Mass Spectrometry Methods for Measuring Protein Stability.

Mass spectrometry is a central technology in the life sciences, providing our most comprehensive account of the molecular inventory of the cell. In parallel with developments in mass spectrometry technologies targeting such assessments of cellular composition, mass spectrometry tools have emerged as versatile probes of biomolecular stability. In this review, we cover recent advancements in this branch of mass spectrometry that target proteins, a centrally important class of macromolecules that accounts for most biochemical functions and drug targets. Our efforts cover tools such as hydrogen-deuterium exchange, chemical cross-linking, ion mobility, collision induced unfolding, and other techniques capable of stability assessments on a proteomic scale. In addition, we focus on a range of application areas where mass spectrometry-driven protein stability measurements have made notable impacts, including studies of membrane proteins, heat shock proteins, amyloidogenic proteins, and biotherapeutics. We conclude by briefly discussing the future of this vibrant and fast-moving area of research.

Mechanistic insights into accelerated α-synuclein aggregation mediated by human microbiome-associated functional amyloids.

The gut microbiome has been shown to have key implications in the pathogenesis of Parkinson's disease (PD). The Escherichia coli functional amyloid CsgA is known to accelerate α-synuclein aggregation in vitro and induce PD symptoms in mice. However, the mechanism governing CsgA-mediated acceleration of α-synuclein aggregation is unclear. Here, we show that CsgA can form stable homodimeric species that correlate with faster α-synuclein amyloid aggregation. Furthermore, we identify and characterize new CsgA homologs encoded by bacteria present in the human microbiome. These CsgA homologs display diverse aggregation kinetics, and they differ in their ability to modulate α-synuclein aggregation. Remarkably, we demonstrate that slowing down CsgA aggregation leads to an increased acceleration of α-synuclein aggregation, suggesting that the intrinsic amyloidogenicity of gut bacterial CsgA homologs affects their ability to accelerate α-synuclein aggregation. Finally, we identify a complex between CsgA and α-synuclein that functions as a platform to accelerate α-synuclein aggregation. Taken together, our work reveals complex interplay between bacterial amyloids and α-synuclein that better informs our understanding of PD causation.

Physics-informed neural network for polarimetric underwater imaging.

Utilizing the polarization analysis in underwater imaging can effectively suppress the scattered light and help to restore target signals in turbid water. Neural network-based solutions can also boost the performance of polarimetric underwater imaging, while most of the existing networks are pure data driven which suffer from ignoring the physical mode. In this paper, we proposed an effective solution that informed the polarimetric physical model and constrains into the well-designed deep neural network. Especially compared with the conventional underwater imaging model, we mathematically transformed the two polarization-dependent parameters to a single parameter, making it easier for the network to converge to a better level. In addition, a polarization perceptual loss is designed and applied to the network to make full use of polarization information on the feature level rather than on the pixel level. Accordingly, the network was able to learn the polarization modulated parameter and to obtain clear de-scattered images. The experimental results verified that the combination of polarization model and neural network was beneficial to improve the image quality and outperformed other existing methods, even in a high turbidity condition.

Towards 3D Bioprinted Spinal Cord Organoids.

Three-dimensional (3D) cultures, so-called organoids, have emerged as an attractive tool for disease modeling and therapeutic innovations. Here, we aim to determine if boundary cap neural crest stem cells (BC) can survive and differentiate in gelatin-based 3D bioprinted bioink scaffolds in order to establish an enabling technology for the fabrication of spinal cord organoids on a chip. BC previously demonstrated the ability to support survival and differentiation of co-implanted or co-cultured cells and supported motor neuron survival in excitotoxically challenged spinal cord slice cultures. We tested different combinations of bioink and cross-linked material, analyzed the survival of BC on the surface and inside the scaffolds, and then tested if human iPSC-derived neural cells (motor neuron precursors and astrocytes) can be printed with the same protocol, which was developed for BC. We showed that this protocol is applicable for human cells. Neural differentiation was more prominent in the peripheral compared to central parts of the printed construct, presumably because of easier access to differentiation-promoting factors in the medium. These findings show that the gelatin-based and enzymatically cross-linked hydrogel is a suitable bioink for building a multicellular, bioprinted spinal cord organoid, but that further measures are still required to achieve uniform neural differentiation.

