Diagnosis and Vulnerability Risk Assessment of Atherosclerotic Plaques Using an Amino Acid-Assembled Near-Infrared Ratiometric Nanoprobe.

To reduce the risk of atherosclerotic disease, it is necessary to not only diagnose the presence of atherosclerotic plaques but also assess the vulnerability risk of plaques. Accurate detection of the reactive oxygen species (ROS) level at plaque sites represents a reliable way to assess the plaque vulnerability. Herein, through a simple one-pot reaction, two near-infrared (NIR) fluorescent dyes, one is ROS responsive and the other is inert to ROS, are coassembled in an amphiphilic amino acid-assembled nanoparticle. In the prepared NIR fluorescent amino acid nanoparticle (named FANP), the fluorescent properties and ROS-responsive behaviors of the two fluorescent dyes are well maintained. Surface camouflage through red blood cell membrane (RBCM) encapsulation endows the finally obtained FANP@RBCM nanoprobe with not only further reduced cytotoxicity and improved biocompatibility but also increased immune escape capability, prolonged blood circulation time, and thus enhanced accumulation at atherosclerotic plaque sites. In vitro and in vivo experiments demonstrate that FANP@RBCM not only works well in probing the occurrence of atherosclerotic plaques but also enables plaque vulnerability assessment through the accurate detection of the ROS level at plaque sites in a reliable ratiometric mode, thereby holding great promise as a versatile tool for the diagnosis and risk assessment of atherosclerotic disease.

Photoactivatable CRISPR/Cas12a Sensors for Biomarkers Imaging and Point-of-Care Diagnostics.

In recent years, the CRISPR/Cas12a-based sensing strategy has shown significant potential for specific target detection due to its rapid and sensitive characteristics. However, the "always active" biosensors are often insufficient to manipulate nucleic acid sensing with high spatiotemporal control. It remains crucial to develop nucleic acid sensing devices that can be activated at the desired time and space by a remotely applied stimulus. Here, we integrated photoactivation with the CRISPR/Cas12a system for DNA and RNA detection, aiming to provide high spatiotemporal control for nucleic acid sensing. By rationally designing the target recognition sequence, this photoactivation CRISPR/Cas12a system could recognize HPV16 and survivin, respectively. We combined the lateral flow assay strip test with the CRISPR/Cas12a system to realize the visualization of nucleic acid cleavage signals, displaying potential instant test application capabilities. Additionally, we also successfully realized the temporary control of its fluorescent sensing activity for survivin by photoactivation in vivo, allowing rapid detection of target nucleic acids and avoiding the risk of contamination from premature leaks during storage. Our strategy suggests that the CRISPR/Cas12a platform can be triggered by photoactivation to sense various targets, expanding the technical toolbox for precise biological and medical analysis. This study represents a significant advancement in nucleic acid sensing and has potential applications in disease diagnosis and treatment.

Allosteric Activator-Regulated CRISPR/Cas12a System Enables Biosensing and Imaging of Intracellular Endogenous and Exogenous Targets.

Sensors designed based on the trans-cleavage activity of CRISPR/Cas12a systems have opened up a new era in the field of biosensing. The current design of CRISPR/Cas12-based sensors in the "on-off-on" mode mainly focuses on programming the activator strand (AS) to indirectly switch the trans-cleavage activity of Cas12a in response to target information. However, this design usually requires the help of additional auxiliary probes to keep the activator strand in an initially "blocked" state. The length design and dosage of the auxiliary probe need to be strictly optimized to ensure the lowest background and the best signal-to-noise ratio. This will inevitably increase the experiment complexity. To solve this problem, we propose using AS after the "RESET" effect to directly regulate the Cas12a enzymatic activity. Initially, the activator strand was rationally designed to be embedded in a hairpin structure to deprive its ability to activate the CRISPR/Cas12a system. When the target is present, target-mediated strand displacement causes the conformation change in the AS, the hairpin structure is opened, and the CRISPR/Cas12a system is reactivated; the switchable structure of AS can be used to regulate the degree of activation of Cas12a according to the target concentration. Due to the advantages of low background and stability, the CRISPR/Cas12a-based strategy can not only image endogenous biomarkers (miR-21) in living cells but also enable long-term and accurate imaging analysis of the process of exogenous virus invasion of cells. Release and replication of virus genome in host cells are indispensable hallmark events of cell infection by virus; sensitive monitoring of them is of great significance to revealing virus infection mechanism and defending against viral diseases.

