Normal Cortex Research Articles

Differential expression of paralog RNA binding proteins establishes a dynamic splicing program required for normal cerebral cortex development.

Sam68 and SLM2 are paralog RNA binding proteins (RBPs) expressed in the cerebral cortex and display similar splicing activities. However, their relative functions during cortical development are unknown. We found that these RBPs exhibit an opposite expression pattern during development. Sam68 expression declines postnatally while SLM2 increases after birth, and this developmental pattern is reinforced by hierarchical control of Sam68 expression by SLM2. Analysis of Sam68:Slm2 double knockout (Sam68:Slm2dko) mice revealed hundreds of exons that respond to joint depletion of these proteins. Moreover, parallel analysis of single and double knockout cortices indicated that exons regulated mainly by SLM2 are characterized by a dynamic splicing pattern during development, whereas Sam68-dependent exons are spliced at relatively constant rates. Dynamic splicing of SLM2-sensitive exons is completely suppressed in the Sam68:Slm2dko developing cortex. Sam68:Slm2dko mice die perinatally with defects in neurogenesis and in neuronal differentiation, and develop a hydrocephalus, consistent with splicing alterations in genes related to these biological processes. Thus, our study reveals that developmental control of separate Sam68 and Slm2 paralog genes encoding homologous RBPs enables the orchestration of a dynamic splicing program needed for brain development and viability, while ensuring a robust redundant mechanism that supports proper cortical development.

Vascular endothelial growth factor and endocan expression in adrenal cortical tumors and their relationship with histopathological prognostic parameters.

The aim of this study was to determine the tissue expressions of vascular endothelial growth factor (VEGF) and endocan in adrenal cortical tumors and the factors associated with them. The study included 6 subjects with adrenocortical adenoma (ACA), 7 subjects with adrenocortical carcinoma (ACC), and 13 control subjects with a normal adrenal cortex. The status of VEGF and endocan expression was determined by the proportions of cells staining on a scale ranging from negative (not staining at all) to strongly positive. VEGF expression was detected in 1 (16.7%) of 6 subjects in the ACA group and in 6 (85.7%) of 7 subjects in the ACC group. VEGF expression was not detected in any of the subjects in the control group. Endocan expression was detected in 6 (100%) of 6 subjects in the ACA group and in 7 (100%) of 7 subjects in the ACC group, while it was detected in only 4 (30.7%) of 13 subjects in the control group. VEGF was expressed with a high frequency in subjects with ACC and with a low frequency in subjects with ACA, but it was not expressed in subjects with normal adrenal cortex tissue. Although endocan was expressed with a higher frequency in subjects with ACC and ACA, it was also expressed in subjects with normal adrenal cortex tissue. The percentage of cells expressed endocan in subjects with ACC was also significantly higher than in subjects with both ACA and normal adrenal cortex.

Identification of Key Genes and Related Drugs of Adrenocortical Carcinoma by Integrated Bioinformatics Analysis.

Adrenocortical carcinoma (ACC) is a malignant carcinoma with an extremely poor prognosis, and its pathogenesis remains to be understood to date, necessitating further investigation. This study aims to discover biomarkers and potential therapeutic agents for ACC through bioinformatics, enhancing clinical diagnosis and treatment strategies. Differentially expressed genes (DEGs) between ACC and normal adrenal cortex were screened out from the GSE19750 and GSE90713 datasets available in the GEO database. An online Venn diagram tool was utilized to identify the common DEGs between the two datasets. The identified DEGs were subjected to functional assessment, pathway enrichment, and identification of hub genes by performing the protein-protein interaction (PPI), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The differences in the expressions of hub genes between ACC and normal adrenal cortex were validated at the GEPIA2 website, and the association of these genes with the overall patient survival was also assessed. Finally, on the QuartataWeb website, drugs related to the identified hub genes were determined. A total of 114 DEGs, 10 hub genes, and 69 known drugs that could interact with these genes were identified. The GO and KEGG analyses revealed a close association of the identified DEGs with cellular signal transduction. The 10 hub genes identified were overexpressed in ACC, in addition to being significantly associated with adverse prognosis in ACC. Three genes and the associated known drugs were identified as potential targets for ACC treatment.

