Probing Alzheimer's pathology: Exploring the next generation of FDDNP analogues for amyloid ß detection.

Fluorescent probes are a powerful tool for imaging amyloid ß (Aß) plaques, the hallmark of Alzheimer's disease (AD). Herein, we report the synthesis and comprehensive characterization of 21 novel probes as well as their optical properties and binding affinities to Aß fibrils. One of these dyes, 1Ae, exhibited several improvements over FDDNP, an established biomarker for Aß- and Tau-aggregates. First, 1Ae had large Stokes shifts (138-213 nm) in various solvents, thereby reducing self-absorption. With a high quantum yield ratio (φ(dichloromethane/methanol) = 104), 1Ae also ensures minimal background emission in aqueous environments and high sensitivity. In addition, compound 1Ae exhibited low micromolar binding affinity to Aß fibrils in vitro (Kd = 1.603 µM), while increasing fluorescence emission (106-fold) compared to emission in buffer alone. Importantly, the selective binding of 1Ae to Aß1-42 fibrils was confirmed by an in cellulo assay, supported by ex vivo fluorescence microscopy of 1Ae on postmortem AD brain sections, allowing unequivocal identification of Aß plaques. The intermolecular interactions of fluorophores with Aß were elucidated by docking studies and molecular dynamics simulations. Density functional theory calculations revealed the unique photophysics of these rod-shaped fluorophores, with a twisted intramolecular charge transfer (TICT) excited state. These results provide valuable insights into the future application of such probes as potential diagnostic tools for AD in vitro and ex vivo such as determination of Aß1-42 in cerebrospinal fluid or blood.

Metabolic Brain Changes Can Predict the Underlying Pathology in Neurodegenerative Brain Disorders: A Case Report of Sporadic Creutzfeldt-Jakob Disease with Concomitant Parkinson's Disease.

The co-occurrence of multiple proteinopathies is being increasingly recognized in neurodegenerative disorders and poses a challenge in differential diagnosis and patient selection for clinical trials. Changes in brain metabolism captured by positron emission tomography (PET) with 18 F-fluorodeoxyglucose (FDG) allow us to differentiate between different neurodegenerative disorders either by visual exploration or by studying disease-specific metabolic networks in individual patients. However, the impact of multiple proteinopathies on brain metabolism and metabolic networks remains unknown due to the absence of pathological studies. In this case study, we present a 67-year-old patient with rapidly progressing dementia clinically diagnosed with probable sporadic Creutzfeldt-Jakob disease (sCJD). However, in addition to the expected pronounced cortical and subcortical hypometabolism characteristic of sCJD, the brain FDG PET revealed an intriguing finding of unexpected relative hypermetabolism in the bilateral putamina, raising suspicions of coexisting Parkinson's disease (PD). Additional investigation of disease-specific metabolic brain networks revealed elevated expression of both CJD-related pattern (CJDRP) and PD-related pattern (PDRP) networks. The patient eventually developed akinetic mutism and passed away seven weeks after symptom onset. Neuropathological examination confirmed neuropathological changes consistent with sCJD and the presence of Lewy bodies confirming PD pathology. Additionally, hyperphosphorylated tau and TDP-43 pathology were observed, a combination of four proteinopathies that had not been previously reported. Overall, this case provides valuable insights into the complex interplay of neurodegenerative pathologies and their impact on metabolic brain changes, emphasizing the role of metabolic brain imaging in evaluating potential presence of multiple proteinopathies.

Sporadic Creutzfeldt-Jakob disease is associated with reorganization of metabolic connectivity in a pathological brain network.

