High-frequency neural activity dysregulation is associated with sleep and psychiatric disorders in BMAL1-deficient animal models.

Sleep disturbance led by BMAL1-deficiency has been recognized both in rodent and non-human primate models. Yet it remained unclear how their diurnal brain oscillations were affected upon BMAL1 ablation and what caused the discrepancy in the quantity of sleep between the two species. Here, we investigated diurnal electroencephalographs of BMAL1-deficient mice and cynomolgus monkeys at young adult age and uncovered a shared defect of dysregulated high-frequency oscillations by Kullback-Leibler divergence analysis. We found beta and gamma oscillations were significantly disturbed in a day versus night manner in BMAL1-deficient monkeys, while in mice the beta band difference was less evident. Notably, the dysregulation of beta oscillations was particularly associated with psychiatric behaviors in BMAL1-deficient monkeys, including the occurrence of self-injuring and delusion-like actions. As such psychiatric phenotypes were challenging to uncover in rodent models, our results offered a unique method to study the correlation between circadian clock dysregulation and psychiatric disorders.

ISX-9 potentiates CaMKIIδ-mediated BMAL1 activation to enhance circadian amplitude.

Circadian dysregulation associates with numerous diseases including metabolic dysfunction, sleep disorder, depression and aging. Given that declined circadian amplitude is a trait commonly found with compromised health, interventions that design in precluding circadian amplitude from dampening will aid to mitigate complex, circadian-related diseases. Here we identify a neurogenic small molecule ISX-9 that is able to support persistent and higher amplitude of circadian oscillations. ISX-9 improves diurnal metabolic rhythms in middle-aged mice. Moreover, the ISX-9-treated mice show better sleep homeostasis with increased delta power during the day time and higher locomotive activity in the dark period. ISX-9 augments CaMKIIδ expression and increases BMAL1 activity via eliciting CaMKIIδ-mediated phosphorylation on BMAL1 residues S513/S515/S516, accordingly composes a positive feedback effect on enhancing circadian amplitude. CaMKIIδ-targeting, and the use of ISX-9 may serve as decent choices for treating circadian-related disorders.

Base-edited cynomolgus monkeys mimic core symptoms of STXBP1 encephalopathy.

Presynaptic syntaxin binding protein 1 (STXBP1) is essential for neurotransmitter release. Heterozygous mutations in this protein cause STXBP1 encephalopathy (STXBP1-E), which is characterized by intellectual disabilities and epilepsies. Since nonhuman primates closely resemble humans, monkey models may advance studies on the pathogenesis and therapeutic treatments of STXBP1-E. We generated cynomolgus monkeys carrying STXBP1 (R292H) mutation through base editing of in vitro fertilized embryos to mimic a clinical condition. The newborn STXBP1-edited monkeys exhibited focal epilepsy, and the animal that survived beyond the first week postpartum presented typical EEG phenotypes. Biochemical analysis of brain biopsy samples showed reduced levels of STXBP1 (MUNC18-1) and SNARE complex proteins. Single-cell sequencing identified one specific cell cluster that may contribute to encephalopathy. Thus, our case report shows that base-edited STXBP1 mutant monkeys are a good animal model for STXBP1-E, and that a base-editing approach is useful for generating primate models of human genetic disorders.

Influenza a virus NS1 resembles a TRAF3-interacting motif to target the RNA sensing-TRAF3-type I IFN axis and impair antiviral innate immunity.

