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Abstract Background Benzyl acetate is an aromatic ester with a jasmine scent. It was discovered in plants and has broad applications in food, cosmetic, and pharmaceutical industries. Its current production predominantly relies on chemical synthesis. In this study, Escherichia coli was engineered to produce benzyl acetate. Results Two biosynthetic routes based on the CoA-dependent β-oxidation pathway were constructed in E. coli for benzyl acetate production. In route I, benzoic acid pathway was extended to produce benzyl alcohol by combining carboxylic acid reductase and endogenous dehydrogenases and/or aldo-keto reductases in E. coli. Benzyl alcohol was then condensed with acetyl-CoA by the alcohol acetyltransferase ATF1 from yeast to form benzyl acetate. In route II, a plant CoA-dependent β-oxidation pathway via benzoyl-CoA was assessed for benzyl alcohol and benzyl acetate production in E. coli. The overexpression of the phosphotransacetylase from Clostridium kluyveri (CkPta) further improved benzyl acetate production in E. coli. Two-phase extractive fermentation in situ was adopted and optimized for benzyl acetate production in a shake flask. The most optimal strain produced 3.0 ± 0.2 g/L benzyl acetate in 48 h by shake-flask fermentation. Conclusions We were able to establish the whole pathway for benzyl acetate based on the CoA-dependent β-oxidation in single strain for the first time. The highest titer for benzyl acetate produced from glucose by E. coli is reported. Moreover, cinnamyl acetate production as an unwanted by-product was very low. Results provided novel information regarding the engineering benzyl acetate production in microorganisms. Graphical abstract

Abstract Background 2-Phenylethanol (2-PE) is one of the most widely used spices. Recently, 2-PE has also been considered a potential aviation fuel booster. However, the lack of scientific understanding of the 2-PE biosynthetic pathway and the cellular response to 2-PE cytotoxicity are the most important obstacles to the efficient biosynthesis of 2-PE. Results Here, metabolic engineering and tolerance engineering strategies were used to improve the production of 2-PE in Komagataella phaffii. First, the endogenous genes encoding the amino acid permease GAP1, aminotransferase AAT2, phenylpyruvate decarboxylase KDC2, and aldehyde dehydrogenase ALD4 involved in the Ehrlich pathway and the 2-PE stress response gene NIT1 in K. phaffii were screened and characterized via comparative transcriptome analysis. Subsequently, metabolic engineering was employed to gradually reconstruct the 2-PE biosynthetic pathway, and the engineered strain S43 was obtained, which produced 2.98 g/L 2-PE in shake flask. Furthermore, transcriptional profiling analyses were utilized to screen for novel potential tolerance elements. Our results demonstrated that cells with knockout of the PDR12 and C4R2I5 genes exhibited a significant increase in 2-PE tolerance. To confirm the practical applications of these results, deletion of the PDR12 and C4R2I5 genes in the hyper 2-PE producing strain S43 dramatically increased the production of 2-PE by 18.12%, and the production was 3.54 g/L. Conclusion This is the highest production of 2-PE produced by K. phaffii via l-phenylalanine conversion. These identified K. phaffii endogenous elements are highly conserved in other yeast species, suggesting that manipulation of these homologues might be a useful strategy for improving aromatic alcohol production. These results also enrich the understanding of aromatic compound biosynthetic pathways and 2-PE tolerance, and provide new elements and strategies for the synthesis of aromatic compounds by microbial cell factories.

Alveolar echinococcosis (AE) is a highly lethal helminth infection. Current chemotherapeutic strategies for AE primarily involve the use of benzimidazoles (BZs) such as mebendazole (MDZ) and albendazole (ABZ), which exhibit limited efficacy. In a previous study, the vaccine of recombinant Echinococcus granulosus P29 (rEgP29) showed significant immunoprotection against E. granulosus in both mice and sheep. In the current study, we utilized hybridoma technology to generate five monoclonal antibodies (mAbs) against P29, among which 4G10F4 mAb exhibited the highest antigen-specific binding capacity. This mAb was selected for further investigation of anti-AE therapy, both in vivo and in vitro. In vitro, 4G10F4 inhibited a noteworthy inhibition of E. multilocularis protoscoleces and primary cells viability through complement-dependent cytotoxicity (CDC) mechanism. In vivo, two experiments were conducted. In the first experiment, mice were intraperitoneally injected with Em protoscoleces, and subsequently treated with 4G10F4 mAb (2.5/5/10 mg/kg) at 12 weeks postinfection once per week for 8 times via tail vein injection. Mice that were treated with 4G10F4 mAb only in dosage of 5mg/kg exhibited a significant lower mean parasite burden (0.89±0.97 g) compared to isotype mAb treated control mice (2.21±1.30 g). In the second experiment, mice were infected through hepatic portal vein and treated with 4G10F4 mAb (5mg/kg) at one week after surgery once per week for 8 times. The numbers of hepatic metacestode lesions of the 4G10F4 treatment group were significantly lower in comparison to the isotype control group. Pathological analysis revealed severe disruption of the inner structure of the metacestode in both experiments, particularly affecting the germinal and laminated layers, resulting in the transformation into infertile vesicles after treatment with 4G10F4. In addition, the safety of 4G10F4 for AE treatment was confirmed through assessment of mouse weight and evaluation of liver and kidney function. This study presents antigen-specific monoclonal antibody immunotherapy as a promising therapeutic approach against E. multilocularis induced AE.

Diacetyl is a flavor compound in wine with a low threshold (1–2 mg/L). It is produced during alcoholic fermentation (AF) Saccharomyces and malolactic fermentation (MLF) initiated by lactic acid bacteria (LAB). Whereas, the environment after AF suppresses the normal metabolism of LAB after AF. Researchs have shown the influence on diacetyl mechanisms of the genes ILV2, ILV6, ILV3, ILV5, BDH1, BDH2, and gene aldB from Lactobacillus plantarum in Saccharomyce uvarum WY1. While we found that the diacetyl contents produced by mutants after MLF (Co-fermentation and Seq-fermentation) were significantly improved compared to AF alone. Moreover, the genes mae1 and mae2 from S. pombe, and gene mleS from L. lactis exhibited significant effect on deacidification in our previous study, but the diacetyl of the mutants showed obvious improvement in this study. Thus the effects of association mutation of genes (ILV2, ILV6, ILV3, ILV5, BDH1, BDH2, aldB, mae1, and mleS) on deacidification, diacetyl and other flavors (organic acids, higher alcohols and esters) metabolism in S. uvarum after AF were detected in the study. Among all the mutants, strains V6AmS, V635mS, and V6B12mS showed the most favorable results. Specifically, the L-malic acid contents decreased to 1.26 g/L, 1.18 g/L, and 1.19 g/L, respectively. Concurrently, diacetyl levels were reduced by 52.56%, 61.84%, and 65.31%. The production of n-propanol increased by 18.84%, 20.89%, and 28.12%, whereas isobutanol levels decreased by 37.01%, 42.36%, and 44.04%, and isoamyl alcohol levels decreased by 19.28%, 19.79%, and 16.74%, compared to the parental strain WY1. Additionally, the concentration of lactate ester in the wine increased to 13.162 mg/L, 14.729 mg/L, and 14.236 mg/L, respectively.

