Personalized Frequency Modulated Transcranial Electrical Stimulation for Associative Memory Enhancement.

Associative memory (AM) is the ability to remember the relationship between previously unrelated items. AM is significantly affected by normal aging and neurodegenerative conditions, thus there is a growing interest in applying non-invasive brain stimulation (NIBS) techniques for AM enhancement. A growing body of studies identifies posterior parietal cortex (PPC) as the most promising cortical target for both transcranial magnetic stimulation (TMS) and transcranial electrical stimulation (tES) to modulate a cortico-hippocampal network that underlines AM. In that sense, theta frequency oscillatory tES protocols, targeted towards the hallmark oscillatory activity within the cortico-hippocampal network, are increasingly coming to prominence. To increase precision and effectiveness, the need for EEG guided individualization of the tES protocols is proposed. Here, we present the study protocol in which two types of personalized oscillatory tES-transcranial alternating current stimulation (tACS) and oscillatory transcranial direct current stimulation (otDCS), both frequency-modulated to the individual theta-band frequency (ITF), are compared to the non-oscillatory transcranial direct current stimulation (tDCS) and to the sham stimulation. The study has cross-over design with four tES conditions (tACS, otDCS, tDCS, sham), and the comprehensive set of neurophysiological (resting state EEG and AM-evoked EEG) and behavioral outcomes, including AM tasks (short-term associative memory, face-word, face-object, object-location), as well as measures of other cognitive functions (cognitive control, verbal fluency, and working memory).

The effects of offline and online prefrontal vs parietal transcranial direct current stimulation (tDCS) on verbal and spatial working memory.

Working memory (WM) is a limited-capacity system or set of processes that enables temporary storage and manipulation of information essential for complex cognitive processes. The WM performance is supported by a widespread neural network in which fronto-parietal functional connections have a pivotal role. Transcranial direct current stimulation (tDCS) is rapidly emerging as a promising tool for understanding the role of various cortical areas and their functional networks on cognitive performance. Here we comprehensively evaluated the effects of tDCS on WM by conducting three cross-over counterbalanced sham-controlled experiments in which we contrasted the effects and interactions of the anodal (i.e. facilitatory) tDCS across anterior-posterior (i.e. DLPFC vs PPC) and left-right (i.e. the lateralization) axes, and across online and offline protocols using both verbal and spatial WM (3-back) tasks as outcomes. In the offline protocols, left DLPFC stimulation affected neither verbal nor spatial WM, while left PPC stimulation increased spatial WM. When applied offline over right DLPFC, tDCS improved verbal WM task and marginally enhanced spatial WM; while when tDCS was applied over the right PPC, facilitatory effects were observed on verbal WM. In the online protocol, tDCS did not modulate WM regardless of the task modality or stimulation loci. In summary, the study did not replicate the left DLPFC tDCS effect on WM, found in some of the previous studies, but demonstrated positive effects of stimulation of the right DLPFC as well as PPC bilaterally. The observed effects varied across modality of the 3-back task, and tDCS protocol applied. The results of this study argue for moving towards targeting the lesser-explored stimulation sites within the fronto-parietal network, such as PPC, to gain a better understanding of the usefulness of tDCS for WM neuromodulation.

Theta-modulated oscillatory transcranial direct current stimulation over posterior parietal cortex improves associative memory.

Associative memory (AM) reflects the ability to remember and retrieve multiple pieces of information bound together thus enabling complex episodic experiences. Despite growing interest in the use of transcranial direct current stimulation (tDCS) for the modulation of AM, there are inconsistent evidence regarding its benefits. An alternative to standard constant tDCS could be the application of frequency-modulated tDCS protocols, that mimic natural function-relevant brain rhythms. Here, we show the effects of anodal tDCS oscillating in theta rhythm (5 Hz; 1.5 ± 0.1 mA) versus constant anodal tDCS and sham over left posterior parietal cortex on cued recall of face-word associations. In a crossover design, each participant completed AM assessment immediately following 20-min theta-oscillatory, constant, and sham tDCS, as well as 1 and 5 days after. Theta oscillatory tDCS increased initial AM performance in comparison to sham, and so did constant tDCS. On the group level, no differences between oscillatory and constant tDCS were observed, but individual-level analysis revealed that some participants responded to theta-oscillatory but not to constant tDCS, and vice versa, which could be attributed to their different physiological modes of action. This study shows the potential of oscillatory tDCS protocols for memory enhancement to produce strong and reliable memory-modulating effects which deserve to be investigated further.

The immediate and delayed effects of single tDCS session over posterior parietal cortex on face-word associative memory.

