Most known cellulase‐associated carbohydrate‐binding modules (CBMs) are attached to the N‐ or C‐terminus of the enzyme or are expressed separately and assembled into multi‐enzyme complexes (for ...example to form cellulosomes), rather than being an insertion into the catalytic domain. Here, by solving the crystal structure, it is shown that MtGlu5 from Meiothermus taiwanensis WR‐220, a GH5‐family endo‐β‐1,4‐glucanase (EC 3.2.1.4), has a bipartite architecture consisting of a Cel5A‐like catalytic domain with a (β/α)8 TIM‐barrel fold and an inserted CBM29‐like noncatalytic domain with a β‐jelly‐roll fold. Deletion of the CBM significantly reduced the catalytic efficiency of MtGlu5, as determined by isothermal titration calorimetry using inactive mutants of full‐length and CBM‐deleted MtGlu5 proteins. Conversely, insertion of the CBM from MtGlu5 into TmCel5A from Thermotoga maritima greatly enhanced the substrate affinity of TmCel5A. Bound sugars observed between two tryptophan side chains in the catalytic domains of active full‐length and CBM‐deleted MtGlu5 suggest an important stacking force. The synergistic action of the catalytic domain and CBM of MtGlu5 in binding to single‐chain polysaccharides was visualized by substrate modeling, in which additional surface tryptophan residues were identified in a cross‐domain groove. Subsequent site‐specific mutagenesis results confirmed the pivotal role of several other tryptophan residues from both domains of MtGlu5 in substrate binding. These findings reveal a way to incorporate a CBM into the catalytic domain of an existing enzyme to make a robust cellulase.
A unique endoglucanase with a carbohydrate‐binding module inserted in the middle of the catalytic domain has been characterized structurally and functionally, providing insights into the mode of action responsible for its enhanced catalytic performance.
Honeysuckle (
Thunb) is a traditional Chinese medicine (TCM) with an antipathogenic activity. MicroRNAs (miRNAs) are small non-coding RNA molecules that are ubiquitously expressed in cells. ...Endogenous miRNA may function as an innate response to block pathogen invasion. The miRNA expression profiles of both mice and humans after the ingestion of honeysuckle were obtained. Fifteen overexpressed miRNAs overlapped and were predicted to be capable of targeting three viruses: dengue virus (DENV), enterovirus 71 (EV71) and SARS-CoV-2. Among them,
was examined to be capable of targeting the EV71 RNA genome by reporter assay and Western blotting. Moreover, honeysuckle-induced
suppression of EV71 RNA and protein expression as well as viral replication were investigated both in vitro and in vivo. We demonstrated that
targeted EV71 at the predicted sequences using luciferase reporter plasmids as well as two infectious replicons (pMP4-y-5 and pTOPO-4643). The suppression of EV71 replication and viral load was demonstrated in two cell lines by luciferase activity, RT-PCR, real-time PCR, Western blotting and plaque assay. Furthermore, EV71-infected suckling mice fed honeysuckle extract or inoculated with
showed decreased clinical scores and a prolonged survival time accompanied with decreased viral RNA, protein expression and virus titer. The ingestion of honeysuckle attenuates EV71 replication and related pathogenesis partially through the upregulation of
expression both in vitro and in vivo. Our previous report and the current findings imply that both honeysuckle and upregulated
can execute a suppressive function against the replication of DENV and EV71. Taken together, this evidence indicates that honeysuckle can induce the expression of
and that this miRNA as well as 11 other miRNAs have great potential to prevent and suppress EV71 replication.
Display omitted
•Mechanism between IGFBPrP1 and autophagy in liver fibrogenesis is poorly understood.•IGFBPrP1 promoted autophagy and contributed to hepatic stellate cell activation.•H19 is an ...autophagy promoter to accelerate IGFBPrP1-induced autophagy.•Mutual regulation occurred between H19 and PI3K/AKT/mTOR pathway in IGFBPrP1-induced hepatic stellate cells activation.•New insights for further studies on the prevention and treatment of liver fibrosis.
Our previous study found that insulin-like growth factor binding protein-associated protein (IGFBPrP1) drives hepatic stellate cells (HSCs) activation, and IGFBPrP1 and transforming growth factor β1 (TGFβ1) likely interact with each other to promote HSCs activation. TGFβ1 reportedly promotes autophagy and contributes to HSCs activation; however, the mechanism between IGFBPrP1 and autophagy in liver fibrogenesis is yet unknown. Moreover, long noncoding RNA (lncRNA) H19 participates in autophagy regulation and plays a crucial function in liver fibrosis.
To define the relationship between IGFBPrP1 and autophagy and the role of H19 in IGFBPrP1-induced hepatic fibrosis.
