Rapid, Portable, and Electricity-free Sample Extraction Method for Enhanced Molecular Diagnostics in Resource-Limited Settings.

The COVID-19 pandemic has highlighted the need for rapid and reliable diagnostics that are accessible in resource-limited settings. To address this pressing issue, we have developed a rapid, portable, and electricity-free method for extracting nucleic acids from respiratory swabs (i.e. nasal, nasopharyngeal and buccal swabs), successfully demonstrating its effectiveness for the detection of SARS-CoV-2 in residual clinical specimens. Unlike traditional approaches, our solution eliminates the need for micropipettes or electrical equipment, making it user-friendly and requiring little to no training. Our method builds upon the principles of magnetic bead extraction and revolves around a low-cost plastic magnetic lid, called SmartLid, in combination with a simple disposable kit containing all required reagents conveniently prealiquoted. Here, we clinically validated the SmartLid sample preparation method in comparison to the gold standard QIAamp Viral RNA Mini Kit from QIAGEN, using 406 clinical isolates, including 161 SARS-CoV-2 positives, using the SARS-CoV-2 RT-qPCR assays developed by the US Centers for Disease Control and Prevention (CDC). The SmartLid method showed an overall sensitivity of 95.03% (95% CI: 90.44-97.83%) and a specificity of 99.59% (95% CI: 97.76-99.99%), with a positive agreement of 97.79% (95% CI: 95.84-98.98%) when compared to QIAGEN's column-based extraction method. There are clear benefits to using the SmartLid sample preparation kit: it enables swift extraction of viral nucleic acids, taking less than 5 min, without sacrificing significant accuracy when compared to more expensive and time-consuming alternatives currently available on the market. Moreover, its simplicity makes it particularly well-suited for the point-of-care where rapid results and portability are crucial. By providing an efficient and accessible means of nucleic acid extraction, our approach aims to introduce a step-change in diagnostic capabilities for resource-limited settings.

The effect of nodal connectivity and strut density within stochastic titanium scaffolds on osteogenesis.

Modern orthopaedic implants use lattice structures that act as 3D scaffolds to enhance bone growth into and around implants. Stochastic scaffolds are of particular interest as they mimic the architecture of trabecular bone and can combine isotropic properties and adjustable structure. The existing research mainly concentrates on controlling the mechanical and biological performance of periodic lattices by adjusting pore size and shape. Still, less is known on how we can control the performance of stochastic lattices through their design parameters: nodal connectivity, strut density and strut thickness. To elucidate this, four lattice structures were evaluated with varied strut densities and connectivity, hence different local geometry and mechanical properties: low apparent modulus, high apparent modulus, and two with near-identical modulus. Pre-osteoblast murine cells were seeded on scaffolds and cultured in vitro for 28 days. Cell adhesion, proliferation and differentiation were evaluated. Additionally, the expression levels of key osteogenic biomarkers were used to assess the effect of each design parameter on the quality of newly formed tissue. The main finding was that increasing connectivity increased the rate of osteoblast maturation, tissue formation and mineralisation. In detail, doubling the connectivity, over fixed strut density, increased collagen type-I by 140%, increased osteopontin by 130% and osteocalcin by 110%. This was attributed to the increased number of acute angles formed by the numerous connected struts, which facilitated the organization of cells and accelerated the cell cycle. Overall, increasing connectivity and adjusting strut density is a novel technique to design stochastic structures which combine a broad range of biomimetic properties and rapid ossification.

Application of aptamers as molecular recognition elements in lateral flow assays.

Owing to their ease in operation and fast turnaround time, lateral flow assays (LFAs) are increasingly being used as point-of-care diagnostic tests for variety of analytes. In a majority of these LFAs, antibodies are used as a molecular recognition element. Antibodies have a number of limitations such as high batch-to-batch variation, poor stability, long development time, difficulty in functionalization and need for ethical approval and cold chain. All these factors pose a great challenge to scale up the antibody-based tests. In recent years, the advent of aptamer technology has made a paradigm shift in the point-of-care diagnostics owing to the various advantages of aptamers over antibodies that favour their adaptability on a variety of sensing platforms including the lateral flow. In this review, we have highlighted the advantages of aptamers over antibodies, suitability of aptamers for lateral flow platforms, different types of aptamer-based LFAs and various labels for aptamer-based LFAs. We have also provided a summary of the applications of aptamer technology in LFAs for analytical applications.

Kir6.2-D323 and SUR2A-Q1336: an intersubunit interaction pairing for allosteric information transfer in the KATP channel complex.

ATP-sensitive potassium (KATP) channels are widely expressed and play key roles in many tissues by coupling metabolic state to membrane excitability. The SUR subunits confer drug and enhanced nucleotide sensitivity to the pore-forming Kir6 subunit, and so information transfer between the subunits must occur. In our previous study, we identified an electrostatic interaction between Kir6 and SUR2 subunits that was key for allosteric information transfer between the regulatory and pore-forming subunit. In this study, we demonstrate a second putative interaction between Kir6.2-D323 and SUR2A-Q1336 using patch clamp electrophysiological recording, where charge swap mutation of the residues on either side of the potential interaction compromise normal channel function. The Kir6.2-D323K mutation gave rise to a constitutively active, glibenclamide and ATP-insensitive KATP complex, further confirming the importance of information transfer between the Kir6 and SUR2 subunits. Sensitivity to modulators was restored when Kir6.2-D323K was co-expressed with a reciprocal charge swap mutant, SUR-Q1336E. Importantly, equivalent interactions have been identified in both Kir6.1 and Kir6.2 suggesting this is a second important interaction between Kir6 and the proximal C terminus of SUR.

