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Small molecule biomarker discovery: Proposed workflow for LC-MS-based clinical research projects
(2023)
Mass spectrometry focusing on small endogenous molecules has become an integral part of biomarker discovery in the pursuit of an in-depth understanding of the pathophysiology of various diseases, ultimately enabling the application of personalized medicine. While LC-MS methods allow researchers to gather vast amounts of data from hundreds or thousands of samples, the successful execution of a study as part of clinical research also requires knowledge transfer with clinicians, involvement of data scientists, and interactions with various stakeholders.
The initial planning phase of a clinical research project involves specifying the scope and design, and engaging relevant experts from different fields. Enrolling subjects and designing trials rely largely on the overall objective of the study and epidemiological considerations, while proper pre-analytical sample handling has immediate implications on the quality of analytical data. Subsequent LC-MS measurements may be conducted in a targeted, semi-targeted, or non-targeted manner, resulting in datasets of varying size and accuracy. Data processing further enhances the quality of data and is a prerequisite for in-silico analysis. Nowadays, the evaluation of such complex datasets relies on a mix of classical statistics and machine learning applications, in combination with other tools, such as pathway analysis and gene set enrichment. Finally, results must be validated before biomarkers can be used as prognostic or diagnostic decision-making tools. Throughout the study, quality control measures should be employed to enhance the reliability of data and increase confidence in the results.
The aim of this graphical review is to provide an overview of the steps to be taken when conducting an LC-MS-based clinical research project to search for small molecule biomarkers.
Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1β. We demonstrate here that the stable ATP analog adenosine 5′-O-(thiotriphosphate) (ATPγS) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5′-promoter region showed weak responses to ATPγS but confered a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3′-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATPγS potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3′-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATPγS. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATPγS, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1β-inducibility. By confocal microscopy we demonstrate that ATPγS increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability.
IL-22 is an immunoregulatory cytokine displaying pathological functions in models of autoimmunity like experimental psoriasis. Understanding molecular mechanisms driving IL-22, together with knowledge on the capacity of current immunosuppressive drugs to target this process, may open an avenue to novel therapeutic options. Here, we sought to characterize regulation of human IL22 gene expression with focus on the established model of Jurkat T cells. Moreover, effects of the prototypic immunosuppressant cyclosporin A (CsA) were investigated. We report that IL-22 induction by TPA/A23187 (T/A) or αCD3 is inhibited by CsA or related FK506. Similar data were obtained with peripheral blood mononuclear cells or purified CD3(+) T cells. IL22 promoter analysis (-1074 to +156 bp) revealed a role of an NF-AT (-95/-91 nt) and a CREB (-194/-190 nt) binding site for gene induction. Indeed, binding of CREB and NF-ATc2, but not c-Rel, under the influence of T/A to those elements could be proven by ChIP. Because CsA has the capability to impair IκB kinase (IKK) complex activation, the IKKα/β inhibitor IKKVII was evaluated. IKKVII likewise reduced IL-22 induction in Jurkat cells and peripheral blood mononuclear cells. Interestingly, transfection of Jurkat cells with siRNA directed against IKKα impaired IL22 gene expression. Data presented suggest that NF-AT, CREB, and IKKα contribute to rapid IL22 gene induction. In particular the crucial role of NF-AT detected herein may form the basis of direct action of CsA on IL-22 expression by T cells, which may contribute to therapeutic efficacy of the drug in autoimmunity.
Background: The transcription factor T-bet is pivotal for initiation of Th1-related immunoactivation. Identification of novel genes directly regulated by T-bet is crucial.
Results: Genome-wide analysis and subsequent experiments revealed that T-bet up-regulates IL-36γ/IL-1F9 in myeloid cells.
Conclusion: IL-1-related IL-36γ is a direct T-bet target in myeloid cells.
