A milder form of NSRP1-associated neurodevelopmental disorder, caused by a missense variant in the nuclear localization signal.

Nuclear Speckle Splicing Regulator Protein 1 (NSRP1) is a splice factor found in nuclear speckles, which are small membrane-free organelles implicated in epigenetic regulation, chromatin organization, DNA repair, and RNA modification. Bi-allelic loss-of-function variants in NSRP1 have recently been identified in patients suffering from a severe neurodevelopmental disorder, presenting with neurodevelopmental delay, epilepsy, microcephaly, hypotonia, and spastic cerebral palsy. Described patients acquired neither independent walking nor speech and often showed anomalies on cerebral MRI. Here we describe the case of a 14-year-old girl with motor and language delay as well as intellectual disability, who presents an ataxic gait but walks without assistance and speaks in short sentences. Whole-genome sequencing revealed the compound heterozygous NSRP1 variants c.114 + 2T > G and c.1595T > A (p.Val532Glu). Functional validation using HEK293T cells transfected with either wild-type or mutated GFP-tagged Nsrp1 suggests that the Val532Glu variant interferes with the function of the nuclear localization signal, and leads to mislocalization of NSRP1 in the cytosol, thus confirming the pathogenicity of the observed variant. This case helps to expand the phenotypic and genetic spectrum associated with pathogenic NSRP1 variants and indicates that this diagnosis should also be suspected in patients with milder phenotypes.

Discovery of antiviral SARS-CoV-2 main protease inhibitors by structure-guided hit-to-lead optimization of carmofur.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) has been targeted for the development of anti-SARS-CoV-2 agents against COVID-19 infection because Mpro processes essential viral polyproteins and plays a key role in SARS-CoV-2 replication. In this study, we report the development of novel SARS-CoV-2 Mpro inhibitors derived from carmofur, a previously identified compound that has shown moderate potency as a covalent inhibitor of SARS-CoV-2 Mpro. To employ a structure-guided drug design strategy, a putative intact binding mode of carmofur at catalytic active site of Mpro was initially predicted by docking simulation. Based on the predicted binding mode, a series of carmofur derivatives aiming to occupy the Mpro substrate binding regions were investigated for structure-activity relationship analysis. As a result, an indole-based derivative, speculated to interact with the S4 binding pocket, 21b (IC50 = 1.5 ± 0.1 µM) was discovered. Its structure was further modified and evaluated in silico by combining docking simulation, free energy perturbation calculation and subpocket interaction analysis to optimize the interactions at the S2 and S4 binding pockets. Among the newly designed novel derivatives, 21h and 21i showed the best inhibitory potencies against Mpro with IC50 values of 0.35 and 0.37 µM, respectively. Moreover, their antiviral activities were confirmed with EC50 values of 20-30 µM in the SARS-CoV-2-infected cell-based assay, suggesting that these novel Mpro inhibitors could be applied as potential lead compounds for the development of substantial anti-SARS-CoV-2 agents.

The Immunosuppressive Potential of Cholesterol Sulfate Through T Cell Microvilli Disruption.

Cholesterol (CL) is required for various biomolecular production processes, including those of cell membrane components. Therefore, to meet these needs, CL is converted into various derivatives. Among these derivatives is cholesterol sulfate (CS), a naturally produced CL derivative by the sulfotransferase family 2B1 (SULT2B1), which is widely present in human plasma. CS is involved in cell membrane stabilization, blood clotting, keratinocyte differentiation, and TCR nanocluster deformation. This study shows that treatment of T cells with CS resulted in the decreased surface expression of some surface T-cell proteins and reduced IL-2 release. Furthermore, T cells treated with CS significantly reduced lipid raft contents and membrane CLs. Surprisingly, using the electron microscope, we also observed that CS led to the disruption of T-cell microvilli, releasing small microvilli particles containing TCRs and other microvillar proteins. However, in vivo, T cells with CS showed aberrant migration to high endothelial venules and limited infiltrating splenic T-cell zones compared with the untreated T cells. Additionally, we observed significant alleviation of atopic dermatitis in mice injected with CS in the animal model. Based on these results, we conclude that CS is an immunosuppressive natural lipid that impairs TCR signaling by disrupting microvillar function in T cells, suggesting its usefulness as a therapeutic agent for alleviating T-cell-mediated hypersensitivity and a potential target for treating autoimmune diseases.

Trogocytic molting of T cell microvilli upregulates T cell receptor surface expression and promotes clonal expansion.

