Oxidative Cascade Iodocyclization of 1,n-Dienes: Synthesis of Iodinated Benzo[b]azepine and Benzo[b]azocine Derivatives.

An oxidative cascade iodocyclization of 1,7- or 1,8-dienes has been realized under mild conditions. By employing I2 as an iodine source, this protocol provides a concise and efficient approach to a great deal of biologically significant iodinated benzo[b]azepine and benzo[b]azocine derivatives in moderate to good yields. The gram-scale synthesis and further transformation of products render the approach practical and attractive. Radical trapping and deuterium-labeling experiments help to understand the mechanism.

Cascade Radical Trifluoromethylthiolation/Cyclization of Dienes To Access SCF3-Containing Medium-Sized Heterocycles.

A novel radical cascade trifluoromethylthiolation/cyclization of dienes (N-alkyl-2-(1-phenylvinyl)aniline derivatives) with AgSCF3 has been developed. This approach provides simple and efficient access to a wide range of SCF3-containing medium-sized rings (7/8/9-membered heterocycles). Preliminary mechanistic studies suggest that the reaction is realized through a silver-assisted radical cascade cyclization process. The large-scale experiment and modification of the product reveal the promising utility of this protocol.

Photo- or Electrochemical Cyclization of Dienes with Diselenides to Access Seleno-Benzo[b]azepines.

A cascade selenylation/cyclization of dienes with diselenides has been realized under visible-light irradiation or electrolysis conditions. Employing O2 or electricity as a "green" oxidant, this protocol provides a green and efficient method for an array of biologically important seleno-benzo[b]azepine derivatives in moderate to good yields. The direct sunlight irradiation and gram-scale reaction render the approach practical and attractive.

Hepatocellular carcinoma detection via targeted enzymatic methyl sequencing of plasma cell-free DNA.

BACKGROUND: Epigenetic variants carried by circulating tumor DNA can be used as biomarkers for early detection of hepatocellular carcinoma (HCC) by noninvasive liquid biopsy. However, traditional methylation analysis method, bisulfite sequencing, with disadvantages of severe DNA damage, is limited in application of low-amount cfDNA analysis. RESULTS: Through mild enzyme-mediated conversion, enzymatic methyl sequencing (EM-seq) is ideal for precise determination of cell-free DNA methylation and provides an opportunity for HCC early detection. EM-seq of methylation control DNA showed that enzymatic conversion of unmethylated C to U was more efficient than bisulfite conversion. Moreover, a relatively large proportion of incomplete converted EM-seq reads contains more than 3 unconverted CH site (CH = CC, CT or CA), which can be removed by filtering to improve accuracy of methylation detection by EM-seq. A cohort of 241 HCC, 76 liver disease, and 279 normal plasma samples were analyzed for methylation value on 1595 CpGs using EM-seq and targeted capture. Model training identified 283 CpGs with significant differences in methylation levels between HCC and non-HCC samples. A HCC screening model based on these markers can efficiently distinguish HCC sample from non-HCC samples, with area under the curve of 0.957 (sensitivity = 90%, specificity = 97%) in the test set, performing well in different stages as well as in serum α-fetoprotein/protein induced by vitamin K absence-II negative samples. CONCLUSION: Filtering of reads with ≥ 3 CHs derived from incomplete conversion can significantly reduce the noise of EM-seq detection. Based on targeted EM-seq analysis of plasma cell-free DNA, our HCC screening model can efficiently distinguish HCC patients from non-HCC individuals with high sensitivity and specificity.

Two-Dimensional Mg2 Si Nanosheet-Enabled Sustained Hydrogen Generation for Improved Repair and Regeneration of Deeply Burned Skin.

Molecular hydrogen holds a high potential for wound healing owing to its anti-inflammatory effect and high biosafety, but commonly used hydrogen administration routes hardly achieve the sustained supply of high-dosage hydrogen, limiting hydrogen therapy efficacy. Here, two-dimensional Mg2 Si nanosheet (MSN) is exploited as a super-persistent hydrogen-releasing nanomaterial with high biocompatibility, and the incorporation of MSN into the chitosan/hyaluronic acid hydrogel (MSN@CS/HA) is developed as a dressing to repair deeply burned skin. The MSN@CS/HA hydrogel dressing can continuously generate hydrogen molecules for about 1 week in the physiological conditions in support of local, long-term, and plentiful hydrogen supply and remarkably promotes the healing and regeneration of deep second-degree and third-degree burn wounds without visible scar and toxic side effect. Mechanistically, a sustained supply of hydrogen molecules induces anti-inflammatory M2 macrophage polarization in time by enhancing CCL2 (chemokine C-C motif ligand 2) expression to promote angiogenesis and reduce fibrosis and also enhances the proliferation and migration capability of skin cells directly and indirectly by locally scavenging overexpressed reactive oxygen species, synergistically favoring wound repair. The proposed synthesis method, therapeutic strategy, and mechanisms will open a window for synthesizing a variety of MSene nanomaterials and developing their various proangiogenesis applications besides wound healing.

