Engineering microbial metabolic homeostasis for chemicals production.

Microbial-based bio-refining promotes the development of a biotechnology revolution to encounter and tackle the enormous challenges in petroleum-based chemical production by biomanufacturing, biocomputing, and biosensing. Nevertheless, microbial metabolic homeostasis is often incompatible with the efficient synthesis of bioproducts mainly due to: inefficient metabolic flow, robust central metabolism, sophisticated metabolic network, and inevitable environmental perturbation. Therefore, this review systematically summarizes how to optimize microbial metabolic homeostasis by strengthening metabolic flux for improving biotransformation turnover, redirecting metabolic direction for rewiring bypass pathway, and reprogramming metabolic network for boosting substrate utilization. Future directions are also proposed for providing constructive guidance on the development of industrial biotechnology.

Identification of 3-ketocapnine reductase activity within the human microbiota.

The microbial synthesis of sulfonolipids within the human body is likely involved in maintaining human health or causing diseases. However, the enzymes responsible for their biosynthesis remain largely unknown. In this study, we identified and verified the role of 3-ketocapnine reductase, the third-step enzyme, in the four-step conversion of l-phosphoserine into sulfobacin B both in vivo and in vitro. This finding builds upon our previous research into sulfonolipid biosynthesis, which focused on the vaginal bacterium Chryseobacterium gleum DSM 16776 and the gut bacterium Alistipes finegoldii DSM 17242. Through comprehensive gene mapping, we demonstrate the widespread presence of potential sulfonolipid biosynthetic genes across diverse bacterial species inhabiting various regions of the human body. These findings shed light on the prevalence of sulfonolipid-like metabolites within the human microbiota, suggesting a potential role for these lipid molecules in influencing the intricate biointeractions within the complex microbial ecosystem of the human body.

Comparative proteomics reveals energy and carbon metabolism changes in Scenedesmus quadricauda mutants induced by heavy-ion beam irradiation.

Microalgae's superior ability to fix carbon dioxide into biomass and high-value bioproducts remains underutilized in biotechnological applications due to a lack of comprehensive understanding of their carbon metabolism and energy conversion. In this work, the strain improvement technique heavy-ion beams (HIB) mutagenesis was employed on the environmentally adaptable microalgae Scenedesmus quadricauda. After several rounds of screening, two contrasting mutants were identified. S-#4 showed low photosynthetic activity and biomass productivity, while S-#26 exhibited adaptability to prolonged high light stress, achieving a 28.34 % increase in biomass yield compared to the wild-type strain. Integrating their photosynthetic characteristics and comparative proteomic analysis revealed that the contrasting protein regulations from central carbon metabolism mainly affects the two mutants' opposite biomass accumulation. Therefore, the divergent regulation of the tricarboxylic acid cycle following HIB mutagenesis could be potential targets for engineering microalgae with superior biomass and high-value products.

Microbial cell factory for butyl butyrate production: Knowledges and perspectives.

Butyl butyrate is a short-chain fatty acid ester (C8) with a fruity aroma. It has broad prospects in the fields of foods, cosmetics and biofuels. At present, butyl butyrate is produced by chemical synthesis in the industry, but it is highly dependent on petroleum-based products. The growing concerns regarding the future scarcity of fossil fuels have been strongly promoted the transition from traditional fossil fuels and products to renewable bioenergy and biochemicals. Therefore, it is necessary to develop a green biochemical technology to replace traditional petroleum-based materials. In recent years, microorganisms such as Escherichia coli and Clostridium have been engineered to serve as cell factories for the sustainable one-pot production of short-chain fatty acid esters, including butyl butyrate. This opinion highlights the recent development in the use of lipases and alcohol acyltransferases (AATs) for butyl butyrate production in microbial fermentation, as well as future perspectives.

Phage tailspike protein coated gold nanoparticles combined with smartphone for rapid bacterial detection and photothermal sterilization.