Although T cell activation is known to involve the internalization of the T cell antigen receptor (TCR), much less is known regarding the release of TCRs following T cell interaction with cognate antigen-presenting cells. In this study, we examine the physiological mechanisms underlying TCR release following T cell activation. We show that T cell activation results in the shedding of TCRs in T cell microvilli, which involves a combined process of trogocytosis and enzymatic vesiculation, leading to the loss of membrane TCRs and microvilli-associated proteins and lipids. Surprisingly, unlike TCR internalization, this event results in the rapid upregulation of surface TCR expression and metabolic reprogramming of cholesterol and fatty acid synthesis to support cell division and survival. These results demonstrate that TCRs are lost through trogocytic 'molting' following T cell activation and highlight this mechanism as an important regulator of clonal expansion.

Ionic-Liquid-Assisted Synthesis of Mixed-Phase Manganese Oxide Nanorods for a High-Performance Aqueous Zinc-Ion Battery.

Aqueous zinc-ion batteries (ZIBs) provide a safer and cost-effective energy storage solution by utilizing nonflammable water-based electrolytes. Although many research efforts are focused on optimizing zinc anode materials, developing suitable cathode materials is still challenging. In this study, one-dimensional, mixed-phase MnO2 nanorods are synthesized using ionic liquid (IL). Here, the IL acts as a structure-directing agent that modifies MnO2 morphology and introduces mixed phases, as confirmed by morphological, structural, and X-ray photoelectron spectroscopy (XPS) studies. The MnO2 nanorods developed by this method are utilized as a cathode material for ZIB application in the coin-cell configuration. As expected, Zn//MnO2 nanorods show a significant increase in their capacity to 347 Wh kg-1 at 100 mA g-1, which is better than bare MnO2 nanowires (207.1 Wh kg-1) synthesized by the chemical precipitation method. The battery is highly rechargeable and maintains good retention of 86% of the initial capacity and 99% Coulombic efficiency after 800 cycles at 1000 mA g-1. The ex situ XPS, X-ray diffraction, and in-depth electrochemical analysis confirm that MnO6 octahedra experience insertion/extraction of Zn2+ with high reversibility. This study suggests the potential use of MnO2 nanorods to develop high-performance and durable battery electrode materials suitable for large-scale applications.

Improving the photoelectrochemical water splitting performance of CuO photocathodes using a protective CuBi2O4 layer.

A heterojunction photocathode of CuO and CuBi2O4 grown on an FTO substrate (FTO/CuO/CuBi2O4) was synthesized using hydrothermal method followed by spin coating and annealing to overcome the bottlenecks encountered by CuO in photoelectrochemical (PEC) water splitting application. The synthesis methods, morphological, structural properties, and composition of each sample under each synthesis condition are discussed in detail. The photocathode with 15 coating layers annealed at 450 °C exhibited the best PEC performance. Moreover, its current density reached 1.23 mA/cm2 under an applied voltage of - 0.6 V versus Ag/AgCl in a neutral electrolyte. Additionally, it exhibited higher stability than the bare CuO thin film. The bonding of CuBi2O4 on CuO resulted in close contact between the two semiconductors, helping the semiconductors support each other to increase the PEC efficiency of the photocathode. CuO acted as the electron-generating layer, and the CuBi2O4 layer helped minimize photocorrosion as well as transport the carriers to the electrode/electrolyte interface to accomplish the hydrogen evolution reaction.

T Cell Immunological Synaptosomes: Definition and Isolation.

In addition to microvilli's role as structural scaffold for TCR clustering, we recently discovered a novel function as message senders. We found that microvilli are separated from the T cell body shortly upon TCR stimulation and vesiculated to form T cell microvilli particles (TMPs), a new type of membrane vesicles. TMPs and synaptic ectosomes, which bud from the synaptic cleft, constitute "T cell immunological synaptosomes (TISs)" and act as conveyors of T cell messages or traits to cognate antigen-presenting cells. In practice, it is almost impossible to distinguish between TMPs and synaptic ectosomes. Here, we describe a newly developed protocol to isolate TISs from activated T cells using antibody-immobilized agarose beads and density gradient ultracentrifugation. We further describe the methods for TIS quantification with flow cytometry and to evaluate TIS efficacy on dendritic cells.

ST3GAL1 and ßII-spectrin pathways control CAR T cell migration to target tumors.

