Diagnosing congenital cytomegalovirus infections using archived dried blood spots: A 15-year observational study, Portugal.

BACKGROUND: Cytomegalovirus (CMV) is a leading cause of congenital infections. Dried blood spots (DBS) collected in the first week of life (Guthrie cards) have been used in the diagnosis of CMV infection outside the three-week window period following birth. The present work summarizes the results of a 15-year observational study in which DBS from 1388 children were used for a late diagnosis of congenital CMV infection. METHODS: Three groups of children were studied: (i) symptomatic (with symptoms at birth or late sequelae) (N = 779); (ii) born to mothers with serological profile of primary CMV infection (N = 75); (iii) without any information (N = 534). A highly sensitive method of DNA extraction (heat-induced) from the DBS was used. CMV DNA was detected by a nested PCR. RESULTS: In total CMV DNA was detected in 7.5% (104/1388) of children. Symptomatic children showed a low rate of CMV DNA detection (6.7%) than children born to mothers with serological profile of primary CMV infection (13.3%) (p = 0.034). Sensorial hearing loss and encephalopathy were the two clinical manifestations with the highest CMV detection rate (18.3% and 11.1%, respectively). Children whose mothers had a confirmed primary infection showed a higher rate of CMV detection (35.3%) when compared with children whose mothers had a not confirmed primary infection (6.9%) (p = 0.007). CONCLUSION: The present work emphasises the importance of testing DBS in symptomatic children even a long time after symptoms onset and in children born to mothers with serologic diagnosis of maternal primary CMV infection when they miss the diagnosis during the three-week window following birth.

Assessing the Polymer Coil-Globule State from the Very First Spectral Modes.

The determination of the coil-globule transition of a polymer is generally based on the reconstruction of scaling laws, implying the need for samples from a rather wide range of different polymer lengths N. The spectral point of view developed in this work allows for a very parsimonious description of all the aspects of the finite-size coil-globule transition on the basis of the first two Rouse (cosine) modes only, shedding new light on polymer theory and reintroducing well-established spectral methods that have been surprisingly neglected in this field so far. Capturing the relevant configuration path features, the proposed approach enables one to determine the state of a polymer without the need of any information about the polymer length or interaction strength. Importantly, we propose an experimental implementation of our analysis that can be easily performed with modern fluorescent imaging techniques and would allow differentiation of coil or globule conformations by simply recording the positions of at least three discernible loci on the polymer.

Diagnosing congenital Cytomegalovirus infection: don't get rid of dried blood spots.

BACKGROUND: Congenital Cytomegalovirus (cCMV) is a serious global public health issue that can cause irreversible fetal and neonatal congenital defects in symptomatic or asymptomatic newborns at birth. In absence of universal cCMV screening, the retrospective diagnosis of cCMV infection in children is only possible by examining Dried Blood Spot (DBS) samples routinely collected at birth and stored for different time spans depending on the newborn screening regulations in force in different countries. In this article, we summarize the arguments in favor of long-term DBS sample storage for detecting cCMV infection. MAIN TEXT: CMV infection is the most common cause of congenital infection resulting in severe defects and anomalies that can be apparent at birth or develop in early childhood. Sensorineural hearing loss is the most frequent consequence of cCMV infection and may have a late onset and progress in the first years of life. The virological diagnosis of cCMV is essential for clinical research and public health practices. In fact, in order to assess the natural history of CMV infection and distinguish between congenital or acquired infection, children should be diagnosed early by analyzing biological samples collected in the first weeks of life (3 weeks by using viral culture and 2 weeks by molecular assays), which, unfortunately, are not always available for asymptomatic or mildly symptomatic children. It now seems possible to overcome this problem since the CMV-DNA present in the blood of congenitally infected newborns can be easily retrieved from the DBS samples on the Guthrie cards routinely collected and stored within 3 days from birth in the neonatal screening program for genetic and congenital diseases. Early collection and long-term storage are inexpensive methods for long-term bio-banking and are the key points of DBS testing for the detection of cCMV. CONCLUSION: DBS sampling is a reliable and inexpensive method for long-term bio-banking, which enables to diagnose known infectious diseases - including cCMV - as well as diseases not jet recognized, therefore their storage sites and long-term storage conditions and durations should be the subject of political decision-making.

