Search Results (27)

Tomato fruit rot severely impacts yield and quality, causing economic losses. This study aimed to identify the pathogenic fungi associated with post-harvest tomato fruit rot and characterize the transcriptomic responses of tomatoes. Pathogens were isolated from diseased tomato fruit tissues and identified using morphology, phylogenetic analysis, and in vitro pathogenicity tests. The genome of Cladosporium oxysporum Co-1 was assembled and annotated. RNA-seq analysis was used to profile transcriptional changes in tomatoes infected with C. oxysporum Co-1, with RT-qPCR validating the RNA-seq data and spectrophotometric assays analyzing the host physiological responses. Three pathogenic fungi were isolated. Colonies of C. oxysporum exhibited a near-circular shape, with colonies transitioning from an olive-green center to gray-green at the edges, and based on ITS, β-tubulin, and EF-1α gene sequences, this isolate exhibited 99% identity with C. oxysporum. The other two fungal isolates were identified as Alternaria alternata and Fusarium incarnatum, respectively, based on morphological and multi-locus sequence analysis. All three strains induced fruit rot and browning in tomatoes, confirming their pathogenicity. The genome size of C. oxysporum Co-1 was 34,515,558 bp, comprising 52 scaffolds with a GC content of 52.82%, and encoding 10,081 protein-coding genes. RNA-seq analysis showed dynamic gene expression changes in tomatoes infected with strain A, with differentially expressed genes enriched in pathogenicity-related pathways. Spectrophotometric assays revealed that peroxidase and superoxide dismutase activities decreased initially followed by an increase post-inoculation with C. oxysporum, indicating that tomatoes defend against pathogen infection through the antioxidant enzyme system. These findings revealed the pathogenic fungi were associated with post-harvest tomato rot disease, provided genomic resources for C. oxysporum, and provided insight into the host’s response to this strain. Full article

Background: Dominantly acting variants in TUBB2B have primarily been associated with cortical dysplasia complex with other brain malformations 7 (CDCBM7), a disorder in which cortical brain abnormalities are typically linked to developmental delay/intellectual disability (DD/ID) and seizures. While the majority of TUBB2B pathogenic variants have been linked to isolated CDCBM7, only one family with CDCBM7 and congenital fibrosis of the extraocular muscles (CFEOM) has been reported so far. We describe a second individual with a severe phenotype of CFEOM combined with CDCBM7 carrying a pathogenic TUBB2B missense variant previously reported in two individuals with isolated CDCBM7. Methods: A trio-based WGS analysis was performed. The structural impact of the identified substitution was assessed by using the UCSF Chimera (v.1.17.3) software and PyMOL docking plugin DockingPie tool. Results: WGS analysis identified a de novo missense TUBB2B variant (p.Ile202Thr, NM_178012.5), previously associated with isolated CDCBM7. Structural analysis and docking simulations revealed that Ile202 contributes to establishing a proper hydrophobic environment required to stabilize GTP/GDP in the β-tubulin pocket. p.Ile202Thr was predicted to disrupt these interactions. Conclusions: Our findings broaden the mutational spectrum of TUBB2B-related CFEOM, targeting a different functional domain of the protein, and further document the occurrence of phenotypic heterogeneity. We also highlight the limitations of exome sequencing in accurately mapping TUBB2B coding exons due to its high sequence homology with TUBB2A and suggest targeted or genome analyses when clinical suspicion is strong. Full article

