A gradient phage-assisted continuous evolution method for screening suppressor tRNAs in Escherichia coli.

Suppressor tRNAs, notable for their capability of reading through the stop codon while maintaining normal peptide synthesis, are promising in treating diseases caused by premature termination codons (PTC). However, the lack of effective engineering methods for suppressor tRNAs has curtailed their application potential. Here, we introduce a directed evolution technology that employs phage-assisted continuous evolution (PACE), combined with gradient biosensors featuring various PTCs in the M13 gene III. Utilizing this novel methodology, we have successfully evolved tRNATrp (UGG) reading through the UGA stop codon in Escherichia coli. Massively parallel sequencing revealed that these mutations predominantly occurred in the anticodon loop. Finally, two suppressor tRNATrp (UGA) mutants exhibited over fivefold increases in readthrough efficiency.

Characterization and safety assessment of bamboo shoot shell cellulose nanofiber: Prepared by acidolysis combined with dynamic high-pressure microfluidization.

The preparation of cellulose nanofiber (CNF) using traditional methods is currently facing challenges due to concerns regarding environmental pollution and safety. Herein, a novel CNF was obtained from bamboo shoot shell (BSS) by low-concentration acid and dynamic high-pressure microfluidization (DHPM) treatment. The resulting CNF was then characterized, followed by in vitro and in vivo safety assessments. Compared to insoluble dietary fiber (IDF), the diameters of HIDF (IDF after low-concentration acid hydrolysis) and CNF were significantly decreased to 167.13 nm and 70.97 nm, respectively. Meanwhile, HIDF and CNF showed a higher crystallinity index (71.32 % and 74.35 %). Structural analysis results indicated the successful removal of lignin and hemicellulose of HIDF and CNF, with CNF demonstrating improved thermostability. In vitro, a high dose of CNF (1500 µg/mL) did not show any signs of cytotoxicity on Caco-2 cells. In vivo, no death was observed in the experimental mice, and there was no significant difference between CNF (1000 mg/kg·bw) and control group in hematological index and histopathological analysis. Overall, this study presents an environmentally friendly method for preparing CNF from BSS while providing evidence regarding its safety through in vitro and in vivo assessments, laying the foundation for its potential application in food.

Directed Evolution of Escherichia coli Nissle 1917 to Utilize Allulose as Sole Carbon Source.

Sugar substitutes are popular due to their akin taste and low calories. However, excessive use of aspartame and erythritol can have varying effects. While D-allulose is presently deemed a secure alternative to sugar, its excessive consumption is not devoid of cellular stress implications. In this study, the evolution of Escherichia coli Nissle 1917 (EcN) is directed to utilize allulose as sole carbon source through a combination of adaptive laboratory evolution (ALE) and fluorescence-activated droplet sorting (FADS) techniques. Employing whole genome sequencing (WGS) and clustered regularly interspaced short palindromic repeats interference (CRISPRi) in conjunction with compensatory expression displayed those genetic mutations in sugar and amino acid metabolic pathways, including glnP, glpF, gmpA, nagE, pgmB, ybaN, etc., increased allulose assimilation. Enzyme-substrate dynamics simulations and deep learning predict enhanced substrate specificity and catalytic efficiency in nagE A247E and pgmB G12R mutants. The findings evince that these mutations hold considerable promise in enhancing allulose uptake and facilitating its conversion into glycolysis, thus signifying the emergence of a novel metabolic pathway for allulose utilization. These revelations bear immense potential for the sustainable utilization of D-allulose in promoting health and well-being.

Self-assembly of maltose-albumin nanoparticles for efficient targeting delivery and therapy in liver cancer.

