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Polyhydroxyalkanoates (PHAs) are microbial polyesters that have the potential to replace nonbiodegradable petroplastics. A real-time in situ PHA quantification method has long been awaited to replace the traditional method, which is time- and labor-consuming. Quantification of PHA in living cells was finally developed from fluorescence intensities generated from the green fluorescence protein (GFP) fused with the

We have described a copper-catalyzed radical 1,2-carbotrifluoromethylselenolation of alkenes using the readily available alkyl halides and (Me4N)SeCF3 salt. Critical to the success is the use of a proline-based N,N,P-ligand to enhance the reducing capability of copper for easy conversion of diverse alkyl halides to the corresponding radicals via a single-electron transfer process. The reaction features a broad substrate scope, including various mono-, di-, and trisubstituted alkenes with many functional groups.

A long-standing objective of metabolic engineering has been to exogenously increase the expression of target genes. In this research, we proposed the permanent RNA replication system using DNA as a template to store genetic information in bacteria. We selected Qβ phage as the RNA replication prototype and made many improvements to achieve target gene expression enhancement directly by increasing mRNA abundance. First, we identified the endogenous gene Rnc, the knockout of which significantly improved the RNA replication efficiency. Second, we elucidated the essential elements for RNA replication and optimized the system to make it more easily applicable. Combined with optimization of the host cell and the system itself, we developed a stable RNA-to-RNA replication tool to directly increase the abundance of the target mRNA and subsequently the target protein. Furthermore, it was proven efficient in enhancing the expression of specific proteins and was demonstrated to be applicable in metabolic engineering. Our system has the potential to be combined with any of the existing methods for increasing gene expression.

Fluorescent materials play an important role in biomedical fields. However, the main types of fluorescent materials suffer from several disadvantages especially the biotoxicity, which largely restrict its wider applications in biological fields. In this study, a highly efficient rare-earth-modified fluorescent material was successfully designed and fabricated based on polyhydroxyalkanoates, which are known as biodegradable and biocompatible materials. A new Functional-PHA polymer was microbially synthesized by engineered Halomonas bluephagenesis and was used as a basal matrix to generate the rare-earth-modified PHA. N-Acetyl-l-cysteine-grafted PHA (NAL-grafted-PHA) was first produced via a UV-initiated thiol-ene click reaction and the rare earth metal ions (Eu3+ and Tb3+) were subsequently chelated onto the NAL-grafted-PHA through the coordination effect. The composite material exhibited intense photoluminescence properties under UV laser excitation, indicating the excellent features as fluorescent material. The enhanced hydrophilicity and superior biocompatibility of rare-earth-chelated PHA were confirmed, suggesting its great potential application value in biomedical fields.

With increasing demand of environmentally friendly materials, development on biobased polymers such as polyhydroxyalkanoate (PHA) is indispensable. An unsaturated PHA, namely, poly(3-hydroxydodecanoate- co-3-hydroxy-9-decenoate), short as P(3HDD- co-3H9D), provides possibilities for functionalization. Two different strategies are explored for synthesis of PHA- graft-graphene nanocomposites with graphene content ranging from 0.2 to 1.5 wt %. Chemical structures of intermediates and products were confirmed by Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR). Uniform dispersion of graphene was observed in formed PHA nanocomposites under scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM). PHA- graft-graphene nanocomposites exhibited higher thermal degradation temperature and enhanced electricity conductivity compared with that of neat PHA. Moreover, lower critical filling content and lower electrical resistivity at same graphene content demonstrated enhanced electrical conductivity of PHA- graft-graphene nanocomposites compared with previously reported blends. The lowest electrical resistivity was 2 Ω·m in sample PHA- graft-graphene nanocomposites with approximately 1.5 wt % graphene content.

