Assay Technology Research Articles

Lactate Transporters Modulate Stromal Cell Remodeling in Myeloproliferative Neoplasms (MPN)

Introduction. Philadelphia-negative myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The diagnostic approach proposed by the World Health Organization (WHO) uses clinical features, bone marrow (BM) morphology, karyotype and molecular genetic tests to classify MPN subtypes. The BM morphology includes cellularity, erythropoiesis, and neutrophil granulopoiesis in context with specific features of megakaryocytes as well as the BM fiber content, especially in early-stage myelofibrosis that present with thrombocytosis and clinically mimic ET. Since alteration of hematopoiesis is constantly associated with profound modifications of the bone marrow (BM), we hypothesize that CD34+ cells from PMF can actively produce lactate and thus rewiring metabolism of immune cells and mesenchymal stromal cells (MSCs). In turn, this could favor immune evasion and remodeling of BM niche. Methods.Cell proliferation was assessed in vitro on primary MSCs, HS-5 cell line, and PBMC cells. Protein expression was performed by Western blot and immunofluorescence assay. The NCBI GEO database was used to select transcriptome datasets and to analyze gene expression in PMF patients. Cytokine detections were performed by Multiplex immunobead assay technology. An adult TPO-mimetic-induced PMF Zebrafish model was used for in vivo evaluation. An immunohistology assay was performed in MPN BM biopsies. Results.CD34+ cells from PMF patients significantly upregulated the lactate monocarboxylate transporters (MCT-1 and -4) compared to healthy CD34+ cells. Consistently, lactate concentration was higher in PMF sera compared to healthy controls. Since PMF patients showed an increased percentage of circulating immunosuppressive cells such as G- and M-MDSC and Treg, we incubated healthy peripheral blood mononucleated cells (PBMNCs) with PMF sera in the presence or absence of MCT1inhibitor (AZD3965) to evaluate the role of lactate in the expansion of these immunological subsets. We found an expansion of both Treg and M-MDSCs, which was reverted after blocking of circulating lactate by AZD3965. In order to study the effect of lactate in BM remodeling, we treated in vitro healthy MSCs with lactate (20 mM) for 24h. Interestingly, lactate induced a modification of extracellular matrix organization, as demonstrated by increased reticulin and collagen deposition and metalloproteases (MMP9, MMP2). Moreover, lactate increased calcium deposit and soluble osteogenic molecular signals (i.e. osteoprotegerin, osteonectin). These results also were obtained after incubation with PMF sera and were reverted after AZD3965 treatment. Since it has been demonstrated the role of cancer-associated fibroblast (CAF) in cancer development through ECM remodeling, we next evaluated the expression of CAF markers in MSCs after lactate exposure. α-SMA, FAP1, and TGFβ were found upregulated and this effect was reverted by AZD3965. Finally, we further confirmed these results in a Zebrafish animal model of PMF. Interestingly, in Zebrafish model we observed a reduction of MCT4 expression that was confirmed in the PBMNCs of the PMF patients. Conclusions. Inhibition of lactate metabolism may represent a strategy to inhibit cancer cells and contribute to restoring the anti-cancer immune response. Therefore, lactate metabolism may represent a promising target to counteract inflammation, osteosclerosis, and fibrosis in PMF patients.

Recent Advances in Surface Plasmon Resonance (SPR) Technology for Detecting Ovarian Cancer Biomarkers

Epithelial Ovarian Cancer (EOC) is a leading cause of cancer-related deaths among women, mainly due to a lack of early detection and screening methods. Advanced immunoassay techniques, such as Luminex and proximity extension assay (PEA) technology, show promise in improving EOC detection by utilizing highly sensitive and specific multiplex panels to detect multiple combinations of biomarkers. However, these advanced immunoassay techniques have certain limitations, especially in validating the performance characteristics such as specificity, sensitivity, limit of detection (LOD), and dynamic range for each EOC biomarker within the panel. Implementing multiplexing in point-of-care (POC) biosensors can enhance EOC biomarker detection, with Surface Plasmon Resonance (SPR) being a versatile option among optical biosensors. There is no study on multiplex SPR biosensors specifically tailored for diagnosing EOC. Recent studies have shown promising results in the single detection of EOC biomarkers using SPR, with LOD for cancer antigen 125 (CA125) at 0.01 U/mL−1 and human epididymis protein 4 (HE4) at 1pM. This study proposes a potential roadmap for scientists and engineers in academia and industry to develop a cost effective yet highly efficient SPR biosensor platform for detecting EOC.

