Pencil-paper on-skin electronics.

Pencils and papers are ubiquitous in our society and have been widely used for writing and drawing, because they are easy to use, low-cost, widely accessible, and disposable. However, their applications in emerging skin-interfaced health monitoring and interventions are still not well explored. Herein, we report a variety of pencil-paper-based on-skin electronic devices, including biophysical (temperature, biopotential) sensors, sweat biochemical (pH, uric acid, glucose) sensors, thermal stimulators, and humidity energy harvesters. Among these devices, pencil-drawn graphite patterns (or combined with other compounds) serve as conductive traces and sensing electrodes, and office-copy papers work as flexible supporting substrates. The enabled devices can perform real-time, continuous, and high-fidelity monitoring of a range of vital biophysical and biochemical signals from human bodies, including skin temperatures, electrocardiograms, electromyograms, alpha, beta, and theta rhythms, instantaneous heart rates, respiratory rates, and sweat pH, uric acid, and glucose, as well as deliver programmed thermal stimulations. Notably, the qualities of recorded signals are comparable to those measured with conventional methods. Moreover, humidity energy harvesters are prepared by creating a gradient distribution of oxygen-containing groups on office-copy papers between pencil-drawn electrodes. One single-unit device (0.87 cm2) can generate a sustained voltage of up to 480 mV for over 2 h from ambient humidity. Furthermore, a self-powered on-skin iontophoretic transdermal drug-delivery system is developed as an on-skin chemical intervention example. In addition, pencil-paper-based antennas, two-dimensional (2D) and three-dimensional (3D) circuits with light-emitting diodes (LEDs) and batteries, reconfigurable assembly and biodegradable electronics (based on water-soluble papers) are explored.

Multiscale porous elastomer substrates for multifunctional on-skin electronics with passive-cooling capabilities.

In addition to mechanical compliance, achieving the full potential of on-skin electronics needs the introduction of other features. For example, substantial progress has been achieved in creating biodegradable, self-healing, or breathable, on-skin electronics. However, the research of making on-skin electronics with passive-cooling capabilities, which can reduce energy consumption and improve user comfort, is still rare. Herein, we report the development of multifunctional on-skin electronics, which can passively cool human bodies without needing any energy consumption. This property is inherited from multiscale porous polystyrene-block-poly(ethylene-ran-butylene)-block-polystyrene (SEBS) supporting substrates. The multiscale pores of SEBS substrates, with characteristic sizes ranging from around 0.2 to 7 µm, can effectively backscatter sunlight to minimize heat absorption but are too small to reflect human-body midinfrared radiation to retain heat dissipation, thereby delivering around 6 °C cooling effects under a solar intensity of 840 W⋅m-2 Other desired properties, rooted in multiscale porous SEBS substrates, include high breathability and outstanding waterproofing. The proof-of-concept bioelectronic devices include electrophysiological sensors, temperature sensors, hydration sensors, pressure sensors, and electrical stimulators, which are made via spray printing of silver nanowires on multiscale porous SEBS substrates. The devices show comparable electrical performances with conventional, rigid, nonporous ones. Also, their applications in cuffless blood pressure measurement, interactive virtual reality, and human-machine interface are demonstrated. Notably, the enabled on-skin devices are dissolvable in several organic solvents and can be recycled to reduce electronic waste and manufacturing cost. Such on-skin electronics can serve as the basis for future multifunctional smart textiles with passive-cooling functionalities.

Implantable Aptamer-Graphene Microtransistors for Real-Time Monitoring of Neurochemical Release in Vivo.

The real-time monitoring of neurochemical release in vivo plays a critical role in understanding the biochemical process of the complex nervous system. Current technologies for such applications, including microdialysis and fast-scan cyclic voltammetry, suffer from limited spatiotemporal resolution or poor selectivity. Here, we report a soft implantable aptamer-graphene microtransistor probe for real-time monitoring of neurochemical release. As a demonstration, we show the monitoring of dopamine with nearly cellular-scale spatial resolution, high selectivity (dopamine sensor >19-fold over norepinephrine), and picomolar sensitivity, simultaneously. Systematic benchtop evaluations, ex vivo experiments, and in vivo studies in mice models highlight the key features and demonstrate the capability of capturing the dopamine release dynamics evoked by pharmacological stimulation, suggesting the potential applications in basic neuroscience studies and studying neurological disease-related processes. The developed system can be easily adapted for monitoring other neurochemicals and drugs by simply replacing the aptamers functionalized on the graphene microtransistors.

