Discovery of new small molecules that can activate distinct programmed cell death pathway is of significant interest as a research tool and for the development of novel therapeutics for pathological ...conditions such as cancer and infectious diseases. The small molecule raptinal was discovered as a pro-apoptotic compound that can rapidly trigger apoptosis by promoting the release of cytochrome c from the mitochondria and subsequently activating the intrinsic apoptotic pathway. As raptinal is very effective at inducing apoptosis in a variety of different cell types in vitro and in vivo, it has been used in many studies investigating cell death as well as the clearance of dying cells. While examining raptinal as an apoptosis inducer, we unexpectedly identified that in addition to its pro-apoptotic activities, raptinal can also inhibit the activity of caspase-activated Pannexin 1 (PANX1), a ubiquitously expressed transmembrane channel that regulates many cell death-associated processes. By implementing numerous biochemical, cell biological and electrophysiological approaches, we discovered that raptinal can simultaneously induce apoptosis and inhibit PANX1 activity. Surprisingly, raptinal was found to inhibit cleavage-activated PANX1 via a mechanism distinct to other well-described PANX1 inhibitors such as carbenoxolone and trovafloxacin. Furthermore, raptinal also interfered with PANX1-regulated apoptotic processes including the release of the 'find-me' signal ATP, the formation of apoptotic cell-derived extracellular vesicles, as well as NLRP3 inflammasome activation. Taken together, these data identify raptinal as the first compound that can simultaneously induce apoptosis and inhibit PANX1 channels. This has broad implications for the use of raptinal in cell death studies as well as in the development new PANX1 inhibitors.
Vascular dementia (VaD) is the second most common form of dementia in the United States and is characterized as a cerebral vessel vascular disease that leads to ischemic episodes. Whereas the ...relationship between caspase-cleaved tau and neurofibrillary tangles (NFTs) in Alzheimer's disease (AD) has been previously described, whether caspase activation and cleavage of tau occurs in VaD is presently unknown. To investigate a potential role for caspase-cleaved tau in VaD, we analyzed seven confirmed cases of VaD by immunohistochemistry utilizing a well-characterized antibody that specifically detects caspase-cleaved tau truncated at Asp421. Application of this antibody (TauC3) revealed consistent labeling within NFTs, dystrophic neurites within plaque-rich regions and corpora amylacea (CA) in the human VaD brain. Labeling of CA by the TauC3 antibody was widespread throughout the hippocampus proper, was significantly higher compared to age matched controls, and co-localized with ubiquitin. Staining of the TauC3 antibody co-localized with MC-1, AT8, and PHF-1 within NFTs. Quantitative analysis indicated that roughly 90% of PHF-1-labeled NFTs contained caspase-cleaved tau. In addition, we documented the presence of active caspase-3 within plaques, blood vessels and pretangle neurons that co-localized with TauC3. Collectively, these data support a role for the activation of caspase-3 and proteolytic cleavage of TauC3 in VaD providing further support for the involvement of this family of proteases in NFT pathology.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The master transcriptional regulator PU.1/Spi-1 engages DNA sites with affinities spanning multiple orders of magnitude. To elucidate this remarkable plasticity, we have characterized 22 ...high-resolution co-crystallographic PU.1/DNA complexes across the addressable affinity range in myeloid gene transactivation. Over a purine-rich core (such as 5ʹ-GGAA-3ʹ) flanked by variable sequences, affinity is negotiated by direct readout on the 5ʹ flank via a critical glutamine (Q226) sidechain and by indirect readout on the 3ʹ flank by sequence-dependent helical flexibility. Direct readout by Q226 dynamically specifies PU.1’s characteristic preference for purines and explains the pathogenic mutation Q226E in Waldenström macroglobulinemia. The structures also reveal how disruption of Q226 mediates strand-specific inhibition by DNA methylation and the recognition of non-canonical sites, including the authentic binding sequence at the CD11b promoter. A re-synthesis of phylogenetic and structural data on the ETS family, considering the centrality of Q226 in PU.1, unifies the model of DNA selection by ETS proteins.
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•High-resolution PU.1 structures unveil the dynamic basis of DNA specificity•Single evolutionary innovation (Q226) determines PU.1’s distinct target profile•Basis of pathogenic Q226E mutation in Waldenström macroglobulinemia•The structures correct the crystallographic record on PU.1/DNA binding
Terrell et al. present 22 high-resolution structures of co-crystal DNA complexes of the master myeloid transcription factor PU.1. They show the role of folded-state dynamics of PU.1 ETS domain in DNA recognition, including strand-specific CpG methylation and recognition of non-canonical targets, over an affinity range of three orders of magnitude.
