Access tunnel engineering to optimize the catalytic cycle of carbohydrate hydrolases with buried active site

The active site of many enzymes is buried inside the protein core and is connected with the surrounding solvent by access tunnels. An emerging approach to optimize these enzymes properties is the engineering of structural features governing the exchange of ligands between the active sites and bulk solvent. However, it is still challenging to redesign the access tunnels of enzymes catalyzing biopolymers like carbohydrate hydrolases because of the extremely complicated substrate structure. In this study, structure-guided saturated mutagenesis was performed to reconstruct all three access tunnels of xylanase S7-xyl from Bacillus halodurans S7, which results in a mutant 254-RL1 with 3.4-fold increase in specific activity. Structural comparison and kinetic analysis revealed that products egress is the rate-limiting step in the catalytic cycle of S7-xyl. The products release tunnel in S7-xyl was experimentally validated, and not the tunnel radius but the length determining the products release efficiency. Application assessment showed that relieving the inhibition of reducing sugars on mutant 254-RL1 could accelerate the hydrolysis efficiency of cellulase on different pretreated lignocellulose materials, representing a good candidate in enzyme cocktails for lignocellulose biodegradation. In addition, the same strategy was successfully utilized to improve the specific activities of three other xylanases with buried active site, suggesting the general application of tunnel engineering to optimize carbohydrate hydrolases with buried active site

Effect and mechanism of chlorogenic acid on cognitive dysfunction in mice by lipopolysaccharide-induced neuroinflammation

BackgroundNeuroinflammation is an important factor causing numerous neurodegenerative pathologies. Inflammation can lead to abnormal neuronal structure and function and even death, followed by cognitive dysfunction. There is growing evidence that chlorogenic acid has anti-inflammatory effects and immunomodulatory activity.PurposeThe aim of this study was to elucidate the potential targets and molecular mechanisms of chlorogenic acid in the treatment of neuroinflammation.MethodsWe used the lipopolysaccharide-induced neuroinflammation mouse model and the lipopolysaccharide-stimulated BV-2 cells in vitro model. Behavioral scores and experiments were used to assess cognitive dysfunction in mice. HE staining and immunohistochemistry were used to assess neuronal damage in the mouse brain. Immunofluorescence detected microglia polarization in mouse brain. Western blot and flow cytometry detected the polarization of BV-2 cells. The migration of BV-2 cells was detected by wound healing assay and transwell assay. Potential targets for chlorogenic acid to exert protective effects were predicted by network pharmacology. These targets were then validated using molecular docking and experiments.ResultsThe results of in vivo experiments showed that chlorogenic acid had an obvious ameliorating effect on neuroinflammation-induced cognitive dysfunction. We found that chlorogenic acid was able to inhibit BV-2 cells M1 polarization and promote BV-2 cells M2 polarization in vitro while also inhibiting the abnormal migration of BV-2 cells. Based on the network pharmacology results, we identified the TNF signaling pathway as a key signaling pathway in which chlorogenic acid exerts anti-neuroinflammatory effects. Among them, Akt1, TNF, MMP9, PTGS2, MAPK1, MAPK14, and RELA are the core targets for chlorogenic acid to function.ConclusionChlorogenic acid can inhibit microglial polarization toward the M1 phenotype and improve neuroinflammation-induced cognitive dysfunction in mice by modulating these key targets in the TNF signaling pathway

Association between the PDE4D gene and ischaemic stroke in the Chinese Han population

A B S T R A C T Recent findings suggests that PDE4D (gene encoding phosphodiesterase 4D) is a stroke-related gene in the Icelandic population, but it is still very controversial as to whether it is a susceptible gene for stroke in other populations. In the present study, we attempted to explore the role of the gene in the pathogenesis of stroke in the Chinese Han population of eastern China. A total of 649 ischaemic stroke patients and 761 unrelated control individuals with no history of stroke or transient ischaemic attack were examined in a case-control study. Four SNPs (single nucleotide polymorphisms) rs152312 (C/T), SNP56 (A/T), SNP83 (C/T) and SNP87 (C/T) with a minor allele frequency over 5 % were genotyped and the corresponding haplotypes were constructed. In an analysis of the combined cardiogenic and carotid stroke group, both the allele (P = 0.0060) and genotype (P = 0.0160) frequencies between cases and controls at SNP83 showed significant differences. However, no difference in haplotype frequencies was observed between cases and controls at rs152312 and SNP56. In the analysis of the small-artery-occlusive stroke group, no difference in allele or genotype frequencies was observed at any marker between cases and controls; the global haplotype frequency in rs152312 and SNP56 had a significant difference between cases and controls (P = 0.0162); the frequency of haplotype C-A was higher in cases than in controls (P = 0.0122). In conclusion, our present findings show that polymorphisms in the PDE4D gene are associated with an increased risk of ischaemic stroke in the Chinese Han population. The present study adds further support to the role of PDE4D in stroke

Artificial trans-encoded small non-coding RNAs specifically silence the selected gene expression in bacteria

