Directed Evolution of a Selective and Sensitive Serotonin Sensor via Machine Learning

Serotonin plays a central role in cognition and is the target of most pharmaceuticals for psychiatric disorders. Existing drugs have limited efficacy; creation of improved versions will require better understanding of serotonergic circuitry, which has been hampered by our inability to monitor serotonin release and transport with high spatial and temporal resolution. We developed and applied a binding-pocket redesign strategy, guided by machine learning, to create a high-performance, soluble, fluorescent serotonin sensor (iSeroSnFR), enabling optical detection of millisecond-scale serotonin transients. We demonstrate that iSeroSnFR can be used to detect serotonin release in freely behaving mice during fear conditioning, social interaction, and sleep/wake transitions. We also developed a robust assay of serotonin transporter function and modulation by drugs. We expect that both machine-learning-guided binding-pocket redesign and iSeroSnFR will have broad utility for the development of other sensors and in vitro and in vivo serotonin detection, respectively

Method for Quantitative Study of Airway Functional Microanatomy Using Micro-Optical Coherence Tomography

We demonstrate the use of a high resolution form of optical coherence tomography, termed micro-OCT (μOCT), for investigating the functional microanatomy of airway epithelia. μOCT captures several key parameters governing the function of the airway surface (airway surface liquid depth, periciliary liquid depth, ciliary function including beat frequency, and mucociliary transport rate) from the same series of images and without exogenous particles or labels, enabling non-invasive study of dynamic phenomena. Additionally, the high resolution of μOCT reveals distinguishable phases of the ciliary stroke pattern and glandular extrusion. Images and functional measurements from primary human bronchial epithelial cell cultures and excised tissue are presented and compared with measurements using existing gold standard methods. Active secretion from mucus glands in tissue, a key parameter of epithelial function, was also observed and quantified

Fluorescent Microangiography Is a Novel and Widely Applicable Technique for Delineating the Renal Microvasculature

Rarefaction of the renal microvasculature correlates with declining kidney function. However, current technologies commonly used for its evaluation are limited by their reliance on endothelial cell antigen expression and assessment in two dimensions. We set out to establish a widely applicable and unbiased optical sectioning method to enable three dimensional imaging and reconstruction of the renal microvessels based on their luminal filling. The kidneys of subtotally nephrectomized (SNx) rats and their sham-operated counterparts were subjected to either routine two-dimensional immunohistochemistry or the novel technique of fluorescent microangiography (FMA). The latter was achieved by perfusion of the kidney with an agarose suspension of fluorescent polystyrene microspheres followed by optical sectioning of 200 µm thick cross-sections using a confocal microscope. The fluorescent microangiography method enabled the three-dimensional reconstruction of virtual microvascular casts and confirmed a reduction in both glomerular and peritubular capillary density in the kidneys of SNx rats, despite an overall increase in glomerular volume. FMA is an uncomplicated technique for evaluating the renal microvasculature that circumvents many of the limitations imposed by conventional analysis of two-dimensional tissue sections

Target 2035-update on the quest for a probe for every protein

Twenty years after the publication of the first draft of the human genome, our knowledge of the human proteome is still fragmented. The challenge of translating the wealth of new knowledge from genomics into new medicines is that proteins, and not genes, are the primary executers of biological function. Therefore, much of how biology works in health and disease must be understood through the lens of protein function. Accordingly, a subset of human proteins has been at the heart of research interests of scientists over the centuries, and we have accumulated varying degrees of knowledge about approximately 65% of the human proteome. Nevertheless, a large proportion of proteins in the human proteome (∼35%) remains uncharacterized, and less than 5% of the human proteome has been successfully targeted for drug discovery. This highlights the profound disconnect between our abilities to obtain genetic information and subsequent development of effective medicines. Target 2035 is an international federation of biomedical scientists from the public and private sectors, which aims to address this gap by developing and applying new technologies to create by year 2035 chemogenomic libraries, chemical probes, and/or biological probes for the entire human proteome

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Immature and Mature Human Astrovirus: Structure, Conformational Changes, and Similarities to Hepatitis E Virus

Human astroviruses (HAstVs) are a major cause of gastroenteritis. HAstV assembles from the structural protein VP90 and undergoes a cascade of proteolytic cleavages. Cleavage to VP70 is required for release of immature particles from cells, and subsequent cleavage by trypsin confers infectivity. We used electron cryomicroscopy and icosahedral image analysis to determine the first experimentally derived, three-dimensional structures of an immature VP70 virion and a fully proteolyzed, infectious virion. Both particles display T=3 icosahedral symmetry and nearly identical solid capsid shells with diameters of ~350Å. Globular spikes emanate from the capsid surface, yielding an overall diameter of ~440Å. While the immature particles display 90 dimeric spikes, the mature capsid only displays 30 spikes, located on the icosahedral 2-fold axes. Loss of the 60 peripentonal spikes likely plays an important role in viral infectivity. In addition, immature HAstV bears a striking resemblance to the structure of hepatitis E virus (HEV)-like particles, as previously predicted from structural similarity of the crystal structure of the astrovirus spike domain with the HEV P-domain [Dong, J., Dong, L., Méndez, E. & Tao, Y. (2011). Crystal structure of the human astrovirus capsid spike. Proc. Natl. Acad. Sci. USA108, 12681–12686]. Similarities between their capsid shells and dimeric spikes and between the sequences of their capsid proteins suggest that these viral families are phylogenetically related and may share common assembly and activation mechanisms. [Display omitted] ► Three‐dimensional electron cryomicroscopy density maps of immature and mature HAstV. ► Mature virions only display 30 of 90 spikes after proteolytic cleavage. ► Immature virions are remarkably similar to HEV