Search Results (20)

This review focuses on the pivotal role of radiotracers in breast cancer imaging, emphasizing their importance in accurate detection, staging, and treatment monitoring. Radiotracers, labeled with radioactive isotopes, are integral to various nuclear imaging techniques, including positron emission tomography (PET) and positron emission mammography (PEM). The most widely used radiotracer in breast cancer imaging is ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG), which highlights areas of increased glucose metabolism, a hallmark of many cancer cells. This allows for the identification of primary tumors and metastatic sites and the assessment of tumor response to therapy. In addition to ¹⁸F-FDG, this review will explore newer radiotracers targeting specific receptors, such as estrogen receptors or HER2, which offer more personalized imaging options. These tracers provide valuable insights into the molecular characteristics of tumors, aiding in tailored treatment strategies. By integrating radiotracers into breast cancer management, clinicians can enhance early disease detection, monitor therapeutic efficacy, and guide interventions, ultimately improving patient outcomes. Ongoing research aimed at developing more specific and sensitive tracers will also be highlighted, underscoring their potential to advance precision medicine in breast cancer care. Full article

Cellular metabolomics provides insights into the metabolic processes occurring within cells and can help researchers understand how these processes are regulated and how they relate to cellular function, health, and disease. In this technical note, we investigated the effects of solvent evaporation equipment and storage condition on high-coverage cellular metabolomics. We previously introduced a robust CIL LC-MS-based cellular metabolomics workflow that encompasses various steps, including cell harvest, metabolic quenching, cell lysis, metabolite extraction, differential chemical isotope labeling, and LC-MS analysis. This workflow has consistently served as the cornerstone of our collaborative research and service projects. As a core facility catering to users with diverse research needs and financial resources, we have encountered scenarios requiring short-term sample storage. For example, the need often arises to transport samples at room temperature from user sites to our core facility. Herein, we present a study in which we compared different solvent evaporation methods (specifically, the nitrogen blowdown evaporator, SpeedVac concentrator, and lyophilizer) and diverse storage conditions (including dried samples stored in a freezer, samples stored in a freezer with methanol, dried samples stored at room temperature, and samples stored at room temperature with methanol). Our findings indicate that the choice of solvent evaporation equipment did not significantly impact the cellular metabolome. However, we observed a noteworthy change in the metabolome after 7 days of storage when cells were stored with methanol, regardless of whether they were kept at −80 °C or room temperature, in contrast to cells that were dried and frozen. Importantly, we detected no significant alterations in cells that were dried and stored at room temperature. In conclusion, to ensure the production of high-quality CIL LC-MS metabolomics results, we strongly recommend that, in situations where low-temperature storage is not feasible, cell samples should be thoroughly dried before storage or shipment at room temperature. Full article

The p38 members of the mitogen-activated protein kinases (MAPKs) family mediate various cellular responses to stress conditions, inflammatory signals, and differentiation factors. They are constitutively active in chronic inflammatory diseases and some cancers. The differences between their transient effects in response to signals and the chronic effect in diseases are not known. The family is composed of four isoforms, of which p38α seems to be abnormally activated in diseases. p38α and p38β are almost identical in sequence, structure, and biochemical and pharmacological properties, and the specific unique effects of each of them, if any, have not yet been revealed. This study aimed to reveal the specific effects induced by p38α and p38β, both when transiently activated in response to stress and when chronically active. This was achieved via large-scale proteomics and phosphoproteomics analyses using stable isotope labeling of two experimental systems: one, mouse embryonic fibroblasts (MEFs) deficient in each of these p38 kinases and harboring either an empty vector or vectors expressing p38α^WT, p38β^WT, or intrinsically active variants of these MAPKs; second, induction of transient stress by exposure of MEFs, p38α^−/−, and p38β^−/− MEFs to anisomycin. Significant differences in the repertoire of the proteome and phosphoproteome between cells expressing active p38α and p38β suggest distinct roles for each kinase. Interestingly, in both cases, the constitutive activation induced adaptations of the cells to the chronic activity so that known substrates of p38 were downregulated. Within the dramatic effect of p38s on the proteome and phosphoproteome, some interesting affected phosphorylation sites were those found in cancer-associated p53 and Hspb1 (HSP27) proteins and in cytoskeleton-associated proteins. Among these, was the stronger direct phosphorylation by p38α of p53-Ser309, which was validated on the Ser315 in human p53. In summary, this study sheds new light on the differences between chronic and transient p38α and p38β signaling and on the specific targets of these two kinases. Full article