The integration of recognition and therapeutic functions in multifunctional biosensors is of great importance in guaranteeing food security and reducing the occurrence of foodborne illness caused by foodborne pathogens. In this study, a biosensor utilizing a "sense-and-treat" approach was developed by integrating phage tailspike protein (TSP) with gold nanoparticles (AuNPs@TSP). The synthesized AuNPs@TSP showed strong binding affinity towards Salmonella typhimurium causing color changes and exhibited effective bactericidal activity when exposed to near-infrared (NIR) irradiation. This biosensor facilitated rapid colorimetric detection of S. typhimurium in 50 min, with a LOD (limit of detection) of 2.53 × 103 CFU/mL output on a smartphone APP after analyzing the red-green-blue (RGB) values from color rendering results. Furthermore, the biosensor displayed high selectivity, rapid response time, and broad applicability when tested with real samples. Moreover, the biosensor exhibited a remarkably efficient antibacterial efficacy of 100 % against S. typhimurium under 808 nm light irradiation for 6 min. This study provides a comprehensive investigation into the potential utilization of biosensors for rapid detection and eradication of foodborne pathogens in food industry.

Construction and Mechanism Exploration of Highly Efficient System for Bacterial Ghosts Preparation Based on Engineered Phage ID52 Lysis Protein E.

Bacterial ghosts (BGs) are hollow bacterial cell envelopes with intact cellular structures, presenting as promising candidates for various biotechnological and biomedical applications. However, the yield and productivity of BGs have encountered limitations, hindering their large-scale preparation and multi-faceted applications of BGs. Further optimization of BGs is needed for the commercial application of BG technology. In this study, we screened out the most effective lysis protein ID52-E-W4A among 13 mutants based on phage ID52 lysis protein E and optimized the liquid culture medium for preparing Escherichia coli Nissle 1917 (EcN). The results revealed a significantly higher lysis rate of ID52-E-W4A compared to that of ID52-E in the 2xYT medium. Furthermore, EcN BGs were cultivated in a fermenter, achieving an initial OD600 as high as 6.0 after optimization, indicating enhanced BG production. Moreover, the yield of ID52-E-W4A-induced BGs reached 67.0%, contrasting with only a 3.1% yield from φX174-E-induced BGs. The extended applicability of the lysis protein ID52-E-W4A was demonstrated through the preparation of Salmonella pullorum ghosts and Salmonella choleraesuis ghosts. Knocking out the molecular chaperone gene slyD and dnaJ revealed that ID52-mediated BGs could still undergo lysis. Conversely, overexpression of integral membrane enzyme gene mraY resulted in the loss of lysis activity for ID52-E, suggesting that the lysis protein ID52-E may no longer rely on SlyD or DnaJ to function, with MraY potentially being the target of ID52-E. This study introduces a novel approach utilizing ID52-E-W4A for recombinant expression, accelerating the BG formation and thereby enhancing BG yield and productivity.

Cofactor engineering in Thermoanaerobacterium aotearoense SCUT27 for maximizing ethanol yield and revealing an enzyme complex with high ferredoxin-NAD+ reductase activity.

Thermoanaerobacterium aotearoense SCUT27 is a prominent producer of biofuels from lignocellulosic materials. To provide sufficient NAD(P)H for ethanol production, redox-related genes, including lactate dehydrogenase (ldh), redox-sensing transcriptional repressor (rex), and hydrogenase (hfsB), were knocked out. However, the growth of strain PRH (Δldh/Δrex/ΔhfsB) was suppressed due to the intracellular redox state imbalance with the increased NADH concentration. Coincidentally, when the Bcd-EtfAB (BCD) complex was overexpressed, the resulting strain PRH-B3 (Δldh/Δrex/ΔhfsB::BCD) grew rapidly and produced ethanol with a high yield. With lignocellulosic hydrolysates, PRH-BA (Δldh/Δrex/ΔhfsB::BCD::adhE) demonstrated high ethanol productivity and yield, reaching levels of 0.45-0.51 g/L/h and 0.46-0.53 g/g sugars, respectively. The study results shed light on the cofactor balance for cell stability and the high ferredoxin-NAD+ reductase activity of the BCD complex under an intracellular low redox state. They also provide an essential reference for developing strains for improved biofuel production.

