Imaging for Predicting Hemorrhagic Transformation of Acute Ischemic Stroke-A Narrative Review.

Hemorrhagic transformation is caused by extravasation of blood products from vessels after acute ischemic stroke. It is an undesirable and potentially devastating complication, which occurs in 10%-40% of clinical cases. Hemorrhagic transformation is classified into four subtypes based on European cooperative acute stroke study II. Predicting hemorrhagic complications at presentation can be useful life saving/altering decisions for the patient. Also, understanding the mechanisms of hemorrhagic transformation can lead to new treatments and intervention measures. We highlighted various imaging techniques that have been used to predict hemorrhagic transformation. Specifically, we looked at the usefulness of perfusion and permeability imaging for hemorrhagic transformation. Use of imaging to predict hemorrhagic transformation could change patient management that may lead to the prevention of hemorrhagic transformation before it occurs. We concluded that the current evidence is not strong enough to rely on these imaging parameters for predicting hemorrhagic transformation and more studies are required.

Prognostic Value of Initial Diagnostic Imaging Findings for Patient Outcomes in Adult Patients with Traumatic Brain Injury: A Systematic Review and Meta-Analysis.

INTRODUCTION: Termed the "silent epidemic," traumatic brain injury (TBI) is one of the greatest global contributors not only to post-traumatic death but also to post-traumatic long-term disability. This systematic review and meta-analysis aims to specifically evaluate the prognostic value of features on initial imaging completed within 24 h of arrival in adult patients with TBI. METHOD: The authors followed the PRISMA 2020 checklist for systematic review and meta-analysis design and reporting. Comprehensive searches of the Medline and Embase databases were carried out. Two independent readers extracted the following demographic, clinical and imaging information using a predetermined data abstraction form. Statistics were performed using Revman 5.4.1 and R version 4.2.0. For pooled data in meta-analysis, forest plots for sensitivity and specificity were created to calculate the diagnostic odds ratio (DOR). Summary receiver operating characteristic (SROC) curves were generated using a bivariate model, and diagnostic accuracy was determined using pooled sensitivity and specificity as well as the area under the receiver operator characteristic curve (AUC). RESULTS: There were 10,733 patients over the 19 studies. Overall, most of the studies included had high levels of bias in multiple, particularly when it came to selection bias in patient sampling, bias in controlling for confounders, and reporting bias, such as in reporting missing data. Only subdural hematoma (SDH) and mortality in all TBI patients had both an AUC with 95% CI not crossing 0.5 and a DOR with 95% CI not crossing 1, at 0.593 (95% CI: 0.556-0.725) and 2.755 (95% CI: 1.474-5.148), respectively. CONCLUSION: In meta-analysis, only SDH with mortality in all TBI patients had a moderate but significant association. Given the small number of studies, additional research focused on initial imaging, particularly for imaging modalities other than NECT, is required in order to confirm the findings of our meta-analysis and to further evaluate the association of imaging findings and outcome.

CT perfusion for Assessment of poor Neurological outcome in Comatose Cardiac Arrest Patients (CANCCAP): protocol for a prospective study.

INTRODUCTION: Cardiac arrest remains one of the most common causes of death with the majority occurring outside of hospitals (out of hospital cardiac arrest). Despite advancements in resuscitation management, approximately 50% of comatose cardiac arrest patients (CCAP) will suffer a severe unsurvivable brain injury. To assess brain injury, a neurological examination is conducted, however, its reliability in predicting outcomes in the first days following cardiac arrest is limited. Non-contrast CT is the most employed scan to assess hypoxic changes, even though it is not sensitive to early hypoxic-ischaemic changes in the brain. CT perfusion (CTP) has shown high sensitivity and specificity in brain death patients, although its use in predicting poor neurological outcome in CCAP has not yet been explored. The purpose of this study is to validate CTP for predicting poor neurological outcome (modified Rankin scale, mRS≥4) at hospital discharge in CCAP. METHODS AND ANALYSIS: The CT Perfusion for Assessment of poor Neurological outcome in Comatose Cardiac Arrest Patients study is a prospective cohort study funded by the Manitoba Medical Research Foundation. Newly admitted CCAP receiving standard Targeted Temperature Management are eligible. Patients undergo a CTP at the same time as the admission standard of care head CT. Admission CTP findings will be compared with the reference standard of an accepted bedside clinical assessment at the time of admission. Deferred consent will be used. The primary outcome is a binary outcome of good neurological status, defined as mRs<4 or poor neurological status (mRs≥4) at hospital discharge. A total of 90 patients will be enrolled. ETHICS AND DISSEMINATION: This study has been approved by the University of Manitoba Health Research Ethics Board. The findings from our study will be disseminated through peer-reviewed journals and presentations at local rounds, national and international conferences. The public will be informed at the end of the study. TRIAL REGISTRATION NUMBER: NCT04323020.

