Noninvasive Detection of Hippocampal Epileptiform Activity on Scalp Electroencephalogram.

Importance: The hippocampus is a highly epileptogenic brain region, yet over 90% of hippocampal epileptiform activity (HEA) cannot be identified on scalp electroencephalogram (EEG) by human experts. Currently, detection of HEA requires intracranial electrodes, which limits our understanding of the role of HEA in brain diseases. Objective: To develop and validate a machine learning algorithm that accurately detects HEA from a standard scalp EEG, without the need for intracranial electrodes. Design, Setting, and Participants: In this diagnostic study, conducted from 2008 to 2021, EEG data were used from patients with temporal lobe epilepsy (TLE) and healthy controls (HCs) to train and validate a deep neural network, HEAnet, to detect HEA on scalp EEG. Participants were evaluated at tertiary-level epilepsy centers at 2 academic hospitals: Massachusetts General Hospital (MGH) or Brigham and Women's Hospital (BWH). Included in the study were patients aged 12 to 78 years with a clinical diagnosis of TLE and HCs without epilepsy. Patients with TLE and HCs with a history of intracranial surgery were excluded from the study. Exposures: Simultaneous intracranial EEG and/or scalp EEG. Main Outcomes and Measures: Performance was assessed using cross-validated areas under the receiver operating characteristic curve (AUC ROC) and precision-recall curve (AUC PR) and additional clinically relevant metrics. Results: HEAnet was trained and validated using data sets that were derived from a convenience sample of 141 eligible participants (97 with TLE and 44 HCs without epilepsy) whose retrospective EEG data were readily available. Data set 1 included the simultaneous scalp EEG and intracranial electrode recordings of 51 patients with TLE (mean [SD] age, 40.7 [15.9] years; 30 men [59%]) at MGH. An automatically generated training data set with 972 095 positive HEA examples was created, in addition to a held-out expert-annotated testing data set with 22 762 positive HEA examples. HEAnet's performance was validated on 2 independent scalp EEG data sets: (1) data set 2 (at MGH; 24 patients with TLE and 20 HCs; mean [SD] age, 42.3 [16.2] years; 17 men [39%]) and (2) data set 3 (at BWH; 22 patients with TLE and 24 HCs; mean [SD] age, 43.0 [14.4] years; 20 men [43%]). For single-event detection of HEA on data set 1, HEAnet achieved a mean (SD) AUC ROC of 0.89 (0.01) and a mean (SD) AUC PR of 0.39 (0.03). On external validation with data sets 2 and 3, HEAnet accurately distinguished TLE from HC (AUC ROC of 0.88 and 0.95, respectively) and predicted epilepsy lateralization with 100% and 92% accuracy, respectively. HEAnet tracked dynamic changes in HEA in response to seizure medication adjustments and performed comparably with human experts in diagnosing TLE from 1-hour scalp EEG recordings, diagnosing TLE in several individuals that experts missed. Without reducing specificity, addition of HEAnet to human expert EEG review increased sensitivity for diagnosing TLE in humans from 50% to 58% to 63% to 67%. Conclusions and Relevance: Results of this diagnostic study suggest that HEAnet provides a novel, noninvasive, quantitative, and clinically relevant biomarker of hippocampal hyperexcitability in humans.

Modifications at the C-terminus to improve pyrrole-imidazole polyamide activity in cell culture.

Pyrrole-imidazole (Py-Im) hairpin polyamides are a class of small molecule DNA minor groove binding compounds that have been shown to modulate endogenous gene expression in cell culture. Gene regulation by polyamides requires efficient cellular uptake and nuclear localization properties for candidate compounds. To further optimize Py-Im polyamides for enhanced potency in cell culture, a focused library of polyamides possessing various modifications at the C-terminus was synthesized and tested. Comparison of polyamide biological activity in two cell lines revealed tolerance for structural modifications and agreement in activity trends between cell lines. The use of an oxime linkage between the polyamide and an aromatic functionality on the C-terminus resulted in a approximately 20-fold increase in the potency of polyamides targeted to the androgen response element (ARE) in LNCaP cells by measuring AR-activated PSA expression.

Modulating hypoxia-inducible transcription by disrupting the HIF-1-DNA interface.

Transcription mediated by hypoxia-inducible factor (HIF-1) contributes to tumor angiogenesis and metastasis but is also involved in activation of cell-death pathways and normal physiological processes. Given the complexity of HIF-1 signaling, it could be advantageous to target a subset of HIF-1 effectors rather than the entire pathway. We compare the genome-wide effects of three molecules that each interfere with the HIF-1-DNA interaction: a polyamide targeted to the hypoxia response element, small interfering RNA targeted to HIF-1alpha, and echinomycin, a DNA-binding natural product with a similar but less specific sequence preference than the polyamide. The polyamide affects a subset of hypoxia-induced genes consistent with its binding site preferences. For comparison, HIF-1alpha siRNA and echinomycin each affect the expression of nearly every gene induced by hypoxia. Remarkably, the total number of genes affected by either polyamide or HIF-1alpha siRNA over a range of thresholds is comparable. The data show that polyamides can be used to affect a subset of a pathway regulated by a transcription factor. In addition, this study offers a unique comparison of three complementary approaches towards exogenous control of endogenous gene expression.

Improved nuclear localization of DNA-binding polyamides.

Regulation of endogenous genes by DNA-binding polyamides requires effective nuclear localization. Previous work employing confocal microscopy to study uptake of fluorophore-labeled polyamides has demonstrated the difficulty of predicting a priori the nuclear uptake of a given polyamide. The data suggest that dye identity influences uptake sufficiently such that a dye-conjugate cannot be used as a proxy for unlabeled analogs. Polyamides capable of nuclear localization unaided by fluorescent dyes are desirable due to size and other limitations of fluorophores. Recently, a polyamide-fluorescein conjugate targeted to the hypoxia response element (HRE) was found to inhibit vascular endothelial growth factor (VEGF) expression in cultured HeLa cells. The current study uses inhibition of VEGF expression as a biological read-out for effective nuclear localization of HRE-targeted polyamides. We synthesized a focused library of non-fluorescent, HRE-targeted polyamides in which the C-terminus 'tail' has been systematically varied. Members of this library bind the HRE with affinities comparable or superior to that of the fluorescein-labeled analog. Although most library members demonstrate modest or no biological activity, two non-fluorescent polyamides are reported with activity rivaling that of the previously reported fluorescein-labeled polyamide. We also show evidence that promoter occupancy by HIF-1, the transcription factor that binds the HRE, is inhibited by HRE-targeted polyamides.