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Abstract Astrocytes play a crucial role in regulating sleep‐wake behavior. However, how astrocytes govern a specific sleep‐arousal circuit remains unknown. Here, the authors show that parafacial zone (PZ) astrocytes responded to sleep‐wake cycles with state‐differential Ca2+ activity, peaking during transitions from sleep to wakefulness. Using chemogenetic and optogenetic approaches, they find that activating PZ astrocytes elicited and sustained wakefulness by prolonging arousal episodes while impeding transitions from wakefulness to non‐rapid eye movement (NREM) sleep. Activation of PZ astrocytes specially induced the elevation of extracellular adenosine through the ATP hydrolysis pathway but not equilibrative nucleoside transporter (ENT) mediated transportation. Strikingly, the rise in adenosine levels induced arousal by activating A1 receptors, suggesting a distinct role for adenosine in the PZ beyond its conventional sleep homeostasis modulation observed in the basal forebrain (BF) and cortex. Moreover, at the circuit level, PZ astrocyte activation induced arousal by suppressing the GABA release from the PZGABA neurons, which promote NREM sleep and project to the parabrachial nucleus (PB). Thus, their study unveils a distinctive arousal‐promoting effect of astrocytes within the PZ through extracellular adenosine and elucidates the underlying mechanism at the neural circuit level.

Summary: Here, we present a protocol for neonatal intracerebroventricular (ICV) delivery of adeno-associated viral vectors (AAVs), achieving gene therapy for a Rett syndrome mouse model. We describe steps for preparing mouse lines, replacing foster mothers, sex typing, and genotyping. We then detail procedures for ICV delivery and validation through immunofluorescent and immunoblot techniques. This protocol is also applicable to preclinical gene therapy research that targets the neonatal mouse brain for other neurodevelopmental disorders.For complete details on the use and execution of this protocol, please refer to Yang et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Mutations in the SRCAP gene are among the genetic alterations identified in autism spectrum disorders (ASD). However, the pathogenic mechanisms remain unclear. In this study, we demonstrate that Srcap+/− mice manifest deficits in social novelty response, as well as increased repetitive behaviors, anxiety, and impairments in learning and memory. Notably, a reduction in parvalbumin-positive neurons is observed in the retrosplenial cortex (RSC) and dentate gyrus (DG) of these mice. Through RNA sequencing, we identify dysregulation in 27 ASD-related genes in Srcap+/− mice. Specifically, we find that Srcap regulates expression of Satb2 via H2A.z in the promoter. Therapeutic intervention via retro-orbital injection of adeno-associated virus (AAV)-Satb2 in neonatal Srcap+/− mice leads to amelioration of the neurodevelopmental and ASD-like abnormalities. Furthermore, the expression of Satb2 only in the RSC of adolescent mice rectifies social novelty impairments. These results underscore the pivotal role of Srcap in neurodevelopment, by regulating Satb2, providing valuable insights for the pathophysiology of ASD.

Abstract Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by core symptoms that consist of social deficits and repetitive behaviors. Unfortunately, no effective medication is available thus far to target the core symptoms of ASD, since the pathogenesis remains largely unknown. To investigate the pathogenesis of the core symptoms in ASD, we constructed Shank1 P1812L-knock-in (KI) mice corresponding to a recurrent ASD-related mutation, SHANK1 P1806L, to achieve construct validity and face validity. Shank1 P1812L-KI heterozygous (HET) mice presented with social deficits and repetitive behaviors without the presence of confounding comorbidities. HET mice also exhibited downregulation of metabotropic glutamate receptor (mGluR1) and associated signals, along with structural abnormalities in the dendritic spines and postsynaptic densities. Combined with findings from Shank1 R882H-KI mice, our study confirms that mGluR1-mediated signaling dysfunction is a pivotal mechanism underlying the core symptoms of ASD. Interestingly, Shank1 P1812L-KI homozygous (HOM) mice manifested behavioral signs of impaired long-term memory rather than autistic-like core traits; thus, their phenotype was markedly different from that of Shank1 P1812L-KI HET mice. Correspondingly, at the molecular level, Shank1 P1812L-KI HOM displayed upregulation of AMPA receptor (GluA2)-related signals. The different patterns of protein changes in HOM and HET mice may explain the differences in behaviors. Our study emphasizes the universality of mGluR1-signaling hypofunction in the pathogenesis of the core symptoms in ASD, providing a potential target for therapeutic drugs. The precise correspondence between genotype and phenotype, as shown in HOM and HET mice, indicates the importance of reproducing disease-related genotypes in mouse models.

Abstract The response rate of pancreatic cancer to chemotherapy or immunotherapy pancreatic cancer is low. Although minimally invasive irreversible electroporation (IRE) ablation is a promising option for irresectable pancreatic cancers, the immunosuppressive tumour microenvironment that characterizes this tumour type enables tumour recurrence. Thus, strengthening endogenous adaptive antitumour immunity is critical for improving the outcome of ablation therapy and post-ablation immune therapy. Here we present a hydrogel microsphere vaccine that amplifies post-ablation anti-cancer immune response via releasing its cargo of FLT3L and CD40L at the relatively lower pH of the tumour bed. The vaccine facilitates migration of the tumour-resident type 1 conventional dendritic cells (cDC1) to the tumour-draining lymph nodes (TdLN), thus initiating the cDC1-mediated antigen cross-presentation cascade, resulting in enhanced endogenous CD8+ T cell response. We show in an orthotopic pancreatic cancer model in male mice that the hydrogel microsphere vaccine transforms the immunologically cold tumour microenvironment into hot in a safe and efficient manner, thus significantly increasing survival and inhibiting the growth of distant metastases.