Detection of SARS-CoV-2 using machine learning-enabled paper-assisted ratiometric fluorescent sensors based on target-induced magnetic DNAzyme.

The development of an advanced analytical platform with regard to SARS-CoV-2 is crucial for public health. Herein, we present a machine learning platform based on paper-assisted ratiometric fluorescent sensors for highly sensitive detection of the SARS-CoV-2 RdRp gene. The assay involves target-induced rolling circle amplification to generate magnetic DNAzyme, which is then detectable using the paper-assisted ratiometric fluorescent sensor. This sensor detects the SARS-CoV-2 RdRp gene with a visible-fluorescence color response. Moreover, leveraging different fluorescence responses, the ResNet algorithm of machine learning assists in accurately identifying fluorescence images and differentiating the concentration of the SARS-CoV-2 RdRp gene with over 99% recognition accuracy. The machine learning platform exhibits exceptional sensitivity and color responsiveness, achieving a limit of detection of 30 fM for the SARS-CoV-2 RdRp gene. The integration of intelligent artificial vision with the paper-assisted ratiometric fluorescent sensor presents a novel approach for the on-site detection of COVID-19 and holds potential for broader use in disease diagnostics in the future.

Resistance to preservatives and the viable but non-culturable state formation of Asaia lannensis in flavored syrups.

Food security is a crucial issue that has caused extensive concern, and the use of food flavors has become prevalent over time. we used the molecular biological techniques, preservative susceptibility testing, viable but non-culturable (VBNC) state induction testing, and a transcriptome analysis to examine the bacterial contamination of favored syrup and identify the causes and develop effective control measures. The results showed that Asaia lannensis WLS1-1 is a microorganism that can spoil food and is a member of the acetic acid bacteria families. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests showed that WLS1-1 was susceptible to potassium sorbate (PS), sodium benzoate (SB), and sodium sulffte (SS) at pH 4.0. It revealed a progressive increase in resistance to these preservatives at increasing pH values. WLS1-1 was resistant to PS, SB and SS with an MIC of 4.0, 2.0 and 0.5 g/L at pH 5.0, respectively. The MIC values exceed the maximum permissible concentrations that can be added. The induction test of the VBNC state demonstrated that WLS1-1 lost its ability to grow after 321 days of PS induction, 229 days of SB induction and 52 days of SS induction combined with low temperature at 4°C. Additionally, laser confocal microscopy and a propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) assay showed that WLS1-1 was still alive after VBNC formation. There were 7.192 ± 0.081 (PS), 5.416 ± 0.149 (SB) and 2.837 ± 0.134 (SS) log10(CFU/mL) of viable bacteria. An analysis of the transcriptome data suggests that Asaia lannensis can enter the VBNC state by regulating oxidative stress and decreasing protein synthesis and metabolic activity in response to low temperature and preservatives. The relative resistance of Asaia lannensis to preservatives and the induction of the VBNC state by preservatives are the primary factors that contribute to the contamination of favored syrup by this bacterium. To our knowledge, this study represents the first evidence of the ability of Asaia lannensis to enter the VBNC state and provides a theoretical foundation for the control of organisms with similar types of activity.

Development of inducible promoters for regulating gene expression in Clostridium tyrobutyricum for biobutanol production.

