Protein kinases play key roles in oncogenic signaling and are a major focus in the development of targeted cancer therapies. Imatinib, a BCR-Abl tyrosine kinase inhibitor, is a successful front-line ...treatment for chronic myelogenous leukemia (CML). However, resistance to imatinib may be acquired by BCR-Abl mutations or hyperactivation of Src family kinases such as Lyn. We have used multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in an imatinib-resistant (MYL-R) and -sensitive (MYL) cell model of CML. Using MIB/MS, expression and activity changes of over 150 kinases were quantitatively measured from various protein kinase families. Statistical analysis of experimental replicates assigned significance to 35 of these kinases, referred to as the MYL-R kinome profile. MIB/MS and immunoblotting confirmed the over-expression and activation of Lyn in MYL-R cells and identified additional kinases with increased (MEK, ERK, IKKα, PKCβ, NEK9) or decreased (Abl, Kit, JNK, ATM, Yes) abundance or activity. Inhibiting Lyn with dasatinib or by shRNA-mediated knockdown reduced the phosphorylation of MEK and IKKα. Because MYL-R cells showed elevated NF-κB signaling relative to MYL cells, as demonstrated by increased IκBα and IL-6 mRNA expression, we tested the effects of an IKK inhibitor (BAY 65-1942). MIB/MS and immunoblotting revealed that BAY 65-1942 increased MEK/ERK signaling and that this increase was prevented by co-treatment with a MEK inhibitor (AZD6244). Furthermore, the combined inhibition of MEK and IKKα resulted in reduced IL-6 mRNA expression, synergistic loss of cell viability and increased apoptosis. Thus, MIB/MS analysis identified MEK and IKKα as important downstream targets of Lyn, suggesting that co-targeting these kinases may provide a unique strategy to inhibit Lyn-dependent imatinib-resistant CML. These results demonstrate the utility of MIB/MS as a tool to identify dysregulated kinases and to interrogate kinome dynamics as cells respond to targeted kinase inhibition.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Over the last decade, there have been significant advances in treatments for anterior ischemic stroke, most notably endovascular thrombectomy (EVT). Despite the success of EVT on overall outcomes, ...intracerebral hemorrhage (ICH) is an important post-procedure complication, often associated with mortality and disability. Hence, predicting the risk of ICH can inform EVT decision making. The ASPECT score is used globally to predict patients' prognosis post-reperfusion therapy. Our objective is to perform a systematic review to collect and synthesize data on the association between ASPECT scores on CT, CTP and DWI-MRI (CT-ASPECT, CTP-ASPECT, and DWI-ASPECT) and the risk of symptomatic ICH after EVT for anterior circulation strokes. We will conduct a broad search of various electronic databases (MEDLINE, EMBASE, CINAHL, PsycINFO, Web of Science, and the Cochrane Database of Systematic Reviews) to identify studies published after January 1st, 2012 (commonly accepted as the beginning of the modern EVT era based on availability of stent-retrievers). Two independent reviewers will screen and include studies evaluating associations between symptomatic ICH after thrombectomy and baseline CT-ASPECT, CTP-ASPECT and DWI-ASPECT scores. Data will be extracted to quantify the risk of sICH after EVT based on the ASPECT scoring.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Kes1, and other oxysterol-binding protein superfamily members, are involved in membrane and lipid trafficking through trans-Golgi network (TGN) and endosomal systems. We demonstrate that Kes1 ...represents a sterol-regulated antagonist of TGN/endosomal phosphatidylinositol-4-phosphate signaling. This regulation modulates TOR activation by amino acids and dampens gene expression driven by Gcn4, the primary transcriptional activator of the general amino acid control regulon. Kes1-mediated repression of Gcn4 transcription factor activity is characterized by nonproductive Gcn4 binding to its target sequences, involves TGN/endosome-derived sphingolipid signaling, and requires activity of the cyclin-dependent kinase 8 (CDK8) module of the enigmatic “large Mediator” complex. These data describe a pathway by which Kes1 integrates lipid metabolism with TORC1 signaling and nitrogen sensing.
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► Kes1 coordinates sterol-binding with PtdIns-4-P signaling on Golgi/endosomes ► Kes1 regulates sphingolipid signaling and activation of TOR by amino acids ► Sphingolipids dampen activity of the nutrient stress transcription factor Gcn4 ► CDK module of large Mediator is required for Gcn4 regulation by sphingolipids
Kes1 regulates endosomal trafficking, integrating lipid metabolism with nutrient sensing to modulate proliferation and maintain cellular homeostasis.
Reversible cerebral vasoconstriction syndrome (RCVS) is a syndrome of recurrent thunderclap headaches and reversible vasoconstriction of the cerebral arteries on neuroimaging within 3 months of ...onset. Initial non-contrast computed tomography (CT) can reveal abnormalities such as ischemic stroke, intracerebral hemorrhage, and subarachnoid hemorrhage (SAH) can be present in patients with RCVS and may delay diagnosis.
