Coupling enzymatic activity and gating in an ancient TRPM chanzyme and its molecular evolution.

Channel enzymes represent a class of ion channels with enzymatic activity directly or indirectly linked to their channel function. We investigated a TRPM2 chanzyme from choanoflagellates that integrates two seemingly incompatible functions into a single peptide: a channel module activated by ADP-ribose with high open probability and an enzyme module (NUDT9-H domain) consuming ADP-ribose at a remarkably slow rate. Using time-resolved cryogenic-electron microscopy, we captured a complete series of structural snapshots of gating and catalytic cycles, revealing the coupling mechanism between channel gating and enzymatic activity. The slow kinetics of the NUDT9-H enzyme module confers a self-regulatory mechanism: ADPR binding triggers NUDT9-H tetramerization, promoting channel opening, while subsequent hydrolysis reduces local ADPR, inducing channel closure. We further demonstrated how the NUDT9-H domain has evolved from a structurally semi-independent ADP-ribose hydrolase module in early species to a fully integrated component of a gating ring essential for channel activation in advanced species.

Human pannexin 1 channel is not phosphorylated by Src tyrosine kinase at Tyr199 and Tyr309.

Protein phosphorylation is one of the major molecular mechanisms regulating protein activity and function throughout the cell. Pannexin 1 (PANX1) is a large-pore channel permeable to ATP and other cellular metabolites. Its tyrosine phosphorylation and subsequent activation have been found to play critical roles in diverse cellular conditions, including neuronal cell death, acute inflammation, and smooth muscle contraction. Specifically, the non-receptor kinase Src has been reported to phosphorylate Tyr198 and Tyr308 of mouse PANX1 (equivalent to Tyr199 and Tyr309 of human PANX1), resulting in channel opening and ATP release. Although the Src-dependent PANX1 activation mechanism has been widely discussed in the literature, independent validation of the tyrosine phosphorylation of PANX1 has been lacking. Here, we show that commercially available antibodies against the two phosphorylation sites mentioned above-which were used to identify endogenous PANX1 phosphorylation at these two sites-are nonspecific and should not be used to interpret results related to PANX1 phosphorylation. We further provide evidence that neither tyrosine residue is a major phosphorylation site for Src kinase in heterologous expression systems. We call on the field to re-examine the existing paradigm of tyrosine phosphorylation-dependent activation of the PANX1 channel.

Human Pannexin 1 Channel is NOT Phosphorylated by Src Tyrosine Kinase at Tyr199 and Tyr309.

Protein phosphorylation is one of the major molecular mechanisms regulating protein activity and function throughout the cell. Pannexin 1 (PANX1) is a large-pore channel permeable to ATP and other cellular metabolites. Its tyrosine phosphorylation and subsequent activation have been found to play critical roles in diverse cellular conditions, including neuronal cell death, acute inflammation, and smooth muscle contraction. Specifically, the non-receptor kinase Src has been reported to phosphorylate Tyr198 and Tyr308 of mouse PANX1 (equivalent to Tyr199 and Tyr309 of human PANX1), resulting in channel opening and ATP release. Although the Src-dependent PANX1 activation mechanism has been widely discussed in the literature, independent validation of the tyrosine phosphorylation of PANX1 has been lacking. Here, we show that commercially available antibodies against the two phosphorylation sites mentioned above-which were used to identify endogenous PANX1 phosphorylation at these two sites-are nonspecific and should not be used to interpret results related to PANX1 phosphorylation. We further provide evidence that neither tyrosine residue is a major phosphorylation site for Src kinase in heterologous expression systems. We call on the field to re-examine the existing paradigm of tyrosine phosphorylation-dependent activation of the PANX1 channel.

A neural mechanism for learning from delayed postingestive feedback.

