Identifications of novel host cell factors that interact with the receptor binding domain of the SARS-CoV-2 spike protein.

SARS-CoV-2 entry into host cells is facilitated by the interaction between the receptor binding domain of its spike protein (CoV2-RBD) and host cell receptor, ACE2, promoting viral membrane fusion. The virus also uses endocytic pathways for entry, but the mediating host factors remain largely unknown. It is also unknown whether mutations in the RBD of SARS-CoV-2 variants promote interactions with additional host factors to promote viral entry. Here, we used the GST pull-down approach to identify novel surface-located host factors that bind to CoV2-RBD. One of these factors, SH3BP4, regulates internalization of CoV2-RBD in an ACE2-independent, but integrin- and clathrin-dependent manner, and mediates SARS-CoV-2 pseudovirus entry, suggesting that SH3BP4 promotes viral entry via the endocytic route. Many of the identified factors, including SH3BP4, ADAM9 and TMEM2, show stronger affinity to CoV2-RBD than to RBD of the less infective SARS-CoV, suggesting SARS-CoV-2-specific utilization. We also found factors preferentially binding to the RBD of the SARS-CoV-2 Delta variant, potentially enhancing its entry. These data identify the repertoire of host cell surface factors that function in the events leading to entry of SARS-CoV-2.

Nitrogen-doped carbon-coated Cu0 activates molecular oxygen for norfloxacin degradation over a wide pH range.

The Fenton-like activated molecular oxygen technology demonstrates significant potential in the treatment of refractory organic pollutants in wastewater, offering promising development prospects. We prepared a N-doped C-coated copper-based catalyst Cu0/NC3-600 through the pyrolysis of Mel-modified Cu-based metal-organic framework (MOF). The results indicate that the degradation of 20 mg/L norfloxacin (NOR) was achieved using 1.0 g/L Cu0/NC3-600 across a wide pH range, with a removal rate exceeding 95 % and total organic carbon (TOC) removals approaching 70 % after 60 min at pH 5-11. The nitrogen doping enhances the electronic structure of the carbon material, facilitating the adsorption of molecular oxygen. Additionally, the formed carbon layer effectively prevent copper leaching,contributing to increased stability to a certain extent. Subsequently, we propose the catalytic reaction mechanism for the Cu0/NC/air system. Under acidic conditions, Cu0/NC3-600 activates molecular oxygen to produce the •O2-, which serves as the primary active species for NOR degradation. While in alkaline conditions, the high-valent copper species Cu3+ is generated in conjunction with •O2-, both working simultaneously for NOR degradation. Furthermore, based on the LC-MS results, we deduced four possible degradation pathways. This work offers a novel perspective on expanding the pH range of copper-based catalysts with excellent ability to activate molecular oxygen for environmental water treatment.

Rapid authentication of red wine by MALDI-MS combined with DART-MS.

A simple, rapid and high-throughput approach was developed for authentication of red wine for the first time, by combining spectral results from matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and direct analysis in real time mass spectrometry (DART-MS). By coupling with orthogonal partial least squares discrimination analysis (OPLS-DA), this approach enabled successful classification of 535 wines from 8 countries, with the correct classification rates of 100% on the calibration set and over 90% on the validation set for almost all countries, and 26 potential characteristic markers selected. Compared to one single technique, this approach allowed detection of more compound ions, and with better fitting and predictive performances. The satisfactory differentiation results of vintages and grape varieties further verified the robustness of the approach. This study demonstrated the feasibility of combining multiple mass spectrometric techniques for wine analysis, which can be extended to other fields or to combinations of other analytical techniques.

TMED10 mediates the trafficking of insulin-like growth factor 2 along the secretory pathway for myoblast differentiation.

