Profiling Strategy Research Articles

Development of a high throughput oxidation profiling strategy for monoclonal antibody products

Oxidation is one of the most common degradation pathways of biopharmaceutics, potentially leading to altered product stability, pharmacokinetics, reduced biological activity and/or an increased immunogenicity. However, it is often insufficiently assessed in early development stages, leaving potential molecule liabilities undiscovered. Aim of the present work was the development of a high throughput oxidation profiling strategy, applicable throughout various stages of biopharmaceutical development. The study demonstrates that the combination of multiple stress assays, including peroxide-based, visible light, and metal-catalyzed oxidation (MCO), enables a comprehensive understanding of a mAb's oxidation susceptibility. The most effective parameters to evaluate oxidation in a high-throughput screening workflow are aggregation, tryptophan oxidation and changes in the hydrophobicity profile of the Fc and Fab subunit measured via Size Exclusion Chromatography, Intrinsic Tryptophan Fluorescence Emission spectroscopy and Reversed-Phase Chromatography subunit analysis, respectively. This oxidation profiling approach is valuable tool to systematically characterize the oxidation susceptibility under relevant conditions, time effective and with minimal sample consumption.

The cost-effectiveness of including liquid biopsy into molecular profiling strategies for newly diagnosed advanced non-squamous non-small cell lung cancer in an Asian population

ObjectivesLiquid biopsy is complementary to tissue biopsy for lung cancer profiling, yet evidence of the cost-effectiveness is limited. This could retard implementation and reimbursement in clinical practice. The aim of this study is to estimate the cost-effectiveness of profiling strategies that include liquid biopsy and to identify the optimal profiling approach for newly diagnosed advanced non-squamous non-small cell lung cancer (NSCLC) in an Asian population using Singapore as an example. Materials and methodsA decision tree and partitioned-survival model was developed from the Singapore healthcare system’s perspective to evaluate the cost-effectiveness of five molecular profiling strategies: either tissue or plasma next-generation sequencing (NGS) alone, a concurrent, and two sequential approaches. Model inputs were informed by local data or published literature. Sensitivity analyses and scenario analyses were undertaken to understand the robustness of the conclusions for decision making. The optimal strategy at different willingness-to-pay (WTP) thresholds was presented by cost-effectiveness acceptability frontier and the expected loss curve. ResultsThe sequential tissue-plasma NGS approach revealed an additional 0.0981 quality adjusted life years (QALYs) for an extra cost of S$3,074 over a 20-year time horizon compared to tissue NGS alone, resulting in an incremental cost-effectiveness ratio (ICER) of S$31,318/QALY and an incremental net monetary benefit of S$1,343 per patient. The findings were sensitive to the costs of pembrolizumab and osimertinib and the probabilities of re-biopsy after tissue NGS. Sequential plasma-tissue NGS and plasma NGS alone were more costly and less effective than alternatives. ConclusionThe sequential tissue-plasma NGS approach generated the highest net monetary benefit and was the optimal testing strategy when WTP was S$45,000/QALY. It retained superiority but understandably with a higher ICER when expensive, non-first line treatments were included. Overall, its routine clinical practice should be proactively considered for newly diagnosed advanced non-squamous NSCLC in an Asian population.

Energy-saving operation in urban rail transit: A deep reinforcement learning approach with speed optimization

The energy consumption of urban rail transit plays a significant role in the operating costs of trains. It is particularly crucial to decrease the energy consumption of the traction power supply in subway systems, as it accounts for approximately half of the total energy consumption of the subway operating organization. To overcome the limitations of traditional real-time speed profile generation methods and the limited exploration capabilities of popular reinforcement learning algorithms in the speed domain, this paper presents the Energy-Saving Maximum Entropy Deep Reinforcement Learning (ES-MEDRL) algorithm. The ES-MEDRL algorithm incorporates Lagrange multipliers and maximum policy entropy as penalties to formulate a novel objective function. This function aims to intensify exploration in the speed domain, minimize train traction energy consumption, and ensure a balance between ride comfort, punctuality, and safety within the subway system. This leads to the optimization of speed profile strategies. To further reduce energy consumption, this paper proposes a secondary optimization strategy for the energy-saving speed profile. This approach involves trading acceptable travel time for improved energy efficiency. To validate the performance of the proposed model and algorithm, numerical experiments are conducted using the Yizhuang Line of the Beijing Metro. The findings demonstrate a minimum 20 % increase in energy efficiency with the ES-MEDRL algorithm compared to manual driving. This algorithm can guide energy-efficient train operations at the planning level.

