Development of protoporphyrinogen oxidase as an efficient selection marker for Agrobacterium tumefaciens-mediated transformation of maize

In this article, we report the isolation of plant protoporphyrinogen oxidase (PPO) genes and the isolation of herbicide-tolerant mutants. Subsequently, an Arabidopsis double mutant (Y426M + S305L) was used to develop a selectable marker system for Agrobacterium tumefaciens-mediated transformation of maize (Zea mays) and to obtain multiple events tolerant to the PPO family of herbicides. Maize transformants were produced via butafenacil selection using a flexible light regime to increase selection pressure. Butafenacil selection per se did not change transgene copy number distribution relative to other selectable marker systems, but the most tolerant events identified in the greenhouse were more likely to contain multiple copies of the introduced mutant PPO gene. To date, more than 2,500 independent transgenic maize events have been produced using butafenacil selection. The high frequency of A. tumefaciens-mediated transformation via PPO selection enabled us to obtain single-copy transgenic maize lines tolerant to field levels of butafenacil.     

Rcl1 depletion impairs 18S pre-rRNA processing at the A1-site and up-regulates a cohort of ribosome biogenesis genes in zebrafish

Yeast Rcl1 is a potential endonuclease that mediates pre-RNA cleavage at the A₂-site to separate 18S rRNA from 5.8S and 25S rRNAs. However, the biological function of Rcl1 in opisthokonta is poorly defined. Moreover, there is no information regarding the exact positions of 18S pre-rRNA processing in zebrafish. Here, we report that zebrafish pre-rRNA harbours three major cleavage sites in the 5′ETS, namely –477nt (A′-site), –97nt (A₀-site) and the 5′ETS and 18S rRNA link (A₁-site), as well as two major cleavage regions within the ITS1, namely 208–218nt (site 2) and 20–33nt (site E). We also demonstrate that depletion of zebrafish Rcl1 mainly impairs cleavage at the A₁-site. Phenotypically, rcl1^–/– mutants exhibit a small liver and exocrine pancreas and die before 15 days post-fertilization. RNA-seq analysis revealed that the most significant event in rcl1^–/– mutants is the up-regulated expression of a cohort of genes related to ribosome biogenesis and tRNA production. Our data demonstrate that Rcl1 is essential for 18S rRNA maturation at the A₁-site and for digestive organogenesis in zebrafish. Rcl1 deficiency, similar to deficiencies in other ribosome biogenesis factors, might trigger a common mechanism to upregulate the expression of genes responsible for ribosome biogenesis.     

The Nicotine-Evoked Locomotor Response: A Behavioral Paradigm for Toxicity Screening in Zebrafish (Danio rerio) Embryos and Eleutheroembryos Exposed to Methylmercury

This study is an adaptation of the nicotine-evoked locomotor response (NLR) assay, which was originally utilized for phenotype-based neurotoxicity screening in zebrafish embryos. Zebrafish embryos do not exhibit spontaneous swimming until roughly 4 days post-fertilization (dpf), however, a robust swimming response can be induced as early as 36 hours post-fertilization (hpf) by means of acute nicotine exposure (30–240μM). Here, the NLR was tested as a tool for early detection of locomotor phenotypes in 36, 48 and 72 hpf mutant zebrafish embryos of the non-touch-responsive maco strain; this assay successfully discriminated mutant embryos from their non-mutant siblings. Then, methylmercury (MeHg) was used as a proof-of-concept neurotoxicant to test the effectiveness of the NLR assay as a screening tool in toxicology. The locomotor effects of MeHg were evaluated in 6 dpf wild type eleutheroembryos exposed to waterborne MeHg (0, 0.01, 0.03 and 0.1μM). Afterwards, the NLR assay was tested in 48 hpf embryos subjected to the same MeHg exposure regimes. Embryos exposed to 0.01 and 0.03μM of MeHg exhibited significant increases in locomotion in both scenarios. These findings suggest that similar locomotor phenotypes observed in free swimming fish can be detected as early as 48 hpf, when locomotion is induced with nicotine.     

The Influence of Malnutrition and Micronutrient Status on Anemic Risk in Children under 3 Years Old in Poor Areas in China

Background

Malnutrition and anemia affect large numbers of young children living in poor areas of China. Multi-micronutrient deficiencies may be related to the prevalence of anemia in different populations, and identifying the risk factors that render children susceptible to anemia is the first step in combating anemia effectively.

