Oxidative stress-resistance assay for screening yeast strains overproducing heterologous proteins

Many natural proteins have been developed into drugs and produced for direct application. Identifying improved hosts to achieve high-level heterologous protein production is a challenge in the study of heterologous protein expression in recombinant yeast. In this study, a novel high-throughput assay to screen such overproducing Saccharomyces cerevisiae strains was systematically developed. The protocol designed was based on screening host strain derivatives with increased superoxide dismutase dependent resistance to oxidative stress. Yeast cells transformed with recombinant plasmid carrying SOD1 gene as a reporter responded exquisitely to oxidative stress induced by elevated concentrations of paraquat. Improved yeast strains resulting from screening clones subjected to genome shuffling through selective pressure argue for a more effective screening system compared with traditonal selection. Moreover, this approach can be employed in general biochemical analysis without utilization of flow cytometry or well plate reader. Therefore, it is expected that the high-throughput assay would make superior strains producing heterologous proteins.     

Regulatory KIR+RA+ T cells accumulate with age and are highly activated during viral respiratory disease

Severe respiratory viral infectious diseases such as influenza and COVID‐19 especially affect the older population. This is partly ascribed to diminished CD8⁺ T‐cell responses a result of aging. The phenotypical diversity of the CD8⁺ T‐cell population has made it difficult to identify the impact of aging on CD8⁺ T‐cell subsets associated with diminished CD8⁺ T‐cell responses. Here we identify a novel human CD8⁺ T‐cell subset characterized by expression of Killer‐cell Immunoglobulin‐like Receptors (KIR⁺) and CD45RA (RA⁺). These KIR⁺RA⁺ T cells accumulated with age in the blood of healthy individuals (20–82 years of age, n = 50), expressed high levels of aging‐related markers of T‐cell regulation, and were functionally capable of suppressing proliferation of other CD8⁺ T cells. Moreover, KIR⁺RA⁺ T cells were a major T‐cell subset becoming activated in older adults suffering from an acute respiratory viral infection (n = 36), including coronavirus and influenza virus infection. In addition, older adults with influenza A infection showed that higher activation status of their KIR⁺RA⁺ T cells associated with longer duration of respiratory symptoms. Together, our data indicate that KIR⁺RA⁺ T cells are a unique human T‐cell subset with regulatory properties that may explain susceptibility to viral respiratory disease at old age.     

The architecture of gene regulatory variation across multiple human tissues: the MuTHER study

While there have been studies exploring regulatory variation in one or more tissues, the complexity of tissue-specificity in multiple primary tissues is not yet well understood. We explore in depth the role of cis-regulatory variation in three human tissues: lymphoblastoid cell lines (LCL), skin, and fat. The samples (156 LCL, 160 skin, 166 fat) were derived simultaneously from a subset of well-phenotyped healthy female twins of the MuTHER resource. We discover an abundance of cis-eQTLs in each tissue similar to previous estimates (858 or 4.7% of genes). In addition, we apply factor analysis (FA) to remove effects of latent variables, thus more than doubling the number of our discoveries (1,822 eQTL genes). The unique study design (Matched Co-Twin Analysis--MCTA) permits immediate replication of eQTLs using co-twins (93%-98%) and validation of the considerable gain in eQTL discovery after FA correction. We highlight the challenges of comparing eQTLs between tissues. After verifying previous significance threshold-based estimates of tissue-specificity, we show their limitations given their dependency on statistical power. We propose that continuous estimates of the proportion of tissue-shared signals and direct comparison of the magnitude of effect on the fold change in expression are essential properties that jointly provide a biologically realistic view of tissue-specificity. Under this framework we demonstrate that 30% of eQTLs are shared among the three tissues studied, while another 29% appear exclusively tissue-specific. However, even among the shared eQTLs, a substantial proportion (10%-20%) have significant differences in the magnitude of fold change between genotypic classes across tissues. Our results underline the need to account for the complexity of eQTL tissue-specificity in an effort to assess consequences of such variants for complex traits.     

Overexpression of AtchyB in Eustoma grandiflorum Shinn Enhances its Tolerance to High-Light Via Zeaxanthin Accumulation

Carotenoids are essential components of the photosynthetic apparatus involved in plant photoprotection. To investigate the protective role of zeaxanthin and the xanthophyll cycle under high-light stress, we increased the capacity for their biosynthesis in Eustoma grandiflorum Shinn by overexpression of a gene (AtchyB) from Arabidopsis thaliana encoding ??-carotene hydroxylase (BCH). This enzyme is involved in the conversion of ??-carotene into zeaxanthin and plays an important role in the carotenoid biosynthetic pathway. Not only was the total carotenoid content of the transgenics enhanced (1.046- to?3.141-fold) but zeaxanthin biosynthesis was also faster and the compound was produced in larger quantities in transgenics (up to 3.344-fold) than in controls upon exposure to high-light stress. Additionally, a greater amount of xanthophyll cycle pigments (1.46- to?2.44-fold) was detected in the transgenics. Under high-light stress, untransformed controls showed obvious growth retardation, while transformants were more tolerant. The net addition of biomass in the transformants was more than that of non-transformants under high-light exposure. Furthermore, a new phenomenon was found: high-light stress induced an apparent periodical accumulation of biomass and zeaxanthin in transformants. Our results supplement data from previous research, and indicate that the periodic enhancement of zeaxanthin formation together with the periodic enlargement of the xanthophyll cycle pool contributes to long-term high-light stress protection and prevents plant damage.     

