Influenza A virus propagation requires the activation of the unfolded protein response and the accumulation of insoluble protein aggregates.

Influenza A virus (IAV) employs multiple strategies to manipulate cellular mechanisms and support proper virion formation and propagation. In this study, we performed a detailed analysis of the interplay between IAV and the host cells' proteostasis throughout the entire infectious cycle. We reveal that IAV infection activates the inositol requiring enzyme 1 (IRE1) branch of the unfolded protein response, and that this activation is important for an efficient infection. We further observed the accumulation of virus-induced insoluble protein aggregates, containing both viral and host proteins, associated with a dysregulation of the host cell RNA metabolism. Our data indicate that this accumulation is important for IAV propagation and favors the final steps of the infection cycle, more specifically the virion assembly. These findings reveal additional mechanisms by which IAV disrupts host proteostasis and uncovers new cellular targets that can be explored for the development of host-directed antiviral strategies.

Daphnia magna Multigeneration Exposure to Carbendazim: Gene Transcription Responses.

The world population is experiencing colossal growth and thus demand for food, leading to an increase in the use of pesticides. Persistent pesticide contamination, such as carbendazim, remains a pressing environmental concern, with potentially long-term impacts on aquatic ecosystems. In the present study, Daphnia magna was exposed to carbendazim (5 µg L-1) for 12 generations, with the aim of assessing gene transcription alterations induced by carbendazim (using a D. magna custom microarray). The results showed that carbendazim caused changes in genes involved in the response to stress, DNA replication/repair, neurotransmission, ATP production, and lipid and carbohydrate metabolism at concentrations already found in the environment. These outcomes support the results of previous studies, in which carbendazim induced genotoxic effects and reproduction impairment (increasing the number of aborted eggs with the decreasing number of neonates produced). The exposure of daphnids to carbendazim did not cause a stable change in gene transcription between generations, with more genes being differentially expressed in the F0 generation than in the F12 generation. This could show some possible daphnid acclimation after 12 generations and is aligned with previous multigenerational studies where few ecotoxicological effects at the individual and populational levels and other subcellular level effects (e.g., biochemical biomarkers) were found.

The hidden RNA code: implications of the RNA epitranscriptome in the context of viral infections.

Emerging evidence highlights the multifaceted roles of the RNA epitranscriptome during viral infections. By modulating the modification landscape of viral and host RNAs, viruses enhance their propagation and elude host surveillance mechanisms. Here, we discuss how specific RNA modifications, in either host or viral RNA molecules, impact the virus-life cycle and host antiviral responses, highlighting the potential of targeting the RNA epitranscriptome for novel antiviral therapies.

Sfl1 is required for Candida albicans biofilm formation under acidic conditions.

Candida albicans is a common Candida species, responsible for infections in various anatomical sites under different environmental conditions, aggravated in the presence of its biofilms. As such, this study aimed to reveal the regulation of C. albicans biofilms under acidic conditions by the transcription factor Sfl1, whose role on biofilm formation is unclear. For that, microbiologic and transcriptomic analyses were performed with the knock-out mutant C. albicans sfl1Δ/sfl1Δ and its parental strain SN76, grown in planktonic and biofilm lifestyles at pH 4 (vaginal pH). The results revealed that despite being a filamentation repressor Sf1 is required for maximal biofilm formation under acidic conditions. Additionally, Sfl1 was found to induce 275 and 126 genes in biofilm and planktonic cells, respectively, with an overlap of 19 genes. The functional distribution of Sfl1 targets was similar in planktonic and biofilm modes but an enrichment of carbohydrate metabolism function was found in biofilm cells, including some genes encoding proteins involved in the biofilm matrix production. Furthermore, this study shows that the regulatory network of Sfl1 in acidic biofilms is complex and includes positive and negative regulation of transcription factors involved in adhesion and biofilm formation, such as Ahr1, Brg1, Tye7, Tec1, Wor1, and some of their targets. Overall, this study shows that Sfl1 is a relevant regulator of C. albicans biofilm formation in acidic environments and contributes to a better understanding of C. albicans virulence under acidic conditions.

