Emerging sexually transmitted viral infections: 2. Review of Zika virus disease.

A sudden increase in the number of newborn infants with microcephaly in Brazil in 2015 brought Zika virus (ZIKV), a less-known infection, to public attention. The rapid increase in the number of cases across the Americas and the devastating complications of infection with ZIKV highlighted the gravity of the situation. Within a relatively short period of time, our knowledge of this infection has significantly increased. This includes the realisation that ZIKV can be sexually transmitted. The aim of the present article is to provide a concise summary on this novel sexually transmitted infection linked to human birth defects and Guillain-Barre Syndrome. According to World Health Organization, individuals living outside areas of ZIKV mosquito transmission where one or both partners have been exposed to ZIKV should abstain from sex or have sex with condoms for at least six months after the last day of possible exposure.

Phenotype of CNTNAP1: a study of patients demonstrating a specific severe congenital hypomyelinating neuropathy with survival beyond infancy.

CHN is genetically heterogeneous and its genetic basis is difficult to determine on features alone. CNTNAP1 encodes CASPR, integral in the paranodal junction high molecular mass complex. Nineteen individuals with biallelic variants have been described in association with severe congenital hypomyelinating neuropathy, respiratory compromise, profound intellectual disability and death within the first year. We report 7 additional patients ascertained through exome sequencing. We identified 9 novel CNTNAP1 variants in 6 families: three missense variants, four nonsense variants, one frameshift variant and one splice site variant. Significant polyhydramnios occurred in 6/7 pregnancies. Severe respiratory compromise was seen in 6/7 (tracheostomy in 5). A complex neurological phenotype was seen in all patients who had marked brain hypomyelination/demyelination and profound developmental delay. Additional neurological findings included cranial nerve compromise: orobulbar dysfunction in 5/7, facial nerve weakness in 4/7 and vocal cord paresis in 5/7. Dystonia occurred in 2/7 patients and limb contractures in 5/7. All had severe gastroesophageal reflux, and a gastrostomy was required in 5/7. In contrast to most previous reports, only one patient died in the first year of life. Protein modelling was performed for all detected CNTNAP1 variants. We propose a genotype-phenotype correlation, whereby hypomorphic missense variants partially ameliorate the phenotype, prolonging survival. This study suggests that biallelic variants in CNTNAP1 cause a distinct recognisable syndrome, which is not caused by other genes associated with CHN. Neonates presenting with this phenotype will benefit from early genetic definition to inform clinical management and enable essential genetic counselling for their families.

A CACNA1D mutation in a patient with persistent hyperinsulinaemic hypoglycaemia, heart defects, and severe hypotonia.

Congenital hyperinsulinaemic hypoglycaemia (HH) can occur in isolation or it may present as part of a wider syndrome. For approximately 40%-50% of individuals with this condition, sequence analysis of the known HH genes identifies a causative mutation. Identifying the underlying genetic aetiology in the remaining cases is important as a genetic diagnosis will inform on recurrence risk, may guide medical management and will provide valuable insights into ß-cell physiology. We sequenced the exome of a child with persistent diazoxide-responsive HH, mild aortic insufficiency, severe hypotonia, and developmental delay as well as the unaffected parents. This analysis identified a de novo mutation, p.G403D, in the proband's CACNA1D gene. CACNA1D encodes the main L-type voltage-gated calcium channel in the pancreatic ß-cell, a key component of the insulin secretion pathway. The p.G403D mutation had been reported previously as an activating mutation in an individual with primary hyper-aldosteronism, neuromuscular abnormalities, and transient hypoglycaemia. Sequence analysis of the CACNA1D gene in 60 further cases with HH did not identify a pathogenic mutation. Identification of an activating CACNA1D mutation in a second patient with congenital HH confirms the aetiological role of CACNA1D mutations in this disorder. A genetic diagnosis is important as treatment with a calcium channel blocker may be an option for the medical management of this patient.

Analysis of cell-free fetal DNA for non-invasive prenatal diagnosis in a family with neonatal diabetes.