BackgroundTelehealth has emerged as a popular channel for providing outpatient services in many countries. However, the majority of telehealth systems focus on operational functions and offer only a sectional patient journey at most. Experiences with incorporating longitudinal real-world medical record data into telehealth are valuable but have not been widely shared. The feasibility and usability of such a telehealth platform, with comprehensive, real-world data via a live feed, for cancer patient care are yet to be studied. ObjectiveThe primary purpose of this study is to understand the feasibility and usability of cancer patient care using a telehealth platform with longitudinal, real-world data via a live feed as a supplement to hospital electronic medical record systems specifically from physician’s perspective. MethodsA telehealth platform was constructed and launched for both physicians and patients. Real-world data were collected and curated using a comprehensive data model. Physician activities on the platform were recorded as system logs and analyzed. In February 2023, a survey was conducted among the platform’s registered physicians to assess the specific areas of patient care and to quantify their before and after experiences, including the number of patients managed, time spent, dropout rate, visit rate, and follow-up data. Descriptive and inferential statistical analyses were performed on the data sets. ResultsOver a period of 15 months, 16,035 unique users (13,888 patients, 1539 friends and family members, and 174 physician groups with 608 individuals) registered on the platform. More than 382,000 messages including text, reminders, and pictures were generated by physicians when communicating with patients. The survey was completed by 78 group leaders (45% of the 174 physician groups). Of the participants, 84% (65.6/78; SD 8.7) reported a positive experience, with efficient communication, remote supervision, quicker response to questions, adverse event prevention, more complete follow-up data, patient risk reduction, cross-organization collaboration, and a reduction in in-person visits. The majority of the participants (59/78, 76% to 76/78, 97.4%) estimated improvements in time spent, number of patients managed, the drop-off rate, and access to medical history, with the average ranging from 57% to 105%. When compared with prior platforms, responses from physicians indicated better experiences in terms of time spent, the drop-off rate, and medical history, while the number of patients managed did not significantly change. ConclusionsThis study suggests that a telehealth platform, equipped with comprehensive, real-world data via a live feed, is feasible and effective for cancer patient care. It enhances inpatient management by improving time efficiencies, reducing drop-off rates, and providing easy access to medical history. Moreover, it fosters a positive experience in physician-patient interactions.

To investigate the dynamic changes in dry matter accumulation in maize after anthesis, we established a logistic model to describe grain filling characteristics (GFC), and analyzed differences between spring and summer maize, and the influence of meteorological factors. The results showed that the logistic model accurately simulated the dynamic changes in grain growth. For spring maize, the fitted hundred-grain weight at maturity was closely related to the average grain filling rate until maturity, days of the active grain filling period, time of the maximum grain filling rate, and duration of the rapid increase in grain weight. For summer maize, it was closely related to the time of the maximum grain filling rate, days of active grain filling period, duration of gradual grain weight, and the rapidly increasing period. The filling characteristics of spring and summer maize differed because of the different meteorological conditions and biological characteristics. The grain filling duration of spring maize was longer than that of summer maize. The maximum grain filling rate of spring maize occurred later than that of summer maize. Temperature and precipitation were the main meteorological factors affecting the hundred-grain weight of spring maize, whereas temperature was the main factor affecting summer maize. The response of spring maize GFC to meteorological factors was more complex than that of summer maize. These results are important for the development of appropriate strategies for improving maize productivity in China.

Abstract The role of follicular T helper (Tfh) cells in humoral response has been considered essential in recent years. Understanding how Tfh cells control complex humoral immunity is critical to developing strategies to improve the efficacy of vaccines against SARS-CoV-2 and other emerging pathogens. However, the immunologic mechanism of Tfh cells in SARS-CoV-2 receptor binding domain (RBD) vaccine strategy is limited. In this study, we expressed and purified recombinant SARS-CoV-2 RBD protein in Drosophila S2 cells for the first time and explored the mechanism of Tfh cells induced by RBD vaccine in humoral immune response. We mapped the dynamic of Tfh cell in lymph node and spleen following RBD vaccination and revealed the relationship between Tfh cells and humoral immune response induced by SARS-CoV-2 RBD vaccine through correlation analysis, blocking of IL-21 signaling pathway, and co-culture of Tfh with memory B cells. Recombinant RBD protein elicited a predominant Tfh1 and Tfh1-17 subset response and strong GC responses in spleen and lymph nodes, especially to enhanced vaccination. IL-21 secreted by Tfh cells affected the development and differentiation of B cells and played a key role in the humoral immune response. These observations will help us further understand the mechanism of protective immune response induced by COVID-19 vaccine and has guiding significance for the development of vaccines against newly emerging mutants.

Abstract Background Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation and concurrent mutations have a poor prognosis. This study aimed to examine anlotinib plus icotinib as a first-line treatment option for advanced NSCLC carrying EGFR mutation with or without concurrent mutations. Methods This phase 2, single-arm, multicenter trial (ClinicalTrials.gov NCT03736837) was performed at five hospitals in China from December 2018 to November 2020. Non-squamous NSCLC cases with EGFR-sensitizing mutations were treated with anlotinib and icotinib. The primary endpoint was progression-free survival (PFS). Secondary endpoints included the objective response rate (ORR), disease control rate (DCR), overall survival (OS), and toxicity. Results Sixty participants were enrolled, including 31 (52%) and 29 (48%) with concurrent mutations and pathogenic concurrent mutations, respectively. The median follow-up was 26.9 (range, 15.0-38.9) months. ORR and DCR were 68.5% and 98.2%, respectively. Median PFS was 15.1 (95%CI: 12.6–17.6) months which met the primary endpoint, median DoR was 13.5 (95%CI: 10.0-17.1) months, and median OS was 30.0 (95%CI: 25.5–34.5) months. Median PFS and OS in patients with pathogenic concurrent mutations were 15.6 (95%CI: 12.5–18.7) months and not reached (95%CI: 17.46 months to not reached), respectively. All patients experienced TRAEs, including 26 (43%) and 1 (1.7%) who had grade ≥ 3 and serious treatment-related adverse events (TRAEs). Conclusions Anlotinib combined with icotinib was effective and well-tolerated as a first-line treatment option for EGFR mutation-positive advanced NSCLC with or without concurrent mutations. Trial registration ClinicalTrials.gov identifier: NCT03736837.