In Situ Protein Microarray for Identifying the Geminivirus-Arabidopsis Interactome.

The protein-protein interactions (PPI) by protein array technology complement other PPI assay technologies such as AP-MS and Y2H. The in situ protein array technology (NAPPA) enables low-cost, rapid, and comprehensive protein detection. It allows standardized and simultaneous assay of a wide range of proteins with a broad range of expression in cells. This technology facilitates the detection of protein-protein interactions within species and between heterologous species such as host-microbe. Here, we described the technique that identified a syntaxin-6 protein-mediated begomovirus infection using an array containing 4600 Arabidopsis genes. The protein microarray assay also identified several other viral protein-host protein interactions.

Adaptation of RiPCA for the Live-Cell Detection of mRNA-Protein Interactions.

RNA-binding proteins (RBPs) act as essential regulators of cell fate decisions, through their ability to bind and regulate the activity of cellular RNAs. For protein-coding mRNAs, RBPs control the localization, stability, degradation, and ultimately translation of mRNAs to impact gene expression. Disruption of the vast network of mRNA-protein interactions has been implicated in many human diseases, and accordingly, targeting these interactions has surfaced as a new frontier in RNA-targeted drug discovery. To catalyze this new field, methods are needed to enable the detection and subsequent screening of mRNA-RBP interactions, particularly in live cells. Using our laboratory's RNA-interaction with Protein-mediated Complementation Assay (RiPCA) technology, herein we describe its application to mRNA-protein interactions and present a guide for the development of future RiPCA assays for structurally diverse classes of mRNA-protein interactions.

High-Throughput Plasma Proteomics to Define the Precursor Multiple Myeloma Proteome and Identify Candidate High-Risk Disease Biomarkers of Progression

Introduction Multiple Myeloma (MM) develops from well-defined precursors Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM), where patients remain stable or may unknowingly rapidly progress. Bone marrow (BM) biopsies are not routine for precursor disease management, and precursor patients are limited to monitoring few proteins within peripheral blood (PB) for signs of progressive disease. Deep proteome profiling of PB circulating proteins may help track disease; however, the dynamic range of the plasma proteome has limited the depth of detection for MS-based proteomics without first depleting abundant proteins and fractionating the samples after digestion to peptides. Technological advancements in multiplex immunoassays with low cross-reactivity and off-target events have enabled plasma profiling for disease stage classification, defining high-risk disease features, and novel therapeutic target discovery. Here, we perform the first comprehensive plasma proteomic profiling study on patients across the MM disease continuum and longitudinal sequential samples from progressive and stable disease. Methods We carried out high-throughput plasma proteomic profiling for approximately 3000 proteins simultaneously using the Olink® Explore 3072 library and Proximity Extension Assay (PEA) technology. Targeted proteins are recognized by multiplexed, matched pairs of antibodies labelled with unique DNA oligonucleotides that upon binding come into proximity, hybridize, and are extended to generate a unique sequence for protein identification with NextGen DNA sequencing. We profiled 423 PB plasma samples from 348 individuals, including MGUS (n=67), SMM (n=179), MM (n=44), and healthy controls (n=58). Sequential samples from patients with progressive disease (n=27) and patients with stable disease with matched clinical follow-up time were also profiled. Precursor defined samples from progressors ranged 1.03-5.88 yrs prior to diagnosis and were untreated in the precursor setting, while MM disease samples were also collected prior to any active disease therapy. Patients had a median clinical follow-up time of 7.05 years. T-tests, ANOVAs, and a linear mixed effect (LME) model were used to identify proteins that change across disease stages, progression status, and time. Results were adjusted for multiple testing using the Benjamini-Hochberg Method. Results We identified 751 significantly dysregulated proteins with the majority upregulated in progressive disease. We captured circulating levels of proteins highly expressed on the surface of plasma cells, including CD38, SDC1, BCMA and SLAMF7, highlighting the utility of PEA technology to monitor clinically-relevant targets for which monoclonal antibodies, antibody-drug conjugates, CAR-T and BiTE therapies are being developed. We identified proteins that significantly distinguished MGUS, SMM, and NDMM from healthy donors (n=222, 423 and 494), where increasing B-cell maturation antigen (BCMA) was a significant classifier and positive control. Consistent with previous findings, baseline BCMA levels were also significantly elevated in SMM-MM progressors vs. SMM non-progressors, further supporting the potential utility of BCMA measurements during routine blood tests of precursor MM patients. Proinflammatory cytokines were also identified, including IL1, IL5, IL6, IL16, and IL18, known to create a BM environment that promotes malignant cell development by suppressing the microenvironment, promoting cellular adhesion, or increasing angiogenesis. Four novel proteins that are vital for calcium homeostasis and integrin-mediated cell adhesion significantly increased from healthy to MM and were also significantly elevated in SMM progressors vs. SMM non-progressors, nominating these proteins as candidate biomarkers of high-risk disease. Conclusion We performed the most comprehensive plasma proteomics study to date, which characterized disease stage proteomes and identified candidate high-risk disease biomarkers in longitudinal progressor samples. Further advancements are underway to validate the accuracy levels of the novel candidates, test the performance of a classification model that recognizes disease stage-specific proteins, and determine how best to integrate proteins into current MM risk stratification models.

