Dorsal Raphe Dopamine Neurons Represent the Experience of Social Isolation.

The motivation to seek social contact may arise from either positive or negative emotional states, as social interaction can be rewarding and social isolation can be aversive. While ventral tegmental area (VTA) dopamine (DA) neurons may mediate social reward, a cellular substrate for the negative affective state of loneliness has remained elusive. Here, we identify a functional role for DA neurons in the dorsal raphe nucleus (DRN), in which we observe synaptic changes following acute social isolation. DRN DA neurons show increased activity upon social contact following isolation, revealed by in vivo calcium imaging. Optogenetic activation of DRN DA neurons increases social preference but causes place avoidance. Furthermore, these neurons are necessary for promoting rebound sociability following an acute period of isolation. Finally, the degree to which these neurons modulate behavior is predicted by social rank, together supporting a role for DRN dopamine neurons in mediating a loneliness-like state. PAPERCLIP.

Regulation of autoantibody activity by the IL-23-TH17 axis determines the onset of autoimmune disease.

The checkpoints and mechanisms that contribute to autoantibody-driven disease are as yet incompletely understood. Here we identified the axis of interleukin 23 (IL-23) and the TH17 subset of helper T cells as a decisive factor that controlled the intrinsic inflammatory activity of autoantibodies and triggered the clinical onset of autoimmune arthritis. By instructing B cells in an IL-22- and IL-21-dependent manner, TH17 cells regulated the expression of ß-galactoside α2,6-sialyltransferase 1 in newly differentiating antibody-producing cells and determined the glycosylation profile and activity of immunoglobulin G (IgG) produced by the plasma cells that subsequently emerged. Asymptomatic humans with rheumatoid arthritis (RA)-specific autoantibodies showed identical changes in the activity and glycosylation of autoreactive IgG antibodies before shifting to the inflammatory phase of RA; thus, our results identify an IL-23-TH17 cell-dependent pathway that controls autoantibody activity and unmasks a preexisting breach in immunotolerance.

Vigorous Exercise in Patients With Congenital Long QT Syndrome: Results of the Prospective, Observational, Multinational LIVE-LQTS Study.

BACKGROUND: Whether vigorous exercise increases risk of ventricular arrhythmias for individuals diagnosed and treated for congenital long QT syndrome (LQTS) remains unknown. METHODS: The National Institutes of Health-funded LIVE-LQTS study (Lifestyle and Exercise in the Long QT Syndrome) prospectively enrolled individuals 8 to 60 years of age with phenotypic and/or genotypic LQTS from 37 sites in 5 countries from May 2015 to February 2019. Participants (or parents) answered physical activity and clinical events surveys every 6 months for 3 years with follow-up completed in February 2022. Vigorous exercise was defined as ≥6 metabolic equivalents for >60 hours per year. A blinded Clinical Events Committee adjudicated the composite end point of sudden death, sudden cardiac arrest, ventricular arrhythmia treated by an implantable cardioverter defibrillator, and likely arrhythmic syncope. A National Death Index search ascertained vital status for those with incomplete follow-up. A noninferiority hypothesis (boundary of 1.5) between vigorous exercisers and others was tested with multivariable Cox regression analysis. RESULTS: Among the 1413 participants (13% <18 years of age, 35% 18-25 years of age, 67% female, 25% with implantable cardioverter defibrillators, 90% genotype positive, 49% with LQT1, 91% were treated with beta-blockers, left cardiac sympathetic denervation, and/or implantable cardioverter defibrillator), 52% participated in vigorous exercise (55% of these competitively). Thirty-seven individuals experienced the composite end point (including one sudden cardiac arrest and one sudden death in the nonvigorous group, one sudden cardiac arrest in the vigorous group) with overall event rates at 3 years of 2.6% in the vigorous and 2.7% in the nonvigorous exercise groups. The unadjusted hazard ratio for experience of events for the vigorous group compared with the nonvigorous group was 0.97 (90% CI, 0.57-1.67), with an adjusted hazard ratio of 1.17 (90% CI, 0.67-2.04). The upper 95% one-sided confidence level extended beyond the 1.5 boundary. Neither vigorous or nonvigorous exercise was found to be superior in any group or subgroup. CONCLUSIONS: Among individuals diagnosed with phenotypic and/or genotypic LQTS who were risk assessed and treated in experienced centers, LQTS-associated cardiac event rates were low and similar between those exercising vigorously and those not exercising vigorously. Consistent with the low event rate, CIs are wide, and noninferiority was not demonstrated. These data further inform shared decision-making discussions between patient and physician about exercise and competitive sports participation. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02549664.