Abstract
Background
Prone positioning (PP) improves oxygenation and respiratory mechanics and is associated with lower mortality in patients with moderate to severe acute respiratory distress ...syndrome (ARDS). Despite this, some patients develop refractory hypoxemia and hypercapnia requiring venovenous extracorporeal membrane oxygenation (VV ECMO) support and are usually cared for in supine position. The physiologic and outcome benefits of routine PP of patients during VV ECMO remains unclear. Hence, we conducted the systematic review and meta-analysis to evaluate the outcome benefits of PP for patients with ARDS being treated with VV ECMO.
Methods
After registration with PROSPERO (CRD42020199723), MEDLINE, EMBASE, Scopus and Cochrane databases were searched for relevant studies that reported PP in more than 10 adult patients supported with VV ECMO from origin to 1 March 2021. Studies were reviewed for quality using appropriate Joanna Briggs Institute (JBI) checklists, and certainty of evidence was assessed using the GRADE approach. The random-effects model (DerSimonian and Laird) was used. The primary outcome of interest was cumulative survival. Secondary outcomes were intensive care unit length of stay (ICU LOS) and ECMO duration. Changes in arterial blood gas (ABG) values, ventilator mechanics and complication rates were also studied.
Results
Of 812 potentially relevant publications, 12 studies (640 patients) met our inclusion criteria. Due to overlapping study populations, 11 studies were included in the final meta-analysis. Cumulative survival in patients that underwent PP was 57% (95% CI 41.9–71.4, high certainty). Patients that underwent PP had longer ICU LOS (+ 14.5 days, 95% CI 3.4–25.7,
p
= 0.01) and ECMO duration (+ 9.6 days, 95% CI 5.5–13.7,
p
< 0.0001). After PP, patients had significantly higher PaO
2
/FiO
2
ratio, lower PaCO
2
and reduced ventilator driving pressure, and no major complications were reported.
Conclusions
PP during VV ECMO appears safe with a cumulative survival of 57% and may result in longer ECMO runs and ICU LOS. However, evidence from appropriately designed randomized trials is needed prior to widespread adoption of PP on VV ECMO.
Acute myeloid leukemia (AML) is an aggressive hematological cancer resulting from uncontrolled proliferation of differentiation-blocked myeloid cells. Seventy percent of AML patients are currently ...not cured with available treatments, highlighting the need of novel therapeutic strategies. A promising target in AML is the mammalian target of rapamycin complex 1 (mTORC1). Clinical inhibition of mTORC1 is limited by its reactivation through compensatory and regulatory feedback loops. Here, we explored a strategy to curtail these drawbacks through inhibition of an important effector of the mTORC1signaling pathway, the eukaryotic initiation factor 4A (eIF4A).
We tested the anti-leukemic effect of a potent and specific eIF4A inhibitor (eIF4Ai), CR-1-31-B, in combination with cytosine arabinoside (araC) or the BCL2 inhibitor venetoclax. We utilized the MOLM-14 human AML cell line to model chemoresistant disease both in vitro and in vivo. In eIF4Ai-treated cells, we assessed for changes in survival, apoptotic priming, de novo protein synthesis, targeted intracellular metabolite content, bioenergetic profile, mitochondrial reactive oxygen species (mtROS) and mitochondrial membrane potential (MMP).
eIF4Ai exhibits anti-leukemia activity in vivo while sparing non-malignant myeloid cells. In vitro, eIF4Ai synergizes with two therapeutic agents in AML, araC and venetoclax. EIF4Ai reduces mitochondrial membrane potential (MMP) and the rate of ATP synthesis from mitochondrial respiration and glycolysis. Furthermore, eIF4i enhanced apoptotic priming while reducing the expression levels of the antiapoptotic factors BCL2, BCL-XL and MCL1. Concomitantly, eIF4Ai decreases intracellular levels of specific metabolic intermediates of the tricarboxylic acid cycle (TCA cycle) and glucose metabolism, while enhancing mtROS. In vitro redox stress contributes to eIF4Ai cytotoxicity, as treatment with a ROS scavenger partially rescued the viability of eIF4A inhibition.
We discovered that chemoresistant MOLM-14 cells rely on eIF4A-dependent cap translation for survival in vitro and in vivo. EIF4A drives an intrinsic metabolic program sustaining bioenergetic and redox homeostasis and regulates the expression of anti-apoptotic proteins. Overall, our work suggests that eIF4A-dependent cap translation contributes to adaptive processes involved in resistance to relevant therapeutic agents in AML.