BACKGROUND AND PURPOSE: Although sporadic Creutzfeldt-Jakob disease (sCJD) is a rare cause of dementia, it is critical to understand its functional networks as the prion protein spread throughout the brain may share similar mechanisms with other more common neurodegenerative disorders. In this study, the metabolic brain network associated with sCJD was investigated and its internal network organization was explored. METHODS: We explored 2-[18 F]fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET) brain scans of 29 sCJD patients, 56 normal controls (NCs) and 46 other dementia patients from two independent centers. sCJD-related pattern (CJDRP) was identified in a cohort of 16 pathologically proven sCJD patients and 16 age-matched NCs using scaled subprofile modeling/principal component analysis and was prospectively validated in an independent cohort of 13 sCJD patients and 20 NCs. The pattern's specificity was tested on other dementia patients and its clinical relevance by clinical correlations. The pattern's internal organization was further studied using graph theory methods. RESULTS: The CJDRP was characterized by relative hypometabolism in the bilateral caudate, thalami, middle and superior frontal gyri, parietal lobe and posterior cingulum in association with relative hypermetabolism in the hippocampi, parahippocampal gyri and cerebellum. The pattern's expression significantly discriminated sCJD from NCs and other dementia patients (p < 0.005; receiver operating characteristic analysis CJD vs. NCs area under the curve [AUC] 0.90-0.96, sCJD vs. Alzheimer's disease AUC 0.78, sCJD vs. behavioral variant of frontotemporal dementia AUC 0.84). The pattern's expression significantly correlated with cognitive, functional decline and disease duration. The metabolic connectivity analysis revealed inefficient information transfer with specific network reorganization. CONCLUSIONS: The CJDRP is a robust metabolic biomarker of sCJD. Due to its excellent clinical correlations it has the potential to monitor disease in emerging disease-modifying trials.

The Cytotoxic Effects of Cannabidiol and Cannabigerol on Glioblastoma Stem Cells May Mostly Involve GPR55 and TRPV1 Signalling.

Glioblastoma (GBM) is one of the most aggressive cancers, comprising 60-70% of all gliomas. The large G-protein-coupled receptor family includes cannabinoid receptors CB1, CB2, GPR55, and non-specific ion receptor protein transporters TRPs. First, we found up-regulated CNR1, GPR55, and TRPV1 expression in glioma patient-derived tissue samples and cell lines compared with non-malignant brain samples. CNR1 and GPR55 did not correlate with glioma grade, whereas TRPV1 negatively correlated with grade and positively correlated with longer overall survival. This suggests a tumour-suppressor role of TRPV1. With respect to markers of GBM stem cells, preferred targets of therapy, TRPV1 and GPR55, but not CNR1, strongly correlated with different sets of stemness gene markers: NOTCH, OLIG2, CD9, TRIM28, and TUFM and CD15, SOX2, OCT4, and ID1, respectively. This is in line with the higher expression of TRPV1 and GPR55 genes in GSCs compared with differentiated GBM cells. Second, in a panel of patient-derived GSCs, we found that CBG and CBD exhibited the highest cytotoxicity at a molar ratio of 3:1. We suggest that this mixture should be tested in experimental animals and clinical studies, in which currently used Δ9-tetrahydrocannabinol (THC) is replaced with efficient and non-psychoactive CBG in adjuvant standard-of-care therapy.

Loss of H3K27me3 expression in canine nerve sheath tumors.

Nerve sheath tumors (NSTs) are characterized by neoplastic proliferation of Schwann cells, perineurial cells, endoneurial and/or epineurial fibroblasts. Diagnosis of NST is often challenging, particularly in distinguishing malignant NST (MNST) from other soft tissue sarcomas, or sometimes between low-grade MNST and benign NST. Recent studies in human pathology have demonstrated loss of trimethylation at lysine 27 of histone 3 (H3K27me3) in a subset of MNSTs using immunohistochemistry. Loss of H3K27me3 expression is rare in other high-grade sarcomas and also appears to be useful in distinguishing benign and low-grade MNSTs from high-grade subsets. In our retrospective study, we performed H3K27me3 immunohistochemistry in 68 canine tumors previously diagnosed as NST. We detected loss of H3K27me3 expression in 25% (n = 17) of all canine NST, including one neurofibroma, whereas 49% (n = 33) of tumors had mosaic loss of expression and 26% (n = 18) retained expression. No statistically significant differences were found between H3K27me3 expression, histopathological features of tumors, and their immunoreactivity for Sox10, claudin-1, GFAP, and Ki67. Because the classification of canine NST is not yet fully established and its correlation with the prognosis and clinical course of the disease is lacking, prospective studies with possible genetic analyses are needed to assess the true diagnostic value of H3K27me3 loss in canine NST.

