An Optimized Miniaturized Filter-Aided Sample Preparation Method for Sensitive Cross-Linking Mass Spectrometry Analysis of Microscale Samples.

Cross-linking mass spectrometry (XL-MS) is a powerful tool for elucidating protein structures and protein-protein interactions (PPIs) at the global scale. However, sensitive XL-MS analysis of mass-limited samples remains challenging, due to serious sample loss during sample preparation of the low-abundance cross-linked peptides. Herein, an optimized miniaturized filter-aided sample preparation (O-MICROFASP) method was presented for sensitive XL-MS analysis of microscale samples. By systematically investigating and optimizing crucial experimental factors, this approach dramatically improves the XL identification of low and submicrogram samples. Compared with the conventional FASP method, more than 7.4 times cross-linked peptides were identified from single-shot analysis of 1 µg DSS cross-linked HeLa cell lysates (440 vs 59). The number of cross-linked peptides identified from 0.5 µg HeLa cell lysates was increased by 58% when further reducing the surface area of the filter to 0.058 mm2 in the microreactor. To deepen the identification coverage of XL-proteome, five different types of cross-linkers were used and each µg of cross-linked HeLa cell lysates was processed by O-MICROFASP integrated with tip-based strong cation exchange (SCX) fractionation. Up to 2741 unique cross-linked peptides were identified from the 5 µg HeLa cell lysates, representing 2579 unique K-K linkages on 1092 proteins. About 96% of intraprotein cross-links were within the maximal distance restraints of 26 Å, and 75% of the identified PPIs reported by the STRING database were with high confidence (scores ≥0.9), confirming the high validity of the identified cross-links for protein structural mapping and PPI analysis. This study demonstrates that O-MICROFASP is a universal and efficient method for proteome-wide XL-MS analysis of microscale samples with high sensitivity and reliability.

Solvothermal Preparation of Crystal Seeds and Anisotropy-Controlled Growth of Silver Nanoplates.

We synthesized silver nanoplates using the solvothermal method and, for the first time, placed them as crystal seeds in a water-based growth solution, thereby successfully achieving the large-scale production of silver nanoplates. The synthesis method enabled independent control of the lateral size and vertical size of the silver nanoplates. More specifically, the lateral size could be adjusted within the range of 565 nm-1.682 µm, while the vertical size was achieved by introducing Cl- as a capping agent and the vertical size was thickened from 18.28 to 40.41 nm.

Dietary crude protein and protein solubility manipulation enhances intestinal nitrogen absorption and mitigates reactive nitrogen emissions through gut microbiota and metabolome reprogramming in sheep.

Dietary nutrient manipulation (e.g. protein fractions) could lower the environmental footprints of ruminants, especially reactive nitrogen (N). This study investigated the impacts of dietary soluble protein (SP) levels with decreased crude protein (CP) on intestinal N absorption, hindgut N metabolism, fecal microbiota and metabolites, and their linkage with N metabolism phenotype. Thirty-two male Hu sheep, with an age of six months and an initial BW of 40.37 ± 1.18 kg, were randomly assigned to four dietary groups. The control diet (CON), aligning with NRC standards, maintained a CP content of 16.7% on a dry matter basis. Conversely, the experimental diets (LPA, LPB, and LPC) featured a 10% reduction in CP compared with CON, accompanied by SP adjustments to 21.2%, 25.9%, and 29.4% of CP, respectively. Our results showed that low-protein diets led to significant reductions in the concentrations of plasma creatinine, ammonia, urea N, and fecal total short-chain fatty acids (SCFA) (P < 0.05). Notably, LPB and LPC exhibited increased total SCFA and propionate concentrations compared with LPA (P < 0.05). The enrichment of the Prevotella genus in fecal microbiota associated with energy metabolism and amino acid (AA) biosynthesis pathways was evident with SP levels in low-protein diets of approximately 25% to 30%. Moreover, LPB and LPC diets demonstrated a decrease in fecal NH 4 + -N and NO 2 - -N contents as well as urease activity, compared with CON (P < 0.05). Concomitantly, reductions in fecal glutamic acid dehydrogenase gene (gdh), nitrite reductase gene (nirS), and nitric oxide reductase gene (norB) abundances were observed (P < 0.05), pointing towards a potential reduction in reactive N production at the source. Of significance, the up-regulation of mRNA abundance of AA and peptide transporters in the small intestine (duodenum, jejunum, and ileum) and the elevated concentration of plasma AA (e.g. arginine, methionine, aspartate, glutamate, etc.) underscored the enhancement of N absorption and N efficiency. In summary, a 10% reduction in CP, coupled with an SP level of approximately 25% to 30%, demonstrated the potential to curtail reactive N emissions through fecal Prevotella enrichment and improve intestinal energy and N utilization efficiency.

