Neurodevelopmental disorder-associated CYFIP2 regulates membraneless organelles and eIF2α phosphorylation via protein interactors and actin cytoskeleton.

De novo variants in the Cytoplasmic FMR1-interacting protein 2 (CYFIP2) have been repeatedly associated with neurodevelopmental disorders and epilepsy, underscoring its critical role in brain development and function. While CYFIP2's role in regulating actin polymerization as part of the WAVE regulatory complex (WRC) is well-established, its additional molecular functions remain relatively unexplored. In this study, we performed unbiased quantitative proteomic analysis, revealing 278 differentially expressed proteins (DEPs) in the forebrain of Cyfip2 knock-out embryonic mice compared to wild-type mice. Unexpectedly, these DEPs, in conjunction with previously identified CYFIP2 brain interactors, included not only other WRC components but also numerous proteins associated with membraneless organelles (MLOs) involved in mRNA processing and translation within cells, including the nucleolus, stress granules, and processing bodies. Additionally, single-cell transcriptomic analysis of the Cyfip2 knock-out forebrain revealed gene expression changes linked to cellular stress responses and MLOs. We also observed morphological changes in MLOs in Cyfip2 knock-out brains and CYFIP2 knock-down cells under basal and stress conditions. Lastly, we demonstrated that CYFIP2 knock-down in cells, potentially through WRC-dependent actin regulation, suppressed the phosphorylation levels of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α), thereby enhancing protein synthesis. These results suggest a physical and functional connection between CYFIP2 and various MLO proteins and also extend CYFIP2's role within the WRC from actin regulation to influencing eIF2α phosphorylation and protein synthesis. With these dual functions, CYFIP2 may fine-tune the balance between MLO formation/dynamics and protein synthesis, a crucial aspect of proper mRNA processing and translation.

CYFIP2 p.Arg87Cys Causes Neurological Defects and Degradation of CYFIP2.

Here, we report the generation and comprehensive characterization of a knockin mouse model for the hotspot p.Arg87Cys variant of the cytoplasmic FMR1-interacting protein 2 (CYFIP2) gene, which was recently identified in individuals diagnosed with West syndrome, a developmental and epileptic encephalopathy. The Cyfip2+/R87C mice recapitulated many neurological and neurobehavioral phenotypes of the patients, including spasmlike movements, microcephaly, and impaired social communication. Age-progressive cytoarchitectural disorganization and gliosis were also identified in the hippocampus of Cyfip2+/R87C mice. Beyond identifying a decrease in CYFIP2 protein levels in the Cyfip2+/R87C brains, we demonstrated that the p.Arg87Cys variant enhances ubiquitination and proteasomal degradation of CYFIP2. ANN NEUROL 2023;93:155-163.

Non-lactational mastitis with multiple sinus wounds treated by integrated traditional Chinese and Western medicine.

OBJECTIVE: Non-lactational mastitis (NLM) is a benign inflammatory disease of the mammary gland, with pain, swelling and redness as the main clinical manifestations. There is no unified and effective standard treatment plan for this disease at present. In addition to breast cancer, non-lactational mastitis is also becoming a presenting complaint in an increasing number of outpatients at the authors' clinic. This case report summarises the treatment and management of a 35-year-old female patient with NLM complicated with multiple sinus wounds after surgery. METHOD: The patient was treated as follows, with: timely debridement according to the local condition of the wound, with manual compression to drain exudate from the sinus wound; selected wound dressings according to their performance and characteristics to fill the sinus tract for drainage and infection control; psychological care of the patient and their family to ensure that patients actively participate in the treatment; family support to the patient to deal with negative emotions; integrated traditional Chinese and Western medicine to prevent/manage infection; dietary care and control; posture management and health education to facilitate the patient's wound healing process. RESULTS: After local management with systemic treatment and management using integrated traditional Chinese and Western medicine, the wound healed after 46 days, with no recurrence during a follow-up period of one year. CONCLUSION: As shown in this case report, the wound should be cut and drained as soon as possible in order to prevent obstruction of the sinus drainage. Modern wound dressings are selected for the 'external' treatment of local wounds. Integrated traditional Chinese and Western medicine may help in systemic therapy of the whole patient.