Properties of cytidine and adenosine deaminases lead to off-target effects for cytosine base editors (CBEs) and adenine base editors (ABEs). Here the authors report that 25 TadA orthologs could be engineered to generate functional ABEs, CBEs or ACBEs via single/double mutations with minimised off-targets.

Hypercompact CRISPR-Cas12f systems have been engineered to generate miniABEs but these have limitations. Here the authors generate Cas12f-derived miniCBEs and develop miniABEs with improved editing and targeting scopes; they use these to correct pathogenic mutations in cell lines and introduce mutations in vivo.

Summary: Strong evidence from human genetic studies associates the thousand and one amino acid kinase 1 (TAOK1) gene with autism spectrum disorder (ASD). In this work, we discovered a de novo frameshifting mutation in TAOK1 within a Chinese ASD cohort. We found that Taok1 haploinsufficiency induces autistic-like behaviors in mice. Importantly, we observed a significant enrichment of Taok1 in the dorsal raphe nucleus (DRN). The haploinsufficiency of Taok1 considerably restrained the activation of DRN neurons during social interactions, leading to the aberrant phosphorylation of numerous proteins. Intriguingly, the genetic deletion of Taok1 in VGlut3-positive neurons of DRN resulted in mice exhibiting autistic-like behaviors. Ultimately, reintroducing wild-type Taok1, but not its kinase-dead variant, into the DRN of adult mice effectively mitigated the autistic-like behaviors associated with Taok1 haploinsufficiency. This work suggests that Taok1, through its influence in the DRN, regulates social interaction behaviors, providing critical insights into the etiology of ASD.

In the original publication [...]

Magnesia-carbon (MgO–C) refractories are used in steelmaking processes as lining materials. We performed lab-scale tests at 1600 °C and a pO2 of 10−15 atm to determine the effect of Al and Si antioxidant level, time and slag composition on the amount and nature of inclusions in the steel and on the corrosion and infiltration of the refractory. In all steel samples, a pick-up of Al, Si and C was observed. For the refractories containing antioxidants, longer holding times seem to increase the number density of inclusions, especially in combination with a slag with lower MgO content. For the given experiments, the beneficial role of antioxidants against slag corrosion was not observed, possibly due to the low pO2, which prevented C oxidation. As both MgAl2O4 and SiC, formed in the refractory, react and dissolve in the slag infiltrating in the refractory, they do not play a protective role.

Rett syndrome is a progressive neurodevelopmental disorder that lacks effective treatments. Although deep-brain stimulation can alleviate some symptoms in Rett model mice, this interventional manipulation requires deliberate surgical operations. Here, we report that electro-acupuncture stimulation (EAS) can ameliorate symptoms of an Mecp2-knockout rat model of Rett syndrome from the remote acupoints Baihui (GV 20), Yongquan (KI 1), and Shenmen (HT 7). We find that EAS not only prolongs the survival time of Rett rats, but also improves their behavior ability, including locomotion, motor coordination, and social interaction. Neural activation was observed in the substantia nigra of the midbrain, corpus striatum, and cerebral cortex of wild-type and Rett model rats, as reflected by the increased expression of the c-Fos protein. Hence, EAS provides a potential promising therapeutic tool for treating neurodevelopmental diseases.

Objective: We aimed to identify the genetic cause of one hydrops fetalis with Noonan syndrome (NS) manifestations including increased nuchal translucency (INT) and ascites through prenatal whole exome sequencing (WES). Case report: The case is a gestational age (GA) 18 fetus of two healthy parents with a normal child. We proceeded the genomic DNA from both fetus amniotic cells and parents to WES and identified a RIT1 mutation (c.268A>G) as the pathogenic cause of the hydrops fetalis by automatic prioritization algorithm after array-comparative genomic hybridization results showing negative. Conclusion: Mutations in RIT1 have been reported as the causes for different fetus structural abnormities in the recent years. This case contributes to the summary delineations of the prenatal NS phenotypes related to RIT1 mutation. In addition, the fast WES application, in this case, has demonstrated its advantage in prenatal disorder diagnosis when conventional karyotyping or chromosomal microarray testing result is negative.

Objective: We investigated a strategy of exome sequencing DNA from the unaffected parents and applied a set of filtering criteria to identify genes where both partners are heterozygous for a potentially pathogenic variant. Case report: We report a non-consanguineous couple who had three daughters, all spontaneous preterm birth at 36 weeks gestation and died in the first period after birth, suspected inborn errors of metabolism. Two days after birth, the first daughter presented with difficulty breathing, cyanosis and died; the second died at 33 days old; the third daughter was isolated under special care and was taken to the mother's room, developed the same symptoms and died after 5 days. Dried blood spot testing screen of 55 congenital metabolic disorders was negative. Conclusion: Heterogenous variant in SLC25A20 gene was found in both parents, contributing to the delineations of the neonatal phenotypes related to SLC25A20 mutation in CACTD.