BACKGROUND: Influenza A virus (IAV) evolves strategies to counteract the host antiviral defense for establishing infection. The influenza A virus (IAV) non-structural protein 1 (NS1) is a key viral factor shown to counteract type I IFN antiviral response mainly through targeting RIG-I signaling. Growing evidence suggests that viral RNA sensors RIG-I, TLR3, and TLR7 function to detect IAV RNA in different cell types to induce type I IFN antiviral response to IAV infection. Yet, it remains unclear if IAV NS1 can exploit a common mechanism to counteract these RNA sensing pathways to type I IFN production at once, then promoting viral propagation in the host. METHODS: Luciferase reporter assays were conducted to determine the effect of NS1 and its mutants on the RIG-I and TLR3 pathways to the activation of the IFN-ß and NF-κB promoters. Coimmunoprecipitation and confocal microscopic analyses were used to the interaction and colocalization between NS1 and TRAF3. Ubiquitination assays were performed to study the effect of NS1 and its mutants on TRAF3 ubiquitination. A recombinant mutant virus carrying NS1 E152A/E153A mutations was generated by reverse genetics for biochemical, ex vivo, and in vivo analyses to explore the importance of NS1 E152/E153 residues in targeting the RNA sensing-TRAF3-type I IFN axis and IAV pathogenicity. RESULTS: Here we report that NS1 subverts the RIG-I, TLR3, and TLR7 pathways to type I IFN production through targeting TRAF3 E3 ubiquitin ligase. NS1 harbors a conserved FTEE motif (a.a. 150-153), in which the E152/E153 residues are critical for binding TRAF3 to block TRAF3 ubiquitination and type I IFN production by these RNA sensing pathways. A recombinant mutant virus carrying NS1 E152A/E153A mutations induces higher type I IFN production ex vivo and in vivo, and exhibits the attenuated phenotype in infected mice, indicating the importance of E152/E153 residues in IAV pathogenicity. CONCLUSIONS: Together our work uncovers a novel mechanism of IAV NS1-mediated immune evasion to promote viral infection through targeting the RNA sensing-TRAF3-type I IFN axis.

SIRT7 Facilitates CENP-A Nucleosome Assembly and Suppresses Intestinal Tumorigenesis.

SIRT7 is a member of the mammalian sirtuins and functions as an NAD+-dependent deacylase. Here we show that SIRT7 deficiency leads to a lowered histone acetyltransferase 1 (HAT1) activity and therefore decreased histone H4K5 and H4K12 acetylation. This in turn causes CENP-A dislocation at the centromere, which further affects chromatin assembly. SIRT7 ablation results in aneuploidy and aging phenotypes, including senescence and nucleolar expansion. Moreover, SIRT7 knockout mice are susceptible to DSS-induced colitis and alcohol-derived epithelial disturbance, revealing a disrupted intestinal epithelial homeostasis. Notably, absence of SIRT7 aggravates the susceptibility of colorectal cancer incidence in APCMin/+ mouse model and elicits further the Wnt signaling. Our findings indicate a tumor suppressive role of SIRT7 in the case of colorectal cancer. Together with the activities in maintaining genome integrity and intestinal homeostasis, activating SIRT7 may serve as a strategy to treat bowel diseases and colorectal cancer.

CDK9 modulates circadian clock by attenuating REV-ERBα activity.

Circadian clock and cell cycle are vital cellular programs acting in a timely-regulated, cyclic manner. The two cellular oscillators are coupled in various ways to facilitate biological processes. Here we report CDK9, a kinase belongs to the CDK family in regulating cell cycle and RNA Pol II activity, can serve as a modulator for circadian clock. We identified CDK inhibitor LY2857785 potently blocked PER2:LUC expression in MEFs from a screen of 17 commonly-used CDK inhibitors. We further analyzed the possible targets of LY2857785 by siRNA approach, and confirmed CDK9 as the main effector. LY2857785 treatment, as well as Cdk9 knock-down, led to lowered expression of Bmal1 in accordance with elevated expression of Rev-Erbα. CDK9 associated with REV-ERBα thus attenuated REV-ERBα binding to the RORE for Bmal1 suppression. To conform the circadian-modulating activity of CDK9 in vivo, we knocked down CDK9 in mice at the anterior hypothalamus covering the central oscillator SCN, and found the respiratory exchange ratio, daily activity and circadian period were altered in the Cdk9-knockdown mice. Together, our finding designated CDK9 as a novel modulator in circadian clock. CDK9 may serve as a vital basis to understand circadian- and cell cycle-misregulated ailments such as cancer.

Cloning of a gene-edited macaque monkey by somatic cell nuclear transfer.

