Distinguishing Isomeric Cyclobutane Thymidine Dimers by Ion Mobility and Tandem Mass Spectrometry.

Irradiation of the major conformation of duplex DNA found in cells (B form) produces cyclobutane pyrimidine dimers (CPDs) from adjacent pyrimidines in a head-to-head orientation (syn) with the C5 substituents in a cis stereochemistry. These CPDs have crucial implications in skin cancer. Irradiation of G-quadruplexes and other non-B DNA conformations in vitro produces, however, CPDs between nonadjacent pyrimidines in nearby loops with syn and head-to-tail orientations (anti) with both cis and trans stereochemistry to yield a mixture of six possible isomers of the T=T dimer. This outcome is further complicated by formation of mixtures of nonadjacent CPDs of C=T, T=C, and C=C, and successful analysis depends on development of specific and sensitive methods. Toward meeting this need, we investigated whether ion mobility mass spectrometry (IMMS) and MS/MS can distinguish the cis,syn and trans,anti T=T CPDs. Ion mobility can afford baseline separation and give relative mobilities that are in accord with predicted cross sections. Complementing this ability to distinguish isomers is MS/MS collisional activation where fragmentation also distinguishes the two isomers and confirms conclusions drawn from ion mobility analysis. The observations offer early support that ion mobility and MS/MS can enable the distinction of DNA photoproduct isomers.

MEN1 deficiency stabilizes PD-L1 and promotes tumor immune evasion of lung cancer.

Multiple Endocrine Neoplasia 1 gene (MEN1), which is known to be a tumor suppressor gene in lung tissues, encodes a 610 amino acid protein menin. Previous research has proven that MEN1 deficiency promotes the malignant progression of lung cancer. However, the biological role of this gene in the immune microenvironment of lung cancer remains unclear. In this study, we found that programmed cell death-ligand 1 (PD-L1) is upregulated in lung-specific KrasG12D mutation-induced lung adenocarcinoma in mice, after Men1 deficiency. Simultaneously, CD8+ and CD3+ T cells are depleted, and their cytotoxic effects are suppressed. In vitro, PD-L1 is inhibited by the overexpression of menin. Mechanistically, we found that MEN1 inactivation promotes the deubiquitinating activity of COP9 signalosome subunit 5 (CSN5) and subsequently increases the level of PD-L1.

Exercise accelerates recruitment of CD8+ T cell to promotes anti-tumor immunity in lung cancer via epinephrine.

BACKGROUND AND PURPOSE: In recent years, there has been extensive research on the role of exercise as an adjunctive therapy for cancer. However, the potential mechanisms underlying the anti-tumor therapy of exercise in lung cancer remain to be fully elucidated. As such, our study aims to confirm whether exercise-induced elevation of epinephrine can accelerate CD8+ T cell recruitment through modulation of chemokines and thus ultimately inhibit tumor progression. METHOD: C57BL/6 mice were subcutaneously inoculated with Lewis lung cancer cells (LLCs) to establish a subcutaneous tumor model. The tumor mice were randomly divided into different groups to performed a moderate-intensity exercise program on a treadmill for 5 consecutive days a week, 45 min a day. The blood samples and tumor tissues were collected after exercise for IHC, RT-qPCR, ELISA and Western blot. In addition, another group of mice received daily epinephrine treatment for two weeks (0.05 mg/mL, 200 µL i.p.) (EPI, n = 8) to replicate the effects of exercise on tumors in vivo. Lewis lung cancer cells were treated with different concentrations of epinephrine (0, 5, 10, 20 µM) to detect the effect of epinephrine on chemokine levels via ELISA and RT-qPCR. RESULTS: This study reveals that both pre- and post-cancer exercise effectively impede the tumor progression. Exercise led to an increase in EPI levels and the infiltration of CD8+ T cell into the lung tumor. Exercise-induced elevation of EPI is involved in the regulation of Ccl5 and Cxcl10 levels further leading to enhanced CD8+ T cell infiltration and ultimately inhibiting tumor progression. CONCLUSION: Exercise training enhance the anti-tumor immunity of lung cancer individuals. These findings will provide valuable insights for the future application of exercise therapy in clinical practice.