[An enrichment strategy based on hydrophobic tagging and reversed-phase chromatographic separation for the analysis of lysine-containing peptides].

Lysine (K) is widely used in the design of lysine-targeted crosslinkers, structural elucidation of protein complexes, and analysis of protein-protein interactions. In "shotgun" proteomics, which is based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), proteins from complex samples are enzymatically digested, generating thousands of peptides and presenting significant challenges for the direct analysis of K-containing peptides. In view of the lack of effective methods for the enrichment of K-containing peptides, this work developed a method which based on a hydrophobic-tag-labeling reagent C10-S-S-NHS and reversed-phase chromatography (termed as HYTARP) to achieve the efficient enrichment and identification of K-containing peptides from complex samples. The C10-S-S-NHS synthesized in this work successfully labeled standard peptides containing various numbers of K and the labeling efficiency achieved up to 96% for HeLa cell protein tryptic digests. By investigating the retention behavior of these labeled peptides in C18 RP column, we found that most K-labeled peptides were eluted once when acetonitrile percentage reached 57.6% (v/v). Further optimization of the elution gradient enabled the efficient separation and enrichment of the K-labeled peptides in HeLa digests via a stepwise elution gradient. The K-labeled peptides accounted for 90% in the enriched peptides, representing an improvement of 35% compared with the number of peptides without the enrichment. The dynamic range of proteins quantified from the enriched K-containing peptides spans 5-6 orders of magnitude, and realized the detection of low-abundance proteins in the complex sample. In summary, the HYTARP strategy offers a straightforward and effective approach for reducing sample complexity and improving the identification coverage of K-containing peptides and low-abundance proteins.

Integrated MICROFASP Method with CZE-Based Fractionation Technique and NanoRPLC-ESI-MS/MS for a Comprehensive Proteomics Analysis of a Submicrogram Sample.

We report a loss-less two-dimensional (2D) separation platform that integrated capillary zone electrophoresis (CZE) fractionation and nanoRPLC-ESI-MS/MS for a comprehensive proteomics analysis of a submicrogram sample. Protein digest was injected into the linear polyacrylamide-coated capillary, followed by CZE separation. The schemes for collecting the fractions were carefully optimized to maximize the protein coverage. The peptide fractions were directly eluted into the autosampler insert vials, followed by the nanoRPLC-ESI-MS/MS analysis without lyophilization and redissolution, thus dramatically minimizing sample loss and potential contamination. The integrated platform generated 30,845 unique peptides and 5231 protein groups from 500 ng of a HeLa protein digest within 11.5 h (90 min CZE fractionation plus 10 h LC-MS analysis). Finally, the developed platform was used to analyze the protein digest prepared by the MICROFASP method with 1 µg of cell lysate as the starting material. Three thousand seven hundred ninety-six (N = 2, RSD = 4.95%) protein groups and 20,577 (N = 2, RSD = 7.89%) peptides were identified from only 200 ng of the resulted tryptic digest within 5.5 h. The results indicated that the combination of the MICROFASP method and the developed CZE/nanoRPLC-MS/MS 2D separation platform enabled comprehensive proteome profiling of a submicrogram biological sample. Data are available via ProteomeXchange with the identifier PXD052735.

Site-specific identification and quantitation of endogenous SUMOylation based on SUMO-specific protease and strong anion exchange chromatography.

