Surface micropatterning of 3D printed PCL scaffolds promotes osteogenic differentiation of BMSCs and regulates macrophage M2 polarization.

Micropatterned structures on the surface of materials possessing biomimetic properties to mimic the extracellular matrix and induce cellular behaviors have been widely studied. However, it is still a major challenge to obtain internally stable and controllable micropatterned 3D scaffolds for bone repair and regeneration. In this study, 3D scaffolds with regular grating arrays using polycaprolactone (PCL) as a matrix material were prepared by combining 3D printing and soft lithography, and the effects of grating micropatterning on osteogenic differentiation of BMSCs and M1/M2 polarization of macrophages were investigated. The results showed that compared with the planar group and the 30um grating spacing group, PCL with a grating spacing of 20um significantly promoted the osteogenic differentiation of BMSCs, induced the polarization of RAW264.7 cells toward M2 type, and suppressed the expression of M1-type pro-inflammatory genes and markers. In conclusion, we successfully constructed PCL-based three-dimensional scaffolds with stable and controllable micrographs (grating arrays) inside, which possess excellent osteogenic properties and promote the formation of an immune microenvironment conducive to osteogenesis. This study is a step forward to the exploration of bone-filling materials affecting cell behavior, and makes a new contribution to the provision of high-quality materials.

Exploring the molecular mechanism of Chlorella vulgaris in response to androstenedione exposure based on genes continuously up-regulated in transcription analysis.

Androstenedione (ADSD) is one of the widely detected androgens in diverse aquatic environments. However, there were few reports on the molecular mechanism of Chlorella vulgaris exposure to ADSD. In our previous research, we have investigated the genes associated with chlorophyll metabolism in Chlorella vulgaris response to ADSD. In this study, we focus on continuously up-regulated genes to explore the mechanism underlying Chlorella vulgaris resistance to ADSD toxicity. Chlorella vulgaris was exposed to ADSD with five concentration gradients. The continuously up-regulated genes were enriched by Series Test of Cluster (STC) analysis and verified by qRT-PCR. Microalgae Super Oxidase Dimutase (SOD) and Microalgae Malonic dialdehyde (MDA), two indicators of oxidative stress, were determined by ELISA after exposure to ADSD. The results showed that ADSD can stimulate the production of extracellular polymeric substances (EPS) and lead to enlargement in the cell body of Chlorella vulgaris. In addition, steroid biosynthesis and oxidoreductase activity processes were consistently up-regulated upon exposure to ADSD. In conclusion, our study highlighted the crucial role of phenotypic modification, hormone synthesis, and redox mechanisms in protecting Chlorella vulgaris cells from the harmful effects of ADSD contamination.

Physicochemical characterization and antioxidant activity of polysaccharides from Chlorella sp. by microwave-assisted enzymatic extraction.

Microwave-assisted enzymatic extraction (MAEE) was used for the separation of polysaccharides from micro-Chlorella. The extraction condition of MAEE was optimized by Box-Behnken design and response surface methodology. Results showed that the optimal condition for the extraction of Chlorella sp. crude polysaccharides (CSCP) was at 50°C for 2.3 h with 380 W of microwave power and 0.31% of enzyme dosage. Under the optimal extraction condition, the extraction yield of CSCP reached 0.72%. Similarly, the α-amylase modification conditions of the CSCP were also optimized, in which the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging rate was used as the response value. The scavenging rate of DPPH free radicals was 17.58% when enzyme dosage was 271 U/g at 51°C for 14 min. Moreover, the enzyme-modified CSCP presented a typical heteropolysaccharide mainly including glucose (48.84%), ribose (13.57%) and mannose (11.30%). MAEE used in this work achieved a high extraction yield of CSCP, which provides an efficient method for the extraction of CSCP from Chlorella sp.

Assessing the potential of Chlorella sp. phycoremediation liquid digestates from brewery wastes mixture integrated with bioproduct production.

Digestates from different anaerobic digesters are promising substrates for microalgal culture, leading to effective wastewater treatment and the production of microalgal biomass. However, further detailed research is needed before they can be used on a large scale. The aims of this study were to investigate the culture of Chlorella sp. in DigestateM from anaerobic fermentation of brewer's grains and brewery wastewater (BWW) and to explore the potential use of the biomass produced under different experimental conditions, including diverse cultivation modes and dilution ratios. Cultivation in DigestateM initiated from 10% (v/v) loading, with 20% BWW, obtained maximum biomass production, reaching 1.36 g L-1 that was 0.27g L-1 higher than 1.09 g L-1 of BG11. In terms of DigestateM remediation, the maximum removal of ammonia nitrogen (NH4 +-N), chemical oxygen demand, total nitrogen, and total phosphorus reached 98.20%, 89.98%, 86.98%, and 71.86%, respectively. The maximum lipid, carbohydrate, and protein contents were 41.60%, 32.44%, and 27.72%, respectively. The growth of Chlorella sp. may be inhibited when the Y(II)-Fv/Fm ratio is less than 0.4.

