Discovery and Characterization of an Acid-Labile Serine-Lysine Cross-Link in Antibody High-Molecular-Weight Species Using a Multipronged Mass Spectrometry Approach.

Characterizing the cross-links responsible for the covalent high-molecular-weight (HMW) species in therapeutic monoclonal antibodies (mAbs) is of great importance as it not only provides a framework for risk assessment but also offers insights for process improvement. However, owing to the complexity and low abundance, identification of novel and unknown cross-links in mAb products can be very challenging. Here, applying a multipronged MS-based approach, we report the discovery of a novel covalent cross-link formed via an imine bond between lysine and serine residues. In particular, this Ser-Lys cross-link was found to be acid-labile and can be easily overlooked by conventional LC-MS techniques operated at low pH. It is worth noting that although imine-based cross-link has been previously reported in collagen protein cross-linking, this is the first time that a Ser-Lys cross-link has been found in a mAb product that contributes to covalent HMW species formation.

Decrypting the Controlled Product Selectivity over Ag-Cu Bimetallic Surface Alloys for Electrochemical CO2 Reduction.

Electrochemical CO2 reduction reaction (ECO2 RR) with controlled product selectivity is realized on Ag-Cu bimetallic surface alloys, with high selectivity towards C2 hydrocarbons/alcohols (≈60 % faradaic efficiency, FE), C1 hydrocarbons/alcohols (≈41 % FE) and CO (≈74 % FE) achieved by tuning surface compositions and applied potentials. In situ spectral investigations and theoretical calculations reveal that surface-composition-dependent d-band center could tune *CO binding strengths, regulating the *CO subsequent reaction pathways and then the product selectivity. Further adjusting the applied potentials will alter the energy of participated electrons, which leads to controlled ECO2 RR selectivity towards desired products. A predominant region map, with an indicator proposed to evaluate the thermodynamic predominance of the *CO subsequent reactions, is then provided as a reliable theoretical guidance for the controllable ECO2 RR product selectivity over bimetallic alloys.

Osmoregulatory strategies of estuarine fish Scatophagus argus in response to environmental salinity changes.

BACKGROUND: Scatophagus argus, an estuarine inhabitant, can rapidly adapt to different salinity environments. However, the knowledge of the molecular mechanisms underlying its strong salinity tolerance remains unclear. The gill, as the main osmoregulatory organ, plays a vital role in the salinity adaptation of the fish, and thus relative studies are constructive to reveal unique osmoregulatory mechanisms in S. argus. RESULTS: In the present study, iTRAQ coupled with nanoLC-MS/MS techniques were employed to explore branchial osmoregulatory mechanisms in S. argus acclimated to different salinities. Among 1,604 identified proteins, 796 differentially expressed proteins (DEPs) were detected. To further assess osmoregulatory strategies in the gills under different salinities, DEPs related to osmoregulatory (22), non-directional (18), hypo- (52), and hypersaline (40) stress responses were selected. Functional annotation analysis of these selected DEPs indicated that the cellular ion regulation (e.g. Na+-K+-ATPase [NKA] and Na+-K+-2Cl- cotransporter 1 [NKCC1]) and ATP synthesis were deeply involved in the osmoregulatory process. As an osmoregulatory protein, NKCC1 expression was inhibited under hyposaline stress but showed the opposite trend in hypersaline conditions. The expression levels of NKA α1 and ß1 were only increased under hypersaline challenge. However, hyposaline treatments could enhance branchial NKA activity, which was inhibited under hypersaline environments, and correspondingly, reduced ATP content was observed in gill tissues exposed to hyposaline conditions, while its contents were increased in hypersaline groups. In vitro experiments indicated that Na+, K+, and Cl- ions were pumped out of branchial cells under hypoosmotic stress, whereas they were absorbed into cells under hyperosmotic conditions. Based on our results, we speculated that NKCC1-mediated Na+ influx was inhibited, and proper Na+ efflux was maintained by improving NKA activity under hyposaline stress, promoting the rapid adaptation of branchial cells to the hyposaline condition. Meanwhile, branchial cells prevented excessive loss of ions by increasing NKA internalization and reducing ATP synthesis. In contrast, excess ions in cells exposed to the hyperosmotic medium were excreted with sufficient energy supply, and reduced NKA activity and enhanced NKCC1-mediated Na+ influx were considered a compensatory regulation. CONCLUSIONS: S. argus exhibited divergent osmoregulatory strategies in the gills when encountering hypoosmotic and hyperosmotic stresses, facilitating effective adaptabilities to a wide range of environmental salinity fluctuation.

