Improving biosynthetic production of pinene through plasmid recombination elimination and pathway optimization.

Pinene is a monoterpene with wide industrial applications, especially as a promising high energy-density jet fuel. Traditional production of pinene on an industrial scale is material consumptive and has a low yield. As an alternative, microbial organisms have been engineered though advanced synthetic biological techniques to produce a variety of heterologous products, including pinene. Here, we investigated the stability of genetic circuits encoding the pinene producing pathway during fermentation and its relationship to the pinene titer. By replacing scar sequences in the genetic elements and modifying the genome of E. coli strain MG1655, plasmid loss caused by serious metabolic burden was eliminated, generating a remarkable increase in the pinene titer. Furthermore, the heterologous mevalonate pathway was analyzed by overexpression of enzymes and intermediates monitoring. Optimized pathway plasmids and strains were combined to increase the pinene titer to 104.6 mg/L.

Quorum-sensing based small RNA regulation for dynamic and tuneable gene expression.

OBJECTIVES: Developing a dynamic regulation strategy is an essential step in establishing an automatic control system for manipulating metabolic fluxes and cellular behaviors. To broaden the extent of the application, a system that can generally control any gene of interest is demanded. RESULTS: Through characterization and optimization, the strategy repressed the immediate expression incrementally from 0 to 90% during culturing. Moreover, by changing single base pair in the lux box of the Plux promoter, the degree of repression of the target genomic gene was tuned to a difference of 70%. This strategy is expected to control metabolic flux without disrupting cell growth. CONCLUSIONS: We engineered bacterial small RNA to develop a pathway-independent strategy that can dynamically repress the expression of any gene at the posttranscription level.

Molecular Dynamics Simulation Reveals Unique Interplays Between a Tarantula Toxin and Lipid Membranes.

Tarantula toxins compose an important class of spider toxins that target ion channels, and some are known to interact with lipid membranes. In this study, we focus on a tarantula toxin, Jingzhaotoxin-III (JZTx-III) that specifically targets the cardiac voltage-gated sodium channel Na[Formula: see text]1.5 and is suspected to be able to interact with lipid membranes. Here, we use an all-atom model and long-term molecular dynamics simulations to investigate the interactions between JZTx-III and lipid membranes of different compositions. Trajectory analyses show that JZTx-III has no substantial interaction with the neutral 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids, but binds to membranes containing negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG). The most intriguing observations in our simulation are the different interactions between the toxin and the membrane in the mixed and pure POPG membrane systems. The POPC/POPG mixed membrane undergoes a phase transition to a rippled phase upon binding of the toxin, while the pure POPG membrane has no apparent change. Moreover, the binding of JZTx-III to both of the mixture and the pure POPG membrane systems induce small conformational changes. The sequence alignment shows that JZTx-III may not partition into the lipid bilayer due to the mutations of a C-terminal hydrophobic residue and some charged residues that affect toxin orientation. Taken together, JZTx-III and lipid membranes have unique effects on each other that may facilitate the specific binding of JZTx-III to Na[Formula: see text]1.5. This computational study also enriches our understanding of the potential complex interactions between spider toxins and lipid membranes.

Heterologous expression and purification of neurotoxic Hainantoxin-III in E. coli.

In the present study, we used Escherichia coli to produce recombinant Hainantoxin-III (rHNTX-III), a 33-amino acid peptic toxin from the tarantula spider Haplopelma hainanum. The toxin has three pairs of disulfide bonds. A pET-HS-HNTX-III vector was constructed and transformed into the E. coli strain SHuffleTM. rHNTX-III was expressed using auto-induction medium. After using a Ni-NTA column, the expressed fusion protein was digested using SUMO protease (ULP1) to remove the HIS-SUMO tag, and then RP-HPLC and ultrafiltration were used for further purification. Then the rHNTX-III was identified by MALDI-TOF/TOF mass spectrometry. The purified rHNTX-III was further analyzed using a whole-cell patch-clamp assay. It was shown that the rHNTX-III was able to block currents generated by human Nav1.7 (hNav1.7) at an IC50 of 225 nM and also have high selectivity for different voltage-gated sodium channels. Therefore, it has very similar activity to the natural one.

