endosome compartment, suggesting that L. pneumophila co-opts the endosomal network of host cells to support its intracellular growth. [ABSTRACT FROM AUTHOR]

Hepatitis delta virus (HDV) infection represents the most severe form of human viral hepatitis; however, the mechanisms underlying its pathology remain incompletely understood. We recently developed an HDV mouse model by injecting adeno-associated viral vectors (AAV) containing replication-competent HBV and HDV genomes. This model replicates many features of human infection, including liver injury. Notably, the extent of liver damage can be diminished with anti-TNF-α treatment. Here, we found that TNF-α is mainly produced by macrophages. Downstream of the TNF-α receptor (TNFR), the receptor-interacting serine/threonine-protein kinase 1 (RIPK1) serves as a cell fate regulator, playing roles in both cell survival and death pathways. In this study, we explored the function of RIPK1 and other host factors in HDV-induced cell death. We determined that the scaffolding function of RIPK1, and not its kinase activity, offers partial protection against HDV-induced apoptosis. A reduction in RIPK1 expression in hepatocytes through CRISPR-Cas9-mediated gene editing significantly intensifies HDV-induced damage. Contrary to our expectations, the protective effect of RIPK1 was not linked to TNF-α or macrophage activation, as their absence did not alter the extent of damage. Intriguingly, in the absence of RIPK1, macrophages confer a protective role. However, in animals unresponsive to type-I IFNs, RIPK1 downregulation did not exacerbate the damage, suggesting RIPK1's role in shielding hepatocytes from type-I IFN-induced cell death. Interestingly, while the damage extent is similar between IFNAR KO and WT mice in terms of transaminase elevation, their cell death mechanisms differ. In conclusion, our findings reveal that HDV-induced type-I IFN production is central to inducing hepatocyte death, and RIPK1's scaffolding function offers protective benefits. Thus, type-I IFN together with TNF-α, contribute to HDV-induced liver damage. These insights may guide the development of novel therapeutic strategies to mitigate HDV-induced liver damage and halt disease progression. Author summary: Hepatitis D is the most aggressive form of viral hepatitis. Our manuscript underscores the complexity of HDV-induced liver damage, where both viral and host factors play significant roles. Previously, we demonstrated that pharmacological inhibition of TNF-α reduced HDV-induced liver damage. This result was corroborated in the present study using TNF-α-deficient mice. Moreover, we reported that the expression of the HDV antigen might have a cytotoxic effect, and HDV replication induces a strong activation of the innate immune system, accompanied by a substantial production of IFN-β. In this study, we discovered that RIPK1, a molecule described as a cell fate modulator acting downstream of TNF-α, plays a protective role during HDV replication. Contrary to our expectations, neither TNF-α nor macrophages (the primary producers of TNF-α), contributed to this protective effect. Instead, it seems type I IFN was involved. Interestingly, the role of type I IFN in HBV-induced liver damage has recently been proposed. Furthermore, our data reveals that several mechanisms of hepatocyte death are at play simultaneously during HDV replication, with apoptosis being one of them. Additional studies are needed to identify other mechanisms involved. Finally, these findings suggest that therapies targeting TNF-α and type-I IFN, or those increasing RIPK1 levels, might be effective in preventing or treating HDV-induced liver damage. [ABSTRACT FROM AUTHOR]

BAK and BAX execute intrinsic apoptosis by permeabilising the mitochondrial outer membrane. Their activity is regulated through interactions with pro-survival BCL-2 family proteins and with non-BCL-2 proteins including the mitochondrial porin VDAC2. VDAC2 is important for bringing both BAK and BAX to mitochondria where they execute their apoptotic function. Despite this important function in apoptosis, while interactions with pro-survival family members are well characterised and have culminated in the development of drugs that target these interfaces to induce cancer cell apoptosis, the interaction between BAK and VDAC2 remains largely undefined. Deep scanning mutagenesis coupled with cysteine linkage identified key residues in the interaction between BAK and VDAC2. Obstructive labelling of specific residues in the BH3 domain or hydrophobic groove of BAK disrupted this interaction. Conversely, mutating specific residues in a cytosol-exposed region of VDAC2 stabilised the interaction with BAK and inhibited BAK apoptotic activity. Thus, this VDAC2–BAK interaction site can potentially be targeted to either inhibit BAK-mediated apoptosis in scenarios where excessive apoptosis contributes to disease or to promote BAK-mediated apoptosis for cancer therapy. BAK and BAX execute apoptotic cell death by increasing the permeability of the mitochondria outer membrane, and this is regulated by interactions with the mitochondrial channel protein VDAC2. This study maps the key residues involved in the BAK:VDAC2 interaction and demonstrate that this interaction can be targeted to either promote or inhibit apoptosis. [ABSTRACT FROM AUTHOR]

Microtubule-targeted agents are commonly used for cancer treatment, though many patients do not benefit. Microtubule-targeted drugs were assumed to elicit anticancer activity via mitotic arrest because they cause cell death following mitotic arrest in cell culture. However, we recently demonstrated that intratumoral paclitaxel concentrations are insufficient to induce mitotic arrest and rather induce chromosomal instability (CIN) via multipolar mitotic spindles. Here, we show in metastatic breast cancer and relevant human cellular models that this mechanism is conserved among clinically useful microtubule poisons. While multipolar divisions typically produce inviable progeny, multipolar spindles can be focused into near-normal bipolar spindles at any stage of mitosis. Using a novel method to quantify the rate of CIN, we demonstrate that cell death positively correlates with net loss of DNA. Spindle focusing decreases CIN and causes resistance to diverse microtubule poisons, which can be counteracted by addition of a drug that increases CIN without affecting spindle polarity. These results demonstrate conserved mechanisms of action and resistance for diverse microtubule-targeted agents. Trial registration: clinicaltrials.gov, NCT03393741. Microtubule-targeted drugs were assumed to elicit anti-cancer activity via mitotic arrest. This study uses experiments in metastatic breast cancer and relevant cellular models to reveal that diverse microtubule poisons instead exert anti-cancer effects due to aberrant transit through mitosis without mitotic arrest. [ABSTRACT FROM AUTHOR]