Polymer coil-globule phase transition is a universal folding principle of Drosophila epigenetic domains.

BACKGROUND: Localized functional domains within chromosomes, known as topologically associating domains (TADs), have been recently highlighted. In Drosophila, TADs are biochemically defined by epigenetic marks, this suggesting that the 3D arrangement may be the "missing link" between epigenetics and gene activity. Recent observations (Boettiger et al. in Nature 529(7586):418-422, 2016) provide access to structural features of these domains with unprecedented resolution thanks to super-resolution experiments. In particular, they give access to the distribution of the radii of gyration for domains of different linear length and associated with different transcriptional activity states: active, inactive or repressed. Intriguingly, the observed scaling laws lack consistent interpretation in polymer physics. RESULTS: We develop a new methodology conceived to extract the best information from such super-resolution data by exploiting the whole distribution of gyration radii, and to place these experimental results on a theoretical framework. We show that the experimental data are compatible with the finite-size behavior of a self-attracting polymer. The same generic polymer model leads to quantitative differences between active, inactive and repressed domains. Active domains behave as pure polymer coils, while inactive and repressed domains both lie at the coil-globule crossover. For the first time, the "color-specificity" of both the persistence length and the mean interaction energy are estimated, leading to important differences between epigenetic states. CONCLUSION: These results point toward a crucial role of criticality to enhance the system responsivity, resulting in both energy transitions and structural rearrangements. We get strong indications that epigenetically induced changes in nucleosome-nucleosome interaction can cause chromatin to shift between different activity states.

The Role of Supercoiling in the Motor Activity of RNA Polymerases.

RNA polymerase (RNAP) is, in its elongation phase, an emblematic example of a molecular motor whose activity is highly sensitive to DNA supercoiling. After a review of DNA supercoiling basic features, we discuss how supercoiling controls polymerase velocity, while being itself modified by polymerase activity. This coupling is supported by single-molecule measurements. Physical modeling allows us to describe quantitatively how supercoiling and torsional constraints mediate a mechanical coupling between adjacent polymerases. On this basis, we obtain a description that may explain the existence and functioning of RNAP convoys.

What people know about congenital CMV: an analysis of a large heterogeneous population through a web-based survey.

BACKGROUND: Congenital CMV (cCMV) infection is a serious public health issue due to both its worldwide prevalence and the severe and permanent impairments it causes. However, awareness of this infection is low in the general population and among pregnant women, and it also seems to be generally disregarded by healthcare providers. The identification of factors behind this inadequate level of knowledge could provide a basis for future preventive measures. This study aimed at evaluating awareness of CMV and cCMV infection and its correlation with socio-demographic variables in a general population. METHODS: The survey was carried out by computer-assisted web interviewing (CAWI). A questionnaire was sent via e-mail to the 70,975 individuals who comprised the whole population (students, administrative staff, teaching staff) of Milan University, Italy in 2015. RESULTS: Out of the 10,190 respondents, 5,351 (52.5 %) had already heard of CMV but only 3,216 (31.8 %) knew that this virus could be implicated in congenital infection. Urine and breastfeeding were the least recognized transmission routes for CMV infection; less than half of respondents accurately identified the right symptoms and sequelae caused by cCMV infection. The correct hygienic measures against cCMV infection were identified in percentages ranging from 55.6 to 75 % depending on the measures proposed but about one in three of interviewees deemed those measures unnecessary in the event of a pregnant woman already being CMV seropositive. From the mean knowledge scores the most complete quality of awareness of CMV turned out to be linked to childbearing-age (25-40 year) and with not having children, even if results for non-parents showed less of them having heard of cCMV than parents. CONCLUSION: Our results indicate a limited and confused awareness of cCMV infection in a large, fairly young and well-educated Italian population.

Theory and simulations of toroidal and rod-like structures in single-molecule DNA condensation.

