Protein Kinase R (PKR) as a Novel dsRNA Sensor in Antiviral Innate Immunity.

Protein kinase R (PKR), a key double-stranded RNA (dsRNA)-activated sensor, is pivotal for cellular responses to diverse stimuli. This protocol delineates a comprehensive methodological framework employing single luciferase assays, yeast assays, immunoblot assays, and quantitative PCR (qPCR) to discern and validate PKR activities and their downstream impacts on NF-κB-activating signaling pathways. These methodologies furnish a systematic approach to unraveling the role of PKR as a dsRNA sensor and effector in antiviral innate immunity, enabling in-depth analyses of dsRNA sensor activities.

Yeast Two-Hybrid Assay for Investigating Antiviral Innate Immunity.

Yeast two-hybrid (YTH) technology is a powerful tool for studying protein interactions and has been widely used in various fields of molecular biology, including the study of antiviral innate immunity. This chapter presents detailed information and experimental procedures for identifying virus-host protein interactions involved in immune regulation using yeast two-hybrid technology.

The fork protection complex promotes parental histone recycling and epigenetic memory.

The inheritance of parental histones across the replication fork is thought to mediate epigenetic memory. Here, we reveal that fission yeast Mrc1 (CLASPIN in humans) binds H3-H4 tetramers and operates as a central coordinator of symmetric parental histone inheritance. Mrc1 mutants in a key connector domain disrupted segregation of parental histones to the lagging strand comparable to Mcm2 histone-binding mutants. Both mutants showed clonal and asymmetric loss of H3K9me-mediated gene silencing. AlphaFold predicted co-chaperoning of H3-H4 tetramers by Mrc1 and Mcm2, with the Mrc1 connector domain bridging histone and Mcm2 binding. Biochemical and functional analysis validated this model and revealed a duality in Mrc1 function: disabling histone binding in the connector domain disrupted lagging-strand recycling while another histone-binding mutation impaired leading strand recycling. We propose that Mrc1 toggles histones between the lagging and leading strand recycling pathways, in part by intra-replisome co-chaperoning, to ensure epigenetic transmission to both daughter cells.

Novel insights on the positive correlation between sense and antisense pairs on gene expression.

A considerable proportion of the eukaryotic genome undergoes transcription, leading to the generation of noncoding RNA molecules that lack protein-coding information and are not subjected to translation. These noncoding RNAs (ncRNAs) are well recognized to have essential roles in several biological processes. Long noncoding RNAs (lncRNAs) represent the most extensive category of ncRNAs found in the human genome. Much research has focused on investigating the roles of cis-acting lncRNAs in the regulation of specific target gene expression. In the majority of instances, the regulation of sense gene expression by its corresponding antisense pair occurs in a negative (discordant) manner, resulting in the suppression of the target genes. The notion that a negative correlation exists between sense and antisense pairings is, however, not universally valid. In fact, several recent studies have reported a positive relationship between corresponding cis antisense pairs within plants, budding yeast, and mammalian cancer cells. The positive (concordant) correlation between anti-sense and sense transcripts leads to an increase in the level of the sense transcript within the same genomic loci. In addition, mechanisms such as altering chromatin structure, the formation of R loops, and the recruitment of transcription factors can either enhance transcription or stabilize sense transcripts through their antisense pairs. The primary objective of this work is to provide a comprehensive understanding of both aspects of antisense regulation, specifically focusing on the positive correlation between sense and antisense transcripts in the context of eukaryotic gene expression, including its implications towards cancer progression. This article is categorized under: RNA Processing > 3' End Processing Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.

Environment-Mediated Vertical Transmission Fostered Uncoupled Phylogenetic Relationships between Longicorn Beetles and Their Symbionts.

