Mechanism Of Cleavage Research Articles

Photocatalyst g-C3N4 for efficient cleavage of lignin C[sbnd]C bonds in micellar aqueous medium

Depolymerizing lignin to prepare aromatic high-value chemicals is beneficial to solve the problem of energy shortage and waste of resources. However, cleavage of lignin CC bonds still faces greater challenges due to the recalcitrance of CC bonds in lignin. Photocatalysis is considered to be a promising way to break lignin CC bonds under mild conditions. Here, the micellar aqueous medium (SDS) composed of sodium lauryl sulfonate and water was prepared. 1, 2-Diphenylethanol (lignin model compound, Dpol) and dioxanesolv poplar lignin can be well dispersed in SDS-8 (mass fraction of 0.08% for sodium lauryl sulfate). Photocatalyst g-C3N4 can efficiently break the lignin CC bonds in SDS-8 and very high photocatalytic efficiency can be obtained. A small amount of acetic acid can further improve photocatalytic efficiency. The main products produced by photocatalytic breaking lignin CC bonds are benzaldehyde and benzoic acid. The mechanism of photocatalytic cleavage of lignin CC bonds mainly follows a photogenerated electron-hole coupled redox mechanism. Thereinto, the hydrogen provided by SDS-8 has powerfully promoted photocatalytic efficiency. Our work provides a new direction for photocatalytic cleavage of lignin CC bonds into aromatic high-value chemicals.

A thermodynamic investigation of amyloid precursor protein processing by human γ-secretase

Human γ-secretase cleaves the transmembrane domains (TMDs) of amyloid precursor protein (APP) into pathologically relevant amyloid-β peptides (Aβs). The detailed mechanisms of the unique endoproteolytic cleavage by the presenilin 1 domain (PS1) of γ-secretase are still poorly understood. Herein, we provide thermodynamic insights into how the α-helical APP TMD is processed by γ-secretase and elucidate the specificity of Aβ48/Aβ49 cleavage using unbiased molecular dynamics and bias-exchange metadynamics simulations. The thermodynamic data show that the unwinding of APP TMD is driven by water hydration in the intracellular pocket of PS1, and the scissile bond T32-L33 or L33-V34 of the APP TMD can slide down and up to interact with D257/D385 to achieve endoproteolysis. In the wild-type system, the L33-V34 scissile bond is more easily hijacked by D257/D385 than T32-L33, resulting in higher Aβ49 cleavage, while the T32N mutation on the APP TMD decreases the energy barrier of the sliding of the scissile bonds and increases the hydrogen bond occupancy for Aβ48 cleavage. In summary, the thermodynamic analysis elucidates possible mechanisms of APP TMD processing by PS1, which might facilitate rational drug design targeting γ-secretase.

Mechanistic classification and benchmarking of polyolefin depolymerization over silica-alumina-based catalysts

Carbon-carbon bond cleavage mechanisms play a key role in the selective deconstruction of alkanes and polyolefins. Here, we show that the product distribution, which encompasses carbon range and formation of unsaturated and isomerization products, serves as a distinctive feature that allows the reaction pathways of different catalysts to be classified. Co, Ni, or Ru nanoparticles immobilized on amorphous silica-alumina, Zeo-Y and ZSM-5, were evaluated as catalysts in the deconstruction of n-hexadecane model substrate with hydrogen to delineate between different mechanisms, i.e., monofunctional- (acid site dominated) or bifunctional-hydrocracking (acid site & metal site) versus hydrogenolysis (metal site dominated), established from the product distributions. The ZSM-5-based catalysts were further studied in the depolymerization of polyethylene. Based on these studies, the catalysts are plotted on an activity-mechanism map that functions as an expandable basis to benchmark catalytic activity and to identify optimal catalysts that afford specific product distributions. The systematic approach reported here should facilitate the acceleration of catalyst discovery for polyolefin depolymerization.

QM/MM Well-Tempered Metadynamics Study of the Mechanism of XBP1 mRNA Cleavage by Inositol Requiring Enzyme 1α RNase.

