Induction of Autophagy by Extract from Corydalis heterocarpa for Skin Anti-Aging.

The extracts of Corydalis heterocarpa, a salt-tolerant plant, exhibit diverse physiological properties, including anti-inflammatory, anticancer, and antiadipogenic effects. However, the anti-aging effects of C. heterocarpa extract (CHE) on human skin cells have not yet been investigated. In the present study, we determined that CHE inhibited senescence-associated ß-galactosidase (SA-ß-gal)-stained senescent human dermal fibroblasts (HDFs). Furthermore, CHE markedly suppressed the expression of major regulatory proteins involved in senescence, including p53, p21, and caveolin-1. Interestingly, CHE promoted autophagic flux, as confirmed by the formation of microtubule-associated protein 1 light chain 3B (LC3B) puncta and lysosomal activity. Notably, using RNA sequencing (RNA-seq), we showed that CHE selectively regulated the gene expression of leucine-rich repeat and sterile alpha motif-containing 1 (LRSAM1), an important regulator of autophagy. The adenosine-monophosphate activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) pathway, which is essential for autophagy regulation, was also modulated by CHE. LRSAM1 depletion not only inhibited LC3B expression but also decreased the autophagy flux induced by CHE. Moreover, the knockdown of LRSAM1 suppressed the reversal of CHE-induced senescence in old HDFs. Collectively, our study has revealed the rejuvenating effects and molecular mechanisms of CHE, suggesting that CHE may be a promising anti-aging agent.

Dysregulated CREB3 cleavage at the nuclear membrane induces karyoptosis-mediated cell death.

Cancer cells often exhibit resistance to apoptotic cell death, but they may be vulnerable to other types of cell death. Elucidating additional mechanisms that govern cancer cell death is crucial for developing new therapies. Our research identified cyclic AMP-responsive element-binding protein 3 (CREB3) as a crucial regulator and initiator of a unique cell death mechanism known as karyoptosis. This process is characterized by nuclear shrinkage, deformation, and the loss of nuclear components following nuclear membrane rupture. We found that the N-terminal domain (aa 1-230) of full-length CREB3 (CREB3-FL), which is anchored to the nuclear inner membrane (INM), interacts with lamins and chromatin DNA. This interaction maintains a balance between the outward force exerted by tightly packed DNA and the inward constraining force, thereby preserving INM integrity. Under endoplasmic reticulum (ER) stress, aberrant cleavage of CREB3-FL at the INM leads to abnormal accumulation of the cleaved form of CREB3 (CREB3-CF). This accumulation disrupts the attachment of CREB3-FL to the INM, resulting in sudden rupture of the nuclear membrane and the onset of karyoptosis. Proteomic studies revealed that CREB3-CF overexpression induces a DNA damage response akin to that caused by UVB irradiation, which is associated with cellular senescence in cancer cells. These findings demonstrated that the dysregulation of CREB3-FL cleavage is a key factor in karyoptotic cell death. Consequently, these findings suggest new therapeutic strategies in cancer treatment that exploit the process of karyoptosis.

A long-term storable gel-laden chip composite built in a multi-well plate enablingin situcell encapsulation for high-throughput liver model.

Hydrogels are widely used as scaffold materials for constructingin vitrothree-dimensional microphysiological systems. However, their high sensitivity to various external cues hinders the development of hydrogel-laden, microscale, and high-throughput chips. Here, we have developed a long-term storable gel-laden chip composite built in a multi-well plate, which enablesin situcell encapsulation and facilitates high-throughput analysis. Through optimized chemical crosslinking and freeze-drying method (C/FD), we have achieved a high-quality of gel-laden chip composite with excellent transparency, uniform porosity, and appropriate swelling and mechanical characteristics. Besides collagen, decellularized extracellular matrix with tissue-specific biochemical compound has been applied as chip composite. As a ready-to-use platform,in situcell encapsulation within the gel has been achieved through capillary force generated during gel reswelling. The liver-mimetic chip composite, comprising HepG2 cells or primary hepatocytes, has demonstrated favorable hepatic functionality and high sensitivity in drug testing. The developed fabrication process with improved stability of gels and storability allows chip composites to be stored at a wide range of temperatures for up to 28 d without any deformation, demonstrating off-the-shelf products. Consequently, this provides an exceptionally simple and long-term storable platform that can be utilized for an efficient tissue-specific modeling and various biomedical applications.

