Harnessing oleaginous yeast to produce omega fatty acids.

Omega fatty acids are important for human health. They are traditionally extracted from animals or plants but can be alternatively produced using oleaginous yeast. Current efforts are producing yeast strains with similar fatty acid distributions and powerful lipogenesis capacity. The next step is to further make the process more competitive.

Exploiting synthetic biology platforms for enhanced biosynthesis of natural products in Yarrowia lipolytica.

With the rapid development of synthetic biology, researchers can design, modify, or even synthesize microorganisms de novo, and microorganisms endowed with unnatural functions can be considered "artificial life" and facilitate the development of functional products. Based on this concept, researchers can solve critical problems related to the insufficient supply of natural products, such as low yields, long production cycles, and cumbersome procedures. Due to its superior performance and unique physiological and biochemical characteristics, Yarrowia lipolytica is a favorable chassis cell used for green biomanufacturing by numerous researchers. This paper mainly reviews the development of synthetic biology techniques for Y. lipolytica and summarizes the recent research progress on the synthesis of natural products in Y. lipolytica. This review will promote the continued innovative development of Y. lipolytica by providing theoretical guidance for research on the biosynthesis of natural products.

A rapid and accurate method for evaluating the degradation of pan-Akt in cells by PROTACs using NanoLuc luciferase.

Proteolysis targeting chimera (PROTAC) is a protein degradation technique that has been increasingly used in the development of new drugs in recent years. Akt is a classical serine/threonine kinase, and its role outside of the kinase has gradually gained attention in recent years, making it one of the proteins targeted by PROTACs. Currently, there are many methods used for the evaluation of intracellular protein degradation, but each has its own advantages or disadvantages. This study aimed to investigate the feasibility of evaluating the degradation of pan-Akt proteins in cells by PROTACs (MS21 and MS170) using the NanoLuc luciferase method. After conducting a thorough comparison between this method and the classical western blot assay in various cells, as well as testing the stability of the experiments between multiple batches, we found that NanoLuc luciferase is a highly accurate, stable, low-cost and easy-to-operate method for the evaluation of intracellular pan-Akt degradation by PROTACs with a short cycle time and high cellular expandability. Given the numerous advantages of this method, it is hypothesized that it could be extended to evaluate the degradation of more target proteins of PROTACs. In summary, the NanoLuc luciferase is a suitable method for early protein degradation screening of PROTAC compounds.

Functional outcome following spontaneous intracerebral hemorrhage between ultrasound guidance endoscopic surgery and conventional craniotomy: A retrospective population-based study.

OBJECTIVES: Ultrasound guidance endoscopic surgery (ES) has been widely used in the treatment of cerebral hemorrhage in recent years, but relevant research articles are still scarce. Our study aims to investigate the effect of ES compared with conventional craniotomy (CC) on the postoperative complications, and prognosis of patients with intracerebral hemorrhage. MATERIALS AND METHODS: The clinical data of 1201 patients with ICH treated in our hospital from January 2017 to January 2020 were collected. The t-test, Chi-squared test and Fisher's exact test were used to analyze the clinical baseline data. Among 1021 spontaneous ICH patients, 193 patients who underwent hematoma evacuation were included in the present analysis. RESULTS: The Glasgow Outcome Scale (GOS) score at 6 months had a favorable prognosis in ES group (p = 0.003). ES group had fewer postoperative complications compared with CC group. Operating time and intraoperative blood loss were significantly lower in ES group than CC group (p = 0.001 and p = 0.002). CONCLUSIONS: Our study revealed that receiving ES improved the prognosis of ICH patients. Additionally, endoscopic surgery diminishes operative time, and intraoperative blood loss and reduces the incidence of postoperative complications.

Efficacy and safety of olmesartan medoxomil­amlodipine besylate tablets (Sevikar®) in older patients with essential hypertension: Subgroup analysis from the Sevikar study.

