The influence of sex on activity in voluntary wheel running, forced treadmill running, and open field testing.

Introduction: Physical activity is commonly used for both measuring and treating dysfunction. While preclinical work has been historically biased towards males, the use of both male and female animals is gaining popularity after multiple NIH initiatives. With increasing inclusion of both sexes, it has become imperative to determine sex differences in common behavioral assays. The purpose of this study was to determine baseline sex differences in 3 activity assays: voluntary wheel running, forced treadmill running, and open field testing. Methods: This was a secondary analysis of sex differences in healthy mice in 3 different assays: Separate mice were used for each assay. Specifically, 16 mice underwent 28 days of voluntary wheel running, 178 mice underwent forced treadmill running, and 88 mice underwent open field testing. Differences between sex across several activity parameters were examined for each assay. Results: In voluntary wheel running, sex differences with larger effect sizes were observed in distance run, running time, and bout duration, with smaller effect size differences in speed, and no difference in total bouts. In forced treadmill running, differences were shown in time to exhaustion, but no difference in max speed attained. In open field, there were sex differences in active time but not in distance and speed in data aggregated over 30 minutes; however, distance and speed in male mice showed a downward trajectory over the final 20 minutes of testing, whereas females maintained the same trajectory. Conclusion: These data suggest that male mice demonstrate comparable activity intensity as female mice but do not match female's duration of activity, especially for volitional tasks. Researchers utilizing these assays should account for sex differences as they could potentially mask true findings in an experiment. Plain English Summary: Physical activity is a common measure to examine function in human subjects with and without disease. Animal models often use measures of physical activity to assess function, yet most of these measures have been done in males only, making interpretation and translation to females and humans difficult. Several measures have been used to measure activity in animals, including those examining voluntary running behavior, maximum capacity, and general activity levels; sex differences between these measures are unclear. We discovered sex differences throughout each of three activity tests. In voluntary running behavior there were large differences between sexes with females running a greater distance and spending more time running. There were small differences in the maximum capacity with females running for a longer period at high intensity. General activity levels showed small differences with females being less active than males. Thus, the greatest differences were found for voluntary running and small differences were found for maximum capacity and general activity levels; differences observed were dependent on the task. Researchers utilizing these assays should account for sex differences as they could potentially mask true findings in an experiment.

Chromosome-scale genome assembly of Poa trivialis and population genomics reveal widespread gene flow in a cool-season grass seed production system.

Poa trivialis (L.) is a cool-season grass species found in various environments worldwide. In addition to being a desired turfgrass species, it is a common weed of agricultural systems and natural areas. As a weed, it is an important contaminant of commercial cool-season grass seed lots, resulting in widespread gene flow facilitated by human activities and causing significant economic losses to farmers. To better understand and manage infestations, we assembled and annotated a haploid genome of P. trivialis and studied troublesome field populations from Oregon, the largest cool-season grass seed producing region in the United States. The genome assembly resulted in 1.35 Gb of DNA sequence distributed among seven chromosome-scale scaffolds, revealing a high content of transposable elements, conserved synteny with Poa annua, and a close relationship with other C3 grasses. A reduced-representation sequencing analysis of field populations revealed limited genetic diversity and suggested potential gene flow and human-assisted dispersal in the region. The genetic resources and insights into P. trivialis provided by this study will improve weed management strategies and enable the development of molecular detection tests for contaminated seed lots to limit seed-mediated gene flow. These resources should also be beneficial for turfgrass breeders seeking to improve desirable traits of commercial P. trivialis varieties and help to guide breeding efforts in other crops to enhance the resiliency of agricultural ecosystems under climate change. Significance Statement: The chromosome-scale assembly of Poa trivialis and population genomic analyses provide crucial insights into the gene flow of weedy populations in agricultural systems and contribute a valuable genomic resource for the plant science community.

The genome of Salmacisia buchloëana, the parasitic puppet master pulling strings of sexual phenotypic monstrosities in buffalograss.