Single cell multi‐omics implicates immune and vascular cells in Alzheimer’s etiology

AbstractBackgroundUnderstanding the genetic etiology of Alzheimer’s disease (AD) is critical to inform effective therapies but remains a challenge. Genetic heritability of late‐onset AD is ∼60‐80%, and genome‐wide association studies (GWAS) have uncovered dozens of noncoding single nucleotide polymorphisms (SNPs) that influence AD risk. Two challenges for the functional interpretation of AD genetic risk are (1) determining the cell types in which SNPs operate and (2) identifying the genes they dysregulate to drive AD pathogenesis. Single‐nucleus sequencing studies of human neurons, microglia, and other brain cell types have begun addressing these challenges. Yet, many AD risk SNPs remain unmapped. One possibility is that they are expressed in immunovascular brain cell types largely missed by current methods.MethodHere, we optimized Vessel Isolation and Nuclei Extraction for Sequencing (VINE‐seq) to capture multimodal gene expression and chromatin accessibility assays across all major brain cell types from 30 cognitively normal, MCI, and AD human prefrontal cortex samples.ResultWe discover early gene dysregulation in immune and vascular cell types along AD progression. We highlight the gene regulatory networks mediating this dysregulation and implicate processes associated with AD pathology and cognitive decline. Finally, we map noncoding AD risk variants to diverse immune and vascular cell types, thereby expanding our understanding of AD etiology.ConclusionDysfunction of diverse human brain immune and vascular cell types mediate the genetic risk for Alzheimer’s disease.

Evaluation of cortical and trabecular bone structure of the mandible in patients using L-Thyroxine

BackgroundLong-term use of L-Thyroxine (LT4), the synthetic thyroxine hormone used for thyroid hormone replacement therapy, is an important risk factor for osteoporosis. The aim of this study was to investigate the differences between mandibular cortical index (MCI) and trabecular bone fractal dimension (FD) values on panoramic radiographs of patients using LT4 and control subjects.MethodsA total of 142 female patients, 71 cases and 71 controls, were analyzed in the study. Ages were matched in case and control groups and the mean age was 36.6 ± 8.2 (18 to 50) years. MCI consisting of C1 (Normal Mandibular Cortex), C2 (Moderately Resorbed Mandibular Cortex) and, C3 (Severely Resorbed Cortex) scores was determined for case and control groups. Fractal analysis was performed using ImageJ on selected regions of interest from the gonial and interdental regions. The box-count method was used to calculate FD values. Wilcoxon signed-rank test, Mann-Whitney U test, and Spearman correlation analysis were applied to compare the measurements. Statistical significance of differences was established at P < 0.05 level.ResultsFD values did not show statistically significant differences between case and control groups (p > 0.05). The mean FD in the right gonial region was 1.38 ± 0.07 in the case group and 1.38 ± 0.08 in the control group (p = 0.715). The mean FD in the right interdental region was 1.37 ± 0.06 in the cases and 1.36 ± 0.06 in the control group (p = 0.373). The mean FD in the left gonial region was 1.39 ± 0.07 in the cases and 1.39 ± 0.07 in the control group (p = 0.865). The mean FD in the left interdental region is 1.37 ± 0.06 in the cases and 1.38 ± 0.05 in the control group (p = 0.369). The most common MCI score was C1, with 62% in the cases and 83.1% in the control group. MCI scores showed a statistically significant difference between cases and controls (p = 0.016, p < 0.05). While the C2 score was higher in the cases, the C1 score was higher in the controls.ConclusionsLT4 use was not associated with the FD of mandibular trabecular bone, but was associated with MCI values of cortical bone. Further studies on larger samples with different imaging modalities and image processing methods are needed.

