Search Results (25)

RNA-seq analysis of the highly differentiated human retinal pigment epithelial (RPE) cell-line ARPE-19, cultured on transwells for ≥4 months, yielded 44,909 genes showing 83.35% alignment with the human reference genome. These included mRNA transcripts of RPE-specific genes and those involved in retinopathies. Monolayers were fed photoreceptor outer segments (POS), designed to be synchronously internalised, mimicking homeostatic RPE activity. Cells were subsequently fixed at 4, 6, 24 and 48 h when POS were previously shown to maximally co-localise with Rab5, Rab7, LAMP/lysosomes and LC3b/autophagic compartments. A comprehensive analysis of differentially expressed genes involved in proteolysis revealed a pattern of gene orchestration consistent with POS breakdown in the autophagy-lysosomal pathway. At 4 h, these included elevated upstream signalling events promoting early stages of cargo transport and endosome maturation compared to RPE without POS exposure. This transcriptional landscape altered from 6 h, transitioning to promoting cargo degradation in autolysosomes by 24–48 h. Longitudinal scrutiny of mRNA transcripts revealed nuanced differences even within linked gene networks. POS exposure also initiated transcriptional upregulation in ubiquitin proteasome and chaperone-mediated systems within 4–6 h, providing evidence of cross-talk with other proteolytic processes. These findings show detailed evidence of transcriptome-level responses to cargo trafficking and processing in RPE cells. Full article

This study identifies and classifies resistance gene analogues (RGAs) in the genomes of Brassica nigra, Sinapis arvensis and Sinapis alba using the RGAugury pipeline. RGAs were categorised into four main classes: receptor-like kinases (RLKs), receptor-like proteins (RLPs), nucleotide-binding leucine-rich repeat (NLR) proteins and transmembrane-coiled-coil (TM-CC) genes. A total of 4499 candidate RGAs were detected, with species-specific proportions. RLKs were the most abundant across all genomes, followed by TM-CCs and RLPs. The sub-classification of RLKs and RLPs identified LRR-RLKs, LRR-RLPs, LysM-RLKs, and LysM-RLPs. Atypical NLRs were more frequent than typical ones in all species. Atypical NLRs were more frequent than typical ones in all species. We explored the relationship between chromosome size and RGA count using regression analysis. In B. nigra and S. arvensis, larger chromosomes generally harboured more RGAs, while S. alba displayed the opposite trend. Exceptions were observed in all species, where some larger chromosomes contained fewer RGAs in B. nigra and S. arvensis, or more RGAs in S. alba. The distribution and density of RGAs across chromosomes were examined. RGA distribution was skewed towards chromosomal ends, with patterns differing across RGA types. Sequence hierarchical pairwise similarity analysis revealed distinct gene clusters, suggesting evolutionary relationships. The study also identified homologous genes among RGAs and non-RGAs in each species, providing insights into disease resistance mechanisms. Finally, RLKs and RLPs were co-localised with reported disease resistance loci in Brassica, indicating significant associations. Phylogenetic analysis of cloned RGAs and QTL-mapped RLKs and RLPs identified distinct clusters, enhancing our understanding of their evolutionary trajectories. These findings provide a comprehensive view of RGA diversity and genomics in these Brassicaceae species, providing valuable insights for future research in plant disease resistance and crop improvement. Full article