The insulin-like growth factor 2 (IGF2) plays critical roles in cell proliferation, migration, differentiation, and survival. Despite its importance, the molecular mechanisms mediating the trafficking of IGF2 along the secretory pathway remain unclear. Here, we utilized a Retention Using Selective Hook system to analyze molecular mechanisms that regulate the secretion of IGF2. We found that a type I transmembrane protein, TMED10, is essential for the secretion of IGF2 and for differentiation of mouse myoblast C2C12 cells. Further analyses indicate that the residues 112-140 in IGF2 are important for the secretion of IGF2 and these residues directly interact with the GOLD domain of TMED10. We then reconstituted the release of IGF2 into COPII vesicles. This assay suggests that TMED10 mediates the packaging of IGF2 into COPII vesicles to be efficiently delivered to the Golgi. Moreover, TMED10 also mediates ER export of TGN-localized cargo receptor, sortilin, which subsequently mediates TGN export of IGF2. These analyses indicate that TMED10 is critical for IGF2 secretion by directly regulating ER export and indirectly regulating TGN export of IGF2, providing insights into trafficking of IGF2 for myoblast differentiation.

Algorithms for de-novo sequencing of peptides by tandem mass spectrometry: A review.

Peptide sequencing is of great significance to fundamental and applied research in the fields such as chemical, biological, medicinal and pharmaceutical sciences. With the rapid development of mass spectrometry and sequencing algorithms, de-novo peptide sequencing using tandem mass spectrometry (MS/MS) has become the main method for determining amino acid sequences of novel and unknown peptides. Advanced algorithms allow the amino acid sequence information to be accurately obtained from MS/MS spectra in short time. In this review, algorithms from exhaustive search to the state-of-art machine learning and neural network for high-throughput and automated de-novo sequencing are introduced and compared. Impacts of datasets on algorithm performance are highlighted. The current limitations and promising direction of de-novo peptide sequencing are also discussed in this review.

Lysyl hydroxylase LH1 promotes confined migration and metastasis of cancer cells by stabilizing Septin2 to enhance actin network.

BACKGROUND: Excessive extracellular matrix deposition and increased stiffness are typical features of solid tumors such as hepatocellular carcinoma (HCC) and pancreatic ductal adenocarcinoma (PDAC). These conditions create confined spaces for tumor cell migration and metastasis. The regulatory mechanism of confined migration remains unclear. METHODS: LC-MS was applied to determine the differentially expressed proteins between HCC tissues and corresponding adjacent tissue. Collective migration and single cell migration microfluidic devices with 6 µm-high confined channels were designed and fabricated to mimic the in vivo confined space. 3D invasion assay was created by Matrigel and Collagen I mixture treat to adherent cells. 3D spheroid formation under various stiffness environment was developed by different substitution percentage GelMA. Immunoprecipitation was performed to pull down the LH1-binding proteins, which were identified by LC-MS. Immunofluorescent staining, FRET, RT-PCR, Western blotting, FRAP, CCK-8, transwell cell migration, wound healing, orthotopic liver injection mouse model and in vivo imaging were used to evaluate the target expression and cellular phenotype. RESULTS: Lysyl hydroxylase 1 (LH1) promoted the confined migration of cancer cells at both collective and single cell levels. In addition, LH1 enhanced cell invasion in a 3D biomimetic model and spheroid formation in stiffer environments. High LH1 expression correlated with poor prognosis of both HCC and PDAC patients, while it also promoted in vivo metastasis. Mechanistically, LH1 bound and stabilized Septin2 (SEPT2) to enhance actin polymerization, depending on the hydroxylase domain. Finally, the subpopulation with high expression of both LH1 and SEPT2 had the poorest prognosis. CONCLUSIONS: LH1 promotes the confined migration and metastasis of cancer cells by stabilizing SEPT2 and thus facilitating actin polymerization.

Boosting electron transport process over multiple channels induced by S-doped carbon and Fe7S8 NPs interface toward high-efficiency antibiotics removal.

