GSK3ß phosphorylates Six1 transcription factor and regulates its APC/CCdh1 mediated proteosomal degradation.

Sine oculis homeobox homolog 1 (Six1) is a developmentally important transcription factor that regulates cellular proliferation, apoptosis, and dissemination during embryogenesis. Six1 overexpression as reported in multiple cancers modulates expression of a repertoire of its target genes causing an increase in proliferation, metastasis and survival of cancer cells. Six1 exists as a cell cycle regulated nuclear phosphoprotein and its cellular turnover is regulated by APC/C (Anaphase promoting complex / Cyclosome) complex mediated proteolysis. However, the kinases that regulate Six1 proteolysis have not been identified and the mechanistic details that cause its overproduction in various cancers are lacking. Here, we report that Six1 is a physiological GSK3ß substrate. GSK3ß interacts with Six1 and phosphorylates it at Ser221 within the conserved consensus sequence in its carboxy terminus. Using pharmacological inhibition, siRNA mediated knockdown and protein overexpression of GSK3ß; we show that GSK3ß regulates Six1 protein stability. Pulse chase analysis of Six1 revealed that GSK3ß regulates its ubiquitin proteolysis such that Six1 phosphomimicking mutant (Six1S221E) for Ser221 site had dramatically increased half-life than its phosphodeficient (Six1S221A) and wild type variants. Furthermore, we demonstrate that GSK3ß rescues Six1 from APC dependent proteolysis by regulating its binding with APC/C co-activator protein Cdh1. Importantly, strong positive correlation exists between GSK3ß and Six1 protein levels throughout the cell cycle and in multiple cancers indicating that GSK3ß activation may in part contribute to Six1 overproduction in a subset of human cancers.

Repurposing Amphotericin B: anti-microbial, molecular docking and molecular dynamics simulation studies suggest inhibition potential of Amphotericin B against MRSA.

Amphotericin B (AMPH) is an anti-fungal drug and this study, for the first time as best of our knowledge, reports the repurposing of the Amphotericin B. The drug was found to show significant antibacterial potential revealed by antimicrobial screening, molecular docking, and mode of action analysis targeting Penicillin Binding Protein 2a (PBP 2a protein) which is target of ß-lactam drugs and is involved in cell wall synthesis. Mode of action analysis showed the drug to have hydrophobic and hydrophilic interactions with both C-terminal, trans-peptidase and non-penicillin binding domain of the protein. Additionally, to evaluate the impact of ligand binding on the protein's conformational dynamics, molecular dynamics (MD) simulations were used. Comparative Dynamical flexibility (RMSF) and Dynamics Cross Correlation (DCCM) followed by MD simulations revealed the complex formation significantly effecting structural dynamics of the enzyme significantly in the non-penicillin binding domain (327-668) and slightly in trans peptidase domain. Radius of gyration assessment further showed ligand binding also decreasing over all compactness of protein. Secondary structure analysis indicated the complex formation changing the conformational integrity in non-penicillin binding domain. Hydrogen bond analysis and MMPBSA, free energy of calculations followed by MD simulations, also complemented the antimicrobial and molecular docking revelations suggesting Amphotericin B to have substantial antibacterial potential.

Serum MicroRNAs as Predictors for HCV Progression and Response to Treatment in Pakistani Patients.

Hepatitis is one of the common liver diseases, imposing a heavy health burden worldwide. Acute hepatitis may develop into chronic hepatitis, progressing to cirrhosis and hepatocellular carcinoma. In the present study, the expression of miRNAs was quantified by real-time PCR, such as miRNA-182, 122, 21, 150, 199, and 222. Along with the control group, HCV was divided into chronic, cirrhosis, and HCC groups. The treated group was also included after the successful treatment of HCV. Biochemical parameters, such as ALT, AST, ALP, bilirubin, viral load, and AFP (HCC), were also evaluated in all of the study groups. We compared the control and diseased groups; these parameters showed significant results (p = 0.000). The viral load was high in HCV but was not detected after treatment. miRNA-182 and miRNA-21 were overexpressed with disease progression, while the expression of miRNA-122 and miRNA-199 was increased compared with the control, but decreased in the cirrhosis stage compared with chronic and HCC. The expression of miRNA-150 was increased in all of the diseased groups compared with the control, but decreased compared with the chronic group. We compared the chronic and treated groups and then all of these miRNAs were down-regulated after treatment. These microRNAs could be used as potential biomarkers for diagnosing different stages of HCV.

