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Background and Purpose: Patients with active cancer face an increased risk of ischemic stroke. Also, stroke may be an initial indicator of cancer. In patients with large vessel occlusion (LVO) stroke treated with thrombectomy, analysis of the clot composition may contribute new insights into the pathological connections between these two conditions., Methods: We compared the content of 64 consecutively retrieved clots from LVO stroke patients with concomitant active cancer and 64 clots from matched-control LVO stroke patients without a history of cancer. Clots were analyzed with respect to histological composition by Martius Scarlet Blue, von Willebrand factor (vWF), citrullinated histone H3 (H3Cit, a biomarker of NETS), CD42b, and CD3 expression by immunohistochemistry. Orbit Image Analysis was used for quantification. Differences between groups were tested using the Mann-Whitney U-test and Chi-square Test., Results: Clots from patients with concomitant cancer had a significantly higher content of vWF (median 26 [IQR13-38]% vs. 10 [4-18]%, p < 0.0001) and H3Cit (median 0.11 [IQR0.02-0.46]% vs. 0.05 [0.00-0.28]% p = 0.027) than controls. The presence of collagen >1% within the retrieved clots was highly indicative of cancer, occurring in 16/64 with active cancer and in 3/64 controls, p = 0.002. After correction for multiple comparisons, the statistical significance for H3Cit was lost. Red and white blood cells, platelets, fibrin, and expression of CD3 and CD42b did not differ between the groups., Conclusions: Clots from LVO patients with concomitant active cancer possess distinct characteristics, indicating an influence of cancer on the innate immune system, fibroblasts, and the vascular endothelium in the formation of LVO clots., (© 2025 The Author(s). European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

The need for biomarkers for acute ischemic stroke (AIS) to understand the mechanisms implicated in pathological clot formation is critical. The levels of the brain natriuretic peptides known as brain natriuretic peptide (BNP) and NT-proBNP have been shown to be increased in patients suffering from heart failure and other heart conditions. We measured their expression in AIS clots of cardioembolic (CE) and large artery atherosclerosis (LAA) etiology, evaluating their location inside the clots, aiming to uncover their possible role in thrombosis. We analyzed 80 thrombi from 80 AIS patients in the RESTORE registry of AIS clots, 40 of which were of CE and 40 of LAA etiology. The localization of BNP and NT-BNP, quantified using immunohistochemistry and immunofluorescence, in AIS-associated white blood cell subtypes was also investigated. We found a statistically significant positive correlation between BNP and NT-proBNP expression levels (Spearman's rho = 0.668 p < 0.0001 *). We did not observe any statistically significant difference between LAA and CE clots in BNP expression (0.66 [0.13-3.54]% vs. 0.53 [0.14-3.07]%, p = 0.923) or in NT-proBNP expression (0.29 [0.11-0.58]% vs. 0.18 [0.05-0.51]%, p = 0.119), although there was a trend of higher NT-proBNP expression in the LAA clots. It was noticeable that BNP was distributed throughout the thrombus and especially within platelet-rich regions. However, NT-proBNP colocalized with neutrophils, macrophages, and T-lymphocytes, suggesting its association with the thrombo-inflammatory process.

Background: Studies during the COVID-19 pandemic have demonstrated an association between COVID-19 virus infection and the development of acute ischemic stroke, particularly large vessel occlusion (LVO). Studying the characteristics and immunohistochemistry of retrieved stroke emboli during mechanical thrombectomy for LVO may offer insights into the pathogenesis of LVO in COVID-19 patients. We examined retrieved COVID-19 emboli from the STRIP, EXCELLENT, and RESTORE registries and compared their characteristics to a control group., Methods: We identified COVID-positive LVO patients from the STRIP, RESTORE, and EXCELLENT studies who underwent mechanical thrombectomy. These patients were matched to a control group controlling for stroke etiology based on Trial of Org 10172 in Acute Stroke Treatment criteria. All clots were stained with Martius Scarlet Blue (MSB) along with immunohistochemistry for interleukin-6 (IL-6), C-reactive protein (CRP), von Willebrand factor (vWF), CD66b, fibrinogen, and citrullinated Histone H3. Clot composition was compared between groups., Results: Nineteen COVID-19-positive patients and 38 controls were included. COVID-19-positive patients had a significantly higher percentage of CRP and vWF. There was no difference in IL-6, fibrin, CD66b, or citrullinated Histone H3 between groups. Based on MSB staining, there was no statistically significant difference regarding the percentage of red blood cells, white blood cells, fibrin, and platelets., Conclusions: Our study found higher concentrations of CRP and vWF in retrieved clots of COVID-19-positive stroke patients compared to COVID-19-negative controls. These findings support the potential role of systemic inflammation as indicated by elevated CRP and endothelial injury as indicated by elevated vWF as precipitating factors in thrombus development in these patients.

