Analysis of the action mechanisms and targets of herbal anticonvulsants highlights opportunities for therapeutic engagement with refractory epilepsy.

Epilepsy is a neurological disorder characterized by spontaneous and recurring seizures. It poses significant therapeutic challenges due to diverse etiology, pathobiology, and pharmacotherapy-resistant variants. The anticonvulsive effects of herbal leads with biocompatibility and toxicity considerations have attracted much interest, inspiring mechanistic analysis with the view of their use for engagement of new targets and combination with antiseizure pharmacotherapies. This article presents a comprehensive overview of the key molecular players and putative action mechanisms of the most common antiepileptic herbals demonstrated in tissue culture and preclinical models. From the review of the literature, it emerges that their effects are mediated via five distinct mechanisms: (1) reduction of membrane excitability through inhibition of cation channels, (2) improvement of mitochondrial functions with antioxidant effects, (3) enhancement in synaptic transmission mediated by GABAA receptors, (4) improvement of immune response with anti-inflammatory action, and (5) suppression of protein synthesis and metabolism. While some of the primary targets and action mechanisms of herbal anticonvulsants (1, 3) are shared with antiseizure pharmacotherapies, herbal leads also engage with distinct mechanisms (2, 4, and 5), suggesting new drug targets and opportunities for their integration with antiseizure medications. Addressing outstanding questions through research and in silico modeling should facilitate the future use of herbals as auxiliary therapy in epilepsy and guide the development of treatment of pharmacoresistant seizures through rigorous trials and regulatory approval.

Neurotoxin-Derived Optical Probes for Biological and Medical Imaging.

The superb specificity and potency of biological toxins targeting various ion channels and receptors are of major interest for the delivery of therapeutics to distinct cell types and subcellular compartments. Fused with reporter proteins or labelled with fluorophores and nanocomposites, animal toxins and their detoxified variants also offer expanding opportunities for visualisation of a range of molecular processes and functions in preclinical models, as well as clinical studies. This article presents state-of-the-art optical probes derived from neurotoxins targeting ion channels, with discussions of their applications in basic and translational biomedical research. It describes the design and production of probes and reviews their applications with advantages and limitations, with prospects for future improvements. Given the advances in imaging tools and expanding research areas benefiting from the use of optical probes, described here resources should assist the discovery process and facilitate high-precision interrogation and therapeutic interventions.

Molecular Biomarkers of Neuronal Injury in Epilepsy Shared with Neurodegenerative Diseases.

In neurodegenerative diseases, changes in neuronal proteins in the cerebrospinal fluid and blood are viewed as potential biomarkers of the primary pathology in the central nervous system (CNS). Recent reports suggest, however, that level of neuronal proteins in fluids also alters in several types of epilepsy in various age groups, including children. With increasing evidence supporting clinical and sub-clinical seizures in Alzheimer's disease, Lewy body dementia, Parkinson's disease, and in other less common neurodegenerative conditions, these findings call into question the specificity of neuronal protein response to neurodegenerative process and urge analysis of the effects of concomitant epilepsy and other comorbidities. In this article, we revisit the evidence for alterations in neuronal proteins in the blood and cerebrospinal fluid associated with epilepsy with and without neurodegenerative diseases. We discuss shared and distinctive characteristics of changes in neuronal markers, review their neurobiological mechanisms, and consider the emerging opportunities and challenges for their future research and diagnostic use.

Brain-derived neurotrophic factor (BDNF) in perinatal depression: Side show or pivotal factor?

Perinatal depression is the most common psychiatric complication of pregnancy, with its detrimental effects on maternal and infant health widely underrated. There is a pressing need for specific molecular biomarkers, with pregnancy-related decline in brain-derived neurotrophic factor (BDNF) in the blood and downregulation of TrkB receptor in the brain reported in clinical and preclinical studies. In this review, we explore the emerging role of BDNF in reproductive biology and discuss evidence suggesting its deficiency as a risk factor for perinatal depression. With the increasing evidence for restoration of serum BDNF levels by antidepressant therapy, the strengthening association of perinatal depression with deficiency of BDNF supports its potential as a surrogate endpoint for preclinical and clinical studies.

Autism-associated CHD8 keeps proliferation of human neural progenitors in check by lengthening the G1 phase of the cell cycle.