Significance: Observations suggest that IL-36γ , besides IFNγ, contributes to T-bet functions in immunopathology
By concerted action in dendritic (DC) and T cells, T-box expressed in T cells (T-bet, Tbx21) is pivotal for initiation and perpetuation of Th1 immunity. Identification of novel T-bet-regulated genes is crucial for further understanding the biology of this transcription factor. By combining siRNA technology with genome-wide mRNA expression analysis, we sought to identify new T-bet-regulated genes in predendritic KG1 cells activated by IL-18. One gene robustly dependent on T-bet was IL-36γ, a recently described novel IL-1 family member. Promoter analysis revealed a T-bet binding site that, along with a κB site, enables efficient IL-36γ induction. Using knock-out animals, IL-36γ reliance on T-bet was extended to murine DC. IL-36γ expression by human myeloid cells was confirmed using monocyte-derived DC and M1 macrophages. The latter model was employed to substantiate dependence of IL-36γ on endogenous T-bet in human primary cells. Ectopic expression of T-bet likewise mediated IL-36γ production in HaCaT keratinocytes that otherwise lack this transcription factor. Additional experiments furthermore revealed that mature IL-36γ has the capability to establish an inflammatory gene expression profile in human primary keratinocytes that displays enhanced mRNA levels for TNFα, CCL20, S100A7, inducible NOS, and IL-36γ itself. Data presented herein shed further light on involvement of T-bet in innate immunity and suggest that IL-36γ, besides IFNγ, may contribute to functions of this transcription factor in immunopathology.
Introduction: Arachidonoyl ethanolamide (AEA) and 2-arachidonoyl glycerol (2-AG) are central lipid mediators of the endocannabinoid system. They are highly relevant due to their involvement in a wide variety of inflammatory, metabolic or malign diseases. Further elucidation of their modes of action and use as biomarkers in an easily accessible matrix, like blood, is restricted by their susceptibility to deviations during blood sampling and physiological co-dependences, which results in high variability of reported concentrations in low ng/mL ranges.
Objectives: The objective of this review is the identification of critical parameters during the pre-analytical phase and proposal of minimum requirements for reliable determination of endocannabinoids (ECs) in blood samples.
Methods: Reported physiological processes influencing the EC concentrations were put into context with published pre-analytical research and stability data from bioanalytical method validation.
Results: The cause for variability in EC concentrations is versatile. In part, they are caused by inter-individual factors like sex, metabolic status and/or diurnal changes. Nevertheless, enzymatic activity in freshly drawn blood samples is the main reason for changing concentrations of AEA and 2-AG, besides additional non-enzymatic isomerization of the latter.
Conclusion: Blood samples for EC analyses require immediate processing at low temperatures (>0 °C) to maintain sample integrity. Standardization of the respective blood tube or anti-coagulant, sampling time point, applied centrifugal force and complete processing time can further decrease variability caused by sample handling. Nevertheless, extensive characterization of study participants is needed to reduce distortion of clinical data caused by co-variables and facilitate research on the endocannabinoid system.
Biological functions of the small leucine-rich proteoglycans: from genetics to signal transduction
(2008)
The small leucine-rich proteoglycan (SLRP) family has significantly expanded in the past decade to now encompass five discrete classes, grouped by common structural and functional properties. Some of these gene products are not classical proteoglycans, whereas others have new and unique features. In addition to being structural proteins, SLRPs constitute a network of signal regulation: being mostly extracellular, they are upstream of multiple signaling cascades. They affect intracellular phosphorylation, a major conduit of information for cellular responses, and modulate distinct pathways, including those driven by bone morphogenetic protein/transforming growth factor β superfamily members, receptor tyrosine kinases such as ErbB family members and the insulin-like growth factor I receptor, and Toll-like receptors. The wealth of mechanistic insights into the molecular and cellular functions of SLRPs has revealed both the sophistication of this family of regulatory proteins and the challenges that remain in uncovering the totality of their functions. This review is focused on novel biological functions of SLRPs with special emphasis on their protein cores, newly described genetic diseases, and signaling events in which SLRPs play key functions.
The transient receptor potential (TRP) ankyrin type 1 (TRPA1) channel is highly expressed in a subset of sensory neurons where it acts as an essential detector of painful stimuli. However, the mechanisms that control the activity of sensory neurons upon TRPA1 activation remain poorly understood. Here, using in situ hybridization and immunostaining, we found TRPA1 to be extensively co-localized with the potassium channel Slack (KNa1.1, Slo2.2, or Kcnt1) in sensory neurons. Mice lacking Slack globally (Slack−/−) or conditionally in sensory neurons (SNS-Slack−/−) demonstrated increased pain behavior after intraplantar injection of the TRPA1 activator allyl isothiocyanate. By contrast, pain behavior induced by the TRP vanilloid 1 (TRPV1) activator capsaicin was normal in Slack-deficient mice. Patch-clamp recordings in sensory neurons and in a HEK cell line transfected with TRPA1 and Slack revealed that Slack-dependent potassium currents (IKS) are modulated in a TRPA1-dependent manner. Taken together, our findings highlight Slack as a modulator of TRPA1-mediated, but not TRPV1-mediated, activation of sensory neurons.
