Combinatorial transcriptomic and genetic dissection of insulin/IGF-1 signaling-regulated longevity in Caenorhabditis elegans.

Classical genetic analysis is invaluable for understanding the genetic interactions underlying specific phenotypes, but requires laborious and subjective experiments to characterize polygenic and quantitative traits. Contrarily, transcriptomic analysis enables the simultaneous and objective identification of multiple genes whose expression changes are associated with specific phenotypes. Here, we conducted transcriptomic analysis of genes crucial for longevity using datasets with daf-2/insulin/IGF-1 receptor mutant Caenorhabditis elegans. Our analysis unraveled multiple epistatic relationships at the transcriptomic level, in addition to verifying genetically established interactions. Our combinatorial analysis also revealed transcriptomic changes associated with longevity conferred by daf-2 mutations. In particular, we demonstrated that the extent of lifespan changes caused by various mutant alleles of the longevity transcription factor daf-16/FOXO matched their effects on transcriptomic changes in daf-2 mutants. We identified specific aging-regulating signaling pathways and subsets of structural and functional RNA elements altered by different genes in daf-2 mutants. Lastly, we elucidated the functional cooperation between several longevity regulators, based on the combination of transcriptomic and molecular genetic analysis. These data suggest that different biological processes coordinately exert their effects on longevity in biological networks. Together our work demonstrates the utility of transcriptomic dissection analysis for identifying important genetic interactions for physiological processes, including aging and longevity.

Exploring the Smoking-Epilepsy Nexus: a systematic review and meta-analysis of observational studies : Smoking and epilepsy.

BACKGROUND: Epilepsy, characterized by recurrent unprovoked seizures, poses significant challenges to affected individuals globally. While several established risk factors for epilepsy exist, the association with cigarette smoking remains debated. This study aims to conduct systematic review and meta-analysis to elucidate the potential association between smoking and the likelihood of epilepsy. METHODS: The search was performed on March 31st, 2023, using the Medline, Embase, Web of Science, Scopus, and ScienceDirect. We included cohort, cross-sectional, and case-control studies in our meta-analysis, conducting subgroup analyses based on smoking history, sex, and epilepsy type to yield specific insights. RESULTS: We identified 2550 studies, of which 17 studies were finally included in this study. The pooled odds ratio of epilepsy was 1.14 (0.96-1.36) in smokers compared to non-smokers. In current smokers compared to non-smokers, the odds ratio was 1.46 (1.13-1.89), while, in former smokers compared to non-smokers, the odds ratio was 1.14 (0.83-1.56). CONCLUSIONS: While the overall association between smoking and epilepsy did not reach statistical significance, a notable association was found among current smokers. The study emphasizes the importance of smoking cessation as a potential preventive measure against epilepsy, especially given the proconvulsive effects of nicotine. Future research should address limitations and explore specific clinical scenarios to enhance our understanding of the complex relationship between cigarette use and epilepsy. SYSTEMATIC REVIEW REGISTRATION: CRD42022342510.

Preclinical and dose-ranging assessment of hESC-derived dopaminergic progenitors for a clinical trial on Parkinson's disease.

Human embryonic stem cell (hESC)-derived midbrain dopaminergic (mDA) cell transplantation is a promising therapeutic strategy for Parkinson's disease (PD). Here, we present the derivation of high-purity mDA progenitors from clinical-grade hESCs on a large scale under rigorous good manufacturing practice (GMP) conditions. We also assessed the toxicity, biodistribution, and tumorigenicity of these cells in immunodeficient rats in good laboratory practice (GLP)-compliant facilities. Various doses of mDA progenitors were transplanted into hemi-parkinsonian rats, and a significant dose-dependent behavioral improvement was observed with a minimal effective dose range of 5,000-10,000 mDA progenitor cells. These results provided insights into determining a low cell dosage (3.15 million cells) for human clinical trials. Based on these results, approval for a phase 1/2a clinical trial for PD cell therapy was obtained from the Ministry of Food and Drug Safety in Korea, and a clinical trial for treating patients with PD has commenced.

Sulfur metabolism-mediated fungal glutathione biosynthesis is essential for oxidative stress resistance and pathogenicity in the plant pathogenic fungus Fusarium graminearum.

IMPORTANCE: Fusarium graminearum is a destructive fungal pathogen that causes Fusarium head blight (FHB) on a wide range of cereal crops. To control fungal diseases, it is essential to comprehend the pathogenic mechanisms that enable fungi to overcome host defenses during infection. Pathogens require an oxidative stress response to overcome host-derived oxidative stress. Here, we identify the underlying mechanisms of the Fgbzip007-mediated oxidative stress response in F. graminearum. ChIP-seq and subsequent genetic analyses revealed that the role of glutathione in pathogenesis is not dependent on antioxidant functions in F. graminearum. Altogether, this study establishes a comprehensive framework for the Fgbzip007 regulon on pathogenicity and oxidative stress responses, offering a new perspective on the role of glutathione in pathogenicity.

