Cnksr2 Loss in Mice Leads to Increased Neural Activity and Behavioral Phenotypes of Epilepsy-Aphasia Syndrome.

Epilepsy Aphasia Syndromes (EAS) are a spectrum of childhood epileptic, cognitive, and language disorders of unknown etiology. CNKSR2 is a strong X-linked candidate gene implicated in EAS; however, there have been no studies of genetic models to dissect how its absence may lead to EAS. Here we develop a novel Cnksr2 KO mouse line and show that male mice exhibit increased neural activity and have spontaneous electrographic seizures. Cnksr2 KO mice also display significantly increased anxiety, impaired learning and memory, and a progressive and dramatic loss of ultrasonic vocalizations. We find that Cnksr2 is expressed in cortical, striatal, and cerebellar regions and is localized at both excitatory and inhibitory postsynapses. Proteomics analysis reveals Cnksr2 anchors key binding partners at synapses, and its loss results in significant alterations of the synaptic proteome, including proteins implicated in epilepsy disorders. Our results validate that loss of CNKSR2 leads to EAS and highlights the roles of Cnksr2 in synaptic organization and neuronal network activity.SIGNIFICANCE STATEMENT Epilepsy Aphasia Syndromes (EAS) are at the severe end of a spectrum of cognitive-behavioral symptoms seen in childhood epilepsies, and they remain an inadequately understood disorder. The prognosis of EAS is frequently poor, and patients have life-long language and cognitive disturbances. Here we describe a genetic mouse model of EAS, based on the KO of the EAS risk gene Cnksr2 We show that these mice exhibit electrophysiological and behavioral phenotypes similar to those of patients, providing an important new model for future studies of EAS. We also provide insights into the molecular disturbances downstream of Cnksr2 loss by using in vivo quantitative proteomics tools.

Anatomical factors influencing temporomandibular joint clicking in young adults: temporomandibular joint structure disorder or lateral pterygoid muscle dysfunction?

Objective: This study aimed to investigate the selected anatomical factors that can potentially influence temporomandibular joint (TMJ) clicking in young adults by assessing TMJ structures and lateral pterygoid muscle (LPM) function using magnetic resonance imaging (MRI). Methods: The patients were divided into four groups: the healthy control group; the clicking on mouth opening group; the clicking on mouth closing group; and the clicking on mouth opening and closing group. Additionally, we used clinical palpation to evaluate the masticatory muscles' functional state and employed MRI using the OCOR-T1WI-FSE-CLOSED, OSAG-PDW-FSE-CLOSED, and OSAG-PDW-FSE-OPEN sequences to analyze the texture of the lateral pterygoid muscle (LPM). Results: The proportion of any articular disc or condylar morphology class did not differ significantly between the TMJ clicking and HC groups. The articular disc position did not differ significantly between the TMJ clicking and HC groups. In the TMJ clicking group, the presence of masticatory muscle dysfunction differed significantly between the clicking and non-clicking sides. Moreover, the LPM accounted for the highest proportion among masticatory muscles with tenderness in all TMJ clicking subgroups (77.78%-100%). Therefore, in the TMJ clicking group, the LPM texture was less defined, more uniform in gray scale, and more similar to local texture (p < 0.0001). Conclusion: The occurrence of TMJ clicking in young adults is unrelated to the TMJ structure but related to the function of masticatory muscles, particularly the LPM.

Proximity analysis of native proteomes reveals phenotypic modifiers in a mouse model of autism and related neurodevelopmental conditions.