We conducted a systematic review and meta-analysis in accordance with the PRISMA guidelines. We aimed to estimate the prevalence of imaging abnormalities on initial non-contrast CT head in adult patients with RCVS.
We searched electronic databases including MEDLINE, EMBASE, and the Cochrane Register of Clinical Trials from inception to August 2, 2022. Eligible studies included articles reporting the prevalence of non-contrast CT abnormalities on initial neuroimaging in patients with RCVS, aged 18 and older. Case series, observational studies and clinical trials were included. Data was extracted directly from included papers using a standardized data charting form.
The search yielded 722 titles with duplicates removed. Twenty studies that included 379 patients with RCVS met inclusion criteria. We classified non-contrast CT abnormalities as either ischemic stroke, ICH, or SAH. We pooled prevalence data using a random effects model with the inverse-variance weighted method. The most common imaging finding was SAH with a pooled prevalence of 24% (95% CI:17%-33%), followed by ICH at 14% (95% CI:8%-22%), and ischemic stroke at 10% (95% CI:7%-14%). The pooled prevalence of any of these imaging abnormalities on initial non-contrast CT was 31% (95% CI:23%-40%). Risk of bias was moderate to very-high-risk for case-series and low-risk for observational studies.
Our review demonstrates that one-third of patients with RCVS will have an abnormality on initial non-contrast CT head, including either an ischemic stroke, ICH, or SAH. These findings highlight the diagnostic challenges of RCVS imaging and contribute to our understanding of this disease.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The metastasis-associated tyrosine phosphatase PRL-3/PTP4A is upregulated in numerous cancers, but the mechanisms modulating PRL-3 activity other than its expression levels have not been ...investigated. Here we report evidence for both Src-dependent tyrosine phosphorylation of PRL-3 and Src-mediated regulation of PRL-3 biological activities. We used structural mutants, pharmacological inhibitors and siRNA to demonstrate Src-dependent phosphorylation of endogenous PRL-3 in SW480 colon cancer cells. We also demonstrated that PRL-3 was not tyrosine phosphorylated in SYF mouse embryo fibroblasts deficient in Src, Yes and Fyn unless Src was re-expressed. Further, we show that platelet-derived growth factor (PDGF) can stimulate PRL-3 phosphorylation in a Src-dependent manner. Finally, we show that PRL-3-induced cell motility, Matrigel invasion and activation of the cytoskeleton-regulating small GTPase RhoC were abrogated in the presence of the phosphodeficient PRL-3 mutant Y53F, or by use of a Src inhibitor. Thus, PRL-3 requires the activity of a Src kinase, likely Src itself, to promote these cancer-associated phenotypes. Our data establish a model for the regulation of PRL-3 by Src that supports the possibility of their coordinate roles in signaling pathways promoting invasion and metastasis, and supports simultaneous use of novel molecularly targeted therapeutics directed at these proteins.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
ObjectiveAdvance consent is a recognised method of obtaining informed consent for participation in research, whereby a potential participant provides consent for future involvement in a study ...contingent on qualifying for the study’s inclusion criteria on a later date. The goal of this study is to map the existing literature on the use of advance consent for enrolment in randomised controlled trials (RCTs) for emergency conditions.DesignScoping review designed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses–Extension for Scoping Reviews guidelines.Data sourcesWe searched electronic databases including MEDLINE, Embase, Web of Science and the Cochrane Register of Clinical Trials from inception to 10 February 2020.Eligibility criteriaEligible studies included articles that discussed or employed the use of advance consent for enrolment in RCTs related to emergency conditions. There were no restrictions on the type of eligible study. Data were extracted directly from included papers using a standardised data charting form. We produced a narrative review including article type and authors’ dispositions towards advance consent.ResultsOur search yielded 1039 titles with duplicates removed. Six articles met inclusion criteria. Three articles discussed the theoretical use of research advance directives in emergency conditions; one article evaluated stakeholders’ perceptions of advance consent; and one article described a method for patients to document their preferences for participation in future research. Only one study employed advance consent to enrol participants into a clinical trial for an emergency condition.ConclusionOur review demonstrates that there has been minimal exploration of advance consent for enrolment in RCTs for emergency conditions. Future studies could aim to assess the acceptability of advance consent to participants, along with the feasibility of enrolling research participants using this method of consent.ProtocolThe protocol for this scoping review was published a priori.
