RNA profiles reveal signatures of future health and disease in pregnancy.

Maternal morbidity and mortality continue to rise, and pre-eclampsia is a major driver of this burden1. Yet the ability to assess underlying pathophysiology before clinical presentation to enable identification of pregnancies at risk remains elusive. Here we demonstrate the ability of plasma cell-free RNA (cfRNA) to reveal patterns of normal pregnancy progression and determine the risk of developing pre-eclampsia months before clinical presentation. Our results centre on comprehensive transcriptome data from eight independent prospectively collected cohorts comprising 1,840 racially diverse pregnancies and retrospective analysis of 2,539 banked plasma samples. The pre-eclampsia data include 524 samples (72 cases and 452 non-cases) from two diverse independent cohorts collected 14.5 weeks (s.d., 4.5 weeks) before delivery. We show that cfRNA signatures from a single blood draw can track pregnancy progression at the placental, maternal and fetal levels and can robustly predict pre-eclampsia, with a sensitivity of 75% and a positive predictive value of 32.3% (s.d., 3%), which is superior to the state-of-the-art method2. cfRNA signatures of normal pregnancy progression and pre-eclampsia are independent of clinical factors, such as maternal age, body mass index and race, which cumulatively account for less than 1% of model variance. Further, the cfRNA signature for pre-eclampsia contains gene features linked to biological processes implicated in the underlying pathophysiology of pre-eclampsia.

International consensus statement on injury and illness reporting in professional road cycling.

INTRODUCTION: Road cycling is a very common recreational and elite sport. To facilitate consistent reporting of injuries and illness in professional road cycling we want to establish terms, definitions, and methods for injury and illness reporting in elite road cycling epidemiology studies, similar to other sports. AIMS/OBJECTIVES: The aim of this study is to establish an international expert statement for injury and illness reporting in professional road cycling. METHODS: We initially conducted a literature review of the injury and illness epidemiology studies in sport and identified popular terms, definitions, and methods which could be applied to road cycling. These terms were then reviewed by our expert panel, with clarification of terms and definitions and additional terms added, if required. The final consensus statement was then agreed by all authors. RESULTS: The relevant definitions for use in professional road cycling epidemiology studies have been agreed. Injury rates should be reported as per 1,000 hours of cycling training, both in and outdoors, and per 1,000 hours of competition as well as per 1,000 hours of non-cycling training. DISCUSSION: We encourage authors to use this expert paper when undertaking injury and illness epidemiology studies in professional road cycling to increase the rigor of the studies as well as allowing comparability between different road cycling studies and epidemiology studies in other sports.

Author Correction: A dynamic three-step mechanism drives the HIV-1 pre-fusion reaction.

In the version of this article initially published, the label above the top right plot in Fig. 3b (HXB2-Alexa Fluor 488) was incorrect. The correct label is 'HXB2-Alexa Fluor 405'. The error has been corrected in the HTML and PDF versions of the article.

A dynamic three-step mechanism drives the HIV-1 pre-fusion reaction.

Little is known about the intermolecular dynamics and stoichiometry of the interactions of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein with its receptors and co-receptors on the host cell surface. Here we analyze time-resolved HIV-1 Env interactions with T-cell surface glycoprotein CD4 (CD4) and C-C chemokine receptor type 5 (CCR5) or C-X-C chemokine receptor type 4 (CXCR4) on the surface of cells, by combining multicolor super-resolution localization microscopy (direct stochastic optical reconstruction microscopy) with fluorescence fluctuation spectroscopy imaging. Utilizing the primary isolate JR-FL and laboratory HXB2 strains, we reveal the time-resolved stoichiometry of CD4 and CCR5 or CXCR4 in the pre-fusion complex with HIV-1 Env. The HIV-1 Env pre-fusion dynamics for both R5- and X4-tropic strains consists of a three-step mechanism, which seems to differ in stoichiometry. Analyses with the monoclonal HIV-1-neutralizing antibody b12 indicate that the mechanism of inhibition differs between JR-FL and HXB2 Env. The molecular insights obtained here identify assemblies of HIV-1 Env with receptors and co-receptors as potential novel targets for inhibitor design.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software.

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Detecting protein aggregation and interaction in live cells: A guide to number and brightness.

The possibility to detect and quantify protein-protein interactions with good spatial and temporal resolutions in live cells is crucial in biology. Number and brightness is a powerful approach to detect both protein aggregation/desegregation dynamics and stoichiometry in live cells. Importantly, this technique can be applied in commercial set ups: both camera based and laser scanning microscopes. It provides pixel-by-pixel information on protein oligomeric states. If performed with two colours, the technique can retrieve the stoichiometry of the reaction under study. In this review, we discuss the strengths and weaknesses of the technique, stressing which are the correct acquisition parameters for a given microscope, the main challenges in analysis, and the limitations of the technique.

nandb-number and brightness in R with a novel automatic detrending algorithm.

SUMMARY: An R package for performing number and brightness image analysis, with the implementation of a novel automatic detrending algorithm. AVAILABILITY AND IMPLEMENTATION: Available at https://github.com/rorynolan/nandb for all platforms. CONTACT: rnolan@well.ox.ac.uk or spadilla@well.ox.ac.uk. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

On the Whereabouts of HIV-1 Cellular Entry and Its Fusion Ports.