K2S2O8/I2-Promoted Electrophilic Selenylative Cyclization To Access Seleno-Benzo[b]azepines.

A novel and simple organoselenium-involved 7-membered cyclization to access diverse seleno-benzo[b]azepines has been developed. This protocol involves an electrophilic cyclization process and is accomplished under mild conditions. Discussion of the mechanism rationalizes the regioselectivity observed in transformation. The studies of further transformation of seleno-benzo[b]azepines and large-scale experiment reveal the promising utility of this methodology.

Engineered Halomonas spp. for production of l-Lysine and cadaverine.

Cadaverine, a derivative of l-lysine, has been used as a monomer for the synthesis of bio-based nylon-5,6. This study engineered Halomonas bluephagenesis TD1.0 by blocking the feedback inhibition, overexpressing the key l-lysine synthesis genes, strengthening the l-lysine export system and increasing the supply of oxaloacetate for production of l-lysine in the supernatant and PHB in the cells. Subsequently, cadaverine biosynthetic pathway was constructed in H. campaniensis LC-9 to improve the efficiency of de novo cadaverine biosynthesis which combines l-lysine producing H. bluephagenesis TDL8-68-259 and cadaverine producing H. campaniensis LC-9-ldcC-lysP. When H. campaniensis LC-9-ldcC-lysP was used as a whole cell catalysis for cadaverine production, the conversion efficiency of l-lysine to cadaverine reached 100% in the presence of 0.05% Triton X-100 for cell membrane permeability enhancement, resulting in 118 g L-1 cadaverine formed in the fermentor. Thus, Halomonas spp. have been successfully constructed for l-lysine and cadaverine production.

Euglena gracilis and Its Aqueous Extract Constructed With Chitosan-Hyaluronic Acid Hydrogel Facilitate Cutaneous Wound Healing in Mice Without Inducing Excessive Inflammatory Response.

Naturally occurring compounds isolated from the microalga Euglena gracilis, such as polysaccharide paramylon, exhibit antimicrobial, anti-viral, antitumor, and anti-inflammatory activities. Whether live E. gracilis cells and its aqueous extract accelerate burn wound healing remains to be investigated. In this study, live E. gracilis cells and its aqueous extract were mixed with chitosan-hyaluronic acid hydrogel (CS/HA) to form cell + CS/HA and extract + CS/HA, which were then smeared onto the deeply burned skin of mice. The efficacy of these mixtures in accelerating wound healing was assessed through wound size reduction measurement, histological and immunofluorescence analyses, and serum pro-inflammatory cytokine level (INF-γ, IL-1ß, and IL-6) determination. The live E. gracilis cells and its aqueous extract were found to facilitate wound healing by enhancing re-epithelization and reducing fibroplasia without stimulating excessive inflammatory response. In conclusion, live E. gracilis cells and its aqueous extract can be potentially used to treat cutaneous wounds.

Engineering Halomonas bluephagenesis for L-Threonine production.

Halophilic Halomonas bluephagenesis (H. bluephagenesis), a chassis for cost-effective Next Generation Industrial Biotechnology (NGIB), was for the first time engineered to successfully produce L-threonine, one of the aspartic family amino acids (AFAAs). Five exogenous genes including thrA*BC, lysC* and rhtC encoding homoserine dehydrogenase mutant at G433R, homoserine kinase, L-threonine synthase, aspartokinase mutant at T344M, S345L and T352I, and export transporter of threonine, respectively, were grouped into two expression modules for transcriptional tuning on plasmid- and chromosome-based systems in H. bluephagenesis, respectively, after pathway tuning debugging. Combined with deletion of import transporter or/and L-threonine dehydrogenase encoded by sstT or/and thd, respectively, the resulting recombinant H. bluephagenesis TDHR3-42-p226 produced 7.5 g/L and 33 g/L L-threonine when grown under open unsterile conditions in shake flasks and in a 7 L bioreactor, respectively. Engineering H. bluephagenesis demonstrates strong potential for production of diverse metabolic chemicals.