TMET-03. REPROGRAMMING OF GLIOBLASTOMA GLUCOSE METABOLISM FUELS TUMOR GROWTH

Abstract Metabolic adaptation can promote oncogenic phenotypes in glioblastoma (GBM), but the metabolic pathways utilized by GBM and how they differ from the pathways utilized in normal cortex are poorly understood. Here, we utilize in vivo stable isotope tracing in mice and patients with GBM to define carbon fate and metabolic rewiring in cancer. We infused uniformly-labeled 13C glucose into mice bearing orthotopic GBM patient-derived xenografts or into patients undergoing surgical resection for likely GBM. Tumor and cortex were physically separated in mice by fluorescence-guided microdissection. In humans, samples of enhancing tumor, non-enhancing tumor and normal cortex were separated intraoperatively using MRI guidance. Samples were subsequently analyzed by liquid chromatography mass spectrometry to determine the downstream metabolic fates of infused 13C glucose. Of eight patients infused with 13C6-glucose, six were confirmed to have GBM while the others had non-GBM high grade gliomas. In mice and in patients, glucose-derived carbon effectively entered glycolysis and labeled glycolytic intermediates equivalently in tumor and cortex. Compared to GBM tissue, cortical tissue preferentially oxidized glucose-derived carbon in the TCA cycle and used glucose-derived carbon to synthesize neurotransmitters. Cortical tissue utilized glucose-derived carbon to synthesize the amino acid and neurotransmitter precursor serine, while GBM tissue derived serine from extracellular sources. By contrast, GBM tissue preferentially utilized glucose-derived carbon for the synthesis of purines, pyrimidines and NAD+/NADH. Using time-course isotope tracing studies in mice with GBM and a novel metabolic flux analysis framework, we quantified absolute metabolic fluxes in vivo and confirmed elevated nucleotide synthesis and serine salvage in GBM. Consistent with these results, eliminating dietary serine in mice slowed GBM PDX growth. These studies are the first measurements of numerous metabolic pathways in humans and indicate that GBMs suppress the physiologic utilization of glucose carbons to fuel biosynthesis, which represents a targetable metabolic liability.

EPCO-50. THE TRANSCRIPTIONAL LANDSCAPE OF KEY INNATE DNA SENSORS WITHIN PRIMARY BRAIN TUMORS

Abstract Primary brain tumors are notoriously resistant to immunotherapy. The efficacy of these treatments potentially requires normal expression of DNA innate sensors within both the immune and tumor compartments. Recently, we found that STING1, involved in the CGAS/STING pathway, is epigenetically silenced in the neoplastic cells of primary brain tumors. This silencing can be traced back to the normal brain. We and others have shown that rescue of STING1 expression can be modulated with epigenetic treatments. The mechanism by which DNA damage in cancer leads to an inflammatory response sheds light on the significance of other DNA sensors. We hypothesize that innate immune silencing within the tumor contributes to immunotherapy resistance and the addition of epigenetic treatments could increase efficacy. We evaluated the transcriptional landscape of DNA innate sensors within normal adult brain and in patient derived tumors including glioblastoma, pilocytic astrocytoma, and diffuse midline gliomas, H3K27M mutant. We found low expression of additional cytosolic DNA sensors including cGAS, AIM2, ZBP1 in normal neuronal as well as tumor cells. Since epigenetic silencing is modulated during normal development, we compared the transcriptional innate profiles from normal cortex brain development to primary brain cancers. DNA innate immune sensor expression correlates with pre-oligodendrocyte progenitor cells and oligodendrocyte progenitor cells. These results suggest the innate immune suppression of these key cytosolic DNA sensors is established early in fetal brain development and retained in the context of primary brain tumors. We are currently exploring the epigenetic profiles of fetal brain and brain tumors to determine how these innate immune expression patterns affect tumorigenesis and response to immunotherapy treatment.

Transcriptomic Characterization of Copper-Binding Proteins for Predicting Prognosis in Glioma.

Copper and copper-binding proteins are key components of tumor progression as they play important roles in tumor invasion and migration, but their associations in gliomas remain unclear. Transcriptomic datasets of glioblastoma, low-grade glioma, and normal brain cortex were derived from the TCGA and GTEX databases. Differentially expressed genes (DEGs) of copper-binding proteins were screened and used to construct a prognostic model based on COX and LASSO regression, which was further validated by the CGGA datasets. The expressions of risk-model genes were selectively confirmed via anatomic feature-based expression analysis and immunohistochemistry. The risk score was stratified by age, gender, WHO grade, IDH1 mutation, MGMT promoter methylation, and 1p/19q codeletion status, and a nomogram was constructed and validated. A total of 21 DEGs of copper-binding proteins were identified and a six-gene risk-score model was constructed, consisting of ANG, F5, IL1A, LOXL1, LOXL2, and STEAP3, which accurately predicted 1-, 3-, and 5-year overall survival rates, with the AUC values of 0.87, 0.88, and 0.82, respectively. The high-risk group had a significantly shorter OS (p < 0.0001) and was associated with old age, wild-type IDH1, a high WHO grade, an unmethylated MGMT promoter, and 1p/19q non-codeletion and had higher levels of immune cell infiltration, cancer-immunity suppressor, and immune checkpoint gene expression as well as a higher TMB. The model based on the genes of copper-binding proteins could contribute to prognosis prediction and provide potential targets against gliomas.