Autocrine regulation of tumor cell repopulation by Hsp70-HMGB1 alarmin complex

BackgroundCancer recurrence is regulated by a variety of factors, among which is the material of dying tumor cells; it is suggested that remaining after anti-cancer therapy tumor cells receive a signal from proteins called damage-associated molecular patterns (DAMPs), one of which is heat shock protein 70 (Hsp70).MethodsTwo models of tumor repopulation were employed, based on minimal population of cancer cells and application of conditioned medium (CM). To deplete the CMs of Hsp70 affinity chromatography on ATP-agarose and immunoprecipitation were used. Cell proliferation and the dynamics of cell growth were measured using MTT assay and xCELLigence technology; cell growth markers were estimated using qPCR and with the aid of ELISA for prostaglandin E detection. Immunoprecipitation followed by mass-spectrometry was employed to identify Hsp70-binding proteins and protein-protein interaction assays were developed to reveal the above protein complexes.ResultsIt was found that CM of dying tumor cells contains tumor regrowth-initiating factors and the removal of one of them, Hsp70, caused a reduction in the relapse-activating capacity. The pull out of Hsp70 alone using ATP-agarose had no effect on repopulation, while the immunodepletion of Hsp70 dramatically reduced its repopulation activity. Using proteomic and immunochemical approaches, we showed that Hsp70 in conditioned medium binds and binds another abundant alarmin, the High Mobility Group B1 (HMGB1) protein; the complex is formed in tumor cells treated with anti-cancer drugs, persists in the cytosol and is further released from dying tumor cells. Recurrence-activating power of Hsp70-HMGB1 complex was proved by the enhanced expression of proliferation markers, Ki67, Aurka and MCM-10 as well as by increase of prostaglandin E production and autophagy activation. Accordingly, dissociating the complex with Hsp70 chaperone inhibitors significantly inhibited the pro-growth effects of the above complex, in both in vitro and in vivo tumor relapse models.ConclusionsThese data led us to suggest that the abundance of the Hsp70-HMGB1 complex in the extracellular matrix may serve as a novel marker of relapse state in cancer patients, while specific targeting of the complex may be promising in the treatment of cancers with a high risk of recurrence.

Novel single nucleotide polymorphism (rs1600485907) of IL-41 gene associated with systemic lupus erythematous