Transcriptomic analysis reveals up-regulated histone genes may play a key role in zebrafish embryo-larvae response to Bisphenol A (BPA) exposure.

Bisphenol A (BPA) can induce complex regulatory mechanisms in many aquatic organisms, and it is difficult to find a suitable analytical method to efficiently enrich key genes responding to BPA exposure. In this study, zebrafish embryo transcriptomic data were obtained from two types of different BPA exposure methods. After BPA exposure, three differential gene enrichment methods were used jointly to identify up-regulated genes or pathways in zebrafish embryo larvae. The results showed that the systemic lupus erythematosus signaling pathway was significantly enriched in all BPA exposure groups. It was also noteworthy that most of the up-regulated genes in systemic lupus erythematosus signaling were histones. In conclusion, this study suggested that autoimmunity signaling was the most common important pathway in zebrafish embryo-larvae response to different BPA exposures, and histones may play a key role in response to low-concentration BPA.

Efficient synthesis of Ala-Tyr by L-amino acid ligase coupled with ATP regeneration system.

The multi-enzyme coupling reaction system has become a promising biomanufacturing platform for biochemical production. Tyr is an essential amino acid, but the limited solubility restricts its use. Tyrosyl dipeptide has been paid more attention due to its higher solubility. In this study, an efficient enzymatic cascade of Ala-Tyr synthesis was developed by a L-amino acid ligase together with polyphosphate kinase (PPK). Two L-amino acid ligases from Bacillus subtilis and Bacillus pumilus were selected and applied for Ala-Tyr synthesis. The L-amino acid ligase from B. subtilis (Bs) was selected and coupled with the PPK from Sulfurovum lithotrophicum (PPKSL) for regenerating ATP to produce Ala-Tyr in one pot. In the optimization system, 40.1 mM Ala-Tyr was produced within 3 h due to efficient ATP regeneration with hexametaphosphate (PolyP(6)) as the phosphate donor. The molar yield was 0.89 mol/mol based on the substrates added, while the productivity of Ala-Tyr achieved 13.4 mM/h, which were the highest yield and productivity ever reported about Ala-Tyr synthesis with L-amino acid ligase.

Early monitoring-to-warning Internet of Things system for emerging infectious diseases via networking of light-triggered point-of-care testing devices.

Early monitoring and warning arrangements are effective ways to distinguish infectious agents and control the spread of epidemic diseases. Current testing technologies, which cannot achieve rapid detection in the field, have a risk of slowing down the response time to the disease. In addition, there is still no epidemic surveillance system, implementing prevention and control measures is slow and inefficient. Motivated by these clinical needs, a sample-to-answer genetic diagnosis platform based on light-controlled capillary modified with a photocleavable linker is first developed, which could perform nucleic acid separation and release by light irradiation in less than 30 seconds. Then, on site polymerase chain reaction was performed in a handheld closed-loop convective system. Test reports are available within 20 min. Because this method is portable, rapid, and easy to operate, it has great potential for point-of-care testing. Additionally, through multiple device networking, a real-time artificial intelligence monitoring system for pathogens was developed on a cloud server. Through data reception, analysis, and visualization, the system can send early warning signals for disease control and prevention. Thus, anti-epidemic measures can be implemented effectively, and deploying and running this system can improve the capabilities for the prevention and control of infectious diseases.

Application of multistage induced electric field for acid hydrolysis of starch in a continuous-flow reactor.

Herein, a multistage induced electric field (IEF) combined with a continuous-flow reactor was utilized to assist the acid hydrolysis of corn, potato, and waxy corn starch for avoiding plate corrosion and heavy metal leakage. It was found that adding IEF stages was beneficial to improve the hydrolysis efficiency. Treating potato, corn, and waxy corn starch via continuous-flow IEF increased the reducing sugar contents up to 78.76 %, 57.86 %, and 66.18 %, respectively. The electrical conductivity of starch grew with the reaction stages, while starch yield demonstrated the opposite trend. Treated starch had higher solubility and gelatinization peak temperature than native starch, with the gelatinization enthalpy showing fluctuations. Meanwhile, the swelling power decreased as the number of IEF stages was increased. Observations of Fourier transform infrared spectroscopy, X-ray diffraction, and scanning electron microscopy indicated that the treated starch became more ordered, and crystalline regions were destroyed to various degrees with pores forming on particle surfaces. These variations could be attributed to acid hydrolysis and IEF.

