PAS Domain Research Articles

Npas4-mediated dopaminergic regulation of safety memory consolidation

Amygdala circuitry encodes associations between conditioned stimuli and aversive unconditioned stimuli and also controls fear expression. However, whether and how non-threatening information for unpaired conditioned stimuli (CS-) is discretely processed remains unknown. The fear expression toward CS- is robust immediately after fear conditioning but then becomes negligible after memory consolidation. The synaptic plasticity of the neural pathway from the lateral to the anterior basal amygdala gates the fear expression of CS-, depending upon neuronal PAS domain protein 4 (Npas4)-mediated dopamine receptor D4 (Drd4) synthesis, which is precluded by stress exposure or corticosterone injection. Herein, we show cellular and molecular mechanisms that regulate the non-threatening (safety) memory consolidation, supporting the fear discrimination.

Discovery of a highly potent NPAS3 heterodimer inhibitor by covalently modifying ARNT

Neuronal PAS domain protein 3 (NPAS3), a basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) family member, is a pivotal transcription factor in neuronal regeneration, development, and related diseases, regulating the expression of downstream genes. Despite several modulators of certain bHLH-PAS family proteins being identified, the NPAS3-targeted compound has yet to be reported. Herein, we discovered a hit compound BI-78D3 that directly blocks the NPAS3-ARNT heterodimer formation by covalently binding to the aryl hydrocarbon receptor nuclear translocator (ARNT) subunit. Further optimization based on the hit scaffold yielded a highly potent Compound 6 with a biochemical EC50 value of 282 ± 61 nM and uncovered the 5-nitrothiazole-2-sulfydryl as a cysteine-targeting covalent warhead. Compound 6 effectively down-regulated NPAS3's transcriptional function by disrupting the interface of NPAS3-ARNT complexes at cellular level. In conclusion, our study identifies the 5-nitrothiazole-2-sulfydryl as a cysteine-modified warhead and provides a strategy that blocks the NPAS3-ARNT heterodimerization by covalently conjugating ARNT Cys336 residue. Compound 6 may serve as a promising chemical probe for exploring NPAS3-related physiological functions.

Clinal variation in PHY (PAS domain) and CRY (CCT domain)-Signs of local adaptation to light quality in Norway spruce.

Detection of the genomic basis of local adaptation to environmental conditions is challenging in forest trees. Phytochromes (PHY) and cryptochromes (CRY) perceive the red (R)/far-red (FR)and blue lightrespectively, thus playing a fundamental role in regulating plant growth and development. PHYO and PHYP from conifers are the equivalents of PHYA/PHYCand PHYB in angiosperms, respectively. Norway spruce shows an adaptive latitudinal cline for shade (low R:FR or FR-enriched light) tolerance and requirement of FR light for its growth. We analyzed the exome capture data that included a uniquely large data set of 1654 Norway spruce trees sampled across many latitudes in Sweden to capture the natural clines for photoperiod and FR light exposure during the growth season. Statistically significant clinal variation was detected in allele and genotype frequencies of missense mutations in coding regions belonging to well-defined functional domains of PHYO (PAS-B),PHYP2 (PASfold-2), CRY1 (CCT1)and CRY2 (CCT2)that strongly correlates with the latitudinal gradient in response to variable light quality in Norway spruce. The missense SNP in PHYO resulting in Asn835Ser, displayed the steepest cline among all other polymorphisms. We propose that these variations in the photoreceptors represent signs of local adaptation to light quality.

Older adults physically inactive in occupational and commuting domains have a higher risk for osteopenia and osteoporosis: A 12-month prospective study.