Construction of key quality indicators for aged care facilities in China: A two-tier Delphi study: Key aged care quality indicators in ACF by Delphi method.

AIM: To construct key quality indicators for aged care facilities in China. BACKGROUND: Evaluating the care quality in aged care facilities is problematic. Evaluation of nursing care quality is important for improving nursing and self-supervision in aged care facilities. However, a few regulations and studies regarding care quality evaluation have been implemented in China. DESIGN AND METHOD: This two-tier Delphi study aimed to achieve consensus on key quality indicators for aged care facilities in China. The entry pool was determined by literature review and research team discussion, followed by a discussion by a panel of experts to establish the items of the Delphi study. Finally, key care quality indicators were established through a two-round Delphi study. This study followed the SQUIRE 2.0 guidelines. RESULTS: The initial 16 quality indicators of the entry pool was developed based on a literature review and a group discussion. Sixteen quality indicators were reduced to eight after the expert discussion. After two rounds of expert consultation, the eight quality indicators became nine, which were then evaluated for importance, formula rationality, and operability using Kendall's harmony coefficients (first round: 0.150, 0.143 and 0.169, respectively; second round: 0.209, 0.159 and 0.173, respectively). CONCLUSIONS: Key quality indicators provide quantifiable evidence for evaluating the care quality in aged care facilities, but their applicability needs continuous improvement. RELEVANCE TO CLINICAL PRACTICE: Nine key quality indicators were selected from numerous indicators for measuring the care quality in aged care facilities, supporting the evaluation of the care quality and self-supervision for aged care facilities. ELDERLY OR PUBLIC CONTRIBUTION: No elderly or public contribution.

Use of physical restraints and its associated risk factors in the aged care facilities: A multicentre cross-sectional study.

AIMS AND OBJECTIVES: To investigate the use of physical restraints in aged care facilities(ACFs)and analyse its associated risk factors. BACKGROUND: Physical restraints have been widely used in ACFs worldwide, but they can cause physical and mental harm to older people. It is important to regulate the use of physical restraint. DESIGN: A cross-sectional observational and correlational multicentre study. METHODS: By convenience sampling method, we selected eight ACFs in four representative regions of Hunan province, China, for this study. The ACF-related information was obtained by interviewing the managers and reviewing records. We conducted investigation and observation on the elderly in the ACFs to understand the use of physical restraints at three different times: 9:30-11:30, 16:00-18:00 and 19:30-21:30 on a working day. The STROBE checklist was followed for this cross-sectional study. RESULTS: This study found that the utilisation rate of physical restraints was 23.2%. The critical risk factors affecting the use of physical restrains include the following: (1) the ratio of nursing staff to the elderly residents; (2)whether there is a dementia care unit at the facility; (3) the number of elderly residents in each room; (4) the elderly residents' age, degree of education, marital status, care dependence and cognitive impairment; (5) whether the elderly has suffered from a stroke or senile dementia; (6) whether the elderly carries medical catheters. CONCLUSION: There is a lack of standardisation in the use of physical restraints in ACFs of central China. Chinese ACFs should develop guidelines and reduction measures to standardise the use of physical restraints, basing on the key factors affecting the use of physical restraints. RELEVANCE TO CLINICAL PRACTICE: The use of physical restraints in ACFs is threatening the safety of the elderly residents. Understanding the implementation of physical restraint in ACFs can provide reference for reducing the use of physical restraint.

Protein interactome and cell-type expression analyses reveal that cytoplasmic FMR1-interacting protein 1 (CYFIP1), but not CYFIP2, associates with astrocytic focal adhesion.

