Building Minimized Epigenetic Clock by iPlex MassARRAY Platform.

Epigenetic clocks are valuable tools for estimating both chronological and biological age by assessing DNA methylation levels at specific CpG dinucleotides. While conventional epigenetic clocks rely on genome-wide methylation data, targeted approaches offer a more efficient alternative. In this study, we explored the feasibility of constructing a minimized epigenetic clock utilizing data acquired through the iPlex MassARRAY technology. The study enrolled a cohort of relatively healthy individuals, and their methylation levels of eight specific CpG dinucleotides in genes SLC12A5, LDB2, FIGN, ACSS3, FHL2, and EPHX3 were evaluated using the iPlex MassARRAY system and the Illumina EPIC array. The methylation level of five studied CpG sites demonstrated significant correlations with chronological age and an acceptable convergence of data obtained by the iPlex MassARRAY and Illumina EPIC array. At the same time, the methylation level of three CpG sites showed a weak relationship with age and exhibited a low concordance between the data obtained from the two technologies. The construction of the epigenetic clock involved the utilization of different machine-learning models, including linear models, deep neural networks (DNN), and gradient-boosted decision trees (GBDT). The results obtained from these models were compared with each other and with the outcomes generated by other well-established epigenetic clocks. In our study, the TabNet architecture (deep tabular data learning architecture) exhibited the best performance (best MAE = 5.99). Although our minimized epigenetic clock yielded slightly higher age prediction errors compared to other epigenetic clocks, it still represents a viable alternative to the genome-wide epigenotyping array.

Design of a Protocol for Soil-Transmitted Helminths (in Light of the Nematode Toxocara canis) DNA Extraction from Feces by Combining Commercially Available Solutions.

One of the main challenges for the mass introduction of the molecular diagnostics of soil-transmitted helminths (STHs) into clinical practice is the lack of a generally recognized effective method for isolating parasitic DNA from fecal samples. In the present study, we assessed the effects of various pretreatment procedures on the efficiency of removing PCR inhibitors and extracting Toxocara canis DNA from feces. We evaluated the effectiveness of four destructive methods (bead beating, the action of temperature-dependent enzymes, freeze-heat cycles, and incubation in a lysis buffer) on the integrity of T. canis eggs and the efficiency of DNA extraction. Also, we evaluated the effects of prewashes and the use of commercial concentrators on DNA extraction from fecal samples contaminated with T. canis eggs. A bead beating procedure was sufficient to destroy the T. canis eggs, while the effects of enzymes and freeze-heat cycles did not lead to a significant destruction of the eggs or the release of Toxocara DNA. Helminth DNA isolation protocols that do not include a bead beating step are not preferred. The preconcentration of STH eggs from feces using a commercial concentrator and subsequent washing can significantly increase the yield of DNA from STHs and reduce PCR inhibition.

Development and application of LAMP assays for the detection of enteric adenoviruses in feces.

Loop-mediated isothermal amplification (LAMP) is an alternative to PCR that is faster and requires fewer resources. Here, we describe two LAMP assays for the detection of human adenoviruses in the feces of children with acute intestinal infections. We designed сolorimetric LAMP (c-LAMP) and real-time LAMP (f-LAMP) with fluorescent probes to detect the DNA of the adenovirus F human adenovirus 40/41 (hAdV40/41) hexon gene. The detection limit of both developed methods was 103 copies/mL, which is comparable to the sensitivity of PCR. The specificities of both c-LAMP and f-LAMP were high, with no false-positive results for clinical samples that do not contain adenovirus F, when testing other viruses and microorganisms. Comparative tests of PCR and LAMP on clinical samples from patients with acute gastroenteritis were carried out. For all samples with a PCR threshold cycle (CT) of up to 36, the PCR and LAMP results completely coincided; however, at low viral loads, the diagnostic sensitivity of LAMP, especially c-LAMP with colorimetric detection, was inferior to that of PCR. The combination of LAMP with modern methods of nucleic acid extraction, both in manual and automatic modes, can reduce the time for a complete study, including extraction of nucleic acid material and amplification, to 60 min. IMPORTANCE In April 2022, several cases of acute hepatitis of unknown origin were reported in children from 12 countries. In many cases, enteric adenovirus or SARS-CoV-2 and adenovirus coinfection were detected. It is known that human adenoviruses can cause different infections of varying severity, from asymptomatic to severe cases with lethal outcomes. There is a need to increase the diagnostic capabilities of clinical laboratories to identify such an underestimated pathogen as adenovirus. Although PCR remains the gold standard for pathogen detection, this method requires specialized equipment and has a long turnaround time to process samples. Previously, LAMP assays for the detection of human adenovirus have been based on measuring the turbidity, the fluorescence of intercalated dyes, or electrophoretic separation. Herein, we present LAMP-based assays with colorimetric or fluorescent detection and perform a detailed assessment of their sensitivity, specificity, and diagnostic performance.