Prohibitin plays a role in the functional plasticity of macrophages.

Immunometabolism plays a crucial role in the activation and functional plasticity of immune cells, which in large determines a variety of health and disease states. Factors that integrate immunometabolism in immune cell signaling and functions are beginning to be identified. Previously, we have reported that two transgenic mouse models, Mito-Ob and mutant Mito-Ob (m-Mito-Ob), overexpressing a pleiotropic protein, prohibitin (PHB) or a mutant form of PHB (Tyr114Phe-PHB or m-PHB), respectively, developed distinct immunometabolic phenotypes. Specifically, the immune phenotype appears to be driven by the monocytic cell lineage. Based on immunophenotyping of their splenocytes, we focused our attention on macrophages and hypothesized that PHB may play a role in regulating the two functionally polarized states, M1 and M2. Here, we report that macrophage polarization to the M1 and M2 phenotypes did not alter PHB protein level, but overexpression of PHB in macrophages differentially affected cytokine production in the two polarized states. Furthermore, we found that mutation of the Tyr114 phosphorylation site in PHB affects ERK and STAT6 signaling, arginase synthesis and activity, and mitochondrial respiration in macrophages indicating an important role of PHB in integrating cell signaling events with cell metabolism. In summary, we have discovered that PHB is a crucial regulator in the functional plasticity of macrophages. These initial studies expect to lay the foundation for future research into the relationship between cell signaling events pertaining to immunometabolism in immune cell functions, which are integral components of immune-related health and disease.

Early diagnosis of mortality using admission CT perfusion in severe traumatic brain injury patients (ACT-TBI): protocol for a prospective cohort study.

INTRODUCTION: Severe traumatic brain injury (TBI) is a catastrophic neurological condition with significant economic burden. Early in-hospital mortality (<48 hours) with severe TBI is estimated at 50%. Several clinical examinations exist to determine brain death; however, most are difficult to elicit in the acute setting in patients with severe TBI. Having a definitive assessment tool would help predict early in-hospital mortality in this population. CT perfusion (CTP) has shown promise diagnosing early in-hospital mortality in patients with severe TBI and other populations. The purpose of this study is to validate admission CTP features of brain death relative to the clinical examination outcome for characterizing early in-hospital mortality in patients with severe TBI. METHODS AND ANALYSIS: The Early Diagnosis of Mortality using Admission CT Perfusion in Severe Traumatic Brain Injury Patients study, is a prospective cohort study in patients with severe TBI funded by a grant from the Canadian Institute of Health Research. Adults aged 18 or older, with evidence of a severe TBI (Glasgow Coma Scale score ≤8 before initial resuscitation) and, on mechanical ventilation at the time of imaging are eligible. Patients will undergo CTP at the time of first imaging on their hospital admission. Admission CTP compares with the reference standard of an accepted bedside clinical assessment for brainstem function. Deferred consent will be used. The primary outcome is a binary outcome of mortality (dead) or survival (not dead) in the first 48 hours of admission. The planned sample size for achieving a sensitivity of 75% and a specificity of 95% with a CI of ±5% is 200 patients. ETHICS AND DISSEMINATION: This study has been approved by the University of Manitoba Health Research Ethics Board. The findings from our study will be disseminated through peer-reviewed journals and presentations at local rounds, national and international conferences. The public will be informed through forums at the end of the study. TRIAL REGISTRATION NUMBER: NCT04318665.