The two members of the cytoplasmic FMR1-interacting protein family, CYFIP1 and CYFIP2, are evolutionarily conserved multifunctional proteins whose defects are associated with distinct types of brain disorders. Even with high sequence homology between CYFIP1 and CYFIP2, several lines of evidence indicate their different functions in the brain; however, the underlying mechanisms remain largely unknown. Here, we performed reciprocal immunoprecipitation experiments using CYFIP1-2 × Myc and CYFIP2-3 × Flag knock-in mice and found that CYFIP1 and CYFIP2 are not significantly co-immunoprecipitated with each other in the knock-in brains compared with negative control wild-type (WT) brains. Moreover, CYFIP1 and CYFIP2 showed different size distributions by size-exclusion chromatography of WT mouse brains. Specifically, mass spectrometry-based analysis of CYFIP1-2 × Myc knock-in brains identified 131 proteins in the CYFIP1 interactome. Comparison of the CYFIP1 interactome with the previously identified brain region- and age-matched CYFIP2 interactome, consisting of 140 proteins, revealed only eight common proteins. Investigations using single-cell RNA-sequencing databases suggested non-neuronal cell- and neuron-enriched expression of Cyfip1 and Cyfip2, respectively. At the protein level, CYFIP1 was detected in both neurons and astrocytes, while CYFIP2 was detected only in neurons, suggesting the predominant expression of CYFIP1 in astrocytes. Bioinformatic characterization of the CYFIP1 interactome, and co-expression analysis of Cyfip1 with astrocytic genes, commonly linked CYFIP1 with focal adhesion proteins. Immunocytochemical analysis and proximity ligation assay suggested partial co-localization of CYFIP1 and focal adhesion proteins in cultured astrocytes. Together, these results suggest a CYFIP1-specific association with astrocytic focal adhesion, which may contribute to the different brain functions and dysfunctions of CYFIP1 and CYFIP2. Cover Image for this issue: https://doi.org/10.1111/jnc.15410.

Echinochrome A Reverses Kidney Abnormality and Reduces Blood Pressure in a Rat Model of Preeclampsia.

We aimed to observe the effects of Echinochrome A (Ech A) on systemic changes using a rat model of preeclampsia. The results showed that an infusion of angiotensin II (Ang II) through an osmotic pump (1 µg/kg/min) on GD 8 increased systolic and diastolic blood pressures and reduced fetal weight and placental weight. The diameters of the glomeruli were expended and glomeruli capillaries were diminished. No change was observed in the heart and liver in the Ang II group, but epithelial structures were disrupted in the uterus. Ech A treatment on GD 14 (100 µg/µL) through the jugular vein reduced systolic and diastolic blood pressures and reversed glomerulus alterations, but the fetal or placental parameters were unaffected. Ech A only partly reversed the effect on the uterus. The mRNA expression of TNF-α was increased and IL-10 and VEGF were reduced in the uterus of the Ang II group, while Ech A restored these changes. A similar trend was observed in the kidney, liver, and heart of this group. Furthermore, Bcl-2 was reduced and Bcl-2/Bax ratios were significantly reduced in the kidney and heart of the Ang II group, while Ech A reversed these changes. We suggest that Ech A modulates inflammation and apoptosis in key systemic organs in Ang II-induced rat preeclampsia and preserves kidney and uterus structures and reduces blood pressure.

Haploinsufficiency of Cyfip2 Causes Lithium-Responsive Prefrontal Dysfunction.