To complete its parasitic lifecycle, Salmacisia buchloëana, a biotrophic fungus, manipulates reproductive organ development, meristem determinacy, and resource allocation in its dioecious plant host, buffalograss (Bouteloua dactyloides; Poaceae). To gain insight into S. buchloëana's ability to manipulate its host, we sequenced and assembled the 20.1 Mb genome of S. buchloëana into 22 chromosome-level pseudomolecules. Phylogenetic analysis suggests that S. buchloëana is nested within the genus Tilletia and diverged from Tilletia caries and Tilletia walkeri ∼40 MYA. We find that S. buchloëana contains a novel chromosome arm with no syntenic relationship to other publicly available Tilletia genomes, and that genes on the novel arm are upregulated upon infection, suggesting that this unique chromosomal segment may have played a critical role in S. buchloëana's evolution and host specificity. Salmacisia buchloëana has one of the largest fractions of serine peptidases (1.53% of the proteome) and one of the highest GC contents (62.3%) in all classified fungi. Analysis of codon base composition indicated that GC content is controlled more by selective constraints than directional mutation, and that S. buchloëana has a unique bias for the serine codon UCG. Finally, we identify 3 inteins within the S. buchloëana genome, 2 of which are located in a gene often used in fungal taxonomy. The genomic and transcriptomic resources generated here will aid plant pathologists and breeders by providing insight into the extracellular components contributing to sex determination in dioecious grasses.

ASIC3 plays a protective role in delayed-onset muscle soreness (DOMS) through muscle acid sensation during exercise.

Immediate exercise-induced pain (IEIP) and DOMS are two types of exercise-induced muscle pain and can act as barriers to exercise. The burning sensation of IEIP occurs during and immediately after intensive exercise, whereas the soreness of DOMS occurs later. Acid-sensing ion channels (ASICs) within muscle afferents are activated by H+ and other chemicals and have been shown to play a role in various chronic muscle pain conditions. Here, we further defined the role of ASICs in IEIP, and also tested if ASIC3 is required for DOMS. After undergoing exhaustive treadmill exercise, exercise-induced muscle pain was assessed in wild-type (WT) and ASIC3-/- mice at baseline via muscle withdrawal threshold (MWT), immediately, and 24 h after exercise. Locomotor movement, grip strength, and repeat exercise performance were tested at baseline and 24 h after exercise to evaluate DOMS. We found that ASIC3-/- had similar baseline muscle pain, locomotor activity, grip strength, and exercise performance as WT mice. WT showed diminished MWT immediately after exercise indicating they developed IEIP, but ASIC3-/- mice did not. At 24 h after baseline exercise, both ASIC3-/- and WT had similarly lower MWT and grip strength, however, ASIC3-/- displayed significantly lower locomotor activity and repeat exercise performance at 24 h time points compared to WT. In addition, ASIC3-/- mice had higher muscle injury as measured by serum lactate dehydrogenase and creatine kinase levels at 24 h after exercise. These results show that ASIC3 is required for IEIP, but not DOMS, and in fact might play a protective role to prevent muscle injury associated with strenuous exercise.

Mitral Valve Echodensities in a Young-Adult Female with Relapsing Polychondritis, Transiently Positive Lupus Anticoagulant, and Systemic Embolism.

Background: Valvular strands seen on echocardiography carry a wide differential diagnosis and may not always have a clear etiology despite taking clinical context into account. The decision of whether to provide anticoagulation for these lesions can be challenging. Case Presentation. A young adult female with an extensive rheumatologic history involving relapsing polychondritis and positive lupus anticoagulant presents to the emergency department with a discolored and painful right toe, as well as right auricular pain and swelling. Initial work-up revealed a possible splenic infarct, vasculitis of the right lower extremity, and mitral valve echodensities on echocardiography, without evidence of infective endocarditis. Due to concern that nonbacterial thrombotic endocarditis may be the cause of the patient's thromboembolic event, her valvular lesions were treated with low molecular weight heparin while awaiting serial imaging. When follow-up echocardiography showed no change in the size of her mitral valve lesions, which would be most consistent with Lambl's excrescences, the care team still faced a decision about which long-term anticoagulation to prescribe. This patient of childbearing age wished to avoid the teratogenicity and long-term monitoring associated with warfarin therapy. Although warfarin was the preferred agent for the patient's rheumatologic comorbidities, she elected to receive enoxaparin therapy for long-term thromboembolism prophylaxis. Conclusions: Even when accounting for clinical context, valvular lesions seen on echocardiography often have uncertain etiology and may require time and serial imaging to determine which treatment to pursue. When long-term anticoagulation is provided for females of childbearing age, shared decision-making with consideration of the patient's personal priorities and comorbidities is essential.