Poly( R-3-hydroxybutyrate- co- R-3-hydroxyhexanoate) (PHBHHx), a family member of microbial polyhydroxyalkanoates (PHA), is a biodegradable and biocompatible material with some hydrophobicity and reasonable strength for packaging and tissue engineering applications. In this study, superhydrophobic PHBHHx is fabricated via a simple nonsolvent-assisted process. The material can absorb all tested hydrophobic solvents and oil up to 6-fold of the material weights from water, permitting applications for cleaning environmental oil or solvent pollutions with convenience of disposal after the usage due to its biodegradability. With an excellent combination of biodegradability and biocompatibility, superhydrophobic PHBHHx films are evaluated for antibioadhesion properities to exploit possible implant usages. Up to 100% reductions for platelet adhesions on the superhydrophobic PHBHHx surfaces are observed compared with that on the control material surfaces. Superhydrophobic biodegradable and biocompatible PHBHHx films demonstrate promising low value and high volume or high value and low volume applications.

Acute myeloid leukemia (AML) is a hematologic malignancy that is characterized by clonal proliferation of myeloid blasts. Despite the progress that has been made in the treatment of various malignant hematopoietic diseases, the effective treatment of AML remains very challenging. Differentiation therapy has emerged as a promising approach for leukemia treatment, and new and effective chemical agents to trigger the differentiation of AML cells, especially drug-resistant cells, are urgently required. Herein, the natural product jungermannenone C, a tetracyclic diterpenoid isolated from liverworts, is reported to induce cell differentiation in AML cells. Interestingly, the unnatural enantiomer of jungermannenone C (1) was found to be more potent than jungermannenone C in inducing cell differentiation. Furthermore, compound 1 targets peroxiredoxins I and II by selectively binding to the conserved cysteine residues and leads to cellular reactive oxygen species accumulation. Accordingly, ent-jungermannenone C (1) shows potential for further investigation as an effective differentiation therapy against AML.

The Lewis acidic β-styryl-B(C6F5)2 reagent 6a undergoes a clean 1,1-carboboration reaction with 1-(PMes2)-2-cyclohexenyl acetylene 9 at 60 °C to give the vicinal P/B-substituted conjugated triene product 10a. At 80 °C this undergoes a stereoselective thermally induced disrotatory electrocyclic ring closure to give the cyclohexadiene-derived P/B system 11. Subsequent TEMPO oxidation gave the substituted phenylene-bridged P/B product 12. Both 11 and 12 are active phosphane/borane frustrated Lewis pairs (FLPs). The FLP 11 reacts in a typical way with phenylacetylene to give the phosphonium/alkynylborate product 13. Compound 12 cleaves dihydrogen at near ambient conditions to give the respective phosphonium/hydridoborate zwitterion 14. Both the FLPs 11 and 12 cooperatively add P/B to the nitrogen atom of nitric oxide (NO) within minutes at room temperature to give the persistent P/B FLPNO• radicals 19 and 21, respectively (both characterized by X-ray diffraction and by EPR spectroscopy). The FLPs 11 and 12 ar...

The C4-bridged unsaturated phosphane/borane frustrated Lewis pairs (P/B FLPs) 4 undergo borane induced phosphane addition to a variety of acetylenic esters or ketones to generate heterocyclic 10-membered intermediates that contain pairs of allyl phosphonium/allenic enolate functionalities. These subsequently undergo phospha-Claisen type rearrangement reactions to give the respective substituted phosphanyl pentadiene products. In two exceptional cases subsequent reactions leading to anomalous phospha-Claisen products were found. One example involved cyclopropane ring formation, and the other carbon-carbon bond activation. Potential mechanistic schemes leading to these products are discussed. Essential examples were characterized by X-ray diffraction.