Recently, Fusarium root rot (FRR)-like symptoms were observed in Uganda’s agroecology zones, prompting the National Agricultural Organisation (NARO) to conduct a disease survey. The survey reports indicated FRR as the second most prevalent root rot disease of common bean in Uganda after Southern blight. Ninety nine Fusarium spp. strains were obtained from samples collected during the surveys. The strains were morphologically and pathogenically characterised and confirmed to cause Fusarium root rot as observed in the field. However, molecular characterization of the strains was not conducted. In this study, therefore, 80 of the strains were characterized using partial sequences of translation elongation factor 1-alpha (TEF-1α) gene, beta tubulin (β tubulin) gene and internal transcribed spacers (ITS) region of ribosomal RNA to determine species diversity. High-quality Sanger sequences from the target genes were compared to the sequences from Fusarium species available in the National Centre for Biotechnology Information coding sequences (NCBI-CDS) database to determine the most likely species the strains belonged. The sequences from our strains were deposited into the NCBI gene bank under ID#288420, 2883276, 2873058 for TEF-1α, β tubulin and ITS respectively. The Fusarium species identified included; F. oxysporum, F. solani, F. equiseti F. delphinoides, F. commune, F. subflagellisporum, F. fabacearum, F. falciforme, F. brevicaudatum, F. serpentimum, F. fredkrugeri and F. brachygibbosum. The diversity of these Fusarium species needs to be taken into consideration when developing breeding programs for management of the disease since currently there is no variety of common bean resistant to FRR in Uganda. Full article

Babesia bigemina is one of the most important etiological agents of bovine babesiosis, a tick-borne disease posing a major threat in the livestock industry globally, including South Africa. Despite the huge economic impact of cattle babesiosis in South Africa, antigenic variation observed among B. bigemina strains worldwide has impeded the successful development of a single vaccine with the potential to eliminate the disease. As such, there is still a dearth of information regarding the conservation of B. bigemina genes encoding functionally important proteins that play a crucial role during the invasion of bovine erythrocytes by merozoites. Fifty blood samples previously collected from cattle in eight provinces of South Africa were genetically tested for the presence of B. bigemina DNA fragments using four nested PCR-based assays. The genes targeted coded for SpeI-AvaI restriction fragment, rhoptry-associated protein 1 (BgRAP-1), apical membrane antigen 1 (BgAMA-1) and β-tubulin (BgβTUB). PCR-generated fragments of randomly selected samples were sequenced. BLAST searches in GenBank were performed with newly determined sequences to search for homologous sequences. Neighbor-joining phylogenies were inferred from aligned, contiguous sequences of BgRAP-1, BgAMA-1 and BgβTUB genes. Nested PCR assays generated single fragments of 170 bp, 472 bp, 765 bp and 302 bp for SpeI-AvaI, BgRAP-1, BgAMA-1 and BgβTUB fragments, respectively. Of the 50 bovine samples tested by nested PCR, 82% (42/50; 95% CI = 69.2–90.2%), 68% (34/50; 95% CI = 54.2–79.2%), 50% (25/50; 95% CI = 36.6–63.4%) and 46% (23/50; 95% CI = 33.0–59.6%) possessed B. bigemina-specific SpeI-AvaI, BgRAP-1, BgAMA-1 and BgβTUB DNA fragments, respectively. The BgRAP-1, BgAMA-1 and BgβTUB sequences of South African B. bigemina isolates shared 98–100% similarity with previously reported sequences of strains originating from cattle in countries other than South Africa. The high genetic conservation observed among geographical isolates of B. bigemina suggests the conserved functional role of BgRAP-1 and BgAMA-1 proteins as potential candidates that could be incorporated in recombinant subunit vaccines. Full article

Phytopathogenic oomycetes, particularly Phytophthora capsici, the causal agent of Phytophthora blight disease in essential vegetables and fruit crops, remains a persistent challenge in the vegetable production industry. However, the core molecular regulators of the pathophysiology and broad-range host characteristics of P. capsici remain unknown. Here, we used transcriptomics and CRISPR-Cas9 technology to functionally characterize the contributions of a novel gene (PcTBCC1) coding for a hypothetical protein with a tubulin-binding cofactor C domain with a putative chloroplast-targeting peptide (cTP) to the pathophysiological development of P. capsici. We observed significant upregulation in the expression of PcTBCC1 during pathogen–host interactions. However, the vegetative growth of the ∆Pctbcc1 strains was not significantly different from the wild-type strains. PcTBCC1 gene replacement significantly compromised the sporulation, pathogenic differentiation, and virulence of P. capsici. At the same time, ∆Pctbcc1 strains were sensitive to cell wall stress-inducing osmolytes. These observations, coupled with the close evolutionary ties between PcTBCC1 and pathogenic oomycetes and algae, partly support the notion that PcTBCC1 is a conserved determinant of pathogenesis. This study provides insights into the significance of tubulin-binding cofactors in P. capsici and underscores the potential of PcTbcc1 as a durable target for developing anti-oomycides to control phytopathogenic oomycetes. Full article

