Spikes and High Frequency Oscillations in Lateral Neocortical Temporal Lobe Epilepsy: Can They Predict the Success Chance of Hippocampus-Sparing Resections?

Purpose: We investigated the distribution of spikes and HFOs recorded during intraoperative electrocorticography (ioECoG) and tried to elaborate a predictive model for postsurgical outcomes of patients with lateral neocortical temporal lobe epilepsy (TLE) whose mesiotemporal structures are left in situ. Methods: We selected patients with temporal lateral neocortical epilepsy focus who underwent ioECoG-tailored resections without amygdalo-hippocampectomies. We visually marked spikes, ripples (80-250 Hz), and fast ripples (FRs; 250-500 Hz) on neocortical and mesiotemporal channels before and after resections. We looked for differences in event rates and resection ratios between good (Engel 1A) and poor outcome groups and performed logistic regression analysis to identify outcome predictors. Results: Fourteen out of 24 included patients had a good outcome. The poor-outcome patients showed higher rates of ripples on neocortical channels distant from the resection in pre- and post-ioECoG than people with good outcomes ( p pre = 0.04, p post = 0.05). Post-ioECoG FRs were found only in poor-outcome patients ( N = 3). A prediction model based on regression analysis showed low rates of mesiotemporal post-ioECoG ripples (OR mesio = 0.13, p mesio = 0.04) and older age at epilepsy onset (OR = 1.76, p = 0.04) to be predictors of good seizure outcome. Conclusion: HFOs in ioECoG may help to inform the neurosurgeon of the hippocampus-sparing resection success chance in patients with lateral neocortical TLE

Genomic structure and alternative splicing of murine R2B receptor protein tyrosine phosphatases (PTPκ, μ, ρ and PCP-2)

BACKGROUND: Four genes designated as PTPRK (PTPκ), PTPRL/U (PCP-2), PTPRM (PTPμ) and PTPRT (PTPρ) code for a subfamily (type R2B) of receptor protein tyrosine phosphatases (RPTPs) uniquely characterized by the presence of an N-terminal MAM domain. These transmembrane molecules have been implicated in homophilic cell adhesion. In the human, the PTPRK gene is located on chromosome 6, PTPRL/U on 1, PTPRM on 18 and PTPRT on 20. In the mouse, the four genes ptprk, ptprl, ptprm and ptprt are located in syntenic regions of chromosomes 10, 4, 17 and 2, respectively. RESULTS: The genomic organization of murine R2B RPTP genes is described. The four genes varied greatly in size ranging from ~64 kb to ~1 Mb, primarily due to proportional differences in intron lengths. Although there were also minor variations in exon length, the number of exons and the phases of exon/intron junctions were highly conserved. In situ hybridization with digoxigenin-labeled cRNA probes was used to localize each of the four R2B transcripts to specific cell types within the murine central nervous system. Phylogenetic analysis of complete sequences indicated that PTPρ and PTPμ were most closely related, followed by PTPκ. The most distant family member was PCP-2. Alignment of RPTP polypeptide sequences predicted putative alternatively spliced exons. PCR experiments revealed that five of these exons were alternatively spliced, and that each of the four phosphatases incorporated them differently. The greatest variability in genomic organization and the majority of alternatively spliced exons were observed in the juxtamembrane domain, a region critical for the regulation of signal transduction. CONCLUSIONS: Comparison of the four R2B RPTP genes revealed virtually identical principles of genomic organization, despite great disparities in gene size due to variations in intron length. Although subtle differences in exon length were also observed, it is likely that functional differences among these genes arise from the specific combinations of exons generated by alternative splicing

Genomic organization and alternative splicing of the human and mouse RPTPρ genes

BACKGROUND: Receptor protein tyrosine phosphatase rho (RPTPρ, gene symbol PTPRT) is a member of the type IIB RPTP family. These transmembrane molecules have been linked to signal transduction, cell adhesion and neurite extension. The extracellular segment contains MAM, Ig-like and fibronectin type III domains, and the intracellular segment contains two phosphatase domains. The human RPTPρ gene is located on chromosome 20q12-13.1, and the mouse gene is located on a syntenic region of chromosome 2. RPTPρ expression is restricted to the central nervous system. RESULTS: The cloning of the mouse cDNA, identification of alternatively spliced exons, detection of an 8 kb 3'-UTR, and the genomic organization of human and mouse RPTPρ genes are described. The two genes are comprised of at least 33 exons. Both RPTPρ genes span over 1 Mbp and are the largest RPTP genes characterized. Exons encoding the extracellular segment through the intracellular juxtamembrane 'wedge' region are widely spaced, with introns ranging from 9.7 to 303.7 kb. In contrast, exons encoding the two phosphatase domains are more tightly clustered, with 15 exons spanning ∼ 60 kb, and introns ranging in size from 0.6 kb to 13.1 kb. Phase 0 introns predominate in the intracellular, and phase 1 in the extracellular segment. CONCLUSIONS: We report the first genomic characterization of a RPTP type IIB gene. Alternatively spliced variants may result in different RPTPρ isoforms. Our findings suggest that RPTPρ extracellular and intracellular segments originated as separate modular proteins that fused into a single transmembrane molecule during a later evolutionary period