Metastability of Protein Solution Structures in the Absence of a Solvent: Rugged Energy Landscape and Glass-like Behavior.

Native ion mobility/mass spectrometry is well-poised to structurally screen proteomes but characterizes protein structures in the absence of a solvent. This raises long-standing unanswered questions about the biological significance of protein structures identified through ion mobility/mass spectrometry. Using newly developed computational and experimental ion mobility/ion mobility/mass spectrometry methods, we investigate the unfolding of the protein ubiquitin in a solvent-free environment. Our data suggest that the folded, solvent-free ubiquitin observed by ion mobility/mass spectrometry exists in a largely native fold with an intact ß-grasp motif and α-helix. The ensemble of folded, solvent-free ubiquitin ions can be partitioned into kinetically stable subpopulations that appear to correspond to the structural heterogeneity of ubiquitin in solution. Time-resolved ion mobility/ion mobility/mass spectrometry measurements show that folded, solvent-free ubiquitin exhibits a strongly stretched-exponential time dependence, which simulations trace to a rugged energy landscape with kinetic traps. Unfolding rate constants are estimated to be approximately 800 to 20,000 times smaller than in the presence of water, effectively quenching the unfolding process on the time scale of typical ion mobility/mass spectrometry measurements. Our proposed unfolding pathway of solvent-free ubiquitin shares substantial characteristics with that established for the presence of solvent, including a polarized transition state with significant native content in the N-terminal ß-hairpin and α-helix. Our experimental and computational data suggest that (1) the energy landscape governing the motions of folded, solvent-free proteins is rugged in analogy to that of glassy systems; (2) large-scale protein motions may at least partially be determined by the amino acid sequence of a polypeptide chain; and (3) solvent facilitates, rather than controls, protein motions.

Angiotensin II type-2 receptor signaling facilitates liver injury repair and regeneration via inactivation of Hippo pathway.

The angiotensin II type 2 receptor (AT2R) is a well-established component of the renin-angiotensin system and is known to counteract classical activation of this system and protect against organ damage. Pharmacological activation of the AT2R has significant therapeutic benefits, including vasodilation, natriuresis, anti-inflammatory activity, and improved insulin sensitivity. However, the precise biological functions of the AT2R in maintaining homeostasis in liver tissue remain largely unexplored. In this study, we found that the AT2R facilitates liver repair and regeneration following acute injury by deactivating Hippo signaling and that interleukin-6 transcriptionally upregulates expression of the AT2R in hepatocytes through STAT3 acting as a transcription activator binding to promoter regions of the AT2R. Subsequently, elevated AT2R levels activate downstream signaling via heterotrimeric G protein Gα12/13-coupled signals to induce Yap activity, thereby contributing to repair and regeneration processes in the liver. Conversely, a deficiency in the AT2R attenuates regeneration of the liver while increasing susceptibility to acetaminophen-induced liver injury. Administration of an AT2R agonist significantly enhances the repair and regeneration capacity of injured liver tissue. Our findings suggest that the AT2R acts as an upstream regulator in the Hippo pathway and is a potential target in the treatment of liver damage.

Heparin versus normal saline for the care of peripheral intravenous catheters in children: A meta-analysis.

AIM: To conduct a meta-analysis to evaluate the role of heparin versus normal saline lock in the care of peripheral intravenous catheters. DESIGN: A meta-analysis. METHODS: This meta-analysis searched nine databases for randomized controlled trials (RCTs) on heparin versus normal saline for the care of peripheral intravenous catheters in children up to April 5, 2023. The quality of included RCTs was evaluated using the risk of bias tool of Cochrane library. RevMan5.3 software was used for data analysis. RESULTS: Ten RCTs with a total of 1255 children were involved. Meta-analysis indicated that heparin lock reduced the incidence of blockage of peripheral intravenous catheter [OR = 2.01, 95% CI (1.42,2.84), p < 0.001], prolonged the duration of peripheral intravenous catheter indwelling[MD = -0.43, 95% CI (-0.75, -0.11), p = 0.008]. There were no statistical differences in the incidence of phlebitis [OR = 1.02, 95% CI (0.59, 1.74), p = 0.95 W]. PUBLIC CONTRIBUTION: Heparin may have more benefits in the nursing care of peripheral intravenous catheters compared with normal saline.

