Yeast Cell Lines Research Articles

Automated Assignment of 15N And 13C Enrichment Levels in Doubly-Labeled Proteins.

Uniform enrichment of 15N and 13C in proteins is commonly employed for 2D heteronuclear NMR measurements of the three-dimensional protein structure. Achieving a high degree of enrichment of both elements is important for obtaining high quality data. Uniform labeling of proteins and glycoproteins expressed in higher organisms (yeast or mammalian cell lines) is more challenging than expression in Escherichia coli, a prokaryote that grows on simple, chemically defined media but does not provide appropriate eukaryotic modifications. It is difficult to achieve complete incorporation of both heavy isotopes, and quality control measures are important for quantitating the level of their enrichment. Mass spectrometry measurements of the isotopic distribution of the intact protein or its proteolytic fragments provide the means to assess the enrichment level. A mass accuracy of 1 ppm or better is shown to be required to distinguish the correct combination of 13C and 15N enrichment due to subtle shifts in peak centroids with differences in the underlying, but unresolved, isotopic fine structure. A simple computer program was developed to optimize the fitting of experimental isotope patterns to statistically derived distributions. This method can determine the isotopic abundance from isotope patterns and isotopologue masses in conventional MS data for peptides, intact proteins, and glycans. For this purpose, MATLAB's isotope simulator, isotopicdist, has been modified to permit the variation of 15N and 13C enrichment levels and to perform a two-dimensional grid search of enrichment levels of both isotopes. We also incorporated an alternate isotope simulator, js-emass, into MATLAB for use in the same fitting program. Herein we benchmark this technique on natural abundance ubiquitin and uniformly [15N,13C]-labeled ubiquitin at both the intact and peptide level, outline considerations for data quality and mass accuracy, and report several improvements we have made to the previously reported analysis of the [15N,13C]-enriched human IgG Fc domain, a glycoprotein that has been expressed in Saccharomyces cerevisiae.

Galactose-1-phosphate inhibits cytochrome c oxidase and causes mitochondrial dysfunction in classic galactosemia

Classic galactosemia is an inborn error of metabolism caused by mutations in the GALT gene resulting in the diminished activity of the galactose-1-phosphate uridyltransferase enzyme. This reduced GALT activity leads to the buildup of the toxic intermediate galactose-1-phosphate and a decrease in ATP levels upon exposure to galactose. In this work, we focused our attention on mitochondrial oxidative phosphorylation in the context of this metabolic disorder. We observed that galactose-1-phosphate accumulation reduced respiratory rates in vivo and changed mitochondrial function and morphology in yeast models of galactosemia. These alterations are harmful to yeast cells since the mitochondrial retrograde response is activated as part of the cellular adaptation to galactose toxicity. In addition, we found that galactose-1-phosphate directly impairs cytochrome c oxidase activity of mitochondrial preparations derived from yeast, rat liver, and human cell lines. These results highlight the evolutionary conservation of this biochemical effect. Finally, we discovered that two compounds - oleic acid and dihydrolipoic acid – that can improve the growth of cell models of mitochondrial diseases, were also able to improve galactose tolerance in this model of galactosemia. These results reveal a new molecular mechanism relevant to the pathophysiology of classic galactosemia - galactose-1-phosphate-dependent mitochondrial dysfunction - and suggest that therapies designed to treat mitochondrial diseases may be repurposed to treat galactosemia.

Design, synthesis, molecular dynamic simulation, DFT analysis, computational pharmacology and decoding the antidiabetic molecular mechanism of sulphonamide-thiazolidin-4-one hybrids

