On-demand low-frequency stimulation for seizure control: efficacy and behavioural implications.

Mesial temporal lobe epilepsy (MTLE), the most common form of focal epilepsy in adults, is often refractory to medication and associated with hippocampal sclerosis. Deep brain stimulation represents an alternative treatment option for drug-resistant patients who are ineligible for resective brain surgery. In clinical practice, closed-loop stimulation at high frequencies is applied to interrupt ongoing seizures, yet has (i) a high incidence of false detections; (ii) the drawback of delayed seizure-suppressive intervention; and (iii) limited success in sclerotic tissue. As an alternative, low-frequency stimulation (LFS) has been explored recently in patients with focal epilepsies. In preclinical epilepsy models, hippocampal LFS successfully prevented seizures when applied continuously. Since it would be advantageous to reduce the stimulation load, we developed a protocol for on-demand LFS. Given the importance of the hippocampus for navigation and memory, we investigated potential consequences of LFS on hippocampal function. To this end, we used the intrahippocampal kainate mouse model, which recapitulates the key features of MTLE, including spontaneous seizure activity and hippocampal sclerosis. Specifically, our online detection algorithm monitored epileptiform activity in hippocampal local field potential recordings and identified short epileptiform bursts preceding focal seizure clusters, triggering hippocampal LFS to stabilize the network state. To probe behavioural performance, we tested the acute influence of LFS on anxiety-like behaviour in the light-dark box test, spatial and non-spatial memory in the object location memory and novel object recognition test, as well as spatial navigation and long-term memory in the Barnes maze. On-demand LFS was almost as effective as continuous LFS in preventing focal seizure clusters but with a significantly lower stimulation load. When we compared the behavioural performance of chronically epileptic mice to healthy controls, we found that both groups were equally mobile, but epileptic mice displayed an increased anxiety level, altered spatial learning strategy and impaired memory performance. Most importantly, with the application of hippocampal LFS before behavioural training and test sessions, we could rule out deleterious effects on cognition and even show an alleviation of deficits in long-term memory recall in chronically epileptic mice. Taken together, our findings may provide a promising alternative to current therapies, overcoming some of their major limitations, and inspire further investigation of LFS for seizure control in focal epilepsy syndromes.

SIROF stabilized PEDOT/PSS allows biocompatible and reversible direct current stimulation capable of driving electrotaxis in cells.

Electrotaxis is a naturally occurring phenomenon in which ionic gradients dictate the directed migration of cells involved in different biological processes such as wound healing, embryonic development, or cancer metastasis. To investigate these processes, direct current (DC) has been used to generate electric fields capable of eliciting an electrotactic response in cells. However, the need for metallic electrodes to deliver said currents has hindered electrotaxis research and the application of DC stimulation as medical therapy. This study aimed to investigate the capability of poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT/PSS) on sputtered iridium oxide film (SIROF) electrodes to generate stable direct currents. The electrochemical properties of PEDOT/PSS allow ions to be released and reabsorbed depending on the polarity of the current flow. SIROF stabilized PEDOT/PSS electrodes demonstrated exceptional stability in voltage and current controlled DC stimulation for periods of up to 12 hours. These electrodes were capable of directing cell migration of the rat prostate cancer cell line MAT-LyLu in a microfluidic chamber without the need for chemical buffers. This material combination shows excellent promise for accelerating electrotaxis research and facilitating the translation of DC stimulation to medical applications thanks to its biocompatibility, ionic charge injection mechanisms, and recharging capabilities in a biological environment.

PARP3, a new therapeutic target to alter Rictor/mTORC2 signaling and tumor progression in BRCA1-associated cancers.