HIV-1 disseminates to diverse tissues through different cell types and establishes long-lived reservoirs. The exact cellular compartment where fusion occurs differs depending on the cell type and mode of viral transmission. This implies that HIV-1 may modulate a number of common host cell factors in different cell types. In this review, we evaluate recent advances on the host cell factors that play an important role in HIV-1 entry and fusion. New insights from restriction factors inhibiting virus-cell fusion in vitro may contribute to the development of future therapeutic interventions. Collectively, novel findings underline the need for potent, host-directed therapies that disrupt the earliest stages of the virus life cycle and preclude the emergence of resistant viral variants.

exampletestr-An easy start to unit testing R packages.

In spite of the utility of unit tests, most R package developers do not write them. exampletestr makes it easier to start writing unit tests by creating shells/skeletons of unit tests based on the examples in the user's package documentation. When completed, these unit tests test whether said examples run  correctly. By combining the functionality of exampletestr with that of covr, having ensured that their examples adequately demonstrate the features of their package, the developer can have much of the work of constructing a comprehensive set of unit tests done for them.

Dynamin-2 Stabilizes the HIV-1 Fusion Pore with a Low Oligomeric State.

One of the key research areas surrounding HIV-1 concerns the regulation of the fusion event that occurs between the virus particle and the host cell during entry. Even if it is universally accepted that the large GTPase dynamin-2 is important during HIV-1 entry, its exact role during the first steps of HIV-1 infection is not well characterized. Here, we have utilized a multidisciplinary approach to study the DNM2 role during fusion of HIV-1 in primary resting CD4 T and TZM-bl cells. We have combined advanced light microscopy and functional cell-based assays to experimentally assess the role of dynamin-2 during these processes. Overall, our data suggest that dynamin-2, as a tetramer, might help to establish hemi-fusion and stabilizes the pore during HIV-1 fusion.

The nature and nurture of cell heterogeneity: accounting for macrophage gene-environment interactions with single-cell RNA-Seq.

BACKGROUND: Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity, despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive, only cataloging heterogeneity at one point in time, and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity, and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages. RESULTS: We introduce the programmable Polaris™ microfluidic lab-on-chip for single-cell sequencing, which performs live-cell imaging while controlling for the culture microenvironment of each cell. Using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of SAMHD1, such as an altered oxidative stress response, have a large paracrine signaling component. Furthermore, we demonstrate single-cell pathway enrichments for cell cycle arrest and APOBEC3G degradation, both associated with the oxidative stress response and altered proteostasis. Interestingly, SAMHD1 and APOBEC3G are both HIV-1 inhibitors ('restriction factors'), with no known co-regulation. CONCLUSION: As single-cell methods continue to mature, so will the ability to move beyond simple 'snapshots' of cell populations towards studying the determinants of population dynamics. By combining single-cell culture, live-cell imaging, and single-cell sequencing, we have demonstrated the ability to study cell phenotypes and microenvironmental influences. It's these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages, for example, react to inflammation and form treatment resistant HIV reservoirs.

The influence of prophylactic antibiotic administration on post-operative morbidity in dental implant surgery. A prospective double blind randomized controlled clinical trial.

OBJECTIVES: A prospective double-blind randomised controlled trial was conducted to test the effect of prophylactic antibiotics on post-operative morbidity and osseointegration of dental implants. MATERIALS AND METHODS: Fifty-five subjects scheduled for implant surgery were enrolled. The patients were randomly assigned to the antibiotic (test group) and placebo (control group). Twenty-seven patients (test group) received 3 g amoxicillin one hour pre-operatively, and 28 patients (control group) received placebo capsules 1 h pre-operatively. No post-operative antibiotics were prescribed. Pain diaries and interference with daily activities diaries were kept by the patients for 1 week post-operatively. Signs of post-operative morbidity (swelling, bruising, suppuration and wound dehiscence) were recorded by the principal investigators at day 2 and day 7 following the operation. Osseointegration was assessed at 2nd stage surgery or 3-4 months post-operatively. RESULTS: The results of this study suggest that the use of prophylactic pre-operative antibiotics may result in higher dental implant survival rates (100% vs. 82%). Five implant failures, one in each of five patients, were reported in the placebo group and none in the antibiotic group (P = 0.0515). No significant differences were found for most of the signs of post-operative morbidity 2 and 7 days post-operatively. Only bruising at 2 days following the operation appeared to be higher in the placebo group (P = 0.0511). Post-operative pain (P = 0.01) and interference with daily activities (P = 0.01) appeared to be significantly lower for the antibiotic group after 7 days. Those patients with implant failure reported higher pain (based on the VAS scores) after 2 days (P = 0.003) and after 7 days (P = 0.0005), higher pain (based on the amount of analgesics used) after 7 days (P = 0.001) and higher interference with daily activities (based on the VAS scores) after 2 days (P = 0.005). CONCLUSIONS: The use of for dental implant surgery may be justified, as it appears to improve implant survival in the short term and also results in less post-operative pain and interference with daily activities. From the results of this study, it appears that prophylactic antibiotics may also be beneficial both in terms of implant survival, especially when the surgical procedure is prolonged due to its difficulty, high number of implants placed or operator's inexperience.