In Silico-Predicted Dynamic Oxlipidomics MS/MS Library: High-Throughput Discovery and Characterization of Unknown Oxidized Lipids.

Nontargeted lipidomics using liquid chromatography-tandem mass spectrometry can detect thousands of molecules in biological samples. However, the annotation of unknown oxidized lipids is limited to the structures present in libraries, restricting the analysis and interpretation of experimental data. Here, we describe Doxlipid, a computational tool for oxidized lipid annotation that predicts a dynamic MS/MS library for every experiment. Doxlipid integrates three key simulation algorithms to predict libraries and covers 32 subclasses of oxidized lipids from the three main classes. In the evaluation, Doxlipid achieves very high prediction and characterization performance and outperforms the current oxidized lipid annotation methods. Doxlipid, combined with a molecular network, further annotates unknown chemical analogs in the same reaction or pathway. We demonstrate the broad utility of Doxlipid by analyzing oxidized lipids in ferroptosis hepatocellular carcinoma, tissue samples, and other biological samples, substantially advancing the discovery of biological pathways at the trace oxidized lipid level.

eccDB: a comprehensive repository for eccDNA-mediated chromatin contacts in multi-species.

SUMMARY: We developed the eccDB database to integrate available resources for extrachromosomal circular DNA (eccDNA) data. eccDB is a comprehensive repository for storing, browsing, searching, and analyzing eccDNAs from multispecies. The database provides regulatory and epigenetic information on eccDNAs, with a focus on analyzing intrachromosomal and interchromosomal interactions to predict their transcriptional regulatory functions. Moreover, eccDB identifies eccDNAs from unknown DNA sequences and analyzes the functional and evolutionary relationships of eccDNAs among different species. Overall, eccDB offers web-based analytical tools and a comprehensive resource for biologists and clinicians to decipher the molecular regulatory mechanisms of eccDNAs. AVAILABILITY AND IMPLEMENTATION: eccDB is freely available at http://www.xiejjlab.bio/eccDB.

A novel protein FNDC3B-267aa encoded by circ0003692 inhibits gastric cancer metastasis via promoting proteasomal degradation of c-Myc.

BACKGROUND: Gastric cancer (GC) ranks fifth in global cancer incidence and third in mortality rate among all cancer types. Circular RNAs (circRNAs) have been extensively demonstrated to regulate multiple malignant biological behaviors in GC. Emerging evidence suggests that several circRNAs derived from FNDC3B play pivotal roles in cancer. However, the role of circFNDC3B in GC remains elusive. METHODS: We initially screened circFNDC3B with translation potential via bioinformatics algorithm prediction. Subsequently, Sanger sequencing, qRT-PCR, RNase R, RNA-FISH and nuclear-cytoplasmic fractionation assays were explored to assess the identification and localization of circ0003692, a circRNA derived from FNDC3B. qRT-PCR and ISH were performed to quantify expression of circ0003692 in human GC tissues and adjacent normal tissues. The protein-encoding ability of circ0003692 was investigated through dual-luciferase reporter assay and LC/MS. The biological behavior of circ0003692 in GC was confirmed via in vivo and in vitro experiments. Additionally, Co-IP and rescue experiments were performed to elucidate the interaction between the encoded protein and c-Myc. RESULTS: We found that circ0003692 was significantly downregulated in GC tissues. Circ0003692 had the potential to encode a novel protein FNDC3B-267aa, which was downregulated in GC cells. We verified that FNDC3B-267aa, rather than circ0003692, inhibited GC migration in vitro and in vivo. Mechanistically, FNDC3B-267aa directly interacted with c-Myc and promoted proteasomal degradation of c-Myc, resulting in the downregulation of c-Myc-Snail/Slug axis. CONCLUSIONS: Our study revealed that the novel protein FNDC3B-267aa encoded by circ0003692 suppressed GC metastasis through binding to c-Myc and enhancing proteasome-mediated degradation of c-Myc. The study offers the potential applications of circ0003692 or FNDC3B-267aa as therapeutic targets for GC.