The Coleoptera Cerambycidae (longicorn beetles) use wood under different states (living healthy, freshly snapped, completely rot, etc.) in a species-specific manner for their larval diet. Larvae of some Cerambycidae groups have mycetomes, accessory organs associated with the midgut that harbor fungal symbiont cells. The symbionts are thought to improve nutrient conditions; however, this has yet to be shown experimentally. To deduce the evolutionary history of this symbiosis, we investigated the characteristics of the mycetomes in the larvae of longicorn beetles collected in Japan. Lepturinae, Necydalinae, and Spondylidinae are the only groups that possess mycetomes, and these three groups' mycetomes and corresponding fungal cells exhibit different characteristics between the groups. However, the phylogenetic relationship of symbiont yeasts does not coincide with that of the corresponding longicorn beetle species, suggesting they have not co-speciated. The imperfect vertical transmission of symbiont yeasts from female to offspring is a mechanism that could accommodate the host-symbiont phylogenetic incongruence. Some Lepturinae species secondarily lost mycetomes. The loss is associated with their diet choice, suggesting that different conditions between feeding habits could have allowed species to discard this organ. We found that symbiont fungi encapsulated in the mycetomes are dispensable for larval growth if sufficient nutrients are given, suggesting that the role of symbiotic fungi could be compensated by the food larvae take. Aegosoma sinicum is a longicorn beetle classified to the subfamily Prioninae, which does not possess mycetomes. However, this species contains a restricted selection of yeast species in the larval gut, suggesting that the symbiosis between longicorn beetles and yeasts emerged before acquiring the mycetomes.

Effects of the combination of red yeast rice-containing commercial Chinese polyherbal preparation with statins for dyslipidemia: a systematic review and meta-analysis.

Background: Significant challenges are associated with the pharmacological management of dyslipidemia, an important risk factor for cardiovascular disease. Limited reliable evidence exists regarding the efficacy of red yeast rice (RYR)-containing commercial Chinese polyherbal preparation (CCPP), despite their widespread use in China. Purpose: We aimed to investigate the efficacy of RYR-containing CCPPs combined with statins in treating dyslipidemia. Methods: Eight databases were searched for relevant randomized controlled trials (RCTs) from database inception date to November 2023. Outcome measures, including low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), triglyceride (TG), clinical efficacy, and adverse reactions, were assessed. The Cochrane Handbook for Systematic Reviews of Interventions was used for quality evaluation, and the meta-analysis was conducted using RevMan 5.3 and Stata 15.1. Results: Thirty-three studies involving 4,098 participants were included. The combination of RYR-containing CCPP, such as Xuezhikang (XZK), Zhibitai (ZBTAI), or Zhibituo (ZBTUO) with statins had a significant effect on the increase in clinical efficacy [RR:1.16, 95%CI (1.13, 1.19), p < 0.00001]. In addition, they also improved blood lipid profile parameters by increasing HDL-C levels [MD:0.21, 95%CI(0.17, 0.25), p < 0.00001], and decreasing TC [MD: 0.60, 95%CI(-0.76, -0.45), p < 0.00001], TG [MD: 0.33, 95%CI(-0.39, -0.26), p < 0.00001] and LDL-C levels [MD: 0.45, 95%CI(-0.54, -0.36), p < 0.00001]. No significant adverse reactions was observed in the RYR-containing CCPPs. Notably, ZBTAI and XZK significantly reduced the incidence of gastrointestinal disturbances and muscular adverse reactions. However, subgroup analyses suggested that the type of CCPPs, dose, and treatment duration might affect the efficacy of RYR-containing CCPPs. Conclusion: RYR-containing CCPPs combined with statins appears to improve lipid profiles and clinical efficacy in patients with dyslipidemia. However, due to the poor quality of the included studies, and some studied showing negative findings was unpublished. The results should be interpreted with caution until further confirmation by well-designed RCTs. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=487402, identifier CRD42023487402.

Effects of yeast culture on growth performance, immune function, antioxidant capacity and hormonal profile in Mongolian ram lambs.