5-Aminolevulinic acid (ALA), an important cell metabolic intermediate useful for cancer treatments or plant growth regulator, was produced by recombinant Escherichia coli expressing the codon optimized mitochondrial 5-aminolevulinic acid synthase (EC: 2.3.1.37, hem1) from Saccharomyces cerevisiae controlled via the plasmid encoding T7 expression system with a T7 RNA polymerase. When a more efficient autoinduced expression approach free of IPTG was applied, the recombinant containing antibiotic-free stabilized plasmid was able to produce 3.6 g/L extracellular ALA in shake flask studies under optimized temperature. A recombinant E. coli expressing synthesis pathways of poly-3-hydroxybutyrate (PHB) and ALA resulted in coproduction of 43% PHB in the cell dry weights and 1.6 g/L extracellular ALA, leading to further reduction on ALA cost as two products were harvested both intracellularly and extracellularly. This was the first study on coproduction of extracellular ALA and intracellular PHB for improving bioprocessing efficiency. The cost of ALA production could be further reduced by employing a Halomonas spp. TD01 able to grow and produce ALA and PHB under continuous and unsterile conditions even though ALA had the highest titer of only 0.7 g/L at the present time.

As a product of a multistep enzymatic reaction, accumulation of poly(3-hydroxybutyrate) (PHB) in Escherichia coli (E. coli) can be achieved by overexpression of the PHB synthesis pathway from a native producer involving three genes phbC, phbA, and phbB. Pathway optimization by adjusting expression levels of the three genes can influence properties of the final product. Here, we reported a semirational approach for highly efficient PHB pathway optimization in E. coli based on a phbCAB operon cloned from the native producer Ralstonia entropha (R. entropha). Rationally designed ribosomal binding site (RBS) libraries with defined strengths for each of the three genes were constructed based on high or low copy number plasmids in a one-pot reaction by an oligo-linker mediated assembly (OLMA) method. Strains with desired properties were evaluated and selected by three different methodologies, including visual selection, high-throughput screening, and detailed in-depth analysis. Applying this approach, strains accumulating 0%-92% PHB contents in cell dry weight (CDW) were achieved. PHB with various weight-average molecular weights (M

Functional polyhydroxyalkanoates (PHAs) allow chemical modifications to widen PHA diversity, promising to increase values of these biodegradable and biocompatible polyesters. Among functional PHAs, unsaturated PHA site chains can be easily grafted to add chemical groups, and to cross-link with other PHA polymer chains. However, it has been very difficult to obtain structurally controllable functional homo-, random, or block PHA. For the first time, a β-oxidation deleted Pseudomonas entomophila was used to successfully synthesize random copolymers of 3-hydroxydodecanoate (3HDD) and 3-hydroxy-9-decenoate (3H9D). Compositions of the random copolymers P(3HDD-co-3H9D) can be adjusted by ratios of dodecanoic acid (DDA) to 9-decenol (9DEO) fed to the culture of P. entomophila. Homopolymer P3H9D was formed when only 9DEO was added to the culture. Diblock copolymers of P3HDD-b-P3H9D were produced by feeding DDA as the first precursor to form a P3HDD block followed by adding 9DEO as the second precursor to form a second P3H9D block. It was demonstrated that random copolymers P(3HDD-co-3H9D) could be crossed-linked under UV-radiation due to the presence of the unsaturated bonds. Thermal and mechanical characterizations of the above homo-, random, and diblock PHA polymers were conducted. It was found that the diblock polymer P3HDD-b-P3H9D increased at least 2-fold on Young's modulus compared with its random copolymers consisting of similar 3HDD/3H9D ratios. This study demonstrates that PHA functionality could be controlled to meet various requirements.

National Ministry of Science and Technology of China [2012CB21500402]; National Natural Science Foundation of China [21276213, 21076171]; State-Key Laboratory of Chemical Engineering of Tsinghua University [SKL-ChE-13A05]

National Natural Science Foundation of China [21076171]; National Ministry of Science and Technology of China [2012CB21500402]; State Key Laboratory of Chemical Engineering of Tsinghua University [SKL-ChE-10A03]

