Inferring epidemiological links from deep sequencing data: a statistical learning approach for human, animal and plant diseases

Pathogen sequence data have been exploited to infer who infected whom, by using empirical and model-based approaches. Most of these approaches exploit one pathogen sequence per infected host (e.g. individual, household, field). However, modern sequencing techniques can reveal the polymorphic nature of within-host populations of pathogens. Thus, these techniques provide a subsample of the pathogen variants that were present in the host at the sampling time. Such data are expected to give more insight on epidemiological links than a single sequence per host. In general, a mechanistic viewpoint to transmission and micro-evolution has been followed to infer epidemiological links from these data. Here, we investigate an alternative approach grounded on statistical learning. The idea consists of learning the structure of epidemiological links with a pseudo-evolutionary model applied to training data obtained from contact tracing, for example, and using this initial stage to infer links for the whole dataset. Such an approach has the potential to be particularly valuable in the case of a risk of erroneous mechanistic assumptions, it is sufficiently parsimonious to allow the handling of big datasets in the future, and it is versatile enough to be applied to very different contexts from animal, human and plant epidemiology. This article is part of the theme issue 'Modelling infectious disease outbreaks in humans, animals and plants: approaches and important themes'. This issue is linked with the subsequent theme issue 'Modelling infectious disease outbreaks in humans, animals and plants: epidemic forecasting and control'

Shear waves elastography for assessment of human Achilles tendon's biomechanical properties: an experimental study

Introduction: Achilles tendon is the most frequently ruptured tendon, but its optimal treatment is increasingly controversial. The mechanical properties of the healing tendon should be studied further. Shear waves elastography (SWE) measures the shear modulus, which is proven to be correlated to elastic modulus in animal tendons. The aim of our study was to study whether the shear moduli of human cadaveric Achilles tendon, given by SWE, were correlated with the apparent elastic moduli of those tendons given by tensile tests. Materials and methods: Fourteen cadaveric lower-limbs were studied. An elastographic study of the Achilles tendon (AT) was first done in clinical-like conditions. SWE was performed at three successive levels (0, 3 and 6 cm from tendon insertion) with elastographic probe oriented parallel to tendon fibers, blindly, for three standardized ankle positions (25° plantar flexion, neutral position, and maximal dorsal flexion). The mean shear moduli were collected through blind offline data-analysis. Then, AT with triceps were harvested. They were subjected to tensile tests. A continuous SWE of the Achilles tendon was performed simultaneously. The apparent elastic modulus was obtained from the experimental stress-strain curve, and correlation with shear modulus (given by SWE) was studied. Results: Average shear moduli of harvested AT, given by SWE made an instant before the tensile tests, were significantly correlated with shear moduli of the same AT made at the same level, previously in clinical-like condition (p < 0.05), only in neutral position. There was a statistical correlation (p < 0.005) and a correlation coefficient R² equal to 0.95 ± 0.05, between shear moduli (SWE) and apparent elastic moduli (tensile tests), for 11 tendons (3 tendons were inoperable due to technical error), before a constant disruption in the correlation curves. Discussion: We demonstrated a significant correlation between SWE of Achilles tendon performed in clinicallike conditions (in neutral position) and SWE performed in harvested tendon. We also found a correlation between SWE performed on harvested tendon and apparent elastic moduli obtained with tensile tests (for 11 specimens). As a consequence, we can suppose that SWE of AT in clinical-like conditions is related to tensile tests. To our knowledge, the ability of SWE to reliably assess biomechanical properties of a tendon or muscle was, so far, only demonstrated in animal models. Conclusion: SWE can provide biomechanical information of the human AT non-invasively

Employing epigenetic memory and native instructive stimuli to stimulate iPS-NLC differentiation