Animals learn the value of foods based on their postingestive effects and thereby develop aversions to foods that are toxic1-6 and preferences to those that are nutritious7-14. However, it remains unclear how the brain is able to assign credit to flavors experienced during a meal with postingestive feedback signals that can arise after a substantial delay. Here, we reveal an unexpected role for postingestive reactivation of neural flavor representations in this temporal credit assignment process. To begin, we leverage the fact that mice learn to associate novel15-18, but not familiar, flavors with delayed gastric malaise signals to investigate how the brain represents flavors that support aversive postingestive learning. Surveying cellular resolution brainwide activation patterns reveals that a network of amygdala regions is unique in being preferentially activated by novel flavors across every stage of the learning process: the initial meal, delayed malaise, and memory retrieval. By combining high-density recordings in the amygdala with optogenetic stimulation of genetically defined hindbrain malaise cells, we find that postingestive malaise signals potently and specifically reactivate amygdalar novel flavor representations from a recent meal. The degree of malaise-driven reactivation of individual neurons predicts strengthening of flavor responses upon memory retrieval, leading to stabilization of the population-level representation of the recently consumed flavor. In contrast, meals without postingestive consequences degrade neural flavor representations as flavors become familiar and safe. Thus, our findings demonstrate that interoceptive reactivation of amygdalar flavor representations provides a neural mechanism to resolve the temporal credit assignment problem inherent to postingestive learning.

Cerebellar contributions to a brainwide network for flexible behavior in mice.

The cerebellum regulates nonmotor behavior, but the routes of influence are not well characterized. Here we report a necessary role for the posterior cerebellum in guiding a reversal learning task through a network of diencephalic and neocortical structures, and in flexibility of free behavior. After chemogenetic inhibition of lobule VI vermis or hemispheric crus I Purkinje cells, mice could learn a water Y-maze but were impaired in ability to reverse their initial choice. To map targets of perturbation, we imaged c-Fos activation in cleared whole brains using light-sheet microscopy. Reversal learning activated diencephalic and associative neocortical regions. Distinctive subsets of structures were altered by perturbation of lobule VI (including thalamus and habenula) and crus I (including hypothalamus and prelimbic/orbital cortex), and both perturbations influenced anterior cingulate and infralimbic cortex. To identify functional networks, we used correlated variation in c-Fos activation within each group. Lobule VI inactivation weakened within-thalamus correlations, while crus I inactivation divided neocortical activity into sensorimotor and associative subnetworks. In both groups, high-throughput automated analysis of whole-body movement revealed deficiencies in across-day behavioral habituation to an open-field environment. Taken together, these experiments reveal brainwide systems for cerebellar influence that affect multiple flexible responses.

Cholinergic interneurons mediate cocaine extinction in male mice through plasticity across medium spiny neuron subtypes.

Cholinergic interneurons (ChINs) in the nucleus accumbens (NAc) have been implicated in the extinction of drug associations, as well as related plasticity in medium spiny neurons (MSNs). However, since most previous work relied on artificial manipulations, whether endogenous acetylcholine signaling relates to drug associations is unclear. Moreover, despite great interest in the opposing effects of dopamine on MSN subtypes, whether ChIN-mediated effects vary by MSN subtype is also unclear. Here, we find that high endogenous acetylcholine event frequency correlates with greater extinction of cocaine-context associations across male mice. Additionally, extinction is associated with a weakening of glutamatergic synapses across MSN subtypes. Manipulating ChIN activity bidirectionally controls both the rate of extinction and the associated plasticity at MSNs. Our findings indicate that NAc ChINs mediate drug-context extinction by reducing glutamatergic synaptic strength across MSN subtypes, and that natural variation in acetylcholine signaling may contribute to individual differences in extinction.

Green Synthesis of Laser-Induced Graphene with Copper Oxide Nanoparticles for Deicing Based on Photo-Electrothermal Effect.

