Mining Public Metagenomes for Environmental Surveillance of Parasites: A Proof of Principle

Parasites often have complex developmental cycles that account for their presence in a variety of difficult-to-analyze matrices, including feces, water, soil, and food. Detection of parasites in these matrices still involves laborious methods. Untargeted sequencing of nucleic acids extracted from those matrices in metagenomic projects may represent an attractive alternative method for unbiased detection of these pathogens. Here, we show how publicly available metagenomic datasets can be mined to detect parasite specific sequences, and generate data useful for environmental surveillance. We use the protozoan parasite Cryptosporidium parvum as a test organism, and show that detection is influenced by the reference sequence chosen. Indeed, the use of the whole genome yields high sensitivity but low specificity, whereas specificity is improved through the use of signature sequences. In conclusion, querying metagenomic datasets for parasites is feasible and relevant, but requires optimization and validation. Nevertheless, this approach provides access to the large, and rapidly increasing, number of datasets from metagenomic and meta-transcriptomic studies, allowing unlocking hitherto idle signals of parasites in our environments.

PINIR: a comprehensive information resource for Pin-II type protease inhibitors

Background Serine protease inhibitors belonging to the Potato type-II Inhibitor family Protease Inhibitors (Pin-II type PIs) are essential plant defense molecules. They are characterized by multiple inhibitory repeat domains, conserved disulfide bond pattern, and a tripeptide reactive center loop. These features of Pin-II type PIs make them potential molecules for protein engineering and designing inhibitors for agricultural and therapeutic applications. However, the diversity in these PIs remains unexplored due to the lack of annotated protein sequences and their functional attributes in the available databases. Results We have developed a database, PINIR (Pin-II type PIs Information Resource), by systematic collection and manual annotation of 415 Pin-II type PI protein sequences. For each PI, the number and position for signature sequences are specified: 695 domains, 75 linkers, 63 reactive center loops, and 10 disulfide bond patterns are identified and mapped. Database analysis revealed novel subcategories of PIs, species-correlated occurrence of inhibitory domains, reactive center loops, and disulfide bond patterns. By analyzing linker regions, we predict that alternative processing at linker regions could generate PI variants in the Solanaceae family. Conclusion PINIR (https://pinir.ncl.res.in) provides a web interface for browsing and analyzing the protein sequences of Pin-II type PIs. Information about signature sequences, spatio-temporal expression, biochemical properties, gene sequences, and literature references are provided. Analysis of PINIR depicts conserved species-specific features of Pin-II type PI protein sequences. Diversity in the sequence of inhibitory domains and reactive loops directs potential applications to engineer Pin-II type PIs. The PINIR database will serve as a comprehensive information resource for further research into Pin-II type PIs.

Biochemical characterization of a recombinant acid phosphatase from Acinetobacter baumannii

Genomic sequence analysis of Acinetobacter baumannii revealed the presence of a putative Acid Phosphatase (AcpA; EC 3.1.3.2). A plasmid construct was made, and recombinant protein (rAcpA) was expressed in E. coli. PAGE analysis (carried out under denaturing/reducing conditions) of nickel-affinity purified protein revealed the presence of a near-homogeneous band of approximately 37 kDa. The identity of the 37 kDa species was verified as rAcpA by proteomic analysis with a molecular mass of 34.6 kDa from the deduced sequence. The dependence of substrate hydrolysis on pH was broad with an optimum observed at 6.0. Kinetic analysis revealed relatively high affinity for PNPP (Km = 90 μM) with Vmax, kcat, and Kcat/Km values of 19.2 pmoles s-1, 4.80 s-1(calculated on the basis of 37 kDa), and 5.30 x 104 M-1s-1, respectively. Sensitivity to a variety of reagents, i.e., detergents, reducing, and chelating agents as well as classic acid phosphatase inhibitors was examined in addition to assessment of hydrolysis of a number of phosphorylated compounds. Removal of phosphate from different phosphorylated compounds is supportive of broad, i.e., ‘nonspecific’ substrate specificity; although, the enzyme appears to prefer phosphotyrosine and/or peptides containing phosphotyrosine in comparison to serine and threonine. Examination of the primary sequence indicated the absence of signature sequences characteristic of Type A, B, and C nonspecific bacterial acid phosphatases.

