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.

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.

Bootstrap Approach To Compare the Slopes of Two Calibrations When Few Standards Are Available.

Comparing the slopes of aqueous-based and standard addition calibration procedures is almost a daily task in analytical laboratories. As usual protocols imply very few standards, sound statistical inference and conclusions are hard to obtain for current classical tests (e.g., the t-test), which may greatly affect decision-making. Thus, there is a need for robust statistics that are not distorted by small samples of experimental values obtained from analytical studies. Several promising alternatives based on bootstrapping are studied in this paper under the typical constraints common in laboratory work. The impact of number of standards, homoscedasticity or heteroscedasticity, three variance patterns, and three error distributions on least-squares fits were considered (in total, 144 simulation scenarios). The Student's t-test is the most valuable procedure when the normality assumption is true and homoscedasticity is present, although it can be highly affected by outliers. A wild bootstrap method leads to average rejection percentages that are closer to the nominal level in almost every situation, and it is recommended for laboratories working with a small number of standards. Finally, it was seen that the Theil-Sen percentile bootstrap statistic is very robust but its rejection percentages depart from the nominal ones (<5%), so its use is not recommended when the number of standards is very small. Finally, a tutorial and free software are given to encourage analytical laboratories to apply bootstrap principles to compare the slopes of two calibration lines.

The crucial role of PNPase in the degradation of small RNAs that are not associated with Hfq.

The transient existence of small RNAs free of binding to the RNA chaperone Hfq is part of the normal dynamic lifecycle of a sRNA. Small RNAs are extremely labile when not associated with Hfq, but the mechanism by which Hfq stabilizes sRNAs has been elusive. In this work we have found that polynucleotide phosphorylase (PNPase) is the major factor involved in the rapid degradation of small RNAs, especially those that are free of binding to Hfq. The levels of MicA, GlmY, RyhB, and SgrS RNAs are drastically increased upon PNPase inactivation in Hfq(-) cells. In the absence of Hfq, all sRNAs are slightly shorter than their full-length species as result of 3'-end trimming. We show that the turnover of Hfq-free small RNAs is growth-phase regulated, and that PNPase activity is particularly important in stationary phase. Indeed, PNPase makes a greater contribution than RNase E, which is commonly believed to be the main enzyme in the decay of small RNAs. Lack of poly(A) polymerase I (PAP I) is also found to affect the rapid degradation of Hfq-free small RNAs, although to a lesser extent. Our data also suggest that when the sRNA is not associated with Hfq, the degradation occurs mainly in a target-independent pathway in which RNase III has a reduced impact. This work demonstrated that small RNAs free of Hfq binding are preferably degraded by PNPase. Overall, our data highlight the impact of 3'-exonucleolytic RNA decay pathways and re-evaluates the degradation mechanisms of Hfq-free small RNAs.

Evaluation of microplastics release from solar water disinfection poly(ethylene terephthalate) and polypropylene containers.

Public health concern associated with the ingestion of microplastics (MPs) released from water packaging materials is increasing. The use of plastic materials for solar disinfection (SODIS) containers has also raised concerns in the SODIS community due to the lack of studies evaluating the presence of MPs in the treated water. In this work, the migration of MPs from poly(ethylene terephthalate, PET) bottles and polypropylene (PP) translucent and transparent jerrycan containers (TJC) into water under natural weathering was investigated using micro-reflectance Fourier Transform Infrared Spectroscopy (µ-FTIR). Containers exposed to sunlight for three months became photodegraded, releasing micro-sized fragments identified as PET, PP and high-density polyethylene (HDPE, from the screw-caps), although with varying degrees of weathering. It is noteworthy that the presence of a clarifying additive in PP formulation did not seem to impact the release of MPs from the containers. The study showed that PP TJC containers released more MPs than PET bottles. Finally, the size of MPs was measured to determine their fate upon ingestion and highlights the need for further studies to understand the safety of these plastic containers for SODIS.

Supervised classification combined with genetic algorithm variable selection for a fast identification of polymeric microdebris using infrared reflectance.

Pollution caused by plastics and, in particular, microplastics has become a source of environmental concern for Society. Their ubiquity, with millions of tons of plastic debris spilled in both land and sea, requires efficient technological improvements in the ways residues are collected, handled, characterized and recycled. For reliable decision-making, dependable chemical information is essential to assess both the nature of the plastics found in the environment and their fate. In this work an efficient method to identify the polymeric composition of microplastic fragments is proposed. It combines infrared reflectance spectra and chemometric methods. A breakthrough result is that the models include polymers weathered under both dry (shoreline) and submerged (in sea water) conditions and, hence, they are very promising as a starting point for eventual practical applications. In addition, no spectral processing is required after the initial measurement. SYNOPSIS: This approach to identify microplastics in aquatic environments combines infrared measurements and multivariate data analysis to fight against (micro)plastic pollution.