Upregulation of tRNA-Ser-AGA-2-1 Promotes Malignant Behavior in Normal Bronchial Cells.

Serine tRNAs (tRNASer) are frequently overexpressed in tumors and associated with poor prognosis and increased risk of recurrence in breast cancer. Impairment of tRNA biogenesis and abundance also impacts proteome homeostasis, and activates protein quality control systems. Herein, we aimed at testing whether increasing tRNASer abundance could foster tumor establishment through activation of the UPR. In order to do so, firstly we confirmed that the expression of tRNA-Ser-AGA-2-1 [hereafter tRNASer(AGA)] was upregulated by 1.79-fold in Stage I NSCLC tumors when compared to normal adjacent tissue. To study the impact of tRNASer(AGA) in early stage tumorigenesis, we induced its upregulation in a non-tumoral bronchial cell line, BEAS-2B. Upregulation of this tRNA increased cellular proliferation and protein synthesis rate, driven by eIF2α dephosphorylation and ATF4 activation downstream of PERK signaling. Futhermore, tRNASer(AGA) enhanced transformation potential in vitro, and promoted the establishment of slow growing tumors with aggressive features in nude mice. Our work highlights the importance of studying tRNA deregulation on early stage tumorigenesis, as they may be potential malignancy and aggressiveness biomarkers.

Steroid-Quinoline Hybrids for Disruption and Reversion of Protein Aggregation Processes.

Reversing protein aggregation within cells may be an important tool to fight protein-misfolding disorders such as Alzheimer's, Parkinson's, and cardiovascular diseases. Here we report the design and synthesis of a family of steroid-quinoline hybrid compounds based on the framework combination approach. This set of hybrid compounds effectively inhibited Aß1-42 self-aggregation in vitro by delaying the exponential growth phase and/or reducing the quantity of fibrils in the steady state. Their disaggregation efficacy was further demonstrated against preaggregated Aß1-42 peptides in cellular assays upon their endocytosis by neuroblastoma cells, as they reverted both the number and the average area of fibrils back to basal levels. The antiaggregation effect of these hybrids was further tested and demonstrated in a cellular model of general protein aggregation expressing a protein aggregation fluorescent sensor. Together, our results show that the new cholesterol-quinoline hybrids possess wide and marked disaggregation capacities and are therefore promising templates for the development of new drugs to deal with conformational disorders.

The Role of MicroRNAs in Proteostasis Decline and Protein Aggregation during Brain and Skeletal Muscle Aging.

Aging can be defined as the progressive deterioration of cellular, tissue, and organismal function over time. Alterations in protein homeostasis, also known as proteostasis, are a hallmark of aging that lead to proteome imbalances and protein aggregation, phenomena that also occur in age-related diseases. Among the various proteostasis regulators, microRNAs (miRNAs) have been reported to play important roles in the post-transcriptional control of genes involved in maintaining proteostasis during the lifespan in several organismal tissues. In this review, we consolidate recently published reports that demonstrate how miRNAs regulate fundamental proteostasis-related processes relevant to tissue aging, with emphasis on the two most studied tissues, brain tissue and skeletal muscle. We also explore an emerging perspective on the role of miRNA regulatory networks in age-related protein aggregation, a known hallmark of aging and age-related diseases, to elucidate potential miRNA candidates for anti-aging diagnostic and therapeutic targets.

Does proteostasis get lost in translation? Implications for protein aggregation across the lifespan.

