Effect of aging and exercise on hTERT expression in thymus tissue of hTERT transgenic bacterial artificial chromosome mice.

Telomere shortening occurs with aging in immune cells and may be related to immunosenescence. Exercise can upregulate telomerase activity and attenuate telomere shortening in immune cells, but it is unknown if exercise impacts other immune tissues such as the thymus. This study aimed to examine human telomerase reverse transcriptase (hTERT) alternative splicing (AS) in response to aging and exercise in thymus tissue. Transgenic mice with a human TERT bacterial artificial chromosome integrated into its genome (hTERT-BAC) were utilized in two different exercise models. Mice of different ages were assigned to an exercise cage (running wheel) or not for 3 weeks prior to thymus tissue excision. Middle-aged mice (16 months) were exposed or not to treadmill running (30 min at 60% maximum speed) prior to thymus collection. hTERT transcript variants were measured by RT-PCR. hTERT transcripts decreased with aging (r = - 0.7511, p < 0.0001) and 3 weeks of wheel running did not counteract this reduction. The ratio of exons 7/8 containing hTERT to total hTERT transcripts increased with aging (r = 0.3669, p = 0.0423) but 3 weeks of voluntary wheel running attenuated this aging-driven effect (r = 0.2013, p = 0.4719). Aging increased the expression of senescence marker p16 with no impact of wheel running. Thymus regeneration transcription factor, Foxn1, went down with age with no impact of wheel running exercise. Acute treadmill exercise did not induce any significant changes in thymus hTERT expression or AS variant ratio (p > 0.05). In summary, thymic hTERT expression is reduced with aging. Exercise counteracted a shift in hTERT AS ratio with age. Our data demonstrate that aging impacts telomerase expression and that exercise impacts dysregulated splicing that occurs with aging.

Central Role of Hypothalamic Circuits for Acupuncture's Anti-Parkinsonian Effects.

Despite clinical data stretching over millennia, the neurobiological basis of the effectiveness of acupuncture in treating diseases of the central nervous system has remained elusive. Here, using an established model of acupuncture treatment in Parkinson's disease (PD) model mice, we show that peripheral acupuncture stimulation activates hypothalamic melanin-concentrating hormone (MCH) neurons via nerve conduction. We further identify two separate neural pathways originating from anatomically and electrophysiologically distinct MCH neuronal subpopulations, projecting to the substantia nigra and hippocampus, respectively. Through chemogenetic manipulation specifically targeting these MCH projections, their respective roles in mediating the acupuncture-induced motor recovery and memory improvements following PD onset are demonstrated, as well as the underlying mechanisms mediating recovery from dopaminergic neurodegeneration, reactive gliosis, and impaired hippocampal synaptic plasticity. Collectively, these MCH neurons constitute not only a circuit-based explanation for the therapeutic effectiveness of traditional acupuncture, but also a potential cellular target for treating both motor and non-motor PD symptoms.

Study of CO2 Adsorption Properties on the SrTiO3(001) Surface with Ambient Pressure XPS.

The adsorption properties of CO2 on the SrTiO3(001) surface were investigated using ambient pressure X-ray photoelectron spectroscopy under elevated pressure and temperature conditions. On the Nb-doped TiO2-enriched (1 × 1) SrTiO3 surface, CO2 adsorption, i.e., the formation of CO3 surface species, occurs first at the oxygen lattice site under 10-6 mbar CO2 at room temperature. The interaction of CO2 molecules with oxygen vacancies begins when the CO2 pressure increases to 0.25 mbar. The adsorbed CO3 species on the Nb-doped SrTiO3 surface increases continuously as the pressure increases but starts to leave the surface as the surface temperature increases, which occurs at approximately 373 K on the defect-free surface. On the undoped TiO2-enriched (1 × 1) SrTiO3 surface, CO2 adsorption also occurs first at the lattice oxygen sites. Both the doped and undoped SrTiO3 surfaces exhibit an enhancement of the CO3 species with the presence of oxygen vacancies, thus indicating the important role of oxygen vacancies in CO2 dissociation. When OH species are removed from the undoped SrTiO3 surface, the CO3 species begin to form under 10-6 mbar at 573 K, thus indicating the critical role of OH in preventing CO2 adsorption. The observed CO2 adsorption properties of the various SrTiO3 surfaces provide valuable information for designing SrTiO3-based CO2 catalysts.