One of the main drivers of autism spectrum disorder is risk alleles within hundreds of genes, which may interact within shared but unknown protein complexes. Here we develop a scalable genome-editing-mediated approach to target 14 high-confidence autism risk genes within the mouse brain for proximity-based endogenous proteomics, achieving the identification of high-specificity spatial proteomes. The resulting native proximity proteomes are enriched for human genes dysregulated in the brain of autistic individuals, and reveal proximity interactions between proteins from high-confidence risk genes with those of lower-confidence that may provide new avenues to prioritize genetic risk. Importantly, the datasets are enriched for shared cellular functions and genetic interactions that may underlie the condition. We test this notion by spatial proteomics and CRISPR-based regulation of expression in two autism models, demonstrating functional interactions that modulate mechanisms of their dysregulation. Together, these results reveal native proteome networks in vivo relevant to autism, providing new inroads for understanding and manipulating the cellular drivers underpinning its etiology.

Immediate implant placement in single mandibular molar with chronic periapical periodontitis.

INTRODUCTION: The present study aims to assess and compare the clinical outcomes of immediate implant placement in the mandibular molar region with or without the presence of chronic periapical periodontitis. MATERIALS AND METHODS: Employing a case-control design, this study encompassed a cohort of patients necessitating implant surgery to supplant a single, failed mandibular molar. Participants exhibiting periapical lesions measuring between > 4 mm and < 8 mm were assigned to the test group, while those without periapical lesions to the control group. Subsequent to flap surgery and tooth extraction, extraction sockets were debrided thoroughly, and implants were immediately implanted (baseline). Permanent restorative procedures were carried out three months post-operation, with follow-up conducted one year post-surgery. During the study period, parameters including implant survival rate, Cone Beam Computer Tomography (CBCT) data, implant stability quotient (ISQ), insertional torque values (ITV), and potential complications were closely monitored. RESULTS: Throughout the yearlong observation period subsequent to implant placement, both groups exhibited a 100% implant survival rate. None of the participants experienced any complications. Both groups demonstrated significant decreases in the height and width of the alveolar bone (P < 0.05). However, there were no statistically discernible differences between corresponding areas in the two groups (P > 0.05). The differences in ITV between the test group (37.94 ± 2.12 N•cm) and the control group (38.55 ± 2.71 N•cm) were not statistically significant at baseline (P > 0.05). A significant rise in ISQ was noted within the same group between baseline and three months post-operation (P < 0.05), while no significant variations in ISQ changes were noted between the two groups (P > 0.05). CONCLUSION: Given the constraints of this investigation, the preliminary clinical outcomes of immediate implant placement in the mandibular molar region with chronic periapical periodontitis do not significantly differ from those observed in instances devoid of chronic periapical periodontitis.

The relationship between the oblique sagittal temporomandibular joint disc position and the volume surface area of the condyle in young TMD adults.

The present study aims to compare the volume surface area of the condyle, the horizontal condylar axial angle and the disc-condyle angle between temporomandibular disorder (TMD) and asymptomatic volunteers, explore and analyze the relationship between the temporomandibular joint (TMJ) disc position in oblique sagittal plane and the volume surface area of the condyle in young adults with TMD symptoms. 84 young adult volunteers were received TMJ examination by Magnetic Resonance Imaging (MRI) and Cone Beam Computed Tomography (CBCT). TMD and asymptomatic volunteers were 42 each. MRI was used to assess the position of TMJ disc in the oblique sagittal plane with the condyle apex method. CBCT data were used for three-dimensional (3D) reconstruction of condyle and the measurements of the horizontal condylar axial angle and the volume surface area of the condyle. The condylar volume surface area of the TMD group was smaller than that of the asymptomatic group (p < 0.05), the disc condyle angle was larger than that of the asymptomatic group (p < 0.05), and no significant difference was found in the horizontal condylar axial angle (p > 0.05). In terms of correlation, the volume surface area of the condyle were negatively correlated with the position of the articular disc in TMD patients (p < 0.05). This significant negative correlation suggests that the possibility of disc displacement can be considered when poor condylar morphology is found.

A Ventromedial Prefrontal-to-Lateral Entorhinal Cortex Pathway Modulates the Gain of Behavioral Responding During Threat.