Transgenic animal models with homologous etiology provide a promising way to pursue the neurobiological substrates of the behavioral deficits in autism spectrum disorder (ASD). Gain-of-function mutations of MECP2 cause MECP2 duplication syndrome, a severe neurological disorder with core symptoms of ASD. However, abnormal brain developments underlying the autistic-like behavioral deficits of MECP2 duplication syndrome are rarely investigated. To this end, a human MECP2 duplication (MECP2-DP) rat model was created by the bacterial artificial chromosome transgenic method. Functional and structural magnetic resonance imaging (MRI) with high-field were performed on 16 male MECP2-DP rats and 15 male wildtype rats at postnatal 28 days, 42 days, and 56 days old. Multimodal fusion analyses guided by locomotor-relevant metrics and social novelty time separately were applied to identify abnormal brain networks associated with diverse behavioral deficits induced by MECP2 duplication. Aberrant functional developments of a core network primarily composed of the dorsal medial prefrontal cortex (dmPFC) and retrosplenial cortex (RSP) were detected to associate with diverse behavioral phenotypes in MECP2-DP rats. Altered developments of gray matter volume were detected in the hippocampus and thalamus. We conclude that gain-of-function mutations of MECP2 induce aberrant functional activities in the default-mode-like network and aberrant volumetric changes in the brain, resulting in autistic-like behavioral deficits. Our results gain critical insights into the biomarker of MECP2 duplication syndrome and the neurobiological underpinnings of the behavioral deficits in ASD.

ObjectiveIn the lipid-rich brain, lipids performed signaling processes associated with the control system of the cell cycle, stress, and inflammatory reactions, as well as maintained brain and cellular homeostasis. The effects of general anesthesia on brain impairment in the elderly were controversial and complex. The study sought to evaluate the effect of lipid metabolism in the brain of aged marmosets and mice under long-term exposure to sevoflurane.MethodsA total of 6 marmosets over 8-year-old and 10 mice aged 18 months were divided into the sevoflurane anesthesia and control groups, respectively. Marmosets in the sevoflurane anesthesia group were exposed to 1.5–2.5% sevoflurane and 100% O2 for 6 h. Mice anesthetized with sevoflurane were exposed to 3% sevoflurane and 60% O2 for 6 h. All prefrontal cortex tissues of marmosets and mice were harvested for the analysis of lipidomics.ResultsCompared to the control group, we found that phosphatidylethanolamine (PE) (18:0/22:5), PE (16:0/22:5), PE (18:2/22:5), PE (14:0/22:5), and PE (18:1/22:5) increased in the prefrontal cortex of marmosets in the sevoflurane group, while triglyceride (TAG)56:5-fatty acid (FA) 20:4, TAG58:10-FA22:6, and TAG60:10-FA22:6 decreased. For aged mice, we indicated that lipid components phosphatidic acid (PA) (18:1/20:2) and TAG52:5-FA20:4 in the sevoflurane group increased, but PE (14:0/22:4), diglyceride (DAG) (16:1/18:2), and lysophosphatidylcholine (LPC) (16:1) + AcO decreased. More deeply, sevoflurane anesthesia resulted in the presence of 70 specific lipids in mice and marmosets. The enriched lipid subclasses were mainly monoacylglycerophosphoethanolamines and five other subclasses.ConclusionSevoflurane caused slight changes in lipid metabolism both in the aged brain of marmosets and mice. However, the pathways of lipid metabolism were not affected. The effects of sevoflurane on lipid metabolism in aged brains may differ among species.

BackgroundSurgery under general anesthesia leads to neural injury, especially in older patients. Sevoflurane anesthesia without surgery for 2 h does not induce neural injury, however, whether prolonger sevoflurane anesthesia without surgery has the same consequence is still unknown.MethodsIn the present study, aged marmosets were exposed to a clinical concentration of sevoflurane (1.5–2%) for 6 h to access the effects of prolonged sevoflurane anesthesia on the levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), Caspase3 activity and myelin formation in the brain.ResultsSevoflurane anesthesia did not alter the expression of IL-6 (120.1 ± 2.21 vs. 120.8 ± 2.25, p = 0.74), TNF-α (189.3 ± 31.35 vs. 218.7 ± 21.47, p = 0.25) and Caspase3 (57.35 ± 1.54 vs. 58.67 ± 1.19, p = 0.53) in the prefrontal cortex (PFC) of aged marmosets. The amount of MBP expression (60.99 ± 6.21 vs. 58.91 ± 2.71, p = 0.77) did not change following sevoflurane exposure.ConclusionSevoflurane anesthesia did not increase the levels of IL-6 and TNF-α, activated the the expression of Caspase3, and induced myelination deficits in the PFC of aged marmosets.

Resolving trajectories of axonal pathways in the primate prefrontal cortex remains crucial to gain insights into higher-order processes of cognition and emotion, which requires a comprehensive map of axonal projections linking demarcated subdivisions of prefrontal cortex and the rest of brain. Here, we report a mesoscale excitatory projectome issued from the ventrolateral prefrontal cortex (vlPFC) to the entire macaque brain by using viral-based genetic axonal tracing in tandem with high-throughput serial two-photon tomography, which demonstrated prominent monosynaptic projections to other prefrontal areas, temporal, limbic, and subcortical areas, relatively weak projections to parietal and insular regions but no projections directly to the occipital lobe. In a common 3D space, we quantitatively validated an atlas of diffusion tractography-derived vlPFC connections with correlative green fluorescent protein-labeled axonal tracing, and observed generally good agreement except a major difference in the posterior projections of inferior fronto-occipital fasciculus. These findings raise an intriguing question as to how neural information passes along long-range association fiber bundles in macaque brains, and call for the caution of using diffusion tractography to map the wiring diagram of brain circuits.