Engineering biosynthesis of polyhydroxyalkanoates (PHA) for diversity and cost reduction.

PHA, a family of natural biopolymers aiming to replace non-degradable plastics for short-term usages, has been developed to include various structures such as short-chain-length (scl) and medium-chain-length (mcl) monomers as well as their copolymers. However, PHA market has been grown slowly since 1980s due to limited variety with good mechanical properties and the high production cost. Here, we review most updated strategies or approaches including metabolic engineering, synthetic biology and morphology engineering on expanding PHA diversity, reducing production cost and enhancing PHA production. The extremophilic Halomonas spp. are taken as examples to show the feasibility and challenges to develop next generation industrial biotechnology (NGIB) for producing PHA more competitively.

A tight cold-inducible switch built by coupling thermosensitive transcriptional and proteolytic regulatory parts.

Natural organisms have evolved intricate regulatory mechanisms that sense and respond to fluctuating environmental temperatures in a heat- or cold-inducible fashion. Unlike dominant heat-inducible switches, very few cold-inducible genetic switches are available in either natural or engineered systems. Moreover, the available cold-inducible switches still have many shortcomings, including high leaky gene expression, small dynamic range (<10-fold) or broad transition temperature (>10°C). To address these problems, a high-performance cold-inducible switch that can tightly control target gene expression is highly desired. Here, we introduce a tight and fast cold-inducible switch that couples two evolved thermosensitive variants, TFts and TEVts, as well as an additional Mycoplasma florum Lon protease (mf-Lon) to effectively turn-off target gene expression via transcriptional and proteolytic mechanisms. We validated the function of the switch in different culture media and various Escherichia coli strains and demonstrated its tightness by regulating two morphogenetic bacterial genes and expressing three heat-unstable recombinant proteins, respectively. Moreover, the additional protease module enabled the cold-inducible switch to actively remove the pre-existing proteins in slow-growing cells. This work establishes a high-performance cold-inducible system for tight and fast control of gene expression which has great potential for basic research, as well as industrial and biomedical applications.

Expression and clinical significance of latent-transforming growth factor beta-binding protein 2 in primary hepatocellular carcinoma.

The present study aimed to explore the expression of latent transforming growth factor ß binding protein 2 (LTBP2) in patients with hepatocellular carcinoma (HCC) and their correlation to clinicopathologial features.Serum levels of LTBP2 in 60 patients with HCC, 35 patients with hepatocellular benign tumors, 60 patients with precancerous lesions of HCC, and 60 healthy volunteers were determined by enzyme-linked immunosorbent assay. The expression levels of LTBP2 at messenger RNA (mRNA) and protein levels in 60 cases of HCC and adjacent tissues were detected by quantitative real-time polymerase chain reaction and immunohisochemistry. Statistical analysis was used to analyze the relationship between LTBP2 and clinical characteristics of patients with HCC.The mRNA and protein levels of LTBP2 were significantly upregulated in HCC tissues compared to adjacent tissues. Additionally, higher serum LTBP2 level was also observed in HCC patients relative to normal controls. Further investigation demonstrated that LTBP2 expression was associated with malignant degree of tumor, tumor progression, tumor differentiation, tumor size, tumor stage and hepatitis virus infection, and has prognostic implications in HCC patients.LTBP2 might be served as a potential biomarker in diagnosis and treatment of HCC.

Synthesis and Characterization of Polyhydroxyalkanoate Organo/Hydrogels.

Synthetic organogels/hydrogels are attracting growing interests due to their potential applications in biomedical fields, organic electronics, and photovoltaics. Photogelation methods for synthesis of organogels/hydrogels have been shown particularly promising because of the high efficiency and simple synthetic procedures. This study synthesized new biodegradable polyhydroxyalkanoates (PHA)-based organogels/hydrogels via UV photo-cross-linking using unsaturated PHA copolymer poly[(R)-3-hydroxyundecanoate-co-(R)-3-hydroxy-10-undecenoate] (PHU10U) with polyethylene glycol dithiol (PDT) as a photo-cross-linker. The PHU10U was synthesized by an engineered Pseudomonas entomophila and characterized via Fourier transform infrared spectroscopy, 1H nuclear magnetic resonance (NMR), and 13C NMR. With decreasing the molar ratio of PHU10U to PDT, both the swelling ratio and pore size were decreased. Meanwhile, increasing densities of the gel networks resulted in a higher compressive modulus. Cell cytotoxicity studies based on the CCK-8 assay on both the PHU10U precursor and PHU10U/PDT hydrogels showed that the novel PHA-based biodegradables acting as hydrogels possess good biocompatibility.

