Age-related changes in the zebrafish and killifish inner ear and lateral line.

Age-related hearing loss (ARHL) is a debilitating disorder for millions worldwide. While there are multiple underlying causes of ARHL, one common factor is loss of sensory hair cells. In mammals, new hair cells are not produced postnatally and do not regenerate after damage, leading to permanent hearing impairment. By contrast, fish produce hair cells throughout life and robustly regenerate these cells after toxic insult. Despite these regenerative abilities, zebrafish show features of ARHL. Here, we show that aged zebrafish of both sexes exhibited significant hair cell loss and decreased cell proliferation in all inner ear epithelia (saccule, lagena, utricle). Ears from aged zebrafish had increased expression of pro-inflammatory genes and significantly more macrophages than ears from young adult animals. Aged zebrafish also had fewer lateral line hair cells and less cell proliferation than young animals, although lateral line hair cells still robustly regenerated following damage. Unlike zebrafish, African turquoise killifish (an emerging aging model) only showed hair cell loss in the saccule of aged males, but both sexes exhibit age-related changes in the lateral line. Our work demonstrates that zebrafish exhibit key features of auditory aging, including hair cell loss and increased inflammation. Further, our finding that aged zebrafish have fewer lateral line hair cells yet retain regenerative capacity, suggests a decoupling of homeostatic hair cell addition from regeneration following acute trauma. Finally, zebrafish and killifish show species-specific strategies for lateral line homeostasis that may inform further comparative research on aging in mechanosensory systems.

The African Turquoise Killifish: A Scalable Vertebrate Model for Aging and Other Complex Phenotypes.

The African turquoise killifish Nothobranchius furzeri is currently the shortest-lived vertebrate that can be bred in captivity. Because of its short life span of only 4-6 months, rapid generation time, high fecundity, and low cost of maintenance, the African turquoise killifish has emerged as an attractive model organism that combines the scalability of invertebrate models with the unique features of vertebrate organisms. A growing community of researchers is using the African turquoise killifish for studies in diverse fields, including aging, organ regeneration, development, "suspended animation," evolution, neuroscience, and disease. A wide range of techniques is now available for killifish research, from genetic manipulations and genomic tools to specialized assays for studying life span, organ biology, response to injury, etc. This protocol collection provides detailed descriptions of the methods that are generally applicable to all killifish laboratories and those that are limited to specific disciplines. Here, we give an overview of the features that render the African turquoise killifish a unique fast-track vertebrate model organism.

High-Energy Ball Milling Promoted Sulfur Immobilization for Constructing High-Performance Na-Storage Carbon Anodes.

Sulfur (S) doping is an effective method for constructing high-performance carbon anodes for sodium-ion batteries. However, traditional designs of S-doped carbon often exhibit low initial Coulombic efficiency (ICE), poor rate capability, and impoverished cycle performance, limiting their practical applications. This study proposes an innovative design strategy to fabricate S-doped carbon using sulfonated sugar molecules as precursors via high-energy ball milling. The results show that the high-energy ball milling can immobilize S for sulfonated sugar molecules by modulating the chemical state of S atoms, thereby creating a S-rich carbon framework with a doping level of 15.5 wt %. In addition, the S atoms are present mainly in the form of C-S bonds, facilitating a stable electrochemical reaction; meanwhile, S atoms expand the spacing between carbon layers and contribute sufficient capacitance-type Na-storage sites. Consequently, the S-doped carbon exhibits a large capacity (>600 mAh g-1), a high ICE (>90%), superior cycling stability (490 mAh g-1 after 1100 cycles at 5 A g-1), and outstanding rate performance (420 mAh g-1 at a high current density of 50 A g-1). Such excellent Na-storage properties of S-doped carbon have rarely been reported in the literatures before.

Breeding and Reproduction of the African Turquoise Killifish Nothobranchius furzeri.

The successful breeding and reproduction of the African turquoise killifish Nothobranchius furzeri in a controlled laboratory setting are required to establish this fish species as a model system for studying vertebrate development and aging. Here, we describe a protocol to care for and hatch African turquoise killifish embryos, raise the juvenile fish to adulthood, and breed this species using sand as the breeding bedding. We also provide suggestions for generating a large quantity of good-quality embryos.

An automated feeding system for the African killifish reveals the impact of diet on lifespan and allows scalable assessment of associative learning.

The African turquoise killifish is an exciting new vertebrate model for aging studies. A significant challenge for any model organism is the control over its diet in space and time. To address this challenge, we created an automated and networked fish feeding system. Our automated feeder is designed to be open-source, easily transferable, and built from widely available components. Compared to manual feeding, our automated system is highly precise and flexible. As a proof of concept for the feeding flexibility of these automated feeders, we define a favorable regimen for growth and fertility for the African killifish and a dietary restriction regimen where both feeding time and quantity are reduced. We show that this dietary restriction regimen extends lifespan in males (but not in females) and impacts the transcriptomes of killifish livers in a sex-specific manner. Moreover, combining our automated feeding system with a video camera, we establish a quantitative associative learning assay to provide an integrative measure of cognitive performance for the killifish. The ability to precisely control food delivery in the killifish opens new areas to assess lifespan and cognitive behavior dynamics and to screen for dietary interventions and drugs in a scalable manner previously impossible with traditional vertebrate model organisms.