FBOX6 ubiquitin ligase complex is involved in the endoplasmic reticulum-associated degradation pathway by mediating the ubiquitination of glycoproteins. FBXO6 interacts with the chitobiose in unfolded N-glycoprotein, pointing glycoproteins toward E2 for ubiquitination. Although the glycoprotein-recognizing mechanism of FBXO6 is well documented, its bona fide interacting glycoproteins are largely unknown. Here we utilized a protein purification approach combined with LC-MS to systematically identify the FBXO6-interacting glycoproteins. Following identification of 39 proteins that specifically interact with FBXO6 in all three different cell lines, 293T, HeLa and Jurkat cells, we compared the protein complex organization between wild-type FBXO6 and its mutant, which fails to recognize glycoproteins. Combining these databases, 29 highly confident glycoproteins that interact with FBXO6 in an N-glycan dependent manner are identified. Our data provide valuable information for the discovery of the interacting glycoproteins of FBXO6 and also demonstrate the potential of these approaches as general platforms for the global discovery of interacting glycoproteins of other FBAs (F-box associated regions) containing F-box proteins.

African trypanosomiasis, caused by the proto zoal pathogen Trypanosoma brucei (T. brucei), is one of the most neglected tropical diseases that are in great need of new drugs. We report the design and synthesis of T. brucei leucyl-tRNA synthetase (TbLeuRS) inhibitors and their structure--activity relationship. Benzoxaborole was used as the core structure and C(6) was modified to achieve improved affinity based on docking results that showed further binding space at this position. Indeed, compounds with C(7) substitutions showed diminished activity due to clash with the eukaryote specific I4ae helix while substitutions at C(6) gave enhanced affinity. TbLeuRS inhibitors with IC(50) as low as 1.6 μM were discovered, and the structure-activity relationship was discussed. The most potent enzyme inhibitors also showed excellent T. brucei parasite growth inhibition activity. This is the first time that TbLeuRS inhibitors are reported, and this study suggests that leucyl-tRNA synthetase (LeuRS) could be a potential target for antiparasitic drug development.

Expression of chirality in the highly ordered assembly structures of various types of natural and synthetic materials is still a mystery. Although surface stresses have been known to widely exist in polymer lamellae, how they affect the twisting chirality is yet an unsolved issue. Here we report inversion of the lamellar twisting chirality of microbial poly(R-3-hydroxybutyrate) copolymers from left-handed to right-handed via copolymerization or blending, with the molecular chirality and the structures in the crystalline core unchanged. We interpret the observed inversion of twisting chirality and the complicated lamellar curvature with a continuum model based on surface elasticity theory. Quantitative simulations reveal that the distribution of the anisotropic surface stresses and the surface elasticity property of the lamellae significantly affect the morphological chirality of lamellar twisting. For different chiral polymers, the same bending direction may correlate to the opposite twisting chirality.

Microbial polyhydroxyalkanoates (PHA) were proposed for the first time as a new type of biofuel. In this paper, poly-R-3-hydroxybutyrate (PHB) and medium chain length PHA (mcl PHA) were, respectively, esterified to become R-3-hydroxybutyrate methyl ester (3HBME) and medium chain length hydroxyalkanoate methyl ester (3HAME) via acid-catalyzed hydrolysis. The recovery percentages of 3HBME and 3HAME were 52 and 65%, respectively. The purities of 3HBME and 3HAME were 97 and 96%, respectively. Combustion heats of 3HBME, 3HAME, ethanol, n-propanol, n-butanol, 0(#) diesel, 90(#) gasoline, and 3HBME-based and 3HAME-based blended fuels were investigated and compared, respectively. It was found that 3HBME and 3HAME had combustion heats valuing 20 and 30 KJ/g, respectively. Ethanol has a combustion heat of 27 KJ/g, while addition of 10% 3HBME or 3HAME enhanced the combustion heat of ethanol to 30 and 35 KJ/g, respectively. The addition of 3HBME or 3HAME into n-propanol and n-butanol led to a slight reduction of their combustion heats. Combustion heats of blended fuels 3HBME/diesel or 3HBME/gasoline and of 3HAME/diesel or 3HAME/gasoline were lower than that of the pure diesel or gasoline. It was roughly estimated that the production cost of PHA-based biofuels should be around US$1200 per ton.