Molecular Characterization, Purification, and Mode of Action of Enterocin KAE01 from Lactic Acid Bacteria and Its In Silico Analysis against MDR/ESBL Pseudomonas aeruginosa.

Bacteriocins are gaining immense importance in therapeutics since they show significant antibacterial potential. This study reports the bacteriocin KAE01 from Enterococcus faecium, along with its characterization, molecular modeling, and antibacterial potency, by targeting the matrix protein of Pseudomonas aeruginosa. The bacteriocin was purified by using ammonium sulfate precipitation and fast protein liquid chromatography (FPLC), and its molecular weight was estimated as 55 kDa by means of SDS-PAGE. The bacteriocin was found to show stability in a wide range of pH values (2.0-10.0) and temperatures (100 °C for 1 h and 121 °C for 15 min). Antimicrobial screening of the purified peptide against different strains of P. aeruginosa showed its significant antibacterial potential. Scanning electron microscopy of bacteriocin-induced bacterial cultures revealed significant changes in the cellular morphology of the pathogens. In silico molecular modeling of KAE01, followed by molecular docking of the matrix protein (qSA) of P. aeruginosa and KAE01, supported the antibacterial potency and SEM findings of this study.

Stress level assessment among health care workers involved in the management of critically ill COVID-19 patients.

BACKGROUND: Intensive care health care workers (HCWs) are frontlines of this crisis as they deal with critically ill COVID-19 patients which can potentially affect their mental well-being and causes different levels of stress. AIM: To determine the prevalence of stress among HCWs involved in the management of critically ill COVID-19 patient, identify the factors associated with stress, and highlight the availability of psychological support provided to HCWs. METHODS: A cross-sectional multicenter, international study using a web-based questionnaire of 27 questions including the Perceived Stress Scale-10 (PSS-10) for assessment of stress level. Questions to identify factors associated with stress, the psychological support provided, and the sociodemographic characteristics were included. RESULTS: We received a total 1649 responses from 59 countries: 550 (34%) were from Europe, 525 (32.36%) from Asia, 283 (17.44%) from Africa, 177 (11%) from America, and 88 (5.42%) from Australia. The average stress level was 22 points on the PSS denoting moderate stress in 1327 (81.8%) respondents, while 239 (14.73%) respondents had a severe level of stress. Female gender, working in high capacity units and remote areas in addition to lack of psychological support, was significantly associated with stress in our study. CONCLUSION: Stress level was moderate to severe among intensive care HCWs during this pandemic, and many factors were associated with stress emphasizing the importance of psychological support during that unprecedented pandemic.

Neuroprotective effects of Bhilawanol and Anacardic acid during glutamate-induced neurotoxicity.

Bhilawanol (Bh) and anacardic acid (AA) are two lipid-soluble compounds mostly found in the nut of Semecarpus anacardium (SA). This herb has many medicinal properties including enhancing learning and memory, yet its active compounds have not been studied for neuroprotective effects. We investigated the neuroprotective effects of Bh and AA against glutamate induced cell death in the adrenal pheochromocytoma cell line of rats (PC12 cells). Cell viability, toxicity and calcium influx were determined by MTT assay, LDH release assay and Fluo-3 imaging while apoptosis was assayed by caspase-3 and Bcl-2 gene expression. Our results showed that Bh and AA treatments significantly increased cell viability, reduced cell toxicity and calcium influx in PC12 cells in addition to suppressing the reactive oxygen species. Furthermore, AA treatment decreased caspase-3 expression level whereas both Bh and AA enhanced the expression of anti-apoptotic gene Bcl-2 in PC12 cells. Both compounds potently inhibited acetylcholinesterase enzyme (AChE) in a dose and time dependent manner. These findings suggest that the traditional use of SA may be explained on the basis of both Bh and AA showing neuroprotective potential due to their effects on enhancing cell viability, reducing cell toxicity most probably by reducing excessive calcium influx and suppression of ROS as well as by decreasing the expression of proapoptotic caspase 3 gene and increasing the expression of antiapoptotic gene Bcl2. Traditional use in enhancing learning and memory was justified in part by inhibition of AChE.