Identification of Essential Genes for the Establishment of Spray-Induced Gene Silencing-Based Disease Control in Fusarium graminearum.

As resistance to chemical fungicides continues to increase inFusarium graminearum, there is a growing need to develop novel disease control strategies. To discover essential genes that could serve as new disease control targets, we selected essential gene candidates that had failed to be deleted in previous studies. Thirteen genes were confirmed to be essential, either by constructing conditional promoter replacement mutants or by employing a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated editing strategy. We synthesized double-stranded RNAs (dsRNAs) targeting these essential genes and analyzed their protective effects in plants using a spray-induced gene silencing (SIGS) method. When dsRNAs targeting Fg10360, Fg13150, and Fg06123 were applied to detached barley leaves prior to fungal inoculation, disease lesions were greatly reduced. Our findings provide evidence of the potential of essential genes identified by a SIGS method to be effective targets for the control of fungal diseases.

Harnessing Liquid Crystal Sensors for High-Throughput Real-Time Detection of Structural Changes in Lysozyme during Refolding Processes.

Despite the rapid advances in process analytical technology, the assessment of protein refolding efficiency has largely relied on off-line protein-specific assays and/or chromatographic procedures such as reversed-phase high-performance liquid chromatography and size exclusion chromatography. Due to the inherent time gap pertaining to traditional methods, exploring optimum refolding conditions for many recombinant proteins, often expressed as insoluble inclusion bodies, has proven challenging. The present study describes a novel protein refolding sensor that utilizes liquid crystals (LCs) to discriminate varying protein structures during unfolding and refolding. An LC layer containing 4-cyano-4'-pentylbiphenyl (5CB) intercalated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) is used as a sensing platform, and its proof-of-concept performance is demonstrated using lysozyme as a model protein. As proteins unfold or refold, a local charge fluctuation at their surfaces modulates their interaction with zwitterionic phospholipid DOPE. This alters the alignment of DOPE molecules at the aqueous/LC interface, affecting the orientational ordering of bulk LC (i.e., homeotropic to planar for refolding and planar to homeotropic for unfolding). Differential polarized optical microscope images of the LC layer are subsequently generated, whose brightness directly linked to conformational changes of lysozyme molecules is quantified by gray scale analysis. Importantly, our LC-based refolding sensor is compatible with diverse refolding milieus for real-time analysis of lysozyme refolding and thus likely to facilitate the refolding studies of many proteins, especially those lacking a method to determine structure-dependent biological activity.

A combined transcriptomic and physiological approach to understanding the adaptive mechanisms to cope with oxidative stress in Fusarium graminearum.

In plant-pathogen interactions, oxidative bursts are crucial for plants to defend themselves against pathogen infections. Rapid production and accumulation of reactive oxygen species kill pathogens directly and cause local cell death, preventing pathogens from spreading to adjacent cells. Meanwhile, the pathogens have developed several mechanisms to tolerate oxidative stress and successfully colonize plant tissues. In this study, we investigated the mechanisms responsible for resistance to oxidative stress by analyzing the transcriptomes of six oxidative stress-sensitive strains of the plant pathogenic fungus Fusarium graminearum. Weighted gene co-expression network analysis identified several pathways related to oxidative stress responses, including the DNA repair system, autophagy, and ubiquitin-mediated proteolysis. We also identified hub genes with high intramodular connectivity in key modules and generated deletion or conditional suppression mutants. Phenotypic characterization of those mutants showed that the deletion of FgHGG4, FgHGG10, and FgHGG13 caused sensitivity to oxidative stress, and further investigation on those genes revealed that transcriptional elongation and DNA damage responses play roles in oxidative stress response and pathogenicity. The suppression of FgHGL7 also led to hypersensitivity to oxidative stress, and we demonstrated that FgHGL7 plays a crucial role in heme biosynthesis and is essential for peroxidase activity. This study increases the understanding of the adaptive mechanisms to cope with oxidative stress in plant pathogenic fungi. IMPORTANCE Fungal pathogens have evolved various mechanisms to overcome host-derived stresses for successful infection. Oxidative stress is a representative defense system induced by the host plant, and fungi have complex response systems to cope with it. Fusarium graminearum is one of the devastating plant pathogenic fungi, and understanding its pathosystem is crucial for disease control. In this study, we investigated adaptive mechanisms for coping with oxidative stress at the transcriptome level using oxidative stress-sensitive strains. In addition, by introducing genetic modification technique such as CRISPR-Cas9 and the conditional gene expression system, we identified pathways/genes required for resistance to oxidative stress and also for virulence. Overall, this study advances the understanding of the oxidative stress response and related mechanisms in plant pathogenic fungi.