Cloning of macaque monkeys by somatic cell nucleus transfer (SCNT) allows the generation of monkeys with uniform genetic backgrounds that are useful for the development of non-human primate models of human diseases. Here, we report the feasibility of this approach by SCNT of fibroblasts from a macaque monkey (Macaca fascicularis), in which a core circadian transcription factor BMAL1 was knocked out by clustered regularly interspaced short palindromic repeat/Cas9 gene editing (see accompanying paper). Out of 325 SCNT embryos transferred into 65 surrogate monkeys, we cloned five macaque monkeys with BMAL1 mutations in both alleles without mosaicism, with nuclear genes identical to that of the fibroblast donor monkey. Further peripheral blood mRNA analysis confirmed the complete absence of the wild-type BMAL1 transcript. This study demonstrates that the SCNT approach could be used to generate cloned monkeys from fibroblasts of a young adult monkeys and paves the way for the development of macaque monkey disease models with uniform genetic backgrounds.

BMAL1 knockout macaque monkeys display reduced sleep and psychiatric disorders.

Circadian disruption is a risk factor for metabolic, psychiatric and age-related disorders, and non-human primate models could help to develop therapeutic treatments. Here, we report the generation of BMAL1 knockout cynomolgus monkeys for circadian-related disorders by CRISPR/Cas9 editing of monkey embryos. These monkeys showed higher nocturnal locomotion and reduced sleep, which was further exacerbated by a constant light regimen. Physiological circadian disruption was reflected by the markedly dampened and arrhythmic blood hormonal levels. Furthermore, BMAL1-deficient monkeys exhibited anxiety and depression, consistent with their stably elevated blood cortisol, and defective sensory processing in auditory oddball tests found in schizophrenia patients. Ablation of BMAL1 up-regulated transcriptional programs toward inflammatory and stress responses, with transcription networks associated with human sleep deprivation, major depressive disorders, and aging. Thus, BMAL1 knockout monkeys are potentially useful for studying the physiological consequences of circadian disturbance, and for developing therapies for circadian and psychiatric disorders.

Neurodegeneration-associated FUS is a novel regulator of circadian gene expression.

BACKGROUND: Circadian rhythms are oscillating physiological and behavioral changes governed by an internal molecular clock, and dysfunctions in circadian rhythms have been associated with ageing and various neurodegenerative diseases. However, the evidence directly connecting the neurodegeneration-associated proteins to circadian control at the molecular level remains sparse. METHODS: Using meta-analysis, synchronized animals and cell lines, cells and tissues from FUS R521C knock-in rats, we examined the role of FUS in circadian gene expression regulation. RESULTS: We found that FUS, an oscillating expressed nuclear protein implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), exerted a novel feedback route to regulate circadian gene expression. Nr1d1-encoded core circadian protein REV-ERBα bound the Fus promoter and regulated the expression of Fus. Meanwhile, FUS was in the same complex as PER/CRY, and repressed the expression of E box-containing core circadian genes, such as Per2, by mediating the promoter occupancy of PSF-HDAC1. Remarkably, a common pathogenic mutant FUS (R521C) showed increased binding to PSF, and caused decreased expression of Per2. CONCLUSIONS: Therefore, we have demonstrated FUS as a modulator of circadian gene expression, and provided novel mechanistic insights into the mutual influence between circadian control and neurodegeneration-associated proteins.

Sleep and circadian abnormalities precede cognitive deficits in R521C FUS knockin rats.

Mutations in fused in sarcoma (Fus) cause familial amyotrophic lateral sclerosis (ALS) and occasionally frontotemporal dementia. Here we report the establishment and characterization of a novel knockin (KI) rat model expressing a Fus point mutation (R521C) via CRISPR/Cas9. The mutant animals developed adult-onset learning and memory behavioral deficits, with reduced spine density in hippocampal neurons. Remarkably, sleep-wake cycle and circadian abnormalities preceded the onset of cognitive deficit. RNA-seq study further demonstrated altered expression of some key sleep and circadian regulators, such as orexin/hypocretin receptor type 2 and casein kinase 1 epsilon, in the mutant rats. Therefore, we have established a rodent model expressing physiological level of a pathogenic mutant FUS, and we found cognitive impairment as a main behavioral deficit at mid age. Furthermore, we have revealed a new role of FUS in sleep and circadian regulation and demonstrated that functional change in FUS could cause sleep-wake and circadian disturbance as early symptoms.

Leaky property of the Dyakonov-like wave at the surface of a semi-infinite metal-dielectric multilayered structure.