De novo mutations (DNMs) in chromodomain helicase DNA binding protein 8 (CHD8) are associated with a specific subtype of autism characterized by enlarged heads and distinct cranial features. The vast majority of these DNMs are heterozygous loss-of-function mutations with high penetrance for autism. CHD8 is a chromatin remodeler that preferentially regulates expression of genes implicated in early development of the cerebral cortex. How CHD8 haploinsufficiency alters the normal developmental trajectory of the brain is poorly understood and debated. Using long-term single-cell imaging, we show that disruption of a single copy of CHD8 in human neural precursor cells (NPCs) markedly shortens the G1 phase of the cell cycle. Consistent with faster progression of CHD8+/- NPCs through G1 and the G1/S checkpoint, we observed increased expression of E cyclins and elevated phosphorylation of Erk in these mutant cells - two central signaling pathways involved in S phase entry. Thus, CHD8 keeps proliferation of NPCs in check by lengthening G1, and mono-allelic disruption of this gene alters cell-cycle timing in a way that favors self-renewing over neurogenic cell divisions. Our findings further predict enlargement of the neural progenitor pool in CHD8+/- developing brains, providing a mechanistic basis for macrocephaly in this autism subtype.

Bioprinting and Preliminary Testing of Highly Reproducible Novel Bioink for Potential Skin Regeneration.

Three-dimensional (3D) bioprinting is considered as a novel approach in biofabricating cell-laden constructs that could potentially be used to promote skin regeneration following injury. In this study, a novel crosslinked chitosan (CH)-genipin (GE) bioink laden with keratinocyte and human dermal fibroblast cells was developed and printed successfully using an extruder-based bioprinter. By altering the composition and degree of CH-GE crosslinking, bioink printability was further assessed and compared with a commercial bioink. Rheological analysis showed that the viscosity of the optimised bioink was in a suitable range that facilitated reproducible and reliable printing by applying low pressures ranging from 20-40 kPa. The application of low printing pressures proved vital for viability of cells loaded within the bioinks. Further characterisation using MTT assay showed that cells were still viable within the printed construct at 93% despite the crosslinking, processing and after subjecting to physiological conditions for seven days. The morphological study of the printed cells showed that they were mobile within the bioink. Furthermore, the multi-layered 3D printed constructs demonstrated excellent self-supportive structures in a consistent manner.

Green fluorescent protein (GFP): is seeing believing and is that enough?

Intracellular compartmentalisation is a significant barrier to the successful nucleocytosolic delivery of biologics. The endocytic system has been shown to be responsible for compartmentalisation, providing an entry point, and trigger(s) for the activation of drug delivery systems. Consequently, many of the technologies used to understand endocytosis have found utility within the field of drug delivery. The use of fluorescent proteins as markers denoting compartmentalisation within the endocytic system has become commonplace. Several of the limitations associated with the use of green fluorescent protein (GFP) within the context of drug delivery have been explored here by asking a series of related questions: (1) Are molecules that regulate fusion to a specific compartment (i.e. Rab- or SNARE-GFP fusions) a good choice of marker for that compartment? (2) How reliable was GFP-marker overexpression when used to define a given endocytic compartment? (3) Can glutathione-s-transferase (GST) fused in frame with GFP (GST-GFP) act as a fluid phase endocytic probe? (4) Was GFP fluorescence a robust indicator of (GFP) protein integrity? This study concluded that there are many appropriate and useful applications for GFP; however, thought and an understanding of the biological and physicochemical character of these markers are required for the generation of meaningful data.

The potential of toxin-based drug delivery systems for enhanced nucleic acid therapeutic delivery.

INTRODUCTION: The potential of gene replacement therapy has been underscored by the market authorization of alipogene tiparvovec (Glybera) and GSK2696273 (Strimvelis) in the EU and recombinant adenovirus-p53 (Gendicine) in China. Common to these systems is the use of attenuated viruses for 'drug' delivery. Whilst viral delivery systems are being developed for siRNA, their application to antisense delivery remains problematic. Non-viral delivery remains experimental, with some notable successes. However, stability and the 'PEG dilemma', balancing toxicity and limited (often liver-tropic) pharmacokinetics/oharmacodynamics, with the membrane destabilizing activity, necessary for nucleocytosolic access and transfection remain a problem. Areas covered: Here we review the use of attenuated protein toxins as a delivery vehicle for nucleic acids, their relationship to the PEG dilemma, and their biological properties with specific reference to their intracellular trafficking. Expert opinion: The possibility of using attenuated toxins as antisense and siRNA delivery systems has been demonstrated in vitro. Systems based upon attenuated anthrax toxin have been shown to have high activity (equivalent to nucleofection) and low toxicity whilst not requiring cationic 'helpers' or condensing agents, divorcing these systems from the problems associated with the PEG dilemma. It remains to be seen whether these systems can operate safely, efficiently and reproducibly, in vivo or in the clinic.

An in vitro evaluation of epigallocatechin gallate (eGCG) as a biocompatible inhibitor of ricin toxin.

