Global urban signatures of phenotypic change in animal and plant populations.

Humans challenge the phenotypic, genetic, and cultural makeup of species by affecting the fitness landscapes on which they evolve. Recent studies show that cities might play a major role in contemporary evolution by accelerating phenotypic changes in wildlife, including animals, plants, fungi, and other organisms. Many studies of ecoevolutionary change have focused on anthropogenic drivers, but none of these studies has specifically examined the role that urbanization plays in ecoevolution or explicitly examined its mechanisms. This paper presents evidence on the mechanisms linking urban development patterns to rapid evolutionary changes for species that play important functional roles in communities and ecosystems. Through a metaanalysis of experimental and observational studies reporting more than 1,600 phenotypic changes in species across multiple regions, we ask whether we can discriminate an urban signature of phenotypic change beyond the established natural baselines and other anthropogenic signals. We then assess the relative impact of five types of urban disturbances including habitat modifications, biotic interactions, habitat heterogeneity, novel disturbances, and social interactions. Our study shows a clear urban signal; rates of phenotypic change are greater in urbanizing systems compared with natural and nonurban anthropogenic systems. By explicitly linking urban development to traits that affect ecosystem function, we can map potential ecoevolutionary implications of emerging patterns of urban agglomerations and uncover insights for maintaining key ecosystem functions upon which the sustainability of human well-being depends.

Automated microscopy for routine malaria diagnosis: a field comparison on Giemsa-stained blood films in Peru.

BACKGROUND: Microscopic examination of Giemsa-stained blood films remains a major form of diagnosis in malaria case management, and is a reference standard for research. However, as with other visualization-based diagnoses, accuracy depends on individual technician performance, making standardization difficult and reliability poor. Automated image recognition based on machine-learning, utilizing convolutional neural networks, offers potential to overcome these drawbacks. A prototype digital microscope device employing an algorithm based on machine-learning, the Autoscope, was assessed for its potential in malaria microscopy. Autoscope was tested in the Iquitos region of Peru in 2016 at two peripheral health facilities, with routine microscopy and PCR as reference standards. The main outcome measures include sensitivity and specificity of diagnosis of malaria from Giemsa-stained blood films, using PCR as reference. METHODS: A cross-sectional, observational trial was conducted at two peripheral primary health facilities in Peru. 700 participants were enrolled with the criteria: (1) age between 5 and 75 years, (2) history of fever in the last 3 days or elevated temperature on admission, (3) informed consent. The main outcome measures included sensitivity and specificity of diagnosis of malaria from Giemsa-stained blood films, using PCR as reference. RESULTS: At the San Juan clinic, sensitivity of Autoscope for diagnosing malaria was 72% (95% CI 64-80%), and specificity was 85% (95% CI 79-90%). Microscopy performance was similar to Autoscope, with sensitivity 68% (95% CI 59-76%) and specificity 100% (95% CI 98-100%). At San Juan, 85% of prepared slides had a minimum of 600 WBCs imaged, thus meeting Autoscope's design assumptions. At the second clinic, Santa Clara, the sensitivity of Autoscope was 52% (95% CI 44-60%) and specificity was 70% (95% CI 64-76%). Microscopy performance at Santa Clara was 42% (95% CI 34-51) and specificity was 97% (95% CI 94-99). Only 39% of slides from Santa Clara met Autoscope's design assumptions regarding WBCs imaged. CONCLUSIONS: Autoscope's diagnostic performance was on par with routine microscopy when slides had adequate blood volume to meet its design assumptions, as represented by results from the San Juan clinic. Autoscope's diagnostic performance was poorer than routine microscopy on slides from the Santa Clara clinic, which generated slides with lower blood volumes. Results of the study reflect both the potential for artificial intelligence to perform tasks currently conducted by highly-trained experts, and the challenges of replicating the adaptiveness of human thought processes.

A microRNA-gated thgRNA platform for multiplexed activation of gene expression in mammalian cells.

To effectively reprogram cellular regulatory networks towards desired phenotypes, it is critical to have the ability to provide precise gene regulation in a spatiotemporal manner. We have previously engineered toehold-gated guide RNA (thgRNA) to enable conditional activation of dCas9-mediated transcriptional upregulation in mammalian cells using synthetic RNA triggers. Here, we demonstrate that microRNA (miR)-gated thgRNAs can be transcribed by type II RNA polymerase to allow multiplexed transcriptional activation using both mRNA and miR. Activation is achieved only by proper miR-mediated processing of the flanking 5' cap and 3' poly A tail and hairpin unblocking by mRNA via strand displacement. This new AND-gate design is exploited to elicit conditional protein degradation based on induced expression of a specific ubiquibody. This new strategy may find many new applications in an RNA-responsive manner.

Deciphering the Design Rules of Toehold-Gated sgRNA for Conditional Activation of Gene Expression and Protein Degradation in Mammalian Cells.