Homogenously dispersed Cu oxide nanoparticles on laser-induced graphene (LIG) were fabricated using a simple two-step laser irradiation. This work emphasized the synergetic photo-electrothermal effect in Cu oxide particles embedded in LIG. Our flexible hybrid composites exhibited high mechanical durability and excellent thermal properties. Moreover, the Cu oxide nanoparticles in the carbon matrix of LIG enhanced the light trapping and multiple electron internal scattering for the electrothermal effect. The best conditions for deicing devices were also studied by controlling the amount of Cu solution. The deicing performance of the sample was demonstrated, and the results indicate that the developed method could be a promising strategy for maintaining lightness, efficiency, excellent thermal performance, and eco-friendly 3D processing capabilities.

Laser-Induced Graphene Heater Pad for De-Icing.

The replacement of electro-thermal material in heaters with lighter and easy-to-process materials has been extensively studied. In this study, we demonstrate that laser-induced graphene (LIG) patterns could be a good candidate for the electro-thermal pad. We fabricated LIG heaters with various thermal patterns on the commercial polyimide films according to laser scanning speed using an ultraviolet pulsed laser. We adopted laser direct writing (LDW) to irradiate on the substrates with computer-aided 2D CAD circuit data under ambient conditions. Our highly conductive and flexible heater was investigated by scanning electron microscopy, transmission electron microscopy, Raman spectroscopy, thermogravimetric analysis, X-ray photoelectron spectroscopy, X-ray diffraction, and Brunauer-Emmett-Teller. The influence of laser scanning speed was evaluated for electrical properties, thermal performance, and durability. Our LIG heater showed promising characteristics such as high porosity, light weight, and small thickness. Furthermore, they demonstrated a rapid response time, reaching equilibrium in less than 3 s, and achieved temperatures up to 190 °C using relatively low DC voltages of approximately 10 V. Our LIG heater can be utilized for human wearable thermal pads and ice protection for industrial applications.

Homologous organization of cerebellar pathways to sensory, motor, and associative forebrain.

Cerebellar outputs take polysynaptic routes to reach the rest of the brain, impeding conventional tracing. Here, we quantify pathways between the cerebellum and forebrain by using transsynaptic tracing viruses and a whole-brain analysis pipeline. With retrograde tracing, we find that most descending paths originate from the somatomotor cortex. Anterograde tracing of ascending paths encompasses most thalamic nuclei, especially ventral posteromedial, lateral posterior, mediodorsal, and reticular nuclei. In the neocortex, sensorimotor regions contain the most labeled neurons, but we find higher densities in associative areas, including orbital, anterior cingulate, prelimbic, and infralimbic cortex. Patterns of ascending expression correlate with c-Fos expression after optogenetic inhibition of Purkinje cells. Our results reveal homologous networks linking single areas of the cerebellar cortex to diverse forebrain targets. We conclude that shared areas of the cerebellum are positioned to provide sensory-motor information to regions implicated in both movement and nonmotor function.

Highly Skin-Conformal Laser-Induced Graphene-Based Human Motion Monitoring Sensor.

Bio-compatible strain sensors based on elastomeric conductive polymer composites play pivotal roles in human monitoring devices. However, fabricating highly sensitive and skin-like (flexible and stretchable) strain sensors with broad working range is still an enormous challenge. Herein, we report on a novel fabrication technology for building elastomeric conductive skin-like composite by mixing polymer solutions. Our e-skin substrates were fabricated according to the weight of polydimethylsiloxane (PDMS) and photosensitive polyimide (PSPI) solutions, which could control substrate color. An e-skin and 3-D flexible strain sensor was developed with the formation of laser induced graphene (LIG) on the skin-like substrates. For a one-step process, Laser direct writing (LDW) was employed to construct superior durable LIG/PDMS/PSPI composites with a closed-pore porous structure. Graphene sheets of LIG coated on the closed-porous structure constitute a deformable conductive path. The LIG integrated with the closed-porous structure intensifies the deformation of the conductive network when tensile strain is applied, which enhances the sensitivity. Our sensor can efficiently monitor not only energetic human motions but also subtle oscillation and physiological signals for intelligent sound sensing. The skin-like strain sensor showed a perfect combination of ultrawide sensing range (120% strain), large sensitivity (gauge factor of ~380), short response time (90 ms) and recovery time (140 ms), as well as superior stability. Our sensor has great potential for innovative applications in wearable health-monitoring devices, robot tactile systems, and human-machine interface systems.