AIMS: An early genetic diagnosis of neonatal diabetes guides clinical management and results in improved treatment in ~ 40% of patients. In the offspring of individuals with neonatal diabetes, a prenatal diagnosis allows accurate estimation of the risk of developing diabetes and, eventually, the most appropriate treatment for the baby. In this study, we performed non-invasive prenatal genetic testing for a fetus at risk of inheriting a paternal KCNJ11 p.R201C mutation causing permanent neonatal diabetes. METHODS: A droplet digital polymerase chain reaction assay was used to detect the presence of the mutation in cell-free circulating DNA (cfDNA) extracted from maternal plasma at 12 and 16 weeks' gestation. RESULTS: The mutation was not detected in the cfDNA samples, suggesting that the fetus had not inherited the KCNJ11 mutation. The fetal DNA fraction was estimated at 6.2% and 10.7%, which is above the detection limit of the assay. The result was confirmed by Sanger sequencing after the baby's birth, confirming that the baby's risk of developing neonatal diabetes was reduced to that of the general population. CONCLUSIONS: We report the first case of non-invasive prenatal testing in a family with neonatal diabetes. A prenatal diagnosis in families at high risk of monogenic diabetes informs both prenatal and postnatal management. Although the clinical impact of this novel technology still needs to be assessed, its implementation in clinical practice (including cases at risk of inheriting mutations from the mother) will likely have a positive impact upon the clinical management of families affected by monogenic diabetes.

Improved genetic testing for monogenic diabetes using targeted next-generation sequencing.

AIMS/HYPOTHESIS: Current genetic tests for diagnosing monogenic diabetes rely on selection of the appropriate gene for analysis according to the patient's phenotype. Next-generation sequencing enables the simultaneous analysis of multiple genes in a single test. Our aim was to develop a targeted next-generation sequencing assay to detect mutations in all known MODY and neonatal diabetes genes. METHODS: We selected 29 genes in which mutations have been reported to cause neonatal diabetes, MODY, maternally inherited diabetes and deafness (MIDD) or familial partial lipodystrophy (FPLD). An exon-capture assay was designed to include coding regions and splice sites. A total of 114 patient samples were tested--32 with known mutations and 82 previously tested for MODY (n = 33) or neonatal diabetes (n = 49) but in whom a mutation had not been found. Sequence data were analysed for the presence of base substitutions, small insertions or deletions (indels) and exonic deletions or duplications. RESULTS: In the 32 positive controls we detected all previously identified variants (34 mutations and 36 polymorphisms), including 55 base substitutions, ten small insertions or deletions and five partial/whole gene deletions/duplications. Previously unidentified mutations were found in five patients with MODY (15%) and nine with neonatal diabetes (18%). Most of these patients (12/14) had mutations in genes that had not previously been tested. CONCLUSIONS/INTERPRETATION: Our novel targeted next-generation sequencing assay provides a highly sensitive method for simultaneous analysis of all monogenic diabetes genes. This single test can detect mutations previously identified by Sanger sequencing or multiplex ligation-dependent probe amplification dosage analysis. The increased number of genes tested led to a higher mutation detection rate.

Utility of therapeutic drug monitoring in the management of HIV-infected pregnant women in receipt of lopinavir.

The pharmacokinetics of antiretroviral drugs in pregnancy is poorly understood. We reviewed the use of therapeutic drug monitoring (TDM) in clinical settings to document plasma concentrations of lopinavir during pregnancy and investigated how clinicians acted upon TDM results. A retrospective review was carried out of all HIV-infected pregnant women taking boosted lopinavir-based highly active antiretroviral therapy (HAART) at five National Health Service (NHS) centres in the UK between May 2004 and March 2007. Seventy-three women in receipt of lopinavir were identified, of whom 89% had plasma lopinavir concentrations above the suggested minimum recommended for wild-type HIV. Initial TDM results prompted dosage change in 10% and assessment of adherence and/or pharmacist review in 11%. TDM was repeated in 29%. TDM can play an important role in the clinical management of HIV-positive pregnant women, allowing informed dose modification and an alternative measure of adherence.

Microdosimetry of inhomogeneous radon progeny distributions in bronchial airways.

A Monte Carlo code, initially developed for the calculation of microdosimetric spectra for alpha particles in cylindrical airways, has been extended to allow the computation (i) of additional microdosimetric parameters and (ii) for realistic exposure conditions in human bronchial airways with respect to surface activity distribution and airway geometry. The objective of the present study was to investigate the effects of non-uniform distributions of radon progeny activities in bronchial airways on cellular energy deposition parameters. Significant variations of hit frequencies, doses and microscopic energy deposition patterns were observed for epithelial cell nuclei, depending strongly on the assumed activity distributions. Thus, epithelial cells located at different positions in a given bronchial airway may experience a wide range of biological responses. The results obtained suggest that the hit frequency may be the primary physical parameter for alpha particles, supplemented by microdosimetric single event spectra, to be related to biological effects for chronic low level exposures.

