The mitochondrial unfolded protein response (mitoUPR) is a stress response pathway that responds to mitochondrial insults by altering gene expression to recover mitochondrial homeostasis. The mitoUPR is mediated by the s...The mitochondrial unfolded protein response (mitoUPR) is a stress response pathway that responds to mitochondrial insults by altering gene expression to recover mitochondrial homeostasis. The mitoUPR is mediated by the stress-activated transcription factor ATFS-1 (activating transcription factor associated with stress 1). Constitutive activation of ATFS-1 increases resistance to exogenous stressors but paradoxically decreases lifespan. In this work, we determined the optimal levels of expression of activated ATFS-1 with respect to lifespan and resistance to stress by treating constitutively active ) worms with different concentrations of RNA interference (RNAi) bacteria targeting . We observed the maximum lifespan of ) worms at full-strength RNAi, which was significantly longer than wild-type lifespan. Under the conditions of maximum lifespan, ) worms did not show enhanced resistance to stress, suggesting a trade-off between stress resistance and longevity. The maximum resistance to stress in ) worms occurred on empty vector. Under these conditions, ) worms are short-lived. This indicates that constitutive activation of ATFS-1 can increase lifespan or enhance resistance to stress but not both, at the same time. Overall, these results demonstrate that constitutively active ATFS-1 can extend lifespan when expressed at low levels and that this lifespan extension is not dependent on the ability of ATFS-1 to enhance resistance to stress.
African trypanosomes are medically important parasites that cause sleeping sickness in humans and nagana in animals. In addition to their pathogenic role, they have emerged as valuable model organisms for studying fundam...African trypanosomes are medically important parasites that cause sleeping sickness in humans and nagana in animals. In addition to their pathogenic role, they have emerged as valuable model organisms for studying fundamental biological processes. Protein tagging is a powerful tool for investigating protein localization and function. In a previous study, we developed two plasmids for rapid and reproducible polymerase chain reaction-based protein tagging in trypanosomes, which enabled the subcellular mapping of 89% of the trypanosome proteome. However, the limited selection of fluorescent protein tags and selectable markers restricted the flexibility of this approach. Here, we present an extended set of >100 plasmids that incorporate universal primer annealing sequences, enabling protein tagging with a range of fluorescent, biochemical and epitope tags, using five different selection markers. We evaluated the suitability of various fluorescent proteins for live and fixed cell imaging, fluorescent movies, and we demonstrate the use of tagging plasmids encoding tandem epitope tags to support expansion microscopy approaches. We show that this series of plasmids is functional in other trypanosomatid parasites, significantly increasing its value. Finally, we developed a new plasmid for tagging glycosylphosphatidylinositol-anchored proteins. We anticipate that this will be an important toolset for investigating trypanosomatid protein localization and function.
It is well accepted that sperm carry an RNA cargo with functions in early embryo development. However, most research has focused on the role of small RNAs, such as microRNAs, transfer RNAs and long non-coding RNAs, while...It is well accepted that sperm carry an RNA cargo with functions in early embryo development. However, most research has focused on the role of small RNAs, such as microRNAs, transfer RNAs and long non-coding RNAs, while protein-coding messenger RNAs (mRNAs) received less attention, even though they represent a substantial amount of the sperm RNA pool. Here, we curated mouse transcriptomic data from mature sperm and selected the most abundant mRNAs (above the 0.7 quantile). The obtained gene list was further filtered using two criteria: (i) mRNAs that are statistically higher in the one-cell embryo compared to the MII oocyte transcriptome, indicative of paternal mRNA contribution after fertilization; and (ii) mRNAs that are found bound to ribosomes in the one-cell embryo, indicative of positive translation in the zygote translatome. Our analysis identified 94 genes that form networks functionally involved in epigenetic chromatin organization, gene expression, RNA processing and translation during zygote genome activation. These findings underscore the significant role of sperm-borne mRNAs in early embryonic development and epigenetic inheritance, highlighting the need for further research to fully understand their functions.