PARP3 has been shown to be a key driver of TGFß-induced epithelial-to-mesenchymal transition (EMT) and stemness in breast cancer cells, emerging as an attractive therapeutic target. Nevertheless, the therapeutic value of PARP3 inhibition has not yet been assessed. Here we investigated the impact of the absence of PARP3 or its inhibition on the tumorigenicity of BRCA1-proficient versus BRCA1-deficient breast cancer cell lines, focusing on the triple-negative breast cancer subtype (TNBC). We show that PARP3 knockdown exacerbates centrosome amplification and genome instability and reduces survival of BRCA1-deficient TNBC cells. Furthermore, we engineered PARP3-/- BRCA1-deficient or BRCA1-proficient TNBC cell lines using the CRISPR/nCas9D10A gene editing technology and demonstrate that the absence of PARP3 selectively suppresses the growth, survival and in vivo tumorigenicity of BRCA1-deficient TNBC cells, mechanistically via effects associated with an altered Rictor/mTORC2 signaling complex resulting from enhanced ubiquitination of Rictor. Accordingly, PARP3 interacts with and ADP-ribosylates GSK3ß, a positive regulator of Rictor ubiquitination and degradation. Importantly, these phenotypes were rescued by re-expression of a wild-type PARP3 but not by a catalytic mutant, demonstrating the importance of PARP3's catalytic activity. Accordingly, reduced survival and compromised Rictor/mTORC2 signaling were also observed using a cell-permeable PARP3-specific inhibitor. We conclude that PARP3 and BRCA1 are synthetic lethal and that targeting PARP3's catalytic activity is a promising therapeutic strategy for BRCA1-associated cancers via the Rictor/mTORC2 signaling pathway.

D-Cateslytin: a new antifungal agent for the treatment of oral Candida albicans associated infections.

The excessive use of antifungal agents, compounded by the shortage of new drugs being introduced into the market, is causing the accumulation of multi-resistance phenotypes in many fungal strains. Consequently, new alternative molecules to conventional antifungal agents are urgently needed to prevent the emergence of fungal resistance. In this context, Cateslytin (Ctl), a natural peptide derived from the processing of Chromogranin A, has already been described as an effective antimicrobial agent against several pathogens including Candida albicans. In the present study, we compared the antimicrobial activity of two conformations of Ctl, L-Ctl and D-Ctl against Candida albicans. Our results show that both D-Ctl and L-Ctl were potent and safe antifungal agents. However, in contrast to L-Ctl, D-Ctl was not degraded by proteases secreted by Candida albicans and was also stable in saliva. Using video microscopy, we also demonstrated that D-Ctl can rapidly enter C. albicans, but is unable to spread within a yeast colony unless from a mother cell to a daughter cell during cellular division. Besides, we revealed that the antifungal activity of D-Ctl could be synergized by voriconazole, an antifungal of reference in the treatment of Candida albicans related infections. In conclusion, D-Ctl can be considered as an effective, safe and stable antifungal and could be used alone or in a combination therapy with voriconazole to treat Candida albicans related diseases including oral candidosis.

D-Cateslytin, a new antimicrobial peptide with therapeutic potential.

The rise of antimicrobial resistant microorganisms constitutes an increasingly serious threat to global public health. As a consequence, the efficacy of conventional antimicrobials is rapidly declining, threatening the ability of healthcare professionals to cure common infections. Over the last two decades host defense peptides have been identified as an attractive source of new antimicrobials. In the present study, we characterized the antibacterial and mechanistic properties of D-Cateslytin (D-Ctl), a new epipeptide derived from L-Cateslytin, where all L-amino acids were replaced by D-amino acids. We demonstrated that D-Ctl emerges as a potent, safe and robust peptide antimicrobial with undetectable susceptibility to resistance. Using Escherichia coli as a model, we reveal that D-Ctl targets the bacterial cell wall leading to the permeabilization of the membrane and the death of the bacteria. Overall, D-Ctl offers many assets that make it an attractive candidate for the biopharmaceutical development of new antimicrobials either as a single therapy or as a combination therapy as D-Ctl also has the remarkable property to potentiate several antimicrobials of reference such as cefotaxime, amoxicillin and methicillin.

Analytical methods to determine electrochemical factors in electrotaxis setups and their implications for experimental design.

Direct current (DC) stimulation can be used to influence the orientation and migratory behavior of cells and results in cellular electrotaxis. Experimental work on such phenomena commonly relies on electrochemical dissolution of silver:silver-chloride (Ag:AgCl) electrodes to provide the stimulation via salt bridges. The strong ionic flow can be expected to influence the cell culture environment. In order to shed more light on which effects that must be considered, and possibly counter balanced, we here characterize a typical DC stimulation system. Silver migration speed was determined by stripping voltammetry. pH variability with stimulation was measured by ratiometric image analysis and conductivity alterations were quantified via two electrode impedance spectroscopy. It could be concluded that pH shifts towards more acidic values, in a linear manner with applied charge, after the buffering capability of the culture medium is exceeded. In contrast, the influence on conductivity was of negligible magnitude. Silver ions could enter the culture chamber at low concentrations long before a clear effect on the viability of the cultured cells could be observed. A design rule of 1cm salt bridge per C of stimulation charge transferred was however sufficient to ensure separation between cells and silver at all times.