The success of the intracellular parasite Toxoplasma gondii in invading host cells relies on the apical complex, a specialized microtubule cytoskeleton structure associated with secretory organelles. The T. gondii genome encodes three isoforms of both α- and β-tubulin, which undergo specific post-translational modifications (PTMs), altering the biochemical and biophysical proprieties of microtubules and modulating their interaction with associated proteins. Tubulin PTMs represent a powerful and evolutionarily conserved mechanism for generating tubulin diversity, forming a biochemical ‘tubulin code’ interpretable by microtubule-interacting factors. T. gondii exhibits various tubulin PTMs, including α-tubulin acetylation, α-tubulin detyrosination, Δ5α-tubulin, Δ2α-tubulin, α- and β-tubulin polyglutamylation, and α- and $\beta$-tubulin methylation. Tubulin glutamylation emerges as a key player in microtubule remodeling in Toxoplasma, regulating stability, dynamics, interaction with motor proteins, and severing enzymes. The balance of tubulin glutamylation is maintained through the coordinated action of polyglutamylases and deglutamylating enzymes. This work reviews and discusses current knowledge on T. gondii tubulin glutamylation. Through in silico identification of protein orthologs, we update the recognition of putative proteins related to glutamylation, contributing to a deeper understanding of its role in T. gondii biology. Full article

The majority of hematopoietic cancers in adults are incurable and exhibit unpredictable remitting-relapsing patterns in response to various therapies. The proto-oncogene c-MYC has been associated with tumorigenesis, especially in hematological neoplasms. Therefore, targeting c-MYC is crucial to find effective, novel treatments for blood malignancies. To date, there are no clinically approved c-MYC inhibitors. In this study, we virtually screened 1578 Food and Drug Administration (FDA)-approved drugs from the ZINC15 database against c-MYC. The top 117 compounds from PyRx-based screening with the best binding affinities to c-MYC were subjected to molecular docking studies with AutoDock 4.2.6. Retinoids consist of synthetic and natural vitamin A derivatives. All-trans-retinoic acid (ATRA) were highly effective in hematological malignancies. In this study, adapalene, a third-generation retinoid usually used to treat acne vulgaris, was selected as a potent c-MYC inhibitor as it robustly bound to c-MYC with a lowest binding energy (LBE) of −7.27 kcal/mol, a predicted inhibition constant (pKi) of 4.69 µM, and a dissociation constant (K_d value) of 3.05 µM. Thus, we examined its impact on multiple myeloma (MM) cells in vitro and evaluated its efficiency in vivo using a xenograft tumor zebrafish model. We demonstrated that adapalene exerted substantial cytotoxicity against a panel of nine MM and two leukemic cell lines, with AMO1 cells being the most susceptible one (IC₅₀ = 1.76 ± 0.39 µM) and, hence, the focus of this work. Adapalene (0.5 × IC₅₀, 1 × IC₅₀, 2 × IC₅₀) decreased c-MYC expression and transcriptional activity in AMO1 cells in a dose-dependent manner. An examination of the cell cycle revealed that adapalene halted the cells in the G₂/M phase and increased the portion of cells in the sub-G₀/G₁ phase after 48 and 72 h, indicating that cells failed to initiate mitosis, and consequently, cell death was triggered. Adapalene also increased the number of p-H3(Ser10) positive AMO1 cells, which is a further proof of its ability to prevent mitotic exit. Confocal imaging demonstrated that adapalene destroyed the tubulin network of U2OS cells stably transfected with a cDNA coding for α-tubulin-GFP, refraining the migration of malignant cells. Furthermore, adapalene induced DNA damage in AMO1 cells. It also induced apoptosis and autophagy, as demonstrated by flow cytometry and western blotting. Finally, adapalene impeded tumor growth in a xenograft tumor zebrafish model. In summary, the discovery of the vitamin A derivative adapalene as a c-MYC inhibitor reveals its potential as an avant-garde treatment for MM. Full article