Homoeologous evolution of the allotetraploid genome of Poa annua L.

BACKGROUND: Poa annua (annual bluegrass) is an allotetraploid turfgrass, an agronomically significant weed, and one of the most widely dispersed plant species on earth. Here, we report the chromosome-scale genome assemblies of P. annua's diploid progenitors, P. infirma and P. supina, and use multi-omic analyses spanning all three species to better understand P. annua's evolutionary novelty. RESULTS: We find that the diploids diverged from their common ancestor 5.5 - 6.3 million years ago and hybridized to form P. annua ≤ 50,000 years ago. The diploid genomes are similar in chromosome structure and most notably distinguished by the divergent evolutionary histories of their transposable elements, leading to a 1.7 × difference in genome size. In allotetraploid P. annua, we find biased movement of retrotransposons from the larger (A) subgenome to the smaller (B) subgenome. We show that P. annua's B subgenome is preferentially accumulating genes and that its genes are more highly expressed. Whole-genome resequencing of several additional P. annua accessions revealed large-scale chromosomal rearrangements characterized by extensive TE-downsizing and evidence to support the Genome Balance Hypothesis. CONCLUSIONS: The divergent evolutions of the diploid progenitors played a central role in conferring onto P. annua its remarkable phenotypic plasticity. We find that plant genes (guided by selection and drift) and transposable elements (mostly guided by host immunity) each respond to polyploidy in unique ways and that P. annua uses whole-genome duplication to purge highly parasitized heterochromatic sequences. The findings and genomic resources presented here will enable the development of homoeolog-specific markers for accelerated weed science and turfgrass breeding.

Chromosome-Scale Genome Assembly and Annotation of Allotetraploid Annual Bluegrass (Poa annua L.).

Poa annua L. is a globally distributed grass with economic and horticultural significance as a weed and as a turfgrass. This dual significance, and its phenotypic plasticity and ecological adaptation, have made P. annua an intriguing plant for genetic and evolutionary studies. Because of the lack of genomic resources and its allotetraploid (2n = 4x = 28) nature, a reference genome sequence would be a valuable asset to better understand the significance and polyploid origin of P. annua. Here we report a genome assembly with scaffolds representing the 14 haploid chromosomes that are 1.78 Gb in length with an N50 of 112 Mb and 96.7% of BUSCO orthologs. Seventy percent of the genome was identified as repetitive elements, 91.0% of which were Copia- or Gypsy-like long-terminal repeats. The genome was annotated with 76,420 genes spanning 13.3% of the 14 chromosomes. The two subgenomes originating from Poa infirma (Knuth) and Poa supina (Schrad) were sufficiently divergent to be distinguishable but syntenic in sequence and annotation with repetitive elements contributing to the expansion of the P. infirma subgenome.

Sleep, circadian rhythm characteristics, and melatonin levels in later life adults with and without coronary artery disease.

STUDY OBJECTIVES: The purpose of this study was to conduct a comprehensive assessment of sleep and circadian rhythms in individuals with and without coronary artery disease (CAD). METHODS: This was a cross-sectional study. Participants were 32 individuals, mean age = 70.9, female 46.9%, 19 with CAD, and 13 without CAD. We assessed sleep quality and 24-hour rest-activity rhythms for 14 days using wrist actigraphy and self-report measures, and circadian rhythm using dim light melatonin onset. RESULTS: Melatonin levels prior to habitual bedtime were significantly lower in individuals with CAD than in those without CAD (median area under the curve = 12.88 vs 26.33 pg/ml × h, P = .049). The median circadian timing measured by dim light melatonin onset was the same for the 2 groups with 20:26 [hours:minutes] for individuals with CAD and 19:53 for the control group (P = .64, r = .14). Compared to the control group, the CAD group had significantly lower amplitude (P = .03, r =-.48), and lower overall rhythmicity (pseudo-F-statistic P = .004, r = -.65) in their 24-hour rest-activity rhythms. CONCLUSIONS: This is one of the first studies to comprehensively assess both sleep and circadian rhythm in individuals with CAD. Compared to non-CAD controls, individuals with CAD had lower levels of melatonin prior to habitual bedtime and a lower 24-hour rest-activity rhythm amplitude and overall rhythmicity. Future studies using larger sample sizes should further investigate the possibility of suppressed circadian rhythmicity in individuals with CAD. CITATION: Moon C, Benson CJ, Albashayreh A, Perkhounkova Y, Burgess HJ. Sleep, circadian rhythm characteristics, and melatonin levels in later life adults with and without coronary artery disease. J Clin Sleep Med. 2023;19(2):283-292.