Background: Myocardial infarction (MI) has been reported as a postinfection sequela of herpes zoster, but with limited data on incidence after zoster and protective effect of the zoster vaccine. This study investigates the risk of developing an MI 30 days postzoster, determines patient-specific risk factors, and investigates the impact of herpes zoster vaccination., Methods: This retrospective cohort study included patients who received care at a Veterans Affairs facility between 2015 and 2020. Time to MI was determined from either 30 days post-zoster infection (zoster cohort) or a primary care appointment (control cohort)., Results: This study assessed a total of 2 165 584 patients. MI within 30 days occurred in 0.34% (n = 244) of the zoster cohort and 0.28% (n = 5782) of the control cohort ( P = .0016). Patients with a documented herpes zoster infection during the study period were 1.35 times more likely to develop an MI within the first 30 days postinfection compared to the control cohort. Patients who received the recombinant zoster vaccine were less likely to have an MI postinfection (odds ratio, 0.82 [95% confidence interval, .74-.92]; P = .0003)., Conclusions: Herpes zoster infection was associated with an increased risk of MI within the first 30 days postinfection. History of prior MI, male sex, age ≥50 years, history of heart failure, peripheral vascular disease, human immunodeficiency virus, prior cerebrovascular accident, and renal disease increased odds of MI 30 days postinfection with herpes zoster. Herpes zoster vaccination decreased the odds of developing an MI in patients aged ≥50 years., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2023.)

Background and Purpose: Post-thrombectomy intracranial hemorrhages (PTIH) are dangerous complications of acute ischemic stroke (AIS) following mechanical thrombectomy. We aimed to investigate if S100b levels in AIS clots removed by mechanical thrombectomy correlated to increased risk of PTIH., Methods: We analyzed 122 thrombi from 80 AIS patients in the RESTORE Registry of AIS clots, selecting an equal number of patients having been pre-treated or not with rtPA (40 each group). Within each subgroup, 20 patients had developed PTIH and 20 patients showed no signs of hemorrhage. Gross photos of each clot were taken and extracted clot area (ECA) was measured using ImageJ. Immunohistochemistry for S100b was performed and Orbit Image Analysis was used for quantification. Immunofluorescence was performed to investigate co-localization between S100b and T-lymphocytes, neutrophils and macrophages. Chi-square or Kruskal-Wallis test were used for statistical analysis., Results: PTIH was associated with higher S100b levels in clots (0.33 [0.08-0.85] vs. 0.07 [0.02-0.27] mm 2 , H1 = 6.021, P = 0.014 * ), but S100b levels were not significantly affected by acute thrombolytic treatment ( P = 0.386). PTIH was also associated with patients having higher NIHSS at admission (20.0 [17.0-23.0] vs. 14.0 [10.5-19.0], H1 = 8.006, P = 0.005) and higher number of passes during thrombectomy (2 [1-4] vs. 1 [1-2.5], H1 = 5.995, P = 0.014 * ). S100b co-localized with neutrophils, macrophages and with T-lymphocytes in the clots., Conclusions: Higher S100b expression in AIS clots, higher NIHSS at admission and higher number of passes during thrombectomy are all associated with PTIH. Further investigation of S100b expression in AIS clots by neutrophils, macrophages and T-lymphocytes could provide insight into the role of S100b in thromboinflammation., Competing Interests: MG and RC were employed by Cerenovus. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rossi, Douglas, Gil, Jabrah, Pandit, Gilvarry, McCarthy, Prendergast, Jood, Redfors, Nordanstig, Ceder, Dunker, Carlqvist, Szikora, Thornton, Tsivgoulis, Psychogios, Tatlisumak, Rentzos and Doyle.)

Background and Aims: Besides the crucial role in the treatment of acute ischemic stroke (AIS), mechanical thrombectomy represents a unique opportunity for researchers to study the retrieved clots, with the possibility of unveiling biological patterns linked to stroke pathophysiology and etiology. We aimed to develop a shotgun proteomic approach to study and compare the proteome of formalin-fixed paraffin-embedded (FFPE) cardioembolic and large artery atherosclerotic (LAA) clots., Methods: We used 16 cardioembolic and 15 LAA FFPE thrombi from 31 AIS patients. The thrombus proteome was analyzed by label-free quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant v1.5.2.8 and Perseus v.1.6.15.0 were used for bioinformatics analysis. Protein classes were identified using the PANTHER database and the STRING database was used to predict protein interactions., Results: We identified 1,581 protein groups as part of the AIS thrombus proteome. Fourteen significantly differentially abundant proteins across the two etiologies were identified. Four proteins involved in the ubiquitin-proteasome pathway, blood coagulation or plasminogen activating cascade were identified as significantly abundant in LAA clots. Ten proteins involved in the ubiquitin proteasome-pathway, cytoskeletal remodeling of platelets, platelet adhesion or blood coagulation were identified as significantly abundant in cardioembolic clots., Conclusion: Our results outlined a set of 14 proteins for a proof-of-principle characterization of cardioembolic and LAA FFPE clots, advancing the proteome profile of AIS human thrombi and understanding the pathophysiology of ischemic stroke., Competing Interests: MG and RM were employed by company Cerenovus. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rossi, Mereuta, Barbachan e Silva, Molina Gil, Douglas, Pandit, Gilvarry, McCarthy, O'Connell, Tierney, Psychogios, Tsivgoulis, Szikora, Tatlisumak, Rentzos, Thornton, Ó Broin and Doyle.)