Small ubiquitin-like modifier (SUMO) modification regulates various eukaryotic cellular processes and plays a pivotal role in interferon (IFN)-mediated antiviral defense. While immunoprecipitation enrichment method is widely used for proteome-wide analysis of endogenous SUMOylation, the inability to target all SUMO forms and high cost of antibodies limited its further application. Herein, we proposed an antibody-free enrichment method based on SUMO-specific protease and strong anion exchange chromatography (SPAX) to globally profile the endogenous SUMOylation. The SUMO1/2/3-modified peptides could be simultaneously enriched by SAX chromatography by utilizing its electrostatic interaction with SUMO1/2/3 remnants, which contained multiple aspartic acids (D) and glutamic acids (E). To remove the co-enriched D/E-containing peptides which might interfere with the detection of low-abundance SUMOylated peptides, SUMO-specific protease was used to cleave the SUMO1/2/3 remnants from enriched SUMOylated peptides. As the deSUMOylated peptides lost SUMO remnants, their interaction with SAX materials became weaker, and the D/E-containing peptides could thus be depleted through the second SAX separation. The SPAX method identified over twice the SUMOylated sites than using SAX method only, greatly improving the identification coverage of endogenous SUMOylated sites. Our strategy was then applied to the site-specific identification and quantification of endogenous SUMOylation in A549 cells stimulated by IFN-γ for the first time. A total of 226 SUMOylated sites on 146 proteins were confidently identified, among which multiple up-regulated sites were involved in IFN-mediated antiviral defense, demonstrating the great promise of SPAX to globally profile and discover endogenous SUMOylation with significant biological functions.

Case report: Nutritionally supported perioperative chemo-immunotherapy for advanced gastric cancer with incomplete pyloric obstruction.

This case describes the benefits of perioperative chemo-immunotherapy for advanced gastric cancer and incomplete pyloric obstruction, supplemented with nutritional support. Early parenteral nutrition to stabilize nutritional status and mitigate nutrition impact symptoms, and in addition, throughout the chemo-immunotherapy perioperative period also maintained oral nutrition support and a tailored dietary plan. Above nutritional support maintained the patient's physical condition during immunotherapy. Eventually, this combination therapy plan leads to a partial response. On the other hand, a combination of therapies that focus more on immune checkpoint inhibitor may be able to mitigate the side effects of chemotherapy. Such findings may yield novel prospects for patients with advanced gastric cancer and incomplete pyloric obstruction, enabling them to achieve better outcomes.

Effect of Adding Al on the Phase Structure and Gettering Performance of TiZrV Non-Evaporable Getter Materials.

Titanium zirconium vanadium (TiZrV) is a widely used non-evaporable getter (NEG) material with the characteristics of a low activation temperature and a large gas absorption capacity. At present, the research on TiZrV getters mainly focuses on the thin-film state, with little research on the bulk state. In this paper, a TiZrV getter was optimized by adding Al, and the phase structure, activation properties, and gettering performance were studied. With the addition of Al, the α-Zr phase and Ti2Zr phase changed into the Ti-Zr phase and Al-Zr, Al-Ti phase. The newly generated phase promoted the diffusion of hydrogen and oxygen atoms. The activation temperature decreased significantly, as shown in the in situ XPS results. The H2 and CO gettering performance of TiZrVAl samples was promoted to 2073 cm3·s-1 and 1912.8 cm3·s-1, increased by 40.7% and 40.3%. This paper provides valuable ideas for optimizing the properties of bulk TiZrV getters.

De novo transcriptome assembly database for 100 tissues from each of seven species of domestic herbivore.

Domesticated herbivores are an important agricultural resource that play a critical role in global food security, particularly as they can adapt to varied environments, including marginal lands. An understanding of the molecular basis of their biology would contribute to better management and sustainable production. Thus, we conducted transcriptome sequencing of 100 to 105 tissues from two females of each of seven species of herbivore (cattle, sheep, goats, sika deer, horses, donkeys, and rabbits) including two breeds of sheep. The quality of raw and trimmed reads was assessed in terms of base quality, GC content, duplication sequence rate, overrepresented k-mers, and quality score distribution with FastQC. The high-quality filtered RNA-seq raw reads were deposited in a public database which provides approximately 54 billion high-quality paired-end sequencing reads in total, with an average mapping rate of ~93.92%. Transcriptome databases represent valuable resources that can be used to study patterns of gene expression, and pathways that are related to key biological processes, including important economic traits in herbivores.

Topological corner state localized bound states in continuum in photonic crystals.