Abstract
Background
While recommended by international societal guidelines in the paediatric population, the use of venoarterial extracorporeal membrane oxygenation (VA ECMO) as mechanical ...circulatory support for refractory septic shock in adults is controversial. We aimed to characterise the outcomes of adults with septic shock requiring VA ECMO, and identify factors associated with survival.
Methods
We searched Pubmed, Embase, Scopus and Cochrane databases from inception until 1st June 2021, and included all relevant publications reporting on > 5 adult patients requiring VA ECMO for septic shock. Study quality and certainty in evidence were assessed using the appropriate Joanna Briggs Institute checklist, and the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) approach, respectively. The primary outcome was survival to hospital discharge, and secondary outcomes included intensive care unit length of stay, duration of ECMO support, complications while on ECMO, and sources of sepsis. Random-effects meta-analysis (DerSimonian and Laird) were conducted.
Data synthesis
We included 14 observational studies with 468 patients in the meta-analysis. Pooled survival was 36.4% (95% confidence interval CI: 23.6%–50.1%). Survival among patients with left ventricular ejection fraction (LVEF) < 20% (62.0%, 95%-CI: 51.6%–72.0%) was significantly higher than those with LVEF > 35% (32.1%, 95%-CI: 8.69%–60.7%,
p
= 0.05). Survival reported in studies from Asia (19.5%, 95%-CI: 13.0%–26.8%) was notably lower than those from Europe (61.0%, 95%-CI: 48.4%–73.0%) and North America (45.5%, 95%-CI: 16.7%–75.8%). GRADE assessment indicated high certainty of evidence for pooled survival.
Conclusions
When treated with VA ECMO, the majority of patients with septic shock and severe sepsis-induced myocardial depression survive. However, VA ECMO has poor outcomes in adults with septic shock without severe left ventricular depression. VA ECMO may be a viable treatment option in carefully selected adult patients with refractory septic shock.
Despite the increasing use of pyrolytic carbon (pyrocarbon) hemiarthroplasty (PyCHA), clinical data reporting on its outcomes remain scarce. To date, no studies have compared the outcomes of stemmed ...PyCHA vs. conventional hemiarthroplasty (HA) and anatomic total shoulder arthroplasty (aTSA) in young patients. The primary aim of this study was to report on the outcomes of the first 159 stemmed PyCHAs performed in New Zealand. The secondary aim was to compare the outcomes of stemmed PyCHA vs. HA and aTSA in patients aged <60 years with osteoarthritis. We hypothesized that stemmed PyCHA would be associated with a low revision rate. We further hypothesized that in young patients, PyCHA would be associated with a lower revision rate and superior functional outcomes compared with HA and aTSA.
Data from the New Zealand National Joint Registry were used to identify patients who underwent PyCHA, HA, and aTSA between January 2000 and July 2022. The total number of revisions in the PyCHA group was determined, and the indications for surgery, reasons for revision, and types of revision were recorded. In patients aged <60 years, a matched-cohort analysis was performed comparing functional outcomes using the Oxford Shoulder Score (OSS). The revision rate of PyCHA was compared with that of HA and aTSA, calculated as revisions per 100 component-years.
In total, 159 cases of stemmed PyCHA were performed and 5 cases underwent revision, resulting in an implant retention rate of 97%. Among patients aged <60 years with shoulder osteoarthritis, 48 underwent PyCHA compared with 150 who underwent HA and 550 who underwent aTSA. Patients treated with aTSA had a superior OSS compared with PyCHA and HA patients. The difference in the OSS between the aTSA and PyCHA groups exceeded the minimal clinically important difference of 4.3. There was no difference in revision rates between the groups.
This study represents the largest cohort of patients treated with PyCHA and is the first to compare stemmed PyCHA with HA and aTSA in young patients. In the short term, PyCHA appear to be a promising implant with an excellent implant retention rate. In patients aged <60 years, the revision rate is comparable between PyCHA and aTSA. However, aTSA remains the implant of choice to optimize early postoperative function. Further studies are required to elucidate the long-term outcomes of PyCHA, particularly how they compare with those of HA and aTSA in young patients.