Early endostatin treatment inhibits metastatic seeding of murine colorectal cancer cells in the liver and their adhesion to endothelial cells

Endostatin, a carboxy-terminal fragment of collagen XVIII, potently inhibits angiogenesis and tumour growth, presumably through induction of apoptosis in endothelial cells and/or inhibition of their migration. Here we have tested how the timing of recombinant human endostatin (rh-E) administration affects its antitumour activity in a liver metastasis model of mouse C26 colorectal carcinoma cells. The effects of rh-E treatment on hepatic tumour load and on early tumour cell seeding were evaluated. Recombinant human endostatin was most effective in reducing intrahepatic tumour growth when administered prior to tumour cell inoculation. Analysis of early tumour cell seeding by using [125I]iododeoxyuridine-labelled C26 cells or by in vivo microscopy showed that rh-E reduced tumour cell seeding in the liver sinusoids. Recombinant human endostatin did not inhibit tumour growth when administered later than 4 days after tumour injection. Pretreatment of human umbilical vein endothelial cells with rh-E in vitro reduced C26 tumour cell adhesion under flow conditions two-fold as assessed by video microscopy and multiphoton laser scanning microscopy. Our results show that rh-E, in addition to antiangiogenic effects, reduces tumour cell adhesion in the liver sinusoids during the very early phases of metastasis formation. These data point towards a previously unknown mode of action of endostatin, that is, its ability to interfere with tumour cell seeding. Such insights may be helpful in the design of trials to improve (surgical) treatment of colorectal carcinoma and liver metastases

Intraoperative electrocorticography using high-frequency oscillations or spikes to tailor epilepsy surgery in the Netherlands (the HFO trial): a randomised, single-blind, adaptive non-inferiority trial

Background Intraoperative electrocorticography is used to tailor epilepsy surgery by analysing interictal spikes or spike patterns that can delineate epileptogenic tissue. High-frequency oscillations (HFOs) on intraoperative electrocorticography have been proposed as a new biomarker of epileptogenic tissue, with higher specificity than spikes. We prospectively tested the non-inferiority of HFO-guided tailoring of epilepsy surgery to spike-guided tailoring on seizure freedom at 1 year.Methods The HFO trial was a randomised, single-blind, adaptive non-inferiority trial at an epilepsy surgery centre (UMC Utrecht) in the Netherlands. We recruited children and adults (no age limits) who had been referred for intraoperative electrocorticography-tailored epilepsy surgery. Participants were randomly allocated (1:1) to either HFO-guided or spike-guided tailoring, using an online randomisation scheme with permuted blocks generated by an independent data manager, stratified by epilepsy type. Treatment allocation was masked to participants and clinicians who documented seizure outcome, but not to the study team or neurosurgeon. Ictiform spike patterns were always considered in surgical decision making. The primary endpoint was seizure outcome after 1 year (dichotomised as seizure freedom [defined as Engel 1A-11 vs seizure recurrence [Engel 1C-4]). We predefined a non-inferiority margin of 10% risk difference. Analysis was by intention to treat, with prespecified subgroup analyses by epilepsy type and for confounders. This completed trial is registered with the Dutch Trial Register, Toetsingonline ABR.NL44527.041.13, and ClinicalTrials.gov, NCT02207673.Findings Between Oct 10, 2014, and Jan 31,2020,78 individuals were enrolled to the study and randomly assigned (39 to HFO-guided tailoring and 39 to spike-guided tailoring). There was no loss to follow-up. Seizure freedom at 1 year occurred in 26 (67%) of 39 participants in the HFO-guided group and 35 (90%) of 39 in the spike-guided group (risk difference -23.5%, 90% CI -39.1 to -7.9; for the 48 patients with temporal lobe epilepsy, the risk difference was -25.5%, -45.1 to -6.0, and for the 30 patients with extratemporal lobe epilepsy it was -20.3%, -46.0 to 5.4). Pathology associated with poor prognosis was identified as a confounding factor, with an adjusted risk difference of-7.9% (90% CI -20.7 to 4.9; adjusted risk difference -12.5%, -31.0 to 5.9, for temporal lobe epilepsy and 5.8%, -7.7 to 19.5, for extratemporal lobe epilepsy). We recorded eight serious adverse events (five in the HFO-guided group and three in the spike-guided group) requiring hospitalisation. No patients died.Interpretation HFO-guided tailoring of epilepsy surgery was not non-inferior to spike-guided tailoring on intraoperative electrocorticography. After adjustment for confounders, HFOs show non-inferiority in extratemporal lobe epilepsy. This trial challenges the clinical value of HFOs as an epilepsy biomarker, especially in temporal lobe epilepsy. Further research is needed to establish whether HFO-guided intraoperative electrocorticography holds promise in extratemporal lobe epilepsy. Copyright (C) 2022 The Author(s). Published by Elsevier Ltd