Current SpCas9 adenine base editors that minimise RNA off-target activities have constrained editing windows. Here the authors use domain insertion of TadA into Cas9 to narrow, expand or shift the editing window with RNA off-target minimization simultaneously

Summary: Autism spectrum disorder (ASD) is a highly heritable neurodevelopmental disorder, causing defects of social interaction and repetitive behaviors. Here, we identify a de novo heterozygous gene-truncating mutation of the Sentrin-specific peptidase1 (SENP1) gene in people with ASD without neurodevelopmental delay. We find that Senp1+/− mice exhibit core autistic-like symptoms such as social deficits and repetitive behaviors but normal learning and memory ability. Moreover, we find that inhibitory and excitatory synaptic functions are severely affected in the retrosplenial agranular (RSA) cortex of Senp1+/− mice. Lack of Senp1 leads to increased SUMOylation and degradation of fragile X mental retardation protein (FMRP), also implicated in syndromic ASD. Importantly, re-introducing SENP1 or FMRP specifically in RSA fully rescues the defects of synaptic function and autistic-like symptoms of Senp1+/− mice. Together, these results demonstrate that disruption of the SENP1-FMRP regulatory axis in the RSA causes autistic symptoms, providing a candidate region for ASD pathophysiology.

Abstract Background CircRNAs are found to affect initiation and progression of several cancer types. However, whether circRNAs are implicated in gallbladder cancer (GBC) progression remains obscure. Methods We perform RNA sequencing in 10 pairs of GBC and para-cancer tissues. CCK8 and clone formation assays are used to evaluate proliferation ability of GBC cells. qPCR and Western blot are used to determine expression of RNAs and proteins, respectively. CircRNA-protein interaction is confirmed by RNA pulldown, RNA immunoprecipitation, and fluorescence in situ hybridization. Results We find that circRNA expression pattern is tremendously changed during GBC development. Among dozens of significantly changed circRNAs, a circRNA generated from the oncogene ERBB2, named as circERBB2, is one of the most significant changes. CircERBB2 promotes GBC proliferation, in vitro and in vivo. Other than being a miRNA sponge, circERBB2 accumulates in the nucleoli and regulates ribosomal DNA transcription, which is one of the rate-limiting steps of ribosome synthesis and cellular proliferation. CircERBB2 regulates nucleolar localization of PA2G4, thereby forming a circERBB2-PA2G4-TIFIA regulatory axis to modulate ribosomal DNA transcription and GBC proliferation. Increased expression of circERBB2 is associated with worse prognosis of GBC patients. Conclusions Our findings demonstrate that circERBB2 serves as an important regulator of cancer cell proliferation and shows the potential to be a new therapeutic target of GBC.

Cytosine base editors are limited by editing scope and potential off-target effects. Here the authors screen diversified lamprey cytidine deaminases along with different protein fusion architectures and present base editors with improved fidelity.

Background: Exposure to anesthetics during early life may impair cognitive functions. However, the underlying mechanisms remain largely unknown. We set out to determine effects of sevoflurane anesthesia on folate metabolism and myelination in young non-human primates, mice and children. Methods: Young rhesus macaque and mice received 2.5 to 3% sevoflurane daily for three days. DNA and RNA sequencing and immunohistochemistry among others were used in the studies. We performed unbiased transcriptome profiling in prefrontal cortex of rhesus macaques and mice after the sevoflurane anesthesia. We constructed a brain blood barrier-crossing AAV-PHP.EB vector to harbor ERMN expression in rescue studies. We measured blood folate levels in children after anesthesia and surgery. Findings: We found that thymidylate synthase (TYMS) gene was downregulated after the sevoflurane anesthesia in both rhesus macaque and mice. There was a reduction in blood folate levels in children after the anesthesia and surgery. Combined with transcriptome and genome-wide DNA methylation analysis, we identified that ERMN was the primary target of the disrupted folate metabolism. Myelination was compromised by the anesthesia in the young mice, which was rescued by systematic administration of folic acid or expression of ERMN in the brain through brain-specific delivery of the adeno-associated virus. Moreover, folic acid and expression of ERMN alleviated the cognitive impairment caused by the sevoflurane anesthesia in the mice. Interpretation: General anesthesia leads to disrupted folate metabolism and subsequently defects in myelination in the developmental brain, and ERMN is the important target affected by the anesthesia via epigenetic mechanisms. Keywords: Sevoflurane, Folate, Methylation, ERMN, Myelination, Neurotoxicity

Abstract Background Amyotrophic lateral sclerosis (ALS) is a late onset neurodegenerative disease with fast progression. ALS has heavy genetic components in which a series of genetic mutations have been identified. In 2013, Mutations of the CREST gene (also known as SS18L1), which functions as a calcium-regulated transcriptional activator, were found in sporadic ALS patients. However, the pathogenic causality and mechanisms of ALS-associated mutations of CREST remain to be determined. Methods In this study, we constructed CREST knockout and Q394X knock-in mice with CRISPR/Cas9 system. Using biochemical and imaging tools, we illustrated core pathological phenotypes in CREST mutant mice and claimed the possible pathogenic mechanisms. Furthermore, we also observed locomotion defects in CREST mutant mice with behavioural tests. Results We demonstrate that ALS-related CREST-Q388X mutation exhibits loss-of-function effects. Importantly, the microglial activation was prevalent in CREST haploinsufficiency mice and Q394X mice mimicking the human CREST Q388X mutation. Furthermore, we showed that both CREST haploinsufficiency and Q394X mice displayed deficits in motor coordination. Finally, we identified the critical role of CREST-BRG1 complex in repressing the expression of immune-related cytokines including Ccl2 and Cxcl10 in neurons, via histone deacetylation, providing the molecular mechanisms underlying inflammatory responses within mice lack of CREST. Conclusion Our findings indicate that elevated inflammatory responses in a subset of ALS may be caused by neuron-derived factors, suggesting potential therapeutic methods through inflammation pathways.