Bone mineral density (BMD) usually decreases with age, and the risk of osteometabolic diseases, such as osteopenia and osteoporosis, increases in older adults. PA is strictly related to BMD. However, the relationship between different domains of PA and bone health in older adults is still unclear, and needs to be further investigated, aiming at the implementation of preventative health measures for this population. Thus, the aim of the current study was to analyze the association between different domains of PA and the risk for osteopenia and osteoporosis in older adults in a 12-month follow-up. Prospective study that included 379 Brazilian community-dwelling older adults, aged ≥ 60years (70 ± 7years, 69% women). Areal bone mineral density (aBMD) total, proximal femur, and lumbar spine were measured by dual energy X-ray absorptiometry (DXA), and PA was self-reported. Binary logistic regression analysis and 95%CI were used to analyze the association between the practice of PA in different domains (at baseline and follow-up) and risk for osteopenia and osteoporosis (follow-up). The risk for osteopenia (total proximal femur or lumbar spine) is higher in older adults who are physically inactive in the occupational domain (OR:3.25; 95%CI:1.24-8.55). In addition, older adults who are physically inactive in the commuting domain (OR:3.43; 95%CI:1.09-10.82) and total PA (OR:5.58; 95%CI:1.57-19.88) present a higher risk for osteoporosis (total proximal femur or lumbar spine) compared to physically active older adults. The risk of osteopenia is higher in older adults who are physically inactive in occupational domain and for osteoporosis the risk is higher in physically inactive in commuting domain and total habitual PA.

Relationships between personality traits, high school sports participation, and physical activity of college students in the United States

Objective: The study aimed to 1) examine relationships between contexts of PA, personality traits, and high school sports participation (sports) and 2) determine significant PA correlates in a college population. Participants: 237 undergraduates at a university in the United States between September 2020 and May 2021 participated. Methods: Participants completed a survey evaluating PA, personality traits, sports, and demographic variables. Pearson partial correlations assessed correlations between different PA domains, personality traits, and sports. Conscientiousness was positively associated with all PA measures (r = .14-.30), except for active transport PA. Sports were related to vigorous and leisure PA. Conscientiousness is related to PA measures and is a significant PA correlate. However, more research is necessary to understand if leisure time PA can enhance Conscientiousness.

Novel mechanism of DNA repair in neurons opens promising avenues for combatting neurodegenerative diseases and brain aging