Essential hypertension is a notable threat for the older (age, ≥65 years) population. However, to the best of our knowledge, a real-world study assessing olmesartan medoxomil-amlodipine besylate (OM-AML) tablets in older Chinese patients with essential hypertension has not been performed. Therefore, the present study aimed to evaluate the efficacy and safety of OM-AML tablets in these patients. A total of 463 older Chinese patients with essential hypertension treated with OM-AML (20/5 mg) tablets (Sevikar®) were analyzed in a prospective, single-arm, multi-center, real-world study. Seated systolic blood pressure (SeSBP) and seated diastolic blood pressure (SeDBP) at baseline, and at week (W)4 and W8 after OM-AML tablet administration were measured. The mean ± standard error change of SeSBP/SeDBP was -10.3±0.8/-4.6±0.5 and -12.5±0.8/-5.6±0.5 mmHg at W4 and W8, respectively. At W4, 74.1 and 26.8% of patients achieved BP target according to the China and American Heart Association (AHA) criteria, while at W8, 78.0 and 38.7% of patients reached these BP targets accordingly. Finally, 76.5 and 80.5% of patients achieved BP response at W4 and W8, respectively. Furthermore, home-measured SeSBP and SeDBP were significantly decreased from W1 to W8 (both P<0.001). Additionally, the satisfaction of both patients and physicians was elevated at W8 compared with at W0 (both P<0.001). The medication possession rate from baseline to W4 and W8 was 95.5 and 92.5%. The most common drug-associated adverse events by system organ classes were nervous system disorder (4.5%), vascular disorder (2.8%), and general disorder and administration site conditions (2.6%), which were generally mild. In conclusion, OM-AML tablets may be considered effective and safe in lowering BP, enabling the achievement of guideline-recommended BP targets in older Chinese patients with essential hypertension.

Engineering Yarrowia lipolytica for Sustainable Production of the Pomegranate Seed Oil-Derived Punicic Acid.

Punicic acid is a conjugated linolenic acid with various biological activities including antiobesity, antioxidant, anticancer, and anti-inflammatory effects. It is often used as a nutraceutical, dietary additive, and animal feed. Currently, punicic acid is primarily extracted from pomegranate seed oil, but it is restricted due to the extended growth cycle, climatic limitations, and low recovery level. There have also been reports on the chemical synthesis of punicic acid, but it resulted in a mixture of structurally similar isomers, requiring additional purification/separation steps. In this study, a comprehensive strategy for the production of punicic acid in Yarrowia lipolytica was implemented by pushing the supply of linoleic acid precursors in a high-oleic oil strain, expressing multiple copies of the fatty acid conjugase gene from Punica granatum, engineering the acyl-editing pathway to improve the phosphatidylcholine pool, and promoting the assembly of punicic acid in the form of triglycerides. The optimal strain with high oil production capacity and a significantly increased punicic acid ratio accumulated 3072.72 mg/L punicic acid, accounting for 6.19% of total fatty acids in fed-batch fermentation, providing a viable, sustainable, and green approach for punicic acid production to substitute plant extraction and chemical synthesis production.

Efficacy and safety of olmesartan medoxomil-amlodipine besylate tablet in Chinese patients with essential hypertension: A prospective, single-arm, multi-center, real-world study.

There lacks real-world study with a large sample size assessing olmesartan medoxomil-amlodipine besylate (OM-AML) tablet. Therefore, this study aimed to evaluate the efficacy and safety of OM-AML tablet in patients with essential hypertension. Totally, 1341 patients from 36 medical centers with essential hypertension who took OM-AML (20/5 mg) tablet were analyzed in the current prospective, single-arm, multi-center, real-world study (SVK study). Seated systolic blood pressure (SeSBP) and seated diastolic blood pressure (SeDBP) at baseline, week (W)4 and W8 were measured. The mean (±SE) change of SeSBP/SeDBP was -10.8 ± 0.4/-6.6 ± 0.3 mmHg at W4 and -12.7 ± 0.5/-7.6 ± 0.3 mmHg at W8, respectively. At W4, 78.8% and 29.0% patients achieved BP target by China and American Heart Association (AHA) criteria; at W8, 84.7% and 36.5% patients reached blood pressure (BP) target by China and AHA criteria, accordingly. Meanwhile, 80.2% and 86.4% patients achieved BP response at W4 and W8, respectively. Home-measured SeSBP and SeDBP decreased from W1 to W8 (both p < .001). Besides, patients' and physicians' satisfaction were elevated at W8 compared with W0 (both p < .001). The medication possession rate was 94.8% from baseline to W4 and 91.3% from baseline to W8. The most common drug-related adverse events were nervous system disorders (4.6%), vascular disorders (2.6%), and general disorders and administration site conditions (2.3%) by system organ class, which were generally mild and manageable. In conclusion, OM-AML tablet is one of the best antihypertensive agents in patients with essential hypertension.

