The RNA-binding protein RBP33 dampens non-productive transcription in trypanosomes.

In-depth analysis of the transcriptomes of several model organisms has revealed that genomes are pervasively transcribed, giving rise to an abundance of non-canonical and mainly antisense RNA polymerase II-derived transcripts that are produced from almost any genomic context. Pervasive RNAs are degraded by surveillance mechanisms, but the repertoire of proteins that control the fate of these non-productive transcripts is still incomplete. Trypanosomes are single-celled eukaryotes that show constitutive RNA polymerase II transcription and in which initiation and termination of transcription occur at a limited number of sites per chromosome. It is not known whether pervasive transcription exists in organisms with unregulated RNA polymerase II activity, and which factors could be involved in the process. We show here that depletion of RBP33 results in overexpression of ∼40% of all annotated genes in the genome, with a marked accumulation of sense and antisense transcripts derived from silenced regions. RBP33 loss does not result in a significant increase in chromatin accessibility. Finally, we have found that transcripts that increase in abundance upon RBP33 knockdown are significantly more stable in RBP33-depleted trypanosomes, and that the exosome complex is responsible for their degradation. Our results provide strong evidence that RBP33 dampens non-productive transcription in trypanosomes.

An RNA-binding protein complex regulates the purine-dependent expression of a nucleobase transporter in trypanosomes.

Post-transcriptional regulation of gene expression is particularly important in trypanosomatid protozoa. RNA-binding proteins (RBPs) regulate mRNA stability and translation, yet information about how RBPs are able to link environmental cues to post-transcriptional control is scarce. In Trypanosoma brucei, we have previously characterized a short RNA stem-loop cis-element (PuRE, Purine Responsive Element) within the 3'-UTR of the NT8 nucleobase transporter mRNA that is necessary and sufficient to confer a strong repression of gene expression in response to purines. In this study, we have identified a protein complex composed of two RNA-binding proteins (PuREBP1 and PuREBP2) that binds to the PuRE in vitro and to NT8 mRNA in vivo. Depletion of PuREBP1 by RNA interference results in the upregulation of just NT8 and the mRNAs encoding the amino acid transporter AATP6 paralogues. Moreover, we found that the PuREBP1/2 complex is associated with only a handful of mRNAs, and that it is responsible for the observed purine-dependent regulation of NT8 expression.

Double Orthogonal Click Reactions for the Development of Antimicrobial Peptide Nanotubes.

A new class of amphipathic cyclic peptides, which assemble in bacteria membranes to form polymeric supramolecular nanotubes giving them antimicrobial properties, is described. The method is based on the use of two orthogonal clickable transformations to incorporate different hydrophobic or hydrophilic moieties in a simple, regioselective, and divergent manner. The resulting cationic amphipathic cyclic peptides described in this article exhibit strong antimicrobial properties with a broad therapeutic window. Our studies suggest that the active form is the nanotube resulted from the parallel stacking of the cyclic peptide precursors. Several techniques, CD, FTIR, fluorescence, and STEM, among others, confirm the nanotube formation.

30 years of parasitology research analysed by text mining.

Bibliometric methods were used to analyse the major research trends, themes and topics over the last 30 years in the parasitology discipline. The tools used were SciMAT, VOSviewer and SWIFT-Review in conjunction with the parasitology literature contained in the MEDLINE, Web of Science, Scopus and Dimensions databases. The analyses show that the major research themes are dynamic and continually changing with time, although some themes identified based on keywords such as malaria, nematode, epidemiology and phylogeny are consistently referenced over time. We note the major impact of countries like Brazil has had on the literature of parasitology research. The increase in recent times of research productivity on 'antiparasitics' is discussed, as well as the change in emphasis on different antiparasitic drugs and insecticides over time. In summary, innovation in parasitology is global, extensive, multidisciplinary, constantly evolving and closely aligned with the availability of technology.

Trypanosomatid parasites rescue heme from endocytosed hemoglobin through lysosomal HRG transporters.