Development of a novel, sensitive translational immunoassay to detect plasma glial fibrillary acidic protein (GFAP) after murine traumatic brain injury.

BACKGROUND: Glial fibrillary acidic protein (GFAP) has emerged as a promising fluid biomarker for several neurological indications including traumatic brain injury (TBI), a leading cause of death and disability worldwide. In humans, serum or plasma GFAP levels can predict brain abnormalities including hemorrhage on computed tomography (CT) scans and magnetic resonance imaging (MRI). However, assays to quantify plasma or serum GFAP in preclinical models are not yet available. METHODS: We developed and validated a novel sensitive GFAP immunoassay assay for mouse plasma on the Meso Scale Discovery immunoassay platform and validated assay performance for robustness, precision, limits of quantification, dilutional linearity, parallelism, recovery, stability, selectivity, and pre-analytical factors. To provide proof-of-concept data for this assay as a translational research tool for TBI and Alzheimer's disease (AD), plasma GFAP was measured in mice exposed to TBI using the Closed Head Impact Model of Engineered Rotational Acceleration (CHIMERA) model and in APP/PS1 mice with normal or reduced levels of plasma high-density lipoprotein (HDL). RESULTS: We performed a partial validation of our novel assay and found its performance by the parameters studied was similar to assays used to quantify human GFAP in clinical neurotrauma blood specimens and to assays used to measure murine GFAP in tissues. Specifically, we demonstrated an intra-assay CV of 5.0%, an inter-assay CV of 7.2%, a lower limit of detection (LLOD) of 9.0 pg/mL, a lower limit of quantification (LLOQ) of 24.8 pg/mL, an upper limit of quantification (ULOQ) of at least 16,533.9 pg/mL, dilution linearity of calibrators from 20 to 200,000 pg/mL with 90-123% recovery, dilution linearity of plasma specimens up to 32-fold with 96-112% recovery, spike recovery of 67-100%, and excellent analyte stability in specimens exposed to up to 7 freeze-thaw cycles, 168 h at 4 °C, 24 h at room temperature (RT), or 30 days at - 20 °C. We also observed elevated plasma GFAP in mice 6 h after TBI and in aged APP/PS1 mice with plasma HDL deficiency. This assay also detects GFAP in serum. CONCLUSIONS: This novel assay is a valuable translational tool that may help to provide insights into the mechanistic pathophysiology of TBI and AD.

Improved Detection of Postoperative Residual Meningioma with [68Ga]Ga-DOTA-TOC PET Imaging Using a High-resolution Research Tomograph PET Scanner.

PURPOSE: PET with somatostatin receptor ligand [68Ga]Ga-DOTA-D-Phe1-Tyr3-octreotide ([68Ga]Ga-DOTA-TOC) is an established method in radiotherapy planning because of the improved detection and delineation of meningioma tissue. We investigated the diagnostic accuracy of supplementary [68Ga]Ga-DOTA-TOC PET in patients with a 3-month postoperative MRI reporting gross-total resection (GTR). EXPERIMENTAL DESIGN: Thirty-seven patients with a histologically proven meningioma and GTR on postoperative MRI were prospectively referred to [68Ga]Ga-DOTA-TOC PET. Detection and volume measurements of [68Ga]Ga-DOTA-TOC-avid lesions in relation to the primary tumor site were recorded. Residual tumor in suspicious lesions suggested by [68Ga]Ga-DOTA-TOC PET was verified by (i) tumor recurrence/progression on subsequent MRI scans according to the Response Assessment of Neuro-Oncology criteria, (ii) subsequent histology, and (iii) follow-up [68Ga]Ga-DOTA-TOC PET scan. RESULTS: Twenty-three PET scans demonstrated [68Ga]Ga-DOTA-TOC-avid lesions suspicious of residual meningioma, where 18 could be verified by (i) tumor progression on subsequent MRI scans (n = 6), (ii) histologic confirmation (n = 3), and (iii) follow-up [68Ga]Ga-DOTA-TOC PET scans confirming the initial PET findings (n = 9) after an overall median follow-up time of 17 months (range, 9-35 months). In contrast, disease recurrence was seen in only 2 of 14 patients without [68Ga]Ga-DOTA-TOC-avid lesions (P < 0.0001). The sensitivity, specificity, and diagnostic accuracy of [68Ga]Ga-DOTA-TOC PET in detecting meningioma residue was 90% [95% confidence interval (CI), 67-99], 92% (95% CI, 62-100), and 90% (95% CI, 74-98; P < 0.0001), respectively. CONCLUSIONS: The majority of patients with GTR on 3-month postoperative MRI may have small unrecognized meningioma residues that can be detected using [68Ga]Ga-DOTA-TOC PET.

