Budget impact of next-generation sequencing for diagnosis of TB drug resistance in Moldova.

BACKGROUND Diagnosing drug resistance is critical for choosing effective TB treatment regimens. Next-generation sequencing (NGS) represents an alternative approach to conventional phenotypic drug susceptibility testing (pDST) for diagnosing TB drug resistance.METHODS We undertook a budget impact analysis estimating the costs of introduction and routine use of NGS in the Moldovan National TB Programme. We conducted an empirical costing study and collated price and operating characteristics for NGS platforms. We examined multiple NGS scenarios in comparison to the current approach (pDST) for pre-treatment drug resistance testing over 2021-2025.RESULTS Annual testing volume ranged from 912 to 1,926 patients. For the pDST scenario, we estimated total costs of US$362,000 (2021 USD) over the 5-year study period. Total costs for NGS scenarios ranged from US$475,000 to US$1,486,000. Lowest cost NGS options involved targeted sequencing as a replacement for pDST, and excluded individuals diagnosed as RIF-susceptible on Xpert® MTB/RIF. For all NGS scenarios, the majority (55-80%) of costs were devoted to reagent kits. Start-up costs of NGS were small relative to routine costs borne each year.CONCLUSION NGS adoption will require expanded resources compared to conventional pDST. Further work is required to better understand the feasibility of NGS in settings such as Moldova.

FIB and MIP: understanding nanoscale porosity in molecularly imprinted polymers via 3D FIB/SEM tomography.

We present combined focused ion beam/scanning electron beam (FIB/SEM) tomography as innovative method for differentiating and visualizing the distribution and connectivity of pores within molecularly imprinted polymers (MIPs) and non-imprinted control polymers (NIPs). FIB/SEM tomography is used in cell biology for elucidating three-dimensional structures such as organelles, but has not yet been extensively applied for visualizing the heterogeneity of nanoscopic pore networks, interconnectivity, and tortuosity in polymers. To our best knowledge, the present study is the first application of this strategy for analyzing the nanoscale porosity of MIPs. MIPs imprinted for propranolol - and the corresponding NIPs - were investigated establishing FIB/SEM tomography as a viable future strategy complementing conventional isotherm studies. For visualizing and understanding the properties of pore networks in detail, polymer particles were stained with osmium tetroxide (OsO4) vapor, and embedded in epoxy resin. Staining with OsO4 provides excellent contrast during high-resolution SEM imaging. After optimizing the threshold to discriminate between the stained polymer matrix, and pores filled with epoxy resin, a 3D model of the sampled volume may be established for deriving not only the pore volume and pore surface area, but also to visualize the interconnectivity and tortuosity of the pores within the sampled polymer volume. Detailed studies using different types of cross-linkers and the effect of hydrolysis on the resulting polymer properties have been investigated. In comparison of MIP and NIP, it could be unambiguously shown that the interconnectivity of the visualized pores in MIPs is significantly higher vs. the non-imprinted polymer, and that the pore volume and pore area is 34% and approx. 35% higher within the MIP matrix. This confirms that the templating process not only induces selective binding sites, but indeed also affects the physical properties of such polymers down to the nanoscale, and that additional chemical modification, e.g., via hydrolysis clearly affects that nature of the polymer.

Low temperature fabrication of biodegradable sugar glass microneedles for transdermal drug delivery applications.

Transdermal drug delivery is limited by the barrier properties of the outer skin layer. Microneedles (MNs) effectively circumvent the skin barrier to offer this route as a potential alternative to oral and parenteral delivery of therapeutics. Biodegradable microneedles offer particular advantages however processing commonly requires elevated temperatures that may adversely affect heat-labile molecules and macromolecules. In this study, solid amorphous sugar glasses containing low residual quantities of water were created by dehydration of trehalose and sucrose sugar combination solutions. Biodegradable sugar glass MNs were fabricated following optimisation of a simple and novel low temperature vacuum deposition micromoulding methodology. These had absolute morphological fidelity to silicon master structures and demonstrated sufficient structural rigidity to efficiently penetrate excised human breast skin. Sugar glass MNs incorporating a marker compound dissolved rapidly and completely in situ releasing dye into deeper skin layers. The biological activity of a model macromolecule was partially retained over extended storage following incorporation into sugar glass. This is the first demonstration that MNs created from amorphous sugar glasses can be used for incorporating and delivering molecules, and potentially biologically active macromolecules, via the transdermal route.

Rapeseed cytoplasm gives advantage in wild relatives and complicates genetically modified crop biocontainment.

Biocontainment methods for genetically modified crops closest to commercial reality (chloroplast transformation, male sterility) would be compromised (in absolute terms) by seed-mediated gene flow leading to chloroplast capture. Even in these circumstances, however, it can be argued that biocontainment still represses transgene movement, with the efficacy depending on the relative frequency of seed- and pollen-mediated gene flow. In this study, we screened for crop-specific chloroplast markers from rapeseed (Brassica napus) amongst sympatric and allopatric populations of wild B. oleracea in natural cliff-top populations and B. rapa in riverside and weedy populations. We found only modest crop chloroplast presence in wild B. oleracea and in weedy B. rapa, but a surprisingly high incidence in sympatric (but not in allopatric) riverside B. rapa populations. Chloroplast inheritance models indicate that elevated crop chloroplast acquisition is best explained if crop cytoplasm confers selective advantage in riverside B. rapa populations. Our results therefore imply that chloroplast transformation may slow transgene recruitment in two settings, but actually accelerate transgene spread in a third. This finding suggests that the appropriateness of chloroplast transformation for biocontainment policy depends on both context and geographical location.