Infiltrating natural killer cells bind, lyse and increase chemotherapy efficacy in glioblastoma stem-like tumorospheres.

Glioblastomas remain the most lethal primary brain tumors. Natural killer (NK) cell-based therapy is a promising immunotherapeutic strategy in the treatment of glioblastomas, since these cells can select and lyse therapy-resistant glioblastoma stem-like cells (GSLCs). Immunotherapy with super-charged NK cells has a potential as antitumor approach since we found their efficiency to kill patient-derived GSLCs in 2D and 3D models, potentially reversing the immunosuppression also seen in the patients. In addition to their potent cytotoxicity, NK cells secrete IFN-γ, upregulate GSLC surface expression of CD54 and MHC class I and increase sensitivity of GSLCs to chemotherapeutic drugs. Moreover, NK cell localization in peri-vascular regions in glioblastoma tissues and their close contact with GSLCs in tumorospheres suggests their ability to infiltrate glioblastoma tumors and target GSLCs. Due to GSLC heterogeneity and plasticity in regards to their stage of differentiation personalized immunotherapeutic strategies should be designed to effectively target glioblastomas.

Upregulation of Cathepsin X in Glioblastoma: Interplay with γ-Enolase and the Effects of Selective Cathepsin X Inhibitors.

Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. Understanding GBM pathobiology and discovering novel therapeutic targets are critical to finding efficient treatments. Upregulation of the lysosomal cysteine carboxypeptidase cathepsin X has been linked to immune dysfunction and neurodegenerative diseases, but its role in cancer and particularly in GBM progression in patients is unknown. In this study, cathepsin X expression and activity were found to be upregulated in human GBM tissues compared to low-grade gliomas and nontumor brain tissues. Cathepsin X was localized in GBM cells as well as in tumor-associated macrophages and microglia. Subsequently, potent irreversible (AMS36) and reversible (Z7) selective cathepsin X inhibitors were tested in vitro. Selective cathepsin X inhibitors decreased the viability of patient-derived GBM cells as well as macrophages and microglia that were cultured in conditioned media of GBM cells. We next examined the expression pattern of neuron-specific enzyme γ-enolase, which is the target of cathepsin X. We found that there was a correlation between high proteolytic activity of cathepsin X and C-terminal cleavage of γ-enolase and that cathepsin X and γ-enolase were colocalized in GBM tissues, preferentially in GBM-associated macrophages and microglia. Taken together, our results on patient-derived material suggest that cathepsin X is involved in GBM progression and is a potential target for therapeutic approaches against GBM.

Brain aggregoma with clonal B-cell perivascular proliferation detected by next-generation sequencing. A case report and review of the literature.

Light-chain deposition disease (LCDD), a rare type of monoclonal immunoglobulin deposition disease, can be presented as systemic or localized, very rarely affecting central nervous system (CNS). Only 10 cases of CNS-LCDD have been described so far. We present an eleventh case of cerebral tumour-like LCDD, called aggregoma, and compare it with previously reported cases. A 49-year-old patient was admitted to the hospital due to a first generalized epileptic seizure. Magnetic resonance imaging (MRI) showed focal lesion in the right occipital lobe. Abundant parenchymal aggregates of pale eosinophilic material were observed, Congo red negative, Thioflavin T moderately positive, and l-light chain positive, but k negative in immunofluorescence with mild perivascular lymphoplasmacytic infiltrates in the intervening brain tissue. Clonality testing by next-generation sequencing showed the monoclonal nature of B-lymphocytes. Electron microscopy showed a finely granular ultrastructure of the aggregates without deposition in the vessel walls. A whole-body workup did not show any extra-cerebral immune dyscrasias.

Large-Scale Transcriptomics-Driven Approach Revealed Overexpression of CRNDE as a Poor Survival Prognosis Biomarker in Glioblastoma.