Neurotensin-specific corticothalamic circuit regulates innate response conflict.

Animals must simultaneously select and balance multiple action contingencies in ambiguous situations: for instance, evading danger during feeding. This has rarely been examined in the context of information selection; despite corticothalamic pathways that mediate sensory attention being relatively well characterized, neural mechanisms filtering conflicting actions remain unclear. Here, we develop a new loom/feed test to observe conflict between naturally induced fear and feeding and identify a novel anterior cingulate cortex (ACC) output to the ventral anterior and ventral lateral thalamus (VA/VL) that adjusts selectivity between these innate actions. Using micro-endoscopy and fiber photometry, we reveal that activity in corticofugal outputs was lowered during unbalanced/singularly occupied periods, as were the resulting decreased thalamic initiation-related signals for less-favored actions, suggesting that the integration of ACC-thalamic firing may directly regulate the output of behavior choices. Accordingly, the optoinhibition of ACC-VA/VL circuits induced high bias toward feeding at the expense of defense. To identify upstream "commander" cortical cells gating this output, we established dual-order tracing (DOT)-translating ribosome affinity purification (TRAP)-a scheme to label upstream neurons with transcriptome analysis-and found a novel population of neurotensin-positive interneurons (ACCNts). The photoexcitation of ACCNts cells indeed caused similarly hyper-selective behaviors. Collectively, this new "corticofugal action filter" scheme suggests that communication in multi-step cingulate circuits may critically influence the summation of motor signals in thalamic outputs, regulating bias between innate action types.

The Surface Chemistry of Methanol on Pd(111) and H-Pd(111) Surfaces: C-O Bond Cleavage and the Effects of Metal Hydride Formation.

Palladium catalysts are frequently employed in processes where methanol is an energy vector or carrier, being useful for the synthesis of methanol from mixtures of carbon dioxide and hydrogen (CO2/H2) or its steam reforming on demand. Results of synchrotron-based ambient pressure X-ray photoelectron spectroscopy for the adsorption of methanol on a Pd(111) model catalyst show a rich surface chemistry and complex phenomena that strongly depend on pressure and temperature. At low pressures (<10-6 Torr) and temperatures (<300 K), CO is the dominant decomposition product. As the pressure increases, cleavage of C-H, O-H, and C-O bonds is observed, and at elevated temperatures (400-600 K) the formation of CO and CHx/C fragments compete on the surface. Thus, existing reaction networks for methanol decomposition must be modified. Furthermore, surface and subsurface hydrogen (coming from PdHx) play a significant role in the stability and removal of CHx and C species.

Protocol for in vivo dual-color fiber photometry in the mouse thalamus.

In vivo calcium imaging of neural activity is an indispensable approach for understanding the mechanisms and functions of neural system. Development of advanced imaging tools and various genetically encoded calcium indicators allows us to simultaneously record the activity of different neural populations. Here, we present a protocol for acquiring neural activity of two discrete neural populations in mice using dual-color fiber photometry. We describe steps for injecting viral constructs and implanting the fiber optic through stereotaxic surgery, calcium signal acquisition, and data analysis. We also describe the incorporation of electroencephalogram and electromyography recordings with dual-color fiber photometry analysis. For complete details on the use and execution of this protocol, please refer to Shin et al.1.

Identification of an Antagonist Targeting G Protein and ß-Arrestin Signaling Pathways of 5-HT7R.

