Riboflavin Accumulation and Characterization of cDNAs Encoding Lumazine Synthase and Riboflavin Synthase in Bitter Melon (Momordica charantia)

Riboflavin (vitamin B₂) is the universal precursor of the coenzymes flavin mononucleotide and flavin adenine dinucleotidecofactors that are essential for the activity of a wide variety of metabolic enzymes in animals, plants, and microbes. Using the RACE PCR approach, cDNAs encoding lumazine synthase (McLS) and riboflavin synthase (McRS), which catalyze the last two steps in the riboflavin biosynthetic pathway, were cloned from bitter melon (Momordica charantia), a popular vegetable crop in Asia. Amino acid sequence alignments indicated that <i>McLS</i> and <i>McRS</i> share high sequence identity with other orthologous genes and carry an N-terminal extension, which is reported to be a plastid-targeting sequence. Organ expression analysis using quantitative real-time RT PCR showed that <i>McLS</i> and <i>McRS</i> were constitutively expressed in <i>M. charantia</i>, with the strongest expression levels observed during the last stage of fruit ripening (stage 6). This correlated with the highest level of riboflavin content, which was detected during ripening stage 6 by HPLC analysis. <i>McLS</i> and <i>McRS</i> were highly expressed in the young leaves and flowers, whereas roots exhibited the highest accumulation of riboflavin. The cloning and characterization of McLS and McRS from <i>M. charantia</i> may aid the metabolic engineering of vitamin B₂ in crops

miR-UL148D inhibits RANTES expression during viral infection.

<p>(A) Genomic location of UL150 and miR-UL148D (upper panel). The predicted mature sequence of miR-UL148D (blue) and its mutated residues at wobble position are shown (red) (bottom panel). (B) HFF cells were infected with Toledo-WT, ToledoΔmiR-UL148D or Toledo-Revertant. After extracting miRNAs from the infected cells, the detection of miR-UL148D was assessed using the RNase protection assay as described under “<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002577#s4" target="_blank">Materials and Methods</a>.” 5S rRNA was presented as a loading control stained by ethidium bromide. IE1 and gB gene expression was analyzed by RT-PCR. (C) Growth curves of Toledo-WT, Toledo-ΔmiR-UL148D and Toledo-Revertant. HFF cells were infected with wild-type, mutant and revertant viruses at an MOI of 2. The total number of cell-free viruses in the supernatants of infected cultures was determined by limiting dilution analyses. (D, E) After HFF cells were infected with Toledo-WT (white bars), ToledoΔmiR-UL148D (light gray bars) and Toledo-Revertant (dark gray bars), culture supernatants were harvested at the indicated post-infection time. The accumulated RANTES in supernatants was quantified by ELISA (D). RANTES mRNA level was detected by qRT-PCR (E). NI indicates non-infected control. Similar data were obtained in three independent experiments and the bars indicate mean ±S.D.</p

Inverse correlation between RANTES mRNA and miR-UL148D expression at a later stage during Toledo infection.

<p>(A) HFF cells were infected with WT-AD169 (triangles) or WT-Toledo (circles). RANTES mRNA at indicated time of post-infection (hpi) was quantified by quantitative RT-PCR and data were normalized to GAPDH mRNA (RANTES/GAPDH). Values represent the average ± S.D. of triplicate experiments. (B) The predicted duplex of the 3′UTR of RANTES and miR-UL148D-WT. The indicated free energy value was calculated using RNA22 (<a href="http://cbcsrv.watson.ibm.com/rna22.html" target="_blank">http://cbcsrv.watson.ibm.com/rna22.html</a>). (C) Alignment of miR-UL148D stem-loop sequence of various HCMV clinical strains. We analyzed the genomic sequences of several HCMV clinical strains from GenBank (Toledo GU937742.1, TB40/E AY446866.1, Merlin AY446894.2, JHC HQ380895.1, VR1814 GU179289.1). The conserved sequence (gray) and non-conserved sequence (blue) is shown. Mature miR-UL148D sequence is highlighted in red. (D) The detection of miR-UL148D in Toledo-infected HFF cells was assessed using the RNase protection assay as described under “<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002577#s4" target="_blank">Materials and Methods</a>.” 5S rRNA bands stained with ethidium bromide are presented as a loading control. Using the same RNA samples, the RANTES mRNA and GAPDH mRNA were measured by RT-PCR (lower panel). NI indicates non-infected control.</p

PNA-based antisense oligonucleotides specific to miR-UL148D revert Toledo-induced inhibition of RANTES production.