Abstract Senescence‐associated microRNAs (SA‐miRNAs) are important molecules for aging regulation. While many aging‐promoting SA‐miRNAs have been identified, confirmed aging‐suppressive SA‐miRNAs are rare, that impeded our full understanding on aging regulation. In this study, we verified that miR‐708 expression is decreased in senescent cells and aged tissues and revealed that miR‐708 overexpression can alleviate cellular senescence and aging performance. About the molecular cascade carrying the aging suppressive action of miR‐708, we unraveled that miR‐708 directly targets the 3′UTR of the disabled 2 (Dab2) gene and inhibits the expression of DAB2. Interestingly, miR‐708‐caused DAB2 downregulation blocks the aberrant mammalian target of rapamycin complex 1 (mTORC1) activation, a driving metabolic event for senescence progression, and restores the impaired autophagy, a downstream event of aberrant mTORC1 activation. We also found that AMP‐activated protein kinase (AMPK) activation can upregulate miR‐708 via the elevation of DICER expression, and miR‐708 inhibitor is able to blunt the antiaging effect of AMPK. In summary, this study characterized miR‐708 as an aging‐suppressive SA‐miRNA for the first time and uncovered a new signaling cascade, in which miR‐708 links the DAB2/mTOR axis and AMPK/DICER axis together. These findings not only demonstrate the potential role of miR‐708 in aging regulation, but also expand the signaling network connecting AMPK and mTORC1.

Abstract Background In synthetic biology, the strength of promoter elements is the basis for precise regulation of target gene transcription levels, which in turn increases the yield of the target product. However, the results of many researches proved that excessive transcription levels of target genes actually reduced the yield of the target product. This phenomenon has been found in studies using different microorganisms as chassis cells, thus, it becomes a bottleneck problem to improve the yield of the target product. Results In this study, promoters PGK1p and TDH3p with different strengths were used to regulate the transcription level of alcohol acetyl transferase encoding gene ATF1. The results demonstrated that the strong promoter TDH3p decreased the production of ethyl acetate. The results of Real-time PCR proved that the transcription level of ATF1 decreased rapidly under the control of TDH3p, and the unfolded protein reaction was activated, which may be the reason for the abnormal production caused by the strong promoter. RNA-sequencing analysis showed that the overexpression of differential gene HSP30 increased the transcriptional abundance of ATF1 gene and production of ethyl acetate. Interestingly, deletion of the heat shock protein family (e.g., Hsp26, Hsp78, Hsp82) decreased the production of ethyl acetate, suggesting that the Hsp family was also involved in the regulation of ATF1 gene transcription. Furthermore, the results proved that the Hsf1, an upstream transcription factor of Hsps, had a positive effect on alleviating the unfolded protein response and that overexpression of Hsf1 reprogramed the pattern of ATF1 gene transcript levels. The combined overexpression of Hsf1 and Hsps further increased the production of ethyl acetate. In addition, kinase Rim15 may be involved in this regulatory pathway. Finally, the regulation effect of Hsf1 on recombinant strains constructed by other promoters was verified, which confirmed the universality of the strategy. Conclusions Our results elucidated the mechanism by which Rim15–Hsf1–Hsps pathway reconstructed the repression of high transcription level stress and increased the production of target products, thereby providing new insights and application strategies for the construction of recombinant strains in synthetic biology.

Abstract Background Highly efficient production of L-phenylalanine (L-Phe) in E. coli has been achieved by multiple rounds of random mutagenesis and modification of key genes of the shikimate (SHIK) and L-Phe branch pathways. In this study, we performed transcriptomic (16, 24 and 48 h) and metabolomic analyses (8, 16, 24, 32,40, and 48 h) based on time sequences in an engineered E. coli strain producing L-Phe, aiming to reveal the overall changes of metabolic activities during the fermentation process. Results The largest biomass increase rate and the highest production rate were seen at 16 h and 24 h of fermentation, respectively reaching 5.9 h−1 and 2.76 g/L/h, while the maximal L-Phe titer of 60 g/L was accumulated after 48 h of fermentation. The DEGs and metabolites involved in the EMP, PP, TCA, SHIIK and L-Phe-branch pathways showed significant differences at different stages of fermentation. Specifically, the significant upregulation of genes encoding rate-limiting enzymes (aroD and yidB) and key genes (aroF, pheA and aspC) pushed more carbon flux toward the L-Phe synthesis. The RIA changes of a number of important metabolites (DAHP, DHS, DHQ, Glu and PPN) enabled the adequate supply of precursors for high-yield L-Phe production. In addition, other genes related to Glc transport and phosphate metabolism increased the absorption of Glc and contributed to rerouting the carbon flux into the L-Phe-branch. Conclusions Transcriptomic and metabolomic analyses of an L-Phe overproducing strain of E. coli confirmed that precursor supply was not a major limiting factor in this strain, whereas the rational distribution of metabolic fluxes was achieved by redistributing the carbon flux (for example, the expression intensity of the genes tyrB, aspC, aroL and aroF/G/H or the activity of these enzymes is increased to some extent), which is the optimal strategy for enhancing L-Phe production.

During malolactic fermentation (MLF) of vinification, the harsh L–malic acid undergoes transformation into the milder L–lactic acid, and via decarboxylation reactions it is catalyzed by malolactic enzymes in LAB. The use of bacterial malolactic starter cultures, which usually present challenges in the industry as the suboptimal conditions after alcoholic fermentation (AF), including nutrient limitations, low temperatures, acidic pH levels, elevated alcohol, and sulfur dioxide concentrations after AF, lead to “stuck” or “sluggish” MLF and spoilage of wines. Saccharomyces uvarum has interesting oenological properties and provides a stronger aromatic intensity than Saccharomyces cerevisiae in AF. In the study, the biological pathways of deacidification were constructed in S. uvarum, which made the S. uvarum carry out the AF and MLF simultaneously, as different genes encoding malolactic enzyme (mleS or mleA), malic enzyme (MAE2), and malate permease (melP or MAE1) from Schizosaccharomyces pombe, Lactococcus lactis, Oenococcus oeni, and Lactobacillus plantarum were heterologously expressed. For further inquiry, the effect of L–malic acid metabolism on the flavor balance in wine, the related flavor substances, higher alcohols, and esters production, were detected. Of all the recombinants, the strains WYm1SN with coexpression of malate permease gene MAE1 from S. pombe and malolactic enzyme gene mleS from L. lactis and WYm1m2 with coexpression of gene MAE1 and malate permease gene MAE2 from S. pombe could reduce the L–malic acid contents to about 1 g/L, and in which the mutant WYm1SN exhibited the best effect on the flavor quality improvement.

