Scavenging ROS: Superoxide Dismutase/Catalase Mimetics by the Use of an Oxidation-Sensitive Nanocarrier/Enzyme Conjugate

Reactive Oxygen Species (ROS) are quintessential inflammatory compounds with oxidizing behavior. We have successfully developed a micellar system with responsiveness at the same time to two of the most important ROS: superoxide and hydrogen peroxide. This allows for an effective and selective capture of the two compounds and, in perspective, for inflammation-responsive drug release. The system is composed of superoxide dismutase (SOD) conjugated to oxidation-sensitive amphiphilic polysulfide/PEG block copolymers; the conjugate combines the SOD reactivity toward superoxide with that of hydrophobic thioethers toward hydrogen peroxide. Specifically, here we have demonstrated how this hybrid system can efficiently convert superoxide into hydrogen peroxide, which is then “mopped-up” by the polysulfides: this <i>modus operandi</i> is functionally analogous to the SOD/catalase combination, with the advantages of (a) being based on a single and more stable system, and (b) a higher overall efficiency due the physical proximity of the two ROS-reactive centers (SOD and polysulfides)

TM domain separation but not α_IIb cytoplasmic domain is required for recruitment and phosphorylation of paxillin.

<p>(A) Recruitment of phosphorylated paxillin (PaxpY31, green) to FA sites (blue) was observed in both wild type (B1) and α_IIb-truncated mutant (B3) but not in the two disulfide-bonded mutants (B2 and B4). Note that recruitment of phosphorylated paxillin was normal in the α_IIb-truncated mutant indicating a dispensable role of the α_IIb cytoplasmic domain in paxillin recruitment. (B) Addition of 1mM DTT restored recruitment of phosphorylated paxillin in both of the disulfide-bonded mutants. White bar: 10μm.</p

All α_IIbβ₃ mutants were successfully expressed with high disulfide-bridge formation efficiency.

<p>(A)Integrin α_IIbβ₃ constructs used in our study. B1: Wild type integrin α_IIbβ₃; B2: Disulfide-bonded α_IIbβ₃(α_IIb_W968C, β₃_I693C); B3: α truncated α_IIbβ₃(α_IIb1-990); B4: α truncated, disulfide-bonded α_IIbβ₃ (α_IIb1-990_W968C, β₃_I693C). (B) Cell lines stably expressing WT and mutated integrin α_IIbβ₃ as evaluated by three different monoclonal antibodies. B1 (WT α_IIbβ₃), B2 (disulfide-bonded α_IIbβ₃ mutant), B3 (α-truncated α_IIbβ₃), and B4 (disulfide-bonded α-truncated α_IIbβ₃). The solid line and dashed line represent untransfected CHO-K1 cells and stable transfectants, respectively. AP3, 7E3, and 10E5 are mAbs targeting different domains of the α_IIbβ₃ heterodimer. (C) Disulfide bonds formed between α and β subunits with high efficiency. Cells labeled with S³⁵ were lysed and subjected to immunoprecipitation by anti-α_IIb mAb 10E5. Immunoprecipitated protein was then resolved by SDS-PAGE and visualized by radioautography. 2mM DTT was used to reduce the disulfide bridge in TM-clasped mutants.</p

Inhibition of PI3K and Src did not affect cell spreading, FAK/paxillin recruitment, or phosphorylation.

<p>(A, B) FAK and paxillin were phosphorylated and recruited to FAs normally in WT (B1) and the α_IIb-truncated mutant (B3) with control treatment (DMSO). TM clasping abolished the events as previously observed; (C, D) Treatment with 1.5μM/mL Wortmannin did not affect recruitment of phosphorylated FAK and paxillin. (E, F) PP1 (15μM/mL) had little to no effect on cell spreading with regard to FAK/paxillin phosphorylation and recruitment. White bar: 10μm.</p

TM domain disassociation is required for cytoplasmic domain dissociation-induced high affinity integrin as measured by soluble ligand binding.

<p>Binding of ligand mimetic mAb PAC-1(A) and fibrinogen (Fbg, B) to B1 through B4 cells in the presence of either EDTA (5mM), Ca²⁺ (5mM), Mn²⁺ (1mM), or Ca²⁺(5mM) with DTT (4mM) as indicated. Error bars represent standard deviation (S.D.) from three independent assays.</p

TM domain separation is required for FA formation, actin fiber organization, FAK activation and recruitment to FA sites in outside-in signaling.