Background: Analysis of cell-free DNA (cfDNA) is promising for broad applications in clinical settings, but with significant bias towards late-stage cancers. Although recent studies have discussed the diverse and degraded nature of cfDNA molecules, little is known about its impact on the practice of cfDNA analysis. Methods: We developed single-strand library preparation and hybrid-capture-based cfDNA sequencing (SLHC-seq) to analysis degraded cfDNA fragments. Next we used SLHC-seq to perform cfDNA profiling in 112 pancreatic cancer patients, and the results were compared with 13 previous reports. Extensive analysis was performed in terms of cfDNA fragments to explore the reasons for higher detection rate of KRAS mutations in the circulation of pancreatic cancers. Findings: By applying the new approach, we achieved higher efficiency in analysis of mutations than previously reported using other detection assays. 791 cancer-specific mutations were detected in plasma of 88% patients with KRAS hotspots detected in 70% of all patients. Only 8 mutations were detected in 28 healthy controls without any known oncogenic or truncating alleles. cfDNA profiling by SLHC-seq was largely consistent with results of tissue-based sequencing. SLHC-seq rescued short or damaged cfDNA fragments along to increase the sensitivity and accuracy of circulating-tumour DNA detection. Interpretation: We found that the small mutant fragments are prevalent in early-stage patients, which provides strong evidence for fragment size-based detection of pancreatic cancer. The new pipeline enhanced our understanding of cfDNA biology and provide new insights for liquid biopsy. Keywords: Pancreatic cancer, Cell-free DNA, Fragmentation, Tissue biopsy, Diagnosis and prognosis

African swine fever (ASF) is a viral disease with a high fatality rate in both domestic pigs and wild boars. ASF has greatly challenged pig-raising countries and also negatively impacted regional and national trade of pork products. To date, ASF has spread throughout Africa, Europe, and Asia. The development of safe and effective ASF vaccines is urgently required for the control of ASF outbreaks. The ASF virus (ASFV), the causative agent of ASF, has a large genome and a complex structure. The functions of nearly half of its viral genes still remain to be explored. Knowledge on the structure and function of ASFV proteins, the mechanism underlying ASFV infection and immunity, and the identification of major immunogenicity genes will contribute to the development of an ASF vaccine. In this context, this paper reviews the available knowledge on the structure, replication, protein function, virulence genes, immune evasion, inactivation, vaccines, control, and diagnosis of ASFV.

Mutations of CNTNAP1 were associated with myelination disorders, suggesting the role of CNTNAP1 in myelination processes. Whether CNTNAP1 may have a role in early cortical neuronal development is largely unknown. In this study, we identified 4 compound heterozygous mutations of CNTNAP1 in 2 Chinese families. Using mouse models, we found that CNTNAP1 is highly expressed in neurons and is located predominantly in MAP2+ neurons during the early developmental stage. Importantly, Cntnap1 deficiency results in aberrant dendritic growth and spine development in vitro and in vivo, and it delayed migration of cortical neurons during early development. Finally, we found that the number of parvalbumin+ neurons in the cortex and hippocampus of Cntnap1–/– mice is strikingly increased by P15, suggesting that excitation/inhibition balance is impaired. Together, this evidence elucidates a critical function of CNTNAP1 in cortical development, providing insights underlying molecular and circuit mechanisms of CNTNAP1-related disease.

Astrocytes respond to and regulate neuronal activity, yet their role in mammalian behavior remains incompletely understood. Especially unclear is whether, and if so how, astrocyte activity regulates contextual fear memory, the dysregulation of which leads to pathological fear-related disorders. We generated GFAP-ChR2-EYFP rats to allow the specific activation of astrocytes in vivo by optogenetics. We found that after memory acquisition within a temporal window, astrocyte activation disrupted memory consolidation and persistently decreased contextual but not cued fear memory accompanied by reduced fear-related anxiety behavior. In vivo microdialysis experiments showed astrocyte photoactivation increased extracellular ATP and adenosine concentrations. Intracerebral blockade of adenosine A1 receptors (A1Rs) reversed the attenuation of fear memory. Furthermore, intracerebral or intraperitoneal injection of A1R agonist mimicked the effects of astrocyte activation. Therefore, our findings provide a deeper understanding of the astrocyte-mediated regulation of fear memory and suggest a new and important therapeutic strategy against pathological fear-related disorders.

