Multiplexed Profiling of Extracellular Vesicles for Biomarker Development

Extracellular vesicles (EVs) are cell-derived membranous particles that play a crucial role in molecular trafficking, intercellular transport and the egress of unwanted proteins. They have been implicated in many diseases including cancer and neurodegeneration. EVs are detected in all bodily fluids, and their protein and nucleic acid content offers a means of assessing the status of the cells from which they originated. As such, they provide opportunities in biomarker discovery for diagnosis, prognosis or the stratification of diseases as well as an objective monitoring of therapies. The simultaneous assaying of multiple EV-derived markers will be required for an impactful practical application, and multiplexing platforms have evolved with the potential to achieve this. Herein, we provide a comprehensive overview of the currently available multiplexing platforms for EV analysis, with a primary focus on miniaturized and integrated devices that offer potential step changes in analytical power, throughput and consistency.

Viral Production in Seawater Filtered Through 0.2-μm Pore-Size Filters: A Hidden Biogeochemical Cycle in a Neglected Realm

Viral production is a key parameter for assessing virus-mediated biogeochemical cycles. One widely used method for the determination of viral production, called the virus reduction assay, reduces viral abundance, while maintaining bacterial abundance, using 0.2-μm pore-size filters. Viral production is estimated from the increase of viral abundance during incubation. We hypothesized that small-cell-sized bacterial communities can pass through 0.2-μm filters and drive viral production, representing a missing fraction of viral production that is missed by the virus reduction assay. Coastal seawater was filtered through 0.2-μm filters and diluted with virus-free seawater. Viral production in the <0.2-μm filtrate was estimated from changes in viral abundance determined through flow cytometry. We found that viruses were produced in the <0.2-μm communities, which were strongly enriched with low nucleic acid content bacteria. Estimated viral production in the <0.2-μm filtrates accounted for up to 43% of total viral production and 10% of dissolved organic carbon production mediated by viral lysis of bacterial cells. By not considering viral production in these <0.2-μm communities, the virus reduction assay may underestimate viral production. Virus–bacteria interactions in <0.2-μm communities may represent a significant and overlooked role of viruses in marine food webs and carbon fluxes.

Picoplankton Diel Variability and Estimated Growth Rates in Epipelagic and Mesopelagic Waters of the Central Red Sea

The diel variability of the abundance and cell size of picoplanktonic groups in the central Red Sea was monitored every 2 h in situ on 4 occasions (once per season) from 2015 to 2016. We distinguished Prochlorococcus, low (LF-Syn) and high (HF-Syn) fluorescence Synechococcus, small (Speuk) and large (Lpeuk) picoeukaryotes and two groups of heterotrophic prokaryotes of low (LNA) and high (HNA) nucleic acid content. The diel variability in abundance was less marked than in cell size and more apparent in autotrophs than heterotrophs. Specific growth rates were estimated by an empirical relationship from measurements obtained in bottle incubations of surface and deep samples collected in the winter compared with in situ variations in cell size over 24 h. Autotrophic picoplankton groups generally grew faster (0.23–0.77 d–1) than heterotrophic prokaryotes (0.12–0.50 d–1). Surface to 100 m depth-weighted specific growth rates displayed a clear seasonal pattern for Prochlorococcus, with maxima in winter (0.77 ± 0.07 d–1) and minima in fall (0.52 ± 0.07 d–1). The two groups of Synechococcus peaked in spring, with slightly higher growth rates of LF-Syn (0.57 ± 0.04 d–1) than HF-Syn (0.43 ± 0.04 d–1). Speuk and Lpeuk showed different seasonal patterns, with lower values of the former (0.27 ± 0.02 and 0.37 ± 0.04 d–1, respectively). HNA consistently outgrew LNA heterotrophic prokaryotes, with a higher growth in the epipelagic (0–200 m, 0.36 ± 0.03 d–1) than in the mesopelagic (200–700 m, 0.26 ± 0.03 d–1), while no differences were found for LNA cells (0.19 ± 0.03 d–1 and 0.17 ± 0.02 d–1, respectively). With all data pooled, the mean diel abundances of autotrophic picoplankton in the upper epipelagic and of HNA cells in the epipelagic and mesopelagic layers were significantly correlated with the specific growth rates estimated from cell size variations. Our high-resolution sampling dataset suggests that changes in growth rates underlie the noticeable seasonality of picoplankton recently described in these tropical waters.