is a Gram-negative bacterium that is associated with periodontitis and acute necrotizing ulcerative gingivitis. utilizes the type IX secretion system (T9SS) to secrete and anchor virulence factors to the cell surface, p...is a Gram-negative bacterium that is associated with periodontitis and acute necrotizing ulcerative gingivitis. utilizes the type IX secretion system (T9SS) to secrete and anchor virulence factors to the cell surface, presumably via C-terminal glycosylation. The identity of the linking sugar and the sites of modification on the cargo are unknown. Here, we employed hidden Markov models to predict cargo proteins in and conducted LC-MS/MS analyses of partially deglycosylated fractions to characterize the C-terminal glycosylation. A total of 80 cargo proteins were predicted based on the presence of a T9SS C-terminal domain (CTD) signal, and these were divided into 48 short CTDs and 32 long CTDs. Cleavage sites for five short and four long CTDs were experimentally determined, and glycosylation was observed at the mature C-terminus of six cargo. Two glycans were identified of delta masses 419.198 and 433.185 Da, corresponding to novel C-terminal amide linkages to N-alanyl dHex-HexNAc and N-alanyl (Me-dHex)-HexNAc, respectively. This indicated that both short and long CTDs supported cleavage and glycosylation. AlphaFold multimer modelling predicted that both kinds of CTDs could bind to the PorV shuttle protein in the same manner, with the conserved CTD motifs interacting with the same sites in PorV.
The small nucleolar RNA (snoRNA) SNORD116 is a small non-coding RNA of interest across multiple biomedical fields of research. Much of the investigation into SNORD116 has been undertaken in the context of the congenital...The small nucleolar RNA (snoRNA) SNORD116 is a small non-coding RNA of interest across multiple biomedical fields of research. Much of the investigation into SNORD116 has been undertaken in the context of the congenital disease Prader-Willi syndrome, wherein SNORD116 expression is lost. However, emerging evidence indicates wider roles in various disease and tissue contexts such as cellular growth, metabolism and signalling. Nevertheless, a conclusive mechanism of action for SNORD116 remains to be established. Here, we review the key findings from these investigations, with the aim of identifying common elements from which to elucidate potential targets and mechanisms of SNORD116. A key recurring element identified is disruption to the insulin/IGF-1 and PI3K/mTOR signalling pathways, contributing to many of the phenotypes associated with SNORD116 modulation explored in this review.
The molecular clock rests upon the assumption that the observed changes among sequences capture the differentiation of lineages, or kinship, as dissimilarity increases with time. Although it has been questioned over the...The molecular clock rests upon the assumption that the observed changes among sequences capture the differentiation of lineages, or kinship, as dissimilarity increases with time. Although it has been questioned over the years, this paradigmatic principle continues to underlie the idea that the polymorphic space of a gene is so vast that it is unattainable in evolutionary time. Thus, the molecular clock has been used to obtain taxonomic annotations, proving to be very effective at delivering testable results. In this article, however, we ask how often this assumption leads to inaccuracies when inferring the lineage of prokaryotic genes. Thus, we open an interesting discussion by simulating, in realistic scenarios, the critical times in which specific 5S rRNA sequences of two distant lineages are exhausting the polymorphic space. We contend that certain genes in one lineage will become increasingly similar to those in another over time, as the space for new variants is finite, mimicking phylogenetic features by convergence or by chance, without implying true kinship.
Applications like drug development need simple and streamlined methods to process samples from 96-well cell culture plates for gene expression measurements. Unfortunately, current options are expensive for such processin...Applications like drug development need simple and streamlined methods to process samples from 96-well cell culture plates for gene expression measurements. Unfortunately, current options are expensive for such processing. Therefore, our aim was to develop a method that would allow streamlined analysis of mRNA from 96-well cell culture plates while being relatively cheap and simple. We developed a method based on the qPCR 'Cells-to-cDNA' approach and validated it against commercially available kits using the same approach or spin columns-based RNA purification. For this purpose, we conducted a series of comparisons of gene expression from peripheral blood mononuclear cells, SK-HEP-1 and U-87 cell cultures in 96-well plates. Our final method involved lysing cells with 25-100 µl solution of 0.5% SDS, 10 mM DTT, 1 mg ml proteinase K dissolved in water, 1 h incubation at 50°C, followed by proteinase K inactivation at 90°C for 5 min and lysate neutralization with 1 : 1 dilution by 20% Tween 20 solution. Reverse transcription and qPCR were carried out using standard methods. This method showed a mean reduction of Ct ± s.d. value by 2.4 ± 1.3 compared with the 'Cells-to-cDNA' kit and by 1.4 ± 0.5 compared with the RNA purification kit with lower variability.