Glial Cell Line-Derived Neurotrophic Factor and Focal Ischemic Stroke.

Focal ischemic stroke (FIS) is a leading cause of human debilitation and death. Following the onset of a FIS, the brain experiences a series of spatiotemporal changes which are exemplified in different pathological processes. One prominent feature of FIS is the development of reactive astrogliosis and glial scar formation in the peri-infarct region (PIR). During the subacute phase, astrocytes in PIR are activated, referred to as reactive astrocytes (RAs), exhibit changes in morphology (hypotrophy), show an increased proliferation capacity, and altered gene expression profile, a phenomenon known as reactive astrogliosis. Subsequently, the morphology of RAs remains stable, and proliferation starts to decline together with the formation of glial scars. Reactive astrogliosis and glial scar formation eventually cause substantial tissue remodeling and changes in permanent structure around the PIR. Glial cell line-derived neurotrophic factor (GDNF) was originally isolated from a rat glioma cell-line and regarded as a potent survival neurotrophic factor. Under normal conditions, GDNF is expressed in neurons but is upregulated in RAs after FIS. This review briefly describes properties of GDNF, its receptor-mediated signaling pathways, as well as recent studies regarding the role of RAs-derived GDNF in neuronal protection and brain recovery. These results provide evidence suggesting an important role of RA-derived GDNF in intrinsic brain repair and recovery after FIS, and thus targeting GDNF in RAs may be effective for stroke therapy.

GLAST-CreERT2 mediated deletion of GDNF increases brain damage and exacerbates long-term stroke outcomes after focal ischemic stroke in mouse model.

Focal ischemic stroke (FIS) is a leading cause of human death. Glial scar formation largely caused by reactive astrogliosis in peri-infarct region (PIR) is the hallmark of FIS. Glial cell-derived neurotrophic factor (GDNF) was originally isolated from a rat glioma cell-line supernatant and is a potent survival neurotrophic factor. Here, using CreERT2 -LoxP recombination technology, we generated inducible and astrocyte-specific GDNF conditional knockout (cKO), that is, GLAST-GDNF-/- cKO mice to investigate the effect of reactive astrocytes (RAs)-derived GDNF on neuronal death, brain damage, oxidative stress and motor function recovery after photothrombosis (PT)-induced FIS. Under non-ischemic conditions, we found that adult GLAST-GDNF-/- cKO mice exhibited significant lower numbers of Brdu+, Ki67+ cells, and DCX+ cells in the dentate gyrus (DG) in hippocampus than GDNF floxed (GDNFf/f ) control (Ctrl) mice, indicating endogenous astrocytic GDNF can promote adult neurogenesis. Under ischemic conditions, GLAST-GDNF-/- cKO mice had a significant increase in infarct volume, hippocampal damage and FJB+ degenerating neurons after PT as compared with the Ctrl mice. GLAST-GDNF-/- cKO mice also had lower densities of Brdu+ and Ki67+ cells in the PIR and exhibited larger behavioral deficits than the Ctrl mice. Mechanistically, GDNF deficiency in astrocytes increased oxidative stress through the downregulation of glucose-6-phosphate dehydrogenase (G6PD) in RAs. In summary, our study indicates that RAs-derived endogenous GDNF plays important roles in reducing brain damage and promoting brain recovery after FIS through neural regeneration and suggests that promoting anti-oxidant mechanism in RAs is a potential strategy in stroke therapy.

Subcellular NAMPT-mediated NAD+ salvage pathways and their roles in bioenergetics and neuronal protection after ischemic injury.