Deep eutectic solvent with Lewis acid for highly efficient biohydrogen production from corn straw.

To boost saccharification and biohydrogen production efficiency from corn straw, Lewis acid enhanced deep eutectic solvent (DES) pretreatment using choline chloride/glycerol was developed. A notable enhancement of the enzymatic hydrolysis efficiency from 26.3 % to 87.0 % was acquired when corn straw was pretreated with aqueous DES at 100 °C for 5 h using 2.0 wt% AlCl3. A maximum biohydrogen yield of 114.8 mL/g total solids (TS) was achieved in the sequential dark fermentation stage, which was 2.1 times higher than that of the raw feedstock (37.1 mL/g TS). The enhanced efficient conversion was ascribed to the effective removal of lignin and hemicellulose, which led to the bio-accessibility of the straw. This work provides new sights for the rational design of efficient AlCl3-aided aqueous DES system toward biohydrogen production from lignocellulosic biomass.

Weighted gene Co-expression network analysis (WGCNA) reveals a set of hub genes related to chlorophyll metabolism process in chlorella (Chlorella vulgaris) response androstenedione.

Androstenedione (ADSD) was the main androgen detected in wastewaters. Chlorella was the most widely used plant in biological wastewater treatment process. In order to understand the toxicological response of chlorella to ADSD contamination, we used the weighted gene co-expression network analysis (WGCNA) method to systematically analyze the gene regulatory networks of chlorella after ADSD treatments. Total of 25 modules was identified from gene co-expression networks, and the turquoise module were selected for GO and KEGG enrichment analysis. Results showed that most hub genes were associated with chloroplast organizations or photosystems processes. Among them, the expressions profiles of hcar, nol, pao and sgr genes were highly correlated to the content fluctuations of chlorophylls after different ADSD treatments. All these results demonstrated that chlorophylls play a key role in preventing cell damage of chlorella caused by ADSD contamination. Besides, we proposed a possible chlorophyll metabolism pathway in chlorella response to ADSD contamination.

Modular Combination of Proteolysis-Responsive Transcription and Spherical Nucleic Acids for Smartphone-Based Colorimetric Detection of Protease Biomarkers.

Sensitive and facile detection of biomarkers is essential for early diagnosis and treatment of diseases. To this end, we here proposed a colorimetric protease assay by the modular combination of proteolysis-responsive transcription and spherical nucleic acids (SNAs). In this assay, target protease-mediated proteolysis triggers the synthesis of RNAs by in vitro transcription, which subsequently results in the aggregation of SNAs with remarkable redshifts in the wavelength of surface plasmon resonance-related absorption. As a proof of concept, this assay achieved the sensitive and specific detection of matrix metalloprotease-2 (MMP-2) with a limit of detection of 3.3 pM. Moreover, the applicability of this colorimetric assay can be expanded to other protease biomarkers (e.g., thrombin and hepatitis C virus NS3/4A) by tuning the target-responsive RNA polymerase module. Furthermore, by the immobilization of SNAs on a glass fiber membrane, a test strip that enables the portable detection of target protease with a smartphone was developed. With the use of a mobile application to capture and process the colorimetric signals, this portable detection system allowed for sensitive evaluation of MMP-2 levels in biological and clinical specimens, highlighting its potential in point-of-care diagnosis of diseases.

Integrated Cascade Biorefinery Processes to Transform Woody Biomass Into Phenolic Monomers and Carbon Quantum Dots.

A novel cascade biorefinery strategy toward phenolic monomers and carbon quantum dots (CQDs) is proposed here via coupling catalytic hydrogenolysis and hydrothermal treatment. Birch wood was first treated with catalytic hydrogenolysis to afford a high yield of monomeric phenols (44.6 wt%), in which 4-propanol guaiacol (10.2 wt%) and 4-propanol syringol (29.7 wt%) were identified as the two major phenolic products with 89% selectivity. An available carbohydrate pulp retaining 82.4% cellulose and 71.6% hemicellulose was also obtained simultaneously, which was further used for the synthesis of CQDs by a one-step hydrothermal process. The as-prepared CQDs exhibited excellent selectivity and detection limits for several heavy metal cations, especially for Fe3+ ions in an aqueous solution. Those cost-efficient CQDs showed great potential in fluorescent sensor in situ environmental analyses. These findings provide a promising path toward developing high-performance sensors on environmental monitoring and a new route for the high value-added utilization of lignocellulosic biomass.

