Mapping conformational changes on bispecific antigen-binding biotherapeutic by covalent labeling and mass spectrometry.

Biotherapeutic's higher order structure (HOS) is a critical determinant of its functional properties and conformational relevance. Here, we evaluated two covalent labeling methods: diethylpyrocarbonate (DEPC)-labeling and fast photooxidation of proteins (FPOP), in conjunction with mass spectrometry (MS), to investigate structural modifications for the new class of immuno-oncological therapy known as bispecific antigen-binding biotherapeutics (BABB). The evaluated techniques unveiled subtle structural changes occurring at the amino acid residue level within the antigen-binding domain under both native and thermal stress conditions, which cannot be detected by conventional biophysical techniques, e.g., near-ultraviolet circular dichroism (NUV-CD). The determined variations in labeling uptake under native and stress conditions, corroborated by binding assays, shed light on the binding effect, and highlighted the potential of covalent-labeling methods to effectively monitor conformational changes that ultimately influence the product quality. Our study provides a foundation for implementing the developed techniques in elucidating the inherent structural characteristics of novel therapeutics and their conformational stability.

Intelligence Sparse Sensor Network for Automatic Early Evaluation of General Movements in Infants.

General movements (GMs) have been widely used for the early clinical evaluation of infant brain development, allowing immediate evaluation of potential development disorders and timely rehabilitation. The infants' general movements can be captured digitally, but the lack of quantitative assessment and well-trained clinical pediatricians presents an obstacle for many years to achieve wider deployment, especially in low-resource settings. There is a high potential to explore wearable sensors for movement analysis due to outstanding privacy, low cost, and easy-to-use features. This work presents a sparse sensor network with soft wireless IMU devices (SWDs) for automatic early evaluation of general movements in infants. The sparse network consisting of only five sensor nodes (SWDs) with robust mechanical properties and excellent biocompatibility continuously and stably captures full-body motion data. The proof-of-the-concept clinical testing with 23 infants showcases outstanding performance in recognizing neonatal activities, confirming the reliability of the system. Taken together with a tiny machine learning algorithm, the system can automatically identify risky infants based on the GMs, with an accuracy of up to 100% (99.9%). The wearable sparse sensor network with an artificial intelligence-based algorithm facilitates intelligent evaluation of infant brain development and early diagnosis of development disorders.

Classification and Analysis of Human Body Movement Characteristics Associated with Acrophobia Induced by Virtual Reality Scenes of Heights.

Acrophobia (fear of heights), a prevalent psychological disorder, elicits profound fear and evokes a range of adverse physiological responses in individuals when exposed to heights, which will lead to a very dangerous state for people in actual heights. In this paper, we explore the behavioral influences in terms of movements in people confronted with virtual reality scenes of extreme heights and develop an acrophobia classification model based on human movement characteristics. To this end, we used wireless miniaturized inertial navigation sensors (WMINS) network to obtain the information of limb movements in the virtual environment. Based on these data, we constructed a series of data feature processing processes, proposed a system model for the classification of acrophobia and non-acrophobia based on human motion feature analysis, and realized the classification recognition of acrophobia and non-acrophobia through the designed integrated learning model. The final accuracy of acrophobia dichotomous classification based on limb motion information reached 94.64%, which has higher accuracy and efficiency compared with other existing research models. Overall, our study demonstrates a strong correlation between people's mental state during fear of heights and their limb movements at that time.

Characterization of bispecific antigen-binding biotherapeutic fragmentation sites using microfluidic capillary electrophoresis coupled to mass spectrometry (mCZE-MS).