Keywords: TRPA1; slack; dorsal root ganglia; pain; mice
The SARS-CoV-2 pandemic has challenged researchers at a global scale. The scientific community’s massive response has resulted in a flood of experiments, analyses, hypotheses, and publications, especially in the field of drug repurposing. However, many of the proposed therapeutic compounds obtained from SARS-CoV-2 specific assays are not in agreement and thus demonstrate the need for a singular source of COVID-19 related information from which a rational selection of drug repurposing candidates can be made. In this paper, we present the COVID-19 PHARMACOME, a comprehensive drug-target-mechanism graph generated from a compilation of 10 separate disease maps and sources of experimental data focused on SARS-CoV-2 / COVID-19 pathophysiology. By applying our systematic approach, we were able to predict the synergistic effect of specific drug pairs, such as Remdesivir and Thioguanosine or Nelfinavir and Raloxifene, on SARS-CoV-2 infection. Experimental validation of our results demonstrate that our graph can be used to not only explore the involved mechanistic pathways, but also to identify novel combinations of drug repurposing candidates.
Type 1 diabetes (T1D) is mainly precipitated by the destruction of insulin-producing β-cells in the pancreatic islets of Langerhans by autoaggressive T cells. The etiology of the disease is still not clear, but besides genetic predisposition the exposure to environmental triggers seems to play a major role. Virus infection of islets has been demonstrated in biopsies of T1D patients, but there is still no firm proof that such an infection indeed results in islet-specific autoimmunity. However, virus infection results in a local inflammation with expression of inflammatory factors, such as cytokines and chemokines that attract and activate immune cells, including potential autoreactive T cells. Many chemokines have been found to be elevated in the serum and expressed by islet cells of T1D patients. In mouse models, it has been demonstrated that β-cells express chemokines involved in the initial recruitment of immune cells to the islets. The bulk load of chemokines is however released by the infiltrating immune cells that also express multiple chemokine receptors. The result is a mutual attraction of antigen-presenting cells and effector immune cells in the local islet microenvironment. Although there is a considerable redundancy within the chemokine ligand-receptor network, a few chemokines, such as CXCL10, seem to play a key role in the T1D pathogenesis. Studies with neutralizing antibodies and investigations in chemokine-deficient mice demonstrated that interfering with certain chemokine ligand-receptor axes might also ameliorate human T1D. However, one important aspect of such a treatment is the time of administration. Blockade of the recruitment of immune cells to the site of autoimmune destruction might not be effective when the disease process is already ongoing. By that time, autoaggressive cells have already arrived in the islet microenvironment and a blockade of migration might even hold them in place leading to accelerated destruction. Thus, an anti-chemokine therapy makes most sense in situations where the cells have not yet migrated to the islets. Such situations include treatment of patients at risk already carrying islet-antigen autoantibodies but are not yet diabetic, islet transplantation recipients, and patients that have undergone a T cell reset as occurring after anti-CD3 antibody treatment.
Sphingosine‐1‐phosphate lyase 1 (S1P lyase or SGPL1) is an essential sphingosine‐1‐phosphate‐degrading enzyme. Its manipulation favors onset and progression of colorectal cancer and others in vivo. Thus, SGPL1 is an important modulator of cancer initiation. However, in established cancer, the impact of retrospective SGPL1 modulation is elusive. Herein, we analyzed how SGPL1 siRNA affects malignancy of the human colorectal cancer cells DLD‐1 and found that in parallel to the reduction of SGPL1 expression levels, migration, invasion, and differentiation status changed. Diminished SGPL1 expression was accompanied with reduced cell migration and cell invasion in scratch assays and transwell assays, whereas metabolic activity and proliferation was not altered. Decreased migration was attended by increased cell–cell‐adhesion through upregulation of E‐cadherin and formation of cadherin‐actin complexes. Spreading cell islets showed lower vimentin abundance in border cells. Furthermore, SGPL1 siRNA treatment induced expression of epithelial cell differentiation markers, such as intestinal alkaline phosphatase and cytokeratin 20. Hence, interference with SGPL1 expression augmented a partial redifferentiation of colorectal cancer cells toward normal colon epithelial cells. Our investigation showed that SGPL1 siRNA influenced tumorigenic activity of established colorectal cancer cells. We therefore suggest SGPL1 as a target for lowering malignant potential of already existing cancer.