Preparative Scale Production of Functional Mouse Aquaporin 4 Using Different Cell-Free Expression Modes

The continuous progress in the structural and functional characterization of aquaporins increasingly attracts attention to study their roles in certain mammalian diseases. Although several structures of aquaporins have already been solved by crystallization, the challenge of producing sufficient amounts of functional proteins still remains. CF (cell free) expression has emerged in recent times as a promising alternative option in order to synthesize large quantities of membrane proteins, and the focus of this report was to evaluate the potential of this technique for the production of eukaryotic aquaporins. We have selected the mouse aquaporin 4 as a representative of mammalian aquaporins. The protein was synthesized in an E. coli extract based cell-free system with two different expression modes, and the efficiencies of two modes were compared. In both, the P-CF (cell-free membrane protein expression as precipitate) mode generating initial aquaporin precipitates as well as in the D-CF (cell-free membrane protein expression in presence of detergent) mode, generating directly detergent solubilized samples, we were able to obtain mg amounts of protein per ml of cell-free reaction. Purified aquaporin samples solubilized in different detergents were reconstituted into liposomes, and analyzed for the water channel activity. The calculated Pf value of proteoliposome samples isolated from the D-CF mode was 133 µm/s at 10°C, which was 5 times higher as that of the control. A reversible inhibitory effect of mercury chloride was observed, which is consistent with previous observations of in vitro reconstituted aquaporin 4. In this study, a fast and convenient protocol was established for functional expression of aquaporins, which could serve as basis for further applications such as water filtration

Strain prioritization and genome mining for enediyne natural products

The enediyne family of natural products has had a profound impact on modern chemistry, biology, and medicine, and yet only 11 enediynes have been structurally characterized to date. Here we report a genome survey of 3,400 actinomycetes, identifying 81 strains that harbor genes encoding the enediyne polyketide synthase cassettes that could be grouped into 28 distinct clades based on phylogenetic analysis. Genome sequencing of 31 representative strains confirmed that each clade harbors a distinct enediyne biosynthetic gene cluster. A genome neighborhood network allows prediction of new structural features and biosynthetic insights that could be exploited for enediyne discovery. We confirmed one clade as new C-1027 producers, with a significantly higher C-1027 titer than the original producer, and discovered a new family of enediyne natural products, the tiancimycins (TNMs), that exhibit potent cytotoxicity against a broad spectrum of cancer cell lines. Our results demonstrate the feasibility of rapid discovery of new enediynes from a large strain collection. IMPORTANCE Recent advances in microbial genomics clearly revealed that the biosynthetic potential of soil actinomycetes to produce enediynes is underappreciated. A great challenge is to develop innovative methods to discover new enediynes and produce them in sufficient quantities for chemical, biological, and clinical investigations. This work demonstrated the feasibility of rapid discovery of new enediynes from a large strain collection. The new C-1027 producers, with a significantly higher C-1027 titer than the original producer, will impact the practical supply of this important drug lead. The TNMs, with their extremely potent cytotoxicity against various cancer cells and their rapid and complete cancer cell killing characteristics, in comparison with the payloads used in FDA-approved antibody-drug conjugates (ADCs), are poised to be exploited as payload candidates for the next generation of anticancer ADCs. Follow-up studies on the other identified hits promise the discovery of new enediynes, radically expanding the chemical space for the enediyne family

Direct and indirect effects of climate on richness drive the latitudinal diversity gradient in forest trees

Data accessibility statement: Full census data are available upon reasonable request from the ForestGEO data portal, http://ctfs.si.edu/datarequest/ We thank Margie Mayfield, three anonymous reviewers and Jacob Weiner for constructive comments on the manuscript. This study was financially supported by the National Key R&D Program of China (2017YFC0506100), the National Natural Science Foundation of China (31622014 and 31570426), and the Fundamental Research Funds for the Central Universities (17lgzd24) to CC. XW was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB3103). DS was supported by the Czech Science Foundation (grant no. 16-26369S). Yves Rosseel provided us valuable suggestions on using the lavaan package conducting SEM analyses. Funding and citation information for each forest plot is available in the Supplementary Information Text 1.Peer reviewedPostprin

Biosynthetic mechanism for mycotoxin fumonisins in the filamentous fungal pathogen Fusarium verticillioides