Clostridium tyrobutyricum is an anaerobe known for its ability to produce short-chain fatty acids, alcohols, and esters. We aimed to develop inducible promoters for fine-tuning gene expression in C. tyrobutyricum. Synthetic inducible promoters were created by employing an Escherichia coli lac operator to regulate the thiolase promoter (PCathl) from Clostridium acetobutylicum, with the best one (LacI-Pto4s) showing a 5.86-fold dynamic range with isopropyl ß- d-thiogalactoside (IPTG) induction. A LT-Pt7 system with a dynamic range of 11.6-fold was then created by combining LacI-Pto4s with a T7 expression system composing of RNA polymerase (T7RNAP) and Pt7lac promoter. Furthermore, two inducible expression systems BgaR-PbgaLA and BgaR-PbgaLB with a dynamic range of ~40-fold were developed by optimizing a lactose-inducible expression system from Clostridium perfringens with modified 5' untranslated region (5' UTR) and ribosome-binding site (RBS). BgaR-PbgaLB was then used to regulate the expressions of a bifunctional aldehyde/alcohol dehydrogenase encoded by adhE2 and butyryl-CoA/acetate Co-A transferase encoded by cat1 in C. tyrobutyricum wild type and Δcat1::adhE2, respectively, demonstrating its efficient inducible gene regulation. The regulated cat1 expression also confirmed that the Cat1-catalyzed reaction was responsible for acetate assimilation in C. tyrobutyricum. The inducible promoters offer new tools for tuning gene expression in C. tyrobutyricum for industrial applications.

Ononin promotes radiosensitivity in lung cancer by inhibiting HIF-1α/VEGF pathway.

BACKGROUND: In our previous study, we provided evidence that Astragalus mongholicus Bunge(AM) and its extracts possess a protective capability against radiation-induced damage, potentially mediated through the reduction of reactive oxygen species (ROS) and nitric oxide (NO). However, we were pleasantly surprised to discover during our experimentation that AM not only offers protection against radiation damage but also exhibits a radiation sensitization effect. This effect may be attributed to a specific small molecule present in AM known as ononin. Currently, radiation sensitizers are predominantly found in nitrazole drugs and nanomaterials, with no existing reports on the radiation sensitization properties of ononin, nor its underlying mechanism. PURPOSE: This study aims to investigate the sensitization effect of the small molecule ononin derived from AM on lung cancer radiotherapy, elucidating its specific molecular mechanism of action. Additionally, the safety profile of combining astragalus small molecule ononin with radiation therapy will be evaluated. METHODS: The effective concentration of ononin was determined through cell survival experiments, and the impact of ononin combined with varying doses of radiation on lung cancer cells was observed using CCK-8 and cell cloning experiments. The apoptotic effect of ononin combined with radiation on lung cancer cells was assessed using Hochester staining, flow cytometry, and WB assay. Additionally, WB and immunofluorescence analysis were conducted to investigate the influence of ononin on HIF-1α/VEGF pathway. Furthermore, Molecular Dynamics Simulation was employed to validate the targeted binding ability of ononin and HIF-1α. A lung cancer cell line was established to investigate the effects of knockdown and overexpression of HIF-1α. Subsequently, the experiment was repeated using tumor bearing nude mice and C57BL/6 mouse models in an in vivo study. Tumor volume was measured using a vernier caliper, while HE, immunohistochemistry, and immunofluorescence techniques were employed to observe the effects of ononin combined with radiation on tumor morphology, proliferation, and apoptosis. Additionally, Immunofluorescence was employed to examine the impact of ononin on HIF-1α/VEGF pathway in vivo, and its effect on liver function in mice was assessed through biochemistry analysis. RESULTS: At a concentration of 25 µM, ononin did not affect the proliferation of lung epithelial cells but inhibited the survival of lung cancer cells. In vitro experiments demonstrated that the combination of ononin and radiation could effectively inhibit the growth of lung cancer cells, induce apoptosis, and suppress the excessive activation of the Hypoxia inducible factor 1 alpha/Vascular endothelial growth factor pathway. In vivo experiments showed that the combination of ononin and radiation reduced the size and proliferation of lung cancer tumors, promoted cancer cell apoptosis, mitigated abnormal activation of the Hypoxia inducible factor 1 alpha pathway, and protected against liver function damage. CONCLUSION: This study provides evidence that the combination of AM and its small molecule ononin can enhance the sensitivity of lung cancer to radiation. Additionally, it has been observed that this combination can specifically target HIF-1α and exert its effects. Notably, ononin exhibits the unique ability to protect liver function from damage while simultaneously enhancing the tumor-killing effects of radiation, thereby demonstrating a synergistic and detoxifying role in tumor radiotherapy. These findings contribute to the establishment of a solid basis for the development of novel radiation sensitizers derived from traditional Chinese medicine.