Effects of IKAP/hELP1 Deficiency on Gene Expression in Differentiating Neuroblastoma Cells: Implications for Familial Dysautonomia

Familial dysautonomia (FD) is a developmental neuropathy of the sensory and autonomous nervous systems. The IKBKAP gene, encoding the IKAP/hELP1 subunit of the RNA polymerase II Elongator complex is mutated in FD patients, leading to a tissue-specific mis-splicing of the gene and to the absence of the protein in neuronal tissues. To elucidate the function of IKAP/hELP1 in the development of neuronal cells, we have downregulated IKBKAP expression in SHSY5Y cells, a neuroblastoma cell line of a neural crest origin. We have previously shown that these cells exhibit abnormal cell adhesion when allowed to differentiate under defined culture conditions on laminin substratum. Here, we report results of a microarray expression analysis of IKAP/hELP1 downregulated cells that were grown on laminin under differentiation or non-differentiation growth conditions. It is shown that under non-differentiation growth conditions, IKAP/hELP1 downregulation affects genes important for early developmental stages of the nervous system, including cell signaling, cell adhesion and neural crest migration. IKAP/hELP1 downregulation during differentiation affects the expression of genes that play a role in late neuronal development, in axonal projection and synapse formation and function. We also show that IKAP/hELP1 deficiency affects the expression of genes involved in calcium metabolism before and after differentiation of the neuroblastoma cells. Hence, our data support IKAP/hELP1 importance in the development and function of neuronal cells and contribute to the understanding of the FD phenotype

Dimerization of Receptor Protein-Tyrosine Phosphatase alpha in living cells

BACKGROUND: Dimerization is an important regulatory mechanism of single membrane-spanning receptors. For instance, activation of receptor protein-tyrosine kinases (RPTKs) involves dimerization. Structural, functional and biochemical studies suggested that the enzymatic counterparts of RPTKs, the receptor protein-tyrosine phosphatases (RPTPs), are inhibited by dimerization, but whether RPTPs actually dimerize in living cells remained to be determined. RESULTS: In order to assess RPTP dimerization, we have assayed Fluorescence Resonance Energy Transfer (FRET) between chimeric proteins of cyan- and yellow-emitting derivatives of green fluorescent protein, fused to RPTPα, using three different techniques: dual wavelength excitation, spectral imaging and fluorescence lifetime imaging. All three techniques suggested that FRET occurred between RPTPα -CFP and -YFP fusion proteins, and thus that RPTPα dimerized in living cells. RPTPα dimerization was constitutive, extensive and specific. RPTPα dimerization was consistent with cross-linking experiments, using a non-cell-permeable chemical cross-linker. Using a panel of deletion mutants, we found that the transmembrane domain was required and sufficient for dimerization. CONCLUSIONS: We demonstrate here that RPTPα dimerized constitutively in living cells, which may be mediated by the transmembrane domain, providing strong support for the model that dimerization is involved in regulation of RPTPs

Absence of repellents in Ustilago maydis induces genes encoding small secreted proteins

The rep1 gene of the maize pathogen Ustilago maydis encodes a pre-pro-protein that is processed in the secretory pathway into 11 peptides. These so-called repellents form amphipathic amyloid fibrils at the surface of aerial hyphae. A SG200 strain in which the rep1 gene is inactivated (∆rep1 strain) is affected in aerial hyphae formation. We here assessed changes in global gene expression as a consequence of the inactivation of the rep1 gene. Microarray analysis revealed that only 31 genes in the ∆rep1 SG200 strain had a fold change in expression of ≥2. Twenty-two of these genes were up-regulated and half of them encode small secreted proteins (SSPs) with unknown functions. Seven of the SSP genes and two other genes that are over-expressed in the ∆rep1 SG200 strain encode proteins that can be classified as secreted cysteine-rich proteins (SCRPs). Interestingly, most of the SCRPs are predicted to form amyloids. The SCRP gene um00792 showed the highest up-regulation in the ∆rep1 strain. Using GFP as a reporter, it was shown that this gene is over-expressed in the layer of hyphae at the medium-air interface. Taken together, it is concluded that inactivation of rep1 hardly affects the expression profile of U. maydis, despite the fact that the mutant strain has a strong reduced ability to form aerial hyphae

Functional Amyloids Composed of Phenol Soluble Modulins Stabilize Staphylococcus aureus Biofilms

Staphylococcus aureus is an opportunistic pathogen that colonizes the skin and mucosal surfaces of mammals. Persistent staphylococcal infections often involve surface-associated communities called biofilms. Here we report the discovery of a novel extracellular fibril structure that promotes S. aureus biofilm integrity. Biochemical and genetic analysis has revealed that these fibers have amyloid-like properties and consist of small peptides called phenol soluble modulins (PSMs). Mutants unable to produce PSMs were susceptible to biofilm disassembly by matrix degrading enzymes and mechanical stress. Previous work has associated PSMs with biofilm disassembly, and we present data showing that soluble PSM peptides disperse biofilms while polymerized peptides do not. This work suggests the PSMs' aggregation into amyloid fibers modulates their biological activity and role in biofilms