Characterization of an expanded set of assays for immunomodulatory proteins using targeted mass spectrometry.

Immunotherapies are revolutionizing cancer care, but many patients do not achieve durable responses and immune-related adverse events are difficult to predict. Quantifying the hundreds of proteins involved in cancer immunity has the potential to provide biomarkers to monitor and predict tumor response. We previously developed robust, multiplexed quantitative assays for immunomodulatory proteins using targeted mass spectrometry, providing measurements that can be performed reproducibly and harmonized across laboratories. Here, we expand upon those efforts in presenting data from a multiplexed immuno-oncology (IO)-3 assay panel targeting 43 peptides representing 39 immune- and inflammation-related proteins. A suite of novel monoclonal antibodies was generated as assay reagents, and the fully characterized antibodies are made available as a resource to the community. The publicly available dataset contains complete characterization of the assay performance, as well as the mass spectrometer parameters and reagent information necessary for implementation of the assay. Quantification of the proteins will provide benefit to correlative studies in clinical trials, identification of new biomarkers, and improve understanding of the immune response in cancer.

Laparoscopic left hemihepatectomy using augmented reality navigation plus ICG fluorescence imaging for hepatolithiasis: a retrospective single-arm cohort study (with video).

BACKGROUND: Laparoscopic left hemihepatectomy (LLH) has been shown to be an effective and safe method for treating hepatolithiasis primarily affecting the left hemiliver. However, this procedure still presents challenges. Due to pathological changes in intrahepatic duct stones, safely dissecting the hilar vessels and determining precise resection boundaries remains difficult, even with fluorescent imaging. Our team proposed a new method of augmented reality navigation (ARN) combined with Indocyanine green (ICG) fluorescence imaging for LLH in hepatolithiasis cases. This study aimed to investigate the feasibility of this combined approach in the procedure. METHODS: Between May 2021 and September 2023, 16 patients with hepatolithiasis who underwent LLH were included. All patients underwent preoperative 3D evaluation and were then guided using ARN and ICG fluorescence imaging during the procedure. Perioperative and short-term postoperative outcomes were assessed to evaluate the safety and efficacy of the method. RESULTS: All 16 patients successfully underwent LLH. The mean operation time was 380.31 ± 92.17 min, with a mean estimated blood loss of 116.25 ± 64.49 ml. ARN successfully aided in guiding hilar vessel dissection in all patients. ICG fluorescence imaging successfully identified liver resection boundaries in 11 patients (68.8%). In the remaining 5 patients (31.3%) where fluorescence imaging failed, virtual liver segment projection (VLSP) successfully identified their resection boundaries. No major complications occurred in any patients. Immediate stone residual rate, stone recurrence rate, and stone extraction rate through the T-tube sinus tract were 12.5%, 6.3%, and 6.3%, respectively. CONCLUSION: The combination of ARN and ICG fluorescence imaging enhances the safety and precision of LLH for hepatolithiasis. Moreover, ARN may serve as a safe and effective tool for identifying precise resection boundaries in cases where ICG fluorescence imaging fails.

Effect of epoxy value on the rheological properties and microcosmic mechanism of WER emulsified asphalt.

Waterborne epoxy resin (WER), a cleaning material with exceptional high-temperature resistance, has attracted much attention to modify emulsified asphalt in the pavement material field. Epoxy value is the critical characteristic index of WER. In this research, three WER with the epoxy values of 0.20 eq/100g, 0.44 eq/100g, and 0.51 eq/100g were utilized as asphalt modifiers. The influence of epoxy value on WER-EA was investigated by comparing the rheological properties of three kinds of WER emulsified asphalt (WER-EA). The modification mechanism of WER-EA has been analyzed using FTIR and SEM. The results demonstrate that different WER-EA resulted in significantly different rheological properties. WER-EA with the epoxy value of 0.20 eq/100g (E20) performed best at high temperatures, with a maximum increase of 17477% in G*/sinδ compared to the neat asphalt and a maximum increase of 66.3% in G*/sinδ compared to the other two WER-EA. WER-EA with 0.44 eq/100g epoxy value (E44) performed best at low temperatures, with a maximum increase in m value of 39.4% and a maximum decrease in S value of 33.3% compared to the other two WER-EA. In addition, the interpenetrating polymer network (IPN) in E20 was observed to be more solid and stable, and IPN in E44 was more uniform. To summarize, lower epoxy value led to a higher degree of WER reaction and higher content of rigid groups, which is more conducive to optimizing the high-temperature property of WER-EA. WER with moderate epoxy value resulted in a low content of polar bonds and thus high content of flexible segments, which helps emulsified asphalt to form a more uniform IPN.

