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Background: Bacillus Calmette-Guérin (BCG) is capable of enhancing the infiltration of immune cells into the tumour. However the temporal dynamics of immune cell patterns in patients receiving BCG instillation remains unclear. Methods: Ninety-six patients who underwent intravesical BCG therapy, comprising 46 responders and 50 non-responders, were retrospectively enroled to explore the evolving immune landscape. This study involved a detailed examination of sequential samples collected before, during, and after BCG treatment to assess BCG's influence on the immune microenvironment, employing techniques such as immunohistochemistry, fluorescent multiplex immunohistochemistry, and mass spectrometry techniques. Results: Our study found that initial BCG instillation leads to enhanced immune cell infiltration, correlating with treatment efficacy, with responders exhibiting more pronounced increases. Non-responders experience a rise in immune cell infiltration and PD-L1 expression during the first instillation, which returns to baseline after treatment. In non-responders, BCG re-challenge fail to further increase immune cell infiltration into the tumour or improve patient outcomes. Strikingly, proteomics data revealed that GBP1 expression was induced by BCG treatment in non-responders. Conclusions: Our findings demonstrated the induction of tumour PD-L1 expression by BCG in non-responders, and therefore provide insights for the combination of BCG and anti-PD1/anti-PD-L1 therapy. [ABSTRACT FROM AUTHOR]

The development of high-throughput omics technology has greatly promoted the development of biomedicine. However, the poor reproducibility of omics techniques limits their application. It is necessary to use standard reference materials of complex RNAs or proteins to test and calibrate the accuracy and reproducibility of omics workflows. The transcriptome and proteome of most cell lines shift during culturing, which limits their applicability as standard samples. In this study, we demonstrated that the human hepatocellular cell line MHCC97H has a very stable transcriptome (r = 0.983~0.997) and proteome (r = 0.966~0.988 for data-dependent acquisition, r = 0.970~0.994 for data-independent acquisition) after 9 subculturing generations, which allows this steady standard sample to be consistently produced on an industrial scale in long term. Moreover, this stability was maintained across labs and platforms. In sum, our study provides omics standard reference material and reference datasets for transcriptomic and proteomics research. This helps to further standardize the workflow and data quality of omics techniques and thus promotes the application of omics technology in precision medicine. [ABSTRACT FROM AUTHOR]

A great number of non-coding RNAs (ncRNAs) account for the majority of the genome. The translation of these ncRNAs has been noted but seriously underestimated due to both technological and theoretical limitations. Based on the development of ribosome profiling (Ribo-seq), full length translating RNA analysis (RNC-seq) and mass spectrometry technology, more and more ncRNAs are being found to be translated in different organism, and some of them can produce functional peptides. While recently, not only individual new functional proteins, but also a new proteome have been experimentally discovered to be encoded by endogenous lncRNAs and circRNAs. These new proteins are of biological significance, suggesting the connection of the translation of ncRNAs to human physiology and diseases. Therefore, an in-depth and systematic understanding of the coding capabilities of ncRNAs is necessary for basic biology and medicine. In this review, we summarize the advances in the field of discovering this new proteome, i.e. "ncRNA-coded" proteins. [ABSTRACT FROM AUTHOR]