In search of novel antidiabetic agents, the study was framed to hybridize and synthesize molecules based on scaffolds of benzenesulfonamide and thiazolidine-4-one, efficiently synthesized (6a-6h) and structurally characterized by FTIR, 1H NMR 13C NMR and HRMS spectroscopy. The antidiabetic activity was assessed based on α-amylase inhibition, glucose uptake assay and PPAR-γ transcription factor assay. Compound 6b (4-(2-((E)-5-((Z)-2-hydroxybenzylidene)-4-oxo-2-(phenylimino) thiazolidin-3-yl) ethyl) benzenesulfonamide) demonstrated promising activity toward α-amylase inhibition having IC50 value of 29.51±1.35 µg/ml. A glucose uptake assay was performed for compounds (6a-6h) to assess the capacity of glucose uptake. Among them, Compound 6b exhibits notable glucose uptake with 88.71% in the yeast cell line. To support the glucose uptake findings of compound 6b, expression of PPAR-γ transcription factor was measured of compound 6b as compared to pioglitazone. Furthermore, induced fit docking was carried out to predict the refined pose of synthesized compounds in the α-amylase binding pocket. MD simulation studies confirmed the stability of compound 6b in the α-amylase binding pocket. Molecules were geometrically optimized by DFT theory using B3LYP 6–31 G (d, p**) basis set, and electronic properties were studied. Network pharmacology analysis for compound 6b identified key pathways involved in T2DM and postprandial hyperglycemia like PI3K-Akt pathway, Type II diabetes mellitus pathway metabolic pathway, and digestion of dietary carbohydrates pathway. These findings provide better insights into further developing benzenesulfonamide-based thiazolidin-4-one hybrids as antidiabetic agents.

A universal system for boosting gene expression in eukaryotic cell-lines

We demonstrate a transcriptional regulatory design algorithm that can boost expression in yeast and mammalian cell lines. The system consists of a simplified transcriptional architecture composed of a minimal core promoter and a synthetic upstream regulatory region (sURS) composed of up to three motifs selected from a list of 41 motifs conserved in the eukaryotic lineage. The sURS system was first characterized using an oligo-library containing 189,990 variants. We validate the resultant expression model using a set of 43 unseen sURS designs. The validation sURS experiments indicate that a generic set of grammar rules for boosting and attenuation may exist in yeast cells. Finally, we demonstrate that this generic set of grammar rules functions similarly in mammalian CHO-K1 and HeLa cells. Consequently, our work provides a design algorithm for boosting the expression of promoters used for expressing industrially relevant proteins in yeast and mammalian cell lines.

Diya – A universal light illumination platform for multiwell plate cultures

Recent progress in protein engineering has established optogenetics as one of the leading external non-invasive stimulation strategies, with many optogenetic tools being designed for invivo operation. Characterization and optimization of these tools require a high-throughput and versatile light delivery system targeting micro-titer culture volumes. Here, we present a universal light illumination platform- Diya, compatible with a wide range of cell culture plates and dishes. Diya hosts specially designed features ensuring active thermal management, homogeneous illumination, and minimal light bleedthrough. It offers light induction programming via a user-friendly custom-designed GUI. Through extensive characterization experiments with multiple optogenetic tools in diverse model organisms (bacteria, yeast, and human cell lines), we show that Diya maintains viable conditions for cell cultures undergoing light induction. Finally, we demonstrate an optogenetic strategy for invivo biomolecular controller operation. With a custom-designed antithetic integral feedback circuit, we exhibit robust perfect adaptation and light-controlled set-point variation using Diya.

Caenorhabditis elegans NSE3 homolog (MAGE-1) is involved in genome stability and acts in inter-sister recombination during meiosis.

Melanoma antigen (MAGE) genes encode for a family of proteins that share a common MAGE homology domain. These genes are conserved in eukaryotes and have been linked to a variety of cellular and developmental processes including ubiquitination and oncogenesis in cancer. Current knowledge on the MAGE family of proteins mainly comes from the analysis of yeast and human cell lines, and their functions have not been reported at an organismal level in animals. Caenorhabditis elegans only encodes 1 known MAGE gene member, mage-1 (NSE3 in yeast), forming part of the SMC-5/6 complex. Here, we characterize the role of mage-1/nse-3 in mitosis and meiosis in C. elegans. mage-1/nse-3 has a role in inter-sister recombination repair during meiotic recombination and for preserving chromosomal integrity upon treatment with a variety of DNA-damaging agents. MAGE-1 directly interacts with NSE-1 and NSE-4. In contrast to smc-5, smc-6, and nse-4 mutants which cause the loss of NSE-1 nuclear localization and strong cytoplasmic accumulation, mage-1/nse-3 mutants have a reduced level of NSE-1::GFP, remnant NSE-1::GFP being partially nuclear but largely cytoplasmic. Our data suggest that MAGE-1 is essential for NSE-1 stability and the proper functioning of the SMC-5/6 complex.