A recent study, titled “A NPAS4-NuA4 Complex Couples Synaptic Activity to DNA Repair,” reveals an exciting new mechanism by which neurons maintain genomic stability in response to external stimuli. Published in Nature on February 23, 2023,1 this paper provides valuable insights into the molecular mechanisms underlying neurodegenerative diseases and brain aging that pave the way for the development of new therapeutic strategies. This commentary reviews the research findings and their potential for future applications. The brain is a highly dynamic and plastic organ that relies on neurons to modify their gene expression under a variety of pathophysiological conditions. Excessive or prolonged neuronal response to external stimuli can lead to DNA damage and genomic instability, thereby being harmful to the brain. Cumulative DNA damage in the neuronal genome is also a hallmark of neurodegeneration and brain aging. Until recently, the molecular mechanism by which neurons repair DNA and maintain genomic stability in response to external stimuli remained unclear. Neuronal PAS Domain Protein 4 (NPAS4) is an activity-induced transcription factor that is selectively expressed in neurons following membrane depolarization and subsequent calcium signaling. Through a series of biochemical and genomic experiments on mice, the researchers first determined that NPAS4 exists as part of a 21-protein complex called NPAS4-NuA4. They then measured DNA damage using γH2AX ChIP-seq, sBLISS-seq, END-seq and SAR-seq, which indicated that NPAS4 preferentially binds to active DNA breaking-induced sites in neurons. These advanced techniques can be used to analyze the binding sites of transcription factors,2 provide whole-genome maps with DNA double-strand breaks,3 monitor DNA terminal excision,4 and localize DNA repair synthesis in vivo and in various cell types.5 Thus, they could contribute greatly to future research in neuroscience and other biomedical fields. In addition, the NPAS4-NuA4 complex serves as a critical mediator in the mechanism underlying neurodegenerative diseases and aging in the brain. Activated in response to neuronal activity driven by changes in sensory experience, NPAS4-NuA4 binds to periodically damaged transcriptional regulatory elements and recruits DNA repair machinery to prevent age-dependent somatic mutation accumulation. Furthermore, impaired NPAS4-NuA4 leads to a range of cellular defects that result in a shortened lifespan, including dysregulation of neuronal activity-dependent transcriptional responses, loss of somatic inhibition of pyramidal neurons, impaired localization of protective repair machinery, and genomic instability. One of the most exciting aspects of this study is its discovery of a link between damage and neuronal activity-dependent regulatory elements and neuronal dysfunction in neurodegenerative diseases. Preventing mutations in the NPAS4-NuA4 complex during neurodevelopment and carefully balancing the proper ratio of excitatory and inhibitory neurons are key to sustaining neuronal survival. This conclusion suggests a new avenue for the development of drugs for Alzheimer's and Parkinson's diseases that target the NPAS4-NuA4 complex and its molecular pathways. The research also provides insight into the complex interplay between gene expression and DNA repair in aging. By targeting the DNA of hippocampal neurons in mice of different ages, researchers found that gene regulatory elements to which NPAS4–NuA4 binds are partially protected against age-dependent accumulation of somatic mutations. This finding suggests that promoting the protection of those recurrently broken sites can help maintain genome integrity and thus prevent mutation. It is worth considering whether all cell types in the body (i.e., beyond neurons) may regulate transcriptional activity, protect vulnerable DNA sites, and maintain genomic stability according to the type of stimulation they receive. There may be activity-dependent protective mechanisms for genome stability that remains unknown and thus need to be explored. Moreover, the unique repair mechanism proposed in this paper was based on mouse models and has not yet been verified in the human nervous system. If this could be explored in humans, the functions and upstream regulatory mechanisms of various protein components of the NPAS4-NuA4 complex could be further clarified to provide a better understanding of the occurrence and development of neurodegeneration. In conclusion, this study demonstrates the complex interplay between neuronal activity, gene expression, and DNA repair in the brain. Its findings broaden our knowledge of the pathogenesis of neurodegenerative diseases and aging in the brain and contribute to the field of neuroscience with important implications for the development of novel therapeutic approaches. Conglin Wang: Writing – original draft. Xintong Ge: Writing – original draft; Writing – review & editing. Wenqiang Xin: Writing – review & editing. Ping Lei: Writing – review & editing. This research was supported by Haihe Laboratory of Cell Ecosystem Innovation Fund (HH22KYZX0048), the National Natural Science Foundation of China (82271401, 82071394, 82072166), Science and Technology Foundation of Tianjin (21JCYBJC01180), and Science and Technology Project of Tianjin Municipal Health Commission (TJWJ2021QN005). The authors declare no conflicts of interest.

Generation of CRISPR-Cas9-mediated knockin mutant models in mice and MEFs for studies of polymorphism in clock genes

The creation of mutant mice has been invaluable for advancing biomedical science, but is too time- and resource-intensive for investigating the full range of mutations and polymorphisms. Cell culture models are therefore an invaluable complement to mouse models, especially for cell-autonomous pathways like the circadian clock. In this study, we quantitatively assessed the use of CRISPR to create cell models in mouse embryonic fibroblasts (MEFs) as compared to mouse models. We generated two point mutations in the clock genes Per1 and Per2 in mice and in MEFs using the same sgRNAs and repair templates for HDR and quantified the frequency of the mutations by digital PCR. The frequency was about an order of magnitude higher in mouse zygotes compared to that in MEFs. However, the mutation frequency in MEFs was still high enough for clonal isolation by simple screening of a few dozen individual cells. The Per mutant cells that we generated provide important new insights into the role of the PAS domain in regulating PER phosphorylation, a key aspect of the circadian clock mechanism. Quantification of the mutation frequency in bulk MEF populations provides a valuable basis for optimizing CRISPR protocols and time/resource planning for generating cell models for further studies.

The circadian rhythm key gene ARNTL2: a novel prognostic biomarker for immunosuppressive tumor microenvironment identification and immunotherapy outcome prediction in human cancers.

