Sequencing by binding rivals SMOR error-corrected sequencing by synthesis technology for accurate detection and quantification of minor (< 0.1%) subpopulation variants.

BACKGROUND: Detecting very minor (< 1%) subpopulations using next-generation sequencing is a critical need for multiple applications, including the detection of drug resistant pathogens and somatic variant detection in oncology. A recently available sequencing approach termed 'sequencing by binding (SBB)' claims to have higher base calling accuracy data "out of the box." This paper evaluates the utility of using SBB for the detection of ultra-rare drug resistant subpopulations in Mycobacterium tuberculosis (Mtb) using a targeted amplicon assay and compares the performance of SBB to single molecule overlapping reads (SMOR) error corrected sequencing by synthesis (SBS) data. RESULTS: SBS displayed an elevated error rate when compared to SMOR error-corrected SBS and SBB techniques. SMOR error-corrected SBS and SBB technologies performed similarly within the linear range studies and error rate studies. CONCLUSIONS: With lower sequencing error rates within SBB sequencing, this technique looks promising for both targeted and unbiased whole genome sequencing, leading to the identification of minor (< 1%) subpopulations without the need for error correction methods.

Phylogeography and transmission of M. tuberculosis in Moldova: A prospective genomic analysis.

BACKGROUND: The incidence of multidrug-resistant tuberculosis (MDR-TB) remains critically high in countries of the former Soviet Union, where >20% of new cases and >50% of previously treated cases have resistance to rifampin and isoniazid. Transmission of resistant strains, as opposed to resistance selected through inadequate treatment of drug-susceptible tuberculosis (TB), is the main driver of incident MDR-TB in these countries. METHODS AND FINDINGS: We conducted a prospective, genomic analysis of all culture-positive TB cases diagnosed in 2018 and 2019 in the Republic of Moldova. We used phylogenetic methods to identify putative transmission clusters; spatial and demographic data were analyzed to further describe local transmission of Mycobacterium tuberculosis. Of 2,236 participants, 779 (36%) had MDR-TB, of whom 386 (50%) had never been treated previously for TB. Moreover, 92% of multidrug-resistant M. tuberculosis strains belonged to putative transmission clusters. Phylogenetic reconstruction identified 3 large clades that were comprised nearly uniformly of MDR-TB: 2 of these clades were of Beijing lineage, and 1 of Ural lineage, and each had additional distinct clade-specific second-line drug resistance mutations and geographic distributions. Spatial and temporal proximity between pairs of cases within a cluster was associated with greater genomic similarity. Our study lasted for only 2 years, a relatively short duration compared with the natural history of TB, and, thus, the ability to infer the full extent of transmission is limited. CONCLUSIONS: The MDR-TB epidemic in Moldova is associated with the local transmission of multiple M. tuberculosis strains, including distinct clades of highly drug-resistant M. tuberculosis with varying geographic distributions and drug resistance profiles. This study demonstrates the role of comprehensive genomic surveillance for understanding the transmission of M. tuberculosis and highlights the urgency of interventions to interrupt transmission of highly drug-resistant M. tuberculosis.

Burkholderia humptydooensis sp. nov., a New Species Related to Burkholderia thailandensis and the Fifth Member of the Burkholderia pseudomallei Complex.

