Nectandrin B significantly increases the lifespan of Drosophila - Nectandrin B for longevity.

Phytochemicals are increasingly recognized in the field of healthy aging as potential therapeutics against various aging-related diseases. Nutmeg, derived from the Myristica fragrans tree, is an example. Nutmeg has been extensively studied and proven to possess antioxidant properties that protect against aging and alleviate serious diseases such as cancer, heart disease, and liver disease. However, the specific active ingredient in nutmeg responsible for these health benefits has not been identified thus far. In this study, we present evidence that Nectandrin B (NecB), a bioactive lignan compound isolated from nutmeg, significantly extended the lifespan of the fruit fly Drosophila melanogaster by as much as 42.6% compared to the control group. NecB also improved age-related symptoms including locomotive deterioration, body weight gain, eye degeneration, and neurodegeneration in aging D. melanogaster. This result represents the most substantial improvement in lifespan observed in animal experiments to date, suggesting that NecB may hold promise as a potential therapeutic agent for promoting longevity and addressing age-related degeneration.

Identification of the Intestinal Microbes Associated with Locomotion.

Given the impact of the gut microbiome on human physiology and aging, it is possible that the gut microbiome may affect locomotion in the same way as the host's own genes. There is not yet any direct evidence linking the gut microbiome to locomotion, though there are some potential connections, such as regular physical activity and the immune system. In this study, we demonstrate that the gut microbiome can contribute differently to locomotion. We remodeled the original gut microbiome of mice through fecal microbiota transplantation (FMT) using human feces and compared the changes in locomotion of the same mice before and three months after FMT. We found that FMT affected locomotion in three different ways: positive, none (the same), and negative. Analysis of the phylogenesis, α-diversities, and ß-diversities of the gut microbiome in the three groups showed that a more diverse group of intestinal microbes was established after FMT in each of the three groups, indicating that the human gut microbiome is more diverse than that of mice. The FMT-remodeled gut microbiome in each group was also different from each other. Fold change and linear correlation analyses identified Lacrimispora indolis, Pseudoflavonifractor phocaeensis, and Alistipes senegalensis in the gut microbiome as positive contributors to locomotion, while Sphingobacterium cibi, Prevotellamassilia timonensis, Parasutterella excrementihominis, Faecalibaculum rodentium, and Muribaculum intestinale were found to have negative effects. This study not only confirms the presence of gut microbiomes that contribute differently to locomotion, but also explains the mixed results in research on the association between the gut microbiome and locomotion.

Regorafenib prevents the development of emphysema in a murine elastase model.

Emphysema is a chronic obstructive lung disease characterized by inflammation and enlargement of the air spaces. Regorafenib, a potential senomorphic drug, exhibited a therapeutic effect in porcine pancreatic elastase (PPE)-induced emphysema in mice. In the current study we examined the preventive role of regorafenib in development of emphysema. Lung function tests and morphometry showed that oral administration of regorafenib (5 mg/kg/day) for seven days after instillation of PPE resulted in attenuation of emphysema. Mechanistically, regorafenib reduced the recruitment of inflammatory cells, particularly macrophages and neutrophils, in bronchoalveolar lavage fluid. In agreement with these findings, measurements using a cytokine array and ELISA showed that expression of inflammatory mediators including interleukin (IL)-1ß, IL-6, and CXCL1/KC, and tissue inhibitor of matrix metalloprotease-1 (TIMP-1), was downregulated. The results of immunohistochemical analysis confirmed that expression of IL-6, CXCL1/KC, and TIMP-1 was reduced in the lung parenchyma. Collectively, the results support the preventive role of regorafenib in development of emphysema in mice and provide mechanistic insights into prevention strategies. [BMB Reports 2023; 56(8): 439-444].

Identification of ulcerative colitis-specific immune cell signatures from public single-cell RNA-seq data.