OBJECTIVE: Genetic variants of the cytoplasmic FMR1-interacting protein 2 (CYFIP2) encoding an actin-regulatory protein are associated with brain disorders, including intellectual disability and epilepsy. However, specific in vivo neuronal defects and potential treatments for CYFIP2-associated brain disorders remain largely unknown. Here, we characterized Cyfip2 heterozygous (Cyfip2+/- ) mice to understand their neurobehavioral phenotypes and the underlying pathological mechanisms. Furthermore, we examined a potential treatment for such phenotypes of the Cyfip2+/- mice and specified a neuronal function mediating its efficacy. METHODS: We performed behavioral analyses of Cyfip2+/- mice. We combined molecular, ultrastructural, and in vitro and in vivo electrophysiological analyses of Cyfip2+/- prefrontal neurons. We also selectively reduced CYFIP2 in the prefrontal cortex (PFC) of mice with virus injections. RESULTS: Adult Cyfip2+/- mice exhibited lithium-responsive abnormal behaviors. We found increased filamentous actin, enlarged dendritic spines, and enhanced excitatory synaptic transmission and excitability in the adult Cyfip2+/- PFC that was restricted to layer 5 (L5) neurons. Consistently, adult Cyfip2+/- mice showed increased seizure susceptibility and auditory steady-state responses from the cortical electroencephalographic recordings. Among the identified prefrontal defects, lithium selectively normalized the hyperexcitability of Cyfip2+/- L5 neurons. RNA sequencing revealed reduced expression of potassium channel genes in the adult Cyfip2+/- PFC. Virus-mediated reduction of CYFIP2 in the PFC was sufficient to induce L5 hyperexcitability and lithium-responsive abnormal behavior. INTERPRETATION: These results suggest that L5-specific prefrontal dysfunction, especially hyperexcitability, underlies both the pathophysiology and the lithium-mediated amelioration of neurobehavioral phenotypes in adult Cyfip2+/- mice, which can be implicated in CYFIP2-associated brain disorders. ANN NEUROL 2020;88:526-543.

Management of Multiple Skeletal Tuberculosis Wounds Complicated With Multiple Deep Sinus Tracts: A Case Report.

BACKGROUND: Tuberculosis (TB) is a leading infectious disease worldwide. It rarely occurs in the scapula and toe joints and is easily misdiagnosed. Without prompt treatment, the associated lesions can spread to surrounding soft tissues such as joint capsules, muscles, tendons, and fascia. In severe cases, the bones and articular surfaces can become significantly damaged; it is not uncommon for deep skeletal TB wounds with sinus tracts to form, which are very difficult to treat. We report our successful wound care management approach for one patient with multiple skeletal TB complicated with multiple deep sinus tracts. CASE: The patient was treated with anti-TB medications, and wound and bone debridement (sharps, surgical) combined with vacuum-shielded drainage (VSD) (Kula, CG Bio Co Ltd, Gyeonggi-do, South Korea) to fill the sinus tract. We removed the caseous (cheese-like) necrotic tissue, purulent drainage, and necrotic tissue at the base of the wound to ensure optimal wound care. Throughout the course of treatment, we selected different types of dressings to maintain moist wound healing and absorb excessive drainage. After 144 days of treatment, the wound and deep sinus tracts completely healed. CONCLUSIONS: Wounds related to skeletal TB with multiple sinus tracts are difficult to manage and heal. We found our wound protocol that included timely debridement and use of VSD was effective for the management of these complex wounds. Specifically, our approach filled the dead space in the sinus tract, removed excessive drainage, promoted the growth of granulation tissue, and overall promoted tissue healing.

Dilated cardiomyopathy mutations in thin-filament regulatory proteins reduce contractility, suppress systolic Ca2+, and activate NFAT and Akt signaling.