Effect of adenosinergic manipulations on amygdala-kindled seizures in mice: Implications for sudden unexpected death in epilepsy.

OBJECTIVE: Sudden unexpected death in epilepsy (SUDEP) results in more years of potential life lost than any neurological condition with the exception of stroke. It is generally agreed that SUDEP happens due to some form of respiratory, cardiac, and electrocerebral dysfunction following a seizure; however, the mechanistic cause of these perturbations is unclear. One possible explanation lies with adenosinergic signaling. Extracellular levels of the inhibitory neuromodulator adenosine rapidly rise during seizures, a countermeasure that is necessary for seizure termination. Previous evidence has suggested that excessive adenosinergic inhibition could increase the risk of SUDEP by silencing brain areas necessary for life, such as the respiratory nuclei of the brainstem. The goal of this investigation was to further clarify the role of adenosine in seizure-induced respiratory and electrocerebral dysfunction. METHODS: To determine the role of adenosine in postictal physiological dysregulation, we pharmacologically manipulated adenosine signaling prior to amygdala-kindled seizures in mice while recording electroencephalogram (EEG), electromyogram, and breathing using whole body plethysmography. The adenosinergic drugs used in this study included selective and nonselective adenosine receptor antagonists and inhibitors of adenosine metabolism. RESULTS: We found that high doses of adenosine receptor antagonists caused some seizures to result in seizure-induced death; however, counterintuitively, animals in these conditions that did not experience seizure-induced death had little or no postictal generalized EEG suppression. Inhibitors of adenosine metabolism had no effect on postictal breathing but did worsen some postictal electrocerebral outcomes. SIGNIFICANCE: The unexpected effect of high doses of adenosine antagonists on seizure-induced death observed in this study may be due to the increase in seizure severity, vasoconstriction, or phosphodiesterase inhibition caused by these drugs at high doses. These findings further clarify the role of adenosine in seizure-induced death and may have implications for the consumption of caffeine in epilepsy patients and the prevention of SUDEP.

Patterns of initial and first-intensifying antidiabetic drug utilization among patients with type 2 diabetes mellitus in Scotland, 2010-2020: A retrospective population-based cohort study.

AIM: To evaluate the utilization and prescribing patterns of antidiabetic drugs (ADDs) for patients with type 2 diabetes mellitus (T2DM) at treatment initiation and first intensification. METHODS: A retrospective cohort study was performed using linked routinely collected data of patients with T2DM who received ADDs between January 2010 and December 2020 in Scotland. The prescribing patterns were quantified using frequency/percentages, absolute/relative change, and trend tests. RESULTS: Overall, 145 909 new ADD users were identified, with approximately 91% (N = 132 382) of patients receiving a single ADD at first treatment initiation. Metformin was the most often prescribed monotherapy (N = 118 737, 89.69%). A total of 50 731 patients (39.40%) who were started on metformin (N = 46 730/118 737, 39.36%) or sulphonylurea (SU; N = 4001/10 029, 39.89%) monotherapy had their treatment intensified with one or more additional ADD. Most initial-metformin (45 963/46 730; 98.36%) and initial-SU users (3894/4001; 97.33%) who added further drugs were intensified with single ADDs. SUs (22 197/45 963; 48.29%) were the most common first-intensifying monotherapy after initial metformin use, but these were replaced by sodium-glucose cotransporter-2 (SGLT2) inhibitors in 2019 (SGLT2 inhibitors: 2039/6065, 33.62% vs. SUs: 1924/6065, 31.72%). Metformin was the most frequently added monotherapy to initial SU use (2924/3894, 75.09%). Although the majority of patients received a single ADD, the use of combination therapy significantly increased over time. Nevertheless, there was a significant increasing trend towards prescribing the newer ADD classes (SGLT2 inhibitors, dipeptidyl peptidase-4 inhibitors) as monotherapy or in combination compared with the older ones (SUs, insulin, thiazolidinediones) at both drug initiation and first intensification. CONCLUSIONS: An overall increasing trend in prescribing the newer ADD classes compared to older ADDs was observed. However, metformin remained the most commonly prescribed first-line ADD, while SGLT2 inhibitors replaced SUs as the most common add-on therapy to initial metformin use in 2019.