A range of in silico methodologies were herein employed to study the unconventional XBP1 mRNA cleavage mechanism performed by the unfolded protein response (UPR) mediator Inositol Requiring Enzyme 1α (IRE1). Using Protein–RNA molecular docking along with a series of extensive restrained/unrestrained atomistic molecular dynamics (MD) simulations, the dynamical behavior of the system was evaluated and a reliable model of the IRE1/XBP1 mRNA complex was constructed. From a series of well-converged quantum mechanics molecular mechanics well-tempered metadynamics (QM/MM WT-MetaD) simulations using the Grimme dispersion interaction corrected semiempirical parametrization method 6 level of theory (PM6-D3) and the AMBER14SB-OL3 force field, the free energy profile of the cleavage mechanism was determined, along with intermediates and transition state structures. The results show two distinct reaction paths based on general acid–general base type mechanisms, with different activation energies that perfectly match observations from experimental mutagenesis data. The study brings unique atomistic insights into the cleavage mechanism of XBP1 mRNA by IRE1 and clarifies the roles of the catalytic residues H910 and Y892. Increased understanding of the details in UPR signaling can assist in the development of new therapeutic agents for its modulation.

Molecular dynamics study on mechanical cleavage mechanisms of GaAs and experimental verification

Laser bars are fast becoming a key device in a range of specific industrial applications. Mechanical cleavage technology is a new and efficient method for fabricating laser bars. However, there is little detailed analysis of the relationship between scratching step and cleavage step. To close this research gap, a molecular dynamics study on cleavage mechanisms of GaAs are reported investigating surface and subsurface damage, and molecular dynamics method could accurately describe the nano-scale processes in semiconductors at atomic scale. Simulation results show that the scratching depth has a significant effect on the scratch quality as compared with other parameters during the scratching process. Then, a series of cleavage experiments were carried out to verify the simulation results and further explore the influence of key scratching parameters on cleavage plane morphology. Experimental results correlate well with the simulations. Consequently, the achieved optimal combination of parameters have been found to be a scratching load of 10 g, scratching speed of 20 mm/s and scratching length of 0.6 mm, which provides direct guidance for the cleavage processing of GaAs materials. Under optimal conditions, the length and surface roughness of the undamaged area can reach 11.77 mm and 0.43 nm respectively.

Autophagy promotes GSDME-mediated pyroptosis via intrinsic and extrinsic apoptotic pathways in cobalt chloride-induced hypoxia reoxygenation-acute kidney injury

Cobalt is a transition element that abundantly exists in the environment. Besides direct hypoxia stress, cobalt ions indirectly induce hypoxia-reoxygenation injury (HRI), the main cause of acute kidney injury (AKI), a life-threatening clinical syndrome characterized by the necrosis of the proximal tubular epithelial cells (PTECs) and inflammation. Pyroptosis, a type of inflammatory programmed cell death, might play an essential role in HRI-AKI. However, whether pyroptosis is involved in cobalt chloride (CoCl2)-induced HRI-AKI remains unknown. Autophagy is a cellular biological process maintaining cell homeostasis that is involved in cell damage in AKI, yet the underlying regulatory mechanism of autophagy on pyroptosis has not been fully understood. In this study, the in vitro and in vivo models of CoCl2-induced HRI-AKI were established with HK-2 cell line and C57BL/6J mouse. Pyroptosis-related markers were detected with western blotting and immunofluorescence assays, and results showed that gasdermin E (GSDME)-mediated pyroptosis was involved in the cell damage in HRI-AKI. Specific chemical inhibitors of caspase 3, caspase 8, and caspase 9 significantly inhibited GSDME-mediated pyroptosis, verifying that GSDME-mediated pyroptosis was induced via the activation of caspase 3/8/9. The western blotting and immunofluorescence assays were adopted to detect the accumulation of the autophagosomes, and results suggested that HRI increased the autophagic level. The effects of autophagy on apoptosis and pyroptosis were evaluated using lentivirus transfection assays to knock down autophagy-specific genes atg5 and fip200, and results demonstrated that autophagy induced GSDME-mediated pyroptosis via apoptotic pathways in HRI-AKI. Our results revealed the involvement of GSDME-mediated pyroptosis in CoCl2-induced HRI-AKI and promoted the understanding of the regulatory mechanism of GSDME cleavage. Our study might provide a potential therapeutic target for HRI-AKI, and will be helpful for the risk evaluation of cobalt exposure.