A multiplexed assay for quantifying immunomodulatory proteins supports correlative studies in immunotherapy clinical trials.

Introduction: Immunotherapy is an effective treatment for a subset of cancer patients, and expanding the benefits of immunotherapy to all cancer patients will require predictive biomarkers of response and immune-related adverse events (irAEs). To support correlative studies in immunotherapy clinical trials, we are developing highly validated assays for quantifying immunomodulatory proteins in human biospecimens. Methods: Here, we developed a panel of novel monoclonal antibodies and incorporated them into a novel, multiplexed, immuno-multiple reaction monitoring mass spectrometry (MRM-MS)-based proteomic assay targeting 49 proteotypic peptides representing 43 immunomodulatory proteins. Results and discussion: The multiplex assay was validated in human tissue and plasma matrices, where the linearity of quantification was >3 orders of magnitude with median interday CVs of 8.7% (tissue) and 10.1% (plasma). Proof-of-principle demonstration of the assay was conducted in plasma samples collected in clinical trials from lymphoma patients receiving an immune checkpoint inhibitor. We provide the assays and novel monoclonal antibodies as a publicly available resource for the biomedical community.

Effects of motor-cognitive interaction based on dual-task gait analysis recognition in middle age to aging people with normal cognition and mild cognitive impairment.

Background: Mild cognitive impairment (MCI) is considered a transitional stage between cognitive normality and dementia among the elderly, and its associated risk of developing Alzheimer's disease (AD) is 10-15 times higher than that of the general population. MCI is an important threshold for the prevention and control of AD, and intervention in the MCI stage may be the most effective strategy to delay the occurrence of AD. Materials and methods: In this study, 68 subjects who met the inclusion criteria were divided into an MCI group (38 subjects) and normal elderly (NE) group (30 subjects). Both groups underwent clinical function assessments (cognitive function, walking function, and activities of daily living) and dual-task three-dimensional gait analysis (walking motor task and walking calculation task). Spatial-temporal parameters were obtained and reduced by principal component analysis, and the key biomechanical indexes were selected. The dual-task cost (DTC) was calculated for intra-group (task factor) and inter-group (group factor) comparisons. Results: The results of the principal component analysis showed that the cadence parameter had the highest weight in all three walking tasks. In addition, there were significant differences in the cadence both walking motor task (WMT) vs. walking task (WT) and walking calculation task (WCT) vs. WT in the MCI group. The cadence in the NE group only showed a significant difference between WMT and WT. The only differences between the MCI group and NE group was DTC cadence in WCT, and no differences were found for cadence in any of the three walking tasks. Conclusion: The results show that dual tasks based on cognitive-motor gait analysis of DTCcadence in MCI have potential value for application in early identification and provide theoretical support to improve the clinical diagnosis of MCI.

Plasmoelectric Potential in Plasmon-Mediated Electrochemistry.

Plasmon-mediated chemical reactions have attracted intensive research interest as a means of achieving desirable reaction yields and selectivity. The energetic charge carriers and elevated local temperature induced by the nonradiative decay of surface plasmons are thought to be responsible for improving reaction outcomes. This study reports that the plasmoelectric potential is another key contributor in plasmon-mediated electrochemistry. Additionally, we disclose a convenient and reliable method for quantifying the specific contributions of the plasmoelectric potential, hot electrons, and photothermal heating to the electroreduction of oxygen at the plasmonic Ag electrode, revealing that the plasmoelectric potential is the dominating nonthermal factor under short-wavelength illumination and moderate electrode bias. This work elucidates novel mechanistic understandings of plasmon-mediated electrochemistry, facilitating high-performance plasmonic electrocatalyst design optimization.