In the field of optics, bound states in the continuum (BICs) are of significant practical importance as they can trap electromagnetic waves spatially, even though their frequency lies within the continuous spectrum. Previous research, however, has shown that BICs localized in optical cavities are highly sensitive to geometric and environmental changes. This sensitivity implies that slight variations can lead to the loss of BICs, necessitating extreme precision in manufacturing, which poses a challenge for practical implementation. To overcome this issue, this study employs topological photonic crystals (PhCs) to engineer topological corner states (TCS) within PhCs. By doing so, it establishes a method for creating topological BICs that are inherently robust against disturbances, thereby enhancing their suitability for real-world applications.

A Method of Reducing Salt Content in Fermented Soy Sauce Improves Its Flavor and Quality.

Most commercially available soy sauce is fermented by high-salt liquid-state (HS) fermentation, which has an excessive salt content and a long fermentation period. In this study, a new salt-reduced fermentation (SR) soy sauce technology involving multiple strains of bacteria was developed to reduce consumers' salt intake. The SR soy sauce was found to have an amino acid nitrogen content of 8.40 g/L and over 80 kinds of flavor substances, which were significantly higher than those of low-salt solid-state fermented soy sauce and approximately equal to HS soy sauce. Compared with HS soy sauce, the salt content of the SR soy sauce was reduced by 59.2%, achieving the salt reduction goal. The proportion of umami amino acids in SR soy sauce reached 32.0% of the total level, enhancing SR soy sauce's quality. Hence, the new fermentation process can decrease salt content and shorten fermentation time.

Early results of high tibial osteotomy versus combined arthroscopic surgery.

Objective: To investigate the early effect of high tibial osteotomy (HTO) compared with combined arthroscopic surgery. Methods: A retrospective study was conducted on patients who underwent HTO at The First Affiliated Hospital of Shandong First Medical University from January 2018 to January 2022. 138 patients (163 knees) with knee osteoarthritis (KOA) treated with HTO were selected. The medial proximal tibial angle (MPTA), joint line convergence angle (JLCA), femoral tibial angle (FTA), hip-knee-ankle (HKA) angle, weight-bearing line (WBL) ratio of the knee joint, opening gap, opening angle, American Knee Society score (KSS), US Hospital for Special Surgery (HSS) score, and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score were measured to determine the different effects between HTO and HTO combined with arthroscopic by logistic regression analysis. Results: Patients with HTO combined with arthroscopic surgery have improved functional scores as well as imaging perspectives compared to preoperative. By multivariate logistic analysis, it was concluded that arthroscopic surgery and gender are influential factors in the outcome of HTO surgery. The postoperative KSS score was 2.702 times more likely to be classified as excellent in the HTO combined with arthroscopy group than in the HTO group [Exp (ß) = 2.702, 95% CI (1.049-6.961), P = 0.039]; the postoperative KSS score was 0.349 times more likely to be classified as excellent in women than in men [Exp (ß) = 0.349, 95% CI (0.138-0.883), P = 0.026]. Conclusion: Better results with HTO combined with arthroscopic surgery. HTO combined with arthroscopy is a better choice in the surgical treatment of KOA.

ATM-Mediated translocation of RanBPM regulates DNA damage response by stabilizing p21 in non-small cell lung cancer cells.

PURPOSE: Platinum-based chemotherapy remains a standard-of-care for most patients with advanced non-small cell lung cancer (NSCLC). DNA damage response (DDR) induced by platinum or Etoposide activated a panel of cell cycle-regulatory proteins including p21 through p53 pathway. Previous studies have reported that RanBPM has been involved in various cellular processes such as DDR by interacting with multiple proteins. However, the underlying mechanism remains unclear. METHODS: NSCLC tissue microarrays were used for assessing the expression of RanBPM by immunohistochemical staining. The roles of RanBPM in the DDR of NSCLC progression was examined in in vitro cell lines and in vivo animal models. The regulation of RanBPM on protein stability and ubiquitination levels were investigated by immunoblots and in vivo ubiquitylation assay. RESULTS: The level of p21 or RanBPM is lower in NSCLC than non-malignant tissues and has a highly positive correlation. Mechanistically, RanBPM protein physically interacts with p21, and RanBPM deubiquitinates p21 by recruiting a deubiquitinase USP11 to maintain protein stability of p21. RanBPM silencing significantly decreased p21 protein level. Conversely, RanBPM overexpression led to the accumulation of endogenous p21 protein regardless of p53 status. Functionally, RanBPM regulates DDR in a p21-dependent manner. Furthermore, DNA damage significantly promoted the nuclear translocation of RanBPM protein through ATM signaling pathways. CONCLUSION: RanBPM is a novel regulator of P21 protein stability, and plays a critical role in the regulation of DDR.