IGFBPrP1 and autophagy were detected in bile duct ligation (BDL)-induced hepatic fibrosis. Adenovirus-mediated IGFBPrP1 was transfected into mouse liver and JS-1 cells with or without LY294002 or rapamycin to examine the effects of IGFBPrP1 on HSCs activation and autophagy as well as the PI3K/AKT/mTOR pathway. lncRNA H19 in liver fibrosis tissues and JS-1 cells induced by IGFBPrP1 were detected, then autophagy and HSCs activation level were detected in JS-1 cells by IGFBPrP1 with H19 overexpression or knowdown.
IGFBPrP1 expression and autophagy level were concomitantly increased in liver tissue with BDL-induced hepatic fibrosis. Furthermore, we found that IGFBPrP1 stimulated autophagy and HSCs activation in vivo and in vitro, and PI3K/AKT/mTOR signaling pathway was involved in the regulation of autophagy by IGFBPrP1. In addition, H19 promoted autophagy by interacting with the PI3K/AKT/mTOR pathway in IGFBPrP1-induced HSCs activation.
IGFBPrP1 promoted autophagy and contributed to HSCs activation via mutual regulation between H19 and the PI3K/AKT/mTOR pathway.
Automatic program repair is a research hotspot in recent years and has made some progress.Most of the existing automatic program repair methods use the test suite to validate patch ...correctness.However, using the test suite to validate a large number of candidate patches will not only bring huge costs, but also lead to the overfitting problem of patches.Therefore, how to improve the efficiency of patch validation and effectively validate patch correctness has become an urgent problem.In order to reduce the cost and improve the patch accuracy, this paper proposes an approach combining two embedding techniques to validate patch correctness.Firstly, this approach uses Doc2 Vec model to calculate the similarity between the patch and the error code, then it uses the classifier based on BERT model to filter out the error patches from the patches screened by the similarity.To evaluate the effectiveness of this approach, experiments are carried out based on five open source Java benchmarks.Experimental results show th
Previous research suggested that insulin-like growth factor binding protein related protein 1 (IGFBPrP1), as a novel mediator, contributes to hepatic fibrogenesis. Matrix metalloproteinases (MMP) and ...tissue inhibitors of metalloproteinases (TIMP) play an essential role in hepatic fibrogenesis by regulating homeostasis and remodeling of the extracellular matrix (ECM). However, the interaction between IGFBPrP1 and MMP/TIMP is not clear. The present study was to knockdown IGFBPrP1 to investigate the correlation between IGFBPrP1 and MMP/TIMP in hepatic fibrosis.
Hepatic fibrosis was induced by thioacetamide (TAA) in mice. Knockdown of IGFBPrP1 expression by ultrasound-targeted microbubble destruction-mediated CMB-shRNA-IGFBPrP1 delivery, or inhibition of the Hedgehog (Hh) pathway by cyclopamine treatment, was performed in TAA-induced liver fibrosis mice. Hepatic fibrosis was determined by hematoxylin and eosin and Sirius red staining. Hepatic expression of IGFBPrP1, α-smooth muscle actin (α-SMA), transforming growth factor β 1 (TGFβ1), collagen I, MMPs/TIMPs, Sonic Hedgehog (Shh), and glioblastoma family transcription factors (Gli1) were investigated by immunohistochemical staining and Western blotting analysis.
We found that hepatic expression of IGFBPrP1, TGFβ1, α-SMA, and collagen I were increased longitudinally in mice with TAA-induced hepatic fibrosis, concomitant with MMP2/TIMP2 and MMP9/TIMP1 imbalance and Hh pathway activation. Knockdown of IGFBPrP1 expression, or inhibition of the Hh pathway, reduced the hepatic expression of IGFBPrP1, TGFβ1, α-SMA, and collagen I and re-established MMP2/TIMP2 and MMP9/TIMP1 balance.
Our findings suggest that IGFBPrP1 knockdown attenuates liver fibrosis by re-establishing MMP2/TIMP2 and MMP9/TIMP1 balance, concomitant with the inhibition of hepatic stellate cell activation, down-regulation of TGFβ1 expression, and degradation of the ECM. Furthermore, the Hh pathway mediates IGFBPrP1 knockdown-induced attenuation of hepatic fibrosis through the regulation of MMPs/TIMPs balance.
K2-capsular
is a hypervirulent pathogen that causes fatal infections. Here, we describe a phage tailspike protein, named K2-2, that specifically depolymerizes the K2 capsular polysaccharide (CPS) of
...into tetrasaccharide repeating units. Nearly half of the products contained
-acetylation, which was thought crucial to the immunogenicity of CPS. The product-bound structures of this trimeric enzyme revealed intersubunit carbohydrate-binding grooves, each accommodating three tetrasaccharide units of K2 CPS. The catalytic residues and the key interactions responsible for K2 CPS recognition were identified and verified by site-directed mutagenesis. Further biophysical and functional characterization, along with the structure of a tetrameric form of K2-2, demonstrated that the formation of intersubunit catalytic center does not require trimerization, which could be nearly completely disrupted by a single-residue mutation in the C-terminal domain. Our findings regarding the assembly and catalysis of K2-2 provide cues for the development of glycoconjugate vaccines against
infection.