Cultured meat with enriched organoleptic properties by regulating cell differentiation.

Research on cultured meat has primarily focused on the mass proliferation or differentiation of muscle cells; thus, the food characteristics of cultured meat remain relatively underexplored. As the quality of meat is determined by its organoleptic properties, cultured meat with similar sensory characteristics to animal-derived meat is highly desirable. In this study, we control the organoleptic and nutritional properties of cultured meat by tailoring the 2D differentiation of primary bovine myoblasts and primary bovine adipose-derived mesenchymal stem cells on gelatin/alginate scaffolds with varying stiffness. We assess the effect of muscle and adipose differentiation quality on the sensory properties of cultured meat. Thereafter, we fabricate cultured meat with similar sensory profiles to that of conventional beef by assembling the muscle and adipose constructs composed of highly differentiated cells. We introduce a strategy to produce cultured meat with enriched food characteristics by regulating cell differentiation with scaffold engineering.

Dysregulation of the Wnt/ß-catenin signaling pathway via Rnf146 upregulation in a VPA-induced mouse model of autism spectrum disorder.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder associated with impaired social behavior and communication, repetitive behaviors, and restricted interests. In addition to genetic factors, environmental factors such as prenatal drug exposure contribute to the development of ASD. However, how those prenatal factors induce behavioral deficits in the adult stage is not clear. To elucidate ASD pathogenesis at the molecular level, we performed a high-resolution mass spectrometry-based quantitative proteomic analysis on the prefrontal cortex (PFC) of mice exposed to valproic acid (VPA) in utero, a widely used animal model of ASD. Differentially expressed proteins (DEPs) in VPA-exposed mice showed significant overlap with ASD risk genes, including differentially expressed genes from the postmortem cortex of ASD patients. Functional annotations of the DEPs revealed significant enrichment in the Wnt/ß-catenin signaling pathway, which is dysregulated by the upregulation of Rnf146 in VPA-exposed mice. Consistently, overexpressing Rnf146 in the PFC impaired social behaviors and altered the Wnt signaling pathway in adult mice. Furthermore, Rnf146-overexpressing PFC neurons showed increased excitatory synaptic transmission, which may underlie impaired social behavior. These results demonstrate that Rnf146 is critical for social behavior and that dysregulation of Rnf146 underlies social deficits in VPA-exposed mice.

Defining regorafenib as a senomorphic drug: therapeutic potential in the age-related lung disease emphysema.

Senescence, a hallmark of aging, is a factor in age-related diseases (ARDs). Therefore, targeting senescence is widely regarded as a practicable method for modulating the effects of aging and ARDs. Here, we report the identification of regorafenib, an inhibitor of multiple receptor tyrosine kinases, as a senescence-attenuating drug. We identified regorafenib by screening an FDA-approved drug library. Treatment with regorafenib at a sublethal dose resulted in effective attenuation of the phenotypes of ßPIX knockdown- and doxorubicin-induced senescence and replicative senescence in IMR-90 cells; cell cycle arrest, and increased SA-ß-Gal staining and senescence-associated secretory phenotypes, particularly increasing the secretion of interleukin 6 (IL-6) and IL-8. Consistent with this result, slower progression of ßPIX depletion-induced senescence was observed in the lungs of mice after treatment with regorafenib. Mechanistically, the results of proteomics analysis in diverse types of senescence indicated that growth differentiation factor 15 and plasminogen activator inhibitor-1 are shared targets of regorafenib. Analysis of arrays for phospho-receptors and kinases identified several receptor tyrosine kinases, including platelet-derived growth factor receptor α and discoidin domain receptor 2, as additional targets of regorafenib and revealed AKT/mTOR, ERK/RSK, and JAK/STAT3 signaling as the major effector pathways. Finally, treatment with regorafenib resulted in attenuation of senescence and amelioration of porcine pancreatic elastase-induced emphysema in mice. Based on these results, regorafenib can be defined as a novel senomorphic drug, suggesting its therapeutic potential in pulmonary emphysema.