Tumorigenesis involves multistep genetic alterations. To elucidate the microRNA (miRNA)–gene interaction network in carcinogenesis, we examined their genome‐wide expression profiles in 96 pairs of ...tumor/non‐tumor tissues from hepatocellular carcinoma (HCC). Comprehensive analysis of the coordinate expression of miRNAs and mRNAs reveals that miR‐122 is under‐expressed in HCC and that increased expression of miR‐122 seed‐matched genes leads to a loss of mitochondrial metabolic function. Furthermore, the miR‐122 secondary targets, which decrease in expression, are good prognostic markers for HCC. Transcriptome profiling data from additional 180 HCC and 40 liver cirrhotic patients in the same cohort were used to confirm the anti‐correlation of miR‐122 primary and secondary target gene sets. The HCC findings can be recapitulated in mouse liver by silencing miR‐122 with antagomir treatment followed by gene‐expression microarray analysis. In vitro miR‐122 data further provided a direct link between induction of miR‐122‐controlled genes and impairment of mitochondrial metabolism. In conclusion, miR‐122 regulates mitochondrial metabolism and its loss may be detrimental to sustaining critical liver function and contribute to morbidity and mortality of liver cancer patients.
Synopsis
Hepatocellular carcinoma (HCC) is one of the most aggressive human malignancies, common in Asia, Africa, and in areas with endemic infections of hepatitis‐B or ‐C viruses (HBV or HCV) (But et al, 2008). Globally, the 5‐year survival rate of HCC is <5% and about 600 000 HCC patients die each year. The high mortality associated with this disease is mainly attributed to the failure to diagnose HCC patients at an early stage and a lack of effective therapies for patients with advanced stage HCC. Understanding the relationships between phenotypic and molecular changes in HCC is, therefore, of paramount importance for the development of improved HCC diagnosis and treatment methods.
In this study, we examined mRNA and microRNA (miRNA)‐expression profiles of tumor and adjacent non‐tumor liver tissue from HCC patients. The patient population was selected from a region of endemic HBV infection, and HBV infection appears to contribute to the etiology of HCC in these patients. A total of 96 HCC patients were included in the study, of which about 88% tested positive for HBV antigen; patients testing positive for HCV antigen were excluded. Among the 220 miRNAs profiled, miR‐122 was the most highly expressed miRNA in liver, and its expression was decreased almost two‐fold in HCC tissue relative to adjacent non‐tumor tissue, confirming earlier observations (Lagos‐Quintana et al, 2002; Kutay et al, 2006; Budhu et al, 2008).
Over 1000 transcripts were correlated and over 1000 transcripts were anti‐correlated with miR‐122 expression. Consistent with the idea that transcripts anti‐correlated with miR‐122 are potential miR‐122 targets, the most highly anti‐correlated transcripts were highly enriched for the presence of the miR‐122 central seed hexamer, CACTCC, in the 3′UTR. Although the complete set of negatively correlated genes was enriched for cell‐cycle genes, the subset of seed‐matched genes had no significant KEGG Pathway annotation, suggesting that miR‐122 is unlikely to directly regulate the cell cycle in these patients. In contrast, transcripts positively correlated with miR‐122 were not enriched for 3′UTR seed matches to miR‐122. Interestingly, these 1042 transcripts were enriched for genes coding for mitochondrially localized proteins and for metabolic functions.
To analyze the impact of loss of miR‐122 in vivo, silencing of miR‐122 was performed by antisense inhibition (anti‐miR‐122) in wild‐type mice (Figure 3). As with the genes negatively correlated with miR‐122 in HCC patients, no significant biological annotation was associated with the seed‐matched genes up‐regulated by anti‐miR‐122 in mouse livers. The most significantly enriched biological annotation for anti‐miR‐122 down‐regulated genes, as for positively correlated genes in HCC, was mitochondrial localization; the down‐regulated mitochondrial genes were enriched for metabolic functions. Putative direct and downstream targets with orthologs on both the human and mouse microarrays showed significant overlap for regulations in the same direction. These overlaps defined sets of putative miR‐122 primary and secondary targets. The results were further extended in the analysis of a separate dataset from 180 HCC, 40 cirrhotic, and 6 normal liver tissue samples (Figure 4), showing anti‐correlation of proposed primary and secondary targets in non‐healthy tissues.
To validate the direct correlation between miR‐122 and some of the primary and secondary targets, we determined the expression of putative targets after transfection of miR‐122 mimetic into PLC/PRF/5 HCC cells, including the putative direct targets SMARCD1 and MAP3K3 (MEKK3), a target described in the literature, CAT‐1 (SLC7A1), and three putative secondary targets, PPARGC1A (PGC‐1α) and succinate dehydrogenase subunits A and B. As expected, the putative direct targets showed reduced expression, whereas the putative secondary target genes showed increased expression in cells over‐expressing miR‐122 (Figure 4).