During routine screening for Burkholderia pseudomallei from water wells in northern Australia in areas where it is endemic, Gram-negative bacteria (strains MSMB43T, MSMB121, and MSMB122) with a similar morphology and biochemical pattern to B. pseudomallei and B. thailandensis were coisolated with B. pseudomallei on Ashdown's selective agar. To determine the exact taxonomic position of these strains and to distinguish them from B. pseudomallei and B. thailandensis, they were subjected to a series of phenotypic and molecular analyses. Biochemical and fatty acid methyl ester analysis was unable to distinguish B. humptydooensis sp. nov. from closely related species. With matrix-assisted laser desorption ionization-time of flight analysis, all isolates grouped together in a cluster separate from other Burkholderia spp. 16S rRNA and recA sequence analyses demonstrated phylogenetic placement for B. humptydooensis sp. nov. in a novel clade within the B. pseudomallei group. Multilocus sequence typing (MLST) analysis of the three isolates in comparison with MLST data from 3,340 B. pseudomallei strains and related taxa revealed a new sequence type (ST318). Genome-to-genome distance calculations and the average nucleotide identity of all isolates to both B. thailandensis and B. pseudomallei, based on whole-genome sequences, also confirmed B. humptydooensis sp. nov. as a novel Burkholderia species within the B. pseudomallei complex. Molecular analyses clearly demonstrated that strains MSMB43T, MSMB121, and MSMB122 belong to a novel Burkholderia species for which the name Burkholderia humptydooensis sp. nov. is proposed, with the type strain MSMB43T (American Type Culture Collection BAA-2767; Belgian Co-ordinated Collections of Microorganisms LMG 29471; DDBJ accession numbers CP013380 to CP013382).IMPORTANCEBurkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. The genus Burkholderia consists of a diverse group of species, with the closest relatives of B. pseudomallei referred to as the B. pseudomallei complex. A proposed novel species, B. humptydooensis sp. nov., was isolated from a bore water sample from the Northern Territory in Australia. B. humptydooensis sp. nov. is phylogenetically distinct from B. pseudomallei and other members of the B. pseudomallei complex, making it the fifth member of this important group of bacteria.

Bilayer Networks within a Hydrogel Shell: A Robust Chassis for Artificial Cells and a Platform for Membrane Studies.

The ability to make artificial lipid bilayers compatible with a wide range of environments, and with sufficient structural rigidity for manual handling, would open up a wealth of opportunities for their more routine use in real-world applications. Although droplet interface bilayers (DIBs) have been demonstrated in a host of laboratory applications, from chemical logic to biosynthesis reaction vessels, their wider use is hampered by a lack of mechanical stability and the largely manual methods employed in their production. Multiphase microfluidics has enabled us to construct hierarchical triple emulsions with a semipermeable shell, in order to form robust, bilayer-bound, droplet networks capable of communication with their external surroundings. These constructs are stable in air, water, and oil environments and overcome a critical obstacle of achieving structural rigidity without compromising environmental interaction. This paves the way for practical application of artificial membranes or droplet networks in diverse areas such as medical applications, drug testing, biophysical studies and their use as synthetic cells.

Burkholderia pseudomallei type G in Western Hemisphere.

Burkholderia pseudomallei isolates from the Western Hemisphere are difficult to differentiate from those from regions in which melioidosis is traditionally endemic. We used internal transcribed spacer typing to determine that B. pseudomallei isolates from the Western Hemisphere are consistently type G. Knowledge of this relationship might be useful for epidemiologic investigations.

Sequencing by Binding rivals error-corrected Sequencing by Synthesis technology for accurate detection and quantification of minor (<0.1%) subpopulation variants.

Detecting very minor (< 1%) subpopulations using next-generation sequencing is a critical need for multiple applications including detection of drug resistant pathogens and somatic variant detection in oncology. To enable these applications, wet lab enhancements and bioinformatic error correction methods have been developed for 'sequencing by synthesis' technology to reduce its inherent sequencing error rate. A recently available sequencing approach termed 'sequencing by binding' claims to have higher base calling accuracy data "out of the box." This paper evaluates the utility of using 'sequencing by binding' for the detection of ultra-rare subpopulations down to 0.001%.

Detection of Burkholderia pseudomallei O-antigen serotypes in near-neighbor species.