Repositioning baloxavir marboxil as VISTA agonist that ameliorates experimental asthma.

V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA), a novel negative checkpoint regulator, plays an essential role in allergic pulmonary inflammation in mice. Treatment with a VISTA agonistic antibody could significantly improve asthma symptoms. Thus, for allergic asthma treatment, VISTA targeting may be a compelling approach. In this study, we examined the functional mechanism of VISTA in allergic pulmonary inflammation and screened the FDA-approved drugs for VISTA agonists. By using mass cytometry (CyTOF), we found that VISTA deficiency primarily increased lung macrophage infiltration in the OVA-induced asthma model, accompanied by an increased proportion of M1 macrophages (CD11b+F4/80+CD86+) and a decreased proportion of M2 macrophages (CD11b+F4/80+CD206+). Further in vitro studies showed that VISTA deficiency promoted M1 polarization and inhibited M2 polarization of bone marrow-derived macrophages (BMDMs). Importantly, we discovered baloxavir marboxil (BXM) as a VISTA agonist by virtual screening of FDA-approved drugs. The surface plasmon resonance (SPR) assays revealed that BXM (KD = 1.07 µM) as well as its active form, baloxavir acid (BXA) (KD = 0.21 µM), could directly bind to VISTA with high affinity. Notably, treatment with BXM significantly ameliorated asthma symptoms, including less lung inflammation, mucus secretion, and the generation of Th2 cytokines (IL-5, IL-13, and IL-4), which were dramatically attenuated by anti-VISTA monoclonal antibody treatment. BXM administration also reduced the pulmonary infiltration of M1 macrophages and raised M2 macrophages. Collectively, our study indicates that VISTA regulates pulmonary inflammation in allergic asthma by regulating macrophage polarization and baloxavir marboxil, and an old drug might be a new treatment for allergic asthma through targeting VISTA.

Curcumae Radix: A Review of Traditional Use, Phytochemistry, Pharmacology, Toxicology and Quality Control.

Curcumae Radix (CuR) is a traditional Chinese medicine that has been used in China for more than 1,000 years. It has the traditional efficacy of activating blood and relieving pain, promoting qi and relieving depression, clearing heart and cooling blood, and promoting gallbladder and removing jaundice. Based on this, many domestic and foreign scholars have conducted systematic studies on its chemical composition, pharmacological effects, toxicity and quality control. Currently, 250 compounds, mainly including terpenoids and curcuminoids, have been isolated and identified from CuR, which has pharmacological activities, including antitumor, anti-inflammatory and analgesic, antidepressant, hepatoprotective, hemostatic, hematopoietic, and treatment of diabetes mellitus. In modern clinical practice, CuR is widely used in the treatment of tumors, breast hyperplasia, hepatitis, and stroke. However, the generation of toxicity and clinical application of CuR and Caryophylli Flos, the determination of the concoction process of artifacts, the determination of specific Quality Marker, and the establishment of the quality control system of CuR, are problems that need to be solved urgently at present.

Refined Electrolyte and Interfacial Chemistry toward Realization of High-Energy Anode-Free Rechargeable Sodium Batteries.

Anode-free rechargeable sodium batteries represent one of the ultimate choices for the 'beyond-lithium' electrochemical storage technology with high energy. Operated based on the sole use of active Na ions from the cathode, the anode-free battery is usually reported with quite a limited cycle life due to unstable electrolyte chemistry that hinders efficient Na plating/stripping at the anode and high-voltage operation of the layered oxide cathode. A rational design of the electrolyte toward improving its compatibility with the electrodes is key to realize the battery. Here, we show that by refining the volume ratio of two conventional linear ether solvents, a binary electrolyte forms a cation solvation structure that facilitates flat, dendrite-free, planar growth of Na metal on the anode current collector and that is adaptive to high-voltage Na (de)intercalation of P2-/O3-type layered oxide cathodes and oxidative decomposition of the Na2C2O4 supplement. Inorganic fluorides, such as NaF, show a major influence on the electroplating pattern of Na metal and effective passivation of plated metal at the anode-electrolyte interface. Anode-free batteries based on the refined electrolyte have demonstrated high coulombic efficiency, long cycle life, and the ability to claim a cell-level specific energy of >300 Wh/kg.