Adoptive transfer of genetically engineered chimeric antigen receptor (CAR) T cells is becoming a promising treatment option for hematological malignancies. However, T cell immunotherapies have mostly failed in individuals with solid tumors. Here, with a CRISPR-Cas9 pooled library, we performed an in vivo targeted loss-of-function screen and identified ST3 ß-galactoside α-2,3-sialyltransferase 1 (ST3GAL1) as a negative regulator of the cancer-specific migration of CAR T cells. Analysis of glycosylated proteins revealed that CD18 is a major effector of ST3GAL1 in activated CD8+ T cells. ST3GAL1-mediated glycosylation induces the spontaneous nonspecific tissue sequestration of T cells by altering lymphocyte function-associated antigen-1 (LFA-1) endocytic recycling. Engineered CAR T cells with enhanced expression of ßII-spectrin, a central LFA-1-associated cytoskeleton molecule, reversed ST3GAL1-mediated nonspecific T cell migration and reduced tumor growth in mice by improving tumor-specific homing of CAR T cells. These findings identify the ST3GAL1-ßII-spectrin axis as a major cell-intrinsic program for cancer-targeting CAR T cell migration and as a promising strategy for effective T cell immunotherapy.

T Cell Microvilli: Finger-Shaped External Structures Linked to the Fate of T Cells.

Microvilli are outer membrane organelles that contain cross-linked filamentous actin. Unlike well-characterized epithelial microvilli, T-cell microvilli are dynamic similar to those of filopodia, which grow and shrink intermittently via the alternate actin-assembly and -disassembly. T-cell microvilli are specialized for sensing Ags on the surface of Ag-presenting cells (APCs). Thus, these finger-shaped microprotrusions contain many signaling-related proteins and can serve as a signaling platforms that induce intracellular signals. However, they are not limited to sensing external information but can provide sites for parts of the cell-body to tear away from the cell. Cells are known to produce many types of extracellular vesicles (EVs), such as exosomes, microvesicles, and membrane particles. T cells also produce EVs, but little is known about under what conditions T cells generate EVs and which types of EVs are released. We discovered that T cells produce few exosomes but release large amounsts of microvilli-derived particles during physical interaction with APCs. Although much is unanswered as to why T cells use the same organelles to sense Ags or to produce EVs, these events can significantly affect T cell fate, including clonal expansion and death. Since TCRs are localized at microvilli tips, this membrane event also raises a new question regarding long-standing paradigm in T cell biology; i.e., surface TCR downmodulation following T cell activation. Since T-cell microvilli particles carry T-cell message to their cognate partner, these particles are termed T-cell immunological synaptosomes (TISs). We discuss the potential physiological role of TISs and their application to immunotherapies.

NSrp70 is a lymphocyte-essential splicing factor that controls thymocyte development.

Alternative pre-mRNA splicing is a critical step to generate multiple transcripts, thereby dramatically enlarging the proteomic diversity. Thus, a common feature of most alternative splicing factor knockout models is lethality. However, little is known about lineage-specific alternative splicing regulators in a physiological setting. Here, we report that NSrp70 is selectively expressed in developing thymocytes, highest at the double-positive (DP) stage. Global splicing and transcriptional profiling revealed that NSrp70 regulates the cell cycle and survival of thymocytes by controlling the alternative processing of various RNA splicing factors, including the oncogenic splicing factor SRSF1. A conditional-knockout of Nsrp1 (NSrp70-cKO) using CD4Cre developed severe defects in T cell maturation to single-positive thymocytes, due to insufficient T cell receptor (TCR) signaling and uncontrolled cell growth and death. Mice displayed severe peripheral lymphopenia and could not optimally control tumor growth. This study establishes a model to address the function of lymphoid-lineage-specific alternative splicing factor NSrp70 in a thymic T cell developmental pathway.

Transgelin-2: A Double-Edged Sword in Immunity and Cancer Metastasis.

Transgelin-2, a small actin-binding protein, is the only transgelin family member expressed in immune cells. In T and B lymphocytes, transgelin-2 is constitutively expressed, but in antigen-presenting cells, it is significantly upregulated upon lipopolysaccharide stimulation. Transgelin-2 acts as a molecular staple to stabilize the actin cytoskeleton, and it competes with cofilin to bind filamentous (F)-actin. This action may enable immune synapse stabilization during T-cell interaction with cognate antigen-presenting cells. Furthermore, transgelin-2 blocks Arp2/3 complex-nucleated actin branching, which is presumably related to small filopodia formation, enhanced phagocytic function, and antigen presentation. Overall, transgelin-2 is an essential part of the molecular armament required for host defense against neoplasms and infectious diseases. However, transgelin-2 acts as a double-edged sword, as its expression is also essential for a wide range of tumor development, including drug resistance and metastasis. Thus, targeting transgelin-2 can also have a therapeutic advantage for cancer treatment; selectively suppressing transgelin-2 expression may prevent multidrug resistance in cancer chemotherapy. Here, we review newly discovered molecular characteristics of transgelin-2 and discuss clinical applications for cancer and immunotherapy.