Background: Doxorubicin, an anthracycline chemotherapeutic known to incur heart damage, decreases heart function in up to 11% of patients. Recent investigations have implicated the Wnt signaling cascade as a key modulator of cardiac tissue repair after myocardial infarction. Wnt upregulation in murine models resulted in stimulation of angiogenesis and suppression of fibrosis after ischemic insult. However, the molecular mechanisms of Wnt in mitigating doxorubicin-induced cardiac insult require further investigation. Identifying cardioprotective mechanisms of Wnt is imperative to reducing debilitating cardiovascular adverse events in oncologic patients undergoing treatment. Methods: Exposing human cardiomyocyte AC16 cells to varying concentrations of Wnt10b and DOX, we observed key metrics of cell viability. To assess the viability and apoptotic rates, we utilized MTT and TUNEL assays. We quantified cell and mitochondrial membrane stability via LDH release and JC-1 staining. To investigate how Wnt10b mitigates doxorubicin-induced apoptosis, we introduced pharmacologic inhibitors of key enzymes involved in apoptosis: FR180204 and SB203580, ERK1/2 and p38 inhibitors. Further, we quantified apoptotic executor enzymes, caspase 3/7, via immunofluorescence. Results: AC16 cells exposed solely to doxorubicin were shrunken with distorted morphology. Cardioprotective effects of Wnt10b were demonstrated via a reduction in apoptosis, from 70.1% to 50.1%. LDH release was also reduced between doxorubicin and combination groups from 2.27-fold to 1.56-fold relative to the healthy AC16 control group. Mitochondrial membrane stability was increased from 0.67-fold in the doxorubicin group to 5.73 in co-treated groups relative to control. Apoptotic protein expression was stifled by Wnt10b, with caspase3/7 expression reduced from 2.4- to 1.3-fold, and both a 20% decrease in p38 and 40% increase in ERK1/2 activity. Conclusion: Our data with the AC16 cell model demonstrates that Wnt10b provides defense mechanisms against doxorubicin-induced cardiotoxicity and apoptosis. Further, we explain a mechanism of this beneficial effect involving the mitochondria through simultaneous suppression of pro-apoptotic p38 and anti-apoptotic ERK1/2 activities. [ABSTRACT FROM AUTHOR]

Disease is a neurodegenerative disorder characterised by the progressive loss of dopaminergic cells of the substantia nigra pars compacta. Even though successful transplantation of dopamine-producing cells into the striatum exhibits favourable effects in animal models and clinical trials; transplanted cell survival is low. Since every transplant elicits an inflammatory response which can affect cell survival and differentiation, we aimed to study in vivo and in vitro the impact of the pro-inflammatory environment on human dopaminergic precursors. We first observed that transplanted human dopaminergic precursors into the striatum of immunosuppressed rats elicited an early and sustained activation of astroglial and microglial cells after 15 days' post-transplant. This long-lasting response was associated with Tumour necrosis factor alpha expression in microglial cells. In vitro, conditioned media from activated BV2 microglial cells increased cell death, decreased Tyrosine hydroxylase-positive cells and induced morphological alterations on human neural stem cells-derived dopaminergic precursors at two differentiation stages: 19 days and 28 days. Those effects were ameliorated by inhibition of Tumour necrosis factor alpha, a cytokine which was previously detected in vivo and in conditioned media from activated BV-2 cells. Our results suggest that a pro-inflammatory environment is sustained after transplantation under immunosuppression, providing a window of opportunity to modify this response to increase transplant survival and differentiation. In addition, our data show that the microglia-derived pro-inflammatory microenvironment has a negative impact on survival and differentiation of dopaminergic precursors. Finally, Tumour necrosis factor alpha plays a key role in these effects, suggesting that this cytokine could be an interesting target to increase the efficacy of human dopaminergic precursors transplantation in Parkinson's Disease. [ABSTRACT FROM AUTHOR]

Accumulation of cytoplasmic inclusions of TAR-DNA binding protein 43 (TDP-43) is seen in both neurons and glia in a range of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Alzheimer's disease (AD). Disease progression involves non-cell autonomous interactions among multiple cell types, including neurons, microglia and astrocytes. We investigated the effects in Drosophila of inducible, glial cell type-specific TDP-43 overexpression, a model that causes TDP-43 protein pathology including loss of nuclear TDP-43 and accumulation of cytoplasmic inclusions. We report that TDP-43 pathology in Drosophila is sufficient to cause progressive loss of each of the 5 glial sub-types. But the effects on organismal survival were most pronounced when TDP-43 pathology was induced in the perineural glia (PNG) or astrocytes. In the case of PNG, this effect is not attributable to loss of the glial population, because ablation of these glia by expression of pro-apoptotic reaper expression has relatively little impact on survival. To uncover underlying mechanisms, we used cell-type-specific nuclear RNA sequencing to characterize the transcriptional changes induced by pathological TDP-43 expression. We identified numerous glial cell-type specific transcriptional changes. Notably, SF2/SRSF1 levels were found to be decreased in both PNG and in astrocytes. We found that further knockdown of SF2/SRSF1 in either PNG or astrocytes lessens the detrimental effects of TDP-43 pathology on lifespan, but extends survival of the glial cells. Thus TDP-43 pathology in astrocytes or PNG causes systemic effects that shorten lifespan and SF2/SRSF1 knockdown rescues the loss of these glia, and also reduces their systemic toxicity to the organism. Author summary: Neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Alzheimer's disease (AD) involve dysfunction in both neuronal and non-neuronal cells in the brain. Each of these disorders involves abnormal accumulation of a protein called TDP-43, which is thought to cause toxic effects that ultimately lead to cell death. We used cell type specific sequencing to characterize the effects of inducing pathological TDP-43 within each of the non-neuronal cell types in Drosophila. This identified changes in expression of a gene called SF2/SRSF1, which has previously been implicated in mediating toxic effects. We found that knocking down the expression of this gene within individual glial cell types was sufficient to prevent the loss of nearby neurons. [ABSTRACT FROM AUTHOR]