Mapping parental DMRs predictive of local and distal methylome remodeling in epigenetic F1 hybrids.

F1 hybrids derived from a cross between two inbred parental lines often display widespread changes in DNA methylation and gene expression patterns relative to their parents. An emerging challenge is to understand how parental epigenomic differences contribute to these events. Here, we generated a large mapping panel of F1 epigenetic hybrids, whose parents are isogenic but variable in their DNA methylation patterns. Using a combination of multi-omic profiling and epigenetic mapping strategies we show that differentially methylated regions in parental pericentromeres act as major reorganizers of hybrid methylomes and transcriptomes, even in the absence of genetic variation. These parental differentially methylated regions are associated with hybrid methylation remodeling events at thousands of target regions throughout the genome, both locally (in cis) and distally (in trans). Many of these distally-induced methylation changes lead to nonadditive expression of nearby genes and associate with phenotypic heterosis. Our study highlights the pleiotropic potential of parental pericentromeres in the functional remodeling of hybrid genomes and phenotypes.

Analysis of The Influence of Promotion Through Digital Marketing and Effectiveness of Marketing Communication on Customer Decisions in Hospitality Industry

This research aims to determine the marketing communication strategies used to increase occupancy. We used qualitative and descriptive research. The researcher employed a case study as the research method. In-depth interviews provided the techniques for collection. The research results revealed that the public relations division, in collaboration with the hotel marketing team, designed and implemented the marketing communication strategy. Public relations is a communication facilitator, especially in communicating hotel products and services through various marketing strategies. This strategy is divided into three, namely the push strategy, which uses distributors or travel agents to encourage customers to use hotel products; the pull strategy, which uses advertising, sales promotions, personal sales, and direct marketing to attract customer interest in hotel products and services, also the profile strategy, which uses public relations to build the hotel's image and reputation to increase customer trust and sales. This research suggests that further research be carried out in quantitative form to obtain a more detailed understanding of the success of each factor that influences marketing communication strategies, such as push, pull, and profile strategies. Meanwhile, from a practical perspective, conducting a more detailed evaluation of each marketing communication tool used by measuring its effectiveness to select the most effective tools in increasing sales or occupancy is recommended.

Abstract 4172: Pathology supervised approaches for ROI placement and segmentation to overcome caveats and pitfalls in high-plex spatial profiling

