Next-generation direct reprogramming.

Tissue repair is significantly compromised in the aging human body resulting in critical disease conditions (such as myocardial infarction or Alzheimer's disease) and imposing a tremendous burden on global health. Reprogramming approaches (partial or direct reprogramming) are considered fruitful in addressing this unmet medical need. However, the efficacy, cellular maturity and specific targeting are still major challenges of direct reprogramming. Here we describe novel approaches in direct reprogramming that address these challenges. Extracellular signaling pathways (Receptor tyrosine kinases, RTK and Receptor Serine/Theronine Kinase, RSTK) and epigenetic marks remain central in rewiring the cellular program to determine the cell fate. We propose that modern protein design technologies (AI-designed minibinders regulating RTKs/RSTK, epigenetic enzymes, or pioneer factors) have potential to solve the aforementioned challenges. An efficient transdifferentiation/direct reprogramming may in the future provide molecular strategies to collectively reduce aging, fibrosis, and degenerative diseases.

Stress-induced reversible cell-cycle arrest requires PRC2/PRC1-mediated control of mitophagy in Drosophila germline stem cells and human iPSCs.

Following acute genotoxic stress, both normal and tumorous stem cells can undergo cell-cycle arrest to avoid apoptosis and later re-enter the cell cycle to regenerate daughter cells. However, the mechanism of protective, reversible proliferative arrest, "quiescence," remains unresolved. Here, we show that mitophagy is a prerequisite for reversible quiescence in both irradiated Drosophila germline stem cells (GSCs) and human induced pluripotent stem cells (hiPSCs). In GSCs, mitofission (Drp1) or mitophagy (Pink1/Parkin) genes are essential to enter quiescence, whereas mitochondrial biogenesis (PGC1α) or fusion (Mfn2) genes are crucial for exiting quiescence. Furthermore, mitophagy-dependent quiescence lies downstream of mTOR- and PRC2-mediated repression and relies on the mitochondrial pool of cyclin E. Mitophagy-dependent reduction of cyclin E in GSCs and in hiPSCs during mTOR inhibition prevents the usual G1/S transition, pushing the cells toward reversible quiescence (G0). This alternative method of G1/S control may present new opportunities for therapeutic purposes.

CandyCollect: at-home saliva sampling for capture of respiratory pathogens.

Streptococcus pyogenes is a major human-specific bacterial pathogen and a common cause of a wide range of symptoms from mild infection such as pharyngitis (commonly called strep throat) to life-threatening invasive infection and post-infectious sequelae. Traditional methods for diagnosis include collecting a sample using a pharyngeal swab, which can cause discomfort and even discourage adults and children from seeking proper testing and treatment in the clinic. Saliva samples are an alternative to pharyngeal swabs. To improve the testing experience for strep throat, we developed a novel lollipop-inspired sampling platform (called CandyCollect) to capture bacteria in saliva. The device can be used in clinics or in the home and shipped back to a lab for analysis, integrating with telemedicine. CandyCollect is designed to capture bacteria on an oxygen plasma treated polystyrene surface embedded with flavoring substances to enhance the experience for children and inform the required time to complete the sampling process. In addition, the open channel structure prevents the tongue from scraping and removing the captured bacteria. The flavoring substances did not affect bacterial capture and the device has a shelf life of at least 2 months (with experiments ongoing to extend the shelf life). We performed a usability study with 17 participants who provided feedback on the device design and the dissolving time of the candy. This technology and advanced processing techniques, including polymerase chain reaction (PCR), will enable user-friendly and effective diagnosis of streptococcal pharyngitis.

AKT inhibition in the central nervous system induces signaling defects resulting in psychiatric symptomatology.