Dilated cardiomyopathy (DCM) is clinically characterized by dilated ventricular cavities and reduced ejection fraction, leading to heart failure and increased thromboembolic risk. Mutations in thin-filament regulatory proteins can cause DCM and have been shown in vitro to reduce contractility and myofilament Ca2+-affinity. In this work we have studied the functional consequences of mutations in cardiac troponin T (R131W), cardiac troponin I (K36Q) and α-tropomyosin (E40K) using adenovirally transduced isolated guinea pig left ventricular cardiomyocytes. We find significantly reduced fractional shortening with reduced systolic Ca2+. Contraction and Ca2+ reuptake times were slowed, which contrast with some findings in murine models of myofilament Ca2+ desensitization. We also observe increased sarcoplasmic reticulum (SR) Ca2+ load and smaller fractional SR Ca2+ release. This corresponds to a reduction in SR Ca2+-ATPase activity and increase in sodium-calcium exchanger activity. We also observe dephosphorylation and nuclear translocation of the nuclear factor of activated T cells (NFAT), with concordant RAC-α-serine/threonine protein kinase (Akt) phosphorylation but no change to extracellular signal-regulated kinase activation in chronically paced cardiomyocytes expressing DCM mutations. These changes in Ca2+ handling and signaling are common to all three mutations, indicating an analogous pathway of disease pathogenesis in thin-filament sarcomeric DCM. Previous work has shown that changes to myofilament Ca2+ sensitivity caused by DCM mutations are qualitatively opposite from hypertrophic cardiomyopathy (HCM) mutations in the same genes. However, we find several common pathways such as increased relaxation times and NFAT activation that are also hallmarks of HCM. This suggests more complex intracellular signaling underpinning DCM, driven by the primary mutation.NEW & NOTEWORTHY Dilated cardiomyopathy (DCM) is a frequently occurring cardiac disorder with a degree of genetic inheritance. We have found that DCM mutations in proteins that regulate the contractile machinery cause alterations to contraction, calcium-handling, and some new signaling pathways that provide stimuli for disease development. We have used guinea pig cells that recapitulate human calcium-handling and introduced the mutations using adenovirus gene transduction to look at the initial triggers of disease before remodeling.

Epilepsy- and intellectual disability-associated CYFIP2 interacts with both actin regulators and RNA-binding proteins in the neonatal mouse forebrain.

Variants of the cytoplasmic FMR1-interacting protein 2 (CYFIP2) gene are associated with early-onset epileptic encephalopathy, intellectual disability, and developmental delay. However, the current understanding of the molecular functions of CYFIP2 is limited to those related to actin dynamics, and thus, the detailed mechanisms of CYFIP2-associated brain disorders remain largely unknown. Here, we isolated the neonatal forebrain CYFIP2 complex using newly generated Cyfip2-3×Flag knock-in mice, and performed mass spectrometry-based analyses to identify proteins in the complex. The CYFIP2 interactome, consisting of 140 proteins, contained not only the expected actin regulators but also 25 RNA-binding proteins (RBPs) including Argonaute proteins. Functionally, overexpression of brain disorder-associated CYFIP2 R87 variants, but not wild-type, inhibited stress granule formation in HeLa cells. Mechanistically, the CYFIP2 R87 variants formed intracellular clusters with Argonaute proteins under both basal and stress conditions, and thereby possibly preventing their assembly into stress granules. Beyond identifying CYFIP2 interactors in vivo, these results may provide novel insights for better understanding the molecular mechanisms of CYFIP2-associated brain disorders.

Exogenous testosterone increases the audience effect in healthy males: evidence for the social status hypothesis.

Several studies have implicated testosterone in the modulation of altruistic behaviours instrumental to advancing social status. Independent studies have also shown that people tend to behave more altruistically when being watched (i.e. audience effect). To date, little is known about whether testosterone could modulate the audience effect. In the current study, we tested the effect of testosterone on altruistic behaviour using a donation task, wherein participants were asked to either accept or reject a monetary transfer to a charity organization accompanying a personal cost either in the presence or absence of an observer. We administered testosterone gel or placebo to healthy young men (n = 140) in a double-blind, placebo-controlled, mixed design. Our results showed that participants were more likely to accept the monetary transfer to the charity when being observed compared to when they completed the task alone. More importantly, this audience effect was amplified among people receiving testosterone versus placebo. Our findings suggest that testosterone administration increases the audience effect and further buttress the social status hypothesis, according to which testosterone promotes status-seeking behaviour in a context-dependent manner.

Impact of glycemic control status on patients with ST-segment elevation myocardial infarction undergoing percutaneous coronary intervention.