Modulation of Insulin Resistance by Silybum marianum Leaves, and its Synergistic Efficacy with Gymnema sylvestre, Momordica charantia, Trigonella-foenum graecum Against Protein Tyrosine Phosphatase 1B

ABSTRACT Prolonged insulin resistance is considered one of the reasons for Type 2 Diabetes Mellitus. Upregulation of Protein tyrosine phosphatase 1B (PTP1B), a negative regulator of insulin signalling, has been well studied as a key regulator in prognosis to insulin resistance. It has been widely studied as a desirable molecular therapeutic target. The study aimed to evaluate the efficacy of leaf extract of the medicinal plants Silybum marianum on the inhibition of PTP1B activity. It also explored the synergistic effect with extracts of Gymnema sylvestre (leaves), Momordica charantia (seeds), and Trigonella foenum graecum (seeds). The S. marianum leaves showed dose-dependent inhibition of PTP1B ranging from 9.48–47.95% (25–1000 μg mL−1). Assay with individual plant extracts showed comparatively lesser inhibition of PTP1B as compared to metformin as a control (38% inhibition). However, a synergistic effect showed nearly 45% PTP1B inhibition (higher than metformin) after the assay was done with selected four plant extracts in combination. The effect of leaf extracts of S. marianum was studied for glucose uptake efficiency in yeast cell lines which was found to be increased by 23% as compared to the control (without extract). Metformin improves glucose upake by yeast cells by ~15–31%. GC-MS analysis revealed 23 phytochemicals, some of which possessed anti-diabetic properties. A dose-dependent increase in antioxidant activity of S. marianum leaves extracts was observed (40–53%). The findings of the study highlighted the presence of various phytochemicals in leaves extracts that are effective against PTP1B inhibition and may help in reinvigorating drug development.

Strategic optimization of conditions for the solubilization of GST-tagged amphipathic helix-containing ciliary proteins overexpressed as inclusion bodies in E. coli

Expression of affinity-tagged recombinant proteins for crystallography, protein–protein interaction, antibody generation, therapeutic applications, etc. mandates the generation of high-yield soluble proteins. Although recent developments suggest the use of yeast, insect, and mammalian cell lines as protein expression platforms, Escherichia coli is still the most popular, due mainly to its ease of growth, feasibility in genetic manipulation and economy. However, some proteins have a spontaneous tendency to form inclusion bodies (IBs) when over-expressed in bacterial expression systems such as E. coli, thus posing a challenge in purification and yield. At times, small peptides undergo degradation during protein production and hence using suitable tags could circumvent the problem. Although several independent techniques have been used to solubilize IBs, these cannot always be applied in a generic sense. Although tagging a GST moiety is known to enhance the solubility of fusion proteins in E. coli, resulting in yields of 10–50 mg/L of the culture, the inherent nature of the protein sequence at times could lead to the formation of IBs. We have been working on a Myc Binding Protein-1 orthologue, viz. Flagellar Associated Protein 174 (FAP174) from the axoneme of Chlamydomonas reinhardtii that binds to an A-Kinase Anchoring Protein 240 (AKAP240) which has been annotated as Flagellar Associated Protein 65 (FAP65). Using an in-silico approach, we have identified two amphipathic helices on FAP65 (CrFAP65AH1 and CrFAP65AH2) that are predicted to bind to FAP174. To test this prediction, we have cloned the GST-tagged peptides, and overexpressed them in E. coli that have resulted in insoluble IBs. The yields of these over-expressed recombinant proteins dropped considerably due to IB formation, indicating aggregation. An integrated approach has been used to solubilize four highly hydrophobic polypeptides, viz. two amphipathic helices and the respective proline variants of FAP65. For solubilizing these polypeptides, variables such as non-denaturing detergents (IGEPAL CA-630), changing the ionic strength of the cell lysis and solubilization buffer, addition of BugBuster®, diluting the cell lysate and sonication were introduced. Our statistically viable results yielded highly soluble and functional polypeptides, indiscreet secondary structures, and a yield of ~ 20 mg/L of the E. coli culture. Our combinatorial strategy using chemical and physical methods to solubilize IBs could prove useful for hydrophobic peptides and proteins with amphipathic helices.