High protein-containing new food by cell powder meat.

Demand for a new protein source to replace meat is increasing to solve various issues such as limited resources and food shortages. Diverse protein sources are being developed, but alternative proteins such as plants or insects need to improve people's perceptions and organoleptic properties. Therefore, cell-based meat research is intensively conducted, and most studies are aimed at scale-up and cost-down via the research of scaffolds and culture media. Here, we proposed a new food by cell powder meat (CPM), which has a high protein content and a meaty flavor. The powder was manufactured 76% more cost-effectively with less serum than the conventional culture medium and without 3D scaffold. Due to its comprehensive characteristics, the potential applicability of CPM in the cell-based meat industry could be expected.

The reverse shock index multiplied by Glasgow coma scale (rSIG) is predictive of mortality in trauma patients according to age.

OBJECTIVE: The role of reverse shock index multiplied Glasgow coma scale (rSIG) in patients post-trauma with traumatic brain injury (TBI) has not yet been defined well. Our study aimed to investigate the predictive performance of rSIG according to age group. METHOD: This is a prospective multi-national and multi-center cohort study using Pan-Asian Trauma Outcome Study registry in Asian-Pacific, conducted on patients post-trauma who visited participating hospitals. The main exposure was low rSIG measured at emergency department. The main outcome was in-hospital mortality. We performed multilevel logistic regression analysis to estimate the association low rSIG and study outcomes. Interaction analysis between rSIG and age group were also conducted. RESULTS: Low rSIG was significantly associated with an increase in in-hospital mortality in patients post-trauma with and without TBI (aOR (95% CI): 1.49 (1.04-2.13) and 1.71 (1.16-2.53), respectively). The ORs for in-hospital mortality differed according to the age group in patients post-trauma with TBI (1.72 (1.44-1.94) for the young group and 1.13 (1.07-1.52) for the old group; p < 0.05). CONCLUSION: Low rSIG is associated with an increase in in-hospital mortality in adult patients post-trauma. However, in patients with TBI, the prediction of mortality is significantly better in younger patient group.

Cntnap2-dependent molecular networks in autism spectrum disorder revealed through an integrative multi-omics analysis.

Autism spectrum disorder (ASD) is a major neurodevelopmental disorder in which patients present with core symptoms of social communication impairment, restricted interest, and repetitive behaviors. Although various studies have been performed to identify ASD-related mechanisms, ASD pathology is still poorly understood. CNTNAP2 genetic variants have been found that represent ASD genetic risk factors, and disruption of Cntnap2 expression has been associated with ASD phenotypes in mice. In this study, we performed an integrative multi-omics analysis by combining quantitative proteometabolomic data obtained with Cntnap2 knockout (KO) mice with multi-omics data obtained from ASD patients and forebrain organoids to elucidate Cntnap2-dependent molecular networks in ASD. To this end, a mass spectrometry-based proteometabolomic analysis of the medial prefrontal cortex in Cntnap2 KO mice led to the identification of Cntnap2-associated molecular features, and these features were assessed in combination with multi-omics data obtained on the prefrontal cortex in ASD patients to identify bona fide ASD cellular processes. Furthermore, a reanalysis of single-cell RNA sequencing data obtained from forebrain organoids derived from patients with CNTNAP2-associated ASD revealed that the aforementioned identified ASD processes were mainly linked to excitatory neurons. On the basis of these data, we constructed Cntnap2-associated ASD network models showing mitochondrial dysfunction, axonal impairment, and synaptic activity. Our results may shed light on the Cntnap2-dependent molecular networks in ASD.