Mega-plasmid found worldwide confers multiple antimicrobial resistance in Salmonella Infantis of broiler origin in Russia.

Plasmids which are the mobile part of the bacterial genome can acquire and carry over genes conferring antimicrobial resistance, thus contributing to rapid adaptation of bacterial community to human-defined environment. In 2014, Israeli scientists have reported a large conjugative mega-plasmid pESI (plasmid for emerging S. Infantis) that provides multiple drug resistance (MDR) of Salmonella Infantis isolated from broilers. Later, very similar pESI-like plasmids have been found in Salmonella isolated from poultry in the United States, Italy, Switzerland, Hungary, and Japan. Here we report detection of pESI-like plasmids in Salmonella Infantis isolated from chicken food products in Russia. Whole genome sequencing of three MDR isolates revealed pESI-like plasmids in all three cases. These plasmids have such typical pESI features as a locus for siderophore yersiniabactin, a cluster of IncI1 conjugative genes, a cluster of type IV pilus genes, and three toxin-antitoxin modules. The pESI-like plasmids carry from two to five resistance genes in each isolate. In total, we observed six antimicrobial resistance genes associated with pESI-like plasmids (aadA1, blaCTX-M-14, dfrA14, sul1, tetA/tetR, tetM). Besides plasmid genes of antimicrobial resistance, all three MDR isolates of S. Infantis harbor a mutation in chromosomal gene gyrA (p.S83Y or p.D87Y) that is associated with resistance to fluoroquinolones. In addition, we performed a comparative bioinformatics meta-analysis of 25 pESI-like plasmids hosted by S. Infantis from the USA, Europe, Latin America, Israel, and Japan. This analysis identified a 173 kB sequence that is common for all pESI-like plasmids and carries virulence operons and toxin-antitoxin modules.

Design and evaluation of microfluidic devices for two-dimensional spatial separations.

Various designs of chips for comprehensive two-dimensional spatial liquid chromatography were investigated. The performance of these chips was initially evaluated using computational fluid dynamics (CFD). A bifurcating distributor with an angle of 140° between branches was implemented in order to achieve a homogeneous velocity field. The cross-sectional area of the channels of the flow distributor was fixed at 0.5 × 0.5 mm, which allows a robust micromilling technique to be used for chip manufacturing. Experiments were performed with chips featuring purposely introduced imperfections in the structure of the bifurcating flow distributor to study its capacity of overcoming potential local clogging. Split peaks were observed when 75% of one of the flow channels was obstructed, in line with the CFD predictions. The main bottlenecks for the performance of the spatial two-dimensional chips were identified, viz. sample injected in the first dimension diverging into the flow distributor and channel discretization (i.e., remixing of first-dimension separation peaks because of finite number of second-dimension channels). Solutions to the former problem were studied by applying a flow resistance in the vertical segments that formed the outlets of the flow distributor and by simulating the presence of constrictions. It was found that a flow resistance of 1.0×10(11) m(-2) reduced the amount of sample diverging into the flow distributor by a factor of 10. The presence of a constriction of 90% of the segment area and 50% of the segment length decreased the diverging flow by a factor of 5. The influence of the linear velocity was significant. Solutions to the channel discretization problem were sought by investigating different designs of spatial two-dimensional chips.

Towards ultra-high peak capacities and peak-production rates using spatial three-dimensional liquid chromatography.

