Biological toxins are a heterogeneous group of compounds that share commonalities with biological and chemical agents. Among them, protein toxins represent a considerable, diverse set. They cover a broad range of molecular weights from less than 1000 Da to more than 150 kDa. This review aims to compare conventional detection methods of protein toxins such as in vitro bioassays with proteomic methods, including immunoassays and mass spectrometry-based techniques and their combination. Special emphasis is given to toxins falling into a group of selected agents, according to the Centers for Disease Control and Prevention, such as Staphylococcal enterotoxins, Bacillus anthracis toxins, Clostridium botulinum toxins, Clostridium perfringens epsilon toxin, ricin from Ricinus communis, Abrin from Abrus precatorius or control of trade in dual-use items in the European Union, including lesser known protein toxins such as Viscumin from Viscum album. The analysis of protein toxins and monitoring for biological threats, i.e., the deliberate spread of infectious microorganisms or toxins through water, food, or the air, requires rapid and reliable methods for the early identification of these agents.

Chronic myeloid leukemia (CML) is a malignant hematopoietic disorder distinguished by the presence of a BCR‑ABL1 fused oncogene with constitutive kinase activity. Targeted CML therapy by specific tyrosine kinase inhibitors (TKIs) leads to a marked improvement in the survival of the patients and their quality of life. However, the development of resistance to TKIs remains a critical issue for a subset of patients. The most common cause of resistance are numerous point mutations in the BCR‑ABL1 gene, followed by less common mutations and multiple mutation-independent mechanisms. Recently, exosomes, which are extracellular vesicles excreted from normal and tumor cells, have been associated with drug resistance and cancer progression. The aim of the present study was to characterize the exosomes released by imatinib‑resistant K562 (K562IR) cells. The K562IR‑derived exosomes were internalized by imatinib‑sensitive K562 cells, which thereby increased their survival in the presence of 2 µM imatinib. The exosomal cargo was subsequently analyzed to identify resistance‑associated markers using a deep label‑free quantification proteomic analysis. There were >3,000 exosomal proteins identified of which, 35 were found to be differentially expressed. From this, a total of 3, namely the membrane proteins, interferon‑induced transmembrane protein 3, CD146 and CD36, were markedly upregulated in the exosomes derived from the K562IR cells, and exhibited surface localization. The upregulation of these proteins was verified in the K562IR exosomes, and also in the K562IR cells. Using flow cytometric analysis, it was possible to further demonstrate the potential of CD146 as a cell surface marker associated with imatinib resistance in K562 cells. Taken together, these results suggested that exosomes and their respective candidate surface proteins could be potential diagnostic markers of TKI drug resistance in CML therapy.

Although smallpox has been known for centuries, the oldest available variola virus strains were isolated in the early 1940s. At that time, large regions of the world were already smallpox-free. Therefore, genetic information of these strains can represent only the very last fraction of a long evolutionary process. Based on the genomes of 48 strains, two clades are differentiated: Clade 1 includes variants of variola major, and clade 2 includes West African and variola minor (Alastrim) strains. Recently, the genome of an almost 400-year-old Lithuanian mummy was determined, which fell basal to all currently sequenced strains of variola virus on phylogenetic trees. Here, we determined two complete variola virus genomes from human tissues kept in a museum in Prague dating back 60 and 160 years, respectively. Moreover, mass spectrometry-based proteomic, chemical, and microscopic examinations were performed. The 60-year-old specimen was most likely an importation from India, a country with endemic smallpox at that time. The genome of the 160-year-old specimen is related to clade 2 West African and variola minor strains. This sequence likely represents a new endemic European variant of variola virus circulating in the midst of the 19th century in Europe.

Clostridium difficile is the causative agent of C. difficile infection (CDI) that could be manifested by diarrhea, pseudomembranous colitis or life-threatening toxic megacolon. The spread of certain strains represents a significant economic burden for health-care. The epidemic successful strains are also associated with severe clinical features of CDI. Therefore, a proteomic study has been conducted that comprises proteomes released from in vitro cultured panel of eight different PCR ribotypes (RTs) and employs the combination of shotgun proteomics and label-free quantification (LFQ) approach. The comparative semi-quantitative analyses enabled investigation of a total of 662 proteins. Both hierarchical clustering and principal component analysis (PCA) created eight distinctive groups. From these quantifiable proteins, 27 were significantly increased in functional annotations. Among them, several known factors connected with virulence were identified, such as toxin A, B, binary toxin, flagellar proteins, and proteins associated with Pro–Pro endopeptidase (PPEP-1) functional complex. Comparative analysis of protein expression showed a higher expression or unique expression of proteins linked to pathogenicity or iron metabolism in RTs 027 and 176 supporting their genetic relatedness and clinical importance at the proteomic level. Moreover, the absence of putative nitroreductase and the abundance of the Abc-type fe3+ transport system protein were observed as biomarkers for the RTs possessing binary toxin genes (027, 176 and 078). Higher expression of selected flagellar proteins clearly distinguished RTs 027, 176, 005 and 012, confirming the pathogenic role of the assembly in CDI. Finally, the histidine synthesis pathway regulating protein complex HisG/HisZ was observed only in isolates possessing the genes for toxin A and B. This study showed the applicability of the LFQ approach and provided the first semi-quantitative insight into the proteomes released from in vitro cultured panel of eight RTs. The observed differences pointed to a new direction for studies focused on the elucidation of the mechanisms underlining the CDI nature.