Reduction of the Influence of Laser Beam Directional Dithering in a Laser Triangulation Displacement Probe.

Directional dithering of a laser beam potentially limits the detection accuracy of a laser triangulation displacement probe. A theoretical analysis indicates that the measurement accuracy will linearly decrease as the laser dithering angle increases. To suppress laser dithering, a scheme for reduction of the influence of laser beam directional dithering in a laser triangulation displacement probe, which consists of a collimated red laser, a laser beam pointing control setup, a receiver lens, and a charge-coupled device, is proposed in this paper. The laser beam pointing control setup is inserted into the source laser beam and the measured object and can separate the source laser beam into two symmetrical laser beams. Hence, at the angle at which the source laser beam dithers, the positional averages of the two laser spots are equal and opposite. Moreover, a virtual linear function method is used to maintain a stable average of the positions of the two spots on the imaging side. Experimental results indicate that with laser beam pointing control, the estimated standard deviation of the fitting error decreases from 0.3531 mm to 0.0100 mm , the repeatability accuracy can be lowered from ±7 mm to ±5 µ m , and the nonlinear error can be reduced from ±6 % FS (full scale) to ±0.16 % FS.

Hepatic stellate cells promote tumor progression by enhancement of immunosuppressive cells in an orthotopic liver tumor mouse model.

The immunosuppressive properties of hepatic stellate cells (HSCs) contribute to the occurrence and development of hepatocellular carcinoma (HCC). The accumulation of cells with immune suppressive activities, such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) is a key mechanism for tumor immune evasion. However, the impact of HSCs on immune cell populations in tumor-bearing hosts is unclear. In this study, we established an orthotopic liver tumor mouse model for studying the complex tumor-host interactions in HCC. The activated HSCs promoted HCC growth not only induced tumor angiogenesis and lymphangiogenesis, but also significantly increased the suppressive immune cell population of Tregs and MDSCs in the spleen, bone marrow, and tumor tissues of the tumor-bearing mice. Murine HCC cell line H22-activated HSCs also expanded the expression of Tregs and MDSCs in vitro. In conclusion, our study suggests a novel role for HSCs in the HCC microenvironment. HSCs can promote HCC progression by enhancement of the immunosuppressive cell population. Targeting HSCs, which is a new concept in adjuvant immunotherapy, may be introduced in the near future to improve the outcome of patients with HCC.

Investigation on uniaxial compression and fracture damage mode of prefabricated parallel double-jointed red sandstone.

As a special type of joint fracture, the fracture evolution characteristics of parallel double joints have important engineering significance for the stability analysis of fractured rock mass. In this work, a new method for calculating stress intensity factor of parallel double-jointed fractures was importantly proposed. Physical uniaxial compression tests were carried out on parallel double jointed red sandstone filled with cement mortar under different geometric parameters, and the macroscopic mechanical properties and failure characteristics of red sandstone are deeply analyzed. The results show that the larger the connectivity rate is, the smaller the peak stress and strain are. The increase of connectivity rate will affect the change rate of transverse strain in the center of rock bridge. The closer the dip angle of the joint is, the lower the peak stress is and the shorter the failure time is. The damage mode of joint tip encroachment affects the lateral displacement of the rock bridge center, and the displacement is always close to the first damage section. The closer the joint tip is to the load, the easier the end-face penetrating cracks occur. The research content can provide basic support for guaranteeing the stability of underground engineering rock mass.

Copper nanoclusters-doped novel carrier with synergistic adsorption-catalytic active sites to enable high-performance dye removal.