Palmitoylation of prohibitin at cysteine 69 facilitates its membrane translocation and interaction with Eps 15 homology domain protein 2 (EHD2).

Plasma membrane translocation of specific cytosolic proteins plays an important role in cell signaling pathways. We have recently shown that prohibitin (PHB) , a protein present in the plasma membranes of various cell types, interacts with Eps 15 homology domain protein 2 (EHD2), a lipid raft protein. However, the mechanism involved in membrane translocation of PHB is not known.We report that PHB undergoes palmitoylation at cysteine 69 (Cys69), and that this palmitoylation is required for PHB's membrane translocation. Furthermore, we demonstrate that membrane translocation of PHB facilitates tyrosine phosphorylation and its interaction with EHD2. Thus, the palmitoylation and membrane translocation of PHB and its interaction with EHD2 may play a role in cell signaling.

Induction of apoptosis in yeast by L-amino acid oxidase from the Malayan pit viper Calloselasma rhodostoma.

Here we report for the first time that L-amino acid oxidase (LAAO), a major component of snake venom, induces apoptosis in yeast. The causative agent for induction of apoptosis has been shown to be hydrogen peroxide, produced by the enzymatic activity of LAAO. However, the addition of catalase, a specific hydrogen peroxide scavenger, does not prevent cell demise completely. Intriguingly, depletion of leucine from the medium by LAAO and the interaction of LAAO with yeast cells are shown to be the major factors responsible for cell demise in the presence of catalase.

Gonadectomy in Mito-Ob mice revealed a sex-dimorphic relationship between prohibitin and sex steroids in adipose tissue biology and glucose homeostasis.

BACKGROUND: Recently, we have developed a novel transgenic mouse model by overexpressing prohibitin (PHB) in adipocytes, which developed obesity due to upregulation of mitochondrial biogenesis in adipocytes, hence named "Mito-Ob." Interestingly, only male Mito-Ob mice developed obesity-related impaired glucose homeostasis and insulin sensitivity, whereas female Mito-Ob mice did not. The observed sex differences in metabolic dysregulation suggest a potential involvement of sex steroids. Thus, the main aim of this study is to investigate the role of sex steroids on the overall phenotype of Mito-Ob mice through gonadectomy, as well as direct effect of sex steroids on adipocytes from Mito-Ob mice in vitro. METHODS: Mito-Ob mice and wild-type CD-1 mice were gonadectomized at 12 weeks of age. Age- and sex-matched sham-operated mice were used as controls. Body weight, white adipose tissue, glucose tolerance, and insulin sensitivity were analyzed 3 months post-surgery. Differentiation of adipocytes isolated from female and male Mito-Ob mice were studied with and without sex steroids. RESULTS: Gonadectomy significantly reduced body weight in Mito-Ob mice compared with sham-operated mice, whereas the opposite trend was observed in wild-type mice. These changes occurred independent of food intake. A corresponding decrease in adipose tissue weight was found in gonadectomized Mito-Ob mice, but depot-specific differences were observed in male and female. Gonadectomy improved glucose tolerance in male wild-type and Mito-Ob mice, but the effect was more pronounced in wild-type mice. Gonadectomy did not alter insulin sensitivity in male Mito-Ob mice, but it was improved in male wild-type mice. In primary cell cultures, testosterone inhibited adipocyte differentiation to a lesser extent in male Mito-Ob preadipocytes compared with the wild-type mice. On the other hand, preadipocytes from female wild-type mice showed better differentiation potential than those from female Mito-Ob mice in the presence of 17ß-estradiol. CONCLUSIONS: PHB requires sex steroids for the development of obese phenotype in Mito-Ob mice, which differentially affect glucose homeostasis and insulin sensitivity in male and female. It appears that PHB plays sex- and adipose depot-specific roles and involves additional factors. In vitro studies suggested that PHB differently influenced adipocyte differentiation in the presence and absence of sex steroids. Overall, this study along with available information in the literature indicated that a multifaceted relationship exists between PHB and sex steroids, which may work in a cell/tissue type- and sex-specific manner.