The effective delivery and targeted release of drugs within tumor cells are critical factors in determining the therapeutic efficacy of nanomedicine. To achieve this objective, a conjugate of maltose (Mal) and bovine serum albumin (BSA) was synthesized by the Maillard reaction and self-assembled into nanoparticles with active-targeting capabilities upon pH/heating induction. This nanoparticle could be effectively loaded with doxorubicin (DOX) to form stable nanodrugs (Mal-BSA/DOX) that were sensitive to low pH or high glutathione (GSH), thereby achieving a rapid drug release (96.82 % within 24 h). In vitro cell experiments indicated that maltose-modified BSA particles efficiently enhance cellular internalization via glucose transporters (GLUT)-mediated endocytosis, resulting in increased intracellular DOX levels and heightened expression of γ-H2AX. Consequently, these results ultimately lead to selective tumor cells death, as evidenced by an IC50 value of 3.83 µg/mL in HepG2 cells compared to 5.87 µg/mL in 293t cells. The efficacy of Mal-BSA/DOX in tumor targeting therapy has been further confirmed by in vivo studies, as it effectively delivered a higher concentration of DOX to tumor tissue. This targeted delivery approach not only reduces the systemic toxicity of DOX but also effectively inhibits tumor growth (TGI, 75.95 %). These findings contribute valuable insights into the advancement of targeting-albumin nanomedicine and further support its potential in tumor treatment.

Curcumin-Loaded Bamboo Shoot Cellulose Nanofibers: Characterization and In Vitro Studies.

Given its high biological and pharmacological activities, curcumin (CUR) offers promising applications in functional foods. However, its low stability and bioavailability have greatly hindered its application in the food industry. The present study prepared cellulose nanofiber (CNF) from bamboo shoot processing byproducts and investigated its potential as a low-cost carrier. Our results showed that CUR was immobilized on CNF surfaces mainly through hydrogen bonding and eventually encapsulated in CNF matrices, forming a CNF-CUR complex with an encapsulation efficiency of 88.34% and a loading capacity of 67.95%. The CUR encapsulated in the complex showed improved stability after thermal and UV light treatments. Moreover, a slow and extended release pattern of CUR in a simulated gastrointestinal tract was observed, which could be appropriately described using the Korsmeyer-Peppas model. These results revealed that CNF is a promising protective carrier for the slow release of CUR, making it a better candidate for functional foods.

Interpretable thoracic pathologic prediction via learning group-disentangled representation.

Deep learning has brought a significant progress in medical image analysis. However, their lack of interpretability might bring high risk for wrong diagnosis with limited clinical knowledge embedding. In other words, we believe it's crucial for humans to interpret how deep learning work for medical analysis, thus appropriately adding knowledge constraints to correct the bias of wrong results. With such purpose, we propose Representation Group-Disentangling Network (RGD-Net) to explain the process of feature extraction and decision making inside deep learning framework, where we completely disentangle feature space of input X-ray images into independent feature groups, and each group would contribute to diagnose of a specific disease. Specifically, we first state problem definition for interpretable prediction with auto-encoder structure. Then, group-disentangled representations are extracted from input X-ray images with the proposed Group-Disentangle Module, which constructs semantic latent space by enforcing semantic consistency of attributes. Afterwards, adversarial constricts on mapping from features to diseases are proposed to prevent model collapse during training. Finally, a novel design of local tuning medical application is proposed based on RGB-Net, which is capable to aid clinicians for reasonable diagnosis. By conducting quantity of experiments on public datasets, RGD-Net have been superior to comparative studies by leveraging potential factors contributing to different diseases. We believe our work could bring interpretability in digging inherent patterns of deep learning on medical image analysis.

CDT-CAD: Context-Aware Deformable Transformers for End-to-End Chest Abnormality Detection on X-Ray Images.