Mitochondrial aconitase suppresses immunity by modulating oxaloacetate and the mitochondrial unfolded protein response.

Accumulating evidence indicates that mitochondria play crucial roles in immunity. However, the role of the mitochondrial Krebs cycle in immunity remains largely unknown, in particular at the organism level. Here we show that mitochondrial aconitase, ACO-2, a Krebs cycle enzyme that catalyzes the conversion of citrate to isocitrate, inhibits immunity against pathogenic bacteria in C. elegans. We find that the genetic inhibition of aco-2 decreases the level of oxaloacetate. This increases the mitochondrial unfolded protein response, subsequently upregulating the transcription factor ATFS-1, which contributes to enhanced immunity against pathogenic bacteria. We show that the genetic inhibition of mammalian ACO2 increases immunity against pathogenic bacteria by modulating the mitochondrial unfolded protein response and oxaloacetate levels in cultured cells. Because mitochondrial aconitase is highly conserved across phyla, a therapeutic strategy targeting ACO2 may eventually help properly control immunity in humans.

Genetic and Transcriptional Regulatory Mechanisms of Lipase Activity in the Plant Pathogenic Fungus Fusarium graminearum.

Lipases, which catalyze the hydrolysis of long-chain triglycerides, diglycerides, and monoglycerides into free fatty acids and glycerol, participate in various biological pathways in fungi. In this study, we examined the biological functions and regulatory mechanisms of fungal lipases via two approaches. First, we performed a systemic functional characterization of 86 putative lipase-encoding genes in the plant-pathogenic fungus Fusarium graminearum. The phenotypes were assayed for vegetative growth, asexual and sexual reproduction, stress responses, pathogenicity, mycotoxin production, and lipase activity. Most mutants were normal in the assessed phenotypes, implying overlapping roles for lipases in F. graminearum. In particular, FgLip1 and Fgl1 were revealed as core extracellular lipases in F. graminearum. Second, we examined the lipase activity of previously constructed transcription factor (TF) mutants of F. graminearum and identified three TFs and one histone acetyltransferase that significantly affect lipase activity. The relative transcript levels of FgLIP1 and FGL1 were markedly reduced or enhanced in these TF mutants. Among them, Gzzc258 was identified as a key lipase regulator that is also involved in the induction of lipase activity during sexual reproduction. To our knowledge, this study is the first comprehensive functional analysis of fungal lipases and provides significant insights into the genetic and regulatory mechanisms underlying lipases in fungi. IMPORTANCE Fusarium graminearum is an economically important plant-pathogenic fungus that causes Fusarium head blight (FHB) on wheat and barley. Here, we constructed a gene knockout mutant library of 86 putative lipase-encoding genes and established a comprehensive phenotypic database of the mutants. Among them, we found that FgLip1 and Fgl1 act as core extracellular lipases in this pathogen. Moreover, several putative transcription factors (TFs) that regulate the lipase activities in F. graminearum were identified. The disruption mutants of F. graminearum-lipase regulatory TFs all showed defects in sexual reproduction, which implies a strong relationship between sexual development and lipase activity in this fungus. These findings provide valuable insights into the genetic mechanisms regulating lipase activity as well as its importance to the developmental stages of this plant-pathogenic fungus.

Two Previously Unrecorded Fungal Species Isolated from Muui Island in Korea.

Fungi are cosmopolitan and they occupy diverse niches as consumers, producers, and decomposers. They play critical roles in the environment by enabling nutrient cycling and generating a plethora of secondary metabolites. This study aimed to identify and characterize fungal strains isolated from diverse sources on Muui Island, Republic of Korea. In 2023, a total of 86 fungal strains were collected and examined. Investigation of the morphological features and phylogenetic analyses of multiple barcode loci identified one putative novel species and two species previously unrecorded in the Republic of Korea: Colletotrichum sp., Colletotrichum guizhouense, and Fusarium brachygibbosum. This study provides a comprehensive description of their molecular phylogenies and morphological characteristics. These findings will contribute to the existing knowledge about fungal species in the Republic of Korea and future research on the fungal diversity on Muui Island.

Systematic transcriptome analysis associated with physiological and chronological aging in Caenorhabditis elegans.