Microplastics in surface water of the Bay of Asunción, Paraguay.

This study identified and quantified microplastics in the Bay of Asunción, Paraguay, and its main tributaries. Surface water samples were sieved in duplicate at six locations using stainless-steel sieves (0.3-4.75 mm range), digested employing the Fenton's reaction (Fe-catalysed H2O2 digestion), and floated using NaCl and NaI. Particles were inspected using a microscope and characterized by IR spectrometry. Microplastics were found in all samples; more abundant (p < 0.05) in water from the bay (13.2 ± 13.4 items·m-3) than from the tributaries (1.0 ± 0.5 items·m-3). Most microplastics were common polymers and their abundance was in the order polypropylene > high-density polyethylene > low-density polyethylene, transparent and white. The results were similar to other regional studies and suggested that their main source was single-use packaging, disposed inadequately due to poor garbage collection.

Weathering-independent differentiation of microplastic polymers by reflectance IR spectrometry and pattern recognition.

The presence and effects of microplastics in the environment is being continuously studied, so the need for a reliable approach to ascertain the polymer/s constituting them has increased. To characterize them, infrared (IR) spectrometry is commonly applied, either reflectance or attenuated total reflectance (ATR). A common problem when considering field samples is their weathering and biofouling, which modify their spectra. Hence, relying on spectral matching between the unknown spectrum and spectral databases is largely defective. In this paper, the use of IR spectra combined with pattern recognition techniques (principal components analysis, classification and regression trees and support vector classification) is explored first time to identify a collection of typical polymers regardless of their ageing. Results show that it is possible to identify them using a reduced suite of spectral wavenumbers with coherent chemical meaning. The models were validated using two datasets containing artificially weathered polymers and field samples.

Development of an analytical procedure to analyze microplastics in edible macroalgae using an enzymatic-oxidative digestion.

Besides being food and a refuge to marine species, macroalgae are a powerful and renewable economic resource. However, they may introduce microplastics (MPs) in the trophic chain. We developed a reliable analytical method to characterize and quantify MPs in common and edible macroalgae. Several digestion methods and filters, along with various measurement options, were studied. A new enzymatic-oxidative protocol with a unique final filtration was selected and validated with a mixture of 5 commercial macroalgae (Undaria pinnatifida spp, Porphyra spp, Ulva spp, Laminaria ochroleuca and Himanthalia elongate). Further, it was shown that washing the macroalgae to release MPs is suboptimal and the potential adhesion of MPs to macroalgae was evaluated. A filter subsampling strategy that scans 33.64 % of its surface reduced the time required to characterize <70 µm particles and fibres directly on the 47 mm diameter filter using an IR microscope (1 sample/day).

Enhancing Free Cyanide Photocatalytic Oxidation by rGO/TiO2 P25 Composites.

Graphene-TiO2 composites have been investigated in various photocatalytic reactions showing successful synergy compared to pristine TiO2. In the present work, graphene oxide (GO) was synthesized by the Hummers method and then reduced graphene oxide-TiO2 composites (rGO/TiO2) were obtained by an in situ GO photoreduction route. X-ray diffraction, FTIR, Raman, UV-vis DRS, and photoluminescence were the main characterization techniques. The obtained composites containing 1 and 3 wt.% rGO were evaluated in the cyanide (50 mg/L) oxidation and Au-cyanide complex (300 mg/L) degradation under UV-A light. The composites showed higher photocatalytic activity than TiO2, mainly with the 1% rGO content. Cyanate and gold nanoparticles, deposited on the photocatalyst's surface, were the main byproducts during the photocatalyst assessment. The improved photocatalytic activity of the composites was attributed to a higher rate of electron transfer and a lower rate of charge recombination due to the chemical interaction of rGO with TiO2.

The poly(A)-dependent degradation pathway of rpsO mRNA is primarily mediated by RNase R.

Polyadenylation is an important factor controlling RNA degradation and RNA quality control mechanisms. In this report we demonstrate for the first time that RNase R has in vivo affinity for polyadenylated RNA and can be a key enzyme involved in poly(A) metabolism. RNase II and PNPase, two major RNA exonucleases present in Escherichia coli, could not account for all the poly(A)-dependent degradation of the rpsO mRNA. RNase II can remove the poly(A) tails but fails to degrade the mRNA as it cannot overcome the RNA termination hairpin, while PNPase plays only a modest role in this degradation. We now demonstrate that in the absence of RNase E, RNase R is the relevant factor in the poly(A)-dependent degradation of the rpsO mRNA. Moreover, we have found that the RNase R inactivation counteracts the extended degradation of this transcript observed in RNase II-deficient cells. Elongated rpsO transcripts harboring increasing poly(A) tails are specifically recognized by RNase R and strongly accumulate in the absence of this exonuclease. The 3' oligo(A) extension may stimulate the binding of RNase R, allowing the complete degradation of the mRNA, as RNase R is not susceptible to RNA secondary structures. Moreover, this regulation is shown to occur despite the presence of PNPase. Similar results were observed with the rpsT mRNA. This report shows that polyadenylation favors in vivo the RNase R-mediated pathways of RNA degradation.