Abstract Background: Tissue-based high-plex platforms evaluate numerous biomarkers in limited tissue sections. Certain tissue features and assay technical limitations may jeopardize the accuracy of this analysis. In this study, we aim to determine tissue specific features that influence image-based region of interest (ROI) selection and segmentation for spatial profiling of carcinomas, and to develop pathology approaches to overcome them. Methodology: A total of 317 samples from different carcinoma types processed with the GeoMx Digital spatial profiling protein assay protocol were assessed to identify deviations from original strategies for ROI placement and segmentation (Table 1). ROI were selected by a pathologist. The initial strategy for all cases was to place rectangles ROIs of 660*785 µm in tumor area and to segment based on morphology biomarkers as indicated in Table 1. Strategies to overcome technical and tissue specific features were recorded and quantified. Results: A total of 2068 ROIs and 4422 AOIs were evaluated. In all the cases, tissue displayed specific features that limited planned ROI placement and segmentation. Table 1 describes these features by tissue type and profiling strategies. A total of 970 ROIs were solved using different annotation type or changes in segmentation strategy, 584 bypassed the limitations by changing the processing protocol; and 322 were not solved due to non-compliance with assay requirements. Conclusions: We identified tissue features that limit the planned ROI selection and segmentation in spatial high-plex profiling, mainly due to tissue processing, distinct histology, type of sample, and presence of adjacent non-neoplastic tissue. A deep understanding of tumor architecture, biology and platform related technical limitations is needed to select areas of analysis for spatial profiling. Table 1. ROI and segmentation strategy methodology and tissue specific features with pathology appro Histological type (Profiling segments) Anal Carcinoma (Tu/TME) Lung NSCLC (Tu/TME) Lung NSCLC TMA (Tu/Tcells/Mϕ) Lung NSCLC TMA (Tu/TME) CRC MT Tu/Immune Breast Ca Tu/Immune/else Pathology Approach (ROIs) Methodology Samples 48 33 80 80 6 70 Biopsy type Incisional (Core/other), Whole tissue Incisional (Core/other)Whole tissue TMA TMA Whole tissue Core Morphology markers/mIF Channels Syto13: FITC/525 PanCK: Cy3/568 CD45: Texas red/615 CD68: Cy5/666 Syto13: FITC/525PanCK: Cy3/568CD20: Texas red/615CD3: Cy5/666 Syto13: FITC/525 PanCK: Cy3/568 CD68: Texas red/615 CD3: Cy5/666 Syto13: FITC/525 PanCK: Cy3/568 CD68: Texas red/615 CD3: Cy5/666 Syto13: FITC/525 PanCK: CD45: Texas red/615 Syto13: FITC/525 PanCK: Cy3/568 CD45: Texas red/615 Strategy for segmentation Tumor (PanCK+) TME (PanCK-) Tumor (PanCK+)TME (PanCK-) Tumor (PanCK+) T cell (CD3+) Mϕ (CD68+) Tumor (PanCK+) TME (PanCK-) Tumor (PanCK+) Immune (CD45+) Tumor (PanCK+) Immune (CD45+) Else (PanCK-CD45-) Total ROIs/AOIs 224/435 144/252 80/218 82/162 944/1573 594/1782 Tissue specific features (ROI number/percentage) Folds (ROIs) 10 1 0 0 9 5 Draw Polygons (25) Adjacent* Non-Tumoral tissue 22 7 9 7 388 40 Draw Polygons (473) Tissue Derived Autofluorescence 3 105 0 1 0 45 Draw Polygons (13). New segment to exclude autofluorescence (141) Sample Size 26 19 1 2 5 138 Polygons (191) Necrosis 13 0 1 1 217 6 Polygons (238) Biological absence of morphology marker 10 0 0 0 0 5 Polygons non segmented for Tu and TME (15) Solid tumors with scant TME 0 21 0 0 0 9 Polygons non segmented for Tu and TME (30) Eosin autofluorescence 0 0 0 0 0 584 Stained new section with Validated PanCK in Cy5/666 channel (584) Failure to reach the minimum number of cell requirements 13 36 22 2 249 43 Excluded segments (322) New scan to select more areas (43) Citation Format: Sharia Hernandez, Claudio J. Arrechedera, Pedro Rocha, Larisa Kostousov, Sean Barnes, Khan Khaja, Luisa M. Solis-Soto. Pathology supervised approaches for ROI placement and segmentation to overcome caveats and pitfalls in high-plex spatial profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4172.

Abstract 7509: Evaluation ofHER-2 expression on circulating tumor cells as a real time biomarker in advanced breast cancer

Abstract Introduction: Breast cancer accounts for 12.5% of all new annual cancer cases worldwide. Cases classified as HER2-positive tend to proliferate, metastasize, and often lead to relapse. HER-2 is the most valid tumor marker, used as a diagnostics and prognostic biomarker in metastatic breast cancer (MBC). Thus, analysis of expression of HER-2 on CTCs can offer a real time dynamic biomarker for treatment options between endocrine therapy versus chemotherapy in advanced breast cancer. Functional assays on CTCs with HER-2 biomarker offer evolving biomarkers especially when the tissue is not accessible and inadequate. Methods: Retrospectively, 179 breast cancer patients' blood was analyzed for the presence of CTCs using the OncoDiscover® platform approved by Drug Controller General of India (CDSCO). Consisting of immunomagnetic multi-components mediated by anti EpCAM antibodies. The isolated cells were immunostained consisting of a nuclear dye DAPI, CK-18, CD45 and HER-2 antibodies. CTCs were identified by the presence of HER-2 + ve, CK 18 + ve, DAPI + ve and CD 45 -ve staining. Validation of HER-2 expression on the CTCs was analyzed based on the linear intensity gradients of the fluorescence signals. CTCs were termed as HER-2 -ve based on weak or no detectable fluorescence signal and termed +ve based on high fluorescence signal. Results: Among the cohort of 179 patient samples, 63.68 % samples showed the presence of CTC (CTC ranged from 1-7). 47.84% of these CTCs showed the expression of HER-2 signal. The mean value for fluorescence intensity for expression of HER-2 in CTCs was found to be 3.23. The observed mean fluorescence intensity for HER-2 expression further emphasizes the robustness of CTCs as a viable source for molecular characterization. Conclusion: Integration of HER-2 analysis on CTCs into the clinical assessment of metastatic breast cancer, may offer a non-invasive, real-time strategy for profiling and possibly paving the way for more precise and tailored therapeutic interventions. Citation Format: Jayant Khandare, Gourishankar Aland, Saloni Andhari, Ganesh Khutale, Prathamesh Patil, Tanvi Deshpande, Meghana Garbhe, Vikas Jadhav, Aravindan Vasudevan, Sreeja Jayant. Evaluation ofHER-2 expression on circulating tumor cells as a real time biomarker in advanced breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7509.