Associative memory (AM), an ability to form and retrieve associations between information units is crucial for everyday functioning and is affected by aging as well as by different neurological conditions. It was shown that rTMS over posterior parietal cortex (PPC) can improve AM of face-word pairs. Therefore, we examined if tDCS will produce comparable effects and explore whether the effect would persist one and five days following the stimulation. Thirty-seven healthy participants took part in cross-over sham-controlled study in which they received 20 min of anodal (1.5 mA) or sham tDCS over left PPC. Following tDCS participants completed face-cued word recall and verbal fluency tasks. A randomly selected subsample (N = 18) has completed follow up memory assessments one and five days after the stimulation. Anodal tDCS facilitated AM performance in comparison to sham with the same trend persisting during the 5-day follow-up period. Additionally, participants with lower AM scores had higher relative gain following anodal tDCS. Anodal tDCS had no effect on the control task (verbal fluency). Results support the existence of a specific enhancing effect on AM produced by facilitatory neuromodulation of the PPC. The effect was more prominent in low-performers and it persisted at least 5 days post-stimulation. These findings support the robustness of tDCS effect on AM and provide a foundation for future research that could lead to its future clinical application.

Discovery of Orally Active Inhibitors of Brahma Homolog (BRM)/SMARCA2 ATPase Activity for the Treatment of Brahma Related Gene 1 (BRG1)/SMARCA4-Mutant Cancers.

SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin subfamily A member 2 (SMARCA2), also known as Brahma homologue (BRM), is a Snf2-family DNA-dependent ATPase. BRM and its close homologue Brahma-related gene 1 (BRG1), also known as SMARCA4, are mutually exclusive ATPases of the large ATP-dependent SWI/SNF chromatin-remodeling complexes involved in transcriptional regulation of gene expression. No small molecules have been reported that modulate SWI/SNF chromatin-remodeling activity via inhibition of its ATPase activity, an important goal given the well-established dependence of BRG1-deficient cancers on BRM. Here, we describe allosteric dual BRM and BRG1 inhibitors that downregulate BRM-dependent gene expression and show antiproliferative activity in a BRG1-mutant-lung-tumor xenograft model upon oral administration. These compounds represent useful tools for understanding the functions of BRM in BRG1-loss-of-function settings and should enable probing the role of SWI/SNF functions more broadly in different cancer contexts and those of other diseases.

Adult skin-derived precursor Schwann cell grafts form growths in the injured spinal cord of Fischer rats.

In this study, GFP+ skin-derived precursor Schwann cells (SKP-SCs) from adult rats were grafted into the injured spinal cord of immunosuppressed rats. Our goal was to improve grafted cell survival in the injured spinal cord, which is typically low. Cells were grafted in hyaluronan-methylcellulose hydrogel (HAMC) or hyaluronan-methylcellulose modified with laminin- and fibronectin-derived peptide sequences (eHAMC). The criteria for selection of hyaluronan was for its shear-thinning properties, making the hydrogel easy to inject, methylcellulose for its inverse thermal gelation, helping to keep grafted cells in situ, and fibronectin and laminin to improve cell attachment and, thus, prevent cell death due to dissociation from substrate molecules (i.e., anoikis). Post-mortem examination revealed large masses of GFP+ SKP-SCs in the spinal cords of rats that received cells in HAMC (5 out of n = 8) and eHAMC (6 out of n = 8). Cell transplantation in eHAMC caused significantly greater spinal lesions compared to lesion and eHAMC only control groups. A parallel study showed similar masses in the contused spinal cord of rats after transplantation of adult GFP+ SKP-SCs without a hydrogel or immunosuppression. These findings suggest that adult GFP+ SKP-SCs, cultured/transplanted under the conditions described here, have a capacity for uncontrolled proliferation. Growth-formation in pre-clinical research has also been documented after transplantation of: human induced pluripotent stem cell-derived neural stem cells (Itakura et al 2015 PLoS One 10 e0116413), embryonic stem cells and embryonic stem cell-derived neurons (Brederlau et al 2006 Stem Cells 24 1433-40; Dressel et al 2008 PLoS One 3 e2622), bone marrow derived mesenchymal stem cells (Jeong et al 2011 Circ. Res. 108 1340-47) and rat nerve-derived SCs following in vitro expansion for >11 passages (Funk et al 2007 Eur. J. Cell Biol. 86 207-19; Langford et al 1988 J. Neurocytology 17 521-9; Morrissey et al 1991 J. Neurosci. 11 2433-42). It is of upmost importance to define the precise culture/transplantation parameters for maintenance of normal cell function and safe and effective use of cell therapy.

Hybrid Crosslinked Methylcellulose Hydrogel: A Predictable and Tunable Platform for Local Drug Delivery.

Design of experiment is used to develop a hybrid methylcellulose hydrogel that combines physical and chemical crosslinks, resulting in an injectable, in situ stiffening, and long-lasting material with predictable swelling and rheological properties. Chemical crosslinking is complete prior to injection, allowing for ease of use and storage. Controlled release of two relevant protein therapeutics and biocompatibility of the hydrogel are demonstrated.

Mathematical model accurately predicts protein release from an affinity-based delivery system.

Affinity-based controlled release modulates the delivery of protein or small molecule therapeutics through transient dissociation/association. To understand which parameters can be used to tune release, we used a mathematical model based on simple binding kinetics. A comprehensive asymptotic analysis revealed three characteristic regimes for therapeutic release from affinity-based systems. These regimes can be controlled by diffusion or unbinding kinetics, and can exhibit release over either a single stage or two stages. This analysis fundamentally changes the way we think of controlling release from affinity-based systems and thereby explains some of the discrepancies in the literature on which parameters influence affinity-based release. The rate of protein release from affinity-based systems is determined by the balance of diffusion of the therapeutic agent through the hydrogel and the dissociation kinetics of the affinity pair. Equations for tuning protein release rate by altering the strength (KD) of the affinity interaction, the concentration of binding ligand in the system, the rate of dissociation (koff) of the complex, and the hydrogel size and geometry, are provided. We validated our model by collapsing the model simulations and the experimental data from a recently described affinity release system, to a single master curve. Importantly, this mathematical analysis can be applied to any single species affinity-based system to determine the parameters required for a desired release profile.

Affinity-based drug delivery systems for tissue repair and regeneration.

Affinity-based release systems use transient interactions to sustain and control the release of a therapeutic from a polymeric matrix. The most common affinity-based systems use heparin-based scaffolds to sustain the release of heparin-binding proteins, such as fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF). However, novel affinity-based systems based on, for example, protein-protein or DNA-protein interactions, are emerging to control the release of an expanding repertoire of therapeutics. Mathematical models of affinity-based systems have provided a thorough understanding of which parameters affect release rate from these systems, and how these release rates can be tuned. In this review, recent affinity-based release systems will be described, including an overview of the various types of affinity interactions used to modulate release, the mechanisms by which release from these systems is tuned, and the time scales of sustained release. This advanced drug delivery paradigm provides tunable and predictable release rates and has expanded the scope of deliverable therapeutics for tissue repair and regeneration.

Affinity-based release of chondroitinase ABC from a modified methylcellulose hydrogel.

Chondroitinase ABC (ChABC) is a promising therapeutic for spinal cord injury as it can degrade the glial scar that is detrimental to regrowth and repair. However, the sustained delivery of bioactive ChABC is a challenge requiring highly invasive methods such as intra-spinal injections, insertion of intrathecal catheters, or implantation of delivery vehicles directly into the tissue. ChABC is thermally unstable, further complicating its delivery. Moreover, there are no commercial antibodies available for its detection. To achieve controlled release, we designed an affinity-based system that sustained the release of bioactive ChABC for at least 7days. ChABC was recombinantly expressed as a fusion protein with Src homology domain 3 (SH3) with an N-terminal histidine (HIS) tag and a C-terminal FLAG tag (ChABC-SH3). Protein purification was achieved using a nickel affinity column and, for the first time, direct quantification of ChABC down to 0.1nM was attained using an in-house HIS/FLAG double tag ELISA. The release of active ChABC-SH3 was sustained from a methylcellulose hydrogel covalently modified with an SH3 binding peptide. The rate of release was tunable by varying either the binding strength of the SH3-protein/SH3-peptide pair or the SH3-peptide to SH3-protein ratio. This innovative system has the potential to be used as a platform technology for the release and detection of other proteins that can be expressed using a similar construct.

Tunable growth factor delivery from injectable hydrogels for tissue engineering.

Current sustained delivery strategies of protein therapeutics are limited by the fragility of the protein, resulting in minimal quantities of bioactive protein delivered. In order to achieve prolonged release of bioactive protein, an affinity-based approach was designed which exploits the specific binding of the Src homology 3 (SH3) domain with short proline-rich peptides. Specifically, methyl cellulose was modified with SH3-binding peptides (MC-peptide) with either a weak affinity or strong affinity for SH3. The release profile of SH3-rhFGF2 fusion protein from hyaluronan MC-SH3 peptide (HAMC-peptide) hydrogels was investigated and compared to unmodified controls. SH3-rhFGF2 release from HAMC-peptide was extended to 10 days using peptides with different binding affinities compared to the 48 h release from unmodified HAMC. This system is capable of delivering additional proteins with tunable rates of release, while maintaining bioactivity, and thus is broadly applicable.

Single-isomer tetrasubstituted olefins from regioselective and stereospecific palladium-catalyzed coupling of beta-chloro-alpha-iodo-alpha,beta-unsaturated esters.

The efficient regioselective and stereospecific synthesis of tetrasubstituted olefins using a mild and convenient method is disclosed. 2-Alkynyl esters are selectively converted to E-beta-chloro-alpha-iodo-alpha,beta-unsaturated esters by exposure to Bu4NI in refluxing dichloroethane. These products are produced cleanly, regio- and stereoselectively, and in high yields. Single-isomer tetrasubstituted olefins bearing four different carbon substituents are then synthesized by sequential palladium-catalyzed coupling reactions. Selectivity results from reactivity differences in the intermediate substrates.