Target-activated transcription for the amplified sensing of protease biomarkers.

Signal amplification is an effective way to achieve sensitive analysis of biomarkers, exhibiting great promise in biomedical research and clinical diagnosis. Inspired by the transcription process, here we present a versatile strategy that enables effective amplification of proteolysis into nucleic acid signal outputs in a homogeneous system. In this strategy, a protease-activatable T7 RNA polymerase is engineered as the signal amplifier and achieves 3 orders of magnitude amplification in signal gain. The versatility of this strategy has been demonstrated by the development of sensitive and selective assays for protease biomarkers, such as matrix metalloproteinase-2 (MMP-2) and thrombin, with sub-picomole sensitivity, which is 4.3 × 103-fold lower than that of the standard peptide-based method. Moreover, the proposed assay has been further applied in the detection of MMP-2 secreted by cancer cells, as well as in the assessment of MMP-2 levels in osteosarcoma tissue samples, providing a general approach for the monitoring of protease biomarkers in clinical diagnosis.

Improving biohydrogen production through dark fermentation of steam-heated acid pretreated Alternanthera philoxeroides by mutant Enterobacter aerogenes ZJU1.

Alternanthera philoxeroides, a notorious invasive aquatic weed, is a typical lignocellulosic feedstock for fermentative biohydrogen production. To improve the dark fermentation performance, steam-heated acid pretreatment and enzymolysis were employed to release reducing sugars from A. philoxeroides, and Enterobacter aerogenes ZJU1 mutagenized by 60Co-γ irradiation was used as the inoculum. Dilute acid accompanied by steam heating significantly disrupted the fiber structures of A. philoxeroides. Scanning electron microscopic images revealed that many pores and fissures were generated in the surface of A. philoxeroides after pretreatment. X-ray diffraction and Fourier transform infrared spectroscopy analyses showed that the pretreatment facilitated the transformation of cellulose I to cellulose II in A. philoxeroides biomass, resulting in the increase of amorphous regions and the decrease of crystallinity. Under the optimum pretreatment condition (1.0 v/v% H2SO4, 135 °C for 15 min), the reducing sugar yield reached 0.354 g/g A. philoxeroides, which was further increased to 0.575 g/g A. philoxeroides after enzymolysis. The biohydrogen yield increased by 59.9% from 38.9 mL/g volatile solids (VS) of raw A. philoxeroides to 62.2 mL/gVS of the pretreated one. As compared to the wild strain, E. aerogenes ZJU1 contributed to an increase of 31.8% in the biohydrogen yield from pretreated A. philoxeroides. Further optimization of bacteria suspensions significantly increased the maximum biohydrogen production rate from 1.42 to 4.64 mL/gVS/h, advanced the biohydrogen production peak, and resulted in an increase of 42.8% in biohydrogen yield to 89.8 mL/gVS.

Functional Titanium Carbide MXenes-Loaded Entropy-Driven RNA Explorer for Long Noncoding RNA PCA3 Imaging in Live Cells.

The visualization of the long noncoding RNA of prostate cancer gene 3 (lncRNA PCA3), a specific biomarker for androgen receptor-positive prostate cancer, in living cells not only directly reflects the gene expression and localization but also offers better insight into its roles in the pathological processes. Here, we loaded an entropy-driven RNA explorer (EDRE) on the TAT peptide-functionalized titanium carbide MXenes (Ti3C2-TAT) for the imaging of nuclear lncRNA PCA3 in live cells. The EDRE was condensed on the Ti3C2-TAT (Ti3C2-TAT@EDRE) by electrostatic interaction. Ti3C2-TAT@EDRE enables the entering of cells and release of TAT peptides and EDRE in the cytoplasm by the glutathione (GSH)-triggered cleavage of the disulfide bonds in Ti3C2-TAT. The released EDRE is delivered into the nucleus by the nucleus-targeted guidance of TAT peptides, and initiated by the target lncRNA PCA3, subsequently leading to the continuous accumulation of fluorescence signals. Consequently, fluorescence analysis of lncRNA PCA3 at low-picomolar concentrations in vitro as well as sensitive live cell imaging of lncRNA PCA3 in the nucleus of androgen receptor-positive LNCaP prostate cancer cells were achieved, providing a versatile strategy for the monitoring of nucleic acid biomarkers in the nucleus of living cells.