Abstract 1943: Discovery and characterization of potential drivers of resistance to the KRAS G12D inhibitor, MRTX1133, in cancer cell line models

Abstract Introduction: KRASG12D is the most common KRAS mutation and is present in approximately 34% of pancreatic cancer, 10-12% of colorectal cancer, 4% of lung adenocarcinoma and in a number of other cancer types. We previously identified MRTX1133, a potent, selective, and non-covalent KRASG12D inhibitor. The advancement of MRTX1133 to clinical trials warrants continued study of the role of the KRASG12D mutation in cancer pathogenesis and progression. Methods: Previous pharmacodynamic and pharmacogenomic profiling in sensitive and partially resistant models identified mechanisms implicated in limiting the anti-tumor response including signaling pathways that induce feedback reactivation and/or bypass KRAS dependence. Thus, cell lines (GP2D and SNU-1033, colorectal cancer; HPAC and AsPC-1, pancreatic cancer) were utilized to study acquired resistance to MRTX1133 following continuous treatment at increasing concentrations over time. These cell lines were confirmed to be stably resistant as determined by evaluation of response to MRTX1133 in vitro and in vivo. These cell lines were extensively profiled to determine underlying mechanisms of resistance utilizing molecular profiling strategies as well as drug combination screens to interrogate pathways that could circumvent resistance. Results: In one of the four generated cell lines with acquired resistance, evidence of secondary KRAS mutations Y96N and H95Q were confirmed by RNAseq and are likely drivers of resistance in this cell line and are consistent with known mechanisms of KRASG12C inhibitor resistance. The three other MRTX1133 resistant cell lines did not show evidence of secondary KRAS mutations and were further evaluated for mechanisms that bypass KRAS dependence. Combinations of MRTX1133 with therapies that target pathways including upstream RTK signaling, mTOR, or AKT broadly enhanced the anti-tumor activity of MRTX1133 in both in vitro cell viability and in vivo efficacy studies in the resistant cell lines. Conclusion: Collectively, these data provide early insight onto potential mechanisms of resistance to selective KRASG12D inhibitors and demonstrate the importance of developing rational combination strategies designed to address drug resistance. In addition, these studies provide a catalogue of potentially druggable vulnerabilities that complement KRAS blockade. Citation Format: Xousaen M. Helu, Ellie Griffiths, Andrew Calinisan, Allan Hebbert, Larry Yan, Natalie Hoffman, David Trinh, Laura Hover, Julio Fernandez-Banet, David M. Briere, Ella Lifset, Peter Olson, James G. Christensen, Jill Hallin. Discovery and characterization of potential drivers of resistance to the KRAS G12D inhibitor, MRTX1133, in cancer cell line models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1943.

