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Abstract Background Genome-scale screening can be applied to efficiently mine for unknown genes with phenotypes of interest or special functions. It is also useful to identify new targets for engineering desirable properties of cell factories. Results Here, we designed a new approach for genome-scale transcription activation using non-homologous end joining (NHEJ)-mediated integration in Yarrowia lipolytica. We utilized this approach to screen for genes that, upon activation, confer phenotypes including improved acetic acid tolerance and xylose metabolism. The candidates were validated using gene overexpression, and functional changes including improved growth performance under multiple stressors and activated pentose metabolism were identified. Conclusions This study provides a simple and effective approach to randomly activate endogenous genes and mine for key targets associated with phenotypes of interest. The specific gene targets identified here will be useful for cell factory construction and biorefining lignocellulose.

Abstract Succinic acid (SA) is an important C4-dicarboxylic acid. Microbial production of SA at low pH results in low purification costs and hence good overall process economics. However, redox imbalances limited SA biosynthesis from glucose via the reductive tricarboxylic acid (TCA) cycle in yeast. Here, we engineer the strictly aerobic yeast Yarrowia lipolytica for efficient SA production without pH control. Introduction of the reductive TCA cycle into the cytosol of a succinate dehydrogenase-disrupted yeast strain causes arrested cell growth. Although adaptive laboratory evolution restores cell growth, limited NADH supply restricts SA production. Reconfiguration of the reductive SA biosynthesis pathway in the mitochondria through coupling the oxidative and reductive TCA cycle for NADH regeneration results in improved SA production. In pilot-scale fermentation, the engineered strain produces 111.9 g/L SA with a yield of 0.79 g/g glucose within 62 h. This study paves the way for industrial production of biobased SA.

Abstract Background β-Farnesene is a sesquiterpene with versatile industrial applications. The production of β-farnesene from waste lipid feedstock is an attractive method for sustainable production and recycling waste oil. Yarrowia lipolytica is an unconventional oleaginous yeast, which can use lipid feedstock and has great potential to synthesize acetyl-CoA-derived chemicals. Results In this study, we engineered Y. lipolytica to produce β-farnesene from lipid feedstock. To direct the flux of acetyl-CoA, which is generated from lipid β-oxidation, to β-farnesene synthesis, the mevalonate synthesis pathway was compartmentalized into peroxisomes. β-Farnesene production was then engineered by the protein engineering of β-farnesene synthase and pathway engineering. The regulation of lipid metabolism by enhancing β-oxidation and eliminating intracellular lipid synthesis was further performed to improve the β-farnesene synthesis. As a result, the final β-farnesene production with bio-engineering reached 35.2 g/L and 31.9 g/L using oleic acid and waste cooking oil, respectively, which are the highest β-farnesene titers reported in Y. lipolytica. Conclusions This study demonstrates that engineered Y. lipolytica could realize the sustainable production of value-added acetyl-CoA-derived chemicals from waste lipid feedstock.

Summary With the rapid development of synthetic biology, the oleaginous yeast Yarrowia lipolytica has become an attractive microorganism for chemical production. To better optimize and reroute metabolic pathways, we have expanded the CRISPR‐based gene expression toolkit of Y. lipolytica. By sorting the integration sites associated with high expression, new neutral integration sites associated with high expression and high integration efficiency were identified. Diverse genetic components, including promoters and terminators, were also characterized to expand the expression range. We found that in addition to promoters, the newly characterized terminators exhibited large variations in gene expression. These genetic components and integration sites were then used to regulate genes involved in the lycopene biosynthesis pathway, and different levels of lycopene production were achieved. The CRISPR‐based gene expression toolkit developed in this study will facilitate the genetic engineering of Y. lipolytica.

Abstract Vitamin D deficiency is a candidate risk factor for osteoporosis, characterized by decreased bone mineral density (BMD). We performed this two-sample Mendelian randomization (MR) analysis to investigate the causal effect of vitamin D on BMD. We extracted 143 single-nucleotide polymorphisms from a recent GWAS on 417,580 participants of European ancestry as instrumental variables, and used summary statistics for BMD at forearm (n = 10,805), femoral neck (n = 49,988), lumbar spine (n = 44,731) and total-body of different age-stages ( 60) (n = 67,358). We explored the direct effect of vitamin D on BMD with an adjusted body mass index (BMI) in a multivariable MR analysis. We found no support for causality of 25-hydroxyvitamin D on BMD at forearm, femoral neck, lumbar spine, and total-body BMD across the lifespan. There was no obvious difference between the total and direct effect of vitamin D on BMD after adjusting for BMI. Our MR analysis provided evidence that genetically determined vitamin D was not causally associated with BMD in the general population. Large-scale randomized controlled trials are warranted to investigate the role of vitamin D supplementation in preventing osteoporosis in the high-risk population.