Multi-Omics Analysis of MCM2 as a Promising Biomarker in Pan-Cancer.

Minichromosome maintenance 2 (MCM2) is a member of the minichromosomal maintenance family of proteins that mainly regulates DNA replication and the cell cycle and is involved in regulating cancer cell proliferation in various cancers. Previous studies have reported that MCM2 plays a pivotal role in cell proliferation and cancer development. However, few articles have systematically reported the pathogenic roles of MCM2 across cancers. Therefore, the present pan-cancer study was conducted. Various computational tools were used to investigate the MCM2 expression level, genetic mutation rate, and regulating mechanism, immune infiltration, tumor diagnosis and prognosis, therapeutic response and drug sensitivity of various cancers. The expression and function of MCM2 were examined by Western blotting and CCK-8 assays. MCM2 was significantly upregulated in almost all cancers and cancer subtypes in The Cancer Genome Atlas and was closely associated with tumor mutation burden, tumor stage, and immune therapy response. Upregulation of MCM2 expression may be correlated with a high level of alterations rate. MCM2 expression was associated with the infiltration of various immune cells and molecules and markedly associated with a poor prognosis. Western blotting and CCK-8 assays revealed that MCM2 expression was significantly upregulated in melanoma cell lines. Our results also suggested that MCM2 promotes cell proliferation in vitro by activating cell proliferation pathways such as the Akt signaling pathways. This study explored the oncogenic role of MCM2 across cancers, provided data on the underlying mechanisms of these cancers for further research and demonstrated that MCM2 may be a promising target for cancer immunotherapy.

Targeted Mass Spectrometry Enables Multiplexed Quantification of Immunomodulatory Proteins in Clinical Biospecimens.

Immunotherapies are revolutionizing cancer care, producing durable responses and potentially cures in a subset of patients. However, response rates are low for most tumors, grade 3/4 toxicities are not uncommon, and our current understanding of tumor immunobiology is incomplete. While hundreds of immunomodulatory proteins in the tumor microenvironment shape the anti-tumor response, few of them can be reliably quantified. To address this need, we developed a multiplex panel of targeted proteomic assays targeting 52 peptides representing 46 proteins using peptide immunoaffinity enrichment coupled to multiple reaction monitoring-mass spectrometry. We validated the assays in tissue and plasma matrices, where performance figures of merit showed over 3 orders of dynamic range and median inter-day CVs of 5.2% (tissue) and 21% (plasma). A feasibility study in clinical biospecimens showed detection of 48/52 peptides in frozen tissue and 38/52 peptides in plasma. The assays are publicly available as a resource for the research community.

A highly annotated database of genes associated with platinum resistance in cancer.

Platinum-based chemotherapy, including cisplatin, carboplatin, and oxaliplatin, is prescribed to 10-20% of all cancer patients. Unfortunately, platinum resistance develops in a significant number of patients and is a determinant of clinical outcome. Extensive research has been conducted to understand and overcome platinum resistance, and mechanisms of resistance can be categorized into several broad biological processes, including (1) regulation of drug entry, exit, accumulation, sequestration, and detoxification, (2) enhanced repair and tolerance of platinum-induced DNA damage, (3) alterations in cell survival pathways, (4) alterations in pleiotropic processes and pathways, and (5) changes in the tumor microenvironment. As a resource to the cancer research community, we provide a comprehensive overview accompanied by a manually curated database of the >900 genes/proteins that have been associated with platinum resistance over the last 30 years of literature. The database is annotated with possible pathways through which the curated genes are related to platinum resistance, types of evidence, and hyperlinks to literature sources. The searchable, downloadable database is available online at http://ptrc-ddr.cptac-data-view.org .