Deep learning methods have achieved great success in medical image analysis domain. However, most of them suffer from slow convergency and high computing cost, which prevents their further widely usage in practical scenarios. Moreover, it has been proved that exploring and embedding context knowledge in deep network can significantly improve accuracy. To emphasize these tips, we present CDT-CAD, i.e., context-aware deformable transformers for end-to-end chest abnormality detection on X-Ray images. CDT-CAD firstly constructs an iterative context-aware feature extractor, which not only enlarges receptive fields to encode multi-scale context information via dilated context encoding blocks, but also captures unique and scalable feature variation patterns in wavelet frequency domain via frequency pooling blocks. Afterwards, a deformable transformer detector on the extracted context features is built to accurately classify disease categories and locate regions, where a small set of key points are sampled, thus leading the detector to focus on informative feature subspace and accelerate convergence speed. Through comparative experiments on Vinbig Chest and Chest Det 10 Datasets, CDT-CAD demonstrates its effectiveness in recognizing chest abnormities and outperforms 1.4% and 6.0% than the existing methods in AP50 and AR on VinBig dateset, and 0.9% and 2.1% on Chest Det-10 dataset, respectively.

Efficient isopropanol-butanol-ethanol (IBE) fermentation by a gene-modified solventogenic Clostridium species under the co-utilization of Fe(III) and butyrate.

To elevate the efficiency of acetone-butanol-ethanol (ABE) fermentation by the wild-type strain WK, an optimal co-utilization system (20 mM Fe3+ and 5 g/L butyrate) was established to bring about a 22.22% increment in the yield of ABE mixtures with a significantly enhanced productivity (0.32 g/L/h). With the heterologous introduction of the secondary alcohol dehydrogenase encoded gene (adh), more than 95% of acetone was eliminated to convert 4.5 g/L isopropanol with corresponding increased butanol and ethanol production by 21.08% and 65.45% in the modified strain WK::adh. Under the optimal condition, strain WK::adh was capable of producing a total of 25.46 g/L IBE biosolvents with an enhanced productivity of 0.35 g/L/h by 45.83% over the original conditions. This work for the first time successfully established a synergetic system of co-utilizing Fe(III) and butyrate to demonstrate a feasible and efficient manner for generating the value-added biofuels through the metabolically engineered solventogenic clostridial strain.

Elimination of carbon catabolite repression through gene-modifying a solventogenic Clostridium sp. strain WK to enhance butanol production from the galactose-rich red seaweed.

With the determination of the Leloir pathway in a solventogenic wild-type strain WK through the transcriptional analysis, two pivotal genes (galK and galT) were systematically co-expressed to demonstrate a significantly enhanced galactose utilization for butanol production with the elimination of carbon catabolite repression (CCR). The gene-modified strain WK-Gal-4 could effectively co-utilize galactose and glucose by directly using an ultrasonication-assisted butyric acid-pretreated Gelidium amansii hydrolysate (BAU) as the substrate, exhibiting the optimal sugar consumption and butanol production from BAU of 20.31 g/L and 7.8 g/L with an increment by 62.35 % and 61.49 % over that by strain WK, respectively. This work for the first time develops a feasible approach to utilizing red algal biomass for butanol fermentation through exploring the metabolic regulation of carbohydrate catabolism, also offering a novel route to develop the future biorefinery using the cost-effective and sustainable marine feedstocks.

Extracellular expression of agarolytic enzymes in Clostridium sp. strain and its application for butanol production from Gelidium amansii.

In this study, Clostridium sp. strain WK-AN1 carrying both genes of agarase (Aga0283) and neoagarobiose hydrolase (NH2780) were successfully constructed to convert agar polysaccharide directly into butanol, contributing to overcome the lack of algal hydrolases in solventogenic clostridia. Through the optimization by the Plackett-Burman design (PBD) and response surface methodology (RSM), a maximal butanol production of 6.42 g/L was achieved from 17.86 g/L agar. Further application of utilizing the butyric acid pretreated Gelidium amansii hydrolysate demonstrated the modified strain obtained the butanol production of 7.83 g/L by 1.63-fold improvement over the wild-type one. This work for the first time establishes a novel route to utilize red algal polysaccharides for butanol fermentation by constructing a solventogenic clostridia-specific secretory expression system for heterologous agarases, which will provide insights for future development of the sustainable third-generation biomass energy.