Aging is associated with changes in a variety of biological processes at the transcriptomic level, including gene expression. Two types of aging occur during a lifetime: chronological and physiological aging. However, dissecting the difference between chronological and physiological ages at the transcriptomic level has been a challenge because of its complexity. We analyzed the transcriptomic features associated with physiological and chronological aging using Caenorhabditis elegans as a model. Many structural and functional transcript elements, such as noncoding RNAs and intron-derived transcripts, were up-regulated with chronological aging. In contrast, mRNAs with many biological functions, including RNA processing, were down-regulated with physiological aging. We also identified an age-dependent increase in the usage of distal 3' splice sites in mRNA transcripts as a biomarker of physiological aging. Our study provides crucial information for dissecting chronological and physiological aging at the transcriptomic level.

Immunosenescence in Caenorhabditis elegans.

Immunosenescence is an age-dependent decline in immune functions and hallmark of aging in diverse species, ranging from invertebrates to mammals. However, identifying the factors responsible for immunosenescence is challenging because of the complexity of immune systems and aging in mammals. The roundworm Caenorhabditis elegans is suitable for understanding immunosenescence because of its simple immune system and rapid aging process. In this review, we discuss the advances in our understanding of immunosenescence in C. elegans. PMK-1/p38 mitogen-activated protein kinase (MAPK), SKN-1/NRF, and ZIP-10/bZIP transcription factor regulate immunosenescence through p38 MAPK and insulin/IGF-1 signaling pathways. Because these factors and pathways are evolutionarily conserved, the findings discussed in this review may help understand the mechanisms underlying immunosenescence and develop new treatment therapy for immunosenescence in humans.

Intron turnover is essential to the development and pathogenicity of the plant pathogenic fungus Fusarium graminearum.

Intron lariats excised during the splicing process are rapidly degraded by RNA lariat debranching enzyme (Dbr1) and several exonucleases. Rapid turnover of lariat RNA is essential to cellular RNA homeostasis. However, the functions of Dbr1 have not been investigated in filamentous fungi. Here, we characterized the molecular functions of Dbr1 in Fusarium graminearum, a major fungal plant pathogen. Deletion of FgDBR1 resulted in pleiotropic defects in hyphal growth, conidiation, sexual reproduction, and virulence. Through transcriptome analysis, we revealed that the deletion mutant exhibited global accumulation of intron lariats and upregulation of ribosome-related genes. Excessive accumulation of lariat RNA led to reduced overall protein synthesis, causing various phenotypic defects in the absence of FgDBR1. The results of this study demonstrate that a compromised intron turnover process affects development and pathogenesis in this fungus and that Dbr1 function is critical to plant pathogenic fungi.

Age-dependent upregulation of Y RNAs in Caenorhabditis elegans.

Y RNA is a conserved small non-coding RNA whose functions in aging remain unknown. Here, we sought to determine the role of C. elegans Y RNA homologs, CeY RNA (CeY) and stem-bulge RNAs (sbRNAs), in aging. We found that the levels of CeY and sbRNAs generally increased during aging. We showed that CeY was downregulated by oxidative and thermal stresses, whereas several sbRNAs were upregulated by oxidative stress. We did not observe lifespan phenotypes by mutations in CeY-coding yrn-1. Future research under various genetic and environmental conditions is required to further evaluate the role of Y RNA in C. elegans aging.

Development of a versatile copper-responsive gene expression system in the plant-pathogenic fungus Fusarium graminearum.

Fusarium graminearum is an important plant-pathogenic fungus that causes Fusarium head blight on wheat and barley, and ear rot on maize worldwide. This fungus has been widely used as a model organism to study various biological processes of plant-pathogenic fungi because of its amenability to genetic manipulation and well-established outcross system. Gene deletion and overexpression/constitutive expression of target genes are tools widely used to investigate the molecular mechanism underlying fungal development, virulence, and secondary metabolite production. However, for fine-tuning gene expression and studying essential genes, a conditional gene expression system is necessary that enables repression or induction of gene expression by modifying external conditions. Until now, only a few conditional expression systems have been developed in plant-pathogenic fungi. This study proposes a new and versatile conditional gene expression system in F. graminearum using the promoter of a copper-responsive gene, designated F. graminearum copper-responsive 1 (FCR1). Transcript levels of FCR1 were found to be greatly affected by copper availability conditions. Moreover, the promoter (PFCR1 ), 1 kb upstream of the FCR1 open reading frame, was sufficient to confer copper-dependent gene expression. Replacement of a green fluorescent protein gene and FgENA5 promoter with a PFCR1 promoter clearly showed that PFCR1 could be used for fine-tuning gene expression in this fungus. We also demonstrated the applicability of this conditional gene expression system to an essential gene study by replacing the promoter of FgIRE1, an essential gene of F. graminearum. This enabled the generation of FgIRE1 suppression mutants, which allowed functional characterization of the gene. This study reported the first conditional gene expression system in F. graminearum using both repression and induction. This system would be a convenient way to precisely control gene expression and will be used to determine the biological functions of various genes, including essential ones.