Glioblastoma is the most common and malignant brain malignancy worldwide, with a 10-year survival of only 0.7%. Aggressive multimodal treatment is not enough to increase life expectancy and provide good quality of life for glioblastoma patients. In addition, despite decades of research, there are no established biomarkers for early disease diagnosis and monitoring of patient response to treatment. High throughput sequencing technologies allow for the identification of unique molecules from large clinically annotated datasets. Thus, the aim of our study was to identify significant molecular changes between short- and long-term glioblastoma survivors by transcriptome RNA sequencing profiling, followed by differential pathway-activation-level analysis. We used data from the publicly available repositories The Cancer Genome Atlas (TCGA; number of annotated cases = 135) and Chinese Glioma Genome Atlas (CGGA; number of annotated cases = 218), and experimental clinically annotated glioblastoma tissue samples from the Institute of Pathology, Faculty of Medicine in Ljubljana corresponding to 2-58 months overall survival (n = 16). We found one differential gene for long noncoding RNA CRNDE whose overexpression showed correlation to poor patient OS. Moreover, we identified overlapping sets of congruently regulated differential genes involved in cell growth, division, and migration, structure and dynamics of extracellular matrix, DNA methylation, and regulation through noncoding RNAs. Gene ontology analysis can provide additional information about the function of protein- and nonprotein-coding genes of interest and the processes in which they are involved. In the future, this can shape the design of more targeted therapeutic approaches.

Cystatin F acts as a mediator of immune suppression in glioblastoma.

PURPOSE: Glioblastoma, the most aggressive type of brain cancer, is composed of heterogeneous populations of differentiated cells, cancer stem cells and immune cells. Cystatin F, an endogenous inhibitor of lysosomal cysteine peptidases, regulates the function of cytotoxic immune cells. The aim of this study was to determine which type of cells expresses cystatin F in glioblastoma and to determine the role of cystatin F during disease progression. METHODS: RT-qPCR and immunohistochemistry were used to determine cystatin F mRNA and protein levels in glioblastoma tissue samples. The internalization of cystatin F was analyzed by Western blotting. Enzyme kinetics, real time invasion and calcein release cytotoxicity assays were used to assess the role of internalized cystatin F. RESULTS: We found that cystatin F was not expressed in non-cancer brain tissues, but that its expression increased with glioma progression. In tumor tissues, extensive staining was observed in cancer stem-like cells and microglia/monocytes, which secrete cystatin F into their microenvironment. In trans activity of cystatin F was confirmed using an in vitro glioblastoma cell model. Internalized cystatin F affected cathepsin L activity in glioblastoma cells and decreased their invasiveness. In addition, we found that cystatin F decreased the susceptibility of glioblastoma cells to the cytotoxic activity of natural killer (NK) cells. CONCLUSIONS: Our data implicate cystatin F as a mediator of immune suppression in glioblastoma. Increased cystatin F mRNA and protein levels in immune, glioblastoma and glioblastoma stem-like cells or trans internalized cystatin F may have an impact on decreased susceptibility of glioblastoma cells to NK cytotoxicity.

Urgent lung transplantation for thymic neoplasm-associated severe constrictive bronchiolitis with bronchiectasis and radiotherapy-induced organizing pneumonia: A case report.

Here, we present the case of a 28-year-old woman who developed severe and progressive thymoma-associated constrictive bronchiolitis with bronchiectasis, despite undergoing thymectomy. The disease was further complicated by radiation-induced organizing pneumonia (RIOP), which developed after adjuvant radiotherapy (RT) for Masaoka stage II thymoma. The patient was successfully treated with an urgent lung transplantation (LTx) for irreversible respiratory failure.

Analysis of miR-9-5p, miR-124-3p, miR-21-5p, miR-138-5p, and miR-1-3p in Glioblastoma Cell Lines and Extracellular Vesicles.

