PAS Domain Research Articles

A role for the circadian transcription factor NPAS2 in the progressive loss of non-rapid eye movement sleep and increased arousal during fentanyl withdrawal in male mice.

Synthetic opioids like fentanyl are contributing to the rise in rates of opioid use disorder and drug overdose deaths. Sleep dysfunction and circadian rhythm disruption may worsen during opioid withdrawal and persist during abstinence. Severe and persistent sleep and circadian alterations are putative factors in opioid craving and relapse. However, very little is known about the impact of fentanyl on sleep architecture and sleep-wake cycles, particularly opioid withdrawal. Further, circadian rhythms regulate sleep-wake cycles, and the circadian transcription factor, neuronal PAS domain 2 (NPAS2) is involved in the modulation of sleep architecture and drug reward. Here, we investigate the role of NPAS2 in fentanyl-induced sleep alterations. To determine the effect of fentanyl administration and withdrawal on sleep architecture, and the role of NPAS2 as a factor in fentanyl-induced sleep changes. Electroencephalography (EEG) and electromyography (EMG) was used to measure non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS) at baseline and following acute and chronic fentanyl administration in wild-type and NPAS2-deficient male mice. Acute and chronic administration of fentanyl led to increased wake and arousal in both wild-type and NPAS2-deficient mice, an effect that was more pronounced in NPAS2-deficient mice. Chronic fentanyl administration led to decreased NREMS, which persisted during withdrawal, progressively decreasing from day 1 to 4 of withdrawal. The impact of fentanyl on NREMS and arousal was more pronounced in NPAS2-deficient mice. Chronic fentanyl disrupts NREMS, leading to a progressive loss of NREMS during subsequent days of withdrawal. Loss of NPAS2 exacerbates the impact of fentanyl on sleep and wake, revealing a potential role for the circadian transcription factor in opioid-induced sleep changes.

EPAS1 Promoter Hypermethylation is a Diagnostic and Prognostic Biomarker for Non-Small Cell Lung Cancer.

Background: The importance of promoter methylation in non-small cell lung cancers (NSCLC) remains to be understood. Thus, we aimed to determine the diagnostic and prognostic value of the methylation of the endothelial PAS domain containing protein-1 (EPAS1) promoter in NSCLC. Methods: EPAS1 promoter methylation levels were quantitated by methylation-specific PCR. Further, we evaluated the expression, promoter methylation, prognostic value, and impact on immune cell infiltration of EPAS1 by analyzing the TCGA database using web-based bioinformatics tools including GEPIA, UALCAN and MethSurv. Results: Our results demonstrated that promoter methylation of EPAS1 downregulated its expression in NSCLC tissues. Additionally, an AUC value of 0.772 indicated that the methylation of the EPAS1 promoter is a potential diagnostic marker for NSCLC. A Kaplan-Meier analysis demonstrated that high methylation levels of CpG sites in the EPAS1 promoter were indicative of poorer overall survival. Further, EPAS1 expression levels were highly correlated with the infiltration of several types of immune cells, including γδ T cells, T follicular helper cells, CD8+ T cells, and CD4+ T-cells. Conclusion: Collectively, our findings suggest that methylation analyses of the EPAS1 promoter could be used as a prognostic biomarker for NSCLC and that EPAS1 potentially plays an important role in immune cell infiltration in NSCLC.

The Histidine Kinase CckA Is Directly Inhibited by a Response Regulator-like Protein in a Negative Feedback Loop.

ABSTRACTIn alphaproteobacteria, the two-component system (TCS) formed by the hybrid histidine kinase CckA, the phosphotransfer protein ChpT, and the response regulator CtrA is widely distributed. In these microorganisms, this system controls diverse functions such as motility, DNA repair, and cell division. In Caulobacterales and Rhizobiales, CckA is regulated by the pseudo- histidine kinase DivL, and the response regulator DivK. However, this regulatory circuit differs for other bacterial groups. For instance, in Rhodobacterales, DivK is absent and DivL consists of only the regulatory PAS domain. In this study, we report that, in Rhodobacter sphaeroides, the kinase activity of CckA is inhibited by Osp, a single domain response regulator (SDRR) protein that directly interacts with the transmitter domain of CckA. In vitro, the kinase activity of CckA was severely inhibited with an equimolar amount of Osp, whereas the phosphatase activity of CckA was not affected. We also found that the expression of osp is activated by CtrA creating a negative feedback loop. However, under growth conditions known to activate the TCS, the increased expression of osp does not parallel Osp accumulation, indicating a complex regulation. Phylogenetic analysis of selected species of Rhodobacterales revealed that Osp is widely distributed in several genera. For most of these species, we found a sequence highly similar to the CtrA-binding site in the control region of osp, suggesting that the TCS CckA/ChpT/CtrA is controlled by a novel regulatory circuit that includes Osp in these bacteria.