Monoclonal antibodies (mAbs) are highly efficacious therapeutics; however, due to their large, dynamic nature, structural perturbations and regional modifications are often difficult to study. Moreover, the homodimeric, symmetrical nature of mAbs makes it difficult to elucidate which heavy chain (HC)-light chain (LC) pairs are responsible for any structural changes, stability concerns, and/or site-specific modifications. Isotopic labeling is an attractive means for selectively incorporating atoms with known mass differences to enable identification/monitoring using techniques such as mass spectrometry (MS) and nuclear magnetic resonance (NMR). However, the isotopic incorporation of atoms into proteins is typically incomplete. Here we present a strategy for incorporating ¹³C-labeling of half antibodies using an Escherichia coli fermentation system. Unlike previous attempts to generate isotopically labeled mAbs, we provide an industry-relevant, high cell density process that yielded >99% ¹³C-incorporation using ¹³C-glucose and ¹³C-celtone. The isotopic incorporation was performed on a half antibody designed with knob-into-hole technology to enable assembly with its native (naturally abundant) counterpart to generate a hybrid bispecific (BsAb) molecule. This work is intended to provide a framework for producing full-length antibodies, of which half are isotopically labeled, in order to study the individual HC-LC pairs. Full article

The rapid source identification and environmental risk assessment (ERA) of hundreds of chemicals of emerging concern (CECs) in river water represent a significant analytical challenge. Herein, a potential solution involving a rapid direct-injection liquid chromatography–tandem mass spectrometry method for the quantitative determination of 102 CECs (151 qualitatively) in river water is presented and applied across six rivers in Germany and Switzerland at high spatial resolution. The method required an injection volume of only 10 µL of filtered sample, with a runtime of 5.5 min including re-equilibration with >10 datapoints per peak per transition (mostly 2 per compound), and 36 stable isotope-labelled standards. Performance was excellent from the low ng/L to µg/L concentration level, with 260 injections possible in any 24 h period. The method was applied in three separate campaigns focusing on the ERA of rivers impacted by wastewater effluent discharges (1 urban area in the Basel city region with 4 rivers, as well as 1 semi-rural and 1 rural area, each focusing on 1 river). Between 25 and 40 compounds were quantified directly in each campaign, and in all cases small tributary rivers showed higher CEC concentrations (e.g., up to ~4000 ng/L in total in the R. Schwarzach, Bavaria, Germany). The source of selected CECs could also be identified and differentiated from other sources at pre- and post- wastewater treatment plant effluent discharge points, as well as the effect of dilution downstream, which occurred over very short distances in all cases. Lastly, ERA for 41 CECs was performed at specific impacted sites, with risk quotients (RQs) at 1 or more sites estimated as high risk (RQ > 10) for 1 pharmaceutical (diclofenac), medium risk (RQ of 1–10) for 3 CECs (carbamazepine, venlafaxine, and sulfamethoxazole), and low risk (RQ = 0.1–1.0) for 7 CECs (i.e., RQ > 0.1 for 11 CECs in total). The application of high-throughput methods like this could enable a better understanding of the risks of CECs, especially in low flow/volume tributary rivers at scale and with high resolution. Full article

Nanoparticles of Human Serum Albumin (NC) labelled with ^99mTc are widely used in Nuclear Medicine and represent the gold-standard for the intraoperative detection of the sentinel lymph node in many kinds of cancer, mainly breast cancer and melanoma. A significant amount of radionuclides can be incorporated into the HSA particle, due to the multiple binding sites, and HSA-based nanocolloid catabolism is a fast and easy process that results in innocuous degradation products. NCs labelled with different isotopes represent an interesting radiopharmaceutical for extending diagnostic accuracy and surgical outcome, but the knowledge of the chemical bond between NCs and isotopes has not been fully elucidated, including information on its strength and specificity. The aim of this study is to investigate and compare the physicochemical characteristics of the bond between NCs and ^99mTc and ⁶⁸Ga isotopes. Commercial kits of HSA-based nanocolloid particles (NanoAlbumon^®) were used. For this purpose, we have primarily studied the kinetic orders of NC radiolabelling. Langmuir isotherms and pH effect on radiolabelling were tested and the stability of the radiometal complex was verified through competition reactions carried out in presence of different ligands. The future goal of our research is the development of inexpensive and instant kits, easily labelled with a wide spectrum of diagnostic and therapeutic isotopes, thus facilitating the availability of versatile and multipurpose radiopharmaceuticals. Full article