Glioblastoma (GBM), the most common primary brain tumor, is a complex and extremely aggressive disease. Despite recent advances in molecular biology, there is a lack of biomarkers, which would improve GBM's diagnosis, prognosis, and therapy. Here, we analyzed by qPCR the expression levels of a set of miRNAs in GBM and lower-grade glioma human tissue samples and performed a survival analysis in silico. We then determined the expression of same miRNAs and their selected target mRNAs in small extracellular vesicles (sEVs) of GBM cell lines. We showed that the expression of miR-21-5p was significantly increased in GBM tissue compared to lower-grade glioma and reference brain tissue, while miR-124-3p and miR-138-5p were overexpressed in reference brain tissue compared to GBM. We also demonstrated that miR-9-5p and miR-124-3p were overexpressed in the sEVs of GBM stem cell lines (NCH421k or NCH644, respectively) compared to the sEVs of all other GBM cell lines and astrocytes. VIM mRNA, a target of miR-124-3p and miR-138-5p, was overexpressed in the sEVs of U251 and U87 GBM cell lines compared to the sEVs of GBM stem cell line and also astrocytes. Our results suggest VIM mRNA, miR-9-5p miRNA, and miR-124-3p miRNA could serve as biomarkers of the sEVs of GBM cells.

CCR5-Mediated Signaling Is Involved in Invasion of Glioblastoma Cells in Its Microenvironment.

The chemokine CCL5/RANTES is a versatile inflammatory mediator, which interacts with the receptor CCR5, promoting cancer cell interactions within the tumor microenvironment. Glioblastoma is a highly invasive tumor, in which CCL5 expression correlates with shorter patient survival. Using immunohistochemistry, we identified CCL5 and CCR5 in a series of glioblastoma samples and cells, including glioblastoma stem cells. CCL5 and CCR5 gene expression were significantly higher in a cohort of 38 glioblastoma samples, compared to low-grade glioma and non-cancerous tissues. The in vitro invasion of patients-derived primary glioblastoma cells and glioblastoma stem cells was dependent on CCL5-induced CCR5 signaling and is strongly inhibited by the small molecule CCR5 antagonist maraviroc. Invasion of these cells, which was enhanced when co-cultured with mesenchymal stem cells (MSCs), was inhibited by maraviroc, suggesting that MSCs release CCR5 ligands. In support of this model, we detected CCL5 and CCR5 in MSC monocultures and glioblastoma-associated MSC in tissue sections. We also found CCR5 expressing macrophages were in close proximity to glioblastoma cells. In conclusion, autocrine and paracrine cross-talk in glioblastoma and, in particular, glioblastoma stem cells with its stromal microenvironment, involves CCR5 and CCL5, contributing to glioblastoma invasion, suggesting the CCL5/CCR5 axis as a potential therapeutic target that can be targeted with repositioned drug maraviroc.

Anti-vimentin, anti-TUFM, anti-NAP1L1 and anti-DPYSL2 nanobodies display cytotoxic effect and reduce glioblastoma cell migration.

BACKGROUND: Glioblastoma is a particularly common and very aggressive primary brain tumour. One of the main causes of therapy failure is the presence of glioblastoma stem cells that are resistant to chemotherapy and radiotherapy, and that have the potential to form new tumours. This study focuses on validation of eight novel antigens, TRIM28, nucleolin, vimentin, nucleosome assembly protein 1-like 1 (NAP1L1), mitochondrial translation elongation factor (EF-TU) (TUFM), dihydropyrimidinase-related protein 2 (DPYSL2), collapsin response mediator protein 1 (CRMP1) and Aly/REF export factor (ALYREF), as putative glioblastoma targets, using nanobodies. METHODS: Expression of these eight antigens was analysed at the cellular level by qPCR, ELISA and immunocytochemistry, and in tissues by immunohistochemistry. The cytotoxic effects of the nanobodies were determined using AlamarBlue and water-soluble tetrazolium tests. Annexin V/propidium iodide tests were used to determine apoptotsis/necrosis of the cells in the presence of the nanobodies. Cell migration assays were performed to determine the effects of the nanobodies on cell migration. RESULTS: NAP1L1 and CRMP1 were significantly overexpressed in glioblastoma stem cells in comparison with astrocytes and glioblastoma cell lines at the mRNA and protein levels. Vimentin, DPYSL2 and ALYREF were overexpressed in glioblastoma cell lines only at the protein level. The functional part of the study examined the cytotoxic effects of the nanobodies on glioblastoma cell lines. Four of the nanobodies were selected in terms of their specificity towards glioblastoma cells and protein overexpression: anti-vimentin (Nb79), anti-NAP1L1 (Nb179), anti-TUFM (Nb225) and anti-DPYSL2 (Nb314). In further experiments to optimise the nanobody treatment schemes, to increase their effects, and to determine their impact on migration of glioblastoma cells, the anti-TUFM nanobody showed large cytotoxic effects on glioblastoma stem cells, while the anti-vimentin, anti-NAP1L1 and anti-DPYSL2 nanobodies were indicated as agents to target mature glioblastoma cells. The anti-vimentin nanobody also had significant effects on migration of mature glioblastoma cells. CONCLUSION: Nb79 (anti-vimentin), Nb179 (anti-NAP1L1), Nb225 (anti-TUFM) and Nb314 (anti-DPYSL2) nanobodies are indicated for further examination for cell targeting. The anti-TUFM nanobody, Nb225, is particularly potent for inhibition of cell growth after long-term exposure of glioblastoma stem cells, with minor effects seen for astrocytes. The anti-vimentin nanobody represents an agent for inhibition of cell migration.