Microtubules (MTs), dynamic polymers of α/β-tubulin heterodimers found in all eukaryotes, are involved in cytoplasm spatial organization, intracellular transport, cell polarity, migration and division, and in cilia biology. MTs functional diversity depends on the differential expression of distinct tubulin isotypes and is amplified by a vast number of different post-translational modifications (PTMs). The addition/removal of PTMs to α- or β-tubulins is catalyzed by specific enzymes and allows combinatory patterns largely enriching the distinct biochemical and biophysical properties of MTs, creating a code read by distinct proteins, including microtubule-associated proteins (MAPs), which allow cellular responses. This review is focused on tubulin-acetylation, whose cellular roles continue to generate debate. We travel through the experimental data pointing to α-tubulin Lys40 acetylation role as being a MT stabilizer and a typical PTM of long lived MTs, to the most recent data, suggesting that Lys40 acetylation enhances MT flexibility and alters the mechanical properties of MTs, preventing MTs from mechanical aging characterized by structural damage. Additionally, we discuss the regulation of tubulin acetyltransferases/desacetylases and their impacts on cell physiology. Finally, we analyze how changes in MT acetylation levels have been found to be a general response to stress and how they are associated with several human pathologies. Full article

In cells, microtubule location, length, and dynamics are regulated by a host of microtubule-associated proteins and enzymes that read where to bind and act based on the microtubule “tubulin code,” which is predominantly encoded in the tubulin carboxy-terminal tail (CTT). Katanin is a highly conserved AAA ATPase enzyme that binds to the tubulin CTTs to remove dimers and sever microtubules. We have previously demonstrated that short CTT peptides are able to inhibit katanin severing. Here, we examine the effects of CTT sequences on this inhibition activity. Specifically, we examine CTT sequences found in nature, alpha1A (TUBA1A), detyrosinated alpha1A, Δ2 alpha1A, beta5 (TUBB/TUBB5), beta2a (TUBB2A), beta3 (TUBB3), and beta4b (TUBB4b). We find that these natural CTTs have distinct abilities to inhibit, most noticeably beta3 CTT cannot inhibit katanin. Two non-native CTT tail constructs are also unable to inhibit, despite having 94% sequence identity with alpha1 or beta5 sequences. Surprisingly, we demonstrate that poly-E and poly-D peptides are capable of inhibiting katanin significantly. An analysis of the hydrophobicity of the CTT constructs indicates that more hydrophobic polypeptides are less inhibitory than more polar polypeptides. These experiments not only demonstrate inhibition, but also likely interaction and targeting of katanin to these various CTTs when they are part of a polymerized microtubule filament. Full article