Metabolic engineering of Yarrowia lipolytica for high-level production of squalene.

Squalene is an important triterpene with a wide range of applications. Given the growing market demand for squalene, the development of microbial cell factories capable of squalene production is considered a sustainable method. This study aimed to investigate the squalene production potential of Yarrowia lipolytica. First, HMG-CoA reductase from Saccharomyces cerevisiae and squalene synthase from Y. lipolytica was co-overexpressed in Y. lipolytica. Second, by enhancing the supply of NADPH in the squalene synthesis pathway, the production of squalene in Y. lipolytica was effectively increased. Furthermore, by constructing an isoprenol utilization pathway and overexpressing YlDGA1, the strain YLSQ9, capable of producing 868.1 mg/L squalene, was obtained. Finally, by optimizing the fermentation conditions, the highest squalene concentration of 1628.2 mg/L (81.0 mg/g DCW) in Y. lipolytica to date was achieved. This study demonstrated the potential for achieving high squalene production using Y. lipolytica.

Building Yarrowia lipolytica Cell Factories for Advanced Biomanufacturing: Challenges and Solutions.

Microbial cell factories have shown great potential for industrial production with the benefit of being environmentally friendly and sustainable. Yarrowia lipolytica is a promising and superior non-model host for biomanufacturing due to its cumulated advantages compared to model microorganisms, such as high fluxes of metabolic precursors (acetyl-CoA and malonyl-CoA) and its naturally hydrophobic microenvironment. However, although diverse compounds have been synthesized in Y. lipolytica cell factories, most of the relevant studies have not reached the level of industrialization and commercialization due to a number of remaining challenges, including unbalanced metabolic flux, conflict between cell growth and product synthesis, and cytotoxic effects. Here, various metabolic engineering strategies for solving the challenges are summarized, which is developing fast and extremely conducive to rational design and reconstruction of robust Y. lipolytica cell factories for advanced biomanufacturing. Finally, future engineering efforts for enhancing the production efficiency of this platform strain are highlighted.

On committor functions in milestoning.

As an optimal one-dimensional reaction coordinate, the committor function not only describes the probability of a trajectory initiated at a phase space point first reaching the product state before reaching the reactant state but also preserves the kinetics when utilized to run a reduced dynamics model. However, calculating the committor function in high-dimensional systems poses significant challenges. In this paper, within the framework of milestoning, exact expressions for committor functions at two levels of coarse graining are given, including committor functions of phase space point to point (CFPP) and milestone to milestone (CFMM). When combined with transition kernels obtained from trajectory analysis, these expressions can be utilized to accurately and efficiently compute the committor functions. Furthermore, based on the calculated committor functions, an adaptive algorithm is developed to gradually refine the transition state region. Finally, two model examples are employed to assess the accuracy of these different formulations of committor functions.

Discovery of novel alkaline-tolerant xylanases from fecal microbiota of dairy cows.

