Morphological, molecular and MALDI-TOF MS identification of ticks and tick-associated pathogens in Vietnam

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported as a promising and reliable tool for arthropod identification, including the identification of alcohol-preserved ticks based on extracted leg protein spectra. In this study, the legs of 361 ticks collected in Vietnam, including 251 Rhiphicephalus sanguineus s.l, 99 Rhipicephalus (Boophilus) microplus, two Amblyomma varanensis, seven Dermacentor auratus, one Dermacentor compactus, and one Amblyomma sp. were submitted for MALDI-TOF MS analyses. Spectral analysis showed intra-species reproducibility and inter-species specificity and the spectra of 329 (91%) specimens were of excellent quality. The blind test of 310 spectra remaining after updating the database with 19 spectra revealed that all were correctly identified with log score values (LSV) ranging from 1.7 to 2.396 with a mean of 1.982 ± 0.142 and a median of 1.971. The DNA of several microorganisms including Anaplasma platys, Anaplasma phagocytophilum, Anaplasma marginale, Ehrlichia rustica, Babesia vogeli, Theileria sinensis, and Theileria orientalis were detected in 25 ticks. Co-infection by A. phagocytophilum and T. sinensis was found in one Rh. (B) microplus.

Evaluating Different Storage Media for Identification of Taenia saginata Proglottids Using MALDI-TOF Mass Spectrometry

Taenia saginata is a helminth that can cause taeniasis in humans and cysticercosis in cattle. A species-specific diagnosis and differentiation from related species (e.g., Taenia solium) is crucial for individual patient management and disease control programs. Diagnostic stool microscopy is limited by low sensitivity and does not allow discrimination between T. saginata and T. solium. Molecular diagnostic approaches are not routinely available outside research laboratories. Recently, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) was proposed as a potentially suitable technique for species-specific helminth diagnosis. However, standardized protocols and commercial databases for parasite identification are currently unavailable, and pre-analytical factors have not yet been assessed. The purpose of this study was to employ MALDI-TOF MS for the identification of T. saginata proglottids obtained from a human patient, and to assess the effects of different sample storage media on the technique’s diagnostic accuracy. We generated T. saginata-specific main spectral profiles and added them to an in-house database for MALDI-TOF MS-based diagnosis of different helminths. Based on protein spectra, T. saginata proglottids could be successfully differentiated from other helminths, as well as bacteria and fungi. Additionally, we analyzed T. saginata proglottids stored in (i) LC–MS grade water; (ii) 0.45% sodium chloride; (iii) 70% ethanol; and (iv) 37% formalin after 2, 4, 6, 8, 12, and 24 weeks of storage. MALDI-TOF MS correctly identified 97.2–99.7% of samples stored in water, sodium chloride, and ethanol, with log-score values ≥2.5, thus indicating reliable species identification. In contrast, no protein spectra were obtained for samples stored in formalin. We conclude that MALDI-TOF-MS can be successfully employed for the identification of T. saginata, and that water, sodium chloride, and ethanol are equally effective storage solutions for prolonged periods of at least 24 weeks.

Phylogenetic relationships among different morphotypes of StY-genomic species Elymus ciliaris and E. amurensis (Poaceae) as a unified macroevolutional complex

Microevolutionary relationships between the Far Eastern StY-genomic species Elymus ciliaris and E. amurensis were studied using a set of experimental methods. No relationship was found between the formal species affiliation of a particular accession and the component composition of protein spectra. The consensus neighbor-joining (NJ) dendrogram based on the variability of ISSR markers showed the features of differentiation among different morphotypes of the two species. Pubescence of leaf blades is not a marker of differences between the studied species. A level of crossability in E. ciliaris s. l. did not allow to study the inheritance of morphological traits. According to the results of sequencing of the nuclear gene GBSS1, the sequences of the St subgenome are the most informative in terms of microevolutionary differentiation. It is proposed to return to the early treatment by N.N. Tsvelev, where E. ciliaris and E. amurensis were considered as subspecies of E. ciliaris s. l.