Competitive Doping Chemistry for Nickel-Rich Layered Oxide Cathode Materials.

Chemical modification of electrode materials by heteroatom dopants is crucial for improving storage performance in rechargeable batteries. Electron configurations of different dopants significantly influence the chemical interactions inbetween and the chemical bonding with the host material, yet the underlying mechanism remains unclear. We revealed competitive doping chemistry of Group IIIA elements (boron and aluminum) taking nickel-rich cathode materials as a model. A notable difference between the atomic radii of B and Al accounts for different spatial configurations of the hybridized orbital in bonding with lattice oxygen. Density functional theory calculations reveal, Al is preferentially bonded to oxygen and vice versa, and shows a much lower diffusion barrier than BIII . In the case of Al-preoccupation, the bulk diffusion of BIII is hindered. In this way, a B-rich surface and Al-rich bulk is formed, which helps to synergistically stabilize the structural evolution and surface chemistry of the cathode.

The expression and significance of leukemia inhibitory factor, interleukin-6 and vascular endothelial growth factor in Chinese patients with endometriosis.

OBJECTIVE: To observe the levels of leukemia inhibitory factor (LIF), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) in blood, peritoneal fluid, ectopic endometrial tissue, and ectopic endometrial stromal cells of patients with endometriosis, and to compare expression of IL-6, LIF and VEGF expression between endometriotic and non-endometriotic patients underwent laparoscopic surgery. METHODS: Thirty-one patients who underwent laparoscopic surgery for endometriosis in our hospital from January 2018 to January 2020 were included in the observation group, and 32 patients who underwent laparoscopic surgery for uterine fibroids, ovarian serous cystadenoma, and ovarian teratomas, were included in the control group. The levels of LIF, IL-6 and VEGF in the blood and peritoneal fluid of the two groups of patients were detected. The levels of LIF, IL-6 and VEGF in ectopic endometrial tissue and self-paired eutopic endometrial tissue, ectopic endometrial stromal cells and self-paired eutopic endometrial stromal cells of patients in the observation group were detected. After treating the primary cultured ectopic endometrial stromal cells with LIF and IL-6 alone or in combination, the changes of VEGF mRNA of ectopic endometrial stromal cells and the VEGF levels in the supernatant were observed. RESULTS: The levels of LIF, IL-6 and VEGF in the blood and peritoneal fluid of the observation group were all higher than those of the control group (P < 0.05), and the levels of LIF, IL-6 and VEGF in the peritoneal fluid of the observation group were significantly higher than those in the blood (P < 0.05). In the observation group, the expression levels of LIF-mRNA and IL-6 mRNA in the ectopic endometrial tissue were higher than those in the self-paired eutopic endometrial tissues (P < 0.05), while the expression level of VEGF mRNA in the ectopic endometrial tissues was lower than that in the self-paired eutopic endometrial tissues (P < 0.05). Besides, the mRNA expression levels of LIF, IL-6 and VEGF in ectopic endometrial stromal cells of the observation group were all higher than those in the self-paired eutopic endometrial stromal cells (P < 0.05). After stimulating ectopic endometrial stromal cells with LIF, IL-6 and LIF + IL-6, respectively, the VEGF levels in the supernatant were all significantly higher than that in the blank control group (P < 0.05). CONCLUSION: The LIF, IL-6 and VEGF levels in blood and peritoneal fluid were increased in patients with endometriosis, and increased LIF and IL-6 in ectopic endometriosis stromal cells can play a synergistic role in increasing the VEGF levels, which may be involved in the occurrence and development of endometriosis.

[In vivo and in vitro Experiment of E74-Like Factor 5 Overexpression Inhibiting the Biological Behavior of Colon Cancer Cells].