Molecular genetic analysis of neural stem cells after space flight and simulated microgravity on earth.

Understanding how stem cells adapt to space flight conditions is fundamental for human space missions and extraterrestrial settlement. We analyzed gene expression in boundary cap neural crest stem cells (BCs), which are attractive for regenerative medicine by their ability to promote proliferation and survival of cocultured and co-implanted cells. BCs were launched to space (space exposed cells) (SEC), onboard sounding rocket MASER 14 as free-floating neurospheres or in a bioprinted scaffold. For comparison, BCs were placed in a random positioning machine (RPM) to simulate microgravity on earth (RPM cells) or were cultured under control conditions in the laboratory. Using next-generation RNA sequencing and data post-processing, we discovered that SEC upregulated genes related to proliferation and survival, whereas RPM cells upregulated genes associated with differentiation and inflammation. Thus, (i) space flight provides unique conditions with distinctly different effects on the properties of BC compared to earth controls, and (ii) the space flight exposure induces postflight properties that reinforce the utility of BC for regenerative medicine and tissue engineering.

Synthesis, pharmacological evaluation and mechanistic study of scutellarin methyl ester -4'-dipeptide conjugates for the treatment of hypoxic-ischemic encephalopathy (HIE) in rat pups.

A series of novel scutellarin methyl ester-4'-dipeptide conjugates exhibiting active transport characteristics and protection against pathological damage caused by hypoxic-ischemic encephalopathy (HIE) were successfully designed and synthesized. The physiochemical properties of the obtained compounds, as well as the Caco-2 cell-based permeability and uptake into hPepT1-MDCK cells were evaluated using various analytical methods. Scutellarin methyl ester-4'-Val-homo-Leu dipeptide (5k) was determined as the optimal candidate with a high apparent permeability coefficient (Papp A to B) of 1.95 ± 0.24 × 10-6 cm/s, low ER (Papp BL to AP/Papp AP to BL) of 0.52 in Caco-2 cells, and high uptake of 25.47 µmol/mg/min in hPepT1-MDCK cells. Comprehensive mechanistic studies demonstrated that pre-treatment of PC12 cells with 5k resulted in more potent anti-oxidative activity, which was manifested by a significant decrease in the malondialdehyde (MDA) and reactive oxygen species (ROS) levels, attenuation of the H2O2-induced apoptotic cell accumulation in the sub-G1 peak, and improvement in the expression of the relevant apoptotic proteins (Bcl-2, Bax, and cleave-caspase-3). Moreover, evaluation of in vivo neuroprotective characteristics in hypoxic-ischemic rat pups revealed that 5k significantly reduced infarction and alleviated the related pathomorphological damage. The compound was also shown to ameliorate the neurological deficit at 48 h as well as to decrease the brain tissue loss at 4 weeks. Conjugate 5k was demonstrated to reduce the amyloid precursor protein (APP) and ß-site APP-converting enzyme-1 (BACE-1) expression. Pharmacokinetic characterization of 5k indicated favorable druggability and pharmacokinetic properties. The conducted docking studies revealed optimal binding of 5k to PepT1. Hydrogen bonding as well as cation-π interactions with the corresponding amino acid residues in the target active site were clearly observed. The obtained results suggest 5k as a potential candidate for anti-HIE therapy, which merits further investigation.

Synthesis, pharmacological evaluation, and mechanistic study of adefovir mixed phosphonate derivatives bearing cholic acid and l-amino acid moieties for the treatment of HBV.

The deficiency of nucleos(t)ide analogues (NAs) as anti-hepatitis B virus (HBV) drugs in clinical use is attributable to their insufficient enrichment in liver and non-target organ toxicity. We aimed to develop potent anti-HBV adefovir derivatives with hepatotrophic properties and reduced nephrotoxicity. A series of adefovir mono l-amino acids, mono cholic acid-drug conjugates were designed and synthesized, and their antiviral activity and uptake in rat primary hepatocytes and Na+-dependent taurocholate co-transporting polypeptide (NTCP)-HEK293 cells were evaluated. We isolated compound 6c as the optimal molecular candidate, with the highest antiviral activity (EC50 0.42 µmol/L, SI 1063.07) and highest cellular uptake in primary hepatocytes and NTCP-HEK293 cells. In-depth mechanistic studies demonstrated that 6c exhibited a lower toxicity in HK-2 cells when compared to adefovir dipivoxil (ADV). This is because 6c cannot be transported by the human renal organic anion transporter 1 (hOAT1). Furthermore, pharmacokinetic characterization and tissue distribution of 6c indicates it has favorable druggability and pharmacokinetic properties. Further docking studies suggested compounds with ursodeoxycholic acid and l-amino acid groups are better at binding to NTCP due to their hydrophilic properties, indicating that 6c is a potential candidate as an anti-HBV therapy and therefore merits further investigation.