Functional classification of genes using the total ancestry method (Yu et al, 2007) identified PPARGC1A (PGC‐1α) as the most connected secondary target. PPARGC1A has been proposed to function as a master regulator of mitochondrial biogenesis (Ventura‐Clapier et al, 2008), suggesting that loss of PPARGC1A expression may contribute to the loss of mitochondrial gene expression correlated with loss of miR‐122 expression. To further validate the link of miR‐122 and PGC‐1α protein, we transfected PLC/PRF/5 cells with miR‐122‐expression vector, and observed an increase in PGC‐1α protein levels. Importantly, transfection of both miR‐122 mimetic and miR‐122‐expression vector significantly reduced the lactate content of PLC/PRF/5 cells, whereas anti‐miR‐122 treatment increased lactate production. Together, the data support the function of miR‐122 in mitochondrial metabolic functions.
Patient survival was not directly associated with miR‐122‐expression levels. However, miR‐122 secondary targets were expressed at significantly higher levels in both tumor and adjacent non‐tumor tissues among survivors as compared with deceased patients, providing supporting evidence for the potential relevance of loss of miR‐122 function in HCC patient morbidity and mortality.
Overall, our findings reveal potentially new biological functions for miR‐122 in liver physiology. We observed decreased expression of miR‐122, a liver‐specific miRNA, in HBV‐associated HCC, and loss of miR‐122 seemed to correlate with the decrease of mitochondrion‐related metabolic pathway gene expression in HCC and in non‐tumor liver tissues, a result that is consistent with the outcome of treatment of mice with anti‐miR‐122 and is of prognostic significance for HCC patients. Further investigation will be conducted to dissect the regulatory function of miR‐122 on mitochondrial metabolism in HCC and to test whether increasing miR‐122 expression can improve mitochondrial function in liver and perhaps in liver tumor tissues. Moreover, these results support the idea that primary targets of a given miRNA may be distributed over a variety of functional categories while resulting in a coordinated secondary response, potentially through synergistic action (Linsley et al, 2007).
A moderate loss of miR‐122 function correlates with up‐regulation of seed‐matched genes and down‐regulation of mitochondrially localized genes in both human hepatocellular carcinoma and in normal mice treated with anti‐miR‐122 antagomir.
Putative direct targets up‐regulated with loss of miR‐122 and secondary targets down‐regulated with loss of miR‐122 are conserved between human beings and mice and are rapidly regulated in vitro in response to miR‐122 over‐ and under‐expression.
Loss of miR‐122 secondary target expression in either tumorous or adjacent non‐tumorous tissue predicts poor survival of heptatocellular carcinoma patients.
Previous studies have demonstrated that temporarily increasing the permeability of the blood-brain barrier using focused ultrasound can reduce β-amyloid plaque load and improve cognitive function in ...animal models of Alzheimer's disease. However, the underlying mechanism and duration for which the effects of one treatment persists for are unknown. Here, we used in vivo two-photon fluorescence microscopy to track changes in β-amyloid plaque sizes in the TgCRND8 mouse model of Alzheimer's disease after one focused ultrasound treatment. We found that one treatment reduced plaques to 62 ± 16% (p ≤ 0.001) of their original volume two days post-sonication; this decrease in size persisted for two weeks. We then sought to evaluate the effectiveness of biweekly focused ultrasound treatments using magnetic resonance imaging-guided focused ultrasound treatments. Three to five biweekly treatments resulted in a 27 ± 7% (p ≤ 0.01) decrease in plaque number and 40 ± 10% (p ≤ 0.01) decrease in plaque surface area compared to untreated littermates. This study demonstrates that one focused ultrasound treatment reduces the size of existing β-amyloid plaques for two weeks, and that repeated biweekly focused ultrasound treatments is an effective method of reducing β-amyloid pathology in moderate-to-late stages of Alzheimer's disease.
Heparanase is a β‐d‐endoglucuronidase that cleaves heparan sulphate, a key component of the ECM and basement membrane. The remodelling of the ECM by heparanase has been proposed to regulate both ...normal physiological and pathological processes, including wound healing, inflammation, tumour angiogenesis and cell migration. Heparanase is also known to exhibit non‐enzymatic functions by regulating cell adhesion, cell signalling and differentiation. In this study, constitutive heparanase‐deficient (Hpse−/−) mice were generated on a C57BL/6 background using the Cre/loxP recombination system, with a complete lack of heparanase mRNA, protein and activity. Although heparanase has been implicated in embryogenesis and development, Hpse−/− mice are anatomically normal and fertile. Interestingly, consistent with the suggested function of heparanase in cell migration, the trafficking of dendritic cells from the skin to the draining lymph nodes was markedly reduced in Hpse−/− mice. Furthermore, the ability of Hpse−/− mice to generate an allergic inflammatory response in the airways, a process that requires dendritic cell migration, was also impaired. These findings establish an important role for heparanase in immunity and identify the enzyme as a potential target for regulation of an immune response.
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