Background: Characterization of the clot occluding the arteries in acute ischemic stroke received ample attention, in terms of elucidating the relationship between the clot composition, its etiology and its amenability for pharmacological treatment and mechanical thrombectomy approaches. Traditional analytical techniques such as conventional 2D histopathology or electron microscopy sample only small parts of the clot. Visualization and analysis in 3D are necessary to depict and comprehend the overall organization of the clot. The aim of this study is to investigate the potential of microCT for characterizing the clot composition, structure, and organization., Methods: In a pilot study, we analyzed with microCT clots retrieved from 14 patients with acute ischemic stroke. The following parameters were analyzed: overall clot density, clot segmentation with various density thresholds, clot volume., Results: Our findings show that human clots are heterogeneous in terms of CT intra-clot density distribution. After fixation in formalin, the clots display a shift toward negative values. On average, we found the mean HU values of red clots retrieved from patients to be -153 HU, with SD = 23.8 HU, for the intermediate clots retrieved from patients -193 HU, SD = 23.7 HU, and for the white clots retrieved from patients -229 HU, SD = 64.8 HU., Conclusion: Our study shows that volumetric and density analysis of the clot opens new perspectives for clot characterization and for a better understanding of thrombus structure and composition., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dumitriu LaGrange, Braunersreuther, Wanke, Berberat, Luthman, Fitzgerald, Doyle, Brina, Reymond, Platon, Muster, Machi, Poletti, Vargas and Lövblad.)

Background and Aim: Mechanical thrombectomy (MT) for large vessel occlusion often requires multiple passes to retrieve the entire thrombus load. In this multi-institutional study we sought to examine the composition of thrombus fragments retrieved with each pass during MT., Methods: Patients who required multiple passes during thrombectomy were included. Histopathological evaluation of thrombus fragments retrieved from each pass was performed using Martius Scarlet Blue staining and the composition of each thrombus component including RBC, fibrin and platelet was determined using image analysis software., Results: 154 patients underwent MT and 868 passes was performed which resulted in 263 thrombus fragments retrieval. The analysis of thrombus components per pass showed higher RBC, lower fibrin and platelet composition in the pass 1 and 2 when compared to pass 3 and passes 4 or more combined (P values <0.05). There were no significant differences between thrombus fragments retrieved in pass 1 and pass 2 in terms of RBC, WBC, fibrin, and platelet composition (P values >0.05). Similarly, when each composition of thrombus fragments retrieved in pass 3 and passes 4 or more combined were compared with each other, no significant difference was noted (P values >0.05)., Conclusion: Our findings confirm that thrombus fragments retrieved with each pass differed significantly in histological content. Fragments in the first passes were associated with lower fibrin and platelet composition compared to fragments retrieved in passes three and four or higher. Also, thrombus fragments retrieved after failed pass were associated with higher fibrin and platelet components.

The composition of ischemic stroke thrombi has gained an increasing amount of interest in recent years. The implementation of endovascular procedures in standard stroke care has granted researchers the unique opportunity to examine patient thrombus material. Increasing evidence indicates that stroke thrombi are complex and heterogenous, consisting of various biochemical (eg, fibrin, von Willebrand Factor, and neutrophil extracellular traps) and cellular (eg, red blood cells, platelets, leukocytes, and bacteria) components. This complex composition may explain therapeutic limitations and also offer novel insights in several aspects of stroke management. Better understanding of thrombus characteristics could, therefore, potentially lead to improvements in the management of patients with stroke. In this review, we provide a comprehensive overview of the lessons learned by examining stroke thrombus composition after endovascular thrombectomy and its potential relevance for thrombectomy success rates, thrombolysis, clinical outcomes, stroke etiology, and radiological imaging.