NAD+ is a cofactor required for glycolysis, tricarboxylic acid cycle, and complex I enzymatic reaction. In mammalian cells, NAD+ is predominantly synthesized through the salvage pathway, where nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme. Previously, we demonstrated that NAMPT exerts a neuroprotective effect in ischemia through the suppression of mitochondrial dysfunction. Mammalian cells maintain distinct NAD+ pools in the cytosol, mitochondria, and nuclei. However, it is unknown whether mitochondria have an intact machinery for NAD+ salvage, and if so, whether it plays a dominant role in bioenergetics, mitochondrial function, and neuronal protection after ischemia. Here, using mouse primary cortical neuron and cortical tissue preparations, and multiple technologies including cytosolic and mitochondrial subfractionation, viral over-expression of transgenes, molecular biology, and confocal microscopy, we provided compelling evidence that neuronal mitochondria possess an intact machinery of NAMPT-mediated NAD+ salvage pathway, and that NAMPT and nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3) are localized in the mitochondrial matrix. By knocking down NMNAT1-3 and NAMPT with siRNA, we found that NMNAT3 has a larger effect on basal and ATP production-related mitochondrial respiration than NMNAT1-2 in primary cultured neurons, while NMNAT1-2 have a larger effect on glycolytic flux than NMNAT3. Using an oxygen glucose deprivation model, we found that mitochondrial, cytoplasmic, and non-subcellular compartmental over-expressions of NAMPT have a comparable effect on neuronal protection and suppression of apoptosis-inducing factor translocation. The current study provides novel insights into the roles of subcellular compartmental NAD+ salvage pathways in NAD+ homeostasis, bioenergetics, and neuronal protection in ischemic conditions.

Reactive astrocytes and therapeutic potential in focal ischemic stroke.

Astrocytes are specialized and the most abundant cell type in the central nervous system (CNS). They play important roles in the physiology of the brain. Astrocytes are also critically involved in many CNS disorders including focal ischemic stroke, the leading cause of brain injury and death in patients. One of the prominent pathological features of a focal ischemic stroke is reactive astrogliosis and glial scar formation. Reactive astrogliosis is accompanied with changes in morphology, proliferation, and gene expression in the reactive astrocytes. This study provides an overview of the most recent advances in astrocytic Ca(2+) signaling, spatial, and temporal dynamics of the morphology and proliferation of reactive astrocytes as well as signaling pathways involved in the reactive astrogliosis after ischemic stroke based on results from experimental studies performed in various animal models. This review also discusses the therapeutic potential of reactive astrocytes in focal ischemic stroke. As reactive astrocytes exhibit high plasticity, we suggest that modulation of local reactive astrocytes is a promising strategy for cell-based stroke therapy.

Histological, cellular and behavioral assessments of stroke outcomes after photothrombosis-induced ischemia in adult mice.

BACKGROUND: Following the onset of focal ischemic stroke, the brain experiences a series of alterations including infarct evolvement, cellular proliferation in the penumbra, and behavioral deficits. However, systematic study on the temporal and spatial dependence of these alterations has not been provided. RESULTS: Using multiple approaches, we assessed stroke outcomes by measuring brain injury, dynamic cellular and glial proliferation, and functional deficits at different times up to two weeks after photothrombosis (PT)-induced ischemic stroke in adult mice. Results from magnetic resonance imaging (MRI) and Nissl staining showed a maximal infarction, and brain edema and swelling 1-3 days after PT. The rate of Bromodeoxyuridine (Brdu)-labeled proliferating cell generation is spatiotemporal dependent in the penumbra, with the highest rate in post ischemic days 3-4, and higher rate of proliferation in the region immediate to the ischemic core than in the distant region. Similar time-dependent generation of proliferating GFAP+ astrocytes and Iba1+ microglia/macrophage were observed in the penumbra. Using behavioral tests, we showed that PT resulted in the largest functional deficits during post ischemic days 2-4. CONCLUSION: Our study demonstrated that first a few days is a critical period that causes brain expansion, cellular proliferation and behavioral deficits in photothrombosis-induced ischemic model, and proliferating astrocytes only have a small contribution to the pools of proliferating cells and reactive astrocytes.