Protein aggregation is a phenomenon of major relevance in neurodegenerative and neuromuscular disorders, cataracts, diabetes and many other diseases. Research has unveiled that proteins also aggregate in multiple tissues during healthy aging yet, the biological and biomedical relevance of this apparently asymptomatic phenomenon remains to be understood. It is known that proteome homeostasis (proteostasis) is maintained by a balanced protein synthesis rate, high protein synthesis accuracy, efficient protein folding and continual tagging of damaged proteins for degradation, suggesting that protein aggregation during healthy aging may be associated with alterations in both protein synthesis and the proteostasis network (PN) pathways. In particular, dysregulation of protein synthesis and alterations in translation fidelity are hypothesized to lead to the production of misfolded proteins which could explain the occurrence of age-related protein aggregation. Nevertheless, some data on this topic is controversial and the biological mechanisms that lead to widespread protein aggregation remain to be elucidated. We review the recent literature about the age-related decline of proteostasis, highlighting the need to build an integrated view of protein synthesis rate, fidelity and quality control pathways in order to better understand the proteome alterations that occur during aging and in age-related diseases.

Emerging Roles of tRNAs in RNA Virus Infections.

Viruses rely on the host cell translation machinery for efficient synthesis of their own proteins. Emerging evidence highlights different roles for host transfer RNAs (tRNAs) in the process of virus replication. For instance, different RNA viruses manipulate host tRNA pools to favor viral protein translation. Interestingly, specific host tRNAs are used as reverse transcription primers and are packaged into retroviral virions. Recent data also demonstrate the formation of tRNA-derived fragments (tRFs) upon infection to facilitate viral replication. Here, we comprehensively discuss how RNA viruses exploit distinct aspects of the host tRNA biology for their benefit. In light of the recent advances in the field, we propose that host tRNA-related pathways and mechanisms represent promising cellular targets for the development of novel antiviral strategies.

Cellular Proteostasis During Influenza A Virus Infection-Friend or Foe?

In order to efficiently replicate, viruses require precise interactions with host components and often hijack the host cellular machinery for their own benefit. Several mechanisms involved in protein synthesis and processing are strongly affected and manipulated by viral infections. A better understanding of the interplay between viruses and their host-cell machinery will likely contribute to the development of novel antiviral strategies. Here, we discuss the current knowledge on the interactions between influenza A virus (IAV), the causative agent for most of the annual respiratory epidemics in humans, and the host cellular proteostasis machinery during infection. We focus on the manipulative capacity of this virus to usurp the cellular protein processing mechanisms and further review the protein quality control mechanisms in the cytosol and in the endoplasmic reticulum that are affected by this virus.

Impact of tRNA Modifications and tRNA-Modifying Enzymes on Proteostasis and Human Disease.

Transfer RNAs (tRNAs) are key players of protein synthesis, as they decode the genetic information organized in mRNA codons, translating them into the code of 20 amino acids. To be fully active, tRNAs undergo extensive post-transcriptional modifications, catalyzed by different tRNA-modifying enzymes. Lack of these modifications increases the level of missense errors and affects codon decoding rate, contributing to protein aggregation with deleterious consequences to the cell. Recent works show that tRNA hypomodification and tRNA-modifying-enzyme deregulation occur in several diseases where proteostasis is affected, namely, neurodegenerative and metabolic diseases. In this review, we discuss the recent findings that correlate aberrant tRNA modification with proteostasis imbalances, in particular in neurological and metabolic disorders, and highlight the association between tRNAs, their modifying enzymes, translational decoding, and disease onset.

Discovery and function of transfer RNA-derived fragments and their role in disease.

Until recently, transfer RNAs (tRNAs) were thought to function in protein translation only. However, recent findings demonstrate that both pre- and mature tRNAs can undergo endonucleolytic cleavage by different ribonucleases originating different types of small non-coding RNAs, known as tRNA-derived fragments (tRFs). tRFs are classified according to their origin and are implicated in various cellular processes, namely apoptosis, protein synthesis control, and RNA interference. Although their functions are still poorly understood, their mechanisms of action vary according to the tRF sub-type. Several tRFs have been associated with cancer, neurodegenerative disorders, and viral infections and growing evidence shows that they may constitute novel molecular targets for modulating pathological processes. Here, we recapitulate the current knowledge of tRF biology, highlight the known functions and mechanisms of action of the different sub-classes of tRFs and discuss their implications in human disease. WIREs RNA 2017, 8:e1423. doi: 10.1002/wrna.1423 For further resources related to this article, please visit the WIREs website.