Introduction: We assessed the correlation between thrombus size before and after mechanical thrombectomy, measured as length by Computed Tomography Angiography/Non-Contrast Computed Tomography (CTA/NCCT) and Extracted Clot Area, ECA, respectively. We also assessed the influence of thrombus size on the number of passes required for clot removal and final recanalization outcome., Materials and Methods: Acute ischaemic stroke (AIS) thrombi retrieved by mechanical thrombectomy from 500 patients and data of clot length by CTA/NCCT were collected from three hospitals in Europe. ECA was obtained by measuring the area of the extracted clot. Non-parametric tests were used for data analysis., Results: A strong positive correlation was found between clot length on CTA/NCCT and ECA (rho = 0.619,N = 500,P < 0.0001*). Vessel size influences clot length on CTA/NCCT (H2 = 98.6, P < 0.0001*) and ECA (H2 = 105.6,P < 0.0001*), but the significant correlation between CTA/NCCT length and ECA was evident in all vessels. Poorer revascularisation outcome was associated with more passes (H5 = 73.1, P < 0.0001*). More passes were required to remove longer clots (CTA/NCCT; H4 = 31.4, P < 0.0001*; ECA; H4 = 50.2, P < 0.0001*). There was no significant main association between recanalization outcome and length on CTA/NCCT or ECA, but medium sized clots (ECA 20-40 mm 2 ) were associated with least passes and highest revascularisation outcome (N = 500, X 2  = 16.2, P < 0.0001*)., Conclusion: Clot length on CTA/NCCT strongly correlates with ECA. Occlusion location influences clot size. More passes are associated with poorer revascularisation outcome and bigger clots. The relationship between size and revascularisation outcome is more complex. Clots of medium ECA take less passes to remove and are associated with better recanalization outcome than both smaller and larger clots., Competing Interests: Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© European Stroke Organisation 2021.)

Background and Purpose: Previous studies have successfully created blood clot analogs for in vitro endovascular device testing using animal blood of various species. Blood components vary greatly among species; therefore, creating clot analogs from human blood is likely a more accurate representation of thrombi formed in the human vasculature., Materials and Methods: Following approval from the Mayo Clinic institutional review board, human whole-blood and platelet donations were obtained from the blood transfusion service. Twelve clot analogs were created by combining different ratios of red blood cells + buffy coat, plasma, and platelets. Thrombin and calcium chloride were added to stimulate coagulation. Clot composition was assessed using histologic and immunohistochemical staining. To assess the similarities of mechanical properties to patient clots, 3 types of clot analogs (soft, elastic, and stiff) were selected for in vitro thrombectomy testing., Results: The range of histopathologic compositions produced is representative of clots removed during thrombectomy procedures. The red blood cell composition ranged from 8.9% to 91.4%, and fibrin composition ranged from 3.1% to 53.4%. Platelets (CD42b) and von Willebrand Factor ranged from 0.5% to 47.1% and 1.0% to 63.4%, respectively. The soft clots had the highest first-pass effect and successful revascularization rates followed by the elastic and stiff clots. Distal embolization events were observed when clot ingestion could not be achieved, requiring device pullback. The incidence rate of distal embolization was the highest for the stiff clots due to the weak clot/device integration., Conclusions: Red blood cell-rich, fibrin-rich, and platelet-rich clot analogs that mimic clots retrieved from patients with acute ischemic stroke were created in vitro. Differing retrieval outcomes were confirmed using in vitro thrombectomy testing in a subset of clots., (© 2021 by American Journal of Neuroradiology.)

Our aim was to assess the utility of a novel machine learning software (Orbit Image Analysis) in the histological quantification of acute ischemic stroke (AIS) clots. We analyzed 50 AIS blood clots retrieved using mechanical thrombectomy procedures. Following H&E staining, quantification of clot components was performed by two different methods: a pathologist using a reference standard method (Adobe Photoshop CC) and an experienced researcher using Orbit Image Analysis. Following quantification, the clots were categorized into 3 types: RBC dominant (≥60% RBCs), Mixed and Fibrin dominant (≥60% Fibrin). Correlations between clot composition and Hounsfield Units density on Computed Tomography (CT) were assessed. There was a significant correlation between the components of clots as quantified by the Orbit Image Analysis algorithm and the reference standard approach (ρ = 0.944**, p < 0.001, n = 150). A significant relationship was found between clot composition (RBC-Rich, Mixed, Fibrin-Rich) and the presence of a Hyperdense artery sign using the algorithmic method (X2(2) = 6.712, p = 0.035*) but not using the reference standard method (X2(2) = 3.924, p = 0.141). Orbit Image Analysis machine learning software can be used for the histological quantification of AIS clots, reproducibly generating composition analyses similar to current reference standard methods., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests; Manuel Stritt is an author of the opensource software Orbit Image Analysis and declares a Non-Financial competing interest, Ray McCarthy and Michael Gilvarry are employees of Cerenovus, Galway, Ireland. This does not alter our adherence to PLOS ONE policies on sharing data and materials. All remaining authors declare no Funding, Employment or Personal financial interests in relation to the work described herein.

Background: Bacteria and bacterial components have been associated with the activation of coagulation factors and initiating the blood clot formation. The aim of this study was to investigate whether bacterial populations are present in clots retrieved from patients that have suffered a large vessel occlusion acute ischemic stroke (AIS)., Materials and Methods: Clot samples were collected from 20 AIS patients who underwent clot retrieval with mechanical thrombectomy. Patient clinical demographic details were noted. Expression of bacterial 16S rDNA was analyzed by standard and real-time polymerase chain reaction (PCR). Gram staining was performed to identify Gram-positive and Gram-negative bacteria., Results: Both the real-time and standard PCR demonstrated no expression of 16S rDNA in any of the 20 clots samples from AIS patients. Gram staining results showed no expression of Gram-positive or Gram-negative bacteria present in the clot samples., Conclusion: Our current study found no bacteria populations in the clots of AIS patients.