Paradoxical Potentiation of Acid-Sensing Ion Channel 3 (ASIC3) by Amiloride via Multiple Mechanisms and Sites Within the Channel.

Acid-Sensing Ion Channels (ASICs) are proton-gated sodium-selective cation channels that have emerged as metabolic and pain sensors in peripheral sensory neurons and contribute to neurotransmission in the CNS. These channels and their related degenerin/epithelial sodium channel (DEG/ENaC) family are often characterized by their sensitivity to amiloride inhibition. However, amiloride can also cause paradoxical potentiation of ASIC currents under certain conditions. Here we characterized and investigated the determinants of paradoxical potentiation by amiloride on ASIC3 channels. While inhibiting currents induced by acidic pH, amiloride potentiated sustained currents at neutral pH activation. These effects were accompanied by alterations in gating properties including (1) an alkaline shift of pH-dependent activation, (2) inhibition of pH-dependent steady-state desensitization (SSD), (3) prolongation of desensitization kinetics, and (4) speeding of recovery from desensitization. Interestingly, extracellular Ca2+ was required for paradoxical potentiation and it diminishes the amiloride-induced inhibition of SSD. Site-directed mutagenesis within the extracellular non-proton ligand-sensing domain (E79A, E423A) demonstrated that these residues were critical in mediating the amiloride-induced inhibition of SSD. However, disruption of the purported amiloride binding site (G445C) within the channel pore blunted both the inhibition and potentiation of amiloride. Together, our results suggest that the myriad of modulatory and blocking effects of amiloride are the result of a complex competitive interaction between amiloride, Ca2+, and protons at probably more than one site in the channel.

Comparison of murine behavioural and physiological responses after forced exercise by electrical shock versus manual prodding.

NEW FINDINGS: What is the central question of this study? Forced treadmill exercise using electrical shock is the most common technique in rodent exercise studies. Here, we examined how the use of electrical shock during forced treadmill exercise affects behavioural and physiological responses in comparison to a novel non-electrical shock technique. What is the main finding and its importance? In comparison to mice that underwent traditional treadmill running induced by electrical shock, mice that underwent forced running using a novel technique involving gentle prodding to induce running showed: (i) higher locomotor activity; (ii) less anxiety-like behaviour; and (iii) altered exercise-induced muscle pain immediately after exercise. ABSTRACT: Animal models of exercise have been useful to understand underlying cellular and molecular mechanisms. Many studies have used methods of exercise that are unduly stressful (e.g., electrical shock to force running), potentially skewing results. Here, we compared physiological and behavioural responses of mice after exercise induced using a prodding technique that avoids electrical shock versus a traditional protocol using electrical shock. We found that exercise performance was similar for both techniques; however, the shock group demonstrated significantly lower locomotor activity and higher anxiety-like behaviour. We also observed divergent effects on muscle pain; the prodding group showed hyperalgesia immediately after exercise, whereas the shock group showed hypoalgesia. Corticosterone concentrations were elevated to a similar extent for both groups. In conclusion, mice that were exercised without shock generated similar maximal exercise performance, but postexercise these mice showed an increase in locomotor activity, less anxiety-like behaviour and altered muscle pain in comparison to mice that exercised with shock. Our data suggest that running of mice without the use of electrical shock is potentially less stressful and might be a better technique to study the physiological and behavioural responses to exercise.

ASICs are required for immediate exercise-induced muscle pain and are downregulated in sensory neurons by exercise training.

