Dnmt3b ablation impairs fracture repair through upregulation of Notch pathway

We previously established that DNA methyltransferase 3b (Dnmt3b) is the sole Dnmt responsive to fracture repair and that Dnmt3b expression is induced in progenitor cells during fracture repair. In the current study, we confirmed that Dnmt3b ablation in mesenchymal progenitor cells (MPCs) resulted in impaired endochondral ossification, delayed fracture repair, and reduced mechanical strength of the newly formed bone in Prx1-Cre;Dnmt3bf/f (Dnmt3bPrx1) mice. Mechanistically, deletion of Dnmt3b in MPCs led to reduced chondrogenic and osteogenic differentiation in vitro. We further identified Rbpjκ as a downstream target of Dnmt3b in MPCs. In fact, we located 2 Dnmt3b binding sites in the murine proximal Rbpjκ promoter and gene body and confirmed Dnmt3b interaction with the 2 binding sites by ChIP assays. Luciferase assays showed functional utilization of the Dnmt3b binding sites in murine C3H10T1/2 cells. Importantly, we showed that the MPC differentiation defect observed in Dnmt3b deficiency cells was due to the upregulation of Rbpjκ, evident by restored MPC differentiation upon Rbpjκ inhibition. Consistent with in vitro findings, Rbpjκ blockage via dual antiplatelet therapy reversed the differentiation defect and accelerated fracture repair in Dnmt3bPrx1 mice. Collectively, our data suggest that Dnmt3b suppresses Notch signaling during MPC differentiation and is necessary for normal fracture repair

Efficient yeast surface-display of novel complex synthetic cellulosomes

Background: The self-assembly of cellulosomes on the surface of yeast is a promising strategy for consolidated bioprocessing to convert cellulose into ethanol in one step. Results: In this study, we developed a novel synthetic cellulosome that anchors to the endogenous yeast cell wall protein a-agglutinin through disulfide bonds. A synthetic scaffoldin ScafAGA3 was constructed using the repeated N-terminus of Aga1p and displayed on the yeast cell surface. Secreted cellulases were then fused with Aga2p to assemble the cellulosome. The display efficiency of the synthetic scaffoldin and the assembly efficiency of each enzyme were much higher than those of the most frequently constructed cellulosome using scaffoldin ScafCipA3 from Clostridium thermocellum. A complex cellulosome with two scaffoldins was also constructed using interactions between the displayed anchoring scaffoldin ScafAGA3 and scaffoldin I ScafCipA3 through disulfide bonds, and the assembly of secreted cellulases to ScafCipA3. The newly designed cellulosomes enabled yeast to directly ferment cellulose into ethanol. Conclusions: This is the first report on the development of complex multiple-component assembly system through disulfide bonds. This strategy could facilitate the construction of yeast cell factories to express synergistic enzymes for use in biotechnology

A multifunctional azobenzene-based polymeric adsorbent for effective water remediation

The efficient removal of trace carcinogenic organic pollutants, such as polycyclic aromatic hydrocarbons (PAHs) and ionic dyes, from water is an important technical challenge. We report a highly effective recyclable multifunctional azobenzene (AZ)-based silica-supported polymeric adsorbent which can simultaneously remove both PAHs and anionic dyes from water to below parts per billion (ppb) level based on multiple interactions such as the hydrophobic effect, [pi]–[pi] stacking and electrostatic interactions, thus providing a new strategy for designer water remediation materials

Management of the endoplasmic reticulum stress by activation of the heat shock response in yeast

In yeast Saccharomyces cerevisiae, accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates the unfolded protein response (UPR), which is mediated by Hac1p. The heat shock response (HSR) mediated by Hsf1p, mainly regulates cytosolic processes and protects the cell from stresses. Here, we find that a constitutive activation of the HSR could increase ER stress resistance in both wild-type and UPR-deficient cells. Activation of HSR decreased UPR activation in the WT (as shown by the decreased HAC1 mRNA splicing). We analyzed the genome-wide transcriptional response in order to propose regulatory mechanisms that govern the interplay between UPR and HSR and followed up for the hypotheses by experiments in vivo and in vitro. Interestingly, we found that the regulation of ER stress response via HSR is (1) only partially dependent on over-expression of Kar2p (ER resident chaperone induced by ER stress); (2) does not involve the increase in protein turnover via the proteasome activity; (3) is related to the oxidative stress response. From the transcription data, we also propose that HSR enhances ER stress resistance mainly through facilitation of protein folding and secretion. We also find that HSR coordinates multiple stress-response pathways, including the repression of the overall transcription and translation