BACKGROUND: The combined effects of diabetes mellitus (DM), admission plasma glucose (APG), and glycated hemoglobin (HbA1c) levels on predicting long-term clinical outcomes in patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (pPCI) are unknown. Therefore, we evaluated their combined effects on long-term clinical outcomes in STEMI patients treated with pPCI. METHODS: In total, 350 consecutive patients with STEMI undergoing pPCI were enrolled. Patients were divided into 3 groups according to DM history and APG and HbA1c levels. The cumulative rates of 24-month all-cause deaths and major adverse cardiac and cerebrovascular events (MACCEs) were calculated. RESULTS: Both the incidence of all-cause deaths and cumulative rates of MACCEs were significantly the lowest in patients without a DM history and admission HbA1c level < 6.5%. DM patients with poor glycemic control or stress hyperglycemia on admission experienced the highest rates of all-cause deaths, MACCEs, and cardiac deaths. Admission HbA1c levels, Triglyceride (TG) levels, hemoglobin levels, DM history, and admission Killip class > 1 correlated with 24-month all-cause death; HbA1c levels on admission, DM history, APG levels, history of stroke, history of coronary heart disease, and TG levels on admission were significantly associated with MACCEs through the 24-month follow-up. The predictive effects of combining DM and APG and HbA1c levels were such that for STEMI patients undergoing pPCI, DM patients with poor glycemic control or with stress hyperglycemia on admission had worse prognosis than other patients. CONCLUSION: Strict control of glycemic status may improve the survival of patients who have both DM and coronary heart diseases.

Triple arginine residues in the proximal C-terminus of TREK K+ channels are critical for biphasic regulation by phosphatidylinositol 4,5-bisphosphate.

TWIK-related two-pore domain K+ channels (TREKs) are activated by acidic intracellular pH (pHi), membrane stretch, temperature, and arachidonic acid (AA). Phosphatidylinositol 4,5-bisphosphate (PIP2) exerts concentration-dependent biphasic regulations, which have been observed: inhibition by high PIP2, activation by partial decrease of PIP2, and inhibition by depletion of PIP2. Consistently, the stimulation of voltage-sensitive PIP2 phosphatase (Dr-VSP) induces initial activation and subsequent inhibition of TREKs. Lys in the proximal C-terminus (pCt) is responsible for the inhibition by high PIP2, which is generated by phosphatidylinositol kinases with ATP; its neutralizing mutation [K330A of human TREK-2 (hTREK-2)] induces tonic high activity, irrespective of ATP. Here we focus on triple successive Arg in pCt (R3-pCt) as a candidate region for the stimulatory regulation by lower PIP2. Their neutralized mutant (R3A-pCt; RRR340-2A and RRR355-7A in hTREK-1 and -2, respectively) showed negligible basal current and was not affected by ATP removal or by Dr-VSP activation. Phosphatidic acid, a phospholipid agonist of TREKs, did not activate R3A-pCt. In contrast, acidic pHi, AA, and high temperature activated R3A-pCt normally, whereas activation by membrane stretch was attenuated. In hTREK-2, combined neutralizations of the inhibitory K330 and R3-pCt (K330A/RRR355-7A) did not recover the suppressed current. In contrast, combined neutralization of pHi-sensing Glu (E332A/R355-7A) induced tonic high current and no further activation by pHi. Interestingly, when the Gly between K330/E332 and R3-pCt was mutated (G334A), hTREK-2 was tonic activated with reversed responses to ATP and acidic pHi. Therefore, we propose that the PIP2-dependent converse regulation of TREKs by Lys and R3-pCt with Gly implies structural flexibility.

Hypertrophic cardiomyopathy mutations increase myofilament Ca2+ buffering, alter intracellular Ca2+ handling, and stimulate Ca2+-dependent signaling.