Fragmentation of therapeutic proteins is a potential critical quality attribute (CQA) that can occur in vivo or during manufacturing or storage due to enzymatic and non-enzymatic degradation pathways, such as hydrolysis, peroxide mediation, and acid/metal catalysis. Characterization of the fragmentation pattern of a therapeutic protein is traditionally accomplished using capillary gel electrophoresis with UV detection under both non-reducing and reducing conditions (nrCGE and rCGE). However, such methods are incompatible with direct coupling to mass spectrometry (MS) due to the use of anionic surfactants, e.g., sodium dodecyl sulfate (SDS). Here, we present a novel method to characterize size-based fragmentation variants of a new biotherapeutic kind using microfluidic ZipChip® capillary zone electrophoresis (mCZE) system interfaced with mass spectrometry (MS) to determine the molecular masses of fragments. A new modality of immuno-oncology therapy, bispecific antigen-binding biotherapeutic, was chosen to investigate its fragmentation pattern using mCZE-MS for the first time, according to our knowledge. Bispecific antigen-binding biotherapeutic samples from different stages of downstream column purification and forced degradation conditions were analyzed. The results were cross-validated with denaturing size-exclusion chromatography-mass spectrometry and conventional rSDS-CGE. In this study, we demonstrated that mCZE-MS could separate and characterize 12-40 kDa bispecific antigen-binding biotherapeutic fragments rapidly (within ≤12 minutes), with higher resolution and better sensitivity than traditional LC-MS methods.

Fibronectin type III domain-containing protein 5 promotes autophagy via the AMPK/mTOR signaling pathway in hepatocellular carcinoma cells, contributing to nab-paclitaxel chemoresistance.

Chemotherapy resistance is a huge challenge in the treatment of hepatocellular carcinoma because resistance to nab-paclitaxel largely affects the efficacy of chemotherapy. An increased expression of fibronectin type III domain-containing protein 5 (FNDC5) in hepatocellular carcinoma cells can predict post-hepatectomy complications in patients with hepatocellular carcinoma and also stimulate proliferation and invasion of hepatocellular carcinoma cells; however, its role in the chemotherapy of hepatocellular carcinoma cells has never been evaluated. Thus, this study aimed to explore whether FNDC5 regulates chemoresistance in hepatocellular carcinoma. We identified by immunohistochemistry that hepatocellular carcinoma tissues had a higher FNDC5 expression than normal tissues adjacent to the cancer cells. Subsequently, knockdown of FNDC5 in hepatocellular carcinoma cells resulted in their diminished resistance to cell death after chemotherapy with nab-paclitaxel. By contrast, overexpression of FNDC5 in hepatocellular carcinoma cells increased the resistance of hepatocellular carcinoma cells to treatment. Moreover, FNDC5 mechanistically promoted autophagy via the AMPK/mTOR signaling pathway, thereby reducing cell death induced by nab-paclitaxel. Finally, we tested our hypothesis by conducting animal experiments. In conclusion, FNDC5 could be used as a biomarker for predicting chemotherapeutic efficacy in hepatocellular carcinoma treated with nab-paclitaxel chemotherapy, and as a therapeutic target to overcome resistance to nab-paclitaxel in hepatocellular carcinoma chemotherapy.

Gated Recurrent Unit Network for Psychological Stress Classification Using Electrocardiograms from Wearable Devices.

In recent years, research on human psychological stress using wearable devices has gradually attracted attention. However, the physical and psychological differences among individuals and the high cost of data collection are the main challenges for further research on this problem. In this work, our aim is to build a model to detect subjects' psychological stress in different states through electrocardiogram (ECG) signals. Therefore, we design a VR high-altitude experiment to induce psychological stress for the subject to obtain the ECG signal dataset. In the experiment, participants wear smart ECG T-shirts with embedded sensors to complete different tasks so as to record their ECG signals synchronously. Considering the temporal continuity of individual psychological stress, a deep, gated recurrent unit (GRU) neural network is developed to capture the mapping relationship between subjects' ECG signals and stress in different states through heart rate variability features at different moments, so as to build a neural network model from the ECG signal to psychological stress detection. The experimental results show that compared with all comparison methods, our method has the best classification performance on the four stress states of resting, VR scene adaptation, VR task and recovery, and it can be a remote stress monitoring solution for some special industries.

Effects of therapeutic hypothermia on the safety of children with severe traumatic brain injury: a systematic review and meta-analysis.

