We previously cloned a 1,3-specific lipase gene from the fungus Rhizomucor miehei and expressed it in methylotrophic yeast Pichia pastoris strain GS115. The enzyme produced (termed RML) was able to catalyze methanolysis of soybean oil and showed strong position specificity. However, the enzyme activity and amount of enzyme produced were not adequate for industrial application. Our goal in the present study was to improve the enzyme properties of RML in order to apply it for the conversion of microalgae oil to biofuel.

Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme that performs multiple physiological activities. Previous studies suggest that PON1 plays an anti-inflammatory role in the cardiovascular system, although its roles in hematopoiesis and adaptive immunity have not been clarified. To investigate the impact of PON1 on the immune system, we generated PON1-knockout (PON1-/-) rats using the CRISPR/Cas9 system. The thymus was smaller in PON1-/- rats than that in wild-type (PON1+/+) rats. Furthermore, analysis of thymocyte development revealed diminished total T cell numbers and a decrease in CD4+, CD8+ and double-positive T cells in peripheral blood and thymus from PON1-/- rats. This may be due to a block in the transition of T cells from the double-negative to the double-positive stage. We also showed that the activation of p38 MAPK phosphorylation contributed to the increased apoptosis and defective T cell development in PON-/- rats. Therefore, our results indicate that PON1 functions as a novel regulator of T cell development.

Background Canonical studies indicate that cytochrome P450 2E1 ( CYP 2E1) plays a critical role in the metabolism of xenobiotics and ultimately participates in tissue damage. CYP 2E1 upregulates in the pathophysiological development of multiple diseases; however, the mechanism of CYP 2E1 upregulation, particularly in heart disease, remains elusive. Methods and Results We found that the level of CYP 2E1 increased in heart tissues from patients with hypertrophic cardiomyopathy; multiple mouse models of heart diseases, including dilated cardiomyopathy, hypertrophic cardiomyopathy, and myocardial ischemia; and HL -1 myocytes under stress. We determined that Myc bound to the CYP 2E1 promoter and activated its transcription by bioinformatics analysis, luciferase activity, and chromatin immunoprecipitation, and Myc expression was modulated by extracellular signal-regulated kinases 1/2 and phosphatidylinositol 3 kinase/protein kinase B pathways under stress or injury in myocardium by signal transduction analysis. In addition, the level of oxidative stress and apoptosis gradually worsened with age in transgenic mice overexpressing CYP 2E1, which was significantly inhibited with CYP 2E1 knockdown. Conclusions Our results demonstrated that CYP 2E1 is likely a sensor of diverse pathophysiological factors and states in the myocardium. Upregulated CYP 2E1 has multiple pathophysiological roles in the heart, including increased oxidative stress and apoptosis as well as energy supply to meet the energy demand of the heart in certain disease states. Our discovery thus provides a basis for a therapeutic strategy for heart diseases targeting Myc and CYP 2E1.

Advances in DNA sequencing technologies have transformed our capacity to perform life science research, decipher the dynamics of complex soil microbial communities and exploit them for plant disease management. However, soil is a complex conglomerate, which makes functional metagenomics studies very challenging.

Fsp27 is a lipid droplet-associated protein almost exclusively expressed in adipocytes where it facilitates unilocular lipid droplet formation. In mice, Fsp27 deficiency is associated with increased basal lipolysis, 'browning' of white fat and a healthy metabolic profile, whereas a patient with congenital CIDEC deficiency manifested an adverse lipodystrophic phenotype. Here we reconcile these data by showing that exposing Fsp27-null mice to a substantial energetic stress by crossing them with ob/ob mice or BATless mice, or feeding them a high-fat diet, results in hepatic steatosis and insulin resistance. We also observe a striking reduction in adipose inflammation and increase in adiponectin levels in all three models. This appears to reflect reduced activation of the inflammasome and less adipocyte death. These findings highlight the importance of Fsp27 in facilitating optimal energy storage in adipocytes and represent a rare example where adipose inflammation and hepatic insulin resistance are disassociated.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive neurodegeneration and cognitive decline. Evodiamine, a main component in Chinese medicine, was found to improve cognitive impairment in AD model mice based on several intensive studies. However, evodiamine has high cytotoxicity and poor bioactivity. In this study, several evodiamine derivatives were synthesized via heterocyclic substitution and amide introduction and screened for cytotoxicity and antioxidant capacity. Under the same concentrations, compound 4c was found to exhibit lower cytotoxicity and higher activity against H2O2 and amyloid β oligomers (AβOs) than evodiamine in vitro and significantly improve the working memory and spatial memory of 3 x Tg and APP/PS1 AD mice. Subsequent RNA sequencing and pathway enrichment analysis showed that 4c affected AD-related genes and the AMPK and insulin signaling pathways. Furthermore, we confirmed that 4c recovered PI3K/AKT/GSK3β/Tau dysfunction in vivo and in vitro. In conclusion, 4c represents a potential lead compound for AD therapy based on the recovery of PI3K/AKT/GSK3β pathway dysfunction.