BACKGROUND: Changes in the expression and activity of the AKT oncogene play an important role in psychiatric disease. We present translational data assessing the role of AKT in psychiatric symptoms. METHODS: (1) We assessed the protein activity of an AKT3 mutant harboring a PH domain mutation (Q60H) detected in a patient with schizophrenia, the corresponding AKT1 mutant (Q61H), and wild-type AKT1 and AKT3 transduced in AKT-null mouse fibroblasts and modeled the Q61H mutation onto the crystal structure of the Akt1 PH domain. (2) We analyzed the results of earlier genome-wide association studies to determine the distribution of schizophrenia-associated single-nucleotide polymorphisms (SNPs) in the AKT3 gene. (3) We analyzed the psychiatric adverse events (AEs) of patients treated with M2698 (p70S6K/AKT1/AKT3 inhibitor) and with other PI3K/AKT/mTOR pathway inhibitors. RESULTS: (1) Proteins encoded by AKT3 (AKT3Q60H) and AKT1 (AKT1Q61H) mutants had lower kinase activity than those encoded by wild-type AKT3 and AKT1, respectively. Molecular modeling of the AKT1-Q61H mutant suggested conformational changes that may reduce the binding of D3-phosphorylated phosphoinositides to the PH domain. (2) We identified multiple SNPs in the AKT3 gene that were strongly associated with schizophrenia (p < 0.5 × 10-8). (3) Psychiatric AEs, mostly insomnia, anxiety, and depression, were noted in 29% of patients treated with M2698. In randomized studies, their incidence was higher in PI3K/AKT/mTOR inhibitor arms compared with placebo arms. All psychiatric AEs were reversible. CONCLUSIONS: Our data elucidate the incidence and mechanisms of psychiatric AEs in patients treated with PI3K/AKT/mTOR inhibitors and emphasize the need for careful monitoring.

The Societal Readiness Thinking Tool: A Practical Resource for Maturing the Societal Readiness of Research Projects.

In this paper, we introduce the Societal Readiness (SR) Thinking Tool to aid researchers and innovators in developing research projects with greater responsiveness to societal values, needs, and expectations. The need for societally-focused approaches to research and innovation-complementary to Technology Readiness (TR) frameworks-is presented. Insights from responsible research and innovation (RRI) concepts and practice, organized across critical stages of project-life cycles are discussed with reference to the development of the SR Thinking Tool. The tool is designed to complement not only shortfalls in TR approaches, but also improve upon other efforts to integrate RRI, sustainability, and design thinking in research and innovation cycles. Operationalization and early-stage user tests of the Tool are reported, along with discussion of potential future iterations and applications.

Chemical Genetic Screen in Drosophila Germline Uncovers Small Molecule Drugs That Sensitize Stem Cells to Insult-Induced Apoptosis.

Cancer stem cells, in contrast to their more differentiated daughter cells, can endure genotoxic insults, escape apoptosis, and cause tumor recurrence. Understanding how normal adult stem cells survive and go to quiescence may help identify druggable pathways that cancer stem cells have co-opted. In this study, we utilize a genetically tractable model for stem cell survival in the Drosophila gonad to screen drug candidates and probe chemical-genetic interactions. Our study employs three levels of small molecule screening: (1) a medium-throughput primary screen in male germline stem cells (GSCs), (2) a secondary screen with irradiation and protein-constrained food in female GSCs, and (3) a tertiary screen in breast cancer organoids in vitro. Herein, we uncover a series of small molecule drug candidates that may sensitize cancer stem cells to apoptosis. Further, we have assessed these small molecules for chemical-genetic interactions in the germline and identified the NF-κB pathway as an essential and druggable pathway in GSC quiescence and viability. Our study demonstrates the power of the Drosophila stem cell niche as a model system for targeted drug discovery.

Pd(II)-Catalyzed Direct Dehydrogenative Mono- and Diolefination of Selenophenes.

Pd(II)-catalyzed dehydrogenative Heck olefination of selenophenes with a broad olefinic substrate scope and high functional group tolerance is demonstrated. Carbonyl-substituted and phenyl-substituted olefins with electron-donating (D) and electron-accepting (A) groups can be regioselectively installed at C2 of the selenophene. The 2-olefinated selenophenes can subsequently undergo a second oxidative olefination to rapidly produce a new class of symmetrical D-π-D or unsymmetrical D-π-A 2,5-diolefinated selenophene materials.

A tripartite cooperative mechanism confers resistance of the protein kinase A catalytic subunit to dephosphorylation.