Notochordal cells (NCs) are linked to a healthy intervertebral disc (IVD), and are considered a promising candidate for cell-based therapies. However, NCs are scarcely available as they are lost early in life, and attempts at in vitro expansion have failed because NCs lose their specific phenotype. The production of notochordal-like cells (NLCs) from human induced pluripotent stem cells (hiPSCs) is a viable alternative. Therefore, this study aimed to build on the tissue-specific epigenetic memory of hiPSCs derived from IVD-progenitor cells (TIE2+-cells) and the instructive capacity of decellularized notochordal cell-derived matrix (dNCM)2 to improve hiPSC differentiation towards mature, healthy matrix-producing NLCs. hiPSCs were generated from TIE2+-IVD cells of three adult donors. As a comparison donor-matched minimally invasive peripheral blood mononuclear (PBM)-derived iPSCs were used. Firstly, hiPSCs were differentiated into mesendodermal progenitors by Wnt pathway activation (N2B27 medium + 3µM CHIR99021)1 for 2 days. Thereafter, the cells were further driven towards the NC-lineage by transfection with synthetic NOTO mRNA1 and matured by switching to a 3D-cell pellet culture in discogenic medium containing 10ng/mL TGF-β1 or 3mg/mL dNCM until day 28. Read-outs included cell morphology, gene and protein expression and matrix deposition. Both TIE2+- and PBM-cell derived hiPSC showed successful differentiation towards mesendodermal progenitors following Wnt-activation on day 2, indicated by the cells moving out of the colonies after CHIR stimulation. Accordingly, a decreased gene expression of pluripotency markers (OCT4, SOX2, NANOG), and upregulation of Wnt and Nodal signaling (LEF1, NODAL) and mesendodermal markers (FOXA2, TBXT) was detected, compared to mTeSR1 controls. This was confirmed by immuno-stains for FOXA2 and TBXT. On day 3, we detected a significant increase in NOTO mRNA levels in all donor lines after transfection compared to untransfected cell pellets. 3D-pellets of all donor lines showed glycosaminoglycan (GAG)- and collagen type II-rich areas after dNCM- but not TGF-β1-treatment on day 28. This was confirmed with the DMMB-assay, showing a significantly increased GAG content in the 3D-pellets treated with dNCM compared to TGF-β1. Next to that, TIE2+-cell derived iPSC pellets contained a significantly higher GAG content after dNCM-treatment compared to the PBM-cell derived hiPSC pellets. Immunohistochemical evaluation showed a heterogeneous cell population including cells positive for chondrogenic- (ACAN, SOX9), NPC/NC- (panKRT, T), and IVD progenitor- markers (CD24, TIE2). In conclusion, using tissue-specific TIE2+-cell derived hiPSCs combined with dNCM-treatment may allow for an improved differentiation capacity indicated by the increased deposition of GAG and collagen type II-rich matrix. However, the obtained cell population is still very heterogeneous and further transcriptome analysis could unravel whether the 3D-pellets contain cells which were successfully driven towards the notochordal-lineage and how these can be enriched based on unique NC-specific markers. Next to that, delineating which epigenetic features are retained after reprogramming of these two cell lines, could shed light on the observed differences in their differentiation capacity. These insights could be used for further optimization of iPS-NLC differentiation and allow for a more purified population of mature, healthy matrix-producing NLCs. This work was funded by Horizon 2020 (no. 825925) and the Dutch Arthritis Society (LLP22). References 1Colombier, P. et al. (2020). NOTO transcription factor directs human induced pluripotent stem cell-derived mesendoderm progenitors to a notochordal fate. Cells, 9(2), 509. 2Bach, Frances C., et al. "Biologic canine and human intervertebral disc repair by notochordal cell-derived matrix: from bench towards bedside." Oncotarget 9.41 (2018): 26507

A novel cell-free mitochondrial fusion assay amenable for high-throughput screenings of fusion modulators

Abstract Background Mitochondria are highly dynamic organelles whose morphology and position within the cell is tightly coupled to metabolic function. There is a limited list of essential proteins that regulate mitochondrial morphology and the mechanisms that govern mitochondrial dynamics are poorly understood. However, recent evidence indicates that the core machinery that governs mitochondrial dynamics is linked within complex intracellular signalling cascades, including apoptotic pathways, cell cycle transitions and nuclear factor kappa B activation. Given the emerging importance of mitochondrial plasticity in cell signalling pathways and metabolism, it is essential that we develop tools to quantitatively analyse the processes of fission and fusion. In terms of mitochondrial fusion, the field currently relies upon on semi-quantitative assays which, even under optimal conditions, are labour-intensive, low-throughput and require complex imaging techniques. Results In order to overcome these technical limitations, we have developed a new, highly quantitative cell-free assay for mitochondrial fusion in mammalian cells. This assay system has allowed us to establish the energetic requirements for mitochondrial fusion. In addition, our data reveal a dependence on active protein phosphorylation for mitochondrial fusion, confirming emerging evidence that mitochondrial fusion is tightly integrated within the global cellular response to signaling events. Indeed, we have shown that cytosol derived from cells stimulated with different triggers either enhance or inhibit the cell-free fusion reaction. Conclusions The adaptation of this system to high-throughput analysis will provide an unprecedented opportunity to identify and characterize novel regulatory factors. In addition, it provides a framework for a detailed mechanistic analysis of the process of mitochondrial fusion and the various axis of regulation that impinge upon this process in a wide range of cellular conditions. See Commentary: http://www.biomedcentral.com/1741-7007/8/9

