Magnetic force spectroscopy is a rapidly developing single molecule technique that found numerous applications at the interface of physics and biology. Since the invention of the first magnetic ...tweezers, a number of modifications were incorporated into the approach that helped relieve the limitations of the original design and amplified its strengths. Inventive molecular biology solutions further advanced the technique by expanding its possible applications. In its present form, the method can be applied to single molecules and live cells without resorting to intense sample irradiation, can be easily multiplexed, accommodates multiple DNAs, displays impressive resolution, and allows a remarkable ease in stretching and twisting macromolecules. In this review, we describe the architecture of magnetic tweezers, key requirements to the experimental design and analysis of data, and outline several applications of the method that illustrate its versatility.
The ability to controllably handle the smallest materials is a fundamental enabling technology for nanoscience. Conventional optical tweezers have proven useful for manipulating microscale objects ...but cannot exert enough force to manipulate dielectric materials smaller than about 100 nm. Recently, several near-field optical trapping techniques have been developed that can provide higher trapping stiffness, but they tend to be limited in their ability to reversibly trap and release smaller materials due to a combination of the extremely high electromagnetic fields and the resulting local temperature rise. Here, we have developed a new form of photonic crystal “nanotweezer” that can trap and release on-command Wilson disease proteins, quantum dots, and 22 nm polymer particles with a temperature rise less than ∼0.3 K, which is below the point where unwanted fluid mechanical effects will prevent trapping or damage biological targets.
We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more ...bases at the 3' and/or 5' end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5' differences and in support of this we report that a 5' isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5' isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes.
Cell envelopes of many bacteria consist of two membranes studded with efflux transporters. Such organization protects bacteria from the environment and gives rise to multidrug resistance. We report a ...kinetic model that accurately describes the permeation properties of this system. The model predicts complex non-linear patterns of drug uptake complete with a bifurcation, which recapitulate the known experimental anomalies. We introduce two kinetic parameters, the efflux and barrier constants, which replace those of Michaelis and Menten for trans-envelope transport. Both compound permeation and efflux display transitions, which delineate regimes of efficient and inefficient efflux. The first transition is related to saturation of the transporter by the compound and the second one behaves as a bifurcation and involves saturation of the outer membrane barrier. The bifurcation was experimentally observed in live bacteria. We further found that active efflux of a drug can be orders of magnitude faster than its diffusion into a cell and that the efficacy of a drug depends both on its transport properties and therapeutic potency. This analysis reveals novel physical principles in the behavior of the cellular envelope, creates a framework for quantification of small molecule permeation into bacteria, and should invigorate structure-activity studies of novel antibiotics.
The CONSORT 2010 statement provides minimum guidelines for reporting randomised trials. Its widespread use has been instrumental in ensuring transparency in the evaluation of new interventions. More ...recently, there has been a growing recognition that interventions involving artificial intelligence (AI) need to undergo rigorous, prospective evaluation to demonstrate impact on health outcomes. The CONSORT-AI (Consolidated Standards of Reporting Trials-Artificial Intelligence) extension is a new reporting guideline for clinical trials evaluating interventions with an AI component. It was developed in parallel with its companion statement for clinical trial protocols: SPIRIT-AI (Standard Protocol Items: Recommendations for Interventional Trials-Artificial Intelligence). Both guidelines were developed through a staged consensus process involving literature review and expert consultation to generate 29 candidate items, which were assessed by an international multi-stakeholder group in a two-stage Delphi survey (103 stakeholders), agreed upon in a two-day consensus meeting (31 stakeholders), and refined through a checklist pilot (34 participants). The CONSORT-AI extension includes 14 new items that were considered sufficiently important for AI interventions that they should be routinely reported in addition to the core CONSORT 2010 items. CONSORT-AI recommends that investigators provide clear descriptions of the AI intervention, including instructions and skills required for use, the setting in which the AI intervention is integrated, the handling of inputs and outputs of the AI intervention, the human–AI interaction and provision of an analysis of error cases. CONSORT-AI will help promote transparency and completeness in reporting clinical trials for AI interventions. It will assist editors and peer reviewers, as well as the general readership, to understand, interpret, and critically appraise the quality of clinical trial design and risk of bias in the reported outcomes.
Reproductive Health Account construction was a long-envisioned dream in the State of Karnataka, India, for capturing inter-actor fund flows in reproductive health. Previous attempts in few states ...lead to successful identification of enablers and disablers within this systemic context. A Reproductive Health Account was constructed using primary reproductive health expenditure data, collected from a pre-estimated sample size of 519 households spread across 15 villages, using probability proportional to size method, from two selected sub-districts Channapatna and Ramanagara, having mediocre performance indices, within Ramanagara District of Karnataka. Secondary data were extracted from public health websites. Expenditures incurred on six types of health services by respondents of reproductive age group (15–49) during financial year, 2017–2018, within two sub-districts was collected over financial year, 2018–2019. Processed data were then converted to four ‘origin to destination’ matrices each capturing fund movement among two actors, based on accounting principles of National Health Account to develop a contextual Reproductive Health Account. Study included four actors namely financial sources, financial agents, health providers and health activities, all pertaining to reproductive health domain. Matrices helped identify a massive 87.23% burden on households, majorly financed by mortgage bearing astronomical interests and sale of meagre assets. Public sector healthcare at 5.47% was found performing unsatisfactorily. Tertiary level was absorbing disproportional amount of 62.93% funds in conjunction with the laboratory and imaging services. Moreover, pharmaceutical bills at 22.97% caused prolonged distress to these households. Government intervention towards absence and shortage of quality infrastructure at the primary and secondary sector levels needs reviewing, for containment of the massive out-of-pocket expenditures.
Plasmonic−fluorescent composite nanoparticles are considered as unique, multifunctional nanoprobes for plasmon- and fluorescence-based imaging and detection. However, their synthesis is challenging ...due to fluorescence quenching of the fluorophore by plasmonic particles and most of the successful methods produce composite particles of large size (diameter > 50 nm), which limit their wider applications. Here we report 20−30 nm diameter plasmonic−fluorescent composite nanoparticles with reasonable fluorescence quantum yield (12−16%). These particles are composed of 3−6 nm diameter Au/Ag cores and fluorescein-incorporated polymeric shells. They have high water solubility, good colloidal stability, stable fluorescence properties, and are amenable in deriving various functional nanoprobes. Different functional nanoprobes are derived from these composites and successfully used for fluorescence-based cell labeling as well as plasmon-based detection applications.
Fluorescent carbon nanoparticles (CNPs) 2−6 nm in size with a quantum yield of about ∼3% were synthesized via nitric acid oxidation of carbon soot, and this approach can be used for milligram-scale ...synthesis of these water-soluble particles. These CNPs are nanocrystalline with a predominantly graphitic structure and show green fluorescence under UV exposure. Nitric acid oxidation induces nitrogen and oxygen incorporation into soot particles, which afforded water solubility and a light-emitting property; the isolation of small particles from a mixture of different sized particles improved the fluorescence quantum yield. These CNPs show encouraging cell-imaging applications. They enter into cells without any further functionalization, and the fluorescence property of these particles can be used for fluorescence-based cell imaging applications.
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