A Phase 0 Study Assessing the Intracranial Activity of a Metabolic Radiosensitizer in Patients with Glioblastoma

Efforts to overcome treatment resistance in glioblastoma (GBM) have been unsuccessful due to tumor heterogeneity and poor intracranial drug penetration. Targeting altered metabolism is a promising approach to improve GBM therapy despite this heterogeneity. Mycophenolate mofetil (MMF) is an inhibitor of purine synthesis that sensitizes GBM to radiation and temozolomide (TMZ) in vitro and in vivo, but its ability to cross the blood brain barrier and inhibit GBM metabolism in patients is unknown. NCT04477200 is a phase 0/1 dose escalation study of MMF combined with radiation and temozolomide in GBM. Here we report the phase 0 results of this study assessing the intracranial activity of MMF. Purine (GTP and IMP) and mycophenolic acid (MPA, the active metabolite of MMF) concentrations were determined using mass spectrometry in flash-frozen tumor (enhancing and non-enhancing) and normal cortex obtained from 8 patients with recurrent GBM who received MMF (500, 1000, 1500 and 2000 mg BID, N = 2 patients each dose level) for 1 week prior to re-resection and 5 control patients who did not receive MMF prior to re-resection. Plasma MPA concentration was similarly quantified to calculate the enhancing tumor, non-enhancing tumor and normal cortex to plasma MPA ratios. Patients who received MMF had a mean MPA concentration of 2.2 ± 0.7 µM in the enhancing tumor samples, 1.2 ± 0.5 µM in the non-enhancing tumor samples and 1.3 ± 0.5 µM in normal cortex. MPA concentration was negligible in control patients. This corresponded to tissue/plasma MPA ratios of 0.31, 0.17 and 0.10 for enhancing tumor, non-enhancing tumor and normal cortex, respectively. The GTP/IMP ratio was decreased by 75% in enhancing tumor in MMF-treated patients compared to untreated controls (p = 0.009), indicating effective target engagement and inhibition of purine synthesis. The GTP/IMP ratio was also decreased in cortex and non-enhancing tumor, though a paucity of control samples prevented statistical analysis. Twice daily MMF treatment yields intracranial drug concentrations above 1 µM and lowers the GTP/IMP ratio in GBMs, consistent with target engagement. As we have previously observed radiosensitization in vitro with MPA concentrations of 1 µM, these data suggest that MMF may achieve adequate CNS penetration for therapeutic benefit. The Phase 1 component of this study to determine the dose limiting toxicity and maximally tolerated dose of MMF when combined with reirradiation in recurrent GBM and radiation and TMZ in newly diagnosed GBM is ongoing.

B-288 The Beginning of the End: AGO2 Protein Identified and Validated as a Novel Biomarker for Multi-cancer Diagnosis Through Bioinformatics Analysis and Immunoassay

Abstract Background Despite significant progress in cancer research, the complexity and heterogeneity of the disease still pose major challenges to accurate diagnosis and effective treatment. Recent advancements in biomedical technologies have led to the identification of reliable potential cancer biomarkers. In this sense, the link between microRNAs (master regulators of gene expression) &amp; cancer is well-established, but there has been little research on the microRNA machinery itself. We aim to investigate whether changes in the expression of genes &amp; proteins involved in the miRNA pathway can be used for multi-cancer detection. Method We analyzed RNASeq data from the TCGA (The Cancer Genome Atlas) and GTEX (The Genotype-Tissue Expression) projects to compare the expression of core components in the miRNA biogenesis pathway, including AGO2, DGCR8, XPO5, RAN, DROSHA, DICER, and TARBP2, in normal and tumorous samples across 22 tissue types, including adrenocortical, AML, breast, colon, lung, liver, esophageal, prostate, pancreas, stomach, thyroid, rectum, uterus and ovarian cancer. We accessed and analyzed the TCGA and GTEx datasets using the UCSC Xena Browser. We also measured the protein concentrations of all these microRNA regulators in 15 normal adrenal cortex, 15 benign adenoma, and 15 adrenocortical cancer tissue homogenate samples using commercial ELISA kits. The Human Protein Argonaute-2 (EIF2C2) ELISA Kit-AE45910HU (Abebio-Co.Ltd) was used for the assay procedure, which was performed according to the manufacturer’s instructions. Statistical analysis was performed using GraphPad Prism, Version 9 (GraphPad Software, CA, USA). A P-value of less than 0.05 was considered statistically significant. Results Our RNASeq analysis results showed that among all microRNA regulators, AGO2 is most highly significantly over-expressed in samples derived from cancer patients compared with normal controls (P &amp;lt; 0.05). In addition, we analysed the protein expression of AGO2 in adrenocortical tissue homogenate samples. Our results demonstrated that the protein expression of AGO2 in adrenocortical carcinoma samples was significantly higher than in normal adrenal cortex and benign adrenocortical tumour samples (P &amp;lt; 0.001). Conclusion Our study highlights the potential role of Argonaute 2 protein (AGO2) in cancer development and progression. AGO2 is a key component of the RNA-induced silencing complex, which regulates gene expression. Our findings show that AGO2 is overexpressed in cancer samples compared to normal controls across a range of cancer types. This suggest that AGO2 could be used as a promising marker for the development of more accurate and specific methods for cancer diagnosis, in combination with other markers. Furthermore, the potential for using AGO2 protein expression in serum samples as a non-invasive diagnostic tool is a promising area for future research. This could be achieved through automated immunoassay, making it a practical diagnostic tool for use in routine clinical practice.