Manipulation of polyhydroxyalkanoate granular sizes in Halomonas bluephagenesis.

Bacterial polyhydroxyalkanoates (PHA) are a family of intracellular polyester granules with sizes ranging from 100 to 500 nm. Due to their small sizes, it has been very difficult to separate the PHA granules from the bacterial broths. This study aims to engineer the PHA size control mechanism to obtain large PHA granular sizes beneficial for the separation. It has been reported that phasin (PhaP) is an amphiphilic protein located on the surface of PHA granules functioning to regulate sizes and numbers of PHA granules in bacterial cells, deletions on PhaPs result in reduced PHA granule number and enhanced granule sizes. Three genes phaP1, phaP2 and phaP3 encoding three PhaP proteins were deleted in various combinations in halophilic bacterium Halomonas bluephagenesis TD01. The phaP1-knockout strain generated much larger PHA granules with almost the same size as their producing cells without significantly affecting the PHA accumulation yet with a reduced PHA molecular weights. In contrast, the phaP2- and phaP3-knockout strains produced slightly larger sizes of PHA granules with increased PHA molecular weights. While PHA accumulation by phaP3-knockout strains showed a significant reduction. All of the PhaP deletion efforts could not form PHA granules larger than a normal size of H. bluephagenesis TD01. It appears that the PHA granular sizes could be limited by bacterial cell sizes. Therefore, genes minC and minD encoding proteins that block formation of cell fission rings (Z-rings) were over-expressed in various phaP deleted H. bluephagenesis TD01, resulting in large cell sizes of H. bluephagenesis TD01 containing PHA granules with sizes of up to 10 µm that has never been observed previously. It can be concluded that PHA granule sizes are limited by the cell sizes. By engineering a large cell morphology large PHA granules can be produced by PhaP deleted mutants.

Chromosome engineering of the TCA cycle in Halomonas bluephagenesis for production of copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV).

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a promising biopolyester with good mechanical properties and biodegradability. Large-scale production of PHBV is still hindered by the high production cost. CRISPR/Cas9 method was used to engineer the TCA cycle in Halomonas bluephagenesis on its chromosome for production of PHBV from glucose as a sole carbon source. Two TCA cycle related genes sdhE and icl encoding succinate dehydrogenase assembly factor 2 and isocitrate lysase were deleted, respectively, in H. bluephagenesis TD08AB containing PHBV synthesis genes on the chromosome, to channel more flux to increase the 3-hydroxyvalerate (3HV) ratio of PHBV. Due to a poor growth behavior of the mutant strains, H. bluephagenesis TY194 equipped with a medium strength Pporin-194 promoter was selected for further studies. The sdhE and/or icl mutant strains of H. bluephagenesis TY194 were constructed to show enhanced cell growth, PHBV synthesis and 3HV molar ratio. Gluconate was used to activate ED pathway and thus TCA cycle to increase 3HV content. H. bluephagenesis TY194 (ΔsdhEΔicl) was found to synthesize 17mol% 3HV in PHBV. Supported by the synergetic function of phosphoenolpyruvate carboxylase and Vitreoscilla hemoglobin encoded by genes ppc and vgb inserted into the chromosome of H. bluephagenesis TY194 (ΔsdhE) serving to enhance TCA cycle activity, a series of strains were generated that could produce PHBV containing 3-18mol% 3HV using glucose as a sole carbon source. Shake flask studies showed that H. bluephagenesis TY194 (ΔsdhE, G7::Pporin-ppc) produced 6.3 g/L cell dry weight (CDW), 65% PHBV in CDW and 25mol% 3HV in PHBV when grown in glucose and gluconate. 25mol% 3HV was the highest reported via chromosomal expression system. PHBV copolymers with different 3HV molar ratios were extracted and characterized. Next-generation industrial biotechnology (NGIB) based on recombinant H. bluephagenesis grown under unsterile and continuous conditions, allows production of P(3HB-0∼25mol% 3HV) in a convenient way with reduced production complexity and cost.