Unraveling the unique butyrate re-assimilation mechanism of Clostridium sp. strain WK and the application of butanol production from red seaweed Gelidium amansii through a distinct acidolytic pretreatment.

Exploration of the algae-derived biobutanol synthesis has become one of the hotspots due to its highly cost-effective and environment-friendly features. In this study, a solventogenic strain Clostridium sp. strain WK produced 13.96 g/L butanol with a maximal yield of 0.41 g/g from glucose in the presence of 24 g/L butyrate. Transcriptional analysis indicated that the acid re-assimilation of this strain was predominantly regulated by genes buk-ptb rather than ctfAB, explaining its special phenotypes including high butyrate tolerance and the pH-independent fermentation. In addition, a butyric acid-mediated hydrolytic system was established for the first time to release a maximal yield of 0.35 g/g reducing sugars from the red algal biomass (Gelidium amansii). Moreover, 4.48 g/L of butanol was finally achieved with a significant enhancement by 29.9 folds. This work reveals an unconventional metabolic pathway for butanol synthesis in strain WK, and demonstrates the feasibility to develop renewable biofuels from marine resources.

Microbial debromination of hexabromocyclododecanes.

Hexabromocyclododecanes (HBCDs), a new sort of brominated flame retardants (BFRs), are globally prevalent and recalcitrant toxic environmental pollutants. HBCDs have been found in many environmental media and even in the human body, leading to serious health concerns. HBCDs are biodegradable in the environment. By now, dozens of bacteria have been discovered with the ability to transform HBCDs. Microbial debromination of HBCDs is via HBr-elimination, HBr-dihaloelimination, and hydrolytic debromination. Biotic transformation of HBCDs yields many hydroxylated and lower brominated compounds which lack assessment of ecological toxicity. Bioremediation of HBCD pollution has only been applied in the laboratory. Here, we review the current knowledge about microbial debromination of HBCDs, aiming to promote the bioremediation applied in HBCD contaminated sites. KEY POINTS: • Microbial debromination of HBCDs is via hydrolytic debromination, HBr-elimination, and HBr-dihaloelimination. • Newly occurred halogenated contaminants such as HBCDs hitch the degradation pathway tamed by previously discharged anthropogenic organohalides. • Strategy that combines bioaugmentation with phytoremediation for bioremediation of HBCD pollution is promising.

Emerging technologies for genetic modification of solventogenic clostridia: From tool to strategy development.

Solventogenic clostridia has been considered as one of the most potential microbial cell factories for biofuel production in the biorefinery industry. However, the inherent shortcomings of clostridia strains such as low productivity, by-products formation and toxic tolerance still strongly restrict the large-scale application. Therefore, concerns regarding the genetic modification of solventogenic clostridia have spurred interests into the development of modern gene-editing tools. In this review, we summarize the latest advances of genetic tools involved in modifying solventogenic clostridia. Following a systematic comparison on their respective characteristics, we then review the corresponding strategies for overcoming the obstacles to the enhanced production. Discussing the progress of other microbial cell factories for solventogenesis, we finally describe the key challenges and trends with valuable recommendations for future large-scale biosolvent industrial application.

Emerging technologies for conversion of sustainable algal biomass into value-added products: A state-of-the-art review.

Concerns regarding high energy demand and gradual depletion of fossil fuels have attracted the desire of seeking renewable and sustainable alternatives. Similar to but better than the first- and second-generation biomass, algae derived third-generation biorefinery aims to generate value-added products by microbial cell factories and has a great potential due to its abundant, carbohydrate-rich and lignin-lacking properties. However, it is crucial to establish an efficient process with higher competitiveness over the current petroleum industry to effectively utilize algal resources. In this review, we summarize the recent technological advances in maximizing the bioavailability of different algal resources. Following an overview of approaches to enhancing the hydrolytic efficiency, we review prominent opportunities involved in microbial conversion into various value-added products including alcohols, organic acids, biogas and other potential industrial products, and also provide key challenges and trends for future insights into developing biorefineries of marine biomass.