Abstract 413: Aneuploid cells depend on the RAF/MEK/ERK pathway and on RNA degradation for overcoming DNA damage and transcriptional burden

Abstract Aneuploidy, a state of karyotype imbalance, is a hallmark of cancer. Understanding the broad implications of aneuploidy at molecular, cellular, and physiological levels is crucial for effectively targeting aneuploid cancer cells. Aneuploidy often results in specific stress responses within cells, which must adapt to for survival and growth. Exploring these adaptive mechanisms to aneuploidy open avenues for targeted cancer therapies. Here, we induced aneuploidy in non-transformed RPE1-hTERT cells, generating multiple stable clones each exhibiting varying degrees of chromosomal imbalances. Employing a comprehensive genomic profiling strategy, we analyzed six isogenic clones using whole-exome and RNA sequencing. We uncovered a complex landscape, where each degree of aneuploidy brought its own set of changes and challenges. Additionally, we explored their cellular dependency landscapes, performing genome-wide CRISPR/Cas9 screenings along with extensive drug screens. As previously reported, we observed and confirmed increased DNA damage and p53 pathway activation in the aneuploid cells. We also found that the aneuploid clones triggered the DNA damage response (DDR), leading to an increased resistance to further DNA damage. Interestingly, we observed that aneuploid cells showed increased activity in the RAF/MEK/ERK pathway. Moreover, these cells demonstrated a heightened sensitivity to a range of clinically significant drugs that target this pathway, in particular in response to both genetic and chemical inhibition of CRAF. The activity of CRAF was found to be functionally connected to the resistance against DNA damage induction, and the inhibition of CRAF making aneuploid cells more susceptible to chemotherapies that induce DNA damage. Next, we observed increased of RNA synthesis and degradation in the aneuploid cells, along with heightened activity in both the nonsense-mediated decay (NMD) and the microRNA-mediated mRNA silencing processes. As a result, aneuploid cells displayed increased sensitivity to the perturbation of several key components of RNA degradation pathways. Furthermore, the dependencies of aneuploid cells on RAF/MEK/ERK activity and on intact RNA degradation were confirmed through examining a broad range of human cancer cell lines, emphasizing their significance in human cancer. Altogether, our results provide a comprehensive resource of karyotypically stable cells representing a range of aneuploidy states, and they reveal previously unidentified weaknesses in aneuploid cells that may be crucial for therapeutic developments. Citation Format: Marica Rosaria Ippolito, Johanna Zerbib Zerbib, Yonatan Eliezer, Eli Reuveni, Sonia Viganò, Giuseppina De Feudis, Anouk Savir Kadmon, Alice Ratti, Sara Martin, Kathrin Laue, Yael Cohen-Sharir, Simone Scorzoni, Francisca Vazquez, Uri Ben-David, Stefano Santaguida. Aneuploid cells depend on the RAF/MEK/ERK pathway and on RNA degradation for overcoming DNA damage and transcriptional burden [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 413.

Abstract 3976: Single-cell TCRɑ/β and TCRγ/δ immune receptor profiling and immunophenotyping using a 96-well plate sorted-cell approach

Abstract Single-cell immune receptor profiling is a revolutionary approach that allows investigators to combine clonotype repertoire identification with paired-chain information and the phenotype of cells (e.g., cell subtype). Single-cell immune receptor profiling can be performed using a medium-throughput approach (1,000-5,000 cells) using microwell arrays or droplet microfluidics technologies. However, these assays are more complicated to run and require expensive reagents and limited sequencing throughput when compared to bulk immune receptor profiling methods. Here, we describe a low-throughput, single-cell immune profiling strategy using sorted cells in 96-well plates. The plate is pre-aliquoted with either T-cell receptor (TCR) ɑ/β or TCR γ/δ primers along with 30 crucial T-cell markers. We perform multiplex RT-PCR amplification and sequencing of the CDR3 regions. The resulting data provides the abundant clonotype counts along with the chain pairing information for these ɑ/β and γ/δ chains, together with the T-cell subtype information using gene-expression profiles. By analyzing the TCR gene rearrangement at the single-cell level, researchers can better understand T cell development, proliferation, and clonality, which are crucial for studying diseases such as cancer, immunodeficiency, and autoimmunity. Furthermore, single-cell TCR sequencing when combined with RNA sequencing datasets, facilitates the identification of γδ T cells. This method provides a standardized tool for identifying potential γδ T cell-based cancer immunotherapies. The technology's cost-effectiveness and ability to analyze clonotypes and immunophenotypes of cells in a single assay make it a valuable tool for unraveling the immune dynamics in various diseases. Citation Format: Alex Chenchik, Tianbing Liu, Mikhail Makhanov, Dongfang Hu, Lester Kobzik, Khadija Ghias, Paul Diehl. Single-cell TCRɑ/β and TCRγ/δ immune receptor profiling and immunophenotyping using a 96-well plate sorted-cell approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3976.