Abstract Producing high value-added products from waste lipid feedstock by microbial cell factory has great advantages to minimize the pollution as well as improve the economic value of wasted oils and fats. Yarrowia lipolytica is a non-conventional oleaginous yeast and can grow on a variety of hydrophobic substrates. In this study, we explored its ability to synthesize α-farnesene, an important sesquiterpene, using lipid feedstock. Based on the α-farnesene production strain, we constructed previously, we identified that Erg12 was the key limiting factor to further increase the α-farnesene production. The α-farnesene production was improved by 35.8% through increasing the copy number of ERG12 and FSERG20 on oleic acid substrate. Expression of heterologous VHb further improved α-farnesene production by 12.7%. Combining metabolic engineering with the optimization of fermentation conditions, the α-farnesene titer and yield reached 10.2 g/L and 0.1 g/g oleic acid, respectively, in fed-batch cultivation. The α-farnesene synthesis ability on waste cooking oil and other edible oils were also explored. Compared with using glucose as carbon source, using lipid substrates obtained higher α-farnesene yield and titer, but lower by-products accumulation, demonstrating the advantage of Y. lipolytica to synthesize high value-added products using lipid feedstock.

IntroductionEndothelial cells play important roles in neurodegenerative diseases caused by diabetes, therefore, we aimed at investigating the mechanisms through which endothelial cells are involved in diabetes development.MethodsSingle cell analysis was performed to identify the major endothelial cell subtypes in cardiovascular tissues that are involved in diabetes development. A cell-cell communication approach was then used to identify ligand-receptor interaction pairs between these cell types. Differential expression analysis between the two experimental groups [standard chow diet group and diabetogenic diet with cholesterol (DDC) group] was used to identify diabetes-related differentially expressed genes (DEGs). The upregulated genes were used to identify candidate ligands or receptors, as well as the corresponding cell types. Cell trajectory inference was performed to identify the stage of cell development and changes in expression of candidate ligands or receptors during cell development. Gene set enrichment analysis (GSEA) was conducted to investigate the biological functions of genes of purpose. Finally, molecular dynamics simulations (MDSs) were used to predict potential drugs with the ability to target the proteins of purpose.ResultsSeven cell types, including five endothelial cell subtypes (EC_1, EC_2, EC_3, EC_4, and EC_EndMT), were identified from endothelial cell-enriched single cell samples from the heart and aorta of mice. Cell-cell communication analysis revealed the potential ligand-receptor interactions between these cell types while five important ligand-receptor-associated genes, including Fn1, Vcam1, Fbn1, Col4a1, and Col4a2, were established by differential expression analysis. Among them, Vcam1 is mainly expressed in EC_EndMT and is involved in interactions between EC_EndMT and other cells. Cell trajectory extrapolation analysis revealed a shift from EC_2/EC_4 to EC_EndMT and a shift from EC_EndMT to EC_3/EC_1 during the progression of diabetes. GSEA analysis revealed that upregulation of VCAM1 may have inhibitory effects on cell growth and energy metabolism.ConclusionEC_EndMT subtypes have a complex role in neurodegenerative diseases caused by diabetes. Through mechanisms involved in cell-cell communication, Vcam1 may play an important role in dysregulation of biological functions of EC_ EndMT. Molecular docking results of the quercetin-VCAM1 complex suggest that quercetin may be an effective drug for targeting this protein.

Abstract Background The coronavirus disease 2019 (COVID-19) pandemic has struck globally and is exerting a devastating toll on humans. The pandemic has led to calls for widespread vitamin D supplementation in public. However, evidence supporting the role of vitamin D in the COVID-19 pandemic remains controversial. Methods We performed a two-sample Mendelian randomization (MR) analysis to analyze the causal effect of the 25-hydroxyvitamin D [25(OH)D] concentration on COVID-19 susceptibility, severity and hospitalization traits by using summary-level GWAS data. The causal associations were estimated with inverse variance weighted (IVW) with fixed effects (IVW-fixed) and random effects (IVW-random), MR-Egger, weighted edian and MR Robust Adjusted Profile Score (MR.RAPS) methods. We further applied the MR Steiger filtering method, MR Pleiotropy RESidual Sum and Outlier (MR-PRESSO) global test and PhenoScanner tool to check and remove single nucleotide polymorphisms (SNPs) that were horizontally pleiotropic. Results We found no evidence to support the causal associations between the serum 25(OH)D concentration and the risk of COVID-19 susceptibility [IVW-fixed: odds ratio (OR) = 0.9049, 95% confidence interval (CI) 0.8197–0.9988, p = 0.0473], severity (IVW-fixed: OR = 1.0298, 95% CI 0.7699–1.3775, p = 0.8432) and hospitalized traits (IVW-fixed: OR = 1.0713, 95% CI 0.8819–1.3013, p = 0.4878) using outlier removed sets at a Bonferroni-corrected p threshold of 0.0167. Sensitivity analyses did not reveal any sign of horizontal pleiotropy. Conclusions Our MR analysis provided precise evidence that genetically lowered serum 25(OH)D concentrations were not causally associated with COVID-19 susceptibility, severity or hospitalized traits. Our study did not provide evidence assessing the role of vitamin D supplementation during the COVID-19 pandemic. High-quality randomized controlled trials are necessary to explore and define the role of vitamin D supplementation in the prevention and treatment of COVID-19.