Pathogenic trypanosomatid parasites are auxotrophic for heme and they must scavenge it from their human host. Trypanosoma brucei (responsible for sleeping sickness) and Leishmania (leishmaniasis) can fulfill heme requirement by receptor-mediated endocytosis of host hemoglobin. However, the mechanism used to transfer hemoglobin-derived heme from the lysosome to the cytosol remains unknown. Here we provide strong evidence that HRG transporters mediate this essential step. In bloodstream T. brucei, TbHRG localizes to the endolysosomal compartment where endocytosed hemoglobin is known to be trafficked. TbHRG overexpression increases cytosolic heme levels whereas its downregulation is lethal for the parasites unless they express the Leishmania orthologue LmHR1. LmHR1, known to be an essential plasma membrane protein responsible for the uptake of free heme in Leishmania, is also present in its acidic compartments which colocalize with endocytosed hemoglobin. Moreover, LmHR1 levels modulated by its overexpression or the abrogation of an LmHR1 allele correlate with the mitochondrial bioavailability of heme from lysosomal hemoglobin. In addition, using heme auxotrophic yeasts we show that TbHRG and LmHR1 transport hemoglobin-derived heme from the digestive vacuole to the cytosol. Collectively, these results show that trypanosomatid parasites rescue heme from endocytosed hemoglobin through endolysosomal HRG transporters, which could constitute novel drug targets.

A short RNA stem-loop is necessary and sufficient for repression of gene expression during early logarithmic phase in trypanosomes.

We have compared the transcriptomes of cultured procyclic Trypanosoma brucei cells in early and late logarithmic phases and found that ∼200 mRNAs were differentially regulated. In late log phase cells, the most upregulated mRNA encoded the nucleobase transporter NT8. The 3' untranslated region (UTR) of NT8 contains a short stem-loop cis-element that is necessary for the regulation of NT8 expression in response to external purine levels. When placed in the 3'-UTR of an unregulated transcript, the cis-element is sufficient to confer regulation in response to purines. To our knowledge, this is the first example of a discrete RNA element that can autonomously regulate gene expression in trypanosomes in response to an external factor and reveals an unprecedented purine-dependent signaling pathway that controls gene expression in eukaryotes.

The essential polysome-associated RNA-binding protein RBP42 targets mRNAs involved in Trypanosoma brucei energy metabolism.

RNA-binding proteins that target mRNA coding regions are emerging as regulators of post-transcriptional processes in eukaryotes. Here we describe a newly identified RNA-binding protein, RBP42, which targets the coding region of mRNAs in the insect form of the African trypanosome, Trypanosoma brucei. RBP42 is an essential protein and associates with polysome-bound mRNAs in the cytoplasm. A global survey of RBP42-bound mRNAs was performed by applying HITS-CLIP technology, which captures protein-RNA interactions in vivo using UV light. Specific RBP42-mRNA interactions, as well as mRNA interactions with a known RNA-binding protein, were purified using specific antibodies. Target RNA sequences were identified and quantified using high-throughput RNA sequencing. Analysis revealed that RBP42 bound mainly within the coding region of mRNAs that encode proteins involved in cellular energy metabolism. Although the mechanism of RBP42's function is unclear at present, we speculate that RBP42 plays a critical role in modulating T. brucei energy metabolism.

Role of the RNA-binding protein ZC3H41 in the regulation of ribosomal protein messenger RNAs in trypanosomes.

BACKGROUND: Trypanosomes are single-celled eukaryotes that rely heavily on post-transcriptional mechanisms to regulate gene expression. RNA-binding proteins play essential roles in regulating the fate, abundance and translation of messenger RNAs (mRNAs). Among these, zinc finger proteins of the cysteine3histidine (CCCH) class have been shown to be key players in cellular processes as diverse as differentiation, regulation of the cell cycle and translation. ZC3H41 is an essential zinc finger protein that has been described as a component of spliced leader RNA granules and nutritional stress granules, but its role in RNA metabolism is unknown. METHODS: Cell cycle analysis in ZC3H41- and Z41AP-depleted cells was carried out using 4',6-diamidino-2-phenylindole staining, microscopic examination and flow cytometry. The identification of ZC3H41 protein partners was done using tandem affinity purification and mass spectrometry. Next-generation sequencing was used to evaluate the effect of ZC3H41 depletion on the transcriptome of procyclic Trypanosoma brucei cells, and also to identify the cohort of mRNAs associated with the ZC3H41/Z41AP complex. Levels of 5S ribosomal RNA (rRNA) species in ZC3H41- and Z41AP-depleted cells were assessed by quantitative reverse transcription-polymerase chain reaction. Surface sensing of translation assays were used to monitor global translation. RESULTS: We showed that depletion of the zinc finger protein ZC3H41 resulted in marked cell cycle defects and abnormal cell morphologies. ZC3H41 was found associated with an essential protein, which we named Z41AP, forming a stable heterodimer, and also with proteins of the poly(A)-binding protein 1 complex. The identification of mRNAs associated with the ZC3H41/Z41AP complex revealed that it is primarily composed of ribosomal protein mRNAs, and that binding to target transcripts is diminished upon nutritional stress. In addition, we observed that mRNAs encoding several proteins involved in the maturation of 5S rRNA are also associated with the ZC3H41/Z41AP complex. Finally, we showed that depletion of either ZC3H41 or Z41AP led to the accumulation of 5S rRNA precursors and a decrease of protein translation. CONCLUSIONS: We propose that ZC3H41 and Z41AP play important roles in controlling the fate of ribosomal components in response to environmental cues.