Prohibitin: A new player in immunometabolism and in linking obesity and inflammation with cancer.

Immunometabolism, which has important implications in cancer biology, has emerged as a major regulator of different immune cell types. Various factors that integrate metabolic switches within immune cells with signal directed program that promote or inhibit their functions remain largely unidentified. Furthermore, sex differences are known to exist in immune functions and cancer incidences in the body and sex steroid hormones are integral component of these differences. However, factors that mediate such differences, and the potential link between the two fundamental aspects of immune cell biology that contributes to sex differences in health and disease remain unexplored. New evidence derived from novel tissue-specific transgenic mouse models of prohibitin (PHB) has revealed its crucial role in sex differences in adipocyte and macrophage functions and a potential role in endocrine-immune crosstalk. This review provides a point of view on the emerging role of PHB in immune functions with special focus on immunometabolism and on the immunomodulatory effects of sex steroids. We propose that PHB plays a crucial role in integrating cell signaling events with metabolic switches, and may serve as a potential target for cancer immunotherapeutic.

Prohibitin: a potential therapeutic target in tyrosine kinase signaling.

Prohibitin is a pleiotropic protein that has roles in fundamental cellular processes, such as cellular proliferation and mitochondrial housekeeping, and in cell- or tissue-specific functions, such as adipogenesis and immune cell functions. The different functions of prohibitin are mediated by its cell compartment-specific attributes, which include acting as an adaptor molecule in membrane signaling, a scaffolding protein in mitochondria, and a transcriptional co-regulator in the nucleus. However, the precise relationship between its distinct cellular localization and diverse functions remain largely unknown. Accumulating evidence suggests that the phosphorylation of prohibitin plays a role in a number of cell signaling pathways and in intracellular trafficking. Herein, we discuss the known and potential importance of the site-specific phosphorylation of prohibitin in regulating these features. We will discuss this in the context of new evidence from tissue-specific transgenic mouse models of prohibitin, including a mutant prohibitin lacking a crucial tyrosine phosphorylation site. We conclude with the opinion that prohibitin can be used as a potential target for tyrosine kinase signal transduction-targeting therapy, including in insulin, growth factors, and immune signaling pathways.

Prohibitin-induced obesity leads to anovulation and polycystic ovary in mice.

Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder and the most common cause of female infertility. However, its etiology and underlying mechanisms remain unclear. Here we report that a transgenic obese mouse (Mito-Ob) developed by overexpressing prohibitin in adipocytes develops polycystic ovaries. Initially, the female Mito-Ob mice were equally fertile to their wild-type littermates. The Mito-Ob mice began to gain weight after puberty, became significantly obese between 3-6 months of age, and ∼25% of them had become infertile by 9 months of age. Despite obesity, female Mito-Ob mice maintained glucose homeostasis and insulin sensitivity similar to their wild-type littermates. Mito-Ob mice showed morphologically distinct polycystic ovaries and elevated estradiol, but normal testosterone and insulin levels. Histological analysis of the ovaries showed signs of impaired follicular dynamics, such as preantral follicular arrest and reduced number, or absence, of corpus luteum. The ovaries of the infertile Mito-Ob mice were closely surrounded by periovarian adipose tissue, suggesting a potential role in anovulation. Collectively, these data suggest that elevated estradiol and obesity per se might lead to anovulation and polycystic ovaries independent of hyperinsulinemia and hyperandrogenism. As obesity often coexists with other abnormalities known to be involved in the development of PCOS such as insulin resistance, compensatory hyperinsulinemia and hyperandrogenism, the precise role of these factors in PCOS remains unclear. Mito-Ob mice provide an opportunity to study the effects of obesity on anovulation and ovarian cyst formation independent of the major drivers of obesity-linked PCOS.