BACKGROUND: Single-cell RNA-seq enabled microscopic studies on tissue microenvironment of many diseases. Inflammatory bowel disease, an autoimmune disease, is involved with various dysfunction of immune cells, for which single-cell RNA-seq may provide us a deeper insight into the causes and mechanism of this complex disease. OBJECTIVE: In this work, we used public single-cell RNA-seq data to study tissue microenvironment around ulcerative colitis, an inflammatory bowel disease causing chronic inflammation and ulcers in large intestine. METHODS: Since not all the datasets provide cell-type annotations, we first identified cell identities to select cell populations of our interest. Differentially expressed genes and gene set enrichment analysis was then performed to infer the polarization/activation state of macrophages and T cells. Cell-to-cell interaction analysis was also performed to discover distinct interactions in ulcerative colitis. RESULTS: Differentially expressed genes analysis of the two datasets confirmed the regulation of CTLA4, IL2RA, and CCL5 genes in the T cell subset and regulation of S100A8/A9, CLEC10A genes in macrophages. Cell-to-cell interaction analysis showed CD4+ T cells and macrophages interact actively to each other. We also identified IL-18 pathway activation in inflammatory macrophages, evidence that CD4+ T cells induce Th1 and Th2 differentiation, and also found that macrophages regulate T cell activation through different ligand-receptor pairs, viz. CD86-CTL4, LGALS9-CD47, SIRPA-CD47, and GRN-TNFRSF1B. CONCLUSION: Analysis of these immune cell subsets may suggest novel strategies for the treatment of inflammatory bowel disease.

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.

Neuregulin-1 reverses anxiety-like behavior and social behavior deficits induced by unilateral micro-injection of CoCl2 into the ventral hippocampus (vHPC).

Neuregulin-1 (NRG1) is an epidermal growth factor family member with essential roles in the developing and adult nervous systems. In recent years, establishing evidence has collectively suggested that NRG1 is a new modulator of central nervous system (CNS) injury and disease, with multifaceted roles in neuroprotection, remyelination, neuroinflammation, and other repair mechanisms. NRG1 signaling exerts its effects via the tyrosine kinase receptors ErbB2-ErbB4. The NRG1/ErbB network in CNS pathology and repair has evolved, primarily in recent years. In the present study, we demonstrated that a unilateral microinjection of CoCl2 into the ventral hippocampus (vHPC) induced hypoxic insult and led to anxiety-related behaviors and deficit sociability in mice. NRG1 treatment significantly alleviated the CoCl2-induced increase of hypoxic-related molecules and behavioral abnormalities. Furthermore, NRG1 reduced the CoCl2-induced neuroinflammation and neuronal deficits in the vHPC or primary hippocampal neurons in mice. Collectively, these results suggest that NRG1 ameliorates hypoxia by alleviating synaptic deficits and behavioral abnormalities of the CoCl2-induced vHPC hypoxic model.

Role of lymphoid lineage cells aberrantly expressing alarmins S100A8/A9 in determining the severity of COVID-19.

BACKGROUND: Alarmins S100A8 and S100A9 are recognized as hallmarks of severe COVID-19 and are primarily produced in myeloid cells, such as monocytes and neutrophils. As single-cell RNA-sequencing (scRNA-seq) data from patients with COVID-19 revealed the expression of S100A8/A9 in lymphoid cells in patients with severe COVID-19. OBJECTIVE: We investigated the characteristics of lymphoid cells expressing S100A8/A9 in COVID-19 patients. METHODS: Publicly available scRNA-seq data from patients with mild (N = 12) or severe (N = 7) COVID-19 were reanalyzed. The data were further divided into the following two groups based on the time of sample collection (from infection-onset): within 6 days (early phase) and after 6 days (late phase). Differential expression and gene set enrichment analyses were performed between S100A8/A9High and S100A8/A9Low lymphoid cells. Finally, cell-cell interaction analysis was performed to investigate the role of lymphoid cells expressing high levels of S100A8/A9 in COVID-19. RESULTS: S100A8/A9 overexpression was observed in lymphoid cells, including B cells, T cells, and NK cells, in patients with severe COVID-19 (compared to patients with mild COVID-19). Cells exhibiting strong interferon/cytokine responses were found to be associated with the severity of COVID-19. Furthermore, differences in S100A8/A9-TLR4/RAGE interactions were confirmed between patients with severe and mild disease. CONCLUSIONS: Lymphoid cells overexpressing S100A8/A9 contribute to the dysregulation of the innate immune response in patients with severe COVID-19, specifically during the early phase of infection. This study fosters a better understanding of the hyper-induction of pro-inflammatory cytokine expression and the generation of a cytokine storm in response to COVID-19 infection.