Structural Basis for Superoxide Activation of Flavobacterium johnsoniae Class I Ribonucleotide Reductase and for Radical Initiation by Its Dimanganese Cofactor.

A ribonucleotide reductase (RNR) from Flavobacterium johnsoniae ( Fj) differs fundamentally from known (subclass a-c) class I RNRs, warranting its assignment to a new subclass, Id. Its ß subunit shares with Ib counterparts the requirements for manganese(II) and superoxide (O2-) for activation, but it does not require the O2--supplying flavoprotein (NrdI) needed in Ib systems, instead scavenging the oxidant from solution. Although Fj ß has tyrosine at the appropriate sequence position (Tyr 104), this residue is not oxidized to a radical upon activation, as occurs in the Ia/b proteins. Rather, Fj ß directly deploys an oxidized dimanganese cofactor for radical initiation. In treatment with one-electron reductants, the cofactor can undergo cooperative three-electron reduction to the II/II state, in contrast to the quantitative univalent reduction to inactive "met" (III/III) forms seen with I(a-c) ßs. This tendency makes Fj ß unusually robust, as the II/II form can readily be reactivated. The structure of the protein rationalizes its distinctive traits. A distortion in a core helix of the ferritin-like architecture renders the active site unusually open, introduces a cavity near the cofactor, and positions a subclass-d-specific Lys residue to shepherd O2- to the Mn2II/II cluster. Relative to the positions of the radical tyrosines in the Ia/b proteins, the unreactive Tyr 104 of Fj ß is held away from the cofactor by a hydrogen bond with a subclass-d-specific Thr residue. Structural comparisons, considered with its uniquely simple mode of activation, suggest that the Id protein might most closely resemble the primordial RNR-ß.

Boundary cap neural crest stem cells promote angiogenesis after transplantation to avulsed dorsal roots in mice and induce migration of endothelial cells in 3D printed scaffolds.

Dorsal root avulsion injuries lead to loss of sensation and to reorganization of blood vessels (BVs) in the injured area. The inability of injured sensory axons to re-enter the spinal cord results in permanent loss of sensation, and often also leads to the development of neuropathic pain. Approaches that restore connection between peripheral sensory axons and their CNS targets are thus urgently need. Previous research has shown that sensory axons from peripherally grafted human sensory neurons are able to enter the spinal cord by growing along BVs which penetrate the CNS from the spinal cord surface. In this study we analysed the distribution of BVs after avulsion injury and how their pattern is affected by implantation at the injury site of boundary cap neural crest stem cells (bNCSCs), a transient cluster of cells, which are located at the boundary between the spinal cord and peripheral nervous system and assist the growth of sensory axons from periphery into the spinal cord during development. The superficial dorsal spinal cord vasculature was examined using intravital microscopy and intravascular BV labelling. bNCSC transplantation increased vascular volume in a non-dose responsive manner, whereas dorsal root avulsion alone did not decrease the vascular volume. To determine whether bNCSC are endowed with angiogenic properties we prepared 3D printed scaffolds, containing bNCSCs together with rings prepared from mouse aorta. We show that bNCSC do induce migration and assembly of endothelial cells in this system. These findings suggest that bNCSC transplant can promote vascularization in vivo and contribute to BV formation in 3D printed scaffolds.

Hidden Real Topology and Unusual Magnetoelectric Responses in Two-Dimensional Antiferromagnets.

