Nanozyme Inhibited Sensor Array for Biothiol Detection and Disease Discrimination Based on Metal Ion-Doped Carbon Dots.

Developing highly active and sensitive nanozymes for biothiol analysis is of vital significance due to their essential roles in disease diagnosis. Herein, two metal ion-doped carbon dots (M-CDs) with high peroxidase-like activity were designed and prepared for biothiol detection and identification through the colorimetric sensor array technique. The two M-CDs can strongly catalyze the decomposition of H2O2, accompanied by color changes of 3,3',5,5'-tetramethylbenzidine (TMB) from colorless to blue, indicating peroxidase-mimicking activities of M-CDs. Compared with pure carbon dots (CDs), M-CDs exhibited enhanced peroxidase-like activity owing to the synergistic effect between metal ions and CDs. However, due to the strong binding affinity between biothiols and metal ions, the catalytic activities of M-CDs could be inhibited by different biothiols to diverse degrees. Therefore, using TMB as a chromogenic substrate in the presence of H2O2, the developed colorimetric sensor array can form differential fingerprints for the three most important biothiols (i.e., cysteine (Cys), homocysteine (Hcy), and glutathione (GSH)), which can be accurately discriminated through pattern recognition methods (i.e., hierarchical clustering analysis (HCA) and principal component analysis (PCA)) with a detection limit of 5 nM. Moreover, the recognition of a single biothiol with various concentrations and biothiol mixtures was also realized. Furthermore, actual samples such as cells and sera can also be well distinguished by the as-fabricated sensor array, demonstrating its potential in disease diagnosis.

DNA Nanoribbon for Efficient Anti-miRNA Peptide Nucleic Acid Delivery and Synergistic Enhancement of Cancer Cell Apoptosis.

Antisense peptide nucleic acid (asPNA), an effective antisense drug, has been employed as a gene therapy agent and a useful tool in molecular biology. Gaining control over the delivery of asPNA to target tissues has been a major hindrance to its wide application in clinical practice. A simple and efficient DNA nanoribbon (DNR)-based drug delivery process has been designed in this study that releases the asPNA agent to inhibit oncogenic microRNAs (miRNAs). Furthermore, we demonstrated how the AS1411 aptamer that binds nucleolin on the cell membranes works as a control mechanism capable of identifying target cancer cells and enhancing the enrichment capacity of DNR. With the biodegradability of DNR, we can efficiently initiate the release of asPNA into the cytoplasm, particularly targeting the intended miR-21 and synergistically increasing programmed cell death 4 (PDCD4) expression to enhance cell apoptosis. We assume that this well-defined delivery mechanism will aid in designing antisense site-specific treatments for various diseases, including cancer.

Peroxidase-Mimetic Copper-Doped Carbon-Dots for Oxidative Stress-Mediated Broad-Spectrum and Efficient Antibacterial Activity.

Carbon dots (CDs) have recently emerged as antibacterial agents and have attracted considerable attention owing to their fascinating merits of small size, facile fabrication, and surface functionalization. Most of them are involved in external light activation or hybridization with other functional nanomaterials. Herein, we present peroxidase-like Cu-doped CDs (Cu-CDs) for in vitro antibacterial applications. The unique peroxidase-mimicking property of the Cu-CDs was demonstrated by tetramethylbenzidine chromogenic assay, electron paramagnetic resonance spectra, and hydroxy radical probe. Escherichia coli and Staphylococcus aureus were chosen as representative gram-negative/positive models against which Cu-CDs exhibited superior antimicrobial activity even at a dosage down to 5 µg/mL. A possible mechanism of action was that the Cu-CDs triggered a catalytic redox reaction of endogenous H2 O2 and glutathione depletion in the bacteria cells, with subsequent oxidative stress and membrane disruption. This work provides a new strategy for the design of microenvironment-responsive antimicrobial nano-agents.

Expression of ribosomal protection protein RppA is regulated by a ribosome-dependent ribo-regulator and two mistranslation products.