Introduction: As effective growth-promoters and immune-modulators, yeast fermented products have shown positive effects in ruminants. To explore the mechanisms of yeast culture promoting growth and regulating immunity, this study investigated the effects of yeast culture, and ß-glucan as one of its main active ingredients, on the growth performance, immune function, antioxidant capacity and hormonal profile in Mongolian ram lambs. Methods: One hundred and five Mongolian ram lambs were randomly assigned to 3 groups, with 35 replicates in each group. The dietary treatments were: total mixed ration (TMR) as the control group, TMR supplemented with 50-70 g/kg yeast culture (YC) or 75 mg/kg ß-glucan. The test period was 137 days. All the sheep were weighed and 6 serum samples were collected in each group on days 0, 30, 60, 90 and 130, respectively. Results: The results showed that both YC and ß-glucan could promote the growth performance with increased average daily gain and decreased feed to weight gain ratio. Moreover, these two feed additives facilitated the immune function by selectively increasing the serum levels of lysozyme, IgG, IgM, INF-γ, TNF-α and some interleukins (IL-1ß, IL-2, IL-6 and IL-8); ameliorated the antioxidant capacity with higher total antioxidant capacity and enzyme activities of catalase and glutathione peroxidase; altered the metabolism-associated hormone levels with higher growth hormone and thyroid hormone T3 but lower cortisol and insulin. Discussion: In conclusion, both YC and ß-glucan could improve the growth performance, immune function and antioxidant capacity, and regulate the serum levels of metabolism-associated hormones, thus exerting effects of promoting growth and improving immune function. Therefore, YC could be considered as a suitable potential alternative strategy to antibiotics and be used as an animal feed additive. This article provides a theoretical basis for the clinical application of such yeast fermented preparations in mutton sheep husbandry.

Zearalenone removal using inactivated yeast embedded in porous modified yam starch aerogels and its application in corn silk tea.

Zearalenone contaminates food and poses a threat to human health. It is vital to develop cost-effective and environmentally-friendly adsorbents for its removal. By screening Sporobolomyces pararoseus (SZ4) and modified yam starch (adsorption capacity (qe) of 1.33 and 0.94 mg/g, respectively), this study prepared a novel composite aerogel adsorbent (P-YSA@SZ410). The compressive strength of P-YSA@SZ410 was 1.35-fold higher than unloaded yeast. It contained several functional groups and three-dimensional interconnected channels, achieving a 0° contact angle within 0.18 s, thereby demonstrating excellent water-absorbent properties. With a qe of 2.96 mg/g at 308 K, the adsorption process of P-YSA@SZ410 was spontaneous, endothermic, and matched pseudo-second-order and Langmuir models. The composite adsorbed zearalenone via electrostatic attraction and hydrogen bonding, maintaining a qe of 2.24 mg/g after five cycles. P-YSA@SZ410 was found to remove zearalenone effectively under various conditions and could be applied to corn silk tea, indicating its great potential as an adsorbent material.

Effect of yeast (Saccharomyces cerevisiae) fermentation on conformational changes in pig liver proteins and their ability to bind to characteristic aldehydes.

This study aimed to explore the potential of a fermentation technology to reduce off-flavour perception and its underlying mechanisms. Results revealed that yeast fermentation (YF) significantly ameliorated the off-flavour of pig liver (p < 0.05). Specifically, YF pre-treatment decreased the relative abundance of α-helix and fluorescence intensity while increasing the surface hydrophobicity and SS level and loosening the microstructure of myofibrillar proteins (MPs) in pig liver. Additionally, the appropriate fermentation treatments enhanced the MP-aldehyde binding capacity by 0.25-1.30 times, demonstrating that YF-induced conformational modifications in pig liver proteins made them more prone to interacting with characteristic aldehydes. Moreover, molecular docking results confirmed that hydrophobic interactions are the primary drivers of MP-aldehyde binding. These findings suggest that YF technology holds immense promise for modulating off-flavour perception in liver products by altering protein conformation.

Understanding the molecular mechanisms of human diseases: the benefits of fission yeasts.