An Outline of Contributing Vaccine Technologies for SARS CoV2 Advancing in Clinical and Preclinical Phase-Trials.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV2) is an RNA virus involving 4 structural and 16 non-structural proteins, and exhibiting high transmission potential and fatality. The emergence of this newly encountered beta coronavirus-SARS CoV2 has brought over 2 million people to death, and more than 10 billion people got infected across the globe as yet. Consequently, the global scientific community has contributed to the synthesis and design of effective immunization technologies to combat this virus. This literature review was intended to gather an update on published reports of the vaccines advancing in the clinical trial phases or preclinical trials, to summarize the foundations and implications of contributing vaccine candidates inferring their impact in the pandemic repression. In addition, this literature review distinctly facilitates an outline of the overall vaccine effectiveness at current doses. The reported data in this review was extracted from research articles, review articles and patents published from January 2020 to July 2021, available on Google Scholar, Pubmed, Pubmed Central, Research Gate, Science direct, and Free Patent Online Database by using combination of keywords. Moreover, some information is retrieved from native web pages of vaccine manufacturing companies' due to progressing research and unavailability of published research papers. Contributing vaccine technologies include: RNA (Ribonucleic acid) vaccines, DNA (Deoxyribonucleic acid) vaccines, viral vector vaccines, protein-based vaccines, inactivated vaccines, viruses-like particles, protein superglue, and live-attenuated vaccines. Some vaccines are prepared by establishing bacterial and yeast cell lines and as self-assembling adenovirus- derived multimeric protein-based self-assembling nanoparticle (ADDOmer). On May 19, WHO has issued an emergency use sanction of Moderna, Pfizer, Sinopharm, AstraZeneca, and Covishield vaccine candidates on account of clinical credibility from experimental data.

MAbs N-glycosylation: Implications for biotechnology and analytics

Monoclonal antibodies (mAbs) are amazingly successful in treating diseases such as various types of cancers, immune disorders, and infections. Currently, mAbs are considered the most versatile therapeutic available. Seminal scientific progress over the last decade on elucidating the functional role of antibody N-glycosylation revealed the significance of this post-translational modification on the stability, half-life, and biological activity of antibodies. Therefore, antibody N-glycosylation became a key aspect of biopharmaceutical mAb production in large-scale manufacturing processes. This Mini-Review summarizes the recent scientific attempts of producing mAbs in glycoengineered animal-, plant-, and yeast cell lines. Furthermore, we also describe novel approaches for analyzing mAb N-glycosylation.

Kinetic monitoring of neuronal stress response to proteostasis dysfunction

Proteostasis dysfunction and activation of the unfolded protein response (UPR) are characteristic of all major neurodegenerative diseases. Nevertheless, although the UPR and proteostasis dysfunction has been studied in great detail in model organisms like yeast and mammalian cell lines, it has not yet been examined in neurons. In this study, we applied a viral vector-mediated expression of a reporter protein based on a UPR transcription factor, ATF4, and time-lapse fluorescent microscopy to elucidate how mouse primary neurons respond to pharmacological and genetic perturbations to neuronal proteostasis. In in vitro models of endoplasmic reticulum (ER) stress and proteasome inhibition, we used the ATF4 reporter to reveal the time course of the neuronal stress response relative to neurite degeneration and asynchronous cell death. We showed how potential neurodegenerative disease co-factors, ER stress and mutant α-synuclein overexpression, impacted neuronal stress response and overall cellular health. This work therefore introduces a viral vector-based reporter that yields a quantifiable readout suitable for non-cell destructive kinetic monitoring of proteostasis dysfunction in neurons by harnessing ATF4 signaling as part of the UPR activation.

Nuclear tRNA export in trypanosomes: a journey full of twists and turns guided by tRNA modifications.

Transfer RNAs play a key role in protein synthesis. Following transcription, tRNAs are extensively processed prior to their departure from the nucleus to become fully functional during translation. This includes removal of 5′ leaders and 3′ trailers by a specific endo- and/or exonuclease, 3′ CCA tail addition, posttranscriptional modifications and in some cases intron removal. In this minireview, the critical factors of nuclear tRNA trafficking are described based on studies in classical models such as yeast and human cell lines. In addition, recent findings and identification of novel regulatory loops of nuclear tRNA trafficking in trypanosomes are discussed with emphasis on tRNA modifications. The comparison between the representatives of opisthokonts and excavates serves here to understand the evolutionary conservation and diversity of nuclear tRNA export mechanisms.