Role and mechanism of the zinc finger protein ZNF580 in foam-cell formation.

Coronary atherosclerotic heart disease poses a significant threat to human health. The pathological basis is atherosclerosis and foam-cell formation is the key factor in the initiation of atherosclerosis. In the present study, foam cell models were established using 50 ng/ml oxidized low-density lipoprotein to stimulate in vitro cultures of THP-1 cells for 72 h. The expression of zinc finger protein 580 (ZNF580), a Cys2-His2 zinc finger protein containing 172 amino acids that was originally cloned by screening a human aortic cDNA library, was measured in foam cells and its interaction with various regulatory factors during foam-cell formation was investigated. Oil red O staining was used to observe cell morphology and intracellular lipid levels. Lentivirus transfection was employed to either overexpress or silence ZNF580 in THP-1 cells, and an inverted fluorescence microscope was used to observe the distribution of ZNF580 immunofluorescence to determine the transfection rate. RNA and total protein were extracted and the expression levels of ZNF580, CD36, peroxisome proliferator-activated receptor-γ (PPAR-γ), ATP-binding cassette transporter A1 (ABCA1) and apolipoprotein E (ApoE) were measured by reverse transcription-quantitative PCR. The protein levels were examined by western blot analysis to evaluate the interaction between ZNF580 and associated regulatory factors. ZNF580 was able to significantly increase the expression levels of ApoE and ABCA1 and significantly decrease the expression levels of CD36 and PPAR-γ, suggesting that ZNF580-mediated inhibition of foam-cell formation is associated with the PPAR-γ-CD36 signalling pathway. Based on these findings, ZNF580 may be a potential therapeutic candidate for the treatment of coronary atherosclerotic heart disease.

Particles and corrected particles of LDL and non-HDL are stronger predicters of coronary lesion in postmenopausal women.

BACKGROUND: The optimum lipid indexes, predicting the coronary lesion in postmenopausal women are not clear. OBJECTIVE: To evaluate the optimum lipid predicter for coronary lesion in routine and advanced lipid tests. METHOD: 300 postmenopausal women were enrolled and assigned into coronary heart disease (CHD) Group (242), and non-CHD Group (58). Routine and advanced lipid indexes were measured with standard laboratory test and nuclear magnetic resonance (NMR) spectroscopy. The correlation and predictivities for CHD of routine and advanced lipid indexes were performed with Logistic regression, Spearman correlation analysis and receiver operating characteristic (ROC). RESULTS: Age (hazard ratio (HR) 2.58, 95% confidence interval (CI) 1.08-5.86, P = 0.03), apolipoprotein B (ApoB) (HR 1.35, 95% CI 1.15-1.59, P < 0.001), corrected particles of low-density lipoprotein (LDL-p-corr) (HR 1.05, 95% CI 1.03-1.06, P < 0.001) and corrected particles of non-high-density lipoprotein (non-HDL-p-corr) (HR 1.02, 95% CI 1.01-1.03, P < 0.001) were the risk factors of CHD. LDL cholesterol (LDL-C), LDL-p, LDL-p-corr, HDL cholesterol (HDL-C), non-HDL cholesterol (non-HDL-C), non-HDL-p and non-HDL-p-corr were in linear correlation with Gensini score. Advanced lipid indexes LDL-p (area under curve (AUC) = 0.750, P = 0.02), LDL-p-corr (AUC = 0.759, P = 0.02), non-HDL-p (AUC = 0.693, P = 0.03) and non-HDL-p-corr (AUC = 0.699, P = 0.03) were more predictive for CHD than the routine ones (LDL-C and non-HDL-C). CONCLUSION: In postmenopausal women, age, ApoB, LDL-p-corr and non-HDL-p-corr were risk factors of CHD. Compared with traditional lipid items, LDL-p, LDL-p-corr, non-HDL-p and non-HDL-p-corr may be better lipid indexes for CHD in postmenopausal women.

Meiotic regulation of the Ndc80 complex composition and function.

This review describes the current models for how the subunit abundance of the Ndc80 complex, a key kinetochore component, is regulated in budding yeast and metazoan meiosis. The past decades of kinetochore research have established the Ndc80 complex to be a key microtubule interactor and a central hub for regulating chromosome segregation. Recent studies further demonstrate that Ndc80 is the limiting kinetochore subunit that dictates the timing of kinetochore activation in budding yeast meiosis. Here, we discuss the molecular circuits that regulate Ndc80 protein synthesis and degradation in budding yeast meiosis and compare the findings with those from metazoans. We envision the regulatory principles discovered in budding yeast to be conserved in metazoans, thereby providing guidance into future investigations on kinetochore regulation in human health and disease.