This study evaluated the serum concentrations of IL-41 and the detection of specific single nucleotide polymorphisms (SNPs) (rs1600485907, rs762398841, and rs575275512) within the IL-41 gene in female subjects diagnosed with systemic lupus erythematosus (SLE). The study sample comprised 124 female patients who had been diagnosed with SLE, with an equivalent number of healthy control volunteers. The levels of serum were determined using the application of an enzyme-linked immunosorbent assay (ELISA) technology, while SNPs were determined by conventional polymerase chain reaction (PCR), and subsequent Sanger sequencing. The results revealed that the mean age of the healthy control group was 31.64 years, whereas the mean age of the group diagnosed with SLE was 33.66 years. The findings of the present investigation indicate that the individuals under examination demonstrated an average disease duration of 9.0 years, whereas the average SLEDAI-2k score for those diagnosed with SLE was 11.0. The concentrations of ESR, CRP, urea, creatinine, C3, and C4 in individuals diagnosed with SLE showed a statistically significant elevation in comparison to the control cohort. The analysis of patients’ anti-nuclear antibodies (ANA) revealed that 89% of individuals have ANA, whereas 95.61% display anti-dsDNA. Significantly elevated levels of serum IL-41 were seen in patients diagnosed with SLE in comparison to healthy controls. A receiver operating characteristic (ROC) analysis was performed on a cohort of patients diagnosed with SLE to assess the diagnostic efficacy of IL-41 in discriminating between SLE patients and non-afflicted persons. The study determined that the specificity of IL-41 was 82.26%, representing the percentage of accurate negative outcomes. In contrast, the sensitivity of IL-41 was found to be 84.68%, indicating the percentage of accurate positive outcomes. The computed value for the area under the curve (AUC) was found to be 0.937. The statistical analysis revealed a significant connection between the existence of both heterozygote and homozygote mutant genotypes of IL-41 (rs1600485907) and an increased vulnerability to the formation of SLE. In conclusion, it has been shown that individuals diagnosed with SLE demonstrate heightened concentrations of IL-41in their circulating blood plasma. Furthermore, a particular genetic variation, specifically a single nucleotide polymorphism (SNP) denoted as rs1600485907 located within the IL-41 gene, has been recognized as a potential susceptibility factor for the onset of this disorder.

Monitoring longitudinal immunological responses to bluetongue virus 17 in experimentally infected sheep

Bluetongue virus (BTV) is an economically important pathogen of ruminant species with worldwide prevalence. While many BTV infections are asymptomatic, animals with symptomatic presentation deteriorate quickly with the sickest succumbing to disease within one week. Animals that survive the infection often require months to recover. The immune response to BTV infection is thought to play a central role in controlling the disease. Key to understanding BTV disease is profiling vertebrate host immunological cellular and cytokine responses. Studies to characterize immune responses in ruminants have been limited by a lack of species-specific reagents and assay technology. Here we assess the longitudinal immunological response to experimental BTV-17-California (CA) infection in sheep using the most up to date assays. We infected a cohort of sheep with BTV-17-CA and longitudinally monitored each animal for clinical disease, viremia and specific immunological parameters (B cells, T cells, monocytes) by RT-qPCR, traditional flow cytometry and/or fluorescent based antibody arrays. BTV-inoculated sheep exhibited clinical signs characteristic of bluetongue virus disease. Circulating virus was demonstrated after 8 days post inoculation (DPI) and remained detectable for the remainder of the time course (24 DPI). A distinct lymphopenia was observed between 7 and 14 DPI that rebounded to mock-inoculated control levels at 17 DPI. In addition, we observed increased expression of pro-inflammatory cytokines after 8 DPI. Taken together, we have established a model of BTV infection in sheep and have successfully monitored the longitudinal vertebrate host immunological response and viral infection progression using a combination of traditional methods and cutting-edge technology.

B-276 Preservation of Proteomic Inflammatory Biomarkers signal in a Saliva Multiomic Buffer at Room Temperature

Abstract Background Saliva represents an attractive biospecimen as it is pain-free, simple for at home collections, and patients can provide multiple samples over time to provide time resolved data. However, few diagnostic tests have been developed using saliva due to poor stability and comparatively low concentration of analytes. Stabilization of biomarkers at ambient temperature in saliva would enable labs to take advantage of the benefits of saliva sample collections, while minimizing the risks posed by the degradation of already scarce analytes. Methods Matched samples of neat serum, saliva, and saliva collected with Saliva Multi-omic Device (SMD) preservative were collected from 30 volunteers (15 male & 15 female) ages 18–64 with no known morbidities. The Olink Inflammation panel was used to examine 92 inflammation-related proteins. The method utilized proximity extension assay technology, wherein antibody probe pairs reacted with their respective target proteins to generate a polymerase chain reaction target sequence through proximity-dependent DNA polymerization. This sequence was measured using real-time polymerase chain reaction and normalized in two steps, producing a semi-quantitative Normalized Protein eXpression (NPX) unit. To evaluate analyte stability specimens were measured initially and then again after 24 h at room temperature (23 ± 2°C). Results Protein expression (NPX) units for 36 of the 92 inflammatory markers had higher concentrations in saliva compared to serum. Serum (78/92) and saliva 78% (72/92) showed similar coverage for the number of markers in the panel where at least 75% of subjects had values exceeding the limit of detection (LoD). SMD preservation buffer improved the average stability of inflammatory protein biomarkers by 19.6% compared to neat saliva and 10.2% compared to serum over 24-hours at room temperature. However, the relative stability of individual between protein markers [DV1] [HM2] varied. For proteins in SMD buffer, initial concentrations of analytes were highly predictive [HM3] of values 24 h later, having Pearson correlation coefficients > 0.7, for 23 of the analytes and greater than 0.5 for 47. The change in protein marker in serum over time was only moderately predictive of the protein’s stability in saliva with or without preservative, having correlations of 0.33 ± 0.20 and 0.47 ± 0.19 (mean ± standard deviation), respectively. Conclusion This study demonstrates potential utility of saliva in proteomic applications utilizing the Olink platform. The SMD preservation buffer stabilized inflammatory biomarkers in saliva samples likely through protease inhibition and antimicrobial action. Preservation of self-collection salivary samples by subjects without cold storage enables noninvasive sampling and reliable measurements even when samples are collected by users at remote locations or under challenging conditions.

