Fine-Grained Lithofacies Types and Sedimentary Model of the Upper Permian Longtan Formation Coal Measures in the Western Guizhou Region, South China.

The upper Permian Longtan Formation is widely distributed in southwestern China and is well known for multilayer coal and high organic shale, with significant shale gas potential that has yet to be fully explored and developed. The Longtan coal-bearing strata are composed of complex lithological assemblages of fine-grained sedimentary rocks such as sandstone, coal, shale, and limestone, which exhibit significant differences from marine shale. To better understand the organic-rich lithofacies, their distribution, and their controlling factors, this study carried out a detailed survey of the outcrop and drill cores in the western Guizhou region and examined the fine-grained lithofacies, their assemblages, and their geochemical characteristics. The results showed that (1) the total organic carbon of the Longtan Formation shale in western Guizhou ranged from 1.44 to 14.79%, with an average of 6.41%, and the organic matter was mainly composed of vitrinite. The mineral composition was mainly clay minerals and brittle minerals; the clay minerals were mainly composed of kaolinite (average 11.13%) and illite/smectite mixed layers (average 26.69%) and the brittle minerals were mainly composed of quartz (average 31.63%) and feldspar (average 12.88%). (2) Eight types of lithofacies were identified, including silty mudstone, muddy siltstone, carbonaceous mudstone, carbonaceous shale, bioclastic-bearing mudstone, bioclastic-bearing sandstone, fine sandstone, and coal seam. (3) The six typical lithofacies assemblages were developed in the Longtan Formation, which represented different sedimentary environments of the marine-continental transitional facies in the study area. The lithofacies assemblages A and C represent sedimentation in the lagoon environment. The lithofacies assemblage B represents peat swamp facies. The lithofacies assemblage D represents a tidal flat facies peat flat-mixed flat-sand flat sedimentary environment. The lithofacies assemblage E and F represent the delta sedimentary environment. (4) The sedimentary model of the Longtan Formation in western Guizhou was predominantly deltaic and tidal flat sedimentary systems. Lithological and lithofacies studies of Longtan fine-grained rocks were used to provide a geological framework for examining the fine grain deposition distribution and shale gas resource evaluation. This study is highly important for understanding the sedimentology and oil and gas exploration in the region, providing a basis for identifying and exploring coal-bearing shale gas potential and a reference for the analysis of shale in the world's continental transitional areas.

Using CIVT-SELEX to Select Aptamers as Genetic Parts to Regulate Gene Circuits in a Cell-Free System.

The complexity of genetic circuits has not seen a significant increase over the last decades, even with the rapid development of synthetic biology tools. One of the bottlenecks is the limited number of orthogonal transcription factor-operator pairs. Researchers have tried to use aptamer-ligand pairs as genetic parts to regulate transcription. However, most aptamers selected using traditional methods cannot be directly applied in gene circuits for transcriptional regulation. To that end, we report a new method called CIVT-SELEX to select DNA aptamers that can not only bind to macromolecule ligands but also undergo significant conformational changes, thus affecting transcription. The single-stranded DNA library with affinity to our example ligand human thrombin protein is first selected and enriched. Then, these ssDNAs are inserted into a genetic circuit and tested in the in vitro transcription screening to obtain the ones with significant inhibitory effects on downstream gene transcription when thrombins are present. These aptamer-thrombin pairs can inhibit the transcription of downstream genes, demonstrating the feasibility and robustness of their use as genetic parts in both linear DNAs and plasmids. We believe that this method can be applied to select aptamers of any target ligands and vastly expand the genetic part library for transcriptional regulation.

Metabolic engineering of Escherichia coli for the production of (R)-α-lipoic acid.