BACKGROUND: Burkholderia pseudomallei is the etiological agent of melioidosis and a CDC category B select agent with no available effective vaccine. Previous immunizations in mice have utilized the lipopolysaccharide (LPS) as a potential vaccine target because it is known as one of the most important antigenic epitopes in B. pseudomallei. Complicating this strategy are the four different B. pseudomallei LPS O-antigen types: A, B, B2, and rough. Sero-crossreactivity is common among O-antigens of Burkholderia species. Here, we identified the presence of multiple B. pseudomallei O-antigen types and sero-crossreactivity in its near-neighbor species. RESULTS: PCR screening of O-antigen biosynthesis genes, phenotypic characterization using SDS-PAGE, and immunoblot analysis showed that majority of B. mallei and B. thailandensis strains contained the typical O-antigen type A. In contrast, most of B. ubonensis and B. thailandensis-like strains expressed the atypical O-antigen types B and B2, respectively. Most B. oklahomensis strains expressed a distinct and non-seroreactive O-antigen type, except strain E0147 which expressed O-antigen type A. O-antigen type B2 was also detected in B. thailandensis 82172, B. ubonensis MSMB108, and Burkholderia sp. MSMB175. Interestingly, B. thailandensis-like MSMB43 contained a novel serotype B positive O-antigen. CONCLUSIONS: This study expands the number of species which express B. pseudomallei O-antigen types. Further work is required to elucidate the full structures and how closely these are to the B. pseudomallei O-antigens, which will ultimately determine the efficacy of the near-neighbor B serotypes for vaccine development.

The Role of Growth Directors in Controlling the Morphology of Hematite Nanorods.

The control of the growth of hematite nanoparticles from iron chloride solutions under hydrothermal conditions in the presence of two different structure promoters has been studied using a range of both structural and spectroscopic techniques including the first report of photo induced force microscopy (PiFM) to map the topographic distribution of the structure-directing agents on the developing nanoparticles. We show that the shape of the nanoparticles can be controlled using the concentration of phosphate ions up to a limit determined to be ~6 × 10-3 mol. Akaganéite (ß-FeOOH) is a major component of the nanoparticles formed in the absence of structure directors but only present in the very early stages (< 8 h) of particle growth when phosphate is present. The PiFM data suggest a correlation between the areas in which phosphate ions are adsorbed and areas where akaganéite persists on the surface. In contrast, goethite (α-FeOOH) is a directly observed precursor of the hematite nanorods when 1,2-diamino propane is present. The PiFM data shows goethite in the center of the developing particles consistent with a mechanism in which the iron hydroxide re-dissolves and precipitates at the nanorod ends as hematite.

Liquid-liquid phase separation: characterisation of a novel device capable of separating particle carrying multiphase flows.

Capillary forces on the microscale are exploited to create a continuous flow liquid-liquid phase separator. Segmented flow regimes of immiscible fluids are generated and subsequently separated into their component phases through an array of high aspect ratio, laser machined, separation ducts (36 microm wide, 130 microm deep) in a planar, integrated, polytetrafluoroethylene (PTFE) microdevice. A controlled pressure differential across the phase separator architecture facilitates the selective passage of the wetting, organic, phase through the separator ducts, enabling separation of microfluidic multiphase flow streams. The reported device is demonstrated to separate water and chloroform segmented flow regimes at flow rates up to 0.4 ml min(-1). Separation efficiency is quantified over a range of flow rates and applied pressure differentials, characterising device behaviour and limits of operation. Experimental measurements and observations are supported by theoretical hydrodynamic and capillary pressure modelling. The influence of material properties and geometric design parameters on phase separation is quantified and optimisation strategies proposed. The novel ability of the membrane free device to separate an organic phase containing suspended microparticulates, from an aqueous phase, is also demonstrated.

Identifying the source of unknown microcystin genes and predicting microcystin variants by comparing genes within uncultured cyanobacterial cells.

While multiple phylogenetic markers have been used in the culture-independent study of microcystin-producing cyanobacteria, in only a few instances have multiple markers been studied within individual cells, and in all cases these studies have been conducted with cultured isolates. Here, we isolate and evaluate large DNA fragments (>6 kb) encompassing two genes involved in microcystin biosynthesis (mcyA2 and mcyB1) and use them to identify the source of gene fragments found in water samples. Further investigation of these gene loci from individual cyanobacterial cells allowed for improved analysis of the genetic diversity within microcystin producers as well as a method to predict microcystin variants for individuals. These efforts have also identified the source of the novel mcyA genotype previously termed Microcystis-like that is pervasive in the Laurentian Great Lakes and they predict the microcystin variant(s) that it produces.

A glimpse of the inner workings of the templated site.

This is the first direct experimental probe, using EXAFS, of the active site within molecularly imprinted polymers and paves the way to a more detailed understanding of the inner workings of molecular imprinting.