Enhancing the synergistic interplay between adsorption and catalytic oxidation to amplify Fenton-like effects remains a pivotal challenge in advancing water pollution remediation strategies. In this study, a suite of novel carriers (SH) composed of silica (SiO2) and hydroxyapatite (HAp) in different ratios were synthesized through an amalgamation of the sol-gel and co-precipitation techniques. Notably, various forms of copper (Cu) species, including Cu2+ ions and Cu nanoclusters (Cu NCs), could be stably incorporated onto the SH surface via meticulous loading and doping techniques. This approach has engendered a new class of Fenton-like catalysts (Cu NCs-SH1-5) characterized by robust acid-base tolerance stability and remarkable recyclability. Compared with the previously reported Cu NCs-HAp, this catalyst with lower Cu species content could achieve better performance in adsorbing and degrading dyes under the aid of hydrogen peroxide (H2O2). The catalyst's dual action sites, specifically the adsorption sites (SiOH, POH, slit pores) and catalytic centers (multivalent Cu species), had clear division of labor and collaborate with each other. Further, reactive oxygen species (ROS) identification and astute electrochemical testing have unveiled the mechanism underpinning the cooperative degradation of dyes by three types of ROS, spawned through electron transfer between the Fenton-like catalyst (Cu NCs-SH) and H2O2. From these insights, the mechanism of synergistic adsorption-catalytic removal was proposed.

18ß-glycyrrhetinic acid inhibits hepatocellular carcinoma development by reversing hepatic stellate cell-mediated immunosuppression in mice.

Hepatic stellate cells (HSCs) have immunosuppressive capabilities and contribute to the occurrence and development of hepatocellular carcinoma (HCC). Thus, activated HSCs may be a suitable target for HCC therapy. Our study used mixed leukocyte reactions (MLR) in vitro to demonstrate that 18ß-glycyrrhetinic acid (GA) could reverse HSC-mediated immunosuppression by reducing T-cell apoptosis and regulatory T (Treg) cells expression, thereby enhancing the ability of T cells to attack tumor cells and attenuating HCC cell invasiveness. Moreover, we established a HCC orthotopic implantation model in immunocompetent C57BL/6 mice, which suggested that GA played a protective role in HCC development by reducing immunosuppression mediated by HSCs in the tumor microenvironment.

A competitive binding-mass spectrometry strategy for high-throughput evaluation of potential critical quality attributes of therapeutic monoclonal antibodies.

Therapeutic monoclonal antibodies (mAbs) have a propensity to host a large number of chemical and enzymatical modifications that need to be properly assessed for their potential impact on target binding. Traditional strategies of assessing the criticality of these attributes often involve a laborious and low-throughput variant enrichment step prior to binding affinity measurement. Here, we developed a novel competitive binding-based enrichment strategy followed by mass spectrometry analysis (namely, competitive binding-MS) to achieve high-throughput evaluation of potential critical quality attributes in therapeutic mAbs. Leveraging the differences in target binding capability under competitive binding conditions, the criticality of multiple mAb attributes can be simultaneously evaluated by quantitative mass spectrometry analysis. The utility of this new workflow was demonstrated in three mAb case studies, where different post-translational modifications occurring within the complementarity-determining regions were successfully interrogated for their impact on antigen binding. As this workflow does not require prior enrichment (e.g., by forced degradation or liquid chromatography fractionation) of the variants, it is particularly valuable during the mAb candidate developability assessment, where fast turn-around time is highly desired to assist candidate selection.Abbreviations: ACN: acetonitrile; ADCC: antibody-dependent cell-mediated cytotoxicity; AEX: anion exchange chromatography; bsAb: bispecific antibody; CDC: complement-dependent cytotoxicity; CDR: complementarity-determining region; CML: carboxymethylation; CQA: critical quality attribute; DDA: data-dependent acquisition; DMSO: dimethyl sulfoxide; DTT: dithiothreitol; FA: formic acid; Fab: Fragment antigen-binding; FcRn: neonatal Fc receptor; HC: heavy chain; HIC: hydrophobic interaction chromatography; IAA: iodoacetamide; IEX: ion exchange chromatography; LC: light chain; mAb monoclonal antibody; msAb: monospecific antibody; MS: mass spectrometry; PBS: phosphate-buffered saline; pI: isoelectric point; PTM: post-translational modification; SCX: strong cation exchange chromatography; SEC: size exclusion chromatography; SPR: surface plasmon resonance; XIC: extracted ion chromatography.

A New Process of Preparing Rice Straw-Reinforced LLDPE Composite.