Energy deposition, cellular radiation effects and lung cancer risk by radon progeny alpha particles.

Slowing down spectra, LET spectra, hit probabilities, and radiation doses were simulated for the interaction of single 218Po and 214Po alpha particles with sensitive basal and secretory cell nuclei in the bronchial epithelium of human and rat lungs for defined exposure conditions. Probabilities per unit track length for transformation, derived from in vitro experiments with C3H 10T1/2 cells, were used to estimate transformation probabilities for randon progeny alpha particles in basal and secretory cells. Different weighting schemes were assumed to relate cellular hit probabilities, doses and transformation probabilities, obtained for different cell depths and airway generations, to lung cancer risk per unit exposure. In vitro transformation and in vivo lung cancer incidence were simulated by a state-vector model which provides a stochastic formulation of dose-rate dependent cellular transitions related to formation of double strand breaks, repair, inactivation, stimulated mitosis and promotion through loss of intercellular communication.

Discharging older patients from home care: who decides and when?

Who is involved in the decisions to discharge patients from home care and how much lead-in time for planning is observed? How does the process of discharging patients from home care differ by reason for discharge? These questions are addressed in this study of 383 Medicare-funded patients discharged from home care agencies in central Ohio.

Regulation of human and mouse procathepsin E gene expression.

Cathepsin E is an intracellular aspartic proteinase that is considered to have a number of physiological roles including antigen processing. Quantitation of procathepsin E mRNA by LightCyclertrade mark technology indicated that the gene was transcribed in lung but not in kidney of both human and mouse origin. In contrast, the transcript was present in mouse spleen and alveolar macrophages but not in the counterpart tissue/cells from humans. Regulation of human and mouse procathepsin E gene expression was shown not to be influenced by the extent of CpG methylation but depended on the recognition of potential binding motifs in each promoter region by transcription factors such as GATA1, PU1 and YY1, as revealed by functional analysis using a series of promoter/luciferase reporter gene fusion constructs. Thus the extent to which the procathepsin E gene is expressed in a particular cell type may depend on the balance between the effects produced by positive-acting, cell-specific transcription factors such as GATA1 and PU1 and the negative influence of the ubiquitous YY1 factor. In this way, the relative abundance and influence of general and cell-specific transcription factors can govern the production of cathepsin E and thereby account for the sporadic cell and tissue distribution of this enzyme in different species.

Human cytomegalovirus mediates cell cycle progression through G(1) into early S phase in terminally differentiated cells.

Terminal differentiation of embryonal carcinoma cells and monocytes has been shown to be important for their permissiveness for human cytomegalovirus (HCMV) infection, even though such terminally differentiated cells have withdrawn from the cell cycle and are, essentially, in G(0) arrest. Recently, data from a number of laboratories have shown that productive infection with HCMV of quiescent fibroblasts held reversibly in G(0) of the cell cycle can result in cell cycle progression, which results eventually in cycle arrest. In contrast to quiescent fibroblasts, the effect of HCMV on cells that have withdrawn irreversibly from the cell cycle due to terminal differentiation has not, so far, been addressed. Here, it is shown that, in cells that have arrested in G(0) as a result of terminal differentiation, HCMV is able to induce cell functions associated with progression of the cell cycle through G(1) into early S phase. This progression is correlated with a direct physical and functional interaction between the HCMV 86 kDa major immediate-early protein (IE86) and the cyclin-dependent kinase inhibitor p21(Cip1).

Modeling energy deposition and cellular radiation effects in human bronchial epithelium by radon progeny alpha particles.

Energy deposition and cellular radiation effects arising from the interaction of single 218Po and 214Po alpha particles with basal and secretory cell nuclei were simulated for different target cell depths in the bronchial epithelium of human airway generations 2, 4, 6, and 10. To relate the random chord lengths of alpha particle tracks through spherical cell nuclei to the resulting biological endpoints, probabilities per unit track length for different cellular radiation effects as functions of LET were derived from in vitro experiments. The radiobiological data employed in the present study were inactivation and mutation (mutant frequency at the HPRT gene) in V79 Chinese hamster cells and inactivation and transformation in C3H 10T1/2 cells. Based on computed LET spectra and relative frequencies of target cells, probabilities for transformation, mutation, and cell killing in basal and secretory cells were computed for a lifetime exposure of 20 WLM. While predicted transformation probabilities were about two orders of magnitude higher than mutation probabilities, they were still about two orders of magnitude lower than inactivation probabilities. Furthermore transformation probabilities for basal cells are generally higher than those for secretory cells, and 214Po alpha particles are primarily responsible for transformations in bronchial target cells.