YAP/Aurora A-mediated ciliogenesis regulates ionizing radiation-induced senescence via Hedgehog pathway in tumor cells.

Primary cilia are antenna-like organelles that play critical roles in sensing and responding to various signals. Nevertheless, the function of primary cilia in cellular response to ionizing radiation (IR) in tumor cells remains unclear. Here, we show that primary cilia are frequently expressed in tumor cells and tissues. Notably, IR promotes cilia formation and elongation in time- and dose-dependent manners. Mechanistic study shows that the suppression of YAP/Aurora A pathway contributes to IR-induced ciliogenesis, which is diminished by Aurora A overexpression. The ciliated tumor cells undergo senescence but not apoptosis in response to IR and the abrogation of cilia formation is sufficient to elevate the lethal effect of IR. Furthermore, we show that IR-induced ciliogenesis leads to the activation of Hedgehog signaling pathway to drive senescence and resist apoptosis, and its blockage enhances cellular radiosensitivity by switching senescence to apoptosis. In summary, this work shows evidence of primary cilia in coordinating cellular response to IR in tumor cells, which may help to supply a novel sensitizing target to improve the outcome of radiotherapy.

Characterization of a cell wall hydrolase with high activity against vegetative cells, spores and biofilm of Bacillus cereus.

Bacillus cereus is a prevalent foodborne pathogen that induces food poisoning symptoms such as vomiting and diarrhea. Its capacity to form spores and biofilm enables it to withstand disinfectants and antimicrobials, leading to persistent contamination during food processing. Consequently, it is necessary to develop novel and efficient antimicrobial agents to control B. cereus, its spores, and biofilms. Peptidoglycan hydrolases have emerged as a promising and eco-friendly alternative owing to their specific lytic activity against pathogenic bacteria. Here, we identified and characterized a Lysozyme-like cell wall hydrolase Lys14579, from the genome of B. cereus ATCC 14579. Recombinant Lys14579 specifically lysed B. cereus without affecting other bacteria. Lys14579 exhibited strong lytic activity against B. cereus, effectively lysing B. cereus cell within 20 min at low concentration (10 µg/mL). It also inhibited the germination of B. cereus spores and prevented biofilm formation at 12.5 µg/mL. Moreover, Lys14579 displayed good antimicrobial stability with negligible hemolysis in mouse red blood cells and no cytotoxicity against RAW264.7 cells. Notably, Lys14579 effectively inhibited B. cereus in boiled rice and minced meat in a dose-dependent manner. Furthermore, bioinformatics analysis and point mutagenesis experiments revealed that Glu-47 was the catalytic site, and Asp-57, Gln-60, Ser-61 and Glu-63 were active-site residues related with the cell wall lytic activity. Taken together, Lys14579 could be a promising biocontrol agent against vegetative cells, spores, and biofilm of B. cereus in food industry.

A nationwide investigation on the characteristics and health risk of trace elements in surface water across China.

Rapid urbanization accelerates the release of anthropogenic heavy metals from local to wider water systems, posing a serious threat to aquatic ecosystems and public health. The characteristics of trace elements were investigated to evaluate the environmental status of surface water in 40 cities of China. The concentrations of 22 elements in surface water ranged from 7.00 × 10-4 to 4.37 × 105 µg/L. The water quality can be classified as "excellent" except Songhuajiang. The levels of As, Cd, Cr, Pb, and Hg are all within the limits permitted by national drinking water quality standards. An obvious regional distribution characteristic was observed, with concentrations of Zn, Mn, Ni, Cu, Co, U, and Cr higher in surface water collected in the north than in the south, while the trends for Cd, Tl, and As are opposite. Notably, Tl shows significant geographical divergences, with the level of surface water collected from the south nine times higher than that from the north. The regional distribution of the mineral, industrial, or agricultural activity might be responsible for the south-to-north difference of these elements. The hazard index (HI) and total cancer risk (TCR) through oral or dermal contact with water-related heavy metals were further calculated. The average HI was 0.54 in the north and 0.29 in the south for adults, while HI for children was relatively higher. The value was 1.01 and 0.55 in the north and south, respectively. TCR in the north is 2.58 × 10-4 and mainly contributed by Cr (88.1 %), while TCR in the south is 4.48 × 10-5 and mainly contributed by As (98.4 %). The research results can provide essential data for effective water resources management and human health protection in China.