Heterotopic ovarian hilus cells of the salpinx: a case report and literature review

Ovarian hilus cells (OHCs), a counterpart of testicular Leydig cells, are usually found in the ovarian poles and produce androstenedione. Their origin remains a matter of debate, although OHCs are assumed to come from the adrenogenital primordium. OHCs are rarely observed around the poles of the ovary, including the mesoovarium, stroma (perisalpinx) of the salpinx, and the wall of paratubal cysts. Their clinical and pathological characteristics are not well-known because of their rarity. Herein, we present a case of ectopic OHCs in a 48-year-old woman. The patient underwent total hysterectomy and bilateral salpingectomy for vaginal bleeding due to multiple leiomyomas. We incidentally found OHCs in the stroma of the infundibulum of the salpinx, just beneath the tubal epithelium. Their size was less than 1 mm, and they were composed of large cells with central round nuclei and abundant clear or granular cytoplasm. OHCs share morphological and immunohistochemical profiles with ectopic adrenal glands, and the differential diagnosis is sometimes difficult. They do not exhibit microscopic encapsulation or the normal adrenal cortex zonation pattern. The patient was discharged and did not show any abnormal findings during 19 months of follow-up. Analyzing the characteristics of testicular Leydig cells will help understand how OHCs develop and why heterotopic OHCs occur in and around the salpinges.

Metabolic heterogeneity in adrenocortical carcinoma impacts patient outcomes.

Spatially resolved metabolomics enables the investigation of tumoral metabolites in situ. Inter- and intratumor heterogeneity are key factors associated with patient outcomes. Adrenocortical carcinoma (ACC) is an exceedingly rare tumor associated with poor survival. Its clinical prognosis is highly variable, but the contributions of tumor metabolic heterogeneity have not been investigated thus far to our knowledge. An in-depth understanding of tumor heterogeneity requires molecular feature-based identification of tumor subpopulations associated with tumor aggressiveness. Here, using spatial metabolomics by high-mass resolution MALDI Fourier transform ion cyclotron resonance mass spectrometry imaging, we assessed metabolic heterogeneity by de novo discovery of metabolic subpopulations and Simpson's diversity index. After identification of tumor subpopulations in 72 patients with ACC, we additionally performed a comparison with 25 tissue sections of normal adrenal cortex to identify their common and unique metabolic subpopulations. We observed variability of ACC tumor heterogeneity and correlation of high metabolic heterogeneity with worse clinical outcome. Moreover, we identified tumor subpopulations that served as independent prognostic factors and, furthermore, discovered 4 associated anticancer drug action pathways. Our research may facilitate comprehensive understanding of the biological implications of tumor subpopulations in ACC and showed that metabolic heterogeneity might impact chemotherapy.