Recently, many small non-coding RNAs (sRNAs) with important regulatory roles have been identified in bacteria. As their eukaryotic counterparts, a major class of bacterial trans-encoded sRNAs acts by basepairing with target mRNAs, resulting in changes in translation and stability of the mRNA. RNA interference (RNAi) has become a powerful gene silencing tool in eukaryotes. However, such an effective RNA silencing tool remains to be developed for prokaryotes. In this study, we described first the use of artificial trans-encoded sRNAs (atsRNAs) for specific gene silencing in bacteria. Based on the common structural characteristics of natural sRNAs in Gram-negative bacteria, we developed the designing principle of atsRNA. Most of the atsRNAs effectively suppressed the expression of exogenous EGFP gene and endogenous uidA gene in Escherichia coli. Further studies demonstrated that the mRNA base pairing region and AU rich Hfq binding site were crucial for the activity of atsRNA. The atsRNA-mediated gene silencing was Hfq dependent. The atsRNAs led to gene silencing and RNase E dependent degradation of target mRNA. We also designed a series of atsRNAs which targeted the toxic genes in Staphyloccocus aureus, but found no significant interfering effect. We established an effective method for specific gene silencing in Gram-negative bacteria

Antibiofilm Activity of an Exopolysaccharide from Marine Bacterium Vibrio sp. QY101

Bacterial exopolysaccharides have always been suggested to play crucial roles in the bacterial initial adhesion and the development of complex architecture in the later stages of bacterial biofilm formation. However, Escherichia coli group II capsular polysaccharide was characterized to exert broad-spectrum biofilm inhibition activity. In this study, we firstly reported that a bacterial exopolysaccharide (A101) not only inhibits biofilm formation of many bacteria but also disrupts established biofilm of some strains. A101 with an average molecular weight of up to 546 KDa, was isolated and purified from the culture supernatant of the marine bacterium Vibrio sp. QY101 by ethanol precipitation, iron-exchange chromatography and gel filtration chromatography. High performance liquid chromatography traces of the hydrolyzed polysaccharides showed that A101 is primarily consisted of galacturonic acid, glucuronic acid, rhamnose and glucosamine. A101 was demonstrated to inhibit biofilm formation by a wide range of Gram-negative and Gram-positive bacteria without antibacterial activity. Furthermore, A101 displayed a significant disruption on the established biofilm produced by Pseudomonas aeruginosa, but not by Staphylococcus aureus. Importantly, A101 increased the aminoglycosides antibiotics' capability of killing P. aeruginosa biofilm. Cell primary attachment to surfaces and intercellular aggregates assays suggested that A101 inhibited cell aggregates of both P. aeruginosa and S. aureus, while the cell-surface interactions inhibition only occurred in S. aureus, and the pre-formed cell aggregates dispersion induced by A101 only occurred in P. aeruginosa. Taken together, these data identify the antibiofilm activity of A101, which may make it potential in the design of new therapeutic strategies for bacterial biofilm-associated infections and limiting biofilm formation on medical indwelling devices. The found of A101 antibiofilm activity may also promote a new recognition about the functions of bacterial exopolysaccharides

The consequences of observing the government-citizen social media interactions during the public health campaign: Effects of comments’ emotions and replying agents

Updated on 27-Aug-2023 This project is funded by a University-level peer-reviewed interdisciplinary funding scheme (the project information is anonymized here for peer review). The full funding information will be disclosed when manuscripts derived from this project are accepted for publication. The government and public health institutions use publicly available social media pages to promote public health campaigns. When such practice helps facilitate the timely communication of policy matters from the government campaign organizers and encourages engagement with the social media users, whether and to what extent the campaign organizers should directly interact (such as replying) with the commenters remains a pressing question. Supporters argue that direct and active interactions with the netizens will create a responsive and competent image of the campaign organizers. In contrast, skeptics say that it would be time-consuming and unrealistic for the campaign organizers to reply to all the comments, and any improper replies will generate unintended, negative consequences. Suppose the campaign organizers decide to interact with the commenters on the open social media pages. In that case, such interactions will be available for all the readers. They would inevitably trigger effects, not only to the commenters whose comments are replied to but also to the massive invisible online users who attend to the pages. Informed by the framework of masspersonal communication (O’Sullivan & Carr, 2018), the present study examines how such public-personal interactions on campaign organizers’ social media pages—the campaign organizers’ directly replying to users’ inquiries as comments to a campaign message—will influence observing audiences’ attitudinal and behavioral consequences, such as the perceived response quality, organizers’ credibility, engagement with the message, and other pro-campaign behavioral outcomes. Campaign organizers are especially making public communications on social media pages; a pressing challenge is whether and how to address users’ nasty comments because these comments will deteriorate observing audiences’ appraisal of the campaign organizers, known as the nasty effect. The campaign organizers’ replies to different emotions of the comments are also likely to produce different consequences. On the other hand, the emergence of social bots will help campaign organizers automatically reply to a large number of users’ comments. Different replying agents might cast different impacts on the observing audiences. Against these backdrops, the present project explores how comment emotions and replying agents in the government-citizen interaction will influence observing audiences’ (i.e., people who are watching an interaction between a commenter and its reply from the governmental institution) consequences during a public health campaign launched by the government via its public health institutions. It implements a survey experiment using a 2 (commenter’s emotions: friendly versus frustrated) x 3 (the replying agent: an AI-powered social bot versus a human social media editor versus an AI-assisted social media editor) factorial design