Targeted Mass Spectrometry Enables Quantification of Novel Pharmacodynamic Biomarkers of ATM Kinase Inhibition.

The ATM serine/threonine kinase (HGNC: ATM) is involved in initiation of repair of DNA double-stranded breaks, and ATM inhibitors are currently being tested as anti-cancer agents in clinical trials, where pharmacodynamic (PD) assays are crucial to help guide dose and scheduling and support mechanism of action studies. To identify and quantify PD biomarkers of ATM inhibition, we developed and analytically validated a 51-plex assay (DDR-2) quantifying protein expression and DNA damage-responsive phosphorylation. The median lower limit of quantification was 1.28 fmol, the linear range was over 3 orders of magnitude, the median inter-assay variability was 11% CV, and 86% of peptides were stable for storage prior to analysis. Use of the assay was demonstrated to quantify signaling following ionizing radiation-induced DNA damage in both immortalized lymphoblast cell lines and primary human peripheral blood mononuclear cells, identifying PD biomarkers for ATM inhibition to support preclinical and clinical studies.

Spatial and temporal analysis of human infection with the avian influenza A (H7N9) virus in China and research on a risk assessment agent-based model.

OBJECTIVES: From 2013 to 2017, the avian influenza A (H7N9) virus frequently infected people in China, which seriously affected the public health of society. This study aimed to analyze the spatial characteristics of human infection with the H7N9 virus in China and assess the risk areas of the epidemic. METHODS: Using kernel density estimation, standard deviation ellipse analysis, spatial and temporal scanning cluster analysis, and Pearson correlation analysis, the spatial characteristics and possible risk factors of the epidemic were studied. Meteorological factors, time (month), and environmental factors were combined to establish an epidemic risk assessment proxy model to assess the risk range of an epidemic. RESULTS: The epidemic situation was significantly correlated with atmospheric pressure, temperature, and daily precipitation (P < 0.05), and there were six temporal and spatial clusters. The fitting accuracy of the epidemic risk assessment agent-based model for lower-risk, low-risk, medium-risk, and high-risk was 0.795, 0.672, 0.853, 0.825, respectively. CONCLUSIONS: This H7N9 epidemic was found to have more outbreaks in winter and spring. It gradually spread to the inland areas of China. This model reflects the risk areas of human infection with the H7N9 virus.

Motor Dual-Tasks for Gait Analysis and Evaluation in Post-Stroke Patients.

Eighteen stroke patients were recruited for this study involving the evaluation of cognition and walking ability and multitask gait analysis. Multitask gait analysis consisted of a single walking task (Task 0), a simple motor dual-task (water-holding, Task 1), and a complex motor dual-task (crossing obstacles, Task 2). The task of crossing obstacles was considered to be equivalent to the combination of a simple walking task and a complex motor task as it involved more nervous system, skeletal movement, and cognitive resources. To eliminate heterogeneity in the results of the gait analysis of the stroke patients, the dual-task gait cost values were calculated for various kinematic parameters. The major differences were observed in the proximal joint angles, especially in the angles of the trunk, pelvis, and hip joints, which were significantly larger in the dual motor tasks than in the single walking task. This research protocol aims to provide a basis for the clinical diagnosis of gait function and an in-depth study of motor control in stroke patients with motor control deficits through the analyses of dual-motor walking tasks.

Temporal and spatial analysis of COVID-19 transmission in China and its influencing factors.