The effects of NAD+ on apoptotic neuronal death and mitochondrial biogenesis and function after glutamate excitotoxicity.

NAD+ is an essential co-enzyme for cellular energy metabolism and is also involved as a substrate for many cellular enzymatic reactions. It has been shown that NAD+ has a beneficial effect on neuronal survival and brain injury in in vitro and in vivo ischemic models. However, the effect of NAD+ on mitochondrial biogenesis and function in ischemia has not been well investigated. In the present study, we used an in vitro glutamate excitotoxicity model of primary cultured cortical neurons to study the effect of NAD+ on apoptotic neuronal death and mitochondrial biogenesis and function. Our results show that supplementation of NAD+ could effectively reduce apoptotic neuronal death, and apoptotic inducing factor translocation after neurons were challenged with excitotoxic glutamate stimulation. Using different approaches including confocal imaging, mitochondrial DNA measurement and Western blot analysis of PGC-1 and NRF-1, we also found that NAD+ could significantly attenuate glutamate-induced mitochondrial fragmentation and the impairment of mitochondrial biogenesis. Furthermore, NAD+ treatment effectively inhibited mitochondrial membrane potential depolarization and NADH redistribution after excitotoxic glutamate stimulation. Taken together, our results demonstrated that NAD+ is capable of inhibiting apoptotic neuronal death after glutamate excitotoxicity via preserving mitochondrial biogenesis and integrity. Our findings provide insights into potential neuroprotective strategies in ischemic stroke.

Dietary NMN supplementation enhances motor and NMJ function in ALS.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease that causes the degeneration of motor neurons in the motor cortex and spinal cord. Patients with ALS experience muscle weakness and atrophy in the limbs which eventually leads to paralysis and death. NAD+ is critical for energy metabolism, such as glycolysis and oxidative phosphorylation, but is also involved in non-metabolic cellular reactions. In the current study, we determined whether the supplementation of nicotinamide mononucleotide (NMN), an NAD+ precursor, in the diet had beneficial impacts on disease progression using a SOD1G93A mouse model of ALS. We found that the ALS mice fed with an NMN-supplemented diet (ALS+NMN mice) had modestly extended lifespan and exhibited delayed motor dysfunction. Using electrophysiology, we studied the effect of NMN on synaptic transmission at neuromuscular junctions (NMJs) in symptomatic of ALS mice (18 weeks old). ALS+NMN mice had larger end-plate potential (EPP) amplitudes and maintained better responses than ALS mice, and also had restored EPP facilitation. While quantal content was not affected by NMN, miniature EPP (mEPP) amplitude and frequency were elevated in ALS+NMN mice. NMN supplementation in diet also improved NMJ morphology, innervation, mitochondrial structure, and reduced reactive astrogliosis in the ventral horn of the lumbar spinal cord. Overall, our results indicate that dietary consumption of NMN can slow motor impairment, enhance NMJ function and improve healthspan of ALS mice.

Pre-B-cell colony-enhancing factor exerts a neuronal protection through its enzymatic activity and the reduction of mitochondrial dysfunction in in vitro ischemic models.