OBJECTIVE: To investigate the effect of E74-like factor 5 (ELF5) overexpression on the growth and invasion ability of colorectal cancer cells and its effect on tumor formation in nude mice. METHODS: Human colorectal cancer SW480 and HT-29 cells were divided into 5 groups: the lentivirus (LV)- GFP group transfected with empty vector LV- GFP, the LV- ELF5 group transfected with recombinant LV- ELF5, the shRNA-NC group transfected with empty vector shRNA-NC, the shRNA- ELF5 group transfected with recombinant shRNA- ELF5, and the control group, not transfected with any vector. Seventy-two h after transfection, the cell supernatant containing lentivirus was collected. The mRNA expression level of ELF5 in each group was examined by real-time fluorescent quantitative PCR (RT-qPCR). The protein expression levels of ELF5, apoptosis-related cleaved Caspase-3/Caspase-3 and cleaved Caspase-9/Caspase-9, and invasion-related E-cadherin and N-cadherin were checked with Western blot. CCK-8 was used to check cell viability. Colony formation experiment was done to evaluate colony formation rate. Flow cytometry was used to assess cell apoptosis. Transwell migration assay was used to examine cell invasion. TUNEL assay was used to examine the apoptosis of tissues cells. Immunohistochemistry test was done to determine the expression of E-cadherin and N-cadherin in tissues. 20 BALB/c nude mice were put into 4 groups (5 in each group): LV- GFP group, shRNA-NC group, LV- ELF5 group, and shRNA- ELF5 group. Recombinant lentiviral SW480 cell supernatants were subcutaneously injected into nude mice to construct nude mice tumorigenesis models and the volume changes of transplanted tumors were monitored. On the 30th day, transplanted tumor tissues from the nude mice were extracted and the tumor mass was measured. Western blot was done to measure the expression of ELF4 protein in the transplanted tumors. TUNEL staining was used to check cell apoptosis in the tissues, and the positive expression of N-cadherin in the transplanted tumor was measured by immunohistochemical tests. RESULTS: Compared with the control group, there was no statistically significant difference in the indicators of the two cell lines in the LV- GFP group and shRNA-NC group. The results of Western blot and RT-qPCR showed that the ELF5 protein and mRNA of the LV- ELF5 group of the two cell lines were up-regulated ( P<0.05, compared with those of the LV- GFP group), and the ELF5 protein and mRNA of the shRNA- ELF5 group were down-regulated ( P<0.05). The ELF5 overexpression system and interference system were successfully constructed. Compared with the LV- GFP group, data from the LV- ELF5 group showed that cell viability and colony formation rate ( P<0.05) were reduced, SW480 and HT-29 cell apoptosis was promoted, cleaved Caspase-3/Caspase-3 and cleaved Caspase-9/Caspase-9 protein expression was up-regulated ( P<0.05), cell invasion was inhibited, and the expression of E-cadherin protein was up-regulated while the expression of N-cadherin protein was down-regulated ( P<0.05). After ELF5 interference, the above-mentioned expression of cells demonstrated an opposite trend ( P<0.05, comparing shRNA- ELF5 group with shRNA-NC group). In vivo experimental results indicated that ELF5 overexpression reduced tumor volume and tumor mass ( P<0.05), promoted cell apoptosis in tissues ( P<0.05), and inhibited N-cadherin protein expression ( P<0.05). When ELF5 expression was inhibited, the above mentioned experimental results showed the opposite trend. CONCLUSION: In vivo and in vitro experiments showed that ELF5 overexpression could promote the apoptosis of colorectal cancer cells and inhibit the growth and invasion of colorectal cancer cells.

Synthesis and evaluation of 1,2,4-oxadiazole derivatives as potential anti-inflammatory agents by inhibiting NF-κB signaling pathway in LPS-stimulated RAW 264.7 cells.

