Dent and Flint maize diversity panels reveal important genetic potential for increasing biomass production.

KEY MESSAGE: Genetic and phenotypic analysis of two complementary maize panels revealed an important variation for biomass yield. Flowering and biomass QTL were discovered by association mapping in both panels. The high whole plant biomass productivity of maize makes it a potential source of energy in animal feeding and biofuel production. The variability and the genetic determinism of traits related to biomass are poorly known. We analyzed two highly diverse panels of Dent and Flint lines representing complementary heterotic groups for Northern Europe. They were genotyped with the 50 k SNP-array and phenotyped as hybrids (crossed to a tester of the complementary pool) in a western European field trial network for traits related to flowering time, plant height, and biomass. The molecular information revealed to be a powerful tool for discovering different levels of structure and relatedness in both panels. This study revealed important variation and potential genetic progress for biomass production, even at constant precocity. Association mapping was run by combining genotypes and phenotypes in a mixed model with a random polygenic effect. This permitted the detection of significant associations, confirming height and flowering time quantitative trait loci (QTL) found in literature. Biomass yield QTL were detected in both panels but were unstable across the environments. Alternative kinship estimator only based on markers unlinked to the tested SNP increased the number of significant associations by around 40% with a satisfying control of the false positive rate. This study gave insights into the variability and the genetic architectures of biomass-related traits in Flint and Dent lines and suggests important potential of these two pools for breeding high biomass yielding hybrid varieties.

Increase in Streptococcus pneumoniae serotype 3 associated parapneumonic pleural effusion/empyema after the introduction of PCV13 in Germany.

INTRODUCTION: Pediatric pneumococcal pneumonia complicated by parapneumonic pleural effusion/empyema (PPE/PE) remains a major concern despite general immunization with pneumococcal conjugate vaccines (PCVs). METHODS: In a nationwide pediatric hospital surveillance study in Germany we identified 584 children <18 years of age with bacteriologically confirmed PPE/PE from October 2010 to June 2018. Streptococcus pneumoniae was identified by culture and/or PCR of blood samples and/or pleural fluid and serotyped. RESULTS: S. pneumoniae was identified in 256 of 584 (43.8%) children by culture (n = 122) and/or PCR (n = 207). The following pneumococcal serotypes were detected in 114 children: serotype 3 (42.1%), 1 (25.4%), 7F (12.3%), 19A (7.9%), other PCV13 serotypes (4.4%) and non-PCV13 serotypes (7.9%). Between October 2010 and June 2014 serotype 1 (38.1%) and serotype 3 (25.4%) were most prevalent, whereas between July 2014 and June 2018 serotype 3 (62.7%) and non-PCV13 serotypes (15.7%) were dominant. Compared to children with other pneumococcal serotypes, children with serotype 3 associated PPE/PE were younger (median 3.2 years [IQR 2.1-4.3 years] vs. median 5.6 years [IQR 3.8-8.2 years]; p < 0.001) and more frequently admitted to intensive care (43 [89.6%] vs. 48 [73.8%]; p = 0.04). Seventy-six of 114 (66.7%) children with pneumococcal PPE/PE had been vaccinated with pneumococcal vaccines. Thirty-nine of 76 (51.3%) had received a vaccine covering the serotype detected. Thirty of these 39 breakthrough cases were age-appropriately vaccinated with PCV13 and considered vaccine failures, including 26 children with serotype 3, three children with serotype 19A and one child with serotype 1. CONCLUSION: Following the introduction of PCV13 in general childhood vaccination we observed a strong emergence of serotype 3 associated PPE/PE in the German pediatric population, including a considerable number of younger children with serotype 3 vaccine breakthrough cases and failures. Future PCVs should not only cover newly emerging serotypes, but also include a more effective component against serotype 3.

Robust detection and identification of multiple oomycetes and fungi in environmental samples by using a novel cleavable padlock probe-based ligation detection assay.