A Collaborative Study: Determination of Mycotoxins in Corn, Peanut Butter, and Wheat Flour Using Stable Isotope Dilution Assay (SIDA) and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

A collaborative study was conducted to evaluate stable isotope dilution assay (SIDA) and LC-MS/MS for the simultaneous determination of aflatoxins B1, B2, G1, and G2; deoxynivalenol; fumonisins B1, B2, and B3; ochratoxin A; HT-2 toxin; T-2 toxin; and zearalenone in foods. Samples were fortified with 12 13C uniformly labeled mycotoxins (13C-IS) corresponding to the native mycotoxins and extracted with acetonitrile/water (50:50 v/v), followed by centrifugation, filtration, and LC-MS/MS analysis. In addition to certified reference materials, the six participating laboratories analyzed corn, peanut butter, and wheat flour fortified with the 12 mycotoxins at concentrations ranging from 1.0 to 1000 ng/g. Using their available LC-MS/MS platform, each laboratory developed in-house instrumental conditions for analysis. The majority of recoveries ranged from 80 to 120% with relative standard derivations (RSDs) <20%. Greater than 90% of the average recoveries of the participating laboratories were in the range of 90-110%, with repeatability RSDr (within laboratory) < 10% and reproducibility RSDR (among laboratory) < 15%. All Z scores of the results of certified reference materials were between -2 and 2. Using 13C-IS eliminated the need for matrix-matched calibration standards for quantitation, simplified sample preparation, and achieved simultaneous identification and quantitation of multiple mycotoxins in a simple LC-MS/MS procedure.

Enhanced hydrogen production of Enterobacter aerogenes mutated by nuclear irradiation.

Nuclear irradiation was used for the first time to generate efficient mutants of hydrogen-producing bacteria Enterobacter aerogenes, which were screened with larger colour circles of more fermentative acid by-products. E. aerogenes cells were mutated by nuclear irradiation of 60Co γ-rays. The screened E. aerogenes ZJU1 mutant with larger colour circles enhanced the hydrogenase activity from 89.8 of the wild strain to 157.4mLH2/(gDWh). The hereditary stability of the E. aerogenes ZJU1 mutant was certified after over ten generations of cultivation. The hydrogen yield of 301mLH2/gglucose with the mutant was higher by 81.8% than that of 166mL/gglucose with the wild strain. The peak hydrogen production rate of 27.2mL/(L·h) with the mutant was higher by 40.9% compared with that of 19.3mL/(L·h) with the wild strain. The mutant produced more acetate and butyrate but less ethanol compared with the wild strain during hydrogen fermentation.

Enhancing hydrogen production of Enterobacter aerogenes by heterologous expression of hydrogenase genes originated from Synechocystis sp.

The hydrogenase genes (hoxEFUYH) of Synechocystis sp. PCC 6803 were cloned and heterologously expressed in Enterobacter aerogenes ATCC13408 for the first time in this study, and the hydrogen yield was significantly enhanced using the recombinant strain. A recombinant plasmid containing the gene in-frame with Glutathione-S-Transferase (GST) gene was transformed into E. aerogenes ATCC13408 to produce a GST-fusion protein. SDS-PAGE and western blot analysis confirm the successful expression of the hox genes. The hydrogenase activity of the recombinant strain is 237.6±9.3ml/(g-DW·h), which is 152% higher than the wild strain. The hydrogen yield of the recombinant strain is 298.3ml/g-glucose, which is 88% higher than the wild strain. During hydrogen fermentation, the recombinant strain produces more acetate and butyrate, but less ethanol. This is corresponding to the NADH metabolism in the cell due to the higher hydrogenase activity with the heterologous expression of hox genes.

Enhanced dark hydrogen fermentation by addition of ferric oxide nanoparticles using Enterobacter aerogenes.

Ferric oxide nanoparticles (FONPs) were used to facilitate dark hydrogen fermentation using Enterobacter aerogenes. The hydrogen yield of glucose increased from 164.5±2.29 to 192.4±1.14mL/g when FONPs concentration increased from 0 to 200mg/L. SEM images of E. aerogenes demonstrated the existence of bacterial nanowire among cells, suggesting FONPs served as electron conduits to enhance electron transfer. TEM showed cellular internalization of FONPs, indicating hydrogenase synthesis and activity was potentially promoted due to the released iron element. When further increasing FONPs concentration to 400mg/L, the hydrogen yield of glucose decreased to 147.2±2.54mL/g. Soluble metabolic products revealed FONPs enhanced acetate pathway of hydrogen production, but weakened ethanol pathway. This shift of metabolic pathways allowed more nicotinamide adenine dinucleotide for reducing proton to hydrogen.