Mass spectrometry (MS)-based quantitative proteomics experiments frequently generate data with missing values, which may profoundly affect downstream analyses. A wide variety of imputation methods have been established to deal with the missing-value issue. To date, however, there is a scarcity of efficient, systematic, and easy-to-handle tools that are tailored for proteomics community. Herein, we developed a user-friendly and powerful stand-alone software, NAguideR, to enable implementation and evaluation of different missing value methods offered by 23 widely used missing-value imputation algorithms. NAguideR further evaluates data imputation results through classic computational criteria and, unprecedentedly, proteomic empirical criteria, such as quantitative consistency between different charge-states of the same peptide, different peptides belonging to the same proteins, and individual proteins participating protein complexes and functional interactions. We applied NAguideR into three label-free proteomic datasets featuring peptide-level, protein-level, and phosphoproteomic variables respectively, all generated by data independent acquisition mass spectrometry (DIA-MS) with substantial biological replicates. The results indicate that NAguideR is able to discriminate the optimal imputation methods that are facilitating DIA-MS experiments over those sub-optimal and low-performance algorithms. NAguideR further provides downloadable tables and figures supporting flexible data analysis and interpretation. NAguideR is freely available at http://www.omicsolution.org/wukong/NAguideR/ and the source code: https://github.com/wangshisheng/NAguideR/.

The glycoprotein spike (S) on the surface of SARS-CoV-2 is a determinant for viral invasion and host immune response. Herein, we characterized the site-specific N-glycosylation of S protein at the level of intact glycopeptides. All 22 potential N-glycosites were identified in the S-protein protomer and were found to be preserved among the 753 SARS-CoV-2 genome sequences. The glycosites exhibited glycoform heterogeneity as expected for a human cell-expressed protein subunit. We identified masses that correspond to 157 N-glycans, primarily of the complex type. In contrast, the insect cell-expressed S protein contained 38 N-glycans, completely of the high-mannose type. Our results revealed that the glycan types were highly determined by the differential processing of N-glycans among human and insect cells, regardless of the glycosites' location. Moreover, the N-glycan compositions were conserved among different sizes of subunits. Our study indicate that the S protein N-glycosylation occurs regularly at each site, albeit the occupied N-glycans were diverse and heterogenous. This N-glycosylation landscape and the differential N-glycan patterns among distinct host cells are expected to shed light on the infection mechanism and present a positive view for the development of vaccines and targeted drugs.

Background: Nanoparticles exhibit great promise for improving the solubility and tissue-specific distribution of chemotherapeutic agents; however, the passive and highly variable enhanced permeability and retention (EPR) effects observed in tumors frequently leads to insufficient delivery of nanodrugs into tumors. The tumor-penetrating peptide iRGD can actively enhance tumor-selective delivery of nanoparticles into tumors by binding to integrin and interacting with tissue-penetrating receptor neuropilin-1. Materials and methods: To improve colorectal cancer treatment, in this study, we prepared a paclitaxel (PTX)-loaded PLGA nanoparticle (PLGA-PTX) and evaluated its tumor-targeting and antitumor activity by co-administration with iRGD. Results: Compared to free PTX, encapsulated PTX retained preferential cytotoxicity toward various colorectal cancer cells while effectively sparing healthy cells. PLGA-PTX treatment resulted in cell cycle arrest at the G2/M phase and apoptosis, leading to inhibition of cancer cell migration and invasion. PLGA-PTX combined with iRGD displayed little enhancement of cytotoxicity in vitro. Despite this, iRGD receptors integrin and neuropilin-1 were found to be primarily overexpressed on abundant tumor vessels in mice bearing colorectal tumors. Consequently, co-administration of nanoparticles with iRGD promoted the selective delivery of nanoparticles into tumor tissues in vivo. Additionally, the combined regimen enhanced the antitumor effects compared to those of each individual reagent. Conclusion: Our findings suggest that PLGA nanoparticles combined with the iRGD peptide provide a promising drug delivery strategy for facilitating active drug accumulation into tumors, given that iRGD receptors are overexpressed on tumor vessels. This co-administration system lacking covalent conjugation provides a more convenient means to combine various therapeutic agents with iRGD to achieve personalized nanotherapy.

Blood–brain barrier (BBB) breakdown and inflammation occurring at the BBB have a key, mainly a deleterious role in the pathophysiology of ischemic stroke. Neddylation is a ubiquitylation-like pathway that is critical in various cellular functions by conjugating neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) to target proteins. However, the roles of neddylation pathway in ischemic stroke remain elusive. Here, we report that NEDD8 conjugation increased during acute phase after ischemic stroke and was present in intravascular and intraparenchymal neutrophils. Inhibition of neddylation by MLN4924, also known as pevonedistat, inactivated cullin-RING E3 ligase (CRL), and reduced brain infarction and improved functional outcomes. MLN4924 treatment induced the accumulation of the CRL substrate neurofibromatosis 1 (NF1). By using virus-mediated NF1 silencing, we show that NF1 knockdown abolished MLN4924-dependent inhibition of neutrophil trafficking. These effects were mediated through activation of endothelial P-selectin and intercellular adhesion molecule-1 (ICAM-1), and blocking antibodies against P-selectin or anti–ICAM-1 antibodies reversed NF1 silencing-induced increase in neutrophil infiltration in MLN4924-treated mice. Furthermore, we found that NF1 silencing blocked MLN4924-afforded BBB protection and neuroprotection through activation of protein kinase C δ (PKCδ), myristoylated alanine-rich C-kinase substrate (MARCKS), and myosin light chain (MLC) in cerebral microvessels after ischemic stroke, and treatment of mice with the PKCδ inhibitor rottlerin reduced this increased BBB permeability. Our study demonstrated that increased neddylation promoted neutrophil trafficking and thus exacerbated injury of the BBB and stroke outcomes. We suggest that the neddylation inhibition may be beneficial in ischemic stroke.

A Bacillus strain, denoted as PY-1, was isolated from the vascular bundle of cotton. Biochemical, physiological and 16S rDNA sequence analysis proved