Exosomes derived from programmed cell death: mechanism and biological significance.

Exosomes are nanoscale extracellular vesicles present in bodily fluids that mediate intercellular communication by transferring bioactive molecules, thereby regulating a range of physiological and pathological processes. Exosomes can be secreted from nearly all cell types, and the biological function of exosomes is heterogeneous and depends on the donor cell type and state. Recent research has revealed that the levels of exosomes released from the endosomal system increase in cells undergoing programmed cell death. These exosomes play crucial roles in diseases, such as inflammation, tumors, and autoimmune diseases. However, there is currently a lack of systematic research on the differences in the biogenesis, secretion mechanisms, and composition of exosomes under different programmed cell death modalities. This review underscores the potential of exosomes as vital mediators of programmed cell death processes, highlighting the interconnection between exosome biosynthesis and the regulatory mechanisms governing cell death processes. Furthermore, we accentuate the prospect of leveraging exosomes for the development of innovative biomarkers and therapeutic strategies across various diseases.

Silica-induced macrophage pyroptosis propels pulmonary fibrosis through coordinated activation of relaxin and osteoclast differentiation signaling to reprogram fibroblasts.

Silica nanoparticle (SiNP) exposure induces severe pulmonary inflammation and fibrosis, but the pathogenesis remains unclear, and effective therapies are currently lacking. To explore the mechanism underlying SiNPs-induced pulmonary fibrosis, we constructed in vivo silica exposure animal models and in vitro models of silica-induced macrophage pyroptosis and fibroblast transdifferentiation. We found that SiNP exposure elicits upregulation of pulmonary proteins associated with pyroptosis, including NLRP3, ASC, IL-1ß, and GSDMD, while the immunofluorescence staining co-localized NLRP3 and GSDMD with macrophage-specific biomarker F4/80 in silica-exposed lung tissues. However, the NLRP3 inhibitor MCC950 and classical anti-fibrosis drug pirfenidone (PFD) were found to be able to alleviate silica-induced collagen deposition in the lungs. In in vitro studies, we exposed the fibroblast to a conditioned medium from silica-induced pyroptotic macrophages and found enhanced expression of α-SMA, suggesting increased transdifferentiation of fibroblast to myofibroblast. In line with in vivo studies, the combined treatment of MCC950 and PFD was demonstrated to inhibit the expression of α-SMA and attenuate fibroblast transdifferentiation. Mechanistically, we adopted high throughput RNA sequencing on fibroblast with different treatments and found activated signaling of relaxin and osteoclast differentiation pathways, where the expression of the dysregulated genes in these two pathways was examined and found to be consistently altered both in vitro and in vivo. Collectively, our study demonstrates that SiNP exposure induces macrophage pyroptosis, which subsequently causes fibroblast transdifferentiation to myofibroblasts, in which the relaxin and osteoclast differentiation signaling pathways play crucial roles. These findings may provide valuable references for developing new therapies for pulmonary fibrosis.

Engineered Exosome for Drug Delivery: Recent Development and Clinical Applications.

Exosomes are nano-sized membrane vesicles that transfer bioactive molecules between cells and modulate various biological processes under physiological and pathological conditions. By applying bioengineering technologies, exosomes can be modified to express specific markers or carry therapeutic cargo and emerge as novel platforms for the treatment of cancer, neurological, cardiovascular, immune, and infectious diseases. However, there are many challenges and uncertainties in the clinical translation of exosomes. This review aims to provide an overview of the recent advances and challenges in the translation of engineered exosomes, with a special focus on the methods and strategies for loading drugs into exosomes, the pros and cons of different loading methods, and the optimization of exosome production based on the drugs to be encapsulated. Moreover, we also summarize the current clinical applications and prospects of engineered exosomes, as well as the potential risks and limitations that need to be addressed in exosome engineering, including the standardization of exosome preparation and engineering protocols, the quality and quantity of exosomes, the control of drug release, and the immunogenicity and cytotoxicity of exosomes. Overall, engineered exosomes represent an exciting frontier in nanomedicine, but they still face challenges in large-scale production, the maintenance of storage stability, and clinical translation. With continuous advances in this field, exosome-based drug formulation could offer great promise for the targeted treatment of human diseases.