Inclusion of deuterated glycopeptides provides increased sequence coverage in hydrogen/deuterium exchange mass spectrometry analysis of SARS-CoV-2 spike glycoprotein.

RATIONALE: Hydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide precise analysis of a protein's conformational dynamics across varied states, such as heat-denatured versus native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX-MS, but one challenge for complete sequence coverage is N-glycosylation sites. The deuteration of peptides post-translationally modified by asparagine-bound glycans (glycopeptides) has not always been identified in previous reports of HDX-MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein. METHODS: We detected deuterated glycopeptides with a Tribrid Orbitrap Eclipse mass spectrometer performing data-dependent acquisition. An MS scan was used to identify precursor ions; if high-energy collision-induced dissociation MS/MS of the precursor indicated oxonium ions diagnostic for complex glycans, then electron transfer low-energy collision-induced dissociation MS/MS scans of the precursor identified the modified asparagine residue and the glycan's mass. As in traditional HDX-MS, the identified glycopeptides were then analyzed at the MS level in samples labeled with D2 O. RESULTS: We report HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric prefusion form, which has 22 predicted N-glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their average isotopic mass shifts from deuteration. Inclusion of the deuterated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural rearrangements. CONCLUSION: Inclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors.

Low-Background CRISPR/Cas12a Sensors for Versatile Live-Cell Biosensing.

The trans-cleavage activity of CRISPR/Cas12a has been widely used in biosensing. However, many CRISPR/Cas12a-based biosensors, especially those that work in "on-off-on" mode, usually suffer from high background and thus impossible intracellular application. Herein, this problem is efficiently overcome by elaborately designing the activator strand (AS) of CRISPR/Cas12a using the "RESET" effect found by our group. The activation ability of the as-designed AS to CRISPR/Cas12a can be easily inhibited, thus assuring a low background for subsequent biosensing applications, which not only benefits the detection sensitivity improvement of CRISPR/Cas12a-based biosensors but also promotes their applications in live cells as well as makes it possible to design high-performance biosensors with greatly improved flexibility, thus achieving the analysis of a wide range of targets. As examples, by using different strategies such as strand displacement, strand cleavage, and aptamer-substrate interaction to reactivate the inhibited enzyme activity, several CRISPR/Cas12a-based biosensing systems are developed for the sensitive and specific detection of different targets, including nucleic acid (miR-21), biological small molecules (ATP), and enzymes (hOGG1), giving the detection limits of 0.96 pM, 8.6 µM, and 8.3 × 10-5 U/mL, respectively. Thanks to the low background, these biosensors are demonstrated to work well for the accurate imaging analysis of different biomolecules in live cells. Moreover, we also demonstrate that these sensing systems can be easily combined with lateral flow assay (LFA), thus holding great potential in point-of-care testing, especially in poorly equipped or nonlaboratory environments.

Erk5 functions in modulation of zebrafish intestinal permeability.

The intestine of zebrafish consists of mucosa, muscularis and serosa. Intestinal epithelial cells (IECs) act as a physical and biochemical barrier to protect against invasion by external commensal bacteria. Cell junction is one of the crucial basis of the barrier function. When cell junctions were disrupted, intestinal permeability would be naturally impeded. Extracellular signal-regulated kinase 5 (ERK5), belonging to the Mitogen-activated protein kinase (MAPK) family, is involved in the normal physiological development of the cardiovascular system and nervous system. But the role of erk5 in intestinal morphogenesis and intestinal function is yet to know. Here, we showed that knockout of the erk5 in zebrafish larvae resulted in intestinal wall hypoplasia, including the thinned intestinal wall, reduced intestinal folds, and disrupted cell junctions. In addition, the intestinal permeability assay demonstrated that knockout of erk5 resulted in increased intestinal permeability. All of these showed that erk5 plays an essential role in the maintenance of intestinal barrier function. Thus, our data indicate that erk5 is a critical effector in intestinal morphogenesis and intestinal function, and dysfunction of erk5 would lead to intestinal diseases.

