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.

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.

Biodegradable biliary stents are promising treatments for biliary benign stenoses. One of the materials considered for their production is polydioxanone (PPDX), which could exhibit a suitable degradation time for use in biodegradable stents. Proper material degradation characteristics, such as sufficient stiffness and disintegration resistance maintained for a clinically relevant period, are necessary to ensure stent safety and efficacy. The hydrolytic degradation of commercially available polydioxanone biliary stents (ELLA-CS, Hradec Králové, Czech Republic) in phosphate-buffered saline (PBS) was studied. During 9 weeks of degradation, structural, physical, and surface changes were monitored using Raman spectroscopy, differential scanning calorimetry, scanning electron microscopy, and tensile and torsion tests. It was found that the changes in mechanical properties are related to the increase in the ratio of amorphous to crystalline phase, the so-called amorphicity. Monitoring the amorphicity using Raman spectroscopy has proven to be an appropriate method to assess polydioxanone biliary stent degradation. At the 1732 cm⁻¹ Raman peak, the normalized shoulder area is less than 9 cm⁻¹ which indicates stent disintegration. The stent disintegration started after 9 weeks of degradation in PBS, which agrees with previous in vitro studies on polydioxanone materials as well as with in vivo studies on polydioxanone biliary stents.

Polydioxanone (PPDX), as an FDA approved polymer in tissue engineering, is an important component of some promising medical devices, e.g., biodegradable stents. The hydrolytic degradation of polydioxanone stents plays a key role in the safety and efficacy of treatment. A new fast and convenient method to quantitatively evaluate the hydrolytic degradation of PPDX stent material was developed. PPDX esophageal stents were degraded in phosphate-buffered saline for 24 weeks. For the first time, the changes in Raman spectra during PPDX biodegradation have been investigated here. The level of PPDX hydrolytic degradation was determined from the Raman spectra by calculating the area under the 1732 cm⁻¹ peak shoulder. Raman spectroscopy, unlike Fourier transform infrared (FT-IR) spectroscopy, is also sensitive enough to monitor the decrease in the dye content in the stents during the degradation. Observation by a scanning electron microscope showed gradually growing cracks, eventually leading to the stent disintegration. The material crystallinity was increasing during the first 16 weeks, suggesting preferential degradation of the amorphous phase. Our results show a new easy and reliable way to evaluate the progression of PPDX hydrolytic degradation. The proposed approach can be useful for further studies on the behavior of PPDX materials, and for clinical practice.

The selection of the best embryo for embryo transfer (ET) is one of the most important steps in IVF (in vitro fertilisation) treatment. Preimplantation genetic testing (PGT) is an invasive method that can greatly facilitate the decision about the best embryo. An alternative way to select the embryo with the greatest implantation potential is by cultivation in a time-lapse system, which can offer several predictive factors. Non-invasive time-lapse monitoring can be used to select quality embryos with high implantation potential under stable culture conditions. The embryo for ET can then be selected based on the determined morphokinetic parameters and morphological features, which according to our results predict a higher implantation potential. This study included a total of 1027 morphologically high-quality embryos (552 normal and 475 abnormal PGT-tested embryos) from 296 patients (01/2016–06/2021). All embryos were cultivated in a time-lapse incubator and PGT biopsy of trophectoderm cells on D5 or D6 was performed. Significant differences were found in the morphological parameters cc2, t5 and tSB and the occurrence of multinucleations in the stage of two-cell and four-cell embryos between the group of genetically normal embryos and abnormal embryos. At the same time, significant differences in the morphological parameters cc2, t5 and tSB and the occurrence of multinucleations in the two-cell and four-cell embryo stage were found between the group of genetically normal embryos that led to clinical pregnancy after ET and the group of abnormal embryos. From the morphokinetic data found in the PGT-A group of normal embryos leading to clinical pregnancy, time intervals were determined based on statistical analysis, which should predict embryos with high implantation potential. Out of a total of 218 euploid embryos, which were transferred into the uterus after thawing (single frozen embryo transfer), clinical pregnancy was confirmed in 119 embryos (54.6%). Our results show that according to the morphokinetic parameters (cc2, t5, tSB) and the occurrence of multinucleations during the first two cell divisions, the best euploid embryo for ET can be selected with high probability.

Coxiella burnetii is the causative agent of the zoonotic disease Q fever. To date, the lipopolysaccharide (LPS) is the only defined and characterized virulence determinant of C. burnetii. In this study, proteome profiles of C. burnetii Nine Mile phase I (RSA 493, NMI) and its isogenic Nine Mile phase II (RSA 439 NMII) isolate with a deep rough LPS were compared on L-929 mouse fibroblasts and in complex (ACCM-2), and defined (ACCM-D) media. Whole proteome extracts were analyzed using a label-free quantification approach. Between 659 and 1,046 C. burnetii proteins of the 2,132 annotated coding sequences (CDS) were identified in any particular experiment. Proteome profiles clustered according to the cultivation conditions used, indicating different regulation patterns. NMI proteome profiles compared to NMII in ACCM-D indicate transition from an exponential to a stationary phase. The levels of regulatory proteins such as RpoS, CsrA2, UspA1, and UspA2 were increased. Comparison of the oxidative stress response of NMI and NMII indicated that ACCM-2 represents a high oxidative stress environment. Expression of peroxidases, superoxide dismutases, as well as thioredoxins was increased for NMI. In contrast, in ACCM-D, only osmoregulation seems to be necessary. Proteome profiles of NMII do not differ and indicate that both axenic media represent similar oxidative stress environments. Deep rough LPS causes changes of the outer membrane stability and fluidity. This might be one reason for the observed differences. Proteins associated with the T4SS and Sec translocon as well as several effector proteins were detectable under all three conditions. Interestingly, none of these putatively secreted proteins are upregulated in ACCM-2 compared to ACCM-D, and L-929 mouse fibroblasts. Curiously, a higher similarity of proteomic patterns (overlapping up- and downregulated proteins) of ACCM-D and bacteria grown in cell culture was observed. Particularly, the proteins involved in a better adaptation or homeostasis in response to the harsh environment of the parasitophorous vacuole were demonstrated for NMI. This semi-quantitative proteomic analysis of C. burnetii compared axenically grown bacteria to those propagated in cell culture.