Mutations in thin filament regulatory proteins that cause hypertrophic cardiomyopathy (HCM) increase myofilament Ca2+ sensitivity. Mouse models exhibit increased Ca2+ buffering and arrhythmias, and we hypothesized that these changes are primary effects of the mutations (independent of compensatory changes) and that increased Ca2+ buffering and altered Ca2+ handling contribute to HCM pathogenesis via activation of Ca2+-dependent signaling. Here, we determined the primary effects of HCM mutations on intracellular Ca2+ handling and Ca2+-dependent signaling in a model system possessing Ca2+-handling mechanisms and contractile protein isoforms closely mirroring the human environment in the absence of potentially confounding remodeling. Using adenovirus, we expressed HCM-causing variants of human troponin-T, troponin-I, and α-tropomyosin (R92Q, R145G, and D175N, respectively) in isolated guinea pig left ventricular cardiomyocytes. After 48 h, each variant had localized to the I-band and comprised ∼50% of the total protein. HCM mutations significantly lowered the Kd of Ca2+ binding, resulting in higher Ca2+ buffering of mutant cardiomyocytes. We observed increased diastolic [Ca2+] and slowed Ca2+ reuptake, coupled with a significant decrease in basal sarcomere length and slowed relaxation. HCM mutant cells had higher sodium/calcium exchanger activity, sarcoplasmic reticulum Ca2+ load, and sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) activity driven by Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of phospholamban. The ryanodine receptor (RyR) leak/load relationship was also increased, driven by CaMKII-mediated RyR phosphorylation. Altered Ca2+ homeostasis also increased signaling via both calcineurin/NFAT and extracellular signal-regulated kinase pathways. Altered myofilament Ca2+ buffering is the primary initiator of signaling cascades, indicating that directly targeting myofilament Ca2+ sensitivity provides an attractive therapeutic approach in HCM.

Shank3 regulates striatal synaptic abundance of Cyld, a deubiquitinase specific for Lys63-linked polyubiquitin chains.

The SH3 and multiple ankyrin repeat domains 3 (Shank3) proteins are core organizers of the postsynaptic density in neuronal excitatory synapses, and their defects cause various neurodevelopmental and neuropsychiatric disorders. Mechanistically, Shank3 directly and indirectly interacts with hundreds of synaptic proteins with diverse functions and potentially exerts its regulatory roles in synaptic development and function via these interactors. However, Shank3-dependent regulation of synaptic abundance has been validated in vivo for only a few Shank3 interactors. Here, using a quantitative proteomic analysis, we identified 136 proteins with altered synaptic abundance in the striatum of Shank3-overexpressing transgenic (TG) mice. By comparing these proteins with those found in a previous analysis of the postsynaptic density of Shank3 knock-out (KO) striatum, we identified and confirmed that cylindromatosis-associated deubiquitinase (Cyld), a deubiquitinase specific for Lys63-linked polyubiquitin chains, was up- and down-regulated in Shank3 TG and KO striatal synapses, respectively. Consistently, we found that the synaptic levels of Lys63-linked polyubiquitin chains were down- and up-regulated in the Shank3 TG and KO striata, respectively. Furthermore, by isolating and analyzing the synaptic Cyld complex, we generated a Cyld interactome consisting of 103 proteins, which may include Cyld substrates. Bioinformatic analyses suggested associations of the Cyld interactome with a few brain disorders and synaptic functions. Taken together, these results suggest that Shank3 regulates the synaptic abundance of Cyld in the mouse striatum and, thereby, potentially modulates the Lys63-linked polyubiquitination of striatal synaptic proteins.

Identification of critical amino acids in the proximal C-terminal of TREK-2 K+ channel for activation by acidic pHi and ATP-dependent inhibition.

TWIK-related two-pore domain K+ channels (TREKs) are regulated by intracellular pH (pHi) and Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Previously, Glu306 in proximal C-terminal (pCt) of mouse TREK-1 was identified as the pHi-sensing residue. The direction of PI(4,5)P2 sensitivity is controversial, and we have recently shown that TREKs are inhibited by intracellular ATP via endogenous PI(4,5)P2 formation. Here we investigate the anionic and cationic residues of pCt for the pHi and ATP-sensitivity in human TREK-2 (hTREK-2). In inside-out patch clamp recordings (ITREK-2,i-o), acidic pHi-induced activation was absent in E332A and was partly attenuated in E335A. Neutralization of cationic Lys (K330A) also eliminated the acidic pHi sensitivity of ITREK-2,i-o. Unlike the inhibition of wild-type (WT) ITREK-2,i-o by intracellular ATP, neither E332A nor K330A was sensitive to ATP. Nevertheless, exogenous PI(4,5)P2 (10 µM) abolished ITREK-2 i-o in all the above mutants as well as in WT, indicating unspecific inhibition by exogenous PI(4,5)P2. In whole-cell recordings of TREK-2 (ITREK-2,w-c), K330A and E332A showed higher or fully active basal activity, showing attenuated or insignificant activation by 2-APB, arachidonic acid, or acidic pHe 6.9. ITREK-1,w-c of WT is largely suppressed by pHe 6.9, and the inhibition is slightly attenuated in K312A and E315A. The results show concerted roles of the oppositely charged Lys and Glu in pCt for the ATP-dependent low basal activity and pHi sensitivity.