Xylo-oligosaccharides (XOS) are considered as a promising type of prebiotics that can be used in foods, feeds, and healthcare products. Xylanases play a key role in the production of XOS from xylan. In this study, we conducted a metagenomic analysis of the fecal microbiota from dairy cows fed with different types of fodders. Despite the diversity in their diets, the main phyla observed in all fecal microbiota were Firmicutes and Bacteroidetes. At the genus level, one group of dairy cows that were fed probiotic fermented herbal mixture-containing fodders displayed decreased abundance of Methanobrevibacter and increased growth of beneficial Akkermansia bacteria. Additionally, this group exhibited a high microbial richness and diversity. Through our analysis, we obtained a comprehensive dataset comprising over 280,000 carbohydrate-active enzyme genes. Among these, we identified a total of 163 potential xylanase genes and subsequently expressed 34 of them in Escherichia coli. Out of the 34 expressed genes, two alkaline xylanases with excellent temperature stability and pH tolerance were obtained. Notably, CDW-xyl-8 exhibited xylanase activity of 96.1 ± 7.5 U/mg protein, with an optimal working temperature of 55 â„ƒ and optimal pH of 8.0. CDW-xyl-16 displayed an activity of 427.3 ± 9.1 U/mg protein with an optimal pH of 8.5 and an optimal temperature at 40 â„ƒ. Bioinformatic analyses and structural modeling suggest that CDW-xyl-8 belongs to GH10 family xylanase, and CDW-xyl-16 is a GH11 family xylanase. Both enzymes have the ability to hydrolyze beechwood xylan and produce XOS. In conclusion, this metagenomic study provides valuable insights into the fecal microbiota composition of dairy cows fed different fodder types, revealing main microbial groups and demonstrating the abundance of xylanases. Furthermore, the characterization of two novel xylanases highlights their potential application in XOS production.

Therapeutic Drug Monitoring-Guided Vancomycin Therapy of a Pediatric Patient after Liver Transplantation: a Case Report.

BACKGROUND: Vancomycin administration is challenging in critically ill patients because of pharmacokinetic changes and requires careful therapeutic drug monitoring (TDM) to guide the appropriate dosing for an effective serum concentration and to avoid toxicity. METHODS: We reported a one-year-old female pediatric patient with a body mass index of 15.4 had successful TDM-guided vancomycin therapy after a living donor liver transplantation for biliary atresia. RESULTS: The patient was admitted to the Intensive Care Unit for sepsis after her second liver transplantation. Even with the administration of the maximum approved vancomycin dosage (40 mg/kg/day), the serum trough levels were less than the recommended therapeutic level. After several adjustments based on TDM, a continuous pump infusion of up to 800 mg/day was needed to reach the desired serum trough concentration of > 10 µg/mL. Sepsis was controlled, and the patient was transferred from the Intensive Care Unit to the general ward and finally discharged home on a regular follow-up plan. CONCLUSIONS: TDM-guided vancomycin continuous infusion may be an effective therapeutic option for pediatric patients after liver transplantation.

Combination of microbial and chemical synthesis for the sustainable production of ß-elemene, a promising plant-extracted anticancer compound.

Beta-elemene, a class of sesquiterpene derived from the Chinese medicinal herb Curcuma wenyujin, is widely used in clinical medicine due to its broad-spectrum antitumor activity. However, the unsustainable plant extraction prompted the search for environmentally friendly strategies for ß-elemene production. In this study, we designed a Yarrowia lipolytica cell factory that can continuously produce germacrene A, which is further converted into ß-elemene with 100% yield through a Cope rearrangement reaction by shifting the temperature to 250°C. First, the productivity of four plant-derived germacrene A synthases was evaluated. After that, the metabolic flux of the precursor to germacrene A was maximized by optimizing the endogenous mevalonate pathway, inhibiting the competing squalene pathway, and expressing germacrene A synthase gene in multiple copies. Finally, the most promising strain achieved the highest ß-elemene titer reported to date with 5.08 g/L. This sustainable and green method has the potential for industrial ß-elemene production.

[Fermentative production of tetraacetyl phytosphingosine: a review].

Tetraacetyl phytosphingosine (TAPS) is an excellent raw material for natural skin care products. Its deacetylation leads to the production of phytosphingosine, which can be further used for synthesizing the moisturizing skin care product ceramide. For this reason, TAPS is widely used in the skin care oriented cosmetics industry. The unconventional yeast Wickerhamomyces ciferrii is the only known microorganism that can naturally secrete TAPS, and it has become the host for the industrial production of TAPS. This review firstly introduces the discovery, functions of TAPS, and the metabolic pathway for TAPS biosynthesis is further introduced. Subsequently, the strategies for increasing the TAPS yield of W. ciferrii, including haploid screening, mutagenesis breeding and metabolic engineering, are summarized. In addition, the prospects of TAPS biomanufacturing by W. ciferrii are discussed in light of the current progresses, challenges, and trends in this field. Finally, guidelines for engineering W. ciferrii cell factory using synthetic biology tools for TAPS production are also presented.