Hydroxychloroquine (HCQ) is a lysosomotropic agent that is commonly used for treating Sjögren's disease (SjD). However, its efficacy is controversial because of the divergent response to the drug among patients. In a subgroup of SjD patients, lysosome-associated membrane protein 3 (LAMP3) is elevated in expression in the salivary glands and promotes lysosomal dysregulation and lysosome-dependent apoptotic cell death. In this study, chloroquine (CQ) and its derivative HCQ were tested for their ability to prevent LAMP3-induced apoptosis, in vitro and on a mouse model of SjD. In addition, efficacy of HCQ treatment was retrospectively compared between high LAMP3 mRNA expression in minor salivary glands and those with LAMP3 mRNA levels comparable with healthy controls. Study results show that CQ treatment stabilized the lysosomal membrane in LAMP3-overexpressing cells via deactivation of cathepsin B, resulting in decreased apoptotic cell death. In mice with established SjD-like phenotype, HCQ treatment also significantly decreased apoptotic cell death and ameliorated salivary gland hypofunction. Retrospective analysis of SjD patients found that HCQ tended to be more effective in improving disease activity index, symptom severity and hypergammaglobulinemia in patients with high LAMP3 expression compared those with normal LAMP3 expression. Taken together, these findings suggested that by determining salivary gland LAMP3 mRNA level, a patient's response to HCQ treatment could be predicted. This finding may provide a novel strategy for guiding the development of more personalized medicine for SjD. [ABSTRACT FROM AUTHOR]

Cancer cells bypass cell death by changing the expression of the BCL-2 family of proteins, which are apoptotic pathway regulators. Upregulation of pro-survival BCL-2 proteins or downregulation of cell death effectors BAX and BAK interferes with the initiation of the intrinsic apoptotic pathway. In normal cells, apoptosis can occur through pro-apoptotic BH3-only proteins interacting and inhibiting pro-survival BCL-2 proteins. When cancer cells over-express pro-survival BCL-2 proteins, a potential remedy is the sequestration of these pro-survival proteins through a class of anti-cancer drugs called BH3 mimetics that bind in the hydrophobic groove of pro-survival BCL-2 proteins. To improve the design of these BH3 mimetics, the packing interface between BH3 domain ligands and pro-survival BCL-2 proteins was analyzed using the Knob-Socket model to identify the amino acid residues responsible for interaction affinity and specificity. A Knob-Socket analysis organizes all the residues in a binding interface into simple 4 residue units: 3-residue sockets defining surfaces on a protein that pack a 4th residue knob from the other protein. In this way, the position and composition of the knobs packing into sockets across the BH3/BCL-2 interface can be classified. A Knob-Socket analysis of 19 BCL-2 protein and BH3 helix co-crystals reveal multiple conserved binding patterns across protein paralogs. Conserved knob residues such as a Gly, Leu, Ala and Glu most likely define binding specificity in the BH3/BCL-2 interface, whereas other residues such as Asp, Asn, and Val are important for forming surface sockets that bind these knobs. These findings can be used to inform the design of BH3 mimetics that are specific to pro-survival BCL-2 proteins for cancer therapeutics. [ABSTRACT FROM AUTHOR]

While both NADPH and (p)ppGpp contain a purine moiety, the enzymatic activity of MESH1 toward NADPH is the cleavage at the 2'-position of ribose, in contrast to the 3'-position in (p)ppGpp. The best-characterized RSH proteins are the multi-domain long RHS proteins involved in the (p)ppGpp synthesis (RelA) and hydrolysis (SpoT). MESH1 can also hydrolyze (p)ppGpp, suggesting a conservation of the biochemical function between MESH1 and SpoT [[19]]. 5 Additional phenotypic analysis of MESH1 knockdown-phenotypic conservations In addition to the ferroptosis survival, MESH1 knockdown also triggers additional phenotypic responses. [Extracted from the article]

Effector proteins secreted by plant pathogens are essential for infection. Cytoplasmic RXLR effectors from oomycetes are characterized by the presence of RXLR and EER motifs that are frequently linked to WY- and/or LWY-domains, folds that are exclusive to this effector family. A related family of secreted candidate effector proteins, carrying WY-domains and the EER motif but lacking the canonical RXLR motif, has recently been described in oomycetes and is mainly found in downy mildew pathogens. Plasmopara viticola is an obligate biotrophic oomycete causing grapevine downy mildew. Here we describe a conserved Pl. viticola secreted candidate non-RXLR effector protein with cell death-inducing activity in Nicotiana species. A similar RXLR effector candidate from the broad host range oomycete pathogen Phytophthora parasitica also induces cell death in Nicotiana. Through comparative tertiary structure modelling, we reveal that both proteins are predicted to carry WY- and LWY-domains. Our work supports the presence of LWY-domains in non-RXLR effectors and suggests that effector candidates with similar domain architecture may exert similar activities. [ABSTRACT FROM AUTHOR]

Background: Topoisomerases are nuclear enzymes that get to the bottom of topological troubles related with DNA all through a range of genetic procedures. More and more studies have shown that topoisomerase-mediated DNA cleavage plays crucial roles in tumor cell death and carcinogenesis. There is however still a lack of comprehensive multi-omics studies related to topoisomerase family genes from a pan-cancer perspective. Methods: In this study, a multiomics pan-cancer analysis of topoisomerase family genes was conducted by integrating over 10,000 multi-dimensional cancer genomic data across 33 cancer types from The Cancer Genome Atlas (TCGA), 481 small molecule drug response data from cancer therapeutics response portal (CTRP) as well as normal tissue data from Genotype-Tissue Expression (GTEx). Finally, overall activity-level analyses of topoisomerase in pan-cancers were performed by gene set variation analysis (GSVA), together with differential expression, clinical relevancy, immune cell infiltration and regulation of cancer-related pathways. Results: Dysregulated gene expression of topoisomerase family were related to genomic changes and abnormal epigenetic modifications. The expression levels of topoisomerase family genes could significantly impact cancer progression, intratumoral heterogeneity, alterations in the immunological condition and regulation of the cancer marker-related pathways, which in turn caused the differences in potential drugs sensitivity and the distinct prognosis of patients. Conclusion: It was anticipated that topoisomerase family genes would become novel prognostic biomarkers for cancer patients and provide new insights for the diagnosis and treatment of tumors. [ABSTRACT FROM AUTHOR]