Background: Therapeutic hypothermia (TH) is effective to treat adult traumatic brain injury (TBI), but there is still controversy about its safety to treat the children with severe TBI. Methods: Clinical studies on TH in children with severe TBI from January 2000 to September 2020 were screened in PubMed, Web of Science, Embase, Cochrane Library, Nature, NCKI, and Wanfang online databases. Data were meta-analyzed by Rev Man 5.3. Differences in mortality, adverse outcomes, duration of Pediatric Intensive Care Unit (PICU), incidence of infection, and incidence of arrhythmia were compared between experimental group and control group. The heterogeneity of the results was evaluated by chi-square test and I2 test in Rev Man 5.3, and publication bias was evaluated by funnel plot. Results: Five articles were included, including 421 children. Cochrane evaluation was B grade or above, and Jadad scale score was over three points. The overall mortality between two groups showed great difference [odds ratio (OR) =1.72, 95% CI: 0.98-3.02, Z=1.87, P=0.04]. The incidence of adverse outcomes (OR =1.39, 95% CI: 0.86-2.25, Z=1.34, P=0.18), the duration of PICU [mean difference (MD) =0.51, 95% CI: -0.33 to 1.35, Z=1.19, P=0.24], the incidence of infection (OR =0.79, 95% CI: 0.51-1.23, Z=1.03, P=0.30), and the incidence of arrhythmia (OR =3.10, 95% CI: 0.77-12.50, Z=1.59, P=0.11) were not considerably different. Discussion: TH significantly reduced overall mortality in children with severe TBI, but did not significantly improve the incidence of adverse outcomes, PICU duration, infection rate, or arrhythmia. These results provided a reference for selecting proper clinical treatment methods for children with severe TBI.

Lactiplantibacillus plantarum HG20 attenuates II type collagen-induced rheumatoid arthritis in rats via anti-inflammatory and inhibition of apoptosis.

AIMS: This study aimed to explore the therapeutic effects of Lactiplantibacillus plantarum HG20 (HG20) on collagen-induced arthritis (CIA) rats and its mechanism. METHODS AND RESULTS: CIA rats were established by injecting bovine type II collagen for 7 days, and treated by intragastric administration HG20 for 21 days. The foot palm temperature and arthritis score were measured once a week. The pathological changes in the knee joint were observed by hematoxylin and eosin staining. The levels of cytokines were detected by enzyme linked immunosorbent assay, and the effects of HG20 on inflammatory and apoptosis pathway of spleen cells were detected by western blot analysis. The results indicated that HG20 reduced the joint swelling degree and foot palm temperature, inhibited the development of joint histopathology, decreased the levels of pro-inflammatory cytokines, down-regulate the expression of pro-inflammatory cytokines by nuclear factor kappa-B pathway, and inhibited the apoptosis of spleen cells by inhibiting phosphatidylinositol 3-kinase/protein kinase B pathway and regulating apoptosis pathways. CONCLUSIONS: HG20 had an adjuvant therapeutic effect on arthritis in CIA rats, and its mechanism might be related to the inflammatory and apoptosis pathway. SIGNIFICANCE AND IMPACT OF STUDY: These results revealed that HG20 could be used as a functional probiotic in the field of food and medical, and which played a potential role in the prevention and treatment of arthritis.

A systematic review and meta-analysis on the curative effects of cardiothoracic surgery for critical patients in the intensive care unit.

BACKGROUND: The aim of this study was to explore the curative effects of cardiothoracic surgery (CTS) for critical patients in the intensive care unit (ICU) using meta-analysis. METHODS: Literature was searched using the following search terms: "cardiothoracic surgery", "intensive care unit", "critical patients", "post-operation", and "curative effects". Rev Man 5.3 was used for meta-analysis. RESULTS: A total of 15 randomized controlled trials (RCTs) were included, all of which had a low risk of bias, indicating medium and high quality. At 30 days after CTS, the number of patients with grade III and IV cardiac function was reduced by 76.84%, and the overall heterogeneity test results revealed that Tau2=0.09, Chi2=17.08, df=5, I2=71%, P=0.004<0.01, Z=7.62, RR =0.33, and 95% CI: 0.24 to 0.43. The incidence of adverse reactions was analyzed in 6 RCTs, and mainly manifested as improper anticoagulation thrombosis and bleeding. The overall heterogeneity test results revealed that Chi2=1.07, df=5, I2=0%, P=0.96, Z=4.93, OR =0.46, 95% CI: 0.34 to 0.63, and P<0.01. The 30-day mortality rate was analyzed in 8 RCTs. The overall analysis using the fixed effects model revealed that there was a notable difference between the experimental group and the baseline, with Z=10.11, OR =0.12, 95% CI: 0.08 to 0.18, and P<0.01. DISCUSSION: CTS can reduce the incidence of adverse events and the mortality rate of critical patients in the ICU, demonstrating high safety.