Subtle sequence differences between two interacting σ54 -dependent regulators lead to different activation mechanisms.

In the strictly anaerobic nitrate reducing bacterium Aromatoleum anaerobium, degradation of 1,3-dihydroxybenzene (1,3-DHB, resorcinol) is controlled by two bacterial enhancer-binding proteins (bEBPs), RedR1 and RedR2, which regulate the transcription of three σ54 -dependent promoters controlling expression of the pathway. RedR1 and RedR2 are identical over their length except for their N-terminal tail which differ in sequence and length (six and eight residues, respectively), a single change in their N-terminal domain (NTD), and nine non-identical residues in their C-terminal domain (CTD). Their NTD is composed of a GAF and a PAS domain connected by a linker helix. We show that each regulator is controlled by a different mechanism: whilst RedR1 responds to the classical NTD-mediated negative regulation that is released by the presence of its effector, RedR2 activity is constitutive and controlled through interaction with BtdS, an integral membrane subunit of hydroxyhydroquinone dehydrogenase carrying out the second step in 1,3-DHB degradation. BtdS sequesters the RedR2 regulator to the membrane through its NTD, where a four-Ile track in the PAS domain, interrupted by a Thr in RedR1, and the N-terminal tail are involved. The presence of 1,3-DHB, which is metabolized to hydroxybenzoquinone, releases RedR2 from the membrane. Most bEBPs assemble into homohexamers to activate transcription; we show that hetero-oligomer formation between RedR1 and RedR2 is favoured over homo-oligomers. However, either an NTD-truncated version of RedR1 or a full-length RedR2 are capable of promoter activation on their own, suggesting they should assemble into homohexamers in vivo. We show that promoter DNA behaves as an allosteric effector through binding the CTD to control ΔNTD-RedR1 multimerization and activity. Overall, the regulation of the 1,3-DHB anaerobic degradation pathway can be described as a novel mode of bEBP activation and assembly.

Impact of Double Covalent Binding of BV in NIR FPs on Their Spectral and Physicochemical Properties.

Understanding the photophysical properties and stability of near-infrared fluorescent proteins (NIR FPs) based on bacterial phytochromes is of great importance for the design of efficient fluorescent probes for use in cells and in vivo. Previously, the natural ligand of NIR FPs biliverdin (BV) has been revealed to be capable of covalent binding to the inherent cysteine residue in the PAS domain (Cys15), and to the cysteine residue introduced into the GAF domain (Cys256), as well as simultaneously with these two residues. Here, based on the spectroscopic analysis of several NIR FPs with both cysteine residues in PAS and GAF domains, we show that the covalent binding of BV simultaneously with two domains is the reason for the higher quantum yield of BV fluorescence in these proteins as a result of rigid fixation of the chromophore in their chromophore-binding pocket. We demonstrate that since the attachment sites are located in different regions of the polypeptide chain forming a figure-of-eight knot, their binding to BV leads to shielding of many sites of proteolytic degradation due to additional stabilization of the entire protein structure. This makes NIR FPs with both cysteine residues in PAS and GAF domains less susceptible to cleavage by intracellular proteases.

A Dual Regulatory Role of the PhoU Protein in Salmonella Typhimurium.