Aryl hydrocarbon receptor nuclear translocator-like 2 (ARNTL2) belongs to the b HLH- PAS domain transcription factor family and is one of the key clock genes that control the circadian rhythm. ARNTL2 plays an important role in human biological functions. However, its role in various tumors, especially in the tumor immune microenvironment (TIME) and immunotherapy, remains unclear. We integrated data from cancer patients from multiple databases, including the Cancer Genome Atlas, Cancer Cell Lineage Encyclopedia, Genotype Tissue Expression, Human Protein Atlas, cBioPortal, TIMER, and ImmuCellAI, with data from a large clinical study, three immunotherapy cohorts, and in vitro experiments to investigate the involvement of ARNTL2 expression in cancer prognosis and immune response. ARNTL2 displayed abnormal expression within most malignant tumors, and is significantly associated with poorer survival and pathologic staging. Through gene-set enrichment analysis (GSEA) and gene-set variation analysis (GSVA), we found that ARNTL2 not only regulates cell cycle-related functions to promote cell proliferation but also regulates autoimmunity-related functions of the innate and adaptive immune systems, and other immune-related signaling pathways. In addition, ARNTL2 overexpression contributes to an immunosuppressive tumor microenvironment that plays a key role in immunosuppression-related features, such as the expression of immunosuppression-related genes and pathways and the number of immunosuppressive-infiltrating cells, including regulatory T cells (Tregs), tumor-associated macrophages (TAMs), and cancer-associated fibroblasts (CAFs). The group of patients with low ARNTL2 expression who received immune checkpoint inhibitors (ICI) therapy had better response rates and longer survival when compared to those with high ARNTL2 expression. The findings of this study suggest that ARNTL2 is a potential human oncogene that plays an important role in tumorigenesis and cancer immunity. Elevated ARNTL2 expression indicates an immunosuppressive tumor microenvironment. Targeting ARNTL2 in combination with ICI therapy could bring more significant therapeutic benefits to patients with cancer. Our study sheds light on the remarkable potential of ARNTL2 in tumor immunity and provides a novel perspective for anti-tumor strategies.

Prediction of Kv11.1 potassium channel PAS-domain variants trafficking via machine learning

Congenital long QT syndrome (LQTS) is characterized by a prolonged QT-interval on an electrocardiogram (ECG). An abnormal prolongation in the QT-interval increases the risk for fatal arrhythmias. Genetic variants in several different cardiac ion channel genes, including KCNH2, are known to cause LQTS. Here, we evaluated whether structure-based molecular dynamics (MD) simulations and machine learning (ML) could improve the identification of missense variants in LQTS-linked genes. To do this, we investigated KCNH2 missense variants in the Kv11.1 channel protein shown to have wild type (WT) like or class II (trafficking-deficient) phenotypes in vitro. We focused on KCNH2 missense variants that disrupt normal Kv11.1 channel protein trafficking, as it is the most common phenotype for LQTS-associated variants. Specifically, we used computational techniques to correlate structural and dynamic changes in the Kv11.1 channel protein PAS domain (PASD) with Kv11.1 channel protein trafficking phenotypes. These simulations unveiled several molecular features, including the numbers of hydrating waters and hydrogen bonding pairs, as well as folding free energy scores, that are predictive of trafficking. We then used statistical and machine learning (ML) (Decision tree (DT), Random forest (RF), and Support vector machine (SVM)) techniques to classify variants using these simulation-derived features. Together with bioinformatics data, such as sequence conservation and folding energies, we were able to predict with reasonable accuracy (≈75%) which KCNH2 variants do not traffic normally. We conclude that structure-based simulations of KCNH2 variants localized to the Kv11.1 channel PASD led to an improvement in classification accuracy. Therefore, this approach should be considered to complement the classification of variant of unknown significance (VUS) in the Kv11.1 channel PASD.