The enhancement of electron transport process on multiple channels of C-Fe and C-S-Fe bonds between dual-reaction centres was investigated for stimulating the antibiotics degradation in Fenton-like processes. Herein, multiple channels structure of sulfur-doped carbon coupled Fe7S8 cluster through C-Fe bond and C-S-Fe bond was constructed through density functional theory (DFT), and S-doped carbon framework coated Fe7S8 nanoparticles (Fe7S8/SC) Fenton-like catalyst was prepared through hydrothermal and subsequent sulfuration process. The DFT calculations revealed that electrons are thermodynamically transferred from carbon to iron along both C-Fe and C-S-Fe bonds. The optimized Fe7S8/SC catalyst exhibited desirable catalytic property for Fenton-like degradation for various antibiotics, the removal of amoxicillin, norfloxacin, and tetracycline hydrochloride reach 98.9%, 97.8%, and 99.3% respectively within 40 min under neutral pH, and catalyst also demonstrated excellent cycle stability after five runs. The excellent degradation effect of antibiotics by Fenton-like catalyst was attributed to the intensified electron transport process by multiple electron transfer channels between dual reaction centres, making FeII easier to regenerate. This study spreads a new route for the enhancement of electron transport process in Fenton-like catalysts by constructing multiple channels.

Performance and mechanism of FeS2/FeSxOy as highly effective Fenton-like catalyst for phenol degradation.

Developing a highly efficient Fenton-like catalyst working in a wide pH range is imperative to accomplish its practical wastewater treatment. Herein, FeS2/FeSxOy catalyst was synthesized by hydrothermal-solvothermal vulcanization with thioacetamide as a sulfur source. Characterization results confirmed FeS2/FeSxOy consisted of pyrite, kornelite, and szomolnokite. FeS2/FeSxOy exhibited superior catalytic activity toward H2O2 activation with more than 96% phenol removal within 5 min in pH 3.0 ∼ 8.0 at 30°C. Radical scavenging experiment and EPR analysis revealed both hydroxyl radicals (·OH) and superoxide anion radicals (O2·-) anticipated in phenol elimination, but ·OH played a dominant role. The detailed degradation experiments and density functional theory (DFT) calculation confirmed the vital role of FeS2 in enhancing phenol abatement. This study not only developed a highly active catalyst for H2O2 activation but also theoretically analyzed the FeS2 function in depth, which provided a guide for designing a highly efficient Fenton-like catalyst.

High-Resolution Micro-object Separation by Rotating Magnetic Chromatography.

The development of new technologies for the separation, selection, and isolation of microparticles such as rare target cells, circulating tumor cells, cancer stem cells, and immune cells has become increasingly important in the last few years. Microparticle separation technologies are usually applied to the analysis of disease-associated cells, but these procedures often face a cell separation problem that is often insufficient for single specific cell analyses. To overcome these limitations, a highly accurate size-based microparticle separation technique, herein called "rotating magnetic chromatography", is proposed in this work. Magnetic nanoparticles, placed in a microfluidic separation channel, are forced to move in well-defined trajectories by an external magnetic field, colliding with microparticles that are in this way separated on the basis of their dimensions with high accuracy and reproducibility. The method was optimized by using fluorescein isothiocyanate-modified polystyrene particles (chosen as a reference standard) and then applied to the analysis of cancer cells like Hep-3B and SK-Hep-1, allowing their fast and high-resolution chromatographic separation as a function of their dimensions. Due to its unmatched sub-micrometer cell separation capabilities, RMC can be considered a break-through technique that can unlock new perspectives in different scientific fields, that is, in medical oncology.

Advances in rapid detection of SARS-CoV-2 by mass spectrometry.

COVID-19 has already been lasting for more than two years and it has been severely affecting the whole world. Still, detection of SARS-CoV-2 remains the frontline approach to combat the pandemic, and the reverse transcription polymerase chain reaction (RT-PCR)-based method is the well recognized detection method for the enormous analytical demands. However, the RT-PCR method typically takes a relatively long time, and can produce false positive and false negative results. Mass spectrometry (MS) is a very commonly used technique with extraordinary sensitivity, specificity and speed, and can produce qualitative and quantitative information of various analytes, which cannot be achieved by RT-PCR. Since the pandemic outbreak, various mass spectrometric approaches have been developed for rapid detection of SARS-CoV-2, including the LC-MS/MS approaches that could allow analysis of several hundred clinical samples per day with one MS system, MALDI-MS approaches that could directly analyze clinical samples for the detection, and efforts for the on-site detection with portable devices. In this review, these mass spectrometric approaches were summarized, and their pros and cons as well as further development were also discussed.