Pathogens exert strong selection on hosts that evolve and deploy different defensive strategies, namely minimizing pathogen exposure (avoidance), directly promoting pathogen elimination (resistance) and/or managing the d...Pathogens exert strong selection on hosts that evolve and deploy different defensive strategies, namely minimizing pathogen exposure (avoidance), directly promoting pathogen elimination (resistance) and/or managing the deleterious effects of illness (disease tolerance). However, how the host response partitions across these processes has not been directly tested in a single host-pathogen system, let alone in the context of known adaptive trajectories resulting from experimental evolution. Here, we compare a population adapted to oral infection with its natural pathogen (BactOral), to its control population to find no evidence for behavioural changes but measurable differences in both resistance and disease tolerance. In BactOral, no differences were detected in bacterial intake or defecation, nor gut cell renewal. However, a measurable relative decrease in bacterial loads correlates with an increase in gut-specific anti-microbial peptide production, pointing to a strengthening in resistance. Additionally, we posit that disease tolerance also contributes to the response of BactOral through a tighter control of self- and pathogen-derived damage caused by bacteria exposure. This study reveals a genetically complex and mechanistically multi-layered response, possibly reflecting the structure of adaptation to infection in natural populations.
Transgenerational epigenetic inheritance (TEI) describes the process whereby distinct epigenetic states are transmitted between generations, resulting in heritable gene expression and phenotypic differences that are inde...Transgenerational epigenetic inheritance (TEI) describes the process whereby distinct epigenetic states are transmitted between generations, resulting in heritable gene expression and phenotypic differences that are independent of DNA sequence variation. Chromatin modifications have been demonstrated to be important in TEI; however, the extent to which they require other signals to establish and maintain epigenetic states is still unclear. Here we investigate whether small non-coding RNAs contribute to different epigenetic states of the Fab2L transgene in triggered by transient long-range chromosomal contacts, which requires Polycomb complex activity to deposit the H3K27me3 modification for long-term TEI. By analysing mutants deficient in small non-coding RNAs, high-throughput sequencing data, long-range chromosomal contacts and gene expression, we demonstrate that small non-coding RNAs do not contribute directly to initiation or maintenance of silencing. However, we uncover an indirect role for microRNA expression in transgene silencing through effects on the Polycomb group gene . Additionally, we show that a commonly used marker gene, (), affects expression, which may be important in interpreting experiments assaying Polycomb function in development. By ruling out a plausible candidate for TEI at the Fab2L transgene, our work highlights the variability in different modes of TEI across species.
The nucleolus, once considered a mere 'ribosome factory', is now recognized as a dynamic hub influencing nearly every aspect of cellular life, from genome organization to stress response and ageing. Despite being a hallm...The nucleolus, once considered a mere 'ribosome factory', is now recognized as a dynamic hub influencing nearly every aspect of cellular life, from genome organization to stress response and ageing. Despite being a hallmark of eukaryotic cells, recent discoveries reveal that even prokaryotes exhibit nucleolus-like structures, hinting at ancient origins for nucleolar functions. This review explores the evolutionary journey of the nucleolus, tracing its roots back to early life and examining its structural and functional diversity across domains. We highlight key nucleolar proteins that play vital roles not only in ribosome production but also in regulating cell cycle, DNA repair and cellular stress, linking nucleolar activity directly to health and disease. Dysfunctions in nucleolar processes are implicated in cancer, ribosomopathies and neurodegenerative disorders, positioning the nucleolus as a critical target for innovative therapeutic strategies. As advanced imaging and molecular techniques unlock deeper insights into both canonical and mysterious non-canonical roles, the nucleolus stands as a model for how cellular microenvironments can evolve to meet complex biological demands. By addressing open questions surrounding the evolution of the nucleolus, its organization and diverse functions, the ideas presented here aim to contribute to the ongoing discussion, challenging traditional paradigms and suggesting new avenues for uncovering the fundamental principles that drive cellular life.