Angiogenesis inhibitor or aggressiveness marker? The function of endostatin in cancer through electrochemical biosensing

This work reports the first electrochemical bioplatform developed for the determination of human endostatin (HE), a biomarker with recognized antiangiogenic potential whose elevated circulating levels have also been associated with the development of aggressive cancers. The developed electroanalytical biotool combines the benefits of using magnetic microparticles for the implementation of sandwich immunoassays and amperometric transduction on disposable carbon electrodes. A limit of detection (LOD) of 34.1 pg mL−1 for HE standards and a selectivity suitable for its foray into the clinical oncology area, are demonstrated. The determination of HE in clinical samples such as lysates and secretomes of colorectal cancer (CRC) cells, plasma, and tissue samples from patients with CRC in different stages, has been faced with satisfactory results showing the ability for discriminating the metastatic capabilities of cells and for identifying and staging CRC patients. The developed bioplatform allows precise quantitative determinations, requiring minimal pre-treatments and sample amounts in only 75 min. In addition, due to the instrumentation and the type of substrates used in the detection step, the biotool is compatible with implementation in multiplexed and/or point-of-need devices, features in which this bioplatform is advantageous with respect to the enzyme linked immunosorbent assay (ELISA) or immunoblotting technologies.

Multiplex genotyping of SNPs in genomic DNA via hydrogel-based assay mediated with MutS and polyethylene glycol

The simultaneous genotyping of multiple single nucleotide polymorphisms (SNPs) in genomic DNA derived from organisms holds significant potential for applications such as precision medicine and food product authentication. However, conventional assay technologies including qPCR-based techniques, microarrays, and hydrogel-based assays face limitations in efficient multiplexing of SNPs, particularly for large-size DNA beyond kilobase scales, due to constraints in multiplex capability, specificity, or sensitivity. In this study, a hydrogel-based multiplex SNP genotyping platform specifically designed for genomic DNA is presented. This platform integrates the ligation detection reaction (LDR) and rolling circle amplification (RCA) techniques within a hydrogel-based multiplex sensing system, enabling adaptable and sensitive SNP genotyping for genomic DNA. To enhance the specificity of the assay, MutS protein and polyethylene glycol are introduced into the protocol, reducing the non-specific ligation and RCA reactions synergistically. With significant specificity improvement of over 10-fold, three types of SNPs within an artificially constructed ∼1000 bp double-stranded DNA (dsDNA) are successfully genotyped with double-digit picomolar sensitivity. Furthermore, the practical applicability of the developed process for the origin identification of raw materials is demonstrated by genotyping three types of SNPs within genomic DNA obtained from two closely related plant species, Korean ginseng (Panax ginseng) and American ginseng (Panax quinquefolius), containing ca. 3.5 gigabase genome size. Of notable significance, this study marks the premiere achievement in PCR-free multiplex genotyping of SNPs in genomic DNA using a single fluorophore.

UBL3 Interaction with α-Synuclein Is Downregulated by Silencing MGST3.