Abstract Background Succinic acid (SA) is a crucial metabolic intermediate and platform chemical. Development of biobased processes to achieve sustainable SA production has attracted more and more attention in biotechnology industry. Yarrowia lipolytica has a strong tricarboxylic acid cycle and tolerates low pH conditions, thus making it a potential platform for SA production. However, its SA titers in glucose media remain low. Results In this study, we screened mitochondrial carriers and C4-dicarboxylic acid transporters to enhance SA secretion in Y. lipolytica. PGC62-SYF-Mae strain with efficient growth and SA production was constructed by optimizing SA biosynthetic pathways and expressing the transporter SpMae1. In fed-batch fermentation, this strain produced 101.4 g/L SA with a productivity of 0.70 g/L/h and a yield of 0.37 g/g glucose, which is the highest SA titer achieved using yeast, with glucose as the sole carbon resource. Conclusion Our results indicated that transporter engineering is a powerful strategy to achieve the efficient secretion of SA in Y. lipolytica, which will promote the industrial production of bio-based SA.

Osteosarcoma, one of the most common malignant bone tumors, is characterized by a high rate of metastasis, and the survival rate of patients with metastatic osteosarcoma is poor. Previous studies have reported that miRNAs often regulate the occurrence and development of various tumors. In this work, we identified miRNA‐542‐5p as a critical miRNA in osteosarcoma by overlapping three Gene Expression Omnibus datasets, and then evaluated miRNA‐542‐5p expression profiles using Gene Expression Omnibus and Sarcoma‐microRNA Expression Database. We used MISIM to investigate miRNAs correlated with miR‐542 and identified potential target genes of miRNA‐542‐5p using miRWalk. Functional and pathway enrichment analyses were performed using The Database for Annotation, Visualization and Integrated Discovery. Protein–protein interaction was performed using Search Tool for the Retrieval of Interacting Genes and Cytoscape. We report that the relative level of miRNA‐542‐5p was significantly higher in osteosarcoma than in healthy bone. Expressions of hsa‐miR‐330 and hsa‐miR‐1202 were found to be strongly correlated with that of miR‐542‐5p. Furthermore, we identified a total of 514 down‐regulated genes as possible targets of miR‐542‐5p. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis demonstrated that the putative target genes of miR‐542‐5p were most enriched in the cell‐cycle process. The differentially expressed genes CDCA5, PARP12 and HSPD1 were found to be hub genes in protein–protein interaction networks. Finally, transfection of the osteosarcoma cell line U2OS with miR‐542‐5p mimics or inhibitor revealed that miR‐542‐5p can promote cell proliferation. In conclusion, our results suggest that miR‐542‐5p may promote osteosarcoma proliferation; thus, this miRNA may have potential as a biomarker for diagnosis and prognosis.

Abstract Background Yarrowia lipolytica, a non-traditional oil yeast, has been widely used as a platform for lipid production. However, the production of other chemicals such as terpenoids in engineered Y. lipolytica is still low. α-Farnesene, a sesquiterpene, can be used in medicine, bioenergy and other fields, and has very high economic value. Here, we used α-farnesene as an example to explore the potential of Y. lipolytica for terpenoid production. Results We constructed libraries of strains overexpressing mevalonate pathway and α-farnesene synthase genes by non-homologous end-joining (NHEJ) mediated integration into the Y. lipolytica chromosome. First, a mevalonate overproduction strain was selected by overexpressing relevant genes and changing the cofactor specificity. Based on this strain, the downstream α-farnesene synthesis pathway was overexpressed by iterative integration. Culture conditions were also optimized. A strain that produced 25.55 g/L α-farnesene was obtained. This is the highest terpenoid titer reported in Y. lipolytica. Conclusions Yarrowia lipolytica is a potentially valuable species for terpenoid production, and NHEJ-mediated modular integration is effective for expression library construction and screening of high-producer strains.