Pharmacists and pharmacy services in COVID-19 literature: A bibliometirc analysis.

Background: The COVID-19 pandemic had an enormous impact on the global economy and healthcare. Pharmacists were vital members of the healthcare system, and they participated in various strategies to reduce the effect of the pandemic. Numerous papers were published discussing their roles during the pandemic. Bibliometric analysis was used to measure the impact of publications on this topic and assessed them qualitatively and quantitatively over a specific time. Objective: Evaluate published literature pertaining to the roles of pharmacists and pharmacy services during the pandemic and identify gaps. Methods: An electronic search was conducted on PubMed database using a specific query. Eligible publications were published in English between January 2020 and January 2022 and discussed the role of pharmacists, pharmacies, and pharmacy departments during the pandemic. Clinical trials, studies on pharmacy education/training, and conference abstracts were excluded. Results: Of 954 records retrieved, 338 (35.4%) from 67 countries were included. Most papers (n = 113; 33.4%) were from the community pharmacy sector, followed by the clinical pharmacy sector (n = 89; 26.3%). Sixty-one (18%) papers were multinational, mostly involving two countries. The average number of citations of the included papers was 6 times (range 0-89). The most common MeSH terms were 'humans', 'hospitals', and 'telemedicine', where the former frequently co-appeared with the terms 'COVID-19' and 'pharmacists.' Conclusions: Results from this study illustrate the innovative and proactive strategies developed by pharmacists during the pandemic. Pharmacists from around the world are encouraged to share their experiences for stronger healthcare systems to counter future pandemics and environmental disasters.

Analysis of cognitive framework and biomedical translation of tissue engineering in otolaryngology.

Tissue engineering is a relatively recent research area aimed at developing artificial tissues that can restore, maintain, or even improve the anatomical and/or functional integrity of injured tissues. Otolaryngology, as a leading surgical specialty in head and neck surgery, is a candidate for the use of these advanced therapies and medicinal products developed. Nevertheless, a knowledge-based analysis of both areas together is still needed. The dataset was retrieved from the Web of Science database from 1900 to 2020. SciMAT software was used to perform the science mapping analysis and the data for the biomedical translation identification was obtained from the iCite platform. Regarding the analysis of the cognitive structure, we find consolidated research lines, such as the generation of cartilage for use as a graft in reconstructive surgery, reconstruction of microtia, or the closure of perforations of the tympanic membrane. This last research area occupies the most relevant clinical translation with the rest of the areas presenting a lower translational level. In conclusion, Tissue engineering is still in an early translational stage in otolaryngology, otology being the field where most advances have been achieved. Therefore, although otolaryngologists should play an active role in translational research in tissue engineering, greater multidisciplinary efforts are required to promote and encourage the translation of potential clinical applications of tissue engineering for routine clinical use.

A long noncoding RNA promotes parasite differentiation in African trypanosomes.