Background and Purpose: Direct aspiration is a recognized technique for revascularization in large-vessel ischemic strokes. There is ongoing debate regarding its efficacy compared with stent retrievers. Every delay in achieving revascularization and a decrease in reperfusion rates reduces the likelihood of patients achieving functional independence. We propose a standardized setup technique for aspiration-first for all anterior circulation thrombectomy procedures for increasing speed and recanalization rates., Materials and Methods: We analyzed 127 consecutive patients treated by a standardized approach to thrombectomy with an intention to perform aspiration-first compared with 127 consecutive patients treated with a stent retriever-first approach. Key time metrics evaluated included groin to first angiogram, first angiogram to reperfusion, groin to first reperfusion, and length of the procedure. The degree of successful recanalization (TICI 2b-3) and the number of passes were compared between the 2 groups., Results: In 127 patients who underwent the standardized technique, the median time from groin puncture to first reperfusion was 18 minutes compared with 26 minutes ( P < .001). The duration of the procedure was shorter compared with the stent retriever group (26 minutes in the aspiration first group versus 47 minutes, P < .001) and required fewer passes (mean, 2.4 versus 3.1; P < .05). A higher proportion of patients had a TICI score of 2b-3 in the aspiration-first group compared with stent retriever group (96.1% versus 85.8%, P < .005)., Conclusions: Our study highlights the increasing speed and recanalization rates achieved with fewer passes in a standardized approach to thrombectomy with an intention to attempt aspiration-first. Any attempt to reduce revascularization time and increase successful recanalization should be used., (© 2019 by American Journal of Neuroradiology.)

Background and Purpose- Nearly 30% of large vessel occlusion acute ischemic stroke clots are from an unknown source. We assessed histological clot composition in a series of patients with large vessel occlusion and investigated correlations between clot composition and stroke pathogenesis. Methods- As part of the multi-institutional STRIP registry (Stroke Thromboembolism Registry of Imaging and Pathology), consecutive emboli retrieved during mechanical thrombectomy were stained using Martius Scarlett Blue and analyzed using machine learning software. We assessed proportions of red blood cells, fibrin, platelets, and white blood cells. Correlations between clot components and stroke pathogenesis (large artery atherosclerosis, cardioembolism, and stroke of undetermined pathogenesis) were assessed using SPSS22. Results- One hundred five patients were included. The proportion of platelet-rich clots (55.0% versus 21.2%; P=0.005) and percentage of platelet content (22.1±4.2% versus 13.9±14.2%; P=0.03) was significantly higher in the large artery atherosclerosis group compared with the cardioembolic group. The proportion of platelet-rich clots (50.0% versus 21.2%; P=0.024) was also significantly higher in the cryptogenic group compared with cardioembolic cases. Large artery atherosclerosis and cryptogenic cases had a similar proportion of platelet-rich clots (55.0% versus 50.0%; P=0.636). There was no significant difference between stroke pathogenesis and the other major clot components. Conclusions- High platelet content of emboli is associated with a large artery atherosclerosis etiology of large vessel occlusion.

Cardiac surgery has been shown to be associated with increased risk of acute ischemic stroke. This report presents a case of a successful mechanical embolectomy procedure to treat a patient for an acute ischemic stroke, which was caused by the cardiac embolization of a 'foreign body' containing debris following a redo sternotomy procedure for mitral valve replacement and tricuspid valve annuloplasty.

Nerve growth factor (NGF) is well characterised as an important pro-survival factor in neuronal cells that can inhibit apoptotic cell death upstream of mitochondrial outer membrane permeabilisation. Here we addressed the question of whether NGF can also protect against apoptosis downstream of caspase activation. NGF treatment promoted a rapid reduction in the level of the p17 subunit of active caspase-3 in PC12 cells that had been induced to undergo apoptosis by various cytotoxins. The mechanism involved TrkA-dependent activation of extracellular signal-regulated kinase (ERK1/2) but not phosphatidylinositol 3-kinase (PI3K)/Akt, and de novo protein synthesis. Involvement of inhibitor of apoptosis proteins (IAPs) and proteasomal degradation were ruled out. In contrast, inhibition of lysosome function using chloroquine and concanamycin A reversed NGF-induced removal of p17. Moreover, in NGF-treated cells, active caspases were found to be localised to lysosomes. The involvement of macroautophagy and chaperone-mediated autophagy were ruled out. Taken together, these findings suggest an anti-apoptotic mechanism by which NGF induces removal of active caspase-3 in a lysosome-dependent manner.