Background and purpose Recovery after intracerebral haemorrhage (ICH) is often slower than ischemic stroke. Despite this, ICH research often quantifies recovery using the same outcome measures ...obtained at the same timepoints as ischemic stroke. The primary objective of this scoping review is to map the existing literature to determine when and how outcomes are being measured in prospective studies of recovery after ICH. Methods We searched MEDLINE, Embase, Cochrane Central Register of Controlled Trials and Web of Science from inception to November 2019, for prospective studies that included patients with ICH. Two investigators independently screened the studies and extracted data around timing and type of outcome assessment. Results Among the 9761 manuscripts reviewed, 395 met inclusion criteria, of which 276 were observational studies and 129 were interventional studies that enrolled 66274 patients. Mortality was assessed in 93% of studies. Functional outcomes were assessed in 85% of studies. The most frequently used functional assessment tool was the modified Rankin Scale (mRS) (60%), followed by the National Institute of Health Stroke Severity Scale (22%) and Barthel Index (21%). The most frequent timepoint at which mortality was assessed was 90 days (41%), followed by 180 days (18%) and 365 days (12%), with 2% beyond 1 year. The most frequent timepoint used for assessing mRS was 90 days (62%), followed by 180 days (21%) and 365 days (17%). Conclusion While most prospective ICH studies report mortality and functional outcomes only at 90 days, a significant proportion do so at 1 year and beyond. Our results support the feasibility of collecting long-term outcome data to optimally assess recovery in ICH.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Equipoise, generally defined as uncertainty about the relative effects of the treatments being compared in a trial, is frequently referenced as an ethical standard for the conduct of randomized ...clinical trials. However, it seems to be defined in several different ways and may be used differently by different individuals. We explored how clinical researchers, chairs of research ethics boards, and philosophers of science define and reason with this term.
We completed semi-structured interviews about clinical trial ethics with 15 clinical researchers, 15 research ethics board chairs, and 15 philosophers of science/bioethicists. Each participant was asked a standardized set of 10 questions, 4 of which were specifically about equipoise. All interviews were conducted telephonically and transcribed. Responses were grouped and analysed via a modified grounded theory method.
Forty-three respondents defined equipoise in 7 logically distinct ways, and 2 respondents could not explicitly define it. The most common definition, offered by 14 respondents (31%), defined "equipoise" as a disagreement at the level of a community of physicians. There was significant variability in definitions offered between and within groups. When asked how they would "operationalize" equipoise - i.e. check or test for its presence - respondents provided 7 alternatives, the most common being in relation to a literature review (15/45, 33%). The vast majority of respondents (35/45, 78%) felt the concept was helpful, though many acknowledged that the lack of a clear definition or operationalization was problematic.
There is significant variation in definitions of equipoise offered by respondents, suggesting that parties within groups and between groups may be referring to different concepts when they reference "equipoise". This non-uniformity may impact fairness and transparency and opens the door to potential ethical problems in the evaluation of clinical trials - for instance, a patient may understand equipoise very differently than the researchers enrolling her in a trial, which could cause her agreement to participate to be based upon false premises.
Under the pandemic conditions created by the novel coronavirus of 2019 (COVID-19), physicians have faced difficult choices allocating scarce resources, including but not limited to critical care beds ...and ventilators. Past experiences with severe acute respiratory syndrome (SARS) and current reports suggest that making these decisions carries a heavy emotional toll for physicians around the world. We sought to explore Canadian physicians' preparedness and attitudes regarding resource allocation decisions.
From April 3 to April 13, 2020, we conducted an 8-question online survey of physicians practicing in the region of Ottawa, Ontario, Canada, organized around 4 themes: physician preparedness for resource rationing, physician preparedness to offer palliative care, attitudes towards resource allocation policy, and approaches to resource allocation decision-making.
We collected 219 responses, of which 165 were used for analysis. The majority (78%) of respondents felt "somewhat" or "a little prepared" to make resource allocation decisions, and 13% felt "not at all prepared." A majority of respondents (63%) expected the provision of palliative care to be "very" or "somewhat difficult." Most respondents (83%) either strongly or somewhat agreed that there should be policy to guide resource allocation. Physicians overwhelmingly agreed on certain factors that would be important in resource allocation, including whether patients were likely to survive, and whether they had dementia and other significant comorbidities. Respondents generally did not feel confident that they would have the social support they needed at the time of making resource allocation decisions.
This rapidly implemented survey suggests that a sample of Canadian physicians feel underprepared to make resource allocation decisions, and desire both more emotional support and clear, transparent, evidence-based policy.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
IntroductionAlthough previously thought to be a rare occurrence, spontaneous recanalisation is not uncommon, with a growing number of reports describing this phenomenon. However, the frequency, time ...course and mechanism of spontaneous recanalisation remain unknown. A better characterisation of these events is essential to ensuring adequate identification and proper future trial design for treatment.ObjectiveTo describe the current body of literature around spontaneous recanalisation following internal carotid occlusion.Methods and analysisWith the assistance of an information specialist, we will search MEDLINE, Embase, Cochrane Central Register for Controlled Trials and Web of Science for studies of adults with spontaneous recanalisation or transient occlusion of the internal carotid artery. Two reviewers will independently collect data on included studies pertaining to publication data, study population information, timepoints of initial presentation, recanalisation and subsequent follow-up.Ethics and disseminationPrimary data will not be collected; therefore, formal ethics is not required. The findings of this study will be disseminated through peer-reviewed publications and presentations at academic conferences.
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