The anti-virulence effect of cranberry active compound proanthocyanins (PACs) on expression of genes in the third-generation cephalosporin-resistant Escherichia coli CTX-M-15 associated with urinary tract infection.

Background: Urinary tract infections (UTIs) are one of the most common infections found in humans, with uropathogenic Escherichia coli (UPEC) being the most common cause. Prevention of UTI is a major global concern due to its recurrent nature, medical cost, and most importantly, the increased antimicrobial resistance among UPEC. The resistance in UPEC is mainly due to the Extended-Spectrum ß-lactamases (ESBL), particularly the E. coli CTXM-15 type which is known for its rapid dissemination worldwide. Treatment options for E. coli CTXM-15 have become limited over recent years because of their multi-drug resistance, hence anti-virulent strategies based on herbal remedies, have considered as a viable option. The cranberry product, Cysticlean® capsules, contain 240 mg of proanthocyanins (PACs), which have been shown to significantly inhibit E. coli adherence, both in vitro and ex vivo, to uroepithelial cells. Method: In this study, the cephalosporin-resistant E. coli isolate NCTC 1553 (E. coli CTXM-15) was analysed by qRT-PCR (quantitative Reverse Transcriptase -Polymerase Chain Reaction) for the expression of virulence factors after treatment with Cysticlean®. qRT-PCR was carried out to detect virulence determinants encoding for toxins SAT, and USP, the iron acquisition system ChuA, the protectins SoxS, KPSM, TraT and RecA, the antibiotic resistance gene CTX-M (encode ß-lactamases), and the transporters IdfB and HcaT. Results: Cysticlean® significantly reduced the expression of all ten selected genes encoding for virulence factors and ß-lactamases. Conclusion: Cranberry product Cysticlean® could represent a practicable alternative option for the prevention of recurrent UTI caused by multi-drug resistant E. coli CTXM-15, as the product acts on multiple bacterial targets.

Development and application of a video-mosaic survey technology to document the status of coral reef communities.

The recent decline in the condition of coral reef communities worldwide has fueled the need to develop innovative assessment tools to document coral abundance and distribution rapidly and effectively. While most monitoring programs rely primarily on data collected in situ by trained divers, digital photographs and video are used increasingly to extract ecological indicators, provide a permanent visual record of reef condition, and reduce the time that divers spend underwater. In this study, we describe the development and application of a video-based reef survey methodology based on an algorithm for image registration and the estimation of image motion and camera trajectory. This technology was used to construct two-dimensional, spatially accurate, high-resolution mosaics of the reef benthos at a scale of up to 400 m(2). The mosaics were analyzed to estimate the size and percent cover of reef organisms and these ecological indicators of reef condition were compared to similar measurements collected by divers to evaluate the potential of the mosaics as monitoring tools. The ecological indicators collected by trained divers compared favorably with those measured directly from the video mosaics. Five out of the eight categories chosen (hard corals, octocorals, Palythoa, algal turf, and sand) showed no significant differences in percent cover based on survey method. Moreover, no significant differences based on survey method were found in the size of coral colonies. Lastly, the capability to extract the same reef location from mosaics collected at different times proved to be an important tool for documenting change in coral abundance as the removal of even small colonies (<10 cm in diameter) was easily documented. The two-dimensional video mosaics constructed in this study can provide repeatable, accurate measurements on the reef-plot scale that can complement measurements on the colony-scale made by divers and surveys conducted at regional scales using remote sensing tools.

Interpretation of hyperspectral remote-sensing imagery by spectrum matching and look-up tables.

A spectrum-matching and look-up-table (LUT) methodology has been developed and evaluated to extract environmental information from remotely sensed hyperspectral imagery. The LUT methodology works as follows. First, a database of remote-sensing reflectance (Rrs) spectra corresponding to various water depths, bottom reflectance spectra, and water-column inherent optical properties (IOPs) is constructed using a special version of the HydroLight radiative transfer numerical model. Second, the measured Rrs spectrum for a particular image pixel is compared with each spectrum in the database, and the closest match to the image spectrum is found using a least-squares minimization. The environmental conditions in nature are then assumed to be the same as the input conditions that generated the closest matching HydroLight-generated database spectrum. The LUT methodology has been evaluated by application to an Ocean Portable Hyperspectral Imaging Low-Light Spectrometer image acquired near Lee Stocking Island, Bahamas, on 17 May 2000. The LUT-retrieved bottom depths were on average within 5% and 0.5 m of independently obtained acoustic depths. The LUT-retrieved bottom classification was in qualitative agreement with diver and video spot classification of bottom types, and the LUT-retrieved IOPs were consistent with IOPs measured at nearby times and locations.