High-activity and excellent-reusability γ-Fe2O3/SiO2 coating on TC4 titanium alloy by plasma electrolytic oxidation for enhanced photo-Fenton degradation.

The immobilized coatings as a kind of promising Fenton-like catalysts with excellent performance and reusability for the efficient degradation of antibiotics and phenol under solar light irradiation is investigated. Herein, the porous γ-Fe2O3/SiO2 immobilized ceramic coating on TC4 titanium alloy as photo-Fenton catalyst was prepared via plasma electrolytic oxidation technology. The as-obtained immobilized coating manifested a remarkable catalytic activity that the removal efficiencies of phenol and various antibiotics could reach more than 92% within 90 min, and presented excellent reusability after six runs in phenol removal. The high activity and excellent reusability of γ-Fe2O3 were attributed to the synergistic effect of multiple pathways to jointly produce abundant •OH, and the combination of γ-Fe2O3 and SiO2 in the coating could effectively reduce iron leaching during the heterogeneous photo-Fenton process, respectively. This work provides a novel strategy for the synthesis of high-performance photo-Fenton catalysts to dispose of wastewater in the future.

Electrospray ionization mass spectrometry with wooden tips: A review.

Electrospray ionization (ESI) is a powerful ionization technique in mass spectrometry (MS). There has been an increasing interest for the new development of ESI technique to extend its applications. ESI-MS with wooden tips (wooden-tip ESI-MS), an ESI technique invented in 2011, enabled not only new applications but also new insights into the ESI mechanism. In this review, the technical aspects of wooden-tip ESI-MS are described, the new features of wooden-tip ESI-MS for sampling and ionization of analytes are highlighted, and the important applications of wooden-tip ESI-MS in various fields in the past 10 years, including food safety, forensic investigation, environmental analysis, biomedical analysis and protein study, are summarized. The perspectives on the further development and applications of wooden-tip ESI-MS are also discussed.

Least absolute shrinkage and selection operator-based prediction of collision cross section values for ion mobility mass spectrometric analysis of lipids.

Collision cross section (CCS) values generated from ion mobility mass spectrometry (IM-MS) have commonly been employed to facilitate lipid identification. However, this is hindered by the limited available lipid standards. Recently, CCS values were predicted by means of computational calculations, though the prediction precision was generally not good and the predicted CCS values of the lipid isomers were almost identical. To address this challenge, a least absolute shrinkage and selection operator (LASSO)-based prediction method was developed for the prediction of lipids' CCS values in this study. In this method, an array of molecular descriptors were screened and optimized to reflect the subtle differences in structures among the different lipid isomers. The use of molecular descriptors together with a wealth of standard CCS values for the lipids (365 in total) significantly improved the accuracy and precision of the LASSO model. Its accuracy was externally validated with median relative errors (MREs) of <1.1% using an independent data set. This approach was demonstrated to allow differentiation of cis/trans and sn-positional isomers. The results also indicated that the LASSO-based prediction method could practically reduce false-positive identifications in IM-MS-based lipidomics.

Revealing the enhancing mechanisms of Fe-Cu bimetallic catalysts for the Fenton-like degradation of phenol.