Effects of various processed methods on chemical constituents and anti-hepatitis activity of Bupleurum scorzonerifolium Willd. by metabonomics and molecular docking.

In this study, a plant metabonomics technique, utilizing ultra-high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI--QTOF), was used to clarify the differences of various processed Bupleurum scorzonerifolium Willd. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to determine the differences in raw and different processed B. scorzonerifolium samples, including vinegar stir-fry, wine stir-fry, honey stir-fry, honey bran stir-fry and charcoal stir-fry. Thus, 39 significant compounds, e.g., saponins, free fatty acids, fatty acid pentitol glycosides, organic acid and linalool glycosides, were clearly or tentatively identified by UPLC-ESI--QTOF-MS/MS fragmentation pathways and by comparison with available reference standards. Most importantly, fatty acid pentitol glycosides were discovered and identified in B. scorzonerifolium for the first time. Furthermore, a HepG2 hepatitis model induced by TNF-α was used to measure the anti-hepatitis effect of raw and processed B. scorzonerifolium in vitro. Molecular docking was used to understand the interaction of key Q-markers with the active sites of the target protein. The results show that the UPLC-QTOF-MS metabolomics coupled with molecular docking is a powerful tool to quickly identify quality control characteristics of B. scorzonerifolium and its products.

Low-polymerization compositional fingerprinting for characterization of Schisandra polysaccharides by hydrophilic interaction liquid chromatography-electrospray mass spectrometry.

A hydrophilic interaction liquid chromatography-negative electrospray-mass spectrometry (HILIC-ESI--MS) coupled with microwave assisted mild acid (MAMA) depolymerization is proposed here for unusual discrimination and characterization of plant polysaccharides: a case study of fruit polysaccharides in Schisandra chinensis and S. sphenanthera (SCP and SSP). The optimized MAMA hydrolysis procedure was proposed for sample preparations of low-polymerization saccharides (Mw < 5000 Da) released in SCP and SSP. In addition, HILIC-MS/MS was employed for elucidation of isomeric glycosidic linkages in terms of 18O labelling. The MAMA hydrolysates showed that the amount of neutral →(4Hex1)n→ moiety is confirmed to be more bigger than that of acidic →(4HexA1)n → in SCP, whereas the amount of acidic →(4HexA1)n→ moiety seems to be more bigger than that of neutral →(4Hex1)n→ in SSP. The resulting low-polymerization compositional fingerprinting (LCF) showed the performance on rapid visualization of SCP and SSP by HILIC-MIM-MS. Principal components analysis (PCA) and hierarchical cluster analysis (HCA) further unveils several key Q-markers (e.g., m/z 503, 369, 665, 827, 989, 1151 and 735) for rapid discrimination of SCP and SSP. This practical study showed that the LCF with PCA and HCA could effectively reflect structural differences and could rapidly achieve discrimination of SCP and SSP.

A dynamic and multilocus metabolic regulation strategy using quorum-sensing-controlled bacterial small RNA.

Metabolic regulation strategies have been developed to redirect metabolic fluxes to production pathways. However, it is difficult to screen out target genes that, when repressed, improve yield without affecting cell growth. Here, we report a strategy using a quorum-sensing system to control small RNA transcription, allowing cell-density-dependent repression of target genes. This strategy is shown with convenient operation, dynamic repression, and availability for simultaneous regulation of multiple genes. The parameters Ai, Am, and RA (3-oxohexanoyl-homoserine lactone [AHL] concentrations at which half of the maximum repression and the maximum repression were reached and value of the maximum repression when AHL was added manually, respectively) are defined and introduced to characterize repression curves, and the variant LuxRI58N is identified as the most suitable tuning factor for shake flask culture. Moreover, it is shown that dynamic overexpression of the Hfq chaperone is the key to combinatorial repression without disruptions on cell growth. To show a broad applicability, the production titers of pinene, pentalenene, and psilocybin are improved by 365.3%, 79.5%, and 302.9%, respectively, by applying combinatorial dynamic repression.