Footprinting Mass Spectrometry of Membrane Proteins: Ferroportin Reconstituted in Saposin A Picodiscs.

Membrane proteins participate in a broad range of cellular processes and represent more than 60% of drug targets. One approach to their structural analyses is mass spectrometry (MS)-based footprinting including hydrogen/deuterium exchange (HDX), fast photochemical oxidation of proteins (FPOP), and residue-specific chemical modification. Studying membrane proteins usually requires their isolation from the native lipid environment, after which they often become unstable. To overcome this problem, we are pursuing a novel methodology of incorporating membrane proteins into saposin A picodiscs for MS footprinting. We apply different footprinting approaches to a model membrane protein, mouse ferroportin, in picodiscs and achieve high coverage that enables the analysis of the ferroportin structure. FPOP footprinting shows extensive labeling of the extramembrane regions of ferroportin and protection at its transmembrane regions, suggesting that the membrane folding of ferroportin is maintained throughout the labeling process. In contrast, an amphipathic reagent, N-ethylmaleimide (NEM), efficiently labels cysteine residues in both extramembrane and transmembrane regions, thereby affording complementary footprinting coverage. Finally, optimization of sample treatment gives a peptic-map of ferroportin in picodiscs with 92% sequence coverage, setting the stage for HDX. These results, taken together, show that picodiscs are a new platform broadly applicable to mass spectrometry studies of membrane proteins.

The catalytic mechanism of vitamin K epoxide reduction in a cellular environment.

Vitamin K epoxide reductases (VKORs) constitute a major family of integral membrane thiol oxidoreductases. In humans, VKOR sustains blood coagulation and bone mineralization through the vitamin K cycle. Previous chemical models assumed that the catalysis of human VKOR (hVKOR) starts from a fully reduced active site. This state, however, constitutes only a minor cellular fraction (5.6%). Thus, the mechanism whereby hVKOR catalysis is carried out in the cellular environment remains largely unknown. Here we use quantitative mass spectrometry (MS) and electrophoretic mobility analyses to show that KO likely forms a covalent complex with a cysteine mutant mimicking hVKOR in a partially oxidized state. Trapping of this potential reaction intermediate suggests that the partially oxidized state is catalytically active in cells. To investigate this activity, we analyze the correlation between the cellular activity and the cellular cysteine status of hVKOR. We find that the partially oxidized hVKOR has considerably lower activity than hVKOR with a fully reduced active site. Although there are more partially oxidized hVKOR than fully reduced hVKOR in cells, these two reactive states contribute about equally to the overall hVKOR activity, and hVKOR catalysis can initiate from either of these states. Overall, the combination of MS quantification and biochemical analyses reveals the catalytic mechanism of this integral membrane enzyme in a cellular environment. Furthermore, these results implicate how hVKOR is inhibited by warfarin, one of the most commonly prescribed drugs.

Phosphorylation-Dependent Conformations of the Disordered Carboxyl-Terminus Domain in the Epidermal Growth Factor Receptor.

The epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, regulates basic cellular functions and is a major target for anticancer therapeutics. The carboxyl-terminus domain is a disordered region of EGFR that contains the tyrosine residues, which undergo autophosphorylation followed by docking of signaling proteins. Local phosphorylation-dependent secondary structure has been identified and is thought to be associated with the signaling cascade. Deciphering and distinguishing the overall conformations, however, have been challenging because of the disordered nature of the carboxyl-terminus domain and resultant lack of well-defined three-dimensional structure for most of the domain. We investigated the overall conformational states of the isolated EGFR carboxyl-terminus domain using single-molecule Förster resonance energy transfer and coarse-grained simulations. Our results suggest that electrostatic interactions between charged residues emerge within the disordered domain upon phosphorylation, producing a looplike conformation. This conformation may enable binding of downstream signaling proteins and potentially reflect a general mechanism in which electrostatics transiently generate functional architectures in disordered regions of a well-folded protein.