BackgroundThis study aimed to explore the association between serum amino acids (AAs) levels and bone mineral density (BMD).MethodsWe performed a two-sample Mendelian randomization (MR) analysis to analyze the associations between the levels of eight AAs and BMD values by using summary-level genome-wide association study (GWAS) data. We applied the MR Steiger filtering method and MR Pleiotropy RESidual Sum and Outlier (MR-PRESSO) global test to check for and remove single nucleotide polymorphisms (SNPs) that were horizontally pleiotropic. The associations were estimated with the inverse variance weighted (IVW), MR-Egger, weighted median and MR Robust Adjusted Profile Score (MR.RAPS) methods.ResultsOur study found that genetically increased isoleucine (Ile) [IVW: effect = 0.1601, 95% confidence interval (CI) = 0.0604 ~ 0.2597, p = 0.0016] and valine (Val) levels (IVW: effect = 0.0953, 95% CI = 0.0251 ~ 0.1655, p = 0.0078) were positively associated with total body BMD (TB-BMD). The results also revealed that genetically increased tyrosine (Tyr) levels were negatively associated with TB-BMD (IVW: effect = -0.1091, 95% CI = -0.1863 ~ -0.0320, p = 0.0055).ConclusionsIn this study, associations between serum AA levels and BMD were established. These findings underscore the important role that serum AAs play in the development of osteoporosis and provide evidence that osteoporosis can be prevented and treated by the intake of certain AAs.

Time-delay chaotic systems with multiple positive Lyapunov exponents have been extensively studied in the field of information security. This paper proposes a new four-dimensional time-delay Lorenz system and its chaotic synchronization through the Lyapunov–Krasovskii theory. The sufficient conditions for the stability of the new chaotic system are obtained by the Routh–Hurwitz criterion, and the control parameters are found to have a significant impact on the speed of synchronous convergence. Furthermore, oscillation circuit simulation is essential for contributing the chaotic system to practical applications. Accordingly, the circuits of the chaotic time-delay system and its coupled synchronous control circuit are innovatively designed by Multisim. Experiment results illustrate the behaviors of various attractors in the new time-delay Lorenz system and the effectiveness of the proposed asymptotic synchronous method.

BackgroundIt remains unclear whether an increased risk of type 2 diabetes (T2D) affects the risk of osteoarthritis (OA).MethodsHere, we used two-sample Mendelian randomization (MR) to obtain non-confounded estimates of the effect of T2D and glycemic traits on hip and knee OA. We identified single-nucleotide polymorphisms (SNPs) strongly associated with T2D, fasting glucose (FG), and 2-h postprandial glucose (2hGlu) from genome-wide association studies (GWAS). We used the MR inverse variance weighted (IVW), the MR–Egger method, the weighted median (WM), and the Robust Adjusted Profile Score (MR.RAPS) to reveal the associations of T2D, FG, and 2hGlu with hip and knee OA risks. Sensitivity analyses were also conducted to verify whether heterogeneity and pleiotropy can bias the MR results.ResultsWe did not find statistically significant causal effects of genetically increased T2D risk, FG, and 2hGlu on hip and knee OA (e.g., T2D and hip OA, MR–Egger OR = 1.1708, 95% CI 0.9469–1.4476, p = 0.1547). It was confirmed that horizontal pleiotropy was unlikely to bias the causality (e.g., T2D and hip OA, MR–Egger, intercept = −0.0105, p = 0.1367). No evidence of heterogeneity was found between the genetic variants (e.g., T2D and hip OA, MR–Egger Q = 30.1362, I2 < 0.0001, p = 0.6104).ConclusionOur MR study did not support causal effects of a genetically increased T2D risk, FG, and 2hGlu on hip and knee OA risk.