Phage-Mediated Explosive Cell Lysis Induces the Formation of a Different Type of O-IMV in Shewanella vesiculosa M7T

Shewanella vesiculosa M7T is a cold-adapted Antarctic bacterium that has a great capacity to secrete membrane vesicles (MVs), making it a potentially excellent model for studying the vesiculation process. S. vesiculosa M7T undergoes a blebbing mechanism to produce different types of MVs, including outer membrane vesicles and outer-inner membrane vesicles (O-IMVs). More recently, other mechanisms have been considered that could lead to the formation of O-IMVs derived from prophage-mediated explosive cell lysis in other bacteria, but it is not clear if they are of the same type. The bacterial growth phase could also have a great impact on the type of MVs, although there are few studies on the subject. In this study, we used high-resolution flow cytometry, transmission electron microscopy, and cryo-electron microscopy (Cryo-EM) analysis to determine the amount and types of MVs S. vesiculosa M7T secreted during different growth phases. We show that MV secretion increases during the transition from the late exponential to the stationary phase. Moreover, prophage-mediated explosive cell lysis is activated in S. vesiculosa M7T, increasing the heterogeneity of both single- and double-layer MVs. The sequenced DNA fragments from the MVs covered the entire genome, confirming this explosive cell lysis mechanism. A different structure and biogenesis mechanisms for the explosive cell lysis-derived double-layered MVs was observed, and we propose to name them explosive O-IMVs, distinguishing them from the blebbing O-IMVs; their separation is a first step to elucidate their different functions. In our study, we used for the first time sorting by flow cytometry and Cryo-EM analyses to isolate bacterial MVs based on their nucleic acid content. Further improvements and implementation of bacterial MV separation techniques is essential to develop more in-depth knowledge of MVs.

“Every cell counts”—experiences with flow cytometry for Austrian drinking water supply

The last decades have seen extensive scientific and technological improvements in many fields of microbiology and molecular biology. Correspondingly, flow cytometry—a rapid, precise and straightforward method for cultivation-independent detection of cells in liquids—has been a major topic in aquatic microbiology and drinking water analysis. Flow cytometry provides information at the single-cell level, including total cell counts, size measurements, nucleic acid content and bacterial viability and activity. While regulatory requirements for water testing rely on cultivation-based methods, flow cytometry can be considered a powerful tool to complement standard procedures.This article provides insights into the methodology and applicability of flow cytometry in the field of microbiological drinking water analysis and presents an overview on several case studies that cover a broad range of different objectives. The later are comprised of a study on flow cytometric characterization of Austrian drinking water resources, of an example for advanced data analysis methods of flow cytometric data, of a study on monitoring microbial regrowth within the distribution network, of an exemplary case of the application of online flow cytometry for high-frequency monitoring and of an introduction to the combination of flow cytometry and sequencing information.Finally, it is argued that due to the high microbiological variability of different water resources, unusual changes of flow cytometric parameters, rather than specific limits, could act as an indicator for further investigation. In this way flow cytometry can provide a good basis for risk assessments in water safety plans. The application of flow cytometry still remains utility-specific and a huge need for standardization of data analysis and interpretation exists in order to achieve a better cooperation of water utilities.

Tracking of the biochemical changes upon pleomorphic adenoma progression using vibrational microspectroscopy

Head and neck tumors can be very challenging to treat because of the risk of problems or complications after surgery. Therefore, prompt and accurate diagnosis is extremely important to drive appropriate treatment decisions, which may reduce the chance of recurrence. This paper presents the original research exploring the feasibility of Fourier transform infrared (FT-IR) and Raman spectroscopy (RS) methods to investigate biochemical alterations upon the development of the pleomorphic adenoma. Principal component analysis (PCA) was used for a detailed assessment of the observed changes and to determine the spectroscopic basis for salivary gland neoplastic pathogenesis. It is implied that within the healthy margin, as opposed to the tumoral tissue, there are parts that differ significantly in lipid content. This observation shed new light on the crucial role of lipids in tissue physiology and tumorigenesis. Thus, a novel approach that eliminates the influence of lipids on the elucidation of biochemical changes is proposed. The performed analysis suggests that the highly heterogeneous healthy margin contains more unsaturated triacylglycerols, while the tumoral section is rich in proteins. The difference in protein content was also observed for these two tissue types, i.e. the healthy tissue possesses more proteins in the anti-parallel β-sheet conformation, whereas the tumoral tissue is dominated by proteins rich in unordered random coils. Furthermore, the pathogenic tissue shows a higher content of carbohydrates and reveals noticeable differences in nucleic acid content. Finally, FT-IR and Raman spectroscopy methods were proposed as very promising methods in the discrimination of tumoral and healthy tissues of the salivary gland.