Filamentous fungi are rich in biologically active polyketides. However, the biosynthetic mechanism of these secondary metabolites is poorly understood. The goal of this Ph.D. study is to elucidate the biosynthetic mechanism for a group of fungal reduced polyketides (including the cholesterol-lowering drug lovastatin, the life-threatening mycotoxin fumonisins, and pathogenesis factor T-toxins) using both genetic and biochemical approaches. In the first project, we established a genetic system that allows, for the first time, specific and functional manipulations of the catalytic domains of FUM1, which is a polyketide synthase (PKS) gene responsible for the 18-carbon polyketide chain of fumonisins produced by corn pathogen Fusarium verticillioides. Using this system, we have obtained fungal mutants containing a chimeric FUM1, in which the ketosynthase (KS) domain had been replaced by the corresponding domain from T-toxins PKS1 or lovastatin LDKS. The mutants containing T-toxin KS produced fumonisins, showing that the heterologous KS is able to functionally interact with six other domains of FUM1. On the other hand, the mutants containing lovastatin KS produced dihydroisocoumarins, which are known to have anti-malarial, antifungal, and anti-tubercular activities and had never been isolated from Fusaria. This work represents the first successful genetic manipulation of fungal PKS genes to produce new, biologically active metabolites in a filamentous fungus. The production of the aromatic metabolites by an engineered non-aromatic type PKS is unprecedented and shows that KS plays a key role in the control of fungal polyketides. In the second project, we used the biochemical approach to determine the function of FUM10 in fumonisin tricarballylic esterifications, which are post-polyketide modifications crucial for the toxins\u27 activity. We have expressed and purified two forms of Fum10p (His6-Fum10p and MBP-Fum10p) from E. coli. After attempts to characterize the obtained enzymes, preliminary data suggested that Fum10p most likely acts as a component of a nonribosomal peptide synthetase-like complex, consisting of Fum10p/Fum7p/Fum14p, together of which is responsible for fumonisin esterifications

Developing a genetic system for functional manipulations of \u3ci\u3eFUM1,\u3c/i\u3e a polyketide synthase gene for the biosynthesis of fumonisins in \u3ci\u3eFusarium verticillioides\u3c/i\u3e

Fumonisins are mycotoxins produced by Fusarium verticillioides, a filamentous fungus that is a widespread pathogen of corn. The biosynthesis of fumonisins is catalyzed by an iterative modular polyketide synthase (PKS). The study of the biosynthetic mechanism for these reduced fungal polyketides has been challenging due to the difficulties in detecting the intermediates with a linear carbon chain and manipulating the 7-domain PKS gene from the filamentous fungus. Here, we described the development of a genetic system for functionally manipulating the methyltransferase domain of FUM1 that is responsible for the assembly of a dimethylated 18-carbon chain. Using a two-stage screening strategy, including both positive and negative screenings, we were able to generate mutant strains with a specifically changed active-site in FUM1. LC-MS analyses indicated that biosynthetic intermediates were detectable in the early stage of culture. The results represent the first functional manipulation of the PKS involved in the biosynthesis of fumonisins

Constitutive Model of Stress-Dependent Seepage in Columnar Jointed Rock Mass

Columnar jointed rock mass (CJRM) is a highly symmetrical natural fractured structure. As the rock mass of the dam foundation of the Baihetan Hydropower Station, the study of its permeability anisotropy is of great significance to engineering safety. Based on the theory of composite mechanics and Goodman’s joint superposition principle, the constitutive model of joints of CJRM is derived according to the Quadrangular prism, the Pentagonal prism and the Hexagonal prism model; combined with Singh’s research results on intermittent joint stress concentration, considering column deflection angles, the joint constitutive model of CJRM in three-dimensional space is established. For the CJRM in the Baihetan dam site area, the Quadrangular prism, the Pentagonal prism and the Hexagonal prism constitutive models were used to calculate the permeability coefficients of CJRM under different deflection angles. The permeability anisotropy characteristics of the three models were compared and verified by numerical simulation results. The results show that the calculation results of the Pentagonal prism model are in good agreement with the numerical simulation results. The variation of permeability coefficient under different confining pressures is compared, and the relationship between permeability coefficient and confining pressure is obtained, which accords with the negative exponential function and conforms to the general rule of joint seepage

Build an Integrated Scene Teaching System for Additive Manufacturing Based on the Integration of Production and Education

Additive manufacturing, also known as 3D printing, is known as one of the subversive intelligent manufacturing technologies that can lead industrial transformation. It has significant advantages in personalized customization and preparation of complex structural components, and is having an important impact on traditional manufacturing process, factory production and processing mode and the entire manufacturing industry chain. At present, the talent gap of additive manufacturing and related specialties in China exceeds 10 million, of which the manufacturing industry has the largest demand for additive manufacturing application talents. It is estimated that the talent gap will be 8 million by 2025. The construction of scenario based teaching system of additive manufacturing integrated courses based on the integration of production and education. At present, it is urgent for universities and vocational education to fully integrate the “research” and “technology” of universities and vocational education institutions with the “production” of enterprises to build a scenario of production and education integration education that suits students. By creating real and vivid scenes for students, Help students get familiar with the background and business operation process of the integrated operation of additive manufacturing, strengthen students’ ability to integrate theoretical knowledge, and apply what they have learned in combination with practice

Fumonisin level in corn-based food and feed from Linxian County, a high-risk area for esophageal cancer in China