Nine new ent-kaurane diterpenoids, isoscoparins D-L (1-9), and an artificial product, the acetonide of rabdoloxin A (10), along with 16 known analogues (11-26), were isolated from the leaves of Isodon scoparius. The new structures were determined by 1D and 2D NMR spectroscopic analysis. Selected compounds were evaluated for their cytotoxicity against NB4, A549, PC-3, MCF-7, and SH-SY5Y cell lines.

We reported previously that NSC606985, a camptothecin analogue, induces apoptosis of acute myeloid leukemia (AML) cells through proteolytic activation of protein kinase Cdelta. Here, we analyzed protein expression profiles of fractionated nuclei, mitochondria, raw endoplasmic reticula, and cytosols of NSC606985-induced apoptotic AML cell line NB4 cells by two-dimensional electrophoresis combined with MALDI-TOF/TOF tandem mass spectrometry. In total, 90 unique deregulated proteins, including 16 compartment-compartment translocated ones, were identified. They contributed to multiple functional activities such as DNA damage repairing, chromosome assembly, mRNA processing, biosynthesis, modification, and degradation of proteins. More interestingly, several increased oxidative stress-related proteins mainly presented in mitochondria, while upregulated glycolysis proteins mainly occurred in the nuclei. With their functional analyses, the possible roles of these deregulated proteins in NSC606985-induced apoptosis were discussed. Collectively, these discoveries would shed new insights for systematically understanding the mechanisms of the camptothecin-induced apoptosis.

To improve the performance of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), gelatin was blended with PHBHHx at different ratios. With increasing gelatin content, the weight loss of gelatin/PHBHHx blend in simulated body fluid at 37 degrees C was accelerated. After 2 months, there was about 15% weight loss in PHBHHx blending with 30% gelatin. Scanning electron microscopy and X-ray diffraction results showed that gelatin blending increased the surface porosity and decreased the crystallinity, which may be responsible for the acceleration of the weight loss. Second harmonic generation results indicated that 10% gelatin blending had less disruption to PHBHHx spatial structure, resulting in better tensile mechanical properties. At the same time, increased surface porosity and decreased crystallinity caused by gelatin incorporation may be beneficial for cell growth compared with pure PHBHHx. All these indicated that gelatin incorporation may improve the performances of PHBHHx to meet the need of different situations during medical implantation.

Real-time atomic force microscopy observation was carried out during crystallization in thin films of chiral poly(R-3-hydroxybutyrate-co-R-3-hydroxyhexanoate) copolymer, and the development details of single lamellae in the banded spherulites are revealed for the first time. The lamellae exhibit complicated growth behaviors: twisting, bending, backward growth, and branching. The lamellae continuously twist to show alternating edge-on and flat-on views along the radii of the spherulites. Giant screw dislocations bring forth to the birth of new lamellae. Interaction between the leading and trailing lamellae contributes to cooperative stacking of the twisting crystals. The lamellae twist before screw dislocations appear, demonstrating that screw dislocations are not causal of twisting. All the observed twisting occurs in the right-handed sense, likely resulting from the chirality of the crystal structure. Increased crystallization temperature results in decreased magnitude of lamellar twisting and bending. ...

Banded spherulites of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-3HV)) random copolymer were prepared by isothermal crystallization at 90 °C for 10 h. Using tapping-mode atomic force microscopy (AFM), the concentric periodic ridges and valleys on the surface of the banded spherulites of this polymer were found to consist of edge-on and flat-on lamellae, respectively. The periodic concentric ridges and valleys observed by AFM corresponded to the periodic extinction rings observed by polarized optical microscopy. AFM measurements showed that the interaction between the AFM probe and the sample surface can be significantly influenced by lamellar orientation.

The reliability of a prediction of achirality of a molecule by inspecting a rotating plane of symmetry is ensured by following the demonstration.