Pre-B-cell colony-enhancing factor (PBEF) is known as a rate-limiting enzyme that converts nicotinamide (NAM) to NMN in the salvage pathway of mammalian NAD⁺ biosynthesis. Previously we found PBEF is exclusively expressed in neurons in the mouse brain; heterozygous PBEF knockout (Pbef⁺/⁻) mice have larger ischemic lesion than wild type mice in photothrombosis-induced ischemia. For the mechanistic study of neuronal protective role of PBEF, we used in vitro oxygen-glucose deprivation (OGD) and glutamate excitotoxicity models of primary cultured neurons in current study. Our results showed that the treatments of neurons with NAM and NAD⁺, the substrate and downstream product of PBEF, respectively, significantly reduced neuronal death after OGD and glutamate excitotoxicity, while treatment of neurons treated with FK866, a PBEF inhibitor, increased neuronal death after OGD. Furthermore, over-expression of human PBEF reduced glutamate excitotoxicity, while over-expression of human PBEF mutants (i.e. H247A and H247E) without enzymatic activity had no effect on neuronal death. We further tested the effect of PBEF on mitochondrial function and biogenesis. Our results show that addition of NAD⁺ and NAM increased mitochondrial biogenesis in neurons after OGD. Over-expression of PBEF in neurons reduced mitochondrial membrane potential depolarization following glutamate stimulation, while over-expression of H247A and H247E did not affect mitochondrial membrane potential depolarization. We conclude that PBEF has a neuroprotective effect in ischemia through its enzymatic activity for NAD⁺ production that can ameliorate mitochondrial dysfunction.

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs).

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of mitochondrial Ca2+ contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Here we present a cell-type specific and mitochondria targeting molecular approach for mitochondrial Ca2+ imaging in astrocytes and neurons in vitro and in vivo. We constructed DNA plasmids encoding mitochondria-targeting genetically encoded Ca2+ indicators (GECIs) GCaMP5G or GCaMP6s (GCaMP5G/6s) with astrocyte- and neuron-specific promoters gfaABC1D and CaMKII and mitochondria-targeting sequence (mito-). For in vitro mitochondrial Ca2+ imaging, the plasmids were transfected in cultured astrocytes and neurons to express GCaMP5G/6s. For in vivo mitochondrial Ca2+ imaging, adeno-associated viral vectors (AAVs) were prepared and injected into the mouse brains to express GCaMP5G/6s in mitochondria in astrocytes and neurons. Our approach provides a useful means to image mitochondrial Ca2+ dynamics in astrocytes and neurons to study the relationship between cytosolic and mitochondrial Ca2+ signaling, as well as astrocyte-neuron interactions.

Acid-Sensing Ion Channels in Glial Cells.

Acid-sensing ion channels (ASICs) are proton-gated cation channels and key mediators of responses to neuronal injury. ASICs exhibit unique patterns of distribution in the brain, with high expression in neurons and low expression in glial cells. While there has been a lot of focus on ASIC in neurons, less is known about the roles of ASICs in glial cells. ASIC1a is expressed in astrocytes and might contribute to synaptic transmission and long-term potentiation. In oligodendrocytes, constitutive activation of ASIC1a participates in demyelinating diseases. ASIC1a, ASIC2a, and ASIC3, found in microglial cells, could mediate the inflammatory response. Under pathological conditions, ASIC dysregulation in glial cells can contribute to disease states. For example, activation of astrocytic ASIC1a may worsen neurodegeneration and glioma staging, activation of microglial ASIC1a and ASIC2a may perpetuate ischemia and inflammation, while oligodendrocytic ASIC1a might be involved in multiple sclerosis. This review concentrates on the unique ASIC components in each of the glial cells and integrates these glial-specific ASICs with their physiological and pathological conditions. Such knowledge provides promising evidence for targeting of ASICs in individual glial cells as a therapeutic strategy for a diverse range of conditions.

Wireless, battery-free push-pull microsystem for membrane-free neurochemical sampling in freely moving animals.

Extensive studies in both animals and humans have demonstrated that high molecular weight neurochemicals, such as neuropeptides and other polypeptide neurochemicals, play critical roles in various neurological disorders. Despite many attempts, existing methods are constrained by detecting neuropeptide release in small animal models during behavior tasks, which leaves the molecular mechanisms underlying many neurological and psychological disorders unresolved. Here, we report a wireless, programmable push-pull microsystem for membrane-free neurochemical sampling with cellular spatial resolution in freely moving animals. In vitro studies demonstrate the sampling of various neurochemicals with high recovery (>80%). Open-field tests reveal that the device implantation does not affect the natural behavior of mice. The probe successfully captures the pharmacologically evoked release of neuropeptide Y in freely moving mice. This wireless push-pull microsystem creates opportunities for neuroscientists to understand where, when, and how the release of neuropeptides modulates diverse behavioral outputs of the brain.

