Epidemiological Studies of Pan-Azole Resistant Aspergillus fumigatus Populations Sampled during Tulip Cultivation Show Clonal Expansion with Acquisition of Multi-Fungicide Resistance as Potential Driver.

Pan-azole resistant isolates are found in clinical and environmental Aspergillus fumigatus (Af) populations. Azole resistance can evolve in both settings, with Af directly targeted by antifungals in patients and, in the environment, Af unintendedly exposed to fungicides used for material preservation and plant disease control. Resistance to non-azole fungicides, including methyl benzimidazole carbamates (MBCs), quinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs), has recently been reported. These fungicide groups are not used in medicine but can play an important role in the further spread of pan-azole resistant genotypes. We investigated the multi-fungicide resistance status and the genetic diversity of Af populations sampled from tulip field soils, tulip peel waste and flower compost heaps using fungicide sensitivity testing and a range of genotyping tools, including STRAf typing and sequencing of fungicide resistant alleles. Two major clones were present in the tulip bulb population. Comparisons with clinical isolates and literature data revealed that several common clonal lineages of TR34/L98H and TR46/Y121F/T289A strains that have expanded successfully in the environment have also acquired resistance to MBC, QoI and/or SDHI fungicides. Strains carrying multiple fungicide resistant alleles have a competitive advantage in environments where residues of multiple fungicides belonging to different modes of action are present.

Lack of an Intron in Cytochrome b and Overexpression of Sterol 14α-Demethylase Indicate a Potential Risk for QoI and DMI Resistance Development in Neophysopella spp. on Grapes.

Asian grapevine leaf rust, caused by Neophysopella meliosmae-myrianthae and N. tropicalis, is often controlled by quinone outside inhibitor (QoI) and demethylation inhibitor (DMI) fungicides in Brazil. Here, we evaluated the sensitivity of 55 Neophysopella spp. isolates to pyraclostrobin (QoI) and tebuconazole (DMI). To elucidate the resistance mechanisms, we analyzed the sequences of the cytochrome b (CYTB) and cytochrome P450 sterol 14α-demethylase (CYP51) target proteins of QoI and DMI fungicides, respectively. The CYP51 expression levels were also determined in a selection of isolates. In leaf disc assays, the mean 50% effective concentration (EC50) value for pyraclostrobin was about 0.040 µg/ml for both species. CYTB sequences were identical among all 55 isolates, which did not contain an intron immediately after codon 143. No amino acid substitution was identified at codons 129, 137, and 143. The mean EC50 value for tebuconazole was 0.62 µg/ml for N. tropicalis and 0.46 µg/ml for N. meliosmae-myrianthae, and no CYP51 sequence variation was identified among isolates of the same species. However, five N. meliosmae-myrianthae isolates grew on leaf discs treated at 10 µg/ml tebuconazole, and these were further exposed to tebuconazole selection pressure. Tebuconazole-adapted laboratory isolates of N. meliosmae-myrianthae showed an eight- to 25-fold increase in resistance after four rounds of selection that was not associated with CYP51 target alterations. In comparison with sensitive isolates, CYP51 expression was induced in the presence of tebuconazole in three out of four tebuconazole-adapted isolates tested. These results suggest a potential risk for QoI and DMI resistance development in Neophysopella spp.

Hfq and RNase R Mediate rRNA Processing and Degradation in a Novel RNA Quality Control Process.