Gene expression is tightly controlled by transcription factors and RNA regulatory elements, including trans-acting small RNAs, cis-regulatory riboswitches and ribosome-dependent ribo-regulators. In the present study, we demonstrated that a ribosome-dependent ribo-regulator and two mistranslation products co-regulate rppA (encoding a ribosomal protection protein) expression in Bacillus thuringiensis BMB171. The leader RNA of the rppA gene controls rppA expression via translation of leader ORF1 resident in its sequence. In the presence of chloramphenicol, a +1 frameshift product (ORF2) and a stop codon readthrough product (ORF3) of ORF1 emerged. ORF3 exerted a negative effect on rppA expression. By contrast, the ORF2 promoted rppA expression. The regulation mode identified in the present study will lead to a deeper understanding of bacterial gene expression.

Scavenger receptor-C acts as a receptor for Bacillus thuringiensis vegetative insecticidal protein Vip3Aa and mediates the internalization of Vip3Aa via endocytosis.

The vegetative insecticidal proteins (Vip), secreted by many Bacillus thuringiensis strains during their vegetative growth stage, are genetically distinct from known insecticidal crystal proteins (ICPs) and represent the second-generation insecticidal toxins. Compared with ICPs, the insecticidal mechanisms of Vip toxins are poorly understood. In particular, there has been no report of a definite receptor of Vip toxins to date. In the present study, we identified the scavenger receptor class C like protein (Sf-SR-C) from the Spodoptera frugiperda (Sf9) cells membrane proteins that bind to the biotin labeled Vip3Aa, via the affinity magnetic bead method coupled with HPLC-MS/MS. We then certified Vip3Aa protoxin could interact with Sf-SR-C in vitro and ex vivo. In addition, downregulation of SR-C expression in Sf9 cells and Spodoptera exigua larvae midgut reduced the toxicity of Vip3Aa to them. Coincidently, heterologous expression of Sf-SR-C in transgenic Drosophila midgut significantly enhanced the virulence of Vip3Aa to the Drosophila larvae. Moreover, the complement control protein domain and MAM domain of Sf-SR-C are involved in the interaction with Vip3Aa protoxin. Furthermore, endocytosis of Vip3Aa mediated by Sf-SR-C correlates with its insecticidal activity. Our results confirmed for the first time that Sf-SR-C acts as a receptor for Vip3Aa protoxin and provides an insight into the mode of action of Vip3Aa that will significantly facilitate the study of its insecticidal mechanism and application.

The opposite roles of agdA and glaA on citric acid production in Aspergillus niger.

Citric acid is produced by an industrial-scale process of fermentation using Aspergillus niger as a microbial cell factory. However, citric acid production was hindered by the non-fermentable isomaltose and insufficient saccharification ability in A. niger when liquefied corn starch was used as a raw material. In this study, A. niger TNA 101ΔagdA was constructed by deletion of the α-glucosidase-encoding agdA gene in A. niger CGMCC 10142 genome using Agrobacterium tumefaciens-mediated transformation. The transformants A. niger OG 1, OG 17, and OG 31 then underwent overexpression of glucoamylase in A. niger TNA 101ΔagdA. The results showed that the α-glucosidase activity of TNA 101ΔagdA was decreased by 62.5 % compared with CGMCC 10142, and isomaltose was almost undetectable in the fermentation broth. The glucoamylase activity of the transformants OG 1 and OG 17 increased by 34.5 and 16.89 % compared with that of TNA 101ΔagdA, respectively. In addition, for the recombinants TNA 101ΔagdA, OG 1 and OG 17, there were no apparent defects in the growth development. Consequently, in comparison with CGMCC 10142, TNA 101ΔagdA and OG 1 decreased the residual reducing sugar by 52.95 and 88.24 %, respectively, and correspondingly increased citric acid production at the end of fermentation by 8.68 and 16.87 %. Citric acid production was further improved by decreasing the non-fermentable residual sugar and increasing utilization rate of corn starch material in A. niger. Besides, the successive saccharification and citric acid fermentation processes were successfully integrated into one step.

Inhibition of oxidative phosphorylation for enhancing citric acid production by Aspergillus niger.