This study investigates innovative approaches to improve the quality and aroma characteristics of Muscat Hamburg wine production by substituting the conventional Saccharomyces cerevisiae yeast with an efficient fermentation strain of Schizosaccharomyces pombe. The typical use of S. cerevisiae in Muscat Hamburg wine often leads to uniformity and prolonged processing times, requiring subsequent malolactic fermentation to degrade excessive malic acid. The study advocates for the replacement of S. cerevisiae with a specific S. pombe strain, Sp-410, isolated from the fermented grains of sauce-flavor Baijiu, a Chinese spirit. Muscat Hamburg wine fermented with the S. pombe strain demonstrates decreased malic acid levels, offering a potential alternative to malolactic fermentation. However, exclusive S. pombe fermentation may result in an overproduction of acetic acid metabolites, leading to a monotonous taste. In response, the study proposes a mixed fermentation approach, combining the S. pombe strain with a Saccharomyces uvarum strain and a non-Saccharomyces yeast, Torulaspora delbrueckii. The optimized mixed fermentation strategies (M:SP+TD and M60SP+TD) involve specific proportions and intervals of inoculation, aiming to enhance the quality and aroma complexity of Muscat Hamburg wine. In conclusion, this research contributes to advancing the production of high-quality Muscat Hamburg wines, utilizing S. pombe as the primary yeast strain and implementing mixed fermentation methodologies.

Abstract Background Adaptive immune response has been thought to play a key role in SARS-CoV-2 infection. The role of B cells, CD4+T, and CD8+T cells are different in vaccine-induced immune response, thus it is imperative to explore the functions and kinetics of adaptive immune response. We collected blood samples from unvaccinated and vaccinated individuals. To assess the mechanisms contributing to protective immunity of CoronaVac vaccines, we mapped the kinetics and durability of humoral and cellular immune responses after primary and boost vaccination with CoronaVac vaccine in different timepoints. Materials and methods We separate PBMC and plasma from blood samples. The differentiation and function of RBD-spcific CD4+T and CD8+T cells were analyzed by flow cytometry and ELISA. Antibodies response was analyzed by ELISA. ELISPOT analysis was perfomed to detected the RBD-spcific memory B cells. CBA analysis was performed to detected the cytokine immune profiles. Graphpad prism 8 and Origin 2021 were used for statistical analysis. Results Vaccine-induced CD4+T cell responses to RBD were more prominent than CD8+T cell responses, and characterized by a predominant Th1 and weak Th17 helper response. CoronaVac vaccine triggered predominant IgG1 antibody response and effectively recalled specific antibodies to RBD protein after booster vaccination. Robust antigen-specific memory B cells were detected (p

Abstract Background Amyrin is an important triterpenoid and precursor to a wide range of cosmetic, pharmaceutical and nutraceutical products. In this study, we metabolically engineered the oleaginous yeast, Yarrowia lipolytica to produce α- and β-amyrin on simple sugar and waste cooking oil. Results We first validated the in vivo enzymatic activity of a multi-functional amyrin synthase (CrMAS) from Catharanthus roseus, by expressing its codon-optimized gene in Y. lipolytica and assayed for amyrins. To increase yield, prevailing genes in the mevalonate pathway, namely HMG1, ERG20, ERG9 and ERG1, were overexpressed singly and in combination to direct flux towards amyrin biosynthesis. By means of a semi-rational protein engineering approach, we augmented the catalytic activity of CrMAS and attained ~ 10-folds higher production level on glucose. When applied together, protein engineering with enhanced precursor supplies resulted in more than 20-folds increase in total amyrins. We also investigated the effects of different fermentation conditions in flask cultures, including temperature, volumetric oxygen mass transfer coefficient and carbon source types. The optimized fermentation condition attained titers of at least 100 mg/L α-amyrin and 20 mg/L β-amyrin. Conclusions The design workflow demonstrated herein is simple and remarkably effective in amplifying triterpenoid biosynthesis in the yeast Y. lipolytica.

Abstract Background Pain is a fearful yet common symptom among lung cancer patients. This multicenter, cross‐sectional study was conducted to examine the current status of pain prevalence and management in lung cancer patients in northern China. Methods A total of 18 hospitals across northern China were selected. Patients with primary lung cancer who visited the outpatient clinic or were admitted in the wards on a preplanned day were invited to complete a questionnaire. Meanwhile, physicians who had experience of treating primary lung cancer patients were also surveyed. Results A total of 533 patients and 197 physicians provided valid responses to the survey, of which 45.4% (242/533) of patients reported pain during the course of disease and 24.2% (129/533) of patients had experienced pain within the past 24 h. The mean average pain intensity by the brief pain inventory was 3.47 ± 1.55. The binary logistic regression analysis showed female gender and stage IV disease were significantly associated with the presence of pain. A total of 74.4% (96/129) of patients reporting pain within 24 h were taking analgesics. The most common reason for patients not using analgesics was that the pain was tolerable (48.2%), while the most common barriers to prescribing opioids as reported by physicians were fear of adverse reactions (43.7%) and fear of addiction (43.1%). Conclusion Despite recognition of the importance of pain control by most physicians and an improvement in cancer pain management, inadequate treatment of cancer pain still exists in lung cancer patients in northern China. High‐quality pain education for both patients and physicians is needed in the future.