Buruli ulcer is a chronic skin disease caused by a toxic lipid mycolactone produced by Mycobacterium ulcerans, which induces local skin tissue destruction and analgesia. However, the cytotoxicity pathway induced by mycolactone remains largely unknown. Here we investigated the mycolactone-induced cell death pathway by screening host factors using a genome-scale lenti-CRISPR mutagenesis assay in human premonocytic THP-1 cells. As a result, 884 genes were identified as candidates causing mycolactone-induced cell death, among which SEC61A1, the α-subunit of the Sec61 translocon complex, was the highest scoring. CRISPR/Cas9 genome editing of SEC61A1 in THP-1 cells suppressed mycolactone-induced endoplasmic reticulum stress, especially eIF2α phosphorylation, and caspase-dependent apoptosis. Although previous studies have reported that mycolactone targets SEC61A1 based on mutation screening and structural analysis in several cell lines, we have reconfirmed that SEC61A1 is a mycolactone target by genome-wide screening in THP-1 cells. These results shed light on the cytotoxicity of mycolactone and suggest that the inhibition of mycolactone activity or SEC61A1 downstream cascades will be a novel therapeutic modality to eliminate the harmful effects of mycolactone in addition to the 8-week antibiotic regimen of rifampicin and clarithromycin. Author summary: Buruli ulcer is a chronic skin disease caused by the bacterium Mycobacterium ulcerans. The disease mainly affects children in West Africa, and the skin ulcers are induced by mycolactone, a toxin produced by the bacteria. The mycolactone diffuses through the skin, killing cells, creating irreversible ulceration, and weakening host immune defenses. However, the cytotoxic pathway induced by mycolactone remains largely unknown. We evaluated the mycolactone-induced cell death pathway by screening host factors using a genome-scale knockout assay in human premonocytic THP-1 cells. We identified 884 genes that are potentially involved in mycolactone-induced cell death, of which SEC61A1, the α-subunit of the Sec61 translocon complex, was the highest ranking. Knockout of SEC61A1 in THP-1 cells resulted in suppression of endoplasmic reticulum stress and caspase-dependent apoptosis induced by mycolactone. These results suggest that SEC61A1 is an essential mediator of mycolactone-induced cell death. [ABSTRACT FROM AUTHOR]

Synthetic lethality in DNA repair pathways is an important strategy for the selective treatment of cancer cells without harming healthy cells and developing cancer-specific drugs. The synthetic lethal interaction between the mismatch repair (MMR) protein, MutL homolog 1 (MLH1), and the mitochondrial base excision repair protein, DNA polymerase γ (Pol γ) was used in this study for the selective treatment of MLH1 deficient cancers. Germline mutations in the MLH1 gene and aberrant MLH1 promoter methylation result in an increased risk of developing many cancers, including nonpolyposis colorectal and endometrial cancers. Because the inhibition of Pol γ in MLH1 deficient cancer cells provides the synthetic lethal selectivity, we conducted a comprehensive small molecule screening from various databases and chemical drug library molecules for novel Pol γ inhibitors that selectively kill MLH1 deficient cancer cells. We characterized these Pol γ inhibitor molecules in vitro and in vivo, and identified 3,3'-[(1,1'-Biphenyl)-4',4'-diyl)bis(azo)]bis[4-amino-1-naphthalenesulfonic acid] (congo red; CR; Zinc 03830554) as a high-affinity binder to the Pol γ protein and potent inhibitor of the Pol γ strand displacement and one-nucleotide incorporation DNA synthesis activities in vitro and in vivo. CR reduced the cell proliferation of MLH1 deficient HCT116 human colon cancer cells and suppressed HCT116 xenograft tumor growth whereas it did not affect the MLH1 proficient cell proliferation and xenograft tumor growth. CR caused mitochondrial dysfunction and cell death by inhibiting Pol γ activity and oxidative mtDNA damage repair, increasing the production of reactive oxygen species and oxidative mtDNA damage in MLH1 deficient cells. This study suggests that the Pol γ inhibitor, CR may be further evaluated for the MLH1 deficient cancers' therapy. [ABSTRACT FROM AUTHOR]

Many adult tissues are composed of differentiated cells and stem cells, each working in a coordinated manner to maintain tissue homeostasis during physiological cell turnover. Old differentiated cells are believed to typically die by apoptosis. Here, we discovered a previously uncharacterized, new phenomenon, which we name erebosis based on the ancient Greek word erebos ("complete darkness"), in the gut enterocytes of adult Drosophila. Cells that undergo erebosis lose cytoskeleton, cell adhesion, organelles and fluorescent proteins, but accumulate Angiotensin-converting enzyme (Ance). Their nuclei become flat and occasionally difficult to detect. Erebotic cells do not have characteristic features of apoptosis, necrosis, or autophagic cell death. Inhibition of apoptosis prevents neither the gut cell turnover nor erebosis. We hypothesize that erebosis is a cell death mechanism for the enterocyte flux to mediate tissue homeostasis in the gut. It has been believed that gut enterocytes continuously die through apoptosis. However, this study shows that gut enterocytes die through a novel cell death mechanism, named erebosis. Erobotic cells lack the characteristic features of apoptotic, necrotic or autophagic cell death; instead they lose their cytoskeleton, cell adhesion and organelles, and their nuclei become flat and indistinct. [ABSTRACT FROM AUTHOR]

Tumors often harbor orders of magnitude more mutations than healthy tissues. The increased number of mutations may be due to an elevated mutation rate or frequent cell death and correspondingly rapid cell turnover, or a combination of the two. It is difficult to disentangle these two mechanisms based on widely available bulk sequencing data, where sequences from individual cells are intermixed and, thus, the cell lineage tree of the tumor cannot be resolved. Here we present a method that can simultaneously estimate the cell turnover rate and the rate of mutations from bulk sequencing data. Our method works by simulating tumor growth and finding the parameters with which the observed data can be reproduced with maximum likelihood. Applying this method to a real tumor sample, we find that both the mutation rate and the frequency of death may be high. Author summary: Tumors frequently harbor an elevated number of mutations, compared to healthy tissue. These extra mutations may be generated either by an increased mutation rate or the presence of cell death resulting in increased cellular turnover and additional cell divisions for tumor growth. Separating the effects of these two factors is a nontrivial problem. Here we present a method which can simultaneously estimate cell turnover rate and genomic mutation rate from bulk sequencing data. Our method is based on the estimation of the parameters of a generative model of tumor growth and mutations. Applying our method to a human hepatocellular carcinoma sample reveals an elevated per cell division mutation rate and high cell turnover. [ABSTRACT FROM AUTHOR]