Creation of an Online Platform for Identification of Microorganisms: Peak Picking or Full-Spectrum Analysis

Identification of microorganisms by MALDI-TOF mass spectrometry is a very efficient method with high throughput, speed, and accuracy. However, it is significantly limited by the absence of a universal database of reference mass spectra. This problem can be solved by creating an Internet platform for open databases of protein spectra of microorganisms. Choosing the optimal mathematical apparatus is the pivotal issue for this task. In our previous study we proposed the geometric approach for processing mass spectrometry data, which represented a mass spectrum as a vector in a multidimensional Euclidean space. This algorithm was implemented in a Jacob4 stand-alone package. We demonstrated its efficiency in delimiting two closely related species of the Bacillus pumilus group. In this study, the geometric approach was realized as R scripts which allowed us to design a Web-based application. We also studied the possibility of using full spectra analysis (FSA) without calculating mass peaks (PPA), which is the logical development of the method. We used 74 microbial strains from the collections of ICiG SB RAS, UNIQEM, IEGM, KMM, and VGM as the models. We demonstrated that the algorithms based on peak-picking and analysis of complete data have accuracy no less than that of Biotyper 3.1 software. We proposed a method for calculating cut-off thresholds based on averaged intraspecific distances. The resulting database, raw data, and the set of R scripts are available online at https://icg-test.mydisk.nsc.ru/s/qj6cfZg57g6qwzN.

Effects of High-Fat Diet Induced Obesity and Fructooligosaccharide Supplementation on Cardiac Protein Expression

The mechanism by which high fat-diet induced obesity affects cardiac protein expression is unclear, and the extent to which this is modulated by prebiotic treatment is not known. These outcomes were assessed in rats initially fed a high-fat diet, then the top 40% weight gain group were randomly allocated to control (CON), high-fat (HF) and HF supplemented with fructooligosaccharide (32 g; HF-FOS) treatments for 12 weeks (n = 10/group). At sacrifice, left ventricles were either frozen or preserved in formalin. Serum was stored for glucose and insulin measurements. Protein spectra was obtained using an Orbitrap analyzer, processed with Sequest and fold changes assessed with Scaffold Q +. Treatment effects for body weights, glucose and insulin were assessed using one-way ANOVA, and the differential protein expression was assessed by a Mann–Whitney U test. The Database for Annotation, Visualization and Integrated Discovery and the Kyoto Encyclopedia of Genes and Genomes identified pathways containing overrepresented proteins. Hematoxylin and eosin sections were graded for hypertrophy and also quantified; differences were identified using Chi-square analyses and Mann-Whitney U tests. HF diet fed rats were significantly (p < 0.05) heavier than CON, and 23 proteins involved in mitochondrial function and lipid metabolism were differentially expressed between HF and CON. Between HF-FOS and HF, 117 proteins involved in contractility, lipid and carbohydrate metabolism were differentially expressed. HF cardiomyocytes were significantly (p < 0.05) more hypertrophic than CON. We conclude that high-fat feeding and FOS are associated with subcellular deviations in cardiac metabolism and contractility, which may influence myocardial function and alter the risk of cardiovascular disease.

Sand fly fauna of Crete and the description of Phlebotomus (Adlerius) creticus n. sp. (Diptera: Psychodidae)

Background The Greek island of Crete is endemic for both visceral leishmaniasis (VL) and recently increasing cutaneous leishmaniasis (CL). This study summarizes published data on the sand fly fauna of Crete, the results of new sand fly samplings and the description of a new sand fly species. Methods All published and recent samplings were carried out using CDC light traps, sticky traps or mouth aspirators. The specific status of Phlebotomus (Adlerius) creticus n. sp., was assessed by morphological analysis, cytochrome b (cytb) sequencing and MALDI-TOF protein profiling. Results Published data revealed the presence of 10 Phlebotomus spp. and 2 Sergentomyia spp. During presented field work, 608 specimens of 8 species of Phlebotomus and one species of Sergentomyia were collected. Both published data and present samplings revealed that the two most common and abundant species were Phlebotomus neglectus, a proven vector of Leishmania infantum causing VL, and Ph. similis, a suspected vector of L. tropica causing CL. In addition, the field surveys revealed the presence of a new species, Ph. (Adlerius) creticus n. sp. Conclusions The identification of the newly described species is based on both molecular and morphological criteria, showing distinct characters of the male genitalia that differentiate it from related species of the subgenus Adlerius as well as species-specific sequence of cytb and protein spectra generated by MALDI-TOF mass spectrometry.