The United States is confronting important challenges at both the early and late stages of science education. At the level of K-12 education, a recent National Research Council report (Successful K-12 STEM Education) proposed a bold restructuring of how science is taught, moving away from memorizing facts and emphasizing hands-on, inquiry-based learning and a deeper understanding of the process of science. At higher levels of training, limited funding for science is leading PhDs to seek training and careers in areas other than research. Might science PhDs play a bigger role in the future of K-12 education, particularly at the high school level? We explore this question by discussing the roles that PhDs can play in high school education and the current and rather extensive barriers to PhDs entering the teaching profession and finally suggest ways to ease the entrance of qualified PhDs into high school education.

The stimuli for neuronal cell death in neurodegenerative disorders are multi-factorial and may include genetic predisposition, environmental factors, cellular stressors such as oxidative stress and free radical production, bioenergy failure, glutamate-induced excitotoxicity, neuroinflammation, disruption of Ca(2+) -regulating systems, mitochondrial dysfunction and misfolded protein accumulation. Cellular stress disrupts functioning of the endoplasmic reticulum (ER), a critical organelle for protein quality control, leading to induction of the unfolded protein response (UPR). ER stress may contribute to neurodegeneration in a range of neurodegenerative disorders. This review summarizes the molecular events occurring during ER stress and the unfolded protein response and it specifically evaluates the evidence suggesting the ER stress response plays a role in neurodegenerative disorders., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Background: The human immune response to mercury is not well characterized despite the body of evidence that suggests that Hg can modulate immune responses, including the induction of autoimmune disease in some mouse models. Dysregulation of cytokine signaling appears to play an important role in the etiology of Hg-induced autoimmunity in animal models., Objectives: In this study, we systematically investigated the human immune response to Hg in vitro in terms of cytokine release., Methods: Human peripheral blood mononuclear cells (PBMCs) were isolated from 20 volunteers who donated blood six separate times. PBMCs were cultured with lipopolysaccharide and concentrations of mercuric chloride (HgCl(2)) up to 200 nM. Seven cytokines representing important pathways in physiologic and pathologic immune responses were measured in supernatants. We used multilevel models to account for the intrinsic clustering in the cytokine data due to experimental design., Results: We found a consistent increase in the release of the proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha, and concurrent decrease in release of the antiinflammatory cytokines interleukin 1-receptor antagonist (IL-1Ra) and IL-10 in human PBMCs treated with subcytotoxic concentrations of HgCl(2). IL-4, IL-17, and interferon-gamma increased in a concentration-response manner. These results were replicated in a second, independently recruited population of 20 different volunteers., Conclusions: Low concentrations of HgCl(2) affect immune function in human cells by dysregulation of cytokine signaling pathways, with the potential to influence diverse health outcomes such as susceptibility to infectious disease or risk of autoimmunity.

Homeobox genes encode transcription factors that regulate embryonic development and postnatal events. Rhox5 (previously called Pem), the founding member of a homeobox gene cluster that we recently identified on the X chromosome, is selectively expressed in granulosa cells in the ovary and other somatic-cell types in other reproductive organs. In this report, we investigate its regulation in granulosa cells in the rat ovary. We found that Rhox5 expression in the ovary is governed by the Rhox5 distal promoter and is expressed at least as early as Day 5 postpartum. Rhox5 mRNA levels are regulated during the ovarian cycle, peaking before ovulation. Deletion analysis revealed a 25-nt element essential for distal promoter transcription in primary granulosa cells. This distal promoter element contains two ETS and one SP1 transcription-factor family binding sites that mutagenesis analysis indicated were essential for high-level transcription. This element was both necessary and sufficient for transcription, because it activated transcription when placed upstream of a heterologous minimal promoter. Cold competition and electrophoretic mobility shift assay studies demonstrated that SP1, SP3, and the ETS family transcription factor GABP bound this element. Dominant-negative forms of GABP and SP3 repressed distal promoter expression in primary rat granulosa, showing that these factors are crucial for Rhox5 expression. Cotransfection of dominant-negative mutants indicated that Rhox5 expression in granulosa cells is regulated by the c-Jun N-terminal protein kinase (JNK, MAPK8) and RAS pathways, which are known to be upstream of ETS family transcription factors. The discovery that Rhox5 expression in granulosa cells is regulated by MAPK pathways and ETS and SP1 family members provides an opportunity to understand how these regulatory pathways and factors collaborate to regulate gene expression during the ovarian cycle.

ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin-like motifs) is a multifunctional protease that is expressed in periovulatory follicles. Herein we show that induction of ADAMTS-1 message in vivo and transcription of the ADAMTS-1 promoter in cultured granulosa cells are dependent on separable but coordinate actions of LH and the progesterone receptor (PR). To analyze the molecular mechanisms by which LH and PR regulate this gene, truncations and site-specific mutants of ADAMTS-1 promoter-luciferase reporter constructs (ADAMTS-1-Luc) were generated and transfected into rat granulosa cell cultures. Three regions of the promoter were found to be important for basal activity, two of which were guanine cytosine-rich binding sites for specificity proteins Sp1/Sp3 and the third bound a nuclear factor 1-like factor. Despite the absence of a consensus PR DNA response element in the proximal ADAMTS-1 promoter, cotransfection of a PRA (or PRB) expression vector stimulated ADAMTS-1 promoter activity, a response that was reduced by the PR antagonist ZK98299. Forskolin plus phorbol myristate acetate also increased promoter activity and, when added to cells cotransfected with PRA, ADAMTS-1 promoter activity increased further. Activation of the ADAMTS-1 promoter by PRA involves functional CAAT enhancer binding protein beta, nuclear factor 1-like factor, and three Sp1/Sp3 binding sites as demonstrated by transfection of mutated promoter constructs. In summary, LH and PRA/B exert distinct but coordinate effects on transactivation of the ADAMTS-1 gene in granulosa cells in vivo and in vitro with PR acting as an inducible coregulator of the ADAMTS-1 gene.

Retinoblastoma (Rb) and family members have been implicated as key regulators of cell proliferation and differentiation. In particular, accumulated data have suggested that the Rb gene product pRb is an important controller of erythroid differentiation. However, current published data are conflicting as to whether the role of pRb in erythroid cells is cell intrinsic or non-cell intrinsic. Here, we have made use of an in vitro erythroid differentiation culture system to determine the cell-intrinsic requirement for pRb in erythroid differentiation. We demonstrate that the loss of pRb function in primary differentiating erythroid cells results in impaired cell cycle exit and terminal differentiation. Furthermore, we have used coculture experiments to establish that this requirement is cell intrinsic. Together, these data unequivocally demonstrate that pRb is required in a cell-intrinsic manner for erythroid differentiation and provide clarification as to its role in erythropoiesis.

ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs-1) is a member of the ADAMTS family of metalloproteases which, together with ADAMTS-4 and ADAMTS-5, has been shown to degrade members of the lectican family of proteoglycans. ADAMTS-1 mRNA is induced in granulosa cells of periovulatory follicles by the luteinizing hormone surge through a progesterone receptor-dependent mechanism. Female progesterone receptor knockout (PRKO) mice are infertile primarily due to ovulatory failure and lack the normal periovulatory induction of ADAMTS-1 mRNA. We therefore investigated the protein localization and function of ADAMTS-1 in ovulating ovaries. Antibodies against two specific peptide regions, the pro-domain and the metalloprotease domain of ADAMTS-1, were generated. Pro-ADAMTS-1 of 110 kDa was identified in mural granulosa cells and appears localized to cytoplasmic secretory vesicles. The mature (85-kDa pro-domain truncated) form accumulated in the extracellular matrix of the cumulus oocyte complex (COC) during the process of matrix expansion. Each form of ADAMTS-1 protein increased >10-fold after the ovulatory luteinizing hormone surge in wild-type but not PRKO mice. Versican is also localized selectively to the ovulating COC matrix and was found to be cleaved yielding a 70-kDa N-terminal fragment immunopositive for the neoepitope DPEAAE generated by ADAMTS-1 and ADAMTS-4 protease activity. This extracellular processing of versican was reduced in ADAMTS-1-deficient PRKO mouse ovaries. These observations suggest that one function of ADAMTS-1 in ovulation is to cleave versican in the expanded COC matrix and that the anovulatory phenotype of PRKO mice is at least partially due to loss of this function.

Early growth response factor (Egr-1) is an inducible zinc finger transcription factor that binds specific GC-rich enhancer elements and impacts female reproduction. These studies document for the first time that FSH rapidly induces Egr-1 expression in granulosa cells of small growing follicles. This response is transient but is reinitiated in preovulatory follicles exposed to the LH analog, human chorionic gonadotropin. Immunohistochemical analysis also showed gonadotropin induced Egr-1 in theca cells. The Egr-1 gene regulatory region responsive to gonadotropin signaling was localized within -164 bp of the transcription initiation site. Binding of Sp1/Sp3 to a proximal GC-box at -64/-46 bp was enhanced by FSH in immature granulosa cells but reduced after human chorionic gonadotropin stimulation of preovulatory follicles despite constant protein expression. This dynamic regulation of Sp1 binding was dependent on gonadotropin-regulated mechanisms that modulate Sp1/3-DNA binding activity. Serum response factor was active in granulosa cells and bound a consensus CArG-box/serum response element site, whereas two putative cAMP response elements within the -164-bp region bound cAMP regulatory element (CRE) binding protein (CREB) and a second cAMP-inducible protein immunologically related to CREB. Transient transfection analyses using Egr-1 promoter-luciferase constructs and site-specific mutations show that the serum response element, GC-box, and CRE-131 are involved in gonadotropin regulation of Egr-1 expression in granulosa cells. Specific kinase inhibitors of Erk or protein kinase A antagonized this induction while exogenously expressed Egr-1 enhanced reporter expression. These observations indicate that the Egr-1 gene is a target of both FSH and LH action that may mediate molecular programs of proliferation and/or differentiation during follicle growth, ovulation, and luteinization.