Emerging evidence suggested that necroptosis has essential functions in many human inflammatory diseases, but the molecular mechanisms of necroptosis remain unclear. Here, we employed SILAC quantitatively dynamic proteomics to compare the protein changes during TNF-α-induced necroptosis at different time points in murine fibrosarcoma L929 cells with caspase-8 deficiency, and then performed the systematical analysis on the signaling networks involved in the progress using bioinformatics methods. Our results showed that a total of 329, 421 and 378 differentially expressed proteins were detected at three stages of necroptosis, respectively. Gene ontology and ingenuity pathway analysis (IPA) revealed that the proteins regulated at early stages of necroptosis (2, 6 h) were mainly involved in mitochondria dysfunction, oxidative phosphorylation and Nrf-2 signaling, while the expression levels of the proteins related to ubiquitin, Nrf-2, and NF-κB pathways were found to have changes at last stages of necroptosis (6, 18 h). Taken together, we demonstrated for the first time that dysfunction of mitochondria and ubiquitin-proteasome signaling contributed to the initiation and execution of necroptosis. These findings may provide clues for the identification of important regulators in necroptosis and the development of novel therapeutic strategies for the related diseases. [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae is a Gram-positive pathogen responsible for pneumonia, otitis media, and meningitis. Manganese and zinc ions are essential for this bacterium, playing regulatory, structural, or catalytic roles as the critical cofactors in the bacterial proteins and metabolic enzymes. Lipoprotein PsaA has been found to mediate Mn and Zn transportation in Streptococcus pneumoniae. In the present work, we conducted a systemic study on the contributions from key amino acids in the metal-binding site of PsaA using various spectroscopic and biochemical methods. Our experimental data indicate that four metal-binding residues contribute unequally to the Mn and Zn binding, and His139 is most important for both the structural stability and metal binding of the protein. PsaA-Mn has a lower thermal stability than PsaA-Zn, possibly due to the different coordination preferences of the metals. Kinetics analysis revealed that PsaA-Mn binding is a fast first-order reaction, whereas PsaA-Zn binding is a slow second-order reaction, implying that PsaA kinetically prefers binding Mn to Zn. The present results provide complementary information for understanding the mechanisms of metal transport and bacterial virulence via lipoproteins in Streptococcus pneumoniae. [ABSTRACT FROM AUTHOR]

The cell-surface lipoprotein SiaA, a component of the SiaABC transporter, acts as the primary receptor for heme in the infamous human pathogen Streptococcus pyogenes. However, little is known about the molecular mechanism of heme binding and release as well as the role of heme-binding ligands that contribute to the uptake of heme into the pathogenic bacteria. The present report aims to clarify the coordination properties of heme iron in SiaA. By substitution of either Met79 or His229 with alanine, the mutant M79A and H229A proteins display significantly decreased heme-binding affinity and substantially increased heme-release rates, as compared with wild-type SiaA protein. Both fluorescence and circular dichroism spectra indicated that heme binding results in alterations in the secondary structure of the protein. Heme release from SiaA is a stepwise process in which heme disassociates firstly from Met79 and then from His229 with distinct conformational changes. His229 may serve as an anchor for heme binding in SiaA and thus may play a major role in the stability of the coordination between heme and the protein. [ABSTRACT FROM AUTHOR]

The removal of Fe(III) from human serum transferrin by chelators is thought to proceed through intermediate species in which the chelator becomes associated with the metal center of the protein. The visible spectral shifts associated with the formation of such intermediates in the wild-type (WT) protein are too small for reliable kinetic data to be obtained. Therefore, studies were undertaken with the recombinant N-terminal lobe variant H249A, a variant showing more pronounced spectral changes. The kinetics of the synergistic anion-exchange reaction between nitrilotriacetate (NTA) and carbonate in variant H249A was studied by stopped-flow spectrophotometry as a model for this process in the WT protein. [ABSTRACT FROM AUTHOR]

Reactions between various apo and metal-bound forms of human serum transferrin (80 kDa) and the recombinant N-lobe (40 kDa) with [Pt(en)Cl2] or cis-[PtCl2(NH3)2] have been investigated in solution via observation of [1H,15N] NMR resonances of the Pt complexes, [1H,13C] resonances of the eCH3 groups of the protein methionine residues, and by chromatographic analysis of single-site methionine mutants. For the whole protein, the preferred Pt binding site appears to be Met256. Additional binding occurs at the other surface-exposed methionine (Met499), which is platinated at a slower rate than Met256. In contrast, binding of similar Pt compounds to the N-lobe of the protein occurs at Met313, rather than Met256. Met313 is buried in the interlobe contact region of intact transferrin. After loss of one chloride ligand from Pt and binding to methionine sulfur of the N-lobe, chelate-ring closure appears to occur with binding to a deprotonated backbone amide nitrogen, and the loss of the other chloride ligand. Such chelate-ring closure was not observed during reactions of the whole protein, even after several days. [ABSTRACT FROM AUTHOR]