OBJECTIVE: To increase the production of (R)-α-lipoic acid directly from octanoic acid using engineered Escherichia coli with the regeneration of S-adenosylmethionine. RESULTS: The biosynthesis of (R)-α-lipoic acid (LA) in E. coli BL21(DE3) is improved by co-expression of lipoate-protein ligase A (LplA) from E. coli MG1655 and lipoate synthase (LipA) from Vibrio vulnificus. The engineered strain produces 20.99 µg l-1 of LA in shake flask cultures. The titers of LA are increased to 169.28 µg l-1 after the optimization of the medium components and fermentation conditions. We find that the [4Fe-4S] cluster is important for the activity of LipA and co-expression of iscSUA promotes the regeneration of the [4Fe-4S] cluster and leads to the highest LA titer of 589.30 µg l-1. CONCLUSION: The method described here can be widely applied for the biosynthesis of (R)-α-lipoic acid and other metabolites.

Biomimetic Asymmetric Composite Dressing by Electrospinning with Aligned Nanofibrous and Micropatterned Structures for Severe Burn Wound Healing.

The surface structure and topography of biomaterials play a crucial role in directing cell behaviors and fates. Meanwhile, asymmetric dressings that mimic the natural skin structure have been identified as an effective strategy for enhancing wound healing. Inspired by the skin structure and the superhydrophobic structure of the lotus leaf, an asymmetric composite dressing was obtained by constructing an asymmetric structure and wettability surface modification on both sides of the sponge based on electrospinning. Among them, the collagen and quaternized chitosan sponge was fabricated by freeze-drying, followed by an aligned poly(ε-caprolactone) (PCL)/gelatin nanofiber hydrophilic inner layer and hierarchical micronanostructure PCL/polystyrene microsphere highly hydrophobic outer layer constructed on each side of the sponge. The proposed asymmetric composite dressing combines topological morphology with the material's properties to effectively prevent bacterial colonization/infection and promote wound healing by directing cellular behavior. In vitro experimental results confirmed that the aligned nanofiber inner layer effectively promotes cell adhesion, proliferation, directed cell growth, and migration. Meanwhile, the sponge has good water absorption and antibacterial properties, while the biomimetic hydrophobic outer layer exhibits strong mechanical properties and resistance to bacterial adhesion. In vivo results showed that the composite dressing can reduce inflammatory response, prevent infection, accelerate angiogenesis and epithelial regeneration, and significantly accelerate the healing of severe burns. Thus, the proposed bionic asymmetric dressing is expected to be a promising candidate for severe burn wound healing.

An injectable double network hydrogel with hemostasis and antibacterial activity for promoting multidrug-resistant bacteria infected wound healing.

Multidrug-resistant bacteria infections frequently occur in wound care due to the excessive use of antibiotics. It can cause scar formation, wound closure delay, multiple organ failure, and high mortality. Here, a double network hydrogel with injectability, hemostasis, and antibacterial activity was developed to prompt multidrug-resistant bacteria infected wound healing. The double network hydrogel is composed of gelatin methacryloyl (GelMA), oxidized dextran (ODex), ε-polylysine (EPL), and bacitracin, and formed through the Schiff-base and UV-initiated crosslinking reaction. The injectable hydrogel with an adhesion effect could adapt to the irregular shape of the wound and possesses good hemostatic ability. The hydrogel presents good flexibility and rapid resilience due to its double network structure, and it can prompt cell proliferation and migration. In particular, the hydrogel has broad-spectrum in vitro antimicrobial activities against S. aureus, E. coli, and methicillin-resistant S. aureus (MRSA), and disrupts E. coli and MRSA biofilms. In vivo results demonstrated that the hydrogel can completely heal MRSA-infected wound in rats within 15 days, through inhibiting the growth of bacteria, accelerating skin tissue reepithelialization, collagen deposition, and angiogenesis, as well as adjusting the expression of CD31, α-SMA, and TNF-α. The findings of this study suggest that the presented hydrogel could enhance multidrug-resistant bacteria infected wound healing and mitigate antimicrobial resistance.

Rapid screening of glycosyltransferases in plants using a linear DNA expression template based cell-free transcription-translation system.