Subacute Sclerosing Panencephalitis Mimicking Posterior Circulation Ischemic Stroke

Abstract Subacute sclerosing panencephalitis (SSPE) is a progressive neurodegenerative disease that develops 2–10 years after a measles infection. The diagnosis is possible through history, electroencephalography findings, and measles antibodies in the cerebrospinal fluid. Magnetic resonance imaging (MRI) is also an assistive tool in the diagnosis. The typical presentation of the disease on MRI is the bilateral, asymmetric lesions in the periventricular and subcortical areas. Brainstem involvement is uncommon. We describe a 12-year-old boy who was initially diagnosed with posterior circulation ischemic stroke with left cerebellar peduncle diffusion restriction, treated with low molecular weight heparin, and later found to have SSPE. His follow-up MRI in 2 months showed massive cerebral cortical and white matter destruction although his first MRI with cerebellar peduncle diffusion restriction initially revealed almost normal cerebral cortex and white matter. Brainstem involvement with diffusion restriction helps expand the radiological spectrum of SSPE.

Astrocyte-derived complement C3 is activated in patients with tuberous sclerosis complex and mediates immune injury: an integrated bioinformatics analysis.

Tuberous sclerosis complex (TSC) is a genetic disorder associated with multiple neurological manifestations. Cortical tubers (CT) are recognized as the hallmark brain lesions of TSC and contribute to neurological and psychiatric symptoms. To understand the molecular mechanism of neuropsychiatric features of TSC, the differentially expressed genes (DEGs) in CT from patients with TSC and those in normal cortex (NC) from participants acting as healthy controls were investigated. The dataset of GSE16969, which had already been published and described (https://onlinelibrary.wiley.com/doi/10.1111/j.1750-3639.2009.00341.x), was downloaded from the Gene Expression Omnibus (GEO), including samples of 4 CT and 4 NC. The R package "limma" was used to screen DEGs in CT and NC. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analyses of the DEGs were conducted using the R package "clusterProfiler". The online software Ingenuity Pathway Analysis (IPA) was used to explore activation/inaction of canonical pathways. The hub gene was selected based on the protein-protein interaction (PPI) network constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database and Cytoscape software. Subsequently, the hub genes at messenger (mRNA) and transcriptional levels were tested. We also explored immune cell-type enrichment using the online database xCell, and assessed the correlation between cell types and C3 expression. Then, we verified the source of C3 by constructing TSC2 knockout cells in the U87 astrocyte cell line. The human neuronal cell line SH-SY5Y was used to examine the effects of excessive complement C3 levels. A total of 455 DEGs were identified. A large number of pathways were involved in the immune response process based on the results of GO, KEGG, and IPA. C3 was identified as a hub gene. Complement C3 was also upregulated in the human CT and peripheral blood. Furthermore, based on the enrichment of functions and signaling pathways, complement C3 played a critical role in immune injury in CT of TSC. In the in vitro experiments, we found that excessive complement C3 was derived from TSC2 knockout U87 cells, and there was an increased intracellular reactive oxygen species (ROS) level in SH-SY5Y cells. Complement C3 is activated in patients with TSC and can mediate immune injury.

Intraoperative glioma characterization using time-resolved fluorescence spectroscopy.

2072 Background: Extent of surgical resection significantly improves progression-free and overall survival in glioma patients. Due to the infiltrative nature of gliomas, tumor margin detection is often difficult, and current intraoperative visualization methods are limited. Time-resolved fluorescence spectroscopy (TRFS) has the potential of differentiating glioma from normal brain tissue, thereby maximizing extent and safety of resection. Methods: Twenty-seven preoperative patients diagnosed with glioma met inclusion criteria for an IRB-approved study aimed to assess the accuracy of TRFS relative to tissue histopathology for differentiating glioma from normal cortex (NC), normal white matter (NWM), and white matter with edema (WME). A multi-channel TRFS system was developed for in vivo brain tissue and tumor characterization during glioma surgery. A custom-designed fiber optic probe was used to deliver ultrafast laser pulses to tissue regions of interest and collect fluorescence signals, with simultaneous registration to intraoperative neuronavigation. The fluorescence light was collected using a photomultiplier tube and digitized across six wavelength bands. Biopsies were taken at each measurement site for evaluation by a blinded neuropathologist to determine the accuracy of TRFS, which was calculated using parameters derived from the recorded fluorescence pulse and used to characterize tissue signatures. Results: During surgical resection of 27 patients, 162TRFS measurements were collected in vivo followed by biopsy of the same tissue. Samples from brain tumors (39 low grade gliomas (LGG, WHO grade I to III), 35 high grade gliomas (HGG, WHO grade IV)) were compared with 47 samples of normal brain tissue (NC and NWM). The time-resolved system was able to differentiate LGG from normal tissue with 97% sensitivity and 93% specificity (93% PPV and 98% NPV). When all tumor grades were tested, the sensitivity dropped to 91% (96% PPV and 86% NPV), which we believe reflects the heterogeneity of HGG. HGG alone showed a sensitivity of 83% and specificity of 93% (91% PPV and 88% NPV). Normal cortex had the fastest decays across all wavelength bands among different tissue types, WME had longer decays compared to other tissue types at all wavelengths, and HGG had a variable but predictably different fluorescence decay pattern. Conclusions: TRFS is an intraoperative tool that can distinguish glioma from normal brain tissue and has the potential to maximize safe surgical resection.