Assembly of TALE-based DNA scaffold for the enhancement of exogenous multi-enzymatic pathway.

Synthetic scaffold systems, which exhibit enzyme clustering effect, have been considered as an important parallel approach for metabolic flux control and pathway enhancement. Here, we described an improved DNA-based scaffold system for synthetic tri-enzymatic pathway in Escherichia coli. With plasmid DNA serving as scaffold and exogenous enzymes fused with rationally designed transcription activator-like effectors (TALEs), our approach successfully clustered three TALE-fused enzymes and significantly increased the production of a mevalonate-producing tri-enzymatic pathway with the optimized scaffold structure and plasmid copy number. These results further suggested the scalability and robustness of the TALE-based scaffold system, and we can assume that it can be used on numerous multi-enzyme metabolic pathways due to its programmable features.

Highly Effective and Low-Cost MicroRNA Detection with CRISPR-Cas9.

MicroRNAs have been reported as related to multiple diseases and have potential applications in diagnosis and therapeutics. However, detection of miRNAs remains improvable, given their complexity, high cost, and low sensitivity as of currently. In this study, we attempt to build a novel platform that detects miRNAs at low cost and high efficacy. This detection system contains isothermal amplification, detecting and reporting process based on rolling circle amplification, CRISPR-Cas9, and split-horseradish peroxidase techniques. It is able to detect trace amount of miRNAs from samples with mere single-base specificity. Moreover, we demonstrated that such scheme can effectively detect target miRNAs in clinical serum samples and significantly distinguish patients of non-small cell lung cancer from healthy volunteers by detecting the previously reported biomarker: circulating let-7a. As the first to use CRISPR-Cas9 in miRNA detection, this method is a promising approach capable of being applied in screening, diagnosing, and prognosticating of multiple diseases.

Edaravone enhances the viability of ischemia/reperfusion flaps.

The purpose of the experiment was to study the efficacy of edaravone in enhancing flap viability after ischemia/reperfusion (IR) and its mechanism. Forty-eight adult male SD rats were randomly divided into 3 groups: control group (n=16), IR group (n=16), and edaravone-treated IR group (n=16). An island flap at left lower abdomen (6.0 cm×3.0 cm in size), fed by the superficial epigastric artery and vein, was created in each rat of all the three groups. The arterial blood flow of flaps in IR group and edaravone-treated IR group was blocked for 10 h, and then the blood perfusion was restored. From 15 min before reperfusion, rats in the edaravone-treated IR group were intraperitoneally injected with edaravone (10 mg/kg), once every 12 h, for 3 days. Rats in the IR group and control group were intraperitoneally injected with saline, with the same method and frequency as the rats in the edaravone-treated IR group. In IR group and edaravone-treated IR group, samples of flaps were harvested after reperfusion of the flaps for 24 h. In the control group, samples of flaps were harvested 34 h after creation of the flaps. The content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) were determined, and changes in organizational structure and infiltration of inflammatory cells were observed by hematoxylin-eosin (HE) staining, apoptotic cells of vascular wall were marked by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, and the apoptotic rate of cells in vascular wall was calculated. The ultrastructural changes of vascular endothelial cells were observed by transmission electron microscopy (TEM). Seven days after the operation, we calculated the flap viability of each group, and marked vessels of flaps by immunohistochemical staining for calculating the average number of subcutaneous vessels. The results showed that the content of MDA, the number of multicore inflammatory cells and apoptotic rate of cells in vascular wall in the edaravone-treated IR group were significantly lower than those in the IR group. The activity of SOD, flap viability and average number of subcutaneous vessels in the edaravone-treated IR group were significantly higher than those in the IR group. All the differences were statistically significant. The ultrastructure injury of vascular endothelial cells in the edaravone-treated IR group was slighter than that in IR group. It was concluded that edaravone can significantly enhance IR flap viability and protect flap vessels, which is related to scavenging oxygen free radicals, reducing the consumption of SOD, reducing the extent of lipid peroxidation and inflammation, and protecting functional structure of vessels in the early stages of reperfusion.