The value of serum neutralizing antibody in evaluating predictability of COVID-19 after recovery and the validation of vaccine.

BACKGROUND: This work aimed to study natural humoral immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: Chemiluminescent immunoassay (CLIA) was used to detect the neutralizing antibody (Nabs) and IgG. RESULTS: Nabs peaked on days 57-96 after symptom onset and remained detected on days 97-132. The Nabs in the 32 patients who were dynamically monitored showed four changing patterns. The titers of Nabs and IgG were correlated, and three modes of relationship were found between them. CONCLUSIONS: Nabs showed a regular change in the course of coronavirus disease 2019 (COVID-19). The detection of Nabs is very important for monitoring the course of COVID-19 and predicting the strength of antibody protection.

Antioxidant Capacity, Inflammatory Response, Carcass Characteristics and Meat Quality of Hu Sheep in Response to Dietary Soluble Protein Levels with Decreased Crude Protein Content.

Manipulating dietary nutrients, especially protein fractions, holds significance in enhancing the antioxidant capacity and immunity function of ruminants. This study investigated the impact of dietary adjustments in soluble protein (SP) levels, in conjunction with a reduction in crude protein (CP) content, on the antioxidant capacity, inflammatory response, carcass characteristics, and meat quality of sheep. This study had four dietary treatments, including a control diet (CON) adhering to NRC standards with a CP content of 16.7% on a dry matter basis and three diets with an approximately 10% reduction in CP content compared to CON with SP levels (% of CP) of 21.2 (SPA), 25.9 (SPB) and 29.4% (SPC), respectively. Thirty-two healthy male Hu sheep, with an initial live weight of 40.37 ± 1.18 kg and age of 6 months, were randomly divided into four groups to receive these respective diets. Our data revealed no significant differences in slaughter performance among treatments (p > 0.05), although low-protein treatments decreased the stomachus compositus index (p < 0.05). Compared with CON, as SP was adjusted to 21.2%, total antioxidant capacity (T-AOC) and catalase (CAT) concentrations were decreased in the serum (p < 0.05), glutathione peroxidase (GSH-Px) content was decreased in jejunum and ileum (p < 0.05), superoxide dismutase (SOD) concentration was reduced in the duodenum (p < 0.05), and malondialdehyde (MDA) content was increased in spleen and ileum (p < 0.05). On the other hand, pro-inflammatory cytokine (IL-1ß, IL-6 and IL-8) contents were upregulated in the serum (p < 0.05), while immunoglobulin (IgA and IgM) contents were reduced in the duodenum (p < 0.05) with SP adjustments. Additionally, the SPB and SPC diets reduced the content of saturated fatty acids and increased the content of polyunsaturated fatty acids compared with CON (p < 0.05), along with retention in the tenderness and water-holding capacity of the longissimus lumborum muscle. In summary, reducing CP by 10% with an SP proportion of ~25-30% improved meat quality without compromising antioxidant capacity and immunity function, while lower SP levels had adverse effects.

Combining sensitivity and robustness: EIT-like characteristic in a 2D topological photonic crystal.

The study of topological photonics has gained significant attention due to its potential application for robust and efficient light manipulation. In this work, we theoretically investigate a two-dimensional photonics crystal that exhibits a topological edge state (TES) and a topological corner state (TCS). Furthermore, we also achieve a coupling between a topological corner state and a trivial cavity (TC), resulting in a phenomenon similar to the electromagnetically induced transparency (EIT) effect. To verify the stability of the EIT-like effect, disorders around TES and TCS are introduced, and the theoretical results show that this structure is immune to the disorders. The achievement of the coupling between topological states can have potential applications in the areas of waveguiding, sensing, and logic gates. It is hoped that this work will contribute to the ongoing efforts in the exploration and utilization of topological photonics.

Multidimensional separation methods for in-depth proteome profiling of low-microgram samples.