Licoricidin Abrogates T-Cell Activation by Modulating PTPN1 Activity and Attenuates Atopic Dermatitis In Vivo.

Licoricidin, the fifth-highest fraction among the isolated 48 molecules from Glycyrrhiza uralensis extracts, has been known as an anti-inflammatory bioactive molecule; however, few studies have shown its inhibitory effect on T-cell activation and atopic dermatitis (AD). This study examined the therapeutic potential of licoricidin in AD by modulating T-cell activation with molecular mechanisms. Licoricidin attenuated the expression of IL-2 mRNA in stimulated T cells without cytotoxicity. Because tyrosine-protein phosphatase nonreceptor type 1 was predicted to interact physically with licoricidin in T cells in silico analysis, the results of tyrosine-protein phosphatase nonreceptor type 1 activity assay and phosphorylation study predicted that licoricidin might abrogate the activity of tyrosine-protein phosphatase nonreceptor type 1 during T-cell activation. Pretreatment with licoricidin controlled the dephosphorylation of Lck on TCR-mediated stimulation. Moreover, licoricidin alleviated the symptoms of dinitrochlorobenzene- and/or mite extract-induced AD, including ear thickness and serum IgE level. Microscopic analysis also showed the effects of licoricidin on the thickness of the dermis and epidermis and infiltration of immune cells. Furthermore, mRNA levels of proinflammatory cytokines were attenuated in the ear lesions of licoricidin-treated AD mice. Therefore, licoricidin has therapeutic potential for treating AD, and its underlying mechanism involves effective modulation of T-cell activation by controlling tyrosine-protein phosphatase nonreceptor type 1 to maintain Lck phosphorylation.

Cell-permeable transgelin-2 as a potent therapeutic for dendritic cell-based cancer immunotherapy.

BACKGROUND: Transgelin-2 is a 22 kDa actin-binding protein that has been proposed to act as an oncogenic factor, capable of contributing to tumorigenesis in a wide range of human malignancies. However, little is known whether this tiny protein also plays an important role in immunity, thereby keeping body from the cancer development and metastasis. Here, we investigated the functions of transgelin-2 in dendritic cell (DC) immunity. Further, we investigated whether the non-viral transduction of cell-permeable transgelin-2 peptide potentially enhance DC-based cancer immunotherapy. METHODS: To understand the functions of transgelin-2 in DCs, we utilized bone marrow-derived DCs (BMDCs) purified from transgelin-2 knockout (Tagln2-/-) mice. To observe the dynamic cellular mechanism of transgelin-2, we utilized confocal microscopy and flow cytometry. To monitor DC migration and cognate T-DC interaction in vivo, we used intravital two-photon microscopy. For the solid and metastasis tumor models, OVA+ B16F10 melanoma were inoculated into the C57BL/6 mice via intravenously (i.v.) and subcutaneously (s.c.), respectively. OTI TCR T cells were used for the adoptive transfer experiments. Cell-permeable, de-ubiquitinated recombinant transgelin-2 was purified from Escherichia coli and applied for DC-based adoptive immunotherapy. RESULTS: We found that transgelin-2 is remarkably expressed in BMDCs during maturation and lipopolysaccharide activation, suggesting that this protein plays a role in DC-based immunity. Although Tagln2-/- BMDCs exhibited no changes in maturation, they showed significant defects in their abilities to home to draining lymph nodes (LNs) and prime T cells to produce antigen-specific T cell clones, and these changes were associated with a failure to suppress tumor growth and metastasis of OVA+ B16F10 melanoma cells in mice. Tagln2-/- BMDCs had defects in filopodia-like membrane protrusion and podosome formation due to the attenuation of the signals that modulate actin remodeling in vitro and formed short, unstable contacts with cognate CD4+ T cells in vivo. Strikingly, non-viral transduction of cell-permeable, de-ubiquitinated recombinant transgelin-2 potentiated DC functions to suppress tumor growth and metastasis. CONCLUSION: This work demonstrates that transgelin-2 is an essential protein for both cancer and immunity. Therefore, transgelin-2 can act as a double-edged sword depending on how we apply this protein to cancer therapy. Engineering and clinical application of this protein may unveil a new era in DC-based cancer immunotherapy. Our findings indicate that cell-permeable transgelin-2 have a potential clinical value as a cancer immunotherapy based on DCs.