The role of EZH1 and EZH2 in development and cancer.

Polycomb Repressive Complex 2 (PRC2) exhibits key roles in mammalian development through its temporospatial repression of gene expression. EZH1 or EZH2 is the catalytic subunit of PRC2 that mediates the mono-, di- and tri-methylation of histone H3 lysine 27 (H3K27me1/2/3), H3K27me2/me3 being a hallmark of facultative heterochromatin. PRC2 is a chromatinmodifying enzyme that is recruited to a limited number of "nucleation sites", spreads H3K27 methylation and fosters chromatin compaction. EZH1 and EZH2 exhibit differences in their expression patterns, levels of histone methyltransferase activity (HMT) in the context of PRC2, and DNA/nucleosome binding activity. This suggests that their roles in heterochromatin formation are disparate. Dysregulation of PRC2 activity leads to aberrant gene expression and is implicated in cancer and developmental diseases. In this review, we discuss the distinct function of PRC2/EZH1 and PRC2/EZH2 in the early and late developmental stages. We then discuss the cancers associated with PRC2/EZH1 and PRC2/EZH2. [BMB Reports 2022; 55(12): 595-601].

Requirement of hepatic pyruvate carboxylase during fasting, high fat, and ketogenic diet.

Pyruvate has two major fates upon entry into mitochondria, the oxidative decarboxylation to acetyl-CoA via the pyruvate decarboxylase complex or the biotin-dependent carboxylation to oxaloacetate via pyruvate carboxylase (Pcx). Here, we have generated mice with a liver-specific KO of pyruvate carboxylase (PcxL-/-) to understand the role of Pcx in hepatic mitochondrial metabolism under disparate physiological states. PcxL-/- mice exhibited a deficit in hepatic gluconeogenesis and enhanced ketogenesis as expected but were able to maintain systemic euglycemia following a 24 h fast. Feeding a high-fat diet to PcxL-/- mice resulted in animals that were resistant to glucose intolerance without affecting body weight. However, we found that PcxL-/- mice fed a ketogenic diet for 1 week became severely hypoglycemic, demonstrating a requirement for hepatic Pcx for long-term glycemia under carbohydrate-limited diets. Additionally, we determined that loss of Pcx was associated with an induction in the abundance of lysine-acetylated proteins in PcxL-/- mice regardless of physiologic state. Furthermore, liver acetyl-proteomics revealed a biased induction in mitochondrial lysine-acetylated proteins. These data show that Pcx is important for maintaining the proper balance of pyruvate metabolism between oxidative and anaplerotic pathways.

Targeted Protein Degradation to Overcome Resistance in Cancer Therapies: PROTAC and N-Degron Pathway.

Extensive progress in understanding the molecular mechanisms of cancer growth and proliferation has led to the remarkable development of drugs that target cancer-driving molecules. Most target molecules are proteins such as kinases and kinase-associated receptors, which have enzymatic activities needed for the signaling cascades of cells. The small molecule inhibitors for these target molecules greatly improved therapeutic efficacy and lowered the systemic toxicity in cancer therapies. However, long-term and high-dosage treatment of small inhibitors for cancer has produced other obstacles, such as resistance to inhibitors. Among recent approaches to overcoming drug resistance to cancers, targeted protein degradation (TPD) such as proteolysis-targeting chimera (PROTAC) technology adopts a distinct mechanism of action by which a target protein is destroyed through the cellular proteolytic system, such as the ubiquitin-proteasome system or autophagy. Here, we review the currently developed PROTACs as the representative TPD molecules for cancer therapy and the N-degrons of the N-degron pathways as the potential TPD ligands.