BACKGROUND: The ability to correctly associate cues and contexts with threat is critical for survival, and the inability to do so can result in threat-related disorders such as posttraumatic stress disorder. The prefrontal cortex (PFC) and hippocampus are well known to play critical roles in cued and contextual threat memory processing. However, the circuits that mediate prefrontal-hippocampal modulation of context discrimination during cued threat processing are less understood. Here, we demonstrate the role of a previously unexplored projection from the ventromedial region of PFC (vmPFC) to the lateral entorhinal cortex (LEC) in modulating the gain of behavior in response to contextual information during threat retrieval and encoding. METHODS: We used optogenetics followed by in vivo calcium imaging in male C57/B6J mice to manipulate and monitor vmPFC-LEC activity in response to threat-associated cues in different contexts. We then investigated the inputs to, and outputs from, vmPFC-LEC cells using Rabies tracing and channelrhodopsin-assisted electrophysiology. RESULTS: vmPFC-LEC cells flexibly and bidirectionally shaped behavior during threat expression, shaping sensitivity to contextual information to increase or decrease the gain of behavioral output in response to a threatening or neutral context, respectively. CONCLUSIONS: Glutamatergic vmPFC-LEC cells are key players in behavioral gain control in response to contextual information during threat processing and may provide a future target for intervention in threat-based disorders.

Identification of new ciliary signaling pathways in the brain and insights into neurological disorders.

Primary cilia are conserved sensory hubs essential for signaling transduction and embryonic development. Ciliary dysfunction causes a variety of developmental syndromes with neurological features and cognitive impairment, whose basis mostly remains unknown. Despite connections to neural function, the primary cilium remains an overlooked organelle in the brain. Most neurons have a primary cilium; however, it is still unclear how this organelle modulates brain architecture and function, given the lack of any systemic dissection of neuronal ciliary signaling. Here, we present the first in vivo glance at the molecular composition of cilia in the mouse brain. We have adapted in vivo BioID (iBioID), targeting the biotin ligase BioID2 to primary cilia in neurons. We identified tissue-specific signaling networks enriched in neuronal cilia, including Eph/Ephrin and GABA receptor signaling pathways. Our iBioID ciliary network presents a wealth of neural ciliary hits that provides new insights into neurological disorders. Our findings are a promising first step in defining the fundamentals of ciliary signaling and their roles in shaping neural circuits and behavior. This work can be extended to pathological conditions of the brain, aiming to identify the molecular pathways disrupted in the brain cilium. Hence, finding novel therapeutic strategies will help uncover and leverage the therapeutic potential of the neuronal cilium.

A new voltammetric sensor based on poly(L-cysteine)/GR composite film modified electrode for the sensitive determination of amaranth in wastewater.

A simple voltammetric sensor was first constructed by dripping graphene (GR) on to the glass carbon electrode (GCE) and subsequently electro-polymerizing L-cysteine film. The sensor had a good voltammetric response to amaranth and linear in the range of 1.0 × 10-8-1.0 × 10-6 mol L-1. The detection limit of amaranth was 4.0 × 10-9 mol L-1 (S/N = 3). Using this method, we successfully obtained satisfactory results of amaranth in confectionery wastewater. This work promoted the potential applications of amino acid materials and GR in environmental pollution science.

Action potential-coupled Rho GTPase signaling drives presynaptic plasticity.