BACKGROUND: The spore germination rate and growth characteristics were compared between the citric acid high-yield strain Aspergillus niger CGMCC 5751 and A. niger ATCC 1015 in media containing antimycin A or DNP. We inferred that differences in citric acid yield might be due to differences in energy metabolism between these strains. To explore the impact of energy metabolism on citric acid production, the changes in intracellular ATP, NADH and NADH/NAD+ were measured at various fermentation stages. In addition, the effects of antimycin A or DNP on energy metabolism and citric acid production was investigated by CGMCC 5751. RESULTS: By comparing the spore germination rate and the extent of growth on PDA plates containing antimycin A or DNP, CGMCC 5751 was shown to be more sensitive to antimycin A than ATCC 1015. The substrate-level phosphorylation of CGMCC 5751 was greater than that of ATCC 1015 on PDA plates with DNP. DNP at tested concentrations had no apparent effect on the growth of CGMCC 5751. There were no apparent effects on the mycelial morphology, the growth of mycelial pellets or the dry cell mass when 0.2 mg L(-1) antimycin A or 0.1 mg L(-1) DNP was added to medium at the 24-h time point. The concentrations of intracellular ATP, NADH and NADH/NAD+ of CGMCC 5751 were notably lower than those of ATCC 1015 at several fermentation stages. Moreover, at 96 h of fermentation, the citric acid production of CGMCC 5751 reached up to 151.67 g L(-1) and 135.78 g L(-1) by adding 0.2 mg L(-1) antimycin A or 0.1 mg L(-1) DNP, respectively, at the 24-h time point of fermentation. Thus, the citric acid production of CGMCC 5751 was increased by 19.89% and 7.32%, respectively. CONCLUSIONS: The concentrations of intracellular ATP, NADH and NADH/NAD+ of the citric acid high-yield strain CGMCC 5751 were notably lower than those of ATCC 1015. The excessive ATP has a strong inhibitory effect on citric acid accumulation by A. niger. Increasing NADH oxidation and appropriately reducing the concentration of intracellular ATP can accelerate glycolysis and the TCA cycle to enhance citric acid yield.

MTHFR and MTRR Genetic Polymorphism of Methotrexate Therapy Outcomes in Early Rheumatoid Arthritis.

Purpose: Methotrexate (MTX) is used as an anchor drug for the treatment of rheumatoid arthritis (RA) and there may be differences in drug action between genotypes. The purpose of this study was to investigate the relationship between clinical efficacy response and disease activity of MTX monotherapy with methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) polymorphisms. Patients and Methods: In the study, a population of 32 patients in East China with early RA fulfilling the diagnostic standards of the American College of Rheumatology (ACR) were enrolled, all of them received MTX monotherapy. Genotyping of patients MTHFR C677T and A1298C, MTRR A66G using tetra-primer ARMS-PCR method and sanger sequencing to verify its accuracy. Results: The distribution of three polymorphic genotypes that were studied is in accordance with the Hardy-Weinberg genetic equilibrium. The patient pathology variables smoke (OR = 0.088, P = 0.037), drink alcohol (OR = 0.039, P = 0.016) and males (OR = 0.088, P = 0.037) were significantly associated with non-response to MTX. Genotype, allele distribution and genetic statistical models were not found to be related to MTX treatment response and disease activity in both the response groups and non-response groups. Conclusion: Our findings suggest that the MTHFR C677T, MTHFR A1298C and MTRR A66G polymorphisms may not predict MTX clinical treatment response and disease activity in patients with early RA. The study revealed that smoke, alcohol, and males were possible influential factors for MTX non-response.

Single-cell RNA-seq of UVB-radiated skin reveals landscape of photoaging-related inflammation and protection by vitamin D.

Ultraviolet rays are a potential threat to nature. It can accelerate skin aging by causing skin damage, cell infiltration, and inflammation. The present study investigated UV-irradiated mouse skin through single-cell sequencing. We observed that UV-irradiated mouse skin mainly induced inflammation of fibroblasts and demonstrated differential gene expression. Cell prediction revealed the significance of macrophages in tissue repair. Furthermore, cell culture studies substantiated vitamin D-induced inhibitory effect on skin inflammation. These findings thus indicate some references for skin photo-protection.