Recently, the real topology has been attracting widespread interest in two dimensions (2D). Here, based on first-principles calculations and theoretical analysis, the monolayer Cr2Se2O (ML-CrSeO) is revealed as the first material example of a 2D antiferromagnetic (AFM) real Chern insulator (RCI) with topologically protected corner states. Unlike previous RCIs, it is found that the real topology of the ML-CrSeO is rooted in one certain mirror subsystem of the two spin channels, and cannot be directly obtained from all the valence bands in each spin channel as commonly believed. In particular, due to antiferromagnetism, the corner modes in ML-CrSeO exhibit strong corner-contrasted spin polarization, leading to spin-corner coupling (SCC). This SCC enables a direct connection between spin space and real space. Consequently, large and switchable net magnetization can be induced in the ML-CrSeO nanodisk by electrostatic means, such as potential step and in-plane electric field, and the corresponding magnetoelectric responses behave like a sign function, distinguished from that of the conventional multiferroic materials. This work considerably broadens the candidate range of RCI materials, and opens up a new direction for topo-spintronics and 2D AFM materials research.

Native ion mobility-mass spectrometry reveals the binding mechanisms of anti-amyloid therapeutic antibodies.

One of the most important attributes of anti-amyloid antibodies is their selective binding to oligomeric and amyloid aggregates. However, current methods of examining the binding specificities of anti-amyloid ß (Aß) antibodies have limited ability to differentiate between complexes that form between antibodies and monomeric or oligomeric Aß species during the dynamic Aß aggregation process. Here, we present a high-resolution native ion-mobility mass spectrometry (nIM-MS) method to investigate complexes formed between a variety of Aß oligomers and three Aß-specific IgGs, namely two antibodies with relatively high conformational specificity (aducanumab and A34) and one antibody with low conformational specificity (crenezumab). We found that crenezumab primarily binds Aß monomers, while aducanumab preferentially binds Aß monomers and dimers and A34 preferentially binds Aß dimers, trimers, and tetrameters. Through collision induced unfolding (CIU) analysis, our data indicate that antibody stability is increased upon Aß binding and, surprisingly, this stabilization involves the Fc region. Together, we conclude that nIM-MS and CIU enable the identification of Aß antibody binding stoichiometries and provide important details regarding antibody binding mechanisms.

Effects of microgravity on neural crest stem cells.

Exposure to microgravity (µg) results in a range of systemic changes in the organism, but may also have beneficial cellular effects. In a previous study we detected increased proliferation capacity and upregulation of genes related to proliferation and survival in boundary cap neural crest stem cells (BC) after MASER14 sounding rocket flight compared to ground-based controls. However, whether these changes were due to µg or hypergravity was not clarified. In the current MASER15 experiment BCs were exposed simultaneously to µg and 1 g conditions provided by an onboard centrifuge. BCs exposed to µg displayed a markedly increased proliferation capacity compared to 1 g on board controls, and genetic analysis of BCs harvested 5 h after flight revealed an upregulation, specifically in µg-exposed BCs, of Zfp462 transcription factor, a key regulator of cell pluripotency and neuronal fate. This was associated with alterations in exosome microRNA content between µg and 1 g exposed MASER15 specimens. Since the specimens from MASER14 were obtained for analysis with 1 week's delay, we examined whether gene expression and exosome content were different compared to the current MASER15 experiments, in which specimens were harvested 5 h after flight. The overall pattern of gene expression was different and Zfp462 expression was down-regulated in MASER14 BC µg compared to directly harvested specimens (MASER15). MicroRNA exosome content was markedly altered in medium harvested with delay compared to directly collected samples. In conclusion, our analysis indicates that even short exposure to µg alters gene expression, leading to increased BC capacity for proliferation and survival, lasting for a long time after µg exposure. With delayed harvest of specimens, a situation which may occur due to special post-flight circumstances, the exosome microRNA content is modified compared to fast specimen harvest, and the direct effects from µg exposure may be partially attenuated, whereas other effects can last for a long time after return to ground conditions.

Photo-crosslinking hydrogel for wound healing in a pilonidal sinus patient after open surgery.