1. The ability of three putative polyamine antagonists to antagonize behavioural changes induced by spermine was assessed. 2. Injection of an excitotoxic dose of spermine (100 microg, i.c.v.) in mice results in the development of a characteristic behavioural profile, which has two temporally distinct phases. The early events include clonic convulsions, and the later, more general excitation, includes tremor and culminates in the development of a fatal tonic convulsion. 3. Co-administration of arcaine (25 microg, i.c.v.) potentiated the early phase effects after spermine injection, but antagonized the development of spermine-induced tonic convulsions. A larger dose of arcaine (50 microg, i.c.v.) given alone resulted in the development of spermine-like body tremor and convulsions. It therefore appears that arcaine is not a pure polyamine antagonist in vivo, but may be a partial agonist. 4. Similarly, 1,10-diaminodecane appeared to act as a partial agonist in vivo, although it was less potent than arcaine. 5. In contrast, diethylenetriamine (DET) effectively inhibited the development of the early effects of spermine, but was ineffective against the spermine-induced CNS excitation and tonic convulsions. 6. It is concluded that none of the putative polyamine antagonists tested behaved as effective polyamine antagonists in vivo, although each produced some antagonism.

1. The involvement of the N-methyl-D-aspartate (NMDA) receptor macrocomplex in the development of spermine-induced CNS excitation in vivo was investigated. 2. Injection of 100 micrograms of spermine into the left lateral cerebral ventricle of female Laca mice (20-25 g) resulted in the development of two distinct phases of CNS excitatory effects which were quantified by a scoring system. 3. The first phase effects occurred within minutes of injection and generally lasted for about 1 h. Most mice showed scratching of the upper body, frequent face washing and some mice developed clonic convulsions. By about 2 h after injection, the second phase of effects began to develop in the form of body tremor which worsened with time and culminated in fatal tonic convulsions, generally within 8 h of injection. 4. Pretreatment of the mice with dizocilpine (0.3 mg kg-1, i.p.) resulted in antagonism of the first phase of spermine-induced effects, but a higher dose (0.3 mg kg-1, (x2), i.p.) was necessary to inhibit the second phase effects. 5. Whereas the glutamate antagonist, 3-((R)-2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (D-CPP) (10, 20 mg kg-1, i.p.), the glycine antagonist 7-chlorokynurenate (10, 30, 50 nmol, i.c.v.), or the polyamine antagonist ifenprodil (30, 60 mg kg-1, i.p.) antagonized the first phase of effects produced by spermine, these agents given as monotherapy, were ineffective against the development of the second phase of effects. 6. Co-administration of ifenprodil with either D-CPP or 7-chlorokynurenate resulted in a dose-dependent antagonism of the development of the second phase of spermine-induced effects. 7. It is concluded that the development of the two temporally distinct phases of spermine-induced effects may be mediated by pharmacologically distinct mechanisms, although the results suggest that the NMDA receptor macrocomplex may be involved in both phases of effects. Furthermore, a moderate dose of D-CPP or 7-chlorokynurenate appears to enhance the inhibitory potential of ifenprodil in vivo.

The aim of the study was to determine whether fatty acid ethyl esters, nonoxidative products of ethanol metabolism selectively present in organs damaged by ethanol abuse, are detectable in the serum after ethanol ingestion. Serum samples of hospital emergency room patients with positive (n = 32) and negative (n = 5) blood ethanol levels were assayed for fatty acid ethyl esters. In a separate study, five healthy subjects received an ethanol dose based on body weight mixed with fruit juice in a 1:2 ratio and administered by measured ingestion. Fatty acid ethyl esters were found in the serum of hospital emergency room patients with positive blood ethanol levels. The concentration of fatty acid ethyl esters in these patients correlated with the concentration of blood ethanol (r = 0.57; 95% confidence interval 0.28 to 0.77; P = 0.0002). In the controlled ethanol ingestion study with five healthy subjects, it was also determined that the serum fatty acid ethyl ester concentration began to decrease within 2 h of the time ethanol ingestion had been stopped. The fatty acid ethyl esters in the serum were bound to lipoprotein and albumin, and there was a higher percentage of saturated fatty acids in the FAEE pool than in the serum free fatty acid and triglyceride pools. These studies indicate that fatty acid ethyl esters, which have been implicated as mediators of ethanol-induced organ toxicity, are present in serum after ethanol ingestion.

During the 1960s, three modalities of treatment aimed at rehabilitation of the drug abuser (methadone maintenance, outpatient drug free treatment, and the residential therapeutic community) were developed. Large amounts of public and private monies have gone to supporting these modalities; little evaluation as to the efficacy of such rehabilitation efforts has been done. This paper attempts to delineate the evaluative research efforts undertaken in the drug abuse field to date. In addition, the findings of an eight-year evaluation of six drug treatment programs in Newark, NJ are presented. The authors propose a paradigm for quick, effective evaluation of drug and alcohol programs at minimal cost.