We investigate the Dyakonov-like surface wave (DLSW) at the interface of a dielectric and a metal-dielectric multilayered (MDM) structure when this MDM structure serves as an elliptic medium according to the effective medium approximation (EMA). Different from the conventional Dyakonov surface waves, we find that this kind of DLSW possesses an unexpected leaky property due to an additional hyperbolic-like wave in the MDM structure, resulting in a significant increase of propagation loss compared to the results estimated by a simple effective model based on the EMA. Moreover, such leaky property is found to be sensitive to the period of the MDM structure. Thus, to diminish this non-negligible leaky loss, one can suppress the amplitude of the leaky component by designing the MDM structure with a smaller period. On the other hand, as the MDM structure sometimes can also support an additional elliptic-like dispersion when it is regarded as a hyperbolic medium, we discuss this condition for completeness. Based on this elliptic-like dispersion, an apparent leaky property is observed in the resultant DLSW. This DLSW propagates with a wider range of propagation direction but suffers from a poor field confinement to the interface it is propagating along.

Determination of dissipative Dyakonov surface waves using a finite element method based eigenvalue algorithm.

A full-vectorial finite element method is developed to analyze the surface waves propagating at the interface between two media which could be dissipative particularly. The dissipative wave possessing a complex-valued propagation constant can be determined precisely for any given propagation direction and thus the property of losses could be thoroughly analyzed. Besides, by applying a special characteristic of the implicit circular block matrix, we reduce the computational consumptions in the analysis. By utilizing this method, the Dyakonov surface wave (DSW) at the interface between a dielectric and a metal-dielectric multilayered (MDM) structure which serves as a hyperbolic medium is discussed. Its propagation loss is smaller for larger period of the MDM structure but its field becomes less confined to the interface.

Physiological links of circadian clock and biological clock of aging.

Circadian rhythms orchestrate biochemical and physiological processes in living organisms to respond the day/night cycle. In mammals, nearly all cells hold self-sustained circadian clocks meanwhile couple the intrinsic rhythms to systemic changes in a hierarchical manner. The suprachiasmatic nucleus (SCN) of the hypothalamus functions as the master pacemaker to initiate daily synchronization according to the photoperiod, in turn determines the phase of peripheral cellular clocks through a variety of signaling relays, including endocrine rhythms and metabolic cycles. With aging, circadian desynchrony occurs at the expense of peripheral metabolic pathologies and central neurodegenerative disorders with sleep symptoms, and genetic ablation of circadian genes in model organisms resembled the aging-related features. Notably, a number of studies have linked longevity nutrient sensing pathways in modulating circadian clocks. Therapeutic strategies that bridge the nutrient sensing pathways and circadian clock might be rational designs to defy aging.

Mutation of kri1l causes definitive hematopoiesis failure via PERK-dependent excessive autophagy induction.

Dysregulation of ribosome biogenesis causes human diseases, such as Diamond-Blackfan anemia, del (5q-) syndrome and bone marrow failure. However, the mechanisms of blood disorders in these diseases remain elusive. Through genetic mapping, molecular cloning and mechanism characterization of the zebrafish mutant cas002, we reveal a novel connection between ribosomal dysfunction and excessive autophagy in the regulation of hematopoietic stem/progenitor cells (HSPCs). cas002 carries a recessive lethal mutation in kri1l gene that encodes an essential component of rRNA small subunit processome. We show that Kri1l is required for normal ribosome biogenesis, expansion of definitive HSPCs and subsequent lineage differentiation. Through live imaging and biochemical studies, we find that loss of Kri1l causes the accumulation of misfolded proteins and excessive PERK activation-dependent autophagy in HSPCs. Blocking autophagy but not inhibiting apoptosis by Bcl2 overexpression can fully rescue hematopoietic defects, but not the lethality of kri1l(cas002) embryos. Treatment with autophagy inhibitors (3-MA and Baf A1) or PERK inhibitor (GSK2656157), or knockdown of beclin1 or perk can markedly restore HSPC proliferation and definitive hematopoietic cell differentiation. These results may provide leads for effective therapeutics that benefit patients with anemia or bone marrow failure caused by ribosome disorders.

Periodic anti-ring back reflectors for hydrogenated amorphous silicon thin-film solar cells.