Exercise training is an effective therapy for many pain-related conditions, and trained athletes have lower pain perception compared with unconditioned people. Some painful conditions, including strenuous exercise, are associated with elevated levels of protons, metabolites, and inflammatory factors, which may activate receptors and/or ion channels, including acid-sensing ion channels (ASICs), on nociceptive sensory neurons. We hypothesized that ASICs are required for immediate exercise-induced muscle pain (IEIP) and that exercise training diminishes IEIP by modulating ASICs within muscle afferents. We found high-intensity interval training (HIIT) reduced IEIP in C57BL/6 mice and diminished ASIC mRNA levels in lumber dorsal root ganglia, and this downregulation of ASICs correlated with improved exercise capacity. Additionally, we found that ASIC3 -/- mice did not develop IEIP; however, the exercise capacity of ASIC3 -/- was similar to wild-type mice. These results suggest that ASICs are required for IEIP and that diminishment of IEIP after exercise training correlates with downregulation of ASICs in sensory neurons.NEW & NOTEWORTHY Exercise performance can be limited by the sensations of muscle fatigue and pain transmitted by muscle afferents. It has been proposed that exercise training abrogates these negative feedback signals. We found that acid-sensing ion channels (ASICs) are required for immediate exercise-induced muscle pain (IEIP). Moreover, exercise training prevented IEIP and was correlated with downregulation of ASICs in sensory neurons.

Multi-state amine sensing by electron transfers in a BODIPY probe.

Amines are ubiquitous in the chemical industry and are present in a wide range of biological processes, motivating the development of amine-sensitive sensors. There are many turn-on amine sensors, however there are no examples of turn-on sensors that utilize the amine's ability to react by single electron transfer (SET). We investigated a new turn-on amine probe with a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophore. BODIPY fluorescence is first preprogrammed into an off state by internal photoinduced electron transfer (PET) to an electron-deficient quinolinium ring, resulting in fluorescence quenching. At low concentrations of aliphatic amine (0 to 10 mM), this PET pathway is shut down by external SET from the amine to the photoexcited charge-transfer state of the probe and the fluorescence is turned on. At high concentrations of amine (50 mM to 1 M), we observed collisional quenching of the BODIPY fluorescence. The probe is selective for aliphatic amines over aromatic amines, and aliphatic thiols or alcohols. The three molecular processes modulate the BODIPY fluorescence in a multi-mechanistic way with two of them producing a direct response to amine concentrations. The totality of the three molecular processes produced the first example of a multi-state and dose-responsive amine sensor.

Gait Speed and Mood, Cognition, and Quality of Life in Older Adults With Atrial Fibrillation.

Background Low gait speed has been linked with impaired mood, cognition, and quality of life (QOL) in older adults. We examined whether low gait speed was associated with impaired mood, cognition, and QOL among older adults with atrial fibrillation (AF). Methods and Results Participants (n=1185) had a diagnosis of AF, aged ≥65 years, CHA2DS2VASc ≥2 and had no contraindications to anticoagulation. Participants completed a 15-foot walk test, and low gait speed was categorized using cutoffs from the Fried Frailty Index. Participants self-reported measures of depressive symptoms (Patient Health Questionnaire 9 ≥10), anxiety symptoms (Generalized Anxiety Disorder 7 ≥10), cognitive impairment (Montreal Cognitive Assessment ≤23), and potentially impaired Atrial Fibrillation Effect Quality-of-Life Questionnaire <80. Participants were on average aged 75.3 (SD: 7.0) years, 48.0% were women, and 85.5% were non-Hispanic white; 85.6% were taking an oral anticoagulant, 26.1% had low gait speed, 8.4% had elevated depressive symptoms, 5.7% had elevated anxiety symptoms, 41.1% were cognitively impaired, and 41.6% had potentially impaired AF-related QOL. Participants with low gait speed were significantly more likely to have elevated depressive symptoms (adjusted odds ratio: 2.1, 95% CI: 1.3-3.4), elevated anxiety symptoms (adjusted odds ratio: 2.2, 95% CI: 1.2-3.9), and cognitive impairment (adjusted odds ratio: 1.5, 95% CI: 1.1-2.1). Impaired AF-related QOL did not differ by gait speed after adjustment for clinical characteristics (adjusted odds ratio: 1.1, 95% CI: 0.8-1.5). Conclusions Twenty-six percent of older adults with AF had low gait speed, and low gait speed was associated with impaired mood and cognition. Further research is needed to determine whether declines in gait speed lead to impaired mood and cognition or whether these conditions develop concurrently.

TMEM16B determines cholecystokinin sensitivity of intestinal vagal afferents of nodose neurons.