OEPR Cloning: an Efficient and Seamless Cloning Strategy for Large- and Multi-Fragments.

Here, an efficient cloning strategy for large DNA fragments and for simultaneous assembly of multiple DNA fragments assembly is presented. This strategy is named OEPR (based on Overlap Extension PCR and Recombination in vivo). OEPR cloning is a seamless, restriction- and ligation-independent method. The method takes advantage of both homologous recombination enzymes in E. coli and overlap PCR. Using OEPR cloning, a long fragment (1-6 kb) or multiple fragments (2-4 fragments) can be easily constructed and simultaneously assembled into a target vector.

Mega primer-mediated molecular cloning strategy for chimaeragenesis and long DNA fragment insertion.

Molecular cloning methods based on primer and overlap-extension PCR are widely used due to their simplicity, reliability, low cost and high efficiency. In this article, an efficient mega primer-mediated (MP) cloning strategy for chimaeragenesis and long DNA fragment insertion is presented. MP cloning is a seamless, restriction/ligation-independent method that requires only three steps: (i) the first PCR for mega primer generation; (ii) the second PCR for exponential amplification mediated by the mega primers and (iii) DpnI digestion and transformation. Most importantly, for chimaeragenesis, genes can be assembled and constructed into the plasmid vector in a single PCR step. By employing this strategy, we successfully inserted four DNA fragments (approximately 500 bp each) into the same vector simultaneously. In conclusion, the strategy proved to be a simple and efficient tool for seamless cloning.

The role of TFIIB-RNA polymerase II interaction in start site selection in yeast cells.

Previous studies have established a critical role of both TFIIB and RNA polymerase II (RNAPII) in start site selection in the yeast Saccharomyces cerevisiae. However, it remains unclear how the TFIIB-RNAPII interaction impacts on this process since such an interaction can potentially influence both preinitiation complex (PIC) stability and conformation. In this study, we further investigate the role of TFIIB in start site selection by characterizing our newly generated TFIIB mutants, two of which exhibit a novel upstream shift of start sites in vivo. We took advantage of an artificial recruitment system in which an RNAPII holoenzyme component is covalently linked to a DNA-binding domain for more direct and stable recruitment. We show that TFIIB mutations can exert their effects on start site selection in such an artificial recruitment system even though it has a relaxed requirement for TFIIB. We further show that these TFIIB mutants have normal affinity for RNAPII and do not alter the promoter melting/scanning step. Finally, we show that overexpressing the genetically isolated TFIIB mutant E62K, which has a reduced affinity for RNAPII, can correct its start site selection defect. We discuss a model in which the TFIIB-RNAPII interaction controls the start site selection process by influencing the conformation of PIC prior to or during PIC assembly, as opposed to PIC stability.

Spatial organization of heterologous metabolic system in vivo based on TALE.

For years, prokaryotic hosts have been widely applied in bio-engineering. However, the confined in vivo enzyme clustering of heterologous metabolic pathways in these organisms often results in low local concentrations of enzymes and substrates, leading to a low productive efficacy. We developed a new method to accelerate a heterologous metabolic system by integrating a transcription activator-like effector (TALE)-based scaffold system into an Escherichia coli chassis. The binding abilities of the TALEs to the artificial DNA scaffold were measured through ChIP-PCR. The effect of the system was determined through a split GFP study and validated through the heterologous production of indole-3-acetic acid (IAA) by incorporating TALE-fused IAA biosynthetic enzymes in E. coli. To the best of our knowledge, we are the first to use the TALE system as a scaffold for the spatial organization of bacterial metabolism. This technique might be used to establish multi-enzymatic reaction programs in a prokaryotic chassis for various applications.