Methods

In this cross-sectional study, a total of 1370 children under 3 years old were selected based on probability proportional to size sampling principles from poor counties of China. Basic characteristics data were collected by questionnaire; then anthropometrics and hemoglobin were measured in the field and anemia prevalence evaluated. Venous blood was drawn from children aged 12–35 months (N = 553) to evaluate micronutrient status. Logistic regression was used to identify the risk factors for children’s anemia.

Results

Among children aged 0–35 months, the prevalence of stunting, low body weight and wasting was 17.5%, 8.6% and 5.1%, respectively, and 25.6% of the children were affected by anemia, with more anemic infants and younger children than older children (P <0.01). There were 26.5%, 12.8%, 14.1% and 20.0% of the children aged 12–35 months affected by iron deficiency, vitamin D deficiency, folic acid deficiency and vitamin B₁₂ deficiency, respectively. For children aged 0–11 months who were breastfed, the mothers’ anemic status was the only factor associated with the child’s anemia (OR = 2.6; 95% CI: 1.2–5.4, P < 0.05). For children aged 12–35 months, multivariate logistic regression indicated that anemia was significantly associated with iron and vitamin B₁₂ deficiency (OR = 5.3; 95% CI: 1.9–14.5, P < 0.01) and monotonous diet (OR = 2.3; 95% CI: 1.1–4.7, P < 0.05) after adjusting for age and gender.

Conclusion

The prevalence of anemia was higher in children under 2 years old and requires urgent intervention. An effective intervention strategy should include iron and vitamin B₁₂ supplements_, improving dietary diversity and controlling breastfeeding mothers'' anemia.     

Disparities and Trends in Indoor Exposure to Secondhand Smoke among U.S. Adolescents: 2000-2009

Introduction

Secondhand smoke (SHS) exposure causes disease and death among nonsmokers. With a plethora of smoke-free legislation implemented and a steady decrease in cigarette consumption noted over the past decade in the U.S., this study assessed trends in indoor SHS exposure among U.S. adolescents in grades 6–12 during 2000–2009.

Methods

Data were obtained from the 2000–2009 National Youth Tobacco Survey – a national survey of U.S. middle and high school students. SHS exposure within an indoor area within the past seven days was self-reported. Trends in indoor SHS exposure during 2000–2009 were assessed overall and by socio-demographic characteristics, using the Wald''s test in a binary logistic regression. Within-group comparisons were performed using chi-squared statistics (p<0.05).

Results

The proportion of U.S. middle and high school students who were exposed to indoor SHS declined from 65.5% in 2000 to 40.5% in 2009 (p<0.05 for linear trend). Significant declines were also observed across all population subgroups. Between 2000 and 2009, prevalence of indoor SHS exposure declined significantly among both middle (58.5% to 34.3%) and high school (71.5% to 45.4%) students. Prevalence of indoor SHS exposure was significantly higher among girls (44.0% in 2009) compared to boys (37.2% in 2009) during each survey year. Similarly, prevalence of indoor SHS exposure during 2000–2009 was highest among non-Hispanic whites (44.2% in 2009) and lowest among non-Hispanic Asians (30.2% in 2009). During each survey year, prevalence was highest among the oldest age group (≥18 years) and lowest among the youngest (9–11 years). Also, prevalence was significantly higher among current cigarette smokers (83.8% in 2009) compared to nonsmokers (34.0% in 2009).

Conclusion

Significant declines in indoor SHS exposure among U.S. middle and high school students occurred during 2000–2009. While the results are encouraging, additional efforts are needed to further reduce youth indoor SHS exposure.     

Major Burden of Severe Anemia from Non-Falciparum Malaria Species in Southern Papua: A Hospital-Based Surveillance Study

Background

The burden of anemia attributable to non-falciparum malarias in regions with Plasmodium co-endemicity is poorly documented. We compared the hematological profile of patients with and without malaria in southern Papua, Indonesia.