Clinical efficacy of escitalopram combined with botulinum toxin A in patients with generalized anxiety disorder and comorbid headache.

BACKGROUND: Generalized anxiety disorder (GAD) is a common mental disorder that happens comorbidly with other diseases. Headache is a common anxiety comorbidity. Previous reports have shown that the selection of therapeutic drugs for GAD patients and comorbid headache is challenging. Therefore, our study aimed to investigate the clinical efficacy of escitalopram combined with botulinum toxin A (BoNT/A) in patients with GAD and comorbid headache and seek an alternative treatment strategy for the comorbidity of GAD and headache. METHODS: A prospective, randomized controlled, double-blind study was performed. The eligible GAD patients with comorbid headache were randomly assigned to the BoNT/A group and the placebo group. All the patients were given oral escitalopram therapy (10-20 mg/day) for the whole duration of the study. The BoNT/A group was given local injections of BoNT/A (50 U per person), whereas the placebo group was given local saline (0.9% NaCl) injections at the beginning and 3 months after the experiments. All participants were followed up for 6 months and relevant information was collected at months 0, 1, 2, 3, and 6. Primary outcomes included the following: (1) the Generalized Anxiety Disorder 7 (GAD-7); (2) the Self-rating Anxiety Scale (SAS); (3) the Hamilton Anxiety Rating Scales (HAMA); (4) days with headache per month; (5) visual analogue scale (VAS). RESULTS: A total of 101 patients (the sex ratio of female to male: 3.39:1) were finally included. Compared with the placebo group, the BoNT/A group showed a significant decrease in GAD-7 scores, SAS scores, HAMA scores, days with headache per month, and VAS scores at months 1, 2, 3, and 6 of follow-up (all p < 0.05). The average time to complete remission of anxiety symptoms (HAMA< 7 points) in the BoNT/A group was less than the placebo group (2 months vs. 3 months). At the same time, the results of the survival analysis showed a clear beneficial effect of BoNT/A relative to placebo on the time to remission of anxiety (log-rank test, p < 0.001). Mean daily doses of escitalopram at the sixth month in the BoNT/A group was smaller than the placebo group (12.5 mg vs. 16.04 mg, p < 0.001). The number of patients who relapsed (HAMA total score ≥ 14 points) at 6 months of follow-up in the BoNT/A group was less than the placebo group (2.2% vs. 14.9%, p < 0.05). The rates of response (HAMA subtraction rate ≥ 50%) were 93.8% for the BoNT/A group and 75.5% for the placebo group (p < 0.05), and the rates of remission (HAMA < 7 points) were 87.5% for the BoNT/A group and 64.2% for the placebo group (p < 0.01) at the sixth month. CONCLUSION: The combination of escitalopram with BoNT/A is a significantly effective intervention in improving clinical efficacy and reducing the recurrence in patients with GAD and comorbid headache, and we believe that this approach will be an additional treatment strategy for future treatment of comorbid headache in GAD. Therefore, we recommend that escitalopram combined with BoNT/A should be given as early as possible in GAD patients and comorbid headache.

Personalized smart voice-based electronic prescription for remote at-home feedback management in cardiovascular disease rehabilitation: a multi-center randomized controlled trial.