<p>2 days before HCMV infection, PNA-control or PNA-anti-miR-UL148D was transfected to HFF. After 48 h of infection, culture media and total RNA were analyzed by ELISA (A) and qRT-PCR (B). Down-regulation of miR-UL148D in the presence of PNA was detected by RNase protection assay (C). NI indicates non-infected control. Similar data were obtained in three independent experiments and the bars indicate mean ±S.D.</p

RANTES-3′UTR is a target of miR-UL148D.

<p>(A) 293T cells were co-transfected with the Renilla luciferase expression vector and firefly luciferase vector expressing normal RANTES-3′UTR (RANTES-3′UTR-WT) and either miR-UL148D, miR-UL148D-mut, or siGFP as a negative control (mock, white bar). The relative luciferase activity was calculated as a ratio of firefly to Renilla luciferase activity. (B) Mutations in the 3′-UTR of RANTES and miR-UL148D. (C) After transfection with RANTES-3′UTR-WT (white bars) or RANTES-3′UTR-mut (gray bars), cells were re-transfected with miR-UL148D-WT or miR-UL148D-mut. The cells were lysed, and firefly luciferase activity was measured and normalized to Renilla luciferase. Data represent the mean ± S.E. of four independent experiments (*statistically significant difference between cells expressing miR-control and those expressing miR-UL148D (P<0.05 by Student's t-test, NS; non-specific).</p

Protection afforded by respirators when performing endotracheal intubation using a direct laryngoscope, GlideScope®, and i-gel® device: A randomized trial

<div><p>Emergency physicians are at risk of infection during invasive procedures, and wearing a respirator can reduce this risk. The aim of this study was to determine whether the protection afforded by a respirator during intubation is affected by the type of airway device used. In this randomized crossover study, 26 emergency physicians underwent quantitative fit tests for a N95 respirator (cup-type or fold-type) before and during intubation with a direct laryngoscope, GlideScope®, or i-gel® airway device. The primary outcome was the fit factor value of the respirator and the secondary outcome was the level of acceptable protection provided (percentage of fit factor scores above 100). Compared with the GlideScope and i-gel device, the fit factor values and level of acceptable protection provided were lower when physicians wore the cup-type respirator while intubating using the direct laryngoscope (200 fit factor [152–200] and 200 fit factor [121.25–200] versus 166 fit factor [70–200], 100% and 100% versus 75%, respectively; all <i>P</i> < 0.001). There were no significant differences in the fit factor value or level of acceptable protection provided when the physicians wore the fold-type respirator while intubating using any of the three airway devices (all <i>P</i> > 0.05). The type of airway device used for endotracheal intubation may influence the protective performance of some types of respirators. Emergency physicians should consider the effects of airway device types on fit factor of N95 respirators, when they perform intubation at risk of infection.</p></div

Demographic characteristics and clinical experience.

<p>Demographic characteristics and clinical experience.</p

Fit factor values for the two types of N95 respirator when used for intubation using three types of airway device.

<p>The fit factor value for the valve type was decreased during intubation. There were significant differences between the fit factor values for the cup-type respirator and the fold-type respirator when intubations were performed using the three different airway devices. ∘Outlier value more than 1.5 times the upper or lower limit.</p

Diagram showing the flow of participants through the study.

<p>Diagram showing the flow of participants through the study.</p

The quantitative fit test was performed using the Porta-Count Plus.

<p>(a) Cup-type respirator, which is preformed to a cup shape. (b) Fold-type respirator, which is flexible and free-folded. (c) Macintosh laryngoscope, which has a size 4 curved blade with a Satin Slip stylet. (d) GlideScope® with a GlideRite rigid stylet. (e) I-gel® airway device.</p