Efficacy and safety of PD-1 inhibitors in recurrent or metastatic nasopharyngeal carcinoma patients after failure of platinum-containing regimens: a systematic review and meta-analysis.

OBJECTIVE: There is a lack of standard salvage treatment options for recurrent or metastatic nasopharyngeal carcinoma (RM-NPC) that has failed platinum-containing regimens. Breakthroughs in immunotherapy have opened up new options for these patients. However, the efficacy and safety of immunotherapy have not been clarified. This study aimed to summarize and assess the efficacy and safety of PD-1 inhibitors in patients with RM-NPC who failed platinum-containing chemotherapy. METHODS: Up to August 25, 2022, clinical trials of PD-1 inhibitors in RM-NPC patients who failed platinum-containing regimens were searched in the PubMed, Embase, Cochrane, and Web of Science databases. Retrieval subject terms included "nasopharyngeal carcinoma", "metastatic", "recurrence", "PD-1", and "PD-L1". The clinical trials eligible for inclusion were systematically reviewed and meta-analyzed. RESULTS: A total of 9 studies including 842 patients with RM-NPC were included in this meta-analysis. The results showed that PD-1 inhibitors had promising efficacy in patients with RM-NPC who failed platinum-containing regimens: objective response rate (ORR) was 24% (95% confidence interval [CI] 21-26%), disease control rate (DCR) was 52% (95% CI 45-58%), 1-year progression-free survival (PFS) rate was 25% (95% CI 18-32%), and 1-year overall survival (OS) rate was 53% (95% CI 37-68%). In terms of treatment-related adverse events (AEs), the incidence of grade ≥ 3 treatment-related AEs was 19% (95% CI 13-24%). In addition, we found that PD-1 inhibitors were more effective in patients with PD-L1 positive than in patients with PD-L1 negative nasopharyngeal carcinoma who had failed platinum-containing regimens (ORR 31% (95%CI 26-35%) vs. 21% (95% CI 17-25%)). CONCLUSION: PD-1 inhibitors may provide a survival benefit for patients with RM-NPC who have failed platinum-containing regimens and have the advantage of a good safety profile, making them a promising treatment option.

Analysis of the N-glycosylation profiles of the spike proteins from the Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2.

N-Glycosylation plays an important role in the structure and function of membrane and secreted proteins. Viral proteins used in cell entry are often extensively glycosylated to assist in protein folding, provide stability, and shield the virus from immune recognition by its host (described as a "glycan shield"). The SARS-CoV-2 spike protein (S) is a prime example, having 22 potential sites of N-glycosylation per protein protomer, as predicted from the primary sequence. In this report, we conducted mass spectrometric analysis of the N-glycosylation profiles of recombinant spike proteins derived from four common SARS-CoV-2 variants classified as Variant of Concern, including Alpha, Beta, Gamma, and Delta along with D614G variant spike as a control. Our data reveal that the amino acid substitutions and deletions between variants impact the abundance and type of glycans on glycosylation sites of the spike protein. Some of the N-glycosylation sequons in S show differences between SARS-CoV-2 variants in the distribution of glycan forms. In comparison with our previously reported site-specific glycan analysis on the S-D614G and its ancestral protein, glycan types on later variants showed high similarity on the site-specific glycan content to S-D614G. Additionally, we applied multiple digestion methods on each sample, and confirmed the results for individual glycosylation sites from different experiment conditions to improve the identification and quantification of glycopeptides. Detailed site-specific glycan analysis of a wide variety of SARS-CoV-2 variants provides useful information toward the understanding of the role of protein glycosylation on viral protein structure and function and development of effective vaccines and therapeutics.

Effects of temperature control on hyperthermia-related cardiac dysfunction in a porcine model of cardiac arrest.