In situ synthesis of copper-ruthenium bimetallic nanoparticles on laser-induced graphene as a peroxidase mimic.

A new type of disposable flexible sensor for hydrogen peroxide (H2O2) detection was developed by in situ synthesis of copper-ruthenium bimetallic nanoparticles on a laser-induced graphene surface (Cu-Ru/LIG). The approach produced Cu-Ru/LIG via a solid phase transfer mechanism which loaded the metal precursor onto LIG, followed by laser scribing without demanding chemical vapor deposition or solution-based reactions. Cu-Ru/LIG showed a high electrocatalytic response toward H2O2 reduction at -0.4 V vs. Ag/AgCl. The sensor also showed good selectivity and reproducibility. This method provides an alternative route to easily synthesize various catalysts on conductive substrates for sensor applications.

The Efficacy of Hypotensive Agents on Intraoperative Bleeding and Recovery Following General Anesthesia for Nasal Surgery: A Network Meta-Analysis.

OBJECTIVES: A systematic review of the literature was conducted to evaluate hypotensive agents in terms of their adverse effects and associations with perioperative morbidity in patients undergoing nasal surgery. METHODS: Two authors independently searched databases (Medline, Scopus, and Cochrane databases) up to February 2020 for randomized controlled trials comparing the perioperative administration of a hypotensive agent with a placebo or other agent. The outcomes of interest for this analysis were intraoperative morbidity, operative time, intraoperative bleeding, hypotension, postoperative nausea/vomiting, and postoperative pain. Both a standard pairwise meta-analysis and network meta-analysis were conducted. RESULTS: Our analysis was based on 37 trials. Treatment networks consisting of six interventions (placebo, clonidine, dexmedetomidine, beta-blockers, opioids, and nitroglycerine) were defined for the network meta-analysis. Dexmedetomidine resulted in the greatest differences in intraoperative bleeding (-0.971; 95% confidence interval [CI], -1.161 to -0.781), intraoperative fentanyl administration (-3.683; 95% CI, -4.848 to -2.518), and postoperative pain (-2.065; 95% CI, -3.170 to -0.960) compared with placebo. The greatest difference in operative time compared with placebo was achieved with clonidine (-0.699; 95% CI, -0.977 to -0.421). All other agents also had beneficial effects on the measured outcomes. Dexmedetomidine was less likely than other agents to cause adverse effects. CONCLUSION: This study demonstrated the superiority of the systemic use of dexmedetomidine as a perioperative hypotensive agent compared with the other five tested agents. However, the other agents were also superior to placebo in improving operative time, intraoperative bleeding, and postoperative pain.

Methylene Blue as a Diagnosis and Screening Tool for Oral Cancer and Precancer.

OBJECTIVES: To evaluate the accuracy of methylene blue (MB) for diagnosing oral cancer and precancer. DATA SOURCES: PubMed, Cochrane Database, Embase, Web of Science, SCOPUS, and Google Scholar. REVIEW METHODS: Two authors working independently reviewed 6 databases from their dates of inception until April 2020. Studies exploring oral mucosal disorders as detected by MB were assessed. True-positive, true-negative, false-positive, and false-negative data were extracted for each study. Methodological quality was evaluated with the Quality Assessment of Diagnostic Accuracy Studies tool (v 2). RESULTS: Seven prospective and retrospective studies (N = 493) were included. The diagnostic odds ratio of MB was 20.017 (95% CI, 10.65-37.63, I2 = 23%). The area under the summary receiver operating characteristic curve was 0.699. Sensitivity was 0.903 (95% CI, 0.84-0.94, I2 = 54%), and specificity was 0.68 (95% CI, 0.60-0.75, I2 = 0%). The correlation between the sensitivity and the false-positive rate was -0.17, indicating an absence of heterogeneity. CONCLUSIONS: Regarding diagnostic accuracy, MB had high sensitivity but low specificity, suggesting that it cannot be recommended as a replacement for the currently used standard of a scalpel biopsy with histologic assessment. Instead, it should be used as an adjunct to conventional assessment because of its low toxicity and price.

