Flexible sites are potential targets for engineering the stability of enzymes. Nevertheless, the success rate of the rigidifying flexible sites (RFS) strategy is still low due to a limited understanding of how to determine the best mutation candidates. In this study, two parallel strategies were applied to identify mutation candidates within the flexible loops of Escherichia coli transketolase (TK). The first was a "back to consensus mutations" approach, and the second was computational design based on ΔΔG calculations in Rosetta. Forty-nine single variants were generated and characterised experimentally. From these, three single-variants I189H, A282P, D143K were found to be more thermostable than wildtype TK. The combination of A282P with H192P, a variant constructed previously, resulted in the best all-round variant with a 3-fold improved half-life at 60 °C, 5-fold increased specific activity at 65 °C, 1.3-fold improved k cat and a T m increased by 5 °C above that of wild type. Based on a statistical analysis of the stability changes for all variants, the qualitative prediction accuracy of the Rosetta program reached 65.3%. Both of the two strategies investigated were useful in guiding mutation candidates to flexible loops, and had the potential to be used for other enzymes.Transketolase (TK), a thiamine diphosphate dependent (ThDP) enzyme, catalyses the reversible transfer of a C2-ketol unit from D-xylulose-5-phosphate to either D-ribose-5-phosphate or D-erythrose-4-phosphate, linking glycolysis to the pentose phosphate pathway in all living cells 1,2 . The stereospecifically controlled carbon-carbon bond forming ability of TK makes it promising as a biocatalyst in industry, for the synthesis of complex carbohydrates and other high-value compounds 3,4 . The use of β -hydroxypyruvate (HPA) as the ketol donor renders the donor-half reaction irreversible, thus increasing the atom efficiency of the reaction favourably for industrial syntheses. Escherichia coli (E. coli) TK converts HPA with a rate of 60 U/mg, significantly higher than the rates of 2 U/mg and 9 U/mg reported for its orthologs from spinach and yeast 5 .Wild-type (WT) E. coli TK has been successfully engineered to have improved and inverted enantioselectivity 6 , as well as an expanded aldol-acceptor substrate range including polar aliphatics 7 , non-polar aliphatics 8 , and heteroaromatics 9,10 . Most recently, E. coli TK has been engineered to synthesize L-gluco-heptulose from L-arabinose, thus transforming a major component of the carbohydrates in sugar beet pulp, into a rare naturally-occurring ketoheptose with potential therapeutic applications in hypoglycaemia and cancer 11 . Additionally, the substrate range accepted by TK has been recently extended to aromatic benzaldehyde derivatives, which opened up potential routes to chiral aromatic amino-alcohols such as chloramphenicol antibiotics, nor-ephedrine, and their analogues with alternative aromatic substituents 12,13 .As a mesophilic enzyme E. coli TK suffers the limitation of low stability t...

No abstract

Traditional energy from fossil fuels like petroleum and coal is limited and contributes to global environmental pollution and climate change. Developing sustainable and eco‐friendly energy is crucial for addressing significant challenges such as climate change, energy dilemma and achieving the long‐term development of human society. Biomass hydrogels, which are easily synthesized and modified, have diverse sources and can be designed for different applications. They are being extensively researched for their applications in artificial intelligence, flexible sensing, biomedicine, and food packaging. The article summarizes recent advances in the preparation and applications of biomass‐based photothermal conversion hydrogels, discussing the light source, photothermal agents, matrix, and preparation methods in detail. It also explores the use of these hydrogels in seawater desalination, photothermal therapy, antibacterial agents, and light‐activated materials, offering new ideas for developing sustainable, efficient, and advanced photothermal conversion biomass hydrogel materials. The article concludes with suggestions for future research, highlighting the challenges and prospects in this field and paving the way for developing of long‐lasting, efficient energy materials.