The outcome of cardiac arrest is worse when there is fever after spontaneous circulation is restored (ROSC). The purpose of this study was to investigate the mechanism of post-ROSC cardiac dysfunction after hyperthermia treatment and the effects of temperature control. Twenty-four male Bama minipigs were randomized into 3 groups (8 per group): CPR + controlled normothermia (CN), CPR + hyperthermia (HT), and CPR + therapeutic mild hypothermia (TMH). Defibrillation was given to pigs with ventricular fibrillation after 8 min of untreated fibrillation. Subsequently, these animals received the post-ROSC treatments of hyperthermia (38 °C), controlled normothermia (37 °C) or hypothermia (33 °C) according to the groups. Hemodynamic parameters, left ventricular ejection fraction, blood samples and myocardial tissues were assessed. At 24 h after the post-ROSC treatments, the pigs treated with hyperthermia showed increments in heart rate and plasma cardiac troponin I, and decreases in mean arterial pressure, cardiac index, and left ventricular ejection fraction, compared to those with the controlled normothermia pigs. However, the deterioration of the above parameters can be attenuated by TMH. The pigs in the TMH group also had a reduced percentage of apoptotic cardiomyocytes, an increased anti-apoptotic Bcl-2/Bax ratio and a decreased caspase-3 activity in myocardium, as compared with both controlled normothermia and hyperthermia pigs. In conclusion, hyperthermia is associated with a worse myocardial dysfunction. TMH improves hyperthermia-induced myocardial dysfunction by attenuating apoptosis in a porcine model of cardiac arrest.

[A glass micropipette vacuum technique of cerebrospinal fluid sampling in C57BL/6 mice].

The purpose of this study was to establish a suitable method for extracting cerebrospinal fluid (CSF) from C57BL/6 mice. A patch clamp electrode puller was used to draw a glass micropipette, and a brain stereotaxic device was used to fix the mouse's head at an angle of 135° from the body. Under a stereoscopic microscope, the skin and muscle tissue on the back of the mouse's head were separated, and the dura mater at the cerebellomedullary cistern was exposed. The glass micropipette (with an angle of 20° to 30° from the dura mater) was used to puncture at a point 1 mm inboard of Y-shaped dorsal vertebral artery for CSF sampling. After the first extraction, the glass micropipette was connected with a 1 mL sterile syringe to form a negative pressure device for the second extraction. The results showed that the successful rate of CSF extraction was 83.33% (30/36). Average CSF extraction amount was (7.16 ± 0.43) µL per mouse. In addition, C57BL/6 mice were given intranasally ferric ammonium citrate (FAC) to establish a model of brain iron accumulation, and the CSF extraction technique established in the present study was used for sampling. The results showed that iron content in the CSF from the normal saline control group was not detected, while the iron content in the CSF from FAC-treated group was (76.24 ± 38.53) µmol/L, and the difference was significant. These results suggest that glass micropipette vacuum technique of CSF sampling established in the present study has the advantages of simplicity, high success rate, large extraction volume, and low bleeding rate, and is suitable for the research on C57BL/6 mouse neurological disease models.

"RESET" Effect: Random Extending Sequences Enhance the Trans-Cleavage Activity of CRISPR/Cas12a.

The trans-cleavage activity of CRISPR/Cas12a has been widely used in biosensing applications. However, the lack of exploration on the fundamental properties of CRISPR/Cas12a not only discourages further in-depth studies of the CRISPR/Cas12a system but also limits the design space of CRISPR/Cas12a-based applications. Herein, a "RESET" effect (random extending sequences enhance trans-cleavage activity) is discovered for the activation of CRISPR/Cas12a trans-cleavage activity. That is, a single-stranded DNA, which is too short to work as the activator, can efficiently activate CRISPR/Cas12a after being extended a random sequence from its 3'-end, even when the random sequence folds into secondary structures. The finding of the "RESET" effect enriches the CRISPR/Cas12a-based sensing strategies. Based on this effect, two CRISPR/Cas12a-based biosensors are designed for the sensitive and specific detection of two biologically important enzymes.

Genotyping-by-sequencing and genome-wide association study reveal genetic diversity and loci controlling agronomic traits in triticale.