Abstract Background Immune checkpoint inhibitors (ICIs) are the standard treatment for advanced lung cancer, but immune‐related adverse events (irAEs) remain poorly understood, especially in a real‐world setting. Methods A multicenter observational study was conducted. Medical records of lung cancer patients treated with ICIs at 26 hospitals from January 1, 2015, to February 28, 2021, were retrieved. Types of ICIs included antiprogrammed cell death 1 or antiprogrammed cell death ligand 1 (PD‐L1) monotherapy, anticytotoxic T‐lymphocyte antigen‐4 monotherapy, or combination therapy. Results In total, 1905 patients with advanced lung cancer were evaluated. The median age was 63 (range 28–87) years, and the male/female ratio was 3.1:1 (1442/463). The primary histological subtype was adenocarcinoma (915). A total of 26.9% (512/1905) of the patients developed 671 irAEs, and 5.8% (110/1905) developed 120 grade 3–5 irAEs. Median duration from ICI initiation to irAEs onset was 56 (range 0–1160) days. The most common irAEs were thyroid dysfunction (7.2%, 138/1905), pneumonitis (6.5%, 124/1905), and dermatological toxicities (6.0%, 115/1905). A total of 162 irAEs were treated with steroids and 11 irAEs led to death. Patients with positive PD‐L1 expression (≥1%) and who received first‐line ICI treatment developed more irAEs. Patients who developed irAEs had a better disease control rate (DCR, 71.3% [365/512] vs. 56.0% [780/1145]; p

In order to increase and stabilize the content of tetramethylpyrazine in liquor, 13 strains of Bacillus were isolated from Maotai Daqu samples, and 5 strains with excellent protease production were selected through the preliminary screening of protease transparent circle test. After a liquid fermentation test, a functional bacterium with high yield of tetramethylpyrazine was screened out. The content of tetramethylpyrazine in the fermentation broth was 12.22 mg/L, which was identified as Bacillus licheniformis GTBL-168. The seed culture solution of this strain was inoculated into the sauce-flavor liquor accumulation culture at 0%, 3%, 5%, and 7% (v/w) to synergize the fermentation with traditional Daqu, and accumulate acetoin and tetramethyl in the glutinous rice grains. The content of pyrazine increased, and the increasing was the largest when the inoculation amount was 7%, and the yield of tetramethylpyrazine increased by 24.88% compared with the control group, the overall changes in physical and chemical indexes were obvious, and the amino nitrogen and total acid increased, the addition of Bacillus licheniformis GTBL-168 had little effect on the microbial structure of the accumulated fermented grains. With the increasing of the inoculation amount, the abundance of Bacillus and other dominant bacteria in the fermented grains also increased. It would lay a foundation for the application of functional bacteria in the process of stacking fermentation.

Abstract Background Central nervous system (CNS) metastases in patients with ALK-positive non-small cell lung cancer (NSCLC) are a cause of substantial morbidity and mortality. Although alectinib had demonstrated promising intracranial efficacy in several clinical trials, data were limited on its CNS activity in real-world settings. Methods In this retrospective study, ALK-positive NSCLC patients with brain metastases (BM) or leptomeningeal metastases (LM) from six hospitals in China were divided into three cohorts based on the treatment history before the administration of alectinib. ALK-TKI-naive patients were enrolled in cohort 1, cohort 2 included patients who experienced intracranial progression with or without extracranial progression after treatment with crizotinib, and cohort 3 included patients who developed progression only in CNS following treatment with other second-generation ALK-TKIs. The definition and evaluation of intracranial and extracranial lesions were based on Response Evaluation Criteria in Solid Tumors version 1.1. Results Sixty-five patients were eligible and included in our study (cohort 1: 20, cohort 2: 32, cohort 3: 13). For the overall population and patients with uncontrolled CNS metastases, similar intracranial response in CNS target lesions was observed: cohort 1: 81.8% and 80%; cohort 2: 76.5% and 86.7%; cohort 3: 42.8% and 33.3%. For patients in these three cohorts, 75% (6/8), 78.6% (11/14), and 83.3% (5/6) were reported to have significant improvement in CNS-related symptoms respectively. The number of patients who were in need of mannitol or corticosteroids decreased remarkably after the treatment of alectinib (p

Abstract Background There is limited data on the clinical outcome, long‐term survival and tolerability of sequential therapy of first‐line crizotinib followed by alectinib in a real‐world setting for Chinese patients with advanced ALK+ NSCLC. Methods The medical records of patients who received sequential therapy with first‐line crizotinib followed by alectinib (no intermittent systemic therapy was allowed between the two ALK‐TKIs) were collected from six centers in China. Combined time treatment to failure (C‐TTF) was defined as the period from the start of crizotinib to the complete discontinuation of alectinib due to any cause. Results A total of 61 patients were included in our study. Fifty‐two patients were switched to alectinib due to disease progression, seven as a result of toxicity, and two due to patient preference. At the time of data cutoff, alectinib treatment was discontinued in 31 patients on account of disease progression while severe adverse events resulted in cessation of alectinib in another two patients. Rebiopsy was conducted in 21 patients following disease progression on alectinib in whom ALK secondary mutation was found in 13 patients. Patients with ALK secondary mutation demonstrated better PFS during treatment with subsequent ALK‐TKIs compared with those without (10.4 vs. 3.1 m, p = 0.0018, HR = 0.08). With a median follow‐up of 34.3 months, C‐TTF was 39.2 months and estimated 5‐year OS was 68.6% in the overall population. Conclusion Sequential therapy with first‐line crizotinib followed by alectinib demonstrated long‐term benefits. Different efficacy in subsequent ALK‐TKI between patients with or without ALK secondary mutation further emphasized the importance of rebiopsy to guide targeted therapy more precisely.

Summary The natural plant product bisabolene serves as a precursor for the production of a wide range of industrially relevant chemicals. However, the low abundance of bisabolene in plants renders its isolation from plant sources non‐economically viable. Therefore, creation of microbial cell factories for bisabolene production supported by synthetic biology and metabolic engineering strategies presents a more competitive and environmentally sustainable method for industrial production of bisabolene. In this proof‐of‐principle study, for the first time, we engineered the oleaginous yeast Yarrowia lipolytica to produce α‐bisabolene, β‐bisabolene and γ‐bisabolene through heterologous expression of the α‐bisabolene synthase from Abies grandis, the β‐bisabolene synthase gene from Zingiber officinale and the γ‐bisabolene synthase gene from Helianthus annuus respectively. Subsequently, two metabolic engineering approaches, including overexpression of the endogenous mevalonate pathway genes and introduction of heterologous multidrug efflux transporters, were employed in order to improve bisabolene production. Furthermore, the fermentation conditions were optimized to maximize bisabolene production by the engineered Y. lipolytica strains from glucose. Finally, we explored the potential of the engineered Y. lipolytica strains for bisabolene production from the waste cooking oil. To our knowledge, this is the first report of bisabolene production in Y. lipolytica using metabolic engineering strategies. These findings provide valuable insights into the engineering of Y. lipolytica for a higher‐level production of bisabolene and its utilization in converting waste cooking oil into various industrially valuable products.