In-Silico Pangenomics of SARS-CoV-2 Isolates Reveal Evidence for Subtle Adaptive Expression Strategies, Continued Clonal Evolution, and Sub-Clonal Emergences, Despite Genome Stability

The devastating SARS-CoV2 pandemic is worsening with relapsing surges, emerging mutants, and increasing mortalities. Despite enormous efforts, it is not clear how SARS-CoV2 adapts and evolves in a clonal background. Laboratory research is hindered by high biosafety demands. However, the rapid sequence availability opened doors for bioinformatics. Using different bioinformatics programs, we investigated 6305 sequences for clonality, expressions strategies, and evolutionary dynamics. Results showed high nucleotide identity of 99.9% among SARS-CoV2 indicating clonal evolution and genome. High sequence identity and phylogenetic tree concordance were obtained with isolates from different regions. In any given tree topology, ~50% of isolates in a country formed country-specific sub-clusters. However, abundances of subtle overexpression strategies were found including transversions, signature-sequences and slippery-structures. Five different short tracks dominated with identical location patterns in all genomes where Slippery-4 AAGAA was the most abundant. Interestingly, transversion and transition substitutions mostly affected the same amino acid residues implying compensatory changes. To ensure these strategies were independent of sequence clonality, we simultaneously examined sequence homology indicators; tandem-repeats, restriction-site, and 3′UTR, 5′ UTR-caps and stem-loop locations in addition to stringent alignment parameters for 100% identity which all confirmed stability. Nevertheless, two rare events; a rearrangement in two SARS-CoV2 isolates against betacoronavirus ancestor and a polymorphism in S gene, were detected. Thus, we report on abundance of transversions, slippery sequences, and ON/OFF molecular structures, implying adaptive expressions had occurred, despite clonal evolution and genome stability. Furthermore, functional validation of the point mutations would provide insights into mechanisms of SARS-CoV2 virulence and adaptation.

Subspecies-specific sequence detection for differentiation of Mycobacterium abscessus complex

Mycobacterium abscessus complex (MABC) is a taxonomic group of rapidly growing, nontuberculous mycobacteria that are found as etiologic agents of various types of infections. They are considered as emerging human pathogens. MABC consists of 3 subspecies—M. abscessus subsp. bolletti, M. abscessus subsp. massiliense and M. abscessus subsp. abscessus. Here we present a novel method for subspecies differentiation of M. abscessus named Subspecies-Specific Sequence Detection (SSSD). This method is based on the presence of signature sequences present within the genomes of each subspecies of MABC. We tested this method against a virtual database of 1505 genome sequences of MABC. Further, we detected signature sequences of MABC in 45 microbiological samples through DNA hybridization. SSSD showed high levels of sensitivity and specificity for differentiation of subspecies of MABC, comparable to those obtained by rpoB sequence typing.

CRISPR 2 PCR and high resolution melting profiling for identification and characterization of clinically-relevant Salmonella enterica subsp. enterica

Background Nontyphoidal Salmonella spp. constitute a major bacterial cause of food poisoning. Each Salmonella serotype causes distinct virulence to humans. Method A small cohort study was conducted to characterize several aspects of Salmonella isolates obtained from stool of diarrheal patients (n = 26) admitted to Phayao Ram Hospital, Phayao province, Thailand. A simple CRISPR 2 molecular analysis was developed to rapidly type Salmonella isolates employing both uniplex and high resolution melting (HRM) curve analysis. Results CRISPR 2 monoplex PCR generated a single Salmonella serotype-specific amplicon, showing S. 4,[5],12:i:- with highest frequency (42%), S. Enteritidis (15%) and S. Stanley (11%); S. Typhimurium was not detected. CRISPR 2 HRM-PCR allowed further classification of S. 4,[5],12:i:- isolates based on their specific CRISPR 2 signature sequences. The highest prevalence of Salmonella infection was during the summer season (April to August). Additional studies were conducted using standard multiplex HRM-PCR typing, which confirmed CRISPR 2 PCR results and, using a machine-learning algorithm, clustered the majority of Salmonella serotypes into six clades; repetitive element-based (ERIC) PCR, which clustered the serotypes into three clades only; antibiogram profiling, which revealed the majority resistant to ampicillin (69%); and test for extended spectrum β-lactamase production (two isolates) and PCR-based detection of bla alleles. Conclusion CRISPR 2 PCR provided a simple assay for detection and identification of clinically-relevant Salmonella serotypes. In conjunction with antibiogram profiling and rapid assay for β-lactamase producers, this approach should facilitate detection and appropriate treatment of Salmonellosis in a local hospital setting. In addition, CRISPR 2 HRM-PCR profiling enabled clustering of S. 4,[5],12:i:-isolates according to CRISPR 2 locus signature sequences, indicative of their different evolutionary trajectories, thereby providing a powerful tool for future epidemiological studies of virulent Salmonella serotypes.