Background/Objectives: Kv2 channels have important conducting and nonconducting functions and are regulated by their co-assembly with ‘silent’ Kv subunits, including Kv9.1. Kv9.1 is co-expressed with Kv2 channels in sensory neurons, and a common allele that changes Ile489 to Val in human Kv9.1 is associated with pain hypersensitivity in patients. The mechanism responsible for this association remains unknown, but we hypothesise that these two variants differ in their regulation of Kv2.1 properties, and this is what we set out to test. Methods: Expression was carried out using HEK293 cells, OHeLa cells, and primary cultures of hippocampal neurons, and the biophysical and trafficking properties of homomeric and heteromeric channels were assessed by confocal fluorescence microscopy and patch clamp analysis. Results: Both Kv9.1Ile and Kv9.1Val were retained within the endoplasmic reticulum when expressed individually, but when co-expressed with Kv2.1, they co-localised with Kv2.1 within the surface clusters. Both variants reduced the surface expression of Kv2.1 channels and the size of channel clusters, with Kv9.1Val producing a greater reduction in surface expression in both the HeLa cells and neurons. They both caused a similar hyperpolarising shift in the voltage dependence of channel activation and inactivation. Concatamers of Kv2.1 and Kv9.1 suggested that both 3:1 and 2:2 ratios of Kv2.1 to Kv9.1 were permitted, although 2:2 resulted in lower surface expression and function. Conclusions: The Ile489Val substitution in Kv9.1 does not disrupt its ability to co-assemble with Kv2 channels, nor its effects on the voltage-dependence of channel gating, but it did produce a greater reduction in the Kv2.1 surface expression, suggesting that this underlies its association with pain hypersensitivity. Full article

Proteins of the PF10950 family feature the DUF2775 domain of unknown function. The most studied are specific tissue (ST) proteins with tandem repeats, which are putative precursors of cyclopeptide alkaloids. Here, we study uncharacterised short ST (SST) proteins with the DUFF2775 domain by analysing 194 sequences from 120 species of 39 taxonomic families in silico. SST proteins have a signal peptide and their size and several other characteristics depend on their individual taxonomic family. Sequence analyses revealed that SST proteins contain two well-conserved regions, one resembling the ST repeat, which could constitute the core of cyclopeptide alkaloids. We studied the unique SST1 gene of Arabidopsis thaliana, which is adjacent to and co-expressed with a gene encoding a protein with a BURP domain, associated with cyclopeptide production. The empirical analysis indicated that the SST1 promoter is mainly activated in the roots, where most of the transcripts accumulate, and that the SST1 protein accumulates in the root vascular cambium. At the cellular level, SST fused to GFP appears in vesicles that co-localise with the endoplasmic reticulum and the vacuole. Thus, SSTs are a new type of PF10950 protein found in core eudicots with two conserved regions that could be involved in root biology. Full article

Genome-wide association studies (GWASs) have revealed numerous loci associated with breast cancer risk, yet the precise causal variants, their impact on molecular mechanisms, and the affected genes often remain elusive. We hypothesised that specific variants exert their influence by affecting cis-regulatory alternative splice elements. An analysis of splicing quantitative trait loci (sQTL) in healthy breast tissue from female individuals identified multiple variants linked to alterations in splicing ratios. Through colocalisation analysis, we pinpointed 43 variants within twelve genes that serve as candidate causal links between sQTL and GWAS findings. In silico splice analysis highlighted a potential mechanism for three genes—FDPS, SGCE, and MRPL11—where variants in proximity to or on the splice site modulate usage, resulting in alternative splice transcripts. Further in vitro/vivo studies are imperative to fully understand how these identified changes contribute to breast oncogenesis. Moreover, investigating their potential as biomarkers for breast cancer risk could enhance screening strategies and early detection methods for breast cancer. Full article

Yeast two-hybrid approaches, which are based on fusion proteins that must co-localise to the nucleus to reconstitute the transcriptional activity of GAL4, have greatly contributed to our understanding of the nitrogen interaction network of cyanobacteria, the main hubs of which are the trimeric PII and the monomeric PipX regulators. The bacterial two-hybrid system, based on the reconstitution in the E. coli cytoplasm of the adenylate cyclase of Bordetella pertussis, should provide a relatively faster and presumably more physiological assay for cyanobacterial proteins than the yeast system. Here, we used the bacterial two-hybrid system to gain additional insights into the cyanobacterial PipX interaction network while simultaneously assessing the advantages and limitations of the two most popular two-hybrid systems. A comprehensive mutational analysis of PipX and bacterial two-hybrid assays were performed to compare the outcomes between yeast and bacterial systems. We detected interactions that were previously recorded in the yeast two-hybrid system as negative, as well as a “false positive”, the self-interaction of PipX, which is rather an indirect interaction that is dependent on PII homologues from the E. coli host, a result confirmed by Western blot analysis with relevant PipX variants. This is, to our knowledge, the first report of the molecular basis of a false positive in the bacterial two-hybrid system. Full article

