Origin of the multi-phasic quenching dynamics in the BLUF domains across the species.

Blue light using flavin (BLUF) photoreceptors respond to light via one of nature's smallest photo-switching domains. Upon photo-activation, the flavin cofactor in the BLUF domain exhibits multi-phasic dynamics, quenched by a proton-coupled electron transfer reaction involving the conserved Tyr and Gln. The dynamic behavior varies drastically across different species, the origin of which remains controversial. Here, we incorporate site-specific fluorinated Trp into three BLUF proteins, i.e., AppA, OaPAC and SyPixD, and characterize the percentages for the Wout, WinNHin and WinNHout conformations using 19F nuclear magnetic resonance spectroscopy. Using femtosecond spectroscopy, we identify that one key WinNHin conformation can introduce a branching one-step proton transfer in AppA and a two-step proton transfer in OaPAC and SyPixD. Correlating the flavin quenching dynamics with the active-site structural heterogeneity, we conclude that the quenching rate is determined by the percentage of WinNHin, which encodes a Tyr-Gln configuration that is not conducive to proton transfer.

An ultrafast phototrigger of the Trp5CN-Trp motif in a ß-hairpin peptide.

Phototriggers are useful molecular tools to initiate reactions in enzymes by light for the purpose of photoenzymatic design and mechanistic investigations. Here, we incorporated the non-natural amino acid 5-cyanotryptophan (W5CN) in a polypeptide scaffold and resolved the photochemical reaction of the W5CN-W motif using femtosecond transient UV/Vis and mid-IR spectroscopy. We identified a marker band of ∼2037 cm-1 from the CN stretch of the electron transfer intermediate W5CN·- in the transient IR measurement and found UV/Vis spectroscopic evidence for the W·+ radical at 580 nm. Through kinetic analysis, we characterized that the charge separation between the excited W5CN and W occurs in 253 ps, with a charge-recombination lifetime of 862 ps. Our study highlights the potential use of the W5CN-W pair as an ultrafast phototrigger to initiate reactions in enzymes that are not light-sensitive, making downstream reactions accessible to femtosecond spectroscopic detection.

N 6-methyladenosine modification contributes to respiratory syncytial virus infection.

Background: Respiratory syncytial virus (RSV) is the second leading cause of death due to lower respiratory tract infections. Effective prevention and treatment measures are lacking, posing a huge socioeconomic burden to the world. N 6-methyladenosine (m6A) is the most common internal modification in messenger RNA and noncoding RNA. Numerous recent studies have shown that the dysregulation of m6A modification is associated with diseases caused by pathogenic viruses. Methods: The changes in m6A modification were evaluated using m6A RNA methylation assay. The differences in gene expression levels of various m6A-modifying enzymes were observed using Quantitative Real-time PCR (qRT-PCR) during RSV infection. The autophagosomes were observed using transmission electron microscopy, and the expression of autophagy-associated protein Microtubule Associated Protein 1 Light Chain 3 Beta Ⅱ/Ⅰ (LC3B Ⅱ/Ⅰ) and Beclin1 in Human Normal Lung Epithelial Cells (BEAS-2B) cells using Western blot during RSV infection. The significantly differentially expressed genes were screened guided by bioinformatics. Their relationship with m6A-modifying enzymes was analyzed through protein-protein interaction network and expression correlation analysis. Results: The m6A abundance decreased and demethylase Fat Mass and Obesity- Associated Protein (FTO) significantly increased during RSV infection after 24 h. We also found that the DNA Damage-Inducible Transcript 3 Protein (DDIT3) level significantly increased during RSV infection after 24 h and observed autophagosomes in BEAS-2B cells. In addition, RSV infection could cause the upregulation of LC3B Ⅱ/Ⅰ and Beclin1. The expression correlation analysis showed that DDIT3 levels were positively correlated with the FTO level, and Methyltransferase Like 3 (METTL3), RNA Binding Motif Protein 15B (RBM15B), YTH Domain-Containing Family Protein 1 (YTHDF1), and levels were negatively correlated with the DDIT3 level. Conclusions: We uncovered a significant role for m6A modification during RSV infection. Also, a correlation was found between m6A and autophagy, providing new ideas for therapeutic advancements in RSV treatment.

A novel traffic accident detection method with comprehensive traffic flow features extraction.

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

Biocrusts mediate the plant community composition of dryland restoration ecosystems.