<p>(A) Clasping of TM domains ablated FA formation and disrupted actin filaments organization. Note that α truncation led to an even distribution of FAs around adherent cells. (B) Treatment with 1mM DTT largely restored cell spreading and FA assembly. Green: focal adhesions (labeled with anti-vinculin antibody); Red: actin filaments (labeled with TRITC-conjugated Phalloidin). White bar: 10μm. (C) Activated FAK (FAKpY397) was recruited normally to FAs in WT (B1) and the α-truncated mutant (B3), but recruitment was abolished by clasping the TM domains (B2 and B4). (D) Treatment with 1mM DTT restored recruitment of phosphorylated FAK to FAs. Green: FAKpY397; Red: actin filaments; Blue: FA marker vinculin. White bar: 10μm.</p

TM separation promotes activation of FAK whereas PI3K regulates Akt and Erk1/2 but not FAK in outside-in signaling.

<p>Cells were seeded on Fbg (20μg/mL)-coated dishes with or without 1mM DTT or with 1.5μM/mL Wortmannin at 37°C for 1h and then lysed and subjected to western blot. Cells seeded on 1% BSA coated dishes were used as control. (A)FAK activation induced by immobilized ligand Fbg is TM domain separation dependent. TM-clasped mutants expressed reduced phosphorylation on Y397. (B) Treatment with Wortmannin ablated Akt and Erk1/2 activation. Removing Wortmannin before cell spreading restored Erk1/2 activation without inducing Akt activation. This may implicate that PI3K but not Akt activity is required for cell survival and proliferation mediated by Erk1/2 in outside-in signaling. (C) Wortmannin treatment did not alter activation of FAK, implicating parallel pathways in outside-in signaling.</p

Asymmetric Total Synthesis of Nannocystin A

Nannocystin A is a novel 21-membered macrolactone isolated from myxobacterium <i>Nanocystis</i> sp. It is a potent elongation factor 1 inhibitor and inhibits cancer cell line growth at nanomolar concentrations. In this work, a concise asymmetric total synthesis of nannocystin A has been developed, which features Sharpless epoxidation, Stille coupling, and final macrolactamization

Data_Sheet_1_Validation of the Chinese Cultural Tightness–Looseness Scale and General Tightness–Looseness Scale.pdf

IntroductionThis study aims to revise the Cultural Tightness–Looseness Scale (CTLS) and General Tightness–Looseness Scale (GTLS), and explore the group heterogeneity of tightness–looseness perception in Chinese populations.MethodsSample 1 (n = 2,388) was used for item analysis and exploratory factor analysis, and sample 2 (n = 2,385) was used for confirmatory factor analysis and latent profile analysis. Sample 3 (n = 512) was used for the reliability test and criterion validity test, among which 162 participants were used for the test–retest reliability examination after a four-week interval. Measurements included the CTLS, GTLS, International Personality Item Pool, Personal Need for Structure Scale, and Campbell Index of Well-Being.ResultsThe revised CTLS contained four items and retained a single-dimensional structure. The revised GTLS consisted of eight items divided into two dimensions: Compliance with Norms and Social Sanctions. Latent profile analysis extracted two profiles on both CTLS and GTLS scores, indicating that the sample can be divided into two subgroups: high and low perception of tightness.DiscussionThe Chinese versions of the CTLS and GTLS can be used as valid and reliable measures of tightness–looseness perception in a Chinese population.</p

Use of In Vitro Systems To Model In Vivo Degradation of Therapeutic Monoclonal Antibodies

Major degradation pathways such as deamidation, isomerization, oxidation, and glycation may be accelerated after administration of antibody therapeutics to the patient. Tracking in vivo product degradation is important because certain post-translational modifications can inactivate the protein and reduce product efficacy. However, in vivo characterization of protein therapeutics is not routinely performed because of technical challenges and limited sample availability. In vitro models offer several potential advantages, including larger sample supplies, simpler and faster methods for sample preparation and analysis, and the potential to distinguish differences in product degradation from differences in product clearance. In this study, we compared the rates of in vivo product degradation using mAb1 recovered from clinical serum samples with the rates of in vitro product degradation using mAb1 recovered from spiked phosphate buffered saline (PBS) and spiked human serum samples to determine if results from the in vitro model systems could be used to predict the in vivo results. The antibody samples were characterized by peptide mapping or intact mass analysis to quantify multiple quality attributes simultaneously, including deamidation, isomerization, oxidation, N-terminal pyroglutamate formation, and glycation. It was clearly demonstrated that both the spiked PBS and spiked serum models were effective in predicting in vivo results for deamidation, isomerization, N-terminal pyroglutamate formation and glycation, whereas only the spiked serum model was effective in predicting in vivo results for oxidation