All the members of the phylum Cnidaria are characterized by the production of venom in specialized structures, the nematocysts. Venom of jellyfish (Medusozoa) and sea anemones (Anthozoa) has been investigated since the 1...All the members of the phylum Cnidaria are characterized by the production of venom in specialized structures, the nematocysts. Venom of jellyfish (Medusozoa) and sea anemones (Anthozoa) has been investigated since the 1970s, revealing a remarkable molecular diversity. Specifically, sea anemones harbour a rich repertoire of neurotoxic peptides, some of which have been developed in drug leads. However, venoms of the vast majority of Anthozoa species remain uncharacterized, particularly in the class Octocorallia. To fill this gap, we applied a proteo-transcriptomic approach to investigate venom composition in , a gorgonian species common in Mediterranean hard-bottom benthic communities. Our results highlighted the peculiarities of the venom of with respect to sea anemones, which is reflected in the presence of several toxins with novel folds, worthy of functional characterization. A comparative genomic survey across the octocoral radiation allowed us to generalize these findings and provided insights into the evolutionary history, molecular diversification patterns and putative adaptive roles of venom toxins. A comparison of whole-body and nematocyst proteomes revealed the presence of different cytolytic toxins inside and outside the nematocysts. Two instances of differential maturation patterns of toxin precursors were also identified, highlighting the intricate regulatory pathways underlying toxin expression.
In the peripheral nervous system, glial cells, known as Schwann cells (SCs), are responsible for supporting and maintaining nerves. One of the most important characteristics of SCs is their remarkable plasticity. In vari...In the peripheral nervous system, glial cells, known as Schwann cells (SCs), are responsible for supporting and maintaining nerves. One of the most important characteristics of SCs is their remarkable plasticity. In various injury contexts, SCs undergo a reprogramming process that generates specialized cells to promote tissue regeneration and repair. However, in pathological conditions, this same plasticity and regenerative potential can be hijacked. Different studies highlight the activation of the epithelial-mesenchymal transition (EMT) as a driver of SC phenotypic plasticity. Although SCs are not epithelial, their neural crest origin makes EMT activation crucial for their ability to adopt repair phenotypes, mirroring the plasticity observed during development. These adaptive processes are essential for regeneration. However, EMT activation in SCs-derived tumours enhances cancer progression and aggressiveness. Furthermore, in the tumour microenvironment (TME), SCs also acquire activated phenotypes that contribute to tumour migration and invasion by activating EMT in cancer cells. In this review, we will discuss how EMT impacts SC plasticity and function from development and tissue regeneration to pathological conditions, such as cancer.
Insects can survive harsh conditions, including Arctic winters, by entering a hormonally induced state of dormancy, known as diapause. Diapause is triggered by environmental cues such as shortening of the photoperiod (le...Insects can survive harsh conditions, including Arctic winters, by entering a hormonally induced state of dormancy, known as diapause. Diapause is triggered by environmental cues such as shortening of the photoperiod (lengthening of the night). The time of entry into diapause depends on the latitude of the insects' habitat, and this applies even within a species: populations living at higher latitudes enter diapause earlier in the year than populations living at lower latitudes. A long-standing question in biology is whether the internal circadian clock, which governs daily behaviour and serves as a reference clock to measure night length, shows similar latitudinal adaptations. To address this question, we examined the onset of diapause and various behavioural and molecular parameters of the circadian clock in the cosmopolitan fly, , a species distributed throughout Europe from the Black Sea (41° N) to Arctic regions (69° N). We found that all clock parameters examined showed the same correlation with latitude as the critical night length for diapause induction. We conclude that the circadian clock has adapted to the latitude and that this may result in the observed latitudinal differences in the onset of diapause.
