Quantitative Proteomic Analysis Reveals the Key Molecular Events Driving Phaeocystis globosa Bloom and Dissipation.

Phaeocystis globosa is a marine-bloom-forming haptophyte with a polymorphic life cycle alternating between free-living cells and a colonial morphotype, that produces high biomass and impacts ecological structure and function. The mechanisms of P. globosa bloom formation have been extensively studied, and various environmental factors are believed to trigger these events. However, little is known about the intrinsic biological processes that drive the bloom process, and the mechanisms underlying P. globosa bloom formation remain enigmatic. Here, we investigated a P. globosa bloom occurring along the Chinese coast and compared the proteomes of in situ P. globosa colonies from bloom and dissipation phases using a tandem mass tag (TMT)-based quantitative proteomic approach. Among the 5540 proteins identified, 191 and 109 proteins displayed higher abundances in the bloom and dissipation phases, respectively. The levels of proteins involved in photosynthesis, pigment metabolism, nitrogen metabolism, and matrix substrate biosynthesis were distinctly different between these two phases. Ambient nitrate is a key trigger of P. globosa bloom formation, while the enhanced light harvest and multiple inorganic carbon-concentrating mechanisms support the prosperousness of colonies in the bloom phase. Additionally, colonies in the bloom phase have greater carbon fixation potential, with more carbon and energy being fixed and flowing toward the colonial matrix biosynthesis. Our study revealed the key biological processes underlying P. globosa blooms and provides new insights into the mechanisms behind bloom formation.

Phosphorylated and Phosphonated Low-Complexity Protein Segments for Biomimetic Mineralization and Repair of Tooth Enamel.

Biomimetic mineralization based on self-assembly has made great progress, providing bottom-up strategies for the construction of new organic-inorganic hybrid materials applied in the treatment of hard tissue defects. Herein, inspired by the cooperative effects of key components in biomineralization microenvironments, a new type of biocompatible peptide scaffold based on flexibly self-assembling low-complexity protein segments (LCPSs) containing phosphate or phosphonate groups is developed. These LCPSs can retard the transformation of amorphous calcium phosphate into hydroxyapatite (HAP), leading to merged mineralization structures. Moreover, the application of phosphonated LCPS over phosphorylated LCPS can prevent hydrolysis by phosphatases that are enriched in extracellular mineralization microenvironments. After being coated on the etched tooth enamel, these LCPSs facilitate the growth of HAP to generate new enamel layers comparable to the natural layers and mitigate the adhesion of Streptococcus mutans. In addition, they can effectively stimulate the differentiation pathways of osteoblasts. These results shed light on the potential biomedical applications of two LCPSs in hard tissue repair.

A novel STING agonist for cancer immunotherapy and a SARS-CoV-2 vaccine adjuvant.

A novel STING agonist, CDGSF, ipsilaterally modified with phosphorothioate and fluorine, was synthesized. The phosphorothioate in CDGSF might be a site for covalent conjugation. Injection of CDGSF generated an immunogenic ("hot") tumor microenvironment to suppress melanoma, more efficiently than dithio CDG. In particular, immunization with SARS-CoV-2 spike protein using CDGSF as an adjuvant elicited an exceptionally high antibody titer and a robust T cell response, overcoming the drawbacks of aluminum hydroxide. These results highlighted the therapeutic potential of CDGSF for cancer immunotherapy and the adjuvant potential of the STING agonist in the SARS-CoV-2 vaccine for the first time.

A host-guest ATP responsive strategy for intracellular delivery of phosphopeptides.

We developed a host-guest ATP responsive strategy for efficient intracellular delivery of phosphopeptides, employing a pegylated arginine clustered calix[5]arene nanocarrier system that has great capability of recognizing the phosphate moieties on peptides and penetrating the cell membrane.

A site-specific branching poly-glutamate tag mediates intracellular protein delivery by cationic lipids.

Intracellular protein delivery is of significance for cellular protein analysis and therapeutic development, but remains challenging technically. Herein, we report a general and highly potent strategy for intracellular protein delivery based on commercially available cationic lipids. In this strategy, a designed double branching poly-glutamate tag is site-specifically attached onto the C-terminal of protein cargos via expressed protein ligation (EPL), which mediates the entrapment of proteins into cationic liposomes driven by electrostatic interaction. The resultant protein-lipid complexes can enter into cytosol with a high efficiency even at the low protein concentration while maintaining protein's biological activity.

Cathepsin L deficiency results in reactive oxygen species (ROS) accumulation and vascular cells activation.