Pilonidal sinus is a chronic infectious disease with large incision and high risk of relapse after surgical management. Therefore, effective intervention strategies are urgently needed to reduce the relapse and shorten the wound healing time. Hydrogels have been widely used in regenerative medicine for its great biocompatibility, however, it remains challenging to integrate the material with wound tissues. Here, we reported a case of pilonidal sinus patient using a novel tissue integration material, Photo-crosslinking hydrogel after open surgery. A 38-year-old man with a pilonidal sinus for ˃5 years underwent open surgery. When the surgery was finished, the wound was injected with hydrogel that was irradiated with a ultraviolet light source until covered and solidified completely. Hydrogel needed to be changed 1-2 times per week. We evaluated the healing time as primary outcome and then followed up for ˃1 year to observe the relapse. The wound healed completely in 46 days after open surgery, which was shorter than that reported in other studies. Meanwhile, no recurrence was detected during follow-up. Photo-crosslinking hydrogel effectively promoted wound healing and has the potential to be easily applied in Pilonidal sinus patients after open surgery.

Room-temperature ferromagnetic CoSe2nanoplates synthesized by chemical vapor deposition.

Among novel two-dimensional materials, transition metal dichalcogenides (TMDs) with 3dmagnetic elements have been extensively researched owing to their unique magnetic, electric, and photoelectric properties. As an important member of TMDs, CoSe2is an interesting material with controversial magnetic properties, hitherto there are few reports related to the magnetism of CoSe2materials. Here, we report the synthesis of CoSe2nanoplates on Al2O3substrates by chemical vapor deposition (CVD). The CVD-grown CoSe2nanoplates exhibit three typical morphologies (regular hexagonal, hexagonal, and pentagonal shapes) and their lateral sizes and thickness of CoSe2nanoplates can reach up to hundreds of microns and several hundred nanometers, respectively. The electric-transport measurement shows a metallic feature of CoSe2nanoplates. Furthermore, the slanted hysteresis loop and nonzero remnant magnetization of the CoSe2nanoplates confirm the ferromagnetism in the temperature range of 5-400 K. This work provides a novel platform for designing CoSe2-based spintronic devices and studying related magnetic mechanisms.

Biodeposition of oysters in an urbanized bay area alleviates the black-malodorous compounds in sediments by altering microbial sulfur and iron metabolism.

The occurrence of the 'black-malodorous phenomenon' in a waterbody is a clear sign of a highly eutrophic bay, the formation of which is associated with microbial sulfur and iron metabolism in the sediments. Oyster farming restoration has been widely studied as an important method for treating eutrophication and related ecological problems. However, few studies focus on the ecosystem-level consequences of oyster farming concerning microbial sulfur and iron cycles in the sediment. Here, we compared the physicochemical features and microbial functions of oyster farms with those of reference areas using the Geochip5.0 technique. Our results showed a significant reduction of acid volatile sulfide (AVS) content associated with oyster farming, thus alleviating the black-malodorous status of Shenzhen Bay in China. Oyster farming created loose and porous sedimentary structures and stimulated the oxidation of black-odorous compounds. Moreover, we observed that the introduction of oysters changed microbial biodiversity significantly based on gyrB gene structure, with typical sulfur- and iron-cycling microbes being enriched. We also demonstrated that microbial abilities involved in sulfur and iron metabolism were greatly increased in oyster farming areas compared with reference areas. Under such circumstances, some cascading processes (AVS uptake and rates of organic matter turnover) were improved, which eventually contributed to black odor reduction. From the microecological perspective, we conclude that the biodeposition of oysters was the key factor for water retention and improvement of microbial metabolism. This study suggests that biodeposition shapes the microbial functional communities in adjacent territories and presumably alleviates the black-malodorous compounds in sediments.

Engineering neoantigen vaccines to improve cancer personalized immunotherapy.

Immunotherapy treatments harnessing the immune system herald a new era of personalized medicine, offering considerable benefits for cancer patients. Over the past years, tumor neoantigens emerged as a rising star in immunotherapy. Neoantigens are tumor-specific antigens arising from somatic mutations, which are proceeded and presented by the major histocompatibility complex on the cell surface. With the advancement of sequencing technology and bioinformatics engineering, the recognition of neoantigens has accelerated and is expected to be incorporated into the clinical routine. Currently, tumor vaccines against neoantigens mainly encompass peptides, DNA, RNA, and dendritic cells, which are extremely specific to individual patients. Due to the high immunogenicity of neoantigens, tumor vaccines could activate and expand antigen-specific CD4+ and CD8+ T cells to intensify anti-tumor immunity. Herein, we introduce the origin and prediction of neoantigens and compare the advantages and disadvantages of multiple types of neoantigen vaccines. Besides, we review the immunizations and the current clinical research status in neoantigen vaccines, and outline strategies for enhancing the efficacy of neoantigen vaccines. Finally, we present the challenges facing the application of neoantigens.