Single-cell sequencing technologies in bladder cancer research: Applications and challenges.

Bladder cancer is among the most common malignant tumors with highly heterogeneous molecular characteristics. Despite advancements of the available therapeutic options, several bladder cancer patients exhibit unsatisfactory clinical outcomes. The lack of specific biomarkers for effective targeted therapy or immunotherapy remains a major obstacle in treating bladder cancer. The rapid development of single-cell techniques is transforming our understanding of the intra-tumoral heterogeneity, thereby providing us with a powerful high-throughput sequencing tool that can reveal tumorigenesis, progression, and invasion in bladder tumors. In this review, we summarise and discuss how single-cell sequencing technologies have been applied in bladder cancer research, to advance our collective knowledge on the heterogeneity of bladder tumor cells, as well as to provide new insights into the complex ecosystem of the tumor microenvironment. The application of single-cell approaches also uncovers the therapeutic resistance mechanism in bladder cancer and facilitates the detection of urinary-exfoliated tumor cells. Moreover, benefiting from the powerful technical advantages of single-cell techniques, several key therapeutic targets and prognostic models of bladder cancer have been identified. It is hoped that this paper can provide novel insights into the precision medicine of bladder cancer.

NupR Responding to Multiple Signals Is a Nucleoside Permease Regulator in Bacillus thuringiensis BMB171.

Nucleoside transport is essential for maintaining intracellular nucleoside and nucleobase homeostasis for living cells. Here, we identified an uncharacterized GntR/HutC family transcriptional regulator, NagR2, renamed NupR (nucleoside permease regulator), that mainly controls nucleoside transport in the Bacillus thuringiensis BMB171 strain. The deletion or overexpression of nupR affected the bacteria's utilization of guanosine, adenosine, uridine, and cytidine rather than thymidine. We further demonstrated that zinc ion is an effector for the NupR, dissociating NupR from its target DNA. Moreover, the expression of nupR is inhibited by NupR, ComK, and PurR, while it is promoted by CcpA. Also, a purine riboswitch located in its 5' noncoding region influences the expression of nupR. Guanine is the ligand of the riboswitch, reducing the expression of nupR by terminating the transcription of nupR in advance. Hence, our results reveal an exquisite regulation mechanism enabling NupR to respond to multiple signals, control genes involved in nucleoside transport, and contribute to nucleoside substance utilization. Overall, this study provides essential clues for future studies exploring the function of the NupR homolog in other bacteria, such as Bacillus cereus, Bacillus anthracis, Klebsiella pneumoniae, and Streptococcus pneumoniae. IMPORTANCE The transport of nucleosides and their homeostasis within the cell are essential for growth and proliferation. Here, we have identified a novel transcription factor, NupR, which, to our knowledge, is the first GntR family transcription factor primarily involved in the regulation of nucleoside transport. Moreover, responding to diverse intracellular signals, NupR regulates nucleoside transport. It is vital for utilizing extracellular nucleosides and maintaining intracellular nucleoside homeostasis. NupR may also be involved in other pathways such as pH homeostasis, molybdenum cofactor biosynthesis, nitrate metabolism, and transport. In addition, nucleosides have various applications, such as antiviral drugs. Thus, the elucidation of the transport mechanism of nucleosides could be helpful for the construction of engineered strains for nucleoside production.

Mitochondria and Lysosomes Participate in Vip3Aa-Induced Spodoptera frugiperda Sf9 Cell Apoptosis.