Development of a new genetic reference material system based on Saccharomyces cerevisiae cells

As an important quality control link of molecular diagnosis, genetic reference materials (RMs) are widely used in various gene detection platforms such as mutation detection, gene quantification, and second generation sequencing. However, contamination, construction, and storage of existing genetic RMs still remain challenges. Here, we established a new genetic RM system based on Saccharomyces cerevisiae. We chose the non-small cell lung cancer (NSCLC) mutation hotspots in Kirsten rat sarcoma viral oncogene (KRAS) and epidermal growth factor receptor (EGFR), using clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR-Cas9) system-mediated gene editing technology, combined with the high homologous recombination efficiency of Saccharomyces cerevisiae. A single copy of the target gene was inserted into the yeast genome, and the inserted target gene was stably inherited with the passage of yeast cells. The copy number calculation for the target gene can replays by cell counting. The RM system was evaluated by sequence, copy number, stability, and homogeneity. In summary, the recombinant yeast cell line has ease of construction and screening, stable genetic characteristics, accurate copy number calculation, and convenient culture and preservation. Our findings may provide new ideas and directions for the research and industrialization of genetic RMs.

Identification and functional characterization of ABCC transporters for Cd tolerance and accumulation in Sedum alfredii Hance

Cd is one of the potential toxic elements (PTEs) exerting great threats on the environment and living organisms and arising extensive attentions worldwide. Sedum alfredii Hance, a Cd hyperaccumulator, is of great importance in studying the mechanisms of Cd hyperaccumulation and has potentials for phytoremediation. ATP-binding cassette sub-family C (ABCC) belongs to the ABC transporter family, which is deemed to closely associate with multiple physiological processes including cellular homeostasis, metal detoxification, and transport of metabolites. In the present work, ten ABCC proteins were identified in S. alfredii Hance, exhibiting uniform domain structure and divergently clustering with those from Arabidopsis. Tissue-specific expression analysis indicated that some SaABCC genes had significantly higher expression in roots (Sa23221 and Sa88F144), stems (Sa13F200 and Sa14F98) and leaves (Sa13F200). Co-expression network analysis using these five SaABCC genes as hub genes produced two clades harboring different edge genes. Transcriptional expression profiles responsive to Cd illustrated a dramatic elevation of Sa14F190 and Sa18F186 genes. Heterologous expression in a Cd-sensitive yeast cell line, we confirmed the functions of Sa14F190 gene encoding ABCC in Cd accumulation. Our study performed a comprehensive analysis of ABCCs in S. alfredii Hance, firstly mapped their tissue-specific expression patterns responsive to Cd stress, and characterized the roles of Sa14F190 genes in Cd accumulation.

RPA1 Gain of Function Causes Human Short Telomere Syndrome with Revertant Somatic Mosaicism