Generating fragments of capsular polysaccharides from pathogenic bacteria with crucial antigenic determinants for vaccine development continues to pose challenges. The significance of the C-terminal region of phage tailspike protein (TSP) in relation to its folding and trimer formation remains largely unexplored. The polysaccharide depolymerase described here demonstrates the ability to depolymerize the K2 CPS of
into tetrasaccharide fragments while retaining the vital
-acetylation modification crucial for immunogenicity. By carefully characterizing the enzyme, elucidating its three-dimensional structures, conducting site-directed mutagenesis, and assessing the antimicrobial efficacy of the mutant enzymes against K2
, we offer valuable insights into the mechanism by which this enzyme recognizes and depolymerizes the K2 CPS. Our findings, particularly the discovery that trimer formation is not required for depolymerizing activity, challenge the current understanding of trimer-dependent TSP activity and highlight the catalytic mechanism of the TSP with an intersubunit catalytic center.
Cellulose nanofibers functionalized with multiple aldehyde group were synthesized as the crosslinker to produce composite self-healing hydrogel and shape memory cryogel from chitosan. The hydrogel ...possessed effective self-healing (∼100% efficiency) and shear-thinning properties. The cryogel had macroporous structure, large water absorption (>4300%), and high compressibility. Both hydrogel and cryogel were injectable. In particular, the cryogel (nanocellulose/chitosan 1:6) revealed thermally induced shape memory, the mechanism of which was elucidated by in situ small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) as changes in orientation of the induced crystalline structure during the shape memory program. The shape memory cryogel with a large size (15 mm × 10 mm × 1.1 mm) injected through a 16 G syringe needle was recoverable in 37 °C water. Moreover, the cryogel was cytocompatible and promoted cell growth. The nanocellulose–chitosan composite hydrogel and cryogel are injectable and degradable biomaterials with adjustable mechanical properties for potential medical applications.
We identify a new amyloidogenic peptide from the glutamine/asparagine-rich region of the FTLD-related protein (TDP-43), which can seed both the full-length and N-terminus-truncated TDP-43. Through ...the microinjection and real-time fluorescence imaging, we also found that this novel peptide could trigger cell apoptosis and initiate TDP-43 aggregation in the cytosol.
The amyloidogenic core in the TAR DNA-binding protein (TDP-43) C-terminal fragment has been characterized with its chemical, biochemical, and structural properties delineated. Various properties of ...the core sequence, including membrane impairment ability and the seeding effect, have also been studied.
Purpose: This study is aimed at investigating the in vivo antitumor activity of a novel cell-impermeable glucuronide prodrug, 9-aminocamptothecin glucuronide (9ACG), and elucidating
the ...synergistically antitumor effects of antiangiogenesis therapy by targeting the tumor microenvironment.
Experimental Design: We analyzed the antitumor effects of 9ACG alone or combined with antiangiogenic monoclonal antibody DC101 on human tumor
xenografts by measuring tumor growth and mouse survival in BALB/c nu/nu nude and NOD/SCID mice. The drug delivery, immune response, and angiogenesis status in treated tumors were assessed by high
performance liquid chromatography, immunohistochemistry, and immunofluorescence assays.
Results: We developed a nontoxic and cell-impermeable glucuronide prodrug, 9ACG, which can only be activated by extracellular β-glucuronidase
to become severely toxic. 9ACG possesses potent antitumor activity against human tumor xenografts in BALB/c nu/nu nude mice but not for tumors implanted in NOD/SCID mice deficient in macrophages and neutrophils, suggesting that these cells
play an important role in activating 9ACG in the tumor microenvironment. Most importantly, antiangiogenic monoclonal antibody
DC101 potentiated single-dose 9ACG antitumor activity and prolonged survival of mice bearing resistant human colon tumor xenografts
by providing strong β-glucuronidase activity and prodrug delivery through enhancing inflammatory cell infiltration and normalizing
tumor vessels in the tumor microenvironment. We also show that inflammatory cells (neutrophils) were highly infiltrated in
advanced human colon cancer tissues compared with normal counterparts.
Conclusions: Our study provides in vivo evidence that 9ACG has potential for prodrug monotherapy or in combination with antiangiognesis treatment for tumors with
infiltration of macrophage or neutrophil inflammatory cells.
PDF