Simultaneous detection and identification of multiple pathogenic microorganisms in complex environmental samples are required in numerous diagnostic fields. Here, we describe the development of a novel, background-free ligation detection (LD) system using a single compound detector probe per target. The detector probes used, referred to as padlock probes (PLPs), are long oligonucleotides containing asymmetric target complementary regions at both their 5' and 3' ends which confer extremely specific target detection. Probes also incorporate a desthiobiotin moiety and an internal endonuclease IV cleavage site. DNA samples are PCR amplified, and the resulting products serve as potential targets for PLP ligation. Upon perfect target hybridization, the PLPs are circularized via enzymatic ligation, captured, and cleaved, allowing only the originally ligated PLPs to be visualized on a universal microarray. Unlike previous procedures, the probes themselves are not amplified, thereby allowing a simple PLP cleavage to yield a background-free assay. We designed and tested nine PLPs targeting several oomycetes and fungi. All of the probes specifically detected their corresponding targets and provided perfect discrimination against closely related nontarget organisms, yielding an assay sensitivity of 1 pg genomic DNA and a dynamic detection range of 10(4). A practical demonstration with samples collected from horticultural water circulation systems was performed to test the robustness of the newly developed multiplex assay. This novel LD system enables highly specific detection and identification of multiple pathogens over a wide range of target concentrations and should be easily adaptable to a variety of applications in environmental microbiology.

Accurate quantification of microorganisms in PCR-inhibiting environmental DNA extracts by a novel internal amplification control approach using Biotrove OpenArrays.

PCR-based detection assays are prone to inhibition by substances present in environmental samples, thereby potentially leading to inaccurate target quantification or false-negative results. Internal amplification controls (IACs) have been developed to help alleviate this problem but are generally applied in a single concentration, thereby yielding less-than-optimal results across the wide range of microbial gene target concentrations possible in environmental samples (J. Hoorfar, B. Malorny, A. Abdulmawjood, N. Cook, M. Wagner, and P. Fach, J. Clin. Microbiol. 42:1863-1868, 2004). Increasing the number of IACs for each quantitative PCR (qPCR) sample individually, however, typically reduces sensitivity and, more importantly, the reliability of quantification. Fortunately, current advances in high-throughput qPCR platforms offer the possibility of multiple reactions for a single sample simultaneously, thereby allowing the implementation of more than one IAC concentration per sample. Here, we describe the development of a novel IAC approach that is specifically designed for the state-of-the-art Biotrove OpenArray platform. Different IAC targets were applied at a range of concentrations, yielding a calibration IAC curve for each individual DNA sample. The developed IACs were optimized, tested, and validated by using more than 5,000 unique qPCR amplifications, allowing accurate quantification of microorganisms when applied to soil DNA extracts containing various levels of PCR-inhibiting compounds. To our knowledge, this is the first study using a suite of IACs at different target concentrations to monitor PCR inhibition across a wide target range, thereby allowing reliable and accurate quantification of microorganisms in PCR-inhibiting DNA extracts. The developed IAC is ideally suited for high-throughput screenings of, for example, ecological and agricultural samples on next-generation qPCR platforms.

Changes in the incidence and bacterial aetiology of paediatric parapneumonic pleural effusions/empyema in Germany, 2010-2017: a nationwide surveillance study.