Phosphorylation of specific residues in the activation loops of AGC kinase group (protein kinase A, G, and C families) is required for activity of most of these kinases, including the catalytic subunit of PKA (PKAc). Although many phosphorylated AGC kinases are sensitive to phosphatase-mediated dephosphorylation, the PKAc activation loop uniquely resists dephosphorylation, rendering it "constitutively" phosphorylated in cells. Previous biophysical experiments and structural modeling have suggested that the N-terminal myristoylation signal and the C-terminal FXXF motif in PKAc regulate its thermal stability and catalysis. Here, using site-directed mutagenesis, molecular modeling, and in cell-free and cell-based systems, we demonstrate that substitutions of either the PKAc myristoylation signal or the FXXF motif only modestly reduce phosphorylation and fail to affect PKAc function in cells. However, we observed that these two sites cooperate with an N-terminal FXXW motif to cooperatively establish phosphatase resistance of PKAc while not affecting kinase-dependent phosphorylation of the activation loop. We noted that this tripartite cooperative mechanism of phosphatase resistance is functionally relevant, as demonstrated by changes in morphology, adhesion, and migration of human airway smooth muscle cells transfected with PKAc variants containing amino acid substitutions in these three sites. These findings establish that three allosteric sites located at the PKAc N and C termini coordinately regulate the phosphatase sensitivity of this enzyme. This cooperative mechanism of phosphatase resistance of AGC kinase opens new perspectives toward therapeutic manipulation of kinase signaling in disease.

Regulation of macrophage migration in ischemic mouse hearts via an AKT2/NBA1/SPK1 pathway.

The role of the AKT2/NBA1/SPK1 signaling cascade in macrophage migration regulation and post-ischemic cardiac remodeling was investigated. We determined that the AKT2/NBA1/SPK1 signaling cascade regulated macrophage migration. A novel role for NBA1 in macrophage migration was discovered. Elevated AKT2 phosphorylation, NBA1, SPK1 (along with phosphorylated SPK1) levels, macrophage recruitment, apoptosis, and fibrosis were found within the infarct area. Atorvastatin had a beneficial effect on cardiac remodeling following myocardial infarction by inhibiting AKT2/NBA1/SPK1-mediated macrophage recruitment, apoptosis, and collagen deposition while increasing angiogenesis in the infarct area. Atorvastatin-related protection of cardiac remodeling following myocardial infarction was abolished in SPK1-KO mice. The AKT2/NAB1/SPK1 pathway is a novel regulating factor of macrophage migration and cardiac remodeling after myocardial infarction.

Synchronously Tailoring Strain Sensitivity and Electrical Stability of Silicone Elastomer Composites by the Synergistic Effect of a Dual Conductive Network.

The use of conductive polymer composites (CPCs) as strain sensors has been widely investigated. A wide range of strain sensitivities and high repeatability are vital for different applications of CPCs. In this study, the relations of the conductive filler network and the strain-sensing behavior and electrical stability under fatigue cycles were studied systematically for the first time based on the conductive polymethylvinylsiloxane (PMVS) composites filled with both carbon nanotubes arrays (CNTAs) and carbon black (CB). It was proved that the composites could be fabricated with large strain-sensing capability and a wide range of strain sensitivities by controlling the volume ratio of CNTA/CB and their amounts. Additionally, the CNTA/CB/PMVS composite with 3 vol % content of fillers showed high sensitivity (GF is 10 at 60% strain), high repeatability (the relative standard deviation (RSD) of the max R/R0 value is 3.58%), and electrical stability under fatigue cycles (value range of R/R0 is 1.62 to 1.82) at the same time due to the synergistic effects of the dual conductive network of CNTAs and CB. This could not be achieved by relying on a single CNTA or CB conductive network. This study may provide guidance for the preparation of high performance CPCs for applications in strain sensors.

Data for effects of lanthanum complex on the thermo-oxidative aging of natural rubber.

Novel mixed antioxidants composed of antioxidant IPPD and lanthanum (La) complex were added as a filler to form natural rubber (NR) composites. By mechanical testing, Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR) and thermogravimetric analysis (TGA), a string of data, including the mechanical properties, the variation of internal groups and the thermal and thermo-oxidative decompositions of NR, was presented in this data article. The data accompanying its research article [1] studied the thermo-oxidative aging properties of NR in detail. The density function theoretical (DFT) calculations were also used as an assistant to study the thermo-oxidative aging mechanism of NR. The data revealed that this new rare-earth antioxidant could indeed enhance the thermo-oxidative aging resistance of NR, which is associated with its different function mechanism from that of the pure antioxidant IPPD.