The parasite Trypanosoma brucei causes African sleeping sickness that is fatal to patients if untreated. Parasite differentiation from a replicative slender form into a quiescent stumpy form promotes host survival and parasite transmission. Long noncoding RNAs (lncRNAs) are known to regulate cell differentiation in other eukaryotes. To determine whether lncRNAs are also involved in parasite differentiation, we used RNA sequencing to survey the T. brucei genome, identifying 1428 previously uncharacterized lncRNA genes. We find that grumpy lncRNA is a key regulator that promotes parasite differentiation into the quiescent stumpy form. This function is promoted by a small nucleolar RNA encoded within the grumpy lncRNA. snoGRUMPY binds to messenger RNAs of at least two stumpy regulatory genes, promoting their expression. grumpy overexpression reduces parasitemia in infected mice. Our analyses suggest that T. brucei lncRNAs modulate parasite-host interactions and provide a mechanism by which grumpy regulates cell differentiation in trypanosomes.

The exosomes of trypanosomes and other protists.

The archaeal exosome contains three heterodimeric RNase PH subunits, forming a hexamer with RNase activity; on top sits a trimer of two different SI domain proteins. In animals and yeast, six different, but related subunits form the RNase PH-like core, but these lack enzyme activity; there are three different Si-domain proteins and enzyme activity is provided by the endo/exonuc lease Rrp44 or-mainly in the nuclear exosome-the Rnase D enzyme Rrp6. Trypanosomes diverged from yeast and mammals very early in eukaryotic evolution. The trypanosome exosome is similar in subunit composition to the human exosome, but instead of being an optional component, trypanosome RRP6 is present in the nucleus and cytoplasm and is required for exosome stability. As in human cells and yeast, the trypanosome exosome has been shown to be required for processing and quality control of rRNA and to be involved in mRNA degradation. Electron microscopy results for a Leishmania exosome suggest that RRP6 is located on the side of the RnasePH ring, interacting with several exosome core proteins. Results of a search for exosome subunits in the genomes of widely diverged protists revealed varied exosome complexity; the Giardia exosome may be as simple as that of Archaea.

The Simpsons did it: Exploring the film trope space and its large scale structure.

Creating a story is a challenging task due to the the complex relations between the parts that make it up, which is why many new stories are built on those cohesive elements or patterns, called tropes that have been shown to work in the past. A trope is a recurring storytelling device or pattern, or sometimes a meta-element, used by the authors to express ideas that the audience can recognize or relate to, such as the Hero's Journey. Discovering tropes and how they cluster in popular works and doing it at scale to generate new plots may benefit writers; in this paper, we analyze them and use a principled procedure to identify trope combinations, or communities, that could possible be successful. The degree of development of these different communities can help us identify areas that are under-developed and, thus, susceptible to such a type of development. To detect these communities, with their associated degree of development and interest, we propose a methodology based on scientometric and complex network analysis techniques. As a secondary objective, we will obtain a general perspective in the trope and films network: the tropesphere. We have used a dataset of 10,766 movies and 25,776 tropes associated with them, together with rating, genres and popularity. Our analysis has shown that not only there are different trope communities associated with specific genres, and that there are significant differences between the rating and popularity of these communities but also there are differences on the level of development between them: emerging/declining, specific, transversal or motor.

The RNA-binding protein TbDRBD3 regulates the stability of a specific subset of mRNAs in trypanosomes.

In trypanosomes, the apparent lack of regulation of RNA polymerase II-dependent transcription initiation poses a challenge to understand how these eukaryotes adjust gene expression to adapt to the contrasting environments they find during their life cycles. Evidence so far indicates that mRNA turnover and translation are the major control points in which regulation is exerted in trypanosomes. However, very little is known about which proteins are involved, and how do they regulate the abundance and translation of different mRNAs in different life stages. In this work, an RNA-binding protein, TbDRBD3, has been identified by affinity chromatography, and its function addressed using RNA interference, microarray analysis and immunoprecipitation of mRNA-protein complexes. The results obtained indicate that TbDRBD3 binds to a subset of developmentally regulated mRNAs encoding membrane proteins, and that this association promotes the stabilization of the target transcripts. These observations raise the possibility that TbDRBD3-mRNA complexes act as a post-transcriptional operon, and provide a framework to interpret how trypanosomes regulate gene expression in the absence of transcriptional control.

The exosomes of trypanosomes and other protists.