Similarities Between Stem Cell Niches in Glioblastoma and Bone Marrow: Rays of Hope for Novel Treatment Strategies.

Glioblastoma is the most aggressive primary brain tumor. Slowly dividing and therapy-resistant glioblastoma stem cells (GSCs) reside in protective peri-arteriolar niches and are held responsible for glioblastoma recurrence. Recently, we showed similarities between GSC niches and hematopoietic stem cell (HSC) niches in bone marrow. Acute myeloid leukemia (AML) cells hijack HSC niches and are transformed into therapy-resistant leukemic stem cells (LSCs). Current clinical trials are focussed on removal of LSCs out of HSC niches to differentiate and to become sensitized to chemotherapy. In the present study, we elaborated further on these similarities by immunohistochemical analyses of 17 biomarkers in paraffin sections of human glioblastoma and human bone marrow. We found all 17 biomarkers to be expressed both in hypoxic peri-arteriolar HSC niches in bone marrow and hypoxic peri-arteriolar GSC niches in glioblastoma. Our findings implicate that GSC niches are being formed in glioblastoma as a copy of HSC niches in bone marrow. These similarities between HSC niches and GSC niches provide a theoretic basis for the development of novel strategies to force GSCs out of their niches, in a similar manner as in AML, to induce GSC differentiation and proliferation to render them more sensitive to anti-glioblastoma therapies.

High FREM2 Gene and Protein Expression Are Associated with Favorable Prognosis of IDH-WT Glioblastomas.

World Health Organization grade IV diffuse gliomas, known as glioblastomas, are the most common malignant brain tumors, and they show poor prognosis. Multimodal treatment of surgery followed by radiation and chemotherapy is not sufficient to increase patient survival, which is 12 to 18 months after diagnosis. Despite extensive research, patient life expectancy has not significantly improved over the last decade. Previously, we identified FREM2 and SPRY1 as genes with differential expression in glioblastoma cell lines compared to nonmalignant astrocytes. In addition, the FREM2 and SPRY1 proteins show specific localization on the surface of glioblastoma cells. In this study, we explored the roles of the FREM2 and SPRY1 genes and their proteins in glioblastoma pathology using human tissue samples. We used proteomic, transcriptomic, and bioinformatics approaches to detect changes at different molecular levels. We demonstrate increased FREM2 protein expression levels in glioblastomas compared to reference samples. At the transcriptomic level, both FREM2 and SPRY1 show increased expression in tissue samples of different glioma grades compared to nonmalignant brain tissue. To broaden our experimental findings, we analyzed The Cancer Genome Atlas glioblastoma patient datasets. We discovered higher FREM2 and SPRY1 gene expression levels in glioblastomas compared to lower grade gliomas and reference samples. In addition, we observed that low FREM2 expression was associated with progression of IDH-mutant low-grade glioma patients. Multivariate analysis showed positive association between FREM2 and favorable prognosis of IDH-wild type glioblastoma. We conclude that FREM2 has an important role in malignant progression of glioblastoma, and we suggest deeper analysis to determine its involvement in glioblastoma pathology.