Most of the time, the deep analysis of a biological sample requires the acquisition of images at different time points, using different modalities and/or different stainings. This information gives morphological, functional, and physiological insights, but the acquired images must be aligned to be able to proceed with the co-localisation analysis. Practically speaking, according to Aristotle’s principle, “The whole is greater than the sum of its parts”, multi-modal image registration is a challenging task that involves fusing complementary signals. In the past few years, several methods for image registration have been described in the literature, but unfortunately, there is not one method that works for all applications. In addition, there is currently no user-friendly solution for aligning images that does not require any computer skills. In this work, DS4H Image Alignment (DS4H-IA), an open-source ImageJ/Fiji plugin for aligning multimodality, immunohistochemistry (IHC), and/or immunofluorescence (IF) 2D microscopy images, designed with the goal of being extremely easy to use, is described. All of the available solutions for aligning 2D microscopy images have also been revised. The DS4H-IA source code; standalone applications for MAC, Linux, and Windows; video tutorials; manual documentation; and sample datasets are publicly available. Full article

Damage-Regulated Autophagy Modulator 1 (DRAM1) is an infection-inducible membrane protein, whose function in the immune response is incompletely understood. Based on previous results in a zebrafish infection model, we have proposed that DRAM1 is a host resistance factor against intracellular mycobacterial infection. To gain insight into the cellular processes underlying DRAM1-mediated host defence, here we studied the interaction of DRAM1 with Mycobacterium marinum in murine RAW264.7 macrophages. We found that, shortly after phagocytosis, DRAM1 localised in a punctate pattern to mycobacteria, which gradually progressed to full DRAM1 envelopment of the bacteria. Within the same time frame, DRAM1-positive mycobacteria colocalised with the LC3 marker for autophagosomes and LysoTracker and LAMP1 markers for (endo)lysosomes. Knockdown analysis revealed that DRAM1 is required for the recruitment of LC3 and for the acidification of mycobacteria-containing vesicles. A reduction in the presence of LAMP1 further suggested reduced fusion of lysosomes with mycobacteria-containing vesicles. Finally, we show that DRAM1 knockdown impairs the ability of macrophages to defend against mycobacterial infection. Together, these results support that DRAM1 promotes the trafficking of mycobacteria through the degradative (auto)phagolysosomal pathway. Considering its prominent effect on host resistance to intracellular infection, DRAM1 is a promising target for therapeutic modulation of the microbicidal capacity of macrophages. Full article

Canola plants suffer severe crop yield and oil content reductions when exposed to water-deficit conditions, especially during the reproductive stages of plant development. There is a pressing need to develop canola cultivars that can perform better under increased water-deficit conditions with changing weather patterns. In this study, we analysed genetic determinants for the main effects of quantitative trait loci (QTL), (Q), and the interaction effects of QTL and Environment (QE) underlying seed yield and related traits utilising 223 doubled haploid (DH) lines of canola in well-watered and water-deficit conditions under a rainout shelter. Moderate water-deficit at the pre-flowering stage reduced the seed yield to 40.8%. Multi-environmental QTL analysis revealed 23 genomic regions associated with days to flower (DTF), plant height (PH) and seed yield (SY) under well-watered and water-deficit conditions. Three seed yield QTL for main effects were identified on chromosomes A09, C03, and C09, while two were related to QE interactions on A02 and C09. Two QTL regions were co-localised to similar genomic regions for flowering time and seed yield (A09) and the second for plant height and chlorophyll content. The A09 QTL was co-located with a previously mapped QTL for carbon isotope discrimination (Δ¹³C) that showed a positive relationship with seed yield in the same population. Opposite allelic effects for plasticity in seed yield were identified due to QE interactions in response to water stress on chromosomes A02 and C09. Our results showed that QTL’s allelic effects for DTF, PH, and SY and their correlation with Δ¹³C are stable across environments (field conditions, previous study) and contrasting water regimes (this study). The QTL and DH lines that showed high yield under well-watered and water-deficit conditions could be used to manipulate water-use efficiency for breeding improved canola cultivars. Full article