Ubiquitin-like 3 (UBL3) is a membrane-anchored protein that plays a crucial role in sorting proteins into small extracellular vesicles. Aggregations of alpha-synuclein (α-syn) are associated with the pathology of neurodegenerative diseases such as Parkinson's disease. Recently, the interaction between UBL3 and α-syn was discovered, with potential implications in clearing excess α-syn from neurons and its role in disease spread. However, the regulator that can mediate the interaction between UBL3 and α-syn remains unclear. In this study, using the split gaussian luciferase complementation assay and RNA interference technology, we identified that QSOX2, HTATIP2, UBE3C, MGST3, NSF, HECTD1, SAE1, and ATG3 were involved in downregulating the interaction between UBL3 and α-syn. Notably, silencing MGST3 had the most significant impact. Immunocytochemistry staining confirmed the impact of MGST3 silencing on the co-localization of UBL3 and α-syn in cells. MGST3 is a part of the antioxidant system, and silencing MGST3 is believed to contribute to oxidative stress. We induced oxidative stress with hydrogen peroxide, observing its effect on the UBL3-α-syn interaction, and showing that 800 µM of H2O2 downregulated this interaction. In conclusion, silencing MGST3 downregulates the interaction between UBL3 and α-syn.

Reducing Lipid Panel Error Allowances to Improve the Accuracy of Cardiovascular Risk Stratification.

The standard lipid panel forms the backbone of atherosclerotic cardiovascular disease risk assessment. Suboptimal analytical performance, along with biological variability, could lead to erroneous risk assessment and management decisions. The current National Cholesterol Education Program (NCEP) performance recommendations have remained unchanged for almost 3 decades despite improvements in assay technology. We investigated the potential extent of risk misclassification when the current recommendations are met and explored the impact of improving analytical performance goals. We extracted lipid panel data for 8506 individuals from the NHANES database and used these to classify subjects into 4 risk groups as recommended by the 2018 US Multisociety guidelines. Analytical bias and imprecision, at the allowable limits, as well as biological variability, were introduced to the measured values to determine the impact on misclassification. Bias and imprecision were systematically reduced to determine the degree of improvement that may be achieved. Using the current performance recommendations, up to 10% of individuals were misclassified into a different risk group. Improving proportional bias by 1%, and fixing imprecision to 3% across all assays reduced misclassifications by up to 10%. The effect of biological variability can be reduced by taking the average of serial sample measurements. The current NCEP recommendations for analytical performance of lipid panel assays allow for an unacceptable degree of misclassification, leading to possible mismanagement of cardiovascular disease risk. Iteratively reducing allowable error can improve this.

Application of a Multiplex Platform to Identify Novel Biomarkers for Pregnancy Location and Viability.

Determining early pregnancy location and viability can be cumbersome, often requiring serial evaluations. This study aimed to identify novel biomarker candidates for pregnancy location and viability using a pseudodiscovery high-throughput technique. This was a case-control study among patients presenting for early pregnancy assessment, including ectopic pregnancies, early pregnancy losses, and viable intrauterine pregnancies. For pregnancy location, ectopic pregnancy was considered "case" and non-ectopic considered "control." For pregnancy viability, viable intrauterine pregnancy was considered "case" and early pregnancy loss + ectopic pregnancy were considered "control." Using Proximity Extension Assay technology from Olink Proteomics, serum levels of 1012 proteins were compared separately for pregnancy location and viability. Receiver operator characteristic curves were generated to determine a biomarker's discriminative abilities. Analysis included 13 ectopic pregnancies, 76 early pregnancy losses, and 27 viable intrauterine pregnancies. For pregnancy location, 18 markers had an area under the curve (AUC) ≥0.80, with three being expressed more in ectopic compared to non-ectopic pregnancies: thyrotropin subunit beta, carbonic anhydrase 3, and DEAD (Asp-Glu-Ala-Asp) box polypeptide 58. For pregnancy viability, two markers had an AUC ≥0.80: lutropin subunit beta and serpin B8. While some of the markers had previously beenimplicated in early pregnancy physiology, others were from pathways not previously explored. Using a high-throughput platform, a large number of proteins were screened as potential biomarkers for pregnancy location and viability, and twenty candidate biomarkers were identified. Further exploration of these proteins may facilitate validation as diagnostic tools for establishing early pregnancy diagnoses.

A Comparative Study on the Lysosomal Cation Channel TMEM175 Using Automated Whole-Cell Patch-Clamp, Lysosomal Patch-Clamp, and Solid Supported Membrane-Based Electrophysiology: Functional Characterization and High-Throughput Screening Assay Development.