In order to successfully tackle the truly complex separation problems arising from areas such as proteomics research, the development of ultra-efficient and fast separation technology is required. In spatial three-dimensional chromatography, components are separated in the space domain with each peak being characterized by its coordinates in a three-dimensional separation body. Spatial three-dimensional (3D-)LC has the potential to offer unprecedented resolving power when orthogonal retention mechanisms are applied, since the total peak capacity is the product of the three individual peak capacities. Due to parallel developments during the second- and third-dimension separations, the analysis time is greatly reduced compared to a coupled-column multi-dimensional LC approach. This communication discusses the different design aspects to create a microfluidic chip for spatial 3D-LC. The use of physical barriers to confine the flow between the individual developments, and flow control by the use of (2)D and (3)D flow distributors is discussed. Furthermore, the in situ synthesis of monolithic stationary phases is demonstrated. Finally, the potential performance of a spatial 3D-LC systems is compared with the performance obtained with state-of-the-art 1D-LC and (coupled-column) 2D-LC approaches via a Pareto-optimization approach. The proposed microfluidic device for 3D-LC featuring 16 (2)D channels and 256 (3)D channels can potentially yield a peak capacity of 8000 in a total analysis time of 10 minutes.

Comprehensive Two-Dimensional Liquid Chromatography with Stationary-Phase-Assisted Modulation Coupled to High-Resolution Mass Spectrometry Applied to Proteome Analysis of Saccharomyces cerevisiae.

Stationary-phase-assisted modulation is used to overcome one of the limitations of contemporary comprehensive two-dimensional liquid chromatography, which arises from the combination of a first-dimension column that is typically narrow and long and a second-dimension column that is wide and short. Shallow gradients at low flow rates are applied in the first dimension, whereas fast analyses (at high flow rates) are required in the second dimension. Limitations of this approach include a low sample capacity of the first-dimension column and a high dilution of the sample in the complete system. Moreover, the relatively high flow rates used for the second dimension make direct (splitless) hyphenation to mass spectrometry difficult. In the present study we demonstrate that stationary-phase-assisted modulation can be implemented in an online comprehensive two-dimensional LC (LC × LC) setup to shift this paradigm. The proposed active modulation makes it possible to choose virtually any combination of first- and second-dimension column diameters without loss in system performance. In the current setup, a 0.30 mm internal diameter first-dimension column with a relatively high loadability is coupled to a 0.075 mm internal diameter second-dimension column. This actively modulated system is coupled to a nanoelectrospray high-resolution mass spectrometer and applied for the separation of the tryptic peptides of a six-protein mixture and for the proteome-wide analyses of yeast from Saccharomyces cerevisiae. In the latter application, about 20000 MS/MS spectra are generated within 24 h analysis time, resulting in the identification of 701 proteins.

Study on the performance of different types of three-dimensional chromatographic systems.

The maximum achievable performance of possible types of three-dimensional chromatographic systems (LC×LC×LC) has been investigated. The Pareto-optimization approach was applied to establish a trade-off between three main objectives (total peak capacity, analysis time and dilution of the sample) and Pareto-front values were obtained. The performances of (x)LC×(x)LC×(x)LC (three-dimensional separation in space), (t)LC×(t)LC×(t)LC (three-dimensional separation in time) and the hybrid (x)LC×(x)LC×(t)LC system were compared mutually and with two-dimensional chromatographic systems. It was found that (x)LC×(x)LC×(x)LC performs best in terms of maximum achievable peak capacity in shortest analysis time. Based on current thin-layer-chromatography performance it should be possible to obtain a peak capacity of 50,000 within 20min. If contemporary column-packing standards can be upheld the achievable limit is approximately 50% higher. However, in an (x)LC×(x)LC×(x)LC chromatographic system analytes remain in the separation domain after the analysis, which complicates the detection. Use of an (x)LC×(x)LC×(t)LC system with elution in the last dimension alleviates the detection problem. The maximum achievable peak capacity in the same analysis time is lower for (x)LC×(x)LC×(t)LC than for (x)LC×(x)LC×(x)LC. Using the same (reasonable) length of the separation domain (e.g. a cube 200×200×200 mm) for both systems, it is possible to achieve peak capacities of 78,000 for (x)LC×(x)LC×(t)LC operated in the gradient mode, which is twice higher than for an (x)LC×(x)LC×(x)LC system. A three-dimensional (three-column) time-based (t)LC×(t)LC×(t)LC system does not greatly improve the performance of (t)LC×(t)LC in terms of (maximum) peak capacity and (minimum) analysis time. Dilution factors in (t)LC×(t)LC×(t)LC are very high. Decreasing the dilution has a detrimental influence on the peak capacity. The trade-off between these objectives is of crucial importance. The influence of several parameters (length of the separation domain, particle size, etc.) on the performance of chromatographic systems was investigated, optimal ranges were found.