Genomic compaction is an essential characteristic of living organisms. Nucleoid-associated proteins (NAPs) are a group of small proteins that play crucial roles in chromosome architecture and affect DNA replication, transcription, and recombination by imposing topological alterations in genomic DNA, thereby modulating global gene expression. EbfC/YbaB was first described as a DNA-binding protein of Borrelia burgdorferi that regulates the expression of surface lipoproteins with roles in virulence. Further studies indicated that this protein binds specifically and non-specifically to DNA and colocalises with nucleoids in this bacterium. The data showed that this protein binds to DNA as a homodimer, although it can form other organised structures. Crystallography analysis indicated that the protein possesses domains responsible for protein–protein interactions and forms a “tweezer” structure probably involved in DNA binding. Moreover, sequence analysis revealed conserved motifs that may be associated with dimerisation. Structural analysis also showed that the tridimensional structure of EbfC/YbaB is highly conserved within the bacterial domain. The DNA-binding activity was observed in different bacterial species, suggesting that this protein can protect DNA during stress conditions. These findings indicate that EbfC/YbaB is a broadly distributed NAP. Here, we present a review of the existing data on this NAP. Full article

Supplemental lighting with light-emitting diode (LED) lamps and/or high-pressure sodium (HPS) lamps was applied to increase the activity of the photosynthetic apparatus and thus productivity of greenhouse cucumber (Cucumis sativus L.) in a high-wire growing system. The colocalisation of the chlorophyll of PSII (located mainly in grana) and carotenoid fluorescence signals in chloroplasts of cucumber leaves was studied under confocal microscopy. Leaf anatomy and some chemical quality traits (dry matter, chlorophyll, carotenoids, total soluble solids, total sugars and nitrate reductase activity) as well as selected chlorophyll fluorescence parameters were also investigated and subjected to the multidimensional principal component analysis together with the data on fruit yield. Under LED lighting, a lower correlation between the occurrence of chlorophyll and carotenoid fluorescence signals was observed, especially in older (lower-located) leaves, which may have resulted from changes in the distribution of carotenoids within chloroplasts and/or relative concentrations of chlorophyll and carotenoids. Compared to toplighting with HPS lamps, most commonly used in commercial greenhouse cucumber production, the application of LED interlighting, especially in combination with LED toplighting, led to the increase in chlorophyll and carotenoid content and photosynthetic performance index in older leaves, which was related to the increased cucumber productivity. Full article

Tospoviruses infect numerous crop species worldwide, causing significant losses throughout the supply chain. As a defence mechanism, plants use RNA interference (RNAi) to generate virus-derived small-interfering RNAs (vsiRNAs), which target viral transcripts for degradation. Small RNA sequencing and in silico analysis of capsicum and N. benthamiana infected by tomato spotted wilt virus (TSWV) or capsicum chlorosis virus (CaCV) demonstrated the presence of abundant vsiRNAs, with host-specific differences evident for each pathosystem. Despite the biogenesis of vsiRNAs in capsicum and N. benthamiana, TSWV and CaCV viral loads were readily detectable. In response to tospovirus infection, the solanaceous host species also generated highly abundant virus-activated small interfering RNAs (vasiRNAs) against many endogenous transcripts, except for an N. benthamiana accession lacking a functional RDR1 gene. Strong enrichment for ribosomal protein-encoding genes and for many genes involved in protein processing in the endoplasmic reticulum suggested co-localisation of viral and endogenous transcripts as a basis for initiating vasiRNA biogenesis. RNA-seq and RT-qPCR-based analyses of target transcript expression revealed an inconsistent role for vasiRNAs in modulating gene expression in N. benthamiana, which may be characteristic of this tospovirus-host pathosystem. Full article