Particulate matter increases beta-amyloid and activated glial cells in hippocampal tissues of transgenic Alzheimer's mouse: Involvement of PARP-1.

Exposure to air pollutants, such as particulate matter (PM), has been implicated in neurodegenerative disorders including Alzheimer's disease (AD). However, direct effects of PM on production of ß-amyloid (Aß), a key pathogenic molecule in AD, and its underlying mechanism are still elusive. Given PM's potential to induce oxidative stress in other tissues, we hypothesized that poly(ADP-ribose) polymerase (PARP-1) might be involved in PM-induced neurotoxicity. To address this, we used an ex vivo model of AD, the organotypic hippocampal slice tissue culture from old (12-14 months-of-age) triple transgenic 3xTg-AD mice. First, we observed that fine PM (aerodynamic diameter < 4 µm) can dose-dependently activate PARP-1 and decrease NAD+ levels in Neuro2A cells. PARP-1 activation did occur under concentrations of PM which did not affect cell viability. Next, we observed that direct treatment of PM increased Aß levels and activated glial cells in the ex vivo hippocampal tissues of 3xTg-AD mice. PM-induced glial activation was most prominent in CA1 region of the hippocampal tissue. Notably, we found that pharmacological inhibition of PARP-1 reversed both PM-induced Aß increase and glial activation, arguing the possible involvement of PARP-1 in PM-induced AD pathogenesis. Our findings suggest that PARP-1 might be a potential molecular target, responsible for mediating negative effects of PM on the brain. Modulating PARP-1 activity could be a promising approach to prevent or alleviate PM-related environmental neurotoxicity which could initiate AD pathogenesis.

Application of first-order nonparaxial scalar theory to determine surface scattering intensity of multilayer optical coatings.

A novel first-order nonparaxial scalar theory for calculating the angular scattering that is caused by the interface roughness in an optical multilayer was proposed. As in the case that the interface roughness is moderate, the analytic expressions of angular-resolved scattering for a typical p-layer design were derived. Notably, these formulas are general because they do not depend on the prior restrictive hypothesis for the correlation degree of the various interfaces in a stack. In order to verify the theory, the formulas in the case of single-surface are presented and are exactly identical to those of the generalized Harvey-Shack theory. Also, their smooth-surface approximations are the same in form as those given by the typical first-order vector perturbation theories and are validated by numerically comparing with the typical vector theory for three representative multilayer design types with slightly rough interfaces. In addition, the usability of the novel theory in the case of moderate roughness is discussed by comparing this theory to the typical theories for optical coatings at different roughness levels.

First-order nonparaxial scalar theory of surface and bulk scattering for high-quality optical coatings.

A novel nonparaxial scalar theory is presented to calculate the angular scattering that is due to interface roughnesses or bulk inhomogeneities in a high-quality optical coating. Based on the empirically modified Beckmann-Kirchhoff surface scatter model, this theory in surface scattering and bulk scattering predicts similar formulas for the angular scattered intensity, and at the same time provides new understanding and insight into multilayer scattering phenomena. It is worth noting that the derived expressions are in the same form as those given by the typical vector methods. Based on comparisons of the surface and bulk models with the corresponding typical models for several multilayer designs, the novel theory is demonstrated to be valid for multilayer coatings even with large incident and scattering angles.