Summary: Short-term memory (STM) is crucial for animals to hold information for a small period of time. Persistent or recurrent neural activity, together with neural oscillations, is known to encode the STM at the cellular level. However, the coding mechanisms at the microcircuitry level remain a mystery. Here, we performed two-photon imaging on behaving mice to monitor the activity of neuronal microcircuitry. We discovered a neuronal subpopulation in the medial prefrontal cortex (mPFC) that exhibited emergent properties in a context-dependent manner underlying a STM-like behavior paradigm. These neuronal subpopulations exclusively comprise excitatory neurons and mainly represent a group of neurons with stronger functional connections. Microcircuitry plasticity was maintained for minutes and was absent in an animal model of Alzheimer’s disease (AD). Thus, these results point to a functional coding mechanism that relies on the emergent behavior of a functionally defined neuronal assembly to encode STM.

Porcine circovirus associated diseases (PCVAD) is a contagious disease of swine caused by porcine circovirus type 2 (PCV2). The capsid protein (Cap) is the sole structural protein and the main antigen of PCV2. Cap is the principal immunogenic protein and induces humoral and cellular immunity. CD154 and GM-CSF are immune adjuvants that enhance responses to vaccines. However, whether these two cellular molecules could produce an enhanced effect in PCV2 vaccines still needs to be further studied. The results of PCR and restriction enzyme showed that the recombinant lentiviral plasmids pCDH-TB-Cap, pCDH-TB-Cap-CD154 and pCDH-TB-Cap were successfully constructed. Western blot and IFA showed that the three fusion proteins TB-Cap, TB-Cap-CD154 and TB-Cap-GM-CSF were stably expressed in CHO-K1 cells. Indirect ELISA assay showed that mice immunized with TB-Cap-CD154 and TB-Cap-GM-CSF fusion proteins produced higher PCV2-specific antibodies than mice immunized with the TB-Cap and a commercial vaccine (p < 0.0001). Moreover, lymphocyte proliferation and flow cytometry showed that the cellular immune response of each immune group was significantly enhanced (p < 0.0001). After PCV2 challenge, the results revealed that the viral loads in serum, lung and kidney of all vaccinated groups were significantly lower than the PBS group (p < 0.0001). The transcription levels of IL-2, IFN-gamma, IL-4 and IL-10 cytokines in the TB-Cap, TB-Cap-CD154 and TB-Cap-GM-CSF groups were significantly higher than those in the PBS and recombinant vaccine groups (p < 0.0001). These results indicated that CD154 and GM-CSF could enhance the ability of TB-Cap protein to induce the body to produce PCV2 specific antibodies and increase the transcription level of cytokines. Thus, CD154 and GM-CSF molecules were a powerful immunoadjuvant for PCV2 subunit vaccines. The novel TB-Cap-CD154 and TB-Cap-GM-CSF subunit vaccine has the potential to be used for the prevention and control of PCVAD.

Abstract Background Methyl-CpG-binding protein-2 (MeCP2) is a critical regulator for neural development. Either loss- or gain-of-function leads to severe neurodevelopmental disorders, such as Rett syndrome (RTT) and autism spectrum disorder (ASD). We set out to screen for MECP2 mutations in patients of ASD and determine whether these autism-related mutations may compromise the proper function of MeCP2. Methods Whole-exome sequencing was performed to screen MECP2 and other ASD candidate genes for 120 patients diagnosed with ASD. The parents of patients who were identified with MECP2 mutation were selected for further Sanger sequencing. Each patient accomplished the case report form including general information and clinical scales applied to assess their clinical features. Mouse cortical neurons and HEK-293 cells were cultured and transfected with MeCP2 wild-type (WT) or mutant to examine the function of autism-associated MeCP2 mutants. HEK-293 cells were used to examine the expression of MeCP2 mutant constructs with Western blot. Mouse cortical neurons were used to analyze neurites and axon outgrowth by immunofluorescence experiments. Results We identified three missense mutations of MECP2 from three autism patients by whole-exome sequencing: p.P152L (c.455C>T), p.P376S (c.1162C>T), and p.R294X (c.880C>T). Among these mutations, p.P152L and p.R294X were de novo mutations, whereas p.P376S was inherited maternally. The diagnosis of RTT was excluded in all three autism patients. Abnormalities of dendritic and axonal growth were found after autism-related MeCP2 mutants were expressed in mouse cortical neurons; suggesting that autism-related MECP2 mutations impair the proper development of neurons. Conclusions Our study identified genetic mutations of the MECP2 gene in autism patients, which were previously considered to be associated primarily with RTT. This finding suggests that loss-of-function mutations of MECP2 may also lead to autism spectrum disorders.

Abstract As an epigenetic modulator of gene expression, Methyl-CpG binding protein 2 (MeCP2) is essential for normal neurological function. Dysfunction of MeCP2 is associated with a variety of neurological disorders. MECP2 gene duplication in human causes neuropsychiatric symptoms such as mental retardation and autism. MeCP2 overexpression in mice results in neurobehavioural disorders, dendritic abnormalities, and synaptic defects. However, how gain of MeCP2 function influences cortical processing of sensory information remains unclear. In this study, we examined visual processing in a mouse model of MECP2 duplication syndrome (MECP2 Tg1 mouse) at 8 and 14 weeks, which were before and after the onset of behavioural symptoms, respectively. In vivo extracellular recordings from primary visual cortex (V1) showed that neurons in Tg1 mice at both adult ages preferred higher spatial frequencies (SFs) than those in wild-type (WT) littermate controls, and the semi-saturation contrasts of neurons were lower in Tg1 mice at 8 weeks but not at 14 weeks. Behavioural experiments showed that the performance for visual detection at high SFs and low contrasts was higher in MECP2 Tg1 mice. Thus, MeCP2 gain-of-function in mice leads to higher visual acuity and contrast sensitivity, both at the levels of cortical response and behavioural performance.