Drug resistance, either intrinsic or acquired, can impair treatment effects and result in increased cell motility and death. MicroRNA-21 (miR-21), a proto-oncogene, may facilitate the development or maintenance of drug resistance in cancer cells. Restoring drug sensitivity can improve therapeutic strategies, a possibility that requires functional evaluation and mechanistic exploration. For miR-21 detection, matched tissue samples from 30 head and neck squamous cell carcinoma (HNSCC) patients and 8 head and neck cancer (HNC) cell lines were obtained. Reverse transcription-PCR to detect expression, MTT and clonogenic assays to evaluate cell proliferation, apoptosis assays, resazurin cell viability assays, western blot and luciferase reporter assays to detect protein expression, and flow cytometry to analyse the cell cycle were adopted. Compared to the corresponding normal control (NC) tissues, 25 cancer tissues had miR-21 upregulation among the 30 matched pair tissues (25/30, 83.8%); furthermore, among the 8 HNC cell lines, miR-21 expression that was notably upregulated in three: UPCI-4B, UMSCC-1, and UPCI-15B. In both the UMSCC-1 and UPCI-4B cell lines, the miR-21 mimic enhanced cell proliferation with reduced apoptosis and increased viability, whereas the miR-21 inhibitor resulted in the opposite effects (all P<0.001); additionally, miR-21 directly targeted the tumour suppressor phosphatase and tensin homologue (PTEN) and inhibited PTEN expression. Furthermore, the miR-21 mimic induced cisplatin resistance, while the miR-21 inhibitor restored cisplatin sensitivity. Overexpression of miR-21 can enhance cell proliferation, reduce apoptosis, and induce drug resistance by inhibiting PTEN expression. Targeting miR-21 may facilitate cancer diagnosis, restore drug sensitivity, and improve therapeutic effects. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic caused by SARS-CoV-2 has resulted in an urgent need for identifying potential therapeutic drugs. In the first half of 2020 tropic antimalarial drugs, such as chloroquine (CQ) or hydroxochloroquine (HCQ) were the focus of tremendous public attention. In the initial periods of the pandemic, many scientific results pointed out that CQ/HCQ could be very effective for patients with severe COVID. While CQ and HCQ have successfully been used against several diseases (such as malaria, autoimmune disease and rheumatic illnesses); long term use of these agents are associated with serious adverse effects (i.e. inducing acute kidney injury, among many others) due to their role in blocking autophagy-dependent self-degradation. Recent experimental and clinical trial data also confirmed that there is no sufficient evidence about the efficient usage of CQ/HCQ against COVID-19. By using systems biology techniques, here we show that the cellular effect of CQ/HCQ on autophagy during endoplasmic reticulum (ER) stress or following SARS-CoV-2 infection results in upregulation of ER stress. By presenting a simple mathematical model, we claim that although CQ/HCQ might be able to ameliorate virus infection, the permanent inhibition of autophagy by CQ/HCQ has serious negative effects on the cell. Since CQ/HCQ promotes apoptotic cell death, here we confirm that addition of CQ/HCQ cannot be really effective even in severe cases. Only a transient treatment seemed to be able to avoid apoptotic cell death, but this type of therapy could not limit virus replication in the infected host. The presented theoretical analysis clearly points out the utility and applicability of systems biology modelling to test the cellular effect of a drug targeting key major processes, such as autophagy and apoptosis. Applying these approaches could decrease the cost of pre-clinical studies and facilitate the selection of promising clinical trials in a timely fashion. [ABSTRACT FROM AUTHOR]

In many human cancers, the rate of cell growth depends crucially on the size of the tumour cell population. Low, zero, or negative growth at low population densities is known as the Allee effect; this effect has been studied extensively in ecology, but so far lacks a good explanation in the cancer setting. Here, we formulate and analyze an individual-based model of cancer, in which cell division rates are increased by the local concentration of an autocrine growth factor produced by the cancer cells themselves. We show, analytically and by simulation, that autocrine signaling suffices to cause both strong and weak Allee effects. Whether low cell densities lead to negative (strong effect) or reduced (weak effect) growth rate depends directly on the ratio of cell death to proliferation, and indirectly on cellular dispersal. Our model is consistent with experimental observations from three patient-derived brain tumor cell lines grown at different densities. We propose that further studying and quantifying population-wide feedback, impacting cell growth, will be central for advancing our understanding of cancer dynamics and treatment, potentially exploiting Allee effects for therapy. Author summary: A common feature of tumour growth is the production, by the cancer cells themselves, of hormones known as growth factors that increase the rate of cell division. This type of signalling makes the growth rate of the tumour depend on the population size in a non-linear manner, and the growth rate might become low or negative for small population sizes. This is known as the Allee effect which has been studied extensively in ecology. We have developed a computational model that can explain the Allee effect in terms of growth factor signalling, and show by mathematical analysis of the model that the magnitude of the Allee effect depends on the ratio of cell death to proliferation, as well as the properties of the growth factor. In addition we show that the model is consistent with experimental observations from three different cell lines derived from the brain tumour glioblastoma. Our findings indicate that the Allee effect can be exploited in order to improve the treatment of glioblastoma patients. [ABSTRACT FROM AUTHOR]

Clostridioides difficile infection (CDI) is the leading cause of nosocomial diarrhea and pseudomembranous colitis in the USA. In addition to these symptoms, patients with CDI can develop severe inflammation and tissue damage, resulting in life-threatening toxic megacolon. CDI is mediated by two large homologous protein toxins, TcdA and TcdB, that bind and hijack receptors to enter host cells where they use glucosyltransferase (GT) enzymes to inactivate Rho family GTPases. GT-dependent intoxication elicits cytopathic changes, cytokine production, and apoptosis. At higher concentrations TcdB induces GT-independent necrosis in cells and tissue by stimulating production of reactive oxygen species via recruitment of the NADPH oxidase complex. Although GT-independent necrosis has been observed in vitro, the relevance of this mechanism during CDI has remained an outstanding question in the field. In this study we generated novel C. difficile toxin mutants in the hypervirulent BI/NAP1/PCR-ribotype 027 R20291 strain to test the hypothesis that GT-independent epithelial damage occurs during CDI. Using the mouse model of CDI, we observed that epithelial damage occurs through a GT-independent process that does not involve immune cell influx. The GT-activity of either toxin was sufficient to cause severe edema and inflammation, yet GT activity of both toxins was necessary to produce severe watery diarrhea. These results demonstrate that both TcdA and TcdB contribute to disease pathogenesis when present. Further, while inactivating GT activity of C. difficile toxins may suppress diarrhea and deleterious GT-dependent immune responses, the potential of severe GT-independent epithelial damage merits consideration when developing toxin-based therapeutics against CDI. Author summary: Clostridioides difficile is an anaerobic spore-forming bacterium that is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis in the USA. This pathogen produces two protein toxins, TcdA and TcdB, that enter host colon cells to cause inflammation, fluid secretion, and cell death. The enzymatic activity of TcdB is a target for novel C. difficile infection (CDI) therapeutics since it is considered the major factor in causing severe CDI. However, necrotic cell death due to non-enzymatic TcdB-host interactions has been reported in cell culture and colonic explant experiments. Here, we generated C. difficile mutant strains with enzyme-inactive toxins to evaluate each toxin's role in an animal model of CDI. We observe an additive role for TcdA and TcdB in disease, and both glucosyltransferase-dependent and independent phenotypes. These findings are expected to inform the development of toxin-based CDI therapeutics. [ABSTRACT FROM AUTHOR]