Enhanced Pavlovian-to-instrumental transfer in internet gaming disorder.

Background and aims: The Pavlovian-to-instrumental transfer (PIT) effect is a phenomenon that Pavlovian conditioned cues that could influence one's instrumental behavior. In several substance and behavioral addictions, such as tobacco use disorder and gambling disorder, addiction-related cues could promote independently trained instrumental drug-seeking/drug-taking behaviors, indicating a specific PIT effect. However, it is unclear whether Internet gaming disorder (IGD) would show a similar change in PIT effects as other addictions. The study aimed to explore the specific PIT effects in IGD. Methods: We administrated a PIT task to individuals with IGD (n = 40) and matched health controls (HCs, n = 50), and compared the magnitude of specific PIT effects between the two groups. The severity of the IGD symptoms was assessed by the Chinese version 9-item Internet Gaming Disorder Scale (IGDS) and the Internet Addiction Test (IAT). Results: We found that: (1) related to the HCs group, the IGD group showed enhanced specific PITgame effects, where gaming-related cues lead to an increased choice rate of gaming-related responses; (2) in the IGD group, the magnitude of specific PITgame effects were positively correlated with IAT scores (rho = 0.39, p = 0.014). Discussion and Conclusions: Individuals with IGD showed enhanced specific PIT effects related to HCs, which were associated with the severity of addictive symptoms. Our results highlighted the incentive salience of gaming-related cues in IGD.

Manufacture and evaluation of a HER2-positive breast cancer immunotoxin 4D5Fv-PE25.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) positive breast cancer is an aggressive subtype, accounting for around 20% of all breast cancers. The development of HER2-targeted therapy has substantially improved patient outcomes. Nevertheless, the increasing rate of side effects and resistance to targeted drugs limit their efficacy in clinical practice. In this study, we designed and synthesized a new immunotoxin, 4D5Fv-PE25, which targets HER2-positive breast cancer, and evaluated its effectiveness in vitro and in vivo. RESULTS: The 4D5Fv-PE25 was expressed in high-density Escherichia coli (E. coli.) using the fermentor method and refined via hydrophobicity, ion exchange, and filtration chromatography, achieving a 56.06% recovery rate. Additionally, the semi-manufactured product with 96% purity was prepared into freeze-dried powder by the lyophilized process. Flow cytometry was used to detect the expression of HER2 in SK-BR-3, BT-474, MDA-MB-231, and MDA-MB-468 breast cancer cell lines. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method was used for cytotoxicity assay, and the half-maximal inhibitory concentration (IC50) of 4D5Fv-PE25 lyophilized products to HER2-positive cell line SK-BR-3 was 12.53 ng/mL. The 4D5Fv-PE25 was injected into xenograft tumor mice via the tail vein on the 1st, 4th, and 8th day, it indicated that the growth of tumor volume was effectively inhibited for 24 days, although the 4D5Fv-PE25 was metabolized within 60 min by measuring the release of 3 H-Thymidine radiation. CONCLUSION: we succeeded in producing the 4D5Fv-PE25 freeze-dried powder using the prokaryotic expression method, and it could be employed as a potential drug for treating HER2-positive breast cancer.

[Corrigendum] FPR2 serves a role in recurrent spontaneous abortion by regulating trophoblast function via the PI3K/AKT signaling pathway.

Subsequently to the publication of the above paper, an interested reader drew to the authors' attention that the '0 h/si-NC + Solvent' and '0 h/si-FPR2 + Solvent' data panels shown in Fig. 4B on p. 7 appeared to contain overlapping sections of data, such that they were potentially derived from the same original source where these panels was intended to show the results from differently performed experiments. After having conducted an independent investigation of the figures in the Editorial Office, it was identified that sections of the 'si-NC + Solvent' and 'si-FPR2 + LY294002' data panels in Fig. 4C also contained overlapping data. After having asked the authors to provide an explanation of these data, they realized that this figure was inadvertently assembled incorrectly. They were, however, able to consult their original data, and the revised version of Fig. 4, containing the correct data panel for the 'si-FPR2 + LY294002' experiment in Fig. 4C and complete data from one of the alternatively performed experiments in Fig. 4B, is shown on the next page. Note that these errors did not adversely affect either the results or the overall conclusions reported in this study. All the authors agree with the publication of this corrigendum, and are grateful to the Editor of Molecular Medicine Reports for allowing them the opportunity to publish this. They also wish to apologize to the readership of the Journal for any inconvenience caused. [Molecular Medicine Reports 24: 838, 2021; DOI: 10.3892/mmr.2021.12478].