In consideration of the limited number of FDA-approved drugs for autism spectrum disorder (ASD), significant efforts have been devoted to identifying novel drug candidates. Among these, 5-HT7R modulators have garnered considerable attention due to their potential in alleviating autism-like behaviors in ASD animal models. In this study, we designed and synthesized biphenyl-3-ylmethylpyrrolidines 3 and biphenyl-3-yl-dihydroimidazoles 4 as 5-HT7R modulators. Through extensive biological tests of 3 and 4 in G protein and ß-arrestin signaling pathways of 5-HT7R, it was determined that 2-(2'-methoxy-[1,1'-biphenyl]-3-yl)-4,5-dihydro-1H-imidazole 4h acted as a 5-HT7R antagonist in both signaling pathways. In in vivo study with Shank3-/- transgenic (TG) mice, the self-grooming behavior test was performed with 4h, resulting in a significant reduction in the duration of self-grooming. In addition, an immunohistochemical experiment with 4h restored reduced neurogenesis in Shank3-/- TG mice, which is confirmed by the quantification of doublecortin (DCX) positive neurons, suggesting the promising therapeutic potential of 4h.

Dynamics of TERT regulation via alternative splicing in stem cells and cancer cells.

Part of the regulation of telomerase activity includes the alternative splicing (AS) of the catalytic subunit telomerase reverse transcriptase (TERT). Although a therapeutic window for telomerase/TERT inhibition exists between cancer cells and somatic cells, stem cells express TERT and rely on telomerase activity for physiological replacement of cells. Therefore, identifying differences in TERT regulation between stem cells and cancer cells is essential for developing telomerase inhibition-based cancer therapies that reduce damage to stem cells. In this study, we measured TERT splice variant expression and telomerase activity in induced pluripotent stem cells (iPSCs), neural progenitor cells (NPCs), and non-small cell lung cancer cells (NSCLC, Calu-6 cells). We observed that a NOVA1-PTBP1-PTBP2 axis regulates TERT alternative splicing (AS) in iPSCs and their differentiation into NPCs. We also found that splice-switching of TERT, which regulates telomerase activity, is induced by different cell densities in stem cells but not cancer cells. Lastly, we identified cell type-specific splicing factors that regulate TERT AS. Overall, our findings represent an important step forward in understanding the regulation of TERT AS in stem cells and cancer cells.

Bioaccumulation of Lead, Cadmium, and Arsenic in a Mining Area and Its Associated Health Effects.

Soil contamination is associated with a high potential for health issues. This study aimed to investigate the bioaccumulation of heavy metals and its associated health impact among residents near a mining area. We performed environmental monitoring by analyzing lead (Pb), cadmium (Cd), and arsenic (As) levels in soil and rice samples, as well as biomonitoring by analyzing blood and urine samples from 58 residents living near the mine. Additionally, concentration trends were investigated among 26 participants in a 2013 study. The Cd and As levels in the soil samples and Cd levels in the rice samples exceeded the criteria for concern. The geometric mean blood Cd level (2.12 µg/L) was two times higher than that in the general population aged > 40 years. The blood Cd level showed decreasing trends from the previous measurements of 4.56-2.25 µg/L, but was still higher than that in the general population. The blood and urine Cd levels were higher in those with a low estimated glomerular filtration rate (eGFR) than in those with normal eGFR. In conclusion, heavy metals from mining areas can accumulate in soil and rice, adversely impacting human health. Continuous environmental monitoring and biomonitoring are required to ensure the safety of residents.

Revealing CO2 dissociation pathways at vicinal copper (997) interfaces.

Size- and shape-tailored copper (Cu) nanocrystals can offer vicinal planes for facile carbon dioxide (CO2) activation. Despite extensive reactivity benchmarks, a correlation between CO2 conversion and morphology structure has not yet been established at vicinal Cu interfaces. Herein, ambient pressure scanning tunneling microscopy reveals step-broken Cu nanocluster evolutions on the Cu(997) surface under 1 mbar CO2(g). The CO2 dissociation reaction produces carbon monoxide (CO) adsorbate and atomic oxygen (O) at Cu step-edges, inducing complicated restructuring of the Cu atoms to compensate for increased surface chemical potential energy at ambient pressure. The CO molecules bound at under-coordinated Cu atoms contribute to the reversible Cu clustering with the pressure gap effect, whereas the dissociated oxygen leads to irreversible Cu faceting geometries. Synchrotron-based ambient pressure X-ray photoelectron spectroscopy identifies the chemical binding energy changes in CO-Cu complexes, which proves the characterized real-space evidence for the step-broken Cu nanoclusters under CO(g) environments. Our in situ surface observations provide a more realistic insight into Cu nanocatalyst designs for efficient CO2 conversion to renewable energy sources during C1 chemical reactions.