A new class of toehold-gated gRNAs (thgRNAs) has been created to provide conditional gene regulation via RNA-mediated activation. However, the detailed design principles remain elusive. Here, we presented an investigation into the design rules for conditional gRNAs by systematically varying the toehold, stem, and flexible loop regions of thgRNA for optimal gene activation in HeLa cells. We determined that nonspecific interactions between the toehold region and the flexible loop are the main driver for the background leak observed in the OFF state. By trimming the toehold length from 15 to 5 nt, the improved thgNT-F design led to a 38-fold increase in the activated ON state with no observable background leak. The same design rule was successfully adapted to target two different regions on the mCherry mRNA with the same impressive fold change. Using the thgRNA to direct conditional protein degradation, we showed up to 8-fold knockdown of a reporter protein through activating expression of a bifunctional ubiquibody GS2-IpaH9.8. This new strategy may find many new applications for cell culture control or cell therapy by removing unwanted proteins in an RNA-responsive manner.

Field evaluation of a prototype tuberculosis lipoarabinomannan lateral flow assay on HIV-positive and HIV-negative patients.

Detection of tuberculosis at the point-of-care (POC) is limited by the low sensitivity of current commercially available tests. We describe a diagnostic accuracy field evaluation of a prototype urine Tuberculosis Lipoarabinomannan Lateral Flow Assay (TB-LAM LFA) in both HIV-positive and HIV-negative patients using fresh samples with sensitivity and specificity as the measures of accuracy. This prototype combines a proprietary concentration system with a sensitive LFA. In a prospective study of 292 patients with suspected pulmonary tuberculosis in Uganda, the clinical sensitivity and specificity was compared against a microbiological reference standard including sputum Xpert MTB/RIF Ultra and solid and liquid culture. TB-LAM LFA had an overall sensitivity of 60% (95%CI 51-69%) and specificity of 80% (95%CI 73-85%). When comparing HIV-positive (N = 86) and HIV-negative (N = 206) patients, there was no significant difference in sensitivity (sensitivity difference 8%, 95%CI -11% to +24%, p = 0.4351) or specificity (specificity difference -9%, 95%CI -24% to +4%, p = 0.2051). Compared to the commercially available Alere Determine TB-LAM Ag test, the TB-LAM LFA prototype had improved sensitivity in both HIV-negative (difference 49%, 95%CI 37% to 59%, p<0.0001) and HIV-positive patients with CD4+ T-cell counts >200cells/µL (difference 59%, 95%CI 32% to 75%, p = 0.0009). This report is the first to show improved performance of a urine TB LAM test for HIV-negative patients in a high TB burden setting. We also offer potential assay refinement solutions that may further improve sensitivity and specificity.

Controlling metabolic flux by toehold-mediated strand displacement.

To maximize desired products in engineered cellular factories it is often necessary to optimize metabolic flux. While a number of works have focused on metabolic pathway enhancement through genetic regulators and synthetic scaffolds, these approaches require time-intensive design and optimization with limited versatility and capacity for scale-up. Recently, nucleic-acid nanotechnology has emerged as an encouraging approach to overcome these limitations and create systems for modular programmable control of metabolic flux. Using toehold-mediated strand displacement (TMSD), nucleic acid constructs can be made into dynamic devices that recognize specific biomolecular triggers for conditional control of gene regulation as well as design of dynamic synthetic scaffolds. This review will consider the various approaches that have been used thus far to control metabolic flux using toehold-gated devices.

Urban driven phenotypic changes: empirical observations and theoretical implications for eco-evolutionary feedback.

Emerging evidence that cities drive micro-evolution raises the question of whether rapid urbanization of Earth might impact ecosystems by causing systemic changes in functional traits that regulate urban ecosystems' productivity and stability. Intraspecific trait variation-variation in organisms' morphological, physiological or behavioural characteristics stemming from genetic variability and phenotypic plasticity-has significant implications for ecological functions such as nutrient cycling and primary productivity. While it is well established that changes in ecological conditions can drive evolutionary change in species' traits that, in turn, can alter ecosystem function, an understanding of the reciprocal and simultaneous processes associated with such interactions is only beginning to emerge. In urban settings, the potential for rapid trait change may be exacerbated by multiple selection pressures operating simultaneously. This paper reviews evidence on mechanisms linking urban development patterns to rapid phenotypic changes, and differentiates phenotypic changes for which there is evidence of micro-evolution versus phenotypic changes which may represent plasticity. Studying how humans mediate phenotypic trait changes through urbanization could shed light on fundamental concepts in ecological and evolutionary theory. It can also contribute to our understanding of eco-evolutionary feedback and provide insights for maintaining ecosystem function over the long term.This article is part of the themed issue 'Human influences on evolution, and the ecological and societal consequences'.