DNA condensation by multivalent cations plays a crucial role in genome packaging in viruses and sperm heads, and has been extensively studied using single-molecule experimental methods. In those experiments, the values of the critical condensation forces have been used to estimate the amplitude of the attractive DNA-DNA interactions. Here, to describe these experiments, we developed an analytical model and a rigid body Langevin dynamics assay to investigate the behavior of a polymer with self-interactions, in the presence of a traction force applied at its extremities. We model self-interactions using a pairwise attractive potential, thereby treating the counterions implicitly. The analytical model allows to accurately predict the equilibrium structures of toroidal and rod-like condensed structures, and the dependence of the critical condensation force on the DNA length. We find that the critical condensation force depends strongly on the length of the DNA, and finite-size effects are important for molecules of length up to 10(5)µm. Our Langevin dynamics simulations show that the force-extension behavior of the rod-like structures is very different from the toroidal ones, so that their presence in experiments should be easily detectable. In double-stranded DNA condensation experiments, the signature of the presence of rod-like structures was not unambiguously detected, suggesting that the polyamines used to condense DNA may protect it from bending sharply as needed in the rod-like structures.

Surveillance of acute flaccid paralysis (AFP) in Lombardy, Northern Italy, from 1997 to 2011 in the context of the national AFP surveillance system.

An Acute Flaccid Paralysis (AFP) surveillance system was set up in Lombardy (Northern Italy) in 1997 in the framework of the national AFP surveillance system, as part of the polio eradication initiative by the World Health Organization (WHO). This surveillance system can now be used to detect Poliovirus (PV) reintroductions from endemic countries. This study aimed at describing the results of the AFP surveillance in Lombardy, from 1997 to 2011.   Overall, 131 AFP cases in Lombardy were reported with a mean annual incidence rate of 0.7/100 000 children <15 years of age (range: 0.3/100 000-1.1/100 000). The sensitivity of the surveillance system was optimal from 2001-2003. The monthly distribution of AFP cases was typical with peaks in November, in January, and in March. The major clinical diagnoses associated with AFP were Guillain-Barré Syndrome (GBS, 40%) and encephalomyelitis/myelitis (13%). According to the virological results, no poliomyelitis cases were caused by wild PV infections, but two Vaccine-Associated Paralytic Paralysis (VAPP) cases were reported in 1997 when the Sabin oral polio vaccine (OPV) was still being administered in Italy. Since a surveillance system is deemed sensitive if at least one case of AFP per 100,000 children <15 years of age is detected each year, our surveillance system needs some improvement and must be maintained until global poliovirus eradication will be declared.

In silico single-molecule manipulation of DNA with rigid body dynamics.

We develop a new powerful method to reproduce in silico single-molecule manipulation experiments. We demonstrate that flexible polymers such as DNA can be simulated using rigid body dynamics thanks to an original implementation of Langevin dynamics in an open source library called Open Dynamics Engine. We moreover implement a global thermostat which accelerates the simulation sampling by two orders of magnitude. We reproduce force-extension as well as rotation-extension curves of reference experimental studies. Finally, we extend the model to simulations where the control parameter is no longer the torsional strain but instead the torque, and predict the expected behavior for this case which is particularly challenging theoretically and experimentally.

DNA topology in chromosomes: a quantitative survey and its physiological implications.

Using a simple geometric model, we propose a general method for computing the linking number of the DNA embedded in chromatin fibers. The relevance of the method is reviewed through the single molecule experiments that have been performed in vitro with magnetic tweezers. We compute the linking number of the DNA in the manifold conformational states of the nucleosome which have been evidenced in these experiments and discuss the functional dynamics of chromosomes in the light of these manifold states.

Protein-DNA electrostatics: toward a new paradigm for protein sliding.

Gene expression and regulation rely on an apparently finely tuned set of reactions between some proteins and DNA. Such DNA-binding proteins have to find specific sequences on very long DNA molecules and they mostly do so in the absence of any active process. It has been rapidly recognized that, to achieve this task, these proteins should be efficient at both searching (i.e., sampling fast relevant parts of DNA) and finding (i.e., recognizing the specific site). A two-mode search and variants of it have been suggested since the 1970s to explain either a fast search or an efficient recognition. Combining these two properties at a phenomenological level is, however, more difficult as they appear to have antagonist roles. To overcome this difficulty, one may simply need to drop the dichotomic view inherent to the two-mode search and look more thoroughly at the set of interactions between DNA-binding proteins and a given DNA segment either specific or nonspecific. This chapter demonstrates that, in doing so in a very generic way, one may indeed find a potential reconciliation between a fast search and an efficient recognition. Although a lot remains to be done, this could be the time for a change of paradigm.