RNA quality control pathways are critical for cell survival. Here, we describe a new surveillance process involved in the degradation of highly structured and stable ribosomal RNAs. The results demonstrated that the RNA chaperone Hfq and the 3'-5' exoribonuclease R mediate the elimination of detrimental rRNA fragments and are required for the correct processing of rRNA precursors. Escherichia coli cells lacking both Hfq and RNase R accumulate a high level of 16S- and 23S-derived rRNA fragments. Hfq and RNase R were also shown to participate in the maturation of 16S and 23S rRNA precursors. This correlates with the fact that in the absence of Hfq and RNase R, there are severe ribosome assembly defects and a sharp reduction in 70S ribosome levels. Hfq and RNase R may act independently or in a complex, as protein interaction studies revealed that these RNA-binding proteins can associate. This is the first demonstration that the well-conserved Hfq and RNase R proteins act on common regulatory pathways, unraveling previously unknown mechanisms of rRNA surveillance with important consequences for translation and cell survival.IMPORTANCE Quality control pathways that oversee the quality of stable RNA molecules are critical for the cell. In this work, we demonstrate, for the first time, a functional link between Hfq and RNase R in the processing and degradation of the highly structured rRNAs. These RNA-binding proteins are required for the maturation of 16S and 23S rRNAs and correct ribosome assembly. Furthermore, they participate in the degradation of rRNAs and clearance of toxic rRNA fragments from the cell. Our studies have also shown that Hfq and RNase R can form a complex. In summary, the cooperation between Hfq and RNase R in metabolic pathways of stable RNAs may represent a broader mechanism of RNA quality control, given the high conservation of these RNA-binding proteins throughout evolution.

The Climate-Driven Genetic Diversity Has a Higher Impact on the Population Structure of Plasmopara viticola Than the Production System or QoI Fungicide Sensitivity in Subtropical Brazil.

Downy mildew, caused by Plasmopara viticola, is the main disease affecting vineyards in subtropical Brazil. Here, we collected 94 P. viticola isolates from four organic and conventional vineyards in the two main grape-growing states of Brazil to evaluate the sensitivity to the quinone outside inhibitor (QoI) azoxystrobin by pheno- and genotyping assays. The impact of location, production system and sensitivity to QoI fungicides on the population genetics and structure of P. viticola was determined using 10 microsatellite markers. Cytochrome b sequencing revealed that 28 and 100% of the isolates from vineyards under organic and conventional management carried the G143A mutation, respectively. The G143A mutation was associated with high levels of azoxystrobin resistance. Three out of the 94 isolates analyzed carried the M125I alteration, not previously described in P. viticola, which was associated with a five-fold reduction in azoxystrobin sensitivity compared to wild-type isolates. Haplotype network analysis based on cytochrome b gene sequences suggested that the Brazilian populations are more closely related to the European than the North American population. A total of six haplotypes were identified, with two of them carrying the G143A mutation. Microsatellite analysis revealed high allelic and genotypic variation among the four populations. Population differentiation analyses indicated that state of origin directly influences the population biology of P. viticola, while production system and QoI sensitivity have little effect. Great genetic diversity, sexual reproduction and high levels of admixture were observed in Rio Grande do Sul State. In contrast, populations in São Paulo State were dominated by a few clonal genotypes, and no admixed genotype was detected between the two genetic pools identified in the state. This study raises the hypothesis that winter weather conditions influence the overwinter survival strategy with profound effects in the population biology of P. viticola.

RNA-Binding Proteins Driving the Regulatory Activity of Small Non-coding RNAs in Bacteria.

Small non-coding RNAs (sRNAs) are critical post-transcriptional regulators of gene expression. Distinct RNA-binding proteins (RBPs) influence the processing, stability and activity of bacterial small RNAs. The vast majority of bacterial sRNAs interact with mRNA targets, affecting mRNA stability and/or its translation rate. The assistance of RNA-binding proteins facilitates and brings accuracy to sRNA-mRNA basepairing and the RNA chaperones Hfq and ProQ are now recognized as the most prominent RNA matchmakers in bacteria. These RBPs exhibit distinct high affinity RNA-binding surfaces, promoting RNA strand interaction between a trans-encoding sRNA and its mRNA target. Nevertheless, some organisms lack ProQ and/or Hfq homologs, suggesting the existence of other RBPs involved in sRNA function. Along this line of thought, the global regulator CsrA was recently shown to facilitate the access of an sRNA to its target mRNA and may represent an additional factor involved in sRNA function. Ribonucleases (RNases) can be considered a class of RNA-binding proteins with nucleolytic activity that are responsible for RNA maturation and/or degradation. Presently RNase E, RNase III, and PNPase appear to be the main players not only in sRNA turnover but also in sRNA processing. Here we review the current knowledge on the most important bacterial RNA-binding proteins affecting sRNA activity and sRNA-mediated networks.