ABSTRACTBacteria utilize two-component regulatory systems to sense and respond to their surroundings. Unlike other two-component systems that directly sense through a sensory domain in the histidine kinase (HK), the PhoB/PhoR two-component system requires additional proteins, including the PstSCAB phosphate transporter and the PhoU protein, to sense phosphate levels. Although PhoU is involved in phosphate signaling by connecting the PstSCAB transporter and PhoR histidine kinase, the mechanism by which PhoU controls expression of pho regulon genes has not yet been clearly understood. Here, we identified PhoU residues required for interacting with PhoR histidine kinase from the intracellular pathogen Salmonella enterica serovar Typhimurium. The PhoU Ala147 residue interacts with the PhoR PAS domain and is involved in repressing pho expression in high phosphate. Unexpectedly, the PhoU Arg184 residue interacts with the PhoR histidine kinase domain and is required for activating pho expression in low Mg2+ by increasing PhoR autophosphorylation, revealing its new function. The substitution of the Arg184 to Gly codon decreased Salmonella virulence both in macrophages and in mice, suggesting that PhoU’s role in promoting PhoR autophosphorylation is required during Salmonella infection.

HCMV-miR-US33-5p promotes apoptosis of aortic vascular smooth muscle cells by targeting EPAS1/SLC3A2 pathway

BackgroundIn patients with acute aortic dissection (AAD), increased vascular smooth muscle cell (VSMC) apoptosis has been found. Human cytomegalovirus (HCMV)-miR-US33-5p was significantly increased in the plasma of patients with AAD. However, the roles of miR-US33-5p in human aortic VSMC (HA-VSMC) apoptosis remain to be elucidated.MethodsIn the current study, cell apoptosis was analyzed by flow cytometry, cell proliferation by CCK-8 assay, and differentially expressed genes by RNA sequencing. Luciferase reporter assay was used for binding analysis between miR-US33-5p and endothelial PAS domain protein 1 (EPAS1), and EPAS1 and amino acid transporter heavy chain, member 2 (SLC3A2). The enrichment degree of SLC3A2 promoter DNA was analyzed by chromatin immunoprecipitation assay. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and immunoblotting were performed for measuring messenger RNA (mRNA) and protein levels, respectively.ResultsIt was found that HCMV infection inhibited proliferation but promoted HA-VSMC apoptosis by upregulating HCMV-miR-US33-5p. Transfection of HCMV-miR-US33-5p mimics the significant effect on several signaling pathways including integrin signaling as shown in the RNA sequencing data. Western blotting analysis confirmed that HCMV-miR-US33-5p mimics suppression of the activity of key factors of the integrin signal pathway including FAK, AKT, CAS, and Rac. Mechanistic study showed that HCMV-miR-US33-5p bound to the 3′-untranslated region of EPAS1 to suppress its expression, leading to suppression of SLC3A2 expression, which ultimately promoted cell apoptosis and inhibited cell proliferation. This was confirmed by the findings that silencing EPAS1 significantly reduced the SLC3A2 expression and inhibited proliferation and key factors of integrin signal pathway.ConclusionsHCMV-miR-US33-5p suppressed proliferation, key factors of integrin signal pathway, and EPAS1/SLC3A2 expression, but promoted HA-VSMC apoptosis. These findings highlighted the importance of HCMV-miR-US33-5p/EPAS1/SCL3A2 signaling and may provide new insights into therapeutic strategies for AAD.

Plant Phytochrome B Assembles an Asymmetric Dimer with Unique Signaling Potential

Phytochromes (Phy) sense red and far‐red light and reversibly interconvert between inactive Pr and active Pfr conformers. Phys typically consist of a photo sensory module (PSM) formed by sequential N‐terminal PAS, GAF, and PHY domains, followed by a C‐terminal histidine kinase‐related domain (HKRD). There are four plant isoforms Phy A through D, and they all contain two additional PAS domains (PAS1 and PAS2) inserted between PSM and HKRD. Despite extensive biochemical and structural studies, there has been a lack of a full‐length structure, hindering our understanding of plant Phy signaling. Here we report a 3.3‐Å cryo‐EM structure of Arabidopsis PhyB dimer. Instead of the widely anticipated parallel association of the two monomers resembling the prokaryotic relatives, Plant PhyB adopts a topologically complex dimeric architecture with the two PSMs in a head‐to‐tail antiparallel form while the two HKRDs in a head‐to‐head parallel form. The PAS2 mediates the antiparallel dimerization of PSM that leads to a parallelogram‐shaped platform. And a preceding ‘modulator’ loop assembles tightly with the PSM of its own protomer. We show that these inter‐ and intra‐protomer connections contribute to stabilizing of dimer and accelerate thermal reversion of Pfr back to Pr. Lean of HKRDs towards the platform results in profound asymmetry of PhyB that might enable distinct signaling potentials to the protomers. We propose that this unique structural dynamism creates an extensive photostate‐sensitive surface for interaction with their downstream signaling partners.