Methylprednisolone as Adjunct to Endovascular Thrombectomy for Large-Vessel Occlusion Stroke

Importance It is uncertain whether intravenous methylprednisolone improves outcomes for patients with acute ischemic stroke due to large-vessel occlusion (LVO) undergoing endovascular thrombectomy. Objective To assess the efficacy and adverse events of adjunctive intravenous low-dose methylprednisolone to endovascular thrombectomy for acute ischemic stroke secondary to LVO. Design, Setting, and Participants This investigator-initiated, randomized, double-blind, placebo-controlled trial was implemented at 82 hospitals in China, enrolling 1680 patients with stroke and proximal intracranial LVO presenting within 24 hours of time last known to be well. Recruitment took place between February 9, 2022, and June 30, 2023, with a final follow-up on September 30, 2023.InterventionsEligible patients were randomly assigned to intravenous methylprednisolone (n = 839) at 2 mg/kg/d or placebo (n = 841) for 3 days adjunctive to endovascular thrombectomy. Main Outcomes and Measures The primary efficacy outcome was disability level at 90 days as measured by the overall distribution of the modified Rankin Scale scores (range, 0 [no symptoms] to 6 [death]). The primary safety outcomes included mortality at 90 days and the incidence of symptomatic intracranial hemorrhage within 48 hours. Results Among 1680 patients randomized (median age, 69 years; 727 female [43.3%]), 1673 (99.6%) completed the trial. The median 90-day modified Rankin Scale score was 3 (IQR, 1-5) in the methylprednisolone group vs 3 (IQR, 1-6) in the placebo group (adjusted generalized odds ratio for a lower level of disability, 1.10 [95% CI, 0.96-1.25]; P = .17). In the methylprednisolone group, there was a lower mortality rate (23.2% vs 28.5%; adjusted risk ratio, 0.84 [95% CI, 0.71-0.98]; P = .03) and a lower rate of symptomatic intracranial hemorrhage (8.6% vs 11.7%; adjusted risk ratio, 0.74 [95% CI, 0.55-0.99]; P = .04) compared with placebo. Conclusions and Relevance Among patients with acute ischemic stroke due to LVO undergoing endovascular thrombectomy, adjunctive methylprednisolone added to endovascular thrombectomy did not significantly improve the degree of overall disability.Trial RegistrationChiCTR.org.cn Identifier: ChiCTR210005172

Secretory pathway engineering enhances secretion of cellobiohydrolase I from Trichoderma reesei in Saccharomyces cerevisiae

Improving the cellulase secretion is beneficial for Saccharomyces cerevisiae used in consolidated bioprocessing (CBP) of cellulosic ethanol. In this study, protein secretory pathway, including protein folding, disulfide bond formation, and protein trafficking and sorting, was modified in S. cerevisiae. The effects of these modifications on the secretion of cellobiohydrolase I (Tr-Cel7A) with its native signal peptide from Trichoderma reesei were investigated. The results showed that overexpression of the protein disulfide isomerase Sc-PDI1 and the plasma membrane targeting soluble N-ethylmaleimide-sensitive factor attachment protein receptor Sc-SSO1, and disruption of the sorting receptor Sc-VPS10 and a Ca2+/Mn2+ ATPase Sc-PMR1, improved respectively the extracellular Tr-Cel7A activities. Among them, disruption of Sc-PMR1 showed better improvement of 162% in the extracellular activity and decreased the glycosylation of Tr-Cel7A. Multiple modifications generally resulted in higher activities. The extracellular activities of the quadruple-modified strain (vps10 Delta/pmr1 Delta/SSO1/PDI1/cel7AF) using p-nitrophenyl-beta-D-cellobioside (pNPC) and phosphoric acid swollen cellulose (PASC) as the substrates, respectively, were 3.9-fold and 1.3-fold higher than that of the reference strain cel7AF. The results indicated that engineering of the protein secretory pathway is an effective approach to improve the Tr-Cel7A secretion in S. cerevisiae. (C) 2013, The Society for Biotechnology, Japan. All rights reserved