Proteomic analysis of amniotic fluid to identify potential targets predicting preterm delivery.

Preterm delivery is a common complication of pregnancy which leads to significant neonatal mortality and morbidity. Identifying predictive markers linked to spontaneous preterm delivery (SPTD) is important for effective treatment and prevention of PTD. To explore potential biomarkers related to SPTD, we performed proteomics analysis in amniotic fluid (AF). In total, we enrolled 30 pregnant women with singleton gestation who underwent clinically indicated amniocentesis at 15-24 weeks of gestation. LC-MS analysis was used to analyze the AF samples of 10 women with SPTD < 34 weeks after cervix cerclage (Preterm group), 10 women with term delivery (TD) ≥ 34 weeks after cervix cerclage (Term group), and 10 women who delivered at term (Normal group). ELISA validation was performed for candidate proteins in a second independent cohort. As a result, we identified 44 differentially expressed proteins (DEPs, P < 0.05) via proteomic analysis, and based on that, 9 primary pathways were also determined in SPTD. Results of the ELISA assay confirmed that the increased concentration of Serpin A1, decreased concentrations of Renin and IGFBP4 were significantly associated with SPTD at ≤34 weeks.

Molecular and physiologic mechanisms of advanced ripening by trunk girdling at early veraison of 'Summer Black' grape.

Although the effects of girdling on grape berry development have been widely studied, the underlying mechanisms are poorly understood, especially at the molecular level. This study investigated the effect of trunk girdling on grape (Vitis L.) berry maturation. Girdling was performed on 5-year-old 'Summer Black' grapevines at early veraison, and transcriptional and physiologic analyses were performed. Trunk girdling promoted sugar accumulation and color development in berries and accelerated berry ripening by 25 days. Genes related to sucrose cleavage and polysaccharide degradation were upregulated at the transcriptional level, which was associated with increased monosaccharide accumulation and berry softening. Anthocyanin biosynthesis and accumulation were also enhanced by trunk girdling through the upregulation of anthocyanin biosynthesis genes including phenylalanine ammonia-lyase and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT). The increased expression of two VvUFGT genes was accompanied by the upregulation of VvMYBA2 under girdling. The upregulation of genes involved in ethylene biosynthesis and hormone (abscisic acid and brassinosteroid) responses and downregulation of genes involved in indoleacetic acid biosynthesis and response may have also promoted berry ripening in the girdling group. A total of 120 differentially expressed transcription factor genes from 29 gene families including MYB, ERF, and MYB-related were identified in the girdling group, which may participate in the regulation of berry development and ripening. These results provide molecular-level insight into the positive effects of trunk girdling on berry development in grapes.

Human placenta-based genome-wide mRNA sequencing to identify TEK/IGF1/CSF1/ANGPT2 as crucial segments in the pathogenesis of pre-eclampsia.