Optical opening of the blood-brain barrier for targeted and ultra-sparse viral infection of cells in mouse cortex

Adeno-associated viruses (AAVs) are used in a wide array of experimental situations for driving expression of biosensors, recombinases, and opto-/chemo-genetic actuators in the brain. However, conventional approaches for minimally invasive, spatially precise, and ultra-sparse AAV-mediated transduction of cells during imaging experiments have remained a significant challenge. Here, we show that intravenous injection of commercially available AAVs at different doses, combined with laser-based perforation of cortical capillaries through a cranial widow, allows for ultra-sparse, titratable, and micron-level precision for delivery of viral vectors with relatively little inflammation or tissue damage. Further, we show the utility of this approach for eliciting sparse expression of GCaMP6, channelrhodopsin, or fluorescent reporters in neurons and astrocytes within specific functional domains in normal and stroke-damaged cortex. This technique represents a facile approach for targeted delivery of viral vectors that should assist in the study of cell types and circuits in the cortex.

RETRACTED: Mechanistic insight into the bioactivity of prodigiosin-entrapped lipid nanoparticles against triple-negative breast, lung and colon cancer cell lines

This research investigates the potentials of prodigiosin(PG) derived from bacteria and its formulations against triple-negative breast (TNB), lung, and colon cancer cells. The PG was extracted from S. marcescens using continuous batch culture, characterized, and formulated into lyophilized parenteral nanoparticles (PNPs). The formulations were characterized with respect to entrapment efficiency (EE), DSC, FT-IR, TEM, and proton nuclear magnetic resonance (1H NMR) spectroscopy. In vitro drug release was evaluated in phosphate buffer (pH 7.4) while acute toxicity, hematological and histopathological studies were performed on rats. The in vitro cytotoxicity was evaluated against TNB (MCF-7), lung (A-549), and colon (HT-29) cancer cell lines. High EE (92.3±12%) and drug release of up to 89.4% within 8h were obtained. DSC thermograms of PG and PG-PNPs showed endothermic peaks indicating amorphous nature. The FT-IR spectrum of PG-PNPs revealed remarkable peaks of pure PG, indicating no strong chemical interaction between the drug and excipients. The TEM micrograph of the PG-PNPs showed nano-sized formulations (20-30nm) whose particles were mostly lamellar and hexagonal structures. The 1H NMR result revealed the chemical structure of PG showing all assigned proton chemical shifts. Toxicity results of the PG and its formulation up to a concentration of 5000mg/kg showed insignificant vacuolar changes of hepatocytes in the liver, with normal renal medulla and cortex in the kidney. The PG and PG-PNPs inhibited the growth of breast, lung, and colon cell lines. The nano-sized lipid formulation (PG-PNPs) showed potential in PG delivery and cancer treatments.

Mass Spectrometry Imaging Reveals Abnormalities in Cardiolipin Composition and Distribution in Astrocytoma Tumor Tissues.