Metabolic processes in fetuses can significantly influence piglet weight at birth. Understanding the genetic determinants of systemic metabolism is crucial for uncovering how genetic and molecular pathways impact biologi...Metabolic processes in fetuses can significantly influence piglet weight at birth. Understanding the genetic determinants of systemic metabolism is crucial for uncovering how genetic and molecular pathways impact biological mechanisms, particularly during the fetal phase. We present data on 1112 plasma metabolites using untargeted ultra-high performance liquid chromatography-tandem mass spectrometry methods, of 260 backcross (BC) fetuses from two sires' breeds at 63 days post-conception. Eight chemical superclasses have been identified, with lipids accounting for the majority of metabolites. Genomic heritability (h²) was estimated for each metabolite, revealing that 50% had h² values below 0.2, with a higher average in the amino acid class compared with the lipid. We annotated 448 significant metabolite quantitative trait loci associated with 10 metabolites, primarily lipids, indicating strong genetic regulation. Additionally, metabolite associations with sex, fetal weight and sire's breed were explored, revealing significant associations for 354 metabolites. Fetal weight influenced the largest number of metabolites, particularly glycerophospholipids and sphingolipids, emphasizing the genetic and metabolic complexity underlying fetal development. These findings enhance our understanding of the genetic regulation of metabolite levels and their associations with key phenotypic traits in fetuses, providing insights into metabolic pathways, potential biomarkers and serving as a baseline dataset for metabolomics studies of fetuses.
Pigment-dispersing factors (PDFs) are neuropeptides that play key roles in controlling the circadian rhythms in various insects, whereas their function remains elusive in other protostomes including tardigrades (water be...Pigment-dispersing factors (PDFs) are neuropeptides that play key roles in controlling the circadian rhythms in various insects, whereas their function remains elusive in other protostomes including tardigrades (water bears). Here we show that the three PDFs of the tardigrade are co-localized in two pairs of inner lobe cells in the brain, whereas only one PDF occurs in four additional cerebral and two extracerebral cells. The axons of the inner lobe cells pass through the contralateral brain hemisphere, descend to the ventral nerve cord and terminate in two pairs of potential release sites in the posteriormost trunk ganglion. Using assays, we demonstrate that all three PDFs and their deorphanized receptor (PDFR) are functional. Widespread localization of PDFR suggests that tardigrade PDFs may act as multifunctional hormones and neuromodulators that control major functions including light detection, neural processing, locomotion, feeding, digestion, osmoregulation, growth, embryonic development and oogenesis/reproduction.
The lung microbiome has recently gained attention for potentially affecting respiratory viral infections, including influenza A virus, respiratory syncytial virus (RSV) and SARS-CoV-2. We will discuss the complexities of...The lung microbiome has recently gained attention for potentially affecting respiratory viral infections, including influenza A virus, respiratory syncytial virus (RSV) and SARS-CoV-2. We will discuss the complexities of the lung microenvironment in the context of viral infections and the use of organ-on-chip (OoC) models in replicating the respiratory tract milieu to aid in understanding the role of temporary microbial colonization. Leveraging the innovative capabilities of OoC, particularly through integrating gut and lung models, opens new avenues to understand the mechanisms linking inter-organ crosstalk and respiratory infections. We will discuss technical aspects of OoC lung models, ranging from the selection of cell substrates for extracellular matrix mimicry, mechanical strain, breathing mechanisms and air-liquid interface to the integration of immune cells and use of microscopy tools for algorithm-based image analysis and systems biology to study viral infection . OoC offers exciting new options to study viral infections across host species and to investigate human cellular physiology at a personalized level. This review bridges the gap between complex biological phenomena and the technical prowess of OoC models, providing a comprehensive roadmap for researchers in the field.
Emerging infectious diseases can have a major impact on fitness of novel hosts, thereby contributing to ongoing species declines. In social insects, collaborative brood care by workers and successful mating of male sexua...Emerging infectious diseases can have a major impact on fitness of novel hosts, thereby contributing to ongoing species declines. In social insects, collaborative brood care by workers and successful mating of male sexuals are key to colony fitness. The microsporidian endoparasite has spread almost globally, shifting across honeybee species and now to bumblebees. However, despite being linked to recent population declines, its possible impact on bumblebee colony fitness remains poorly understood. Here, we show that infections can significantly impact worker feeding glands, as well as longevity, sperm quality and mating abilities of drones. In the laboratory, workers and drones were either exposed to the parasite or not. Then, parasite infection rates and loads, as well as lethal and sublethal parameters, were assessed. Infected drones revealed higher parasite infection rates and spore titres, as well as mortality compared with female workers, suggesting sex-specific susceptibility. Furthermore, infections impaired feeding glands, affected sperm traits and altered mating behaviour, all of which are key to colony fitness. Our findings provide a mechanistic explanation on how contributes to the ongoing decline of wild bumblebee populations, calling for respective mitigation measures.