Pre-eclampsia is a pregnancy-specific disease commonly occurring in late pregnancy and has always been threatening maternal and fetal lives, yet the etiology and pathogenesis of pre-eclampsia are still uncertain. To depict the overall changes of genes at the genome-wide level and identify potential biomarkers for early diagnosis of pre-eclampsia, we conducted this study by collecting placenta samples donated by six pregnancy women, among whom three healthy women were included as controls and three women were diagnosed with pre-eclampsia. The placental sample tissues were then subjected to high-throughput sequencing. Furthermore, we proceeded with bioinformatics analysis and formulated the hypothesis of pre-eclampsia development and verified the potential targets of pre-eclampsia by immunohistochemistry. Demographically, we found that the baseline characteristics of study subjects were highly homogeneous except for gestational weeks and blood pressure, where the blood pressure was higher and gestational weeks were shorter in the pre-eclampsia group (systolic blood pressure 123.33 ± 4.62 vs. 148.67 ± 3.79 mmHg, p = 0.046; diastolic blood pressure 79.00 ± 5.20 vs. 88.33 ± 2.89 mmHg, p = 0.068; gestational weeks 39.33 ± 1.03 vs. 35.76 ± 2.41, p = 0.050). Specific pathological changes were identified, shown as syncytial knots, fibrinoid necrosis, perivillous fibrin deposition, and vasculitis. For high-throughput sequencing, a total of 1,891 dysregulated genes were determined, of which 960 genes were downregulated and 931 genes were upregulated. The bioinformatics analysis indicated that these genes, with different molecular functions in different parts of cells, were primarily responsible for endothelium development and vascular process in the circulatory system, and more than 10 signaling pathways were involved. By focusing on the PI3K-Akt signaling pathway, Rap1 signaling pathway, and disease enrichment analysis item pre-eclampsia, TEK, CSF1, IGF1, and ANGPT2 were identified to promote the development of pre-eclampsia. After confirming the placental expression of these genes at the protein level, we proposed the pathogenesis of pre-eclampsia as follows: the downregulation of TEK, CSF1, IGF1, and ANGPT2 may inhibit trophoblast proliferation and affect the remodeling of spiral arteries, causing maternal and fetal malperfusion and impeding nutrient exchange, thereby leading to clinical manifestations of pre-eclampsia.

Suppression of FPR2 expression inhibits inflammation in preeclampsia by improving the biological functions of trophoblast via NF-κB pathway.

PURPOSE: The dysfunction of trophoblast during inflammation plays an important role in PE. Formyl peptide receptor 2 (FPR2) plays crucial roles in the development of inflammation-associated disease. This present study aimed to explore the effect of FPR2 on a trophoblast cellular model of preeclampsia. METHODS: The expression of FPR2 in placenta was detected by immunohistochemical staining and western blotting. Transfection of siRNA was used to knockdown FPR2 in HTR-8/SVneo cells. Inflammatory cytokines were detected by ELISA. CCK8, Transwell, wound healing, FACS and tube formation assays were performed to observe the abilities of cell proliferation, migration, invasion, apoptosis and angiogenesis. Western blotting was implemented to clarify that NF-κB signaling pathway was downstream of FPR2. RESULTS: The expression levels of FPR2 were higher in placental tissues of patients with PE. Knockdown of FPR2 expression by siFPR2 or inhibition of its activity by WRW4 decreased the release of proinflammatory cytokines in HTR8/SVneo cells treated with LPS. Knockdown of FPR2 expression or inhibition of its activity further reversed the LPS-induced attenuation of the proliferation, migration, invasion and angiogenesis and increase in apoptosis in HTR8/SVneo cells. Moreover, the NF-κB signaling pathway was activated in both placental tissues of patients with PE and LPS-treated HTR8/SVneo cells. However, the activation was attenuated when FPR2 was knocked down or inhibited. CONCLUSION: Suppression of FPR2 expression alleviated the effects of inflammation induced by LPS on trophoblasts via the NF-κB signaling pathway, which provided a novel and potential strategy for the treatment of PE.

Predicting the risk of fetal macrosomia at pregnancy in Shandong province: a case-control study.

BACKGROUND AND AIM: Macrosomia is used to describe an infant born with excessively high weight, and it brings lots of unexpected risks in clinical work. Macrosomia causes considerable challenges for both physicians and pregnant women. Our objectives were to identify factors in gravida to be associated with the risk of macrosomia, to guide clinical prevention and treatment. METHODS: The study assessed risk factors of macrosomia by comparison with normal birth weight neonates, and a case-control study was conducted at Shandong Provincial Maternity and Child Healthcare Hospital. We followed and selected the relevant indicators of gravida who gave birth to macrosomia or normal infants, and applied statistical analysis to identify clinical indicators related to macrosomia. RESULTS: Maternal blood glucose (OR 3.88 (1.07, 14.15)), history of abnormal conception (OR 18.44 (1.05, 322.89)), situation of menarche (OR 13.53 (1.28, 142.66)), and menstrual cycle of gravida (OR 13.24 (1.17, 150.24)) were significant influencing factors of macrosomia, but did not appear in the univariate analysis. Adding gestational age at delivery (OR 4.00 (1.45, 11.09)), triglyceride (OR 0.01 (<0.01, 0.40)), and MBI (OR 46.35 (2.08, >99.99)) of pregnant women, the area under the curve (AUC) curve was drawn for forecasting the risk of macrosomia, and the value of AUC was 0.9174. The triglyceride blood index of pregnant women was the only one that was inversely proportional to the probability of giving birth to macrosomic infants. The low-density lipoprotein (LDL) (OR 0.29 (0.12, 0.72)) and total cholesterol (OR 0.39 (0.20, 0.75)) were important factors in univariate analysis, and both of them were negative correlation factors of macrosomia. All influencing factors in multivariate analysis were selected for drawing a receiver operating characteristic (ROC) curve, and the value of the AUC was 0.9174. CONCLUSIONS: This analysis could therefore accurately predict the risk of pregnant women who would deliver macrosomic infants.