Post- and Pre-Radiolabeling Assays for anti Thymidine Cyclobutane Dimers as Intrinsic Photoprobes of Various Types of G-Quadruplexes, Reverse Hoogsteen Hairpins, and Other Non-B DNA Structures.

G-quadruplexes are thought to play an important role in gene regulation and telomere maintenance, but developing probes for their presence and location is challenging due to their transitory and highly dynamic nature. The majority of probes for G-quadruplexes have relied on antibody or small-molecule binding agents, many of which can also alter the dynamics and relative populations of G-quadruplexes. Recently, it was discovered that ultraviolet B (UVB) irradiation of human telomeric DNA and various G-quadruplex forming sequences found in human promoters, as well as reverse Hoogsteen hairpins, produces a unique class of non-adjacent anti cyclobutane pyrimidine dimers (CPDs). Therefore, one can envision using a pulse of UVB light to irreversibly trap these non-B DNA structures via anti CPD formation without perturbing their dynamics, after which the anti CPDs can be identified and mapped. As a first step toward this goal, we report radioactive post- and pre-labeling assays for the detection of non-adjacent CPDs and illustrate their use in detecting trans,anti T=(T) CPD formation in a human telomeric DNA sequence. Both assays make use of snake venom phosphodiesterase (SVP) to degrade the trans,anti T=(T) CPD-containing DNA to the tetranucleotide pTT=(pTT) corresponding to CPD formation between the underlined T's of two separate dinucleotides while degrading the adjacent syn TT CPDs to the trinucleotide pGT=T. In the post-labeling assay, calf intestinal phosphodiesterase is used to dephosphorylate the tetranucleotides, which are then rephosphorylated with kinase and [32P]-ATP to produce radiolabeled mono- and diphosphorylated tetranucleotides. The tetranucleotides are confirmed to be non-adjacent CPDs by 254 nm photoreversion to the dinucleotide p*TT. In the pre-labeling assay, radiolabeled phosphates are introduced into non-adjacent CPD-forming sites by ligation prior to irradiation, thereby eliminating the dephosphorylation and rephosphorylation steps. The assays are also demonstrated to detect the stereoisomeric cis,anti T=(T) CPD.

Perspective on the potential of tandem-ion mobility /mass spectrometry methods for structural proteomics applications.

Cellular processes are usually carried out collectively by the entirety of all proteins present in a biological cell, i.e. the proteome. Mass spectrometry-based methods have proven particularly successful in identifying and quantifying the constituent proteins of proteomes, including different molecular forms of a protein. Nevertheless, protein sequences alone do not reveal the function or dysfunction of the identified proteins. A straightforward way to assign function or dysfunction to proteins is characterization of their structures and dynamics. However, a method capable to characterize detailed structures of proteins and protein complexes in a large-scale, systematic manner within the context of cellular processes does not yet exist. Here, we discuss the potential of tandem-ion mobility / mass spectrometry (tandem-IM/MS) methods to provide such ability. We highlight the capability of these methods using two case studies on the protein systems ubiquitin and avidin using the tandem-TIMS/MS technology developed in our laboratory and discuss these results in the context of other developments in the broader field of tandem-IM/MS.

Differentiation of Isomeric, Nonseparable Carbohydrates Using Tandem-Trapped Ion Mobility Spectrometry-Mass Spectrometry.