Recent evidence suggests a link between cathepsin L (CTSL) and vascular diseases. However, its contribution to reactive oxygen species (ROS) homeostasis in the vasculature remains unknown. p66shc is a redox enzyme implicated in mitochondrial ROS generation and translation of oxidative signals. In this study, we explored the relationship between CTSL and oxidative damage in vasculature and whether the oxidative damage is mediated by p66shc.Carotid arteries from aged mice (24 months old) showed a reduction in CTSL expression compared with young wild-type mice (4 months old). Local knockdown of CTSL in carotid arteries of young mice by adenoviral vector encoding the short hairpin RNA targeting CTSL leading to premature vascular aging, as shown by mitochondrial disruption, increased ß-galactosidase-positive cells, reduced telomerase activity, and up-regulation of p66shc. Knockdown of CTSL decreased the expression of mitochondrial oxidative phosphorylation (OXPHOS) complexes I, III, and IV, leading to increased mitochondrial ROS and hyperpolarization of the mitochondrial membrane in vitro. Furthermore, knockdown of CTSL also stimulated ROS production and senescence in vascular cells, accompanied by the up-regulation of p66shc.However, p66shc knockdown blunted the alteration in ROS production, and senescence in CTSL knockdown vascular cells. This study suggests that CTSL knockdown partially induces vascular cells damage via increased ROS production and up-regulation of p66shc.

High Glucose Induces Down-Regulated GRIM-19 Expression to Activate STAT3 Signaling and Promote Cell Proliferation in Cell Culture.

Recent studies indicated that Gene Associated with Retinoid-IFN-Induced Mortality 19 (GRIM-19), a newly discovered mitochondria-related protein, can regulate mitochondrial function and modulate cell viability possibly via interacting with STAT3 signal. In the present study we sought to test: 1) whether GRIM-19 is involved in high glucose (HG) induced altered cell metabolism in both cancer and cardiac cells, 2) whether GRIM-19/STAT3 signaling pathway plays a role in HG induced biological effects, especially whether AMPK activity could be involved. Our data showed that HG enhanced cell proliferation of both HeLa and H9C2 cells, which was closely associated with down-regulated GRIM-19 expression and increased phosphorylated STAT3 level. We showed that GRIM-19 knock-down alone in normal glucose cultured cells can also result in an increase in phosphorylated STAT3 level and enhanced proliferation capability, whereas GRIM-19 over-expression can abolished HG induced STAT3 activation and enhanced cell proliferation. Importantly, both down-regulated or over-expression of GRIM-19 increased lactate production in both HeLa and H9C2 cells. The activated STAT3 was responsible for increased cell proliferation as either AG-490, an inhibitor of JAK2, or siRNA targeting STAT3 can attenuate cell proliferation increased by HG. In addition, HG increased lactate acid levels in HeLa cells, which was also observed when GRIM-19 was genetically manipulated. However, HG did not affect the lactate levels in H9C2 cells. Of note, over-expression of GRIM-19 and silencing of STAT3 both increased lactate production in H9C2 cells. As expected, HG resulted in significant decreases in phosphorylated AMPKα levels in H9C2 cells, but not in HeLa cells. Interestingy, activation of AMPKα by metformin was associated with a reversal of the suppressed GRIM-19 expression in H9C2 cells, the fold of changes in GRIM-19 expression by metformin were much less in HeLa cells. Metformin did not affect the phosphorylated STAT3 lelvels, however, decreased its levels in H9C2, especially in the setting of HG culture. Not like HG alone which resulted in no changes in lactate acid in H9C2 cells, metformin can increase lactate acid levels in H9C2 cells. Increased lactate induced by metformin was also observed in HeLa cells.

An effective biphase system accelerates hesperidinase-catalyzed conversion of rutin to isoquercitrin.

Isoquercitrin is a rare, natural ingredient with several biological activities that is a key precursor for the synthesis of enzymatically modified isoquercitrin (EMIQ). The enzymatic production of isoquercitrin from rutin catalyzed by hesperidinase is feasible; however, the bioprocess is hindered by low substrate concentration and a long reaction time. Thus, a novel biphase system consisting of [Bmim][BF4]:glycine-sodium hydroxide (pH 9) (10:90, v/v) and glyceryl triacetate (1:1, v/v) was initially established for isoquercitrin production. The biotransformation product was identified using liquid chromatography-mass spectrometry, and the bonding mechanism of the enzyme and substrate was inferred using circular dichroism spectra and kinetic parameters. The highest rutin conversion of 99.5% and isoquercitrin yield of 93.9% were obtained after 3 h. The reaction route is environmentally benign and mild, and the biphase system could be reused. The substrate concentration was increased 2.6-fold, the reaction time was reduced to three tenths the original time. The three-dimensional structure of hesperidinase was changed in the biphase system, which α-helix and random content were reduced and ß-sheet content was increased. Thus, the developed biphase system can effectively strengthen the hesperidinase-catalyzed synthesis of isoquercitrin with high yield.