Degradation and expansion are current threats associated with drylands. During natural or artificial restoration, dryland ecosystems tend to contain a unique community, namely, biocrusts. Studies have shown that biocrusts serve multiple functions and have the potential to accelerate or inhibit degradation. However, the mechanisms by which biocrusts mediate dryland plant community structure and composition remain largely unknown. In this study, we performed a series of pot experiments under greenhouse conditions at three disturbance levels (strong, moderate and no disturbance) and at three rainfall gradients (- 50 %, normal, + 50 %) for different biocrust types. Our objective was to examine how biocrusts influence the introduction and establishment of different functional forms of plant species during the different succession stages of dryland recovery. Our results showed that biocrusts have significantly different effects on the seed germination and survival of the tested plant species. Although strong disturbances to the biocrusts and increasing rainfall alleviated the inhibitory effects of biocrusts on the perennial herb (S. glareosa) and subshrub (C. latens) species, these factors could only promote the temporary survival of these two plants in the revegetated area. These results indicate that biocrusts in revegetated systems play important sieving functions during plant species establishment. Additionally, the survival of S. viridis, a plant species with small seeds adapted to the current habitat, was promoted in the community, but two plant species that are currently unsuitable, S. glareosa and C. latens, were filtered out by the biocrusts. Our conclusions suggest that biocrusts play an ecological filtering role in plant species introduction and establishment in revegetated ecosystems and demonstrate the scientific significance of biocrusts in maintaining the health and stability of dryland ecosystems.

The nature of proton-coupled electron transfer in a blue light using flavin domain.

Proton-coupled electron transfer (PCET) is key to the activation of the blue light using flavin (BLUF) domain photoreceptors. Here, to elucidate the photocycle of the central FMN-Gln-Tyr motif in the BLUF domain of OaPAC, we eliminated the intrinsic interfering W90 in the mutant design. We integrated the stretched exponential function into the target analysis to account for the dynamic heterogeneity arising from the active-site solvation relaxation and the flexible H-bonding network as shown in the molecular dynamics simulation results, facilitating a simplified expression of the kinetics model. We find that, in both the functional wild-type (WT) and the nonfunctional Q48E and Q48A, forward PCET happens in the range of 105 ps to 344 ps, with a kinetic isotope effect (KIE) measured to be ∼1.8 to 2.4, suggesting that the nature of the forward PCET is concerted. Remarkably, only WT proceeds with an ultrafast reverse PCET process (31 ps, KIE = 4.0), characterized by an inverted kinetics of the intermediate FMNH˙. Our results reveal that the reverse PCET is driven by proton transfer via an intervening imidic Gln.

Probing Intermolecular Interactions of Amyloidogenic Fragments of SOD1 by Site-Specific Tryptophan and Its Noncanonical Derivative.

Transient amyloid intermediates are likely to be cytotoxic and play an essential role in amyloid-associated neurodegenerative diseases. Characterization of their structural and dynamic evolution is the key to elucidating the molecular mechanism of amyloid formation. Here, combining circular dichroism (CD), exciton couplet theory, and Fourier transform infrared spectroscopy with site-specific tryptophan (Trp) and its noncanonical derivative 5-cyano-tryptochan (Trp5CN), we developed a method to monitor strand-to-strand tertiary and sheet-to-sheet quaternary interactions in the aggregation cascades of an amyloidogenic fragment from protein SOD128-38 (with the sequence KVKVWGSIKGL). We found that the exciton couplet generated from the Bb band of Trp can be used as a probe for side chain interactions. Its sensitivity can be further improved by four times with the incorporation of Trp5CN. We further observed a red-shift of ∼2 cm-1 and a broadening of ∼2 cm-1 in the IR band generated from the CN stretch during the aggregation, which we attributed to the transition from a corkscrew-like structure to a cross-linked intermediate phase. We show here that the integration of optical methods with unique aromatic side chain-related probes is able to elucidate amyloid intermolecular interactions and even capture elusive transient intermediates on and off the amyloid assembling pathway.

One-pot fluorescent assay for sensitive detection of APOBEC3A activity.

We reported a one-pot fluorescence-based assay to quantitively detect A3A activity combined with cytosine deamination and uracil excision. After deamination by A3A and USER enzyme treatment, the fluorescent turn-on effect at 520 nm was observed, which can be used to evaluate the A3A activity and screen inhibitors.

The Leptinotarsa forkhead transcription factor O exerts a key function during larval-pupal-adult transition.