Prediction of mammalian tissue-specific CLOCK–BMAL1 binding to E-box DNA motifs

The Brain and Muscle ARNTL-Like 1 protein (BMAL1) forms a heterodimer with either Circadian Locomotor Output Cycles Kaput (CLOCK) or Neuronal PAS domain protein 2 (NPAS2) to act as a master regulator of the mammalian circadian clock gene network. The dimer binds to E-box gene regulatory elements on DNA, activating downstream transcription of clock genes. Identification of transcription factor binding sites and genomic features that correlate to DNA binding by BMAL1 is a challenging problem, given that CLOCK–BMAL1 or NPAS2–BMAL1 bind to several distinct binding motifs (CANNTG) on DNA. Using three different types of tissue-specific machine learning models with features based on (1) DNA sequence, (2) DNA sequence plus DNA shape, and (3) DNA sequence and shape plus histone modifications, we developed an interpretable predictive model of genome-wide BMAL1 binding to E-box motifs and dissected the mechanisms underlying BMAL1–DNA binding. Our results indicated that histone modifications, the local shape of the DNA, and the flanking sequence of the E-box motif are sufficient predictive features for BMAL1–DNA binding. Our models also provide mechanistic insights into tissue specificity of DNA binding by BMAL1.

Comprehensive analyses reveal the prognosis and biological function roles of chromatin regulators in lung adenocarcinoma.

The present study explored the prognosis and biological function roles of chromatin regulators (CRs) in patients with lung adenocarcinoma (LUAD). Using transcriptome profile and clinical follow-up data of LUAD dataset, we explored the molecular classification, developed, and validated a CR prognostic model, built an individual risk scoring system in LUAD, and compared the clinical and molecular characteristics between different subtypes and risk stratifications. We investigated the chemotherapy sensitivity and predicted potential immunotherapy response. Lastly, we collected the clinical samples and validated the prognosis and potential function role of NAPS2. Our study indicated that LUAD patients could be classified into two subtypes that had obviously different clinical background and molecular features. We constructed a prognostic model with eight CR genes, which was well validated in several other population cohort. We built high- and low-risk stratifications for LUAD patients. Patients from high-risk group were totally different from low-risk groups in clinical, biological function, gene mutation, microenvironment, and immune infiltration levels. We idented several potential molecular compounds for high-risk group treatment. We predicted that high-risk group may have poor immunotherapy response. We finally found that Neuronal PAS Domain Protein 2 (NPAS2) involved in the progression of LUAD via regulating cell adhesion. Our study indicated that CR involved in the progression of LUAD and affect their prognosis. Different therapeutic strategies should be developed for different molecular subtypes and risk stratifications. Our comprehensive analyses uncover specific determinants of CRs in LUAD and provides implications for investigating disease-associated CRs.

Exploratory Transcriptomic Profiling Reveals the Role of Gut Microbiota in Vascular Dementia.

Stroke is the second most common cause of cognitive impairment and dementia. Vascular dementia (VaD), a cognitive impairment following a stroke, is common and significantly impacts the quality of life. We recently demonstrated via gut microbe transplant studies that the gut microbe-dependent trimethylamine-N-oxide (TMAO) pathway impacts stroke severity, both infarct size and long-term cognitive outcomes. However, the molecular mechanisms that underly the role of the microbiome in VaD have not been explored in depth. To address this issue, we performed a comprehensive RNA-sequencing analysis to identify differentially expressed (DE) genes in the ischemic cerebral cortex of mouse brains at pre-stroke and post-stroke day 1 and day 3. A total of 4016, 3752 and 7861 DE genes were identified at pre-stroke and post-stroke day 1 and day 3, respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated pathways of neurodegeneration in multiple diseases, chemokine signaling, calcium signaling, and IL-17 signaling as the key enriched pathways. Inflammatory response genes interleukin-1 beta (Il-1β), chemokines (C-X-C motif chemokine ligand 10 (Cxcl10), chemokine ligand 2 (Ccl2)), and immune system genes (S100 calcium binding protein 8 (S100a8), lipocalin-2 (Lcn2)) were among the most significantly upregulated genes. Hypocretin neuropeptide precursor (Hcrt), a neuropeptide, and transcription factors such as neuronal PAS domain protein 4 (Npas4), GATA binding protein 3 (Gata3), and paired box 7 (Pax7) were among the most significantly downregulated genes. In conclusion, our results indicate that higher plasma TMAO levels induce differential mRNA expression profiles in the ischemic brain tissue in our pre-clinical stroke model, and the predicted pathways provide the molecular basis for regulating the TMAO-enhanced neuroinflammatory response in the brain.