To develop a heterogeneous Fenton-like catalyst with desirable activity and reusability remains a great challenge for the practical degradation of environmental remediation. Herein, we demonstrate a dendritic Fe-Cu bimetallic catalyst consisted of a Cu/Fe3O4 shell and a FeCu core (E100). In comparisons of single Cu, Fe and Fe3O4, E100 performs far better performance for the Fenton-like degradation of phenol, and its dominant Fenton-like active centers are Fe species under acidic pH or Cu species under neutral pH. Particularly, Cu-based Fenton-like reactions are greatly accelerated by galvanic micro-cells effects that come from the special co-existence of Cu/Fe3O4 shell, and subsequently, owing to the Cu leaching from the shell, the inner FeCu core of E100 is able to be exposed and further strengthen Fe-based Fenton-like reactions. Overall, the appropriate synergistic effects endow E100 with superior catalytic activity and reusability than other catalysts. Our work pushes forward a step for understanding the catalytic mechanism of Fe-Cu bimetallic catalysts and provides new sights for fabricating efficient Fenton-like catalysts for environmental remediation.

A distinct giant coat protein complex II vesicle population in Arabidopsis thaliana.

Plants live as sessile organisms with large-scale gene duplication events and subsequent paralogue divergence during evolution. Notably, plant paralogues are expressed tissue-specifically and fine-tuned by phytohormones during various developmental processes. The coat protein complex II (COPII) is a highly conserved vesiculation machinery mediating protein transport from the endoplasmic reticulum to the Golgi apparatus in eukaryotes1. Intriguingly, Arabidopsis COPII paralogues greatly outnumber those in yeast and mammals2-6. However, the functional diversity and underlying mechanism of distinct COPII paralogues in regulating protein endoplasmic reticulum export and coping with various adverse environmental stresses are poorly understood. Here we characterize a novel population of COPII vesicles produced in response to abscisic acid, a key phytohormone regulating abiotic stress responses in plants. These hormone-induced giant COPII vesicles are regulated by an Arabidopsis-specific COPII paralogue and carry stress-related channels/transporters for alleviating stresses. This study thus provides a new mechanism underlying abscisic acid-induced stress responses via the giant COPII vesicles and answers a long-standing question on the evolutionary significance of gene duplications in Arabidopsis.

Dual-Mode Sensing Platform for Electrochemiluminescence and Colorimetry Detection Based on a Closed Bipolar Electrode.

Development of sensors uniting different sensing principles is in line with the concept of reliable, comprehensive, and diversified equipment construction. However, the current exploration in this field is obstructed by compromise of reaction conditions and inevitable mutual interference arising from different sensing modes. This work reported a closed bipolar electrode (c-BPE) strategy for dual-modality detection or dual-target detection. To this end, a c-BPE sensing platform installed in physically separated anode and cathode compartments was well designed and carefully optimized. If luminol was present in the anode section and Prussian blue (PB) was at the cathode part, single stimulation could realize electrochemiluminescence (ECL) from luminol at the anode and conversion of PB to Prussian white (PW) at the cathode. The latter reaction helped elevate the ECL signal and also prepared for colorimetric detection as color change from PW to PB under the trigger of oxidant (like H2O2) was used to track the content of the oxidant. Thus, dual signals were obtained for dual-modality detection of single target or the detection of different targets was realized at different poles. Detection of glucose was carried out to validate the application for dual-modality detection, while VLDL/AChE and NADH/H2O2 assays illustrated the potential of dual-target detection. The proposed platform possesses outstanding sensing performance including selectivity, repeatability, long-term stability, accuracy, and so forth. This work implements a breakthrough in designing dual-mode sensors and is expected to present a rational basis for development of a diversified sensing platform.

An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking.

The fidelity of protein transport in the secretory pathway relies on the accurate sorting of proteins to their correct destinations. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on specific sorting machinery to be enriched into transport vesicles. Here, we used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry (MS) analyses of the isolated vesicles revealed cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner. We found that two of them, FAM84B (also known as LRAT domain containing 2 or LRATD2) and PRRC1, contain proline-rich domains and regulate anterograde trafficking. Further analyses revealed that PRRC1 is recruited to endoplasmic reticulum (ER) exit sites, interacts with the inner COPII coat, and its absence increases membrane association of COPII. In addition, we uncovered cargo proteins that depend on GTP hydrolysis to be captured into vesicles. Comparing control cells with cells depleted of the cargo receptors, SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Our results indicate that the vesicle formation assay in combination with quantitative MS analysis is a robust and powerful tool to uncover novel factors that mediate vesicular trafficking and to uncover cargo clients of specific cellular factors.