BackgroundCurrently, many targeted drugs are approved for treatment of ALK fusion non-small cell lung cancer. However, it has been previously assumed that patients with 5′ non-oncogenic kinase (5′ NOK) fusion detected by DNA next-generation sequencing (NGS) would not benefit from ALK inhibitors because of lack of an intact kinase domain.Case descriptionA novel 5′ NOK fusion form, ALK-CYP27C1 (A19:C5), was detected by DNA NGS in surgical tissue specimens of a patient with recurrent lung adenosquamous carcinoma. The patient achieved 29 months of progression-free survival with ensartinib treatment. The results of RNA NGS from the same operative tissue identified EML4-ALK (E13:A20) fusion variant type I.ConclusionThis is the first case to provide real-world evidence of effective treatment of a patient with the 5′ NOK fusion form at the DNA level but functional EML4-ALK at the RNA level, illustrating the need for RNA testing in 5′ NOK patients.

Abstract Background In biological cells, promoters drive gene expression by specific binding of RNA polymerase. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for optimizing biosynthetic pathways in metabolic engineering has recently become an active area of research. Results In this study, we systematically detected and characterized the common promoter elements in the unconventional yeast Yarrowia lipolytica, and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. The results indicate that the hybrid promoter strength can be fine-tuned by promoter elements, namely, upstream activation sequences (UAS), TATA box and core promoter. Notably, the UASs of Saccharomyces cerevisiae promoters were reported for the first time to be functionally transferred to Y. lipolytica. Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in Y. lipolytica. By expressing the key pathway gene, ScARO10, with the promoter library, 1.1–30.3 folds increase in the isoamyl alcohol titer over that of the control strain Y. lipolytica Po1g KU70∆ was achieved. Interestingly, the highest titer increase was attained with a weak promoter P UAS1B4-EXPm to express ScARO10. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in Y. lipolytica. Conclusion We envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement.

Itaconic acid (IA) is a high-value organic acid with a plethora of industrial applications. In this study, we seek to develop a microbial cell factory that could utilize waste cooking oil (WCO) as raw material for circular and cost-effective production of the abovementioned biochemical. Specifically, we expressed cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus in either the cytosol or peroxisome of Yarrowia lipolytica and assayed for production of IA on WCO. To further improve production yield, the 10 genes involved in the production pathway of acetyl-CoA, an intermediate metabolite necessary for the synthesis of cis-aconitic acid, were individually overexpressed and investigated for their impact on IA production. To minimize off-target flux channeling, we had also knocked out genes related to competing pathways in the peroxisome. Impressively, IA titer up to 54.55 g/L was achieved in our engineered Y. lipolytica in a 5 L bioreactor using WCO as the sole carbon source.

Two-years-old Sequoia sempervirens seedlings were foliar sprayed once and twice with chlorocholine chloride (CCC) at 0, 100, 500, 1 000, 1 500 and 2 000 mg.l-1. The purpose was to investigate the effect of CCC on the growth and photosynthetic activity of S. sempervirens seedlings under natural cooling and drought in autumn and winter. The findings showed that the increments of plant height and crown diameter were significantly decreased with the increase of chlorocholine chloride concentration, and the increment of root collar diameter, net photosynthetic rate, actual photochemical quantum yield and photosynthetic electron transport rate showed the trend of increasing first and then decreasing, and reached the maximum at concentrations of 1 000~2 000 mg.l-1. There was not a significant difference between two applications and single application. It suggests that 1 000~2 000 mg.l-1 chlorocholine chloride can protect the photosynthetic activity of S. sempervirens seedlings and alleviate the stress induced by low temperatures and drought in autumn and winter.

Distilled jujube liquor is an alcoholic beverage made from jujube, which has a unique flavor and a sweet taste. The purpose of this study was to explore the effect of mixed fermentation on the quality of distilled jujube liquor by comparing the performance of mixed fermentation between S. cerevisiae, Pichia pastoris and Lactobacillus. The results showed that there were significant differences in the quality of the jujube liquor between the combined strains. Moreover, Lactobacillus increased and P. pastoris reduced the total acid content. The results from an E-nose showed that the contents of methyl, alcohol, aldehyde, and ketone substances in the test bottle decreased significantly after decanting, while the contents of inorganic sulfide and organic sulfide increased. Fifty flavor compounds were detected, including nineteen esters, twelve alcohols, seven ketones, six aldehydes, three alkenes, one furan, one pyridine, and one acid. There were no significant differences in the type or content of flavor compounds. However, PLS-DA showed differences among the samples. Eighteen volatile organic compounds with variable importance in projection values > 1 were obtained. There were sensory differences among the four samples. Compared with the sample fermented with only S. cerevisiae, the samples co-fermented with Lactobacillus or with P. pastoris had an obvious bitter taste and mellow taste, respectively. The sample fermented by all three strains had a prominent fruity flavor. Except for the sample fermented with only S. cerevisiae, the jujube flavor was weakened to varying degrees in all samples. Co-fermentation could be a valuable method to improve the flavor quality of distilled jujube liquor. This study revealed the effects of different mixed fermentation modes on the sensory flavor of distilled jujube liquor and provided a theoretical basis for the establishment of special mixed fermentation agents for distilled jujube liquor in the future.