Somatostatin Receptor-Targeted Radiopeptide Therapy in Treatment-Refractory Meningioma: Individual Patient Data Meta-analysis.

Somatostatin receptor (SSTR)-targeted peptide receptor radionuclide therapy (PRRT) represents a promising approach for treatment-refractory meningiomas. Methods: We performed an individual patient data meta-analysis, including all published data on meningioma patients treated with SSTR-targeted PRRT. The main outcomes were toxicity, response to treatment, progression-free survival (PFS), and overall survival (OS). We applied the Kaplan-Meier method to estimate survival probabilities and report incidence rates per 100 person-years. We applied Cox proportional hazards models to determine the effect of covariates. Results: We screened 537 papers and identified 6 eligible cohort studies. We included a total of 111 patients who had treatment-refractory meningioma and received SSTR-targeted PRRT. Disease control was achieved in 63% of patients. The 6-mo PFS rates were 94%, 48%, and 0% for World Health Organization grades I, II, and III, respectively. The risk of disease progression decreased by 13% per 1,000-MBq increase in the total applied activity. The 1-y OS rates were 88%, 71%, and 52% for World Health Organization grades I, II, and III, respectively. The risk of death decreased by 17% per 1,000-MBq increase in the total applied activity. The main side effects comprised transient hematotoxicity, such as anemia in 22% of patients, leukopenia in 13%, lymphocytopenia in 24%, and thrombocytopenia in 17%. Conclusion: To our knowledge, this individual patient data meta-analysis represents the most comprehensive analysis of the benefits of and adverse events associated with SSTR-targeted PRRT for treatment-refractory meningioma. The treatment was well tolerated, achieved disease control in most cases, and showed promising results regarding PFS and OS.

PET imaging of meningioma with 18F-FLT: a predictor of tumour progression.

We have previously reported that PET with 3'-deoxy-3'-18F-fluorothymidine (18F-FLT) provides a non-invasive assessment of cell proliferation in vivo in meningiomas. The purpose of this prospective study was to evaluate the potential of 18F-FLT PET in predicting subsequent tumour progression in asymptomatic meningiomas. Forty-three adult patients harbouring 46 MRI-presumed (n = 40) and residual meningiomas from previous surgery (n = 6) underwent a 60-min dynamic 18F-FLT PET scan prior to radiological surveillance. Maximum and mean tumour-to-blood ratios (TBRmax, TBRmean) of tracer radioactivity were calculated. Tumour progression was defined according to the latest published trial end-point criteria for bidimensional (2D) and corresponding yet exploratory volumetric measurements from the Response Assessment of Neuro-Oncology (RANO) workgroup. Independent-sample t-test, Pearson correlation coefficient, Cox regression, and receiver operating characteristic (ROC) curve analyses were used whenever appropriate. The median follow-up time after 18F-FLT PET imaging was 18 months (range 5-33.5 months). A high concordance rate (91%) was found with regard to disease progression using 2D-RANO (n = 11) versus volumetric criteria (n = 10). Using 2D-RANO criteria, 18F-FLT uptake was significantly increased in patients with progressive disease, compared to patients with stable disease (TBRmax, 5.5 ± 1.3 versus 3.6 ± 1.1, P < 0.0001; TBRmean, 3.5 ± 0.8 versus 2.4 ± 0.7, P < 0.0001). ROC analysis yielded optimal thresholds of 4.4 for TBRmax [sensitivity 82%, specificity 77%, accuracy 78%, and area under curve (AUC) 0.871; P < 0.0001] and 2.8 for TBRmean (sensitivity 82%, specificity 77%, accuracy 78%, AUC 0.848; P = 0.001) for early differentiation of patients with progressive disease from patients with stable disease. Upon excluding patients with residual meningioma or patients with stable disease with less than 12 months follow-up, the thresholds remained unchanged with similar diagnostic accuracies. Moreover, positive correlations were found between absolute and relative tumour growth rates and 18F-FLT uptake (r < 0.513, P < 0.015) that remained similar when excluding patients with residual meningioma or patients with stable disease and shorter follow-up period. Diagnostic accuracies were slightly inferior at 76% when assessing disease progression using volumetric criteria, while the thresholds remained unchanged. Multivariate analysis revealed that TBRmax was the only independent predictor of tumour progression (P < 0.046), while age, gender, baseline tumour size, tumour location, peritumoural oedema, and residual meningioma had no influence. The study reveals that 18F-FLT PET is a promising surrogate imaging biomarker for predicting subsequent tumour progression in treatment-naïve and asymptomatic residual meningiomas.