Transcriptomic analysis in the striatum reveals the involvement of Nurr1 in the social behavior of prenatally valproic acid-exposed male mice.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that exhibits neurobehavioral deficits characterized by abnormalities in social interactions, deficits in communication as well as restricted interests, and repetitive behaviors. The basal ganglia is one of the brain regions implicated as dysfunctional in ASD. In particular, the defects in corticostriatal function have been reported to be involved in the pathogenesis of ASD. Surface deformation of the striatum in the brains of patients with ASD and their correlation with behavioral symptoms was reported in magnetic resonance imaging (MRI) studies. We demonstrated that prenatal valproic acid (VPA) exposure induced synaptic and molecular changes and decreased neuronal activity in the striatum. Using RNA sequencing (RNA-Seq), we analyzed transcriptome alterations in striatal tissues from 10-week-old prenatally VPA-exposed BALB/c male mice. Among the upregulated genes, Nurr1 was significantly upregulated in striatal tissues from prenatally VPA-exposed mice. Viral knockdown of Nurr1 by shRNA significantly rescued the reduction in dendritic spine density and the number of mature dendritic spines in the striatum and markedly improved social deficits in prenatally VPA-exposed mice. In addition, treatment with amodiaquine, which is a known ligand for Nurr1, mimicked the social deficits and synaptic abnormalities in saline-exposed mice as observed in prenatally VPA-exposed mice. Furthermore, PatDp+/- mice, a commonly used ASD genetic mouse model, also showed increased levels of Nurr1 in the striatum. Taken together, these results suggest that the increase in Nurr1 expression in the striatum is a mechanism related to the changes in synaptic deficits and behavioral phenotypes of the VPA-induced ASD mouse model.

MarkerCount: A stable, count-based cell type identifier for single-cell RNA-seq experiments.

Cell type identification is a key step toward downstream analysis of single cell RNA-seq experiments. Although the primary objective is to identify known cell populations, good identifiers should also recognize unknown clusters which may represent a previously unidentified subpopulation of a known cell type or tumor cells of an unknown phenotype. Herein, we present MarkerCount, which utilizes the number of expressed markers, regardless of their expression level. MarkerCount works in both reference- and marker-based mode, where the latter utilizes existing lists of markers, while the former uses a pre-annotated dataset to find markers to be used for cell type identification. In both modes, MarkerCount first utilizes the "marker count" to identify cell populations and, after rejecting uncertain cells, reassigns cell type and/or makes corrections in cluster-basis. The performance of MarkerCount was evaluated and compared with existing identifiers, both marker- and reference-based, that can be customized using publicly available datasets and marker databases. The results show that MarkerCount performs better in the identification of known populations as well as of unknown ones, when compared to other reference- and marker-based cell type identifiers for most of the datasets analyzed.

Kinesin-1-dependent transport of the ßPIX/GIT complex in neuronal cells.

Proper targeting of the ßPAK-interacting exchange factor (ßPIX)/G protein-coupled receptor kinase-interacting target protein (GIT) complex into distinct cellular compartments is essential for its diverse functions including neurite extension and synaptogenesis. However, the mechanism for translocation of this complex is still unknown. In the present study, we reported that the conventional kinesin, called kinesin-1, can transport the ßPIX/GIT complex. Additionally, ßPIX bind to KIF5A, a neuronal isoform of kinesin-1 heavy chain, but not KIF1 and KIF3. Mapping analysis revealed that the tail of KIF5s and LZ domain of ßPIX were the respective binding domains. Silencing KIF5A or the expression of a variety of mutant forms of KIF5A inhibited ßPIX targeting the neurite tips in PC12 cells. Furthermore, truncated mutants of ßPIX without LZ domain did not interact with KIF5A, and were unable to target the neurite tips in PC12 cells. These results defined kinesin-1 as a motor protein of ßPIX, and may provide new insights into ßPIX/GIT complex-dependent neuronal pathophysiology. [BMB Reports 2021; 54(7): 380-385].