Methods and Findings

Clinical and laboratory data were linked for all patients presenting to a referral hospital between April 2004 and December 2012. Data were available on patient demographics, malaria diagnosis, hemoglobin concentration, and clinical outcome, but other potential causes of anemia could not be identified reliably. Of 922,120 patient episodes (837,989 as outpatients and 84,131 as inpatients), a total of 219,845 (23.8%) were associated with a hemoglobin measurement, of whom 67,696 (30.8%) had malaria. Patients with P. malariae infection had the lowest hemoglobin concentration (n = 1,608, mean = 8.93 [95% CI 8.81–9.06]), followed by those with mixed species infections (n = 8,645, mean = 9.22 [95% CI 9.16–9.28]), P. falciparum (n = 37,554, mean = 9.47 [95% CI 9.44–9.50]), and P. vivax (n = 19,858, mean = 9.53 [95% CI 9.49–9.57]); p-value for all comparisons <0.001. Severe anemia (hemoglobin <5 g/dl) was present in 8,151 (3.7%) patients. Compared to patients without malaria, those with mixed Plasmodium infection were at greatest risk of severe anemia (adjusted odds ratio [AOR] 3.25 [95% CI 2.99–3.54]); AORs for severe anaemia associated with P. falciparum, P. vivax, and P. malariae were 2.11 (95% CI 2.00–2.23), 1.87 (95% CI 1.74–2.01), and 2.18 (95% CI 1.76–2.67), respectively, p<0.001. Overall, 12.2% (95% CI 11.2%–13.3%) of severe anemia was attributable to non-falciparum infections compared with 15.1% (95% CI 13.9%–16.3%) for P. falciparum monoinfections. Patients with severe anemia had an increased risk of death (AOR = 5.80 [95% CI 5.17–6.50]; p<0.001). Not all patients had a hemoglobin measurement, thus limitations of the study include the potential for selection bias, and possible residual confounding in multivariable analyses.

Conclusions

In Papua P. vivax is the dominant cause of severe anemia in early infancy, mixed P. vivax/P. falciparum infections are associated with a greater hematological impairment than either species alone, and in adulthood P. malariae, although rare, is associated with the lowest hemoglobin concentration. These findings highlight the public health importance of integrated genus-wide malaria control strategies in areas of Plasmodium co-endemicity. Please see later in the article for the Editors'' Summary     

Peripheral Glia Have a Pivotal Role in the Initial Response to Axon Degeneration of Peripheral Sensory Neurons in Zebrafish

Axon degeneration is a feature of many peripheral neuropathies. Understanding the organismal response to this degeneration may aid in identifying new therapeutic targets for treatment. Using a transgenic zebrafish line expressing a bacterial nitroreductase (Ntr)/mCherry fusion protein in the peripheral sensory neurons of the V, VII, IX, and X cranial nerves, we were able to induce and visualize the pathology of axon degeneration in vivo. Exposure of 4 days post fertilization Ntr larvae to the prodrug metronidazole (Met), which Ntr metabolizes into cytotoxic metabolites, resulted in dose-dependent cell death and axon degeneration. This was limited to the Ntr-expressing sensory neurons, as neighboring glia and motor axons were unaffected. Cell death was rapid, becoming apparent 3–4 hours after Met treatment, and was followed by phagocytosis of soma and axon debris by cells within the nerves and ganglia beginning at 4–5 hours of exposure. Although neutrophils appear to be activated in response to the degenerating neurons, they did not accumulate at the sites of degeneration. In contrast, macrophages were found to be attracted to the sites of the degenerating axons, where they phagocytosed debris. We demonstrated that peripheral glia are critical for both the phagocytosis and inflammatory response to degenerating neurons: mutants that lack all peripheral glia (foxD3^−/−; Ntr) exhibit a much reduced reaction to axonal degeneration, resulting in a dramatic decrease in the clearance of debris, and impaired macrophage recruitment. Overall, these results show that this zebrafish model of peripheral sensory axon degeneration exhibits many aspects common to peripheral neuropathies and that peripheral glia play an important role in the initial response to this process.     