Integrated hand-held electrochemical sensor for multicomponent detection in urine.

Electrochemical sensors have shown great advantage and application potential in point-of-care testing (POCT) related scenarios. However, some fatal problems plague its widespread utilization, which include the susceptibility of sensors to interference in real samples (e.g. pH), the contradiction between the limited objects detectable for most sensors and the requirement of multi-target analysis in most cases, and the complicated procedures in sensor preparation as well as in routine use. This paper contributed a tip-like electrochemical sensor prototype. By integrated with a commercial pipettor, it fulfilled semi-automatic assay procedure of sampling, detection and rinsing, thus saving operational time and manual work. The tip sensor owns the property of simple fabrication and is free from any modification of extra bio/chem materials. Moreover, built on multiple electrochemical signal outputs including open circuit potential, peak current and potential of specific electrochemical reaction, this work established a novel multi-component sensing strategy, wherein detection of uric acid (UA), urea and pH in urine samples was realized by using one single working electrode. The detection range for the above targets is 5.0~600 µM for UA, 4.0~8.0 for pH and 0.5~7.0 mM for urea with the detection limits (S/N = 3) of 0.05 µM for UA and 5.4 µM for urea, and the sensitivity of pH assay is 73 mV/pH. Notably, as variation of sample pH has impact on electrochemical analysis, the pH-related parameter was introduced for calibration to diminish such interference. The developed tip sensor and the novel sensing strategy may open a new window for electrochemical technology and broaden its application in POCT.

Data storage using peptide sequences.

Humankind is generating digital data at an exponential rate. These data are typically stored using electronic, magnetic or optical devices, which require large physical spaces and cannot last for a very long time. Here we report the use of peptide sequences for data storage, which can be durable and of high storage density. With the selection of suitable constitutive amino acids, designs of address codes and error-correction schemes to protect the order and integrity of the stored data, optimization of the analytical protocol and development of a software to effectively recover peptide sequences from the tandem mass spectra, we demonstrated the feasibility of this method by successfully storing and retrieving a text file and the music file Silent Night with 40 and 511 18-mer peptides respectively. This method for the first time links data storage with the peptide synthesis industry and proteomics techniques, and is expected to stimulate the development of relevant fields.

Interdomain flexibility and interfacial integrity of ß-lactamase inhibitory protein (BLIP) modulate its binding to class A ß-lactamases.

ß-Lactamase inhibitory protein (BLIP) consists of a tandem repeat of αß domains conjugated by an interdomain loop and can effectively bind and inactivate class A ß-lactamases, which are responsible for resistance of bacteria to ß-lactam antibiotics. The varied ability of BLIP to bind different ß-lactamases and the structural determinants for significant enhancement of BLIP variants with a point mutation are poorly understood. Here, we investigated the conformational dynamics of BLIP upon binding to three clinically prevalent class A ß-lactamases (TEM1, SHV1, and PC1) with dissociation constants between subnanomolar and micromolar. Hydrogen deuterium exchange mass spectrometry revealed that the flexibility of the interdomain region was significantly suppressed upon strong binding to TEM1, but was not significantly changed upon weak binding to SHV1 or PC1. E73M and K74G mutations in the interdomain region improved binding affinity toward SHV1 and PC1, respectively, showing significantly increased flexibility of the interdomain region compared to the wild-type and favorable conformational changes upon binding. In contrast, more rigidity of the interfacial loop 135-145 was observed in these BLIP mutants in both free and bound states. Consistently, molecular dynamics simulations of BLIP exhibited drastic changes in the flexibility of the loop 135-145 in all complexes. Our results indicated for the first time that higher flexibility of the interdomain linker, as well as more rigidity of the interfacial loop 135-145, could be desirable determinants for enhancing inhibition of BLIP to class A ß-lactamases. Together, these findings provide unique insights into the design of enhanced inhibitors.