The lysosomal cation channel TMEM175 is a Parkinson's disease-related protein and a promising drug target. Unlike whole-cell automated patch-clamp (APC), lysosomal patch-clamp (LPC) facilitates physiological conditions, but is not yet suitable for high-throughput screening (HTS) applications. Here, we apply solid supported membrane-based electrophysiology (SSME), which enables both direct access to lysosomes and high-throughput electrophysiological recordings. In SSME, ion translocation mediated by TMEM175 is stimulated using a concentration gradient at a resting potential of 0 mV. The concentration-dependent K+ response exhibited an I/c curve with two distinct slopes, indicating the existence of two conducting states. We measured H+ fluxes with a permeability ratio of PH/PK = 48,500, which matches literature findings from patch-clamp studies, validating the SSME approach. Additionally, TMEM175 displayed a high pH dependence. Decreasing cytosolic pH inhibited both K+ and H+ conductivity of TMEM175. Conversely, lysosomal pH and pH gradients did not have major effects on TMEM175. Finally, we developed HTS assays for drug screening and evaluated tool compounds (4-AP, Zn as inhibitors; DCPIB, arachidonic acid, SC-79 as enhancers) using SSME and APC. Additionally, we recorded EC50 data for eight blinded TMEM175 enhancers and compared the results across all three assay technologies, including LPC, discussing their advantages and disadvantages.

Aptamers Targeting the PD-1/PD-L1 Axis: A Perspective.

Aptamers have emerged in recent years as alternatives to antibodies or small molecules to interfere with the immune check points by blocking the PD-1/PD-L1 interactions and represent an interesting perspective for immuno-oncology. Aptamers are RNA or DNA nucleotides able to bind to a target with high affinity, with the target ranging from small molecules to proteins and up to cells. Aptamers are identified by the SELEX method that can be modified for specific purposes. The range of applications of aptamers covers therapy as well as new alternative assay technologies similar to ELISA. Aptamers' limited plasma stability can be managed using delivery strategies. The goal of this Perspective is to give an overview of the current development of aptamers targeting the most studied immune checkpoint modulators, PD-1 and PD-L1, and analogous strategies with aptamers for other immuno-related targets.

Effects of Short- And Medium-Term Exposures to Lower Air Temperature on 71 Novel Biomarkers of Subclinical Inflammation: Results from the KORA F4 Study.

Increasing evidence has revealed that exposure to low temperatures is linked to a higher risk of chronic diseases and death; however, the mechanisms underlying the observed associations are still poorly understood. We performed a cross-sectional analysis with 1115 participants from the population-based KORA F4 study, which was conducted in Augsburg, Germany, from 2006 to 2008. Seventy-one inflammation-related protein biomarkers were analyzed in serum using proximity extension assay technology. We employed generalized additive models to explore short- and medium-term effects of air temperature on biomarkers of subclinical inflammation at cumulative lags of 0-1 days, 2-6 days, 0-13 days, 0-27 days, and 0-55 days. We found that short- and medium-term exposures to lower air temperature were associated with higher levels in 64 biomarkers of subclinical inflammation, such as Protein S100-A12 (EN-RAGE), Interleukin-6 (IL-6), Interleukin-10 (IL-10), C-C motif chemokine 28 (CCL28), and Neurotrophin-3 (NT-3). More pronounced associations between lower air temperature and higher biomarker of subclinical inflammation were observed among older participants, people with cardiovascular disease or prediabetes/diabetes, and people exposed to higher levels of air pollution (PM2.5, NO2, and O3). Our findings provide intriguing insight into how low air temperature may cause adverse health effects by activating inflammatory pathways.

Paraneoplastic or not? Sirtuin-2 in anti-N-methyl-D-aspartate receptor encephalitis.