Plants have an extensively large number of enzymes including glycosyltransferases that are important in the biosynthesis of natural products. However, it is time-consuming and challenging to study these enzymes and only a small percentage of them have been well-characterized. Here, we report a rapid method to screen plant glycosyltransferases using a linear DNA expression template (LET) based cell-free transcription-translation system (TX-TL). As a proof of concept, we amplified and tested glycosyltransferases from Arabidopsis thaliana and showed that the catalytic activity results of these glycosyltransferases from LET-based-TX-TL were consistent with previous studies. We then chose a local medicinal plant Anoectochilus roxburghii, acquired its transcriptome sequences, and applied this method to study its glycosyltransferases. We rapidly expressed all the putative UDP-glucose glycosyltransferases using LET-based-TX-TL and discovered 6 unreported active glycosyltransferases which can catalyze the glycosylation of quercetin into isoquercitrin. Thus, LET-based-TX-TL was shown to be a powerful tool for researchers to rapidly screen plant glycosyltransferases for the first time.

Smartphone-based reading system integrated with phycocyanin-enhanced latex nanospheres immunoassay for on-site determination of aflatoxin B1 in foodstuffs.

Traditional methods for aflatoxin B1 (AFB1) detection are complex, time-consuming, labor-intensive, and high cost. Moreover, they require sophisticated large-scale instrumentation, which limits their on-site rapid detection. Herein, phycocyanin fluorescent nanospheres based on fluorescence immunochromatographic assay were developed for quantitative detection of AFB1 at parts-per-billion (ppb) levels in foodstuffs. Phycocyanin and anti-AFB1 monoclonal antibodies were coupled on the surface of latex nanospheres to amplify the fluorescence signal and improve the sensitivity. The fluorescence intensity was measured by a self-developed smartphone-based reading system. Under the optimal conditions, this approach achieved quantitative point-of-care detection of AFB1 within 25 min. The calibration curve for AFB1 was linear in the range of 0.2-48 ppb, and the limit of detection was 0.16 ppb. The practical applicability of the proposed approach was demonstrated by the determination of AFB1 in naturally contaminated samples, and the results were consistent with HPLC detection.

Preparation of aminated fish scale collagen and oxidized sodium alginate hybrid hydrogel for enhanced full-thickness wound healing.

Acute full-thickness wounds require a more extended healing period, thus increasing the risk of infection. Severe infection frequently resulted in wound ulceration, necrosis, and even life-threatening complications. Here, a hybrid hydrogel comprising aminated collagen (AC), oxidized sodium alginate (OSA), and antimicrobial peptides (polymyxin B sulfate and bacitracin) was developed to enhance full-thickness wound healing. The AC with low immunogenicity and high biocompatibility was made from marine fish scales, which are eco-friendly, low-cost, and sustainable. The cross-linked hydrogel was formed by a Schiff base reaction without any catalysts and additional procedures. As expected, the presented hybrid hydrogel can effectively against E. coli and S. aureus, as well as promote cell growth and angiogenesis in vitro. In addition, the hydrogel can promote full-thickness wound healing in a rat model through accelerating reepithelialization, collagen deposition, and angiogenesis. Our work demonstrated that the hybrid hydrogel has promising applications in the field of wound healing, which would prompt the utilization of marine fish resources during food processing.

Asymmetric Wettable Composite Wound Dressing Prepared by Electrospinning with Bioinspired Micropatterning Enhances Diabetic Wound Healing.

Wound dressings with asymmetric wettability surfaces can effectively prevent bacterial colonization and tissue dehydration and have shown great potential for diabetic wound healing applications. However, the construction of a highly hydrophobic outer surface with high biocompatibility and permeability is still the major challenge in the preparation of asymmetric wettable dressings. Inspired by the superhydrophobic surface structures existing in nature, an asymmetric wettable composite wound dressing with a highly hydrophobic outer layer was successfully prepared for diabetic wound healing in this study. The hydrophobic outer layer was fabricated by the electrospinning of poly(ε-caprolactone) (PCL) on a micron-pore-size nylon mesh template, and the hydrophilic inner layer was obtained by the electrospinning of pioglitazone-incorporated gelatin (Gel-pio). The hydrophobic outer layer of the dressing with a hierarchical micro-nanostructure exhibits excellent ability to waterproof and prevent bacterial adhesion, whereas the hydrophilic inner layer can promote cell proliferation, migration, and angiogenesis by its nanofiber structure and biocompatible gelatin composition. The presented dressing has good mechanical properties, permeability, and high biocompatibility. More importantly, the results of full-thickness skin wound model evaluation on db/db mice (type 2 diabetes) and STZ rats (type 1 diabetes) indicate that the developed dressing can promote wound healing by stimulating cell proliferation, angiogenesis, collagen deposition, and re-epithelialization. The findings of this study suggest that the bioinspired asymmetric wettable composite wound dressing can be used as a promising candidate for diabetic wound healing.