KEY MESSAGE: The genetic diversity and loci underlying agronomic traits were analyzed by the reads coverage and genome-wide association study based genotyping-by-sequencing in a diverse population consisting of 199 accessions. Triticale (× Triticosecale Wittmack) is an economically important grain forage and energy crop planted worldwide for its high biomass. Little is known about the genetic diversity and loci underlying agronomic traits in triticale. We performed genotyping-by-sequencing of 199 cultivars and mapped reads to the A, B, D, and R genomes for karyotype analysis. These cultivars could mostly be grouped into five types. Some chromosome abnormalities occurred with high frequency, such as 2D (2R) substitution, deletion of the long arm of chromosome 2D or the short arm of 5R, and translocation of the long arms of 7D/7A, the short arms of 6D/6A, or the long arms of 1D/1A. We chose only widely planted hexaploid triticale cultivars (153) for genome-wide association study. These cultivars could be divided into nine distinct groups, and the linkage disequilibrium decay was 25.4 kb in this population. We identified 253 significant marker-trait associations (MTAs) on 20 chromosomes, except 7R. Twenty-one reliable MTAs were identified repeatedly over two environments. We predicted 16 putative candidate genes involved in plant growth and development using the genome sequences of wheat and rye. These results provide a basis for understanding the genetic mechanisms of agronomic traits and will benefit the breeding of improved hexaploid triticale.

General toxicity and genotoxicity of altertoxin I: A novel 28-day multiendpoint assessment in male Sprague-Dawley rats.

The mycotoxin altertoxin I (ATX-I) is one of secondary metabolites produced by Alternaria fungi and is frequently detected as food and feed contaminants. Little is known about the genotoxicity of the ATX-I. In order to evaluate potential genotoxicity and general toxicity of ATX-I, the novel 28-day multiendpoint (Pig-a assay + micronucleus [MN] test + comet assay) genotoxicity platform was applied. Male Sprague-Dawley (SD) rats were randomized to five groups (six rats per group), that is, a positive control group (N-ethyl-N-nitrosourea [ENU], 40 mg/kg.bw/d), two solvent control groups (PBS and corn oil), and two ATX-I-treated groups (low-dose group [1.10 µg/kg.bw/d] and high-dose group [5.51 µg/kg.bw/d]). Treatments were administered by oral gavage to male SD rats for 28 consecutive days. Histopathological damages in the liver, kidney, and spleen were observed, but without significant changes in hematological and serum biochemical parameters. Genotoxic endpoints indicated that ATX-I could cause DNA damage. To summarize, in a relatively low-dose range, ATX-I may not have direct genotoxicity in vivo but could induce liver, kidney, and spleen damage.

Oxidative Cleavage-Based Three-Dimensional DNA Biosensor for Ratiometric Detection of Hypochlorous Acid and Myeloperoxidase.

Methods to detect and quantify disease biomarkers with high specificity and sensitivity in biological fluids play a key role in enabling clinical diagnosis, including point-of-care testing. Myeloperoxidase (MPO) is an emerging biomarker for the detection of inflammation, neurodegenerative diseases, and cardiovascular disease, where excess MPO can lead to oxidative damage to biomolecules in homeostatic systems. While numerous methods have been developed for MPO analysis, most techniques are challenging in clinical applications due to the lack of amplification methods, high cost, or other practical drawbacks. Enzyme-linked immunosorbent assays are currently used for the quantification of MPO in clinical practice, which is often limited by the availability of antibodies with high affinity and specificity and the significant nonspecific binding of antibodies to the analytical surface. In contrast, nucleic acid-based biosensors are of interest because of their simplicity, fast response time, low cost, high sensitivity, and low background signal, but detection targets are limited to nucleic acids and non-nucleic acid biomarkers are rare. Recent studies reveal that the modification of a genome in the form of phosphorothioate is specifically sensitive to the oxidative effects of the MPO/H2O2/Cl- system. We developed an oxidative cleavage-based three-dimensional DNA biosensor for rapid, ratiometric detection of HOCl and MPO in a "one-pot" method, which is simple, stable, sensitive, specific, and time-saving and does not require a complex reaction process, such as PCR and enzyme involvement. The constructed biosensor has also been successfully used for MPO detection in complex samples. This strategy is therefore of great value in disease diagnosis and biomedical research.

DNA nanolantern-based split aptamer probes for in situ ATP imaging in living cells and lighting up mitochondria.

