Mechanisms of Peroxynitrous Acid and Methyl Peroxynitrite, ROONO (R = H, Me), Rearrangements: A Conformation-Dependent Homolytic Dissociation

The O−O bond breaking reactions of peroxynitrous acid and methyl peroxynitrite, ROONO (R = H, Me), were investigated theoretically using the (U)CCSD/6-31+G*, (U)CCSD(T)/6-31+G*//(U)CCSD/6-31+G*, and CBS-QB3 methods. The OONO dihedral angle has a remarkably large influence on the barriers for cleavage of the O−O bonds, which influences the subsequent radical recombination to yield nitrates (RONO2). A barrier of ca. 18−19 kcal/mol is predicted for RO−ONO dissociation involving a 2A1-like NO2 fragment in transition states beginning from a cis-OONO conformation. This pathway is significantly favored relative to a 2B2-like transition state with a trans-ONOO conformation; the latter has a barrier of 33−34 kcal/mol. Notably, the favored cis-OONO pathway is “electronically correct” (because 2A1 NO2 is a N-centered radical), but “geometrically incorrect” for subsequent N−O bond formation to yield RONO2. The imperfect initial orientation of RO/NO2 for N−O bond formation rationalizes some escape of free radicals, in competition with low-barrier RO• and NO2 orientational motions followed by near-barrierless collapse to RONO2. For HOONO, the pathway for HONO2 formation may include a hydrogen-bonded intermediate, •OH···ONO•, earlier proposed as a source of one-electron processes occurring after O−O bond cleavage. The cis-ONOO rearrangement barrier is in accord with the experimental free energy of activation (18 ± 1 kcal/mol) for the rearrangement of peroxynitrous acid (HOONO) into nitric acid (HNO3). MeOONO has a similar rearrangement mechanism, although the pathways for its rearrangement lack any hydrogen-bonded intermediates

Data_Sheet_1_Impact of 25-Hydroxy Vitamin D on White Matter Hyperintensity in Elderly Patients: A Systematic Review and Meta-Analysis.docx

Some studies show that low serum vitamin D levels are associated with white matter hyperintensity (WMH), while other studies report no association. This meta-analysis aimed to investigate the presence of an association between serum 25-hydroxy vitamin D [25(OH)D] levels and WMH. PubMed, Embase, the Cochrane Library, CNKI, WANFANG, and VIP were searched for available papers published up to December 2020. The outcomes were the odds ratios (ORs) with 95% confidence intervals (CIs) for the association between different vitamin D statuses and WMH. All meta-analyses were performed using a random-effects model. Five studies (4393 patients) were included. Compared with sufficient 25(OH)D levels, 25(OH)D deficiency was not associated with WMH (OR = 1.67, 95%CI: 0.92–3.04; I2 = 70.2%, Pheterogeneity = 0.009), nor was 25(OH)D insufficiency (OR = 1.21, 95%CI: 0.89–1.65; I2 = 48.1%, Pheterogeneity = 0.103). A decrease of 25 nmol/L in 25(OH)D levels was associated with WMH (OR = 1.83, 95%CI: 1.34-2.49; I2 = 0%, Pheterogeneity= 0.512). The sensitivity analyses showed that the results were robust. 25(OH)D deficiency and insufficiency are not associated with WMH. A decrease of 25 nmol/L in 25(OH)D levels was associated with WMH, but this result will have to be confirmed. Prospective trials, both cross-sectional and longitudinal, are necessary to examine the association between 25(OH)D levels and WMH.</p

Dissection of Protein Interactomics Highlights MicroRNA Synergy

<div><p>Despite a large amount of microRNAs (miRNAs) have been validated to play crucial roles in human biology and disease, there is little systematic insight into the nature and scale of the potential synergistic interactions executed by miRNAs themselves. Here we established an integrated parameter synergy score to determine miRNA synergy, by combining the two mechanisms for miRNA-miRNA interactions, miRNA-mediated gene co-regulation and functional association between target gene products, into one single parameter. Receiver operating characteristic (ROC) analysis indicated that synergy score accurately identified the gene ontology-defined miRNA synergy (AUC = 0.9415, <i>p</i><0.001). Only a very small portion of the random miRNA-miRNA combinations generated potent synergy, implying poor expectancy of widespread synergy. However, targeting more key genes made two miRNAs more likely to act synergistically. Compared to other miRNAs, miR-21 was a highly exceptional case due to frequent appearance in the top synergistic miRNA pairs. This result highlighted its essential role in coordinating or strengthening physiological and pathological functions of other miRNAs. The synergistic effect of miR-21 and miR-1 were functionally validated for their significant influences on myocardial apoptosis, cardiac hypertrophy and fibrosis. The novel approach established in this study enables easy and effective identification of condition-restricted potent miRNA synergy simply by concentrating the available protein interactomics and miRNA-target interaction data into a single parameter synergy score. Our results may be important for understanding synergistic gene regulation by miRNAs and may have significant implications for miRNA combination therapy of cardiovascular disease.</p></div