Correlations between the symptoms of insomnia, depression, sleep quality, antitumor necrosis factor therapy, and disrupted circadian clock gene expression in inflammatory bowel disease.

Inflammatory bowel disease (IBD) might be accompanied by emotional disturbances. Circadian rhythm genes, such as brain and muscle ARNT‑Like 1 (BMAL1), circadian locomotor output cycles kaput (CLOCK), neuronal PAS domain protein 2 (NPAS2), or nuclear receptor subfamily 1 group D member 1 (NR1D1) are related to inflammation and psychiatric symptoms that might modulate their expression. The study aimed to compare the expression of the BMAL1, CLOCK, NPAS2, NR1D1 mRNA in IBD patients and healthy controls (HCs). We evaluated the association between the gene expression and the disease severity, antitumor necrosis factor (TNF) therapy, sleep quality, insomnia, and depression. A total of 81 IBD patients and 44 HCs were recruited and classified according to the disease activity and IBD type (ulcerative colitis [UC] or Crohn disease [CD]). The participants filled out questionnaires assessing their sleep quality, daytime sleepiness, insomnia, and depression. Venous blood samples were collected, and the IBD patients on the anti‑TNF therapy had their blood drawn before and after 14 weeks of the treatment. In comparison with HCs, the IBD group had decreased expression of all studied genes apart from the BMAL1 gene. UC individuals with exacerbation had decreased expression of the CLOCK and the NPAS2 genes, as compared with the remission group. UC severity negatively correlated with the CLOCK, NPAS2, and NR1D1 mRNA levels. The IBD participants with depression symptoms had a decreased expression of the CLOCK and the NR1D1 genes, as compared with those without mood disturbances. Poor sleep quality was associated with a decreased expression of the NR1D1 gene. Biologic treatment decreased the expression of the BMAL1 gene. Disruption of the clock gene expression might constitute a molecular background of sleep disorders and depression in IBD and might contribute to UC exacerbation.

ZnO Nanorods Create a Hypoxic State with Induction of HIF-1 and EPAS1, Autophagy, and Mitophagy in Cancer and Non-Cancer Cells.

Nanomaterials are gaining increasing attention as innovative materials in medicine. Among nanomaterials, zinc oxide (ZnO) nanostructures are particularly appealing because of their opto-electrical, antimicrobial, and photochemical properties. Although ZnO is recognized as a safe material and the Zn ion (Zn2+) concentration is strictly regulated at a cellular and systemic level, different studies have demonstrated cellular toxicity of ZnO nanoparticles (ZnO-NPs) and ZnO nanorods (ZnO-NRs). Recently, ZnO-NP toxicity has been shown to depend on the intracellular accumulation of ROS, activation of autophagy and mitophagy, as well as stabilization and accumulation of hypoxia-inducible factor-1α (HIF-1α) protein. However, if the same pathway is also activated by ZnO-NRs and how non-cancer cells respond to ZnO-NR treatment, are still unknown. To answer to these questions, we treated epithelial HaCaT and breast cancer MCF-7 cells with different ZnO-NR concentrations. Our results showed that ZnO-NR treatments increased cell death through ROS accumulation, HIF-1α and endothelial PAS domain protein 1 (EPAS1) activation, and induction of autophagy and mitophagy in both cell lines. These results, while on one side, confirmed that ZnO-NRs can be used to reduce cancer growth, on the other side, raised some concerns on the activation of a hypoxic response in normal cells that, in the long run, could induce cellular transformation.

Theanine, a Tea-Leaf-Specific Amino Acid, Alleviates Stress through Modulation of Npas4 Expression in Group-Housed Older Mice.