Vip3Aa, a soluble protein produced by certain Bacillus thuringiensis strains, is capable of inducing apoptosis in Sf9 cells. However, the apoptosis mechanism triggered by Vip3Aa is unclear. In this study, we found that Vip3Aa induces mitochondrial dysfunction, as evidenced by signs of collapse of mitochondrial membrane potential, accumulation of reactive oxygen species, release of cytochrome c, and caspase-9 and -3 activation. Meanwhile, our results indicated that Vip3Aa reduces the ability of lysosomes in Sf9 cells to retain acridine orange. Moreover, pretreatment with Z-Phe-Tyr-CHO (a cathepsin L inhibitor) or pepstatin (a cathepsin D inhibitor) increased Sf9 cell viability, reduced cytochrome c release, and decreased caspase-9 and -3 activity. In conclusion, our findings suggested that Vip3Aa promotes Sf9 cell apoptosis by mitochondrial dysfunction, and lysosomes also play a vital role in the action of Vip3Aa.

Fibroblast Growth Factor Receptor, a Novel Receptor for Vegetative Insecticidal Protein Vip3Aa.

Vegetative insecticidal proteins (Vips), which are secreted by some Bacillus thuringiensis strains during vegetative growth, exhibit high virulence to many pests. Vip3A proteins have been used commercially both in some bio-insecticides and in transgenic crops; however, compared with insecticidal crystal proteins, the mechanism of action of Vip3A is still unclear. In this work, we indicated that the fibroblast growth factor receptor-like protein (Sf-FGFR) from the membrane of Sf9 cells could bind to Vip3Aa. The interaction between Vip3Aa and Sf-FGFR was confirmed by pull-down assays and dot blotting experiment in vitro. The binding affinity between Vip3Aa and extracellular regions of Sf-FGFR (GST-FGFR-N) was determined by microscale thermophoresis assay (MST). Moreover, Vip3Aa-Flag could be co-immunoprecipitated with Sf-FGFR-V5 ex vivo. Furthermore, knockdown of Sf-FGFR gene in Sf9 cells resulted in reducing the mortality of those cells to Vip3Aa. In summary, our data indicated that Sf-FGFR is a novel receptor for Vip3Aa.

NagRBt Is a Pleiotropic and Dual Transcriptional Regulator in Bacillus thuringiensis.

NagR, belonging to the GntR/HutC family, is a negative regulator that directly represses the nagP and nagAB genes, which are involved in GlcNAc transport and utilization in Bacillus subtilis. Our previous work confirmed that the chitinase B gene (chiB) of Bacillus thuringiensis strain Bti75 is also negatively controlled by YvoABt, the ortholog of NagR from B. subtilis. In this work, we investigated its regulatory network in Bti75 and found that YvoABt is an N-acetylglucosamine utilization regulator primarily involved in GlcNAc catabolism; therefore YvoABt is renamed as NagRBt. The RNA-seq data revealed that 27 genes were upregulated and 14 genes were downregulated in the ΔnagR mutant compared with the wild-type strain. The regulon (exponential phase) was characterized by RNA-seq, bioinformatics software, electrophoretic mobility shift assays, and quantitative real-time reverse transcription PCR. In the Bti75 genome, 19 genes that were directly regulated and 30 genes that were indirectly regulated by NagRBt were identified. We compiled in silico, in vitro, and in vivo evidence that NagRBt behaves as a repressor and activator to directly or indirectly influence major biological processes involved in amino sugar metabolism, nucleotide metabolism, fatty acid metabolism, phosphotransferase system, and the Embden-Meyerhof-Parnas pathway.

Complete genome sequence of Bacillus thuringiensis L-7601, a wild strain with high production of melanin.

Bacillus thuringiensis L-7601 (B. thuringiensis L-7601), belonging to Bacillus thuringiensis subsp. dendrolimus serotype H4a4b, is a wild-type strain which has the ability to produce melanin during the exponential phase of growth. The melanin produced is an excellent UV protective agent for the crystal insecticidal proteins. Here, we report the complete genome of B. thuringiensis L-7601 including one 5,790,408 bp chromosome and three plasmids. 6,519 CDSs and 150 RNA genes, including 106 tRNA genes, 39 rRNA genes and 5 ncRNA genes, were identified from the whole genome. In addition, our results indicated that homogentisic acid pathway is the melanogenic pathway in B. thuringiensis and accumulation of melanin is the consequence of hmgA frameshift mutant.