Structural and Mechanistic Insights into Chain Release of the Polyene PKS Thioesterase Domain

Polyketides serve as rich source of therapeutically relevant drug leads. The manipulation of polyketide synthases (PKSs) for generating derivatives with improved activities usually results in substantially reduced yields. Growing evidence suggests that type I PKS thioesterase (TE) domains are key bottlenecks in the biosynthesis of polyene antibiotics, such as pimaricin and amphotericin, and their unnatural derivatives. Herein, we elucidate the structure of the 26-membered macrolide-complexed TE domain from the pimaricin pathway (Pim TE), which specifies a spacious bifunnel-shaped substrate channel with a highly hydrophobic cleft proximal to the catalytic triad and a hydrophilic loop I region specific for the cyclization of amphiphilic polyene macrolide. Notably, the natural intermediate with C12-COOH is stabilized by a hydrogen-bond network, as well as by interactions between the polyene moiety and the hydrophobic cleft. Moreover, the bottleneck in processing the unnatural intermediate with C12-CH3 is attributed to the unstable and mismatched docking of the curved substrate in the channel. Aided by an in vitro assay with a fully elongated linear polyene intermediate as the substrate, multiple strategies were adopted, herein, to engineer Pim TE, including introducing H-bond donors, enhancing hydrophobic interactions, and modifying the catalytic center. Efficient TE mutations with increased substrate conversion up to 39.2% in vitro were further conducted in vivo, with a titer increase as high as 37.1% for the less toxic decarboxylated pimaricin derivatives with C12-CH3. Our work uncovers the mechanism of TE-catalyzed polyene macrolide formation and highlights TE domains as targets for PKS manipulation for titer increases in engineered unnatural polyketide derivatives

Structural and Mechanistic Insights into Chain Release of the Polyene PKS Thioesterase Domain

Polyketides serve as rich source of therapeutically relevant drug leads. The manipulation of polyketide synthases (PKSs) for generating derivatives with improved activities usually results in substantially reduced yields. Growing evidence suggests that type I PKS thioesterase (TE) domains are key bottlenecks in the biosynthesis of polyene antibiotics, such as pimaricin and amphotericin, and their unnatural derivatives. Herein, we elucidate the structure of the 26-membered macrolide-complexed TE domain from the pimaricin pathway (Pim TE), which specifies a spacious bifunnel-shaped substrate channel with a highly hydrophobic cleft proximal to the catalytic triad and a hydrophilic loop I region specific for the cyclization of amphiphilic polyene macrolide. Notably, the natural intermediate with C12-COOH is stabilized by a hydrogen-bond network, as well as by interactions between the polyene moiety and the hydrophobic cleft. Moreover, the bottleneck in processing the unnatural intermediate with C12-CH3 is attributed to the unstable and mismatched docking of the curved substrate in the channel. Aided by an in vitro assay with a fully elongated linear polyene intermediate as the substrate, multiple strategies were adopted, herein, to engineer Pim TE, including introducing H-bond donors, enhancing hydrophobic interactions, and modifying the catalytic center. Efficient TE mutations with increased substrate conversion up to 39.2% in vitro were further conducted in vivo, with a titer increase as high as 37.1% for the less toxic decarboxylated pimaricin derivatives with C12-CH3. Our work uncovers the mechanism of TE-catalyzed polyene macrolide formation and highlights TE domains as targets for PKS manipulation for titer increases in engineered unnatural polyketide derivatives

Structural and Mechanistic Insights into Chain Release of the Polyene PKS Thioesterase Domain

Polyketides serve as rich source of therapeutically relevant drug leads. The manipulation of polyketide synthases (PKSs) for generating derivatives with improved activities usually results in substantially reduced yields. Growing evidence suggests that type I PKS thioesterase (TE) domains are key bottlenecks in the biosynthesis of polyene antibiotics, such as pimaricin and amphotericin, and their unnatural derivatives. Herein, we elucidate the structure of the 26-membered macrolide-complexed TE domain from the pimaricin pathway (Pim TE), which specifies a spacious bifunnel-shaped substrate channel with a highly hydrophobic cleft proximal to the catalytic triad and a hydrophilic loop I region specific for the cyclization of amphiphilic polyene macrolide. Notably, the natural intermediate with C12-COOH is stabilized by a hydrogen-bond network, as well as by interactions between the polyene moiety and the hydrophobic cleft. Moreover, the bottleneck in processing the unnatural intermediate with C12-CH3 is attributed to the unstable and mismatched docking of the curved substrate in the channel. Aided by an in vitro assay with a fully elongated linear polyene intermediate as the substrate, multiple strategies were adopted, herein, to engineer Pim TE, including introducing H-bond donors, enhancing hydrophobic interactions, and modifying the catalytic center. Efficient TE mutations with increased substrate conversion up to 39.2% in vitro were further conducted in vivo, with a titer increase as high as 37.1% for the less toxic decarboxylated pimaricin derivatives with C12-CH3. Our work uncovers the mechanism of TE-catalyzed polyene macrolide formation and highlights TE domains as targets for PKS manipulation for titer increases in engineered unnatural polyketide derivatives