In contrast to their postsynaptic counterparts, the contributions of activity-dependent cytoskeletal signaling to presynaptic plasticity remain controversial and poorly understood. To identify and evaluate these signaling pathways, we conducted a proteomic analysis of the presynaptic cytomatrix using in vivo biotin identification (iBioID). The resultant proteome was heavily enriched for actin cytoskeleton regulators, including Rac1, a Rho GTPase that activates the Arp2/3 complex to nucleate branched actin filaments. Strikingly, we find Rac1 and Arp2/3 are closely associated with synaptic vesicle membranes in adult mice. Using three independent approaches to alter presynaptic Rac1 activity (genetic knockout, spatially restricted inhibition, and temporal optogenetic manipulation), we discover that this pathway negatively regulates synaptic vesicle replenishment at both excitatory and inhibitory synapses, bidirectionally sculpting short-term synaptic depression. Finally, we use two-photon fluorescence lifetime imaging to show that presynaptic Rac1 activation is coupled to action potentials by voltage-gated calcium influx. Thus, this study uncovers a previously unrecognized mechanism of actin-regulated short-term presynaptic plasticity that is conserved across excitatory and inhibitory terminals. It also provides a new proteomic framework for better understanding presynaptic physiology, along with a blueprint of experimental strategies to isolate the presynaptic effects of ubiquitously expressed proteins.

Essential role for InSyn1 in dystroglycan complex integrity and cognitive behaviors in mice.

Human mutations in the dystroglycan complex (DGC) result in not only muscular dystrophy but also cognitive impairments. However, the molecular architecture critical for the synaptic organization of the DGC in neurons remains elusive. Here, we report Inhibitory Synaptic protein 1 (InSyn1) is a critical component of the DGC whose loss alters the composition of the GABAergic synapses, excitatory/inhibitory balance in vitro and in vivo, and cognitive behavior. Association of InSyn1 with DGC subunits is required for InSyn1 synaptic localization. InSyn1 null neurons also show a significant reduction in DGC and GABA receptor distribution as well as abnormal neuronal network activity. Moreover, InSyn1 null mice exhibit elevated neuronal firing patterns in the hippocampus and deficits in fear conditioning memory. Our results support the dysregulation of the DGC at inhibitory synapses and altered neuronal network activity and specific cognitive tasks via loss of a novel component, InSyn1.

Solid-phase extraction of seventeen alternative flame retardants in water as determined by ultra-high-performance liquid chromatography-tandem mass spectrometry.

Flame retardants have evoked public concerns owing to their extensive usage in consumer products and potential adverse effects on human health. In this study, a rapid and sensitive solid-phase extraction-ultra-high-performance liquid chromatography-tandem mass spectrometry (SPE-UPLC-MS/MS) method was developed to determine hexabromocyclododecane (HBCD), tetrabromobisphenol-A (TBBPA), six bromophenols (BPs), and nine organophosphate flame retardants (OPFRs) in water. Because of the differences in elution conditions and ionization modes for group 1 (HBCD, TBBPA, and the BPs) and group 2 (OPFRs), we had to run them twice under the different conditions to analyse group 1 and group 2 using UPLC-MS/MS. The method detection limits were 0.1-2.5 ng/L, linearity range was 0.1-100.0 ng/L for group 1 (HBCD, TBBPA, and the BPs). The method detection limit was 0.10 ng/L, and the linearity range was 0.25-250 ng/L for the OPFRs. First, the pH values of the water samples were adjusted to the range of 2-3. Then, the acidified water samples were extracted by hydrophilic-lipophilic-balance solid phase extraction (HLB-SPE) cartridges, which were eluted with 12 mL of acetonitrile. Finally, the recoveries of HBCD, TBBPA, and the BPs were 76.2-98.1%, and the relative standard deviations (RSDs, n = 5) were 2.0-28.5%. Regarding the OPFRs, the recoveries were 72.4-110.3%, and the RSDs were 0.6-6.9%. The stability experiment showed that the concentration differences were less than 15%, meeting the requirement for quality control samples. This proposed method was successfully applied to surface water, ground water, raw water, finished water, tap water, and bottled water samples.

Plug-and-Play Protein Modification Using Homology-Independent Universal Genome Engineering.