The role of model organisms such as yeasts in life science research is crucial. Although the baker's yeast (Saccharomyces cerevisiae) is the most popular model among yeasts, the contribution of the fission yeasts (Schizosaccharomyces) to life science is also indisputable. Since both types of yeasts share several thousands of common orthologous genes with humans, they provide a simple research platform to investigate many fundamental molecular mechanisms and functions, thereby contributing to the understanding of the background of human diseases. In this review, we would like to highlight the many advantages of fission yeasts over budding yeasts. The usefulness of fission yeasts in virus research is shown as an example, presenting the most important research results related to the Human Immunodeficiency Virus Type 1 (HIV-1) Vpr protein. Besides, the potential role of fission yeasts in the study of prion biology is also discussed. Furthermore, we are keen to promote the uprising model yeast Schizosaccharomyces japonicus, which is a dimorphic species in the fission yeast genus. We propose the hyphal growth of S. japonicus as an unusual opportunity as a model to study the invadopodia of human cancer cells since the two seemingly different cell types can be compared along fundamental features. Here we also collect the latest laboratory protocols and bioinformatics tools for the fission yeasts to highlight the many possibilities available to the research community. In addition, we present several limiting factors that everyone should be aware of when working with yeast models.

Involvement of lipid-translocating exporter family proteins in determination of myriocin sensitivity in budding yeast.

Myriocin is an inhibitor of serine palmitoyltransferase involved in the initial biosynthetic step for sphingolipids, and causes potent growth inhibition in eukaryotic cells. In budding yeast, Rsb1, Rta1, Pug1, and Ylr046c are known as the Lipid-Translocating Exporter (LTE) family and believed to contribute to export of various cytotoxic lipophilic compounds. It was reported that Rsb1 is a transporter responsible for export of intracellularly accumulated long-chain bases, which alleviate the cytotoxicity. In this study, it was found that LTE family genes are involved in determination of myriocin sensitivity in yeast. Analyses of effects of deletion and overexpression of LTE family genes suggested that all LTEs contribute to suppression of cytotoxicity of myriocin. It was confirmed that RSB1 overexpression suppressed reduction in complex sphingolipid levels caused by myriocin treatment, possibly exporting myriocin to outside of the cell. These results suggested that LTE family genes function as a defense mechanism against myriocin.

Physiological responses contributing to multiple stress tolerance in Pichia kudriavzevii with potential enhancement for ethanol fermentation.

Economically feasible ethanol production requires efficient hydrolysis of lignocellulosic biomass and high-temperature processing to enable simultaneous saccharification and fermentation. During the lignocellulolysic hydrolysate, the yeast must encounter with a multiple of inhibitors such as heat and furfural. To solve this problem, a potential fermentative yeast strain that tolerated simultaneous multistress and enhance ethanol concentration was investigated. Twenty yeast isolates were classified into two major yeast species, namely Pichia kudriavzevii (twelve isolates) and Candida tropicalis (eight isolates). All P. kudriavzevii isolates were able to grow at high temperature (45 °C) and exhibited stress tolerance toward furfural. Among P. kudriavzevii isolates, NUCG-S3 presented the highest specific growth rate under each stress condition of heat and furfural, and multistress. Morphological changes in P. kudriavzevii isolates (NUCG-S2, NUCG-S3, NUKL-P1, NUKL-P3, and NUOR-J1) showed alteration in mean cell length and width compared to the non-stress condition. Ethanol production by glucose was also determined. The yeast strain, NUCG-S3, gave the highest ethanol concentrations at 99.46 ± 0.82, 62.23 ± 0.96, and 65.80 ± 0.62 g/l (P < 0.05) under temperature of 30 °C, 40 °C, and 42 °C, respectively. The tolerant isolated yeast NUCG-S3 achieved ethanol production of 53.58 ± 3.36 and 48.06 ± 3.31 g/l (P < 0.05) in the presence of 15 mM furfural and multistress (42 °C with 15 mM furfural), respectively. Based on the results of the present study, the novel thermos and furfural-tolerant yeast strain P. kudriavzevii NUCG-S3 showed promise as a highly proficient yeast for high-temperature ethanol fermentation.

Autophagy of Glycogen Is Non-Selective in Komagataella phaffii.