Effects of Conformational Alteration Induced by d‑/l‑Isonucleoside Incorporation in siRNA on Their Stability in Serum and Silencing Activity

We report here that all of the d- or l-isonucleoside (isoNA) modified siRNAs investigated showed the characteristic A-form conformation in the circular dichroism (CD) spectra compared to native siRNA. The d-isoNA modification had less influence on the thermal stability of siRNAs, but all l-isoNA modification displayed a significant tendency to decrease the thermal stability of siRNA. It was also found that the stabilities of d-/l-isoNA modified siMek1 in serum were different and d-isoNA modification was more potent, i.e., increase of serum stability of siRNA, than l-isoNA modification. When d-isoNA incorporated at position 4 and position 5 at antisense strand of siMek1 showed obvious improvement on serum stability, however, l-isoNA incorporated at positions 11 and 12 at antisense strand and position 9 at sense strand made the siMek1 duplex formed very unstable in serum. The silencing activities of modified siMek1s with d-/l-isoNA at position 1 of antisense strand also dropped dramatically; however, the modification at 3′-terminal of the sense strand with d- or l-isoNA significantly enhanced the silencing activity targeting the antisense strand as reporter and minimized the passenger strand-specific off-target effect. IsoNA modified in the seed area of siMek1, <b>siMek1 A04D</b> and <b>siMek1 A05L</b>, showed similar activity to the native one and better target selectivity. In the case of modification at the position near the cleavage area, it was found that d- or l-isoNA modified sense strand at position 8, 9, or 15 of siMek1 could retain the silencing activities targeting the antisense strand as reporter. Especially, both <b>siMek1 S15D</b> and <b>siMek1 S15L</b> showed good silencing activity and high target selectivity compared to native siMek1. The effects of conformational alteration of such isoNA modification of siRNA on their stability in serum and silencing activity are discussed based on computer simulation. Systematic investigation of the relationship between modified siRNA conformation and their physical and biological properties should provide a useful guideline for chemical modification and optimization of siRNA for further clinical application

Activities of two <i>phz‘</i>-<i>’lacZ</i> transcriptional or translational fusions in strain M18 and its two mutants.

<p>The <i>phz1‘-’lacZ</i> and <i>phz2‘-’lacZ</i> transcriptional or translational fusion in plasmids pME6522 (A) or pME6015 (B). Their transcriptional (C) or translational activities (D) in wild type strain <i>Pseudomonas</i> sp. M18 (square), <i>phzA1-G1</i> inactivated mutant M18ΔP1 (triangle) and <i>phzA2-G2</i> inactivated mutant M18ΔP2 (circle). Symbols: open, <i>phz1‘-’lacZ</i> fusion; solid, <i>phz2‘-’lacZ</i> fusion. All experiments were performed in triplicate, and each value is presented as the average ± standard deviation.</p

Self-Assembled R848/Chlorin e6 Nanoparticles for Combinatorial Immunotherapy of Head and Neck Squamous Cell Carcinoma

Effective therapeutic approaches against head and neck squamous cell carcinoma (HNSCC), which is a prevalent malignant tumor, are currently lacking. Although immunotherapy has emerged as a promising strategy for treating HNSCC, its efficacy is severely limited by the immunosuppressive tumor microenvironment. Self-delivery nanoparticles, comprising Toll-like receptor (TLR) agonists and photosensitizers, were successfully developed to address this challenge in combinatorial immunotherapy for HNSCC treatment. After intravenous administration, these nanoparticles efficiently accumulated in tumors, enhancing the bioavailability of the agonist and triggering the TLR signaling pathway to remodel the immunosuppressive tumor microenvironment. Simultaneously, the photosensitizers induced immunogenic cell death and activated the stimulator of the interferon gene (STING) pathway in tumor cells via photodynamic therapy. The development of tumors was significantly inhibited by enhancing the antitumor immunity induced by the nanoparticles. This study provided a new paradigm for HNSCC immunotherapy, involving the synergistic regulation of the immune microenvironment with photodynamic therapy

Identification of functional MDAs for nine complex human diseases by co-considering direct and indirect interactions.

<p>Highly plausible MDAs were highlighted. COPD: chronic obstructive pulmonary disease.</p