To reduce the pollution resulting from discarding waste plastic film and burning straw, a new method of preparing straw-reinforced LLDPE composites was developed to utilize these wastes. The straws were first laid parallel on an LLDPE film and then rolled up. The rolls containing long straws were laid into a mat and then hot-pressed into a long straw composite board (the mass of straw accounted for 60%). Slope-cutting the straw, grinding the straw, and twisting the roll were designed to improve the physical and mechanical properties of long straw composites. Among them, slope-cutting the straw combined with twisting the roll provided the best properties. Compared to the extruded straw particle composite, the composite prepared with the new method improved the tensile strength, bending strength, impact strength, and water resistance by 358%, 151%, 416%, and 81%, respectively. Slope-cutting exposed more inner surface at the end of the straw. Scanning electron microscope observations showed that the straw inner surface was more tightly bonded with the LLDPE matrix than the outer surface. Meanwhile, the integrity of the straw was retained as much as possible, and thus greatly improved the performance of the resulting composites. Dynamic mechanical analysis, differential scanning calorimetry, and thermogravimetric analysis show that the viscous deformation of the composites prepared by the new method was reduced and the rigidity was increased, and the combination of straw and LLDPE forms a dense composite material with good interfacial bonding. It greatly slowed down the degree of its pyrolysis.

Development of a chromatography-free method for high-throughput MS-based bioanalysis of therapeutic monoclonal antibodies.

Aim: Our objective was to test the feasibility of developing an LC-free, MS-based approach for high-throughput bioanalysis of humanized therapeutic monoclonal antibodies. Methodology: A universal tryptic peptide from human IgG1, IgG3 and IgG4 was selected as the surrogate peptide for quantitation. After tryptic digestion, the surrogate peptide was fractionated via solid-phase extraction before being subjected to direct infusion-based MS/MS analysis. A high-resolution, multiplexed (MSX = 2) parallel reaction monitoring method was developed for data acquisition. Results & conclusion: This proof-of-concept study demonstrated the feasibility of achieving high-throughput MS-based bioanalysis of monoclonal antibodies using an LC-free workflow with sensitivity comparable to conventional LC-MS/MS-based methods.

Post-Column Denaturation-Assisted Native Size-Exclusion Chromatography-Mass Spectrometry for Rapid and In-Depth Characterization of High Molecular Weight Variants in Therapeutic Monoclonal Antibodies.

The high molecular weight (HMW) size variants present in therapeutic monoclonal antibody (mAb) samples need to be closely monitored and characterized due to their impact on product safety and efficacy. Because of the complexity and often low abundances in final drug substance (DS) samples, characterization of such HMW species is challenging and traditionally requires offline enrichment of the HMW species followed by analysis using various analytical tools. Here, we report the development of a postcolumn denaturation-assisted native SEC-MS method that allows rapid and in-depth characterization of mAb HMW species directly from unfractionated DS samples. This method not only provides high-confidence identification of HMW complexes based on accurate mass measurement of both the intact assembly and the constituent subunits but also allows in-depth analysis of the interaction nature and location. In addition, using the extracted ion chromatograms, derived from high-quality, native-like mass spectra, the elution profiles of each noncovalent and/or nondissociable complex can be readily reconstructed, facilitating the comprehension of a complex HMW profile. The utility of this novel method was demonstrated in different applications, ranging from enriched HMW characterization at late stage development, comparability assessment due to process changes, and forced degradation study of coformulated mAbs. As this method does not require prior enrichment, it is thus desirable for providing both rapid and in-depth characterization of HMW species during the development of therapeutic mAbs.

Preparation of Selenocysteine-Containing Forms of Human SELENOK and SELENOS.

Selenoprotein K (SELENOK) and Selenoprotein S (SELENOS) are the members of the endoplasmic-reticulum-associated degradation (ERAD) complex, which is responsible for translocating misfolded proteins from the endoplasmic reticulum (ER) to the cytosol for degradation. Besides its involvement in the ERAD, SELENOK was shown to bind and stabilize the palmitoyl transferase DHHC6, and thus contributes to palmitoylation. SELENOK and SELENOS reside in the ER membrane by the way of a single transmembrane helix. Both contain an intrinsically disordered region with a selenocysteine (Sec) located one or two residues away from the C-terminus. Here, we describe the preparation of the Sec-containing forms of SELENOS and SELENOK. SELENOK, which contains no native cysteines, was prepared in an E. coli cysteine auxotroph strain by exploiting the codon and the insertion machinery of Cys for the incorporation of Sec. In contrast, the preparation of SELENOS, which contains functionally important cysteine residues, relied on E. coli's native Sec incorporation mechanism.

Quantification of Membrane Protein-Detergent Complex Interactions.