Impact of Curcumin on Nucleic Acids in Diethanolamine Induced Toxicity in Mus musculus

Tremendous hike in the use of chemicals in every consumables worldwide, led researchers to investigate the effects of various products and their ingredients that are being used in routine. Diethanolamine (DEA) is one such organic compound used in various industries and several personal care products that are being used daily. To evaluate DEA toxicity on nucleic acid content, Swiss strain male albino mice were chosen as animal model for in vivo experiments. Mice were exposed to DEA (110, 165, 330 mg/kg body weight/day) for 30 days. Animals were sorted into nine different groups, each containing 10 animals per group. In untreated control groups of animals no significant changes in nucleic acid content were noted whereas in DEA exposed animals, nucleic acid content decreased significantly (p<0.05) in a dose-dependent manner. However, more decrease were noted in high dose (330 mg/kg body weight/day) exposed animals as compared to other groups of animals. For the mitigation of the toxicity generated by DEA, curcumin, a miraculous antioxidant, which is an active component of turmeric was used. Curcumin (10, 20, 30 mg/kg body weight/day) was orally administered for 30 days along with the high dose of DEA. After completion of treatment, animals were humanly sacrificed and liver was quickly isolated for further biochemical evaluations. In animals exposed to curcumin for 30 days the nucleic acid content increased significantly (p<0.05) as compared to DEA-HD treated groups of animals. The effect was dose-dependent. The ameliorative effect of curcumin might be due to its high antioxidant potency.

Cell Population Data and Serum Polyclonal Immunoglobulin Free Light Chains in the Assessment of COVID-19 Severity

Distinguishing between severe and nonsevere COVID-19 to ensure adequate healthcare quality and efficiency is a challenge for the healthcare system. The aim of this study was to assess the usefulness of CBC parameters together with analysis of FLC serum concentration in risk stratification of COVID-19. Materials and methods: CBC was analyzed in 735 COVID ICU, COVID non-ICU, and non-COVID ICU cases. FLC concentration was analyzed in 133 of them. Results: COVID ICU had neutrophils and lymphocytes with the greatest size, granularity, and nucleic acid content. Significant differences in concentrations of κ and λ FLCs were shown between COVID ICU and COVID non-ICU. However, no difference was found in the κ/λ ratio between these groups, and the ratio stayed within the reference value, which indicates the presence of polyclonal FLCs. FLC κ measurement has significant power to distinguish between severe COVID-19 and nonsevere COVID-19 (AUC = 0.7669), with a sensitivity of 86.67% and specificity of 93.33%. The κ coefficients’ odds ratio of 3.0401 was estimated. Conclusion: It can be concluded that the results obtained from the measure of free light immunoglobulin concentration in serum are useful in distinguishing between severe and nonsevere COVID-19.

Platelets and tumor-associated RNA transfer

Until recently the nucleic acid content of platelets was considered to be fully determined by their progenitor megakaryocyte. However, it is now well understood that additional mediators (e.g. cancer cells) can intervene, thereby influencing the RNA repertoire of platelets. Platelets are highly dynamic cells, able to communicate and influence their environment. For instance, platelets have been involved in various steps of cancer development and progression by supporting tumor growth, survival and dissemination. Cancer cells can directly and/or indirectly influence the platelet RNA content, resulting in tumor-mediated 'education' of platelets. Alterations in the tumor-educated platelet (TEP) RNA profile have been described as a novel source of potential biomarkers. Individual platelet RNA biomarkers as well as complex RNA signatures may be used for early-detection of cancer and treatment monitoring. Here we review the RNA transfer occurring between cancer cells and platelets. We explore the potential use of platelet RNA biomarkers as a liquid biopsy biosource, and discuss methods to evaluate the transcriptomic content of platelets.