Primary cilium participates in radiation-induced bystander effects through TGF-ß1 signaling.

Many studies have indicated that tumor growth factor-beta (TGF-ß) signaling mediates radiation-induced bystander effects (RIBEs). The primary cilium (PC) coordinates several signaling pathways including TGF-ß signaling to regulate diverse cellular processes. But whether the PC participates in TGF-ß induced RIBEs remains unclear. The cellular levels of TGF-ß1 were detected by western blot analysis and the secretion of TGF-ß1 was measured by ELISA kit. The ciliogenesis was altered by CytoD treatment, STIL siRNA transfection, IFT88 siRNA transfection, or KIF3a siRNA transfection, separately, and was detected by western blot analysis and immunofluorescence staining. G0 /G1 phase cells were arrested by serum starvation and S phase cells were induced by double thymidine block. The TGF-ß1 signaling was interfered by LY2109761, a TGF-ß receptor 1 (TßR1) inhibitor, or TGF-ß1 neutral antibody. The DNA damages were induced by TGF-ß1 or radiated conditional medium (RCM) from irradiated cells and were reflected by p21 expression, 53BP1 foci, and γH2AX foci. Compared with unirradiated control, both A549 and Beas-2B cells expressed and secreted more TGF-ß1 after carbon ion beam or X-ray irradiation. RCM collected from irradiated cells or TGF-ß1 treatment caused an increase of DNA damage in cocultured unirradiated Beas-2B cells while blockage of TGF-ß signaling by TßR1 inhibitor or TGF-ß1 neutral antibody alleviates this phenomenon. IFT88 siRNA or KIF3a siRNA impaired PC formation resulted in an aggravated DNA damage in bystander cells, while elevated PC formation by CytoD or STIL siRNA resulted in a decrease of DNA damage. Furthermore, TGF-ß1 induced more DNA damages in S phases cells which showed lower PC formation rate and less DNA damages in G0 /G1 phase cells which showed higher PC formation rate. This study demonstrates the particular role of primary cilia during RCM induced DNA damages through TGF-ß1 signaling restriction and thereby provides a functional link between primary cilia and RIBEs.

Specific separation and sensitive detection of foodborne pathogens by phage-derived bacterial-binding protein-nano magnetic beads coupled with smartphone-assisted paper sensor.

Foodborne pathogen infection poses a significant threat to public health and is considered as one of the most serious hazards in global food safety. Herein, a sensitive and efficient method for on-site monitoring of foodborne pathogens was developed by using a smartphone-assisted paper-sensor combined with phage-derived bacterial-binding proteins-nano magnetic beads (PBPs-MBs). PBPs including tail fiber protein (TFP:gp13), cell-wall binding domain (CBD) of endolysin and tailspike protein (TSP) coated on the surface of MBs were applied for rapid separation and enrichment of targeted bacteria (Escherichia coli O157:H7, Staphylococcus aureus and Salmonella typhimurium, respectively) from food samples in 20 min before detection on paper-based sensors. The paper-based sensor was loaded with the lytic agent (polymyxin B) to induce bacterial lysis and release specific endogenous enzymes. Subsequently, three distinct chromogenic substrates were hydrolyzed by their corresponding enzymes, resulting in characteristic color changes on the paper, respectively. In addition, a smartphone APP for red-green-blue (RGB) color analysis of paper was able to directly detect three foodborne pathogens. As a result, the limit of detection (LOD) values for three foodborne pathogens were found to be 2.44 × 102, 2.68 × 104 and 4.62 × 103 CFU/mL, respectively, which were much lower than other studies (106-108 CFU/mL) based on enzymes. Moreover, the feasibility of this approach was further assessed through the successful detection of targeted bacteria in real samples with satisfactory recovery rates. In conclusion, this smartphone-assisted biosensor offers promising application potential for point-of-care testing (POCT) of foodborne pathogens in resource-scarce areas.