A detailed insight into drug delivery from PEDOT based on analytical methods: effects and side effects.

The possibility to release drugs from conducting polymers, like polypyrrole or poly(3,4-ethylenedioxythiophene) (PEDOT), has been described and investigated for a variety of different substances during the last years, showing a wide interest in these release systems. A point that has not been looked at so far however is the possibility of other substances, next to the intended ones, leaving the polymer film under the high voltage excursions during redox sweeping. In this study we target this weakness of commonly used detection methods by implementing a high precision analytical method (high-performance liquid chromatography) that allows a separation and subsequently a detailed quantification of all possible release products. We could identify a significantly more complex release behavior for a PEDOT:Dex system than has been assumed so far, revealing the active release of the monomer upon redox activation. The released EDOT could thereby be shown to result from the bulk material, causing a considerable loss of polymer (>10% during six release events) that could partly account for the observed degradation or delamination effects of drug-eluting coatings. The monomer leakage was found to be substantially higher for a PEDOT:Dex film compared to a PEDOT:PSS sample. This finding indicates an overestimation of drug release if side products are mistaken for the actual drug mass. Moreover the full picture of released substances implements the need for further studies to reduce the monomer leakage and identify possible adverse effects, especially in the perspective of releasing an anti-inflammatory substance for attenuation of the foreign body reaction toward implanted electrodes.

PARP3 affects the relative contribution of homologous recombination and nonhomologous end-joining pathways.

The repair of toxic double-strand breaks (DSB) is critical for the maintenance of genome integrity. The major mechanisms that cope with DSB are: homologous recombination (HR) and classical or alternative nonhomologous end joining (C-NHEJ versus A-EJ). Because these pathways compete for the repair of DSB, the choice of the appropriate repair pathway is pivotal. Among the mechanisms that influence this choice, deoxyribonucleic acid (DNA) end resection plays a critical role by driving cells to HR, while accurate C-NHEJ is suppressed. Furthermore, end resection promotes error-prone A-EJ. Increasing evidence define Poly(ADP-ribose) polymerase 3 (PARP3, also known as ARTD3) as an important player in cellular response to DSB. In this work, we reveal a specific feature of PARP3 that together with Ku80 limits DNA end resection and thereby helps in making the choice between HR and NHEJ pathways. PARP3 interacts with and PARylates Ku70/Ku80. The depletion of PARP3 impairs the recruitment of YFP-Ku80 to laser-induced DNA damage sites and induces an imbalance between BRCA1 and 53BP1. Both events result in compromised accurate C-NHEJ and a concomitant increase in DNA end resection. Nevertheless, HR is significantly reduced upon PARP3 silencing while the enhanced end resection causes mutagenic deletions during A-EJ. As a result, the absence of PARP3 confers hypersensitivity to anti-tumoral drugs generating DSB.

Poly(ADP-ribose) polymerase 3 (PARP3), a newcomer in cellular response to DNA damage and mitotic progression.

The ADP ribosyl transferase [poly(ADP-ribose) polymerase] ARTD3(PARP3) is a newly characterized member of the ARTD(PARP) family that catalyzes the reaction of ADP ribosylation, a key posttranslational modification of proteins involved in different signaling pathways from DNA damage to energy metabolism and organismal memory. This enzyme shares high structural similarities with the DNA repair enzymes PARP1 and PARP2 and accordingly has been found to catalyse poly(ADP ribose) synthesis. However, relatively little is known about its in vivo cellular properties. By combining biochemical studies with the generation and characterization of loss-of-function human and mouse models, we describe PARP3 as a newcomer in genome integrity and mitotic progression. We report a particular role of PARP3 in cellular response to double-strand breaks, most likely in concert with PARP1. We identify PARP3 as a critical player in the stabilization of the mitotic spindle and in telomere integrity notably by associating and regulating the mitotic components NuMA and tankyrase 1. Both functions open stimulating prospects for specifically targeting PARP3 in cancer therapy.