Multiple mitochondrial dysfunction syndromes (MMDS) presents as complex mitochondrial damage, thus impairing a variety of metabolic pathways. Heart dysplasia has been reported in MMDS patients; however, the specific clinical symptoms and pathogenesis remain unclear. More urgently, there is a lack of an animal model to aid research. Therefore, we selected a reported MMDS causal gene, Isca1, and established an animal model of MMDS complicated with cardiac dysplasia.

The GGGGCC (G4C2) repeat expansion in the human open reading frame 72 on chromosome 9, C9orf72, is the most common cause of amyotrophic lateral sclerosis (ALS). Studies in transgenic mouse models have linked the pathogenic mechanism of G4C2 repeat expansion to RNA foci or the accumulation of unnatural dipeptide repeats in neurons. However, only one of the existing transgenic mouse lines developed typical ALS.

Plastin 3 (PLS3), a protein involved in formation of filamentous actin (F-actin) bundles, is important in human bone health. Recent studies identify PLS3 as a novel bone regulator and PLS3 mutations can lead to a rare monogenic early-onset osteoporosis. However, the mechanism of PLS3 mutation leading to osteoporosis is unknown, and its effective treatment strategies have not been established. Here, we have constructed a novel rat model with clinically relevant hemizygous E10-16del mutation in PLS3 (PLS3E10-16del/0) that recapitulates the osteoporotic phenotypes with obviously thinner cortical thickness, significant decreases in yield load, maximum load, and breaking load of femora at 3, 6, 9 months old compared to wild-type rats. Histomorphometric analysis indicates a significantly lower mineral apposition rate in PLS3E10-16del/0 rats. Treatment with alendronate (1.0 µg/kg/day) or teriparatide (40 µg/kg five times weekly) for 8 weeks significantly improves bone mass and bone microarchitecture, and bone strength is significantly increased after teriparatide treatment (p＜0.05). Thus, our results indicate that PLS3 plays an important role in the regulation of bone microstructure and bone strength, and we provide a novel animal model for the study of X-linked early-onset osteoporosis. Alendronate and teriparatide treatment could be a potential treatment for early-onset osteoporosis induced by PLS3 mutation.

Leptin receptor, which is encoded by the diabetes (db) gene and is highly expressed in the choroid plexus, regulatesenergy homeostasis, the balance between food intake and energy expenditure, fertility and bone mass. Here, using CRISPR/Cas9 technology, we created the leptin receptor knockout rat. Homozygous leptin receptor null rats are characterized by obesity, hyperphagia, hyperglycemia, glucose intolerance, hyperinsulinemia and dyslipidemia. Due to long-term poor glycemic control, the leptin receptor knockout rats also develop some diabetic complications such as pancreatic, hepatic and renal lesions. In addition, the leptin receptor knockout rats show a significant decrease in bone volume and bone mineral density of the femur compared with their wild-type littermates. Our model has rescued some deficiency of the existing rodent models, such as the transient hyperglycemia of db/db mice in the C57BL/6J genetic background and the delayed onset of glucose intolerance in the Zucker rats, and it is proven to be a useful animal model for biomedical and pharmacological research on obesity and diabetes.

Microbial cell surface display technology is a powerful tool for displaying proteins on the surfaces of cells. However, few anchoring proteins can be employed for the display of target proteins on the cell surface of the environmentally benign Gram-positive bacterium Bacillus subtilis. In this study, bioinformatics tools were used to screen all of the encoded proteins of B. subtilis for potential anchoring proteins. A green fluorescent protein (eGFP) reporter system was constructed to evaluate the cell-display efficiency of the selected membrane proteins and their promoters. The anchoring protein SpoIIIJ demonstrated the strongest anchoring activity of all of the selected anchoring proteins from Bacillus spp. cells. A linker was designed to link the anchoring protein SpoIIIJ and eGFP, which had the ability to increase the expression of the fusion protein by 58.32%. Two bio-remediated related proteins (the organophosphorus hydrolase OPHC2 and the metal binding protein CadR) were successfully expressed on the cell surfaces of Bacillus spp. using this system. Therefore, our results suggest that this microbial surface display system may be useful for the expression of target proteins on the cell surfaces and has potential applications in the bioremediation of environmental pollution.

TSPAN7 is related to various neurological disorders including autism spectrum disorder (ASD). However, the underlying synaptic mechanism of TSPAN7 in ASD is still unclear. Here, we showed that Tspan7 knockout rats exhibited ASD-like and ID-like behavioral phenotypes, brain structure alterations including decreased hippocampal and cortical volume, and related pathological changes including reduced hippocampal neurons number, neuronal complexity, dendritic spines, and synapse-associated proteins. Then, we found that TSPAN7 deletion interrupted the integrin β1/FAK/SRC signal pathway that was followed by the down-regulation of PSD95, SYN, and GluR1/2, which are key synaptic integrity-related proteins. Furthermore, reactivation of SRC restored the expression of synaptic integrity-related proteins in primary neurons of TSPAN7 knockout brains. Taken together, our results suggested that TSPAN7 knockout caused ASD-like and ID-like behaviors in rats and impaired neuronal synapses possibly through the down-regulation of the integrin β1/FAK/SRC signal pathway, which might be a new mechanism on regulation of synaptic proteins expression and on ASD pathogenesis by mutated TSPAN7. These findings provide novel insights into the role of TSPAN7 in psychiatric diseases and highlight integrin β1/FAK/SRC as a potential target for ASD therapy.