Dyskeratosis congenita (DC) is a short telomere syndrome with bone marrow failure (BMF), mucocutaneous fragility and predisposition to malignancy due to inherited mutations in telomere associated genes. The genetic cause remains unknown in 10-20% of DC patients. Here, we describe a new DC candidate gene, RPA1, which encodes the largest subunit of the Replication Protein A (RPA) complex, a single stranded DNA (ssDNA) binding protein essential for DNA replication, damage repair and telomere maintenance. The patient presented at the age of 5 years with pancytopenia and hypocellular marrow with morphology resembling refractory cytopenia of childhood, mucocutaneous triad and a congenital retinal anomaly. Further workup revealed telomeres in blood below 1st percentile, normal female karyotype and a negative chromosomal breakage test. After excluding known genetic causes for DC, family whole exome sequencing identified a de novoRPA1 c.718G>A (p.E240K) mutation with a predicted pathogenic CADD score of 23 and absent in >140,000 gnomAD population controls. The mutation had 50% allelic frequency in fibroblasts in comparison to a reduced mutation burden of 27% in bone marrow (BM) cells. Using bulk and single cell sequencing of BM, we identified 2 somatic compensatory events that likely explain patient's stable hematologic phenotype over a period of 20 years: i) an acquired RPA1 p.E579X mutation (in cis with p.E240K) was found at 10% frequency, which resulted in RNA degradation of the mutated allele as confirmed by RNA sequencing; ii) a founder clone with uniparental isodisomy on chromosome 17p encompassing RPA1 locus causing loss of germline E240K mutation. Analysis of serial BM samples using SNP arrays and deep sequencing demonstrated UPD17p clonal expansion concurrent with a decline of germline p.E240K and increase of acquired p.E579X mutation. This is consistent with a toxic, likely gain-of-function (GOF) effect of germline p.E240K mutation leading to acquired loss-of-function (LOF) escape mechanisms. RPA1 is essential to all DNA metabolism transactions that encounter ssDNA, including binding to telomeric 3' overhangs during late S-phase to unfold G-quadruplexes and facilitate telomerase activity. Several RPA1 genetic variants impart telomere shortening and an S-phase defect in yeast and human cell lines. However, the molecular mechanisms are not well understood and thus far, RPA1 gene has not been linked to human disease. In an iPSC-based disease model with homozygous RPA1 p.E240K knock-in, we discovered severe telomere shortening in RPA1 mutant iPSCs and iPSC derived hematopoietic progenitors (HPs) compared to WT counterparts. Furthermore, decreased erythroid differentiation capacity was noted in RPA1-mutant HPs. Because the germline p.E240K mutation is located in DNA binding domain A of RPA1, we performed electromobility shift assay to assess the DNA binding affinity of mutant RPA1 protein. The p.E240K heterotrimeric RPA complex shows increased ssDNA binding compared to wild type RPA. In summary, we describe the first human disease manifesting as short telomere syndrome secondary to a de novoRPA1 mutation with likely GOF effect, which forces the development of multiple revertant mosaic events, similar to the newly described SAMD9/SAMD9L disorders. Our studies also have implications for the mechanistic understanding of the role of RPA1 in telomere maintenance and hematopoiesis. Disclosures Niemeyer: Celgene: Consultancy; Novartis: Consultancy.

Treatment of murine colitis by Saccharomyces boulardii secreting atrial natriuretic peptide.

Inflammatory bowel disease is a lifelong disorder that involves chronic inflammation in the small and large intestines. Current therapies, including aminosalicylates, corticosteroids, and anti-inflammatory biologics, can only alleviate the symptoms and often cause adverse effects with long-term usage. Engineered probiotics provide an alternative approach to treat inflammatory bowel disease in a self-renewable and local delivery fashion. In this work, we utilized a yeast probiotic Saccharomyces boulardii for this purpose. We developed a robust method to integrate recombinant genes into the Ty elements of S. boulardii. Stable yeast cell lines that secreted various anti-inflammatory proteins, including IL-10, TNFR1-ECD, alkaline phosphatase, and atrial natriuretic peptide (ANP), were successfully created and investigated for their efficacies to the DSS-induced colitis in mice through oral administration. While IL-10, TNFR1-ECD, and alkaline phosphatase did not show therapeutic effects, the ANP-secreting S. boulardii effectively ameliorated the mouse conditions as reflected by the improvements in body weight, disease activity index, and survival rate. A post-mortem examination revealed that the ANP-treated mice exhibited significant downregulations of TNF-α and IL-1β and an upregulation of IL-6 in colon tissues. This observation is consistent with the previous reports showing that TNF-α and IL-1β are responsible for initiating the pathogenesis, whereas IL-6 plays a protective role in colitis. Overall, we demonstrated that S. boulardii is a safe and robust vehicle for recombinant protein delivery in the gastrointestinal tract, and ANP is a potential anti-inflammatory drug for colitis treatment. KEY MESSAGES: Recombinant genes can be robustly integrated into the transposable elements of S. boulardii. Oral administration of S. boulardii secreting IL-10 or TNF-α inhibitor did not exert therapeutic effects for DSS-induced colitis in mice. Atrial natriuretic peptide-secreting S. boulardii effectively ameliorated the murine colitis as reflected by improved body weight, disease activity index, and survival rate. The ANP-treated mice exhibited decreased mRNA levels of TNF-α and IL-1β and an increased mRNA level of IL-6 in colon tissues.

Variants in Homologous Recombination Genes EXO1 and RAD51 Related with Premature Ovarian Insufficiency.