OBJECTIVES: Parapneumonic pleural effusions/empyema (PPE/PE) are severe complications of community-acquired pneumonia. We investigated the bacterial aetiology and incidence of paediatric PPE/PE in Germany after the introduction of universal pneumococcal conjugate vaccine (PCV) immunization for infants. METHODS: Children <18 years of age hospitalized with pneumonia-associated PPE/PE necessitating pleural drainage or persisting >7 days were reported to the German Surveillance Unit for Rare Diseases in Childhood between October 2010 and June 2017. All bacteria detected in blood or pleural fluid (by culture/PCR) were included, with serotyping for Streptococcus pneumoniae. RESULTS: The median age of all 1447 PPE/PE patients was 5 years (interquartile range 3-10). In 488 of the 1447 children with PPE/PE (34%), 541 bacteria (>40 species) were detected. Aerobic gram-positive cocci accounted for 469 of 541 bacteria detected (87%); these were most frequently Streptococcus pneumoniae (41%), Streptococcus pyogenes (19%) and Staphylococcus aureus (6%). Serotype 3 accounted for 45% of 78 serotyped S. pneumoniae strains. Annual PPE/PE incidence varied between 14 (95%CI 12-16) and 18 (95%CI 16-21) PPE/PE per million children. Incidence of S. pneumoniae PPE/PE decreased from 3.5 (95%CI 2.5-4.6) per million children in 2010/11 to 1.5 (95%CI 0.9-2.4) in 2013/14 (p 0.002), followed by a re-increase to 2.2 (95%CI 1.5-3.2) by 2016/17 (p 0.205). CONCLUSIONS: In the era of widespread PCV immunization, cases of paediatric PPE/PE were still caused mainly by S. pneumoniae and, increasingly, by S. pyogenes. The re-increase in the incidence of PPE/PE overall and in S. pneumoniae-associated PPE/PE indicates ongoing changes in the bacterial aetiology and requires further surveillance.

An immortalized rat ventral mesencephalic cell line, RTC4, is protective in a rodent model of stroke.

One therapeutic approach to stroke is the transplantation of cells capable of trophic support, reinnervation, and/or regeneration. Previously, we have described the use of novel truncated isoforms of SV40 large T antigen to generate unique cell lines from several primary rodent tissue types. Here we describe the generation of two cell lines, RTC3 and RTC4, derived from primary mesencephalic tissue using a fragment of mutant T antigen, T155c (cDNA) expressed from the RSV promoter. Both lines expressed the glial markers vimentin and S100beta, but not the neuronal markers NeuN, MAP2, or beta-III-tubulin. A screen for secreted trophic factors revealed substantially elevated levels of platelet-derived growth factor (PDGF) in RTC4, but not RTC3 cells. When transplanted into rat cortex, RTC4 cells survived for at least 22 days and expressed PDGF. Because PDGF has been reported to reduce ischemic injury, we examined the protective functions of RTC4 cells in an animal model of stroke. RTC4 or RTC3 cells, or vehicle, were injected into rat cortex 15-20 min prior to a 60-min middle cerebral artery ligation. Forty-eight hours later, animals were sacrificed and the stroke volume was assessed by triphenyl-tetrazolium chloride (TTC) staining. Compared to vehicle or RTC3 cells, transplanted RTC4 cells significantly reduced stroke volume. Overall, we generated a cell line with glial properties that produces PDGF and reduces ischemic injury in a rat model of stroke.

Virtopsy: fatal stab wounds to the skull--the relevance of ante-mortem and post-mortem radiological data in case reconstructions.

Homicides with a survival of several days are not uncommon in forensic routine work. Reconstructions of these cases by autopsy alone are very difficult and may occasionally lead to unsatisfying results. For the medico-legal reconstruction of these cases, ante-mortem and post-mortem radiological imaging should always be included in the expertise. We report on a case of fatal penetrating stab wounds to the skull in which a case reconstruction was only possible by combining the radiological ante- and post-mortem data with the autopsy findings.

Isolation and properties of cyclic AMP-dependent protein kinase from Dictyostelium discoideum.

Cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) in Dictyostelium discoideum was shown to be developmentally controlled. No activity was measured in vegetative cells, but activity increased rapidly during differentiation. A simple procedure for the isolation of the catalytic subunit of the kinase from aggregating cells is presented. The cyclic AMP-dependent holoenzyme could be reconstituted by adding purified D. discoideum cyclic AMP-binding protein. Molecular weight, kinetic parameters, pH dependence and affinity for cyclic AMP were determined for the enzyme. Most properties are similar to those of cyclic AMP-dependent kinase from mammalian cells.

A soluble factor and GTP gamma S are required for Dictyostelium discoideum guanylate cyclase activity.