Accurate and specific analysis of adenosine triphosphate (ATP) expression levels in living cells can provide valuable information for understanding cell metabolism, physiological activities and pathologic mechanisms. Herein, DNA nanolantern-based split aptamer nanoprobes are prepared and demonstrated to work well for in situ analysis of ATP expression in living cells. The nanoprobes, which carry multiple split aptamer units on the surface, are easily and inexpensively prepared by a "one-pot" assembly reaction of four short oligonucleotide strands. A series of characterization experiments verify that the nanoprobes have good monodispersity, strong biostability, high cell internalization efficiency, and fluorescence resonance energy transfer (FRET)-based ratiometric response to ATP in the concentration range covering the entire intracellular ATP expression level. By changing the intracellular ATP level via different treatments, the nanoprobes are demonstrated to show excellent performance in intracellular ATP expression analysis, giving a highly ATP concentration-dependent ratiometric fluorescence signal output. ATP-induced formation of large-sized DNA aggregates not only amplifies the FRET signal output, but also makes in situ ATP-imaging analysis in living cells possible. In situ responsive crosslinking of nanoprobes also makes them capable of lighting up the mitochondria of living cells. By simply changing the split aptamer sequence, the proposed DNA nanolantern-based split aptamer strategy might be easily extended to other targets.

A method for measuring the experimental resolution of laboratory assays (clinical biochemical, blood count, immunological, and qPCR) to evaluate analytical performance.

BACKGROUND: The measurement method for experimental resolution and related data to evaluate analytical performance is poorly explored in clinical research. We established a method to measure the experimental resolution of clinical tests, including biochemical tests, automatic hematology analyzer methods, immunoassays, chemical experiments, and qPCR, to evaluate their analytical performance. METHODS: Serially diluted samples in equal proportions were measured, and correlation analysis was performed between the relative concentration and the measured value. Results were accepted for p ≤ 0.01 of the correlation coefficient. The minimum concentration gradient (eg, 10%) was defined as the experimental resolution. For this method, the smaller the value, the higher the experimental resolution and the better the analytical performance. RESULTS: The experimental resolution of the most common biochemical indices reached 10%, with some even reaching 1%. The results of most counting experiments showed experimental resolution up to 10%, whereas the experimental resolution of the classical chemical assays reached 1%. Unexpectedly, the experimental resolution of more sensitive assays, such as immunoassays was only 25% when using the manual method and 10% for qPCR. CONCLUSION: This study established a method for measuring the experimental resolution of laboratory assays and provides a new index for evaluating the reliability of methods in clinical laboratories.

1α,25-Dihydroxyvitamin D3 prevents renal oxidative damage via the PARP1/SIRT1/NOX4 pathway in Zucker diabetic fatty rats.

Diabetic nephropathy (DN) is one of the most important renal complications associated with diabetes, and the mechanisms are yet to be fully understood. To date, few studies have shown the antioxidant effects of 1α,25-dihydroxyvitamin-D3 [1,25(OH)2D3] on hyperglycemia-induced renal injury. The aim of the present study was to explore the potential mechanism by which 1,25(OH)2D3 reduced oxidative stress in diabetic rat kidneys. In this study, we established a vitamin D-deficient spontaneous diabetes model: 5-6 wk of age Zucker diabetic fatty (ZDF) rats were treated with or without 1,25(OH)2D3 for 7 wk, age-matched Zucker lean rats served as control. Results showed that ZDF rats treated with 1,25(OH)2D3 had decreased body mass, food intake, water intake, and urine volume. 1,25(OH)2D3 ameliorated urine glucose, blood glucose and abnormal glucose tolerance. Additionally, 1,25(OH)2D3 significantly lowered microalbuminuria, decreased the glomerular basement membrane thickness, and in some degree inhibited glomerular hypertrophy, mesangial expansion, and tubular dilatation. Furthermore, 1,25(OH)2D3 attenuated renal oxidative damage, as reflected by the levels of malondialdehyde, reduced glutathione, 4-hydroxynonenal, 8-hydroxy-2'-deoxyguanosine, and reactive oxygen species production, and notably inhibited poly(ADP-ribose) polymerase-1 (PARP1), activated sirtuin 1 (SIRT1), and decreased the expression of NADPH oxidase 4 (NOX4). Of interest, the abovementioned proteins could be involved in the antioxidant mechanism of 1,25(OH)2D3 in diabetic rat kidneys. Our study showed that oxidative stress might be a major contributor to DN pathogenesis and uncovered the antioxidant role of 1,25(OH)2D3 in diabetic nephropathy that was associated with the PARP1/SIRT1/ NOX4 pathway.