Akt kinase C-terminal modifications control activation loop dephosphorylation and enhance insulin response.

The Akt protein kinase, also known as protein kinase B, plays key roles in insulin receptor signalling and regulates cell growth, survival and metabolism. Recently, we described a mechanism to enhance Akt phosphorylation that restricts access of cellular phosphatases to the Akt activation loop (Thr(308) in Akt1 or protein kinase B isoform alpha) in an ATP-dependent manner. In the present paper, we describe a distinct mechanism to control Thr(308) dephosphorylation and thus Akt deactivation that depends on intramolecular interactions of Akt C-terminal sequences with its kinase domain. Modifications of amino acids surrounding the Akt1 C-terminal mTORC2 (mammalian target of rapamycin complex 2) phosphorylation site (Ser(473)) increased phosphatase resistance of the phosphorylated activation loop (pThr(308)) and amplified Akt phosphorylation. Furthermore, the phosphatase-resistant Akt was refractory to ceramide-dependent dephosphorylation and amplified insulin-dependent Thr(308) phosphorylation in a regulated fashion. Collectively, these results suggest that the Akt C-terminal hydrophobic groove is a target for the development of agents that enhance Akt phosphorylation by insulin.

miR-146a targets Fos expression in human cardiac cells.

miR-146a is a microRNA whose transcript levels are induced in the heart upon activation of NF-κB, a transcription factor induced by pro-inflammatory molecules (such as TNF-α) that is strongly related to the pathogenesis of cardiac disorders. The main goal of this study consisted of studying new roles of miR-146a in cardiac pathological processes caused by the pro-inflammatory cytokine TNF-α. Our results demonstrate that miR-146a transcript levels were sharply increased in cardiac ventricular tissue of transgenic mice with specific overexpression of TNF-α in the heart, and also in a cardiomyocyte cell line of human origin (AC16) exposed to TNF-α. Among all the in silico predicted miR-146a target genes, Fos mRNA and protein levels notably decreased after TNF-α treatment or miR-146a overexpression. These changes correlated with a diminution in the DNA-binding activity of AP-1, the Fos-containing transcription factor complex. Interestingly, AP-1 inhibition was accompanied by a reduction in matrix metalloproteinase (MMP)-9 mRNA levels in human cardiac cells. The specific regulation of this MMP by miR-146a was further confirmed at the secretion and enzymatic activity levels, as well as after anti-miR-mediated miR-146a inhibition. The results reported here demonstrate that Fos is a direct target of miR-146a activity and that downregulation of the Fos-AP-1 pathway by miR-146a has the capacity to inhibit MMP-9 activity. Given that MMP-9 is an AP-1 target gene involved in cardiac remodeling, myocardial dysfunction and progression of heart failure, these findings suggest that miR-146a might be a new and promising therapeutic tool for treating cardiac disorders associated with enhanced inflammation in the heart.

Evaluation of a simulation-based workshop on clinical performance for emergency physicians and nurses.

BACKGROUND: Simulation-based medical education has been growing rapidly and becomes one of the most popular teaching methods for improving patient safety and patient care. The Simulation Subcommittee of the Hong Kong College of Emergency Medicine organized an educational program emphasizing the team training, clinical decision-making and communication skills. This study aimed to evaluate the attitude of the participants toward a new training program and the change in the knowledge on clinical performance in emergency physicians and nurses after attending the educational program. METHODS: A course evaluation form was filled in by the participants at the end of the workshop. An assessment of 20 multiple-choice questions with 5 options was administered to the participants before and after the 2-day simulation-based training workshop. RESULTS: A total of 72 doctors and nurses working in the Accident and Emergency Department were enrolled. The average pretest and posttest scores were 12 and 14.3 respectively. The percentage improvement in the mean score of the pretest and posttest was 11.5%. The Chi-square test showed significant improvement in the pretest and posttest score grading (P=0.00). Paired t-test revealed significant difference between the mean scores of the pretest and posttest (P=0.00). CONCLUSIONS: Participants had positive attitude toward this new training program. Significant improvement of the knowledge on clinical performance in healthcare professionals in the Accident and Emergency Department was observed after the participation in this simulation-based educational program.