Phosphorylation of SU(VAR)3–9 by the Chromosomal Kinase JIL-1

The histone methyltransferase SU(VAR)3–9 plays an important role in the formation of heterochromatin within the eukaryotic nucleus. Several studies have shown that the formation of condensed chromatin is highly regulated during development, suggesting that SU(VAR)3–9''s activity is regulated as well. However, no mechanism by which this may be achieved has been reported so far. As we and others had shown previously that the N-terminus of SU(VAR)3–9 plays an important role for its activity, we purified interaction partners from Drosophila embryo nuclear extract using as bait a GST fusion protein containing the SU(VAR)3–9 N-terminus. Among several other proteins known to bind Su(VAR)3–9 we isolated the chromosomal kinase JIL-1 as a strong interactor. We show that SU(VAR)3–9 is a substrate for JIL-1 in vitro as well as in vivo and map the site of phosphorylation. These findings may provide a molecular explanation for the observed genetic interaction between SU(VAR)3–9 and JIL-1.     

Labeled microRNA pull-down assay system: an experimental approach for high-throughput identification of microRNA-target mRNAs

We developed a simple, direct and cost-effective approach to search for the most likely target genes of a known microRNA (miRNA) in vitro. We term this method ‘labeled miRNA pull-down (LAMP)’ assay system. Briefly, the pre-miRNA is labeled with digoxigenin (DIG), mixed with cell extracts and immunoprecipitated by anti-DIG antiserum. When the DIG-labeled miRNA and bound mRNA complex are obtained, the total cDNAs are then subcloned and sequenced, or RT–PCR-amplified, to search for the putative target genes of a known miRNA. After successfully identifying the known target genes of Caenorhabditis elegans miRNAs lin-4 and let-7 and zebrafish let-7, we applied LAMP to find the unknown target gene of zebrafish miR-1, which resulted in the identification of hand2. We then confirmed hand2 as a novel target gene of miR-1 by whole-mount in situ hybridization and luciferase reporter gene assay. We further validated this target gene by microarray analysis, and the results showed that hand2 is the top-scoring among 302 predicted putative target genes. We concluded that LAMP is an experimental approach for high-throughput identification of the target gene of known miRNAs from both C. elegans and zebrafish, yielding fewer false positive results than those produced by using only the bioinformatics approach.     

Zebrafish: as an integrative model for twenty-first century toxicity testing

The zebrafish embryo is a useful small model for investigating vertebrate development because of its transparency, low cost, transgenic and morpholino capabilities, conservation of cell signaling, and concordance with mammalian developmental phenotypes. From these advantages, the zebrafish embryo has been considered as an alternative model for traditional in vivo developmental toxicity screening. The use of this organism in conjunction with traditional in vivo developmental toxicity testing has the potential to reduce cost and increase throughput of testing the chemical universe, prioritize chemicals for targeted toxicity testing, generate predictive models of developmental toxicants, and elucidate mechanisms and adverse outcome pathways for abnormal development. This review gives an overview of the zebrafish embryo for pre dictive toxicology and 21st century toxicity testing. Developmental eye defects were selected as an example to evaluate data from the U.S. Environmental Protection Agency's ToxCast program comparing responses in zebrafish embryos with those from pregnant rats and rabbits for a subset of 24 environmental chemicals across >600 in vitro assay targets. Cross-species comparisons implied a common basis for biological pathways associated with neuronal defects, extracellular matrix remodeling, and mitotic arrest.     

A MIV-150/Zinc Acetate Gel Inhibits SHIV-RT Infection in Macaque Vaginal Explants

To extend our observations that single or repeated application of a gel containing the NNRTI MIV-150 (M) and zinc acetate dihydrate (ZA) in carrageenan (CG) (MZC) inhibits vaginal transmission of simian/human immunodeficiency virus (SHIV)-RT in macaques, we evaluated safety and anti-SHIV-RT activity of MZC and related gel formulations ex vivo in macaque mucosal explants. In addition, safety was further evaluated in human ectocervical explants. The gels did not induce mucosal toxicity. A single ex vivo exposure to diluted MZC (1∶30, 1∶100) and MC (1∶30, the only dilution tested), but not to ZC gel, up to 4 days prior to viral challenge, significantly inhibited SHIV-RT infection in macaque vaginal mucosa. MZC''s activity was not affected by seminal plasma. The antiviral activity of unformulated MIV-150 was not enhanced in the presence of ZA, suggesting that the antiviral activity of MZC was mediated predominantly by MIV-150. In vivo administration of MZC and CG significantly inhibited ex vivo SHIV-RT infection (51–62% inhibition relative to baselines) of vaginal (but not cervical) mucosa collected 24 h post last gel exposure, indicating barrier effect of CG. Although the inhibitory effect of MZC (65–74%) did not significantly differ from CG (32–45%), it was within the range of protection (∼75%) against vaginal SHIV-RT challenge 24 h after gel dosing. Overall, the data suggest that evaluation of candidate microbicides in macaque explants can inform macaque efficacy and clinical studies design. The data support advancing MZC gel for clinical evaluation.     