Thermal Proteome Profiling Strategy Identifies CNPY3 as a Cellular Target of Gambogic Acid for Inducing Prostate Cancer Pyroptosis.

There is an urgent requirement to acquire a comprehensive comprehension of novel therapeutic targets for prostate cancer to facilitate the development of medications with innovative mechanisms. In this study, we identified gambogic acid (GBA) as a specific pyroptosis inducer in prostatic cancer cells. By using a thermal proteome profiling (TPP) strategy, we revealed that GBA induces pyroptosis by directly targeting the canopy FGF signaling regulator (CNPY3), which was previously considered "undruggable". Moreover, through the utilization of the APEX2-based proximity labeling method, we found that GBA recruited delactatease SIRT1, resulting in the elimination of lysine lactylation (Kla) on CNPY3. Of note, SIRT1-mediated delactylation influenced the cellular localization of CNPY3 to promote lysosome rupture for triggering pyroptosis. Taken together, our study identified CNPY3 as a distinctive cellular target for pyroptosis induction and its potential application in prostate cancer therapy.

Proteins à la carte: riboproteogenomic exploration of bacterial N-terminal proteoform expression.

With the emerging theme of genes within genes comprising the existence of alternative open reading frames (ORFs) generated by translation initiation at in-frame start codons, mechanisms that control the relative utilization of annotated and alternative TIS need to be unraveled and our molecular understanding of resulting proteoforms broadened. Utilizing complementary ribosome profiling strategies to map ORF boundaries, we uncovered dual-encoding ORFs generated by in-frame TIS usage in Salmonella. Besides demonstrating that alternative TIS usage may generate proteoforms with different characteristics, such as differential localization and specialized function, quantitative aspects of conditional retapamulin-assisted ribosome profiling (Ribo-RET) translation initiation maps offer unprecedented insights into the relative utilization of annotated and alternative TIS, enabling the exploration of gene regulatory mechanisms that control TIS usage and, consequently, the translation of N-terminal proteoform pairs.

Proteomic characterization of extracellular vesicles in programmed cell death.

Programmed cell death (PCD) is a fundamental biological process that plays a critical role in cell development, differentiation, and homeostasis. The secretion and uptake of extracellular vesicles (EVs) is one of the important regulatory mechanisms for PCD. EVs are natural membrane structures secreted by cells that contain a variety of proteins, lipids, nucleic acids, and other bioactive molecules. Due to their important roles in intercellular communication and disease progression, there is great interest in studying EVs and their cargo. Different protein components are sorted and packaged in EVs, allowing EVs to perform their functions. The study of EV proteomics helps us understand the role of PCD in the development of diseases. Meanwhile, proteomics is a powerful tool for studying the composition and function of EVs, which assists in the identification, quantification, and profiling of protein components of EVs, and provides insight into the molecular mechanisms involved in PCD and related diseases. In this review, we summarize the characteristics of EV proteomics in different types of PCD, compare different proteomic profiling strategies for EVs, and discuss the impact of EV proteomics on cell function and regulation during PCD, to understand its role in the pathogenesis of related diseases.

Label-free plasmonic spectral profiling of serum DNA

Genetic and epigenetic modifications are linked to the activation of oncogenes and inactivation of tumor suppressor genes. Likewise, the associated molecular alternations can best inform precision medicine for personalized tumor treatment. Therefore, performing characterization of genetic and epigenetic alternations at the molecular level represents a crucial step in early diagnosis and/or therapeutics of cancer. However, the prevailing methods for DNA analysis involve a series of tedious and complicated steps, in which important genetic and epigenetic information could be lost or altered. To provide a potential approach for non-invasive, direct, and efficient DNA analysis, herein, we present a promising strategy for label-free molecular profiling of serum DNA in its pristine form by fusing surface-enhanced Raman spectroscopy with machine learning on a superior plasmonic nanostructured platform. Using DNA methylation and single-point mutation as two case studies, the presented strategy allows a well-balanced sensitive and specific detection of epigenetic and genetic changes at the single-nucleotide level in serum. We envision the presented label-free strategy could serve as a versatile tool for direct molecular profiling in pristine forms of a wide range of biological markers and aid biomedical diagnostics as well as therapeutics.