Laser-Induced Biochar Formation through 355 nm Pulsed Laser Irradiation of Wood, and Application to Eco-Friendly pH Sensors.

Due to the limited availability of agricultural land, pH sensing is becoming more and more important these days to produce efficient agricultural products. Therefore, to fabricate eco-friendly and disposable sensors, the black carbon, which is called biochar, is formed by irradiation of a UV pulsed laser having a wavelength of 355 nm onto wood and applying the resulting material as a pH sensor. The surfaces of three types of wood (beech, cork oak, and ash) were converted to the graphitic structure after UV laser irradiation; their morphologies were investigated. In addition, since the content of lignin, an organic polymer, is different for each wood, optimal laser irradiation conditions (laser fluence) needed to form these woods into pH sensors were considered. Depending on the degree of oil-like material generated after laser irradiation, a disposable pH sensor that can be used from one to three times is fabricated; due to the environmental characteristics of wood and biochar, the sensor shows high availability in that it can be easily discarded after use on agricultural land. After that, it can be used as filter in soil. Our wood-based pH sensor sensitively measures sequential changes from pH 4 to pH 10 and shows a very linear change of △R/R, indicating its potential for use in agriculture.

Fabrication of UV Laser-Induced Porous Graphene Patterns with Nanospheres and Their Optical and Electrical Characteristics.

Many studies have been conducted to fabricate unique structures on flexible substrates and to apply such structures to a variety of fields. However, it is difficult to produce unique structures such as multilayer, nanospheres and porous patterns on a flexible substrate. We present a facile method of nanospheres based on laser-induced porous graphene (LIPG), by using laser-induced plasma (LIP). We fabricated these patterns from commercial polyimide (PI) film, with a 355 nm pulsed laser. For a simple one-step process, we used laser direct writing (LDW), under ambient conditions. We irradiated the PI film at a defocused plane -4 mm away from the focal plane, for high pulse overlap rate. The effect of the laser scanning speed was investigated by FE-SEM, to observe morphological characterization. Moreover, we confirmed the pattern characteristics by optical microscope, Raman spectroscopy and electrical experiments. The results suggested that we could modulate the conductivity and structural color by controlling the laser scanning speed. In this work, when the speed of the laser is 20 mm/s and the fluence is 5.28 mJ/cm2, the structural color is most outstanding. Furthermore, we applied these unique characteristics to various colorful patterns by controlling focal plane.

Urban particulate matter regulates tight junction proteins by inducing oxidative stress via the Akt signal pathway in human nasal epithelial cells.

Recent studies have revealed that increased reactive oxidative stress (ROS) induced by particulate matter (PM) affects tight junction (TJ) functions; however, the molecular mechanisms underlying this effect have not been evaluated fully. Cultured human epithelial cells obtained from inferior turbinate tissues were exposed to an urban PM (UPM) standard reference material (SRM 1648a). Intracellular ROS level and expression of proinflammatory cytokines and TJ proteins were examined. Expression level of phosphorylated (p)-Akt, p38, p65 were compared between exposed and unexposed cells. Cells were pretreated with the ROS scavenger N-acetylcysteine (NAC) or Akt inhibitor MK-2206 before exposure to determine whether the changes in cellular ROS and TJ protein expression could be reversed. Exposure to UPM significantly increased ROS levels and inflammatory cytokine expression levels, and decreased expression of TJ proteins zonula occludins (ZO)-1, occludin, claudin-1, and E-cadherin. UPM exposure increased p-Akt, p-p38, and p65 expression levels, and NAC pretreatment reversed these effects. Akt inhibition decreased UPM-induced ROS formation and p38 and p65 protein phosphorylation, and restored the decreased ZO-1 and E-cadherin expression. Akt inhibition and ROS scavenging may provide targets for maintaining epithelial integrity by restoring decreased TJ protein expression during exposure to UPM.