The archaeal exosome contains three heterodimeric RNase PH subunits, forming a hexamer with RNase activity; on top sits a trimer of two different SI domain proteins. In animals and yeast, six different, but related subunits form the RNase PH-like core, but these lack enzyme activity; there are three different SI-domain proteins and enzyme activity is providedby the endo/exonuclease Rrp44 or--mainly in the nuclear exosome--the Rnase D enzyme Rrp6. Trypanosomes diverged from yeast and mammals very early in eukaryotic evolution. The trypanosome exosome is similar in subunit composition to the human exosome, but instead of being an optional component, trypanosome RRP6 is present in the nucleus and cytoplasm and is required for exosome stability. As in human cells and yeast, the trypanosome exosome has been shown to be required for processing and quality control of rRNA and to be involved in mRNA degradation. Electron microscopy results for a Leishmania exosome suggest that RRP6 is located on the side of the RnasePH ring, interacting with several exosome core proteins. Results of a search for exosome subunits in the genomes of widely diverged protists revealed varied exosome complexity; the Giardia exosome may be as simple as that of Archaea.

Effect of antiseptic gels in the microbiologic colonization of the suture threads after oral surgery.

Three different bioadhesive gels were evaluated in a double-blind randomized clinical trial in which microbial growth in the suture thread was assessed following post-surgical application of the aforementioned gels. Also assessed in this trial were, the intensity of post-surgical pain as well as the degree of healing of the patients' surgical wounds. A total of 21 patients (with 42 wisdom teeth) participated in this trial. Chlorhexidine gel, chlorhexidine-chitosan gel, and hyaluronic acid gel were evaluated, with a neutral water-based gel serving as the control agent. The aerobic and facultative anaerobic bacterial recovery on blood agar was lower in the placebo group than in the experimental groups. The most significant difference (p = 0.04) was observed in the chlorhexidine-chitosan group. in which the growth of Blood Agar and Mitis Salivarius Agar was significantly higher than in the placebo group. The intensity of post-surgical pain was very similar among all the groups. Significantly better healing rates were observed in the patients treated with chlorhexidine-chitosan gel when compared with those who used the placebo gel (p = 0.03), and in particular when compared with those patients who used hyaluronic acid gel (p = 0.01). Through our microbiological analyses, we were able to conclude that none of the bioadhesive gels tested resulted in beneficial reductions in the bacterial/fungal populations. However, the healing rates of patients who were treated with chlorhexidine-chitosan were better than those of the patients who used either the placebo gel or the hyaluronic acid gel.

Depletion of dimeric all-alpha dUTPase induces DNA strand breaks and impairs cell cycle progression in Trypanosoma brucei.

The enzyme deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is responsible for the control of intracellular levels of dUTP thus controlling the incorporation of uracil into DNA during replication. Trypanosomes and certain eubacteria contain a dimeric dUTP-dUDPase belonging to the recently described superfamily of all-alpha NTP pyrophosphatases which bears no resemblance with typical eukaryotic trimeric dUTPases and presents unique properties regarding substrate specificity and product inhibition. While the biological trimeric enzymes have been studied in detail and the human enzyme has been proposed as a promising novel target for anticancer chemotherapeutic strategies, little is known regarding the biological function of dimeric proteins. Here, we show that in Trypanosoma brucei, the dimeric dUTPase is a nuclear enzyme and that down-regulation of activity by RNAi greatly reduces cell proliferation and increases the intracellular levels of dUTP. Defects in growth could be partially reverted by the addition of exogenous thymidine. dUTPase-depleted cells presented hypersensitivity to methotrexate, a drug that increases the intracellular pools of dUTP, and enhanced uracil-DNA glycosylase activity, the first step in base excision repair. The knockdown of activity produces numerous DNA strand breaks and defects in both S and G2/M progression. Multiple parasites with a single enlarged nucleus were visualized together with an enhanced population of anucleated cells. We conclude that dimeric dUTPases are strongly involved in the control of dUTP incorporation and that adequate levels of enzyme are indispensable for efficient cell cycle progression and DNA replication.

Triphenyltin derivatives of sulfanylcarboxylic esters.