The PACS (phosphofurin acidic cluster sorting protein) proteins are membrane trafficking regulators, required for maintaining cellular homeostasis and preventing disease states. Mutations in human and cause human neuro...The PACS (phosphofurin acidic cluster sorting protein) proteins are membrane trafficking regulators, required for maintaining cellular homeostasis and preventing disease states. Mutations in human and cause human neurodevelopmental disorders, characterized by epileptic seizures and neurodevelopmental delay. In vertebrates, functional analysis of PACS proteins is complicated by the presence of two paralogues which can compensate for the loss of each other. Here, we characterize the unique fly homologue of human PACS proteins. We demonstrate that Drosophila PACS (dPACS) is required for cell division in dividing spermatocytes and neuroblasts. In primary spermatocytes, dPACS colocalizes with GOLPH3 at the Golgi stacks and is essential for maintaining Golgi architecture. In dividing cells, dPACS is necessary for central spindle stability and contractile ring constriction. dPACS and GOLPH3 proteins form a complex and are mutually dependent for localization to the cleavage site. We propose that dPACS, by associating with GOLPH3, mediates the flow of vesicle trafficking that supports furrow ingression during cytokinesis. Furthermore, loss of dPACS leads to defects in tubulin acetylation and severe bang sensitivity, a phenotype associated with seizures in flies. Together our findings suggest that a Drosophila disease model may contribute to understanding the molecular mechanisms underpinning human PACS syndromes.
Antimicrobial resistance (AMR) is one of the top global health threats. In 2019, AMR was associated with 4.95 million deaths, of which 1.97 million were caused by drug-resistant infections directly. The main subset of AM...Antimicrobial resistance (AMR) is one of the top global health threats. In 2019, AMR was associated with 4.95 million deaths, of which 1.97 million were caused by drug-resistant infections directly. The main subset of AMR is antibiotic resistance, that is, the resistance of bacteria to antibiotic treatment. Traditional and most commonly used antibiotic susceptibility tests are based on the detection of bacterial growth and its inhibition in the presence of an antimicrobial. These tests typically take over 1-2 days to perform, so empirical therapy schemes are often administered before proper testing. Rapid tests for AMR are necessary to optimize the treatment of bacterial infection. Here, we combine the MTT test with Raman spectroscopy to provide a 1.5 h long test for minimal inhibitory concentration determination. Several and strains were tested with three types of antibiotics, including ampicillin from penicillin family, kanamycin from aminoglycoside family and levofloxacin from fluoroquinolone family. The test provided the same minimal inhibitory concentrations as traditional Etest confirming its robustness.
Secretory cargos are exported from the ER via COPII-coated vesicles that have an inner matrix of Sec23/Sec24 heterotetramers and an outer cage of Sec13/Sec31 heterotetramers. In addition to COPII, Sec13 is part of the nu...Secretory cargos are exported from the ER via COPII-coated vesicles that have an inner matrix of Sec23/Sec24 heterotetramers and an outer cage of Sec13/Sec31 heterotetramers. In addition to COPII, Sec13 is part of the nuclear pore complex (NPC) and the regulatory SEA/GATOR complex in eukaryotes, which typically have one Sec13 orthologue. The kinetoplastid parasite has two paralogues: TbSec13.1, an accepted component of both COPII and the NPC, and TbSec13.2. Little is known about TbSec13.2, but others have proposed that it, and its orthologue in the distantly related diplonemid , operate exclusively in the SEA/GATOR complex, and that this represents an evolutionary diversification of function unique to the euglenozoan protists. Using RNAi silencing in trypanosomes, we show both TbSec13s are essential. Knockdown of each dramatically and equally delays transport of GPI-anchored secretory cargo, indicating roles for both in COPII-mediated trafficking from the ER. Immunofluorescence and proximity labelling studies confirm that both TbSec13.1 and TbSec13.2 co-localize with TbSec24.1 to ER exit sites, and thus are functional components of the COPII machinery. Our findings indicate that TbSec13.2 function is not restricted to the SEA/GATOR complex in trypanosomes.