The Circadian Transcription Factor NPAS2 Modulates Fentanyl-mediated Withdrawal and Hyperalgesia but Not Tolerance, in a Sex-dependent Manner.

Prescription opioids are the mainstay treatment for severe pain. Unfortunately, their chronic use can cause misuse, addiction, and overdoses death. Risks for these life-threatening outcomes are increased by side-effects which develop upon long-term opioid use, including analgesic tolerance, opioid-induced hypersensitivity, and physical dependence. In addition, altered circadian rhythmicity and disruption of sleep patterns are also a common trait in patients on opioid therapy. Prior research suggested intricate bidirectional interactions between circadian rhythmicity, the pathophysiology of pain, and opioid-mediated analgesia and side-effects. Circadian rhythms are generated by the rhythmic expression of clock genes such as the circadian transcription factor Neuronal PAS domain protein 2 (Npas2) that is expressed in DRG, spinal cord and brain structures involved in pain modulation and opioid analgesia. However, whether it is involved in opioid side-effects was never investigated. Thus, the present study was designed to explore the possible role of NPAS2 signaling in fentanyl-induced side-effects. Using male and female mutant mice with non-functional NPAS2 protein (NPAS2-/-), we performed behavioral studies to evaluate the impact of the absence of NPAS2 signaling on behavioral models of fentanyl-induced analgesia, tolerance, hyperalgesia, and dependence. Our findings show that thermal pain thresholds, acute analgesia, and tolerance development to a fixed dose of fentanyl were unchanged in male and female NPAS2-deficient mice. However, we found that female but not male NPAS2-defficient mice displayed a decreased potency of high doses of fentanyl after tolerance development. In addition, female NPAS2-defficient mice showed markedly more symptoms of physical dependence when withdrawal was precipitated by naloxone, which was not observed in male mice. Conversely, while female NPAS2-defficient mice developed similar fentanyl-induced hypersensitivity, male NPAS2-defficient mice had higher fentanyl-induced hypersensitivity, when compared to matched-sex WT littermate controls. Together, our findings suggest sex-specific effects of NPAS2 signaling in the regulation of fentanyl-induced tolerance, hyperalgesia, and dependence. Grant support from K01DA038654; R21DA041872; R01HL150432. Prescription opioids are the mainstay treatment for severe pain. Unfortunately, their chronic use can cause misuse, addiction, and overdoses death. Risks for these life-threatening outcomes are increased by side-effects which develop upon long-term opioid use, including analgesic tolerance, opioid-induced hypersensitivity, and physical dependence. In addition, altered circadian rhythmicity and disruption of sleep patterns are also a common trait in patients on opioid therapy. Prior research suggested intricate bidirectional interactions between circadian rhythmicity, the pathophysiology of pain, and opioid-mediated analgesia and side-effects. Circadian rhythms are generated by the rhythmic expression of clock genes such as the circadian transcription factor Neuronal PAS domain protein 2 (Npas2) that is expressed in DRG, spinal cord and brain structures involved in pain modulation and opioid analgesia. However, whether it is involved in opioid side-effects was never investigated. Thus, the present study was designed to explore the possible role of NPAS2 signaling in fentanyl-induced side-effects. Using male and female mutant mice with non-functional NPAS2 protein (NPAS2-/-), we performed behavioral studies to evaluate the impact of the absence of NPAS2 signaling on behavioral models of fentanyl-induced analgesia, tolerance, hyperalgesia, and dependence. Our findings show that thermal pain thresholds, acute analgesia, and tolerance development to a fixed dose of fentanyl were unchanged in male and female NPAS2-deficient mice. However, we found that female but not male NPAS2-defficient mice displayed a decreased potency of high doses of fentanyl after tolerance development. In addition, female NPAS2-defficient mice showed markedly more symptoms of physical dependence when withdrawal was precipitated by naloxone, which was not observed in male mice. Conversely, while female NPAS2-defficient mice developed similar fentanyl-induced hypersensitivity, male NPAS2-defficient mice had higher fentanyl-induced hypersensitivity, when compared to matched-sex WT littermate controls. Together, our findings suggest sex-specific effects of NPAS2 signaling in the regulation of fentanyl-induced tolerance, hyperalgesia, and dependence. Grant support from K01DA038654; R21DA041872; R01HL150432.