Advanced assessment through intact glycopeptide analysis of Infliximab's biologics and biosimilar.

Characterization of therapeutic monoclonal antibodies (mAbs) represents a major challenge for analytical sciences due to their heterogeneity associated with post-translational modifications (PTMs). The protein glycosylation requires comprehensive identification, which could influence on the mAbs' structure and their function. Here, we demonstrated high-resolution tandem mass spectrometry with an ultra-high-performance liquid chromatography for characterization and comparison between biologics and biosimilar of infliximab at an advanced level. Comparing the N- and O-glycopeptides profiles, a total of 49 and 54 glycopeptides was identified for each product of the biologics and biosimilar, respectively. We also discovered one novel N-glycosylation site at the light chain from both biopharmaceuticals and one novel O-glycopeptide at the heavy chain from only biosimilar. Site-specific glycopeptide analysis process will be a robust and useful technique for evaluating therapeutic mAbs and complex glycoprotein products.

Transcriptome and Proteome Co-Profiling Offers an Understanding of Pre-Harvest Sprouting (PHS) Molecular Mechanisms in Wheat (Triticum aestivum).

While wheat (Triticum aestivum L.) is a widely grown and enjoyed crop, the diverse and complex global situation and climate are exacerbating the instability of its supply. In particular, pre-harvest sprouting (PHS) is one of the major abiotic stresses that frequently occurs due to irregular climate conditions, causing serious damage to wheat and its quality. In this study, transcriptomic analysis with RNA-seq and proteomic analysis with LC-MS/MS were performed in PHS-treated spikes from two wheat cultivars presenting PHS sensitivity and tolerance, respectively. A total of 13,154 differentially expressed genes (DEGs) and 706 differentially expressed proteins (DEPs) were identified in four comparison groups between the susceptible/tolerant cultivars. Gene function and correlation analysis were performed to determine the co-profiled genes and proteins affected by PHS treatment. In the functional annotation of each comparative group, similar functions were confirmed in each cultivar under PHS treatment; however, in Keumgang PHS+7 (K7) vs. Woori PHS+7 (W7), functional annotations presented clear differences in the "spliceosome" and "proteasome" pathways. In addition, our results indicate that alternative splicing and ubiquitin-proteasome support the regulation of germination and seed dormancy. This study provides an advanced understanding of the functions involved in transcription and translation related to PHS mechanisms, thus enabling specific proposals for the further analysis of germination and seed dormancy mechanisms and pathways in wheat.

Investigation of the Hydrogen Sulfide Signaling Pathway in Schwann Cells during Peripheral Nerve Degeneration: Multi-Omics Approaches.

N-ethylmaleimide (NEM) inhibits peripheral nerve degeneration (PND) by targeting Schwann cells in a hydrogen sulfide (H2S)-pathway-dependent manner, but the underlying molecular and pharmacological mechanisms are unclear. We investigated the effect of NEM, an α,ß-unsaturated carboxyl compound, on H2S signaling in in vitro- and ex vivo-dedifferentiated Schwann cells using global proteomics (LC-MS) and transcriptomics (whole-genome and small RNA-sequencing (RNA-seq)) methods. The multi-omics analyses identified several genes and proteins related to oxidative stress, such as Sod1, Gnao1, Stx4, Hmox2, Srxn1, and Edn1. The responses to oxidative stress were transcriptionally regulated by several transcription factors, such as Atf3, Fos, Rela, and Smad2. In a functional enrichment analysis, cell cycle, oxidative stress, and lipid/cholesterol metabolism were enriched, implicating H2S signaling in Schwann cell dedifferentiation, proliferation, and myelination. NEM-induced changes in the H2S signaling pathway affect oxidative stress, lipid metabolism, and the cell cycle in Schwann cells. Therefore, regulation of the H2S signaling pathway by NEM during PND could prevent Schwann cell demyelination, dedifferentiation, and proliferation.