OBJECTIVES: The purpose of this study was to explore the temporal and spatial characteristics of COVID-19 transmission and its influencing factors in China, from January to October 2020. METHODS: About 81,000 COVID-19 confirmed case data, Baidu migration index data, air pollutants, meteorological data, and government response strictness index data were collected from 31 provincial-level regions (excluding Hong Kong, Macao, and Taiwan) and 337 prefecture-level cities. The spatio-temporal characteristics of COVID-19 were explored using spatial autocorrelation, hot spot, and spatio-temporal scanning statistics. At the same time, Spearman rank correlation analysis and multiple linear regression were used to explore the relationship between influencing factors and confirmed COVID-19 cases. RESULTS: The distribution of COVID-19 in China tends to be stable over time, with spatial correlation and prominent clustering regions. Spatio-temporal scanning analysis showed that most COVID-19 high-incidence months were from January to March at the beginning of the epidemic, and the area with the highest aggregation risk was Hubei Province (RR=491.57) which was 491.57 times the aggregation risk of other regions. Among the meteorological variables, the daily average temperature, wind speed, precipitation, and new COVID-19 cases were negatively correlated. The air pollution concentration and migration index were positively correlated with new confirmed cases, and the government response strict index was strongly negatively correlated with confirmed COVID-19 cases. CONCLUSIONS: Environmental temperature has a certain inhibitory effect on the transmission of COVID-19; the air pollution concentration and migration index have a certain promoting effect on the transmission of COVID-19. The strict government response index indicates that the greater the intensity of government intervention, the fewer COVID-19 cases will occur.

Multiomic analysis identifies CPT1A as a potential therapeutic target in platinum-refractory, high-grade serous ovarian cancer.

Resistance to platinum compounds is a major determinant of patient survival in high-grade serous ovarian cancer (HGSOC). To understand mechanisms of platinum resistance and identify potential therapeutic targets in resistant HGSOC, we generated a data resource composed of dynamic (±carboplatin) protein, post-translational modification, and RNA sequencing (RNA-seq) profiles from intra-patient cell line pairs derived from 3 HGSOC patients before and after acquiring platinum resistance. These profiles reveal extensive responses to carboplatin that differ between sensitive and resistant cells. Higher fatty acid oxidation (FAO) pathway expression is associated with platinum resistance, and both pharmacologic inhibition and CRISPR knockout of carnitine palmitoyltransferase 1A (CPT1A), which represents a rate limiting step of FAO, sensitize HGSOC cells to platinum. The results are further validated in patient-derived xenograft models, indicating that CPT1A is a candidate therapeutic target to overcome platinum resistance. All multiomic data can be queried via an intuitive gene-query user interface (https://sites.google.com/view/ptrc-cell-line).

All-trans retinoic acid enhances the effect of Fra-1 to inhibit cell proliferation and metabolism in cervical cancer.

OBJECTIVES: This study on all-trans retinoic acid was designed to explore its effect on the ability of Fra-1 to cervical cancer cell development. The results show that all-trans retinoic acid enhances the effect of Fra-1 on inhibiting cervical cancer proliferation and the glucose consumption, its effect on the loss of mitochondrial membrane potential, on the decreasing of lactic acid as well as ATP, and also influences the expression of MDM2/P53/P21 and LDHA. RESULTS: The results show that the expression of Fra-1 is higher in all-trans retinoic acid-treated cervical cancer. Flow cytometry and kit detection show that all-trans retinoic acid can enhance the ability of Fra-1 to lose the mitochondrial membrane potential, inhibit the glucose consumption and the production of lactic acid as well as ATP. CCK8 and colony formation assays indicate that all-trans retinoic acid enhances the ability of Fra-1 to inhibit cell proliferation. In addition, through Western blot analysis, it was determined that P53 and P21 were up-regulated, and MDM2 and LDHA were down-regulated. CONCLUSION: The overall results of the study strongly suggest that all-trans retinoic acid enhances the effect of Fra-1 on inhibiting cervical cancer proliferation and metabolism in vitro, and also influences the expression of MDM2/P53/P21 and LDHA.

A dataset describing a suite of novel antibody reagents for the RAS signaling network.