Abstract Background Limonene is an important biologically active natural product widely used in the food, cosmetic, nutraceutical and pharmaceutical industries. However, the low abundance of limonene in plants renders their isolation from plant sources non-economically viable. Therefore, engineering microbes into microbial factories for producing limonene is fast becoming an attractive alternative approach that can overcome the aforementioned bottleneck to meet the needs of industries and make limonene production more sustainable and environmentally friendly. Results In this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was successfully engineered to produce both d-limonene and l-limonene by introducing the heterologous d-limonene synthase from Citrus limon and l-limonene synthase from Mentha spicata, respectively. However, only 0.124 mg/L d-limonene and 0.126 mg/L l-limonene were produced. To improve the limonene production by the engineered yeast Y. lipolytica strain, ten genes involved in the mevalonate-dependent isoprenoid pathway were overexpressed individually to investigate their effects on limonene titer. Hydroxymethylglutaryl-CoA reductase (HMGR) was found to be the key rate-limiting enzyme in the mevalonate (MVA) pathway for the improving limonene synthesis in Y. lipolytica. Through the overexpression of HMGR gene, the titers of d-limonene and l-limonene were increased to 0.256 mg/L and 0.316 mg/L, respectively. Subsequently, the fermentation conditions were optimized to maximize limonene production by the engineered Y. lipolytica strains from glucose, and the final titers of d-limonene and l-limonene were improved to 2.369 mg/L and 2.471 mg/L, respectively. Furthermore, fed-batch fermentation of the engineered strains Po1g KdHR and Po1g KlHR was used to enhance limonene production in shake flasks and the titers achieved for d-limonene and l-limonene were 11.705 mg/L (0.443 mg/g) and 11.088 mg/L (0.385 mg/g), respectively. Finally, the potential of using waste cooking oil as a carbon source for limonene biosynthesis from the engineered Y. lipolytica strains was investigated. We showed that d-limonene and l-limonene were successfully produced at the respective titers of 2.514 mg/L and 2.723 mg/L under the optimal cultivation condition, where 70% of waste cooking oil was added as the carbon source, representing a 20-fold increase in limonene titer compared to that before strain and fermentation optimization. Conclusions This study represents the first report on the development of a new and efficient process to convert waste cooking oil into d-limonene and l-limonene by exploiting metabolically engineered Y. lipolytica strains for fermentation. The results obtained in this study lay the foundation for more future applications of Y. lipolytica in converting waste cooking oil into various industrially valuable products.

Shikimic acid (SA) is a key intermediate in the aromatic amino-acid biosynthetic pathway, as well as an important precursor for synthesizing many valuable antiviral drugs. The asymmetric reduction of 3-dehydroshikimic acid (DHS) to SA is catalyzed by shikimate dehydrogenase (AroE) using NADPH as the cofactor; however, the intracellular NADPH supply limits the biosynthetic capability of SA. Glucose dehydrogenase (GDH) is an efficient enzyme which is typically used for NAD(P)H regeneration in biocatalytic processes. In this study, a series of NADPH self-sufficient whole-cell biocatalysts were constructed, and the biocatalyst co-expressing Bmgdh–aroE showed the highest conversion rate for the reduction of DHS to SA. Then, the preparation of whole-cell biocatalysts by fed-batch fermentation without supplementing antibiotics was developed on the basis of the growth-coupled l-serine auxotroph. After optimizing the whole-cell biocatalytic conditions, a titer of 81.6 g/L SA was obtained from the supernatant of fermentative broth in 98.4% yield (mol/mol) from DHS with a productivity of 40.8 g/L/h, and cofactor NADP+ or NADPH was not exogenously supplemented during the whole biocatalytic process. The efficient relay-race synthesis of SA from glucose by coupling microbial fermentation with a biocatalytic process was finally achieved. This work provides an effective strategy for the biosynthesis of fine chemicals that are difficult to obtain through de novo biosynthesis from renewable feedstocks, as well as for biocatalytic studies that strictly rely on NAD(P)H regeneration.

Abstract Fatty acid ethyl esters (FAEEs) are fatty acid‐derived molecules and serve as an important form of biodiesel. The oleaginous yeast Yarrowia lipolytica is considered an ideal host platform for the production of fatty acid‐derived products due to its excellent lipid accumulation capacity. In this proof‐of‐principle study, several metabolic engineering strategies were applied for the overproduction of FAEE biodiesel in Y. lipolytica. Here, chromosome‐based co‐overexpression of two heterologous genes, namely, PDC1 (encoding pyruvate decarboxylase) and ADH1 (encoding alcohol dehydrogenase) from Saccharomyces cerevisiae, and the endogenous GAPDH (encoding glyceraldehyde‐3‐phosphate dehydrogenase) gene of Y. lipolytica resulted in successful biosynthesis of ethanol at 70.8 mg/L in Y. lipolytica. The engineered Y. lipolytica strain expressing the ethanol synthetic pathway together with a heterologous wax ester synthase (MhWS) exhibited the highest FAEE titer of 360.8 mg/L, which is 3.8‐fold higher than that of the control strain when 2% exogenous ethanol was added to the culture medium of Y. lipolytica. Furthermore, a synthetic microbial consortium comprising an engineered Y. lipolytica strain that heterologously expressed MhWS and a S. cerevisiae strain that could provide ethanol as a substrate for the production of the final product in the final engineered Y. lipolytica strain was created in this study. Finally, this synthetic consortium produced FAEE biodiesel at a titer of 4.8 mg/L under the optimum coculture conditions.

Abstract Background Microbial biofuel production provides a promising sustainable alternative to fossil fuels. 1-Butanol is recognized as an advanced biofuel and is gaining attention as an ideal green replacement for gasoline. In this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was first engineered with a heterologous CoA-dependent pathway and an endogenous pathway, respectively. Results The co-overexpression of two heterologous genes ETR1 and EutE resulted in the production of 1-butanol at a concentration of 65 μg/L. Through the overexpression of multiple 1-butanol pathway genes, the titer was increased to 92 μg/L. Cofactor engineering through endogenous overexpression of a glyceraldehyde-3-phosphate dehydrogenase and a malate dehydrogenase further led to titer improvements to 121 μg/L and 110 μg/L, respectively. In addition, the presence of an endogenous 1-butanol production pathway and a gene involved in the regulation of 1-butanol production was successfully identified in Y. lipolytica. The highest titer of 123.0 mg/L was obtained through this endogenous route by combining a pathway gene overexpression strategy. Conclusions This study represents the first report on 1-butanol biosynthesis in Y. lipolytica. The results obtained in this work lay the foundation for future engineering of the pathways to optimize 1-butanol production in Y. lipolytica.

Silicon (Si) is a macroelement in plants. The biological effects and mitigation mechanisms of silicon under environmental stress have become hot topics. The main objectives of this study were to elucidate the roles of Si in alleviating the effects on the phenotype, micromorphology and anatomy of the leaves of rice seedlings under acid rain stress. The results indicated that the combined or single effects of Si and simulated acid rain (SAR) stress on rice roots depended on the concentration of Si and the intensity of the SAR stress. The combined or single effects of the moderate concentration of Si (2.0 mM) and light SAR (pH 4.0) enhanced the growth of the rice leaves and the development of the mesophyll cells, and the combined effects were stronger than those of the single treatments. The high concentration of Si (4.0 mM) and severe SAR (pH 3.0 or 2.0) exerted deleterious effects. The incorporation of Si (2.0 or 4.0 mM) into SAR at pH values of 3.0 or 2.0 promoted rice leaf growth, decreased necrosis spots, maintained the structure and function of the mesophyll cells, increased the epicuticular wax content and wart-like protuberance (WP) density, and improved the stomatal characteristics of the leaves of rice seedlings more than the SAR only treatments. The alleviatory effects observed with a moderate concentration of Si (2.0 mM) were better than the effects obtained with the high concentration of Si (4.0 mM). The alleviatory effects were due to the enhancement of the mechanical barriers in the leaf epidermis.