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.

MDGA1 negatively regulates amyloid precursor protein-mediated synapse inhibition in the hippocampus.

Balanced synaptic inhibition, controlled by multiple synaptic adhesion proteins, is critical for proper brain function. MDGA1 (meprin, A-5 protein, and receptor protein-tyrosine phosphatase mu [MAM] domain-containing glycosylphosphatidylinositol anchor protein 1) suppresses synaptic inhibition in mammalian neurons, yet the molecular mechanisms underlying MDGA1-mediated negative regulation of GABAergic synapses remain unresolved. Here, we show that the MDGA1 MAM domain directly interacts with the extension domain of amyloid precursor protein (APP). Strikingly, MDGA1-mediated synaptic disinhibition requires the MDGA1 MAM domain and is prominent at distal dendrites of hippocampal CA1 pyramidal neurons. Down-regulation of APP in presynaptic GABAergic interneurons specifically suppressed GABAergic, but not glutamatergic, synaptic transmission strength and inputs onto both the somatic and dendritic compartments of hippocampal CA1 pyramidal neurons. Moreover, APP deletion manifested differential effects in somatostatin- and parvalbumin-positive interneurons in the hippocampal CA1, resulting in distinct alterations in inhibitory synapse numbers, transmission, and excitability. The infusion of MDGA1 MAM protein mimicked postsynaptic MDGA1 gain-of-function phenotypes that involve the presence of presynaptic APP. The overexpression of MDGA1 wild type or MAM, but not MAM-deleted MDGA1, in the hippocampal CA1 impaired novel object-recognition memory in mice. Thus, our results establish unique roles of APP-MDGA1 complexes in hippocampal neural circuits, providing unprecedented insight into trans-synaptic mechanisms underlying differential tuning of neuronal compartment-specific synaptic inhibition.

Comparison of protein characterization using In solution and S-Trap digestion methods for proteomics.

Protein extraction and digestion are important analytical steps in the study of proteomics. The use of sodium dodecyl sulfate (SDS) buffer makes it possible to effectively analyze various proteins. Its use was evaluated using the S-Trap digestion method and compared to the traditional In solution digestion method. Differences in protein composition were examined for each protein preparation method. S-Trap digestion followed by SDS buffer extraction clearly increased the number of identified proteins, including more mitochondrial and membrane-related proteins. The S-Trap digestion method with 5% SDS buffer was applied to the pellet remaining from the removal of RIPA buffer-soluble proteins, which identified more extracellular space proteins than the conventional S-Trap digestion method. S-Trap digestion of the pellet was particularly advantageous for identifying proteins located inside multilayer membranes.

Amphipathic Small Molecule AZT Compound Displays Potent Inhibitory Effects in Cancer Cell Proliferation.

Cancer has been identified as a leading cause of death worldwide, and the increasing number of cancer cases threatens to shorten the average life expectancy of people. Recently, we reported a 3-azido-3-deoxythymidine (AZT)-based amphipathic small molecule, ADG-2e that revealed a notable potency against tumor metastasis. To evaluate the anticancer potential of ADG-2e, we assessed its anticancer potency in vitro and in vivo. Anticancer screening of ADG-2e against cervical cancer cells, HeLa CCL2, and BT549 mammary gland ductal carcinoma showed significant inhibition of cancer cell proliferation. Furthermore, mechanistic investigations revealed that cancer cell death presumably proceeded through an oncosis mechanistic pathway because ADG-2e treated cells showed severe damage on the plasma membrane, a loss of membrane integrity, and leakage of α-tubulin and ß-actin. Finally, evaluation of the antitumorigenic potential of ADG-2e in mouse xenograft models revealed that this compound potentially inhibits cancer cell proliferation. Collectively, these findings suggest that ADG-2e can evolve as an anticancer agent, which may represent a model for nucleoside-based small molecule anticancer drug discovery.