Alternative polyadenylation mechanism links secreted phosphoprotein 1 gene to glioblastoma

Secreted phosphoprotein 1 (SPP1), also known as osteopontin (OPN), is a multifunctional protein expressed in diverse normal tissues, and functionally is involved in cellular matrix and signaling processes. Many studies have linked SPP1 to pathophysiological conditions including cancer. The aim of this study is to evaluate the 3'UTR length of SPP1 gene in glioblastoma cell line. 3' Rapid Amplification of cDNA End (3'-RACE) was used to determine the 3' end of SPP1 gene. APAatlas data base, GEPIA web server, and miRcode were also used to extract related information and bioinformatic analysis part. In this study we show that SPP1 gene undergoes Alternative cleavage and Polyadenylation (APA) mechanism, by which it generates two 3' termini, longer isoform and shorter isoform, in glioblastoma derived cell line, U87-MG. Further bioinformatic analysis reveals that SPP1 alternative 3'UTR (aUTR), which is absent in shorter isoform, is targeted by two families of microRNAs-miR-181abcd/4262 and miR-154/872. These miRNAs also target and perhaps negatively regulate NAP1L1 and ENAH genes that are involved in cell proliferation and cell polarity, respectively. Relative expression difference (RED), obtained from RNA-seq data of diverse normal tissues, representing APA usage appears to be negatively correlated with expression of NAP1L1 and ENAH, emphasizing co-expression of SPP1 longer isoform with these two genes, indicating miRNA sponge function of aUTR (longer 3'UTR). Bioinformatic analysis also shows that in normal brain tissue longer APA isoform of SPP1 is expressed; however shorter isoform appears to be expressed in cancer condition. Together, this study reveals that SPP1 APA isoforms have different pattern in normal and cancerous conditions, which can be considered as a diagnostic and prognostic marker in cancers.

Enhanced Dihydrogen Activation by Mononuclear Iridium(II) Compounds: A Mechanistic Study.

The organometallic chemistry of 4d and 5d transition metals has been vastly dominated by closed‐shell states. The reactivity of their metalloradical species is though remarkable, albeit yet poorly understood and with limited mechanistic investigations available. In this work we report the synthesis and characterization of two mononuclear IrII species, including the first dinitrogen adduct. These compounds activate dihydrogen at a dissimilar rate, in the latter case several orders of magnitude faster than its IrI precursor. A combined experimental/computational investigation to ascertain the mechanism of this transformation in IrII compounds is reported.

Dealloying of Ni- and Fe-Based Alloys in Boiling Caustic Environments and the Parameters Influencing the Geometry of a Dealloyed Layer