Exclusive Production of Gentamicin C1a from Micromonospora purpurea by Metabolic Engineering.

Gentamicin C1a is an important precursor to the synthesis of etimicin, a potent antibiotic. Wild type Micromonospora purpurea Gb1008 produces gentamicin C1a, besides four other gentamicin C components: C1, C2, C2a, and C2b. While the previously reported engineered strain M. purpurea GK1101 can produce relatively high titers of C1a by blocking the genK pathway, a small amount of undesirable C2b is still being synthesized in cells. Gene genL (orf6255) is reported to be responsible for converting C1a to C2b and C2 to C1 in Micromonospora echinospora ATCC15835. In this work, we identify the genL that is also responsible for the same methylation in Micromonospora purpurea. Based on M. purpurea GK1101, we construct a new strain with genL inactivated and show that no C2b is produced in this strain. Therefore, we successfully engineer a strain of M. purpurea that solely produces gentamicin C1a. This strain can potentially be used in the industrial production of C1a for the synthesis of etimicin.

Construction of Incoherent Feedforward Loop Circuits in a Cell-Free System and in Cells.

Cells utilize transcriptional regulation networks to respond to environmental signals. Network motifs, such as feedforward loops, play essential roles in these regulatory networks. In this work, we construct two different functional and modular incoherent type 1 feedforward loop circuits in a cell-free transcription-translation system and in cells. With the help of mathematical modeling and the cell-free system, we can streamline the design-build-test cycles of the circuits, in which we characterize and optimize these circuits in vitro to confirm that they function as expected before implementing them in vivo. We show that the performance of these circuits from in vitro studies closely recapitulates those from in vivo experiments. We demonstrate that these feedforward loops show dynamic response and pulse-like behavior both in vitro and in vivo. These novel feedforward loop network motifs can be incorporated in more complicated biological circuits as detectors or responders.

On-chip membrane protein cell-free expression enables development of a direct binding assay: A curious case of potassium channel KcsA-Kv1.3.

Despite the significant role integral membrane proteins (IMPs) play in the drug discovery process, it remains extremely challenging to express, purify, and in vitro stabilize them for detailed biophysical analyses. Cell-free transcription-translation systems have emerged as a promising alternative for producing complex proteins, but they are still not a viable option for expressing IMPs due to improper post-translational folding of these proteins. We have studied key factors influencing in vitro folding of cell-free-expressed IMPs, particularly oligomeric proteins (i.e., ion channels). Using a chimeric ion channel, KcsA-Kv1.3 (K-K), as a model IMP, we have investigated several physiochemical determinants including artificial bilayer environments (i.e., lipid, detergent) for K-K in vitro stabilization. We observed that fusion of a 'superfolder' green fluorescent protein (sfGFP) to K-K as a protein expression reporter not only improves the protein yield, but surprisingly facilitates the K-K tetramer formation, probably by enhancing the solubility of monomeric K-K. Additionally, anionic lipids (i.e., DMPG) were found to be essential for the correct folding of cell-free-expressed monomeric K-K into tetramer, underscoring the importance of lipid-protein interaction in maintaining structural-functional integrity of ion channels. We further developed methods to integrate cell-free-expressed IMPs directly onto a biosensor chip. We employed a solid-supported lipid bilayer onto the surface plasmon resonance (SPR) chip to insert nascent K-K in a membrane. In a different approach, an anti-GFP-functionalized surface was used to capture in situ expressed K-K via its sfGFP tag. Interestingly, only the K-K-functionalized capture surface prepared by the latter strategy was able to interact with K-K's small binding partners. This generalizable approach can be further extended to other membrane proteins for developing direct binding assays involving small ligands.