Simple sequence repeats reveal uneven distribution of genetic diversity in chloroplast genomes of Brassica oleracea L. and (n = 9) wild relatives.

Diversity in the chloroplast genome of 171 accessions representing the Brassica 'C' (n = 9) genome, including domesticated and wild B. oleracea and nine inter-fertile related wild species, was investigated using six chloroplast SSR (microsatellite) markers. The lack of diversity detected among 105 cultivated and wild accessions of B. oleracea contrasted starkly with that found within its wild relatives. The vast majority of B. oleracea accessions shared a single haplotype, whereas as many as six haplotypes were detected in two wild species, B. villosa Biv. and B. cretica Lam.. The SSRs proved to be highly polymorphic across haplotypes, with calculated genetic diversity values (H) of 0.23-0.87. In total, 23 different haplotypes were detected in C genome species, with an additional five haplotypes detected in B. rapa L. (A genome n = 10) and another in B. nigra L. (B genome, n = 8). The low chloroplast diversity of B. oleracea is not suggestive of multiple domestication events. The predominant B. oleracea haplotype was also common in B. incana Ten. and present in low frequencies in B. villosa, B. macrocarpa Guss, B. rupestris Raf. and B. cretica. The chloroplast SSRs reveal a wealth of diversity within wild Brassica species that will facilitate further evolutionary and phylogeographic studies of this important crop genus.

Fitness of hybrids between rapeseed (Brassica napus) and wild Brassica rapa in natural habitats.

Fitness of hybrids between genetically modified (GM) crops and wild relatives influences the likelihood of ecological harm. We measured fitness components in spontaneous (non-GM) rapeseed x Brassica rapa hybrids in natural populations. The F1 hybrids yielded 46.9% seed output of B. rapa, were 16.9% as effective as males on B. rapa and exhibited increased self-pollination. Assuming 100% GM rapeseed cultivation, we conservatively predict < 7000 second-generation transgenic hybrids annually in the United Kingdom (i.e. approximately 20% of F1 hybrids). Conversely, whilst reduced hybrid fitness improves feasibility of bio-containment, stage projection matrices suggests broad scope for some transgenes to offset this effect by enhancing fitness.

Molecular imprinted polymer sensors: implications for therapeutics.

A biosensor is a sensor that uses biological selectivity to limit its perception to particular key molecules and can be defined as an analytical device possessing a biological or biologically derived sensing element integrated with or associated closely with a physicochemical transducer. In the future it is likely that a number of key developments in therapeutic monitoring and intelligent drug delivery will rely on real-time feedback information in order to deliver an appropriate response. However due to issues of integration and the fragility and unreliability of the bio-molecule, biosensors are currently unable to fulfil this role. Molecular imprinted polymers are viable alternatives to both antibodies and enzymes and this review considers the current position of molecular imprinted polymer sensing.

How many species of cichlid fishes are there in African lakes?

The endemic cichlid fishes of Lakes Malawi, Tanganyika and Victoria are textbook examples of explosive speciation and adaptive radiation, and their study promises to yield important insights into these processes. Accurate estimates of species richness of lineages in these lakes, and elsewhere, will be a necessary prerequisite for a thorough comparative analysis of the intrinsic and extrinsic factors influencing rates of diversification. This review presents recent findings on the discoveries of new species and species flocks and critically appraises the relevant evidence on species richness from recent studies of polymorphism and assortative mating, generally using behavioural and molecular methods. Within the haplochromines, the most species-rich lineage, there are few reported cases of postzygotic isolation, and these are generally among allopatric taxa that are likely to have diverged a relatively long time in the past. However, many taxa, including many which occur sympatrically and do not interbreed in nature, produce viable, fertile hybrids. Prezygotic barriers are more important, and persist in laboratory conditions in which environmental factors have been controlled, indicating the primary importance of direct mate preferences. Studies to date indicate that estimates of alpha (within-site) diversity appear to be robust. Although within-species colour polymorphisms are common, these have been taken into account in previous estimates of species richness. However, overall estimates of species richness in Lakes Malawi and Victoria are heavily dependent on the assignation of species status to allopatric populations differing in male colour. Appropriate methods for testing the specific status of allopatric cichlid taxa are reviewed and preliminary results presented.

Pharmaceutical applications for molecularly imprinted polymers.

Molecular imprinting is a means of introducing sites of specific molecular arrangement into an otherwise uniform polymeric matrix. This is achieved by formation of a pre-polymerisation complex between complementary monomers and the template molecule. Subsequent polymerisation in the presence of a crosslinker, in a porogenic environment, results in the production of a macroporous polymer capable of specific molecular recognition. This paper considers potential roles for molecularly imprinted polymers within a pharmaceutical remit. Applications including controlled release, drug monitoring devices and biological receptor mimetics are discussed. Histamine and ephedrine molecularly imprinted polymers (MIPs) were studied as potential biological receptor mimics whilst a propranolol MIP was investigated for its use as a rate attenuating selective excipient in a transdermal controlled release device. Preliminary studies concerning the preparation of a theophylline selective transcutaneous monitoring device, using a theophylline MIP, are also described.