Group rearing is a common housing condition, but group-housed older mice show increased adrenal hypertrophy, a marker of stress. However, the ingestion of theanine, an amino acid unique to tea leaves, suppressed stress. We aimed to elucidate the mechanism of theanine's stress-reducing effects using group-reared older mice. The expression of repressor element 1 silencing transcription factor (REST), which represses excitability-related genes, was increased in the hippocampus of group-reared older mice, whereas the expression of neuronal PAS domain protein 4 (Npas4), which is involved in the regulation of excitation and inhibition in the brain, was lower in the hippocampus of older group-reared mice than in same-aged two-to-a-house mice. That is, the expression patterns of REST and Npas4 were found to be just inversely correlated. On the other hand, the expression levels of the glucocorticoid receptor and DNA methyltransferase, which suppress Npas4 transcription, were higher in the older group-housed mice. In mice fed theanine, the stress response was reduced and Npas4 expression tended to be increased. These results suggest that Npas4 expression was suppressed by the increased expression of REST and Npas4 downregulators in the group-fed older mice, but that theanine avoids the decrease in Npas4 expression by suppressing the expression of Npas4 transcriptional repressors.

KidA, a novel interactor of the cyanobacterial circadian oscillator that tunes its period.

Composed of only three proteins, KaiA, KaiB, and KaiC, the cyanobacterial circadian clock is the simplest known biochemical oscillator that can be reconstituted in vitro. While the molecular mechanism of the three proteins has been studied in depth, how they are connected to the rest of the cellular physiology remains unclear. In order to identify previously unknown players in the broader network of the circadian system, we performed a coimmunoprecipitation (co-IP)/mass spec experiment on S. elongatus PCC 7942 cell lysate. Using KaiB in its nighttime form (fold-switched KaiB) as bait, we identified a novel clock interactor protein we named Kai-Interacting Diguanylate cyclase A (KidA), which is predicted to have four N-terminal PAS domains and C-terminal GGDEF/EAL domains. We found that KidA shortens the period of the in vivo rhythms in a dose-dependent manner and alters the entrainment to light-dark cycles. We found that an N-terminal triple-PAS domain fragment is sufficient for the period-shortening phenotype. Consistently, adding purified triple-PAS fragment to the in vitro Kai oscillator reconstituted the dose-dependent shortening of the period. Our in vitro co-IP showed that the N-terminal PAS domains of KidA can directly and specifically bind to the fold-switched form of KaiB. To understand the potential mechanism, we used a computational modeling approach where a fold-switched KaiB binder was added to the Kai oscillator model. The results showed that KidA can tune the period by shifting the equilibrium of KaiB fold-switching. The discovery of KidA suggests a potential role of PAS domain in prokaryotic timekeeping, which has been well documented only in eukaryotic clock systems. Future studies could unveil more potential of KidA, such as integration of time-of-day information and other signals via multiple PAS domains and regulation of cellular processes via cyclic-di-GMP.

In vitro assessment of Neuronal PAS domain 2 mitigating compounds for scarless wound healing.

The core circadian gene Neuronal PAS domain 2 (NPAS2) is expressed in dermal fibroblasts and has been shown to play a critical role in regulating collagen synthesis during wound healing. We have performed high throughput drug screening to identify genes responsible for downregulation of Npas2 while maintaining cell viability. From this, five FDA-approved hit compounds were shown to suppress Npas2 expression in fibroblasts. In this study, we hypothesize that the therapeutic suppression of Npas2 by hit compounds will have two effects: (1) attenuated excessive collagen deposition and (2) accelerated dermal wound healing without hypertrophic scarring. To test the effects of each hit compound (named Dwn1, 2, 3, 4, and 5), primary adult human dermal fibroblasts (HDFa) were treated with either 0, 0.1, 1, or 10 μM of a single hit compound. HDFa behaviors were assessed by picrosirius red staining and quantitative RT-PCR for in vitro collagen synthesis, cell viability assay, in vitro fibroblast-to-myofibroblast differentiation test, and cell migration assays. Dwn1 and Dwn2 were found to significantly affect collagen synthesis and cell migration without any cytotoxicity. Dwn3, Dwn4, and Dwn5 did not affect collagen synthesis and were thereby eliminated from further consideration for their role in mitigation of gene expression or myofibroblast differentiation. Dwn1 also attenuated myofibroblast differentiation on HDFa. Dwn1 and Dwn2 may serve as possible therapeutic agents for future studies related to skin wound healing.