Immune checkpoint blockade (ICB) therapies, such as immune checkpoint inhibitors against programmed death ligand-1 (PD-L1), have not been successful in treating patients with pancreatic ductal adenocarcinoma (PDAC). Despite the critical role of PD-L1 in various types of cancers, the regulatory mechanism of PD-L1 expression on the cell surface of PDAC is poorly understood. Therefore, uncovering potential modulators of cell surface localisation of PD-L1 may provide a new strategy to improve ICB therapy in patients with PDAC. Here, we examined the role of ezrin/radixin/moesin (ERM) family scaffold proteins that crosslink transmembrane proteins with the actin cytoskeleton in the surface localisation of PD-L1 in KP-2 cells, a human PDAC cell line. Our results demonstrated the abundant protein expression of PD-L1, ezrin, and radixin, but not moesin, as well as their colocalisation in the plasma membrane. Interestingly, immunoprecipitation analysis detected the molecular interaction of PD-L1 with ezrin and radixin. Moreover, gene silencing of ezrin moderately decreased the mRNA and cell surface expression of PD-L1, while that of radixin greatly decreased the surface expression of PD-L1 without altering the mRNA levels. Thus, radixin and ezrin differentially modulate the cell surface localisation of PD-L1 in KP-2 cells, highlighting a potential therapeutic target to improve the current ICB therapy in PDAC. Full article

Background: Community-acquired pneumonia (CAP) is an acute disease condition with a high risk of rapid deteriorations. We analysed the influence of genetics on cytokine regulation to obtain a better understanding of patient’s heterogeneity. Methods: For up to N = 389 genotyped participants of the PROGRESS study of hospitalised CAP patients, we performed a genome-wide association study of ten cytokines IL-1β, IL-6, IL-8, IL-10, IL-12, MCP-1 (MCAF), MIP-1α (CCL3), VEGF, VCAM-1, and ICAM-1. Consecutive secondary analyses were performed to identify independent hits and corresponding causal variants. Results: 102 SNPs from 14 loci showed genome-wide significant associations with five of the cytokines. The most interesting associations were found at 6p21.1 for VEGF (p = 1.58 × 10⁻²⁰), at 17q21.32 (p = 1.51 × 10⁻⁹) and at 10p12.1 (p = 2.76 × 10⁻⁹) for IL-1β, at 10p13 for MIP-1α (CCL3) (p = 2.28 × 10⁻⁹), and at 9q34.12 for IL-10 (p = 4.52 × 10⁻⁸). Functionally plausible genes could be assigned to the majority of loci including genes involved in cytokine secretion, granulocyte function, and cilial kinetics. Conclusion: This is the first context-specific genetic association study of blood cytokine concentrations in CAP patients revealing numerous biologically plausible candidate genes. Two of the loci were also associated with atherosclerosis with probable common or consecutive pathomechanisms. Full article

Trypanosome brucei, the causative agent of African sleeping sickness, harbours a highly ordered, subpellicular microtubule cytoskeleton that defines many aspects of morphology, motility and virulence. This array of microtubules is associated with a large number of proteins involved in its regulation. Employing proximity-dependent biotinylation assay (BioID) using the well characterised cytoskeleton-associated protein CAP5.5 as a probe, we identified CAP50 (Tb927.11.2610). This protein colocalises with the subpellicular cytoskeleton microtubules but not with the flagellum. Depletion by RNAi results in defects in cytokinesis, morphology and partial disorganisation of microtubule arrays. Published proteomics data indicate a possible association of CAP50 with two other, yet uncharacterised, cytoskeletal proteins, CAP52 (Tb927.6.5070) and CAP42 (Tb927.4.1300), which were therefore included in our analysis. We show that their depletion causes phenotypes similar to those described for CAP50 and that they are essential for cellular integrity. Full article