Altered glucocorticoid metabolism in girls with central obesity.

BACKGROUND: Dysregulation of glucocorticoid metabolism is known to be a causative factor of obesity. However, only a few studies have evaluated the enzymatic activities involved in glucocorticoid metabolism in the pediatric population. OBJECTIVE: To examine whether circulating glucocorticoid metabolites and their ratios reflecting the activities of metabolic enzyme are associated with obesity and body composition in girls. METHODS: A total of 227 girls aged 7-13 years (131 control, 45 overweight, 51 obese) were enrolled in this study. Serum concentrations of glucocorticoids (11-deoxycortisol, cortisol, tetrahydrocortisol [THF], allo-THF, allo-dihydrocortisol [allo-DHF], and cortisone) were evaluated by gas chromatography-mass spectrometry. Enzyme activities corresponding to the ratios of cortisol and cortisone to their respective precursors and metabolites were also assessed. RESULTS: Serum levels of allo-THF were significantly higher in obese girls compared with those in overweight and control girls (P = 0.018); however, concentrations of other cortisol metabolites were not significantly different between the groups studied. According to the severity of obesity, increasing trends in the metabolic ratios reflecting the activity of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) [(cortisol + allo-DHF + allo-THF + THF)/cortisone], relative 5α/5ß-reductase [allo-THF/THF] activity, and 3α-HSD [allo-THF/allo-DHF] activity, were noted (P-for-trend <0.05). Body fat percentage and waist-to-height ratio positively correlated with the activities of 11ß-HSD1 and 3α-HSD (P < 0.05). Following covariate control, girls with central obesity demonstrated significantly higher metabolic ratios reflecting 11ß-HSD1, relative 5α/5ß-reductase, and 3α-HSD activities (P < 0.05). CONCLUSIONS: We found an altered glucocorticoid metabolism suggesting increased production of cortisol by 11ß-HSD1 and increased metabolic clearance of cortisol catalyzed by 3α-HSD in girls with central obesity.

Diacetyl odor shortens longevity conferred by food deprivation in C. elegans via downregulation of DAF-16/FOXO.

Dietary restriction extends lifespan in various organisms by reducing the levels of both nutrients and non-nutritional food-derived cues. However, the identity of specific food-derived chemical cues that alter lifespan remains unclear. Here, we identified several volatile attractants that decreased the longevity on food deprivation, a dietary restriction regimen in Caenorhabditis elegans. In particular, we found that the odor of diacetyl decreased the activity of DAF-16/FOXO, a life-extending transcription factor acting downstream of insulin/IGF-1 signaling. We then demonstrated that the odor of lactic acid bacteria, which produce diacetyl, reduced the nuclear accumulation of DAF-16/FOXO. Unexpectedly, we showed that the odor of diacetyl decreased longevity independently of two established diacetyl receptors, ODR-10 and SRI-14, in sensory neurons. Thus, diacetyl, a food-derived odorant, may shorten food deprivation-induced longevity via decreasing the activity of DAF-16/FOXO through binding to unidentified receptors.

Effect of Protein Surface Charge Distribution on Protein-Polyelectrolyte Complexation.

Charge anisotropy or the presence of charge patches at protein surfaces has long been thought to shift the coacervation curves of proteins and has been used to explain the ability of some proteins to coacervate on the "wrong side" of their isoelectric point. This work makes use of a panel of engineered superfolder green fluorescent protein mutants with varying surface charge distributions but equivalent net charge and a suite of strong and weak polyelectrolytes to explore this concept. A patchiness parameter, which assessed the charge correlation between points on the surface of the protein, was used to quantify the patchiness of the designed mutants. Complexation between the polyelectrolytes and proteins showed that the mutant with the largest patchiness parameter was the most likely to form complexes, while the smallest was the least likely to do so. The patchiness parameter was found to correlate well with the phase behavior of the protein-polymer mixtures, where both macrophase separation and the formation of soluble aggregates were promoted by increasing the patchiness depending on the polyelectrolyte with which the protein was mixed. Increasing total charge and increasing strength of the polyelectrolyte promote interactions for oppositely charged polyelectrolytes, while charge regulation is also key to interactions for similarly charged polyelectrolytes, which must interact selectively with oppositely charged patches.