Enhanced T cell effector function upon differentiation of CD8+ and CD4+ T cells in the presence of BioE-1197 through potential off target ARK family inhibition

Abstract We sought to evaluate PAS domain containing kinase (PASK) inhibition during T cell differentiation using the previously described PASK inhibitor BioE-1197. Notably, CD8+, Th1, and Th2 T cells activated and expanded in the presence of BioE-1197 display elevated lineage specific cytokine production upon restimulation compared to control differentiated T cells, suggesting BioE-1197 may prove to be a powerful means to enhance T cell function. Curiously however, BioE-1197 mediated these effects even in PASK KO T cells suggesting its mechanism of action was independent of PASK. BLAST alignment of the PASK kinase domain revealed members of the AMPK related kinases (ARK) family as potential targets of BioE-1197. ARKs phosphorylate class IIa histone deacetylases (HDACs) in response to activation. To assess whether BioE-1197 inhibits ARK family member activity, we activated and expanded CD8+ T cells with or without BioE-1197. Control differentiated CD8s show elevated phosphorylated and total HDAC7 over time after activation. However, BioE-1197 differentiated cells do not exhibit elevated phosphorylation or total expression of HDAC7 after activation. Concurrent reduction of phosphorylation and total HDAC7 is a phenotype previously reported in cells treated with ARK family kinase inhibitors, suggesting BioE-1197 may be functioning to inhibit an ARK family member(s). Ongoing work aims to definitively demonstrate that the ability of BioE-1197 to enhance effector function is via inhibition of ARK family members and the subsequent regulation of HDAC7. Overall these studies identify BioE-1197 as a novel agent to enhance effector T cell function and suggests potential utility in tumor immunotherapy and enhancing vaccine efficacy. Supported by grants from NIH (P41EB028239-03, R01AI077610-10) and the Bloomberg-Kimmel Institute for Cancer Immunotherapy.

Hysteretic hERG channel gating current recorded at physiological temperature

Cardiac hERG channels comprise at least two subunits, hERG 1a and hERG 1b, and drive cardiac action potential repolarization. hERG 1a subunits contain a cytoplasmic PAS domain that is absent in hERG 1b. The hERG 1a PAS domain regulates voltage sensor domain (VSD) movement, but hERG VSD behavior and its regulation by the hERG 1a PAS domain have not been studied at physiological temperatures. We recorded gating charge from homomeric hERG 1a and heteromeric hERG 1a/1b channels at near physiological temperatures (36 ± 1 °C) using pulse durations comparable in length to the human ventricular action potential. The voltage dependence of deactivation was hyperpolarized relative to activation, reflecting VSD relaxation at positive potentials. These data suggest that relaxation (hysteresis) works to delay pore closure during repolarization. Interestingly, hERG 1a VSD deactivation displayed a double Boltzmann distribution, but hERG 1a/1b deactivation displayed a single Boltzmann. Disabling the hERG 1a PAS domain using a PAS-targeting antibody similarly transformed hERG 1a deactivation from a double to a single Boltzmann, highlighting the contribution of the PAS in regulating VSD movement. These data represent, to our knowledge, the first recordings of hERG gating charge at physiological temperature and demonstrate that VSD relaxation (hysteresis) is present in hERG channels at physiological temperature.

Designing Stable Bacillus anthracis Antigens with a View to Recombinant Anthrax Vaccine Development.

Anthrax is a disease caused by Bacillus anthracis that affects mammals, including humans. Recombinant B. anthracis protective antigen (rPA) is the most common basis for modern anthrax vaccine candidates. However, this protein is characterised by low stability due to proteolysis and deamidation. Here, for the first time, two modification variants leading to full-size rPA stabilisation have been implemented simultaneously, through deamidation-prone asparagine residues substitution and by inactivation of proteolysis sites. Obtained modified rPA (rPA83m) has been demonstrated to be stable in various temperature conditions. Additionally, rPA1+2 containing PA domains I and II and rPA3+4 containing domains III and IV, including the same modifications, have been shown to be stable as well. These antigens can serve as the basis for a vaccine, since the protective properties of PA can be attributed to individual PA domains. The stability of each of three modified anthrax antigens has been considerably improved in compositions with tobacco mosaic virus-based spherical particles (SPs). rPA1+2/rPA3+4/rPA83m in compositions with SPs have maintained their antigenic specificity even after 40 days of incubation at +37 °C. Considering previously proven adjuvant properties and safety of SPs, their compositions with rPA83m/rPA1+2/rPA3+4 in any combinations might be suitable as a basis for new-generation anthrax vaccines.