Exercise as a Therapy to Maintain Telomere Function and Prevent Cellular Senescence.

Exercise transiently impacts the expression, regulation, and activity of TERT/telomerase to maintain telomeres and protect the genome from insults. By protecting the telomeres (chromosome ends) and the genome, telomerase promotes cellular survival and prevents cellular senescence. By increasing cellular resiliency, via the actions of telomerase and TERT, exercise promotes healthy aging.

Formation of Graphene on Gold-Nickel Surface Alloys.

Nickel (Ni)-catalyzed growth of a single- or rotated-graphene layer is a well-established process above 800 K. In this report, a Au-catalyzed, low-temperature, and facile route at 500 K for graphene formation is described. The substantially lower temperature is enabled by the presence of a surface alloy of Au atoms embedded within Ni(111), which catalyzes the outward segregation of carbon atoms buried in the Ni bulk at temperatures as low as 400-450 K. The resulting surface-bound carbon in turn coalesces into graphene above 450-500 K. Control experiments on a Ni(111) surface show no evidence of carbon segregation or graphene formation at these temperatures. Graphene is identified by its out-of-plane optical phonon mode at 750 cm-1 and its longitudinal/transverse optical phonon modes at 1470 cm-1 while surface carbon is identified by its C-Ni stretch mode at 540 cm-1, as probed by high-resolution electron energy-loss spectroscopy. Dispersion measurements of the phonon modes confirm the presence of graphene. Graphene formation is observed to be maximum at 0.4 ML Au coverage. The results of these systematic molecular-level investigations open the door to graphene synthesis at the low temperatures required for integration with complementary metal-oxide-semiconductor processes.

A brainstem-to-mediodorsal thalamic pathway mediates sound-induced arousal from slow-wave sleep.

Auditory-induced arousal is a defense mechanism of animals against potential dangers. Although the thalamus is the neural substrate that relays sensory information to the cortex, its function is reduced during slow-wave sleep (SWS), also known as deep sleep. Despite this, animals are capable of waking up in response to external sensory stimuli, suggesting the existence of neural circuits that are involved in this response. Here, we report that kainate-class-type ionotropic glutamate receptor subunit 4 (GRIK4)-positive mediodorsal (MD) thalamic neurons act as a neural substrate for arousals from SWS. These neurons become active during arousal from SWS and their photoactivation can induce arousal from SWS. Moreover, we show that these neurons are influenced by glutamatergic neurons in the brainstem, the activity of which increases during auditory-induced arousals. These results suggest that this brainstem-MD pathway can mediate wakefulness from SWS.

Intronic Cis-Element DR8 in hTERT Is Bound by Splicing Factor SF3B4 and Regulates hTERT Splicing in Non-Small Cell Lung Cancer.

Splicing of the hTERT gene to produce the full-length (FL) transcript is necessary for telomerase enzyme activity and telomere-dependent cellular immortality in the majority of human tumors, including non-small cell lung cancer (NSCLC) cells. The molecular machinery to splice hTERT to the FL isoform remains mostly unknown. Previously, we reported that an intron 8 cis-element termed "direct repeat 8" (DR8) promotes FL hTERT splicing, telomerase, and telomere length maintenance when bound by NOVA1 and PTBP1 in NSCLC cells. However, some NSCLC cells and patient tumor samples lack NOVA1 expression. This leaves a gap in knowledge about the splicing factors and cis-elements that promote telomerase in the NOVA1-negative context. We report that DR8 regulates FL hTERT splicing in the NOVA1-negative and -positive lung cancer contexts. We identified splicing factor 3b subunit 4 (SF3B4) as an RNA trans-factor whose expression is increased in lung adenocarcinoma (LUAD) tumors compared with adjacent normal tissue and predicts poor LUAD patient survival. In contrast to normal lung epithelial cells, which continued to grow with partial reductions of SF3B4 protein, SF3B4 knockdown reduced hTERT splicing, telomerase activity, telomere length, and cell growth in lung cancer cells. SF3B4 was also demonstrated to bind the DR8 region of hTERT pre-mRNA in both NOVA1-negative and -positive NSCLC cells. These findings provide evidence that DR8 is a critical binding hub for trans-factors to regulate FL hTERT splicing in NSCLC cells. These studies help define mechanisms of gene regulation important to the generation of telomerase activity during carcinogenesis. IMPLICATIONS: Manipulation of a core spliceosome protein reduces telomerase/hTERT splicing in lung cancer cells and results in slowed cancer cell growth and cell death, revealing a potential therapeutic strategy.