Tweetable abstract Multidimensional separation methods with improved sensitivity and peak capacity and throughput allow in-depth proteome profiling of low-microgram samples.

CircRNA-02191 regulating unsaturated fatty acid synthesis by adsorbing miR-145 to enhance CD36 expression in bovine mammary gland.

CD36 functions as a receptor for long-chain fatty acids, promoting the absorption and transport of long-chain unsaturated fatty acids. However, the regulatory influence of upstream circRNAs or miRNAs on its expression in cow mammary gland remains unclear. Herein, we performed high-throughput sequencing to screen for differentially expressed miRNAs and mRNAs in bovine mammary tissue during the late-lactation and the dry period to screen and conducted bioinformatics analysis to identify 420 miRNA/mRNA pairs, including miR-145/CD36. Experimental results indicate that miR-145 can directly target CD36 and inhibit its expression. Additionally, the circRNA-02191 sequence is predicted to contain a miR-145 binding site. As shown by dual luciferase reporter system detection, circRNA-02191 bound to miR-145 and its overexpression significantly reduced the expression of miR-145. Furthermore, the overexpression of miR-145 inhibited triglyceride accumulation, while circRNA-02191 enhanced the expression of the miR-145 target gene CD36. The above results indicate that circRNA-02191 can regulate triglyceride and fatty acid components by binding miR-145 and subsequently alleviating the inhibitory effect of miR-145 on the expression of CD36. Taken together, these findings present a novel approach to improve milk quality by analyzing the regulatory effect and mechanism regulating the circ02191/miR-145/CD36 pathway on fatty acid synthesis in the mammary gland of dairy cows.

A Facile and High-Sensitivity Method for Determining Proteinogenic Amino Acid Enantiomers by Integrating Chiral Phosphinate Derivatizing, 31P NMR and Parallel Reaction Monitoring.

Here, we have documented a new protocol to determine d/l-amino acids by derivatizing amino acids via a chiral phosphinate. (RP)-l-Menthyl phenylphosphinate was able to bond both primary and secondary amines, as well as improve the sensitivity of analytes in MS. Eighteen pairs of amino acids were successfully labeled except for Cys which has a thiol group on the side chain, and the chirality of amino acids can be discriminated by 31P NMR. Seventeen pairs of amino acids were separated by a C18 column within 45 min of elution, and resolution values ranged from 2.01 to 10.76. The lowest limit of detection was 10 pM acquired at parallel reaction monitoring, in which two factors collectively contributed that the ability of protonation of phosphine oxide and the sensitivity of parallel reaction monitoring. Chiral phosphine oxides might be a promising tool in future chiral metabolomics.

Implantation of skin-derived precursor Schwann cells improves erectile function in a bilateral cavernous nerve injury rat model.

BACKGROUND: This study was conducted to investigate the therapeutic potential of the skin-derived precursor Schwann cells for the treatment of erectile dysfunction in a rat model of bilateral cavernous nerve injury. RESULTS: The skin-derived precursor Schwann cells-treatment significantly restored erectile functions, accelerated the recovery of endothelial and smooth muscle tissues in the penis, and promoted nerve repair. The expression of p-Smad2/3 decreased after the treatment, which indicated significantly reduced fibrosis in the corpus cavernosum. CONCLUSIONS: Implantation of skin-derived precursor Schwann cells is an effective therapeutic strategy for treating erectile dysfunction induced by bilateral cavernous nerve injury.

RéSUMé: CONTEXTE: Cette étude a été menée pour étudier le potentiel thérapeutique des cellules de Schwann dérivées de la peau pour le traiter la dysfonction érectile survenue dans un modèle de lésion bilatérale du nerf caverneux chez le rat. RéSULTATS: Le traitement par des cellules de Schwann dérivées de la peau a significativement restauré les fonctions érectiles, accéléré la récupération des tissus endothéliaux et des tissues musculaires lisses du pénis, et a favorisé la réparation nerveuse. L'expression de p-Smad2/3 a diminué après le traitement, ce qui indique une fibrose significativement réduite dans le corps caverneux. CONCLUSION: L'implantation de cellules de Schwann dérivées de la peau est une stratégie thérapeutique efficace pour traiter la dysfonction érectile induite par une lésion bilatérale du nerf caverneux.