Abstract Background Classical strain engineering methods often have limitations in altering multigenetic cellular phenotypes. Here we try to improve Saccharomyces cerevisiae ethanol tolerance and productivity by reprogramming its transcription profile through rewiring its key transcription component RNA polymerase II (RNAP II), which plays a central role in synthesizing mRNAs. This is the first report on using directed evolution method to engineer RNAP II to alter S. cerevisiae strain phenotypes. Results Error-prone PCR was employed to engineer the subunit Rpb7 of RNAP II to improve yeast ethanol tolerance and production. Based on previous studies and the presumption that improved ethanol resistance would lead to enhanced ethanol production, we first isolated variant M1 with much improved resistance towards 8 and 10% ethanol. The ethanol titers of M1 was ~122 g/L (96.58% of the theoretical yield) under laboratory very high gravity (VHG) fermentation, 40% increase as compared to the control. DNA microarray assay showed that 369 genes had differential expression in M1 after 12 h VHG fermentation, which are involved in glycolysis, alcoholic fermentation, oxidative stress response, etc. Conclusions This is the first study to demonstrate the possibility of engineering eukaryotic RNAP to alter global transcription profile and improve strain phenotypes. Targeting subunit Rpb7 of RNAP II was able to bring differential expression in hundreds of genes in S. cerevisiae, which finally led to improvement in yeast ethanol tolerance and production.

Accumulated genetic evidences indicate that the contactin associated protein-like (CNTNAP) family is implicated in autism spectrum disorders (ASD). In this study, we identified genetic mutations in the CNTNAP3 gene from Chinese Han ASD cohorts and Simons Simplex Collections. We found that CNTNAP3 interacted with synaptic adhesion proteins Neuroligin1 and Neuroligin2, as well as scaffolding proteins PSD95 and Gephyrin. Significantly, we found that CNTNAP3 played an opposite role in controlling the development of excitatory and inhibitory synapses in vitro and in vivo, in which ASD mutants exhibited loss-of-function effects. In this study, we showed that the male Cntnap3-null mice exhibited deficits in social interaction， spatial learning and prominent repetitive behaviors. These evidences elucidate the pivotal role of CNTNAP3 in synapse development and social behaviors, providing mechanistic insights into ASD.

African swine fever is a highly contagious global disease caused by the African swine fever virus. Since African swine fever (ASF) was introduced to Georgia in 2007, it has spread to many Eurasian countries at an extremely fast speed. It has recently spread to China and other major pig-producing countries in southeast Asia, threatening global pork production and food security. As there is no available vaccine at present, prevention and control must be carried out based on early detection and strict biosecurity measures. Early detection should be based on the rapid identification of the disease on the spot, followed by laboratory diagnosis, which is essential for disease control. In this review, we introduced the prevalence, transmission routes, eradication control strategies, and diagnostic methods of ASF. We reviewed the various methods of diagnosing ASF, focusing on their technical characteristics and clinical test results. Finally, we give some prospects for improving the diagnosis strategy in the future.

Objective: The objective of this study is to evaluate novel variations in a Chinese family of Han ethnicity, with arrhythmogenic right ventricular cardiomyopathy (ARVC) by the powerful technology of whole-exome sequencing (WES). Methods: Genomic DNA from representative family members was extracted and processed for WES via standardized methods. The exome sequence data were analyzed by Genome Analysis Toolkit software. Sanger sequencing was performed on DNA from the proband for genotype confirmation and from the other family members for familial co-segregation analysis. Results: A rare single nucleotide variant c.1592T>G (p.Phe531Cys) in exon 10 of desmoglein-2 (DSG2) was identified by WES in the affected individuals but not in the healthy control, which was confirmed by Sanger sequencing, suggesting that the mutation probably was a causal mutation for the familial disorder of ARVC. Conclusion: We identified a rare disease-causing mutation of the DSG2 gene associated with familial ARVC. The results might contribute to the early genetic analysis in inherited cardiomyopathies.

ABSTRACT MicroRNAs (miRNAs) are critical for both development and function of the central nervous system. Significant evidence suggests that abnormal expression of miRNAs is associated with neurodevelopmental disorders. MeCP2 protein is an epigenetic regulator repressing or activating gene transcription by binding to methylated DNA. Both loss-of-function and gain-of-function mutations in the MECP2 gene lead to neurodevelopmental disorders such as Rett syndrome, autism and MECP2 duplication syndrome. In this study, we demonstrate that miR-130a inhibits neurite outgrowth and reduces dendritic spine density as well as dendritic complexity. Bioinformatics analyses, cell cultures and biochemical experiments indicate that miR-130a targets MECP2 and down-regulates MeCP2 protein expression. Furthermore, expression of the wild-type MeCP2, but not a loss-of-function mutant, rescues the miR-130a-induced phenotype. Our study uncovers the MECP2 gene as a previous unknown target for miR-130a, supporting that miR-130a may play a role in neurodevelopment by regulating MeCP2. Together with data from other groups, our work suggests that a feedback regulatory mechanism involving both miR-130a and MeCP2 may serve to ensure their appropriate expression and function in neural development.