Forkhead box O (FoxO) protein, a major downstream transcription factor of insulin/insulin-like growth factor signaling/target of rapamycin pathway (IIS/TOR), is involved in the regulation of larval growth and the determination of organ size. FoxO also interacts with 20-hydroxyecdysone (20E) and juvenile hormone (JH) signal transduction pathways, and hence is critical for larval development in holometabolans. However, whether FoxO plays a critical role during larval metamorphosis needs to be further determined in Leptinotarsa decemlineata. We found that 20E stimulated the expression of LdFoxO. RNA interference (RNAi)-aided knockdown of LdFoxO at the third-instar stage repressed 20E signaling and reduced larval weight. Although the resultant larvae survived through the third-fourth instar ecdysis, around 70% of the LdFoxO depleted moribund beetles developmentally arrested at prepupae stage. These LdFoxO depleted beetles were completely wrapped in the larval exuviae, gradually darkened and finally died. Moreover, approximately 12% of the LdFoxO RNAi beetles died as pharate adults. Ingestion of either 20E or JH by the LdFoxO depletion beetles excessively rescued the corresponding hormonal signals, but could not alleviate larval performance and restore defective phenotypes. Therefore, FoxO plays an important role in regulation of larval-pupal-adult transformation in L. decemlineata, in addition to mediation of IIS/TOR pathway and stimulation of ecdysteroidogenesis.

A far-red emissive two-photon fluorescent probe for quantification of uracil in genomic DNA.

We report a new method for dU detection in genomic DNA combined with UNG excision and fluorescent probe labeling. UNG can remove uracil bases to introduce abasic sites, which can react with NRNO to produce intense fluorescence because of the inhibition of the PET effect. It can also cause the polymerase extension to stop to provide details of dU site information.

Correction: Small-molecule-based human genome G4 profiling reveals potential gene regulation activity.

Correction for 'Small-molecule-based human genome G4 profiling reveals potential gene regulation activity' by Weiwu Zeng et al., Chem. Commun., 2019, 55, 2269-2272, DOI: 10.1039/C8CC10052G.

Small-molecule-based human genome G4 profiling reveals potential gene regulation activity.

Small-molecule-based G4 isolation from genomic DNA has enabled the identification of a total of 51 446 PQSs (potential G-quadruplex sites). Incubating cells with PDP for 3 h to 72 h resulted in the differential expression of a variety of genes, indicating a potential function of G4s in gene regulation.

Vascular reactivity screen of Chinese medicine danhong injection identifies Danshensu as a NO-independent but PGI2-mediated relaxation factor.

Danhong injection (DHI) is the most prescribed injection form of Chinese medicine for the treatment of cardiovascular diseases such as atherosclerosis, angina pectoris, and stroke in China. However, its active components and action mechanisms remain poorly defined. We hypothesized that DHI contains active components that could prevent and restore endothelial dysfunction by improving vascular relaxation activity. DHI increased vasorelaxation in vivo and ex vivo of rat aortas. Vascular reactivity screen identified that danshensu was the major relaxation factor in DHI. DHI-mediated endothelial-dependent vasorelaxation was independent on nitric oxide/endothelial nitric oxide synthase but was via prostacyclin pathway by increasing cyclooxygenase (COX)-2 gene expression and prostacyclin production. Our results revealed a previously unknown endothelium-dependent vasorelaxation mechanism by danshensu and together with previously reported activity on ion channels of vascular smooth muscle cells, demonstrated that its dual actions contribute to a multicomponent Chinese herbal medicine that synergistically targets different pathways to achieve its well-documented cardiovascular protective effects.

Anti-inflammatory activity of baicalein in LPS-stimulated RAW264.7 macrophages via estrogen receptor and NF-κB-dependent pathways.

Baicalein has been used for many years as a popular antiviral and antibacterial in China. Recent investigations revealed that baicalein also has anti-inflammatory activities. Our results indicated that baicalein increases ERE-luciferase activity in an estrogen receptor (ER)-dependent manner when either ERα or ERß were coexpressed in Hela cells. This study examined whether baicalein exerts an anti-inflammatory effect in RAW264.7 cells through an estrogen receptor-dependent pathway and through regulation of NF-ĸB activation. In lipopolysaccharide (LPS)-induced RAW264.7 cells, baicalein exerts anti-inflammatory effects by inhibiting iNOS, COX-2, and TNF-α mRNA expression; NO production; as well as inflammatory cytokine (IL-1ß, PGE2, and TNF-α) production through an ER-dependent pathway. These effects are accompanied with the inhibition of the transcription factor NF-ĸB activation and IκBα phosphorylation. We therefore conclude that baicalein inhibits LPS-induced inflammatory cytokine production via regulation of the NF-ĸB pathway and estrogen-like activity, suggesting that it may be useful for preventing inflammation-related diseases.