Molecular targets of cisplatin in HeLa cells explored through competitive activity-based protein profiling strategy

Cisplatin is widely used as anticancer drugs, and DNA is considered as the main target. Considering its high affinity towards cysteines and the important role of cystine containing proteins, we applied a competitive activity-based protein profiling strategy to identify protein cysteines that bind with cisplatin in HeLa cells. Living cells were treated with cisplatin at cytotoxic concentrations, then the protein was extracted. After labeling with desthiobiotin iodoacetamide (DBIA) probe, protein was precipitated, digested and isotopically labeled, subsequently the peptides were combined, and the biotinylated cysteine-containing peptides were enriched and quantified by LC-MS/MS. A total of 3571 peptides which originated from 1871 proteins were identified using the DBIA probe. Among them, 46 proteins were screened as targets, including proteins that have been identified as binding proteins by previous study. A novel cisplatin target, calpain-1 (CAPN1), was identified and validated as binding with cisplatin in vitro.

Liquid chromatography-mass spectrometry-based strategy for systematic profiling of chemical components and associated quantitative analysis of quality markers in Qi-Wei-Tong-Bi oral liquid.

Qi-Wei-Tong-Bi oral liquid (QWTB), a famous Chinese medicine preparation composed of seven crude drugs has a good therapeutic effect on rheumatoid arthritis and is widely used in China.However, its chemical composition and quality control have not been comprehensively and systematically investigated. In this study, high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was employed for its chemical profiling. As a result, 100 components were chemically characterized. Additionally, high-performance liquid chromatography coupled with a quadrupole linear ion trap mass spectrometry method was developed to simultaneously quantify nine bioactive components (hyperoside, ononin, quercetin, sinomenine, magnoflorine, gallic acid, protocatechuic acid, monotropein, and cyclo-(Pro-Tyr)) in multiple-reaction monitoring mode. After successful validation in terms of linearity, precision, repeatability, and recovery, the assay method was applied for the determination of 10 batches of QWTB. The results showed that QWTB was enriched in sinomenine and magnoflorine with the highest amount up to hundreds or even thousands of µg/mL, while quercetin, ononin, cyclo-(Pro-Tyr), and hyperoside were much lower with the lowest content below 10µg/mL. This study work would help to reveal the chemical profiling and provide a valuable and reliable approach for quality evaluation and even pharmacodynamic material basis studies of QWTB.

Sulfonium-Based Activity-Based Probes for Lysine-Selective Protein Profiling under Alkaline Conditions

AbstractDue to their strong nucleophilicities, nucleophilic lysine and cysteine residues can be easily recognized and modified by electrophilic groups, thus, acting as the targets for covalent ligands or drugs. Therefore, the development of site-specific protein-modification chemistry for various nucleophilic residues has been explored to label proteins selectively for many biological and therapeutic applications. In this study, we constructed a series of sulfonium-based small molecules to react with the amine group of lysine residues by utilizing the strong electrophilicity of sulfonium, resulting in lysine-selective labeling via the formation of classical amide bonds under alkaline conditions (pH 9.0–11.0). After systematic optimization of the labeling conditions, this strategy was utilized for protein labeling across various bacteria’s lysates. Finally, combined with the activity-based protein profiling (ABPP) strategy, we successfully identified and analyzed hundreds of labeled lysine residues in the bacterial proteome.

N-Cinnamoylpyrrole-derived alkaloids from the genus Piper as promising agents for ischemic stroke by targeting eEF1A1