Continuous molecular enrichment in microfluidic systems.

Highly efficient molecular extractions in continuous flow microfluidic systems are demonstrated utilising the rapid mixing properties of biphasic segmented flow in conjunction with suspended micro-particulate adsorbents. A continuous flow technique providing potential for continual on-line sample enrichment, purification and clean-up in chemical synthesis, and sample preparation.

Assays for the rapid and specific identification of North American Yersinia pestis and the common laboratory strain CO92.

We present TaqMan-minor groove binding (MGB) assays for an SNP that separates the Yersinia pestis strain CO92 from all other strains and for another SNP that separates North American strains from all other global strains.

The use of FTIR and NMR spectroscopies to study prepolymerisation interactions in nitrogen heterocycles.

A detailed investigation into the functional groups responsible for the formation of a noncovalent complex between 2-aminopyridine (template) and methacrylic acid (functional monomer) has been carried out using FTIR spectroscopy and confirmed by (1)H NMR spectroscopic data. The approach adopted to confirm the mechanism of interaction was the analysis of the template plus the structurally similar 2-methylaminopyridine and 2-dimethylaminopyridine. A 1:1 stoichiometry of complexation was determined by Job plot analysis following titration, with FTIR results complementing those of the (1)H NMR study. The strength of interaction between 2-aminopyridine and the functional monomer measured through band shifts by FTIR spectroscopy was compared with such interactions for the isomers 3- and 4-aminopyridine. This comparison identified a clear correlation between template pK(a), degree of interaction and subsequent nonspecific binding in the nonimprinted polymer. Using FTIR spectroscopy it was also possible to observe the effect of temperature on the prepolymerisation solution. IR spectra showed that lower temperatures led to more stabilized interactions of the hydrogen-bonded complex. The potential advantages of FTIR spectroscopy compared with (1)H NMR spectroscopy in studying prepolymerisation solutions have been identified.

Lack of genetic differentiation in aggressive and secondary bark beetles (Coleoptera: Curculionidae, Scolytinae) from Arizona.

Bark beetles (Coleoptera: Curculionidae, Scolytinae) play an important role as disturbance agents in ponderosa pine (Pinus ponderosa Douglas ex Lawson) forests of Arizona. However, from 2001 to 2003, elevated bark beetle activity caused unprecedented levels of ponderosa pine mortality. A better understanding of the population structure of these species will facilitate analysis of their dispersal patterns and improve management strategies. Here, we use fluorescently labeled amplified fragment length polymorphism (fAFLP) analysis to resolve genetic variation among and within sampling locations in northcentral Arizona of Ips pini (Say), Dendroctonus brevicomis LeConte, and D. frontalis Zimmermann. We generated genetic fingerprints for >500 beetle specimens and analyzed genetic diversity. For all species, gene flow estimates among sampling locations were high, and significant population subdivision was not discernible across a large portion of ponderosa pine forests in Arizona. However, a weak relationship was detected with I. pini population structure and elevation. Because of the lack of genetic differentiation detected throughout the large study area, our findings suggest these insects are capable of long distance dispersal and exhibit a high degree of gene flow across a broad region. We conclude that our results are consistent with strong dispersal patterns and large population sizes of all three species.

Development of Immunoassays for Burkholderia pseudomallei Typical and Atypical Lipopolysaccharide Strain Typing.