Pearls and Pitfalls in Interpretation of 68Ga-DOTATOC PET Imaging.

DOTA-D-Phe-Tyr-octreotide labeled with Ga (Ga-DOTATOC) is the commonly used PET tracer for imaging meningioma because of its high affinity to somatostatin receptor subtype 2 (SSTR2) and an established imaging modality for planning radiation and radionuclide therapy. However, SSTR2 is not an exclusive marker for meningioma, and not all meningiomas express high levels of SSTR2. The SSTR2 expression has been reported in other intracranial tumors, for example, glioma, pituitary adenoma, medullablastoma, primitive neuroectodermal tumors, and hemangioblastoma leading to a significant risk of misinterpretation of PET/CT findings. We present 2 cases with similar Ga-DOTATOC uptakes in 2 distinct etiologies, for example, cerebral lymphoma and meningioma.

Pharmacokinetic analysis of [68Ga]Ga-DOTA-TOC PET in meningiomas for assessment of in vivo somatostatin receptor subtype 2.

PURPOSE: DOTA-D-Phe1-Tyr3-octreotide with gallium-68 ([68Ga]Ga-DOTA-TOC) is one of the PET tracers that forms the basis for peptide receptor radionuclide therapy based on somatostatin receptor subtype 2 (SSTR2) expression in meningiomas. Yet, the quantitative relationship between [68Ga]Ga-DOTA-TOC accumulation and SSTR2 is unknown. We conducted a correlative analysis of a range of [68Ga]Ga-DOTA-TOC PET metric(s) as imaging surrogate(s) of the receptor binding in meningiomas by correlating the PET results with SSTR2 expression from surgical specimens. We additionally investigated possible influences of secondary biological factors such as vascularization, inflammation and proliferation. METHODS: Fifteen patients with MRI-presumed or recurrent meningiomas underwent a 60-min dynamic [68Ga]Ga-DOTA-TOC PET/CT before surgery. The PET data comprised maximum and mean standardized uptake values (SUVmax, SUVmean) with and without normalization to reference regions, and quantitative measurements derived from kinetic modelling using a reversible two-tissue compartment model with the fractional blood volume (VB). Expressions of SSTR2 and proliferation (Ki-67, phosphohistone-H3, proliferating cell nuclear antigen) were determined by immunohistochemistry and/or quantitative polymerase chain reaction (qPCR), while biomarkers of vascularization (vascular endothelial growth factor A (VEGFA), endothelial marker CD34) and inflammation (cytokine interleukin-18, microglia/macrophage-specific marker CD68) by qPCR. RESULTS: Histopathology revealed 12 World Health Organization (WHO) grade I and three WHO grade II meningiomas showing no link to SSTR2. The majority of [68Ga]Ga-DOTA-TOC PET metrics showed significant associations with SSTR2 protein, while all PET metrics were positively correlated with SSTR2 mRNA with the best results for mean tumour-to-blood ratio (TBRmean) (r = 0.757, P = 0.001) and SUVmean (r = 0.714, P = 0.003). Significant positive correlations were also found between [68Ga]Ga-DOTA-TOC PET metrics, and VEGFA and VB. SSTR2 mRNA was moderately correlated with VEGFA (r = 0.539, P = 0.038). Neither [68Ga]Ga-DOTA-TOC PET metrics nor SSTR2 were correlated with proliferation or inflammation. CONCLUSION: [68Ga]Ga-DOTA-TOC accumulation in meningiomas is associated with SSTR2 binding and vascularization with TBRmean being the best PET metric for assessing SSTR2.