FPR2 serves a role in recurrent spontaneous abortion by regulating trophoblast function via the PI3K/AKT signaling pathway.

Recurrent spontaneous abortion (RSA) effects both the physical and mental health of women of reproductive age. Trophoblast dysfunction may result in RSA due to shallow placental implantation. The mechanisms underlying formyl peptide receptor 2 (FPR2) on the biological functions of trophoblasts remain to be elucidated. The present study aimed to explore the potential functions of FPR2, a G protein­coupled receptor, in placental trophoblasts. The location and expression levels of FPR2 in the villi tissue of patients with RSA were detected using immunohistochemical staining, reverse transcription­quantitative PCR and western blotting. Following the transfection of small interfering RNA targeting FPR2 in HTR­8/SVneo cells, a Cell Counting Kit­8 assay was used to determine the levels of cell viability. Flow cytometry was used to examine the levels of cell apoptosis and gap closure and Transwell assays were carried out to evaluate the levels of cell migration and invasion. A tube formation assay was performed to detect the levels of capillary­like structure formation. Western blotting was used to detect the expression levels of proteins in the associated signaling pathways. The expression of FPR2 was present in villi trophoblasts and was markedly increased in patients with RSA. The levels of trophoblast invasion, migration and tube formation were markedly increased following FPR2 knockdown, whereas the levels of apoptosis were markedly decreased. In addition, FPR2 knockdown caused an increase in the phosphorylation levels of AKT and PI3K. Thus, FPR2 may be involved in the regulation of trophoblast function via the PI3K/AKT signaling pathway. The results of the present study provided a theoretical basis for the use of FPR2 as a target for the treatment of trophoblast­associated diseases, such as RSA.

Chemical conjugation of FITC to track silica nanoparticles in vivo and in vitro: An emerging method to assess the reproductive toxicity of industrial nanomaterials.

Previous studies have demonstrated that silica nanoparticle (SiNP) exposure induces pulmonary and cardiovascular diseases, yet their transportation and degradation in vivo have not been fully elucidated. From the perspective of reproduction, this study was implemented to examine the uterine accumulation of SiNP and explore its reproductive toxicity and pathogenic mechanisms. First, we coupled FITC onto SiNPs and intratracheally instilled them into pregnant mice on the fifth gestational day, and the toxic effect of SiNP was evaluated in vitro and in vivo. It was found that SiNP penetrated the trophoblast membrane, leading to apoptosis and suppression of cell proliferation, tube formation, and invasion in a dose-dependent manner. Mechanistically, SiNP dysregulated the expression of Scd1, Slc27a1, and Cpt1a, and induced over synthesis and efflux obstruction of fatty acid through the PPARγ signaling pathway. The downregulation of Caspase-3 triggered apoptosis of trophoblast, which was causally associated with intracellular fatty acid accumulation as revealed by the correlation analysis. Besides, SiNP induced uterine inflammation in vivo, which aggravated with the observation prolongation within 24 h. Overall, SiNPs were visualized by coupling with FITC, and the uterine accumulation of SiNP induced fatty acid metabolic disorder, biological dysfunction, and trophoblast apoptosis, which were mediated in part by the PPARγ signaling pathway. These findings would contribute to understanding the environmental impacts of SiNP better, as well as the development of control measures for environmental pollution.

Effect of RvD1/FPR2 on inflammatory response in chorioamnionitis.