Nickel alloys and stainless have been the material of choice for a wide range of industrial applications like power and petrochemical plants that involve exposure to corrosive environments. For instance, Alloy 800 (Fe-30Ni-20Cr) has been used as the tubing material in CANDU and Siemens steam generators (SGs) for decades with excellent in-service performance. However, despite their good resistance to general to localize corrosion, Ni- and Fe-based alloys are susceptible to environmentally assisted cracking (EAC) in certain environments, such as hot caustic aqueous solutions. Laboratory experiments have shown that these materials are susceptible to dealloying in hot caustic environments due to the selective removal of Fe, Cr and Mo (less noble elements). As a result of dealloying, a brittle nanoporous film forms on the surface that is enriched in Ni (the more noble element). This film has been proposed to act as a precursor to stress corrosion cracking (SCC) by a film-induced cleavage (FIC) mechanism, where the brittle film injects a high speed/energy crack to the substrate material.The fast kinetics of dealloying requires a connected percolating pathway of less noble element atoms to exist within the alloy that extends from the surface into the material. The presence of this pathway depends on the composition of the alloy. The parting limit defines the minimum amount of the more reactive element(s) in a particular alloy system that makes it susceptible to dealloying. Below the parting limit composition, dealloying will be suppressed by surface diffusion of the more noble element, even at very oxidizing potentials, and the surface of the material will show a passive-like behavior (general dissolution would eventually occur at high anodic overpotentials). Besides the alloy composition, dealloying highly depends on the electrode potential. Below a threshold potential (dealloying critical potential), surface diffusion of the more noble element will overcome the dissolution of the less noble element(s) and suppress dealloying.In this study, we examine different factors that affect dealloying and the geometry of a dealloyed layer through electrochemical measurements and nano-scale characterization techniques. Several engineering Ni- and Fe-based alloys were tested in deaerated boiling caustic solutions to understand the effect of alloy composition on the susceptibility to dealloying. Results indicate that an increase in Ni content will shift the dealloying critical potential to more positive potentials and subsequently the resistance to dealloying. Although Ni is the alloying element that provides nobility and resistance in hot caustic environments, our results show that the thermodynamic and kinetic behaviour, and the content of the less noble elements play an important role in these environments. A comparison between different alloys with similar Ni-content but different Cr and Mo contents shows the beneficial and detrimental role of Cr and Mo in dealloying, respectively. The air-formed chromia-based oxide on the surface of alloys with high Cr-content slowly dissolves in the caustic environment, delays the dissolution of less noble elements, and increases the resistance to dealloying.Changes in electrode potential and time of exposure to the caustic environments, alter the geometry of the dealloyed layer. Results show an increase in the diameter and coarsening of ligaments with increasing time of exposure to the caustic environment. Coarsening of ligaments is believed to impair the ability of a dealloyed layer to inject a micro-crack to the underlying material and initiate SCC. Also, results show that an increase in anodic overpotential of only a few millivolts in the vicinity of dealloying critical potential would change the geometry of the dealloyed layer significantly. The surface diffusion of the more noble element (i.e. Ni) cannot keep up with the fast removal of atoms of the less noble elements (i.e. Cr, Mo, and Fe) at more positive electrode potentials compared to the dealloying critical potential which results in a weak bonding at the film/metal interface. The weak bonding may not be able to support the transmission of a high-speed crack to the substrate material, reducing the likelihood of SCC initiation. Figure 1

Efficiently and selectively photocatalytic cleavage of C[sbnd]C bond by C3N4 nanosheets: Defect-enhanced engineering and rational reaction route

Photocatalytic cleavage of CC bond in lignin under mild conditions transforms solar energy into high value-added aromatic compounds. However, the limited understanding of cleavage mechanisms and the catalyst structure-performance relation obstruct the rational design of efficient photocatalysts. Reported herein is the construction of porous carbon nitride (C3N4) with carbon vacancies as heterogeneous photocatalysts via a sequential self-assembly and diol-induced thermal exfoliation. In the photocatalytic cleavage of βO4 ether bonds, carbon vacancies in C3N4 can serve as the trapping site to suppress the recombination of photogenerated charge, and endow the C3N4 with high level of N and thus make it easier for the oxidation of Cβ–H, contributing to the enhancement of performance. Furthermore, the experimental result and in situ ATR-SEIRAS spectra unveil that O2 could facilitate the cleavage of CC bond but is not indispensable in this process, which brings new fundamental understanding of βO4 bonds cleavage.

A novel approach to inulin depolymerization: A Monte Carlo based model

• Monte Carlo model for inulin depolymerization. • End-biting/internal random cleavage mechanism was adopted. • The detailed molecular weight distribution can be simulated. • The model can distinguish between two scission mechanisms. • The obtained kinetic parameters are in line with literature. Modelling the depolymerization of natural and synthetic polymers is still a challenge in the field of chemical reaction engineering, as the classical approaches based on complex consecutive/parallel reaction mechanisms show limited capabilities in predicting phenomena that nature would do statistically. A Kinetic Monte Carlo model is proposed, which assumes that polymers can be cleaved following two parallel reaction mechanisms: (i) end-biting (i.e., unzipping), (ii) internal random cleavage. The model was studied via a systematic parametric investigation, and it showed high flexibility in predicting detailed product distribution and concentration profiles of monomers as a function of reaction time. The model was applied to a industrially interesting case: inulin hydrolysis, giving mostly fructose as reaction product. A very good fit was obtained and the model was capable to explain the often-overlooked details observed in the experimental results, e.g., the sigmoidal kinetics in the early stages of the hydrolysis and its dependence on experimental conditions.