BackgroundAssociations between inflammatory bowel disease (IBD) [including ulcerative colitis (UC) and Crohn’s disease (CD)] and ankylosing spondylitis (AS) were discovered in observational studies, but no evidence supported the causal relationship between the two diseases.MethodsWe employed two-sample Mendelian randomization (MR) to estimate the unconfounded bidirectional causal associations between IBD (including UC and CD) and AS. We selected single-nucleotide polymorphisms (SNPs) from genome-wide association studies (GWAS) after strictly assessing the quality of the studies in the IEU GWAS database. Sensitivity analyses were also conducted to verify whether heterogeneity and pleiotropy can bias the MR results.ResultsWe found positive causal effects of genetically increased UC, CD, and IBD risk on AS (e.g., UC and AS, IVW OR: 1.0256, 95% CI: 1.0130∼1.0385, p = 6.43E-05). However, we did not find significant causal associations of AS with UC, CD, or IBD (e.g., AS and UC, IVW OR: 1.1858, 95% CI: 0.8639∼1.6278, p = 0.2916). The sensitivity analysis also confirmed that horizontal pleiotropy was unlikely to bias the causality (e.g., UC and AS, MR-Egger: intercept p = 0.1326). The leave-one-out analysis also demonstrated that the observed links were not driven by SNP. No evidence of heterogeneity was found between the genetic variants (e.g., UC and AS, MR-Egger: Q statistic = 43.1297, I2

Abstract. The Asia-Pacific region includes countries with diverse cultural, demographic, and socio-political backgrounds. Countries such as Japan have very high life expectancy and an aged population. China and India, with a combined population over 2.7 billion, will experience a huge wave of ageing population with subsequent osteoporotic injuries. Australia will experience a similar increase in the osteoporotic fracture burden, and is leading the region by establishing a national hip fracture registry with governmental guidelines and outcome monitoring. While it is impossible to compare fragility hip fracture care in every Asia-Pacific country, this review of 4 major nations gives insight into the challenges facing diverse systems. They are united by the pursuit of internationally accepted standards of timely surgery, combined orthogeriatric care, and secondary fracture prevention strategies.

This paper investigates the effect of type and dosage of multiwalled carbon nanotubes (MWCNTs) on the mechanical and self-sensing properties of engineered cementitious composites (ECCs). Two types of MWCNTs (MWCNTa and MWCNTb) were employed. The tensile and flexural strengths of CNT-reinforced ECCs were improved compared with normal ECCs, while the ultimate tensile strain and midspan deflection were reduced. Compared with the dosage of MWCNTs, the type had less effect on these properties. The percolation threshold was around 0.3 wt.%. ECCs containing MWCNTs had good self-sensing ability under different loading conditions. When the midspan deflection increased from 0.1 to 0.6 mm, the fractional change in resistivity reached 9%. The dosage of MWCNTs had a significant effect on the self-sensing ability. As the MWCNT content increased, the amplitude of fractional change in resistivity decreased.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enter human cells by binding its spike protein receptor-binding domain (S-RBD) with human angiotensin-converting enzyme 2 (hACE2). In the present work, a novel drug-exploring view (food taste: umami) to interfere SARS-CoV-2 S-RBD and one of its variants (Omicron) into human body was explored by an in-silico study. Their interfering interaction and potential mechanism have been illuminated: 1) 34 umami molecules presenting low binding score (< −7 kcal/mol) in molecular docking bonded with active residues of SARS-CoV-2 (Omicron) S-RBD/hACE2 protein by intermolecular interaction (hydrogen bond, hydrophobic interaction and salt bridge, mainly); 2) 4 umami molecules classified in nucleotides and peptides formed relatively stable protein-ligand contacts during molecular dynamic simulation; 3) 3 critical residues (His34, Glu37, and Asp406) remained positive effects in maintaining the ligand-protein structural stability. Taken together, these 4 umami molecules could be supportive as orally bioactive agents for preventing SARS-CoV-2 (Omicron) S-RBD/hACE2 interaction which needs further experimental assays to prove.

Grade variation on interstate highways affects the roadway geometric design, vehicle performance and driver behavior, thus possibly exerting an unexpected effect on vehicular speed. Hence, determining the internal relationship between grade and speed is important and useful for drivers, traffic regulators and other traffic participants. However, the problem with performing this research is the lack of large-scale gradient and speed data. Google Earth (GE) provides an application programming interface for extracting elevation data worldwide. The elevation dataset from GE can be easily converted to grade data. In addition, our team has collected and stored speed series data for different freeways over several years. Based on the above obtainable grade and speed datasets, we conducted research on the effect of grades on free flow speeds from two perspectives. First, the influence of grades on speed was analyzed from both quantitative and qualitative aspects. The analysis of the distributions of four typical types of speeds demonstrated a decreasing tendency as the speed increased. Steeper grades generated a more intense speed fluctuation in terms of the four types of speeds. Second, a model based on the Student's t-test was developed to evaluate the level of significant difference among speed series under neighboring grades. The Student's t-test demonstrated that adjacent grades do not significantly influence the speeds. In summary, speeds under different grades showed obviously different tendencies. The findings of this study can help transport authorities set more reasonable speed limits and improve the geometric design of interstates with grade variation constraints.