The satiety effects and metabolic actions of cholecystokinin (CCK) have been recognized as potential therapeutic targets in obesity for decades. We identified a potentially novel Ca2+-activated chloride (Cl-) current (CaCC) that is induced by CCK in intestinal vagal afferents of nodose neurons. The CaCC subunit Anoctamin 2 (Ano2/TMEM16B) is the dominant contributor to this current. Its expression is reduced, as is CCK current activity in obese mice on a high-fat diet (HFD). Reduced expression of TMEM16B in the heterozygote KO of the channel in sensory neurons results in an obese phenotype with a loss of CCK sensitivity in intestinal nodose neurons, a loss of CCK-induced satiety, and metabolic changes, including decreased energy expenditure. The effect on energy expenditure is further supported by evidence in rats showing that CCK enhances sympathetic nerve activity and thermogenesis in brown adipose tissue, and these effects are abrogated by a HFD and vagotomy. Our findings reveal that Ano2/TMEM16B is a Ca2+-activated chloride channel in vagal afferents of nodose neurons and a major determinant of CCK-induced satiety, body weight control, and energy expenditure, making it a potential therapeutic target in obesity.

Acid Sensing Ion Channel 1a (ASIC1a) Mediates Activity-induced Pain by Modulation of Heteromeric ASIC Channel Kinetics.

Chronic muscle pain is acutely worsened by exercise. Acid sensing ion channels (ASIC) are heteromeric channels expressed in muscle sensory neurons that detect decreases in pH. We have previously shown ASIC3 is important in activity-induced hyperalgesia. However, ASICs form heteromers with ASIC1a being a key component in sensory neurons. Therefore, we studied the role of ASIC1a in mice using behavioral pharmacology and genetic deletion in a model of activity-induced hyperalgesia. We found ASIC1a-/- mice developed mechanical hyperalgesia similar to wild-type mice, but antagonism of ASIC1a, with psalmotoxin, prevented development of mechanical hyperalgesia in wild-type mice, but not in ASIC1a-/- mice. To explain this discrepancy, we then performed electrophysiology studies of ASICs and examined the effects of psalmotoxin on ASIC heteromers. We expressed ASIC1a, 2 and 3 heteromers or ASIC1 and 3 heteromers in CHO cells, and examined the effects of psalmotoxin on pH sensitivity. Psalmotoxin significantly altered the properties of ASIC hetomeric channels. Specifically, in ASIC1a/2/3 heteromers, psalmotoxin slowed the kinetics of desensitization, slowed the recovery from desensitization, and inhibited pH-dependent steady-state desensitization, but had no effect on pH-evoked current amplitudes. We found a different pattern in ASIC1a/3 heteromers. There was a significant leftward shift in the pH dose response of steady-state desensitization and decrease in pH-evoked current amplitudes. These results suggest that blockade of ASIC1a modulates the kinetics of heteromeric ASICs to prevent development of activity-induced hyperalgesia. These data suggest ASIC1a is a key subunit in heteromeric ASICs and may be a pharmacological target for treatment of musculoskeletal pain.

Inchworm movement of two rings switching onto a thread by biased Brownian diffusion represent a three-body problem.

The coordinated motion of many individual components underpins the operation of all machines. However, despite generations of experience in engineering, understanding the motion of three or more coupled components remains a challenge, known since the time of Newton as the "three-body problem." Here, we describe, quantify, and simulate a molecular three-body problem of threading two molecular rings onto a linear molecular thread. Specifically, we use voltage-triggered reduction of a tetrazine-based thread to capture two cyanostar macrocycles and form a [3]pseudorotaxane product. As a consequence of the noncovalent coupling between the cyanostar rings, we find the threading occurs by an unexpected and rare inchworm-like motion where one ring follows the other. The mechanism was derived from controls, analysis of cyclic voltammetry (CV) traces, and Brownian dynamics simulations. CVs from two noncovalently interacting rings match that of two covalently linked rings designed to thread via the inchworm pathway, and they deviate considerably from the CV of a macrocycle designed to thread via a stepwise pathway. Time-dependent electrochemistry provides estimates of rate constants for threading. Experimentally derived parameters (energy wells, barriers, diffusion coefficients) helped determine likely pathways of motion with rate-kinetics and Brownian dynamics simulations. Simulations verified intercomponent coupling could be separated into ring-thread interactions for kinetics, and ring-ring interactions for thermodynamics to reduce the three-body problem to a two-body one. Our findings provide a basis for high-throughput design of molecular machinery with multiple components undergoing coupled motion.