Correction to: Isolation and Analysis of Bacterial Ribosomes Through Sucrose Gradient Ultracentrifugation.

This chapter was inadvertently published without including the author Cátia Bárria. The correct authorship for this chapter should have been Ricardo F. dos Santos, Cátia Bárria, Cecília M. Arraiano, and José M. Andrade. And the sentence before the final sentence in the acknowledgement section should have been printed as "R.F.dS. is recipient of an FCT Doctoral fellowship (PD/BD/105733/2014) and Cátia Bárria is recipient of a FCT Post-doctoral grant PTDC/BIA-BQM/28479/2017)". These corrections have been updated in the chapter.

RNA Structure Analysis by Chemical Probing with DMS and CMCT.

RNA structure is important for understanding RNA function and stability within a cell. Chemical probing is a well-established and convenient method to evaluate the structure of an RNA. Several structure-sensitive chemicals can differentiate paired and unpaired nucleotides. This chapter specifically addresses the use of DMS and CMCT. Although exhibiting different affinities, the combination of these two chemical reagents enables screening of all four nucleobases. DMS and CMCT are only reactive with exposed unpaired nucleotides. We have used this method to analyze the effect of the RNA chaperone Hfq on the conformation of the 16S rRNA. The strategy here described may be applied for the study of many other RNA-binding proteins and RNAs.

Isolation and Analysis of Bacterial Ribosomes Through Sucrose Gradient Ultracentrifugation.

Ribosomes are large macromolecular complexes responsible for the translation process. During the course of ribosome biogenesis and protein synthesis, extra-ribosomal factors interact with the ribosome or its subunits to assist in these vital processes. Here we describe a method to isolate and analyze not only bacterial ribosomes but also their associated factors, providing insights into translation regulation. This detailed protocol allows the separation and monitoring of the ribosomal species and their interacting partners along a sucrose density gradient. Simultaneously, fractionation of the gradient allows for the recovery of 70S ribosomes and its subunits enabling a wide range of downstream applications. This protocol can be easily adapted to ribosome-related studies in other species or for separating other macromolecular complexes.

New molecular interactions broaden the functions of the RNA chaperone Hfq.

The RNA chaperone Hfq is an important bacterial post-transcriptional regulator. Most studies on Hfq are focused on the role of this protein on small non-coding RNAs (sRNAs) and messenger RNAs (mRNAs). The most well-characterized function of Hfq is its role as RNA matchmaker, promoting the base-pairing between sRNAs and their mRNA targets. However, novel substrates and previous unrecognized functions of Hfq have now been identified, which expanded the regulatory spectrum of this protein. Hfq was recently found to bind rRNA and act as a new ribosome biogenesis factor, affecting rRNA processing, ribosome assembly, translational efficiency and translational fidelity. Hfq was also found to bind tRNAs, which could provide an additional mechanism for its role on the accuracy of protein synthesis. The list of substrates does not include RNA exclusively since Hfq was shown to bind DNA, playing an important role in DNA compaction. Additionally, Hfq is also capable to establish many protein-protein interactions. Overall, the functions of the RNA-binding protein Hfq have been expanded beyond its function in small RNA-mediated regulation. The identification of additional substrates and new functions provides alternative explanations for the importance of the chaperone Hfq as a global regulator.

Major 3'-5' Exoribonucleases in the Metabolism of Coding and Non-coding RNA.

3'-5' exoribonucleases are key enzymes in the degradation of superfluous or aberrant RNAs and in the maturation of precursor RNAs into their functional forms. The major bacterial 3'-5' exoribonucleases responsible for both these activities are PNPase, RNase II and RNase R. These enzymes are of ancient nature with widespread distribution. In eukaryotes, PNPase and RNase II/RNase R enzymes can be found in the cytosol and in mitochondria and chloroplasts; RNase II/RNase R-like enzymes are also found in the nucleus. Humans express one PNPase (PNPT1) and three RNase II/RNase R family members (Dis3, Dis3L and Dis3L2). These enzymes take part in a multitude of RNA surveillance mechanisms that are critical for translation accuracy. Although active against a wide range of both coding and non-coding RNAs, the different 3'-5' exoribonucleases exhibit distinct substrate affinities. The latest studies on these RNA degradative enzymes have contributed to the identification of additional homologue proteins, the uncovering of novel RNA degradation pathways, and to a better comprehension of several disease-related processes and response to stress, amongst many other exciting findings. Here, we provide a comprehensive and up-to-date overview on the function, structure, regulation and substrate preference of the key 3'-5' exoribonucleases involved in RNA metabolism.