In this study, a series of compounds with 1,2,4-oxadiazole core was designed and synthesized for the optimization of JC01, an anti-inflammatory hit identified from our in-house compound library using NF-κB pathway luciferase assay and NO production assay. All the synthetic compounds 1-29 have been screened for their anti-inflammatory effects by evaluating their inhibition against LPS-induced NO release, and compound 17 exhibited the highest activity. Western blotting and immunofluorescence analysis revealed that 17 prominently inhibited LPS-induced activation of NF-κB in RAW264.7 cells and blocked the phosphorylation of p65. Consistent with these results, it was found that 17 prevented the nuclear translocation of NF-κB induced by LPS. These data highlighted 17 as a promising anti-inflammatory agent by inhibiting NF-κB activity.

Differentiating Westlake Longjing tea from the first- and second-grade producing regions using ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry-based untargeted metabolomics in combination with chemometrics.

There are numerous articles published for geographical discrimination of tea. However, few research works focused on the authentication and traceability of Westlake Longjing green tea from the first- and second-grade producing regions because the tea trees are planted in a limited growing zone with identical cultivate condition. In this work, a comprehensive analytical strategy was proposed by ultrahigh performance liquid chromatography-quadrupole time-of-flight mass spectrometry-based untargeted metabolomics coupled with chemometrics. The automatic untargeted data analysis strategy was introduced to screen metabolites that expressed significantly among different regions. Chromatographic features of metabolites can be automatically and efficiently extracted and registered. Meanwhile, those that were valuable for geographical origin discrimination were screened based on statistical analysis and contents in samples. Metabolite identification was performed based on high-resolution mass values and tandem mass spectra of screened peaks. Twenty metabolites were identified, based on which the two-way encoding partial least squares discrimination analysis was built for geographical origin prediction. Monte Caro simulation results indicated that prediction accuracy was up to 99%. Our strategy can be applicable for practical applications in the quality control of Westlake Longjing green tea.

Two new naphthoate derivatives from Morinda officinalis.

Two new naphthoate derivatives, including a symmetrical dimer (1) and a monomer (2), were separated from the roots of Morinda officinalis var. hirsuta. Their structures were characterized on the basis of spectroscopic means especially MS and NMR methods. Biological evaluations revealed that the two compounds did not show inhibition against both cholinesterases AChE and BChE, while the dimer (1) did exhibit moderate growth inhibitory activity toward one human osteosarcoma cell line U2OS with an IC50 value of 18.5 ± 1.1 µM.

Functional characterization of a liverworts bHLH transcription factor involved in the regulation of bisbibenzyls and flavonoids biosynthesis.

BACKGROUND: The basic helix-loop-helix (bHLH) transcription factors (TFs), as one of the largest families of TFs, play important roles in the regulation of many secondary metabolites including flavonoids. Their involvement in flavonoids synthesis is well established in vascular plants, but not as yet in the bryophytes. In liverworts, both bisbibenzyls and flavonoids are derived through the phenylpropanoids pathway and share several upstream enzymes. RESULTS: In this study, we cloned and characterized the function of PabHLH1, a bHLH family protein encoded by the liverworts species Plagiochasma appendiculatum. PabHLH1 is phylogenetically related to the IIIf subfamily bHLHs involved in flavonoids biosynthesis. A transient expression experiment showed that PabHLH1 is deposited in the nucleus and cytoplasm, while the yeast one hybrid assay showed that it has transactivational activity. When PabHLH1 was overexpressed in P. appendiculatum thallus, a positive correlation was established between the content of bibenzyls and flavonoids and the transcriptional abundance of corresponding genes involved in the biosynthesis pathway of these compounds. The heterologous expression of PabHLH1 in Arabidopsis thaliana resulted in the activation of flavonoids and anthocyanins synthesis, involving the up-regulation of structural genes acting both early and late in the flavonoids synthesis pathway. The transcription level of PabHLH1 in P. appendiculatum thallus responded positively to stress induced by either exposure to UV radiation or treatment with salicylic acid. CONCLUSION: PabHLH1 was involved in the regulation of the biosynthesis of flavonoids as well as bibenzyls in liverworts and stimulated the accumulation of the flavonols and anthocyanins in Arabidopsis.