Cellular senescence causes irreversible growth arrest of cells. Prolonged accumulation of senescent cells in tissues leads to increased detrimental effects due to senescence associated secretory phenotype (SASP). Recent findings suggest that elimination of senescent cells has a beneficial effect on organismal aging and lifespan. In this study, using a validated replicative senescent human dermal fibroblasts (HDFs) model, we showed that elimination of senescent cells is possible through the activation of an apoptotic mechanism. We have shown in this replicative senescence model, that cell senescence is associated with DNA damage and cell cycle arrest (p21, p53 markers). We have shown that Silybum marianum flower extract (SMFE) is a safe and selective senolytic agent targeting only senescent cells. The elimination of the cells is induced through the activation of apoptotic pathway confirmed by annexin V/propidium iodide and caspase-3/PARP staining. Moreover, SMFE suppresses the expression of SASP factors such as IL-6 and MMP-1 in senescent HDFs. In a co-culture model of senescent and young fibroblasts, we demonstrated that senescent cells impaired the proliferative capacities of young cells. Interestingly, when the co-culture is treated with SMFE, the cell proliferation rate of young cells is increased due to the decrease of the senescent burden. Moreover, we demonstrated in vitro that senescent fibroblasts trigger senescent process in normal keratinocytes through a paracrine effect. Indeed, the conditioned medium of senescent HDFs treated with SMFE reduced the level of senescence-associated beta-galactosidase (SA-β-Gal), p16INK4A and SASP factors in keratinocytes compared with CM of senescent HDFs. These results indicate that SMFE can prevent premature aging due to senescence and even reprograms aged skin. Indeed, thanks to its senolytic and senomorphic properties SMFE is a candidate for anti-senescence strategies. [ABSTRACT FROM AUTHOR]

Cytomegalovirus (CMV) infection has a major impact on the T-cell pool, which is thought to be associated with ageing of the immune system. The effect on the T-cell pool has been interpreted as an effect of CMV on non-CMV specific T-cells. However, it remains unclear whether the effect of CMV could simply be explained by the presence of large, immunodominant, CMV-specific memory CD8+ T-cell populations. These have been suggested to establish through gradual accumulation of long-lived cells. However, little is known about their maintenance. We investigated the effect of CMV infection on T-cell dynamics in healthy older adults, and aimed to unravel the mechanisms of maintenance of large numbers of CMV-specific CD8+ T-cells. We studied the expression of senescence, proliferation, and apoptosis markers and quantified the in vivo dynamics of CMV-specific and other memory T-cell populations using in vivo deuterium labelling. Increased expression of late-stage differentiation markers by CD8+ T-cells of CMV+ versus CMV- individuals was not solely explained by the presence of large, immunodominant CMV-specific CD8+ T-cell populations. The lifespans of circulating CMV-specific CD8+ T-cells did not differ significantly from those of bulk memory CD8+ T-cells, and the lifespans of bulk memory CD8+ T-cells did not differ significantly between CMV- and CMV+ individuals. Memory CD4+ T-cells of CMV+ individuals showed increased expression of late-stage differentiation markers and decreased Ki-67 expression. Overall, the expression of senescence markers on T-cell populations correlated positively with their expected in vivo lifespan. Together, this work suggests that i) large, immunodominant CMV-specific CD8+ T-cell populations do not explain the phenotypical differences between CMV+ and CMV- individuals, ii) CMV infection hardly affects the dynamics of the T-cell pool, and iii) large numbers of CMV-specific CD8+ T-cells are not due to longer lifespans of these cells. Author summary: Most people are infected with cytomegalovirus (CMV). Once infected, CMV stays in our body for the rest of our life. It rarely causes severe disease, thanks to T-cells that keep the virus at bay. CMV induces exceptionally large T-cell numbers. While typically no more than 1% of our T-cells recognize a certain virus, for CMV this can be as much as 50%. CMV is therefore used in vaccines to induce high T-cell numbers to other viruses or bacteria. A possible downside of CMV is that it is thought to lead to faster ageing of the immune system. We have studied whether the exceptionally large number of CMV-specific T-cells is due to a lack of cell death of these cells, and whether CMV infection affects T-cells to other viruses. We found that the changes in the T-cell pool of CMV-infected individuals cannot be explained by the presence of large numbers of CMV-specific T-cells. This suggests that CMV infection may also affect T-cells specific for other viruses. We also found that CMV-specific T-cells live as long as T-cells specific for other viruses. These insights are important for our understanding of the effects of CMV infection and for the development of CMV-based vaccines. [ABSTRACT FROM AUTHOR]

Background: The manual processes used for risk assessments are not scaling to the amount of data available. Although automated approaches appear promising, they must be transparent in a public policy setting. Objective: Our goal is to create an automated approach that moves beyond retrieval to the extraction step of the information synthesis process, where evidence is characterized as supporting, refuting, or neutral with respect to a given outcome. Methods: We combine knowledge resources and natural language processing to resolve coordinated ellipses and thus avoid surface level differences between concepts in an ontology and outcomes in an abstract. As with a systematic review, the search criterion, and inclusion and exclusion criterion are explicit. Results: The system scales to 482K abstracts on 27 chemicals. Results for three endpoints that are critical for cancer risk assessments show that refuting evidence (where the outcome decreased) was higher for cell proliferation (45.9%), and general cell changes (37.7%) than for cell death (25.0%). Moreover, cell death was the only end point where supporting claims were the majority (61.3%). If the number of abstracts that measure an outcome was used as a proxy for association there would be a stronger association with cell proliferation than cell death (20/27 chemicals). However, if the amount of supporting evidence was used (where the outcome increased) the conclusion would change for 21/27 chemicals (20 from proliferation to death and 1 from death to proliferation). Conclusions: We provide decision makers with a visual representation of supporting, neutral, and refuting evidence whilst maintaining the reproducibility and transparency needed for public policy. Our findings show that results from the retrieval step where the number of abstracts that measure an outcome are reported can be misleading if not accompanied with results from the extraction step where the directionality of the outcome is established. [ABSTRACT FROM AUTHOR]