Native Mass Spectrometry, Ion Mobility, Electron-Capture Dissociation, and Modeling Provide Structural Information for Gas-Phase Apolipoprotein E Oligomers.

Apolipoprotein E (apoE) is an essential protein in lipid and cholesterol metabolism. Although the three common isoforms in humans differ only at two sites, their consequences in Alzheimer's disease (AD) are dramatically different: only the ε4 allele is a major genetic risk factor for late-onset Alzheimer's disease. The isoforms exist as a mixture of oligomers, primarily tetramer, at low µM concentrations in a lipid-free environment. This self-association is involved in equilibrium with the lipid-free state, and the oligomerization interface overlaps with the lipid-binding region. Elucidation of apoE wild-type (WT) structures at an oligomeric state, however, has not yet been achieved. To address this need, we used native electrospray ionization and mass spectrometry (native MS) coupled with ion mobility (IM) to examine the monomer and tetramer of the three WT isoforms. Although collision-induced unfolding (CIU) cannot distinguish the WT isoforms, the monomeric mutant (MM) of apoE3 shows higher stability when submitted to CIU than the WT monomer. From ion-mobility measurements, we obtained the collision cross section and built a coarse-grained model for the tetramer. Application of electron-capture dissociation (ECD) to the tetramer causes unfolding starting from the C-terminal domain, in good agreement with solution denaturation data, and provides additional support for the C4 symmetry structure of the tetramer.

Reconstitution of RNA Polymerase I Upstream Activating Factor and the Roles of Histones H3 and H4 in Complex Assembly.

RNA polymerase I (Pol I) transcription in Saccharomyces cerevisiae requires four separate factors that recruit Pol I to the promoter to form a pre-initiation complex. Upstream Activating Factor (UAF) is one of two multi-subunit complexes that regulate pre-initiation complex formation by binding to the ribosomal DNA promoter and by stimulating recruitment of downstream Pol I factors. UAF is composed of Rrn9, Rrn5, Rrn10, Uaf30, and histones H3 and H4. We developed a recombinant Escherichia coli-based system to coexpress and purify transcriptionally active UAF complex and to investigate the importance of each subunit in complex formation. We found that no single subunit is required for UAF assembly, including histones H3 and H4. We also demonstrate that histone H3 is able to interact with each UAF-specific subunit, and show that there are at least two copies of histone H3 and one copy of H4 present in the complex. Together, our results provide a new model suggesting that UAF contains a hybrid H3-H4 tetramer-like subcomplex.

Membrane Protein Structure in Live Cells: Methodology for Studying Drug Interaction by Mass Spectrometry-Based Footprinting.

Mass spectrometry-based footprinting is an emerging approach for studying protein structure. Because integral membrane proteins are difficult targets for conventional structural biology, we recently developed a mass spectrometry (MS) footprinting method to probe membrane protein-drug interactions in live cells. This method can detect structural differences between apo and drug-bound states of membrane proteins, with the changes inferred from MS quantification of the cysteine modification pattern, generated by residue-specific chemical labeling. Here, we describe the experimental design, interpretation, advantages, and limitations of using cysteine footprinting by taking as an example the interaction of warfarin with vitamin K epoxide reductase, a human membrane protein. Compared with other structural methods, footprinting of proteins in live cells produces structural information for the near native state. Knowledge of cellular conformational states is a necessary complement to the high-resolution structures obtained from purified proteins in vitro. Thus, the MS footprinting method is broadly applicable in membrane protein biology. Future directions include probing flexible motions of membrane proteins and their interaction interface in live cells, which are often beyond the reach of conventional structural methods.

Laser-Initiated Radical Trifluoromethylation of Peptides and Proteins: Application to Mass-Spectrometry-Based Protein Footprinting.