The reaction of 3-(aryl)-2-sulfanylpropenoic acids [H2xspa; x: p=3-phenyl-, f=3-(2-furyl)-, t=3-(2-thienyl)-] with methanol or ethanol gave the corresponding methyl (Hxspme) or ethyl (Hxspee) esters. The reaction of these esters (HL) with triphenyltin(IV) hydroxide gave compounds of the type [SnPh3L], which were isolated and characterized as solids by elemental analysis, IR spectroscopy and mass spectrometry and in solution by multinuclear (1H, 13C and 119Sn) NMR spectroscopy. The structures of [SnPh3(pspme)], [SnPh3(fspme)] and [SnPh3(fspee)] were determined by X-ray diffractometry and the antimicrobial activity against E. coli, S. aureus, B. subtilis, P. aeruginosa, Resistant P. aeruginosa (a strain resistant to 'carbapenem'), and C. albicans was tested and the in vitro cytotoxic activity against the HeLa-229, A2780 and A2780cis cell lines was determined for all compounds.

The subcellular localisation of trypanosome RRP6 and its association with the exosome.

The exosome, a complex of 3'-exoribonucleases and associated proteins, is involved in the degradation of eukaryotic mRNAs in the cytoplasm, and has RNA processing and quality control functions in the nucleus. In yeast, the nuclear exosome differs from the cytoplasmic one in that it contains an additional non-essential component, Rrp6p. In contrast, a small proportion of human RRP6 has been shown to localise to the cytoplasm as well. When we purified the Trypanosoma brucei exosome from cytosolic extracts we found RRP6, apparently in stoichiometric amounts. We here confirm that RRP6 is in the trypanosome cytoplasm and nucleus. The level of RRP6 was unaffected by depletion of core exosome subunits by RNA interference and over-expression of tagged RRP6 was possible, indicating that RRP6 can be present independent of exosome association.

Urgencias protésicas por agresiones a los diferentes elementos componentes del sistema estomatognático

Resumen: Se realizó un estudio observacional, descriptivo y transversal en la clínica estomatológica de Güines, República de Cuba, desde Septiembre de 2018 a Octubre del 2020 con el objetivo de identificar las principales lesiones según el elemento agredido por las cuales los pacientes solicitan atención inmediata. De un total de 9750 pacientes, se obtuvo una población objeto de estudio de 320 pacientes portadores de prótesis con agresiones al órgano paraprotésico. Se realizó el interrogatorio, el examen bucal y de la articulación témporomandibular. Los datos se recogieron en una planilla. Se usó X2 y Duncan para el análisis estadístico. La mucosa resulto ser el elemento más agredido (73,44%) mientras que los estados inflamatorios (2,50 %), el dolor periodontal (2,81 %), la estomatitis subprótesis grado II (36,88 %), las mialgias (5,94 %) y las combinaciones de síntomas y signos articulares (5,94 %) fueron las principales lesiones por las que se solicitó tratamiento.

Resumo: Foi realizado um estudo observacional, descritivo e transversal na Clínica de Estomatologia Güines, de setembro de 2018 a outubro de 2020, com o objetivo de identificar as principais lesões de acordo com o elemento agredido para o qual os pacientes solicitam atendimento imediato. pacientes, foi obtida uma população de estudo de 320 pacientes com próteses com ataques ao órgão paraprotético. O interrogatório, o exame oral e a articulação temporomandibular foram realizados. Os dados foram coletados em planilha e X2 e Duncan foram utilizados para análise estatística. A mucosa foi o elemento mais afetado (73,44%) enquanto estados inflamatórios (2,50%), dor periodontal (2,81%), estomatite subprotética grau II (36,88%), mialgias (5,94%) e combinações de sintomas e sinais articulares ( 5,94%) foram as principais lesões para as quais os pacientes solicitaram atendimento de urgência.

Abstract: An observational, descriptive and cross-sectional study was carried out in the Güines stomatological clinic, from September 2018 to October 2020 with the aim of identifying the main injuries according to the assaulted element for which patients request immediate attention. Out of a total of 9,750 patients, a study population of 320 patients with prostheses with attacks on the paraprosthetic organ was obtained. The interrogation, the oral examination and the temporomandibular joint were carried out. Data were collected on a spreadsheet and X2 and Duncan were used for statistical analysis. The mucosa was the most affected element (73.44%) while inflammatory states (2.50%), periodontal pain (2.81%), grade II sub-prosthetic stomatitis (36.88%), myalgias (5.94%) and combinations of joint symptoms and signs (5.94%) were the main injuries for which patients requested emergency treatment.