Acidolysis as a biorefinery approach to producing advanced bioenergy from macroalgal biomass: A state-of-the-art review.

Facing fossil fuels consumption and its accompanying environmental pollution, macroalgae, as a major part of the third-generation (3G) biomass, has great potential for bioenergy development due to its species-abundant, renewable and carbohydrate-rich properties. Diluted acid treatment is one of the most effective approaches to releasing fermentable sugars from macroalgal biomass in a short period, but the optimal conditions need to be explored to maximize the hydrolytic yield for the subsequent microbial conversion. Therefore, this review aims to summarize the latest advances in various acids and other auxiliary methods adopted to increase the hydrolytic efficiency of macroalgae. Following an overview of the strategies of different algal types, methods involved in the bioconversion of biofuels and microbial fuel cells (MFC) from algal hydrolysates are also described. For the 3G biorefinery development, the review further discusses key challenges and trends for future utilizing marine biomass to achieve the large-scale industrial production.

Biobutanol production from sulfuric acid-pretreated red algal biomass by a newly isolated Clostridium sp. strain WK.

Marine biomass, especially the algal biomass, is currently considered to be one of the most potential candidates for biofuels conversion during the development of biomass utilization. In this study, a diluted sulfuric acid pretreatment method was established for biobutanol from red algal biomass Gelidium amansii using a newly isolated Clostridium sp. strain WK. Under the optimal condition of 2% sulfuric acid treated in 20 Min at 131 °C, the maximal hydrolysis percentage of biomass can reach up to 80.95%, and the biobutanol production was obtained to be 3.46 g/L with a yield of 0.20 g/g after the fermentation of biomass hydrolysate. This result demonstrated a 12.5-fold enhancement of conversion efficiency compared with the untreated control, which provides a new and efficient way to develop the biobutanol industry by utilizing the abundant, low cost, and carbohydrate-rich algal biomass.

Enhanced bioconversion of hemicellulosic biomass by microbial consortium for biobutanol production with bioaugmentation strategy.

As a renewable and sustainable source for next-generation biofuel production, lignocellulosic biomass can be effectively utilized in environmentally friendly manner. In this study, a stable, xylan-utilizing, anaerobic microbial consortium MC1 enriched from mangrove sediments was established, and it was taxonomically identified that the genera Ruminococcus and Clostridium from this community played a crucial role in the substrate utilization. In addition, a butanol-producing Clostridium sp. strain WST was introduced via the bioaugmentation process, which resulted in the conversion of xylan to biobutanol up to 10.8 g/L, significantly improving the butanol yield up to 0.54 g/g by 98-fold. When this system was further applied to other xylan-rich biomass, 1.09 g/L of butanol could be achieved from 20 g/L of corn cob. These results provide another new method to efficiently convert xylan, the main hemicellulose from lignocellulosic biomass into biofuels through a low-cost and eco-friendly manner.

The Draft Genome Sequence of a Novel High-Efficient Butanol-Producing Bacterium Clostridium Diolis Strain WST.

A wild-type solventogenic strain Clostridium diolis WST, isolated from mangrove sediments, was characterized to produce high amount of butanol and acetone with negligible level of ethanol and acids from glucose via a unique acetone-butanol (AB) fermentation pathway. Through the genomic sequencing, the assembled draft genome of strain WST is calculated to be 5.85 Mb with a GC content of 29.69% and contains 5263 genes that contribute to the annotation of 5049 protein-coding sequences. Within these annotated genes, the butanol dehydrogenase gene (bdh) was determined to be in a higher amount from strain WST compared to other Clostridial strains, which is positively related to its high-efficient production of butanol. Therefore, we present a draft genome sequence analysis of strain WST in this article that should facilitate to further understand the solventogenic mechanism of this special microorganism.