High performance graphene oxide based rubber composites.

In this paper, graphene oxide/styrene-butadiene rubber (GO/SBR) composites with complete exfoliation of GO sheets were prepared by aqueous-phase mixing of GO colloid with SBR latex and a small loading of butadiene-styrene-vinyl-pyridine rubber (VPR) latex, followed by their co-coagulation. During co-coagulation, VPR not only plays a key role in the prevention of aggregation of GO sheets but also acts as an interface-bridge between GO and SBR. The results demonstrated that the mechanical properties of the GO/SBR composite with 2.0 vol.% GO is comparable with those of the SBR composite reinforced with 13.1 vol.% of carbon black (CB), with a low mass density and a good gas barrier ability to boot. The present work also showed that GO-silica/SBR composite exhibited outstanding wear resistance and low-rolling resistance which make GO-silica/SBR very competitive for the green tire application, opening up enormous opportunities to prepare high performance rubber composites for future engineering applications.

A truncated fragment of Src protein kinase generated by calpain-mediated cleavage is a mediator of neuronal death in excitotoxicity.

Excitotoxicity resulting from overstimulation of glutamate receptors is a major cause of neuronal death in cerebral ischemic stroke. The overstimulated ionotropic glutamate receptors exert their neurotoxic effects in part by overactivation of calpains, which induce neuronal death by catalyzing limited proteolysis of specific cellular proteins. Here, we report that in cultured cortical neurons and in vivo in a rat model of focal ischemic stroke, the tyrosine kinase Src is cleaved by calpains at a site in the N-terminal unique domain. This generates a truncated Src fragment of ~52 kDa, which we localized predominantly to the cytosol. A cell membrane-permeable fusion peptide derived from the unique domain of Src prevents calpain from cleaving Src in neurons and protects against excitotoxic neuronal death. To explore the role of the truncated Src fragment in neuronal death, we expressed a recombinant truncated Src fragment in cultured neurons and examined how it affects neuronal survival. Expression of this fragment, which lacks the myristoylation motif and unique domain, was sufficient to induce neuronal death. Furthermore, inactivation of the prosurvival kinase Akt is a key step in its neurotoxic signaling pathway. Because Src maintains neuronal survival, our results implicate calpain cleavage as a molecular switch converting Src from a promoter of cell survival to a mediator of neuronal death in excitotoxicity. Besides unveiling a new pathological action of Src, our discovery of the neurotoxic action of the truncated Src fragment suggests new therapeutic strategies with the potential to minimize brain damage in ischemic stroke.

Induced overexpression of Na(+)/Ca(2+) exchanger does not aggravate myocardial dysfunction induced by transverse aortic constriction.

BACKGROUND: Alterations in expression and activity of cardiac Na(+)/Ca(2+) exchanger (NCX1) have been implicated in the pathogenesis of heart failure. METHODS AND RESULTS: Using transgenic mice in which expression of rat NCX1 was induced at 5 weeks of age, we performed transverse aortic constriction (TAC) at 8 weeks and examined cardiac and myocyte function at 15-18 weeks after TAC (age 23-26 weeks). TAC induced left ventricular (LV) and myocyte hypertrophy and increased myocardial fibrosis in both wild-type (WT) and NCX1-overexpressed mice. NCX1 and phosphorylated ryanodine receptor expression was increased by TAC, whereas sarco(endo)plasmic reticulum Ca(2+)-ATPase levels were decreased by TAC. Action potential duration was prolonged by TAC, but to a greater extent in NCX1 myocytes. Na(+)/Ca(2+) exchange current was similar between WT-TAC and WT-sham myocytes, but was higher in NCX1-TAC myocytes. Both myocyte contraction and [Ca(2+)](i) transient amplitudes were reduced in WT-TAC myocytes, but restored to WT-sham levels in NCX1-TAC myocytes. Despite improvement in single myocyte contractility and Ca(2+) dynamics, induced NCX1 overexpression in TAC animals did not ameliorate LV hypertrophy, increase ejection fraction, or enhance inotropic (maximal first derivative of LV pressure rise, +dP/dt) responses to isoproterenol. CONCLUSIONS: In pressure-overload hypertrophy, induced overexpression of NCX1 corrected myocyte contractile and [Ca(2+)](i) transient abnormalities but did not aggravate or improve myocardial dysfunction.