Chorioamnionitis (CAM), as a common intrauterine infectious disease, is the leading cause of premature birth, stillbirth, neonatal infection and sepsis. The formyl peptide receptor 2 (FPR2) is a member of GPCRs widely distributed in a variety of tissues and is associated with many inflammatory diseases. With the discovery of FPR2 in human placenta, the possibility of exploring the function of FPR2 in obstetrics is evolving. The Resolvin D1 (RvD1) plays an important role in the resolution of inflammation by combining with FPR2. In this study, we evaluated the role of FPR2 and RvD1 in CAM, not only in the human placenta but also in mouse models. The expression of FPR2 increased in the placenta of CAM patients and the downstream PPARγ/NF-κB signalling changed accordingly. Moreover, Fpr2-/- mice were highly susceptible to LPS, displaying a worse CAM symptom, compared with WT mice. By establishing a model of trophoblast inflammation in vitro, it was confirmed that RvD1 rescued the effect of LPS on inflammation by combining with FPR2 and its downstream PPARγ/NF-κB pathway. Otherwise, RvD1 improved the preterm labour in a mouse model of CAM induced by LPS. Altogether, these findings show that RvD1 alleviated the inflammation of trophoblast in vivo and in vitro through FPR2/PPARγ/NF-κB pathway, suggesting RvD1/FPR2 might be a novel therapeutic strategy to alleviate CAM.

Molecular and physiological responses in roots of two full-sib poplars uncover mechanisms that contribute to differences in partial submergence tolerance.

Poplar is a major afforestation tree species in flood-prone areas. Here, we compared molecular and physiological responses in the roots of two full-sib poplar clones, LS1 (flood-tolerant) and LS2 (flood-susceptive), subjected to stagnant flooding using transcript and metabolite profiling. LS1 displayed less phenotypic damage and superior leaf gas exchange and plant growth compared with those of LS2. We concluded that three characteristics might contribute to the differences in flood tolerance between LS1 and LS2. First, fermentation was initiated through lactic dehydrogenation in LS1 roots under flooding and subsequently dominated by alcohol fermentation. However, lactic dehydrogenase was persistently active in flooded LS2. Second, 13 differentially expressed genes associated with energy and O2 consumption processes under soil flooding had lower transcript levels in LS1 than those in LS2, which might contribute to better energy-/O2-saving abilities and behaviours in flood-tolerant LS1 than those in flood-susceptible LS2 under hypoxic stress. Third, LS1 possessed increased reactive oxygen species scavenging abilities compared with those of LS2 under edaphic flooding. Our data are a valuable contribution to understanding the mechanisms involved in the flood tolerance of poplar.

Screening and optimization of low-cost medium for Pseudomonas putida Rs-198 culture using RSM

The plant growth-promoting rhizobacterial strain Pseudomonas putida Rs-198 was isolated from salinized soils from Xinjiang Province. We optimized the composition of the low-cost medium of P. putida Rs-198 based on its bacterial concentration, as well as its phosphate-dissolving and indole acetic acid (IAA)-producing capabilities using the response surface methodology (RSM), and a mathematical model was developed to show the effect of each medium component and its interactions on phosphate dissolution and IAA production. The model predicted a maximum phosphate concentration in medium containing 63.23 mg/L inorganic phosphate with 49.22 g/L corn flour, 14.63 g/L soybean meal, 2.03 g/L K2HPO4, 0.19 g/L MnSO4 and 5.00 g/L NaCl. The maximum IAA concentration (18.73 mg/L) was predicted in medium containing 52.41 g/L corn flour, 15.82 g/L soybean meal, 2.40 g/L K2HPO4, 0.17 g/L MnSO4 and 5.00 g/L NaCl. These predicted values were also verified through experiments, with a cell density of 10(13) cfu/mL, phosphate dissolution of 64.33 mg/L, and IAA concentration of 18.08 mg/L. The excellent correlation between predicted and measured values of each model justifies the validity of both the response models. The study aims to provide a basis for industrialized fermentation using P. putida Rs-198.

Screening and optimization of low-cost medium for Pseudomonas putida Rs-198 culture using RSM.