MG53 is dispensable for T-tubule maturation but critical for maintaining T-tubule integrity following cardiac stress.

The cardiac transverse (T)-tubule membrane system is the safeguard for cardiac function and undergoes dramatic remodeling in response to cardiac stress. However, the mechanism by which cardiomyocytes repair damaged T-tubule network remains unclear. In the present study, we tested the hypothesis that MG53, a muscle-specific membrane repair protein, antagonizes T-tubule damage to protect against maladaptive remodeling and thereby loss of excitation-contraction coupling and cardiac function. Using MG53-knockout (MG53-KO) mice, we first established that deficiency of MG53 had no impact on maturation of the T-tubule network in developing hearts. Additionally, MG53 ablation did not influence T-tubule integrity in unstressed adult hearts as late as 10months of age. Following left ventricular pressure overload-induced cardiac stress, MG53 protein levels were increased by approximately three-fold in wild-type mice, indicating that pathological stress induces a significant upregulation of MG53. MG53-deficient mice had worsened T-tubule disruption and pronounced dysregulation of Ca2+ handling properties, including decreased Ca2+ transient amplitude and prolonged time to peak and decay. Moreover, MG53 deficiency exacerbated cardiac hypertrophy and dysfunction and decreased survival following cardiac stress. Our data suggest MG53 is not required for T-tubule development and maintenance in normal physiology. However, MG53 is essential to preserve T-tubule integrity and thereby Ca2+ handling properties and cardiac function under pathological cardiac stress.

The volume-regulated anion channel (LRRC8) in nodose neurons is sensitive to acidic pH.

The leucine rich repeat containing protein 8A (LRRC8A), or SWELL1, is an essential component of the volume-regulated anion channel (VRAC) that is activated by cell swelling and ionic strength. We report here for the first time to our knowledge its expression in a primary cell culture of nodose ganglia neurons and its localization in the soma, neurites, and neuronal membrane. We show that this neuronal VRAC/SWELL1 senses low external pH (pHo) in addition to hypoosmolarity. A robust sustained chloride current is seen in 77% of isolated nodose neurons following brief exposures to extracellular acid pH. Its activation involves proton efflux, intracellular alkalinity, and an increase in NOX-derived H2O2. The molecular identity of both the hypoosmolarity-induced and acid pHo-conditioned VRAC as LRRC8A (SWELL1) was confirmed by Cre-flox-mediated KO, shRNA-mediated knockdown, and CRISPR/Cas9-mediated LRRC8A deletion in HEK cells and in primary nodose neuronal cultures. Activation of VRAC by low pHo reduces neuronal injury during simulated ischemia and N-methyl-D-aspartate-induced (NMDA-induced) apoptosis. These results identify the VRAC (LRRC8A) as a dual sensor of hypoosmolarity and low pHo in vagal afferent neurons and define the mechanisms of its activation and its neuroprotective potential.

Extreme Stabilization and Redox Switching of Organic Anions and Radical Anions by Large-Cavity, CH Hydrogen-Bonding Cyanostar Macrocycles.

Encapsulation of unstable guests is a powerful way to enhance their stability. The lifetimes of organic anions and their radicals produced by reduction are typically short on account of reactivity with oxygen while their larger sizes preclude use of traditional anion receptors. Here we demonstrate the encapsulation and noncovalent stabilization of organic radical anions by C-H hydrogen bonding in π-stacked pairs of cyanostar macrocycles having large cavities. Using electrogenerated tetrazine radical anions, we observe significant extension of their lifetimes, facile molecular switching, and extremely large stabilization energies. The guests form threaded pseudorotaxanes. Complexation extends the radical lifetimes from 2 h to over 20 days without altering its electronic structure. Electrochemical studies show tetrazines thread inside a pair of cyanostar macrocycles following voltage-driven reduction (+e-) of the tetrazine at -1.00 V and that the complex disassembles after reoxidation (-e-) at -0.05 V. This reoxidation is shifted 830 mV relative to the free tetrazine radical indicating it is stabilized by an unexpectedly large -80 kJ mol-1. The stabilization is general as shown using a dithiadiazolyl anion. This finding opens up a new approach to capturing and studying unstable anions and a radical anions when encapsulated by size-complementary anion receptors.