Reinvestigation of clopidogrel bioactivation unveils new cytochrome P450-catalyzed thioester cleavage mechanism

The serendipitous prodrug clopidogrel (CPG, M0) is the mainstay antiplatelet drug in clinical use. The thiophene moiety of CPG undergoes ring opening to form the active metabolite (M13) through two steps of cytochrome P450 (CYP)-catalyzed oxidation. The stable intermediate resulting from the first oxidation, 2-oxo-CPG (M2), is proposed to be oxidized to form an S-oxide intermediate (M11), which proceeds with a hydrolytic pathway to yield a sulfenic acid (M12) and subsequently the bioreduced active metabolite (M13). To test the long-standing pathway of M2 to M13 via M11, we have chemically synthesized M11 but found it does not undergo the proposed hydrolytic activation in various conditions including in liver microsomal incubations. To seek an alternative mechanism, 18O tracing studies were performed with both H218O and 18O2, and LC-MS studies show that the carboxylate product moiety acquires its O-atom from oxygen instead of water, which rules out M11 as the bioactivation intermediate. To explain the 18O tracing results, a one-step Baeyer-Villiger-like mechanism is proposed for the CYP-dependent thioester cleavage, which features the incorporation of the two O-atoms of O2 into the two product moieties of carboxylate and sulfenic acid. The research presented herein provides a biochemical basis for delineating the clinical pharmacology of a mainstay treatment and expands our understanding of CYP catalysis.

Advanced microstructural characterization of transgranular Pb-caustic SCC in Alloy 690TT

Alloy 690TT was tested at 340 °C in mildly caustic environments containing lead (Pb) to investigate the transgranular cracking mechanism of Pb-induced stress corrosion cracking (PbSCC). Advanced electron microscopy characterization revealed that the fracture surfaces exhibited evidence of cleavage and regions with significant enrichment of metallic Ni, which maintained the same orientation as the original grain matrix. Moreover, there are some indications that crystallographic cracking along {100} and {110} planes is possible at high pH in Pb-containing environment. These experimental observations are consistent with a form of film-induced cleavage mechanism, assisted by de-alloying and exacerbated by planar slip in the matrix.

Structure of Human TMPRSS2 in Complex with SARS-CoV-2 Spike Glycoprotein and Implications for Potential Therapeutics.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 520 million people around the globe resulting in more than 6.2 million as of May 2022. Understanding the cell entry mechanism of SARS-CoV-2 and its entire repertoire is a high priority for developing improved therapeutics. The SARS-CoV-2 spike glycoprotein (S-protein) engages with host receptor ACE2 for adhesion and serine proteases furin and TMPRSS2 for proteolytic activation and subsequent entry. Recent studies have highlighted the molecular details of furin and S-protein interaction. However, the structural and molecular interplay between TMPRSS2 and S-protein remains enigmatic. Here, using biochemical, structural, computational, and molecular dynamics approaches, we investigated how TMPRSS2 recognizes and activates the S-protein to facilitate viral entry. First, we identified three potential TMPRSS2 cleavage sites in the S2 domain of S-protein (S2′, T1, and T2) and reported the structure of TMPRSS2 with its individual catalytic triad. By employing computational modeling and structural analyses, we modeled the macromolecular structure of TMPRSS2 in complex with S-protein, which incited the mechanism of S-protein processing or cleavage for a new path of viral entry. On the basis of structure-guided drug screening, we also report the potential TMPRSS2 inhibitors and their structural interaction in blocking TMPRSS2 activity, which could impede the interaction with the spike protein. These findings reveal the role of TMPRSS2 in the activation of SARS-CoV-2 for its entry and insight into possible intervention strategies.

High-throughput UPLC-Q-TOF-MS/MS coupled with multivariable data processing approach for the rapid screening and characterization of chemical constituents and potential bioactive compounds from DangguiShaoyao San.