The catechin, epigallocatechin gallate (eGCG), found in green tea, has inhibitory activity against a number of protein toxins and was investigated in relation to its impact upon ricin toxin (RT) in vitro. The IC(50) for RT was 0.08±0.004 ng/mL whereas the IC(50) for RT+100 µM eGCG was 3.02±0.572 ng/mL, indicating that eGCG mediated a significant (p<0.0001) reduction in ricin toxicity. This experiment was repeated in the human macrophage cell line THP-1 and IC(50) values were obtained for RT (0.54±0.024 ng/mL) and RT+100 µM eGCG (0.68±0.235 ng/mL) again using 100 µM eGCG and was significant (p=0.0013). The documented reduction in ricin toxicity mediated by eGCG was found to be eGCG concentration dependent, with 80 and 100 µg/mL (i.e. 178 and 223 µM respectively) of eGCG mediating a significant (p=0.0472 and 0.0232) reduction in ricin toxicity at 20 and 4 ng/ml of RT in Vero and THP-1 cells (respectively). When viability was measured in THP-1 cells by propidium iodide exclusion (as opposed to the MTT assays used previously) 10 ng/mL and 5 ng/mL of RT was used. The addition of 1000 µM and 100 µM eGCG mediated a significant (p=0.0015 and <0.0001 respectively) reduction in ricin toxicity relative to an identical concentration of ricin with 1 µg eGCG. Further, eGCG (100 µM) was found to reduce the binding of RT B chain to lactose-conjugated Sepharose as well as significantly (p=0.0039) reduce the uptake of RT B chain in Vero cells. This data suggests that eGCG may provide a starting point to refine biocompatible substances that can reduce the lethality of ricin.

Disarmed anthrax toxin delivers antisense oligonucleotides and siRNA with high efficiency and low toxicity.

Inefficient cytosolic delivery and vector toxicity contribute to the limited use of antisense oligonucleotides (ASOs) and siRNA as therapeutics. As anthrax toxin (Atx) accesses the cytosol, the purpose of this study was to evaluate the potential of disarmed Atx to deliver either ASOs or siRNA. We hypothesized that this delivery strategy would facilitate improved transfection efficiency while eliminating the toxicity seen for many vectors due to membrane destabilization. Atx complex formation with ASOs or siRNA was achieved via the in-frame fusion of either Saccharomyces cerevisiae GAL4 or Homo sapien sapien PKR (respectively) to a truncation of Atx lethal factor (LFn), which were used with Atx protective antigen (PA). Western immunoblotting confirmed the production of: LFN-GAL4, LFn-PKR and PA which were detected at ~45.9 kDa, ~37 kDa, and ~83 kDa respectively and small angle neutron scattering confirmed the ability of PA to form an annular structure with a radius of gyration of 7.0 ± 1.0 nm when placed in serum. In order to form a complex with LFn-GAL4, ASOs were engineered to contain a double-stranded region, and a cell free in vitro translation assay demonstrated that no loss of antisense activity above 30 pmol ASO was evident. The in vitro toxicity of both PA:LFn-GAL4:ASO and PA:LFn-PKR:siRNA complexes was low (IC50>100 µg/mL in HeLa and Vero cells) and subcellular fractionation in conjunction with microscopy confirmed the detection of LFn-GAL4 or LFn-PKR in the cytosol. Syntaxin5 (Synt5) was used as a model target gene to determine pharmacological activity. The PA:LFn-GAL4:ASO complexes had transfection efficiency approximately equivalent to Nucleofection® over a variety of ASO concentrations (24h post-transfection) and during a 72 h time course. In HeLa cells, at 200 pmol ASO (with PA:LFN-GAL4), 5.4 ± 2.0% Synt5 expression was evident relative to an untreated control after 24h. Using 200 pmol ASOs, Nucleofection® reduced Synt5 expression to 8.1 ± 2.1% after 24h. PA:LFn-GAL4:ASO transfection of non- or terminally-differentiated THP-1 cells and Vero cells resulted in 35.2 ± 19.1%, 36.4 ± 1.8% and 22.9 ± 6.9% (respectively) Synt5 expression after treatment with 200 pmol of ASO and demonstrated versatility. Nucleofection® with Stealth RNAi™ siRNA reduced HeLa Synt5 levels to 4.6 ± 6.1% whereas treatment with the PA:LFn-PKR:siRNA resulted in 8.5 ± 3.4% Synt5 expression after 24h (HeLa cells). These studies report for the first time an ASO and RNAi delivery system based upon protein toxin architecture that is devoid of polycations. This system may utilize regulated membrane back-fusion for the cytosolic delivery of ASOs and siRNA, which would account for the lack of toxicity observed. High delivery efficiency suggests further in vivo evaluation is warranted.