Conserved and highly expressed tRNA derived fragments in zebrafish.

BACKGROUND: Small non-coding RNAs (sncRNAs) are a class of transcripts implicated in several eukaryotic regulatory mechanisms, namely gene silencing and chromatin regulation. Despite significant progress in their identification by next generation sequencing (NGS) we are still far from understanding their full diversity and functional repertoire. RESULTS: Here we report the identification of tRNA derived fragments (tRFs) by NGS of the sncRNA fraction of zebrafish. The tRFs identified are 18-30 nt long, are derived from specific 5' and 3' processing of mature tRNAs and are differentially expressed during development and in differentiated tissues, suggesting that they are likely produced by specific processing rather than random degradation of tRNAs. We further show that a highly expressed tRF (5'tRF-Pro(CGG)) is cleaved in vitro by Dicer and has silencing ability, indicating that it can enter the RNAi pathway. A computational analysis of zebrafish tRFs shows that they are conserved among vertebrates and mining of publicly available datasets reveals that some 5'tRFs are differentially expressed in disease conditions, namely during infection and colorectal cancer. CONCLUSIONS: tRFs constitute a class of conserved regulatory RNAs in vertebrates and may be involved in mechanisms of genome regulation and in some diseases.

Ethanol exposure induces upregulation of specific microRNAs in zebrafish embryos.

Prenatal exposure to ethanol leads to a myriad of developmental disorders known as fetal alcohol spectrum disorder, often characterized by growth and mental retardation, central nervous system damage, and specific craniofacial dysmorphic features. The mechanisms of ethanol toxicity are not fully understood, but exposure during development affects the expression of several genes involved in cell cycle control, apoptosis, and transcriptional regulation. MicroRNAs (miRNAs) are implicated in some of these processes, however, it is not yet clear if they are involved in ethanol-induced toxicity. In order to clarify this question, we have exposed zebrafish embryos to ethanol and evaluated whether a miRNA deregulation signature could be obtained. Zebrafish embryos were exposed to 1 and 1.5% of ethanol from 4 h postfertilization (hpf) to 24 hpf. The miRNA expression profiles obtained reveal significant miRNA deregulation and show that both ethanol concentrations upregulate miR-153a, miR-725, miR-30d, let-7k, miR-100, miR-738, and miR-732. Putative gene targets of deregulated miRNAs are involved in cell cycle control, apoptosis, and transcription, which are the main processes affected by ethanol toxicity. The conservation of affected mechanisms among vertebrates leads us to postulate that similar miRNA deregulation occurs in humans, highlighting a relevant role of miRNAs in ethanol toxicology.

Next-generation sequencing of miRNAs with Roche 454 GS-FLX technology: steps for a successful application.

MicroRNAs (miRNAs) are a class of small RNAs (sRNAs) of approximately 22 nucleotides in length that control eukaryotic gene expression at the translational level. They regulate a wide variety of biological processes, namely developmental timing, cell differentiation, cell proliferation, the immune response, and infection. Their identification is essential to understand eukaryotic biology. Their small size, low abundance, and high instability complicated early identification, however new generation genome sequencing approaches, such as the Roche 454 Pyrosequencer, allow for both miRNA identification and for generating miRNA profiles in a given sample. This technique avoids cloning steps in bacteria and is a fast and bias-minimized tool to discover novel miRNAs and other sRNAs on a genome-wide scale. Prior to sequencing, cDNA libraries are built for each sample using total RNA as starter material. Each cDNA library can be tagged with specific identifier sequences that allow sequencing different samples in the same chip run. Here, we describe the protocols for the construction of sRNA cDNA libraries for 454 sequencing, and we include tips for overcoming problems often encountered during cDNA library preparation.