Effects of Huatan Tongluo decoction on vascular endothelial growth factor receptor 2 expression in synovial tissues of rats with collagen-induced arthritis.

OBJECTIVE: To determine the therapeutic effect and potential mechanism of Huatan Tongluo decoction on rats with collagen-induced arthritis. METHODS: Forty specific pathogen-free Wistar rats were selected, and 10 were randomly selected as the control (group 1). The remaining rats were injected intradermally with emulsified type II bovine collagen at the tail base and back, followed by a booster 7 d post first immunization. After establishing collagen-induced arthritis (CIA), rats were randomly divided into three groups (n = 10). The rats were treated orally for 30 d as follows: group 1, saline; group 2, model (saline); group 3, tripterygium polyglycoside (TP; 7.81 mg/kg, positive control); group 4, Huatan Tongluo decoction (HTTL; 7.5 g/kg). Body weight, ankle swelling and arthritis index were measured over the course of the study. The rats were sacrificed 30 d after treatment. Morphological changes in the synovium were observed by hematoxylin and eosin staining. Pannus formation and synovial thickness in the left ankle were observed by color Doppler ultrasoundVascular endothelial growth factor (VEGF) and VEGFR2 protein levels were measured by immunohistochemistry. VEGF/VEGFR2 mRNA levels were measured by real-time quantitative polymerase chain reaction. RESULTS: Compared with the model group, a significantly lower arthritis index was observed in the positive control group (P < 0.05) and HTTL group (P < 0.01), after treatment. Both positive control and HTTL reduced intra-articular pannus formation and synovial thickening. Furthermore, VEGF mRNA, and VEGFR2 protein and mRNA levels were significantly downregulated (P < 0.05) in the treatment groups. CONCLUSION: Inhibition of the expression of VEGF and VEGFR2 in synovial tissues and the formation of pannus and synovial hyperplasia may be part of the mechanism of HTTL for relieving the symptoms of rheumatoid arthritis in CIA rats.

LncRNA ZEB2-AS1 promotes pancreatic cancer cell growth and invasion through regulating the miR-204/HMGB1 axis.

Recently, lncRNA ZEB2-AS1 was identified as a lncRNA that promoted cancer progression. However, the biological function and the underlying mechanism of ZEB2-AS1 in pancreatic cancer had not been reported. In the current study, we revealed that the expression level of ZEB2-AS1 was elevated in pancreatic cancer cell lines and tissues. ZEB2-AS1 inhibition decreased cell growth and invasion in pancreatic cancer. Mechanismly, ZEB2-AS1 exerted as a ceRNA and negatively regulated miR-204 expression. In addition, HMGB1 was identified as a down-stream target of miR-204. The miR-204/HMGB1 axis mediated ZEB2-AS1's effect on pancreatic cancer. Our findings revealed that lncRNA ZEB2-AS1 may be a candidate prognostic biomarker and a target for new therapies in pancreatic cancer patients.

Engineering of Halomonas bluephagenesis for low cost production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose.

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] is one of the most promising biomaterials expected to be used in a wide range of scenarios. However, its large-scale production is still hindered by the high cost. Here we report the engineering of Halomonas bluephagenesis as a low-cost platform for non-sterile and continuous fermentative production of P(3HB-co-4HB) from glucose. Two interrelated 4-hydroxybutyrate (4HB) biosynthesis pathways were constructed to guarantee 4HB monomer supply for P(3HB-co-4HB) synthesis by working in concert with 3-hydroxybutyrate (3HB) pathway. Interestingly, only 0.17 mol% 4HB in the copolymer was obtained during shake flask studies. Pathway debugging using structurally related carbon source located the failure as insufficient 4HB accumulation. Further whole genome sequencing and comparative genomic analysis identified multiple orthologs of succinate semialdehyde dehydrogenase (gabD) that may compete with 4HB synthesis flux in H. bluephagenesis. Accordingly, combinatory gene-knockout strains were constructed and characterized, through which the molar fraction of 4HB was increased by 24-fold in shake flask studies. The best-performing strain was grown on glucose as the single carbon source for 60 h under non-sterile conditions in a 7-L bioreactor, reaching 26.3 g/L of dry cell mass containing 60.5% P(3HB-co-17.04 mol%4HB). Besides, 4HB molar fraction in the copolymer can be tuned from 13 mol% to 25 mol% by controlling the residual glucose concentration in the cultures. This is the first study to achieve the production of P(3HB-co-4HB) from only glucose using Halomonas.