Visualization of differential GPCR crosstalk in DRD1-DRD2 heterodimer upon different dopamine levels.

Dopaminergic signaling is regulated by transient micromolar (phasic) and background nanomolar (tonic) dopamine releases in the brain. These dopamine signals can be differentially translated by dopamine receptor type 1 and type 2, DRD1 and DRD2, which are G protein-coupled receptors (GPCRs). In response to dopamine, DRD1 and DRD2 are known to mediate opposite functions on cAMP production via Gs and Gi protein signaling. Interestingly, they can form a heterodimer. However, receptor crosstalk between DRD1-DRD2 heterodimers has not been directly measured, but it was only inferred from measuring downstream signaling pathways. Here we develop fluorescent protein-based multicolor biosensors which can monitor individual activation states of DRD1 and DRD2, and apply them to directly monitor the functional crosstalk between DRD1-DRD2 heterodimers in live cells. Utilizing these powerful tools, we surprisingly discover differential crosstalk in the DRD1-DRD2 heterodimers upon different dopamine (DA) levels: DRD1 activation is selectively inhibited at micromolar DA levels, while DRD2 is inhibited only by nanomolar DA concentration, implying a novel function of the DRD1-DRD2 heterodimer upon different DA levels. Our results imply differential receptor crosstalk and novel functions of the DRD1-DRD2 heterodimer in response to physiological dopamine levels from nanomolar to micromolar dopamine concentrations.

Acute Exercise Regulates hTERT Gene Expression and Alternative Splicing in the hTERT-BAC Transgenic Mouse Model.

INTRODUCTION: Aerobic exercise maintains telomere length through increased human telomerase reverse transcriptase (hTERT) expression and telomerase enzyme activity. The impact of acute exercise on hTERT alternative splicing (AS) is unknown. PURPOSE: This study aimed to examine hTERT AS in response to acute treadmill running. METHODS: A bacterial artificial chromosome mouse model containing the 54-kilobase hTERT gene locus inserted into its genome (hTERT-BAC) was utilized. The gastrocnemius, left ventricle, and brain were excised before (Pre), upon cessation (Post), and during recovery (1, 24, 48, and 72 h; n = 5/time point) from treadmill running (30 min at 60% maximum speed). Full-length (FL) hTERT and the "minus beta" (-ß) AS variant (skips exons 7 and 8 and does not code for active telomerase) were measured by gel-based and droplet digital reverse transcription-polymerase chain reaction methods. SF3B4 and SRSF2 protein expression were measured by Western blotting. RESULTS: Compared with Pre, FL hTERT increased at Post before decreasing during recovery in the gastrocnemius (48 and 72 h; P ≤ 0.001) and left ventricle (24 h; P = 0.004). The percentage of FL hTERT in the gastrocnemius also increased during recovery (1 and 72 h; P ≤ 0.017), whereas a decrease was observed in the left ventricle (1, 24, and 48 h; P ≤ 0.041). hTERT decreased in the brain (48 h), whereas FL hTERT percentage remained unaltered. SF3B4 protein expression decreased throughout recovery in the gastrocnemius and tended to be associated with FL hTERT (r = -0.348, P = 0.075) and -ß in opposite directions (r = 0.345, P = 0.067). CONCLUSIONS: Endurance exercise increased hTERT gene expression, and altered FL hTERT splicing in contractile tissues and may maintain telomere length necessary to improve the function and health of the organism.

Neural correlates of multidimensional motor outputs in an excitatory parafascicular-zona incerta circuit.