Comprehensive Analysis of the Glycan Complement of SARS-CoV-2 Spike Proteins Using Signature Ions-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation (EThcD) Mass Spectrometry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic of coronavirus disease 2019 (COVID-19). The spike protein expressed on the surface of this virus is highly glycosylated and plays an essential role during the process of infection. We conducted a comprehensive mass spectrometric analysis of the N-glycosylation profiles of the SARS-CoV-2 spike proteins using signature ions-triggered electron-transfer/higher-energy collision dissociation (EThcD) mass spectrometry. The patterns of N-glycosylation within the recombinant ectodomain and S1 subunit of the SARS-CoV-2 spike protein were characterized using this approach. Significant variations were observed in the distribution of glycan types as well as the specific individual glycans on the modification sites of the ectodomain and subunit proteins. The relative abundance of sialylated glycans in the S1 subunit compared to the full-length protein could indicate differences in the global structure and function of these two species. In addition, we compared N-glycan profiles of the recombinant spike proteins produced from different expression systems, including human embryonic kidney (HEK 293) cells and Spodoptera frugiperda (SF9) insect cells. These results provide useful information for the study of the interactions of SARS-CoV-2 viral proteins and for the development of effective vaccines and therapeutics.

An integrative overview of physiological and proteomic changes of cytokinin-induced potato (Solanum tuberosum L.) tuber development in vitro.

Potato tuberization is a complicated biological process regulated by multiple phytohormones, in particular cytokinins (CKs). The information available on the molecular mechanisms regulating tuber development by CKs remains largely unclear. Physiological results initially indicated that low 6-benzylaminopurine (BAP) concentration (3 mg l-1 ) advanced the tuberization beginning time and promoted tuber formation. A comparative proteomics approach was applied to investigate the proteome change of tuber development by two-dimensional gel electrophoresis in vitro, subjected to exogenous BAP treatments (0, 3, 6 and 13 mg l-1 ). Quantitative image analysis showed a total of 83 protein spots with significantly altered abundance (>2.5-fold, P < 0.05), and 55 differentially abundant proteins were identified by MALDI-TOF/TOF MS. Among these proteins, 22 proteins exhibited up-regulation with the increase of exogenous BAP concentration, and 31 proteins were upregulated at 3 mg l-1 BAP whereas being downregulated at higher BAP concentrations. These proteins were involved in metabolism and bioenergy, storage, redox homeostasis, cell defense and rescue, transcription and translation, chaperones, signaling and transport. The favorable effects of low BAP concentrations on tuber development were found in various cellular processes, mainly including the stimulation of starch and storage protein accumulation, the enhancement of the glycolysis pathway and ATP synthesis, the cellular homeostasis maintenance, the activation of pathogen defense, the higher efficiency of transcription and translation, as well as the enhanced metabolite transport. However, higher BAP concentration, especially 13 mg l-1 , showed disadvantageous effects. The proposed hypothetical model would explain the interaction of these proteins associated with CK-induced tuber development in vitro.

Highly Integrated, Biostable, and Self-Powered DNA Motor Enabling Autonomous Operation in Living Bodies.

An ultimate goal of synthetic DNA motor studies is to mimic natural protein motors in biological systems. Here, we rationally designed a highly integrated and biostable DNA motor system with high potential for living body operation, through simple assembly of a Mn2+-dependent DNAzyme-powered DNA motor with a degradable MnO2 nanosheet. The motor system shows outstanding high integration and improved biostability. High integration confers the motor system with the ability to deliver all the core components to the target sites as a whole, thus, enabling precise control of the spatiotemporal distribution of these components and achieving high local concentrations. At the target sites, reduction of the MnO2 nanosheet by intracellular glutathione (GSH) not only releases the DNA motor, which can then be initiated by the intracellular target, but also produces Mn2+ in situ to power the autonomous and progressive operation of the DNA motor. Interestingly, the resultant consumption of GSH in turn protects the DNA motor from destruction by physiological GSH, thus, conferring our motor system with improved biostability, reduced false-positive outputs, and consequently, an increased potential to be applied in a living body. As a proof of concept, the highly integrated DNA motor system was demonstrated to work well for amplified imaging detection of survivin mRNA (mRNA), an important tumor biomarker, in both living cancer cells and living tumor-bearing mice. This work reveals concepts and strategies promoting synthetic DNA motor applications in biological systems.