Machine learning based anti-cancer drug response prediction and search for predictor genes using cancer cell line gene expression.

Although many models have been proposed to accurately predict the response of drugs in cell lines recent years, understanding the genome related to drug response is also the key for completing oncology precision medicine. In this paper, based on the cancer cell line gene expression and the drug response data, we established a reliable and accurate drug response prediction model and found predictor genes for some drugs of interest. To this end, we first performed pre-selection of genes based on the Pearson correlation coefficient and then used ElasticNet regression model for drug response prediction and fine gene selection. To find more reliable set of predictor genes, we performed regression twice for each drug, one with IC50 and the other with area under the curve (AUC) (or activity area). For the 12 drugs we tested, the predictive performance in terms of Pearson correlation coefficient exceeded 0.6 and the highest one was 17-AAG for which Pearson correlation coefficient was 0.811 for IC50 and 0.81 for AUC. We identify common predictor genes for IC50 and AUC, with which the performance was similar to those with genes separately found for IC50 and AUC, but with much smaller number of predictor genes. By using only common predictor genes, the highest performance was AZD6244 (0.8016 for IC50, 0.7945 for AUC) with 321 predictor genes.

Neuregulin 1/ErbB4/Akt signaling attenuates cytotoxicity mediated by the APP-CT31 fragment of amyloid precursor protein.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by neuronal and synaptic loss. The cytoplasmic tail of amyloid precursor protein (APP) undergoes sequential cleavage at a specific intracellular caspase site to generate the cytoplasmic terminal 31 (CT31) fragment. The APP-CT31 fragment is a potent inducer of apoptosis. The cytotoxicity of APP-CT31 in SH-SY5Y cells was evaluated by the lactate dehydrogenase (LDH) assay. TUNEL staining was used to detect apoptotic signals in SH-SY5Y cells and primary cortical neurons. The expression of apoptosis-related proteins, such as p53, PUMA (p53 up-regulated modulator of apoptosis), and cleaved was investigated by immunofluorescence analysis and Western blotting. In this study, we investigated the neuroprotective effect of neuregulin 1 (NRG1) against cytotoxicity induced by APP-CT31. Our data showed that CT31 induced cytotoxicity and apoptosis in SH-SY5Y cells and primary cortical neurons. NRG1 attenuated the neurotoxicity induced by the expression of APP-CT31. We also showed that APP-CT31 altered the expression of p53 and cleaved caspase 3. However, treatment with NRG1 rescued the APP-CT31-induced upregulation of p53 and cleaved caspase 3 expression. The protective effect of NRG1 was abrogated by inhibition of the ErbB4 receptor and Akt. These results indicate an important role of ErbB4/Akt signaling in NRG1-mediated neuroprotection, suggesting that endogenous NRG1/ErbB4 signaling represents a valuable therapeutic target in AD.

Neuregulin-1 inhibits CoCl2-induced upregulation of excitatory amino acid carrier 1 expression and oxidative stress in SH-SY5Y cells and the hippocampus of mice.

Excitatory amino acid carrier 1 (EAAC1) is an important subtype of excitatory amino acid transporters (EAATs) and is the route for neuronal cysteine uptake. CoCl2 is not only a hypoxia-mimetic reagent but also an oxidative stress inducer. Here, we found that CoCl2 induced significant EAAC1 overexpression in SH-SY5Y cells and the hippocampus of mice. Transient transfection of EAAC1 reduced CoCl2-induced cytotoxicity in SH-SY5Y cells. Based on this result, upregulation of EAAC1 expression by CoCl2 is thought to represent a compensatory response against oxidative stress in an acute hypoxic state. We further demonstrated that pretreatment with Neuregulin-1 (NRG1) rescued CoCl2-induced upregulation of EAAC1 and tau expression. NRG1 plays a protective role in the CoCl2-induced accumulation of reactive oxygen species (ROS) and reduction in antioxidative enzyme (SOD and GPx) activity. Moreover, NRG1 attenuated CoCl2-induced apoptosis and cell death. NRG1 inhibited the CoCl2-induced release of cleaved caspase-3 and reduction in Bcl-XL levels. Our novel finding suggests that NRG1 may play a protective role in hypoxia through the inhibition of oxidative stress and thereby maintain normal EAAC1 expression levels.