N-glycosylation of the SARS-CoV-2 spike protein at Asn331 and Asn343 is involved in spike-ACE2 binding, virus entry, and regulation of IL-6.

The coronavirus disease 2019 (COVID-19) pandemic is an ongoing global public health crisis. The causative agent, the SARS-CoV-2 virus, enters host cells via molecular interactions between the viral spike protein and the host cell ACE2 surface protein. The SARS-CoV-2 spike protein is extensively decorated with up to 66 N-linked glycans. Glycosylation of viral proteins is known to function in immune evasion strategies but may also function in the molecular events of viral entry into host cells. Here, we show that N-glycosylation at Asn331 and Asn343 of SARS-CoV-2 spike protein is required for it to bind to ACE2 and for the entry of pseudovirus harboring the SARS-CoV-2 spike protein into cells. Interestingly, high-content glycan binding screening data have shown that N-glycosylation of Asn331 and Asn343 of the RBD is important for binding to the specific glycan molecule G4GN (Galß-1,4 GlcNAc), which is critical for spike-RBD-ACE2 binding. Furthermore, IL-6 was identified through antibody array analysis of conditioned media of the corresponding pseudovirus assay. Mutation of N-glycosylation of Asn331 and Asn343 sites of the spike receptor-binding domain (RBD) significantly reduced the transcriptional upregulation of pro-inflammatory signaling molecule IL-6. In addition, IL-6 levels correlated with spike protein levels in COVID-19 patients' serum. These findings establish the importance of RBD glycosylation in SARS-CoV-2 pathogenesis, which can be exploited for the development of novel therapeutics for COVID-19.

Elementary mechanisms of calmodulin regulation of NaV1.5 producing divergent arrhythmogenic phenotypes.

In cardiomyocytes, NaV1.5 channels mediate initiation and fast propagation of action potentials. The Ca2+-binding protein calmodulin (CaM) serves as a de facto subunit of NaV1.5. Genetic studies and atomic structures suggest that this interaction is pathophysiologically critical, as human mutations within the NaV1.5 carboxy-terminus that disrupt CaM binding are linked to distinct forms of life-threatening arrhythmias, including long QT syndrome 3, a "gain-of-function" defect, and Brugada syndrome, a "loss-of-function" phenotype. Yet, how a common disruption in CaM binding engenders divergent effects on NaV1.5 gating is not fully understood, though vital for elucidating arrhythmogenic mechanisms and for developing new therapies. Here, using extensive single-channel analysis, we find that the disruption of Ca2+-free CaM preassociation with NaV1.5 exerts two disparate effects: 1) a decrease in the peak open probability and 2) an increase in persistent NaV openings. Mechanistically, these effects arise from a CaM-dependent switch in the NaV inactivation mechanism. Specifically, CaM-bound channels preferentially inactivate from the open state, while those devoid of CaM exhibit enhanced closed-state inactivation. Further enriching this scheme, for certain mutant NaV1.5, local Ca2+ fluctuations elicit a rapid recruitment of CaM that reverses the increase in persistent Na current, a factor that may promote beat-to-beat variability in late Na current. In all, these findings identify the elementary mechanism of CaM regulation of NaV1.5 and, in so doing, unravel a noncanonical role for CaM in tuning ion channel gating. Furthermore, our results furnish an in-depth molecular framework for understanding complex arrhythmogenic phenotypes of NaV1.5 channelopathies.

Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms.

Voltage-gated sodium channels, NaVs, are responsible for the rapid rise of action potentials in excitable tissues. NaV channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-NaV nanobodies (Nbs), Nb17 and Nb82, that recognize the NaV1.4 (skeletal muscle) and NaV1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of NaV1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human NaV isoform has been challenging because they usually show undesired crossreactivity for different NaV isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNaV1.4 or CTNaV1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNaV1.4 and CTNaV1.5 with high affinity (KD ∼ 40-60 nM). In addition, as proof of concept, we show that Nb82 could detect NaV1.4 and NaV1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo NaV1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-NaV reagents to capture NaVs from cell lysates and as molecular visualization agents for NaVs.