Cross-sectional and longitudinal associations of active travel, organised sport and physical education with accelerometer-assessed moderate-to-vigorous physical activity in young people: the International Children\u2019s Accelerometry Database

BackgroundPhysical activity (PA) declines during childhood. Important sources of PA are active travel, organised sport and physical education (PE), but it is unclear how these domain-specific PA sources contribute to (changes in) daily moderate-to-vigorous PA (MVPA) in young people. This study aimed to examine (1) the cross-sectional association between domain-specific physical activity (i.e., active travel, organised sport and PE) and daily minutes in accelerometer-assessed MVPA; and (2) the longitudinal association between domain-specific physical activity at baseline and change in daily minutes in MVPA.MethodsParticipants (baseline age 11.3 ± .1.2 years) were drawn from three studies in the International Children’s Accelerometry Database. The contribution of self-reported standardised active travel, organised sport and PE to accelerometer-measured daily minutes in MVPA was examined using linear regression. In cross-sectional analyses, MVPA was regressed on each PA domain in separate models, adjusted for study, age, sex, maternal education, season, and monitor wear time. In longitudinal analyses, change in MVPA was regressed on each of the baseline PA domains, additionally adjusting for changes in season and wear time, follow-up duration, and baseline MVPA. R-squared was used to compare variance explained by each PA domain.ResultsIn the cross-sectional analyses (n = 3871), organised sport (standardised β = 3.81, 95% confidence interval [95%CI] = 3.06, 4.56) and active travel (β = 3.46, 95%CI = 2.73, 4.19) contributed more to daily MVPA than PE (β = 0.82, 95%CI = -0.02, 1.66). Compared to the base model which included only covariates (R2 = 21.5%), organised sport (absolute change: + 1.9%) and active travel (+ 1.7%) models explained more of the variance than the PE model (± < 0.1%). Associations followed a similar pattern in the longitudinal analyses (n = 2302), but none of the PA domains predicted change in MVPA (organised sport: standardised β = 0.85, 95%CI = -0.03, 1.72; active travel: β = 0.68, 95%CI = -0.14, 1.50; PE: β = 0.02, 95%CI = -0.87, 0.91).ConclusionsA multi-sectoral approach covering a wide range of PA domains should be promoted to minimise the age-related decline in MVPA during childhood.

Cyclin C-Cdk8 Kinase Phosphorylation of Rim15 Prevents the Aberrant Activation of Stress Response Genes.

Cells facing adverse environmental cues respond by inducing signal transduction pathways resulting in transcriptional reprograming. In the budding yeast Saccharomyces cerevisiae, nutrient deprivation stimulates stress response gene (SRG) transcription critical for entry into either quiescence or gametogenesis depending on the cell type. The induction of a subset of SRGs require nuclear translocation of the conserved serine-threonine kinase Rim15. However, Rim15 is also present in unstressed nuclei suggesting that additional activities are required to constrain its activity in the absence of stress. Here we show that Rim15 is directly phosphorylated by cyclin C-Cdk8, the conserved kinase module of the Mediator complex. Several results indicate that Cdk8-dependent phosphorylation prevents Rim15 activation in unstressed cells. First, Cdk8 does not control Rim15 subcellular localization and rim15∆ is epistatic to cdk8∆ with respect to SRG transcription and the execution of starvation programs required for viability. Next, Cdk8 phosphorylates a residue in the conserved PAS domain in vitro. This modification appears important as introducing a phosphomimetic at Cdk8 target residues reduces Rim15 activity. Moreover, the Rim15 phosphomimetic only compromises cell viability in stresses that induce cyclin C destruction as well as entrance into meiosis. Taken together, these findings suggest a model in which Cdk8 phosphorylation contributes to Rim15 repression whilst it cycles through the nucleus. Cyclin C destruction in response to stress inactivates Cdk8 which in turn stimulates Rim15 to maximize SRG transcription and cell survival.