Early-life stress induces EAAC1 expression reduction and attention-deficit and depressive behaviors in adolescent rats.

Neonatal maternal separation (NMS), as an early-life stress (ELS), is a risk factor to develop emotional disorders. However, the exact mechanisms remain to be defined. In the present study, we investigated the mechanisms involved in developing emotional disorders caused by NMS. First, we confirmed that NMS provoked impulsive behavior, orienting and nonselective attention-deficit, abnormal grooming, and depressive-like behaviors in adolescence. Excitatory amino acid carrier 1 (EAAC1) is an excitatory amino acid transporter expressed specifically by neurons and is the route for the neuronal uptake of glutamate/aspartate/cysteine. Compared with that in the normal control group, EAAC1 expression was remarkably reduced in the ventral hippocampus and cerebral cortex in the NMS group. Additionally, EAAC1 expression was reduced in parvalbumin-positive hippocampal GABAergic neurons in the NMS group. We also found that EAAC1-knockout (EAAC1-/-) mice exhibited impulsive-like, nonselective attention-deficit, and depressive-like behaviors compared with WT mice in adolescence, characteristics similar to those of the NMS behavior phenotype. Taken together, our results revealed that ELS induced a reduction in EAAC1 expression, suggesting that reduced EAAC1 expression is involved in the pathophysiology of attention-deficit and depressive behaviors in adolescence caused by NMS.

Integrin-mediated adhesions in regulation of cellular senescence.

Bioinformatic and functional data link integrin-mediated cell adhesion to cellular senescence; however, the significance of and molecular mechanisms behind these connections are unknown. We now report that the focal adhesion-localized ßPAK-interacting exchange factor (ßPIX)-G protein-coupled receptor kinase interacting protein (GIT) complex controls cellular senescence in vitro and in vivo. ßPIX and GIT levels decline with age. ßPIX knockdown induces cellular senescence, which was prevented by reexpression. Loss of ßPIX induced calpain cleavage of the endocytic adapter amphiphysin 1 to suppress clathrin-mediated endocytosis (CME); direct competition of GIT1/2 for the calpain-binding site on paxillin mediates this effect. Decreased CME and thus integrin endocytosis induced abnormal integrin signaling, with elevated reactive oxygen species production. Blocking integrin signaling inhibited senescence in human fibroblasts and mouse lungs in vivo. These results reveal a central role for integrin signaling in cellular senescence, potentially identifying a new therapeutic direction.

Braille Display for Portable Device Using Flip-Latch Structured Electromagnetic Actuator.

We propose an electromagnetic-based braille display that can represent two-dimensional information. The key principle is a flip-latch structure, which allows satisfying requirements of both protrusion force for braille recognition and low power consumption. A magnet-inserted flip-latch has an eccentric shape, and is driven by and flips over the protruded voice coil and pushing the braille pin. Then it acts as a latch to lock and maintain the pin protrusion without additional energy consumption. We manufacture braille display modules and arrange them into a braille display with a total of 192 pins (16 columns and 12 rows). The pin-to-pin spacing is 2.5 mm, and the thickness of the display is about 5.5 mm. Each pin can switch states in 5 ms of operating time with 1W of power. In this paper, we describe the design and operating mechanism of the proposed actuator and perform operation tests to obtain stable driving conditions for the display. Finally, applications and limitations of the proposed braille display are analyzed.