Germacrane-type sesquiterpenoids with cytotoxic activity from Sigesbeckia orientalis.

Eleven new highly oxygenated germacrane-type sesquiterpenoids (1-11) and 16 known analogues (12-27) were isolated from the aerial parts of Sigesbeckia orientalis. Their structures, including absolute configurations, were determined by comprehensive spectroscopic methods especially NMR and ECD analyses. Compounds 13, 21 and 23 possessing an 8-methacryloxy group showed stronger in vitro cytotoxicity against human A549 and MDA-MB-231 cancer cell lines than other co-metabolites, with IC50 values ranging from 6.02 to 10.77 µM comparable to the positive control adriamycin.

Identification of chalcone isomerase in the basal land plants reveals an ancient evolution of enzymatic cyclization activity for synthesis of flavonoids.

Flavonoids ubiquitously distribute to the terrestrial plants and chalcone isomerase (CHI)-catalyzed intramolecular and stereospecific cyclization of chalcones is a committed step in the production of flavonoids. However, so far the bona fide CHIs are found only in vascular plants, and their origin and evolution remains elusive. We conducted transcriptomic and/or genomic sequence search, subsequent phylogenetic analysis, and detailed biochemical and genetic characterization to explore the potential existence of CHI proteins in the basal bryophyte liverwort species and the lycophyte Selaginella moellendorffii. We found that both liverwort and Selaginella species possess canonical CHI-fold proteins that cluster with their corresponding higher plant counterparts. Among them, some members exhibited bona fide CHI activity, which catalyze stereospecific cyclization of both 6'-hydroxychalcone and 6'-deoxychalcone, yielding corresponding 5-hydroxy and 5-deoxyflavanones, resembling the typical type II CHIs currently known to be 'specific' for legume plants. Expressing those primitive bona fide CHIs in the Arabidopsis chi mutant restores the seed coat transparent testa phenotype and the accumulation of flavonoids. These findings, in contrast to our current understanding of the evolution of enzymatic CHIs, suggest that emergence of the bona fide type II CHIs is an ancient evolution event that occurred before the divergence of liverwort lineages.

Design, Synthesis, and Antiproliferative Evaluation of Novel Coumarin/2-Cyanoacryloyl Hybrids as Apoptosis Inducing Agents by Activation of Caspase-Dependent Pathway.

A series of novel coumarin/2-cyanoacryloyl hybrids were prepared and evaluated for their in vitro anticancer activity. Among them, two analogs 5p and 5q showed promising antiproliferative activity against a panel of cancer cell lines, including A549, H157, HepG2, MCF7, MG63, and U2OS. Particularly, 5q showed the most potent activity towards MG63 cells with an IC50 value of 5.06 ± 0.25 µM. Morphological observation and 4,6-diamidino-2-phenylindole (DAPI) staining assay showed that 5q-treated MG63 cells displayed significant apoptosis characteristics. Moreover, flow cytometric detection of phosphatidylserine externalization revealed that 5q induced MG63 apoptosis in a dose-dependent manner. Real-time PCR and western blot assay further confirmed that 5q had strong effects to induce MG63 cell apoptosis, suggesting that the action was associated with down-regulation of the anti-apoptotic protein Bcl-2, upregulation of pro-apoptotic protein Bax, and induced activation of caspase-3, 8, and 9. The present results provide a new chemotype for anticancer drug development and continuing investigation into candidates with coumarin/2-cyanoacryloyl scaffold is warranted.

Design and synthesis of pregnenolone/2-cyanoacryloyl conjugates with dual NF-κB inhibitory and anti-proliferative activities.