Protein phosphorylation is a crucial post-translational modification that plays an important role in the regulation of cellular signaling processes. Site-specific quantitation of phosphorylation levels can help decipher the physiological functions of phosphorylation modifications under diverse physiological statuses. However, quantitative analysis of protein phosphorylation degrees is still a challenging task due to its dynamic nature and the lack of an internal standard simultaneously available for the samples differently prepared for various phosphorylation extents. In this study, stable-isotope dimethyl labeling coupled with phosphatase dephosphorylation (DM + deP) was tried to determine the site-specific degrees of phosphorylation in proteins. Firstly, quantitation accuracy of the (DM + deP) approach was confirmed using synthetic peptides of various simulated phosphorylation degrees. Afterwards, it was applied to evaluate the phosphorylation stoichiometry of milk caseins. The phosphorylation degree of Ser130 on α-S1-casein was also validated by absolute quantification with the corresponding synthetic phosphorylated and nonphosphorylated peptides under a selected reaction monitoring (SRM) mode. Moreover, this (DM + deP) method was used to detect the phosphorylation degree change of Ser82 on the Hsp27 protein of HepG2 cells caused by tert-butyl hydroperoxide (t-BHP) treatment. The results showed that the absolute phosphorylation degree obtained from the (DM + deP) approach was comparable with the relative quantitation resulting from stable-isotope dimethyl labeling coupled with TiO₂ enrichment. This study suggested that the (DM + deP) approach is promising for absolute quantification of site-specific degrees of phosphorylation in proteins, and it may provide more convincing information than the relative quantification method. Full article

The high-resolution structural study of huntingtin exon-1 (HttEx1) has long been hampered by its intrinsic properties. In addition to being prone to aggregate, HttEx1 contains low-complexity regions (LCRs) and is intrinsically disordered, ruling out several standard structural biology approaches. Here, we use a cell-free (CF) protein expression system to robustly and rapidly synthesize (sub-) pathological HttEx1. The open nature of the CF reaction allows the application of different isotopic labeling schemes, making HttEx1 amenable for nuclear magnetic resonance studies. While uniform and selective labeling facilitate the sequential assignment of HttEx1, combining CF expression with nonsense suppression allows the site-specific incorporation of a single labeled residue, making possible the detailed investigation of the LCRs. To optimize CF suppression yields, we analyze the expression and suppression kinetics, revealing that high concentrations of loaded suppressor tRNA have a negative impact on the final reaction yield. The optimized CF protein expression and suppression system is very versatile and well suited to produce challenging proteins with LCRs in order to enable the characterization of their structure and dynamics. Full article

Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are molecular imaging strategies that typically use radioactively labeled ligands to selectively visualize molecular targets. The nanomolar sensitivity of PET and SPECT combined with the high specificity and affinity of monoclonal antibodies have shown great potential in oncology imaging. Over the past decades a wide range of radio-isotopes have been developed into immuno-SPECT/PET imaging agents, made possible by novel conjugation strategies (e.g., site-specific labeling, click chemistry) and optimization and development of novel radiochemistry procedures. In addition, new strategies such as pretargeting and the use of antibody fragments have entered the field of immuno-PET/SPECT expanding the range of imaging applications. Non-invasive imaging techniques revealing tumor antigen biodistribution, expression and heterogeneity have the potential to contribute to disease diagnosis, therapy selection, patient stratification and therapy response prediction achieving personalized treatments for each patient and therefore assisting in clinical decision making. Full article

Chikungunya virus (CHIKV) and Zika virus (ZIKV) are emerging arboviruses that pose a worldwide threat to human health. Currently, neither vaccine nor antiviral treatment to control their infections is available. As the skin is a major viral entry site for arboviruses in the human host, we determined the global proteomic profile of CHIKV and ZIKV infections in human skin fibroblasts using stable isotope labelling by amino acids in cell culture (SILAC)-based mass spectrometry analysis. We show that the expressions of the interferon-stimulated proteins MX1, IFIT1, IFIT3 and ISG15, as well as expressions of defense response proteins DDX58, STAT1, OAS3, EIF2AK2, and SAMHD1 were significantly upregulated in these cells upon infection with either virus. Exogenous expression of IFITs proteins markedly inhibited CHIKV and ZIKV replication which, accordingly, was restored following the abrogation of IFIT1 or IFIT3. Overexpression of SAMHD1 in cutaneous cells or pretreatment of cells with the virus-like particles containing SAMHD1 restriction factor Vpx resulted in a strong increase or inhibition, respectively, in both CHIKV and ZIKV replication. Moreover, silencing of SAMHD1 by specific SAMHD1-siRNA resulted in a marked decrease in viral RNA levels. Together, these results suggest that IFITs are involved in the restriction of replication of CHIKV and ZIKV and provide, as yet unreported, evidence for a proviral role of SAMHD1 in arbovirus infection of human skin cells. Full article