Analysis of endogenous protein localization, function, and dynamics is fundamental to the study of all cells, including the diversity of cell types in the brain. However, current approaches are often low throughput and resource intensive. Here, we describe a CRISPR-Cas9-based homology-independent universal genome engineering (HiUGE) method for endogenous protein manipulation that is straightforward, scalable, and highly flexible in terms of genomic target and application. HiUGE employs adeno-associated virus (AAV) vectors of autonomous insertional sequences (payloads) encoding diverse functional modifications that can integrate into virtually any genomic target loci specified by easily assembled gene-specific guide-RNA (GS-gRNA) vectors. We demonstrate that universal HiUGE donors enable rapid alterations of proteins in vitro or in vivo for protein labeling and dynamic visualization, neural-circuit-specific protein modification, subcellular rerouting and sequestration, and truncation-based structure-function analysis. Thus, the "plug-and-play" nature of HiUGE enables high-throughput and modular analysis of mechanisms driving protein functions in cellular neurobiology.

Electrochemical behavior of amaranth and its sensitive determination based on Pd-doped polyelectrolyte functionalized graphene modified electrode.

In this work, poly(sodium p-styrenesulfonate) (PSS)-functionalized graphene supported palladium nanoparticles (Pd) composites were fabricated with simple ultrasonic bath method. The morphology and structure of PSS-GR-Pd composites were characterized using UV-vis absorption spectra, X-ray diffraction and Transmission Electron Microscopy. By combining the merits of the PSS-GR and Pd NPs, a new electrochemical sensor was erected to detect amaranth based on the PSS-GR-Pd nanocomposites. The electrochemical behavior of amaranth was investigated systematically in 0.1molL-1 phosphate buffer solution (PBS 2.0). At the optimum parameter, Ipa was found to be linearly dependent on the concentrations of amaranth (1×10-7-9×10-6molL-1). The detection limit was 7nM (S/N=3) and sensitivity was 5.85µAµM-1. Finally, this system was utilized for determining amaranth in soft drink using the standard addition method.

Enrichment of GABAA Receptor α-Subunits on the Axonal Initial Segment Shows Regional Differences.

Although it is generally recognized that certain α-subunits of γ-aminobutyric acid type A receptors (GABAARs) form enriched clusters on the axonal initial segment (AIS), the degree to which these clusters vary in different brain areas is not well known. In the current study, we quantified the density, size, and enrichment ratio of fluorescently labeled α1-, α2-, or α3-subunits aggregates co-localized with the AIS-marker ankyrin G and compared them to aggregates in non-AIS locations among different brain areas including hippocampal subfields, basal lateral amygdala (BLA), prefrontal cortex (PFC), and sensory cortex (CTX). We found regional differences in the enrichment of GABAAR α-subunits on the AIS. Significant enrichment was identified in the CA3 of hippocampus for α1-subunits, in the CA1, CA3, and BLA for α2-subunits, and in the BLA for α3-subunits. Using α-subunit knock-out (KO) mice, we found that BLA enrichment of α2- and α3-subunits were physiologically independent of each other, as the enrichment of one subunit was unaffected by the genomic deletion of the other. To further investigate the unique pattern of α-subunit enrichment in the BLA, we examined the association of α2- and α3-subunits with the presynaptic vesicular GABA transporter (vGAT) and the anchoring protein gephyrin (Geph). As expected, both α2- and α3-subunits on the AIS within the BLA received prominent GABAergic innervation from vGAT-positive terminals. Further, we found that the association of α2- and α3-subunits with Geph was weaker in AIS versus non-AIS locations, suggesting that Geph might be playing a lesser role in the enrichment of α2- and α3-subunits on the AIS. Overall, these observations suggest that GABAARs on the AIS differ in subunit composition across brain regions. As with somatodendritic GABAARs, the distinctive expression pattern of AIS-located GABAAR α-subunits in the BLA, and other brain areas, likely contribute to unique forms of GABAergic inhibitory transmission and pharmacological profiles seen in different brain areas.

A Simple and Sensitive Voltammetric Method for the Determination of Orange II Based on a Functionalized Graphene-Modified Electrode.