Overexpression of GLT1 in fps1DeltagpdDelta mutant for optimum ethanol formation by Saccharomyces cerevisiae

Glycerol is the main byproduct produced under anaerobic ethanol fermentations by Saccharomyces cerevisiae and consumes a considerable amount of substrate. To verify the metabolic phenotype predications for increasing ethanol formation, two engineered S. cerevisiae KAM-14, KAM-15 strains were constructed for possible redirection of glycerol carbon flux into ethanol by overexpression of GLT1 in the fps1DeltagpdDelta mutant. The engineered strains KAM-14 and KAM-15 compared to the control strain KAM-2, produced 12.24% and 10.42% higher ethanol, 39.72% and 31.03% lower glycerol yield during anaerobic batch fermentations, respectively. The maximum specific growth rates of KAM-14 and KAM-15 were found to be relatively lower than that of KAM-2 during the exponential growth phase. In the meantime, the biomass concentrations of both KAM-14 and KAM-15 were similar to KAM-2. Acetate and pyruvate concentrations of KAM-14 and KAM-15 were greatly decreased comparing to those of KAM-2, respectively. These experimental results approved the metabolic pathway strategies to improve ethanol formation.     

Variability of gene expression profiles in human blood and lymphoblastoid cell lines

Background

Infection of plants by pathogens and the subsequent disease development involves substantial changes in the biochemistry and physiology of both partners. Analysis of genes that are expressed during these interactions represents a powerful strategy to obtain insights into the molecular events underlying these changes. We have employed expressed sequence tag (EST) analysis to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe oryzae and fungal genes involved in infectious growth within the host during a compatible interaction.

Results

A cDNA library was constructed with RNA from rice leaves (Oryza sativa cv. Hwacheong) infected with M. oryzae strain KJ201. To enrich for fungal genes, subtraction library using PCR-based suppression subtractive hybridization was constructed with RNA from infected rice leaves as a tester and that from uninfected rice leaves as the driver. A total of 4,148 clones from two libraries were sequenced to generate 2,302 non-redundant ESTs. Of these, 712 and 1,562 ESTs could be identified to encode fungal and rice genes, respectively. To predict gene function, Gene Ontology (GO) analysis was applied, with 31% and 32% of rice and fungal ESTs being assigned to GO terms, respectively. One hundred uniESTs were found to be specific to fungal infection EST. More than 80 full-length fungal cDNA sequences were used to validate ab initio annotated gene model of M. oryzae genome sequence.

Conclusion

This study shows the power of ESTs to refine genome annotation and functional characterization. Results of this work have advanced our understanding of the molecular mechanisms underpinning fungal-plant interactions and formed the basis for new hypothesis.     

Generation of medaka gene knockout models by target-selected mutagenesis

We have established a reverse genetics approach for the routine generation of medaka (Oryzias latipes) gene knockouts. A cryopreserved library of N-ethyl-N-nitrosourea (ENU) mutagenized fish was screened by high-throughput resequencing for induced point mutations. Nonsense and splice site mutations were retrieved for the Blm, Sirt1, Parkin and p53 genes and functional characterization of p53 mutants indicated a complete knockout of p53 function. The current cryopreserved resource is expected to contain knockouts for most medaka genes.     

MicroRNA expression in abdominal and gluteal adipose tissue is associated with mRNA expression levels and partly genetically driven

To understand how miRNAs contribute to the molecular phenotype of adipose tissues and related traits, we performed global miRNA expression profiling in subcutaneous abdominal and gluteal adipose tissue of 70 human subjects and characterised which miRNAs were differentially expressed between these tissues. We found that 12% of the miRNAs were significantly differentially expressed between abdominal and gluteal adipose tissue (FDR adjusted p<0.05) in the primary study, of which 59 replicated in a follow-up study of 40 additional subjects. Further, 14 miRNAs were found to be associated with metabolic syndrome case-control status in abdominal tissue and three of these replicated (primary study: FDR adjusted p<0.05, replication: p<0.05 and directionally consistent effect). Genome-wide genotyping was performed in the 70 subjects to enable miRNA expression quantitative trait loci (eQTL) analysis. Candidate miRNA eQTLs were followed-up in the additional 40 subjects and six significant, independent cis-located miRNA eQTLs (primary study: p<0.001; replication: p<0.05 and directionally consistent effect) were identified. Finally, global mRNA expression profiling was performed in both tissues to enable association analysis between miRNA and target mRNA expression levels. We find 22% miRNAs in abdominal and 9% miRNAs in gluteal adipose tissue with expression levels significantly associated with the expression of corresponding target mRNAs (FDR adjusted p<0.05). Taken together, our results indicate a clear difference in the miRNA molecular phenotypic profile of abdominal and gluteal adipose tissue, that the expressions of some miRNAs are influenced by cis-located genetic variants and that miRNAs are associated with expression levels of their predicted mRNA targets.     

Target-selected mutagenesis of the rat

The rat is one of the most extensively studied model organisms, and with its genome being sequenced, tools to manipulate gene function in vivo have become increasingly important. We here report proof of principle for target-selected mutagenesis as a reverse genetic or knockout approach for the rat.