Chikungunya virus (CHIKV) and Zika virus (ZIKV) are emerging arboviruses that pose a worldwide threat to human health. Currently, neither vaccine nor antiviral treatment to control their infections is available. As the skin is a major viral entry site for arboviruses in the human host, we determined the global proteomic profile of CHIKV and ZIKV infections in human skin fibroblasts using Stable Isotope Labelling by Amino acids in Cell culture (SILAC)-based mass-spectrometry analysis. We show that the expression of the interferon-stimulated proteins MX1, IFIT1, IFIT3 and ISG15, as well as expression of defense response proteins DDX58, STAT1, OAS3, EIF2AK2 and SAMHD1 was significantly up-regulated in these cells upon infection with either virus. Exogenous expression of IFITs proteins markedly inhibited CHIKV and ZIKV replication which, accordingly, was restored following the abrogation of IFIT1 or IFIT3. Overexpression of SAMHD1 in cutaneous cells, or pretreatment of cells with the virus-like particles containing SAMHD1 restriction factor Vpx, resulted in a strong increase or inhibition, respectively, of both CHIKV and ZIKV replication. Moreover, silencing of SAMHD1 by specific SAMHD1-siRNA resulted in a marked decrease of viral RNA levels. Together, these results suggest that IFITs are involved in the restriction of replication of CHIKV and ZIKV and provide, as yet unreported, evidence for a proviral role of SAMHD1 in arbovirus infection of human skin cells. Full article

Amyloid diseases, including neurodegenerative diseases such as Alzheimer’s and Parkinson’s, are linked to a poorly understood progression of protein misfolding and aggregation events that culminate in tissue-selective deposition and human pathology. Elucidation of the mechanistic details of protein aggregation and the structural features of the aggregates is critical for a comprehensive understanding of the mechanisms of protein oligomerization and fibrillization. Vibrational spectroscopies, such as Fourier transform infrared (FTIR) and Raman, are powerful tools that are sensitive to the secondary structure of proteins and have been widely used to investigate protein misfolding and aggregation. We address the application of the vibrational approaches in recent studies of conformational dynamics and structural characteristics of protein oligomers and amyloid fibrils. In particular, introduction of isotope labelled carbonyl into a peptide backbone, and incorporation of the extrinsic unnatural amino acids with vibrational moieties on the side chain, have greatly expanded the ability of vibrational spectroscopy to obtain site-specific structural and dynamic information. The applications of these methods in recent studies of protein aggregation are also reviewed. Full article

Inteins are self-splicing polypeptides with an ability to excise themselves from flanking host protein regions with remarkable precision; in the process, they ligate flanked host protein fragments. Inteins are distributed sporadically across all three domains of life (bacteria, archaea, and unicellular eukaryotes). However, their apparent localized distribution in DNA replication, repair, and recombination proteins (the 3Rs), particularly in bacteria and archaea, is enigmatic. Our understanding of the localized distribution of inteins in the 3Rs, and their possible regulatory role in such distribution, is still only partial. Nevertheless, understanding the chemistry of post-translational self-splicing of inteins has opened up opportunities for protein chemists to modify, manipulate, and bioengineer proteins. Protein-splicing technology is adapted to a wide range of applications, starting with untagged protein purification, site-specific protein labeling, protein biotinylation, isotope incorporation, peptide cyclization, as an antimicrobial target, and so on. This review is focused on the chemistry of splicing; the localized distribution of inteins, particularly in the 3Rs and their possible role in regulating host protein function; and finally, the use of protein-splicing technology in various protein engineering applications. Full article

A variety of nuclear magnetic resonance (NMR) applications have been developed for structure-based drug discovery (SBDD). NMR provides many advantages over other methods, such as the ability to directly observe chemical compounds and target biomolecules, and to be used for ligand-based and protein-based approaches. NMR can also provide important information about the interactions in a protein-ligand complex, such as structure, dynamics, and affinity, even when the interaction is too weak to be detected by ELISA or fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) or to be crystalized. In this study, we reviewed current NMR techniques. We focused on recent progress in NMR measurement and sample preparation techniques that have expanded the potential of NMR-based SBDD, such as fluorine NMR (¹⁹F-NMR) screening, structure modeling of weak complexes, and site-specific isotope labeling of challenging targets. Full article

MnO_x-CeO₂ mixed oxides are considered efficient oxidation catalysts superior to the corresponding single oxides. Although these oxides have been the subject of numerous studies, their fundamental performance indicators, such as turnover frequency (TOF) or specific activity, are scarcely reported. The purpose of the present work is to investigate the effect of catalyst composition on the concentration of active sites and intrinsic activity in ethanol oxidation by the employment of temperature-programmed desorption and oxidation of isotopically-labelled ethanol, ¹²CH₃¹³CH₂OH. The transformation pathways of preadsorbed ethanol in the absence of gaseous oxygen refer to dehydrogenation to acetaldehyde followed by its dissociation combined with oxidation by lattice oxygen. In the presence of gaseous oxygen, lattice oxygen is rapidly restored and the main products are acetaldehyde, CO₂, and water. CO₂ forms less easily on mixed oxides than on pure MnO_x. The TOF of ethanol oxidation has been calculated assuming that the amount of adsorbed ethanol and CO₂ produced during temperature-programmed oxidation (TPO) is a reliable indicator of the concentration of the active sites. Full article