Burkholderia pseudomallei is the causative agent of melioidosis, a severe infection endemic to many tropical regions. Lipopolysaccharide (LPS) is recognized as an important virulence factor used by B. pseudomallei Isolates of B. pseudomallei have been shown to express one of four different types of LPS (typical LPS, atypical LPS types B and B2, and rough LPS) and in vitro studies have demonstrated that LPS types may impact disease severity. The association between LPS types and clinical manifestations, however, is still unknown, in part because an effective method for LPS type identification is not available. Thus, we developed antigen capture immunoassays capable of distinguishing between the LPS types. Mice were injected with B or B2 LPS for atypical LPS-specific monoclonal antibody (mAb) isolation; only two mAbs (3A2 and 5B4) were isolated from mice immunized with B2 LPS. Immunoblot analysis and surface plasmon resonance demonstrated that 3A2 and 5B4 are reactive with both B2 and B LPS where 3A2 was shown to possess higher affinity. Assays were then developed using capsular polysaccharide-specific mAb 4C4 for bacterial capture and 4C7 (previously shown to bind typical LPS) or 3A2 mAbs for typical or atypical LPS strain detection, respectively. The evaluations performed with 197 strains of Burkholderia and non-Burkholderia species showed that the assays are reactive to B. pseudomallei and Burkholderia mallei strains and have an accuracy of 98.8% (zero false positives and two false negatives) for LPS typing. The results suggest that the assays are effective and applicable for B. pseudomallei LPS typing.

Novel biphasic separations utilising highly selective molecularly imprinted polymers as biorecognition solvent extraction agents.

Molecularly imprinted polymers (MIPs) represent a class of artificial receptors that promise an environmentally robust alternative to naturally occurring biorecognition elements of biosensing devices and systems. However, in general, the performance of conventional MIPs in aqueous environments is poor. In the study reported here, this limitation has been addressed by the novel application of MIPs as a solvent extraction solid phase in a biphasic solvent system. This paper describes a previously unreported use of MIPs as solvent extraction reagents, their successful application to aqueous sample media and the opportunities for utilisation of this unique system in novel biosensing and separation procedures. This study demonstrates the development of a novel biphasic solvent system utilising MIP in the extracting phase to enhance both efficiency and selectivity of a simple two phase liquid extraction. Monodisperse propranolol imprinted polymer microspheres [p(divinylbenzene-co-methacrylic acid)] were prepared by precipitation polymerisation. Initially, the affinity of the polymers for (R,S)-propranolol was assessed by established techniques whereby the MIP demonstrated greater affinity for the template than did the non-imprinted control polymer (NIP). Importantly, MIP performance was also assessed using the novel dual solvent system. The depletion of (R,S)-propranolol from the aqueous phase into the polymer containing organic phase was determined. When compared to control extractions containing no polymer the presence of MIP in the extracting solvent phase resulted in an increased extraction of (R,S)-propranolol from the aqueous phase. Importantly, this extraction was significantly greater in the presence of MIP when compared to NIP. This unique principle generates opportunities for MIP based extractions and chemical enrichments in industrial applications, offering commercial, ecological and practical advantages to traditional solvent extraction techniques. The technique is readily transferable to analytical microsystems utilising MIP recognition elements generating promising opportunities for MIP based sensing of aqueous sample media.

Concentration dependent atrazine-atrazine complex formation promotes selectivity in atrazine imprinted polymers.

An atrazine (ATR) molecularly imprinted polymer (MIP) was prepared using a non-covalent strategy. The affinity and selectivity of the polymer was initially evaluated under non-equilibrium conditions and the polymer was shown to possess good template selectivity. The selectivity of the polymer was further investigated under equilibrium conditions and over a range of concentrations using Scatchard plots and Hill plots and by assessing distribution coefficients and normalised selectivity values. It was observed that both selectivity and affinity were dependent on the concentration of the ligand and that unusually selectivity and affinity were better at higher atrazine concentrations. It was concluded that this phenomenon resulted from the formation of atrazine-atrazine complexes during the pre-polymerisation stage and during rebinding and that the polymer demonstrated improved atrazine affinity when the conditions favoured complex formation.

From 3D to 2D: a review of the molecular imprinting of proteins.

Molecular imprinting is a generic technology that allows for the introduction of sites of specific molecular affinity into otherwise homogeneous polymeric matrices. Commonly this technique has been shown to be effective when targeting small molecules of molecular weight <1500, while extending the technique to larger molecules such as proteins has proven difficult. A number of key inherent problems in protein imprinting have been identified, including permanent entrapment, poor mass transfer, denaturation, and heterogeneity in binding pocket affinity, which have been addressed using a variety of approaches. This review focuses on protein imprinting in its various forms, ranging from conventional bulk techniques to novel thin film and monolayer surface imprinting approaches.