Coordinated regulation of cardiac Na(+)/Ca (2+) exchanger and Na (+)-K (+)-ATPase by phospholemman (FXYD1).

Phospholemman (PLM) is the founding member of the FXYD family of regulators of ion transport. PLM is a 72-amino acid protein consisting of the signature PFXYD motif in the extracellular N terminus, a single transmembrane (TM) domain, and a C-terminal cytoplasmic tail containing three phosphorylation sites. In the heart, PLM co-localizes and co-immunoprecipitates with Na(+)-K(+)-ATPase, Na(+)/Ca(2+) exchanger, and L-type Ca(2+) channel. The TM domain of PLM interacts with TM9 of the α-subunit of Na(+)-K(+)-ATPase, while its cytoplasmic tail interacts with two small regions (spanning residues 248-252 and 300-304) of the proximal intracellular loop of Na(+)/Ca(2+) exchanger. Under stress, catecholamine stimulation phosphorylates PLM at serine(68), resulting in relief of inhibition of Na(+)-K(+)-ATPase by decreasing K(m) for Na(+) and increasing V(max), and simultaneous inhibition of Na(+)/Ca(2+) exchanger. Enhanced Na(+)-K(+)-ATPase activity lowers intracellular Na(+), thereby minimizing Ca(2+) overload and risks of arrhythmias. Inhibition of Na(+)/Ca(2+) exchanger reduces Ca(2+) efflux, thereby preserving contractility. Thus, the coordinated actions of PLM during stress serve to minimize arrhythmogenesis and maintain inotropy. In acute cardiac ischemia and chronic heart failure, either expression or phosphorylation of PLM or both are altered. PLM regulates important ion transporters in the heart and offers a tempting target for development of drugs to treat heart failure.

Cardioprotection of controlled and cardiac-specific over-expression of A(2A)-adenosine receptor in the pressure overload.

Adenosine binds to three G protein-coupled receptors (R) located on the cardiomyocyte (A(1)-R, A(2A)-R and A(3)-R) and provides cardiac protection during both ischemic and load-induced stress. While the role of adenosine receptor-subtypes has been well defined in the setting of ischemia-reperfusion, far less is known regarding their roles in protecting the heart during other forms of cardiac stress. Because of its ability to increase cardiac contractility and heart rate, we hypothesized that enhanced signaling through A(2A)-R would protect the heart during the stress of transverse aortic constriction (TAC). Using a cardiac-specific and inducible promoter, we selectively over-expressed A(2A)-R in FVB mice. Echocardiograms were obtained at baseline, 2, 4, 8, 12, 14 weeks and hearts were harvested at 14 weeks, when WT mice developed a significant decrease in cardiac function, an increase in end systolic and diastolic dimensions, a higher heart weight to body weight ratio (HW/BW), and marked fibrosis when compared with sham-operated WT. More importantly, these changes were significantly attenuated by over expression of the A(2A)-R. Furthermore, WT mice also demonstrated marked increases in the hypertrophic genes ß-myosin heavy chain (ß-MHC), and atrial natriuretic factor (ANF)--changes that are mediated by activation of the transcription factor GATA-4. Levels of the mRNAs encoding ß-MHC, ANP, and GATA-4 were significantly lower in myocardium from A(2A)-R TG mice after TAC when compared with WT and sham-operated controls. In addition, three inflammatory factors genes encoding cysteine dioxygenase, complement component 3, and serine peptidase inhibitor, member 3N, were enhanced in WT TAC mice, but their expression was suppressed in A(2A)-R TG mice. A(2A)-R over-expression is protective against pressure-induced heart failure secondary to TAC. These cardioprotective effects are associated with attenuation of GATA-4 expression and inflammatory factors. The A(2A)-R may provide a novel new target for pharmacologic therapy in patients with cardiovascular disease.