[Detection of serum desmoglein antibody level using enzyme-linked immunosorbent assay (ELISA) for monitoring disease activity in patients with pemphigus vulgaris].

OBJECTIVE: Pemphigus is an autoimmune blistering disease of skin and mucous membranes. Pemphigus vulgaris (PV) is a major subtype of pemphigus, which is histologically characterized by suprabasal acantholysis. The major antigen in PV is desmosomal glycoproteins desmoglein (Dsg) 3. The autoantibodies against Dsg3 cause loss of adhesion between keratinocytes. Some PV patients also have circulating anti-Dsg1 autoantibodies. Enzyme-linked immunosorbent assays (ELISAs) with recombinant Dsg3 and Dsg1 are highly sensitive and specific for detecting anti-Dsg3 and anti-Dsg1 autoantibodies in PV patients. To evaluate the role of desmosomal glycoproteins desmoglein (Dsg3) ELISA and Dsg1 ELISA for detecting anti-Dsg3 and anti-Dsg1 autoantibodies in monitoring disease activity in Pemphigus vulgaris patients. METHODS: Twenty PV patients with long-term follow-up were included. We tested their serial sera with modified Dsg3 ELISA (MESACUP Desmoglein TEST "Dsg3", Medical & Biological Laboratories Co. LTD.), Dsg1 ELISA(MESACUP Desmoglein TEST "Dsg1", Medical & Biological Laboratories Co. LTD.) and indirect immunofluorescence (IIF). Then we analyzed the correlation between Dsg3 ELISA index values, Dsg1 ELISA index values, IIF titres and disease activity scores (ABSIS) along the time course. RESULTS: There were significant correlations between Dsg3 ELISA index values, Dsg1 ELISA index values, IIF titres and disease activity scores (both skin scores and oral scores) (P<0.01) along the time course. Significant differences of Dsg3 ELISA index values, Dsg1 ELISA index values and IIF titres between active time-point group and clinical remission time-point group were also observed (P<0.01). We found that Dsg3 ELISA index values, Dsg1 ELISA index values and IIF titres fluctuated in parallel with disease activity, and ELISA index values were superior to IIF titres. CONCLUSION: Dsg3 ELISA index values fluctuating in parallel with disease activity are useful to monitor disease activity, predict flares or relapses and plan the schedules for tapering the drugs.

Coronary flow velocity pattern and recovery of regional left ventricular function: the relationship observed in patients with reperfused acute myocardial infarction.

Coronary flow velocity pattern (CFVP) recorded within 3 days of percutaneous coronary intervention (PCI) has been reported to be useful in predicting left ventricular (LV) function. The aim of this prospective study was to investigate, via transthoracic Doppler echocardiography, whether the relationship between CFVP and recovery of LV function persists. Our study group comprised 37 patients with 1st anterior-wall acute myocardial infarction who underwent successful PCI for lesions in the left anterior descending coronary artery (LAD). The CFVP in the LAD was recorded at 24-48 hours, 7 days, and 4 weeks after PCI. Myocardial contrast echocardiography was performed at 24-48 hours after PCI. The diastolic deceleration time (DDT) at each stage correlated significantly with the regional LV wall-motion score index at 6-month follow-up (r=-0.58 at 24-48 hr, -0.57 at day 7, and -0.50 at week 4; P <0.01 for all). The mean DDT increased over time. Optimal cutoff values for DDT to predict regional LV wall-motion score indices of <2.0 were 327 ms at 24-48 hours (sensitivity, 0.78; specificity, 0.64), 495 ms at day 7 (sensitivity, 0.75; specificity, 0.69), and 525 ms at week 4 (sensitivity, 0.83; specificity, 0.69). The DDT at 24-48 hours significantly correlated, better than the peak creatine kinase value, with reperfusion (r=0.68, P <0.01) as defined by myocardial contrast echocardiography. In conclusion, CFVP in the LAD can be used, within 4 weeks after PCI, to predict the recovery of regional LV function in patients with reperfused anterior-wall acute myocardial infarction.