Twenty-five novel pregnenolone/2-cyanoacryloyl conjugates (6-30) were designed and prepared, with the aim of developing novel anticancer drugs with dual NF-κB inhibitory and anti-proliferative activities. Compounds 22 and 27-30 showed inhibition against TNF-α-induced NF-κB activation in luciferase assay, which was confirmed by Western blotting. Among them, compound 30 showed potent NF-κB inhibitory activity (IC50=2.5µM) and anti-proliferative against MCF-7, A549, H157, and HL-60 cell lines (IC50=6.5-36.2µM). The present study indicated that pregnenolone/2-cyanoacryloyl conjugate I can server asa novel scaffold for developing NF-κB inhibitors and anti-proliferative agents in cancer chemotherapy.

[Study on NIR Model Transfer between Similar Kinds of Fruits Based on Slope/Bias Algorithm].

In order to expand the application range of the model for a single kind of fruits in the portable near infrared instrument, this paper comes up with a new method for the soluble solid content (SSC) model transfer between different kinds of fruits. This method is focusing on the idea of model transfer between different instruments. Based on the similar physical and chemical properties of apples, peaches and pears, such as the range of SSC content, fruit size and the thickness of peel, a simple Slope/Bias algorithm is applied to the transfer of apple SSC partial least square (PLS) model. After that, it can be used to predict pear & peach SSC value with very little extra samples. It's more convenient and costs less by using this method. For pear samples, by using extra 35 standard samples to transfer apple SSC model, RMSEP reduced from 1.009 °Brix to 0.565 °Brix. For peaches, extra 40 standard samples led to a significant reduce of RMSEP from 1.726 °Brix to 0.677 °Brix after model transfer. To validate the feasibility of this model transfer method, both pear and peach SSC models were tested using the same Slope/Bias algorithm model transfer respectively. A pear SSC model was firstly set up and then transferred with Slope/Bias method. Taking 30 standard apples as samples, RMSEP value reached 0.597°Brix, while taking 40 standard peaches as samples, RMSEP value reached 0.689°Brix. The peach SSC model was transferred in the same way. For apples, using 35 standard samples, RMSEP value reached 0.654°Brix, and for pears, using 30 standard samples, RMSEP value reached 0.439°Brix. These results show that slope/bias algorithm can be used to transfer model between similar kinds of fruits such as apples, pears and peaches. The paper provides innovative ideas for the model transfer among similar kinds of materials, so that the portable near infrared instruments can be used more conveniently and widely.

Production of Bioactive Recombinant Bovine Chymosin in Tobacco Plants.

Chymosin (also known as rennin) plays an essential role in the coagulation of milk in the cheese industry. Chymosin is traditionally extracted from the rumen of calves and is of high cost. Here, we present an alternative method to producing bovine chymosin from transgenic tobacco plants. The CYM gene, which encodes a preprochymosin from bovine, was introduced into the tobacco nuclear genome under control of the viral 35S cauliflower mosaic promoter. The integration and transcription of the foreign gene were confirmed with Southern blotting and reverse transcription PCR (RT-PCR) analyses, respectively. Immunoblotting analyses were performed to demonstrate expression of chymosin, and the expression level was quantified by enzyme-linked immunosorbent assay (ELISA). The results indicated recombinant bovine chymosin was successfully expressed at an average level of 83.5 ng/g fresh weight, which is 0.52% of the total soluble protein. The tobacco-derived chymosin exhibited similar native milk coagulation bioactivity as the commercial product extracted from bovine rumen.

Stable Expression of Basic Fibroblast Growth Factor in Chloroplasts of Tobacco.

Basic fibroblast growth factor (bFGF) is a multifunctional factor in acceleration of cell proliferation, differentiation and transference, and therefore widely used in clinical applications. In this study, expression vector pWX-Nt03 harboring a codon-optimized bFGF gene was constructed and introduced into the tobacco chloroplasts by particle bombardment. After four rounds of selection, bFGF was proved to integrate into the chloroplast genome of regenerated plants and two of four transgenic plants were confirmed to be homoplastomic by PCR and Southern hybridization. ELISA assay indicated that bFGF represented approximately 0.1% of total soluble protein in the leaves of transplastomic tobacco plants. This is the first report of bFGF expression via chloroplast transformation in model plant, providing an additional option for the production of chloroplast-produced therapeutic proteins.