Recovery from Anemia in Patients with Severe Aortic Stenosis Undergoing Transcatheter Aortic Valve Implantation – Prevalence,Predictors and Clinical Outcome

Introduction

Preoperative anemia is common in patients with severe aortic stenosis undergoing transcatheter aortic valve implantation (TAVI) and has been linked to a poorer outcome – including a higher 1-year mortality. The aim of this study was to investigate the impact of successful TAVI on baseline anemia.

Methods

A total of 253 patients who survived at least 1 year following TAVI were included in this study. The prevalence, predictors and clinical outcome of hemoglobin (Hb)-recovery were assessed.

Results

The prevalence of baseline anemia was 49% (n = 124) – recovery from anemia occurred in 40% of the anemic patients (n = 49) at 1 year after TAVI with an increase in mean Hb-level of 1.35 g/dL from baseline. This increase was not related to an improvement in renal function. At multivariate analysis, a high peak gradient (OR 4.82, P = 0.003) was shown to be an independent predictor for Hb-recovery, while blood transfusion (OR 0.31, P = 0.038) and chronic kidney disease (CKD, OR 0.33, P = 0.043) were identified as negative predictors at, respectively, one and two years after TAVI. When compared to patients without baseline anemia, those anemic patients with Hb-recovery had a similar functional improvement (OR 0.98, P = 0.975), whereas those without Hb-recovery had a significantly lower likelihood of functional improvement with ≧2 NYHA classes (OR 0.49, P = 0.034) and a higher likelihood of re-hospitalization within the first year after TAVI (OR 1.91, P = 0.024).

Conclusion

Recovery from anemia occurs in 40% of anemic patients at 1 year after TAVI – mainly in those with a high gradient and without CKD. Blood transfusion was found to have a transient adverse effect on this Hb-recovery. Finally, anemic patients without Hb-recovery experience less functional improvement and have a higher re-hospitalization rate within the first year after TAVI.     

Inhibitors of neutrophil recruitment identified using transgenic zebrafish to screen a natural product library

Cell migration is fundamental to the inflammatory response, but uncontrolled cell migration and excess recruitment of neutrophils and other leukocytes can cause damage to the tissue. Here we describe the use of an in vivo model – the Tg(mpx:GFP)ⁱ¹¹⁴ zebrafish line, in which neutrophils are labelled by green fluorescent protein (GFP) – to screen a natural product library for compounds that can affect neutrophil migratory behaviour. Among 1040 fungal extracts screened, two were found to inhibit neutrophil migration completely. Subfractionation of these extracts identified sterigmatocystin and antibiotic PF1052 as the active components. Using the EZ-TAXIScan chemotaxis assay, both compounds were also found to have a dosage-dependent inhibitory effect on murine neutrophil migration. Furthermore, neutrophils treated with PF1052 failed to form pseudopods and appeared round in shape, suggesting a defect in PI3-kinase (PI3K) signalling. We generated a transgenic neutrophil-specific PtdIns(3,4,5)P₃ (PIP3) reporter zebrafish line, which revealed that PF1052 does not affect the activation of PI3K at the plasma membrane. In human neutrophils, PF1052 neither induced apoptosis nor blocked AKT phosphorylation. In conclusion, we have identified an antibiotic from a natural product library with potent anti-inflammatory properties, and have established the utility of the mpx:GFP transgenic zebrafish for high-throughput in vivo screens for novel inhibitors of neutrophil migration.KEY WORDS: Neutrophil, Recruitment, Migration, Drug screen, Zebrafish     

Severe Anemia in Papua New Guinean Children from a Malaria-Endemic Area: A Case-Control Etiologic Study

Background

There are few detailed etiologic studies of severe anemia in children from malaria-endemic areas and none in those countries with holoendemic transmission of multiple Plasmodium species.