A 14-bp deletion in bovine EPAS1 gene is associated with carcass traits

EPAS1 (Endothelial PAS Domain Protein 1) gene is well-known for its function in plateau hypoxia adaptability. It encodes HIF-2α, which involved in the induction of genes regulated by oxygen and then affects multiple physiological processes such as angiogenesis and energy metabolism. All of these indicate it may affect the development of animals. In this study, a 14-bp deletion in EPAS1 gene was uncovered in Shandong black cattle population (n = 502). Two genotypes (II and ID) were found and the frequency of the homozygous II genotype is higher than the heterozygous ID genotype. This population is consisted with HWE (p > 0.05). And more importantly, the 14-bp deletion was associated with outside flat (p = 0.003), brisket (p = 0.001), and knuckle (p = 0.032). These findings suggested that the 14-bp deletion is significantly associated with carcass traits, which could be served as a molecular marker applied to cow breeding.

Phosphorylation chemistry of the Bordetella PlrSR TCS and its contribution to bacterial persistence in the lower respiratory tract.

Bordetella species cause lower respiratory tract infections in mammals. B. pertussis and B. bronchiseptica are the causative agents of whooping cough and kennel cough, respectively. The current acellular vaccine for B. pertussis protects against disease but does not prevent transmission or colonization. Cases of pertussis are on the rise even in areas of high vaccination. The PlrSR two-component system, is required for persistence in the mouse lung. A partial plrS deletion strain and a plrS H521Q strain cannot survive past 3 days in the lung, suggesting PlrSR works in a phosphorylation-dependent mechanism. We characterized the biochemistry of B. bronchiseptica PlrSR and found that both proteins function as a canonical two-component system. His521 was essential and Glu522 was critical for PlrS autophosphorylation. Asn525 was essential for phosphatase activity. The PAS domain was critical for both PlrS autophosphorylation and phosphatase activities. PlrS could both phosphotransfer to and exert phosphatase activity toward PlrR. Unexpectedly, PlrR formed a tetramer when unphosphorylated and a dimer upon phosphorylation. Finally, we demonstrated the importance of PlrS phosphatase activity for persistence within the murine lung. By characterizing PlrSR we hope to guide future in vivo investigation for development of new vaccines and therapeutics.

Destabilization of HIF-1a by Diabetes, Oxidative Stress, Obesity and Other Related Disorders

One of the most fundamental molecular processes in response to hypoxia is the activation and stabilization of a transcriptional factor called hypoxia induced factor 1a (HIF-1a), which is responsible for the regulation of many downstream effector genes. Multiple key biological pathways such as proliferation, energy metabolism, invasion, and metastasis are governed by these genes. This article discusses the role of hypoxia-inducible factor 1a (HIF-1a) in metabolic and pathological processes, particularly in adipose tissue, oxidative stress, inflammation, diabetes and cancer. HIF1A is a basic helix-loophelix PAS domain containing protein, and is considered as the master transcriptional regulator of cellular and developmental response to hypoxia. HIF-1a regulates the expression of genes involved in angiogenesis, glucose metabolism, inflammation and oxidative stress. In obesity, adipose tissue hypoxia leads to increased expression of HIF-1a, which can lead to chronic inflammation and adipose tissue dysfunction. Another field that HIF-1a is also involved in cancer pathogenesis pathways, such as proliferation, invasion, angiogenesis, and metastasis, and is considered a potential therapeutic target for metabolic/genetic diseases and cancer. Direct and indirect HIF-1 inhibitors have been identified, but only a few have entered clinical trials due to their multiple side effects.