ObjectiveSpinocerebellar ataxia type 3 (SCA3) is the most commonly occurring type of autosomal dominant spinocerebellar ataxia. The present study aims to investigate progressive changes in white matter (WM) fiber in asymptomatic and symptomatic patients with SCA3.MethodsA total of 62 participants were included in this study. Among them, 16 were asymptomatic mutation carriers (pre-SCA3), 22 were SCA3 patients with clinical symptoms, and 24 were normal controls (NC). Group comparison of tract-based spatial statistics was performed to identify microstructural abnormalities at different SCA3 disease stages.ResultsDecreased fractional anisotropy (FA) and increased mean diffusivity (MD) were found in the left inferior cerebellar peduncle and superior cerebellar peduncle (SCP) in the pre-SCA3 group compared with NC. The symptomatic SCA3 group showed brain-wide WM tracts impairment in both supratentorial and infratentorial networks, and the mean FA value of the WM skeleton showed a significantly negative correlation with the International Cooperative Ataxia Rating Scale (ICARS) scores. Specifically, FA of the bilateral posterior limb of the internal capsule negatively correlated with SCA3 disease duration. We also found that FA values in the right medial lemniscus and SCP negatively correlated with ICARS scores, whereas FA in the right posterior thalamic radiation positively correlated with Montreal Cognitive Assessment scores. In addition, MD in the middle cerebellar peduncle, left anterior limb of internal capsule, external capsule, and superior corona radiate positively correlated with ICARS scores in SCA3 patients.ConclusionWM microstructural changes are present even in the asymptomatic stages of SCA3. In individuals in which the disease has progressed to the symptomatic stage, the integrity of WM fibers across the whole brain is affected. Furthermore, abnormalities in WM tracts are closely related to SCA3 disease severity, including movement disorder and cognitive dysfunction. These findings can deepen our understanding of the neural basis of SCA3 dysfunction.

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMR1 gene that inactivate expression of the gene product, the fragile X mental retardation 1 protein (FMRP). In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology to generate Fmr1 knockout (KO) rats by disruption of the fourth exon of the Fmr1 gene. Western blotting analysis confirmed that the FMRP was absent from the brains of the Fmr1 KO rats (Fmr1exon4-KO). Electrophysiological analysis revealed that the theta-burst stimulation (TBS)–induced long-term potentiation (LTP) and the low-frequency stimulus (LFS)–induced long-term depression (LTD) were decreased in the hippocampal Schaffer collateral pathway of the Fmr1exon4-KO rats. Short-term plasticity, measured as the paired-pulse ratio, remained normal in the KO rats. The synaptic strength mediated by the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) was also impaired. Consistent with previous reports, the Fmr1exon4-KO rats demonstrated an enhanced 3,5-dihydroxyphenylglycine (DHPG)–induced LTD in the present study, and this enhancement is insensitive to protein translation. In addition, the Fmr1exon4-KO rats showed deficits in the probe trial in the Morris water maze test. These results demonstrate that deletion of the Fmr1 gene in rats specifically impairs long-term synaptic plasticity and hippocampus-dependent learning in a manner resembling the key symptoms of FXS. Furthermore, the Fmr1exon4-KO rats displayed impaired social interaction and macroorchidism, the results consistent with those observed in patients with FXS. Thus, Fmr1exon4-KO rats constitute a novel rat model of FXS that complements existing mouse models.

Cognitive defects in autism spectrum disorder (ASD) include socialization and communication: key behavioral capacities that separate humans from other species. Here, we analyze gene expression in the prefrontal cortex of 63 autism patients and control individuals, as well as 62 chimpanzees and macaques, from natal to adult age. We show that among all aberrant expression changes seen in ASD brains, a single aberrant expression pattern overrepresented in genes involved synaptic-related pathways is enriched in nucleotide variants linked to autism. Furthermore, only this pattern contains an excess of developmental expression features unique to humans, thus resulting in the disruption of human-specific developmental programs in autism. Several members of the early growth response (EGR) transcription factor family can be implicated in regulation of this aberrant developmental change. Our study draws a connection between the genetic risk architecture of autism and molecular features of cortical development unique to humans.

Intellectual disability (ID) is a heterogeneous disorder caused by chromosomal abnormalities, monogenic factors and environmental factors. 22q13 deletion syndrome is a genetic disorder characterized by severe ID. Although the frequency of 22q13 deletions in ID is unclear, it is believed to be largely underestimated. To address this issue, we used Affymetrix Human SNP 6.0 array to detect the 22q13 deletions in 234 Chinese unexplained ID patients and 103 controls. After the Quality Control (QC) test of raw data, 22q13 deletions were found in four out of 230 cases (1.7%), while absent in parents of the cases and 101 controls. A review of genome-wide microarray studies in ID was performed and the frequency of 22q13 deletions from the literatures was 0.24%, much lower than our report. The overlapping region shared by all 4 cases encompasses the gene SHANK3. A heterozygous de novo nonsense mutation Y1015X of SHANK3 was identified in one ID patient. Cortical neurons were prepared from embryonic mice and were transfected with a control plasmid, shank3 wild-type (WT) or mutant plasmids. Overexpression of the Y1015 mutant in neurons significantly affected neurite outgrowth compared with shank3 WT. These findings suggest that 22q13 deletions may be a more frequent cause for Chinese ID patients than previously thought, and the SHANK3 gene is involved in the neurite development.