Bacterial Response to Oxidative Stress and RNA Oxidation.

Bacteria have to cope with oxidative stress caused by distinct Reactive Oxygen Species (ROS), derived not only from normal aerobic metabolism but also from oxidants present in their environments. The major ROS include superoxide O2 -, hydrogen peroxide H2O2 and radical hydroxide HO•. To protect cells under oxidative stress, bacteria induce the expression of several genes, namely the SoxRS, OxyR and PerR regulons. Cells are able to tolerate a certain number of free radicals, but high levels of ROS result in the oxidation of several biomolecules. Strikingly, RNA is particularly susceptible to this common chemical damage. Oxidation of RNA causes the formation of strand breaks, elimination of bases or insertion of mutagenic lesions in the nucleobases. The most common modification is 8-hydroxyguanosine (8-oxo-G), an oxidized form of guanosine. The structure and function of virtually all RNA species (mRNA, rRNA, tRNA, sRNA) can be affected by RNA oxidation, leading to translational defects with harmful consequences for cell survival. However, bacteria have evolved RNA quality control pathways to eliminate oxidized RNA, involving RNA-binding proteins like the members of the MutT/Nudix family and the ribonuclease PNPase. Here we summarize the current knowledge on the bacterial stress response to RNA oxidation, namely we present the different ROS responsible for this chemical damage and describe the main strategies employed by bacteria to fight oxidative stress and control RNA damage.

Development of a fast and efficient method to analyze microplastics in planktonic samples.

Microplastics (MPs) affect plankton (a basis of the trophic chain) and planktivorous fish can ingest them through food confusion or by trophic transmission. Consensus to determine MPs in plankton is lacking and, so, three digestion treatments were evaluated: Alkaline (potassium hydroxide) and enzymatic (protease plus lipase) digestions, both combined with a hydrogen peroxide stage; and an oxidative method using a surfactant (sodium dodecyl sulfate) plus hydrogen peroxide. The alkaline method using potassium hydroxide was found to damage polystyrene. MPs were identified with a stereomicroscope and characterized by reflectance infrared microscopy in semi-automatic mode (using dedicated multi-well aluminium plates). Analytical recoveries for polypropylene, polystyrene, polyethylene, polyamide, polyvinyl chloride and polyethylene terephthalate were higher than 75%, 82% and 83% for the alkaline, enzymatic and oxidative treatments, respectively. The enzymatic method was successfully validated in a European interlaboratory exercise and the oxidative method was demonstrated to be a reliable, fast and cheaper alternative.

PNPase is a key player in the regulation of small RNAs that control the expression of outer membrane proteins.

In this report, we demonstrate that exonucleolytic turnover is much more important in the regulation of sRNA levels than was previously recognized. For the first time, PNPase is introduced as a major regulatory feature controlling the levels of the small noncoding RNAs MicA and RybB, which are required for the accurate expression of outer membrane proteins (OMPs). In the absence of PNPase, the pattern of OMPs is changed. In stationary phase, MicA RNA levels are increased in the PNPase mutant, leading to a decrease in the levels of its target ompA mRNA and the respective protein. This growth phase regulation represents a novel pathway of control. We have evaluated other ribonucleases in the control of MicA RNA, and we showed that degradation by PNPase surpasses the effect of endonucleolytic cleavages by RNase E. RybB was also destabilized by PNPase. This work highlights a new role for PNPase in the degradation of small noncoding RNAs and opens the way to evaluate striking similarities between bacteria and eukaryotes.

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.

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.

Standardization of the minimum information for publication of infrared-related data when microplastics are characterized.

Infrared spectrometry (IR) became a workhorse to characterize microplastics (MPs) worldwide. However, reports on the experimental conditions to measure them decreased alarmingly. As complete, relevant information on the instrumental setup determining IR spectra is crucial for scientific reproducibility, ca. 50% of the papers that reported FTIR to measure MPs were evaluated and it was found that most studies cannot be replicated due to missing experimental details. To ameliorate this, the most critical parameters influencing IR spectra are depicted, their impact when matching a spectrum against databases exemplified, and, following efforts from other scientific fields, a minimum information for publication of IR-related data on MPs characterization (MIPIR-MP) is proposed, along with a brief, simple paragraph to resume the most critical information to be reported. This can be used to improve the worrying figures that point out to a reproducibility crisis in the field, as disclosed by the survey.

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.

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.

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.