Danggui Shaoyao San (DSS), a herbal formula, has been widely used for decades in China to treat senile dementia and dysmenorrhea. Here, an integrative high-throughput UPLC-Q-TOF-MS/MS method coupled with a multivariable data processing approach was established for rapidly screening and identifying chemical constituents and potential bioactive compounds from DSS. Through the comparison with mass fragment ions, relevant literature, and in-house reference material database coupled with MS cleavage mechanism, 150 chemical constituents, mainly including triterpenoids, flavonoids, phathalides, and organic acids, were tentatively characterized. Most of them were identified for the first time. Then, principal component analysis was used to evaluate the differences in chemical profiles between groups, whereas the variable importance of the projection (VIP) spectrum (VIP > 1) and the trend plot of orthogonal partial least squares discriminant analysis were applied to intuitively screen the candidate variables present only in the dosed group. Consequently, by comparison with all the characterized components in vitro, 23 potential bioactive compounds were successfully identified, comprising 5 triterpenoids, 4 phathalides, 4 flavonoids, 4 organic acids, 3 lactones, and 3 other compounds, which were present in various medicinal materials, reflecting a synergistic mechanism. This work developed a rapid, reliable, and robust approach for comprehensive characterization of the chemical components and potential bioactive compounds of DSS, providing solid data for further research on pharmacodynamic substances and pharmacological mechanisms of DSS.

In vitro DNA interaction, topoisomerase I/II Inhibition and cytotoxic properties of polymeric copper(II) complex bridged with perchlorate ion containing N4-type schiff base ligand

Cu(II) complex containing p-Cl-isonitrosoacetophenone based on N4-type Schiff base ligand was synthesized and characterized by elemental analysis, FTIR, 1H NMR, 13C NMR and UV-Visible. The crystal structure of the synthesized complex was determined by single crystal X-ray analysis. In the crystal structure, the intermolecular hydrogen bonds link the one dimensional polymers into a network structure, in which they may be effective in the stabilization of the structure. The binding profile of the complex with CT-DNA was carried out by absorption spectra and fluorescence measurements. The experimental results were revealed that binding of the complex with CT-DNA via intercalation. The concentration and time dependent DNA cleavage studies including cleavage mechanism of the complex was performed by employing gel electrophoresis assay, where the complex has been found to cleave supercoiled DNA with high efficiency. Topoisomerase I and IIα inhibition assays with Cu(II) complex were performed. Complex showed strong inhibition against both enzymes at 5 µM. The cytotoxic activity of the complex was performed on human cell lines, breast cancer MDA-MB-231, lung carcinoma A549, colorectal adenocarcinoma HT29, neuroblastoma SH-SY5Y and normal cell line HEK-293 by MTT assay. The complex was exhibited remarkably good cytotoxic potential on cancer cell lines HT29 and SH-SY5Y.

Fast pyrolysis of guaiacyl-syringyl (GS) type milled wood lignin: Product characteristics and CH4 formation mechanism study

It is anticipated that the insight into the demethylation and mechanism of CH4 formation from natural lignin using in-situ diffuse reflectance infrared Fourier transform spectroscopy (in-situ FTIR) combined with two-dimensional perturbation correlation infrared spectroscopy (2D-PCIS) and density functional theory (DFT) calculation analysis would contribute to a deeper insight of bond cleavage mechanism of lignin pyrolysis. Herein, GS-type lignin (poplar MWL) was characterized by Fourier transform infrared spectroscopy (FTIR) and heteronuclear Single-Quantum Correlation Nuclear Magnetic Resonance (HSQC), and its pyrolysis at different temperatures was performed in a lab-scale fixed-bed reactor. The biochar, gaseous and liquid products were qualitative, and quantitative analysis of gases and bio-oil is demonstrated using gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS). The key of CH4 formation is the homolytic cleavage of the methoxyl functional group generating methyl radical and further verified via in-situ FTIR combined with 2D-PCIS and DFT calculation. The study established a new methodology based on multiple factor analysis to evaluate the CH4 formation mechanism in GS-type milled wood lignin at the molecular level, which is of positive significance for increasing lignin valorization and improving the environment.