The plant growth-promoting rhizobacterial strain Pseudomonas putida Rs-198 was isolated from salinized soils from Xinjiang Province. We optimized the composition of the low-cost medium of P. putida Rs-198 based on its bacterial concentration, as well as its phosphate-dissolving and indole acetic acid (IAA)-producing capabilities using the response surface methodology (RSM), and a mathematical model was developed to show the effect of each medium component and its interactions on phosphate dissolution and IAA production. The model predicted a maximum phosphate concentration in medium containing 63.23 mg/L inorganic phosphate with 49.22 g/L corn flour, 14.63 g/L soybean meal, 2.03 g/L K2HPO4, 0.19 g/L MnSO4 and 5.00 g/L NaCl. The maximum IAA concentration (18.73 mg/L) was predicted in medium containing 52.41 g/L corn flour, 15.82 g/L soybean meal, 2.40 g/L K2HPO4, 0.17 g/L MnSO4 and 5.00 g/L NaCl. These predicted values were also verified through experiments, with a cell density of 10(13) cfu/mL, phosphate dissolution of 64.33 mg/L, and IAA concentration of 18.08 mg/L. The excellent correlation between predicted and measured values of each model justifies the validity of both the response models. The study aims to provide a basis for industrialized fermentation using P. putida Rs-198.

Directed evolution and structural prediction of cellobiohydrolase II from the thermophilic fungus Chaetomium thermophilum.

Cellulases can be engineered with enhanced properties for broad use in scientific and industrial applications. In this study, the wild-type cbh2 gene of the thermophilic fungus Chaetomium thermophilum encoding cellobiohydrolase II (CBHII) was mutagenized through in vitro directed evolution. The resulting Pichia pastoris yeast library was screened, and two transformants were selected for enhanced CBHII activities that were not attributed to increased gene copy numbers. The optimum fermentation times of the two mutant transformants were shortened to 4-5 days after methanol induction compared to 6 days for the wild-type. The optimum reaction temperature (60 °C) and pH level (5 or 6) of the mutant CBHII proteins, designated CBHIIX16 and CBHIIX305, were higher than those of wild-type CBHII (50 °C and pH 4). Kept at 80 °C for 1 h, CBHIIX16 and CBHIIX305 retained >50% of their activities, while the wild-type CBHII lost all activity. Sequence analysis of CBHIIX16 and CBHIIX305 revealed that they contained five and six mutated amino acids, respectively. Structural modeling confirmed the presence of carbohydrate binding type-1 and catalytic domains, where the hydrogen bond numbers between the 227th and 203rd amino acids were increased, which perhaps contributed to the elevated enzyme stability. Therefore, the two CBHII mutants selected for increased enzymatic activities also demonstrated elevated optimum reaction temperature and pH levels and enhanced thermal stability. These properties may be beneficial in practical applications for CBHII.

Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in thermophilic fungus Thermomyces lanuginosus.

OBJECTIVE: To establish a stable transformation system of the thermophilic fungus Thermomyces lanuginosus for its insertional mutagenesis. METHODS: Agrobacterium tumefaciens-mediated transformation (ATMT) was applied to establish transformation system of T. lanuginosus. Southern blotting of hph gene and cloning of transforming DNA (T-DNA) flanking sequences were used to determine insert number and site of T-DNA in the fungal genome, respectively. RESULTS: A reliable transformation method is established for T. lanuginosus. Specifically, pre-germinating spores of T. lanuginosus used at co-cultivated period was a prerequisite. T. lanuginosus germinating spores co-cultivated with Agrobacterium tumefaciens at 28 degrees C for 48 h achieved the highest transformation efficiency. Addition of Acetosyringone (AS) during pre-culture of A. tumefaciens and co-cultivation of T. lanuginosus germinating spores with A. tumefaciens was essentially required, and the best results were obtained with AS at the concentration of 500 microM. Southern blotting analysis showed that majority of transformants (79.2%) contained a single insertion of T-DNA. Thermal asymmetric interlaced PCR (TAIL-PCR) analysis showed random insertion of T-DNA in the fungal genome. Using the transformation system, some stable phenotypic mutants of T. lanuginosus were obtained. CONCLUSION: We report, for the first time, a simple and efficient method for transforming T. lanuginosus by using ATMT. This approach provides a tool for insertional mutagenesis gene tagging in this thermophilic fungus.