Glycogen is an important reserve polysaccharide from bacteria to human. It is organized in glycogen granules that also contain several proteins involved in their metabolism. Glycogen granules can be mobilized in mammalian lysosomes and yeast vacuoles. They are delivered to these organelles by macroautophagy (hereafter autophagy). However, whether this is a selective or a non-selective process remains a matter of debate. It was proposed to be selective and called "glycophagy" (for selective autophagy of glycogen) in the mouse liver. However, the evidence of this selectivity is lacking in other glycogen-rich organs, such as the heart and skeletal muscle, which both are heavily impacted by the aberrant lysosomal accumulation of glycogen in Pompe disease. We recently developed the Komagataella phaffii yeast as a simple model to study the relationship of glycogen and autophagy. Using this model, we showed that cytosolic glycogen granules are delivered to the vacuole by non-selective autophagy, at least during nitrogen starvation. We speculate that this type of autophagy might be responsible for the lysosomal glycogen turnover in non-hepatic mammalian tissues.

Engineering a versatile yeast platform for sesquiterpene production from glucose or methanol.

Natural sesquiterpene are valuable compounds with diverse applications in industries, such as cosmetics and energy. Microbial synthesis offers a promising way for sesquiterpene production. Methanol, can be synthesized from CO2 and solar energy, serves as a sustainable carbon source. However, it is still a challenge to utilize methanol for the synthesis of value-added compounds. Pichia pastoris (syn. Komagataella phaffii), known for its efficient utilization of glucose and methanol, has been widely used in protein synthesis. With advancements in technology, P. pastoris is gradually engineered for chemicals production. Here, we successfully achieved the synthesis of α-bisabolene in P. pastoris with dual carbon sources by expressing the α-bisabolene synthase gene under constitutive promoters. We systematically analyzed the effects of different steps in the mevalonate (MVA) pathway when methanol or glucose was used as the carbon source. Our finding revealed that the sesquiterpene synthase module significantly increased the production when methanol was used. While the metabolic modules MK and PMK greatly improved carbon source utilization, cell growth, and titer when glucose was used. Additionally, we demonstrated the synthesis of ß-farnesene from dual carbon source by replacing the α-bisabolene synthase with a ß-farnesene synthase. This study establishes a platform strain that is capable to synthesize sesquiterpene from different carbon sources in P. pastoris. Moreover, it paves the way for the development of P. pastoris as a high-efficiency microbial cell factory for producing various chemicals, and lays foundation for large-scale synthesis of high value-added chemicals efficiently from methanol in P. pastoris.

Methods to Monitor Nucleophagy in Yeast.

The selective degradation of nuclear components via autophagy, termed nucleophagy, is an essential process observed from yeasts to mammals and crucial for maintaining nucleus homeostasis and regulating nucleus functions. In the budding yeast Saccharomyces cerevisiae, nucleophagy occurs in two different manners: one involves autophagosome formation for the sequestration and vacuolar transport of nucleus-derived vesicles (NDVs), and the other proceeds with the invagination of the vacuolar membrane for the uptake of NDVs into the vacuole, termed macronucleophagy and micronucleophagy, respectively. This chapter describes methods to analyze and quantify activities of these nucleophagy pathways in yeast.

Fluorescence Microscopy and Immunoblotting for Mitophagy in Budding Yeast.

Selective removal of excess or damaged mitochondria is an evolutionarily conserved process that contributes to mitochondrial quality and quantity control. This catabolic event relies on autophagy, a membrane trafficking system that sequesters cytoplasmic constituents into double membrane-bound autophagosomes and delivers them to lysosomes (vacuoles in yeast) for hydrolytic degradation and is thus termed mitophagy. Dysregulation of mitophagy is associated with various diseases, highlighting its physiological relevance. In budding yeast, the pro-mitophagic single-pass membrane protein Atg32 is upregulated under prolonged respiration or nutrient starvation, anchored on the surface of mitochondria, and activated to recruit the autophagy machinery for the formation of autophagosomes surrounding mitochondria. In this chapter, we provide protocols to assess Atg32-mediated mitophagy using fluorescence microscopy and immunoblotting.

Screening of protein-based inhibitors for the intracellular domain of epidermal growth factor receptor by directed evolution using the yeast Gγ recruitment system.