A nuclear receptor heterodimer, CgPPAR2-CgRXR, acts as a regulator of carotenoid metabolism in Crassostrea gigas

Nuclear receptors (NRs) are mostly ligand-activated transcription factors in animals and play essential roles in metabolism and homeostasis. The NR heterodimer composed of PPAR/RXR (peroxisome proliferator-activated receptor/retinoidXreceptor) is considered a key regulator of lipid metabolism in vertebrate. However, in molluscs, how this heterodimer is involved in carotenoid metabolism remains unclear. To elucidate how this heterodimer regulates carotenoid metabolism, we identified a PPAR gene in C. gigas, designated as CgPPAR2 (LOC105323212), and functionally characterized it using two-hybrid and reporter systems. CgPPAR2 is a direct orthologue of vertebrate PPARs and the second PPAR gene identified in C. gigas genome in addition to CgPPAR1 (LOC105317849). The results demonstrated that CgPPAR2 protein can form heterodimer with C. gigas RXR (CgRXR), and then regulate carotenoid metabolism by controlling carotenoid cleavage oxygenases with different carotenoid cleavage efficiencies. This regulation can be affected by retinoid ligands, i.e., carotenoid derivatives, validating a negative feedback regulation mechanism of carotenoid cleavage for retinoid production. Besides, organotins may disrupt this regulatory process through the mediation of CgPPAR2/CgRXR heterodimer. This is the first report of PPAR/RXR heterodimer regulating carotenoid metabolism in mollusks, contributing to a better understanding of the evolution and conservation of this nuclear receptor heterodimer.

Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9.

Class 2 CRISPR effectors Cas9 and Cas12 may have evolved from nucleases in IS200/IS605 transposons. IscB is about two-fifths the size of Cas9 but shares a similar domain organization. The associated ωRNA plays the combined role of CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) to guide double-stranded DNA (dsDNA) cleavage. Here we report a 2.78-angstrom cryo-electron microscopy structure of IscB-ωRNA bound to a dsDNA target, revealing the architectural and mechanistic similarities between IscB and Cas9 ribonucleoproteins. Target-adjacent motif recognition, R-loop formation, and DNA cleavage mechanisms are explained at high resolution. ωRNA plays the equivalent function of REC domains in Cas9 and contacts the RNA-DNA heteroduplex. The IscB-specific PLMP domain is dispensable for RNA-guided DNA cleavage. The transition from ancestral IscB to Cas9 involved dwarfing the ωRNA and introducing protein domain replacements.

Diagnostic Fragmentation Pathways for Identification of Phthalate Metabolites in Nontargeted Analysis Studies.

Phthalates have been studied due to their linkages with adverse developmental effects; however, metabolites of this class of compounds are undercharacterized and are poorly captured by traditional targeted analysis. In this study, we developed a nontargeted analysis approach for identifying and classifying phthalate metabolites based on a comprehensive study of their fragmentation pathways in electrospray ionization (ESI) quadrupole-time-of-flight mass spectrometry (QTOF-MS). This approach identifies molecular features in the data as phthalate metabolites via the detection of three structurally significant fragment ions. Then phthalate metabolites are classified into four types based on the presence of additional fragment ions specific to each type. Cleavage mechanisms for each class of phthalate metabolite are proposed based on fragmentation patterns generated at various collision energies (CE). All of the tested phthalate metabolites including oxidative and nonoxidative metabolites produced a fragment ion at m/z 121.0295, representing the deprotonated benzoate ion [C6H5COO]-. Most tested phthalate metabolites can produce a specific ion at m/z 147.0088, the deprotonated o-phthalic anhydride ion. However, phthalate carboxylate metabolites can only produce the [M-H-R]- ion at m/z 165.0193 and do not produce the fragment at m/z 147.0088. Other phthalate oxidative metabolites (hydroxyl- and oxo-) follow a different fragmentation pathway than nonoxidative metabolites. With this workflow, eight unknown phthalate metabolites were putatively identified in pooled urine, with one identified as a previously unreported metabolite by a combination of the MS/MS spectrum and the predicted retention time. Method detection limits for phthalate metabolites in urine were also estimated.