BackgroundIschemic stroke (IS) is a serious cerebrovascular disease characterized by significantly elevated mortality and disability rates, and the treatments available for this disease are limited. Neuroinflammation and oxidative stress are deemed the major causes of cerebral ischemic injury. N-Cinnamoylpyrrole alkaloids form a small group of natural products from the genus Piper and have not been extensively analyzed pharmacologically. Thus, identifying the effect and mechanism of N-cinnamoylpyrrole-derived alkaloids on IS is worthwhile. PurposeThe present research aimed to explore the antineuroinflammatory and antioxidative stress effects of N-cinnamoylpyrrole-derived alkaloids isolated from the genus Piper and to explain the effects and mechanism on IS. MethodsN-cinnamoylpyrrole-derived alkaloids were isolated from Piper boehmeriaefolium var. tonkinense and Piper sarmentosum and identified by various chromatographic methods. Lipopolysaccharide (LPS)-induced BV-2 microglia and a mouse model intracerebroventricularly injected with LPS were used to evaluate the antineuroinflammatory and antioxidative stress effects. Oxygen‒glucose deprivation/reperfusion (OGD/R) and transient middle cerebral artery occlusion (tMCAO) models were used to evaluate the effect of PB-1 on IS. To elucidate the fundamental mechanism, the functional target of PB-1 was identified by affinity-based protein profiling (ABPP) strategy and verified by cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS), and circular dichroism (CD) analyses. The effect of PB-1 on the NF-κB and NRF2 signaling pathways was subsequently evaluated via western blotting and immunofluorescence staining. ResultsThe results showed that N-cinnamoylpyrrole-derived alkaloids significantly affected neuroinflammation and oxidative stress. The representative compound, PB-1 not only inhibited neuroinflammation and oxidative stress induced by LPS or OGD/R insult, but also alleviated cerebral ischemic injury induced by tMCAO. Further molecular mechanism research found that PB-1 promoted antineuroinflammatory and antioxidative stress activities via the NF-κB and NRF2 signaling pathways by targeting eEF1A1. ConclusionOur research initially unveiled that the therapeutic impact of PB-1 on cerebral ischemic injury might rely on its ability to target eEF1A1, leading to antineuroinflammatory and antioxidative stress effects. The novel discovery highlights eEF1A1 as a potential target for IS treatment and shows that PB-1, as a lead compound that targets eEF1A1, may be a promising therapeutic agent for IS.

Rationale and Design of a Phase 2, Double-blind, Placebo-Controlled, Randomized Trial Evaluating AMP Kinase-Activation by Metformin in Focal Segmental Glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS), the most common primary glomerular disease leading to end-stage kidney disease (ESKD), is characterized by podocyte injury and depletion, whereas minimal change disease (MCD) has better outcomes despite podocyte injury. Identifying mechanisms capable of preventing podocytopenia during injury could transform FSGS to an "MCD-like" state. Preclinical data have reported conversion of an MCD-like injury to one with podocytopenia and FSGS by inhibition of AMP-kinase (AMPK) in podocytes. Conversely, in FSGS, AMPK-activation using metformin (MF) mitigated podocytopenia and azotemia. Observational studies also support beneficial effects of MF on proteinuria and chronic kidney disease (CKD) outcomes in diabetes. A randomized controlled trial (RCT) to test MF in podocyte injury with FSGS has not yet been conducted. We report the rationale and design of phase 2, double-blind, placebo-controlled RCT evaluating the efficacy and safety of MF as adjunctive therapy in FSGS. By randomizing 30 patients with biopsy-confirmed FSGS to MF or placebo (along with standard immunosuppression), we will study mechanistic biomarkers that correlate with podocyte injury or depletion and evaluate outcomes after 6 months. We specifically integrate novel urine, blood, and tissue markers as surrogates for FSGS progression along with unbiased profiling strategies. Our phase 2 trial will provide insight into the potential efficacy and safety of MF as adjunctive therapy in FSGS-a crucial step to developing a larger phase 3 study. The mechanistic assays here will guide the design of other FSGS trials and contribute to understanding AMPK activation as a potential therapeutic target in FSGS. By repurposing an inexpensive agent, our results will have implications for FSGS treatment in resource-poor settings.

Primer on Adhesive Arachnoiditis

This article explores the resurgence of adhesive arachnoiditis (AA), a previously rare disorder, in the 21st century. Historically linked to tuberculosis and other infections, AA’s re-emergence is attributed to advancements in MRI technology, aiding in improved diagnosis and an increase in spinal procedures, possibly leading to iatrogenic cases. Complications of AA include chronic pain and motor dysfunction, significantly impacting quality of life. Treatment involves a multidisciplinary approach, including pharmacological and osteopathic treatments, possible surgical interventions, and psychological support. The article provides an in-depth look at AA’s epidemiology, clinical profile, causes, diagnosis, and treatment strategies, highlighting its complex nature and the necessity for heightened awareness among medical practitioners.