The RNA-binding protein Hfq is important for ribosome biogenesis and affects translation fidelity.

Ribosome biogenesis is a complex process involving multiple factors. Here, we show that the widely conserved RNA chaperone Hfq, which can regulate sRNA-mRNA basepairing, plays a critical role in rRNA processing and ribosome assembly in Escherichia coli Hfq binds the 17S rRNA precursor and facilitates its correct processing and folding to mature 16S rRNA Hfq assists ribosome assembly and associates with pre-30S particles but not with mature 30S subunits. Inactivation of Hfq strikingly decreases the pool of mature 70S ribosomes. The reduction in ribosome levels depends on residues located in the distal face of Hfq but not on residues found in the proximal and rim surfaces which govern interactions with the sRNAs. Our results indicate that Hfq-mediated regulation of ribosomes is independent of its function as sRNA-regulator. Furthermore, we observed that inactivation of Hfq compromises translation efficiency and fidelity, both features of aberrantly assembled ribosomes. Our work expands the functions of the Sm-like protein Hfq beyond its function in small RNA-mediated regulation and unveils a novel role of Hfq as crucial in ribosome biogenesis and translation.

The Role of Ribonucleases and sRNAs in the Virulence of Foodborne Pathogens.

Contaminated food is the source of many severe infections in humans. Recent advances in food science have discovered new foodborne pathogens and progressed in characterizing their biology, life cycle, and infection processes. All this knowledge has been contributing to prevent food contamination, and to develop new therapeutics to treat the infections caused by these pathogens. RNA metabolism is a crucial biological process and has an enormous potential to offer new strategies to fight foodborne pathogens. In this review, we will summarize what is known about the role of bacterial ribonucleases and sRNAs in the virulence of several foodborne pathogens and how can we use that knowledge to prevent infection.

Development of a birthweight standard and comparison with currently used standards. What is a 10th centile?

INTRODUCTION: Fetal growth charts are often used in clinical practice. It is important to understand the usefulness and the pitfalls associated with these tools. Without validation, it is difficult to ascertain if the cutoffs we intend are the ones we actually select. We developed a national standard for birthweight (BW) and compared it with other published reference values. STUDY DESIGN: Multicenter retrospective study. We collected data on live births, including first trimester ultrasound and pathology, from 23 to 42 weeks' gestational age (GA). We used a variation of the lambda (λ), mu (µ), and sigma (σ) method (LMS) to construct and smooth predicted centiles. GA data was plotted and modeled in days from 24 to 42 weeks. Resulting centiles were validated and compared with other published and widely used reference values. Data from both BW and estimated fetal weight was used to validate the model. RESULTS: Data on 661,338 births were collected from 22 institutions, including 71,515 cases with first trimester ultrasound. We excluded preterm cesarean section from analysis, because of a significant bias (up to 18%) on BW and used exclusively first trimester ultrasound dates from 34 to 42 weeks. The standard compares favorably with tables currently in use, both ultrasound and birthweight based. CONCLUSION: The use of first trimester ultrasound limits variability by minimizing some random error sources, such as data introduction and GA errors, while allowing better precision (GA in days). This results in a narrower range in the extreme centiles than other charts. Validation with estimates of fetal weight are sound in second and early third trimester fetuses, because that will be a "real world" usage of this standard. While there are similarities between our series and some international/foreign growth charts, other are unfit to characterize our population. This reinforces the need for validation of standards, and sound methodological practices when doing so.

The role of RNase R in trans-translation and ribosomal quality control.