RAS genes are frequently mutated in cancer and have for decades eluded effective therapeutic attack. The National Cancer Institute's RAS Initiative has a focus on understanding pathways and discovering therapies for RAS-driven cancers. Part of these efforts is the generation of novel reagents to enable the quantification of RAS network proteins. Here we present a dataset describing the development, validation (following consensus principles developed by the broader research community), and distribution of 104 monoclonal antibodies (mAbs) enabling detection of 27 phosphopeptides and 69 unmodified peptides from 20 proteins in the RAS network. The dataset characterizes the utility of the antibodies in a variety of applications, including Western blotting, immunoprecipitation, protein array, immunohistochemistry, and targeted mass spectrometry. All antibodies and characterization data are publicly available through the CPTAC Antibody Portal, Panorama Public Repository, and/or PRIDE databases. These reagents will aid researchers in discerning pathways and measuring expression changes in the RAS signaling network.

DNA Replication Stress Phosphoproteome Profiles Reveal Novel Functional Phosphorylation Sites on Xrs2 in Saccharomyces cerevisiae.

In response to replication stress, a phospho-signaling cascade is activated and required for coordination of DNA repair and replication of damaged templates (intra-S-phase checkpoint) . How phospho-signaling coordinates the DNA replication stress response is largely unknown. We employed state-of-the-art liquid chromatography tandem-mass spectrometry (LC-MS/MS) approaches to generate high-coverage and quantitative proteomic and phospho-proteomic profiles during replication stress in yeast, induced by continuous exposure to the DNA alkylating agent methyl methanesulfonate (MMS) . We identified 32,057 unique peptides representing the products of 4296 genes and 22,061 unique phosphopeptides representing the products of 3183 genes. A total of 542 phosphopeptides (mapping to 339 genes) demonstrated an abundance change of greater than or equal to twofold in response to MMS. The screen enabled detection of nearly all of the proteins known to be involved in the DNA damage response, as well as many novel MMS-induced phosphorylations. We assessed the functional importance of a subset of key phosphosites by engineering a panel of phosphosite mutants in which an amino acid substitution prevents phosphorylation. In total, we successfully mutated 15 MMS-responsive phosphorylation sites in seven representative genes including APN1 (base excision repair); CTF4 and TOF1 (checkpoint and sister-chromatid cohesion); MPH1 (resolution of homologous recombination intermediates); RAD50 and XRS2 (MRX complex); and RAD18 (PRR). All of these phosphorylation site mutants exhibited MMS sensitivity, indicating an important role in protecting cells from DNA damage. In particular, we identified MMS-induced phosphorylation sites on Xrs2 that are required for MMS resistance in the absence of the MRX activator, Sae2, and that affect telomere maintenance.

Novel connections between DNA replication, telomere homeostasis, and the DNA damage response revealed by a genome-wide screen for TEL1/ATM interactions in Saccharomyces cerevisiae.

Tel1 is the budding yeast ortholog of the mammalian tumor suppressor and DNA damage response (DDR) kinase ATM. However, tel1-Δ cells, unlike ATM-deficient cells, do not exhibit sensitivity to DNA-damaging agents, but do display shortened (but stably maintained) telomere lengths. Neither the extent to which Tel1p functions in the DDR nor the mechanism by which Tel1 contributes to telomere metabolism is well understood. To address the first question, we present the results from a comprehensive genome-wide screen for genetic interactions with tel1-Δ that cause sensitivity to methyl methanesulfonate (MMS) and/or ionizing radiation, along with follow-up characterizations of the 13 interactions yielded by this screen. Surprisingly, many of the tel1-Δ interactions that confer DNA damage sensitivity also exacerbate the short telomere phenotype, suggesting a connection between these two phenomena. Restoration of normal telomere length in the tel1-Δ xxx-Δ mutants results in only minor suppression of the DNA damage sensitivity, demonstrating that the sensitivity of these mutants must also involve mechanisms independent of telomere length. In support of a model for increased replication stress in the tel1-Δ xxx-Δ mutants, we show that depletion of dNTP pools through pretreatment with hydroxyurea renders tel1-Δ cells (but not wild type) MMS-sensitive, demonstrating that, under certain conditions, Tel1p does indeed play a critical role in the DDR.