Amoeba of Dictyostelium discoideum show a rapid, transient cGMP synthesis in response to chemotactic stimulation. Using Mg(2+)-GTP as a substrate, guanylate cyclase (E.C. 4.6.1.2.) activity is found exclusively in the particulate fraction of Dictyostelium cells. Here we show that the activity is dependent on the presence of the non-hydrolysable GTP-analogue GTP gamma S, which itself is only a poor substrate for the enzyme under the prevailing conditions. Evidence is presented that a transient exposure of the enzyme to GTP gamma S is sufficient to constitutively activate the enzyme. GTP gamma S-dependent activity is found to require a factor that can be separated from the enzyme by washing the particulate fraction with low salt buffer. Addition of the soluble cell fraction to these washed membranes restores enzyme activity.

A 2020 vision for American health care.

We enter this century with an unprecedented federal budget surplus-$4.6 trillion over the next 10 years. A substantial portion of the surplus comes from savings in the health care sector. The 1997 Balanced Budget Act cut payments to Medicare providers and raised the premiums for individual beneficiaries, but we overshot the mark. Instead of balancing the budget, we generated a huge surplus. We underestimated the magnitude of Medicare savings. Medicare savings over the period from 1998 to 2007 represent an estimated 15% of the total budget surplus. Fifteen percent of the 10-year budget surplus from 2001 to 2010 comes to $680 billion. We also underestimated the drop-off in Medicaid coverage, as welfare reform took hold. In the year 2000 Medicare and Medicaid outlays were an estimated $104 billion less than projected just 5 years ago-representing an estimated 45% of the budget surplus this year, or about $1 trillion of the 10-year surplus.

Health-compromising behaviors: why do adolescents smoke or drink?: identifying underlying risk and protective factors.

OBJECTIVES: To better understand the motivation for adolescent smoking and drinking and to identify the underlying risk and protective factors associated with these behaviors among adolescents. DESIGN: Cross-sectional, school-based survey of students in grades 5 through 12. PARTICIPANTS: A nationally representative sample of 2574 boys and 2939 girls in grades 7 through 12 from 297 public, private, and parochial schools across the United States who participated in The Commonwealth Fund Survey of the Health of Adolescent Girls and Boys in 1997. MAIN OUTCOME MEASURES: Sex-specific adjusted relative risks (RRs) and 95% confidence intervals (CIs) comparing self-reported regular smokers and regular drinkers by risk and protective factors with adolescents reporting none of these behaviors. RESULTS: Adolescent boys and girls were equally likely to be regular smokers (11.2%). The prevalence rate of regular drinking was only slightly higher for boys (22.4%) than it was for girls (19.3%). The rates of both health-risk behaviors were significantly higher for those reporting risk factors, and the strengths of associations varied by sex. Sex differences also emerged in motivation for engaging in these behaviors. When we adjusted for demographic characteristics, exposure to childhood abuse (RR, 4.1; 95% CI, 2.4-7.0) and stressful life events (RR, 2.4; 95% CI, 1.1-5.4) were strongly associated with increased risk for boys' regular smoking. Similar associations were found for regular drinking. For girls, a history of abuse (RR, 1.8; 95% CI, 1.1-2.8), violence within the family (RR, 2.2; 95% CI, 1.6-3.2), depressive symptoms (RR, 1.6; 95% CI, 1.0-2.4), and stressful life events (RR, 3.1; 95% CI, 1.8-5.6) were significantly associated with increased risk for regular smoking. Similar associations were again found for regular drinking. Parental support was protective against both health-risk behaviors for both sexes. Participation in extracurricular activities was associated with lower risk for regular smoking for boys (RR, 0.4; 95% CI, 0.2-0.7) and for girls (RR, 0.3; 95% CI, 0.2-0.5); however, there was no significant association between drinking behavior and participation in activities. CONCLUSIONS: The increased risk for regular smoking and regular drinking among adolescents with a history of abuse, family violence, depressive symptoms, and stressful life events suggests that routine screening for abuse, violence, and other family experiences should be an essential component of adolescent health care visits. Effective prevention programs to reduce smoking and drinking among adolescents should recognize that health-risk behaviors may be associated with other negative life experiences and that the strength of these associations differs by sex.