A simple and sensitive voltammetric sensor for Orange II was developed, based on a poly(sodium p-styrenesulfonate)-functionalized graphene-modified glassy carbon electrode. This voltammetric sensor showed strong accumulation ability and an excellent voltammetric response for Orange II. The electrochemical behavior of Orange II was systematically investigated in a pH 7.0 phosphate buffer solution. By linear sweep voltammetry, under optimum conditions, a good linear relationship was obtained between peak currents and Orange II concentrations in the wider range of 3 × 10(-8) to 5 × 10(-6) mol/L, with an LOD of 1 × 10(-8) mol/L. In addition, the proposed Orange II sensor was successfully applied to real food samples with satisfactory recovery.

Modulation of anxiety and fear via distinct intrahippocampal circuits.

Recent findings indicate a high level of specialization at the level of microcircuits and cell populations within brain structures with regards to the control of fear and anxiety. The hippocampus, however, has been treated as a unitary structure in anxiety and fear research despite mounting evidence that different hippocampal subregions have specialized roles in other cognitive domains. Using novel cell-type- and region-specific conditional knockouts of the GABAA receptor α2 subunit, we demonstrate that inhibition of the principal neurons of the dentate gyrus or CA3 via α2-containing GABAA receptors (α2GABAARs) is required to suppress anxiety, while the inhibition of CA1 pyramidal neurons is required to suppress fear responses. We further show that the diazepam-modulation of hippocampal theta activity shows certain parallels with our behavioral findings, suggesting a possible mechanism for the observed behavioral effects. Thus, our findings demonstrate a double dissociation in the regulation of anxiety versus fear by hippocampal microcircuitry.

Lack of neuronal nitric oxide synthase results in attention deficit hyperactivity disorder-like behaviors in mice.

Nitric oxide (NO) is an important molecule for the proper development and function of the central nervous system. In this study, we investigated the behavioral alterations in the neuronal NO synthase knockout mice (NOS1 KO) with a deficient NO production mechanism in the brain, characterizing it as a potential rodent model for attention deficit hyperactivity disorder (ADHD). NOS1 KO exhibited higher locomotor activity than their wildtype counterparts in a novel environment, as measured by open field (OF) test. In a 2-way active avoidance paradigm (TWAA), we found sex-dependent effects, where male KO displayed deficits in avoidance and escape behavior, sustained higher incidences of shuttle crossings, and higher incidences of intertrial interval crossings, suggesting learning, and/or performance impairments. On the other hand, female KO demonstrated few deficits in TWAA. Molsidomine (MSD), a NO donor, rescued TWAA deficits in male KO when acutely administered before training. In a passive avoidance paradigm, KO of both sexes displayed significantly shorter step-through latencies after training. Further, abnormal spontaneous motor activity rhythms were found in the KO during the dark phase of the day, indicating dysregulation of rhythmic activities. These data indicate that NOS1 KO mimics certain ADHD-like behaviors and could potentially serve as a novel rodent model for ADHD.

Electrochemical behavior of the insecticide pymetrozine at an electrochemically pretreated glassy carbon electrode and its analytical application.

A simple and sensitive electrochemical method for the determination of the insecticide pymetrozine was proposed using a simple electrochemically pretreated glassy carbon electrode (EPGCE). Compared with the bare GCE, the electrochemical response signal of pymetrozine at the EPGCE showed a significant increase. The electrochemical behavior of pymetrozine was investigated systematically and some dynamics were investigated in detail for the first time. The results indicate that the reaction of pymetrozine on the EPGCE is a two electron and two proton process, which is controlled by both diffusion and adsorption. Under optimum conditions, a good linear relationship was obtained between peak currents and pymetrozine concentrations in the range of 1 × 10-7 to 5 × 10-6 mol L-1 with a detection limit of 8 × 10-8 mol L-1 (S/N = 3). The proposed method was applied to the determination of pymetrozine in real samples with satisfactory recovery results. Finally, the degradation of pymetrozine was also studied in natural soil.