Premature ovarian insufficiency (POI) is characterized by cessation of menstruation before 40 years of age and elevated serum level of FSH (>25 IU/L). Recent studies have found a few causative genes responsible for POI enriched in meiotic recombination and DNA damage repair pathways. To investigate the role of variations in homologous recombination genes played in POI pathogenesis. The whole exome sequencing was performed in 50 POI patients with primary amenorrhea. Functional characterizations of the novel variants were carried out in budding yeast and human cell line. We identified 8 missense variants in 7 homologous recombination genes, including EXO1, RAD51, RMI1, MSH5, MSH2, MSH6, and MLH1. The mutation p.Thr52Ser in EXO1 impaired the meiotic process of budding yeast and p.Glu68Gly in RAD51-altered protein localization in human cells, both of them impaired the efficiency of homologous recombination repair for DNA double-stranded breaks in human cells. Our study first linked the variants of EXO1 and RAD51 with POI and further highlighted the role of DNA repair genes in ovarian dysgenesis.

CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos.

Early embryonic development is driven exclusively by maternal gene products deposited into the oocyte. Although critical in establishing early developmental programs, maternal gene functions have remained elusive due to a paucity of techniques for their systematic disruption and assessment. CRISPR-Cas13 systems have recently been employed to degrade RNA in yeast, plants, and mammalian cell lines. However, no systematic study of the potential of Cas13 has been carried out in an animal system. Here, we show that CRISPR-RfxCas13d (CasRx) is an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos. We demonstrate that zygotically expressed and maternally provided transcripts are efficiently targeted, resulting in a 76% average decrease in transcript levels and recapitulation of well-known embryonic phenotypes. Moreover, we show that this system can be used in medaka, killifish, and mouse embryos. Altogether, our results demonstrate that CRISPR-RfxCas13d is an efficient knockdown platform to interrogate gene function in animal embryos.

Inhibition of histone acetyltransferase GCN5 extends lifespan in both yeast and human cell lines.

Histone acetyltransferases (HATs) are important enzymes that transfer acetyl groups onto histones and thereby regulate both gene expression and chromosomal structures. Previous work has shown that the activation of sirtuins, which are histone deacetylases, can extend lifespan. This suggests that inhibiting HATs may have a similar beneficial effect. In the present study, we utilized a range of HAT inhibitors or heterozygous Gcn5 and Ngg1 mutants to demonstrate marked yeast life extension. In human cell lines, HAT inhibitors and selective RNAi‐mediated Gcn5 or Ngg1 knockdown reduced the levels of aging markers and promoted proliferation in senescent cells. Furthermore, this observed lifespan extension was associated with the acetylation of histone H3 rather than that of H4. Specifically, it was dependent upon H3K9Ac and H3K18Ac modifications. We also found that the ability of caloric restriction to prolong lifespan is Gcn5‐, Ngg1‐, H3K9‐, and H3K18‐dependent. Transcriptome analysis revealed that these changes were similar to those associated with heat shock and were inversely correlated with the gene expression profiles of aged yeast and aged worms. Through a bioinformatic analysis, we also found that HAT inhibition activated subtelomeric genes in human cell lines. Together, our results suggest that inhibiting the HAT Gcn5 may be an effective means of increasing longevity.

PaperClip DNA Assembly: Reduce, Reuse, Recycle.

Creating DNA constructs is a basic and fundamental step in molecular and synthetic biology. While prices for gene synthesis are decreasing, it is still more economical in most cases to assemble constructs from a library of components (Parts). Many methods for DNA assembly are available, but most require either a fixed and inflexible format for the construct, with all Parts first being cloned in specific donor plasmids, or remaking Parts with new homology ends for each specific assembly reaction, requiring large numbers of single-use oligonucleotides. PaperClip assembly allows Parts stored in any format (linear PCR products or synthetic DNA, or cloned in any plasmid) to be used in totally flexible assembly reactions; up to 11 parts can be assembled in a single reaction, in any order, to give a linear or circular construct, and the oligonucleotides required in the assembly process can be reused in any subsequent assembly. In addition to constructing plasmids for bacterial transformation, PaperClip is also well suited to generate linear products for direct transfection of yeast, mammalian, or cyanobacterial cell lines. Thus, PaperClip offers a simple, flexible, and economical route to multipart assembly of constructs for a wide variety of purposes.