The expression of proteins in Escherichia coli is often essential for their characterization, modification, and subsequent application. Gene sequence is the major factor contributing expression. In this study, we used the expression data from 6438 heterologous proteins under the same expression condition in E. coli to construct a deep learning classifier for screening high- and low-expression proteins. In conjunction with conserved residue analysis to minimize functional disruption, a mutation predictor for enhanced protein expression (MPEPE) was proposed to identify mutations conducive to protein expression. MPEPE identified mutation sites in laccase 13B22 and the glucose dehydrogenase FAD-AtGDH, that significantly increased both expression levels and activity of these proteins. Additionally, a significant correlation of 0.46 between the predicted high level expression propensity with the constructed models and the protein abundance of endogenous genes in E. coli was also been detected. Therefore, the study provides foundational insights into the relationship between specific amino acid usage, codon usage, and protein expression, and is essential for research and industrial applications.

Plastic polymers are widely used in agriculture, industry, and our daily life because of their convenient and economic properties. However, pollution caused by plastic polymers, especially polyethylene (PE), affects both animal and human health when they aggregate in the environment, as they are not easily degraded under natural conditions. In this study, Enterobacter sp. D1 was isolated from the guts of wax moth (Galleria mellonella). Microbial colonies formed around a PE film after 14 days of cultivation with D1. Roughness, depressions, and cracks were detected on the surface of the PE film by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Fourier transform infrared spectroscopy (FTIR) showed the presence of carbonyl functional groups and ether groups on the PE film that was treated with D1. Liquid chromatography-tandem mass spectrometry (LC-MS) also revealed that the contents of certain alcohols, esters, and acids were increased as a result of the D1 treatment, indicating that oxidation reaction occurred on the surface of the PE film treated with D1 bacteria. These observations confirmed that D1 bacteria has an ability to degrade PE.

Poly(ethylene terephthalate) (PET) is used widely by human beings, but is very difficult to degrade. Up to now, the PET degradation effect of PETase from Ideonella sakaiensis 201-F6 (IsPETase) variants with low stability and activity was not ideal. In this study, a mutation design tool, Premuse, was developed to integrate the sequence alignment and quantitative selection of the preferred mutations based on natural sequence evolution. Ten single point mutants were selected from 1486 homologous sequences using Premuse, and then two mutations (W159H and F229Y) with improved stability were screened from them. The derived double point mutant, W159H/F229Y, exhibited a strikingly enhanced enzymatic performance. Its Tm and catalytic efficiency values (kcat/Km) respectively increased by 10.4 °C and 2.0-fold using p-NPP as the substrate compared with wild type. The degradation activity for amorphous PET was increased by almost 40-fold in comparison with wild type at 40 °C in 24 h. Additionally, the variant could catalyze biodegradation of PET bottle preform at a mean rate of 23.4 mgPET/h/mgenzyme. This study allowed us to design the mutation more efficiently, and provides a tool for achieving biodegradation of PET pollution under mild natural environments.

Multiple mitochondrial dysfunction (MMD) can lead to complex damage of mitochondrial structure and function, which then lead to the serious damage of various metabolic pathways including cerebral abnormalities. However, the effects of MMD on heart, a highly mitochondria-dependent tissue, are still unclear. In this study, we use iron-sulfur cluster assembly 1 (Isca1), which has been shown to cause MMD syndromes type 5 (MMDS5), to verify the above scientific question.

Hundreds of pathogenic variants of mitochondrial DNA (mtDNA) have been reported to cause mitochondrial diseases, which still lack effective treatments. It is a huge challenge to install these mutations one by one. We repurposed the DddA-derived cytosine base editor to incorporate a premature stop codon in the mtProtein-coding genes to ablate mitochondrial proteins encoded in the mtDNA (mtProteins) instead of installing pathogenic variants and generated a library of both cell and rat resources with mtProtein depletion. In vitro, we depleted 12 of 13 mtProtein-coding genes with high efficiency and specificity, resulting in decreased mtProtein levels and impaired oxidative phosphorylation. Moreover, we generated six conditional knockout rat strains to ablate mtProteins using Cre/loxP system. Mitochondrially encoded ATP synthase membrane subunit 8 and NADH:ubiquinone oxidoreductase core subunit 1 were specifically depleted in heart cells or neurons, resulting in heart failure or abnormal brain development. Our work provides cell and rat resources for studying the function of mtProtein-coding genes and therapeutic strategies.