Epithelial tissue geometry directs emergence of bioelectric field and pattern of proliferation.

Patterns of proliferation are templated by both gradients of mechanical stress as well as by gradients in membrane voltage (Vm), which is defined as the electric potential difference between the cytoplasm and the extracellular medium. Either gradient could regulate the emergence of the other, or they could arise independently and synergistically affect proliferation within a tissue. Here, we examined the relationship between endogenous patterns of mechanical stress and the generation of bioelectric gradients in mammary epithelial tissues. We observed that the mechanical stress gradients in the tissues presaged gradients in both proliferation and depolarization, consistent with previous reports correlating depolarization with proliferation. Furthermore, disrupting the Vm gradient blocked the emergence of patterned proliferation. We found that the bioelectric gradient formed downstream of mechanical stresses within the tissues and depended on connexin-43 (Cx43) hemichannels, which opened preferentially in cells located in regions of high mechanical stress. Activation of Cx43 hemichannels was necessary for nuclear localization of Yap/Taz and induction of proliferation. Together, these results suggest that mechanotransduction triggers the formation of bioelectric gradients across a tissue, which are further translated into transcriptional changes that template patterns of growth.

Direct Fabrication of Ultra-Sensitive Humidity Sensor Based on Hair-Like Laser-Induced Graphene Patterns.

Three-dimensional (3-D) porous graphitic structures have great potential for sensing applications due to their conductive carbon networks and large surface area. In this work, we present a method for facile fabrication of hair-like laser induced graphene (LIG) patterns using a laser scribing system equipped with a 355 nm pulsed laser. The polyimide (PI) film was positioned on a defocused plane and irradiated at a slow scanning speed using a misaligned laser beam. These patterns have the advantages of a large surface area and abundant oxidation groups. We have applied the hair-like LIG patterns to a humidity sensor. The humidity sensor showed good sensitivity characteristics and a large amount of electronic carriers can be stored.

Flexible and Highly Sensitive Strain Sensor Based on Laser-Induced Graphene Pattern Fabricated by 355 nm Pulsed Laser.

A laser-induced-graphene (LIG) pattern fabricated using a 355 nm pulsed laser was applied to a strain sensor. Structural analysis and functional evaluation of the LIG strain sensor were performed by Raman spectroscopy, scanning electron microscopy (SEM) imaging, and electrical-mechanical coupled testing. The electrical characteristics of the sensor with respect to laser fluence and focal length were evaluated. The sensor responded sensitively to small deformations, had a high gauge factor of ~160, and underwent mechanical fracture at 30% tensile strain. In addition, we have applied the LIG sensor, which has high sensitivity, a simple manufacturing process, and good durability, to human finger motion monitoring.

Sound tuning of amygdala plasticity in auditory fear conditioning.

Various auditory tones have been used as conditioned stimuli (CS) for fear conditioning, but researchers have largely neglected the effect that different types of auditory tones may have on fear memory processing. Here, we report that at lateral amygdala (LA) synapses (a storage site for fear memory), conditioning with different types of auditory CSs (2.8 kHz tone, white noise, FM tone) recruits distinct forms of long-term potentiation (LTP) and inserts calcium permeable AMPA receptor (CP-AMPAR) for variable periods. White noise or FM tone conditioning produced brief insertion (<6 hr after conditioning) of CP-AMPARs, whereas 2.8 kHz tone conditioning induced more persistent insertion (≥6 hr). Consistently, conditioned fear to 2.8 kHz tone but not to white noise or FM tones was erased by reconsolidation-update (which depends on the insertion of CP-AMPARs at LA synapses) when it was performed 6 hr after conditioning. Our data suggest that conditioning with different auditory CSs recruits distinct forms of LA synaptic plasticity, resulting in more malleable fear memory to some tones than to others.