Overstaged Rectal Cancer by MRI due to Fibrosis Induced by Tattoo Marker.

Endoscopic colorectal tattooing with carbon-based dyes is commonly employed in order to assist with later localization of the lesion. Although carbon is thought to be nontoxic, there usually is some inflammatory reaction with fibrosis and granuloma formation after tissue injection. The aim of this report is to alert to a possible underestimated, late consequence of colorectal carbon-based marker tattooing, namely pronounced fibrosis at the site of the injection that could lead to a blurring and misinterpretation of changes evaluated by radiological techniques. We describe a case of cT stage overestimation due to fibrosis of the rectal wall and perirectal fat, induced by carbon-based dye injection in a 66-year-old patient. In our case it was an overestimation of MR evaluation in the case of early invasive carcinoma. Although there have been some studies on tissue effect of carbon-based dyes, the possible scenario consequence of cancer stage overestimation due to fibrosis has not yet been described. Such a mistake could lead to inappropriate overtreatment. Clinicians must be aware of the possible consequences of dye injection and resultant overestimation of T stage of colorectal cancer. More histological studies concerning histological changes after carbon-based marker tattooing are needed to establish the extent of its significance.

Zika Virus-Associated Micrencephaly: A Thorough Description of Neuropathologic Findings in the Fetal Central Nervous System.

CONTEXT: -The 2015 outbreak of Zika virus in Brazil resulted in a 20-times increased prevalence of congenital microcephaly in stillborns and neonates and was instrumental in raising the suspicion of a causal association between Zika virus and microcephaly. OBJECTIVE: -To provide a comprehensive description of the neuropathologic features of congenital Zika virus infection. DESIGN: -Autopsy evaluation of the brain from a fetus of 32 weeks and 6 days of gestation, with a prenatal diagnosis of microcephaly associated with polymerase chain reaction-confirmed, fetal, Zika virus infection. RESULTS: -Multiple severe pathology findings were present. These included lissencephaly, except for the occipital lobes, where some pachygyria was observed. Also present was reduction and thinning of white matter, ventriculomegaly of the lateral ventricles, and coalescent calcifications in the cortical-subcortical white matter border associated with glioneuronal outbursting into the subarachnoid space above and heterotopias below. There were small, scattered calcifications in the basal ganglia, with fewer in the white matter and germinal matrix, and none in the cerebellum and brainstem. The cerebellum and pontine base were atrophic because of Wallerian degeneration or maldevelopment of descending tracts and pontocerebellar connections. CONCLUSION: -Our findings are in agreement with neuroimaging of Zika virus-associated fetal and infant micrencephalic brains and, to some extent, with neuroimaging of other intrauterine infections causing microcephaly.

Zika Virus Disrupts Phospho-TBK1 Localization and Mitosis in Human Neuroepithelial Stem Cells and Radial Glia.

The mechanisms underlying Zika virus (ZIKV)-related microcephaly and other neurodevelopment defects remain poorly understood. Here, we describe the derivation and characterization, including single-cell RNA-seq, of neocortical and spinal cord neuroepithelial stem (NES) cells to model early human neurodevelopment and ZIKV-related neuropathogenesis. By analyzing human NES cells, organotypic fetal brain slices, and a ZIKV-infected micrencephalic brain, we show that ZIKV infects both neocortical and spinal NES cells as well as their fetal homolog, radial glial cells (RGCs), causing disrupted mitoses, supernumerary centrosomes, structural disorganization, and cell death. ZIKV infection of NES cells and RGCs causes centrosomal depletion and mitochondrial sequestration of phospho-TBK1 during mitosis. We also found that nucleoside analogs inhibit ZIKV replication in NES cells, protecting them from ZIKV-induced pTBK1 relocalization and cell death. We established a model system of human neural stem cells to reveal cellular and molecular mechanisms underlying neurodevelopmental defects associated with ZIKV infection and its potential treatment.