Efeito de herbicidas e fungicidas nas curvas de progresso e quantificação de danos causados pela ferrugem asiática da soja / Effect of herbicides and fungicides on progress curves and damage quantification caused by soybean rust

O fungo Phakopsora pachyrhizi Sydow & Sydow representa grande ameaça à sojicultura nas principais regiões produtoras do mundo, onde significativas perdas foram relatadas. Na quantificação de danos causados, muitas são as variáveis a serem estudadas, como área abaixo da curva de progresso da doença (AACPD), absorção de luz da área foliar sadia (HAA) e duração da área foliar sadia (HAD), além das curvas de progresso da doença. Neste trabalho, objetivou-se verificar a influência de diferentes herbicidas e fungicidas no progresso da doença, bem como suas implicações nas variáveis referentes à área foliar sadia e à produtividade da cultura. Os tratamentos testados em duas cultivares ('MG/BR46 Conquista' e 'BRS Valiosa RR') foram: 1. Testemunha; 2. Testemunha com herbicidas; 3. Herbicidas e pyraclostrobin (V4) + pyraclostrobin + epoxiconazole (R2 e R5.1); 4. Herbicidas e pyraclostrobin (V4) + epoxiconazole (R2 e R5.1); e 5. Herbicidas (V4) + pyraclostrobin + epoxiconazole (R2 e R5.1). Os herbicidas utilizados em 'MG/BR-46 Conquista' foram sethoxydim, bentazon e chlorimuron-ethyl, sendo utilizado glyphosate na 'BRS Valiosa RR'. O modelo logístico foi o que melhor se adequou à severidade média das avaliações e à curva de progresso da doença. Para o terço inferior, posição do dossel mais propícia à doença, o modelo logístico com taxa variável foi o que mais se ajustou aos dados. A pulverização de pyraclostrobin + epoxiconazole reduziu a taxa de desenvolvimento de P. pachyrhizi em relação à epoxiconazole. A utilização da mistura influenciou as variáveis AACPD, HAA e HAD, sendo estas consideradas apropriadas para a quantificação dos danos provocados pela ferrugem asiática. Na 'MG/BR-46 Conquista', a ação dos herbicidas afetaram temporariamente a área foliar, atingindo indiretamente as variáveis HAA e HAD.

Phakopsora pachyrhizi Sydow & Sydow is the worst threat for soybean crop in the most important growing regions around the world, where great losses were observed. Many variables are studied in the quantification of damage such as area under the disease progress curve (AUDPC), healthy leaf area absorption (HAA), healthy leaf area duration (HAD), in addition to disease progress curves. The present research aimed to evaluate the effect of several herbicides and fungicides on the progress of the disease and its implications for variables related to healthy leaf area and yield. The treatments were evaluated in two cultivars (MG/BR-46 Conquista e BRS Valiosa RR): 1. Control; 2. Control with herbicides; 3. Herbicides and pyraclostrobin (V4) + pyraclostrobin+ epoxiconazole (R2 and R5.1), 4. Herbicides and pyraclostrobin (V4) + epoxiconazole (R2 and R5.1). 5. Herbicides (V4) + pyraclostrobin + epoxiconazole (R2 and R5.1). The herbicides used in MG/BR-46 Conquista were sethoxydim, bentazon and chlorimuron-ethyl; in BRS Valiosa RR, glyphosate was used. The logistic was the model that best adapted to the mean severity ratings and the disease progress curve. At the bottom of canopy, position more favorable to this disease, the logistic model with variable rate was the model that better explain the data. The spraying of strobilurin's group associated with triazole (pyraclostrobin + epoxiconazole) reduced the rate of asian rust development, in relation to the group of ergosterol biosynthesis inhibitors fungicide (epoxiconazole). The pyraclostrobin + epoxiconazole influenced the variables AUDPC, HAA and HAD, and these can be considered suitable to quantify damage caused by this disease. In MG/BR-46 Conquista, the herbicides action affects temporarily the leaf area, affecting indirectly the HAA and HAD variables.