Study of biofilms by V. cholerae strains on the surfaces of biotic and abiotic substrates using mass spectrometry

Objective: identification and comparative study of protein spectra of Vibrio cholerae biofilms by MALDI-ToF mass spectrometry.Materials and methods: V. cholerae O1 strains isolated from different sources were studied. Methods: bacteriological, mass spectrometry.Results: mass spectrometric analysis showed that it was impossible to identify strains from “plankton” samples and from chitin and plastic plates. After culturing the biofilm and plankton strains, all of them were assigned to the V. cholerae species with a Score above 2,300. A сomparative analysis of MALDI-ToF mass spectra of different variants of V. cholera cultures revealed differences, while general peaks with different intensity were maintained.Conclusions: all samples of V. cholerae cultures taken for analysis aft er culture on a nutrient medium were reliably identified with a high Score. As a result of comparative analysis of protein mass spectra by the m/z value and relative intensity of peaks, no proteins characteristic only for plankton cultures or biofilms of V. cholerae were detected. The stability of the phenotypic properties of V. cholerae regardless of its form of existence and the high reliability of the MALDI-ToF mass spectrometry method for identifying both planktonic and biofilm forms of V. cholerae with the preservation of the research protocol and bacteriological culturation.

Parallel factor analysis enables quantification and identification of highly-convolved data independent-acquired protein spectra

High-throughput data-independent acquisition (DIA) is the method of choice for quantitative proteomics, combining the best practices of targeted and shotgun proteomics approaches. The resultant DIA spectra are, however, highly convolved and with no direct precursor-fragment correspondence, complicating the analysis of biological samples. Here we present PARADIAS (PARAllel factor analysis of Data Independent Acquired Spectra), a GPU-powered unsupervised multiway factor analysis framework that deconvolves multispectral scans to individual analyte spectra, chromatographic profiles, and sample abundances, using the PARAFAC tensor decomposition method based on variation of informative spectral features. The deconvolved spectra can be annotated with traditional database search engines or used as a high-quality input for de novo sequencing methods. We demonstrate that spectral libraries generated with PARADIAS substantially reduce the false discovery rate underlying the validation of spectral quantification. PARADIAS covers up to 33 times more total ion current than library-based approaches, which typically use less than 5 % of total recorded ions, thus allowing the quantification and identification of signals from unexplored DIA spectra.

Discrimination among Cryptococcus deneoformans, C. neoformans and interspecies hybrids using MALDI-TOF Mass Spectrometry

ABSTRACTBackgroundDifferentiation of the species within the Cryptococcus neoformans complex (C. deneoformans, C. neoformans and C. neoformans interspecies hybrids –C. deneoformans x C. neoformans-) is important to define the epidemiology of the infection.ObjectivesIn this study we attempted the discrimination of three C. neoformans species using MALDI-TOF MS coupled with an in-house library.MethodsAll Cryptococcus spp. isolates were identified by AFLP markers. An in-house database was constructed 26 well characterized C. deneoformans, C. neoformans and interspecies hybrids. Forty-four Cryptococcus spp. isolates were blindly identified using MALDI-TOF MS (Bruker Daltonics) and the expanded library. Their protein spectra were also submitted to hierarchical clustering and the resulting species were verified via Partial Least Squares Differential Analysis (PLS-DA) and Support-Vector Machine (SVM).ResultsMALDI-TOF MS coupled with the in-house library allowed 100% correct identification of C. deneoformans and C. neoformans but misidentified the interspecies hybrids. The same level of discrimination among C. deneoformans and C. neoformans was achieved applying SVM. The application of the PLS-DA and SVM algorithms in a two-step analysis allowed 96.95% and 96.55% correct discrimination of C. neoformans from the interspecies hybrids, respectively. Besides, PCA analysis prior to SVM provided 98.45% correct discrimination of the 3 species analysed in a one-step analysis.ConclusionsOur results indicate that MALDI-TOF MS could be a rapid and reliable tool for the correct discrimination of C. deneoformans and C. neoformans. The correct identification of the interspecies hybrids could only be achieved by hierarchical clustering with other protein spectra from the same species.