Corrigendum to "Use of the nonavalent HPV vaccine in individuals previously fully or partially vaccinated with bivalent or quadrivalent HPV vaccines" [Vaccine 34 (2016) 757–761]

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Estimation of the individual residual risk of cervical cancer after vaccination with the nonavalent HPV vaccine

Background: The nonavalent HPV (9vHPV) vaccine is indicated for active immunisation of individuals from the age of 9 years against cervical, vulvar, vaginal and anal premalignant lesions and cancers causally related to vaccine HPV high risk types 16, 18, 31, 33, 45, 52 and 58, and to the HPV low risk types 6 and 11, causing genital warts. Objective: To estimate the lifetime risk (up to the age of 75 years) for developing cervical cancer after vaccinating a HPV naive girl (e.g. 9 to 12 years old) with the 9vHPV vaccine in the hypothetical absence of cervical cancer screening. Methods: We built Monte Carlo simulation models using historical pre-screening age-specific cancer incidence data and current mortality data from Denmark, Finland, Norway, Sweden and the UK. Estimates of genotype contribution fractions and vaccine efficacy were used to estimate the residual lifetime risk after vaccination assuming lifelong protection. Results: We estimated that, in the hypothetical absence of cervical screening and assuming lifelong protection, 9vHPV vaccination reduced the lifetime cervical cancer and mortality risks 7-fold with a residual lifetime cancer risks ranging from 1/572 (UK) to 1/238 (Denmark) and mortality risks ranging from 1/1488 (UK) to 1/851 (Denmark). After decades of repetitive cervical screenings, the lifetime cervical cancer and mortality risks was reduced between 2- and 4-fold depending on the country. Conclusion: Our simulations demonstrate how evidence can be generated to support decision-making by individual healthcare seekers regarding cervical cancer prevention

Shear wear elastography of the human Achilles tendon: a cadaveric study of factors influencing the repeatability

1. Introduction Achilles tendinopathy is a frequent disease, which can lead to tendon rupture (Hess 2010). Its treatment is controversial, partly due to the actual lack of reliable diagnostic tools in clinical practice. Shear waves elastography (SWE) is a new mode of ultrasound imaging, allowing real-time and in vivo assessment of tendon stiffness. It may facilitate tendinopathy management as this pathology is usually associated with an increase of tendon stiffness (Hess 2010; Aubry et al. 2013). The reproducibility of SWE is good in tissues such as breast (Evans et al. 2010) or liver (Muller et al. 2009), but it varies among different published studies (with variable protocols) on human Achilles tendon (AT). The aim of this study was to assess the reproducibility of human cadaveric AT SWE and to study the influencing factors, in real and optimal conditions 2. Methods 2.1. ‘Clinical practice-like’ protocol Twelve lower limbs of fresh frozen human cadavers (age = 84 ± 5 years, mean ± SD) were examined. The ATs were assessed with SWE (Aixplorer®, Supersonic Imagine, Aix-en-Provence, France), randomly and blindly by three operators. Longitudinal and axial slices were performed at three successive levels (0, 3, and 6 cm from the calcaneal insertion), in three ankle positions (position #1: neutral position, position #2: maximal dorsal flexion, position #3: 25° plantar flexion), maintained by custom-made splints (Figure 1). The longitudinal measurement at the 2nd level was made five times by every operator. The probe was held by the operator and stationed lightly on top of a generous amount of coupling gel, perpendicularly to the skin, motionless during 10 s (time of acquisition of a video clip of data). Data processing was performed blindly by one operator, who positioned the region of interest (ROI) inside the tendon area, in which the mean shear modulus was defined. Repeatability and reproducibility were calculated. 2.2. Comparison to measurements in optimal conditions Then, the limbs were surgically prepared: the ATs (with bone insertions) were sampled and mounted in a testing machine. A standardized load (10 N) was applied, and three SWE measurements were done for each tendon sample, in order to assess the reproducibility. 3. Results and discussion Mean shear modulus values were equal to 290.4 kPa (SD: 49.8), 392.0 kPa (SD: 67.6) and 126.7 kPa (SD: 40.6) for positions #1, 2 and 3, and 47.3 kPa (SD: 18.8) for the tendon samples. In longitudinal slices, our ‘clinical practice-like’ protocol reproducibility was equal to 22.1, 20.7, and 33.4% for ankle positions #1, #2, and #3, and the repeatability was equal to 15.8, 18.5, and 16.3%, respectively. The reproducibility of the tendon samples SWE was equal to 12.8%. Our results are consistent with Aubry, who demonstrated that shear modulus increased during dorsal flexion of the ankle (Aubry et al. 2013). The repeatability of SWE in tendons has been reported as ‘good’ to ‘very good’ in animal tendons through increasing loads (Peltz et al. 2013), but varies among different studies, in human AT in vivo. Turan (Turan et al. 2013) reported intra-observer agreement of 0.77, and inter-observer agreement of 0.79, while Aubry (Aubry et al. 2013) found lower inter-observer reproducibility (intra-class coefficient (ICC) equal to 0.46 and less), as well as Peltz (Peltz et al. 2013) for the repeatability (ICC = 0.42). Our results were better for the repeatability than the reproducibility, as well as for the isolated tendon SWE’s repeatability (considered as the reference value). It can be explained by the time needed to achieve a complete measurement session with three operators, which is longer than a repeatability study session (one operator). Indeed, AT has viscoelastic properties (Brum et al. 2014), such as relaxation (once the tendon has been installed on the splint), which leads to a decreasing of tendon stiffness over time. In our study, we found a mean 23% decrease of shear modulus after 20 min of installation, for position #1, 29% for position #2 (Figure 2), and 19% for position three. 4. Conclusions The repeatability of AT SWE is higher in maximal dorsal flexion, but values decrease faster over time in that position, making it necessary to take every SWE measurements after a constant time once the patient has been positioned. These findings were a prerequisite for the realization of another study, in order to validate SWE in human tendon. This study, which compares shear modulus (SWE) with apparent elastic modulus obtained by simultaneous tensile tests, will be presented soon. Acknowledgements The authors are grateful to the ParisTech BiomecAM chair program on subject-specific musculoskeletal modeling for funding (with the support of ParisTech and Yves Cotrel Foundations, Société Générale, Proteor, and Covea)