Pulling chromatin apart: Unstacking or Unwrapping?

BACKGROUND: Understanding the mechanical properties of chromatin is an essential step towards deciphering the physical rules of gene regulation. In the past ten years, many single molecule experiments have been carried out, and high resolution measurements of the chromatin fiber stiffness are now available. Simulations have been used in order to link those measurements with structural cues, but so far no clear agreement among different groups has been reached. RESULTS: We revisit here some of the most precise experimental results obtained with carefully reconstituted fibers. CONCLUSIONS: We show that the mechanical properties of the chromatin fiber can be quantitatively accounted for by the stiffness of the DNA molecule and the 3D structure of the chromatin fiber.

Extraction of DNA from dried blood in the diagnosis of congenital CMV infection.

Viral DNA detection in dried blood spotted on filter paper, dried blood spots (DBS), is valuable in the diagnosis of viral infections, with at the moment congenital cytomegalovirus (CMV) being the most common application. CMV detection in clinical samples taken within the first 2-3 weeks after birth differentiates congenital CMV infection from the in general harmless postnatal acquired cytomegalovirus infection. DBS render the possibility to diagnose congenital CMV infection retrospectively, e.g., when late-onset hearing loss, the most frequently encountered symptom of congenital CMV infection, becomes manifest. Additionally, CMV DNA detection in DBS can be of usage in recently advocated newborn screening on congenital CMV infection. The procedure of CMV DNA detection in DBS consists of two separate steps: (1) DNA extraction from the DBS, followed by (2) CMV DNA amplification. Here, we describe two efficient methods for the extraction of DNA from DBS. Sensitivity, specificity, and applicability of the methods for high-throughput usage are discussed.

On the topology of chromatin fibres.

The ability of cells to pack, use and duplicate DNA remains one of the most fascinating questions in biology. To understand DNA organization and dynamics, it is important to consider the physical and topological constraints acting on it. In the eukaryotic cell nucleus, DNA is organized by proteins acting as spools on which DNA can be wrapped. These proteins can subsequently interact and form a structure called the chromatin fibre. Using a simple geometric model, we propose a general method for computing topological properties (twist, writhe and linking number) of the DNA embedded in those fibres. The relevance of the method is reviewed through the analysis of magnetic tweezers single molecule experiments that revealed unexpected properties of the chromatin fibre. Possible biological implications of these results are discussed.

Effective interaction between charged nanoparticles and DNA.

We investigate the effective interaction mediated by salt ions between charged nanoparticles (NPs) and DNA. DNA is modeled as an infinite cylinder with a constant surface charge in an implicit solvent. Monte Carlo simulations are used to compute the free energy of the system described in the framework of the primitive model of electrolytes, which accounts for excluded volumes of salt ions. A mean-field Poisson-Boltzmann theory also allows us to compute the free energy and provides us with explicit formulae for its main characteristics (position and depth of the minimum). We intend here to identify the physical parameters that have a major impact on the NP-DNA interaction, in an attempt to evaluate physico-chemical properties which could play a role in genotoxicity or, which could be exploited for therapeutic use. Thus, we investigate the influence on the effective interaction of: the shape of the nanoparticle, the magnitude of the nanoparticle charge and its distribution, the value of the pH of the solution, the magnitude of Van der Waals interactions depending on the nature of the constitutive material of the NP (metal vs. dielectric). We show that for positively charged concave NPs the effective interaction is repulsive at short distance, so that it presents a minimum at distance from the DNA. This short-range repulsion is specific to indented particles and is a robust property that holds for a large range of materials and charge densities.

Linker histones incorporation maintains chromatin fiber plasticity.

Genomic DNA in eukaryotic cells is organized in supercoiled chromatin fibers, which undergo dynamic changes during such DNA metabolic processes as transcription or replication. Indeed, DNA-translocating enzymes like polymerases produce physical constraints in vivo. We used single-molecule micromanipulation by magnetic tweezers to study the response of chromatin to mechanical constraints in the same range as those encountered in vivo. We had previously shown that under positive torsional constraints, nucleosomes can undergo a reversible chiral transition toward a state of positive topology. We demonstrate here that chromatin fibers comprising linker histones present a torsional plasticity similar to that of naked nucleosome arrays. Chromatosomes can undergo a reversible chiral transition toward a state of positive torsion (reverse chromatosome) without loss of linker histones.