Liquid Biopsy in Pre-Metastatic Niche: From Molecular Mechanism to Clinical Application.

Metastatic dissemination represents a hallmark of cancer that is responsible for the high mortality rate. Recently, emerging evidence demonstrates a time-series event-pre-metastatic niche (PMN) has a profound impact on cancer metastasis. Exosomes, cell-free DNA (cfDNA), circulating tumor cells (CTC), and tumor microenvironment components, as critical components in PMN establishment, could be monitored by liquid biopsy. Intensive studies based on the molecular profile of liquid biopsy have made it a viable alternative to tissue biopsy. Meanwhile, the complex molecular mechanism and intercellular interaction are great challenges for applying liquid biopsy in clinical practice. This article reviews the cellular and molecular components involved in the establishment of the PMN and the promotion of metastasis, as well as the mechanisms of their interactions. Better knowledge of the characteristics of the PMN may facilitate the application of liquid biopsy for clinical diagnosis, prognosis, and treatment.

Roles of circulating tumor DNA in PD-1/PD-L1 immune checkpoint Inhibitors: Current evidence and future directions.

Circulating tumor DNA (ctDNA) sequencing holds considerable promise for early diagnosis and detection of surveillance and minimal residual disease. Blood ctDNA monitors specific cancers by detecting the alterations found in cancer cells, such as apoptosis and necrosis. Due to the short half-life, ctDNA reflects the actual burden of other treatments on tumors. In addition, ctDNA might be preferable to monitor tumor development and treatment compared with invasive tissue biopsy. ctDNA-based liquid biopsy brings remarkable strength to targeted therapy and precision medicine. Notably, multiple ctDNA analysis platforms have been broadly applied in clinical immunotherapy. Through targeted sequencing, early variations in ctDNA could predict response to immune checkpoint inhibitor (ICI). Several studies have demonstrated a correlation between ctDNA kinetics and anti-PD1 antibodies. The need for further research and development remains, although this biomarker holds significant prospects for early cancer detection. This review focuses on describing the basis of ctDNA and its current utilities in oncology and immunotherapy, either for clinical management or early detection, highlighting its advantages and inherent limitations.

Property evaluation on the spontaneous polarity for nanopowders.

For the tourmaline fine powders, in view of their low polarized charge density and easily occurred neutralization, a new evaluation model on the spontaneous polarity was proposed. By adjusting the temperature and applying electric field, the polarized charge could be measured. On this basis, a portable evaluation device was designed and assembled into four parts: Voltage Input Unit, Temperature Control Unit, Sample Loading Unit, and Charge Detection Unit. Using the designed device, the property evaluation on the spontaneous polarity of tourmaline fine powders was carried out. The spontaneous polarization intensity was finally achieved. After experimental verification, the method had the characteristics of easy operation and high accuracy.

Strain-induced quantum phase transition in the C3Sc4 monolayer: towards multiple gapless fermions.

Most two dimensional (2D) topological materials host only one kind of fermionic state. However, realizing multiple gapless fermions in a single 2D material is rarely reported. Furthermore, researchers face challenges in regulating various gapless fermion transitions using specific methods. Herein, we perform a study based on the first-principles calculation to investigate the electronic structures and the related fermionic states of strained 2D C3Sc4. C3Sc4 is an ideal system in the ground state with twelve Dirac points. The dynamical, mechanical, and thermal stabilities of the proposed C3Sc4 monolayer are demonstrated in detail. Interestingly, under the condition of 9.5% biaxial tensile strain, gapless and quadratic Weyl fermionic states are observed at the Γ point. A gapless and massless pseudospin-1 fermion appears at the Γ point in the 2D C3Sc4 system under 13% biaxial tensile strain and with hole doping. The Fermi velocity of this massless pseudospin-1 fermion is 2.1 × 105 m s-1, comparable to that of well-known 2D gapless topological materials. The results indicate that 2D C3Sc4 is an ideal playground to explore interesting behaviors of quantum phase transitions and rich gapless fermionic states and also reveal its potential applications in high-speed nano-devices.