NAD+ Metabolism and Diseases with Motor Dysfunction.

Neurodegenerative diseases result in the progressive deterioration of the nervous system, with motor and cognitive impairments being the two most observable problems. Motor dysfunction could be caused by motor neuron diseases (MNDs) characterized by the loss of motor neurons, such as amyotrophic lateral sclerosis and Charcot-Marie-Tooth disease, or other neurodegenerative diseases with the destruction of brain areas that affect movement, such as Parkinson's disease and Huntington's disease. Nicotinamide adenine dinucleotide (NAD+) is one of the most abundant metabolites in the human body and is involved with numerous cellular processes, including energy metabolism, circadian clock, and DNA repair. NAD+ can be reversibly oxidized-reduced or directly consumed by NAD+-dependent proteins. NAD+ is synthesized in cells via three different paths: the de novo, Preiss-Handler, or NAD+ salvage pathways, with the salvage pathway being the primary producer of NAD+ in mammalian cells. NAD+ metabolism is being investigated for a role in the development of neurodegenerative diseases. In this review, we discuss cellular NAD+ homeostasis, looking at NAD+ biosynthesis and consumption, with a focus on the NAD+ salvage pathway. Then, we examine the research, including human clinical trials, focused on the involvement of NAD+ in MNDs and other neurodegenerative diseases with motor dysfunction.

Astroglia Abnormalities in Post-stroke Mood Disorders.

Stroke is the leading cause of human death and disability. After a stroke, many patients may have some physical disability, including difficulties in moving, speaking, and seeing, but patients may also exhibit changes in mood manifested by depression, anxiety, and cognitive changes which we call post-stroke mood disorders (PSMDs). Astrocytes are the most diverse and numerous glial cell type in the central nervous system (CNS). They provide structural, nutritional, and metabolic support to neurons and regulate synaptic activity under normal conditions. Astrocytes are also critically involved in focal ischemic stroke (FIS). They undergo many changes after FIS. These changes may affect acute neuronal death and brain damage as well as brain recovery and PSMD in the chronic phase after FIS. Studies using postmortem brain specimens and animal models of FIS suggest that astrocytes/reactive astrocytes are involved in PSMD. This chapter provides an overview of recent advances in the molecular base of astrocyte in PSMD. As astrocytes exhibit high plasticity after FIS, we suggest that targeting local astrocytes may be a promising strategy for PSMD therapy.

Metabolomic and transcriptional profiling reveals bioenergetic stress and activation of cell death and inflammatory pathways in vivo after neuronal deletion of NAMPT.

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the NAD+ salvage pathway. Our previous study demonstrated that deletion of NAMPT gene in projection neurons using Thy1-NAMPT-/- conditional knockout (cKO) mice causes neuronal degeneration, muscle atrophy, neuromuscular junction abnormalities, paralysis and eventually death. Here we conducted a combined metabolomic and transcriptional profiling study in vivo in an attempt to further investigate the mechanism of neuronal degeneration at metabolite and mRNA levels after NAMPT deletion. Here using steady-state metabolomics, we demonstrate that deletion of NAMPT causes a significant decrease of NAD+ metabolome and bioenergetics, a buildup of metabolic intermediates upstream of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in glycolysis, and an increase of oxidative stress. RNA-seq shows that NAMPT deletion leads to the increase of mRNA levels of enzymes in NAD metabolism, in particular PARP family of NAD+ consumption enzymes, as well as glycolytic genes Glut1, Hk2 and PFBFK3 before GAPDH. GO, KEGG and GSEA analyses show the activations of apoptosis, inflammation and immune responsive pathways and the inhibition of neuronal/synaptic function in the cKO mice. The current study suggests that increased oxidative stress, apoptosis and neuroinflammation contribute to neurodegeneration and mouse death as a direct consequence of bioenergetic stress after NAMPT deletion.

The effect of NAMPT deletion in projection neurons on the function and structure of neuromuscular junction (NMJ) in mice.