Organic bioelectronics based on conjugated polymers as the active electronic material have been shown to operate efficiently at the biointerface. Their translation into a commercial medical device will hinge on their long-term operation in vivo. This will require the device to be subjected to clinically approved sterilization techniques without deterioration in its physical and electronic properties. To date, there remains a gap in the literature addressing the impact of this critical preoperative procedure on the properties of conjugated polymers. This study aims to address this gap by assessing the physical and electronic properties of a sterilized porous bioelectronic patch having polyaniline as the conjugated polymer. The patch was sterilized by autoclave, ethylene oxide, and gamma (γ-) irradiation at 15, 25, and 50 kGy doses. Autoclaving resulted in cracking and macroscopic degradation of the patch, while patches sterilized by γ-irradiation at 50 kGy exhibited reduced mechanical and electronic properties, attributed to chain scission and nonuniform cross-linking caused by high dose irradiation. Ethylene oxide and γ-irradiation at 15 and 25 kGy sterilization appeared to be the most effective at maintaining the mechanical and electronic properties of the patch and inducing a minimal immune response as revealed by a receding fibrotic capsule after 4 week implantation. Our findings pave the way toward closing the gap for the translation of organic bioelectronic devices from acute to long-term in vivo models.

Interaction of conjugated polymers with liposomes is an attractive approach that benefits from both systems’ characteristics such as electroactivity and enhanced interaction with cells. Conjugated polymer‐liposome complexes have been investigated for bioimaging, drug delivery, and photothermal therapy. Their fabrication has largely been achieved by multistep procedures that require first the synthesis and processing of the conjugated polymer. Here, a new one step fabrication approach is reported based on in situ polymerization of a conjugated monomer precursor around liposomes. Polyaniline (PANI) doped with phytic acid is synthesized via oxidative polymerization in the presence of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphatidylcholine (DOPC) vesicles to produce a conductive aqueous suspension of Liposome‐PANI complexes. PANI interacts with liposomes without disrupting the bilayer as shown using differential scanning calorimetry and fluorescence quenching studies of the hydrophobic Nile red probe. The electronic conductivity of the Liposome‐PANI complexes, which stems from the doped PANI accessible on the liposome surface, is confirmed using conductive atomic force microscopy and electrochemical impedance spectroscopy. Further, short‐term in vitro cell studies show that the complexes colocalize with the cell membrane without reducing cell proliferation. This study presents a novel fabrication route to conductive suspensions of conjugated polymer‐liposome complexes suitable for potential applications at the biointerface.

No abstract

With the rapidly increasing of automobiles, traffic accidents are gradually becoming more frequent. This creates a great need for effective traffic anomaly detection algorithms. Existing methods shed light on directly inferring the abnormalities from traffic flow, which is short in features extraction and representation of traffic flows. In this paper, we propose three new traffic flow features, namely the road congestion, the traffic intensity, and the traffic state instability, for more comprehensive traffic status representation and anomaly detection. Residual analysis, quadratic discrimination, multi-resolution wavelet analysis are integrated for the extraction of the aforementioned features, which will be applied for the downstream tasks of traffic anomaly detection. Experimental results reveal that accident identification based on the proposed features is more effective than the raw traffic flow, which is supposed to provide an alternative approach for further applications and studies.

Translation into the clinic of organic bioelectronic devices having conjugated polymers as the active material will hinge on their long-term operation in vivo. This will require the device to be subject to clinically approved sterilization techniques without a deterioration in its physical and electronic properties. To date, there remains a gap in the literature addressing the impact of this critical pre-operative procedure on the properties of conjugated polymers. This study aims to address this gap by assessing the physical and electronic properties of a sterilized porous bioelectronic patch having polyaniline as the conjugated polymer. The patch was sterilized by autoclave, ethylene oxide and gamma (γ-) irradiation at 15, 25, and 50 kGy doses. Autoclaving resulted in cracking and macroscopic degradation of the patch, while patches sterilized by γ-irradiation at 50 kGy exhibited reduced mechanical and electronic properties, attributed to chain scission and non-uniform crosslinking caused by the high dose irradiation. Ethylene oxide and γ-irradiation at 15 and 25 kGy sterilization appeared to be the most effective at maintaining the mechanical and electronic properties of the patch, as well as inducing a minimal immune response as revealed by a receding fibrotic capsule after 4 weeks implantation. Our findings pave the way towards closing the gap for the translation of organic bioelectronic devices from acute to long-term in vivo models.