Rib Cartilage Assessment Relative to the Healthy Ear in Young Children with Microtia Guiding Operative Timing.

BACKGROUND: The optimal age at which to initiate for auricular reconstruction is controversial. Rib cartilage growth is closely related to age and determines the feasibility and outcomes of auricular reconstruction. We developed a method to guide the timing of auricular reconstruction in children with microtia ranging in age from 5 to 10 years. METHODS: Rib cartilage and the healthy ear were assessed using low-dose multi-slice computed tomography. The lengths of the eighth rib cartilage and the helix of the healthy ear (from the helical crus to the joint of the helix and the earlobe) were measured. Surgery was performed when the two lengths were approximately equal. RESULTS: The preoperative eighth rib measurements significantly correlated with the intraoperative measurements (P < 0.05). From 5 to 10 years of age, eighth rib growth was not linear. In 76 (62.8%) of 121 patients, the eighth rib length was approximately equal to the helix length in the healthy ear; satisfactory outcomes were achieved in these patients. In 18 (14.9%) patients, the eighth rib was slightly shorter than the helix, helix fabrication was accomplished by adjusting the length of the helical crus of stent, and satisfactory outcomes were also achieved. Acceptable outcomes were achieved in 17 (14.0%) patients in whom helix fabrication was accomplished by cartilage splicing. In 9 (7.4%) patients with insufficient rib cartilage length, the operation was delayed. In one (0.8%) patient with insufficient rib cartilage length, which left no cartilage for helix splicing, the result was unsatisfactory. CONCLUSIONS: Eighth rib cartilage growth is variable. Rib cartilage assessment relative to the healthy ear can guide auricular reconstruction and personalize treatment in young patients with microtia.

An efficient strategy for heterologous expression and purification of active peptide hainantoxin-IV.

Hainantoxin-IV (HNTX-IV) from the venom of the spider Selenocosmia hainana is a potent antagonist that specifically inhibits the tetrodotoxin-sensitive (TTX-S) sodium channels. The toxin peptide consists of 35 amino acids and adopts a typical inhibitory cystine knot (ICK) motif. To obtain adequate HNTX-IV peptides for further insight into the structure-activity relationships of the toxin, a novel strategy including cloning, expression and purification was developed in an E. coli expression system. For this purpose, a seamless restriction-free (RF) cloning method was employed for the construction of an expression vector to avoid introducing unwanted sequences into the target gene. Furthermore, the solubility of recombinant HNTX-IV could be promoted efficiently by the combination of a glutathione S-transferase (GST) tag and a small ubiquitin-related modifier (SUMO) tag. Finally, an affinity-chromatography-free purification strategy was developed by cut-off dialysis tubing combined with trichloroacetic acid (TCA) extraction. Further HPLC purification yielded recombinant, tag-free HNTX-IV with high yield and purity. The molecular weight of recombinant HNTX-IV (rHNTX-IV) is identical to its theoretical value according to Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) analysis. The recombinant toxin has similar activity (IC50 value of 120 nM) on the tetrodotoxin-sensitive (TTX-S) sodium channels in adult rat dorsal root ganglion (DRG) neurons to native toxins. In the report, an efficient and cost-effective strategy for producing rHNTX-IV was developed, which paved the way for the further study of structure-activity relationships of rHNTX-IV and its pharmaceutical applications.

Screening for voltage-gated sodium channel interacting peptides.

The voltage-gated sodium channel (VGSC) interacting peptide is of special interest for both basic research and pharmaceutical purposes. In this study, we established a yeast-two-hybrid based strategy to detect the interaction(s) between neurotoxic peptide and the extracellular region of VGSC. Using a previously reported neurotoxin JZTX-III as a model molecule, we demonstrated that the interactions between JZTX-III and the extracellular regions of its target hNav1.5 are detectable and the detected interactions are directly related to its activity. We further applied this strategy to the screening of VGSC interacting peptides. Using the extracellular region of hNav1.5 as the bait, we identified a novel sodium channel inhibitor SSCM-1 from a random peptide library. This peptide selectively inhibits hNav1.5 currents in the whole-cell patch clamp assays. This strategy might be used for the large scale screening for target-specific interacting peptides of VGSCs or other ion channels.