Nuclear rupture at sites of high curvature compromises retention of DNA repair factors.

The nucleus is physically linked to the cytoskeleton, adhesions, and extracellular matrix-all of which sustain forces, but their relationships to DNA damage are obscure. We show that nuclear rupture with cytoplasmic mislocalization of multiple DNA repair factors correlates with high nuclear curvature imposed by an external probe or by cell attachment to either aligned collagen fibers or stiff matrix. Mislocalization is greatly enhanced by lamin A depletion, requires hours for nuclear reentry, and correlates with an increase in pan-nucleoplasmic foci of the DNA damage marker γH2AX. Excess DNA damage is rescued in ruptured nuclei by cooverexpression of multiple DNA repair factors as well as by soft matrix or inhibition of actomyosin tension. Increased contractility has the opposite effect, and stiff tumors with low lamin A indeed exhibit increased nuclear curvature, more frequent nuclear rupture, and excess DNA damage. Additional stresses likely play a role, but the data suggest high curvature promotes nuclear rupture, which compromises retention of DNA repair factors and favors sustained damage

Changing climate and outbreaks of forest pest insects in a cold northern country, Finland

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Estimating the delay between host infection and disease (incubation period) and assessing its significance to the epidemiology of plant diseases.

Knowledge of the incubation period of infectious diseases (time between host infection and expression of disease symptoms) is crucial to our epidemiological understanding and the design of appropriate prevention and control policies. Plant diseases cause substantial damage to agricultural and arboricultural systems, but there is still very little information about how the incubation period varies within host populations. In this paper, we focus on the incubation period of soilborne plant pathogens, which are difficult to detect as they spread and infect the hosts underground and above-ground symptoms occur considerably later. We conducted experiments on Rhizoctonia solani in sugar beet, as an example patho-system, and used modelling approaches to estimate the incubation period distribution and demonstrate the impact of differing estimations on our epidemiological understanding of plant diseases. We present measurements of the incubation period obtained in field conditions, fit alternative probability models to the data, and show that the incubation period distribution changes with host age. By simulating spatially-explicit epidemiological models with different incubation-period distributions, we study the conditions for a significant time lag between epidemics of cryptic infection and the associated epidemics of symptomatic disease. We examine the sensitivity of this lag to differing distributional assumptions about the incubation period (i.e. exponential versus Gamma). We demonstrate that accurate information about the incubation period distribution of a pathosystem can be critical in assessing the true scale of pathogen invasion behind early disease symptoms in the field; likewise, it can be central to model-based prediction of epidemic risk and evaluation of disease management strategies. Our results highlight that reliance on observation of disease symptoms can cause significant delay in detection of soil-borne pathogen epidemics and mislead practitioners and epidemiologists about the timing, extent, and viability of disease control measures for limiting economic loss.ML thanks the Institut Technique français de la Betterave industrielle (ITB) for funding this project. CAG and JANF were funded by the UK’s Biotechnology and Biological Sciences Research Council (BBSRC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript