NOS1AP polymorphisms reduce NOS1 activity and interact with prolonged repolarization in arrhythmogenesis

Aims  NOS1AP single-nucleotide polymorphisms (SNPs) correlate with QT prolongation and cardiac sudden death in patients affected by long QT syndrome type 1 (LQT1). NOS1AP targets NOS1 to intracellular effectors. We hypothesize that NOS1AP SNPs cause NOS1 dysfunction and this may converge with prolonged action-potential duration (APD) to facilitate arrhythmias. Here we test (i) the effects of NOS1 inhibition and their interaction with prolonged APD in a guinea pig cardiomyocyte (GP-CMs) LQT1 model; (ii) whether pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from LQT1 patients differing for NOS1AP variants and mutation penetrance display a phenotype compatible with NOS1 deficiency. Methods and results  In GP-CMs, NOS1 was inhibited by S-Methyl-L-thiocitrulline acetate (SMTC) or Vinyl-L-NIO hydrochloride (L-VNIO); LQT1 was mimicked by IKs blockade (JNJ303) and β-adrenergic stimulation (isoproterenol). hiPSC-CMs were obtained from symptomatic (S) and asymptomatic (AS) KCNQ1-A341V carriers, harbouring the minor and major alleles of NOS1AP SNPs (rs16847548 and rs4657139), respectively. In GP-CMs, NOS1 inhibition prolonged APD, enhanced ICaL and INaL, slowed Ca2+ decay, and induced delayed afterdepolarizations. Under action-potential clamp, switching to shorter APD suppressed ‘transient inward current’ events induced by NOS1 inhibition and reduced cytosolic Ca2+. In S (vs. AS) hiPSC-CMs, APD was longer and ICaL larger; NOS1AP and NOS1 expression and co-localization were decreased. Conclusion  The minor NOS1AP alleles are associated with NOS1 loss of function. The latter likely contributes to APD prolongation in LQT1 and converges with it to perturb Ca2+ handling. This establishes a mechanistic link between NOS1AP SNPs and aggravation of the arrhythmia phenotype in prolonged repolarization syndromes

Optical modulation of excitation-contraction coupling in human-induced pluripotent stem cell-derived cardiomyocytes

Non-genetic photostimulation is a novel and rapidly growing multidisciplinary field that aims to induce light-sensitivity in living systems by exploiting exogeneous phototransducers. Here, we propose an intramembrane photoswitch, based on an azobenzene derivative (Ziapin2), for optical pacing of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The light-mediated stimulation process has been studied by applying several techniques to detect the effect on the cell properties. In particular, we recorded changes in membrane capacitance, in membrane potential (V-m), andmodulation of intracellular Ca2+ dynamics. Finally, cell contractility was analyzed using a custom MATLAB algorithm. Photostimulation of intramembrane Ziapin2 causes a transient V-m hyperpolarization followed by a delayed depolarization and action potential firing. The observed initial electrical modulation nicely correlates with changes in Ca2+ dynamics and contraction rate. This work represents the proof of principle that Ziapin2 can modulate electrical activity and contractility in hiPSC-CMs, opening up a future development in cardiac physiology

Calmodulinopathy: Functional Effects of CALM Mutations and Their Relationship With Clinical Phenotypes

In spite of the widespread role of calmodulin (CaM) in cellular signaling, CaM mutations lead specifically to cardiac manifestations, characterized by remarkable electrical instability and a high incidence of sudden death at young age. Penetrance of the mutations is surprisingly high, thus postulating a high degree of functional dominance. According to the clinical patterns, arrhythmogenesis in CaM mutations can be attributed, in the majority of cases, to either prolonged repolarization (as in long-QT syndrome, LQTS phenotype), or to instability of the intracellular Ca2+ store (as in catecholamine-induced tachycardias, CPVT phenotype). This review discusses how mutations affect CaM signaling function and how this may relate to the distinct arrhythmia phenotypes/mechanisms observed in patients; this involves mechanistic interpretation of negative dominance and mutation-specific CaM-target interactions. Knowledge of the mechanisms involved may allow critical approach to clinical manifestations and aid in the development of therapeutic strategies for “calmodulinopathies,” a recently identified nosological entity

Chitosan gated organic transistors printed on ethyl cellulose as a versatile platform for edible electronics and bioelectronics

Edible electronics is an emerging research field targeting electronic devices that can be safely ingested and directly digested or metabolized by the human body. As such, it paves the way to a whole new family of applications, ranging from ingestible medical devices and biosensors, to smart labelling for food quality monitoring and anti-counterfeiting. Being a newborn research field, many challenges need to be addressed to realize fully edible electronic components. In particular, an extended library of edible electronic materials is required, with suitable electronic properties depending on the target device and compatible with large-area printing processes, to allow scalable and cost-effective manufacturing. In this work, we propose a platform for future low-voltage edible transistors and circuits that comprises an edible chitosan gating medium and inkjet printed inert gold electrodes, compatible with low thermal budget edible substrates, such as ethylcellulose. We report the compatibility of the platform, characterized by critical channel features as low as 10 µm, with different inkjet printed carbon-based semiconductors, including biocompatible polymers present in the picograms range per device. A complementary organic inverter is also demonstrated with the same platform as a proof-of-principle logic gate. The presented results offer a promising approach to future low-voltage edible active circuitry, as well as a testbed for non-toxic printable semiconductors

Cryptanalysis of Dedicated Cryptographic Hash Functions

In this thesis we study the security of a number of dedicated cryptographic hash functions against cryptanalytic attacks. We begin with an introduction to what cryptographic hash functions are and what they are used for. This is followed by strict definitions of the security properties often required from cryptographic hash functions. FSB hashes are a class of hash functions derived from a coding theory problem. We attack FSB by modeling the compression function of the hash by a matrix in GF(2). We show that collisions and preimages can easily be found in FSB with the proposed security parameters. We describe a meet-in-the-middle attack against the FORK-256 hash function. The attack requires 2^112.8 operations to find a collision, which is a 38000-fold improvement over the expected 2^128 operations. We then present a method for finding slid pairs for the compression function of SHA-1; pairs of inputs and messages that produce closely related outputs in the compression function. We also cryptanalyse two block ciphers based on the compression function of MD5, MDC-MD5 and the Kaliski-Robshaw "Crab" encryption algorithm. VSH is a hash function based on problems in number theory that are believed to be hard. The original proposal only claims collision resistance; we demonstrate that VSH does not meet the other hash function requirements of preimage resistance, one-wayness, and collision resistance of truncated variants. To explore more general cryptanalytic attacks, we discuss the d-Monomial test, a statistical test that has been found to be effective in distinguishing iterated Boolean circuits from real random functions. The test is applied to the SHA and MD5 hash functions. We present a new hash function proposal, LASH, and its initial cryptanalysis.The LASH design is based on a simple underlying primitive, and some of its security can be shown to be related to lattice problems

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Arrhythmias and Heart Rate:Mechanisms and Significance of a Relationship

The occurrence of arrhythmia is often related to basic heart rate. Prognostic significance is associated with such a relationship; furthermore, heart rate modulation may result as an ancillary effect of therapy, or be considered as a therapeutic tool. This review discusses the cellular mechanisms underlying arrhythmia occurrence during tachycardia or bradycardia, considering rate changes per se or as a mirror of autonomic modulation. Besides the influence of steady-state heart rate, dynamic aspects of changes in rate and autonomic balance are considered. The discussion leads to the conclusion that the prognostic significance of arrhythmia relationship with heart rate, and the consequence of heart rate on arrhythmogenesis, may vary according to the substrate present in the specific case and should be considered accordingly.</p

A novel computerized assessment of manual spatial exploration in unilateral spatial neglect

Unilateral spatial neglect is a neuropsychological syndrome commonly observed after stroke and defined by the inability to attend or respond to contralesional stimuli. Typically, symptoms are assessed using clinical tests that rely upon visual/perceptual abilities. However, neglect may affect high-level representations controlling attention in other modalities as well. Here we developed a novel manual exploration test using a touch screen computer to quantify spatial search behaviour without visual input. Twelve chronic stroke patients with left neglect and 27 patients without neglect (based on clinical tests) completed our task. Four of the 12 “neglect” patients exhibited clear signs of neglect on our task as compared to “non-neglect” patients and healthy controls, and six other patients (from both groups) also demonstrated signs of neglect compared to healthy controls only. While some patients made asymmetrical responses on only one task, generally, patients with the strongest neglect performed poorly on multiple tasks. This suggests that representations associated with different modalities may be affected separately, but that severe forms of neglect are more likely related to damage in a common underlying representation. Our manual exploration task is easy to administer and can be added to standard neglect screenings to better measure symptom severity

Optical excitation of organic semiconductors as a highly selective strategy to induce vascular regeneration and tissue repair

Therapeutic neovascularization represents a promising strategy to rescue the vascular network and restore organ function in cardiovascular disorders (CVDs), including acute myocardial infarction, heart failure, peripheral artery disease, and brain stroke. Endothelial colony forming cells (ECFCs), which are mobilized in circulation upon an ischemic insult, are commonly regarded as the most suitable cellular tool to achieve therapeutic neovascularization. ECFCs can be genetically or pharmacologically manipulated to enhance their vasoreparative potential by boosting specific pro-angiogenic signalling pathways. However, optical stimulation represents the most reliable approach to control cellular activity because of its high selectivity and unprecedented spatio-temporal resolution. Herein, we discuss a novel strategy to drive ECFC angiogenic activity in ischemic tissues by combining geneless optical excitation with photosensitive organic semiconductors. We describe how photoexcitation of the conducting polymer poly(3-hexylthiophene-2,5-diyl), also known as P3HT, stimulates extracellular Ca2+ entry through Transient Receptor Potential Vanilloid 1 (TRPV1) channels upon the production of hydrogen peroxide (H2O2) in the cleft between the nanomaterial and the cell membrane. H2O2-induced TRPV1-dependent Ca2+ entry stimulates ECFC proliferation and tube formation, thereby providing the proof-of-concept that photoexcitation of organic semiconductors may offer a reliable strategy to stimulate ECFCs-dependent neovascularization in CVDs