A functionalized Sup35NM nanofibril-assisted oriented antibody capture in lateral flow immunoassay for sensitive detection of dengue type II NS1.

Rapid and sensitive dengue non-structural protein 1 (NS1) detection assay is essential for the treatment of disease and currently releases high medical cost burdens. To address the limitations of conventional LFIA strips, we have developed an improved Sup35NM-Z-based LFIA that immobilizes antibodies on cellulose membranes in an orientated manner to increase the sensitivity of LFIA strips. A dual-functional Sup35NM nanofibril was fabricated by fusion with the antibody binding domain; resultant nanofibril from the amyloid Sup35NM was sprayed on the T-line to orientate the capture antibody and produces fluorescence signals. Antibody binding analysis showed that self-assembly of the Sup35NM monomer does not affect the binding activity of the Z-domain with the antibody. The NS1 for DENV-2 infection was chosen as a model target antigen to assess the feasibility of the Sup35NM-Z-domain-based LFIA platform. Under optimal conditions, the Sup35NM-Z-domain-based LFIA detected NS1 within 15 min with a detection limit of 1.29 ng/ml, while the detection limit of traditional LFIA with the same concentration of anti-NS1-Ab1 on the T-line by conventional physical adsorption was 2.20 ng/ml, 1.7 times higher than that of Sup35NM-Z-domain-based LFIA. As compared to traditional LFIAs, the Sup35NM-Z-based LFIA had a wide detection range of 1.29-625 ng/mL. The LFIA's clinical performance in identifying NS1 was also assessed using 15 clinical samples. The LFIA accurately recognized positive and negative samples, equal to 86.7% accuracy. The developed Sup35NM-Z-domain-based LFIA in this study offers great potential for the identification of target markers because of its greatly improved sensitivity and wider detection range.

Regulation of Circulating miR-342-3p Alleviates the Radiation-Induced Immune System Injury.

Ionizing radiation in space, radiation devices or nuclear disasters are major threats to human health and public security. Expanding countermeasures for dealing with accidental or occupational radiation exposure is crucial for the protection of radiation injuries. Circulating microRNAs (miRNAs) have emerged as promising radiation biomarkers in recent years. However, the origin, distribution and functions of radiosensitive circulating miRNAs remain unclear, which obstructs their clinical applications in the future. In this study, we found that mmu-miR-342-3p (miR-342) in mouse serum presents a stable and significant decrease after X-ray total-body irradiation (TBI). Focusing on this miRNA, we investigated the influences of circulating miR-342 on the radiation-induced injury. Through tail vein injection of Cy5-labeled synthetic miR-342, we found the exogenous miR-342-Cy5 was mainly enriched in metabolic and immune organs. Besides, the bioinformatic analysis predicted that miR-342 might involve in immune-related processes or pathways. Further, mice were tail vein injected with synthetic miR-342 mimetics (Ago-miR-342) after irradiation to upregulate the level of miR-342 in circulating blood. The results showed that the upregulation of circulating miR-342 alleviated the radiation-induced depletion of CD3+CD4+ T lymphocytes and influenced the levels of IL-2 and IL-6 in irradiated mice. Moreover, the injection of Ago-miR-342 improved the survival rates of mice with acute radiation injury. Our findings demonstrate that upregulation of circulating miR-342 alleviates the radiation-induced immune system injury, which provides us new insights into the functions of circulating miRNAs and the prospect as the targets for mitigation of radiation injuries.

Simulated microgravity-induced oxidative stress and loss of osteogenic potential of osteoblasts can be prevented by protection of primary cilia.