Large and periodic anti-ring arrays are fabricated by using a monolayer of polymer/nanosphere hybrid technique and applied as back reflectors in substrate-type hydrogenated amorphous silicon (a-Si:H) thin-film solar cells. The structure of each anti-ring comprises a nanodome centered inside a nanohole. The excitation of Bloch wave surface plasmon polaritons is observed in the Ag-coated anti-ring arrays. The nanodomes of the anti-ring arrays turn out to enhance large-angle light scattering and increase the effective optical path in the solar cell. The resulting efficiency of an ultrathin a-Si:H (thickness: 150 nm) solar cell is enhanced by 39% compared to that with a flat back reflector and by 13% compared to that with a nanohole back reflector.

Enhancing bright-field image of microorganisms by local plasmon of Ag nanoparticle array.

Inspecting biological cells with bright-field light microscopy often engenders a challenge, owing to their optical transparency. We show that imaging contrast can be greatly enhanced as yeast cells are placed on a silver nanoparticle array. Its near- and far-field traits, revealed by electrodynamic simulations, illustrate that the enhancement is attributed to the sensitivity of its plasmonic characteristics to the attached cells. This study demonstrates that the silver nanoparticle array can serve as the agent for concurrently enhancing Raman scattering and imaging contrast of microorganisms for identification and examination.

SIRT1 and other sirtuins in metabolism.

Sirtuins such as SIRT1 are conserved protein NAD(+)-dependent deacylases and thus their function is intrinsically linked to cellular metabolism. Over the past two decades, accumulating evidence has indicated that sirtuins are not only important energy status sensors but also protect cells against metabolic stresses. Sirtuins regulate the aging process and are themselves regulated by diet and environmental stress. The versatile functions of sirtuins including, more specifically, SIRT1 are supported by their diverse cellular location allowing cells to sense changes in energy levels in the nucleus, cytoplasm, and mitochondrion. SIRT1 plays a critical role in metabolic health by deacetylating many target proteins in numerous tissues, including liver, muscle, adipose tissue, heart, and endothelium. This sirtuin also exerts important systemic effects via the hypothalamus. This review will cover these topics and suggest that strategies to maintain sirtuin activity may be on the horizon to forestall diseases of aging.

SIRT1 mediates central circadian control in the SCN by a mechanism that decays with aging.

SIRT1 is a NAD(+)-dependent protein deacetylase that governs many physiological pathways, including circadian rhythm in peripheral tissues. Here, we show that SIRT1 in the brain governs central circadian control by activating the transcription of the two major circadian regulators, BMAL1 and CLOCK. This activation comprises an amplifying circadian loop involving SIRT1, PGC-1α, and Nampt. In aged wild-type mice, SIRT1 levels in the suprachiasmatic nucleus are decreased, as are those of BMAL1 and PER2, giving rise to a longer intrinsic period, a more disrupted activity pattern, and an inability to adapt to changes in the light entrainment schedule. Young mice lacking brain SIRT1 phenocopy these aging-dependent circadian changes, whereas mice that overexpress SIRT1 in the brain are protected from the effects of aging. Our findings indicate that SIRT1 activates the central pacemaker to maintain robust circadian control in young animals, and a decay in this activity may play an important role in aging.

DnaK functions as a central hub in the E. coli chaperone network.

Cellular chaperone networks prevent potentially toxic protein aggregation and ensure proteome integrity. Here, we used Escherichia coli as a model to understand the organization of these networks, focusing on the cooperation of the DnaK system with the upstream chaperone Trigger factor (TF) and the downstream GroEL. Quantitative proteomics revealed that DnaK interacts with at least ~700 mostly cytosolic proteins, including ~180 relatively aggregation-prone proteins that utilize DnaK extensively during and after initial folding. Upon deletion of TF, DnaK interacts increasingly with ribosomal and other small, basic proteins, while its association with large multidomain proteins is reduced. DnaK also functions prominently in stabilizing proteins for subsequent folding by GroEL. These proteins accumulate on DnaK upon GroEL depletion and are then degraded, thus defining DnaK as a central organizer of the chaperone network. Combined loss of DnaK and TF causes proteostasis collapse with disruption of GroEL function, defective ribosomal biogenesis, and extensive aggregation of large proteins.