Silicon (Si) has an important function in reducing the damage of environmental stress on plants. Acid rain is a serious abiotic stress factor, and Si can alleviate the stress induced by acid rain on plants. Based on these assumptions, we investigated the effects of silicon on the growth, root phenotype, mineral element contents, hydrogen peroxide (H2O2) and antioxidative enzymes of rice (Oryza sativa L.) seedling roots under simulated acid rain (SAR) stress. The results showed that the combined or single effects of Si and/or SAR on rice roots depend on the concentration of Si and the pH of the SAR. The combined or single effects of a low or moderate concentration of Si (1.0 or 2.0 mM) and light SAR (pH 4.0) enhanced the growth of rice roots, and the combined effects were stronger than those of the single treatment. A high concentration of Si (4.0 mM) or severe SAR (pH 2.0) exerted deleterious effects. The incorporation of Si (1.0, 2.0 or 4.0 mM) into SAR with pH 3.0 or 2.0 promoted the rice root growth, decreased the H2O2 content, increased the Si concentration and the superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX) activities, maintained the balance of mineral element (K, Ca, Mg, Fe, Zn, and Cu) concentrations in the roots of rice seedlings compared with SAR alone. The alleviatory effects observed with a moderate concentration of Si (2.0 mM) were better than the effects obtained with a low or high concentration of Si (1.0 or 4.0 mM). The observed effects were due to disruptions in the absorption and utilization of mineral nutrients and impacts on the activity of antioxidant enzymes in roots, and this conclusion suggests that the degree of rice root damage caused by acid rain might be attributed to not only acid rain but also the level of Si in the soil.

The crop intercepted photosynthetically active radiation (PAR) and radiation use efficiency (RUE) vary markedly in different intercropping systems. The HHLA (horizontally homogeneous leaf area) and ERCRT (extended row crop radiation transmission) models have been established to calculate the intercepted PAR for intercrops. However, there is still a lack of study on the intercepted PAR and RUE under different intercropping configurations using different models. To evaluate the intercepted PAR and RUE in maize and soybean under different intercropping systems, we tested different strip intercropping configurations (SI1, SI2, and SI3 based on ERCRT model) and a row intercropping configurations (RI based on HHLA model) in comparison to monoculture. Our results showed that the intercepted PAR and RUE of intercropping systems were all higher than those of monoculture. The soybean intercepted PAR in strip intercropping was 1.35 times greater than that in row intercropping. In row intercropping (RI), the lack of soybean intercepted PAR resulted in a significant reduction of soybean dry matter. Therefore, it is not the recommended configuration for soybean. In strip intercropping patterns, with the distance between maize strip increased by 0.2 m, the intercepted PAR of soybean increased by 20%. The SI2 (maize row spacing at 0.4 m and the distance between maize strip at 1.6 m) was the recommended configuration to achieve the highest value of intercepted PAR and RUE among tested strip intercropping configurations. The method of dry matter estimation using intercepted PAR and RUE is useful in simulated experiments. The simulated value was verified in comparison with experimental data, which confirmed the credibility of the simulation model. Moreover, it also provides help in the development of functional-structural plant model (FSPM).

Higher order singular value decomposition (HOSVD) is an efficient data-driven decomposition technique, and shows the salient ability in the representation of high-dimensional data and feature extraction. In addition, fuzzy reasoning can solve the uncertainties of the source images' contributions to the fused image and is easy to apply. Motivated by the advantages mentioned above, a new HOSVD and fuzzy reasoning-based multi-focus image fusion method is proposed. Firstly, sub-tensor is constructed by two image patches separately from the two multi-focus images and HOSVD is employed to obtain the decomposition coefficients of image patches. Secondly, the weighted average fusion rule based on fuzzy reasoning is proposed for fusing the decomposition coefficients, and fuzzy reasoning rule is designed based on average energy, regional energy and match degree. Finally, the fused image is achieved by the inversing HOSVD. Experimental results indicate that the proposed method performs better than other methods both visually and quantitatively.

Alveolar echinococcosis is considered to be one of the most potentially lethal parasitic zoonotic diseases. However, the molecular mechanisms by which Echinococcus multilocularis interacts with hosts are poorly understood, hindering the prevention and treatment of this disease. Due to the great advantages of cell culture systems for molecular research, numerous attempts have been made to establish primary cell cultures for E. multilocularis. In this study we developed a simple, rapid, and economical method that allows E. multilocularis metacestode tissue blocks to generate daughter vesicles without the continuous presence of host feeder cells in a regular medium. We performed anaerobic, hypoxic (1% O2), normoxic, and semi-anaerobic (in sealed tubes) cultures and found that E. multilocularis metacestode tissues can produce daughter vesicles only in the sealed tubes after 4 wk of incubation. The daughter vesicles cultivated in this system were remarkably enlarged under anaerobic conditions after 8 days of culture, whereas vesicles cultured under hypoxic (1% O2) and normoxic conditions showed only a mild increase in volume. Our in vitro cultivated vesicles showed strong viability and could be used to test antiparasitic drugs, isolate primary cells, and infect animals. [ABSTRACT FROM AUTHOR]

Reinforcing fibers play a key role in improving the tribological properties of friction polymer composites. Cellulose reinforcing fibers were extracted from rapeseed straw waste and prepared as friction composites based on wet granulation, and the effects of heat and rapeseed straw fiber (RSF) additions on the tribological properties were systematically investigated. The addition of RSF induced an important transformation of the major wear mechanism to abrasive wear, especially at 9 wt%, where the abundant tribo-films contributed to the stability of the friction coefficients (fade rate 6.8%, recovery rate 106.6%), resulting in a 37.5% increase in wear resistance compared to no addition. Furthermore, XRD, FTIR and SEM analyses showed that the silane-ethanol solution modification contributed to the good performance of RSF. Highlights • Rapeseed straw fiber (RSF) and granulation technology for polymer composites. • Addition of RSF transforms adhesive and fatigue wear into abrasive wear. • Friction models are developed to clarify the wear mechanism transformation. • A 37.5% increase in wear resistance was achieved when RSF was added at 9 wt%. [ABSTRACT FROM AUTHOR]