Objective: To investigate the quality and efficacy of remote at-home rehabilitation for patients with cardiovascular disease (CVD) using personalized smart voice-based electronic prescription, and further explore the standardized health management mode of remote family cardiac rehabilitation. Trial design: A multicenter, randomized (1:1), non-blind, parallel controlled study. Methods: A total of 171 patients with CVD who were admitted to 18 medical institutions in China from April 2021 to October 2022 were randomly divided into a treatment group (86 cases) and a control group (85 cases) in a non-blinded experiment, based on the sequence of enrollment. The control group received routine at-home rehabilitation training, and the treatment group received remote feedback-based at-home cardiac rehabilitation management based on routine at-home rehabilitation training. The primary outcome was the difference in VO2peak (mL/min/kg) after 12 weeks. A linear mixed model was developed with follow-up as the dependent variable. Age and baseline data were utilized as covariates, whereas hospital and patient characteristics were adjusted as random-effect variables. As the linear mixed model can accommodate missing data under the assumption of random missing data, there was no substitute missing value for quantitative data. Results: A total of 171 participants, with 86 in the experimental group and 85 in the control group, were included in the main analysis. The analysis, which used linear mixing model, revealed significant differences in cardiopulmonary function indexes (VO2/kg peak, VO2peak, AT, METs, and maximum resistance) at different follow-up time (0, 4, and 12 weeks) in the experimental group (p < 0.05). In the control group, there was no significant difference in cardiopulmonary values at different follow-up time (0, 4, and 12 weeks; p > 0.05). VO2/kg peak (LS mean 1.49, 95%CI 0.09-2.89, p = 0.037) and other indicators of cardiopulmonary function (p < 0.05) were significantly different between the experimental group and the control group at week 12. The results were comparable in the complete case analysis. Conclusion: The remote home cardiac rehabilitation management mode using personalized smart voice-based electronic prescription provides several benefits to patients, including improvements in muscle strength, endurance, cardiopulmonary function, and aerobic metabolism. It also helps reduce risk factors for cardiovascular disease and enhances patients' self-management abilities and treatment compliance.Clinical trial registration: http://www.chictr.org.cn, identifier ChiCTR2100044063.

Reprogramming the fatty acid metabolism of Yarrowia lipolytica to produce the customized omega-6 polyunsaturated fatty acids.

Omega-6 polyunsaturated fatty acids (ω6-PUFAs), such as γ-linolenic acid (GLA), dihomo-γ-linolenic acid (DGLA) and arachidonic acid (ARA), are indispensable nutrients for human health. Harnessing the lipogenesis pathway of Yarrowia lipolytica creates a potential platform for producing customized ω6-PUFAs. This study explored the optimal biosynthetic pathways for customized production of ω6-PUFAs in Y. lipolytica via either the Δ6 pathway from Mortierella alpina or the Δ8 pathway from Isochrysis galbana. Subsequently, the proportion of ω6-PUFAs in total fatty acids (TFAs) was effectively increased by bolstering the provision of precursors for fatty acid biosynthesis and carriers for fatty acid desaturation, as well as preventing fatty acid degradation. Finally, the proportions of GLA, DGLA and ARA synthesized by customized strains accounted for 22.58%, 46.65% and 11.30% of TFAs, and the corresponding titers reached 386.59, 832.00 and 191.76 mg/L in shake-flask fermentation, respectively. This work provides valuable insights into the production of functional ω6-PUFAs.

Combining orthogonal plant and non-plant fatty acid biosynthesis pathways for efficient production of microbial oil enriched in nervonic acid in Yarrowia lipolytica.

Nervonic acid has proven efficacy in brain development and the prevention of neurodegenerative diseases. Here, an alternative and sustainable strategy for nervonic acid-enriched plant oil production was established. Different ß-ketoacyl-CoA synthases and heterologous Δ15 desaturase were co-expressed, combined with the deletion of the ß-oxidation pathway to construct orthogonal plant and non-plant nervonic acid biosynthesis pathways in Yarrowia lipolytica. A "block-pull-restrain" strategy was further applied to improve the supply of stearic acid as the precursor of the non-plant pathway. Then, lysophosphatidic acid acyltransferase from Malania oleifera (MoLpaat) was identified, which showed specificity for nervonic acid. Endogenous LPAAT was exchanged by MoLPAAT resulted in 17.10 % nervonic acid accumulation. Finally, lipid metabolism was engineered and cofactor supply was increased to boost the lipid accumulation in a stable null-hyphal strain. The final strain produced 57.84 g/L oils with 23.44 % nervonic acid in fed-batch fermentation, which has the potential to substitute nervonic acid-enriched plant oil.