Structural basis for SdgB- and SdgA-mediated glycosylation of staphylococcal adhesive proteins.

The initiation of infection of host tissues by Staphylococcus aureus requires a family of staphylococcal adhesive proteins containing serine-aspartate repeat (SDR) domains, such as ClfA. The O-linked glycosylation of the long-chain SDR domain mediated by SdgB and SdgA is a key virulence factor that protects the adhesive SDR proteins against host proteolytic attack in order to promote successful tissue colonization, and has also been implicated in staphylococcal agglutination, which leads to sepsis and an immunodominant epitope for a strong antibody response. Despite the biological significance of these two glycosyltransferases involved in pathogenicity and avoidance of the host innate immune response, their structures and the molecular basis of their activity have not been investigated. This study reports the crystal structures of SdgB and SdgA from S. aureus as well as multiple structures of SdgB in complex with its substrates (for example UDP, N-acetylglucosamine or SDR peptides), products (glycosylated SDR peptides) or phosphate ions. Together with biophysical and biochemical analyses, this structural work uncovered the novel mechanism by which SdgB and SdgA carry out the glycosyl-transfer process to the long SDR region in SDR proteins. SdgB undergoes dynamic changes in its structure such as a transition from an open to a closed conformation upon ligand binding and takes diverse forms, both as a homodimer and as a heterodimer with SdgA. Overall, these findings not only elucidate the putative role of the three domains of SdgB in recognizing donor and acceptor substrates, but also provide new mechanistic insights into glycosylation of the SDR domain, which can serve as a starting point for the development of antibacterial drugs against staphylococcal infections.

Design and synthesis of a DNA-encoded combinatorial library of bicyclic peptoids.

Here we describe the design and synthesis of a DNA-encoded library of bicyclic peptoids. We show that our solid-phase strategy is facile and DNA-compatible, yielding a structurally diverse combinatorial library of bicyclic peptoids of various ring sizes. We also demonstrate that affinity-based screening of a DNA-encoded library of bicyclic peptoids enables to efficiently identify high-affinity ligands for a target protein. Given their highly constraint structures, as well as increased cell permeability and proteolytic stability relative to native peptides, bicyclic peptoids could be an excellent source of protein capture agents. As such, our DNA-encoded library of bicyclic peptoids will serve as versatile tools that facilitate the generation of potent ligands against many challenging targets, such as intracellular protein-protein interactions.

Identification of Nucleolin as a Novel AEG-1-Interacting Protein in Breast Cancer via Interactome Profiling.

Breast cancer is one of the most common malignant diseases worldwide. Astrocyte elevated gene-1 (AEG-1) is upregulated in breast cancer and regulates breast cancer cell proliferation and invasion. However, the molecular mechanisms by which AEG-1 promotes breast cancer have yet to be fully elucidated. In order to delineate the function of AEG-1 in breast cancer development, we mapped the AEG-1 interactome via affinity purification followed by LC-MS/MS. We identified nucleolin (NCL) as a novel AEG-1 interacting protein, and co-immunoprecipitation experiments validated the interaction between AEG-1 and NCL in breast cancer cells. The silencing of NCL markedly reduced not only migration/invasion, but also the proliferation induced by the ectopic expression of AEG-1. Further, we found that the ectopic expression of AEG-1 induced the tyrosine phosphorylation of c-Met, and NCL knockdown markedly reduced this AEG-1 mediated phosphorylation. Taken together, our report identifies NCL as a novel mediator of the oncogenic function of AEG-1, and suggests that c-Met could be associated with the oncogenic function of the AEG-1-NCL complex in the context of breast cancer.