Analysis of protein-protein interactions during cytomegalovirus infection.

A key area in the study of infection by cytomegalovirus (CMV), or that of any other virus, is to gain an understanding of the manner in which viral proteins interact with those of the host cell. The most widely used method to identify interactions between viral and cellular proteins in the infected cell is that of co-immunoprecipitation; lysates from infected cells are treated with antibody which recognises, say, a viral protein of interest, and the resulting immune complexes are then screened for the presence of a cellular protein of interest: the presence of the second protein in the immune complexes is indicative of an interaction between the two proteins in vivo. However, such interaction need not necessarily be direct, as immunoprecipitation of a viral protein that could interact with a single component of a multiprotein complex might be expected to co-precipitate all the proteins in that complex. Therefore assays might be said to demonstrate protein-protein associations in the cell, rather than direct interactions. The resolving power of co-immunoprecipitations is also limited in other respects. First, the success of the assay relies heavily on the quality of the immunological reagents used; it is also possible that antibody binding will actually disrupt the interaction to be studied.

A consistent set of neutron kerma coefficients from thermal to 150 MeV for biologically important materials.

Neutron cross sections for nonelastic and elastic reactions on a range of elements have been evaluated for incident energies up to 150 MeV. These cross sections agree well with experimental cross section data for charged-particle production as well as neutron and photon production. Therefore they can be used to determine kerma coefficients for calculations of energy deposition by neutrons in matter. Methods used to evaluate the neutron cross sections above 20 MeV, using nuclear model calculations and experimental data, are described. Below 20 MeV, the evaluated cross sections from the ENDF/B-VI library are adopted. Comparisons are shown between the evaluated charged-particle production cross sections and measured data. Kerma coefficients are derived from the neutron cross sections, for major isotopes of H, C, N, O, Al, Si, P, Ca, Fe, Cu, W, Pb, and for ICRU-muscle, A-150 tissue-equivalent plastic, and other compounds important for treatment planning and dosimetry. Numerous comparisons are made between our kerma coefficients and experimental kerma coefficient data, to validate our results, and agreement is found to be good. An important quantity in neutron dosimetry is the kerma coefficient ratio of ICRU-muscle to A-150 plastic. When this ratio is calculated from our kerma coefficient data, and averaged over the neutron energy spectra for higher-energy clinical therapy beams [three p (68) + Be beams, and a d (48.5) + Be beam], a value of 0.94 +/- 0.03 is obtained. Kerma ratios for water to A-150 plastic, and carbon to oxygen, are also compared with measurements where available.

Nuclear data for radiotherapy: presentation of a new ICRU report and IAEA initiatives.

An ICRU report entitled "Nuclear Data for Neutron and Proton Radiotherapy and for Radiation Protection" is in preparation. The present paper presents an overview of this report, along with examples of some of the results obtained for evaluated nuclear cross sections and kerma coefficients. These cross sections are evaluated using a combination of measured data and the GNASH nuclear model code for elements of importance for biological, dosimetric, beam modification and shielding purposes. In the case of hydrogen both R-matrix and phase-shift scattering theories are used. In the report neutron cross sections and kerma coefficients will be presented up to 150 MeV and proton cross sections up to 250 MeV. An IAEA Consultants' Meeting was also convened to examine the "Status of Nuclear Data needed for Radiation Therapy and Existing Data Development Activities in Member States". Recommendations were made regarding future endeavours.

Functional analysis of the Huntington's disease (HD) gene promoter.

The basis for the highly specific neuronal vulnerability seen in Huntington's disease (HD) has not been determined. Recent studies have demonstrated that variation in HD protein expression occurs in the striatum, with affected regions showing increased HD immunoreactivity. Experiments in HD and SCA1 transgenic mice suggest a correlation between phenotypic severity and expression of the mutant transgene. To gain insights into control of HD gene expression, and to investigate the possibility of cell-cell differences in transcription, we have analysed the 5' upstream region of the HD gene in a neuronal (SK-N-SH) and a non-neuronal (JEG3) cell line. Reporter gene assays demonstrated the presence of a key positive-acting region apparently arising from two Sp1 sites in a tandem repeat acting synergistically. This site is polymorphic, and a single Sp1 site is associated with reduced levels of transcription. These experiments also reveal differences in control of expression between neuronal and non-neuronal cell lines.