Nicotinamide adenine dinucleotide (NAD+) plays a critical role in energy metabolism and bioenergetic homeostasis. Most NAD+ in mammalian cells is synthesized via the NAD+ salvage pathway, where nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme, converting nicotinamide into nicotinamide mononucleotide (NMN). Using a Thy1-Nampt-/- projection neuron conditional knockout (cKO) mouse, we studied the impact of NAMPT on synaptic vesicle cycling in the neuromuscular junction (NMJ), end-plate structure of NMJs and muscle contractility of semitendinosus muscles. Loss of NAMPT impaired synaptic vesicle endocytosis/exocytosis in the NMJs. The cKO mice also had motor endplates with significantly reduced area and thickness. When the cKO mice were treated with NMN, vesicle endocytosis/exocytosis was improved and endplate morphology was restored. Electrical stimulation induced muscle contraction was significantly impacted in the cKO mice in a frequency dependent manner. The cKO mice were unresponsive to high frequency stimulation (100 Hz), while the NMN-treated cKO mice responded similarly to the control mice. Transmission electron microscopy (TEM) revealed sarcomere misalignment and changes to mitochondrial morphology in the cKO mice, with NMN treatment restoring sarcomere alignment but not mitochondrial morphology. This study demonstrates that neuronal NAMPT is important for pre-/post-synaptic NMJ function, and maintaining skeletal muscular function and structure.

Photothrombosis ischemia stimulates a sustained astrocytic Ca2+ signaling in vivo.

Although there is significant information concerning the consequences of cerebral ischemia on neuronal function, relatively little is known about functional responses of astrocytes, the predominant glial-cell type in the central nervous system. In this study, we asked whether focal ischemia would impact astrocytic Ca(2+) signaling, a characteristic form of excitability in this cell type. In vivo Ca(2+) imaging of cortical astrocytes was performed using two-photon (2-P) microscopy during the acute phase of photothrombosis-induced ischemia initiated by green light illumination of circulating Rose Bengal. Although whisker evoked potentials were reduced by over 90% within minutes of photothrombosis, astrocytes in the ischemic core remained structurally intact for a few hours. In vivo Ca(2+) imaging showed that an increase in transient Ca(2+) signals in astrocytes within 20 min of ischemia. These Ca(2+) signals were synchronized and propagated as waves amongst the glial network. Pharmacological manipulations demonstrated that these Ca(2+) signals were dependent on activation of metabotropic glutamate receptor 5 (mGluR5) and metabotropic gamma-aminobutyric acid receptor (GABA(B)R) but not by P2 purinergic receptor or A1 adenosine receptor. Selective inhibition of Ca(2+) in astrocytes with BAPTA significantly reduced the infarct volume, demonstrating that the enhanced astrocytic Ca(2+) signal contributes to neuronal damage presumably through Ca(2+)-dependent release of glial glutamate. Because astrocytes offer multiple functions in close communication with neurons and vasculature, the ischemia-induced increase in astrocytic Ca(2+) signaling may represent an initial attempt for these cells to communicate with neurons or provide feed back regulation to the vasculature.

Improving aquaporin Z expression in Escherichia coli by fusion partners and subsequent condition optimization.

Aquaporin Z (AqpZ), a typical orthodox aquaporin with six transmembrane domains, was expressed as a fusion protein with TrxA in E. coli in our previous work. In the present study, three fusion partners (DsbA, GST and MBP) were employed to improve the expression level of this channel protein in E. coli. The result showed that, compared with the expression level of TrxA-AqpZ, five- to 40-fold increase in the productivity of AqpZ with fusion proteins was achieved by employing these different fusion partners, and MBP was the most efficient fusion partner to increase the expression level. By using E. coli C43 (DE3)/pMAL-AqpZ, the effects of different expression conditions were investigated systematically to improve the expression level of MBP-AqpZ in E. coli. The high productivity of MBP-AqpZ (200 mg/l) was achieved under optimized conditions. The present work provides a novel approach to improve the expression level of membrane proteins in E. coli.