Listening to parents. A national survey of parents with young children.

OBJECTIVE: To document the child-rearing needs and pediatric health care experiences of parents with children from birth to 3 years old. DESIGN: A nationally representatives sample of 2017 parents with children younger than 3 years using a 25-minute structured telephone questionnaire. Interviews were completed by 68% of the screened eligible respondents. The margin of sampling error for results at the 95% confidence level was +/- 3 percentage points. RESULTS: Seventy-six percent of children younger than 3 years were reported by parents to be in excellent health; 88% had a regular source of pediatric health care. Seventy-one percent of parents who received special pediatric services rated their child's physician as excellent in providing good health care. Seventy-nine percent of parents reported they could use more information in at least 1 of 6 areas of child rearing, and 53% wanted information in at least 3 areas. Forty-two percent had talked with their child's physician about "nonmedical" concerns; 39% of parents read to or looked at a picture book with their child on a daily basis; 51% of parents set daily routines for meals, naps, and bedtime. Breast-feeding and reading to the child on a daily basis were much more likely if a physician encouraged parents to do so. CONCLUSIONS: Most parents view the pediatric health care system as meeting the physical health needs of their young children. Parents want more information and support on child-rearing concerns, yet pediatric clinicians often fail to discuss nonmedical questions with them. The interventions of pediatric clinicians can positively affect parental behavior. Pediatric practices should consider creative ways to reconstitute and augment their current services and systems of care.

Development of a multiplex AmpliDet RNA assay for simultaneous detection and typing of potato virus Y isolates.

A multiplex AmpliDet RNA assay was developed for the specific detection of potato virus Y (PVY), and for the differentiation of the PVY(N), PVY(O/C) strains and the tuber necrotic isolates (PVY(NTN)). The assay is based on the generic amplification of a region within the coat protein coding region of all known PVY isolates by nucleic acid sequence-based amplification (NASBA) and strain-specific detection by molecular beacons. PVY(NTN)-specific diagnosis is achieved by detecting PVY(N) and PVY(O)-specific sequences flanking a recombination site that is associated with the tuber necrotic pathotype. The assay exhibited good specificity toward the various PVY strains in both single and mixed infections. The technique was validated by the use of 47 PVY isolates originating from six countries. The results of the AmpliDet RNA assay were identical or consistent with those of biological characterisation in the decisive majority of cases.

Development of a multiplex AmpliDet RNA for the simultaneous detection of Potato leafroll virus and Potato virus Y in potato tubers.

A novel isothermal multiplex AmpliDet RNA system is described for the simultaneous amplification and detection of Potato leafroll virus (PLRV) and Potato virus Y (PVY) in seed potatoes. The risk of contamination by carry-over during diagnostic screening is eliminated by performing the reaction in a single closed tube. The viruses present in a sample are identified using differently coloured molecular beacons directed to a selected virus-specific sequence within the amplicon formed during amplification. With this system, as little as 10 fg of purified PLRV or PVY can be detected. The presence of both viruses in a sample is detected by the multiplex assay within a high range of virus concentrations. The reliability of the multiplex assay was compared with the enzyme-linked immunosorbent assay for detection of PLRV- or PVY-antigens in potato tubers. The multiplex assay detected clearly the viruses present originally in the potato tubers in all samples, demonstrating its potential for routine diagnostic work and high-throughput screening.

Direct detection of potato leafroll virus in potato tubers by immunocapture and the isothermal nucleic acid amplification method NASBA.

NASBA, an isothermal amplification method for nucleic acids, was applied to the detection of RNA of potato leafroll virus (PLRV) in a single enzymatic reaction at 41 degrees C. A set of primers was selected from the coat protein open reading frame sequence of PLRV to allow amplification of viral RNA. The NASBA reaction products were visualized after electrophoresis by ethidium bromide or acridine orange staining. The specificity of the amplification products was validated by Northern blot analysis with a PLRV-specific 32P-labelled oligonucleotide probe. The procedure was coupled to immunocapture of PLRV virions from tuber extracts by immobilized antibodies in microtubes. It was possible to discriminate readily by this method between uninfected and primarily PLRV-infected potato tubers. NASBA is suitable for the direct detection of PLRV in potato tubers from primarily infected plants, offering the potential to considerably simplify the inspection of seed-potatoes for virus infection.

Multiplex RT-PCR detection of four aphid-borne strawberry viruses in Fragaria spp. in combination with a plant mRNA specific internal control.

The principal aphid-borne viruses infecting Strawberry (Fragaria spp.) Strawberry crinkle virus (SCV), Strawberry mild yellow edge virus (SMYEV), Strawberry mottle virus (SMoV) and Strawberry vein banding virus (SVBV) can cause serious crop losses. In this paper, a multiplex reverse transcriptase polymerase chain reaction (RT-PCR) method is described for the simultaneous detection of all four viruses in combination with a plant mRNA specific internal control which can be used as an indicator of the effectiveness of the extraction and RT-PCR. In total, 18 strawberry isolates infected naturally were analysed by this method. Every combination of RNA virus was able to be detected and a full complement of all four viruses were found together in three isolates, all taken from wild strawberry (Fragaria chiloensis (L.) Duch.) in Chile. The upper detection limit for the four viruses was at an extract dilution of 1/200. The broad applicability of the RNA specific internal control primers-which produced a PCR fragment of the expected size in 25 of 27 plant species tested-combined with improvements, made in extraction methods described provides potentially a standard method for comparable RT-PCR analyses in a wide variety of plant species.

Universal coverage: building on Medicare and employer financing.

Universal coverage requires a strategy to sever the link between insurance coverage and employment in order to build a more stable insurance base. Universality also will require subsidies to make coverage affordable. Given these realities, the authors evaluate different financing proposals--employer and individual premium mandates and taxes--in terms of equity, affordability, and administrative and political criteria. An expansion of Medicare offers an insurance alternative that takes advantage of existing administrative structures of national scope.

Market failure? Individual insurance markets for older Americans.

This study examines the viability of tax credits and nongroup markets for covering uninsured adults ages fifty to sixty-four. We find that adults in this age group covered by nongroup plans tend to be healthier and wealthier than the average for their peers, yet more of them go without care and experience high medical bills relative to their incomes. Individual-market premiums rise steeply with age in most states and are well above employer-group rates. Costs are likely to be unaffordable for most uninsured older adults, even with large tax credits or in states with community rating. These findings indicate a need to include risk and age pooling to reach the uninsured in this age group.

Embraceable you: how employers influence health plan enrollment.

Based on data from a 1999 national survey of 1,939 randomly selected employers, this paper examines the policies that affect the percentage of workers eligible for and enrolled in a firm's health plan. In 1994, 14 percent of employees worked for a firm offering cash-back payments, but fewer than 1 percent worked for a firm with income-related premiums or deductibles. The strongest determinants of eligibility rates are the waiting time for new employees before they are deemed eligible, and eligibility standards for part-time workers. The primary determinants of the take-up rate are lowest monthly employee contribution for single coverage, and the percentage of the workforce earning less than $20,000 per year.

Insurance matters for low-income adults: results from a five-state survey.

Using survey data from 2,000 low-income adult respondents in each of five states, this DataWatch assesses how uninsured, low-income adults differ from low-income adults who have public or private insurance and how Medicaid expansions have affected insurance coverage patterns across states with different eligibility policies. Findings show that the proportion of low-income uninsured adults is two to three times higher in states that have not expanded Medicaid eligibility beyond relatively low welfare levels. Compared with persons who have either Medicaid or private insurance, uninsured persons report more difficulties getting needed care, are less likely to have a regular provider, and rate the care they do receive as lower quality.