An Automated Kinematic Measurement System for Sagittal Plane Murine Head Impacts.

Mild traumatic brain injuries are typically caused by nonpenetrating head impacts that accelerate the skull and result in deformation of the brain within the skull. The shear and compressive strains caused by these deformations damage neural and vascular structures and impair their function. Accurate head acceleration measurements are necessary to define the nature of the insult to the brain. A novel murine head tracking system was developed to improve the accuracy and efficiency of kinematic measurements obtained with high-speed videography. A three-dimensional (3D)-printed marker carrier was designed for rigid fixation to the upper jaw and incisors with an elastic strap around the snout. The system was evaluated by impacting cadaveric mice with the closed head impact model of engineered rotational acceleration (CHIMERA) system using an energy of 0.7 J (5.29 m/s). We compared the performance of the head-marker system to the previously used skin-tracking method and documented significant improvements in measurement repeatability (aggregate coefficient of variation (CV) within raters from 15.8 to 1.5 and between raters from 15.5 to 1.5), agreement (aggregate percentage error from 24.9 to 8.7), and temporal response (aggregate temporal curve agreement from 0.668 to 0.941). Additionally, the new system allows for automated software tracking, which dramatically decreases the analysis time required (74% reduction). This novel head tracking system for mice offers an efficient, reliable, and real-time method to measure head kinematics during high-speed impacts using CHIMERA or other rodent or small mammal head impact models.

In vivo imaging of cell proliferation in meningioma using 3'-deoxy-3'-[18F]fluorothymidine PET/MRI.

PURPOSE: Positron emission tomography (PET) with 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) provides a noninvasive assessment of tumour proliferation in vivo and could be a valuable imaging modality for assessing malignancy in meningiomas. We investigated a range of static and dynamic [18F]FLT metrics by correlating the findings with cellular biomarkers of proliferation and angiogenesis. METHODS: Seventeen prospectively recruited adult patients with intracranial meningiomas underwent a 60-min dynamic [18F]FLT PET following surgery. Maximum and mean standardized uptake values (SUVmax, SUVmean) with and without normalization to healthy brain tissue and blood radioactivity obtained from 40 to 60 min summed dynamic images (PET40-60) and ~ 60-min blood samples were calculated. Kinetic modelling using a two-tissue reversible compartmental model with a fractioned blood volume (VB) was performed to determine the total distribution volume (VT). Expressions of proliferation and angiogenesis with key parameters including Ki-67 index, phosphohistone-H3 (phh3), MKI67, thymidine kinase 1 (TK1), proliferating cell nuclear antigen (PCNA), Kirsten RAt Sarcoma viral oncogene homolog (KRAS), TIMP metallopeptidase inhibitor 3 (TIMP3), and vascular endothelial growth factor A (VEGFA) were determined by immunohistochemistry and/or quantitative polymerase chain reaction. RESULTS: Immunohistochemistry revealed 13 World Health Organization (WHO) grade I and four WHO grade II meningiomas. SUVmax and SUVmean normalized to blood radioactivity from PET40-60 and blood sampling, and VT were able to significantly differentiate between WHO grades with the best results for maximum and mean tumour-to-whole-blood ratios (sensitivity 100%, specificity 94-95%, accuracy 99%; P = 0.003). Static [18F]FLT metrics were significantly correlated with proliferative biomarkers, especially Ki-67 index, phh3, and TK1, while no correlations were found with VEGFA or VB. Using Ki-67 index with a threshold > 4%, the majority of [18F]FLT metrics showed a high ability to identify aggressive meningiomas with SUVmean demonstrating the best performance (sensitivity 80%, specificity 81%, accuracy 80%; P = 0.024). CONCLUSION: [18F]FLT PET could be a useful imaging modality for assessing cellular proliferation in meningiomas.