[Auricular reconstruction for microtia with modified Nagata method].

OBJECTIVE: To investigate a method for total reconstruction of auricle. METHODS: 90 patients (101 ears) with congenital microtia underwent two-stage operations for auricular reconstruction. The first stage involved fabrication and grafting of autologous costal cartilage, removing the remnant ear cartilage, embedding the framework into local flap of the mastoid region, transferring the remnant ear lobule flap to link to the inferior framework. The second stage was creating an auriculocephalic sulcus. The reconstruction was performed 4 - 12 months after the first surgery. Skin incision was made 5 mm lateral side of the posterior margin of the auricle. The ear framework carrying a thick ear fascia was separated from the side of the head, the frames of the costal cartilage banked at the first operation were harvested, shaved and transplanted to the posterior wall of the concha with sutures; adjust stand position and angle, so that made the ear shape, position, axis, close to the healthy ear, and auriculocephalic angle was slightly larger than the contralateral ear. Two random flap was designed with superior on the root of the helix and in the inferior-posterior direction of the inferior mastoid area, two flapes were elevated and transplanted to posterior auricular sulcus to cover the grafted cartilage. Skin graft was performed in the remaining raw surface. RESULTS: A total of 90 patients were operated, all of 101 constructed ears achieved satisfied or near satisfied shapes. Five cases of partial skin flap necrosis were caused by pedicle impairment. Exposure of cartilage framework happened in two cases. The auriculocephalic sulcus of four cases diminished after the second stage operation. Three month to two-year follow-up of 67 patients showed that the reconstructed ears were satisfied with the results, including good shapes and steady auriculocephalic angles. CONCLUSIONS: The method is a simple, safe and reliable method for total aural reconstruction.

[Reconstruction of segmental ear helix defect with post-auricular skin flap].

OBJECTIVE: To explore the method of repairing segmental ear helix defect. METHODS: Twenty-one patients with segmental ear helix defect were repaired with post-auricular skin flap. In the first stage operation, ear helix defect was assessed, including the anterior and posterior area defect. According to the defect, post-auricular skin flap was designed and transplanted to repair the defect. Six weeks later, the pedicle of the post-auricular skin flap was cut off, elevated, and folded to form the helix. The secondary defect was directly sutured or repaired with skin graft. RESULTS: Twenty-one patients were treated with this method. In two to 12 months follow-up, all flaps survived and reconstructed ear helices were in good shape. The reconstructed ears were in symmetry to the healthy ones. CONCLUSION: The method is safe and effective for the correction of segmental ear helix defect.

[Creation of auriculocephalic sulcus with two random skin flaps from mastoid area combined with skin graft].

OBJECTIVE: To investigate the method for creation of auriculocephalic sulcus. METHODS: The reconstruction was performed 4-12 months after the first surgery. Skin incision was made 5mm posterior to the outer margin of the auricle. The ear framework was elevated with a thick fascia at the deep surface. The costal cartilage banked at the first operation was shaved and transplanted to the deep surface of the concha with sutures. The position and angle of the ear framework was adjusted to be familiar to the healthy ear. The auriculocephalic angle was slightly larger than that in the contralateral ear. Two flaps were designed at the upper and lower area of reconstructed ear and rotated to cover the cartilage. The wound at the donor site was closed with skin graft. RESULTS: A total of 72 patients were treated. All the flaps survived completely. 51 patients were followed up for 3-24 months with satisfactory results. The auriculocephalic sulcus maintained at about 20-30 degree. CONCLUSIONS: It is a simple, safe and reliable method to create a auriculocephalic sulcus with two random skin flaps from mastoid area combined with skin graft.