Tunable Transcriptional Interference at the Endogenous Alcohol Dehydrogenase Gene Locus in Drosophila melanogaster.

Neighboring sequences of a gene can influence its expression. In the phenomenon known as transcriptional interference, transcription at one region in the genome can repress transcription at a nearby region in cis Transcriptional interference occurs at a number of eukaryotic loci, including the alcohol dehydrogenase (Adh) gene in Drosophila melanogasterAdh is regulated by two promoters, which are distinct in their developmental timing of activation. It has been shown using transgene insertion that when the promoter distal from the Adh start codon is deleted, transcription from the proximal promoter becomes de-regulated. As a result, the Adh proximal promoter, which is normally active only during the early larval stages, becomes abnormally activated in adults. Whether this type of regulation occurs in the endogenous Adh context, however, remains unclear. Here, we employed the CRISPR/Cas9 system to edit the endogenous Adh locus and found that removal of the distal promoter also resulted in the untimely expression of the proximal promoter-driven mRNA isoform in adults, albeit at lower levels than previously reported. Importantly, transcription from the distal promoter was sufficient to repress proximal transcription in larvae, and the degree of this repression was dependent on the degree of distal promoter activity. Finally, upregulation of the distal Adh transcript led to the enrichment of histone 3 lysine 36 trimethylation over the Adh proximal promoter. We conclude that the endogenous Adh locus is developmentally regulated by transcriptional interference in a tunable manner.

Aurora B-dependent Ndc80 degradation regulates kinetochore composition in meiosis.

The kinetochore complex is a conserved machinery that connects chromosomes to spindle microtubules. During meiosis, the kinetochore is restructured to accommodate a specialized chromosome segregation pattern. In budding yeast, meiotic kinetochore remodeling is mediated by the temporal changes in the abundance of a single subunit called Ndc80. We previously described the regulatory events that control the timely synthesis of Ndc80. Here, we report that Ndc80 turnover is also tightly regulated in meiosis: Ndc80 degradation is active in meiotic prophase, but not in metaphase I. Ndc80 degradation depends on the ubiquitin ligase APCAma1 and is mediated by the proteasome. Importantly, Aurora B-dependent Ndc80 phosphorylation, a mark that has been previously implicated in correcting erroneous microtubule-kinetochore attachments, is essential for Ndc80 degradation in a microtubule-independent manner. The N terminus of Ndc80, including a 27-residue sequence and Aurora B phosphorylation sites, is both necessary and sufficient for kinetochore protein degradation. Finally, defects in Ndc80 turnover predispose meiotic cells to chromosome mis-segregation. Our study elucidates the mechanism by which meiotic cells modulate their kinetochore composition through regulated Ndc80 degradation, and demonstrates that Aurora B-dependent regulation of kinetochores extends beyond altering microtubule attachments.

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis.

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene NDC80 during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in other yeast strains and growth conditions.

Transcription of a 5' extended mRNA isoform directs dynamic chromatin changes and interference of a downstream promoter.

Cell differentiation programs require dynamic regulation of gene expression. During meiotic prophase in Saccharomyces cerevisiae, expression of the kinetochore complex subunit Ndc80 is downregulated by a 5' extended long undecoded NDC80 transcript isoform. Here we demonstrate a transcriptional interference mechanism that is responsible for inhibiting expression of the coding NDC80 mRNA isoform. Transcription from a distal NDC80 promoter directs Set1-dependent histone H3K4 dimethylation and Set2-dependent H3K36 trimethylation to establish a repressive chromatin state in the downstream canonical NDC80 promoter. As a consequence, NDC80 expression is repressed during meiotic prophase. The transcriptional mechanism described here is rapidly reversible, adaptable to fine-tune gene expression, and relies on Set2 and the Set3 histone deacetylase complex. Thus, expression of a 5' extended mRNA isoform causes transcriptional interference at the downstream promoter. We demonstrate that this is an effective mechanism to promote dynamic changes in gene expression during cell differentiation.

Kinetochore inactivation by expression of a repressive mRNA.

Differentiation programs such as meiosis depend on extensive gene regulation to mediate cellular morphogenesis. Meiosis requires transient removal of the outer kinetochore, the complex that connects microtubules to chromosomes. How the meiotic gene expression program temporally restricts kinetochore function is unknown. We discovered that in budding yeast, kinetochore inactivation occurs by reducing the abundance of a limiting subunit, Ndc80. Furthermore, we uncovered an integrated mechanism that acts at the transcriptional and translational level to repress NDC80 expression. Central to this mechanism is the developmentally controlled transcription of an alternate NDC80 mRNA isoform, which itself cannot produce protein due to regulatory upstream ORFs in its extended 5' leader. Instead, transcription of this isoform represses the canonical NDC80 mRNA expression in cis, thereby inhibiting Ndc80 protein synthesis. This model of gene regulation raises the intriguing notion that transcription of an mRNA, despite carrying a canonical coding sequence, can directly cause gene repression.