Increased severity of the CHIMERA model induces acute vascular injury, sub-acute deficits in memory recall, and chronic white matter gliosis.

Traumatic brain injury (TBI) is a leading cause of death and disability in modern societies. Diffuse axonal and vascular injury are nearly universal consequences of mechanical energy impacting the head and contribute to disability throughout the injury severity spectrum. CHIMERA (Closed Head Impact Model of Engineered Rotational Acceleration) is a non-surgical, impact-acceleration model of rodent TBI that reliably produces diffuse axonal injury characterized by white matter gliosis and axonal damage. At impact energies up to 0.7 joules, which result in mild TBI in mice, CHIMERA does not produce detectable vascular or grey matter injury. This study was designed to expand CHIMERA's capacity to induce more severe injuries, including vascular damage and grey matter gliosis. This was made possible by designing a physical interface positioned between the piston and animal's head to allow higher impact energies to be transmitted to the head without causing skull fracture. Here, we assessed interface-assisted single CHIMERA TBI at 2.5 joules in wild-type mice using a study design that spanned 6 h-60 d time points. Injured animals displayed robust acute neurological deficits, elevated plasma total tau and neurofilament-light levels, transiently increased proinflammatory cytokines in brain tissue, blood-brain barrier (BBB) leakage and microstructural vascular abnormalities, and grey matter microgliosis. Memory deficits were evident at 30 d and resolved by 60 d. Intriguingly, white matter injury was not remarkable at acute time points but evolved over time, with white matter gliosis being most extensive at 60 d. Interface-assisted CHIMERA thus enables experimental modeling of distinct endophenotypes of TBI that include acute vascular and grey matter injury in addition to chronic evolution of white matter damage, similar to the natural history of human TBI.

Recurrent glioblastoma versus late posttreatment changes: diagnostic accuracy of O-(2-[18F]fluoroethyl)-L-tyrosine positron emission tomography (18F-FET PET).

BACKGROUND: Diagnostic accuracy in previous studies of O-(2-[18F]-fluoroethyl)-L-tyrosine (18F-FET) PET in patients with suspected recurrent glioma may be influenced by prolonged dynamic PET acquisitions, heterogeneous populations, different non-standard-of-care therapies, and PET scans performed at different time points post radiotherapy. We investigated the diagnostic accuracy of a 20-minute 18F-FET PET scan in MRI-suspected recurrent glioblastoma 6 months after standard radiotherapy and its ability to prognosticate overall survival (OS). METHODS: In total, 146 glioblastoma patients with 168 18F-FET PET scans were reviewed retrospectively. Patients with MRI responses to bevacizumab or undergoing re-irradiation or immunotherapy after 18F-FET PET were excluded. Maximum and mean tumor-to-background ratios (TBRmax, TBRmean) and biological tumor volume (BTV) were recorded and verified by histopathology or clinical/radiological follow-up. Thresholds of 18F-FET parameters were determined by receiver operating characteristic (ROC) analysis. Prognostic factors were investigated in Cox proportional hazards models. RESULTS: Surgery was performed after 104 18F-FET PET scans, while clinical/radiological surveillance was used following 64, identifying 152 glioblastoma recurrences and 16 posttreatment changes. ROC analysis yielded thresholds of 2.0 for TBRmax, 1.8 for TBRmean, and 0.55 cm3 for BTV in differentiating recurrent glioblastoma from posttreatment changes with the best performance of TBRmax (sensitivity 99%, specificity 94%; P < 0.0001) followed by BTV (sensitivity 98%, specificity 94%; P < 0.0001). Using these thresholds, 166 18F-FET PET scans were correctly classified. Increasing BTV was associated with shorter OS (P < 0.0001). CONCLUSION: A 20-minute 18F-FET PET scan is a powerful tool to distinguish posttreatment changes from recurrent glioblastoma 6-month postradiotherapy, and predicts OS.