Temporal analysis of protein lysine acetylation during adipocyte differentiation.

The post-translational modification of protein by acetylation has been emerging as a prevalent modification in enzymes that catalyze intermediary metabolism. However, the dynamics of protein acetylation during adipocyte differentiation that involves a major shift in cellular metabolism is not known. In this study, we investigated the temporal changes in acetylation during adipocyte differentiation. Almost all acetylated proteins identified showed a sequential change in acetylation during the differentiation process. While the majority of the acetylated proteins showed a sequential upregulation during adipocyte differentiation, in a few proteins a sequential downregulation of protein acetylation was also observed. Our findings suggest that a wide-ranging temporal change in protein acetylation occurs during adipocyte differentiation including differentially expressed proteins signifying an important role in adipocyte differentiation.

Mutually exclusive acetylation and ubiquitylation among enzymes involved in glucose metabolism.

The posttranslational modification (PTM) in protein occurs in a regiospecific manner. In addition, the most commonly occurring PTMs involve similar residues in proteins such as acetylation, ubiquitylation, methylation and sumoylation at the lysine residue and phosphorylation and O-GlcNAc modification at serine/threonine residues. Thus, the possibility of modification sites where two such PTMs may occur in a mutually exclusive manner (ME-PTM) is much higher than known. A recent surge in the identification and the mapping of the commonly occurring PTMs in proteins has revealed that this is indeed the case. However, in what way such ME-PTM sites are regulated and what could be their relevance in the coordinated network of protein function remains to be known. To gain such potential insights in a biological context, we analyzed two most prevalent PTMs on the lysine residue by acetylation and ubiquitylation along with the most abundant PTM in proteins by phosphorylation among enzymes involved in glucose metabolism, a fundamental process in biology. The analysis of the PTM data sets has revealed two important clues that may be intrinsically associated with their regulation and function. First, the most commonly occurring PTMs by phosphorylation, acetylation and ubiquitylation are widespread and clustered in most of the enzymes involved in glucose metabolism; and the prevalence of phosphorylation sites correlates with the number of acetylation and ubiquitylation sites including the ME-modification sites. Second, the prevalence of ME-acetylation/ubiquitylation sites is exceptionally high among enzymes involved in glucose metabolism and have distinct pattern among the subset of enzymes of glucose metabolism such as glycolysis, tricarboxylic acid (TCA) cycle, glycogen synthesis, and the irreversible steps of gluconeogenesis. We hypothesize that phosphorylation including tyrosine phosphorylation plays an important role in the regulation of ME-acetylation/ubiquitylation sites and their similar pattern among the subset of functionally related proteins allows their coordinated regulation in the normal physiology. Similarly their coordinated dysregulation may underlie the disease processes such as reprogrammed metabolism in cancer, obesity, type 2 diabetes, and cardiovascular diseases. Our hypothesis provides an opportunity to understand the regulation of ME-PTMs in proteins and their relevance at the network level and is open for experimental validation.

The ubiquitin pathway: an emerging drug target in cancer therapy.

Ubiquitination is a highly ordered multistep enzymatic process, carried out by a well-defined systematic ubiquitin pathway, which is required for maintaining appropriate levels and functional activities of various cellular proteins. Targeted regulation of proteins by ubiquitin pathway controls numerous cellular processes including cell proliferation, signal transduction, apoptosis, transcriptional regulation, receptor modulation as well as endocytosis. Significant progress has been made in recent years in not only understanding the structure, function and important regulatory roles of ubiquitin network but also the alterations of ubiquitin pathway in various human diseases including cancer. Based on the progress made, it is now possible to target specifically various components involved in the ubiquitin pathway such as E3 ubiquitin ligases, deubiquitinases and proteosome for potential anticancer therapies. Here we review the potential drug targets available in the ubiquitin system and the small molecule inhibitors that can target these components in the pathway, which can be developed into novel anticancer therapeutics in the near future.

Akt-mediated phosphorylation of CDK2 regulates its dual role in cell cycle progression and apoptosis.