Background: Immune checkpoint inhibition is effective in several cancers. Expression of programmed death-ligand 1 (PD-L1) on circulating tumor or immune effector cells could provide insights into selection of patients for immune checkpoint inhibition. Methods: Whole blood was collected at serial timepoints from metastatic breast cancer patients and healthy donors for circulating tumor cell (CTC) and platelet PD-L1 analysis with a phycoerythrin-labeled anti-human PD-L1 monoclonal antibody (Biolegend clone 29E.2A3) using the CellSearch® assay. CTC PD-L1 was considered positive if detected on at least 1% of the cells; platelet PD-L1 was considered positive if ≥100 platelets per CellSearch frame expressed PD-L1. Results: A total of 207 specimens from 124 metastatic breast cancer patients were collected. 52/124 (42%) samples at timepoint-1 (at or close to time of progressive disease) had ≥5 CTC/7.5ml whole blood. Of those, 21 (40%) had positive CTC PD-L1. In addition, platelet PD-L1 expression was observed in 35/124 (28%) at timepoint-1. Platelet PD-L1 was not detected in more than 70 specimens from 12 healthy donors. Platelet PD-L1 was associated with ≥5 CTC/7.5ml whole blood (p = 0.0002), less likely in patients with higher red blood cell counts (OR = 0.72, p<0.001) and a history of smoking tobacco (OR = 0.76, p<0.001). Platelet PD-L1 staining was not associated with tumor marker status, recent procedures or treatments, platelet-affecting drugs, or CTC PD-L1 expression. Conclusion: PD-L1 expression was found in metastatic breast cancer patients on both CTC and platelets in an independent fashion. Inter-patient platelet PD-L1 expression was highly heterogeneous suggesting that it is a biological event associated with cancer in some but not all patients. Taken together, our data suggest that CTC and platelet PD-L1 expression could play a role in predicting which patients should receive immune checkpoint inhibition and as a pharmacodynamics biomarker during treatment. [ABSTRACT FROM AUTHOR]

NK2 homeobox 1 (NKX2-1) is a thyroid transcription factor essential for proper thyroid formation and maintaining its physiological function. In thyroid cancer, NKX2-1 expression decreases in parallel with declined differentiation. However, the molecular pathways and mechanisms connecting NKX2-1 to thyroid cancer phenotypes are largely unknown. This study aimed to examine the effects of NKX2-1 re-expression on dedifferentiated thyroid cancer cell death and explore the underlying mechanisms. A human papillary thyroid carcinoma cell line lacking NKX2-1 expression was infected with an adenoviral vector containing Nkx2-1. Cell viability decreased after Nkx2-1 transduction and apoptosis and necrosis were detected. Arginase 2 (ARG2), regulator of G protein signaling 4 (RGS4), and RGS5 mRNA expression was greatly increased in Nkx2-1-transducted cells. After suppressing these genes by siRNA, cell death, apoptosis, and necrosis decreased in RGS4 knockdown cells. These findings demonstrated that cell death was induced via apoptosis and necrosis by NKX2-1 re-expression and involves RGS4. [ABSTRACT FROM AUTHOR]

Background: For their functions of insulin biosynthesis and glucose- and fatty acid- mediated insulin secretion, Langerhans β-cells require an intracellular milieu rich in oxygen. This requirement makes β-cells, with their constitutively low antioxidative defense, susceptible to the oxidative stress. Although much progress has been made in identifying its molecular basis in experimental systems, whether the oxidative stress due to excessive fatty acids plays a crucial role in the Langerhans cell degeneration in primates is still debated. Methods: Focusing on Hsp70.1, which has dual functions as molecular chaperone and lysosomal stabilizer, the mechanism of lipotoxicity to Langerhans cells was studied using macaque monkeys after the consecutive injections of the lipid peroxidation product 'hydroxynonenal'. Based on the 'calpain-cathepsin hypothesis' formulated in 1998, calpain activation, Hsp70.1 cleavage, and lysosomal integrity were studied by immunofluorescence histochemistry, electron microscopy, and Western blotting. Results: Light microscopy showed more abundant vacuole formation in the hydroxynonenal-treated islet cells than the control cells. Electron microscopy showed that vacuolar changes, which were identified as enlarged rough ER, occurred mainly in β-cells followed by δ-cells. Intriguingly, both cell types showed a marked decrease in insulin and somatostatin granules. Furthermore, they exhibited marked increases in peroxisomes, autophagosomes/autolysosomes, lysosomal and peroxisomal membrane rupture/permeabilization, and mitochondrial degeneration. Disrupted peroxisomes were often localized in the close vicinity of degenerating mitochondria or autolysosomes. Immunofluorescence histochemical analysis showed an increased co-localization of activated μ-calpain and Hsp70.1 with the extralysosomal release of cathepsin B. Western blotting showed increases in μ-calpain activation, Hsp70.1 cleavage, and expression of the hydroxynonenal receptor GPR109A. Conclusions: Taken together, these data implicate hydroxynonenal in both oxidation of Hsp70.1 and activation of μ-calpain. The calpain-mediated cleavage of the carbonylated Hsp70.1, may cause lysosomal membrane rupture/permeabilization. The low defense of primate Langerhans cells against hydroxynonenal and peroxisomally-generated hydrogen peroxide, was presumably overwhelmed to facilitate cell degeneration. [ABSTRACT FROM AUTHOR]