The level of mycotoxin fumonisins in corn-based food and feed collected from Linxian County, a high-risk area for esophageal cancer in China, has been analyzed using high-performance liquid chromatographic coupled with evaporative laser scattering detector (HPLC-ELSD). A total of 104 corn kernel samples were obtained from local households, granaries, wholesale markets (central markets), and retail markets (stores and supermarkets). Fumonisin B1 (FB1) was detected in the samples from households, granaries, central markets, and stores, with a positive rate of 61.5%, 50%, 33.3%, and 17%, respectively. No fumonisin was detected in samples from the supermarket. The highest FB1 levels (0.30–3.20 μg/ g; mean, 1.42 μg/g) were found in samples from the granary, followed by household (0.25–1.80 μg/g; mean, 0.73 μg/g), central market (0.25–1.10 μg/g; mean, 0.51 μg/g), and store (0.22–0.34 μg/g; mean, 0.28 μg/g). Among the 80 corn kernel samples collected from local households, 18 of 24 (75.0%) moldy samples contained high levels of FB1 (0.28–3.30 μg/ g; mean, 1.58 μg/g), and 20 of 56 (35.7%) apparently healthy samples contained low levels of FB1 (0.21–0.82 μg/g; mean, 0.46 μg/g). As the central market plays an important role in trade of corn-based food and feed in China, a total of 115 cornbased food and feed samples were collected from the local central market. The highest FB1 levels (0.30–3.13 μg/g; mean, 1.50 μg/g) were found in feed, followed by unprocessed food (0.31–0.63 μg/g; mean, 0.47 μg/g) and processed food (0.21– 0.28 μg/g; mean, 0.25 μg/g). The positive incidence of FB1 in feed, unprocessed, and processed food were 53.6%, 33.3% and 17.9%, respectively. In conclusion, the results showed that corn-based food and feed from Linxian County contained low level of FB1 (\u3c2 μg/g) in general, but efforts should be made to control the fumonisin contamination in corn kernels stored in granaries and households

Functional replacement of the ketosynthase domain of \u3ci\u3eFUM1\u3c/i\u3e for the biosynthesis of fumonisins, a group of fungal reduced polyketides

The genetic manipulation of the biosynthesis of fungal reduced polyketides has been challenging due to the lack of knowledge on the biosynthetic mechanism, the difficulties in the detection of the acyclic, non-aromatic metabolites, and the complexity in genetically manipulating filamentous fungi. Fumonisins are a group of economically important mycotoxins that contaminate maize-based food and feed products worldwide. Fumonisins contain a linear dimethylated C18 chain that is synthesized by Fum1p, which is a single module polyketide synthase (PKS). Using a genetic system that allows the specific manipulation of PKS domains in filamentous fungus Fusarium verticillioides, we replaced the KS domain of fumonisin FUM1 with the KS domain of T-toxin PKS1 from Cochliobolus heterostrophus. Although PKS1 synthesizes different polyketides, the F. verticillioides strain carrying the chimeric PKS produced fumonisins. This represents the first successful domain swapping in PKSs for fungal reduced polyketides and suggests that KS domain alone may not be sufficient to control the product’s structure. To further test if the whole fumonisin PKS could be functionally replaced by a PKS that has a similar domain architecture, we replaced entire FUM1 with PKS1. This strain did not produce any fumonisin or new metabolites, suggesting that the intrinsic interactions between the intact PKS and downstream enzymes in the biosynthetic pathway may play a role in the control of fungal reduced polyketides

Deoxidized gulose moiety attenuates the pulmonary toxicity of 6'-deoxy-bleomycin Z without effect on its antitumor activity

Bleomycins (BLMs) are broad-spectrum antitumor drugs, but the dose-dependent lung toxicity has restricted their therapeutic applications. Many efforts have contributed to develop novel BLM analogues, but mainly focused on single functional domain owing to the structural complexity of BLM. Benefit from the engineered production of two novel analogues 6'-deoxy-BLM Z (6'-DO-BLM Z) and BLM Z, they together with clinical BLM-sulfate comprised a good model with varied sugar or C-terminal domain in any two of them, allowing us to study their structure-activity relationships pairwise. Our investigations suggested the biological activities of BLM or its analogues are mainly depended on the C-terminal amine, while the changed C-terminal amine endowed BLM Z with much higher pulmonary toxicity comparing to BLM-sulfate, whereas the deoxidized gulose unit with same C-terminal amine evidently attenuated the pulmonary toxicity of 6'-DO-BLM Z without effect on antitumor activity. Further mechanistic studies revealed that the alleviation of pulmonary toxicity in 6'-DO-BLM Z by a slight change in the sugar moiety could attribute to the decrease of ROS production and thereby reduce the subsequent caspase-1 activity and resulting inflammatory response. Therefore, the synergistic modifications on C-terminal amine and sugar moiety provide new insights to efficiently develop potential BLM candidate with good clinical performance