Sudden unexpected death in epilepsy.

PURPOSE OF REVIEW: Sudden unexpected death in epilepsy (SUDEP) is a leading cause of death in patients with epilepsy. This review highlights the recent literature regarding epidemiology on a global scale, putative mechanisms and thoughts towards intervention and prevention. RECENT FINDINGS: Recently, numerous population-based studies have examined the incidence of SUDEP in many countries. Remarkably, incidence is quite consistent across these studies, and is commensurate with the recent estimates of about 1.2 per 1000 patient years. These studies further continue to support that incidence is similar across the ages and that comparable factors portend heightened risk for SUDEP. Fervent research in patients and animal studies continues to hone the understanding of potential mechanisms for SUDEP, especially those regarding seizure-induced respiratory dysregulation. Many of these studies and others have begun to lay out a path towards identification of improved treatment and prevention means. However, continued efforts are needed to educate medical professionals about SUDEP risk and the need to disclose this to patients. SUMMARY: SUDEP is a devastating potential outcome of epilepsy. More is continually learned about risk and mechanisms from clinical and preclinical studies. This knowledge can hopefully be leveraged into preventive measures in the near future.

Guidelines for mouse and human DC functional assays.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Recent studies have provided evidence for an increasing number of phenotypically distinct conventional DC (cDC) subsets that on one hand exhibit a certain functional plasticity, but on the other hand are characterized by their tissue- and context-dependent functional specialization. Here, we describe a selection of assays for the functional characterization of mouse and human cDC. The first two protocols illustrate analysis of cDC endocytosis and metabolism, followed by guidelines for transcriptomic and proteomic characterization of cDC populations. Then, a larger group of assays describes the characterization of cDC migration in vitro, ex vivo, and in vivo. The final guidelines measure cDC inflammasome and antigen (cross)-presentation activity. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

Meta-analysis of factors associated with antidiabetic drug prescribing for type 2 diabetes mellitus.

BACKGROUND: There is a lack of consensus on prescribing alternatives to initial metformin therapy and intensification therapy for type 2 diabetes mellitus (T2DM) management. This review aimed to identify/quantify factors associated with prescribing of specific antidiabetic drug classes for T2DM. METHODS: Five databases (Medline/PubMed, Embase, Scopus, Web of Science) were searched using the synonyms of each concept (patients with T2DM, antidiabetic drugs and factors influencing prescribing) in both free text and Medical Subject Heading (MeSH) forms. Quantitative observational studies evaluating factors associated with antidiabetic prescribing of metformin, sulfonylurea, thiazolidinedione, Dipeptidyl-peptidase 4 inhibitors (DPP4-I), sodium glucose transporter 2 inhibitors (SGLT2-I), Glucagon-Like peptide receptor agonist (GLP1-RA) and insulin in outpatient settings and published from January 2009 to January 2021 were included. Quality assessment was performed using a Newcastle-Ottawa scale. The validation was done for 20% of identified studies. The pooled estimate was measured using a three-level random-effect meta-analysis model based on odds ratio [95% confidence interval]. Age, sex, body mass index (BMI), glycaemic control (HbA1c) and kidney-related problems were quantified. RESULTS: Of 2331 identified studies, 40 met the selection criteria. Of which, 36 and 31 studies included sex and age, respectively, while 20 studies examined baseline BMI, HbA1c and kidney-related problems. The majority of studies (77.5%, 31/40) were rated as good and despite that the overall heterogeneity for each studied factor was more than 75%, it is mostly related to within-study variance. Older age was significantly associated with higher sulfonylurea prescription (1.51 [1.29-1.76]), yet lower prescribing of metformin (0.70 [0.60-0.82]), SGLT2-I (0.57 [0.42-0.79]) and GLP1-RA (0.52 [0.40-0.69]); while higher baseline BMI showed opposite significant results (sulfonylurea: 0.76 [0.62-0.93], metformin: 1.22 [1.08-1.37], SGLT2-I: 1.88 [1.33-2.68], and GLP1-RA: 2.35 [1.54-3.59]). Both higher baseline HbA1c and having kidney-related problems were significantly associated with lower metformin prescription (0.74 [0.57-0.97], 0.39 [0.25-0.61]), but more insulin prescriptions (2.41 [1.87-3.10], 1.52 [1.10-2.10]). Also, DPP4-I prescriptions were higher for patients with kidney-related problems (1.37 [1.06-1.79]) yet lower among patients with higher HbA1c (0.82 [0.68-0.99]). Sex was significantly associated with GLP1-RA and thiazolidinedione prescribing (F:M; 1.38 [1.19-1.60] and 0.91 [0.84-0.98]). CONCLUSION: Several factors were identified as potential determinants of antidiabetic drug prescribing. The magnitude and significance of each factor differed by antidiabetic class. Patient's age and baseline BMI had the most significant association with the choice of four out of the seven studied antidiabetic drugs followed by the baseline HbA1c and kidney-related problems which had an impact on three studied antidiabetic drugs, whereas sex had the least impact on prescribing decision as it was associated with GLP1-RA and thiazolidinedione only.