Gene expression not only depends on the rate of transcription but is also largely controlled at the post-transcriptional level. Translation rate and mRNA decay greatly influence the final protein levels. Surveillance mechanisms are essential to ensure the quality of the RNA and proteins produced. Trans-translation is one of the most important systems in the quality control of bacterial translation. This process guarantees the destruction of abnormal proteins and also leads to degradation of the respective defective RNAs through the action of Ribonuclease R (RNase R). This exoribonuclease hydrolyzes RNAs starting from their 3' end. Besides its involvement in trans-translation, RNase R also participates in the quality control of rRNA molecules involved in ribosomal biogenesis. RNase R is thus emerging as a key factor in ensuring translation accuracy. This review focuses on issues related to the quality control of translation, with special emphasis on the role of RNase R.

Gestational age and fetal growth assessment among obstetricians.

OBJECTIVE: We aimed to characterize gestational age assessment and fetal growth evaluation among obstetricians. METHODS: Observational, cross-sectional study. We applied a questionnaire to obstetrics specialists and residents, during a national congress on obstetrics. RESULTS: Almost all 179 respondents correct gestational age in the first trimester by ultrasound, but 63% only if there is a difference of 2-9 days. Ultrasound at 11-13 weeks was considered more accurate than at 8-10 weeks by 81%, with a higher proportion of specialists choosing correctly the last answer (p = 0.05). One-third of the respondents did not correctly point the error associated with the ultrasound estimation of fetal weight (EFW). Of the 88% who use a growth table, only 32% were able to identify it by publication/author. Ninety-eight percent identify fetal growth restriction risk (FGR) with centiles (10th in 76%) and 73% of doctors diagnose FGR without other pathological findings (10th in 49%). 44% finds that a low EFW centile maintenance (4th to 3rd) is more worrisome than the crossing of two quartiles (75th to 24th). CONCLUSIONS: The role of ultrasound in gestational age assessment and use of EFW use for FGR classification was disparate among participants. EFW and respective centiles may be over relied upon.

The role of RNases in the regulation of small RNAs.

Ribonucleases (RNases) are key factors in the control of biological processes, since they modulate the processing, degradation and quality control of RNAs. This review gives many illustrative examples of the role of RNases in the regulation of small RNAs (sRNAs). RNase E and PNPase have been shown to degrade the free pool of sRNAs. RNase E can also be recruited to cleave mRNAs when they are interacting with sRNAs. RNase III cleaves double-stranded structures, and can cut both the sRNA and its RNA target when they are hybridized. Overall, ribonucleases act as conductors in the control of sRNAs. Therefore, it is very important to further understand their role in the post-transcriptional control of gene expression.

Feasibility and reproducibility of transvaginal, transabdominal, and 3D volume reconstruction sonography for measurement of the corpus callosum at different gestational ages.

OBJECTIVE: To compare the feasibility and reproducibility of transvaginal and transabdominal approaches, and 3D volume reconstruction sonography for measurement of corpus callosum (CC) length at different gestational ages. METHODS: Forty-six normal fetuses were examined by 2D and 3D ultrasound at 23-25, 27-28 and 31-32 weeks of gestation. Direct mid-sagittal views were obtained by either a transabdominal and/or transvaginal approach. 3D reconstructed mid-sagittal views were obtained by 3D multiplanar manipulations and Volume Contrast Imaging in the C-plane technique (VCI-C) from volumes acquired in axial planes. RESULTS: The CC could be measured in 91% of transvaginal acquisitions, in 52% of transabdominal acquisitions, in 92% of multiplanar reconstructions, and in 86% of VCI-C reconstructions. The success rate was independent of gestational age for transvaginal acquisition and slightly dependent on gestational age for 3D reconstruction techniques. Transabdominal acquisition was dependent on gestational age and fetal presentation. Inter- and intra-observer agreement was slightly better for measurements obtained from direct mid-sagittal views with either transvaginal or transabdominal acquisition than in views obtained by volume reconstruction. The reproducibility of measurements taken in reconstructed mid-sagittal views decreased with gestational age. CONCLUSION: 3D volume reconstruction techniques allow visualization and measurement of the CC in a high percentage of cases, with good reproducibility.