NMDAR and LGI1 encephalitis are important types of autoimmune encephalitis (AE) with significant morbidity. In this study, we used a proteomic approach in search for novel clinically relevant biomarkers in these types of encephalitides. Swedish and Czech tertiary neuroimmunology centers collaborated in this retrospective exploratory study. Fifty-eight cerebrospinal fluid samples of 28 patients with AE (14 definite NMDAR, 14 with definite LGI1 encephalitis) and 30 controls were included. CSF samples were analyzed using proximity extension assay technology (Olink® Target 96 Inflammation panel). For each CSF sample, 92 proteins were measured. Clinical variables were retrospectively collected and correlations with protein levels were statistically analyzed. Patients and controls differed significantly in the following 18 biomarkers: TNFRSF9, TNFRSF12, TNFRSF14, TNF-ß, TNF-α, IL7, IL10, IL-12B, IFN-γ, CD5, CD6, CASP-8, MMP-1, CXCL-8, CXCL-10, CXCL-11, IL-20RA, Sirtuin-2 (SIRT2). In LGI1 encephalitis, no clinically useful association was found between biomarkers and clinical variables. In the NMDAR encephalitis group, SIRT2, TNF-ß and CD5 were significantly associated with ovarian teratoma. For SIRT2, this was true even for the first patients' CSF sample (SIRT2 without vs. with tumor [mean ± SD]: 2.2 ± 0.29 vs. 2.88 ± 0.48; p=0.007, 95% CI [-1.15, -0.22]; point-biserial correlation: rpb=0.66, p=0.011). SIRT2 was positively correlated with age (rpb=0.39, p=0.018) and total hospital days (r=0.55, p=<0.001). Sirtuin-2 should be investigated as a biomarker of paraneoplastic etiology in NMDAR encephalitis.

Comparison and Research Progress of Protein Detection Technology for Cultural Relic Materials

The analysis of historical materials is an essential component of cultural heritage conservation. Protein was one of the most important and pervasive organic substances in ancient human societies. Through the qualitative and quantitative examination of protein-based materials, it is possible to clarify their source and functions of substances in cultural heritages, investigate the manufacturing technology of cultural heritage, and identify their deterioration mechanism. On the basis of these analyses, corresponding measurements are therefore feasible. Currently, mass spectrometry, chromatography, spectroscopy, nuclear magnetic, proteomics, and immunoassay are used to analyse protein materials. Proteomics techniques and enzyme-linked immunosorbent assay (ELISA) technology are two of the most common methods for detecting ancient proteins. This article discusses the evolution of protein component detection in ancient materials, as well as the implementation of proteomics and ELISA techniques for the analysis of proteins. In addition, the characteristics of these two techniques were contrasted in order to propose the most recent analytical techniques and the direction of future research.

Recent progress on the detection of animal-derived food stimulants using mass spectrometry-based techniques.

The misuse of animal-derived stimulants in food is becoming increasingly common, and mass spectrometry (MS) is used extensively for their detection and analysis. There is a growing demand for abused-substances detection, highlighting the need for systematic studies on the advantages of MS-based methods in detecting animal-derived stimulants. We reviewed the application of chromatography-mass spectrometry to the screening and detection of food stimulants of animal origin. Specifically, we analyzed four common animal sources of synthetic steroids, β-receptor agonists, zearalenol (ZAL), and glucocorticoids. We also explored the potential of using chromatography-mass spectrometry to detect and analyze animal-derived foods. We searched and screened the Web of Science and Google Scholar databases until April 2023. Our inclusion criteria included a publication year within the last 5 years, publication language of English, and the research fields of food analysis, environmental chemistry, and polymer science. Our keywords were "mass spectrometry," "anabolic androgenic steroids," "β-2agonists," "glucocorticoids," "zearalenone," and "doping." Although traditional techniques such as thin-layer chromatography and enzyme-linked immunoassays are simple, fast, and suitable for the initial screening of bulk products, they are limited by their relatively high detection limits. Among the methods based on MS, gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry are the most widely used for detecting food doping agents of animal origin. However, a sensitive method with high repeatability and a short analysis time for a large number of samples is still required. Advances in MS have enabled the detection of extremely low concentrations of these substances. Combining different techniques, such as high-resolution mass spectrometry, ultra-high performance liquid chromatography-tandem mass spectrometry, gas chromatography-combustion-isotope ratio mass spectrometry, ultra-high performance liquid chromatography-high resolution mass spectrometry, and two-dimensional chromatography, offers significant advantages for detecting trace illicit drugs in animal-derived foods. Due to advances in assay technology and sample preparation methods, sample collection and storage methods such as dried blood spots, dried urine spots, and volumetric absorptive microsampling are increasingly accepted because of their increased stability and cost-effectiveness. MS significantly improves the efficiency of detecting doping agents of animal origin. With the continuous development of MS technology, its application in the fields of doping detection and the analysis of doping agents of animal origin is expected to become more extensive.