Bruchids are the most important pests of leguminous seeds in the world. In this study, the focus was done on Callosobruchus maculatus, a serious pest of Vigna unguiculata seeds. As no efficient control methods preventing collateral effects on beneficials currently exist, this study investigated whether RNA interference (RNAi) could provide a new biotechnological and selective tool for bruchids control. Three principal objectives were followed including (i) the identification of all RNAi machinery core components and a key protein to silence in C. maculatus genome (c.f., dicer-2, argonaute-2, R2D2, and laccase 1), (ii) the identification of suitable reference gene for RT-qPCR analyses, and (iii) the micro-injection of dsRNA coding for laccase 1 to adults of C. maculatus to assess gene expression levels by RT-qPCR and potentially related mortalities. Phylogenetical analyses performed from transcriptomic information successfully identified all necessary proteins in the RNAi mechanism and also the open reading frame of laccase 1 in C. maculatus. A new reference gene was identified (i.e., alpha-tubuline 1) and coupled with glutiathone S transferase for RT-qPCR analyses. Double-stranded RNAs coding for laccase 1 and green fluorescent protein (control) were produced and 400 ng of each dsRNA were micro-injected into C. maculatus adults. RT-qPCR analyses revealed a stable significant decrease in laccase 1 expression in about 80% of adults treated with laccase 1 dsRNA after three days post-injection. No significant mortalities were observed which is probably related to the non-exposure of adults to anti-nutritional factors that are usually regulated by laccase. Further research should focus either on the feeding larval stage which is directly exposed to anti-nutritional factors, or on other target genes to induce dead phenotypes. This study is the first gene silencing report on a bruchid species and supports RNAi as a potential future method of control. Full article

Microtubules (MTs) are dynamic components of the cell cytoskeleton involved in several cellular functions, such as structural support, migration and intracellular trafficking. Despite their high similarity, MTs have functional heterogeneity that is generated by the incorporation into the MT lattice of different tubulin gene products and by their post-translational modifications (PTMs). Such regulations, besides modulating the tubulin composition of MTs, create on their surface a “biochemical code” that is translated, through the action of protein effectors, into specific MT-based functions. This code, known as “tubulin code”, plays an important role in neuronal cells, whose highly specialized morphologies and activities depend on the correct functioning of the MT cytoskeleton and on its interplay with a myriad of MT-interacting proteins. In recent years, a growing number of mutations in genes encoding for tubulins, MT-interacting proteins and enzymes that post-translationally modify MTs, which are the main players of the tubulin code, have been linked to neurodegenerative processes or abnormalities in neural migration, differentiation and connectivity. Nevertheless, the exact molecular mechanisms through which the cell writes and, downstream, MT-interacting proteins decipher the tubulin code are still largely uncharted. The purpose of this review is to describe the molecular determinants and the readout mechanisms of the tubulin code, and briefly elucidate how they coordinate MT behavior during critical neuronal events, such as neuron migration, maturation and axonal transport. Full article

Background: Twelve separate streams of empirical data make a strong case for cannabis-induced accelerated aging including hormonal, mitochondriopathic, cardiovascular, hepatotoxic, immunological, genotoxic, epigenotoxic, disruption of chromosomal physiology, congenital anomalies, cancers including inheritable tumorigenesis, telomerase inhibition and elevated mortality. Methods: Results from a recently published longitudinal epigenomic screen were analyzed with regard to the results of recent large epidemiological studies of the causal impacts of cannabis. We also integrate theoretical syntheses with prior studies into these combined epigenomic and epidemiological results. Results: Cannabis dependence not only recapitulates many of the key features of aging, but is characterized by both age-defining and age-generating illnesses including immunomodulation, hepatic inflammation, many psychiatric syndromes with a neuroinflammatory basis, genotoxicity and epigenotoxicity. DNA breaks, chromosomal breakage-fusion-bridge morphologies and likely cycles, and altered intergenerational DNA methylation and disruption of both the histone and tubulin codes in the context of increased clinical congenital anomalies, cancers and heritable tumors imply widespread disruption of the genome and epigenome. Modern epigenomic clocks indicate that, in cannabis-dependent patients, cannabis advances cellular DNA methylation age by 25–30% at age 30 years. Data have implications not only for somatic but also stem cell and germ line tissues including post-fertilization zygotes. This effect is likely increases with the square of chronological age. Conclusion: Recent epigenomic studies of cannabis exposure provide many explanations for the broad spectrum of cannabis-related teratogenicity and carcinogenicity and appear to account for many epidemiologically observed findings. Further research is indicated on the role of cannabinoids in the aging process both developmentally and longitudinally, from stem cell to germ cell to blastocystoids to embryoid bodies and beyond. Full article