A recessive variant in SIM2 in a child with complex craniofacial anomalies and global developmental delay

Rare deletions and duplications on the long arm of Chromosome 21 have previously been reported in many patients with craniofacial and developmental phenotypes. However, this Down Syndrome Critical Region (DSCR) contains multiple genes, making identifying a single causative gene difficult. Here, we report a case of a boy with bicoronal craniosynostosis, facial dysmorphism, developmental delay, and intellectual impairment who was found by whole genome sequencing to have a homozygous missense mutation in the Single-Minded Homolog 2 (SIM2) gene (c.461 A > G, p.Tyr154Cys) within the DSCR. SIM2 encodes an essential bHLH and PAS domain transcription factor expressed during fetal brain development and acts as a master regulator of neurogenesis. This variant is globally very rare, segregates in the family, and is predicted to be highly deleterious by in silico analysis, 3D molecular modeling of protein structure, and functional analysis of zebrafish models. Zebrafish expressing the human SIM2p.Y154C variant displayed a progressed microcephaly-like phenotype and head shape abnormalities. When combined with careful phenotyping of the patient vis-à-vis previously reported cases harboring structural variants in this critical 21q22 region, the data support a pathogenic role of SIM2 in this complex syndrome and demonstrates the utility of next-generation sequencing in prioritizing genes in contiguous deletions/duplications syndromes and diagnosing microarray-negative patients in the craniofacial clinic.

Functional upregulation of hERG1b ion channels by direct interactions with the hERG1a N-terminal PAS domain and N-linker regions

A major physiological role of hERG1 (human Ether-á-go-go-Related Gene) potassium channels is to repolarize cardiac action potentials. Two isoforms, hERG1a and hERG1b, associate to form the native cardiac IKr current in vivo. Inherited mutations in hERG1a or hERG1b cause prolonged cardiac repolarization, Long QT Syndrome and sudden death arrhythmia. hERG1a subunits assemble with and enhance the number of hERG1b subunits at the plasma membrane, but the mechanism for the increase in hERG1b by hERG1a is not well understood.

Intranasal calcitonin gene-related peptide administration impairs fear memory retention in mice through the PKD/p-HDAC5/Npas4 pathway

The calcitonin gene-related peptide (CGRP) suppresses fear memory retention in mice. Although intracerebroventricular administration of CGRP alters the fear memory processes, making it a promising therapeutic strategy for post-traumatic stress disorder (PTSD), direct brain injection into patients is not practical. Therefore, we propose that intranasal application may be an effective way to deliver CGRP to the brain. This study tested whether CGRP nasal administration exerts the same effect as intracerebroventricular administration using C57BL6J mice. The amount of CGRP in the cerebrospinal fluid and hippocampus 30 min after nasal administration of CGRP was significantly higher when compared with saline. Intranasal CGRP also elicited photophobic behaviors similar to intracerebroventricular injection. Moreover, intranasal CGRP decreased fear memory retention but did not affect reactivation and extinction of fear memory. We found intranasal CGRP significantly increased the expression of protein kinase D (PKD), phosphorylated histone deacetylase 5 (p-HDAC5) and neuronal PAS domain protein 4 (Npas4) in the hippocampus. CGRP-mediated impairment of fear memory and Npas4 expression increases were attenuated significantly by the CGRP receptor antagonist BIBN4096. Together, our data demonstrate that intranasal CGRP delivery activates the PKD/p-HDAC5/Npas4 pathway, decreases fear memory retention.

Circadian transcription factor NPAS2 and the NAD+ -dependent deacetylase SIRT1 interact in the mouse nucleus accumbens and regulate reward.