Here, we show that CDK2, an S-phase cyclin-dependent kinase, is a novel target for Akt during cell cycle progression and apoptosis. Akt phosphorylates CDK2 at threonine 39 residue both in vitro and in vivo. Although CDK2 threonine 39 phosphorylation mediated by Akt enhances cyclin-A binding, it is dispensable for its basal binding and the kinase activity. In addition, for the first time, we report a transient nucleo-cytoplasmic shuttling of Akt during specific stages of the cell cycle, in particular during the late S and G2 phases. The Akt that is re-localized to the nucleus phosphorylates CDK2 and causes the temporary cytoplasmic localization of the CDK2-cyclin-A complex. The CDK2 cytoplasmic redistribution is required for cell progression from S to G2-M phase, because the CDK2 T39A mutant, which lacks the phosphorylation site and is defective in cytoplasmic localization, severely affects cell cycle progression at the transition from S to G2-M. Interestingly, we also show that the Akt/CDK2 pathway is constitutively activated by some anticancer drugs, such as methotrexate and docetaxel, and under these conditions it promotes, rather than represses, cell death. Thus, the constitutive activation of the Akt/CDK2 pathway and changed subcellular localization promotes apoptosis. By contrast, the transient, physiological Akt/CDK2 activation is necessary for cell cycle progression.

Cell survival, cell death and cell cycle pathways are interconnected: implications for cancer therapy.

The partial cross-utilization of molecules and pathways involved in opposing processes like cell survival, proliferation and cell death, assures that mutations within one signaling cascade will also affect the other opposite process at least to some extent, thus contributing to homeostatic regulatory circuits. This review highlights some of the connections between opposite-acting pathways. Thus, we discuss the role of cyclins in the apoptotic process, and in the regulation of cell proliferation. CDKs and their inhibitors like the INK4-family (p16(Ink4a), p15(Ink4b), p18(Ink4c), p19(Ink4d)), and the Cip1/Waf1/Kip1-2-family (p21(Cip1/Waf1), p27(Kip1), p57(Kip2)) are shown both in the context of proliferation regulators and as contributors to the apoptotic machinery. Bcl2-family members (i.e. Bcl2, Bcl-X(L) Mcl-1(L); Bax, Bok/Mtd, Bak, and Bcl-X(S); Bad, Bid, Bim(EL), Bmf, Mcl-1(S)) are highlighted both for their apoptosis-regulating capacity and also for their effect on the cell cycle progression. The PI3-K/Akt cell survival pathway is shown as regulator of cell metabolism and cell survival, but examples are also provided where aberrant activity of the pathway may contribute to the induction of apoptosis. Myc/Mad/Max proteins are shown both as a powerful S-phase driving complex and as apoptosis-sensitizers. We also discuss multifunctional proteins like p53 and Rb (RBL1/p107, RBL2/p130) both in the context of G1-S transition and as apoptotic triggers. Finally, we reflect on novel therapeutic approaches that would involve redirecting over-active survival and proliferation pathways towards induction of apoptosis in cancer cells.

Mechanisms of cell death induction by L-amino acid oxidase, a major component of ophidian venom.

L-amino acid oxidase (LAAO) from the Malayan pit viper induces both necrosis and apoptosis in Jurkat cells. Cell death by necrosis is attributed to H2O2 produced by oxidation of alpha-amino acids. In the presence of catalase that effectively scavenges H2O2, a switch to apoptosis is observed. The major factors contributing to apoptosis are proposed to be: (i) generation of toxic intermediates from fetal calf serum (ii) binding and internalization of LAAO. The latter process appears to be mediated by the glycan moiety of the enzyme as desialylation reduces cytotoxicity. D-amino acid oxidase (DAAO), which catalyzes the same reaction as LAAO but lacks glycosylation, triggers necrosis as a consequence of H2O2 production but not apoptosis in the presence of catalase. Thus induction of cell death by LAAO appears to involve both the generation of H2O2 and the molecular interaction of the glycan moiety of the enzyme with structures at the cell surface.