Transcription within condensed chromatin: Steric hindrance facilitates elongation.

During eukaryotic transcription, RNA-polymerase activity generates torsional stress in DNA, having a negative impact on the elongation process. Using our previous studies of chromatin fiber structure and conformational transitions, we suggest that this torsional stress can be alleviated, thanks to a tradeoff between the fiber twist and nucleosome conformational transitions into an activated state named "reversome". Our model enlightens the origin of polymerase pauses, and leads to the counterintuitive conclusion that chromatin-organized compaction might facilitate polymerase progression. Indeed, in a compact and well-structured chromatin loop, steric hindrance between nucleosomes enforces sequential transitions, thus ensuring that the polymerase always meets a permissive nucleosomal state.

Pp65 antigenemia, plasma real-time PCR and DBS test in symptomatic and asymptomatic cytomegalovirus congenitally infected newborns.

BACKGROUND: Many congenitally cytomegalovirus-infected (cCMV) neonates are at risk for severe consequences, even if they are asymptomatic at birth. The assessment of the viral load in neonatal blood could help in identifying the babies at risk of sequelae. METHODS: In the present study, we elaborated the results obtained on blood samples collected in the first two weeks of life from 22 symptomatic and 48 asymptomatic newborns with cCMV diagnosed through urine testing. We evaluated the performances of two quantitative methods (pp65 antigenemia test and plasma Real-time PCR) and the semi-quantitative results of dried blood sample (DBS) test in the aim of identifying a valid method for measuring viral load. RESULTS: Plasma qPCR and DBS tests were positive in 100% of cases, antigenemia in 81%. Only the latter test gave quantitatively different results in symptomatic versus asymptomatic children. qPCR values of 103 copies/ml were found in 52% of newborn. "Strong" DBS test positivity cases had higher median values of both pp65 positive PBL and DNA copies/ml than cases with a "weak" positivity. CONCLUSIONS: As expected antigenemia test was less sensitive than molecular tests and DBS test performed better on samples with higher rates of pp65 positive PBL and higher numbers of DNA copies/ml. The prognostic significance of the results of these tests will be evaluated on completion of the ongoing collection of follow-up data of these children.

Nonspecific DNA-protein interaction: why proteins can diffuse along DNA.

Recent single molecule experiments have reported that DNA binding proteins (DNA-BPs) can diffuse along DNA. This suggests that interactions between proteins and DNA play a role during the target search even far from their specific site on DNA. Here we show by means of Monte Carlo simulations and analytical calculations that there is a counterintuitive repulsion between the two oppositely charged macromolecules at a nanometer range. For the concave shape of DNA-BPs, and for realistic protein charge densities, we find that the DNA-protein interaction free energy has a minimum at a finite surface-to-surface separation, in which proteins can easily slide. When a protein encounters its target, the free energy barrier is completely counterbalanced by the H-bond interaction, thus enabling the sequence recognition.

External quality assessment of cytomegalovirus DNA detection on dried blood spots.

BACKGROUND: Testing for viral DNA in neonatal blood dried on paper (DBS) has proved a valid means of diagnosing congenital CMV infection with both clinical and epidemiological relevance. To assess the quality of the detection of CMV-DNA on DBS in laboratories performing this test a proficiency panel consisting of nine samples with two blood spots on each filter paper was produced and distributed. Six samples were derived from whole blood, negative for CMV DNA and antibody, and spiked with cell-grown CMV Towne in various concentrations (7.3 x 102 - 9.6 x 105 copies/ml), one was a CMV positive clinical specimen (3.9 x 106 copies/ml), and two samples were CMV-negative whole blood. RESULTS: The 27 responding laboratories from 14 countries submitted 33 datasets obtained by means of conventional PCR (n = 5) or real-time PCR (n = 28) technologies. A correct positive result was reported in at least 91% of datasets in samples with a viral load of 8.8 x 104 copies/ml or higher. However only 59% and 12% identified the 9.4 x 103 and 7.3 x 102 copies/ml samples, respectively, correctly as positive. False positive results were reported by 9% of laboratories and in 11% of datasets. CONCLUSION: These results indicate a clear need for improvement of methods as sensitivity and false-positivity still appear to be a major problem in a considerable number of laboratories.