Methodology/Principal Findings

We examined associates of severe anemia in 143 well-characterized Papua New Guinean (PNG) children aged 0.5–10 years with hemoglobin concentration <50 g/L (median [inter-quartile range] 39 [33]–[44] g/L) and 120 matched healthy children (113 [107–119] g/L) in a case-control cross-sectional study. A range of socio-demographic, behavioural, anthropometric, clinical and laboratory (including genetic) variables were incorporated in multivariate models with severe anemia as dependent variable. Consistent with a likely trophic effect of chloroquine or amodiaquine on parvovirus B19 (B19V) replication, B19V PCR/IgM positivity had the highest odds ratio (95% confidence interval) of 75.8 (15.4–526), followed by P. falciparum infection (19.4 (6.7–62.6)), vitamin A deficiency (13.5 (5.4–37.7)), body mass index-for-age z-score <2.0 (8.4 (2.7–27.0)) and incomplete vaccination (2.94 (1.3–7.2)). P. vivax infection was inversely associated (0.12 (0.02–0.47), reflecting early acquisition of immunity and/or a lack of reticulocytes for parasite invasion. After imputation of missing data, iron deficiency was a weak positive predictor (6.4% of population attributable risk).

Conclusions/Significance

These data show that severe anemia is multifactorial in PNG children, strongly associated with under-nutrition and certain common infections, and potentially preventable through vitamin A supplementation and improved nutrition, completion of vaccination schedules, and intermittent preventive antimalarial treatment using non-chloroquine/amodiaquine-based regimens.     

Ubiquitin recognition by FAAP20 expands the complex interface beyond the canonical UBZ domain

FAAP20 is an integral component of the Fanconi anemia core complex that mediates the repair of DNA interstrand crosslinks. The ubiquitin-binding capacity of the FAAP20 UBZ is required for recruitment of the Fanconi anemia complex to interstrand DNA crosslink sites and for interaction with the translesion synthesis machinery. Although the UBZ–ubiquitin interaction is thought to be exclusively encapsulated within the ββα module of UBZ, we show that the FAAP20–ubiquitin interaction extends beyond such a canonical zinc-finger motif. Instead, ubiquitin binding by FAAP20 is accompanied by transforming a disordered tail C-terminal to the UBZ of FAAP20 into a rigid, extended β-loop that latches onto the complex interface of the FAAP20 UBZ and ubiquitin, with the invariant C-terminal tryptophan emanating toward I44^Ub for enhanced binding specificity and affinity. Substitution of the C-terminal tryptophan with alanine in FAAP20 not only abolishes FAAP20–ubiquitin binding in vitro, but also causes profound cellular hypersensitivity to DNA interstrand crosslink lesions in vivo, highlighting the indispensable role of the C-terminal tail of FAAP20, beyond the compact zinc finger module, toward ubiquitin recognition and Fanconi anemia complex-mediated DNA interstrand crosslink repair.     

Xgrip109: A γ Tubulin–Associated Protein with an Essential Role in γ Tubulin Ring Complex (γTuRC) Assembly and Centrosome Function

Previous studies indicate that γ tubulin ring complex (γTuRC) can nucleate microtubule assembly and may be important in centrosome formation. γTuRC contains approximately eight subunits, which we refer to as Xenopus gamma ring proteins (Xgrips), in addition to γ tubulin. We found that one γTuRC subunit, Xgrip109, is a highly conserved protein, with homologues present in yeast, rice, flies, zebrafish, mice, and humans. The yeast Xgrip109 homologue, Spc98, is a spindle–pole body component that interacts with γ tubulin. In vertebrates, Xgrip109 identifies two families of related proteins. Xgrip109 and Spc98 have more homology to one family than the other. We show that Xgrip109 is a centrosomal protein that directly interacts with γ tubulin. We have developed a complementation assay for centrosome formation using demembranated Xenopus sperm and Xenopus egg extract. Using this assay, we show that Xgrip109 is necessary for the reassembly of salt-disrupted γTuRC and for the recruitment of γ tubulin to the centrosome. Xgrip109, therefore, is essential for the formation of a functional centrosome.