Cucurbitariaceae has a high biodiversity worldwide on various hosts and is distributed in tropical and temperate regions. Woody litters collected in Changchun, Jilin Province, China, revealed a distinct collection of fungi in the family Cucurbitariaceae based on morphological and molecular data. Phylogenetic analyses of the concatenated matrix of the internal transcribed spacer (ITS) region, the large subunit (LSU) of ribosomal DNA, the RNA polymerase II subunit (rpb2), the translation elongation factor 1-alpha (tef1-α) and β-tubulin (β-tub) genes indicated that the isolates represent Allocucurbitaria and Parafenestella species based on maximum likelihood (ML), maximum parsimony (MP) and Bayesian analysis (BPP). We report four novel species: Allocucurbitaria mori, Parafenestella changchunensis, P. ulmi and P. ulmicola. The importance of five DNA markers for species-level identification in Cucurbitariaceae was determined by Assemble Species by Automatic Partitioning (ASAP) analyses. The protein-coding gene β-tub is determined to be the best marker for species level identification in Cucurbitariaceae. Full article

Actin and tubulin proteins from Trichomonas vaginalis are crucial for morphogenesis and mitosis. This parasite has 10 and 11 genes coding bonafide actin and tubulin proteins, respectively. Hence, the goal of this work was to analyze these actin and tubulin genes, their expression at the mRNA and protein levels, and their parasite localization in intercellular interaction and cytokinesis. Representative bonafide actin (tvact1) and tubulin (tvtubα1) genes were cloned into and expressed in Escherichia coli. The recombinant proteins TvACT1r and TvTUBα1r were affinity purified and used as antigens to produce polyclonal antibodies. These antibodies were used in 1DE and 2DE WB and indirect immunofluorescence assays (IFA). By IFA, actin was detected as a ring on the periphery of ameboid, ovoid, and cold-induced cyst-like parasites, on pseudopods of amoeboid parasites, and in cytoplasmic extensions (filopodia) in cell–cell interactions. Tubulin was detected in the axostyle, flagellum, undulating membrane, and paradesmose during mitosis. Paradesmose was observed by IFA mainly during cytokinesis. By scanning electron microscopy, a tubulin-containing nanotubular structure similar to the tunneling nanotubes (TNTs) was also detected in the last stage of cytokinesis. In conclusion, actin and tubulin are multigene families differentially expressed that play important roles in intercellular interactions and cytokinesis. Full article

Essential cellular processes of microtubule disassembly and protein degradation, which span lengths from tens of $\mathsf{\mu }$m to nm, are mediated by specialized molecular machines with similar hexameric structure and function. Our molecular simulations at atomistic and coarse-grained scales show that both the microtubule-severing protein spastin and the caseinolytic protease ClpY, accomplish spectacular unfolding of their diverse substrates, a microtubule lattice and dihydrofolate reductase (DHFR), by taking advantage of mechanical anisotropy in these proteins. Unfolding of wild-type DHFR requires disruption of mechanically strong $\beta$-sheet interfaces near each terminal, which yields branched pathways associated with unzipping along soft directions and shearing along strong directions. By contrast, unfolding of circular permutant DHFR variants involves single pathways due to softer mechanical interfaces near terminals, but translocation hindrance can arise from mechanical resistance of partially unfolded intermediates stabilized by $\beta$-sheets. For spastin, optimal severing action initiated by pulling on a tubulin subunit is achieved through specific orientation of the machine versus the substrate (microtubule lattice). Moreover, changes in the strength of the interactions between spastin and a microtubule filament, which can be driven by the tubulin code, lead to drastically different outcomes for the integrity of the hexameric structure of the machine. Full article