BIOLOGICAL ANTIARRHYTHMICS-SODIUM CHANNEL INTERACTING PROTEINS.

Voltage gated Na channels (NaV) are essential for excitation of tissues. Mutations in NaVs cause a spectrum of human disease from autism and epilepsy to cardiac arrhythmias to skeletal myotonias. The carboxyl termini (CT) of NaV channels are hotspots for disease-causing mutations and are richly invested with protein interaction sites. We have focused on the regulation of NaV by two proteins that bind in this region: calmodulin (CaM) and non-secreted fibroblast growth factors (iFGF or FHF). CaM regulates NaV gating, mediating Ca2+-dependent inactivation (CDI) in a channel isoform-specific manner, while Ca2+-free CaM (apo-CaM) binding broadly regulates NaV opening and suppresses the arrhythmogenic late Na current (INa-L). FHFs inhibit CDI, in NaV isoforms that exhibit this property, and potently suppress INa-L, the latter requiring the amino terminus of the FHF. A peptide comprised of the first 39 amino acids of FHF1A is sufficient to inhibit INa-L, constituting a credible specific antiarrhythmic.

Effects of Interfacial Shear on Particle Aggregation at an Oil/Water Interface.

Using a Stokesian dynamics simulation, the microstructure of particle aggregates at an oil/water interface with an applied Couette flow is studied. The results of the aggregation are consistent with previously published experimental work demonstrating multiple regimes of behavior based on the relative strength of shear and capillary forces. In previous work, densification of aggregates at low shear rates was theorized to occur due to short time scale fragmentation/reaggregation of aggregates with rigid particle bonds. In simulations, densification is observed at low shear rates but occurs due to local reorganization of particles due to capillary torques over long time scales. Moderate shear rates create mobile bonds between particles at shorter time scales, allowing aggregates to fragment without reaggregation into smaller isolated clusters, consistent with prior experimental work. At the highest shear rates, aggregation is inhibited completely.

The effect of time-of-day and circadian phase on vulnerability to seizure-induced death in two mouse models.

KEY POINTS: Sudden unexpected death in epilepsy (SUDEP) is the leading cause of premature death in patients with refractory epilepsy. SUDEP typically occurs during the night, although the reason for this is unclear. We found that, in normally entrained mice, time-of-day alters vulnerability to seizure-induced death. We found that, in free-running mice, circadian phase alters the vulnerability to seizure-induced death. These findings suggest that circadian rhythmicity may be responsible for the increased night-time prevalence of SUDEP ABSTRACT: Sudden unexpected death in epilepsy (SUDEP) is the leading cause of epilepsy-related death. SUDEP typically occurs during the night following a seizure. Many aspects of mammalian physiology are regulated by circadian rhythms in ways that might make seizures occuring during the night more dangerous. Using two mouse models of seizure-induced death, we demonstrate that time-of-day and circadian rhythms alter vulnerability to seizure-induced death. We exposed normally entrained DBA/1 mice to a potentially seizure-inducing acoustic stimulus at different times of day and compared the characteristics and outcomes of the seizures. Time-of-day did not alter the probability of a seizure but it did alter the probability of seizure-induced death. To determine whether circadian rhythms alter vulnerability to seizure-induced death, we induced maximal electroshock seizures in free-running C57BL/6J mice at different circadian time points at the same time as measuring breathing via whole body plethysmography. Circadian phase did not affect seizure severity but it did alter postictal respiratory outcomes and the probability of seizure-induced death. By contrast to our expectations, in entrained and free-running mice, vulnerability to seizure-induced death was greatest during the night and subjective night, respectively. These findings suggest that circadian rhythmicity may be responsible for the increased night-time prevalence of SUDEP and that the underlying mechanism is phase conserved between nocturnal and diurnal mammals. All of the seizures in the present study were induced during wakefulness, indicating that the effect of time point on vulnerability to seizure-induced death was not the result of sleep. Understanding why SUDEP occurs more frequently during the night may inform future preventative countermeasures.