Folic Acid Functionalized Carbon Dot/Polypyrrole Nanoparticles for Specific Bioimaging and Photothermal Therapy.

Polypyrrole nanoparticles (PPy-NPs) with excellent near-infrared absorption are commonly used as photothermal therapy (PTT) agents; however, PTT using PPy-NPs has a limitation in that it is difficult to maximize their therapeutic effect because of the lack of specific targeting. In this study, to overcome the difficulty of targeting, folic acid functionalized carbon dots (FA-CDs) with bright green fluorescence properties were combined with carboxylated PPy-NPs via the EDC/NHS coupling reaction to yield a PTT imaging agent. The synthesized FA-CD/PPy-NPs with excellent photostability performed folate receptor (FR) positive HeLa cancer cell imaging by green fluorescence signals of FA-CDs and exhibited high cell viability (above 90%) even at 500 µg/mL. The viability of HeLa cells incubated with 200 µg/mL FA-CD/PPy-NPs was dramatically decreased to 25.02 ± 1.85% by NIR laser irradiation, through photothermal therapeutic effects of FA-CD/PPy-NPs with high photothermal conversion efficiency (η = 40.80 ± 1.54%). The cancer cell death by FA-CD/PPy-NPs was confirmed by fluorescence imaging of FA-CDs as well as live/dead cell staining assay (calcein-AM/PI). These results demonstrate that the FA-CD/PPy-NPs can be utilized as multifunctional theranostic agents for specific bioimaging and treatment of FR-positive cancer cells.

The Comparison of Clinical Variables Among Competitive Employment, Partial Employment, and Unemployment Group in Schizophrenic Outpatients / 신경정신의학

Objectives@#The purpose of this research was to examine differences between clinical variables among outpatients with schizophrenia in competitive and partial employed and unemployed groups. @*Methods@#The study subjects were 168 outpatients with schizophrenia, and these were divided into three groups, that is, competitive (n=20), partial (n=28), and unemployed (n=120) groups, based on employment status. Job and psychopathology data were collected by interview and using self-report questionnaires (self-stigma, will to recover, family attitude, perceived social support, insight into disease, self-esteem, hopelessness, and attitude to drugs). @*Results@#The proportion of male patients in the competitive employment group was greater than in the partial or unemployed groups. Education level was significantly higher, and the number of hospitalizations was significantly lower in the competitive group than in the unemployed group.Levels of self-stigma, will to recover, and perceived social support were significantly higher, and levels of self-esteem and hopelessness were significantly lower in the competitive and partial employment groups than in the unemployed group. Patients in these two groups also reported a more positive family attitude, more insight into the disease, and a more positive attitude toward drugs than patients in the unemployed group. @*Conclusion@#Most clinical variables were similar in the competitive and partial employment groups.The findings of this study suggest environmental and systematic factors are more important for the employment of outpatients with schizophrenia than disease-associated factors.

Cervical Sagittal Imbalance after Cervical Laminoplasty in Elderly Patients.

PURPOSE: To determine the effect of age on sagittal cervical alignment after cervical laminoplasty procedure so as to identify the group with the greatest degree of variation. Study Setting. Single-center retrospective chart review in a tertiary referral hospital. Outcome Measures. The sagittal vertical axis (SVA) (C2-7), T1 slope, and cervical lordosis. METHODS: We included patients who underwent cervical laminoplasty between 2014 and 2018 and divided 60 consecutive patients into two groups using the cut-off age of 65 years. The Paired t-test and Mann-Whitney U test were used to compare changes between preoperative radiographic cervical sagittal parameters and those 1 year after surgery. RESULTS: Mean patient ages in the older and younger groups were 71 years and 52 years, respectively. The difference of C2-7 SVA was greater in the older group. CONCLUSION: Postoperative cervical balance can be worse when laminoplasty is performed in elderly patients.

Structural Characteristics, Binding Partners and Related Diseases of the Calponin Homology (CH) Domain.

The calponin homology (CH) domain is one of the most common modules in various actin-binding proteins and is characterized by an α-helical fold. The CH domain plays important regulatory roles in both cytoskeletal dynamics and signaling. The CH domain is required for stability and organization of the actin cytoskeleton, calcium mobilization and activation of downstream pathways. The CH domain has recently garnered increased attention due to its importance in the onset of different diseases, such as cancers and asthma. However, many roles of the CH domain in various protein functions and corresponding diseases are still unclear. Here, we review current knowledge about the structural features, interactome and related diseases of the CH domain.