Oxidative stress has been considered to be closely related to spaceflight-induced bone loss; however, mechanism is elusive and there are no effective countermeasures. Using cultured rat calvarial osteoblasts exposed to microgravity simulated by a random positioning machine, this study addressed the hypotheses that microgravity-induced shortening of primary cilia leads to oxidative stress and that primary cilium protection prevents oxidative stress and osteogenesis loss. Microgravity was found to induce oxidative stress (as represented by increased levels of reactive oxygen species (ROS) and malondialdehyde production, and decreased activities of antioxidant enzymes), which was perfectly replicated in osteoblasts growing in NG with abrogated primary cilia (created by transfection of an interfering RNA), suggesting the possibility that shortening of primary cilia leads to oxidative stress. Oxidative stress was accompanied by mitochondrial dysfunction (represented by increased mitochondrial ROS and decreased mitochondrial membrane potential) and intracellular Ca2+ overload, and the latter was found to be caused by increased activity of Ca2+ channel transient receptor potential vanilloid 4 (TRPV4), as also evidenced by TRPV4 agonist GSK1016790A-elicited Ca2+ influx. Supplementation of HC-067047, a specific antagonist of TRPV4, attenuated microgravity-induced mitochondrial dysfunction, oxidative stress, and osteogenesis loss. Although TRPV4 was found localized in primary cilia and expressed at low levels in NG, microgravity-induced shortening of primary cilia led to increased TRPV4 levels and Ca2+ influx. When primary cilia were protected by miR-129-3p overexpression or supplementation with a natural flavonoid moslosooflavone, microgravity-induced increased TRPV4 expression, mitochondrial dysfunction, oxidative stress, and osteogenesis loss were all prevented. Our data revealed a new mechanism that primary cilia function as a controller for TRPV4 expression. Microgravity-induced injury on primary cilia leads to increased expression and overactive channel of TRPV4, causing intracellular Ca2+ overload and oxidative stress, and primary cilium protection could be an effective countermeasure against microgravity-induced oxidative stress and loss of osteogenic potential of osteoblasts.

Metabolic engineering of Thermoanaerobacterium aotearoense strain SCUT27 for biofuels production from sucrose and molasses.

BACKGROUND: Sucrose-rich sugarcane trash surpasses 28 million tons globally per year. Effective biorefinery systems could convert these biomasses to bioproducts, such as bioethanol from sugarcane sucrose in Brazil. Thermophilic microbes for biofuels have attracted great attention due to their higher fermentation temperature and wide substrate spectrum. However, few thermophiles using sucrose or molasses for biofuels production was reported. Thermoanaerobacterium aotearoense SCUT27 has been considered as an efficient ethanol producer, but it cannot directly utilize sucrose. In this study, various sucrose metabolic pathways were introduced and analyzed in Thermoanaerobaterium. RESULTS: The sucrose-6-phosphate hydrolase (scrB), which was from a screened strain Thermoanaerobacterium thermosaccharolyticum G3-1 was overexpressed in T. aotearoense SCUT27 and endowed this strain with the ability to utilize sucrose. In addition, overexpression of the sucrose-specific PTS system (scrA) from Clostridium acetobutylicum accelerated the sucrose transport. To strengthen the alcohols production and substrates metabolism, the redox-sensing transcriptional repressor (rex) in T. aotearoense was further knocked out. Moreover, with the gene arginine repressor (argR) deleted, the ethanologenic mutant P8S10 showed great inhibitors-tolerance and finally accumulated ~ 34 g/L ethanol (a yield of 0.39 g/g sugars) from pretreated cane molasses in 5 L tank by fed-batch fermentation. When introducing butanol synthetic pathway, 3.22 g/L butanol was produced by P8SB4 with a yield of 0.44 g alcohols/g sugars at 50℃. This study demonstrated the potential application of T. aotearoense SCUT27 for ethanol and butanol production from low cost cane molasses. CONCLUSIONS: Our work provided strategies for sucrose utilization in thermophiles and improved biofuels production as well as stress tolerances of T. aotearoense SCUT27, demonstrating the potential application of the strain for cost-effective biofuels production from sucrose-based feedstocks.