The parafascicular nucleus (Pf) in medial thalamus is interconnected with prefrontal cortex and basal ganglia. Though much research has determined its importance in cognitive regulation of behaviour, its projections to regions in subthalamus remain less known. Such connections include those to zona incerta (ZI), located immediately dorsal to subthalamic nuclei (STN) regulating motor output, and whose role in a motor context is only beginning to be investigated. We thus examined circuits from parafascicular (Pf) thalamus to ZI, and its activity during locomotion and spontaneous behaviours in mice. We found that a distinct group of CaMKIIα-positive excitatory parafascicular neurons, separated from VGLUT2-positive excitatory neurons, project widely into ZI, more than adjacent STN. Our results from fibre photometry and decoding with general linear model (GLM) indicate that PF-ZI pathways do not specifically correlate with amount of locomotion or movement velocity, but instead show more specified activity during relative directional changes of movements observed in turning, sniffing behaviours. These results hint at the PF-ZI pathway having a distinct role in directing action specificity and have implications for subcortical bases in dimensional control of behaviours.

Discovery of G Protein-Biased Antagonists against 5-HT7R.

5-HT7R belongs to a family of G protein-coupled receptors and is associated with a variety of physiological processes in the central nervous system via the activation of the neurotransmitter serotonin (5-HT). To develop selective and biased 5-HT7R ligands, we designed and synthesized a series of pyrazolyl-diazepanes 2 and pyrazolyl-piperazines 3, which were evaluated for binding affinities to 5-HTR subtypes and functional selectivity for G protein and ß-arrestin signaling pathways of 5-HT7R. Among them, 1-(3-(3-chlorophenyl)-1H-pyrazol-4-yl)-1,4-diazepane 2c showed the best binding affinity for 5-HT7R and selectivity over other 5-HTR subtypes. It was also revealed as a G protein-biased antagonist. The self-grooming behavior test was performed with 2c in vivo with Shank3-/- transgenic (TG) mice, wherein 2c significantly reduced self-grooming duration time to the level of wild-type mice. The results suggest that 5-HT7R could be a potential therapeutic target for treating autism spectrum disorder stereotypy.

Discovery of G Protein-Biased Ligands against 5-HT7R.

There has been significant attention concerning the biased agonism of G protein-coupled receptors (GPCRs), and it has resulted in various pharmacological benefits. 5-HT7R belongs to a GPCR, and it is a promising pharmaceutical target for the treatment of neurodevelopmental and neuropsychiatric disorders. Based on our previous research, we synthesized a series of 6-chloro-2'-methoxy biphenyl derivatives 1, 2, and 3 with a variety of amine scaffolds. These compounds were evaluated for their binding affinities to 5-HTR subtypes and their functional selectivity toward the Gs protein and the ß-arrestin signaling pathways of 5-HT7R. Among them, 2-(6-chloro-2'-methoxy-[1,1'-biphenyl]-3-yl)-N-ethylethan-1-amine, 2b, was found to be a G-protein-biased ligand of 5-HT7R. In an in vivo study with Shank3 transgenic mice, the self-grooming behavior test was performed with 2b, which increased the duration of self-grooming. The experiments further suggested that 5-HT7R is associated with autism spectrum disorders (ASDs) and could be a therapeutic target for the treatment of stereotypy in ASDs.

How Rh surface breaks CO2 molecules under ambient pressure.

Utilization of carbon dioxide (CO2) molecules leads to increased interest in the sustainable synthesis of methane (CH4) or methanol (CH3OH). The representative reaction intermediate consisting of a carbonyl or formate group determines yields of the fuel source during catalytic reactions. However, their selective initial surface reaction processes have been assumed without a fundamental understanding at the molecular level. Here, we report direct observations of spontaneous CO2 dissociation over the model rhodium (Rh) catalyst at 0.1 mbar CO2. The linear geometry of CO2 gas molecules turns into a chemically active bent-structure at the interface, which allows non-uniform charge transfers between chemisorbed CO2 and surface Rh atoms. By combining scanning tunneling microscopy, X-ray photoelectron spectroscopy at near-ambient pressure, and computational calculations, we reveal strong evidence for chemical bond cleavage of O‒CO* with ordered intermediates structure formation of (2 × 2)-CO on an atomically flat Rh(111) surface at room temperature.