Substance use disorders are associated with disruptions to both circadian rhythms and cellular metabolic state. At the molecular level, the circadian molecular clock and cellular metabolic state may be interconnected through interactions with the nicotinamide adenine dinucleotide (NAD+ )-dependent deacetylase, sirtuin 1 (SIRT1). In the nucleus accumbens (NAc), a region important for reward, both SIRT1 and the circadian transcription factor neuronal PAS domain protein 2 (NPAS2) are highly enriched, and both are regulated by the metabolic cofactor NAD+ . Substances of abuse, like cocaine, greatly disrupt cellular metabolism and promote oxidative stress; however, their effects on NAD+ in the brain remain unclear. Interestingly, cocaine also induces NAc expression of both NPAS2 and SIRT1, and both have independently been shown to regulate cocaine reward in mice. However, whether NPAS2 and SIRT1 interact in the NAc and/or whether together they regulate reward is unknown. Here, we demonstrate diurnal expression of Npas2, Sirt1 and NAD+ in the NAc, which is altered by cocaine-induced upregulation. Additionally, co-immunoprecipitation reveals NPAS2 and SIRT1 interact in the NAc, and cross-analysis of NPAS2 and SIRT1 chromatin immunoprecipitation sequencing reveals several reward-relevant and metabolic-related pathways enriched among shared gene targets. Notably, NAc-specific Npas2 knock-down or a functional Npas2 mutation in mice attenuates SIRT1-mediated increases in cocaine preference. Together, our data reveal an interaction between NPAS2 and SIRT1 in the NAc, which may serve to integrate cocaine's effects on circadian and metabolic factors, leading to regulation of drug reward.

Therapeutic downregulation of neuronal PAS domain 2 (Npas2) promotes surgical skin wound healing.

Attempts to minimize scarring remain among the most difficult challenges facing surgeons, despite the use of optimal wound closure techniques. Previously, we reported improved healing of dermal excisional wounds in circadian clock neuronal PAS domain 2 (Npas2)-null mice. In this study, we performed high-throughput drug screening to identify a compound that downregulates Npas2 activity. The hit compound (Dwn1) suppressed circadian Npas2 expression, increased murine dermal fibroblast cell migration, and decreased collagen synthesis in vitro. Based on the in vitro results, Dwn1 was topically applied to iatrogenic full-thickness dorsal cutaneous wounds in a murine model. The Dwn1-treated dermal wounds healed faster with favorable mechanical strength and developed less granulation tissue than the controls. The expression of type I collagen, Tgfβ1, and α-smooth muscle actin was significantly decreased in Dwn1-treated wounds, suggesting that hypertrophic scarring and myofibroblast differentiation are attenuated by Dwn1 treatment. NPAS2 may represent an important target for therapeutic approaches to optimal surgical wound management.

Phylogenetic analysis of phytochrome A gene from Lablab purpureus (L.) Sweet

BackgroundPhytochromes are the best characterized photoreceptors that perceive Red (R)/Far-Red (FR) signals and mediate key developmental responses in plants. It is well established that photoperiodic control of flowering is regulated by PHY A (phytochrome A) gene. So far, the members of PHY A gene family remains unexplored in Lablab purpureus, and therefore, their functions are still not deciphered. PHYA3 is the homologue of phytochrome A and known to be involved in dominant suppression of flowering under long day conditions by downregulating florigens in Glycine max. The present study is the first effort to identify and characterize any photoreceptor gene (PHYA3, in this study) in Lablab purpureus and decipher its phylogeny with related legumes. ResultsPHYA3 was amplified in Lablab purpureus cv GNIB-21 (photo-insensitive and determinate) by utilizing primers designed from GmPHYA3 locus of Glycine max. This study was successful in partially characterizing PHYA3 in Lablab purpureus (LprPHYA3) which is 2 kb longer and belongs to exon 1 region of PHYA3 gene. Phylogenetic analysis of the nucleotide and protein sequences of PHYA genes through MEGA X delineated the conservation and evolution of Lablab purpureus PHYA3 (LprPHYA3) probably from PHYA genes of Vigna unguiculata, Glycine max and Vigna angularis. A conserved basic helix-loop-helix motif bHLH69 was predicted having DNA binding property. Domain analysis of GmPHYA protein and predicted partial protein sequence corresponding to exon-1 of LprPHYA3 revealed the presence of conserved domains (GAF and PAS domains) in Lablab purpureus similar to Glycine max. ConclusionPartial characterization of LprPHYA3 would facilitate the identification of complete gene in Lablab purpureus utilizing sequence information from phylogenetically related species of Fabaceae. This would allow screening of allelic variants for LprPHYA3 locus and their role in photoperiod responsive flowering. The present study could aid in modulating photoperiod responsive flowering in Lablab purpureus and other related legumes in near future through genome editing.