Effect of collagen and EPS components on the viscoelasticity of Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa is an opportunistic pathogen that causes thousands of deaths every year in part due to its ability to form biofilms composed of bacteria embedded in a matrix of self-secreted extracellular polysaccharides (EPS), e-DNA, and proteins. In chronic wounds, biofilms are exposed to the host extracellular matrix, of which collagen is a major component. How bacterial EPS interacts with host collagen and whether this interaction affects biofilm viscoelasticity is not well understood. Since physical disruption of biofilms is often used in their removal, knowledge of collagen's effects on biofilm viscoelasticity may enable new treatment strategies that are better tuned to biofilms growing in host environments. In this work, biofilms are grown in the presence of different concentrations of collagen that mimic in vivo conditions. In order to explore collagen's interaction with EPS, nine strains of P. aeruginosa with different patterns of EPS production were used to grow biofilms. Particle tracking microrheology was used to characterize the mechanical development of biofilms over two days. Collagen is found to decrease biofilm compliance and increase relative elasticity regardless of the EPS present in the system. However, this effect is minimized when biofilms overproduce EPS. Collagen appears to become a de facto component of the EPS, through binding to bacteria or physical entanglement.

Bilobal architecture is a requirement for calmodulin signaling to CaV1.3 channels.

Calmodulin (CaM) regulation of voltage-gated calcium (CaV) channels is a powerful Ca2+ feedback mechanism that adjusts Ca2+ influx, affording rich mechanistic insights into Ca2+ decoding. CaM possesses a dual-lobed architecture, a salient feature of the myriad Ca2+-sensing proteins, where two homologous lobes that recognize similar targets hint at redundant signaling mechanisms. Here, by tethering CaM lobes, we demonstrate that bilobal architecture is obligatory for signaling to CaV channels. With one lobe bound, CaV carboxy tail rearranges itself, resulting in a preinhibited configuration precluded from Ca2+ feedback. Reconstitution of two lobes, even as separate molecules, relieves preinhibition and restores Ca2+ feedback. CaV channels thus detect the coincident binding of two Ca2+-free lobes to promote channel opening, a molecular implementation of a logical NOR operation that processes spatiotemporal Ca2+ signals bifurcated by CaM lobes. Overall, a unified scheme of CaV channel regulation by CaM now emerges, and our findings highlight the versatility of CaM to perform exquisite Ca2+ computations.

Enhancement of human iPSC-derived cardiomyocyte maturation by chemical conditioning in a 3D environment.

Recent advances in the understanding and use of pluripotent stem cells have produced major changes in approaches to the diagnosis and treatment of human disease. An obstacle to the use of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for regenerative medicine, disease modeling and drug discovery is their immature state relative to adult myocardium. We show the effects of a combination of biochemical factors, thyroid hormone, dexamethasone, and insulin-like growth factor-1 (TDI) on the maturation of hiPSC-CMs in 3D cardiac microtissues (CMTs) that recapitulate aspects of the native myocardium. Based on a comparison of the gene expression profiles and the structural, ultrastructural, and electrophysiological properties of hiPSC-CMs in monolayers and CMTs, and measurements of the mechanical and pharmacological properties of CMTs, we find that TDI treatment in a 3D tissue context yields a higher fidelity adult cardiac phenotype, including sarcoplasmic reticulum function and contractile properties consistent with promotion of the maturation of hiPSC derived cardiomyocytes.

Structural and Functional Dynamics of Soil Microbes following Spruce Beetle Infestation.