Cerebral Microvascular Injury: A Potentially Treatable Endophenotype of Traumatic Brain Injury-Induced Neurodegeneration.

Traumatic brain injury (TBI) is one the most common human afflictions, contributing to long-term disability in survivors. Emerging data indicate that functional improvement or deterioration can occur years after TBI. In this regard, TBI is recognized as risk factor for late-life neurodegenerative disorders. TBI encompasses a heterogeneous disease process in which diverse injury subtypes and multiple molecular mechanisms overlap. To develop precision medicine approaches where specific pathobiological processes are targeted by mechanistically appropriate therapies, techniques to identify and measure these subtypes are needed. Traumatic microvascular injury is a common but relatively understudied TBI endophenotype. In this review, we describe evidence of microvascular dysfunction in human and animal TBI, explore the role of vascular dysfunction in neurodegenerative disease, and discuss potential opportunities for vascular-directed therapies in ameliorating TBI-related neurodegeneration. We discuss the therapeutic potential of vascular-directed therapies in TBI and the use and limitations of preclinical models to explore these therapies.

Repetitive closed-head impact model of engineered rotational acceleration (CHIMERA) injury in rats increases impulsivity, decreases dopaminergic innervation in the olfactory tubercle and generates white matter inflammation, tau phosphorylation and degeneration.

Traumatic brain injury (TBI) affects at least 3 M people annually. In humans, repetitive mild TBI (rmTBI) can lead to increased impulsivity and may be associated with chronic traumatic encephalopathy. To better understand the relationship between repetitive TBI (rTBI), impulsivity and neuropathology, we used CHIMERA (Closed-Head Injury Model of Engineered Rotational Acceleration) to deliver five TBIs to rats, which were continuously assessed for trait impulsivity using the delay discounting task and for neuropathology at endpoint. Compared to sham controls, rats with rTBI displayed progressive impairment in impulsive choice. Histological analyses revealed reduced dopaminergic innervation from the ventral tegmental area to the olfactory tubercle, consistent with altered impulsivity neurocircuitry. Consistent with diffuse axonal injury generated by CHIMERA, white matter inflammation, tau immunoreactivity and degeneration were observed in the optic tract and corpus callosum. Finally, pronounced grey matter microgliosis was observed in the olfactory tubercle. Our results provide insight into the mechanisms by which rTBI leads to post-traumatic psychiatric-like symptoms in a novel rat TBI platform.

A novel mechanism for the anticancer activity of aspirin and salicylates.

Epidemiological studies indicate that long­term aspirin usage reduces the incidence of colorectal cancer (CRC) and may protect against other non­CRC associated adenocarcinomas, including oesophageal cancer. A number of hypotheses have been proposed with respect to the molecular action of aspirin and other non­steroidal anti­inflammatory drugs in cancer development. The mechanism by which aspirin exhibits toxicity to CRC has been previously investigated by synthesising novel analogues and derivatives of aspirin in an effort to identify functionally significant moieties. Herein, an early effect of aspirin and aspirin­like analogues against the SW480 CRC cell line was investigated, with a particular focus on critical molecules in the epidermal growth factor (EGF) pathway. The present authors proposed that aspirin, diaspirin and analogues, and diflunisal (a salicylic acid derivative) may rapidly perturb EGF and EGF receptor (EGFR) internalisation. Upon longer incubations, the diaspirins and thioaspirins may inhibit EGFR phosphorylation at Tyr1045 and Tyr1173. It was additionally demonstrated, using a qualitative approach, that EGF internalisation in the SW480 cell line may be directed to endosomes by fumaryldiaspirin using early endosome antigen 1 as an early endosomal marker and that EGF internalisation may also be perturbed in oesophageal cell lines, suggestive of an effect not only restricted to CRC cells. Taken together and in light of our previous findings that the aspirin­like analogues can affect cyclin D1 expression and nuclear factor­κB localisation, it was hypothesized that aspirin and aspirin analogues significantly and swiftly perturb the EGFR axis and that the protective activity of aspirin may in part be explained by perturbed EGFR internalisation and activation. These findings may also have implications in understanding the inhibitory effect of aspirin and salicylates on wound healing, given the critical role of EGF in the response to tissue trauma.