Described is a novel, laser-initiated radical trifluoromethylation for protein footprinting and its broad residue coverage. . CF3 reacts with 18 of the 20 common amino acids, including Gly, Ala, Ser, Thr, Asp, and Glu, which are relatively silent with regard to . OH. This new approach to footprinting is a bridge between trifluoromethylation in materials and medicinal chemistry and structural biology and biotechnology. Its application to a membrane protein and to myoglobin show that the approach is sensitive to protein conformational change and solvent accessibility.

Three dimensional approach to investigating biological effects along energetic ion beam pathways.

Heavy ion beams have many exciting applications, including radiotherapy of deep-seated tumors and simulation tests of space irradiation for astronauts. These beams often use a feature that concentrates the energy deposition largely along the end of the energy pathway, leading to different distributions of biological effects along the axial direction. Currently, there is relatively little information regarding the radial directional difference of biological effects along the heavy ion paths. This study utilized a filter membrane that was quantatively applied with cells to demonstrate a 3D distribution model of irradiation on biological effects in living organisms. Some results have indicated that there is excitatory effect on the non-irradiated regions with energetic ions, which may give new insights into the distribution of biological effects along the paths of heavy ion beams with mid-high energy.

Hybrid Methods Reveal Multiple Flexibly Linked DNA Polymerases within the Bacteriophage T7 Replisome.

The physical organization of DNA enzymes at a replication fork enables efficient copying of two antiparallel DNA strands, yet dynamic protein interactions within the replication complex complicate replisome structural studies. We employed a combination of crystallographic, native mass spectrometry and small-angle X-ray scattering experiments to capture alternative structures of a model replication system encoded by bacteriophage T7. Two molecules of DNA polymerase bind the ring-shaped primase-helicase in a conserved orientation and provide structural insight into how the acidic C-terminal tail of the primase-helicase contacts the DNA polymerase to facilitate loading of the polymerase onto DNA. A third DNA polymerase binds the ring in an offset manner that may enable polymerase exchange during replication. Alternative polymerase binding modes are also detected by small-angle X-ray scattering with DNA substrates present. Our collective results unveil complex motions within T7 replisome higher-order structures that are underpinned by multivalent protein-protein interactions with functional implications.

Warfarin traps human vitamin K epoxide reductase in an intermediate state during electron transfer.

Although warfarin is the most widely used anticoagulant worldwide, the mechanism by which warfarin inhibits its target, human vitamin K epoxide reductase (hVKOR), remains unclear. Here we show that warfarin blocks a dynamic electron-transfer process in hVKOR. A major fraction of cellular hVKOR is in an intermediate redox state containing a Cys51-Cys132 disulfide, a characteristic accommodated by a four-transmembrane-helix structure of hVKOR. Warfarin selectively inhibits this major cellular form of hVKOR, whereas disruption of the Cys51-Cys132 disulfide impairs warfarin binding and causes warfarin resistance. Relying on binding interactions identified by cysteine alkylation footprinting and mass spectrometry coupled with mutagenesis analysis, we conducted structure simulations, which revealed a closed warfarin-binding pocket stabilized by the Cys51-Cys132 linkage. Understanding the selective warfarin inhibition of a specific redox state of hVKOR should enable the rational design of drugs that exploit the redox chemistry and associated conformational changes in hVKOR.

Incorporation of a Reporter Peptide in FPOP Compensates for Adventitious Scavengers and Permits Time-Dependent Measurements.

Incorporation of a reporter peptide in solutions submitted to fast photochemical oxidation of proteins (FPOP) allows for the correction of adventitious scavengers and enables the normalization and comparison of time-dependent results. Reporters will also be useful in differential experiments to control for the inclusion of a radical-reactive species. This incorporation provides a simple and quick check of radical dosage and allows comparison of FPOP results from day-to-day and lab-to-lab. Use of a reporter peptide in the FPOP workflow requires no additional measurements or spectrometers while building a more quantitative FPOP platform. It requires only measurement of the extent of reporter-peptide modification in a LC/MS/MS run, which is performed by using either data-dependent scanning or an inclusion list. Graphical Abstract ᅟ.