Protein-based therapeutics, including antibodies and antibody-like-proteins, have increasingly attracted attention due to their high specificity compared to small-molecular drugs. The Gγ recruitment system, one of the in vivo yeast two-hybrid systems for detecting protein-protein interactions, has been previously developed using yeast signal transduction machinery. In this study, we modified the Gγ recruitment system to screen the protein mutants that efficiently bind to the intracellular domain of the epidermal growth factor receptor L858R mutant (cytoEGFRL858R). Using the modified platform, we performed in vivo directed evolution for growth factor receptor-bound protein 2 (Grb2) and its truncated variant containing only the Src-homology 2 (SH2) domain, successfully identifying several mutants that more strongly bound to cytoEGFRL858R than their parental proteins. Some of them contained novel beneficial mutations (F108Y and Q144H) and specifically bound to the recombinant cytosolic phosphorylated EGFR in vitro, highlighting the utility of the evolutionary platform.

Dynamic Live Cell Imaging of Budding Yeast Meiosis.

For over a century, major advances in understanding meiosis have come from the use of microscopy-based methods. Studies using the budding yeast, Saccharomyces cerevisiae, have made important contributions to our understanding of meiosis because of the facility with which budding yeast can be manipulated as a genetic model organism. In contrast, imaging-based approaches with budding yeast have been constrained by the small size of its chromosomes. The advent of advances in fluorescent chromosome tagging techniques has made it possible to use yeast more effectively for imaging-based approaches as well. This protocol describes live cell imaging methods that can be used to monitor chromosome movements throughout meiosis in living yeast cells.

Analysis of transcriptional response in haploid and diploid Schizosaccharomyces pombe under genotoxic stress.

Whole genome duplications are implicated in genome instability and tumorigenesis. Human and yeast polyploids exhibit increased replication stress and chromosomal instability, both hallmarks of cancer. In this study, we investigate the transcriptional response of Schizosaccharomyces pombe to increased ploidy generally, and in response to treatment with the genotoxin methyl methanesulfonate (MMS). We find that treatment of MMS induces upregulation of genes involved in general response to genotoxins, in addition to cell cycle regulatory genes. Downregulated genes are enriched in transport and sexual reproductive pathways. We find that the diploid response to MMS is muted compared to the haploid response, although the enriched pathways remain largely the same. Overall, our data suggests that the global S. pombe transcriptome doubles in response to increased ploidy but undergoes modest transcriptional changes in both unperturbed and genotoxic stress conditions.

Renal Impairment of Proximal Tubular Injury Caused by Red Yeast Rice Supplement: Report of 2 Cases.

Introduction: Drug-induced tubulointerstitial injury is a common cause of renal impairment. Since the mechanisms of drug-induced tubular injury are diverse, various treatment approaches are needed according to the pathogenesis. Renal biopsy is indispensable to determine not only the pathological diagnosis, but also the underlying mechanism, and to guide appropriate treatment. Most recently, one of the red yeast supplements has been widely highlighted as a novel cause of tubular damage, mainly in Japan and Asia. However, neither detailed pathological findings nor the mechanism of renal impairment has been sufficiently reported. Case Presentation: Two cases of renal impairment after taking red yeast supplement internally are presented. Both cases showed renal dysfunction with low uric acid, potassium, and phosphorus levels, characteristic features of Fanconi syndrome. The renal biopsy findings of both cases showed severe injury to the proximal tubules with mild inflammatory cell infiltration. The proximal tubules exhibited diffuse loss of the brush border, flattening, and tubular lumen dilation. Immunofluorescence showed no deposition of immunoglobulin and complement in the glomeruli and tubules. Electron microscopic findings indicated proximal tubular damage without crystal deposition. Moreover, immunohistochemistry using the proximal tubular marker CD10 and a marker for distal tubules including the loop of Henle, E-cadherin, collectively demonstrated that the focus of renal injury in both cases was mainly the proximal tubules. Conclusions: The red yeast rice supplement itself, its metabolized product, or other unknown contaminant components might directly induce proximal tubulopathy rather than an allergic reaction-related tubulointerstitial nephritis.