Cardiolipin (CL) is a mitochondrial lipid with diverse roles in cellular respiration, signaling, and organelle membrane structure. CL content and composition are essential for proper mitochondrial function. Deranged mitochondrial energy production and signaling are key components of glial cell cancers and altered CL molecular species have been observed in mouse brain glial cell xenograft tumors. The objective of this study was to describe CL structural diversity trends in human astrocytoma tumors of varying grades and correlate these trends with histological regions within the heterogeneous astrocytoma microenvironment. To this aim, we applied desorption electrospray ionization coupled with high field asymmetric ion mobility mass spectrometry (DESI-FAIMS-MS) to map CL molecular species in human normal cortex (N = 29), lower-grade astrocytoma (N = 19), and glioblastoma (N = 28) tissues. With this platform, we detected 46 CL species and 12 monolysocardiolipin species from normal cortex samples. CL profiles detected from glioblastoma tissues lacked diversity and abundance of longer chain polyunsaturated fatty acid containing CL species when compared to CL detected from normal and lower-grade tumors. CL profiles correlated with trends in tumor viability and tumor infiltration. Structural characterization of the CL species by tandem MS experiments revealed differences in fatty acid and double bond isomer composition among astrocytoma tissues compared with normal cortex and glioblastoma tissues. The GlioVis platform was used to analyze astrocytoma gene expression data from the CGGA dataset. Decreased expression of several mitochondrial respiratory enzyme encoding-genes was observed for higher-grade versus lower-grade tumors, however no significant difference was observed for cardiolipin synthesis enzyme CRLS1.

Combining methionine-PET and MRI fluid-attenuated inversion-recovery mismatch to determine glioma molecular subtype.

11 C-methionine (MET)-PET is a useful tool in neuro-oncology. The T2-fluid-attenuated inversion recovery (FLAIR) mismatch sign on MRI is a characteristic finding in lower grade gliomas with isocitrate dehydrogenase (IDH) mutations and the absence of the 1p/19q codeletion; however, the T2-FLAIR mismatch sign has low sensitivity in differentiating gliomas and does not aid in identifying glioblastomas with IDH mutations. We therefore investigated the efficacy of the combination of the T2-FLAIR mismatch sign and MET-PET for accurately determining the molecular subtype of gliomas of all grades. The present study comprised 208 adult patients diagnosed with supratentorial glioma confirmed by molecular genetics and histopathology. The ratio of the maximum lesion MET accumulation to the mean normal frontal cortex MET accumulation (T/N) was measured. The presence or absence of the T2-FLAIR mismatch sign was determined. The presence or absence of the T2-FLAIR mismatch sign and the MET T/N ratio were compared between glioma subtypes to evaluate individual and combined utility in identifying gliomas with IDH mutations and no 1p/19q codeletion (IDHmut-Noncodel) or gliomas with IDH mutations (IDHmut). The addition of MET-PET to MRI for the presence of the T2-FLAIR mismatch sign improved diagnostic accuracy, with the area under the curve values increasing from .852 to .871 for IDHmut-Noncodel and from .688 to .808 for IDHmut. The combination of the T2-FLAIR mismatch sign and MET-PET may provide improved diagnostic utility in differentiating gliomas according to molecular subtype, particularly in determining IDH mutation status.

Diagnostic performance of multiparametric magnetic resonance imaging in the differentiation of clear cell renal cell cancer.

This study aimed to evaluate the diagnostic performance of multiparametric magnetic resonance imaging (mpMRI) in the differentiation of renal cell carcinoma (RCC) subtypes. This is a retrospective diagnostic performance study, in which the diagnostic performances of mpMRI features were evaluated to differentiate clear cell RCC (ccRCC) from non-clear cell RCC (non-ccRCC). Adult patients who were evaluated using a 3-Tesla dynamic contrast-enhanced mpMRI before undergoing partial or radical nephrectomy for possible malignant renal tumors were included in the study. Signal intensity change percentages (SICP) between contrast-enhanced phases and pre-administration period for both the tumor and normal renal cortex, and tumor-to-cortex enhancement index (TCEI); tumor apparent diffusion coefficient (ADC) values; tumor-to-cortex ADC ratio; and a scale which was developed according to the tumor signal intensities on the axial fat-suppressed T2-weighted Half-Fourier Acquisition Single-shot Turbo spin Echo (HASTE) images were used in ROC analysis to estimate the presence of ccRCC in the patients. The reference test positivity was the histopathologic examination of the surgical specimens. Ninety-eight tumors from 91 patients were included in the study, and 59 of them were ccRCC, 29 were pRCC, and 10 were chRCC. The mpMRI features that had the three highest sensitivity rates were excretory phase SICP, T2-weighted HASTE scale score, and corticomedullary phase TCEI (93.2%, 91.5%, and 86.4%, respectively). However, those with the three highest specificity rates were nephrographic phase TCEI, excretory phase TCEI, and tumor ADC value (94.9%, 94.9%, and 89.7%, respectively). Several parameters on mpMRI showed an acceptable performance to differentiate ccRCC from non-ccRCC.