Although fundamentally significant in structural, chemical, and membrane biology, the interfacial protein-detergent complex (PDC) interactions have been modestly examined because of the complicated behavior of both detergents and membrane proteins in aqueous phase. Membrane proteins are prone to unproductive aggregation resulting from poor detergent solvation, but the participating forces in this phenomenon remain ambiguous. Here, we show that using rational membrane protein design, targeted chemical modification, and steady-state fluorescence polarization spectroscopy, the detergent desolvation of membrane proteins can be quantitatively evaluated. We demonstrate that depleting the detergent in the sample well produced a two-state transition of membrane proteins between a fully detergent-solvated state and a detergent-desolvated state, the nature of which depended on the interfacial PDC interactions. Using a panel of six membrane proteins of varying hydrophobic topography, structural fingerprint, and charge distribution on the solvent-accessible surface, we provide direct experimental evidence for the contributions of the electrostatic and hydrophobic interactions to the protein solvation properties. Moreover, all-atom molecular dynamics simulations report the major contribution of the hydrophobic forces exerted at the PDC interface. This semiquantitative approach might be extended in the future to include studies of the interfacial PDC interactions of other challenging membrane protein systems of unknown structure. This would have practical importance in protein extraction, solubilization, stabilization, and crystallization.

1,1-Disubstituted olefin synthesis via Ni-catalyzed Markovnikov hydroalkylation of alkynes with alkyl halides.

A Ni-catalyzed Markovnikov hydroalkylation of alkynes with alkyl halides is described. The reaction proceeds smoothly without the use of sensitive organometallic reagents and shows good functional-group compatibility, enabling the efficient synthesis of a variety of 1,1-disubstituted olefins. It also provides a straightforward approach for the modification of complex organic molecules.

Cu-catalyzed cross-coupling reactions of epoxides with organoboron compounds.

A copper-catalyzed cross-coupling reaction of epoxides with arylboronates is described. This reaction is not limited to aromatic epoxides, because aliphatic epoxides are also suitable substrates. In addition, N-sulfonyl aziridines can be successfully converted into the products. This reaction provides convenient access to ß-phenethyl alcohols, which are valuable synthetic intermediates.

Selenoprotein K form an intermolecular diselenide bond with unusually high redox potential.

Selenoprotein K (SelK) is a membrane protein involved in antioxidant defense, calcium regulation and the ER-associated protein degradation pathway. We found that SelK exhibits a peroxidase activity with a rate that is low but within the range of other peroxidases. Notably, SelK reduced hydrophobic substrates, such as phospholipid hydroperoxides, which damage membranes. Thus, SelK might be involved in membrane repair or related pathways. SelK was also found to contain a diselenide bond-the first intramolecular bond of that kind reported for a selenoprotein. The redox potential of SelK was -257 mV, significantly higher than that of diselenide bonds in small molecules or proteins. Consequently, SelK can be reduced by thioredoxin reductase. These finding are essential for understanding SelK activity and function.

Efficacy of mesenchymal stem cells derived from human adipose tissue in inhibition of hepatocellular carcinoma cells in vitro.

Hepatocellular carcinoma (HCC) is often diagnosed at an advanced stage, and over the past several decades, many researchers have worked to develop novel effective therapies for HCC patients. The functional contributions of mesenchymal stem cells to human malignancies, including HCC growth and progression, are controversial, and the potential mechanisms underlying these effects are not clear. The aim of this study was to investigate the effect of adipose-derived mesenchymal stem cells (ADSCs) on the growth of HCC cells. In this study, a conditioned medium from ADSCs (ADSC-CM) efficiently inhibited HCC cell proliferation and division, and induced HCC cell death through the downregulation of Akt signaling. These findings indicated that the ADSC-CM could inhibit HCC growth. Thus, the ADSC-CM is a good candidate for the treatment of HCC patients for whom no effective therapy is available.

Preconcentration with membrane cell and adsorptive polarographic determination of formaldehyde in air.

The present paper describes a procedure that formaldehyde in air was preconcentrated in a membrane cell and its content was determined by adsorptive polarography. First the formaldehyde in air samples was preconcentrated in a membrane cell using water, then the formaldehyde reacted with 2,4-dinitrophenyl hydrazine to form 2,4-dinitrophenyl hydrazone, which can be adsorbed at the mercury electrode and yields a sensitive adsorptive polarographic wave. Over the range 6.0x10(-10)-5.0x10(-6) M, the peak currents are linearly proportional to the concentration of formaldehyde, the detection limit is 2.0x10(-10) M.