Several studies have revealed a correlation between chronic inflammation and nicotinamide adenine dinucleotide (NAD+) metabolism, but the precise mechanism involved is unknown. Here, we report that the genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the salvage pathway of NAD+ biosynthesis, reduced oxidative stress, inflammation, and keratinocyte DNA damage, hyperproliferation, and cell death in zebrafish models of chronic skin inflammation, while all these effects were reversed by NAD+ supplementation. Similarly, genetic and pharmacological inhibition of poly(ADP-ribose) (PAR) polymerase 1 (Parp1), overexpression of PAR glycohydrolase, inhibition of apoptosis-inducing factor 1, inhibition of NADPH oxidases, and reactive oxygen species (ROS) scavenging all phenocopied the effects of Nampt inhibition. Pharmacological inhibition of NADPH oxidases/NAMPT/PARP/AIFM1 axis decreased the expression of pathology-associated genes in human organotypic 3D skin models of psoriasis. Consistently, an aberrant induction of NAMPT and PARP activity, together with AIFM1 nuclear translocation, was observed in lesional skin from psoriasis patients. In conclusion, hyperactivation of PARP1 in response to ROS-induced DNA damage, fueled by NAMPT-derived NAD+, mediates skin inflammation through parthanatos cell death. A study of chronic skin inflammation reveals that hyperactivation of PARP1 in response to ROS-induced DNA damage, fueled by NAMPT-derived NAD+, mediates skin inflammation via parthanatos cell death, identifying NAMPT, PARP1 and AIFM1 as novel therapeutic targets for psoriasis. [ABSTRACT FROM AUTHOR]

The purpose of this study was to explore potential mechanisms of cytotoxicity towards HeLa and HT29 cells displayed by Pediocin PA-1. We did this by carrying out sequence alignments and 3D modelling of related bacteriocins which have been studied in greater detail: Microcin E492, Enterocin AB heterodimer and Divercin V41. Microcin E492 interacts with Toll-Like Receptor 4 in order to activate an apoptosis reaction, sequence alignment showed a high homology between Pediocin PA-1 and Microcin E492 whereas 3D modelling showed Pediocin PA-1 interacting with TLR-4 in a way reminiscent of Microcin E492. Furthermore, Pediocin PA-1 had the highest homology with the Enterocin heterodimer, particularly chain A; Enterocin has also shown to cause an apoptotic response in cancer cells. Based on this we are led to strongly believe Pediocin PA-1 interacts with TLRs in order to cause cell death. If this is the case, it would explain the difference in cytotoxicity towards HeLa over HT29 cells, due to difference in expression of particular TLRs. Overall, we believe Pediocin PA-1 exhibits a dual effect which is dose dependant, like that of Microcin. Unfortunately, due to the COVID-19 pandemic, we were unable to carry out experiments in the lab, and the unavailability of important data meant we were unable to provide and validate out solid conclusions, but rather suggestions. However, bioinformatic analysis is still able to provide information regarding structure and sequence analysis to draw plausible and evidence based conclusions. We have been able to highlight interesting findings and how these could be translated into future research and therapeutics in order to improve the quality of treatment and life of cancer patients. [ABSTRACT FROM AUTHOR]

Background: Gastric cancer (GC) is a multifactorial disease with high mortality. Anti-HER2 therapy is a promising strategy in GC treatment and trastuzumab was approved by FDA (Food and Drug Administration) as the first and the second line of treatment of the disease. Purpose: The aim of the study was to examine the effectiveness of a combination of etoposide with trastuzumab or pertuzumab in AGS gastric cancer cells and breast cancer cells such as MCF-7, MDA-MB-231 and HCC1954. Methods and findings: The cytotoxic effects of the tested compounds against gastric and breast cancer cells were checked by MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) assay. The anti-proliferative potential was analyzed by the incorporation of [3H]-thymidine into DNA. Fluorescent microscopy and flow cytometry was used to demonstrate the effect of the compounds on apoptosis. The mitochondrial membrane potential, and the activity of caspase-8 and caspase-9 were assessed. Autophagosomes and autolysosomes formation was checked by flow cytometry. The concentrations of Beclin-1, LC3A and LC3B were performed using ELISA. The expression of LC3A/B was also determined. The results from our study proved that the combination of etoposide with anti-HER2 antibodies was not cytotoxic against breast cancer cells, whereas the combination of etoposide with anti-HER2 antibodies decreased viability and DNA biosynthesis in gastric cancer cells. The interaction of etoposide with pertuzumab or trastuzumab induced programmed cell death via extrinsic and intrinsic apoptotic pathways in AGS gastric cancer cells, but did not affect autophagy, where a decrease of Beclin-1, LC3A and LC3B was observed in comparison with the untreated control. Conclusions: The study demonstrated that etoposide (12.5 μM) with pertuzumab represent a promising strategy in gastric cancer treatment, but further in vivo examinations are also required. [ABSTRACT FROM AUTHOR]

In plants, nucleotide-binding domain and leucine-rich repeat (NLR)-containing proteins can form receptor networks to confer hypersensitive cell death and innate immunity. One class of NLRs, known as NLR required for cell death (NRCs), are central nodes in a complex network that protects against multiple pathogens and comprises up to half of the NLRome of solanaceous plants. Given the prevalence of this NLR network, we hypothesised that pathogens convergently evolved to secrete effectors that target NRC activities. To test this, we screened a library of 165 bacterial, oomycete, nematode, and aphid effectors for their capacity to suppress the cell death response triggered by the NRC-dependent disease resistance proteins Prf and Rpi-blb2. Among 5 of the identified suppressors, 1 cyst nematode protein and 1 oomycete protein suppress the activity of autoimmune mutants of NRC2 and NRC3, but not NRC4, indicating that they specifically counteract a subset of NRC proteins independently of their sensor NLR partners. Whereas the cyst nematode effector SPRYSEC15 binds the nucleotide-binding domain of NRC2 and NRC3, the oomycete effector AVRcap1b suppresses the response of these NRCs via the membrane trafficking-associated protein NbTOL9a (Target of Myb 1-like protein 9a). We conclude that plant pathogens have evolved to counteract central nodes of the NRC immune receptor network through different mechanisms. Coevolution with pathogen effectors may have driven NRC diversification into functionally redundant nodes in a massively expanded NLR network. Plant pathogens have evolved to counteract their hosts' immune systems. A screen for pathogen effectors that suppress sensor NLR-mediated cell death in tobacco identifies effectors from a cyst nematode and an oomycete that suppress the NRC branch of the immune network to inhibit the immune-related cell death response. [ABSTRACT FROM AUTHOR]