As the range of bark beetles expands into new forests and woodlands, the need to understand their effects on multiple trophic levels becomes increasingly important. To date, much attention has been paid to the aboveground processes affected by bark beetle infestation, with a focus on photoautotrophs and ecosystem level processes. However, indirect effects of bark beetle on belowground processes, especially the structure and function of soil microbiota remains largely a black box. Our study examined the impacts of bark beetle-induced tree mortality on soil microbial community structure and function using high-throughput sequencing of the soil bacterial and fungal communities and measurements of extracellular enzyme activities. The results suggest bark beetle infestation affected edaphic conditions through increased soil water content, pH, electrical conductivity, and carbon/nitrogen ratio and altered bulk and rhizosphere soil microbial community structure and function. Finally, increased enzymatic activity suggests heightened microbial decomposition following bark beetle infestation. With this increase in enzymatic activity, nutrients trapped in organic substrates may become accessible to seedlings and potentially alter the trajectory of forest regeneration. Our results indicate the need for incorporation of microbial processes into ecosystem level models.IMPORTANCE Belowground impacts of bark beetle infestation have not been explored as thoroughly as their aboveground counterparts. In order to accurately model impacts of bark beetle-induced tree mortality on carbon and nutrient cycling and forest regeneration, the intricacies of soil microbial communities must be examined. In this study, we investigated the structure and function of soil bacterial and fungal communities following bark beetle infestation. Our results show bark beetle infestation to impact soil conditions, as well as soil microbial community structure and function.

Role of the gut microbiome in mediating standard metabolic rate after dietary shifts in the viviparous cockroach, Diploptera punctata.

Diet may be a significant determinant of insect gut microbiome composition. However, the extent to which dietary shifts shape both the composition and relevant functions of insect gut microbiomes, and ultimately impact host energy balance (i.e. metabolic phenotype), is not well understood. We investigated the impacts of diet switching on Diploptera punctata females maintained on a dog food (DF) diet relative to those fed a comparatively sub-optimal cellulose-amended dog food (CADF) diet for 4 weeks. After this period, dietary shift resulted in a significantly higher average mass-specific standard metabolic rate (SMR) in CADF-fed females compared with DF-fed females. We also uncovered significant 13C-enrichment in DF-fed insect samples relative to CADF-fed insect samples and lowered bacterial essential amino acid (EAA) provisioning in CADF-fed samples. Differences in SMR and EAA provisioning were not accompanied by significant differences in overall microbiome composition between the two groups. However, cellulolytic and nitrogen-fixing bacterial families dominant in wild omnivorous cockroaches and wood-feeding termites were significantly enriched in CADF-fed females than in DF-fed females, at the end of the study. We propose that these changes in microbiome composition after dietary shifts are associated with changes in EAA provisioning and possibly SMR. Further studies are needed to comprehensively understand the relative importance of gut microbial functions among the complexity of factors known to underscore SMR responses in insects under varying dietary conditions.

Desmin Phosphorylation Triggers Preamyloid Oligomers Formation and Myocyte Dysfunction in Acquired Heart Failure.

RATIONALE: Disrupted proteostasis is one major pathological trait that heart failure (HF) shares with other organ proteinopathies, such as Alzheimer and Parkinson diseases. Yet, differently from the latter, whether and how cardiac preamyloid oligomers (PAOs) develop in acquired forms of HF is unclear. OBJECTIVE: We previously reported a rise in monophosphorylated, aggregate-prone desmin in canine and human HF. We now tested whether monophosphorylated desmin acts as the seed nucleating PAOs formation and determined whether positron emission tomography is able to detect myocardial PAOs in nongenetic HF. METHODS AND RESULTS: Here, we first show that toxic cardiac PAOs accumulate in the myocardium of mice subjected to transverse aortic constriction and that PAOs comigrate with the cytoskeletal protein desmin in this well-established model of acquired HF. We confirm this evidence in cardiac extracts from human ischemic and nonischemic HF. We also demonstrate that Ser31 phosphorylated desmin aggregates extensively in cultured cardiomyocytes. Lastly, we were able to detect the in vivo accumulation of cardiac PAOs using positron emission tomography for the first time in acquired HF. CONCLUSIONS: Ser31 phosphorylated desmin is a likely candidate seed for the nucleation process leading to cardiac PAOs deposition. Desmin post-translational processing and misfolding constitute a new, attractive avenue for the diagnosis and treatment of the cardiac accumulation of toxic PAOs that can now be measured by positron emission tomography in acquired HF.