Quaternary DS-SSMA Communications Employing Generalized Detector over Rayleigh Fading Channels

This paper deals with an employment of the complex modulators and generalized receivers constructed based on the generalized approach to signal processing (GASP) in noise in quaternary direct-sequence spread-spectrum multiple-access (DS-SSMA) communication systems with complex signature sequences in flat Rayleigh fading. Owing to availability of large sets of complex spreading sequences possessing perfect correlation characteristics, the interest to use the complex spreading sequences in DS-SSMA communication systems has increased. The complex spreading sequences investigated in the present paper are based on the recently introduced orthogonal unified complex Hadamard transform (UCHN) sequences. In the present paper, the complex processing in modulators and generalized receivers is also employed with the purpose to take advantage of the correlation properties of complex signature sequences. We derive the average bit error rate (BER) for quarternary synchronous communications system based on the generalized approach to signal processing in noise first, and then the BER for quaternary asynchronous ones is evaluated using the characteristic function approach. We presented some results based on the Gaussian approximation method for asynchronous communication systems constructed on the basis of the generalized approach to signal processing in noise Computer modeling results demonstrate that the communication systems using UCHT spreading sequences perform generally better than the GOLD sequences and the 4-phase family A sequences. Comparative analysis between the asynchronous communication systems constructed on the basis of the generalized approach to signal processing in noise demonstrates superiority over the asynchronous communication systems employing the correlation receiver.

Nef Alleles derived from HIV-Positive Patients with Pulmonary Hypertension Affect Production and Function of Exosomes in Endothelial Colony Forming Cells

Background: The HIV positive population has a > 300 fold higher risk for developing pulmonary hypertension (PH) as compared to the general population. PH is a disease characterized by elevated blood pressure in the pulmonary vasculature and is currently with no cure. Experiments with primates found that Nef, an HIV encoded protein secreted in exosomes from HIV+ cells, plays an important role in the development of HIV-PH. HIV-Nef+ exosomes induce endothelial dysfunction, including premature senescence of endothelial colony forming cells (ECFCs). We hypothesize that exosome functions and concentrations in HIV+ patients may vary based on Nef-mutations, and thus potentially define a predictor for the risk of developing PH. Methods: Nef exosomes were isolated from HEK293? cells transfected with molecular constructs containing HIV Nef signature sequences recovered from patients with HIV-PH. The Nef exosomes were then used to treat ECFC. We measured cell proliferation (by MTT assay), apoptosis (cleaved caspase-3 by Western blot), and cell senescence (p16ink4A by flow cytometry) in ECFCs treated with Nef exosomes. LM-10-Nanosight was used to quantify exosome concentrations in patient plasma. Results: ECFC treated with Nef exosomes induced a 30% reduction in cell proliferation (p<0.05, MTT). Western blot analysis demonstrated no significant change in apoptotic signaling, and flow cytometric analysis of p16ink4A showed increased senescence in Nef exosome-treated ECFCs versus mock control. “Nef PH-transfected cells showed that decreased proliferation was dependent on specific mutations of Nef. Conclusion: Expression of Nef in cells results in increased exosome production and treatment of ECFC with these exosomes resulted in decreased proliferation, which inducting the cells towards senescence based on signature sequences in Nef.

Resolution and Cooccurrence Patterns of Gardnerella leopoldii, G. swidsinskii, G. piotii, and G. vaginalis within the Vaginal Microbiome

ABSTRACT Gardnerella vaginalis is a hallmark of vaginal dysbiosis, but it is found in the microbiomes of women with and without vaginal symptoms and those who do not have microbiologically defined dysbiosis. G. vaginalis encompasses diverse taxa differing in attributes that are potentially important for virulence, and there is evidence that clades or subgroups within the species are differentially associated with clinical outcomes. The G. vaginalis species description was recently emended, and three new species within the genus were defined (G. leopoldii, G. swidsinskii, and G. piotii). 16S rRNA sequences for the four Gardnerella species are all >98.5% identical, and no signature sequences differentiate them. We demonstrated that Gardnerella species can be resolved using partial chaperonin 60 (cpn60) sequences, with pairwise percent identities of 87.1 to 97.8% among the type strains. Pairwise cooccurrence patterns of Gardnerella spp. in the vaginal microbiomes of 413 reproductive aged Canadian women were investigated, and several significant cooccurrences of species were identified. Abundance of G. vaginalis and G. swidsinskii was associated with vaginal symptoms of abnormal odor and discharge. cpn60 barcode sequencing can provide a rapid assessment of the relative abundance of Gardnerella spp. in microbiome samples, providing a powerful method of elucidating associations between these diverse organisms and clinical outcomes. Researchers should consider using cpn60 instead of 16S RNA for better resolution of these important organisms.