Carbohydrates play important roles in biological processes, but their identification remains a significant analytical problem. While mass spectrometry has increasingly enabled the elucidation of carbohydrates, current approaches are limited in their abilities to differentiate isomeric carbohydrates when these are not separated prior to tandem-mass spectrometry analysis. This analytical challenge takes on increased relevance because of the pervasive presence of isomeric carbohydrates in biological systems. Here, we demonstrate that TIMS2-MS2 workflows enabled by tandem-trapped ion mobility spectrometry-mass spectrometry (tTIMS/MS) provide a general approach to differentiate isomeric, nonseparated carbohydrates. Our analysis shows that (1) cross sections measured by TIMS are sufficiently precise and robust for ion identification; (2) fragment ion cross sections from TIMS2 analysis can be analytically exploited to identify carbohydrate precursors even if the precursor ions are not separated by TIMS; (3) low-abundant fragment ions can be exploited to identify carbohydrate precursors even if the precursor ions are not separated by IMS. (4) MS2 analysis of fragment ions produced by TIMS2 can be used to validate and/or further characterize carbohydrate structures. Taken together, our analysis underlines the opportunities that tandem-ion mobility spectrometry/MS methods offer for the characterization of mixtures of isomeric carbohydrates.

Molecular Characterization of a B Cell Adaptor for Phosphoinositide 3-Kinase Homolog in Lamprey (Lampetra japonica) and Its Function in the Immune Response.

Human B cell adaptor for phosphoinositide 3-kinase (BCAP) is identified as an adaptor protein expressed in B cells and plays a critical immunomodulatory role in B cell receptor signaling and humoral immune response. In the current study, a homolog of BCAP (Lja-BCAP) was identified in Lampetra japonica. The open reading frame of Lja-BCAP contains 2181bp nucleotides and encodes a protein of 726 amino acids. After being stimulated by mixed bacteria, the mRNA and protein expression levels of Lja-BCAP and the activation levels of tyrosine kinases increased significantly in peripheral blood lymphocytes, gills and supraneural myeloid bodies, respectively. However, after the knockdown of Lja-BCAP by RNAi in vivo, the activation of tyrosine kinases was inhibited in the above tissues, which indicated that Lja-BCAP participated in the anti-bacterial immune response of lampreys. After lipopolysaccharide (LPS) stimulation, the expression of Lja-BCAP in peripheral blood lymphocytes, gills and supraneural myeloid bodies were significantly up-regulated 2.5, 2.2, and 11.1 times (p < 0.05) compared to the control group, respectively; while after phytohemagglutinin (PHA) stimulation, the up-regulation of Lja-BCAP was only detected in peripheral blood lymphocytes. The above results show that Lja-BCAP mainly participates in the LPS-mediated immune response of lampreys.

A Transferable, Sample-Independent Calibration Procedure for Trapped Ion Mobility Spectrometry (TIMS).

Ion mobility spectrometry-mass spectrometry (IMS-MS) determines momentum transfer cross sections of ions to elucidate their structures. Recent IMS methods employ electrodynamic fields or nonstationary buffer gases to separate ions. These methods require a calibration procedure to determine ion mobilities from the experimental data. This applies in particular to trapped IMS (TIMS), a novel IMS method with reported high resolving powers. Here, we report the first systematic assessment of the accuracy and the limitations of mobility calibration in TIMS. Our data show that the currently used TIMS calibration approach reproduces drift tube mobilities to approximately 1% (95th percentile). Furthermore, we develop a transferable and sample-independent calibration procedure for TIMS. The central aspects of our approach are (1) a calibration function derived from a solution to the Boltzmann transport equation and (2) calibration constants based on a Taylor expansion of instrument properties (TEIP). The key advantage of our calibration approach over current ones is its transferability: one equation and one set of parameters are sufficient to calibrate ion mobilities for various instrument settings, compound classes, or charge states. Our approach is transferable over time and sufficiently accurate (∼1-2%) for structure-elucidation purposes. While we develop our calibration procedure specifically for TIMS, the approach we take is generic in nature and can be applied to other IMS systems.